PimCell. microfluidic construction kit ADVANCED CELL CULTURE CATALOGUE & APPLICATION NOTES

Transcription

1 2018 PimCell microfluidic construction kit ADVANCED CELL CULTURE CATALOGUE & APPLICATION NOTES

2 PimCell microfluidic construction kit PimCell line is a group of related cell culture microfluidic products developed and manufactured by PimBio B.V. (The Netherlands). With our plug-and-play modular microfluidic technology you will significantly accelerate the development of your own 3D cell culture products which comply with microscopic slide and microtiter plate standards. Advantages of PimCell plug & play microfluidic technology highly reproducible adapted to existing lab equipment and automation comply with microscopic slide standards meeting all of the requirements of the ANSI/SLAS standard economical access to the fields of organ-on-a-chip technology adapted to every desired cell culture scaffolding short learning curve static and dynamic cell culture excellent imaging simple maintenance About PimBio B.V. PimBio B.V. is a privately held company founded in 2014 to develop new and promising microfluidic devices for bioengineering of physiologically relevant living tissue for different applications. The development of a platform technology producing 3D physiologically relevant in vitro models will enable the generation of fully differentiated living tissue types and whole organs which can be used in scientific research, regenerative medicine and industry. PimBio has the vision to operate as a market leader in the manufacturing of microfluidic devices, by investing in quality, innovation and capacity. PimBio supports the 3Rs, the guiding principles of reduction, refinement and replacement of animal experiments. Having a basis in The Netherlands with our partner network extending into West Europe we can provide existing processes and manufacturing methods for in vitro applications. All our products are manufactured and packaged in clean rooms by our partners certified according to ISO 9001 and ISO The essence for PimBio is to work together with customers on innovative and reliable solutions in the complete trajectory from idea to manufacturing. The mission of PimBio is to provide research and industrial community a unique combination of micro technologies, materials, microfluidics knowledge and customer application know-how, which enables PimBio to offer customers innovative and sustainable solutions with 3Rs potential to improve people s lives. PIMBIO B.V. 1

3 PimCell microfluidic miniplate M5 M5 is our first PimCell microfluidic plate s model. This model is a microscopy-slide-sized and microfluidics based cell culture miniplate that enables culturing and screening of an unlimited number of physiologically relevant organ and tissue models. All PimCell microfluidic devices are based on the the ANSI/SLAS microtiter plate standards, robotand pipette operatable and support a wide range of assays. The miniplates are made from a thermoplastic medical grade polymer which is completely nonpolar and amorphous. For the highest quality images, the optical properties of the polymer bottom are comparable to those of glass. It has a very low autofluorescence, a very low permeability for water vapour and a low capacity for the absorption of water. PimCell miniplates are compatible with all microscopy and readout equipment, including confocal microscopy and luminescent readers. The PimCell microfluidic miniplate M5 contains three independent cell culture units (chips) on a slide of standard microscopy format. Each unit has one open access cell culture chamber and two separated flow channels with high fluidic resistance central parts which can allow to perform gravity driven perfusion of microfluidic channels (please see our Application Notes for more information). Each flow channel has one inlet and one outlet connected to own inlet/outlet port. A part of wall of the high fluidic resistance part of each channel is open into the open access cell culture chamber. The main utility of the high resistance part is to reduce the flow rate of cell culture media during long-term experiments with unidirectional flow in the microfluidic channels. The microfluidic channels are adapted for microvascular in vitro tissue engineering. Technical Features: - product number: application: one-use R&D only - available: unsterile - sterilize with 70% ethanol - material: Zeonor - polymer bottom: Zeonor foil 188 µm - cell culture chamber 1.43mmX1.43mmX0.4mm with 0.86 µl volume - short microfluidic channel has 140 µm depth, 1.5 mm width and 2.87 µl volume - long microfluidic channel has 140 µm depth, 1.5 mm width and 4.58 µl volume - high fluidic resistance part has 25 µm depth, 250 µm width, 1.43 mm length and about 9nL volume - one well has 59 µl volume - oil sensitive - can be used with silicone immersion oil only! Fig. 1: Top view of PimCell microfluidic miniplate M5 with three cell culture units (chips) per miniplate. One cell culture unit has one open access cell culture chamber, one short microfluidic channel and one long microfluidic channel PIMBIO B.V. 2

4 Triple 500 µl reservoirs with luer interface These reusable triple media reservoirs with a volume of 500 µl per single reservoir has a standard male luer interface to connect to an incorporated female luer port of a PimCell miniplate. Covering with sterile breathable adhesive film can reduce problems associated with media evaporation and contamination risk. Technical Features: - product number: application: R&D cell culture only - depth 2 cm and volume 500 µl (25 µl per 1 mm depth) - available: unsterile - autoclavable C 20 min - suitable for PimCell miniplates with female luer ports - material: polypropylene (opaque) - 1 unit consists of 24 pieces Fig. 2: Triple media reservoirs PIMBIO B.V. 3

5 Syringe connector Our syringe connector allows to couple male luer interfaces of a syringe with tubing deriving from microfluidic channels. Technical Features: - product number: application: R&D cell culture only - available: unsterile - autoclavable C 20 min - suitable for syringe pumps - material: clear polycarbonate - 1 unit consists of 20 pieces Fig. 3: A transparent syringe connector (connected to a male luer fluid connector) PIMBIO B.V. 4

6 Silicone tubes Our silicone tubing (ID: 1.5 mm; OD 3.0 mm, 1 m) is biologically inert and does not inhibit cell culture. It stays flexible down to -50 C and maintains its chemical, mechanical and electrical performance up to +200 C. The tubing meets the European Pharmacopoeia requirements for silicone elastomer for closures and tubing, USP Class VI biological tests and ISO biocompatibility (systemic toxicity, irritation, sensitization, cytotoxicity, hemocompatibility and Ames test). It meets FDA and BfR positive lists as well as food grade tests according to EC directives. You can use silicone tubes to connect connectors from respective interfaces or to connect hard plastic tubes like PTFE tubings with pumps. Technical Features: - product number: application: R&D cell culture only - available: unsterile - autoclavable C 20 min - suitable for syringe pumps - material: silicone - 1 unit consists of 5 m tubing Fig. 4: Silicone tubing PIMBIO B.V. 5

7 Male luer plugs Our transparent male luer plugs enable you to close the female luer interfaces on our miniplates. With the help of these plugs, you can introduce cell culture scaffolding seeded with cells inside our open top cell culture chamber or move liquid through the female luer interface into the fluidic channels on chip and safely close the fluidic interfaces to avoid a contamination risk. Clear polycarbonate allows you to directly visualise cell culture inside our cell culture chamber. Technical Features: - product number: application: R&D cell culture only - available: unsterile - autoclavable C 20 min - material: clear polycarbonate - 1 unit consists of 40 pieces Fig. 5: Male luer plugs PIMBIO B.V. 6

8 Male luer fluid connectors Male luer fluid connectors allow to link the fluid ports on top of the miniplates with those of adjusted slides, with typical laboratory equipment such as syringes or with standard tubes. Technical Features: - product number: application: R&D cell culture only - available: unsterile - autoclavable C 20 min - material: clear polycarbonate - 1 unit consists of 40 pieces Fig. 6: Male luer fluid connectors PIMBIO B.V. 7

9 Application notes With our plug-and-play PimCell microfluidic construction kit you can open new ways to significantly accelerate development of your own OOC products which comply with worldwide accepted microscopic slide and microtiter plate standards. These application notes can help you to run you experiments with our 3D cell culture system and describe first basic steps to start with microfluidic PimCell miniplates. This part introduces how to operate PimCell miniplates for advanced cell culture. Using our microfluidic construction kit you can create either static or dynamic cell culture. Option 1 Static cell culture (without flow inside microfluidic system) Fig. 7: A bottom view of a microfluidic cell culture unit of PimCell microfluidic miniplate M5. A short microfluidic channel (AB) has one inlet port (A) and one outlet port (B). A long microfluidic channel (DE) has also one inlet port (D) and one outlet port (E). These two microfluidic channels direct communicate through an open access cell culture chamber (C) Fig. 8: A scheme of cell culture without flow between a cell culture chamber (C) and microfluidic channels (AB and DE) PIMBIO B.V. 8

10 Option 2 Dynamic (gravity driven) cell culture with flow along the cell culture chamber Fig. 9: A scheme of 3D cell culture with flow (white arrows) inside microfluidic channels (AB and DE) along the cell culture chamber (C) Fig. 10: A bottom view of high fluidic resistance central parts (FG and HI) of microfluidic channels. These two microfluidic channels allow long-term gravity driven unidirectional perfusion without any additional equipment like a pump or a perfusion rocker platform (for more information please see Application Notes). PIMBIO B.V. 9

11 Static cell culture (Option 1) The most simple way to use PimCell miniplates for 2D cell culture is to introduce cell culture media and cells into the cell culture chamber of one microfluidic unit of PimCell miniplate. This Application Note can be easy adapted for 3D cell culture just by introducing a 3D cell culture scaffolding like Matrigel into an open access cell culture chamber. 1. Preparation steps 1.1 Sterilization Our cell culture products which direct contact with cell culture like miniplates, reservoirs and connectors are produced in cleanroom facilities and clean areas to prevent physical impurities and contamination. The materials are not sterilized when delivered. Our materials are compatible with ethanol (EtOH) sterilization. Sterilise unsterile products using 70% EtOH. It is highly recommended to rinse sterilized thoroughly with sterile water, buffer or media before cell culturing to remove EtOH residues. 1.2 PimCell coating PimCell microfluidic miniplate M5 is made from a thermoplastic medical grade polymer which is biocompatible and permits direct cell growth but it is very hydrophobic and should be coated for efficient adhesion of most cells. All coating solutions can be calculated for a specific amount of coating material per area (µg/cm²) recommended by the manufacturer s reference. Dilute a coating solution to the desired concentration according to the manufacturer s specifications or references. For the coating setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 coating solution one sterile plastic 1.0 ml insulin syringe one sheet of sterile (para)film Don t use glass or metal syringes to avoid miniplate damaging Work under sterile conditions Gently connect an insulin syringe loaded with 100 µl coating solution to the well of the open access cell culture chamber Gently introduce the coating solution into microfluidic system by pushing the syringe s plunger Fig. 11: Male luer slip of an insulin syringe connected to female luer slip of the well of the open access cell culture chamber PIMBIO B.V. 10

12 1.2.2 The solution will appear inside inlet/outlet wells of microfluidic channels Disconnect the syringe and cover the wells with sterile (para)film or breathable adhesive film to avoid evaporation and incubate according to the manufacturer s specifications or references Fig. 12: A well of one microfluidic channel filled with a coating solution After incubation, remove the (para)film from the wells and gently aspirate the microfluidic system through inlets or outlets ports completely using the connected to the cell culture chamber syringe by pulling its plunger. Avoid aspiration through the open access cell culture chamber to protect integrity of its coating Add 50 µl ultra-pure water or PBS per each well of the microfluidic unit and gently aspirate the microfluidic system through inlets or outlets ports completely using a syringe by pulling its plunger Repeat Step three times to rinse the microfluidic system. Aspirate all microfluidic system completely and let it dry at room temperature. PimCell microfluidic system is ready to use. Store under sterile conditions and use as soon as possible Fig. 13: Insulin syringe with a standard male luer slip can be easy connected to incorporated female luers of PimCell miniplate 1.3 Degasification Place PimCell microfluidic miniplate M5 at 37 0 C for 24h before PimCell miniplate loading (e.g. into a cell culture incubator) At a higher temperature plastic material can take up less gas than at a lower temperature. If plastic materials like olefin have been stored at room temperature, the gas (air) that is solved in plastic will be released from plastic material when heated up in the incubator. Air bubbles will then emerge PIMBIO B.V. 11

13 inside microfluidic channels or tubing and close the flow pathway. Therefore, you have to prevent this effect by degassing all the parts involved beforehand. Each time you take the system out of the incubator, this process will start over. Take care that you work as fast as possible at room temperature to constant keep the temperature of the system at plus 37 0 C. Never leave the microfluidic system outside the incubator for more than 15 minutes. Always warm media and PBS in a 37 0 C water bath before opening. This will avoid the potential contamination from air being drawn into a cold bottle of media/pbs upon opening. 2. 2D Cell Culture for Option 1 For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 one sterile plastic 1.0 ml insulin syringe two triple media reservoirs transparent sterile breathable adhesive film cells and cell culture media 2.1 Calculate number of cells per ml cell suspension Prepare cell suspension and cell culture media according to the manufacturer s specifications or reference 2.2 Place one degassed and coated PimCell miniplate, two triple media reservoirs, one syringe and one sheet of sterile adhesive film into a sterile laminar flow cabinet and unpack all materials 2.3 Connect a syringe loaded with 100 µl of cell culture media to the cell culture chamber and gently introduce the cell culture media into the microfluidic system by pushing the syringe s plunger Fig. 14: Cell culture media introduction into microfluidic system 2.4 Gently but tightly connect reservoirs to all wells of the microfluidic channels Tip! All models of PimCell microfluidic miniplates have a standard top part with a footprint of 96-well microtiter plate according the ANSI/SLAS standards. PimCell M5 model has one well per each cell culture unit which is not connected to its microfluidic system. Don t use this well of PimCell M5 model and a part of a triple media reservoir above this well PIMBIO B.V. 12

14 2.5 Gently add 20 µl cell suspension to the cell culture chamber media reservoir via the wall above the cell culture media Cover all reservoirs with the breathable adhesive film Incubate at 37 0 C and 5% CO 2 for half an hour for cell attachment Fig. 15: Cell suspension introduction 2.6 After cell attachment, remove the breathable adhesive film from the cell culture chamber media reservoir and gently add 200 µl cell-free media via the wall above the cell suspension Don t trap air bubbles Avoid pointing the pipet tip directly onto the channel s inlet 2.7 After that, tightly cover the cell culture chamber reservoir with the sterile breathable adhesive film and incubate at 37 0 C and 5% CO 2 If you want to cultivate the cells for more than one day under static conditions, a complete media exchange has to be made every 24 hours 3. Medium exchange 3.1 For media changing remove the breathable adhesive film and aspirate the old media from all reservoirs Avoid pointing the pipet tip directly onto microfluidic channel s inlets to prevent aspiration of media from microfluidic channels 3.2 After that, gently add 240 µl of fresh media into the cell culture chamber reservoir via the wall above microfluidic channel s openings Don t trap air bubbles Fig. 16: Media aspiration Avoid pointing the pipet tip directly onto the channel s openings Fig. 17: Media introduction into an empty reservoir PIMBIO B.V. 13

15 3.3 Add 190 µl of fresh media into the reservoirs of the microfluidic channels via the wall above microfluidic channel s openings. Gravity driven flow from the reservoir of the cell culture chamber into the reservoirs of the microfluidic channels will refresh all media inside microfluidic channels during media level equilibration Cover the reservoirs with the sterile breathable adhesive film and incubate at 37 0 C and 5% CO D Cell Culture for Option 1 The most simple way to adapt Option 1 to 3D cell culture is to introduce desired 3D cell culture scaffolding (e.g. Matrigel) direct into a cell culture chamber. Suggestion: Since two microfluidic channels of one cell culture unit can be easy divided by 3D cell culture scaffold we have different options for coating of microfluidic channels: 1) coating before scaffold introduction; 2) coating after scaffold introduction; 3) no coating at all since 3D scaffold offers excellent cell attachment surface. It is possible to use different coatings in different microfluidic channels of one cell culture unit. For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 two sterile plastic 1.0 ml insulin syringes two triple reservoirs sterile breathable adhesive film 3D cell culture scaffolding (e.g. Matrigel) cells and cell culture medium 4.1 Prepare desired scaffolding/cell mix and cell culture medium according to the manufacturer s specifications or references 4.2 Place one sterilized and degassed PimCell miniplate, sterilized reservoirs, two sterile syringes and a sterile breathable adhesive film into a sterile laminar flow cabinet and unpack all materials 4.3 Gently introduce 1 µl of scaffolding or scaffolding/cell mix into the cell culture chamber of warm PimCell miniplate direct above the central part of the cell culture chamber to fill it with the cell culture scaffolding Fig. 18: Cell culture scaffolding introduction PIMBIO B.V. 14

16 Tip! Let op, use only warm miniplates to stimulate direct polymerisation of a temperature sensitive scaffolding like Matrigel. The polymerisation should occur direct on the miniplate before liquid scaffolding can escape from the cell culture chamber into the microfluidic channels and close it Fig. 19: Polymerized scaffolding inside the cell culture chamber 4.4 After that, very gently add 40 µl of cell culture media into the cell culture chamber s well via the wall of the well above the polymerized scaffolding or scaffolding/cell mix Don t trap air bubbles Avoid pointing the pipet tip directly onto the scaffolding Fig. 20: Media introduction via the wall of a cell culture chamber above the polymerized scaffolding 4.5 Add 50 µl cell culture media to the inlet reservoir of the short microfluidic channel (see AB of Fig.7). Connect an empty syringe to the outlet port of the short microfluidic channel and very gently aspirate media from the outlet well through the microfluidic channel by pulling the plunger of the syringe until media appears inside the outlet well. Let op, push the syringe s plunder very gently and slowly to avoid the hydrogel disconnection from the bottom of the cell culture chamber by hard flow inside the high resistance central part of each microfluidic channel Tip! In most cases cell culture scaffolding/plastic interactions are non-covalent and this means that scaffolding have very fragile connection with plastic surfaces of microfluidic miniplates. Be very gently with medium introduction into microfluidic system loaded with a cell culture scaffolding to avoid the scaffolding dissociation from the microfluidic pathway. Never push media inside a microfluidic channel and use aspiration method only! 4.6 Disconnect the syringe from the short microfluidic channel 4.7 Repeat Step 4.5 with the long microfluidic channel (see DE of Fig.7) 4.8 Disconnect the syringe from the long microfluidic channel and connect all wells to the media reservoirs 4.9 Add 200 µl of the cell culture media per one media reservoir via the walls above microfluidic system openings Cover the caps with the breathable adhesive film to prevent contamination and reduce media evaporation. Incubate at 37 0 C and 5% CO 2 PIMBIO B.V. 15

17 Dynamic cell culture (Option 2) with gravity-driven flow along the cell culture chamber Gravity is the driving force that accomplishes the transfer of fluids. Gravity-driven flow is movement of fluids from place to place (e.g. from one media reservoir to another) due to hydrostatic pressure exerted by a difference in height (relative to gravitational field) between these two places. The most simple way to use PimCell miniplates for Option 2 is creation a hydrostatic pressure difference ( P) between an inlet and an outlet reservoir of a microfluidic channel to establish gravity-driven flow of cell culture media through the high resistance central part of the microfluidic channel. Fig. 21: A schema of gravity-driven flow inside a microfluidic channel due to hydrostatic pressure difference between two media reservoirs Considering a pressure driven Poiseuille flow in a long, straight and rigid channel, the volume flow rate Q is determined by the ratio of the driving pressure difference ΔP and the hydraulic resistance R (of the high resistance part of PimCell microfluidic channel in our case): Q = P/R With a given driving pressure difference ΔP, the volume flow rate Q is based on the hydraulic resistance R. The higher the hydraulic resistance R, the lower the volume flow rate Q. The flow rate and fluidic shear stress of the high resistance central part of the microfluidic channel next to the open access cell culture chamber can be controlled by modifying the hydraulic resistance R. The hydraulic resistance of a straight channel (the high resistance flow channel) with rectangular Rrec cross-sections is: where w, h and L are the width, height, and length of the straight channel, and η is the fluidic viscosity. When the fluidic viscosity is 0,01 dynes/cm 2 s (like cell suspension) the hydraulic resistance of the high resistance flow channel will be about 4,68 x10 6 (please see Table 1 with estimated flow rates). PIMBIO B.V. 16

18 P [cm] Q [µl/hour] Q [µl/day] Days of stable perfusion per mm of P drop 1 0,07 1,84 13,5 0,8 0,06 1,47 16,9 0,6 0,04 1,10 22,6 0,4 0,03 0,73 33,9 0,2 0,01 0,36 67,8 0,1 0,007 0,18 135,6 Total days of gravity-driven flow 290 Table 1: Estimated flow rates of gravity-driven perfusion by hydrostatic pressure drop of media with the fluidic viscosity of 0,01 dynes/cm 2 s. Low flow rates inside the high resistance flow channel allow sufficient media refreshment inside this channel since the volume of the high resistance flow channel is only about 9 nl. 1. Preparation steps are general for 2D and 3D cell culture 1.1 PimCell coating See Steps of Option Sterilisation Sterilise the unsterile materials using 70% EtOH. It is highly recommended to rinse the products thoroughly with sterile water, buffer or media before cell culturing to remove EtOH residues. 1.3 Degasification Place PimCell microfluidic miniplate M5 and flow accessories at 37 0 C for 24h before PimCell miniplate loading (e.g. into a cell culture incubator) 2. Cell culture for Option 2 For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 sterile plastic 1.0 ml insulin syringe(s) two triple media reservoirs one sheet of breathable adhesive film cells, cell culture media and scaffolding (e.g. Matrigel) 1 Prepare desired scaffolding/cell mix and cell culture media according to the manufacturer s specifications or references 1.1 Place degassed and sterilized PimCell miniplate, sterilized reservoirs, sterile syringe(s) and sterile breathable adhesive film into a sterile laminar flow cabinet and unpack all materials 1.2 For 3D cell culture preparation see Steps of Option Add appropriate amount of cell culture media to each media reservoir to create desired hydrostatic pressure difference and gravity-driven flow between reservoirs 1.4 Cover all media reservoirs with the breathable adhesive film to prevent media evaporation from the microfluidic channels during incubation PIMBIO B.V. 17

19 FAQ 1 Q: Can I use PimCell miniplate to create a body-on-a-chip system? A: Yes, it is possible to interconnect different cell culture units using our male luer connectors. 12 interconnected cell culture units of four PimCell miniplates M5 could create coculture of 12 different in vitro models. 2 Q: How can I use PimCell miniplate for anaerobic cell culture? A: PimCell miniplate is a closed and gas impermeable system when you replace our silicone tubing with a gas impermeable tubing. There are different options known e.g.: 1) completely fill the flow pathway with an anaerobic cell culture media under anaerobic conditions and pump the anaerobic media through the microfluidic system; 2) cover anaerobic cell culture media in all reservoirs with a special oil to create an anaerobic barrier between air and media. 3 Q: How can I use PimCell miniplate for explants cultures? A: The most simple way to use PimCell miniplates for explant cultures is to use an open access cell culture chamber as a perfusion chamber: 1) place explant into a cell culture chamber; 2) connect the chamber to a pump via our pump fluid connector; and 3) start perfusion of a culture media through the chamber into microfluidic channels. Tip! - Cut a small piece of an explant out using a sterile biopsy punch with 1.5 mm hole - When you have a permanent moving explant like intestinal mucosa you can cut a 4.0 mm diameter out from a highly porous membrane (using a punch with 4.0 mm hole) to cover & fix the moving explant inside the cell culture chamber by fixing the membrane with a transparent male luer connector. 4 Q: I need a system for live visualization of epithelial-endothelial interactions (2D/2D). How can I use PimCell miniplate to create such in vitro model? A: You can create a 2D membrane between microfluidic channels and a cell culture chamber using coating technology e.g.: 1) coat an open access cell culture chamber with a hydrogel like Collagen type I which could create a very thin membrane between microfluidic channels and the cell culture chamber; 2) seed e.g. blood endothelia cells into the microfluidic channels and epithelial cells e.g. respiratory epithelial cells into the cell culture chamber. Additionally, the open cell culture chamber allows to create an air-liquid interface. PIMBIO B.V. 18

20 Disclaimer Whilst we endeavour to ensure that the information in this catalogue is correct, PimBio B.V. reserves the right not to be responsible for the correctness, completeness or quality of the information provided. Liability claims regarding damage caused by the use of any information provided, including any kind of information which is incomplete or incorrect, will therefore be rejected. All offers are not-binding and without obligation. Parts of the pages or the complete publication including all offers and information might be extended, changed or partly or completely deleted by PimBio B.V. without separate announcement. All products and services offered within this catalogue are intended for R&D use only. PimBio B.V. Kloosterstraat AB Oss The Netherlands info@pimbio.com PIMBIO B.V. 19