Corning Storage Bottles Selection and Use Guide

Transcription

1 Corning Storage Bottles Selection and Use Guide Contents Glass Screw Cap Storage Bottles Plastic Screw Cap Storage Bottles Caps and Accessories Care and Use Glassware Safety Precautions Ordering Information

2 Corning Storage Bottles Corning offers a wide variety of glass and plastic screw cap bottles designed to meet all of your storage needs. This guide will help you select the best bottle to meet these needs. The following table shows the major differences between Corning s glass and plastic storage bottles. Comparison of Corning Glass and Plastic Storage Bottles Corning offers PYREX storage bottles in a wide variety of sizes, shapes and styles to meet all of your storage needs. Composition of Code 7740 Glass Chemicals % (approx.) SiO 2 81% B 2 O 3 13% Na 2 O 3% Al 2 O 3 2% K 2 O 1% Corning uses Code 7740 Type I, Class A borosilicate glass for all of its glass storage bottles. This glass conforms to federal specification DD-G-54 lb and ASTM E-438 (except for K 2 O content). This glass also meets the U.S. Pharmacopoeia specs for Type I Borosilicate Glass. PYREX and PYREXPLUS Glass Bottles Corning and Costar Plastic Bottles Reusable: easy to clean and sterilize; very durable Disposable: Sterile and ready to use; very for long life convenient, no washing or clean up required Excellent chemical resistance: good for acids, alkalis Lower chemical resistance: good for media and solvents but also media and aqueous solutions and aqueous solutions Excellent temperature resistance: bottles can be Lower temperature resistance: only repeatedly sterilized polycarbonate bottles can be autoclaved Nine sizes: 25 ml to 10L Five sizes: 125 ml to 1L Glass Screw Cap Storage Bottles PYREX reusable storage bottles are designed for heavy duty storage of reagents, sterile tissue culture media and sera, biological fluids and other aqueous and nonaqueous solutions. All PYREX borosilicate glass bottles offer: Code 7740 Type 1, Class A borosilicate glass for superior chemical and thermal resistance, able to withstand repeated sterilization cycles or freezing down to -70 C. Linerless, one-piece autoclavable polypropylene plug seal caps and drip-free pouring rings Permanent enamel marking spot and graduations A wide range of optional caps for venting and aseptic liquid collection or transfer see Caps and Accessories on page 5. Choice of traditional round storage bottles or space saving square storage bottles Comparison of PYREX and PYREXPLUS Glass Storage Bottles PYREX Round Clear PYREXPLUS PYREX PYREX Glass and Low Actinic Round Glass Square Glass Wide Mouth Storage Bottles Storage Bottles Storage Bottles Glass Storage Bottles Bottle Material Code 7740 Code 7740 borosilicate Code 7740 Code 7740 borosilicate glass glass with PVC coating borosilicate glass borosilicate glass Cap Color Orange Green Orange Orange Cap Material Polypropylene Polypropylene Polypropylene Polypropylene (Maximum. Temp.) (140º) (140º) (140º) (140º) Autoclavable ( 121ºC, 15 psi) Yes Yes Yes Yes Maximum Recommended 230º 80º 230º 230º Working Temp. ( C), Bottle only Minimum Recommended -70º* 10º* -70º* -70º* Working Temp. (ºC)* Use with Corning Recommended Recommended Not Recommended NA Vacuum Filters Available sizes: 25 ml, 50 ml, 100 ml, 100 ml, 250 ml, 100 ml, 250 ml, 500 ml, 1L & 2L 250 ml, 500 ml, 1L, 500 ml, 1L, 2L, 500 ml & 1L, 2L, 5L & 10L 5L & 10L * Bottle performance in freezers depends on both the temperature and contents in the bottle. It is strongly recommended that a trial run be performed under actual conditions to test the suitability of the bottles for frozen storage. Extreme service at 490 C. 2

3 Corning s round borosilicate glass storage bottles are available clear for general storage (left), with low actinic red stain for light sensitive reagents (middle), or with a tough PVC coating (PYREXPLUS, right) for extra layer of safety against breakage and spills. PYREX Round Glass Storage Bottles Available with either clear glass for routine storage applications or low actinic red glass for storing light sensitive materials Nine sizes to choose from: 25 ml, 50 ml, 100 ml, 250 ml, 500 ml, 1L, 2L, 5L and 10L 100 ml through 2L sizes can be used with Corning Bottle Top Vacuum Filters. Caution: Bottles larger than 2L should NOT be used with bottle top filter units, or in other applications involving vacuum pressure, as breakage may occur. PYREXPLUS Round Glass Storage Bottles Bottles have a protective PVC coating to help prevent glass from shattering and to reduce spills. Seven sizes to choose from: 100 ml, 250 ml, 500 ml, 1L, 2L, 5L and 10L. Autoclavable at 121 C and resistant to thermal shock. Caution: Do not place these bottles over direct heat or flame or heat above 121 C moist heat or 110 C dry heat. Come with autoclavable GL45 green polypropylene plug seal caps with drip-free pouring rings Teal enameled graduations and marking spot. Covered by U.S. Patent # 4,940,613. Ideal for use with Corning Bottle Top Vacuum Filters or other mild vacuum applications PYREX Square Glass Storage Bottles Easier to handle and require less space (13 to 20%) on the shelf or in the autoclave Four sizes to choose from: 100 ml, 250 ml, 500 ml and 1L. Caution: Square bottles should NOT be used with bottle top filter units, or in other applications involving vacuum pressure, as breakage may occur. Square PYREX storage bottles save on space in refrigerators and autoclaves. 3

4 Corning Storage Bottles PYREX wide mouth storage bottles have extra wide neck opening for use with funnels and for easier access and pouring. PYREX Wide Mouth Glass Storage Bottles Three sizes to choose from: 500 ml, 1L and 2L Neck opening is 5 times larger than on GL45 threaded bottles much easier to add or remove powders and easier to clean. Plastic Screw Cap Storage Bottles Corning s disposable polystyrene and polycarbonate storage bottles are designed for researchers who want to store sterile tissue culture media and sera, buffers, biological fluids and other aqueous solutions in convenient, disposable bottles All plastic storage bottles offer: Convenience sterile, ready to use with no clean up after 45 mm diameter caps provide an air tight seal and help minimize contamination and leaks Choice of three styles: Corning square polycarbonate bottles are break resistant and autoclavable or microwavable Corning easy grip round polystyrene bottles have a low profile for easier gripping and greater stability Costar traditional round bottles Corning offers round polystyrene storage bottles in two designs (above left and middle) as well as new square polycarbonate storage bottles (above right). 4

5 Comparison of Polystyrene and Polycarbonate Storage Bottles Costar Round Polystyrene Corning Easy Grip Round Corning Square Polycarbonate Storage Bottles Polystyrene Storage Bottles Storage Bottles Bottle Material Polystyrene Polystyrene Polycarbonate Cap Color Red Orange Orange Cap Material (Maximum High Density High Density Polypropylene Temperature, C) Polyethylene (121º) Polyethylene (121º) (140º) Autoclavable No No Yes, once (121ºC, 15 psi, 20 minutes) Maximum Recommended 70º 70º 130º Working Temperature (C) Minimum Recommended -20º* -20º* -80º* Working Temperature (C)* Use with Corning Recommended Recommended Not Recommended Vacuum Filters Available sizes: 125 ml, 250 ml, 500 ml 150 ml, 250 ml, 500 ml 150 ml, 250 ml, 500 ml and 1L and 1L and 1L Autoclaving will reduce mechanical strength. * Bottle performance in freezers depends on both the temperature and contents in the bottle. It is strongly recommended that a trial run be performed under actual conditions to test the suitability of the bottles for frozen storage. Caps and Accessories Corning offers a variety of cap colors and styles for PYREX storage bottles. Septum caps provide syringe access to solutions without sample contamination. Bottles with these autoclavable dispensing caps are ideal for aseptic liquid transfer and collection. All PYREX storage bottles come with polypropylene caps that are fully autoclavable at temperatures up to 140ºC. These one piece caps seal using an integral plug that is designed to mold against the inside rims of glass bottles. While these one piece caps also fit the plastic storage bottles, they will not provide a gas tight seal. Optional plug seal gray caps have a single vent in their centers consisting of a 0.22 µm PTFE membrane that is heat sealed on the inside to the cap. This membrane permits automatic gas exchange, allowing pressure equalization during autoclaving to insure sterility of solutions in the bottle. The hydrophobic PTFE membrane material will prevent liquids from wetting through. Optional red caps with polybutylterephthalate (PBT) coated rubber liners are available for applications requiring temperatures as high as 180ºC. These two piece caps use a traditional gasket design to seal against the rim. The optional red PBT high temperature pouring rings should always be used in conjunction with these caps for applications above 140º C. 5

6 Corning Storage Bottles Corning offers reusable PBT septum caps for storage bottles (not available for wide mouth bottles) with a choice of silicone septa or PTFE faced silicone septa. The replaceable septa are ordered separately. Dispensing caps (Corning Cat. No ) with two 15.4 cm stainless steel tube ports can easily convert storage bottles with GL45 threads for use in automated cell culture or assay systems requiring aseptic liquid dispensing or collection. These reusable caps can be used on all Corning glass and plastic bottles with 45 mm diameter necks. Care and Use Cleaning PYREX Storage Bottles Wash bottles as quickly as possible after use. Bottles contaminated with blood clots, culture media, etc., should be sterilized before cleaning. If a thorough cleaning is not immediately possible, then soak bottles in water. If bottles are not cleaned immediately, it may become difficult or impossible to remove the material later. Place the glassware in a large container filled with water for soaking. The bottles should then be rinsed in tap water, scrubbed with detergent and rinsed again. Most new glassware is slightly alkaline in reaction. For critical cell culture work, new bottles should be soaked several hours in acid water (a 1% solution of hydrochloric or nitric acid) before washing. Rinsing After cleaning, rinse glass bottles with running tap water. Allow the water to run into and over them for a short time, then partly fill each piece with water, thoroughly shake and empty at least six times. The final rinse should be done using deionized, distilled or other source of good quality water. PYREXPLUS Labware Use and Care Do not place PYREXPLUS labware over direct heat or open flame. The recommended temperature use range for PYREXPLUS labware is 10º C to 80º C. Do not continuously expose PYREXPLUS labware to heat above 80º C. Do not expose to dry heat in a dishwasher above 110ºC (230ºF). Drying time should not exceed 15 minutes at 110ºC (230ºF). Do not steam autoclave above 121ºC (250ºF). Sterilizing time should not exceed 15 minutes. Do not refrigerate below -20ºC (-4ºF). Do not remove the protective coating. Do not use a vessel on which the coating is hardened, darkened or otherwise damaged. Do not allow prolonged or repeated exposure of the coating to strong acids or solvents. Do not use a vessel once the glass is broken. Immediately transfer the contents of a broken vessel to an approved container and properly dispose of the broken vessel. Do not incinerate discarded vessels. Place in proper disposal containers. Proper care and handling of PYREX and PYREXPLUS labware will greatly increase its life and increase the safety of your workplace. Visit for additional information on the use and care of your PYREX and PYREXPLUS glassware. 6

7 Glassware Safety Precautions These products are intended for use by persons knowledgeable in safe laboratory practices. Failure can result from surface damage, improper pressure or temperature, or use with incompatible chemicals. WARNING: To avoid breakage and possible injury, Corning recommends the following: Do not heat glassware that is scratched, etched, cracked or nicked, it will be prone to breakage. Do not use any implement inside a glass vessel that can scratch or chip the glass. Do not use PYREX or PYREXPLUS bottles over direct heat such as a flame or on a hot plate. Do not put hot bottles onto cold or wet surfaces, or cold bottles onto hot surfaces. The bottles may break due to the thermal shock caused by the temperature change. Extra care to prevent thermal shock must be used when removing bottles from ultra low temperature freezers (-70º to -135ºC). Immediately rinse the entire bottle under cold running water until thawing begins. Do not place bottles directly from the freezer into warm water baths. Because of variations in conditions, Corning cannot guarantee glassware against breakage and personal injury in vacuum or pressure applications. Do not use reagent bottles larger than 1L or laboratory bottles larger than 2L with bottle top filter units, or in vacuum applications. Do not use any bottle in pressure applications. Adequate safety precautions should always be taken when handling or using glassware to protect against breakage and personal injury. Bottles should NOT have caps tightened immediately after autoclaving because the vacuum resulting from cooling can cause breakage. This statement does not apply to the vented cap ( LTMC). The vented cap (Cat. No LTMC) is NOT recommended for use on the 5L and 10L bottles and 1397 SERIES BOTTLES ARE NOT RECOMMENDED FOR VACUUM USE. This physical properties chart is intended to be used as a general guide only. The mechanical strength, color, appearance and dimensional stability of plastic labware are affected to varying degrees by the chemicals they contact. Specific operating conditions, especially temperature, will also affect the chemical resistance. PYREXPLUS media bottles (Cat. No series) have a protective plastic coating for extra protection in vacuum applications and can be autoclaved. PYREX Media Bottles and Vacuum Safety Round PYREX media storage bottles (Cat. No series), 2L and smaller, are ideal for room temperature vacuum filtration with Corning Bottle Top Filters or aspiration applications. For additional protection during vacuum application PYREXPLUS media bottles with a protective PVC coating (Cat. No series) are highly recommended. Square PYREX media storage bottles (Cat. No series) should not be used with bottle top filter units, or in other applications involving vacuum, as breakage may occur because of their shape. Because of variations in conditions or damage from usage, such as scratches or uneven heating, Corning cannot guarantee any glassware against breakage under vacuum or pressure. All glass containers used in vacuum work must be securely and adequately taped or shielded to restrain flying glass in the event of an implosion. Adequate precautions should be taken to protect personnel doing such work including using gloves and safety glasses, goggles, or a face shield. For additional safety information visit 7

8 Corning Storage Bottles Physical properties of Corning Glass and Plastic Bottles C H E M I C A L R E S I S T A N C E Thermal Recommended Not Recommended Sterilization Durability PYREX Glass Almost all chemicals Hydrofluoric acid, hot Autoclave or 490 C* Bottles phosphoric acid, and dry heat strong, hot alkali PYREXPLUS Weak acids, strong alkali, oils, Hydrofluoric acid, hot Autoclave 110 C Glass Bottles, alcohols phosphoric acid, strong, hot PVC coated alkali, aldehydes, ketones, strong acids, and chlorinated solvents Polycarbonate Weak acids, weak alkali, alcohols Strong acids, strong alkali, Autoclave once 135 C Plastic Bottles ketones, aliphatic, aromatic and chlorinated hydrocarbons Polystyrene Weak acids, strong alkali, alcohols Strong acids, ketones, Do not autoclave 80 C Plastic Bottles aliphatic, aromatic and chlorinated hydrocarbons Polypropylene Acids, alkali, solvents, oils alcohols Aromatic and chlorinated Autoclave 140ºC Caps & Rings hydrocarbons, oxidizing acids Polyethylene Acids, alkali, aldehydes, solvents, Aliphatic, aromatic, and Do not autoclave 121ºC Caps alcohol, oils chlorinated hydrocarbons, oxidizing acids PBT Caps and Weak acids, weak alkali, alcohols, Strong acids, strong alkali, Autoclave or 180ºC ETFE Rings esters, ether, benzene, mineral oil and ketones dry heat Sterilization time should not exceed 15 minutes at 121 C (250 F). Drying time should not exceed 15 minutes at 110 C (230 F). *Thermal up shock or down shock may cause breakage. Ordering Information Glass Bottles 1395 Laboratory Storage Bottle Capacity Thread Approx. Diameter Grad. Range Grad. Interval Qty/ Cat. No. (ml) Size x Height (mm) (ml) (ml) Cs GL x GL32 46 x GL45 56 x GL45 70 x GL45 86 x L 1000 GL x L 2000 GL x L 5000 GL x L GL x Square Laboratory Storage Bottle Capacity Thread Approx. Diameter Grad. Range Grad. Interval Qty/ Cat. No. (ml) Size x Height (mm) (ml) (ml) Cs GL32 50 x GL45 64 x GL45 78 x L 1000 GL45 94 x

9 Ordering Information (continued) 1397 GLS 80 Wide Mouth Bottle Approx. Grad. Grad. Capacity Thread Diameter x Range Interval Qty/ Cat. No. (ml) Size Height (mm) (ml) (ml) Cs GLS x L 1000 GLS x L 2000 GLS x Low Actinic Bottle Approx. Grad. Grad. Capacity Thread Diameter x Range Interval Qty/ Cat. No. (ml) Size Height (mm) (ml) (ml) Cs GL x GL32 46 x GL45 56 x GL45 70 x GL45 86 x L 1000 GL x L 2000 GL x L 5000 GL x L 10,000 GL x PYREXPLUS Bottle Capacity Thread Approx. Diameter Grad. Range Grad. Interval Qty/ Cat. No. (ml) Size x Height (mm) (ml) (ml) Cs GL45 56 x GL45 70 x GL45 86 x L 1000 GL x L 2000 GL x Plastic Storage Bottles Capacity Neck Dia. Qty/ Qty/ Cat. No. (ml) Shape Bottle Material (mm) Bag Cs Square Polycarbonate Square Polycarbonate Square Polycarbonate Square Polycarbonate Easy Grip Round Polystyrene Easy Grip Round Polystyrene Easy Grip Round Polystyrene Easy Grip Round Polystyrene Round Polystyrene Round Polystyrene Round Polystyrene Round Polystyrene

10 Corning Storage Bottles Ordering Information (continued) Caps and Accessories Thread Cat. No Description Color Material Fits Seal Qty/Cs HTSC Open Top Cap Red PBT GL25 Requires Septa SS Silicone Septa White Silicone GL25 Requires Cap TS PTFE Faced White Silicone/ GL25 Requires Cap 10 Silicone Septa PTFE LTC Cap Orange PP GL32 Plug Seal HTSC Open Top Cap Red PBT GL32 Requires Septa SS Silicone Septa White Silicone GL32 Requires Cap TS PTFE Faced White Silicone/ GL32 Requires Cap 10 Silicone Septa PTFE LTR Pouring Ring Clear PP GL32 NA LTC Cap Orange PP GL45 Plug Seal LTC1 Cap Purple PP GL45 Plug Seal LTC2 Cap Light Gray PP GL45 Plug Seal LTC3 Cap Green PP GL45 Plug Seal LTMC Vented Cap with Gray PP GL45 Plug Seal µm Pore Hydrophobic PTFE Membrane HTSC* High Temperature Red PBT GL45 PTFE/ 10 Cap Silicone Liner * Cap with Orange PP GL45 Rim Seal cm SS Tube Ports HTSC* Open Top Cap Red PBT GL45 Requires Septa SS* Silicone Septa White Silicone GL45 Requires Cap TS* PTFE Faced White Silicone/ GL45 Requires Cap 10 Silicone Septa PTFE LTR Pouring Ring Clear PP GL45 NA HTR High Temp. Red ETFE GL45 NA 50 Pouring Ring LTC Cap Orange PP GLS80 Plug Seal 10 PP = Polypropylene, PBT = Polybutylterephthalate, PTFE = Polytetrafluoroethylene, ETFE = Ethylenetetrafluoroethylene, SS = Stainless Steel *Can be used with Corning and Costar plastic storage bottles with 45mm necks. 10

11 Bottle Top Vacuum Filters Individually packaged, sterile and certified nonpyrogenic Adaptors are color coded by membrane type Fit most glass and plastic media storage bottles with GL45 caps Do not use on square glass bottles or glass bottles larger than 2L Volume Neck Size Pore Size Color-Coded Cat. No. Membrane (ml) (mm) (µm) Adapter Qty/Cs 150 ml Capacity, 50 mm Diameter Membrane CA Orange CA Orange PES Yellow ml Capacity, 70 mm Diameter Membrane NY Red CA Orange CA Orange PES Yellow 12 1,000 ml Capacity, 90 mm Diameter Membrane CA 1, Orange PES 1, Yellow 12 PES = Polyethersulfone, CA = Cellulose Acetate, CN = Cellulose Nitrate, NY = Nylon. 11

12 Corning Filter Selection and Use Guide Innovative Products for Filtration Choosing a filter doesn t have to be complicated. Corning has simplified the process. Just follow these four easy steps: Step 1: Match your application with the best pore size. Step 2: Select the best membrane and housing material for your application. Filter membrane characteristics Filter housing characteristics Chemical compatibility Step 3: Select the correct membrane diameter to optimize flow rate and throughput. Step 4: Choose the best filter design for your application. Disposable syringe/disc filters Disposable plastic vacuum filters Reusable glass vacuum filters Spin-X disposable centrifuge filters Using a filter should not be difficult either. Corning has included helpful hints to help you improve your filter performance: Effect of pore size Effect of membrane area Effect of temperature Effect of prefiltration Effect of pressure differential Safety Precautions References Step 1: Match your application with the best pore size. The pore size is usually determined by your application or objective. Routine laboratory sterilization of most media, buffers, biological fluids and gases is usually done with 0.2 or 0.22 µm pore filter membranes. Clarification and prefiltration of solutions and solvents is best accomplished with 0.45 µm or larger filter membranes. Prefiltration to improve filter performance can also be accomplished by the use of glass fiber prefilters supplied with some Corning filter units. Use Table 1 to match your applications with a recommended membrane and pore size. Table 1. Selecting the Pore Size Application Pore Size (µm) Membrane Availability Sterilization and Ultracleaning 0.20 to 0.22 All Membranes except Teflon of Aqueous Solutions Ultracleaning of Solvents (HPLC) 0.20 to 0.22 RC,* Teflon, Nylon Clarification of Aqueous Solutions 0.45 All Membranes except Teflon Clarification of Solvents (HPLC) 0.45 RC, Teflon, Nylon Coarse Particle Removal 0.8 SFCA,* glass fiber prefilters *RC = Regenerated Cellulose; SFCA = Surfactant-Free Cellulose Acetate 1

13 Percent µg/cm 2 ml/min/cm 2 ml/min/cm Flow Rate - High Purity Water 0 Corning New PES Corning CA Corning CN Membrane Corning New PES Corning CA Corning CN Membrane 1.80 Corning NY Flow Rate - 10% Calf Serum in MEM Percent Water - Extractable Material Corning New PES Corning CA Corning CN Membrane lgg BSA Corning New PES Corning CA Corning CN Membrane Corning NY Corning NY Relative Protein Binding Corning NY PES: polyethersulfone; CA: cellulose acetate; CN: cellulose nitrate; NY: nylon Figure 1. Important Performance Characteristics of Corning Filter Membranes Step 2: Select the best membrane and housing material for your application. Corning Filter Membranes Your filter unit must be fully compatible with the chemical characteristics of your sample. Some filter membranes contain non-toxic wetting agents that may interfere with some applications. Other membranes may bind proteins or other macromolecules leading to premature filter clogging or loss of valuable samples. Therefore, it is very important to understand their characteristics and the potential effects filter membranes can have on the solutions they contact. The four graphs ( Figure 1, left) compare the flow rates, levels of extractable materials, and relative amounts of protein binding of four of the most popular membranes used in Corning filters. Combining this with the information from Tables 2 and 3 will help you choose the best Corning membranes for your applications: Table 2. Characteristics of Corning Filter Membranes Cellulose Cellulose Polyether- Regenerated Teflon Nitrate Acetate Nylon sulfone cellulose (PTFE) Wetting Yes Yes No, naturally No Yes Does Agents hydrophilic not wet Protein Very High Very Low Low to Very low Low NA Binding Moderate DNA High Very low Very high Very low Low NA Binding Chemical Low Low Moderate Low Very high Very high Resistance to high Cellulose acetate (CA) membranes have a very low binding affinity for most macromolecules and are especially recommended for applications requiring low protein binding, such as filtering culture media containing sera. However, both cellulose acetate and cellulose nitrate membranes are naturally hydrophobic and have small amounts (less than 1%) of non-toxic wetting agents added during manufacture to ensure proper wetting of the membrane. If desired, these agents can be easily removed prior to use by filtering a small amount of warm purified water through the membrane or filter unit. Surfactant free cellulose acetate membranes, with very low levels of extractables, are available on some Corning syringe filters. Cellulose nitrate (CN) membranes are recommended for filtering solutions where protein binding is not a concern. They are recommended for use in general laboratory applications such as buffer filtration. Corning s cellulose nitrate membranes are Triton X-100-free and noncytotoxic. Nylon membranes are naturally hydrophilic and are recommended for applications requiring very low extractables since they do not contain any wetting agents, detergents or surfactants. Their greater chemical resistance makes them better for filtering more aggressive solutions, such as alcohols and DMSO. However, like cellulose nitrate membranes, they may bind greater amounts of proteins and other macromolecules than do the cellulose acetate or PES membranes. They are recommended for filtering protein-free culture media. Polyethersulfone (PES) membranes are highly recommended for filtering cell culture media. PES has both very low protein binding and extractables. PES also demonstrates faster flow rates than cellulosic or nylon membranes. Regenerated cellulose (RC) membranes are hydrophilic and have very good chemical resistance to solvents, including DMSO. They are used to ultraclean and de-gas solvents and mobile phases used in HPLC applications. Teflon (PTFE; polytetrafluorethylene) membranes are naturally and permanently hydrophobic. They are ideal for filtering gases, including humidified air. The 2

14 extreme chemical resistance of Teflon membranes makes them very useful for filtering solvents or other aggressive chemicals for which other membranes are unsuitable. Because of their hydrophobicity, Teflon membranes must be prewetted with a solvent, such as ethanol, before aqueous solutions can be filtered. Glass fiber filters are used a depth filter for prefiltration of solutions. They have very high particle loading capacity and are ideal for prefiltering dirty solutions and difficult to filters biological fluids such as sera. Corning Filter Housing Materials The filter housing materials, as well as the filter membrane must be compatible with the solutions being filters Polystyrene (PS) is used in the filter funnels and storage bottles for all of the Corning plastic vacuum filters. This plastic polymer should only be used in filtering and storing nonaggressive aqueous solutions and biological fluids. Refer to Table 3 for more chemical compatibility information. Acrylic copolymer (AC) and Polyvinyl chloride (PVC) are used in some of the Corning syringe filter housings. These plastics should only be used in filtering nonaggressive aqueous solutions and biological fluids. Refer to Table 3 for more chemical compatibility information. Polypropylene (PP) is used in the Spin-X centrifuge filters and some of the syringe and disc filter housings. This plastic polymer has very good resistance to many solvents, refer to Table 3 for more chemical compatibility information. Borosilicate glass (Pyrex and PyrexPlus brand) is very resistance to virtually all laboratory solvents. However, it can be attacked by hydrofluoric or hot phosphoric acid etched by hot alkali. The coating of PyrexPlus brand labware is designed to resist leakage resulting from a brief chemical exposure that might occur if the vessel is broken. Prolonged and/or repeated chemical exposure of the coating to aldehydes, ketones, chlorinated solvents and concentrated acids should be avoided. Chemical Compatibility The mechanical strength, color, appearance, and dimensional stability of Corning filters are affected to varying degrees by the chemicals with which they come into contact. Specific operating conditions, especially temperature and length of exposure, will also affect their chemical resistance. Table 3 provides a general Guideline for the chemical resistance of Corning filter membranes and housings. Table 3. Chemical Resistance Guide for Corning Filters* Filter Membranes Housing materials Chemical Class CN CA NY PES RC PTFE PS PP AC PYR PVC Weak Acids Strong Acids Alcohols Aldehydes Aliphatic Amines Aromatic Amines Bases Esters Hydrocarbons Ketones Key: 1 = Recommended; 2 = May be suitable for some applications, a trial run is recommended; 3 = Not recommended; CN = cellulose nitrate; CA = cellulose acetate; NY = nylon; PYR = Pyrex Glass; PES = polyethersulfone; RC = regenerated cellulose; PS = polystyrene; PTFE = polytetrafluorethylene (Teflon); PP = polypropylene; PVC = polyvinylchlorides; AC = Acrylic copolymer. 3 * This information has been developed from a combination of laboratory tests, technical publications, or material suppliers. It is believed to be reliable. Due to conditions outside of Corning s control, such as variability in temperatures, concentrations, duration of exposure and storage conditions, no warranty is given or is to be implied with respect to this information.

15 Step 3: Select the correct membrane diameter to optimize flow rate and throughput. The third step is selecting a filter that will have enough volume capacity or throughput to process your entire sample quickly and efficiently. This is primarily determined by the effective surface area of the membrane. Table 4 shows the relationship between filter diameter, effective filtration surface area and expected throughput volumes. The lower values are typical of viscous or particle-laden solutions; the higher values are typical of buffers or serum-free medium. Table 4. Typical Expected Throughput Volumes Filter Diameter Effective Filter Expected and Description Area (cm 2 ) Throughput (ml)* 4 mm syringe/disc mm syringe/disc µstar syringe filter mm syringe/disc mm syringe/disc mm disc mm vacuum system mm vacuum system mm vacuum system mm vacuum system *These values assume an aqueous solution and a 0.2 micron membrane. Solutions containing sera or other proteinaceous materials will be at the lower end of the range. Use of prefilters may extend the throughput % above the values shown. Step 4: Choose the best filter design for your application. Corning offers three basic filter types: positive pressure-driven syringe and disc filters, Spin-X centrifuge tube filters driven by centrifugation, and vacuum-driven filters. The vacuum driven filters offer several different designs and styles in both reusable glass and disposable plastic products Corning Syringe Filters µstar Syringe Filters Syringe/Disc filters The smaller conventional Corning syringe disc-type filters (4, 15, and 25-26mm diameter) are used with syringes which serves as both the fluid reservoir and the pressure source. They are 100% integrity tested. The HPLC certified nonsterile syringe filters are available with nylon, regenerated cellulose or Teflon (PTFE) membranes in polypropylene housing for extra chemical resistance. The sterile tissue culture tested syringe filters are available in PES, regenerated cellulose (ideal for use with DMSO-containing solutions) or surfactant-free cellulose acetate membranes in either polypropylene or acrylic copolymer housings. The larger 50mm diameter disc filter has a Teflon (PTFE) membrane and polypropylene housing with hose barb connectors. This product is ideal for filtering aggressive solvents or gases and applications requiring sterile venting of gases. Because they have a hydrophobic (will not pass aqueous solutions) membrane, they are also ideal for protecting vacuum lines and pumps. In addition, the unique µstar syringe filters have a bi-directional flow pattern that eliminates the priming and air lock effects of conventional syringe tip filters. These filters have either cellulose nitrate or cellulose acetate filters in a PVC housing. µstar filters utilize all of the membrane surface area for a working volume up to 100 ml while minimizing fluid retention to less than 30 µl with an air purge. Integrity tested with a maximum operating pressure of 100 psi, the µstar filter is designed for sterilization and clarification of aqueous solutions including: media additives, serum, antibiotics, biological fluids, and virus suspensions. 4

16 Corning Disposable Plastic Vacuum Filters These sterile filters are available in four styles: complete filter/storage systems, bottle top filters, centrifuge tube top filters, or the new one-piece filter systems. Corning filters feature adapters that are color-coded by membrane type for easy product identification. Angled hose connector simplifies vacuum line attachment. Four membranes are available to meet all of your filtration needs: cellulose acetate, cellulose nitrate, nylon, or polyethersulfone. Corning filter/storage systems consist of a polystyrene filter funnel joined by an adapter ring to a removable polystyrene storage bottle with a separate sterile polyethylene cap. Receiver bottles feature easy grip sides for improved handling. Additional Corning polystyrene receiver/storage bottles can be ordered separately to increase throughput. Corning Filter/Storage Systems Corning Bottle Top Filters Corning 115 ml One-piece Vacuum Filter Corning 150 ml Centrifuge Tube Top Filter Corning bottle top filters have the same polystyrene filter funnel designs and capacities as the filter systems, but the adapter ring is designed for threading onto a glass bottle supplied by the user. Select either the 33 mm thread design for standard narrow glass mouth media bottles or the 45 mm design for Pyrex Media Bottles or PyrexPlus Media bottles. See Safety Precautions (below) for recommendations on using these products with glass bottles. 150 ml centrifuge tube top filters feature a 150 ml polystyrene filter funnel with a 50 mm diameter cellulose acetate membrane attached to a 50mL polypropylene centrifuge tube to minimize unnecessary transfers by filtering directly into centrifuge tube. 115 ml one-piece vacuum filters are completely self-contained with a separate pour spout for convenience. They are available with either cellulose acetate or nylon filter membranes in a polystyrene housing. They have a low center of gravity and wide base for stability; separate pour spout to remove the filtrate minimizing chances for contamination. 5

17 BP 1760 BP 1755 BP 1825 Corning Filterware Reusable Glass Vacuum Filters Corning Filterware reusable glass vacuum filters are available with a choice of 47 mm or 90 mm fritted glass filters in an all-glass filter apparatus with a 40/35 glass joint connecting the filter support base to the filter flask. These units are ideal for: HPLC Mobile Phase Filtration Analysis of Particulate or Bacterial Contamination General Laboratory Microfiltration Similar Corning Filterware reusable glass vacuum filters that attach to standard glass filter flasks using a rubber stopper instead of a ground glass joint are also available with either 47 mm or 90 mm diameter fritted glass filters. Pyrex Glass Filter Vacuum flasks for use with the above Corning Filterware fritted glass filters are available in two styles: standard Pyrex filter flasks are blown in special molds, in shapes designed to give maximum mechanical strength; or PyrexPlus filter flasks that features a protective PVC coating for longer product life and safety. The protective coating helps prevent glass from shattering and reduces spills and is autoclavable (121 C) and resistant to thermal shock. Corning Spin-X Centrifuge Tube Filters Spin-X centrifuge tube filters consist of a membrane-containing (either cellulose acetate or nylon) filter unit within a polypropylene centrifuge tube. They filter small sample volumes by centrifugation for bacteria removal, particle removal, HPLC sample preparation, removal of cells from media and purification of DNA from agarose and polyacrylamide gels. (See Corning Technical Bulletin: Spin-X purification of DNA from agarose gels.) Corning Spin-X Centrifuge Tube Filters Improving Filter Performance Getting the best performance from your filtration products requires two very important steps: selecting the right products for the job, and then using these products effectively. The first part of this brochure covers the steps required to select the right filter for your applications; this section will help you optimize the filtration process by keying on the two most important areas: maximizing filter flow rate and throughput or capacity. The flow rate and throughput of filters are dependent on many variables. Some variables, such as temperature, pressure, and especially, the characteristics of the sample, require special attention. 6

18 Table 5. Corning Filter Designs Filter Available Diameters Membrane Pore Design Sterile (mm) materials Sizes (µm) Special Features Syringe Filters Some 4, 15, 25, RC, PES, SFCA, 0.2, 0.45, Ideal for small volume pressure filtration and 26 NY, & PTFE and 0.8 µstar Syringe Yes Not CA and CN 0.22, 0.45, Ideal for sterilizing aqueous solutions and Filters applicable and 0.8 biological fluids Disc filters Yes 50 PTFE 0.2 Ideal for filtering solvents and gases Vacuum Filter Yes 50, 70 PES, CA, CN, 0.2 (NY only) Easy grip bottles for storing filtrate Storage Systems and 90 and Nylon 0.22 and 0.45 (PES, CA, CN) Bottle Top Yes 50, 70 PES, CA, CN, 0.2 (NY only) Two neck widths to fit most glass bottles Vacuum Filters and 90 and Nylon 0.22 and 0.45 (PES, CA, CN) Tube Top Yes 50 CA 0.22 and 0.45 Minimizes unnecessary transfers by Vacuum Filters filtering into a 50 ml centrifuge tube One piece Yes 60 CA and CN 0.22 and 0.45 Very economical with separate pour spout Vacuum Filters Spin-X Some 7.7 CA and Nylon 0.22 and 0.45 Ideal for purifying DNA from agarose gels Centrifuge Filters Glass Vacuum No 47 and 90 Fritted glass Not Ideal for filtering solvents for HPLC Filters applicable applications Key: CN = cellulose nitrate; CA = cellulose acetate; PES = polyethersulfone; RC = regenerated cellulose; PTFE = polytetrafluoroethylene (Teflon); SFCA = surfactant-free cellulose acetate. Effect of Pore Size The pore size of filter membranes is usually dictated by the requirements of the filter application rather than the desired flow rate. Larger pore membranes usually have both faster flow rates and greater capacity before pore clogging slows the flow. Figure 2 indicates the effect of pore size on filter performance. As expected, the initial flow rate (steep part of the curve) of the.45 µm filter was approximately twice that of the.22 µm filter, although its capacity or throughput prior to clogging (the area at the plateau) was only about 20% greater. Figure 2. Effect of Pore Size on Performance Volume Filtered (ml) Maximum flow rate.45 µm pores.22 µm pores Approaching total capacity Time Required (seconds) Test Conditions: Medium containing 10% fetal bovine serum was filtered using cellulose acetate membranes at 23 C and 600 mm Hg vacuum. 7

19 Effect of Membrane Area The easiest and most practical way to increase filter flow rate is to increase the effective surface area of the filter membrane. Corning offers both syringe and vacuum filter units with a choice of membrane diameters that give a wide range of flow rates and throughputs (See Table 4). Effect of Fluid Temperature For most applications, filtering solutions at room temperature is fine. Usually increasing the temperature of a solution will increase the flow rate. For example, increasing the temperature of cell culture medium from 4 C to 37 C resulted in a doubling of the flow rate. This is most likely due to a decrease in the viscosity of the medium. However, in some cases, filtration at lower temperatures may increase the overall throughput, especially with protein and lipid containing solutions such as serum. Effect of Pressure Differential For vacuum driven filtration a pressure differential (vacuum) of 400 mm Hg (7.73 psig) is good. Increasing the pressure differential further will slightly increase the flow rate, but it may also result in excess foaming as the gases in the filtrate come out of solution as bubbles. This is especially a problem with filtering bicarbonatebuffered cell culture media. The dissolved carbon dioxide in the medium will evolve quickly at higher-pressure differentials leading to a rise in ph and excessive foaming if serum proteins are present. Effect of Prefiltration A simple way to improve filter performance is to pretreat your solution. High speed centrifugation will remove most suspended particle and reduce filter clogging, extending both flow rate and throughput. (Corning 250 and 500 ml centrifuge bottles are ideal for centrifuging larger liquid volumes.) Prefiltration through a glass fiber pad or depth filter will also reduce particle load and premature membrane clogging. The use of a glass fiber prefilter has been demonstrated to more than double the throughput when filtering calf serum. These glass fiber prefilters are included with all Corning vacuum filter systems and bottle top filters. For particularly difficult to filter solutions, it may be helpful to first prefilter the solution through a larger pore membrane filter. Safety Precautions Corning filter units are intended for use by persons knowledgeable in safe laboratory practices. Safety is one of the most critical concerns of any lab. Because of variations in conditions, Corning cannot guarantee any glassware or plasticware against breakage under vacuum or pressure. Failure can result from surface damage, improper pressure or temperature, or use with incompatible chemicals. Adequate precautions should always be taken to protect personnel doing such work. To help improve lab safety, Corning has compiled these common sense suggestions concerning the safe use of filtration products: Use of vacuum-driven filters on glass or plastic bottles may cause personal injury if they implode during use. Eye protection is strongly recommended whenever glass or plastic vessels are used under partial vacuum negative pressure to guard against these injuries. Only bottles specifically designed for these applications should be used. Never use the 45 mm threaded bottle top filters on Pyrex or PyrexPlus brand Media Bottles larger than 2 liter capacity or that are square. Use of bottle top filters with PyrexPlus Media Bottles (with plastic safety coatings) is highly recommended for vacuum filtration. Never use the 33 mm threaded bottle top filters on standard glass media bottles that are larger than 500 ml or on bottles that are not cylindrical. 8

20 Never use plastic roller bottles as substitute receiver bottles during vacuum filtration. Do not use a bottle for vacuum applications if it is not designed to withstand a vacuum; if the bottle is scratched, chipped or cracked; if the bottle is clamped in such a way as to induce stress; or if the bottle is being hand held. Care must be taken when using syringe filters with small syringes (5 ml or less) as the pressures generated may exceed the 75 psi limit, causing a possible membrane or housing failure. Loss of valuable contents and personal injury may result. If clogging causes slower flow rates, we recommend that you replace filters rather than increase the pressure. References Brock, T.S. Membrane Filtration: A User s Guide and Reference Manual. Science Tech, Inc. Madison, WI, 1883, 381 pp. Lukaszewicz, R.C. and Fisher, R. Mechanisms of Membrane Filtration for Particulate and Microbial Retention in Critical Applications. Pharmaceutical Technology, Vol. 5: June Walsh, R.L. and Coles, M.E. Binding of IgG and other Proteins to Microfilters. Clin. Chem. 26(3): , Ordering Information Corning Syringe Filters The Corning syringe filter units are of the conventional, round design with products ranging from 3 mm to 50 mm in diameter. The 50mm diameter filter has a hydrophobic PTFE membrane in a polypropylene housing with two hose barbs. It is designed for sterile venting, protecting vacuum lines and pumps, or filtering gases and non-aqueous solvents. Corning Diam. Filter Pore Membrane Housing Inlet/ List Per Cat. No. (mm) Size (µm) Material Material Sterile Outlet Pkg. Qty/Cs Case (US$) RC PP Yes LL/LS Ind 50 $ RC PP Yes LL/LS Ind PTFE PP No LL/LS Bulk RC PP Yes LL/LS Ind RC PP Yes LL/LS Ind PTFE PP No LL/LS Bulk SFCA-PF AC Yes LL/LS Ind SFCA AC Yes LL/LS Ind SFCA AC Yes LL/LS Ind SFCA AC Yes LL/LS Ind RC PP Yes LL/LS Ind RC PP Yes LL/LS Ind NY PP Yes LL/LS Ind NY PP Yes LL/LS Ind PTFE PP No LL/LS Bulk PTFE PP Yes HB/HB Ind PES AC Yes LL/LS Ind PTFE PP No LL/LS Bulk PTFE PP No LL/LS Bulk 50 $90.00 PP = Polypropylene, AC = Acrylic Copolymer, LL = Luer Lock-Female, LS = Luer Slip/Male, HB = Hose Barb, NY = Nylon, PES = Polyethersulfone, PTFE = Teflon, RC = Regenerated Cellulose, SFCA = Surfactant Free Cellulose Acetate, SFCA-PF = Surfactant Free Cellulose Acetate with Prefilter 9

21 Corning µstar Syringe Filters µstar units have a bi-directional flow pattern that eliminates the priming and air lock effects of conventional syringe tip filters. µstar utilizes all of the membrane surface area for a working volume up to 100 ml while minimizing fluid retention to less than 30 µl with an air purge. Integrity tested with a maximum operating pressure of 100 psi, the µstar, a Class II medical device, is designed for sterilization and clarification of aqueous solutions including: media additives, serum, antibiotics, biological fluids, radio-tracers, and virus suspensions. Sterile, certified non-pyrogenic. Corning Pore List Per Cat. No. Membrane Color Size (µm) Qty/Cs Case (US$) 8110 CA Blue $ CA Clear CA Green CN Smoke CA - cellulose acetate, CN - cellulose nitrate Corning Spin-X Centrifuge Tube Filters Spin-X centrifuge tube filters consist of a membrane-containing filter unit within a centrifuge tube. They filter by centrifugation for bacteria removal, particle removal, HPLC sample preparation, removal of cells from media and DNA removal from agarose or acrylamide gels. Maximum Relative Centrifugal Force (RCF) is 16,000. Unless noted, product is sterile and certified non-pyrogenic. Corning Membrane Working Pore Size Tube Size List Per Cat. No. Material Volume (µl) (µm) (ml) Qty/Cs Case (US$) 8160 CA $ * CA CA * CA * NY * NY * Indicates that the product is non-sterile and is not certified non-pyrogenic. CA = cellulose acetate, NY = nylon Corning Bottle Top Filters Available in 33 mm and 45 mm neck sizes. The 150mL filter is available in CA, CN, and NY in both 0.2 µm and.45 µm pore sizes. The PES is available in only 0.22 µm pore size. Filters are color-coded by membrane type. Individually packaged, sterile and certified non-pyrogenic. Prefilters included. 10 Corning Membrane Volume Neck Size Pore Size Color-Coded List Per Cat. No. Material (ml) (mm) (µm) Adapter Qty/Cs Case (US$) CA Orange 48 $ CA Orange 48 $ CA Orange 48 $ CA Orange 48 $ PES Yellow 48 $ PES Yellow 48 $ CN Blue 48 $ CN Blue 48 $ NY Red 48 $ CN Blue 48 $ CN Blue 48 $ NY Red 48 $ PES = polyethersulfone, CA = cellulose acetate, CN = cellulose nitrate, NY = nylon

22 Corning Vacuum Filter Systems Corning filters feature adapters that are color-coded by membrane type for easy product identification. Angled hose connector simplifies vacuum line attachment. Receiver bottles feature easy grip sides for improved handling. Individually packaged sterile, certified non-pyrogenic. Caps for receiver bottles are supplied sterile and individually packaged. Prefilters included. Corning 150 ml Vacuum Filter Systems with 50 mm Diameter Membranes Corning Membrane Funnel/Bottle Pore Size Color-Coded List Per Cat. No. Material Volume (ml) (µm) Adapter Qty/Cs Case (US$) PES 150/ Yellow 12 $ CA 150/ Orange CA 150/ Orange CN 150/ Blue CN 150/ Blue NY 150/ Red NY 150/ Red PES = polyethersulfone, CA = cellulose acetate, CN = cellulose nitrate, NY = nylon Corning 250 ml Vacuum Filter Systems with 50mm Diameter Membranes Corning Membrane Funnel/Bottle Pore Size Color-Coded List Per Cat. No. Material Volume (ml) (µm) Adapter Qty/Cs Case (US$) CN 250/ Blue 12 $ CN 250/ Blue CA 250/ Orange CA 250/ Orange NY 250/ Red NY 250/ Red PES 250/ Yellow PES = polyethersulfone, CA = cellulose acetate, CN = cellulose nitrate, NY = nylon Corning 500 ml Vacuum Filter Systems with 70 mm Diameter Membranes Corning Membrane Funnel/Bottle Pore Size Color-Coded List Per Cat. No. Material Volume (ml) (µm) Adapter Qty/Cs Case (US$) NY 500/ Red 12 $ CN 500/ Blue CN 500/ Blue CA 500/ Orange CA 500/ Orange NY 500/ Red PES 500/ Yellow PES = polyethersulfone, CA = cellulose acetate, CN = cellulose nitrate, NY = nylon 11

23 Corning 1000 ml Vacuum Filter Systems with 90mm Diameter Membranes Corning Membrane Funnel/Bottle Pore Size Color-Coded List Per Cat. No. Material Volume (ml) (µm) Adapter Qty/Cs Case (US$) CN 1000/ Blue 12 $ C N 1000/ Blue NY 1000/ Red CA 1000/ Orange CA 1000/ Orange NY 1000/ Red ES 1000/ Yellow CA 500*/ Orange CA 500*/ Orange * 500 ml Funnel with 70 mm membrane PES = polyethersulfone, CA = cellulose acetate, CN = cellulose nitrate, NY = nylon Corning 150 ml Tube Top Filter with 50mm Membranes Minimizes unnecessary transfers by filtering directly into centrifuge tube. Includes two (2) centrifuge tube stands with each case. The polypropylene tube is supplied with an individually wrapped cap for storage. Individually packaged, sterile, certified non-pyrogenic. Corning Membrane Funnel/Bottle Pore Size List Per Cat. No. Material Volume (ml) (µm) Qty/Cs Case (US$) CA 150/ $ CA 150/ CA = cellulose acetate Corning 115 ml One-piece Vacuum Filters with 60mm Membrane Diameter Low center of gravity and wide base for stability; separate pour spout to remove filtered sample which minimizes contamination. Individually packaged, sterile, certified non-pyrogenic. Corning Membrane Funnel/Bottle Pore Size List Per Cat. No. Material Volume (ml) (µm) Qty/Cs Case (US$) CA $ CA CN CN CA = cellulose acetate, CN = cellulose nitrate 12

24 Corning Filterware Reusable Glass Vacuum Filters Available with a choice of 47 mm or 90 mm fritted glass filters in an all-glass filter apparatus with a 40/35 glass joint connecting the filter support base of 47 mm or 90 mm and a filter flask. Corning Qty. Per List Per Cat. No. Exp Description Case Case (US$) BP All-Glass Filter Apparatus, 47mm, 300 ml Funnel, 1 liter Flask 1 $ BP All-Glass Filter Apparatus, 47mm, 500 ml Funnel, 2 liter Flask BP All-Glass Filter Apparatus, 47mm, 1000 ml Funnel, 4 liter Flask BP All-Glass Filter Apparatus, 90mm, 1000 ml Funnel, 4 liter Flask BP Funnel/Glass Support Assembly with 47 mm glass frit BP Funnel/Glass Support Assembly with 9 0mm glass frit BP Filter Flasks 1000mL filter flask with #8 Rubber Stopper Joint, #2 Hose Connector BP mL filter flask with #8 Rubber Stopper Joint, #2 Hose Connector BP ml filter flask, #8 Rubber Stopper Joint, #2 Hose Connector in the Exp field denotes a Corning EXPRESS item. For additional information, refer to the catalog or the ordering section of the... MISSING TEXT 13

25 Transwell Permeable Supports Selection and Use Guide Table of Contents Introduction Selecting the Right Transwell Membrane and Pore Size Selecting the Right Transwell System Using Transwell Permeable Supports Helpful Hints General Directions for Use Ordering Information Polyester (PET) Transwell Inserts Collagen-Coated Transwell-COL Inserts Netwell Inserts and Accessories Polycarbonate Membrane Transwell Inserts Snapwell Inserts HTS Transwell-24 Well Permeable Supports , 12, and 24 Well Cell Culture Plates HTS Transwell-96 Well Permeable Supports

26 SEM of the surface of a 0.4 µm pore polycarbonate membrane SEM of a PTFE membrane showing its pore structure The polyester Transwell-Clear inserts in a 6 well plate show the clarity of the membrane Introduction Cell and tissue culture techniques are becoming increasingly important for basic and applied life science research. The development of new culture vessels and cell attachment substrates is currently being driven by the need to produce an environment that resembles the in vivo state as closely as possible to enable the growth of specialized cell types. Consequently, using permeable supports with microporous membranes have become a standard method for culturing these cells. These permeable supports have allowed significant improvements in culturing polarized cells since these permeable supports permit cells to uptake and secrete molecules on both their basal and apical surfaces and thereby carry out metabolic activities in a more natural fashion. Membrane filters have been used as cell growth substrates since the transfilter metanephric induction studies of Grobstein (Nature, 172: ; 1953). Adapted over the years to a variety of cell types and numerous applications, permeable supports treated for cell growth are now recognized as providing significant advantages over solid, impermeable cell growth substrates. For epithelial and other cell types, the use of permeable supports in vitro allows cells to be grown and studied in a polarized state under more natural conditions. Cellular differentiation can also proceed to higher levels resulting in cells that morphologically and functionally better represent their in vitro counterparts. Cellular functions such as transport, absorption and secretion can also be studied since cells grown on permeable supports provide convenient, independent access to apical and basolateral plasma membrane domains. The use of permeable support systems for cell culture has proven to be an invaluable tool in the cell biology laboratory. Selecting the Right Transwell Membrane and Pore Size Transwell permeable supports are available in three membrane materials: polycarbonate (PC), polyester (PET), and collagen-coated polytetrafluoroethylene (PTFE). See Table 1 for additional information on these membrane characteristics. Polyester Transwell-Clear inserts feature a microscopically transparent polyester membrane that is tissue culture treated for optimal cell attachment and growth. Transwell-Clear inserts provide better cell visibility under phase contrast microscopy and allow assessment of cell viability and monolayer formation. Polycarbonate Transwell inserts are available in a variety of pore sizes ranging from 0.4 µm to 8.0 µm. All are treated for optimal cell attachment. Transwell-COL inserts have a transparent (when wet), collagen-treated PTFE membrane that promotes cell attachment and spreading and allow cells to be visualized during culture. The Transwell-COL membrane has an equimolar mixture of types I and III collagen derived from bovine placentas. Unlike traditional coating techniques that result in occluding film layers, Corning s proprietary coating process results in a biologically stable collagen that covers every fibril of the filter matrix, thereby retaining the porosity of the membrane. Table 1. Characteristics of Transwell Membranes These 12 mm diameter Transwell inserts have a polycarbonate membrane Polyester Polycarbonate Collagen-coated Characteristics (PET) (PC) (PTFE) Optical properties Clear Translucent Clear when wet Cell visibility Good Poor Cell outlines Tissue culture treated Yes Yes No Membrane thickness 10 µm 10 µm 50 µm Matrix/ECM coatable Yes Yes Yes Collagen treated No No Yes Available Pore Sizes (µm) 0.4, 1.0, 3.0, , 3.0, 5.0, , 3.0 2

27 24.5 mm Transwell-COL insert being placed into a 6 well microplate. Selecting Pore Sizes Selecting the correct pore size for experiments using Transwell permeable supports is also very important. Table 2 reviews common permeable support applications along with recommended pore sizes. The smallest pore size Transwell membranes (0.4 µm) are primarily used in drug transport studies and in studies requiring the formation of a fully differentiated monolayer of cells. Cell invasion, chemotaxis and motility studies are usually done in Transwell membranes with 3.0 µm or larger pores. The ability of cells to migrate through pores of a membrane is dependent on the cell line used and the culture conditions, as well as the pore size. Cell migration will not occur with pores smaller than 3.0 µm. For critical experiments, Corning suggests experimenting with appropriate controls with a range of pore sizes to determine which size works best with your cell cultures and your specific application. As an alternative, follow recommendations in published scientific literature. For additional application and use information, please refer to the Transwell Bibliography on the Technical Information section of the Corning Life Sciences web site ( that lists over 800 literature references using Transwell permeable supports. Chemical Compatibility All of the Transwell membranes are compatible with histological fixatives including methanol and formaldehyde. The polyester Transwell membranes have the best overall chemical resistance. These membranes (but not the polystyrene housings) are compatible with many alcohols, amines, esters, ethers, ketones, oils and some solvents including many halogenated hydrocarbons and DMSO, but are not recommended for use with strong acids and bases. 75 mm Transwell insert and 100 mm dish bottom Snapwell inserts are designed for use with diffusion or Ussing chambers. Pore Density Of the three types of Transwell membranes, only the collagen-coated PTFE membrane does not have a defined pore density because it is a tortuous path membrane. The two membranes with nominally defined pore densities are polycarbonate and polyester. The 0.4 µm polyester Transwell membranes do not have as high a pore density as the polycarbonate Transwell membranes but have better optical clarity as a result. The nominal pore densities for Corning polycarbonate and polyester membranes are given in Table 3. Selecting the Right Transwell Plates Transwell permeable support units come in three basic designs: Traditional Transwell plate inserts that are used individually in 6, 12, and 24 well plates or 100 mm dishes; HTS Transwell-24 and HTS Transwell-96 insert plates that are mounted in special holders to allow for automation and ease of handling; Snapwell inserts for use in diffusion or Ussing chambers. More detailed information on each of these products is found below and in the ordering section. Traditional Transwell Permeable Supports Transwell inserts are available in four membrane diameters: 6.5 mm (24 well plate), 12 mm (12 well plate), 24 mm (6 well plate) and 75 mm (100 mm dish) formats. See Table 4 for cell growth areas provided by these sizes. Several membrane types and a large selection of pore sizes are available with each of these units. The patented self-centering design prevents medium from wicking between the sides of the insert and the well wall. The hanging design keeps the Transwell membrane about a millimeter off the bottom of the well. This prevents co-cultured cell monolayers in the bottom of the well from being scratched or disturbed when the insert is moved. Windows or openings in the sides of the Transwell insert allow access to the bottom compartment. 3

28 Table 2. Permeable Support Application Selection Chart SEM of macrophages on a Transwell polycarbonate membrane (Courtesy of B. Wetzel, S. Wahl, E. Westbrook and L. Altma, NIH, Bethesda, MD). Recommended Applications Pore Sizes Transport and Permeability Studies 0.4, 1.0, 3.0 µm Macromolecules, ions, water, small molecular weight solutes, hormones, growth factors, etc. Cell Polarity 0.4, 1.0, 3.0 µm Polarized distribution of ion channels, enzymes, transport proteins, receptors, lipids Sorting and targeting Polarity development and maintenance Synthesis and assembly of tight junctions Endocytosis 0.4, 1.0, 3.0 µm Protein turnover Membrane recycling Receptor-ligand interactions for growth factors, hormones, antibodies, viruses, toxins Drug Transport 0.4, 1.0, 3.0 µm Localization of receptors and polarity of drug responses Drug effects on vascular permeability Drug transport across epithelial (Caco-2 cells) and endothelial barriers Drug transport across brain microvessel endothelial cells Metastatic Potential and Invasion 5.0, 8.0 µm Tumor invasion and metastasis Clonogenic assays Invasion inhibitors Extracellular matrix effects Chemotaxis/Motility Studies 3.0, 5.0, 8.0 µm Phagocytosis Chemotactic and haptotactic response of formed elements in blood Migration of tissue macrophages Co-culture 0.4, 1.0, 3.0 µm Cell-cell interactions Cell-substrate interactions Tumor heterogeneity Cell-matrix interactions Feeder layers and feeder layers for stem cell cultures Microbial Pathogenesis 0.4, 1.0, 3.0 µm Virus, bacteria, parasite attachment to host cell plasma membrane Invasion and penetration of epithelial barriers Microbial receptors Drug effects on microbial receptors Tissue Remodeling 0.4, 1.0, 3.0 µm Wound healing Angiogenesis Re-epithelialization Inflammation In Vitro Fertilization 0.4, 1.0, 3.0 µm Granulosa cell culture Steroidogenesis Endocrine and paracrine factors affecting granulosa differentiation, blastocyst hatching from zona pellucida, substrate attachment, and trophoblast cell outgrowth 4

29 Table 3. Nominal Pore Densities for Transwell Polyester and Polycarbonate Membranes Nominal Pore Density* Pore Size* Polycarbonate Membrane Polyester Membrane 0.4 µm 1 x 10 8 pores/cm 2 4 x 10 6 pores/cm µm Not available 1.6 x 10 6 pores/cm µm 2 x 10 6 pores/cm 2 2 x 10 6 pores/cm µm 4 x 10 5 pores/cm 2 Not available 8.0 µm 1 x 10 5 pores/cm 2 1 x 10 5 pores/cm 2 * Values are reported as nominal and may vary due to inherent variability of the manufacturing process. To ensure success, we recommend that researchers validate their methods independent from our reported values. Table 4. Transwell Permeable Support Growth Areas Transwell Insert Multiple Well Plate Insert Membrane Diameter* or Dish Style Growth Area* 4.26 mm 96 well cm mm 24 well 0.33 cm 2 12 mm 12 well 1.12 cm 2 24 mm 6 well 4.67 cm 2 75 mm 100 mm dish 44 cm 2 * Values are reported as nominal and may vary due to inherent variability of our manufacturing process. To insure success, we recommend that researchers validate their methods independent from our reported values. HTS Transwell Permeable Support Plates The HTS Transwell Permeable Support Plates are arrays of either 24 or 96 individual Transwell inserts connected by a rigid, robotics-friendly holder that enables all of the Transwell-24 or Transwell-96 inserts to be handled as a single unit. This makes HTS Transwell systems ideal tools for running automated, high throughput drug transport (Caco-2 cells) cell toxicity studies or cell migation and invasion studies. The HTS-96 System is ideal for high throughput transport studies. HTS Transwell Systems are designed for use with robotics. The HTS Transwell-96 Well Permeable Supports (U.S. Patent No. 6,943,009) have multiple components: a 96 well insert support plate with a choice of either 1.0 µm or 8.0 µm pore polyester, or 0.4 µm, 3.0 µm, or 5.0 µm pore polycarbonate membranes; a Reservoir Plate with a removable media stabilizer for feeding cultures; a 96 well Receiver Plate for use in assays (black or clear); and two lids to minimize evaporation and protect against contamination. Each well insert has a cm2 membrane area and large apical and basolateral access ports for feeding and sampling. The HTS Transwell-24 Well Permeable Support is available with a treated polycarbonate membrane in either 0.4 µm or 3.0 µm pore sizes or a 0.4 µm pore polyester membrane and provides an excellent substrate for cell attachment, growth, and differentiation. An open culture reservoir plate is used to reduce liquid handling during cell feeding (medium can be exchanged all at once). Once the cell layers are confluent, the HTS Transwell-24 insert is transferred to a standard Corning 24 well microplate for running experiments. Snapwell Inserts The Snapwell insert (U.S. Patent No. 5,272,083) is a modified Transwell culture insert that contains a 12 mm diameter tissue culture treated polycarbonate or clear polyester membrane supported by a detachable ring. Available in 0.4 or 3.0 µm pore sizes, these inserts are primarily used for transport and electrophysiological studies. Once cells are grown to confluence, this ring-supported membrane can be placed into either vertical or horizontal diffusion or Ussing chambers. Chambers are available from Harvard Apparatus: 5

30 Transwell insert Upper compartment Microporous membrane Lower compartment The porous bottom of the insert provides independent access to both sides of a cell monolayer giving researchers a versatile tool to study cell transport and other metabolic activities in vitro. Using Transwell Permeable Supports Helpful Hints 1. Cell morphology and cell densities on permeable supports are influenced by filter pore size. 2. Larger pore sizes may permit some cell types to migrate through the pores on the permeable support. 3. Cells grown on permeable supports are often sensitive to initial seeding density for good cell attachment. On first use, try bracketing a range of seeding densities for optimum growth (typically 10 3 to 10 5 cells/cm 2 ). 4. Cell attachment and spreading may be improved by preincubating permeable supports in medium prior to seeding. 5. Cells requiring extracellular matrix coatings on plastic substrates may also require them on permeable supports. 6. The Transwell-Clear insert contains a transparent tissue culture treated polyester membrane that allows easier viewing of cells using phase contrast microscopy. 7. The Transwell-COL insert contains a PTFE membrane that has been treated with an equimolar mixture of types I and III bovine placental collagens. This results in a biologically stabilized collagen matrix covering the fibrils of the filter membrane. These Transwell inserts are excellent for the growth of cells requiring a biological coating. General Directions for Use 1. Transwell inserts are used by first adding medium to the multiple well plate well, then adding the Transwell insert, and then adding the medium and cells to the inside compartment of the Transwell insert. Recommended medium volumes are shown in Table An initial equilibrium period may be used to improve cell attachment by adding medium to the multiple well plate well and then to the Transwell insert. The plate should then be incubated for at least one hour or even overnight at the same temperature that will be used to grow the cells. The cells are then added in fresh medium to the Transwell insert and returned to the incubator. 3. The medium level can be checked periodically and fresh medium added as required. 4. Transwell inserts have three openings for standard pipette tips to allow samples to be added or removed from the lower compartment. 6

31 Table 5. Recommended Transwell Permeable Support Medium Volumes Add the medium to the culture plate first, then add the medium and cells to the Transwell insert. The three side wall openings for pipette tip access can be seen in this 24 mm polycarbonate membrane Transwell insert. Transwell Insert Multiple Well Volume Volume Added Insert membrane Plate or Added per to Inside of Diameter* Growth Area* Dish Type Plate Well Transwell Insert 4.26 mm cm 2 96 well ml ml 6.5 mm 0.33 cm 2 24 well 0.6 ml 0.1 ml 12 mm 1.12 cm 2 12 well 1.5 ml 0.5 ml 24 mm 4.67 cm 2 6 well 2.6 ml 1.5 ml 75 mm 44 cm mm dish 13 ml 9 ml * Values are reported as nominal and may vary due to inherent variability of the manufacturing process. To ensure success, we recommend that researchers validate their methods independent from our reported values. 5. Cell monolayers may be fixed and stained while in the Transwell insert using standard cytological techniques. Avoid using solvents that dissolve polystyrene or the polycarbonate or polyester membrane materials. Processing steps may be carried out by sequentially moving the Transwell insert through a series of multiple well plate wells containing the appropriate reagents. Protocols for fixing and staining Transwell inserts are available on the Corning Life Sciences web site: techdocs/transwell_staining_protocol.pdf. 6. If it is necessary to remove cells from Transwell membranes, we recommend rinsing both the Transwell insert and the plate well. Then the dissociating solution should be added to both the well and the Transwell insert and incubated until the cells begin to come off. A protocol, Trypsinization Procedure for Transwell Inserts ( for removing cells from Transwell inserts is available in the technical section of the Corning Life Sciences web site. 7. Corning recommends using a Micromatic 8-channel Aspirator (Cat. No. 6113, www. cadencescience.com) for removing medium from HTS Transwell-96 systems. These aspirators are designed to remove medium and solutions from the upper wells without damaging the sensitive cell monolayers. 8. The polycarbonate or polyester membrane with the fixed and stained cells attached may be removed from the Transwell insert by carefully cutting around the membrane edges with a scalpel. 9. The collagen-coated PTFE membrane is fragile and requires careful handling during removal. A wetted cellulosic membrane filter should be placed in direct contact with the underside of the Transwell insert membrane before it is cut out with a scalpel. The wetted, more rigid, cellulosic filter will serve as a support for the collagen-coated membrane. Ordering Information Fixed and crystal violet stained CHO-K1 cells on a 3 µm PET membrane. 24 mm Transwell-Clear Insert Polyester (PET) Membrane Transwell-Clear Inserts Transwell-Clear inserts feature a thin, microscopically transparent polyester membrane that is tissue culture treated for optimal cell attachment and growth. Transwell-Clear inserts provide excellent cell visibility under phase contrast microscopy and allow assessment of cell viability and monolayer formation. Transwell-Clear inserts are available sterile and preloaded in 6, 12 and 24 multiple well plates. All plates come with lids. Cat. Membrane Growth Surface Membrane Inner Inserts/ No. Diameter* (mm) Area* (cm 2 ) Pore Size (µm) Packaging Case inserts/24 well plate inserts/24 well plate inserts/12 well plate inserts/12 well plate inserts/6 well plate inserts/6 well plate 24 * Values are reported as nominal and may vary due to inherent variability of the manufacturing process. To insure success, we recommend that researchers validate their methods independent from our reported values. 7

32 Collagen-Coated Transwell -COL Inserts Transwell-COL inserts have a transparent when wet, collagen-treated PTFE membrane that promotes cell attachment and spreading and allows cells to be visualized during culture. The Transwell-COL membrane has an equimolar mixture of types I and III collagen derived from bovine placentas. Unlike traditional coating techniques that result in occluding film layers, Corning s proprietary coating process results in a biologically stable collagen that covers every fibril of the filter matrix, thereby retaining the porosity of the membrane. Transwell- COL inserts are sterile and individually blister packed. Appropriate multiple well plates are included in each case. All plates come with lids. Membrane Growth Membrane Multiple Cat. Diameter* Surface Pore Size Inner Well Inserts/ No. (mm) Area* (cm 2 ) (µm) Packaging Plate Case Individually wrapped 2-24 well Individually wrapped 2-24 well Individually wrapped 2-12 well Individually wrapped 2-12 well Individually wrapped 4-6 well Individually wrapped 4-6 well 24 * Values are reported as nominal and may vary due to inherent variability of the manufacturing process. To insure success, we recommend that researchers validate their methods independent from our reported values. Netwell Inserts Netwell Inserts and Accessories Costar Netwell inserts have polyester mesh bottoms attached to polystyrene inserts Used as tissue carriers, supports and strainers for culture of small organs, tissue slices or explants at the air-media interface Provide coarse filtration of tissue homogenates, cell suspensions and microcarriers Handy carrier for immunocytochemical staining of tissue slices (see accessories below) Available in two mesh sizes and diameters Preloaded in 6- or 12-well microplates 24 mm Netwell inserts fit in Corning 50 ml plastic centrifuge tubes Membrane Polyester Diameter Membrane Inner Cat. No. (mm) Mesh Size (µm) Color Packaging Qty/Cs n/a 12/plate n/a 12/plate n/a 6/plate n/a 6/plate 48 Netwell Reagent Tray 3517 n/a n/a black n/a n/a n/a white n/a 200 Netwell Carrier n/a n/a n/a n/a n/a n/a 8 8

33 Polycarbonate Membrane Transwell Inserts These Transwell inserts feature a thin, translucent polycarbonate membrane available in six pore sizes ranging from 0.4 µm to 8.0 µm. All are treated for optimal cell attachment. They are supplied sterile and come preloaded in multiple well plates or dishes. The polycarbonate membrane is compatible with most organic fixatives and stains. All plates come with lids. 12 mm Polycarbonate Transwell Insert Membrane Growth Membrane Cat. Diameter* Surface Pore Size Inner Inserts/ No. (mm) Area* (cm 2 ) (µm) Packaging Case inserts/24 well plate inserts/24 well plate inserts/24 well plate inserts/24 well plate inserts/12 well plate inserts/12 well plate inserts/6 well plate inserts/6 well plate inserts/6 well plate insert/100 mm dish insert/100 mm dish 12 * Values are reported as nominal and may vary due to inherent variability of the manufacturing process. To insure success, we recommend that researchers validate their methods independent from our reported values. 75 mm Polycarbonate Transwell Insert Snapwell Inserts with polycarbonate (lower) and polyester (upper) membranes Snapwell Inserts The Snapwell insert (U.S. Patent No. 5,272,083) is a modified Transwell culture insert that contains a 12 mm diameter tissue culture treated membrane supported by a detachable ring. Once cells are grown to confluence, this ring-supported membrane can be placed into either vertical or horizontal diffusion or Ussing chambers. Chambers are available from Harvard Apparatus: Snapwell inserts are provided sterile and preloaded in 6 well plates. All plates come with lids. Membrane Growth Membrane Cat. Diameter Surface Pore Size Membrane Inner Inserts/ No. (µm)* Area* (cm 2 ) (µm) Material Packaging Case mm Polycarbonate 6 inserts/6 well plate mm Clear Polyester 6 inserts/6 well plate mm Polycarbonate 6 inserts/6 well plate 24 * Values are reported as nominal and may vary due to inherent variability of the manufacturing process. To insure success, we recommend that researchers validate their methods independent from our reported values. 9

34 HTS Transwell-24 System showing both the culture reservoir and the 24 well microplate. HTS Transwell -24 Well Permeable Supports The HTS Transwell-24 System has an array of 24 wells with permeable inserts connected by a rigid, robotics-friendly tray that enables all 24 Transwell inserts to be handled as a single unit. The individually packaged product consists of two individually wrapped HTS Transwell-24 units loaded into open reservoirs and includes two 24 well plates. The bulk packaged products consist of 12 HTS Transwell-24 units loaded into 24 well plates only. Open reservoirs can be purchased separately. Choice of either 0.4 µm polyester membrane or 0.4 µm and 3.0 µm pore polycarbonate membrane Cell growth area is 0.33 cm 2 /well Choice of either individual or bulk packaging HTS Transwell-24 Systems are all tissue culture treated and sterile Cat. Membrane Pore Size No. Description Material (µm) Qty/Cs 3396 HTS Transwell-24 System: insert tray in a reservoir PC plate with lid, 1/pack; plus a separate 24 well receiver plate with lid, 1/pack 3379 HTS Transwell-24 System: insert tray in a reservoir PET plate with lid, 1/pack; plus a separate 24 well receiver plate with lid, 1/pack 3397 HTS Transwell-24 System, Bulk Packed: insert trays PC in 24 well plates with lids, 12/pack 3378 HTS Transwell-24 System, Bulk Packed: insert trays PET in 24 well plates with lids, 12/pack 3398 HTS Transwell-24 System: insert tray in a reservoir PC plate with lid, 1/pack; plus a separate 24 well receiver plate with lid, 1/pack 3399 HTS Transwell-24 System, Bulk Packed: insert trays PC in 24 well plates with lids, 12/pack 3395 HTS Transwell-24 Reservoir (Feeder) Plate and lid, NA NA 48 not treated, 12/pack 6, 12, and 24 Well Cell Culture Plates These multiple well plates are treated for optimal cell attachment, sterilized by gamma radiation and are certified nonpyrogenic. All plates have a uniform footprint and a raised bead to aid in stacking. Alphanumeric labels provide individual well identification. The 6.5, 12, and 24.5 mm Transwell inserts are designed to automatically center themselves when placed into the appropriate culture plate. Corning offers a variety of multiple well plate designs and sizes. Cat. Number Well Growth No. of Wells Diameter* (mm) Surface Area* (cm 2 ) Qty/Pk Qty/Cs * Values are reported as nominal and may vary due to inherent variability of the manufacturing process. To insure success, we recommend that researchers validate their methods independent from our reported values. 10

35 HTS Transwell-96 System showing the culture reservoir with removable media stabilizer (top), the 96 well insert tray (middle) and the 96 well receiver plate (bottom). HTS Transwell -96 Well Permeable Support Systems and Plates The HTS Transwell-96 Well Permeable Support has an array of 96 wells with permeable inserts connected by a rigid, robotics-friendly tray that enables all 96 inserts to be handled as a single unit. HTS Transwell-96 insert membranes are all tissue culture treated and sterile Choice of either a polyester (PET) membrane (1.0 µm, 8.0 µm pore sizes) or polycarbonate (PC) membranes (0.4 µm, 3.0 µm, 5.0 µm pore sizes) Cell growth area is cm 2 /well which is 20 to 50% greater than competitive devices Large apical and basolateral access ports for easier filling and sampling Removable media stabilizer reduces media spills during handling Automation optimized design with multichannel feeder ports, improved gripping surface and standard bar coding The HTS Transwell-96 Systems (0.4 µm PC and 1.0 µm PET) are packaged with the 96 well insert plate in a reservoir plate and includes the 96 well receiver plate with lid. The HTS Transwell-96 Well Plates (3.0 µm and 5.0 µm PC, 8.0 µm PET) are packaged with the 96 well insert plate in a 96 well receiver plate with lid. Reservoir plates and black 96 well receiver plates may be purchased separately. Membrane Cat. Pore Size Qty/ Qty/ No. Description (µm) Membrane Pk Cs 3381 HTS Transwell-96 System, reservoir and receiver plates 0.4 PC 1 1 with 2 lids 3391 HTS Transwell-96 System, reservoir and receiver plates 0.4 PC 5 5 with 2 lids 3380 HTS Transwell-96 System, reservoir and receiver plates 1.0 PET 1 1 with 2 lids 3392 HTS Transwell-96 System, reservoir and receiver plates 1.0 PET 5 5 with 2 lids 3385 HTS-Transwell-96 Well Plate, receiver plate and lid, 3.0 PC 1 2 individual 3386 HTS-Transwell-96 Well Plate, receiver plate and lid, 3.0 PC 4 8 bulk 3387 HTS-Transwell-96 Well Plate, receiver plate and lid, 5.0 PC 4 8 bulk 3388 HTS-Transwell-96 Well Plate, receiver plate and lid, 5.0 PC 1 2 individual 3374 HTS-Transwell-96 Well Plate, receiver plate and lid, 8.0 PET 1 2 individual 3384 HTS-Transwell-96 Well Plate, receiver plate and lid, 8.0 PET 4 8 bulk 3382 HTS Transwell-96 receiver plate with lid, tissue n/a n/a culture treated 3383 HTS Transwell-96 reservoir plate with removable media n/a n/a stabilizer and lid, not treated 3583 HTS Transwell-96 black receiver plate with lid, n/a n/a tissue culture treated 11

36 Corning Roller Bottles Selection and Use Guide Innovative Products for Biotechnology Life Sciences Table of Contents Introduction Corning Disposable Plastic Roller Bottles Corning PYREX Glass Reusable Roller Bottles Tips on Solving Growth Problems in Roller Bottles Technical Assistance References

37 Standard Corning polystyrene roller bottles are surface modified for improved cell attachment using a traditional gas-plasma treatment. This generates highly energetic oxygen ions which graft onto the surface polystyrene chains so that the bottle surface becomes hydrophilic and negatively charged so cells can better attach.. Polystyrene roller bottles are also available with a patented Corning CellBIND Surface treatment that uses a novel microwave plasma process for treating the culture surface. This process improves cell attachment by incorporating significantly more oxygen into the cell culture surface than the traditional plasma treatment, rendering it more hydrophilic (wettable) and increasing the stability of the surface. The Corning CellBIND cell culture surface: Improves cell attachment leading to increased cell growth and yields Results in more consistent and even cell attachment Helps adapt cells to reduced serum or serum-free conditions Reduces premature cell detachment from confluent cultures Corning Smooth Wall Polystyrene Roller Bottles The 850 cm 2 roller bottles are graduated to 2000 ml in 50 ml increments; the 1750 cm 2 bottles are graduated to 1500 ml in 50 ml increments. These roller bottles are available with three nominal growth areas. Actual growth areas may be greater depending on the amount of medium used: 490 cm 2 (bottles are cm in diameter, cm in length including cap); 850 cm 2 (bottles are cm in diameter, cm in length including cap); 1750 cm 2 (bottles are cm in diameter, cm in length including cap). Two surfaces are available: The standard Corning tissue culture treated surface The new patented Corning CellBIND surface treatment Three caps are available: The traditional plug seal cap design is available on the 490 cm 2 roller bottle; Easy Grip caps are designed for more comfortable manual handling, have half turn on/off seating, smooth, rounded edges, and large, easy-to-grip knurls. They are not vented. The Easy Grip cap design is also available with filtered vents. Vent caps feature a 0.2 µm membrane sealed into the Easy Grip cap to permit sterile gas exchange when working with bottles in open cell culture systems. Two packaging options are available: Packed two per clear plastic bag; Bulk packed 5, 20, or 22 per bag. Corning Surface Qty/ Qty/ Cat. No. Area (cm 2 ) Cap Style Surface Graduations Pk Cs Plug Seal Tissue Culture Treated No Easy Grip Corning CellBIND Surface Yes Easy Grip Tissue Culture Treated Yes Easy Grip Vent Tissue Culture Treated Yes Easy Grip Vent Corning CellBIND Surface Yes Easy Grip Tissue Culture Treated Yes Easy Grip Tissue Culture Treated Yes Easy Grip Corning CellBIND Surface Yes Easy Grip Tissue Culture Treated Yes

38 Corning Expanded Surface Polystyrene Roller Bottles Although these bottles have the same approximate outer dimensions of the 850 cm 2 roller bottles, the ribbed design provides twice the growth surface while maintaining the same exterior dimensions. Two smooth window areas that run the length of the bottle provide microscopic viewing as well as pouring surfaces. The bottles are graduated from 100 to 1000 ml in 100 ml increments. Two surfaces are available: The standard Corning tissue culture treated surface The patented Corning CellBIND surface treatment Two caps are available: Easy Grip caps are designed for more comfortable manual handling, have half turn on/off seating, smooth, rounded edges, and large, easy-to-grip knurls. They are not vented. The Easy Grip cap design is also available with filtered vents. Vent caps feature a 0.2 µm membrane sealed into the Easy Grip cap to permit sterile gas exchange when working with bottles in open cell culture systems. Two packaging options are available: Packed two per clear plastic bag Bulk packed either 5 or 20 per bag Corning Surface Cat. No. Area (cm 2 ) Cap Style Surface Qty/Pk Qty/Cs Easy Grip Tissue Culture Treated Easy Grip Vent Tissue Culture Treated Easy Grip Tissue Culture Treated Easy Grip Tissue Culture Treated Easy Grip Corning CellBIND Surface Easy Grip Vent Tissue Culture Treated Corning PYREX Glass Reusable Roller Bottles Corning PYREX glass reusable roller bottles are manufactured from borosilicate glass for optical clarity and mechanical strength. They are designed to withstand repeated wet or dry sterilization. PYREX Glass Roller Bottles with 38 mm Screw Cap Bottles are supplied with 38 mm deep skirted, rubber-lined, phenolic screw caps. Corning Cell Growth Dimensions Qty/ Cat. No. Description Area (cm 2 ) O.D. x Height (mm) Cs Roller Bottle x Roller Bottle x Roller Bottle x Roller Bottle x CAP Cap, Phenolic G.P.I n/a n/a 1 4

39 PYREX Glass Roller Bottles with 45 mm Screw Cap Bottles have linerless, one-piece orange polypropylene plug seal caps with GL45 threads and drip-free pouring rings. A wide range of optional caps are available including: colored caps for ease of sorting and identification or vented membrane caps with 0.22 µm PTFE hydrophobic membranes for gas exchange. Corning Cell Growth Dimensions Qty/ Cat. No. Description Area (cm 2 ) O.D. x Height (mm) Cs Roller Bottle x Roller Bottle x Roller Bottle x Roller Bottle x PYREX Glass Roller Bottle with 51 mm Screw Cap Wider mouth simplifies cell harvesting. Bottles are supplied with 51 mm phenolic, rubberlined caps. Corning Cell Growth Dimensions Qty/ Cat. No. Description Area (cm 2 ) O.D. x Height (mm) Cs Roller Bottle x Roller Bottle x Roller Bottle x CAP Phenolic Cap G.P.I n/a n/a 1 Tips on Solving Growth Problems in Roller Bottles The constant movement of the medium across the surface of the bottle, slow though it appears, can make it more difficult for cells to attach and grow in roller bottles compared to stationary vessels such as flasks and dishes. The constant motion of the medium can also lead to a more stressful cell environment than is found in stationary culture systems. Consequently, any technique-related issues that reduce the attachment ability of cells is magnified and clearly stands out (Freshney, 1994). This section will focus on identifying (and correcting) some of the common sources of growth problems in roller bottles. Please note than many of these growth problems are not readily observed during routine microscopic observation of live cultures. The occurrence and extent of these problems are best observed when sample cultures are first fixed and stained (1% crystal violet was used for the photos in this article) prior to observation. Figure 1. The bottle on the left was rotated too fast resulting in uneven distribution of cells to the ends of the bottle. The bottle on the right was rotated at the correct speed. The cells in these bottles were fixed and stained with crystal violet to show their density and distribution. Uneven Cell Attachment and Clumping One of the most commonly encountered problems using roller bottles is difficulty getting the cells to attach and form an even monolayer in the bottle. Rotating bottles at inappropriate speeds is a common cause of attachment problems. If the bottles are rotated too quickly for cells to easily attach, areas of heavy cell growth often appear as circular bands towards both ends of the bottles (See Figure 1). This is because the medium flow is slightly slower at the ends than in the middle of the bottles. Rotating bottles too fast may also result in large clumps of cells. This results from the tendency of cells to form clumps since they find it easier to adhere to each other than to the surface of the roller bottle. Eventually these clumps become large enough that they can attach to the bottle surface. A recommended starting speed for initiating roller bottle cultures is 0.5 to 1.0 revolutions per minute (rpm) to start. However, if cells have difficulty attaching, slower speeds (0.1 to 0.4 rpm) should be used until the cells are attached. 5

40 Another possible solution to cell attachment problems in roller bottles is the Corning CellBIND Surface. The first novel cell culture surface treatment in over 20 years, this surface is designed to improve cell attachment under difficult conditions, such as growth roller bottles. It is also useful for growing difficult cells such as primary cultures or transfected cells over expressing proteins. Developed by Corning scientists, this patented technology (U.S. Patent 6,617,152) uses a microwave plasma process for treating the culture surface. This process improves cell attachment by incorporating significantly more oxygen into the cell culture surface than traditional plasma or corona discharge treatments, rendering it more hydrophilic (wettable) and increasing the stability of the surface. Unlike biological coatings, the Corning CellBIND surface is a nonbiological surface that requires no special handling or storage. Because the polymer is treated, rather than coated, the surface is more consistent and stable. This enhanced cell performance has already led to a major biotechnology company choosing Corning roller bottles with the Corning CellBIND surface for producing a new FDA approved protein therapeutic. Table 1. Recommended Media Volumes Corning Plastic Roller Bottles (area) Recommended media volumes 490 cm to 150 ml 850 cm to 255 ml 1700 cm to 510 ml 1750 cm to 525 ml Corning PYREX Glass Roller Bottles (area) Recommended media volumes cm to 200 ml 840 cm to 255 ml 1170 cm to 350 ml 1330 cm to 400 ml 1585 cm to 475 ml 1585 cm to 475 ml Figure 2. Most of the cells have attached to the bottom end of this bottle as a result of it being stood on end for too long immediately after seeding it with cells. Cell damage during subculturing, or incomplete inactivation or removal of dissociating enzymes can also make it more difficult for cells to attach and result in banding or clumping. The protein-based cell receptors used to initiate cell attachment become damaged by the dissociating procedures and must be replaced before the cells can reattach. Poorly regulated incubator temperatures (temperatures that are too high or too low) will also make it more difficult for cells to evenly attach to roller bottles. Using too small a volume of medium, or a medium that contains insufficient attachment factors (serum-free media for example) can have very similar effects. Approximately 0.2 to 0.3 ml of medium per square centimeter of growth area is a good recommended starting volume, depending on the cell line used and the frequency of feeding (See Table 1). It may be necessary to adjust feeding frequency (medium changes) to optimize cell growth. If the bottles are initially rotated too slowly, or if they slip or stop turning even for a short time during the initial cell attachment period, uneven longitudinal bands of cell growth may appear. Cleaning the rollers on the roller apparatus should alleviate slipping bottles. If necessary, rubber bands can be placed around the ends of the bottles to improve traction. Bands of heavy growth at just one end of the bottle are often the result of the roller apparatus not being level. This causes an increased amount of medium and cells at the end of the bottle that is lower (see Figure 2). Furthermore, the longer it takes the cells to attach, the more time there is for them to gradually roll down the length of the bottle to the lower end before attaching. Standing a bottle on end for too long after initially seeding it with cells can have a similar effect. 6

41 Figure 3. Two clear bands caused by debris in the medium scraping away cells as the bottle was rotated. Clear Bands Occasionally clear circular bands will occur on roller bottles where the cells appear to have been swept away (See Figure 3). While small pieces of rolling debris or large cell clumps can cause this to occur, one of the most common causes is the short-term presence of bubbles in the initial cell inoculum. These bubbles, when in contact with the sides of the slowly rotating bottle, can act as miniature plows, scraping off the cells as they begin to attach. Avoid bubble formation by carefully pouring medium down the sides of the bottles, or pipetting it directly into the bottom of the bottles. Cell suspensions used for inoculating roller bottles should be prepared to ensure they are bubble-free. Direction of bottle rotation Cells are pushed off surface by the bubble Air bubble Streaking Condensation (essentially pure water) falling onto exposed cells can cause some unusual patterns and events. This problem usually occurs in roller bottles that have been removed from an incubator and are standing upright at cooler room temperatures awaiting processing. Due to temperature differences, water vapor will condense on the inside of the cap. The resulting droplets may then coalesce and run down the sides of the bottle across the cells that are now only covered by a very thin film of medium. These cells will then undergo a strong osmotic shock. If they have formed a confluent monolayer, they may tear or pull apart from each other along the path the water takes, creating a visible dagger-like streak (See Figure 4). Cells that have not reached confluency may round up and float off into the medium, leaving behind a long clear streak devoid of cells. Figure 4. A clear streak free of cells resulting from cap condensation running down the cell sheet while the bottle was temporally stored upright awaiting processing. Figure 5. A cell monolayer damaged by scraping with a pipette (left). Cells peeling away from the surface of the bottle (right). Peeling Heavily confluent cell monolayers (especially fibroblasts) will occasionally start to peel away from the surface of the roller bottle (See Figure 5). This often results, not from surface treatment failure, but from the formation of a flexible sheet of tightly interconnected cells and cell-manufactured extracellular matrix. Over time, mechanical stresses can develop in the cell sheet from cellular movements and contractions that may then cause the cell sheet to tear or pull away from the roller bottle. Physical damage from pipetting directly onto the cell sheet, tearing it with the end of the pipette, or other manipulations to the cell sheet may also initiate cell sheet peeling. Corning recommends tryhing the Corning CellBIND Surface if peeling occurs. Unusual Growth Patterns Static electrical charges that build up on roller bottles can also adversely affect cell attachment. This problem occurs more frequently when the relative humidity is very low during the winter (or year round in some lab locations). Wiping the vessels with a clean damp towel, increasing the room humidity, or using commercially available antistatic devices may eliminate or greatly reduce this problem. Extra care should be used to avoid rubbing roller bottles against the packaging when removing them from their plastic bags as this can increase the static charge. The way in which medium is added to a roller bottle can also cause unusual growth patterns due to the differential binding of serum attachment proteins in the medium to the wall of the bottle. 7

42 Poor Growth Many problems with the growth, maintenance, or performance of cell cultures are eventually traced back to the medium in which the cells are cultured. These medium-related problems are often slow to develop and are rarely obvious, usually requiring significant effort to track down and eliminate. Unless heavily contaminated, good culture medium is not visibly different in appearance from defective culture medium. The only good way to determine medium quality is to attempt to grow cells with it; this is the basic quality control procedure used by reliable commercial media manufacturers. Cell cultures respond to deficient or toxic media in different ways depending upon both the nature and the degree of the problem. These responses can range from minor changes in growth rate or cell attachment, to changes in the rates of production of viruses or bioproducts, or even the total loss of the culture. Determining if the medium is responsible for a problem is relatively easy; simply test the suspected batch against a sample proven to be effective. Determining why the medium is defective is extremely difficult due to the numerous reagents and complex steps involved. Therefore, time and energy is better spent preventing media problems than trying to find and fix them later; management by prevention is the key to successful media production. The following sections will discuss three of the most common problem areas encountered using culture media. Buffers Many growth problems result when customers do not supply the CO 2 levels required by the bicarbonate-based buffering system of the medium they are using, resulting in poor ph control. Media buffered with low levels of sodium bicarbonate, such as found in Eagle s Minimal Essential Media (MEM) buffered with Hanks salts (0.350 g/l sodium bicarbonate), are designed for use in sealed (air tight) culture vessels in incubators without elevated CO 2 levels. This buffer system is often used in industrial scale production using roller bottles since it does not require a CO 2 incubator. However, Eagle s MEM buffered with Earle s salts (2.2 g/l sodium bicarbonate) requires open culture vessels (dishes, plates or flasks with loose or vented caps) in a humidified incubator capable of maintaining 5% to 7% CO 2. Table 2 gives the bicarbonate levels found in some commonly used cell culture media. Usually the higher the level of sodium bicarbonate, the higher the level of CO 2 that must be supplied for optimum buffering capacity. Table 2. Bicarbonate Levels in Some Commonly Used Mammalian Cell Culture Media Sodium bicarbonate Extra CO 2 levels (g/l) required Eagle s Minimal Essential Medium (MEM) with Earle s salts 2.2 Yes Eagle s MEM with Hanks salts 0.35 No Medium 199 with Earle s salts 2.2 Yes Medium 199 with Hanks salts 0.35 No MEM α Medium 2.2 Yes Dulbecco s Modified Eagle s Medium (DMEM) 3.7 Yes DMEM/F to Yes Ham s F Yes MCDB 131 Medium Yes McCoy s 5A 2.2 Yes RPMI Yes CMRL 1066 Medium 2.2 Yes Leibovitz s L-15 Medium None No Often, the above bicarbonate-based buffer systems are supplemented with the addition of HEPES, a widely used organic buffer. The use of this buffer can lead to additional problems upon exposure of the medium to fluorescent light (see next section below). 8

43 Fluorescent Light-induced Toxicity The deleterious effect of fluorescent light on culture media may be the single most overlooked source of chemically induced cytotoxicity. It is very important to store media and cells growing in culture vessels in the dark away from sources of fluorescent light that will interact with light sensitive media components (riboflavin, tryptophan and HEPES). These interactions result in the production of hydrogen peroxide and free radicals that are directly toxic to cells. This well-documented problem is often ignored when there are cell growth issues (Wang, 1976, Wang and Nixon, 1978). Since the toxic effects of improperly stored media slowly increase with time, this problem is particularly difficult to identify. Besides direct cytotoxicity, other light-induced damaging effects include genetic damage (increase in mutation rates and chromosomal aberrations). Figure 6. Photomicrographs (1000x) of VERO cells stained with Hoechst dye. DNA containing nuclei and mycoplasma stain brightly under ultraviolet light allowing the clean culture on the left (a) to easily be distinguished from infected culture on the right (b). (Photomicrographs courtesy of Bionique Testing Laboratories, Inc.) Mycoplasma Although the problem of cell culture contamination is beyond the scope of this brochure, it is important to draw attention to another potential and widespread source of mysterious cell attachment and growth problems. (For more detailed information on the problem of cell culture contamination, refer to Lincoln and Gabridge, 1998; Ryan, 1994; Rottem and Barile, 1993; McGarrity, 1982; McGarrity, 1976.) Due to the very high densities they can achieve in cell culture (up to 10 8 /ml), mycoplasmas (unlike other contaminants such as bacteria and fungi) cause serious adverse effects on cell cultures without clouding the medium or being visible under the microscope (See Figure 6). Mycoplasmas often grow attached to the cell membrane; as a result, a single cell may have several hundred mycoplasma on its membrane which greatly affects the ability to attach and grow. An ongoing mycoplasma screening program is an essential requirement for all cell culture labs working with cell lines (Lincoln and Gabridge, 1998). Without such a program, mycoplasma contamination, along with the associated problems and embarrassment, is likely to occur at some point. Technical Assistance For additional technical support and product information, please call or Technical and product information is also available on the Corning Life Sciences web site at 9