Protocol USD2991 (1) Pall SoloHill Small-Scale Microcarrier Screening Studies Using Six-Well Plates and Snap-Top Tubes

Protocol USD2991 (1) Pall SoloHill Small-Scale Microcarrier Screening Studies Using Six-Well Plates and Snap-Top Tubes

Introduction Static cell culture experiments in multi-well plates and snap-top tubes are useful starting points to assess microcarrier compatibility with cells. Often, cells grow to the highest densities on microcarriers with the best attachment kinetics. The following protocol provides a framework to begin the Pall SoloHill microcarrier screening process, as attachment kinetics are dependent upon cell type as well as microcarrier and environmental conditions (e.g., media composition). The goal of this screening study is to familiarize investigators with microcarrier handling and the visualization of cells on microcarriers, as well as to provide quantitative measures of cell attachment and growth. This protocol is a precursor to experiments aimed at optimization of conditions in small-scale stir vessels and subsequent transfer of developed protocols into larger bioreactor formats. An important parameter to consider when performing screening studies is the composition and concentration of serum and other proteins in the medium during attachment. Serum contains proteins that can facilitate cell attachment to microcarriers, but it also contains albumin and other proteins which could potentially act as inhibitors of attachment at elevated concentrations. Proteins commonly used in serum-free media formulations can also influence attachment kinetics and efficiency. The sensitivity of cell attachment to serum and/or other proteins is cell type specific. For some cell types, attachment is completely independent of protein concentration. Therefore, it follows that protein concentrations should be closely monitored during screening studies. In the case of serum-free cultures, proteins and other molecules that function as serum replacements may also influence cell attachment. Manipulating concentrations of these components during attachment studies may be also prove useful when attempting to optimize cell attachment to microcarriers. For serum-containing cultures, it is a good strategy to perform screening studies twice. The first round of screening should be performed at standard serum concentrations (i.e. 5%). Afterwards, a second round of screening can be repeated at low serum levels for the microcarrier candidates that did not perform well in the first study, as the poor performance may be the result of serum sensitivity, and not cell-microcarrier compatibility. As a general guideline, low serum attachment is typically performed between 0.05% and 0.5% serum, but not completely serum-free, as the serum does contain factors that can enhance cell attachment and prevent cell damage. Although performing cell attachment under low serum conditions adds an extra step to the cell culture process, it has the potential reward of enhancing cell growth, as high cell densities often depend on good attachment kinetics. A similar strategy can be used for serum-free or chemically-defined systems. Procedure 1. Selection of Sample Container Prepare multiple replicates of each sample for each cell-microcarrier condition to be tested. These replicates allow one to harvest the cells at various time points over four to five days. Obtain a suitable container for each sample to be evaluated. Such containers include cell culture plates (see Table 1 for details regarding cell culture plates) and snap-top tubes (microfuge or 15 ml tubes are examples). The optimal cell culture plates to use are those with hydrophobic surfaces. The hydrophobic nature of these surfaces promotes cell attachment to microcarriers. Corning CoStar u Ultra-Low Attachment cell culture plates can be used for this purpose. If cost savings are of interest, a standard non-tissue culture-treated plate can be modified with a hydrophobic coating such as poly (2-hydroxyethyl, methylacrylate; phema, Sigma Aldrich P3932). If the experiment is limited to a of couple days, a standard non-tissue culture-treated plate can be used. 2

Cell culture plates are convenient for visualizing cells on microcarriers using a microscope and can accommodate larger culture volumes than a microfuge tube. Plates should be used if the goal of the experiment is to visualize cells for microcarrier compatibility. However, when analyzing many samples at different time points, tubes allow for easier sample handling and assaying. Tubes should be used when quantification is needed, such as assessing attachment kinetics and cell growth. If the goal of the study is to access attachment kinetics, analyze early time points (i.e. 15, 30, 45, 60, 90, 120, 180 minutes, etc.). Table 1 Multi-well plates for microcarrier assessment Catalog # Wells per Plate Media Volume (ml) Growth Area (cm 2 ) Well Diameter (mm) BD Falcon u 351146* 6 2 9.5 34.8 BD Falcon 353224** 6 2 9.5 34.8 Corning CoStar 3471 6 2 9.5 34.8 BD Falcon 351143* 12 1.3 3.8 22.1 BD Falcon 353225** 12 1.3 3.8 22.1 BD Falcon 351147* 24 0.9 1.9 15.6 BD Falcon 353225** 24 0.9 1.9 15.6 Corning CoStar 3473 24 0.9 1.9 15.6 Note: Other equivalent brands of plates (i.e., Thermo Scientific Nunc) may be used. * non-treated ** tissue culture-treated ultra-low attachment 2. Microcarrier Surface Area Considerations To ensure the most accurate and reproducible experimental results, each sample should contain an equivalent microcarrier surface area, medium volume, and cell number. Since the surface area to mass ratio varies depending on the microcarrier type, some samples may contain a larger microcarrier mass (milligrams) than others to maintain constant surface area across all samples (see Table 2). Table 2 Example of Material Requirements to Maintain Consistent Surface Area for Different Microcarrier Types Microcarriers with diameter Microcarriers with diameter Microcarriers with diameter range 160-200 μm range 90-150 μm range of 125-212 μm Relative Density Range 1.090-1.150 1.022-1.030 1.022-1.030 Nominal Surface Area 515 480 360 per Gram (cm 2 ) Total Sample Volume (ml) 2 2 2 Volume of 25 mg/ml Microcarrier 0.777 0.833 1.111 Slurry per Sample (ml) Microcarrier Mass (mg) 19.4 20.8 27.8 Total Surface Area per 10 10 10 Sample (cm 2 ) * Denotes all microcarriers types contained in the standard Pall SoloHill microcarrier starter kit: Hillex II, Plastic, Plastic Plus, Collagen-Coated, FACT III, ProNectin F- Coated, Glass-Coated. Equivalent microcarrier surface areas are achieved by adding various quantities of microcarriers to each well. www.pall.com/biopharm 3

3. Microcarrier Stock Preparation To assist in the delivery of microcarriers to each sample, prepare a stock of microcarriers at 25 mg/ml in water by autoclaving for 30 minutes at 121 C. If sterility (as defined by end user validation) is maintained, this stock solution can be used for up to 3 months. Some liquid may evaporate during autoclaving. Replace this liquid to maintain a 25 mg/ml suspension concentration. Alternately, after autoclaving, aseptically aspirate the water, being careful not to aspirate microcarriers, and resuspend the microcarriers in basic (non-serum containing) media at a concentration of 25 mg/ml. This strategy has the benefit of eliminating the need to aspirate water in each individual sample container. Do not store microcarriers for extended periods of time in media. Depending on experimental goals, a more concentrated or dilute stock might be preferred. One stock suspension should be prepared for each microcarrier type. 4. Microcarrier Stock Volume Requirements Initial attachment studies should be performed at a microcarrier suspension concentration of 5 cm 2 /ml. When using microfuge tubes, each sample will contain 1 ml of media; therefore, each sample will require 5 cm 2 of microcarrier surface area. Calculate the volume of microcarrier stock required to deliver this surface area by using the following equation: V M = V SC S A M C M V M - Target microcarrier stock solution volume (in ml) V s - Sample volume (1 ml for snap-top tubes) C s - Sample microcarrier concentration (5 cm 2 /ml) A M - Microcarrier surface area (cm 2 /g) C M - Microcarrier stock solution concentration (0.025 g/ml) If the goal of the experiment is to assess attachment kinetics, it is beneficial to use a higher microcarrier concentration, such as 10 cm 2 /ml. This assures more cells are present, which improves cell counting accuracy. In these cases, replace the 5 cm 2 /ml above with the actual concentration to determine the volume of stock material to add. When using cell culture plates, a larger volume sample can be tested depending on the size of the well. If a larger volume is to be tested, replace the 1 ml sample volume with the actual sample volume in the above calculation. 5. Microcarrier Addition to Sample Container Aseptically suspend the microcarrier stock via agitation or pipetting, and then dispense the appropriate volume of microcarrier stock suspension into each sample. Allow the microcarriers to settle to the bottom of the sample. Once settled, aseptically aspirate the water, being careful not to aspirate microcarriers. If a mistake occurs when performing this step on a cell culture plate, remove microcarriers from well and discard, rinse wells with sterile phosphate buffered saline (PBS) or water and re-aliquot microcarriers into the freshly cleaned wells. If dispensing a microcarrier stock suspended in basal media, there is no need to aspirate the media as would be performed if the stock were prepared in water. 4

6. Sample Volume Considerations Before adding any material to the sample container, consider how much volume of each material will be required to fill the container to its proper final volume. For instance, a 1 ml snap-top microfuge tube will probably have volume requirements broken down as follows: 50 µl (5%) FBS or equivalent serum (subject to change depending on the target serum concentration) 50 µl (5%) cell slurry used to seed or inoculate the sample with cells 900 µl (90%) basal media 7. Media Addition to Sample Container Add an appropriate volume of medium to each sample such that the final sample volume is on target and consistent with the targeted microcarrier suspension concentration. Redistribute the microcarriers by gentle agitation, or rocking the six-well plate or gently inverting the tube. If performing attachment at low protein/serum concentrations, only add basal medium at this step. The very low amounts of serum required for cell attachment will be added later with the feed cell slurry. If performing attachment at standard serum/protein concentrations, add both basal medium and serum at this step. If the goal of the experiment is to culture cells for longer than one day, and attachment is being performed under low-protein conditions, serum or other protein supplements should be added after attachment to bring the solution to the normal protein concentrations (i.e. 5%.) This is highly dependent on the conditions tested, but generally will occur 1-4 hours after inoculation or once 80-90% of the cells have attached. It is important to minimize this time period if possible, so that cell health isn t adversely affected by the absence of serum. 8. Feed Cell Slurry Preparation The cell feed must be prepared in a manner to achieve a uniform, single-cell suspension. Perform a cell count (or nuclei count) on this single-cell suspension so that that the cell slurry concentration is known. Calculate the volume of cell slurry required to supply the appropriate number of cells to each sample by using the following equation. Then, dilute this volume to 5% of the total sample volume with serum-containing medium. V c = V SC S N i C C V c - Target cell slurry volume (in ml) V s - Sample volume (1 ml for snap-top tubes) C s - Sample microcarrier concentration (5 cm 2 /ml) N i - Cell seeding density (20,000 cells/cm 2 ) C c - Cell slurry concentration (cells/ml) One strategy to obtain a uniform single-cell suspension of feed material is to gently pipette suspension of trypsinized cells up and down. For low-serum attachment conditions, the serum present during attachment will be provided via the feed cell slurry. Therefore, it is important to prepare this material at the appropriate serum concentration, taking into account the 20x dilution when this material is added to the sample. For standard-serum attachment conditions, prepare the feed cell slurry at the target serum concentration. If the experimental goal is to evaluate attachment kinetics, cells should be seeded at 200,000 cells/cm 2 to facilitate accurate cell counting. www.pall.com/biopharm 5

9. Cell Addition to Sample Container Acclimate the microcarriers and medium in a 37 C cell culture incubator for at least 20 minutes. This helps equilibrate the ph at favorable conditions before cells are added. Once equilibrated, add the required volume (5% of total volume) of feed cell slurry to each sample. 10. Sample Mixing and Incubation Carefully agitate multi-well plate for 10 to 15 seconds with a side-to-side and a back-and-forth motion or gently invert the tubes to ensure a thorough mixing of the microcarriers and cells. In the case of the cell culture plate, this will also prevent the media from splashing onto the plate cover or between wells. Incubate at 37 C. Agitate every 15 minutes for the first hour or longer if attachment kinetics are slower than anticipated. Perform media exchanges as necessary after attachment has occurred if samples are to be cultured for a long period of time. Analysis Cell Visualization for Biocompatibility 1. Cell attachment and spreading can be observed at 100x magnification on an inverted microscope at various time intervals and qualitative assessment of the attachment and spreading can be performed. Cells can be visualized at the edges or circumference of the microcarriers as rounded (initial attachment phase), gumdropshaped (early spreading) or flattened (completely spread). Figure 1 Cell Shape a) Rounded b) Gumdrop c) Flattened Ability to visualize spread cells on microcarriers is dependent upon cell characteristics. For example, fibroblast-like cells tend to be flatter than epithelial cells when they are spread on surfaces and are more difficult to visualize without using special techniques to aid in visualization. Since samples are only intermittently mixed, cell attachment to microcarriers can be uneven. When evaluating the performance of each sample, it is more important to consider the percentage of cells that have attached and morphology of such cells. A general benchmark for good performance is 90% cell attachment to microcarriers within the first hour of incubation. Cell/microcarrier conditions that perform well by this criterion should be further evaluated in small-scale stirred vessels, which allow for better control of cell attachment and growth uniformity. 2. Several techniques can be used to enhance visualization of cells on microcarriers if needed: Fluorescence techniques (pre-loading of cells with fluorescent dyes prior to binding) DAPI staining after fixation with 4% paraformaldehyde Acridine orange Direct visualization by phase microscopy Cell tracker staining for Hillex II products 6

Cell-microcarrier compatibility can be evaluated visually over the course of up to one week. Visual observation of the cells under a microscope will indicate whether cells are healthy and expanding when exposed to the microcarrier surface coating. Unhealthy cells will generally detach from the microcarrier surface and die in suspension without dividing. Cell Counts Several methods can be used to successfully harvest cells for counting. The simplest technique is to aspirate the media from the sample, being careful not to shear cells. Then remove cell-laden microcarriers from sample. Wash the remaining microcarriers with phosphate buffered saline (PBS), aspirate, and add trypsin. Allow cells to incubate in trypsin until they dissociate from the microcarrier surface (generally 10 minutes at 21 C, depending on cell type), then pipette to obtain a single-cell suspension. Cells can then be counted with a hemocytometer using trypan blue. Microcarriers generally do not get under the cover slip, so they will not interfere with the count. Keep track of all the reagent volumes used as well as the medium volume removed so that the cell count can be adjusted with the appropriate dilution/concentration factor. If evaluating cell attachment kinetics, repeat the above procedure, but save the aspirated medium in separate container to count in addition. The percentage of cells attached at each time point will be the cells counted on the microcarriers divided by the total number of cells added to the well. To close the mass balance, perform a cell count on the aspirated media. The cell counts on the microcarriers as well as the cell counts of the aspirated medium should add up to equal the number of cells originally added to the sample. If performing the experiment on a cell culture plate, it may be easier to first transfer the sample from the plate to a microfuge tube before beginning the harvest procedure described above. Once cells have attached to microcarriers, they will begin to spread and proliferate. As time progresses, these new cells will impact the accuracy of cell count assay used to evaluate attachment kinetics. Depending on the growth rate of the cell type, the attachment kinetics generally cannot be evaluated in this format past 24 hours. As an alternative to aspiration, the sample can be passed through a cell strainer to separate cells bound to microcarriers from cells in suspension. Nuclei Counts Allow microcarriers to settle to the bottom of the sample. Aspirate the media from the sample, being careful not to remove cell-laden microcarriers from sample. Add crystal violet solution to each sample. Allow cells to incubate in crystal violet until they dissociate from the microcarrier surface. This process can be as quick as one minute with vortexing or last up to one hour at 37 C, depending on cell type. After the incubation period, vortex the sample vigorously for at least one minute to complete the nuclei release process. Nuclei can then be counted with a hemocytometer. Microcarriers generally do not get under the cover slip, so they will not interfere with the count. Keep track of all the reagent volumes used as well as the media volume removed so that the nuclei count can be adjusted with the appropriate dilution/concentration factor. If evaluating cell attachment kinetics, repeat the above procedure, but save the aspirated media in separate container to count in addition. The percentage of cells attached at each time point will be the nuclei counted on the microcarriers divided by the total number of cells (or nuclei) added to the well. To close the mass balance, perform a nuclei count on the aspirated medium. The nuclei counts on the microcarriers as well as the nuclei counts of the aspirated media should add up to equal the number of cells (or nuclei) originally added to the sample. www.pall.com/biopharm 7

If the sample plate contains many other samples, it may be easier to first transfer the sample from the plate to a microfuge tube before beginning the harvest procedure described above. Once cells have attached to microcarriers, they will begin to spread and proliferate. As time progresses, these new cells will impact the accuracy of cell and nuclei count assays used to evaluate attachment kinetics. Depending on the growth rate of the cell type, the attachment kinetics generally cannot be evaluated past 24 hours. The composition of Crystal Violet solution used for quantifying nuclei is 1 mg/ml crystal violet and 0.5% Triton u X-100 in 0.1 M citric acid. The concentration of Triton can be optimized for different cell types, as higher Triton concentration can dissolve nuclei, while lower concentration tends to leave behind too much debris. As an alternative to aspiration, the sample can be passed through a cell strainer to separate cells bound microcarriers from cells in suspension. For questions or additional information, please contact our Technical Support Team: microcarriers@pall.com. Visit us on the Web at www.pall.com/cellculture E-mail us at microcarriers@pall.com Customer Ordering Pall Life Sciences 4370 Varsity Drive, Suite B Ann Arbor, MI 48108 microcarriers@pall.com 734-973-2956 phone Corporate Headquarters Port Washington, NY, USA +1.800.717.7255 toll free (USA) +1.516.484.5400 phone biopharm@pall.com e-mail European Headquarters Fribourg, Switzerland +41 (0)26 350 53 00 phone LifeSciences.EU@pall.com e-mail Asia-Pacific Headquarters Singapore +65 6389 6500 phone sgcustomerservice@pall.com e-mail International Offices Pall Corporation has offices and plants throughout the world in locations such as: Argentina, Australia, Austria, Belgium, Brazil, Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, Mexico, the Netherlands, New Zealand, Norway, Poland, Puerto Rico, Russia, ingapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, the United Kingdom, the United States, and Venezuela. Distributors in all major industrial areas of the world. To locate the Pall office or distributor nearest you, visit www.pall.com/contact The information provided in this literature was reviewed for accuracy at the time of publication. Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly. 2017, Pall Corporation. Pall, and Hillex are trademarks of Pall Corporation. indicates a trademark registered in the USA and TM indicates a common law trademark. Filtration.Separation. Solution. is a service mark of Pall Corporation. ucostar is a registered trademark of Corning, Inc.; Falcon is a Trademark of BD Biosciences; Triton is a trademark of The Dow Chemical Company. 10/17, PDF, GN17.06937 USD2991 (1)