Laboratory Techniques on Microcarrier Culture

Laboratory Techniques on Microcarrier Culture Laboratory Manual Prof. Wei-Shou Hu 1990 c b t. u mn.edu

Cell and Tissue Reactor Engineering 2000 University of Minnesota Introduction: Section 1: Section 2: Section 3: Section 4: Section 5: Section 6: Section 7: Section 8: Section 9: Section 10: Section 11: Section 12: Section 13: Section 14: (Video) Preparation of microcarrier spinner flasks (Video Text) Preparation of microcarrier from stock suspension (Video Text) Trypsinization of cells from tissue culture flasks (Video Text) Inoculation of cells into microcarrier culture (Video Text) Setup of a microcarrier fermentor (Video Text) Cell attachment (Video) Fixing and staining of cells on microcarrier (Video Text) Cell counting (Video Text) Morphology change during growth (Video) Changing medium (Video) Aeration through silicone rubber tubing (Video Text) Sparging (Video) Detachment of cells from microcarriers by trypsinization (Video Text) Morphology and viability of plastic glass or macroporous microcarriers (Video Text)

Cell and Tissue Reactor Engineering 2000 University of Minnesota Microcarrier Culture Techniques I. PREPARATION OF MICROCARRIER SPINNER FLASKS A. Preparing and Sterilizing Spinner Flasks II. PREPARATION OF MICROCARRIER FROM STOCK SUSPENSION 1. EXAMPLE: CALCULATION OF MICROCARRIER AMOUNT FOR CULTIVATING VERO CELLS B. Preparing Microcarrier Stock Suspension C. Preparing Microcarriers in Spinners III. TRYPSINIZATION OF CELLS FROM TISSUE CULTURE FLASKS 1. EXAMPLE: HOW MANY CELLS ARE NEEDED FOR INOCULATING A MICROCARRIER CULTURE? 2. EXAMPLE : HOW MANY CELLS CAN BE ATTAINED FROM FLASKS? B. Cell trypsinization from T-Flasks or Roller Bottles IV. INOCULATION OF CELLS INTO MICROCARRIER CULTURE A. Inoculation of Cells Onto Microcarriers B. Monitoring Cell Attachment Progress V. SET-UP OF MICROCARRIER FERMENTOR VI. FIXING AND STAINING CELLS ON MICROCARRIERS A. Staining Microcarrier Cells VII. CELL COUNTING 1. EXAMPLE: ERROR IN DETERMINING CELL CONCENTRATION IN A MICROCARRIER CULTURE B. Nuclei Counting C. Microcarrier Concentrations Other Than 5 g/l D. Cell Counting by Nuclei Number VIII. AERATION THROUGH SILICONE RUBBER TUBING IX. MEASUREMENT OF OXYGEN CONSUMPTION RATE USING A BLOOD-GAS ANALYZER A. Measurement of Oxygen Consumption Rate in a Sealed Vessel

X. MORPHOLOGY ON PLASTIC AND GLASS MICROCARRIERS A. Viable stain of cells on microcarriers or macroporous beads XI. DETACHMENT OF CELLS FROM MICROCARRIERS BY TRYPSINIZATION A. Decision Making Chart for Trypsinization B. Trypsinization of Cells from Cytodex 3 Microcarriers C. 10.3 Cleaning the Spinner Flasks XII. APPENDIX: COMPOSITION OF SOLUTIONS A. Trypan Blue 0.1% in PBS B. Microcarrier Culture Counting Stain C. Fixative Stain D. Washing and Incubation Buffer for Trypsinization of Microcarrier Cultures E. Ethidium bromide (EB) Stock Solution F. FDA Stock Solution

I. PREPARATION OF MICROCARRIER SPINNER FLASKS Before starting a microcarrier culture, the spinner flasks and the microcarrier stock suspensions should be prepared. Many different types of spinner flasks can be used. A spinner flask has one or two side arms. The side arm is used for inoculation and sampling; in many cases it is also used for aeration. The Techne spinner is equipped with a ball shaped stirrer which rotates at the bottom of the flask. Another mechanism of mixing (needed to suspend the microcarriers) is to use a magnetic stirring bar that rests at the bottom of the stirrer. In other cases, the magnetic stirring bar is suspended and is away from the bottom. When a magnetic stirring bar is used, it is sometimes equipped with two blades made of Teflon. In some instances the blades are pitched with an angle to the vertical axis; in other cases, the blades are vertically placed. In general, pitched blades are preferable to vertical ones, although they are not readily available and may require special order. When an impeller (usually a magnetic stirring bar) is equipped with blades, it is essential to leave enough clearance between the tip of the blade and the wall of the flask (too small a clearance may crush some cells). In general, it is important to take apart microcarrier flasks and thoroughly clean them. A flask should be siliconized prior to use. Siliconization can be achieved by rinsing with Sigmacote 1 (Sigma Chemical Company). A number of other surface-treating agents are available; they are mostly organosilane and are readily available from chemical distributors and hardware stores. The siliconization procedure involves taking about 1 ml of the solution, pouring it into the spinner flask, and spreading it all over the glass surface. The solution can then be transferred to the next spinner and reused several times (Sigmacote is reusable if kept free from moisture). A siliconized flask should then be rinsed thoroughly and put through the regular glassware cleaning procedure. The impeller and remaining parts of the spinner flask can then be reassembled; however, it is important to make sure that enough space is left between the bottom of the suspended impeller and the bottom of the flask. A 1.0-1.5 cm distance is generally adequate for a 250 ml flask. However, this varies according to agitation conditions and the geometry of the flask. Too small a clearance will cause cell destruction; too large a clearance will results in a cone of settled microcarriers at the bottom. The assembled spinner flask should be tested on a magnetic stirrer to ensure that it stirs gently without wobbling. 1 Sigmacote is a silicon solution prepared in heptane and it forms a tight, microscopically thin film on glass, which reduces microcarrier adhesion to the glass.

It is necessary to leave the cap loosened or have an aeration port that allows the air in the flask to be exchanged with the ambient air. The head space of most spinners has a volume of 200 ml which contains approximately 1.65 mmoles of oxygen, if we neglect the CO 2 and assume that the oxygen content is 0.21. 1atm 0.21 0.21 N (mole) = atm L (0.82) ( 273 + 27) K K = 0.00165 mole = 1.65 mmole If the cell concentration is 10 6 cells/ml, the volume of the culture is 100 ml, and the specific oxygen consumption rate is 10-10 mmole/cell hr. Then the oxygen demand is then: 10-10 10 6 100 = 0.01mmole/hr In 24 h the cells will consume 15% of the oxygen in the head space. This can severely decrease the oxygen transfer rate in the spinner. So, the possibility of oxygen starvation in a spinner cannot be overlooked. The fully assembled flask can then be autoclaved for 20 min at 121 C in a dry cycle. A. Preparing and Sterilizing Spinner Flasks Procedure: 1. Separate impeller and flask. If the spinner has not been siliconized, do so now: a. Dry spinner completely b. Transfer 1 ml of Sigmacote to the spinner; swirl to rinse the entire spinner flask surface c. Reuse the solution in the next spinner; discard when necessary d. Set aside and leave for several hours to overnight e. Rinse flasks thoroughly with distilled water f. Allow flasks to dry; proceed with standard glassware sterilization 2. Wash the impeller separately; if necessary, take it apart to get rid of entrapped beads. 3. Adjust impeller height so that it clears between 1.0 and 1.5 cm from the bottom of the bottle. 4. Test on a magnetic stirrer to ensure that the spinner turns smoothly (the impeller should be centered, and it should stir gently without wobbling as it turns). 5. Fill one of the side arms or the aeration port about halfway with glass wool. 6. Cover side arms with aluminum foil.

7. Cover the cap so that it is also lined with the aluminum foil. 8. Attach autoclave tape to the flask. 9. Autoclave 20 min at 121 o C on dry cycle. 10. Don t remove aluminum foil until use. II. PREPARATION OF MICROCARRIER FROM STOCK SUSPENSION Most microcarriers from commercial sources are provided as dry beads. The addition of dry beads directly into fermentors or spinners for sterilization is usually not appropriate. They are usually washed, resuspended in PBS and then autoclaved. In a laboratory they are often kept in stock suspension until use. The volume of dextran-based microcarriers increases after being hydrated. The hydrated volume is very dependent on the ionic strength of the solution in which they are suspended. The hydration step can be done in any flask or beaker. The beaker is siliconized prior to use in order to reduce the amount of microcarriers sticking to the wall. After adding the beads and PBS to the beaker, use a glass rod to stir gently. Most types of microcarriers are readily suspended in the solution; however, some beads, especially those based on polystyrene or protein coated ones, may agglomerate or even float at the top of the liquid. This usually can be handled easily by continued stirring. Alternatively a small amount of surface active agent such as Tween- 20 can be added to facilitate wetting the beads. The washing step can be easily done on a coarse grade sintered glass funnel that has about 5 times the volume of the packed hydrated beads. After transferring the bead suspension from the beaker to the sintered glass funnel, vacuum is applied to drain the liquid. PBS is then added to the sintered-glass funnel to suspend the beads and the washing step is repeated (Washing with 30 volumes of PBS is usually sufficient). The beads are then transferred to another beaker. The volume of the suspension is brought to a level that gives the desired final microcarrier concentration in the stock suspension. It is easier to carry out this step on a toploading balance, if one is available. Some microcarrier preparations have too wide a particle size or density distribution. At times it may be desirable to carry out the washing steps in a beaker. The debris of those particles with too small of a diameter or particles with too low densities settle at much slower rates and are removed by suction after the majority of beads have settled. The appropriate concentration of microcarriers in suspension is, of course, dependent on the final concentration to be used in the culture. Normally a convenient concentration is one which give approximately 20-30% settled bead volume. For dextran-based microcarriers this is equivalent to 10 to 15 g/l. For polystyrene based microcarriers this is approximately 120 to 200 g/l.

Suspend and resuspend the microcarrier in phosphate buffer saline before autoclaving. The microcarrier stock suspension is then autoclaved. The conditions for autoclaving vary with the size of container used. 500 ml to 1000 ml bottles with 10 g/l of dextran microcarriers are normally used. An autoclave setting of 20 min. at 121 o C is sufficient under these conditions. At times the microcarrier suspension is transferred to a fermentor and sterilized in situ. Under such conditions, care should be taken to avoid vigorous boiling of the liquid. Vigorous boiling may cause microcarriers to stick to the fermentor interior above the liquid level or even to some piping system. It is a nuisance for cleaning at the very least and may even contaminate the flow stream which comes in contact with the pipings Since microcarrier beads are suspended in PBS, they are usually rinsed with tissue culture medium to displace PBS before use. Otherwise, the PBS carried over with the microcarrier can dilute the cell culture medium significantly. How much microcarrier concentration should be used in a culture? This is largely dictated by the desired total surface area for cell growth and the maximum cell concentration. In general, the higher the microcarrier concentration, the higher the maximum cell concentration that can be achieved. However, there are some practical constraints. Too high a settled bead volume can be problematic in mixing and suspending the microcarriers. A 40-50% packed bead volume is roughly the upper limit. At high cell concentrations, oxygen transferred can be limiting. Furthermore, for many cell types, if cell concentration is higher than 3 x 10 6 /ml, the medium almost needs to be replenished at least 50-80% daily. To ease the maintenance of the culture it is better to use continuous perfusion. A microcarrier concentration which gives rise to a settled bead volume of 5-15% is relatively convenient. Interstitial liquid occupies 20-50% of the volume (referred to as void volume ) in packed or settled beads, depending on the particle size distribution and on how tightly the beads are packed (close packed monodispersed spheres have 0.27 void volume). Thus the surface area available for growth is: 6 V' (1- ε) = A (2.1) d where V is the packed volume, ε is the fraction of void volume, and d is the mean diameter of the bead. A is the surface area available for growth. The amount of beads (m, gram, milligram) needed is best estimated by examining the settled bead volume in the stock suspension. Keeping in mind that Equation 2.1 is a first approximation (due to errors in estimating ε and d) and that the mean diameter values given in commercial literature are not always reliable. For solid beads such as polystyrene-based ones (as opposed to porous beads such as dextran based ones) the amount of beads (m, in mg or g) can be estimated from the desired surface area.

1 m = A Πd 2 Πd 6 ρ = [total surface area] 3 1 surface area per [volume per bead][density] bead (2.2) where d and ρ are mean values. Most solid beads (polystyrene, hollow glass beads) have densities that range from 1.05 to 1.25. Even with the same lot of microcarriers, sometimes a distribution of microcarrier density is observed. Thus, Equation 2.2 is only used as a first approximation. 1. EXAMPLE: CALCULATION OF MICROCARRIER AMOUNT FOR CULTIVATING VERO CELLS The calculation of the amount of microcarriers needed in a 1 liter culture to produce 4 10 9 cells is shown below. When Vero cells are grown to confluence as a monolayer on a 500 cm 2 roller bottle, the total cell number is approximately 6 to 7 10 7. Thus, the confluent density is 1.2 to 1.6 10 5 cells cm 2. To produce 4 10 9 cells, one needs 3.3 to 4 10 4 cm 2. If the density of the polystyrene beads is 1.2 g/cm 3 and the mean diameter is 150 µm (0.015 cm), then 4 2 0.015cm g = 4 10 (cm ) 1.2 = 80g (2.3) 6 cm m 3 Note: This amount is almost 16-fold that required for dextran based microcarriers. For small quantities, washing can be done in dispensable centrifuge tubes. Microcarrier beads settle to the bottom of the centrifuge tube quickly. Or, as an alternative, centrifugation can be used. In general, the microcarrier suspension can be centrifuged at about 500 r.p.m. As soon as the centrifuge reaches the peak speed, it can be turned off. The supernatant is then sucked off. When using a pipette suck from the side of the centrifuge tube and from the top of the liquid surface to minimize the amount of microcarriers sucked off with the supernatant. After gently filling the centrifuge tube with medium, one may recap and invert the centrifuge tube to suspend beads. Avoid the formation of any foam in these manipulations. Depending on the quantity of the beads, washing with medium once or twice is sufficient.

When pouring in the medium, it is easier to tilt the spinner back so that the beads don t settle on the side arm. Leave enough volume for the inoculation of cells later. Growth medium is then added to the spinner flask to the desired total volume. The entire flask is then incubated at 37 o C in an appropriate carbon dioxide environment (if no CO 2 environment is available, use medium with a low ph). This step is important to allow the temperature and ph to equilibrate before the inoculation. B. Preparing Microcarrier Stock Suspension Materials: 1. Microcarriers (dry form from vendor) 2. 500 ml or 1 liter beaker, presiliconized with Sigmacote 3. PBS 4. 250 ml or 500 ml sintered-glass funnel (coarse grain; medium grain would be okay too, but not fine). 5. 500 ml or 1 liter screw-cap Pyrex bottle 6. Large plastic funnel Procedure: 1. Weigh the desired amount of microcarriers using a balance (usually 5 g dextran-based beads or 50-100 g polystyrene beads to prepare a 500 ml bottle). Also record the weight of the empty beaker. 2. Transfer to a beaker. Polystyrene beads don t change volume; dextran beads will swell (5 g will swell to about 900-1100 ml after hydrated with PBS). 3. Add PBS to about 30% total final volume. 4. Stir with a glass or metal rod to disperse the beads. 5. No unhydrated or unwetted beads should float at the top of the liquid. 6. Transfer the hydrated beads to the sinter-glass funnel and fill it. Turn on the vacuum pump. As the liquid level in the funnel decreases, keep transferring the microcarrier suspension into the funnel until all beads have been transferred. 7. After the liquid is drained, turn off vacuum pump. Refill the funnel with PBS; use a stirring rod to suspend beads. 8. Turn on vacuum pump again. 9. Repeat steps 7-8 until a total of approximately 30 times the bead volume (i.e. about 3 liters for 5 g of dextran-based or 50-80 g of polystyrene-based beads) is used. Turn off vacuum pump. 10. Scrape bead cake off; transfer to beaker. 11. Add some PBS to suspend the residual microcarriers on the funnel; transfer to the beaker.

12. Place the beaker containing microcarriers on the balance. Add PBS to the beaker until the appropriate weight is reached (assume 1cc of PBS is 1 g). 13. Place a plastic funnel (plastic is less sticky to microcarriers than glass) on the mouth of the 500 ml or 1 liter bottle; place the bottle on a balance. 14. Stir the bead suspension vigorously until it is well mixed. 15. Pour the bead suspension into the bottle until the desired weight is reached. Note: If no toploading balance is available, premark the desired liquid level on two opposite sides of the beaker or the bottle and fill up PBS to that level; the error is only a few percent. 16. Label the bottle (lot no., concentration of beads, date). 17. Screw the cap loosely on the bottle. 18. Autoclave at 121 o C for 20 min; use slow exhaust cycle. 19. After autoclaving, remove the bottles and tighten the cap. The cap is only to be loosened again in a laminar flow hood when ready for use. C. Preparing Microcarriers in Spinners Materials: 1. Spinner flasks, sterile 2. Culture medium, approx. 120 ml/250 ml bottles, sterile 3. 50 ml centrifuge tubes, sterile 4. Microcarrier stock solution, 10 g/l in PBS, sterile 5. Pasteur pipet 6. Phosphate buffer saline, (PBS) 1X concentration, sterile 7. Pipetter, sterile (plastic) 8. Magnetic stirrer and stir plate Procedure: Transferring Microcarriers to Test Tube 1. Mix microcarrier stock suspension by swirling the bottle. 2. Transfer an appropriate amount of the mixed microcarrier suspension to a 50 ml centrifuge tube. 3. Centrifuge microcarrier beads at 500 rpm for a few seconds (or let settle by gravity). 4. Suck off supernatant with a pasteur pipette in vacuum. When approaching microcarrier cake, place the tip of pasteur pipette at the top of liquid level along the centrifuge wall so beads are not sucked off. Replace PBS in the Beads with Medium 5. Fill centrifuge tube containing the beads with medium containing no fetal calf serum.

6. Invert tube to suspend beads; do not cause foaming. 7. Centrifuge at 500 rpm for a few seconds. 8. Suck off medium, as in step 4. 9. Repeat steps 5-8 once if necessary. Preparing Microcarrier-Medium Mixture for Inoculation 10. Suck off any residual water in the spinner flask to be used for cultivation. 11. Fill centrifuge tube that contains microcarriers from step 9 with culture medium (including serum and/or growth factors). 12. Invert tube to suspend beads; do not cause foaming. 13. Pour entire contents of tube (beads and medium) into the spinner flask; do not let the solution touch the sides of the mouth. 14. Add more medium to the spinner flask to 75-90% of the final culture volume. The remaining 10 or 25% will allow for addition of inoculum (cells and medium). 15. Place spinner flask containing beads in the 37 o C carbon dioxide incubator for at least 30 minutes (for temperature and ph equilibrium). 16. Note: Depending on the NaHCO 3 concentration used, ph may rise while not in the CO 2 incubator. To avoid this, either: a. Inject some sterile CO 2 into the spinner flask. or b. Use medium that has a ph of about 6.8 (orange color) in step 11. III. TRYPSINIZATION OF CELLS FROM TISSUE CULTURE FLASKS For most laboratory operations, the cells needed for inoculation are obtained from either T-flasks or roller bottles. The trypsinization procedure for microcarrier inoculation is basically the same as that used for typical cell propagation except that it is highly desirable to generate as many single cells (as opposed to cell clumps) as possible. If a large fraction of cells form clumps, the resulting cell distribution on microcarriers tends to be more skewed. In general, the best results can be obtained by subculturing the cells in roller bottles or T-flasks a few days before the actual inoculation so that subconfluent cells can be used for trypsinization and inoculation. For better results, any clumps observed on the T-flask or the roller bottles should be removed before trypsinization. Removal of the cell clumps can usually be done by simple suction. Before trypsinizing cells from T-flasks or roller bottles, it is important to know: (1) how many cells are needed for inoculation, and (2) how many cells can be obtained from each flask or roller bottle. Cell density (cells/cm 2 ) and cell concentration (cells/ml) can be estimated from T-flask and roller bottle when the cells are

subcultured. If no prior knowledge is available, 10 4 cells/cm 2 and 10 5 cells/ml are good starting points. Another constraint is the cell to bead ratio. Statistically one needs to provide at least 4-5 cells or clumps per bead to reduce the fraction of empty beads to near zero. In the attachment process, one clump with many cells is only a single attachment unit. 1. EXAMPLE: HOW MANY CELLS ARE NEEDED FOR INOCULATING A MICROCARRIER CULTURE? Assume that we want to inoculate 10 5 cells to 2 x 10 4 beads. 0n average, each bead acquires 5 cells, however, if the10 5 cells are in the form of 2 x 10 4 units of 5 cell clumps, the average bead will acquire one clump. Under such conditions a significant portion of beads will have no clump (or no cells) while others may have 2-3 clumps (10-15 cells). Thus, the cell distribution on microcarriers is inferior to the case of a single cell suspension. In the inoculation process, some cells inevitably form clumps that can behave as single units of attachment; therefore, inoculum is usually better at 8-12 cells/bead. For microcarriers with a mean diameter in the range of 150-170 µm at 10% settled bead volume, 3.5 to 4.5 x 10 5 cell/ml is usually adequate to meet the criteria of 10 4 cells/cm 2, 10 5 cells/ml, and 8 cells/bead. 2. EXAMPLE : HOW MANY CELLS CAN BE ATTAINED FROM FLASKS? In a confluent monolayer of large cells (e.g. normal diploid embryonic foreskin fibroblast), the cell density is approximately 4 x 10 4 cells/cm 2. This density can increase by another 20-30% if medium is replenished regularly after confluence. In some small cells (e.g. Vero and swine intestine cells), the monolayer density is about 10 to 15 x 10 4 /cm 2. If cells are trypsinized at a subconfluent stage near the end of their exponential growth, one can use 1.5 to 2 x 10 7 cells/500 cm 2 roller bottle for large cells and 6 to 8 x 10 7 cells/500 cm 2 roller bottle for small cells as an estimate. The standard trypsinization procedure used in our laboratory usually involves washing with PBS once or twice. Trypsin solution is then added to the flask. In general, 0.02% EDTA is included in the trypsin solution. Following the incubation period after addition of trypsin solution, moderate force is applied to the T-flasks or the roller bottles to facilitate cell detachment from the surface. After trypsinization, at least twice as much volume of serum medium is added to the flask to stop the trypsinization. The cell clumps are gently dispersed by shearing them with a pipette. To avoid generating foam in the cell suspension, one should not shoot air into the liquid during the repeated pipetting. Separate the cells by centrifugation before inoculation. If the volume of

the cell suspension needed for inoculation is relatively high (e.g. more than 5% of the total culture volume), centrifugation is beneficial in reducing the amount of trypsin being carried over to the new culture. If a centrifugation step is used to pellet the cells, it is necessary to disperse the cells gently after centrifugation to make sure single-cell suspension is generated again before inoculation. The trypsinization step should not be started until the culture vessel and medium are ready, and temperature and ph are equilibrated. This minimizes the time cells are kept in suspension after trypsinization. If cells are not to be inoculated immediately after trypsinization and resuspension, the cell suspension should be kept on ice. B. Cell trypsinization from T-Flasks or Roller Bottles 1. Materials: a. 1X trypsin solution containing 0.02% EDTA b. Culture media with serum c. 1X PBS, sterile d. Pipettes, sterile e. Pasteur pipettes, sterile f. Vacuum pump g. New T-flasks, sterile h. Trypan blue solution 2. Procedure: a. Loosen the caps of flasks, roller bottles, PBS, and media bottles. b. Suck off media from flask/roller bottle. c. Add approximately 5 ml PBS per T-75 flask; approximately 20 ml per roller bottle. Add PBS to the T-flasks along the wall, opposite to the cell attachment side, or add to the bottom of the roller bottle. d. Wash with PBS by tilting the flask or rolling the bottle to disperse over cells. e. Suck off PBS with Pasteur pipettes or 10 ml pipettes. Suck off the liquid from the bottom f. of T-flask or roller bottle, or from the side of the T-flask that has no cells, to avoid sucking off cells. g. Suck off any big cell clumps with a Pasteur pipette. h. Add 1 ml trypsin solution to a T-75 (about 0.5 ml for a T-25, 2 ml for a T-150 and 4-5 ml for roller bottles). 3. Quickly: a. Disperse trypsin over cells. b. Place T-flask in incubator or roller bottles in a roller at 37 C for 2-4 min. c. At the end of 2-3 min, check in the microscope to see if cells are rounding up. If so, cells on the side of the flask will slide off. If not, incubate for a few more minutes. d. To stop the trypsin reaction, add medium (containing serum).

e. Disperse cell clumps into single cells by shearing them with a pipette: suck up most of the volume without creating air bubbles. To ensure that most cells are detached from the surface, shoot the cell suspension out along the wall on which cells grow. Make sure not to submerge the pipette tip in the cell suspension. This avoids shooting air into the suspension, which causes foaming. f. Now the trypsinized cells are ready for inoculation to spinners, or other roller bottles and T-flasks. IV. INOCULATION OF CELLS INTO MICROCARRIER CULTURE Before inoculation, the microcarrier spinner flask is removed from the incubator and placed on a magnetic stirrer in the laminar flow hood. Slow agitation is used to ensure that all the microcarriers are kept in suspension. The balance amount of the medium is added to the cell suspension. As a routine practice, 80% of final culture is employed before inoculation. For a 100 ml culture with 5 g/l of dextran-based microcarriers, 0.5 g of beads are suspended in 80 ml of medium in the spinner flasks and placed in the incubator to equilibrate ph and temperature. If 8 ml of trypsinized cells suspended at 5 x 10 6 cells/ml are needed for inoculation, the balance of medium is 12 ml. The cell suspension is gently dispersed again to ensure that the cells are in single-cell suspension. The cell suspension can then be poured into the spinner flasks while the microcarriers are in suspension. This process must be done as quickly as possible to avoid changes in temperature and ph. Some cell types can be cultivated either as suspension cells or as adherent cells on microcarriers. For these types of cells, it is advantageous to grow the seed culture as suspension cells. These suspension cells can be used for direct inoculation. This eliminates the trypsinization step. After inoculation, the spinner flask is placed back on the magnetic stirrer in the incubator. In general, a minimum speed is required to keep the microcarriers in suspension. Agitating too fast may affect the cell attachment rate after inoculation or even be detrimental to cells. If the agitation rate is too slow or if the impeller is placed too high above the bottom, a cone of microcarriers may form at the bottom of the flask. If the impeller is placed too low or is too large (not enough clearance between the tip of the impeller and the wall of the flask), one may see crushed beads in the suspension. A. Inoculation of Cells Onto Microcarriers Procedure: 1. Calculate the amount of cell suspension (see section 3) to give the desired inoculum cell concentration. 2. Transfer the amount of cells needed for inoculation to a sterile test tube. Add culture medium to the test tube to make up the volume to the final volume of the spinner. 3. Disperse cells gently by pipetting.

4. Quickly inoculate cells while spinner is stirring. This ensures even cell distribution on the microcarriers. 5. Place spinners in a 37 C, carbon dioxide incubator. Use a stirring speed approximately 10% higher than that required to suspend the microcarriers. B. Monitoring Cell Attachment Progress 1. After 2 h, check to see if cells have attached to microcarriers: While the spinner flask is stirring on a magnetic stir plate, use a plastic pipette to remove a sample (0.2 ml for a 5 g/l of Cytodex microcarriers; adjust proportionally for other microcarrier concentrations). Place the sample into a well of the 24 well plate. Look at the sample under the microscope. 2. If the cells still have not attached after 3-4 h, they probably never will. Note any dead, floating cells. V. SET-UP OF MICROCARRIER FERMENTOR Animal cells are sensitive to excessive mechanical agitation, so a low agitation rate is usually used in cell culture. Agitation in mammalian cell culture mainly serves to suspend cells or microcarriers and achieve fluid mixing rather than to disperse air bubbles for enhancement of oxygen transfer, as in microbial culture. Microcarriers and cell culture require somewhat different agitation than free cell suspensions. The diameter of microcarriers is in the range of 150-200 um as opposed to 10-20 um for free cells. The terminal settling velocity of microcarriers in cell culture medium is almost two orders of magnitude higher than that of free cells. This implies that there is a much higher relative velocity between microcarriers and fluid than between free cells and fluid; therefore, cells on microcarriers experience a higher fluid shear force. The impeller-agitated stirred-tank reactor is still the most widely used in microcarrier cultures. The liquid-height to tank-diameter ratio is typically slightly higher than one. A marine-type impeller or paddles with pitched blades are preferred to turbines. Typically, the center of the agitator is placed at a distance from the vessel bottom equal to approximately 40% of the total liquid height. The impeller-diameter to tank-diameter ratio is usually higher for a microcarrier culture vessel than for a microbial fermentor. The relatively fast settling velocity of microcarriers is best illustrated by stopping the agitation in a microcarrier culture. A microcarrier-free zone is quickly established at the top of the vessel after agitation stops. A minimum agitation required to keep microcarriers completely in suspension should be used at the beginning of the culture. The impeller speed shown for this confluent culture is somewhat higher than that which should be used for a culture in its initial growth period. With a marine impeller or pitched blades, the fluid can be pumped either upward or downward. There appears to be no difference in cell growth whether the fluid was pumped either upward or downward; however, pumping upward is seen more often.

An easy way to design an impeller is to use a glass jar with an impeller attached to an agitator. The blades of the impeller can be modified by attaching plastic plates to them. Agitation rate can also be adjusted. The movement of microcarriers under different agitation conditions can then be visualized by using microcarriers stained with trypan blue. VI. FIXING AND STAINING CELLS ON MICROCARRIERS During the cultivation period, it is necessary to sample the culture periodically for cell count and microscopic observation. It is also highly desirable to preserve these samples from day to day, so that samples from different days can be compared for morphology. 24-well plates are convenient for preparing samples for microscopic observation. The amount of sample required for microscopic observation is, of course, dependent on the concentration of microcarrier used. Too large a sample results in thick layers, which interfere with viewing the cells. Observation should first be made without staining. This allows one to detect unattached cells before they are removed during fixing and staining. Most healthy microcarrier culture do not have many cells in the suspension; exceptions are those cases in which cells also grow in suspension such as CHO or 293 cells which have been adapted to suspension growth. The fixing and staining steps are usually combined using a fixative/staining solution. Drops of the staining solution are added to the well. The suspension is allowed to sit for a few seconds; PBS is then added to fill the well. A short period is necessary to allow the microcarriers to settle to the bottom before suction is applied to remove approximately 70% of the solution from the well. In removing the solution, it is easier to apply pipette tips to the wall of the well to minimize the number of microcarriers that are sucked off. The PBS washing step can be repeated until the color of the solution is light enough for microscopic observation. A green filter can be used in the inverted microscope for easier observation. Most of the dead cells falling from the microcarriers are spherical. When excessive mechanical force is used, you may see elongated cells with one end clinging to the microcarrier and the other end in suspension. In microscopic examination, one should pay attention to cell distribution on microcarriers. The cell distribution on microcarriers should be relatively uniform. In other words, the number of cells per bead should not span over a very large range. In particular, one should not observe one population of densely populated beads and another population of sparsely populated or even bare beads. If this has occurred, the inoculation procedure should be improved. A. Staining Microcarrier Cells Materials:

1. Plastic 1 ml pipettes 2. 24-well plate 3. Magnetic stirrer 4. Staining Solution :Crystal Violet 0.5% in ethanol 40% and PBS 60% 5. Phosphate buffer saline (PBS) 6. Pasteur pipettes and suction bulb Procedure: 1. For a 5 g/1 culture, remove a 0.2 ml sample of microcarrier culture while it is spinning, and place it in a well of the 24-well plate (0.05 ml for a 96 well plate). *The following steps can be performed on an open bench: 2. Add 2-3 drops of fixative stain into well; swirl to mix. 3. Wait 1 minute to fix cells. 4. Fill well to the top with nonsterile PBS. 5. Wait 2-3 minutes for beads to settle. 6. Suck off PBS and staining solution with vacuum. Move the Pasteur pipette down along the wall and the surface of the liquid to avoid sucking out the beads (leave a small amount of liquid so that cells do not dry out). 7. If necessary, repeat Steps 4-6 until the solution is clear enough for microscopic observation. 8. Use green filter on microscope for viewing. Samples may be kept in 24-well plates as long as they are kept in PBS. For photographing, however, the samples should be photographed as soon as possible. VII. CELL COUNTING The cell concentration on the microcarriers is typically measured by counting nuclei. Before the sample is withdrawn from the spinner culture, make sure that the culture is well mixed. A 1-2 ml sample is usually sufficient for cell counting. For an accurate measurement, all microcarriers in the pipette must be released into the test tube for cell counting. This can be achieved by holding the pipette vertically as the microcarrier suspension is released from the pipette. When using a fermentor, take the sample by withdrawing fluid from the sampling tube. When taking samples from the sampling tube, use a low flow rate to avoid exerting excessive shear force on the cells in the sampling tube. As a general practice, whenever a sample is taken for cell count, another sample is placed in a 24-well plate for microscopic observation. The culture supernatant must be removed before adding nuclei counting solution. Microcarriers settle to the bottom of the tube quickly, even without centrifugation. However, the boundary between the supernatant and the microcarriers is usually not sharp, making subsequent removal of supernatant a bit difficult. Centrifuging at about 500 rpm for 30-60 seconds forms a packed bed of microcarrier beads at the bottom of

the tube. After centrifugation, a sharp boundary is seen between the surface of packed microcarriers and the supernatant. Vacuum suction is then applied to remove the supernatant. Care should be taken to avoid sucking off any microcarriers: Place the tip of the Pasteur pipette on the interface between air and liquid, and aspirate the liquid along the wall. For an accurate measurement, try to remove as much liquid as possible, leaving only the packed bed of microcarrier beads at the bottom of the centrifuge tube. The crystal violet/citric acid counting solution is then added to the beads to disperse them from the bottom of the tube. Triton X-100 (0.1%) can be added to the crystal violet/citric acid counting solution to aid dissolving the plasma membrane of the cells. Be careful to avoid leaving unsubmerged beads on the wall of the test tube. The test tube is then screw-capped and incubated at 37 C for approximately 1 hour or overnight. This incubation period stains the cell nuclei and also loosens it from cellular material. Nuclei are released from microcarriers by gentle shearing with a Pasteur pipette or a 200 ml pipette tip. A relatively gentle pipetting up and down through the Pasteur pipette will usually release all nuclei. Too fast a flow rate may damage the nuclei and render the counting difficult. Foaming can occur if air bubbles are sucked into the Pasteur pipette or if air is blown into the liquid when releasing the nuclei suspension from the pipette. In general, one should also avoid generating foam during repeated pipetting. Pipetting 4 to 6 times is usually sufficient to release all nuclei from microcarriers; however, it will vary with cell type and growth stage. After this shearing step. it is advisable to place a drop of the sheared nuclei microcarrier mixture on a glass slide to observe under a microscope. This can help ensure that all nuclei have been released from the microcarriers, improving counting accuracy. After releasing all nuclei into the counting solution, one is ready to perform a cell count using the hemacytometer. In general, nuclei stay in the suspension relatively well. However, if the nuclei suspension has been sitting for some time, mix it gently before placing it in the hemacytometer. Usually a drop of the suspension (including microcarrier beads and nuclei) is placed on the edge of the counting chamber by capillary action of a Pasteur pipette. The clearance between the cover slide and the hemacytometer is relatively small and does not allow the microcarrier to pass through. Thus, nuclei migrate with the counting liquid into the hemacytometer counting chamber while the microcarriers remain outside. The nuclei are ovoid and purple. At times, one sees a large amount of cell debris. This should not cause concern unless a large number of broken nuclei are also observed. It is usually an indication that too much shear force was applied; therefore, the count may not be accurate. This procedure of releasing nuclei from microcarriers is generally applicable to different cell types. In some cases, cells are only poorly attached to microcarriers and fall off easily. Some of those cells may appear

as intact cells with stained nuclei inside even after repeated shearing. In other cases, cells on microcarriers may have formed multi-layers or even clumps. This may cause difficulty in releasing all of the nuclei from microcarriers. Therefore, it is important to observe microcarrier and nuclei suspension under a microscope after the shearing step. A question that needs to be addressed is how much counting solution should be added to the packed microcarrier beads. In general, a constant volumetric ratio of counting solution to the packed bead volume should be maintained. 1. EXAMPLE: ERROR IN DETERMINING CELL CONCENTRATION IN A MICROCARRIER CULTURE Consider the case of a culture concentration of 5 g/l of Cytodex 1 microcarriers. In a 1 ml sample, there are approximately 0.12 ml packed beads and 0.88 ml supernatant. The 0.12 ml packed beads contains approximately 0.085 ml solid microcarriers and 0.035 ml interstitial liquid. All of the supernatant is sucked off after centrifugation; however, the interstitial liquid is still retained in the packed beads. Counting solution (1 ml) is then added and nuclei are released. After release, the nuclei are evenly distributed, not in 1 ml but in 1.035 ml of the total liquid. These nuclei are then counted in the hemacytometer. The concentration obtained in the hemacytometer count is taken as the concentration of nuclei (or cells/ml) in the reactor. Therefore, approximately a 3-4% error occurs in such a counting procedure. This error is negligible under most circumstances; however, it becomes significant at higher microcarrier concentrations in which the volume of counting solution was not proportionally increased. Since there are many ways to define the cell concentration, it is important to be consistent. B. Nuclei Counting Sampling and Releasing Nuclei from Microcarrier Materials: 1. Plastic pipettes, 1 ml 2. Centrifuge tubes, 15 ml 3. Counting stain (crystal violet 0.1% in citric acid 0.1 M) + 0.1% Triton X-100 (optional) 4. Pasteur pipettes and suction bulb 5. Eppendorf tubes 6. Vacuum pump 7. Magnetic stirrer Procedure:

1. For a culture with 5 g/l of microcarriers, remove 1 ml of culture while it is stirring on the magnetic stirrer with a 1 ml plastic pipette. 2. Hold the pipette straight down while releasing the sample into a 15 ml centrifuge tube. 3. Centrifuge for 30 sec. to 1 min at 500 rpm. 4. If no supernatant is to be saved, go to (b) a. If supernatant is to be saved for chemical analysis (glucose, glutamine, lactate, etc.), use a Pasteur pipette with a bulb to remove most of the supernatant. Be careful not to suck off beads or to disturb the boundary between beads and liquid. Transfer the supernatant to sample tube (such as Eppendorf tube). b. Attach a Pasteur pipette to vacuum. Hold both the pipette and tube straight. Move pipette down along the wall and about 0.2 mm above the liquid level. Liquid will be sucked off through the meniscus. [This allows most liquid to be withdrawn without disturbing the beads.] 5. Add 1 ml of counting solution by squirting it straight down all at once, so that it thoroughly mixes with the microcarriers. 6. Incubate for 1 h at 37 C, or for 12-24 h at room temperature. Make sure the cap is on tight to avoid evaporation. 7. Shear cell nuclei solution using a Pasteur pipette in a vertical position. Gently pipette the solution up and down a few times without generating air bubbles. 8. Count the nuclei (see 8.3). C. Microcarrier Concentrations Other Than 5 g/l When a different microcarrier concentration is used, adjust the amount of counting solution proportionally. The reason for making such an adjustment is explained below using the following symbols: n: cells/mg beads V: total sample volume taken (ml) c: microcarrier concentration, mg beads/ml sample ρ: density of beads ε: void fraction in settled microcarriers (ml interstitial space/ml settled bead volume) 1/((1-ε)ρ): settled volume of microcarrier, m1 packed vol/g beads X: cells/ml culture

In general we are interested in determining the cell concentration in the culture, which can be expressed as nc = x (7.1) If a sample of volume, V, is taken, after removing supernatant, the packed volume of microcarriers in sample is cv(1/((1- ε) ρ) and the number of cells on microcarriers is ncv. After adding the counting solution with volume (V'), the total volume becomes V' + cv(l/((l- ε) ρ)) (7.1) There is a residual liquid in the interstitial space of the packed beads. Thus, total liquid volume after adding counting solution is Vt = V' + ε cv(l/((1- ε) ρ) (7.2) The cell concentration in counting solution is the total number of cells divided by the total liquid volume ncv X' = (7.3) V t Because V and V t are not equal, there is some error. For c = 5 g/l, we usually take V = 1 ml and V' = 1 ml. The second term in Eq. 7.2 is relatively small, so V is almost the same as V'. However, if microcarrier concentration, c, is large, the second term becomes large and the error can be very significant. Therefore it is advisable to keep the volume ratio (counting solution to microcarriers) constant when comparing growth kinetics at different microcarrier concentrations.

If c = 2.5 g/l, we take V = 2 ml and V c = 1 ml. X and X' is then related by a dilution factor. Therefore, X = cell conc/ml number counting soln = dilution factor of cells in four grids 10 4 4 *Dilution factor = 1 if c = 5 g/l and V c = 1 ml; dilution factor is 0.5 if c = 2.5 g/l and V c = 1 ml. (Both are for 1 ml sample.) D. Cell Counting by Nuclei Number Materials: 1. Neubauer hemacytometer with cover slip 2. Pasteur pipette with suction bulb Procedure: 1. Shear cells off from microcarriers (sample from Step 6, page 24). a. Hold sample test tube and a Pasteur pipette (with a pipettor) straight up. b. Gently suck up most of the volume. c. Return volume vertically without sticking pipette into the liquid so no air bubbles form. When releasing, do not allow volume to touch the centrifuge wall because the microcarriers may stick. 2. Repeat Step 1 approximately 4-6 times. 3. To check if shearing was done properly, view a few drops of the solution on a glass slide; make sure that cell nuclei have detached from the microcarrier beads. 4. Prepare hemacytometer: a. Clean hemacytometer with tissue lens paper. b. Dampen ridges on hemacytometer with water. c. Slide clean cover slip over ridges to ensure a tight fit. 5. Shake tube gently by tapping the test tube with finger on the test tube wall. 6. Place Pasteur pipette into the nuclei suspension and allow the liquid to enter it by capillary action. 7. Apply solution to the edge of the chamber, which then fills by capillary action. Reload the pipette and fill the second chamber. 8. Count nuclei in the four outer grids. Nuclei are solid, smooth and oval. 9. To calculate cell count per ml:

X = cell conc/ml number counting soln = dilution factor of cells in four grids 10 4 4 10. The total count in the four outer grids should be less than 400. If it is necessary, make an appropriate dilution with the counting solution. VIII. AERATION THROUGH SILICONE RUBBER TUBING Sparging is often avoided in microcarrier culture, especially in small-scale operations. Instead, the oxygen and carbon dioxide supply is achieved by diffusion through silicone rubber tubing, which is wrapped around the vessel on stainless steel support rods. In most reported cases, the silicone rubber tubing is simply anchored inside the fermentor, either on the baffle plates or on tubes or piping. Many factors (wall thickness, outer-to-inner diameter ratio, etc.) affect the permeability of the silicone rubber tubing to oxygen. The dissolved oxygen concentration should be maintained above approximately 30% saturation with air (~0.055 mm or ~50 mmhg) The oxygen transfer rate (combining transfer through silicone tubing and the liquid surface aeration) should both meet the cells' consumption demand and be maintained at 30% saturation. If permeability (or amount of oxygen transferred across tubing using air at 1 atm) and the peak oxygen consumption rate of the culture are both known, one can calculate the length (L) of tubing required: * Pˆ L(C - Cl )V = OUR V = qoxv (8.1) Pˆ = pπd where Pˆ is the product of permeability (p) and mean perimeter of the tubing ( π d ) and is a characteristic of tubing. The other symbols are: C * : dissolved oxygen at saturation with air (mmole/l) C : dissolved oxygen level to be maintained in the culture (mmole/l) l OUR: oxygen uptake rate (mmole/l) q o : specific oxygen consumption rate (mmole/cell) x: cell concentration (cells/l) V: culture volume (L)

The above equation does not take surface aeration into account. If no data on q o is available, one can use an average value of typical oxygen consumption (5 x 10-11 to 5 x 10-10 mmole/cell hr). Alternately one can measure the actual oxygen consumption rate of cells at different growth stages. The oxygen transfer rate of the reactor vessel with a known amount of silicone tubing can also be measured easily. The rate of change in dissolved concentration in a reactor is the result of transfer into the liquid and the consumption by cells as described by Eq. 8.2 V dc dt l = K L a(c * - C)V - q o xv (8.2) where aeration) (1/hr). The first term on the right-hand side accounts for oxygen transfer, and the second term for consumption. Eliminating the transfer term by performing the oxygen consumption rate measurement in a K L a is the apparent volumetric oxygen transfer coefficient (combining surface and silicone rubber tubing sealed vessel without any gas phase, one can measure OUR by following the change in C. l Figure 8.1 is a schematic diagram of a set-up for the oxygen uptake rate measurements. The device for oxygen uptake measurement is a 80 cm3 custom made spinner flask with a tightly sealed rubber stopper. A dissolved oxygen electrode is inserted through the stopper to the flask. Prior to the experiment, the D.O. electrode is calibrated under the experimental conditions to be used. During the experiment, the flask is placed in a constant temperature room or incubator. Care should be taken to ensure that the stopper of the flask is tightly sealed so that no gas bubbles enter the flask during the experiment. The cell suspension inside the flask is stirred with a suspended magnetic stirring bar. The analogue output of the dissolved oxygen electrode can be either converted to digital signals and stored in a computer or sent to a chart recorder. The dissolved oxygen electrode should have a 90% response time of no more than two minutes. The response time is usually defined as the Figure 8.1 time period in which the output of the electrode reaches 90% of the new steady-state value after a step change in dissolved oxygen from 0% to 100% saturation with air. Oxygen should decrease at a constant rate, except for the initial few data points and the period in which the oxygen concentration is very low. The specific oxygen consumption is then