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Because serum (FBS) s not used, certain elements will need to be added to the media, such as transferrin (binds iron), insulin, albumin. b. Same media is used throughout the process. c. If you choose to make the media, you will need a media prep room. 2. Cho cell growth a. These cells will be adapted for growth in suspension and do not need to adhere to a solid substrate (ie, microcarrier) b. The cells have very strict growth requirements and will grow slowly, have poor production or die if not treated properly. For instance, the cells will not grow well at densities less than 10^5/ ml and will not grow to densities higher than 10^7/ ml. c. At all times, the temperature will need to be maintained at 37 C with 5% CO2 to provide buffering capability with bicarbonate in the media and adequate dissolved oxygen. In the larger steel reactors, this will require heat removal. d. Upon thawing, the 1 ml frozen stock will be inoculated into 10 mls media (not all of the cells survive freezing and thawing). 3. Seed train vs production reactor a. In the seed train, the priority is to expand the cell mass by maintaining a healthy cell population in the exponential growth phase. As soon as they reach the end of the exponential phase, dilute them into a larger volume of fresh media. The cells will be secreting antibody during this time, but that will not be harvested during seed train production. Early seed train reactors can include T-‐flasks, roller bottles, wave bioreactors (disposables), etc. These reactors are operated as batch or fed-‐batch (to replace consumed nutrients) – there is no outlet because this would result in cell loss. b. In the steel production bioreactor, the priority is to use the cell mass to maximize yield of antibody. We are no longer concerned with propagating a healthy cell population. Thus, cells will be seeded into the reactor, allowed to grow until waste products accumulate and the cells begin to die. The process is terminated when a sufficient number of cells have dies that the live cells are no longer producing high levels of antibody. Note, these larger reactors will require “steaming in place” to sterilize prior to use.
tyrpsin.) for the second cell line. check to insure that the floors are being frequently wet mopped with a disinfectant solution. Contaminated culture flasks must be removed from the incubator immediately. Iodine can be used instead) E. Incubate and observe for colony formation. The glass cultureware. F. After the manipulation of cells. Face mask. The split ratio of each passage must not be changed except under special circumstances. gloves. H. The sterile room will be used for sterile operations only and will be kept as uncluttered as possible. C. It will not. Once each month check for sterility within the hood by placing open blood agar plates in the hood for one (1) hour. It must not be stored with water or PBS as its content. must be cleaned promptly. after removing the medium. H. GENERAL RULES FOR MEDIA PREPARATION UPDATED 2/22/02 A. B.CELL CULTURE TECHNIQUES GENERAL RULES FOR MANIPULATING ANIMAL CELLS A. but apparently is not anymore. etc. Filter used in media preperation must be kept in a secure location. (Wescodyne was once available through U-Stores. Bottles should always be wiped with a solution of Wescodyne and left for approximately 15 minutes prior to the addition of serum to the medium. D. UV lamp must be left on overnight in the hood. G. and clean lab coat will always be worn during media preparation. Lights in the laminar flow hood room should be turned off and the door closed. Spent medium must not be left on the bench. In the daytime the door to the laminar flow hood room may be kept open but the door to 260B should also be kept closed. make sure the sterile room is cleaned up. filters must be stored where the user can be confident that the packaging will not be comprimised. if the hood is working properly. Never use the sample bottle of medium or buffer for two different cell lines. F. A contaminated filter can ruin an entire batch of media. C. in any way. E. The passge of cells must be recorded. autoclaved and disposed of properly. be used as a storage room. The glassware should be stored clean and dry. During media dispensing only things which are essential to the operation should be placed in the hood area. This is to minimize the disruption of sterile air flow in the most critical areas. Never handle two different cell lines in the hood simultaneously. At the end of each work day. D. B. the hood must be cleaned (remove the medium designated for the first cell line being handled) before introducing the materials (medium. flasks. it must be recorded. When it is changed. 12 . Microrganisms may glow when liquid is present. only an occasional colony should be observed. G. It must either frozen for later analysis or be disposed of properly after cultivation.
If sterile PBS is desired. 10X stock solution is not sterilized and stored at room temperature. 3. Making 1 L of 1X solution from 10X stock: 1. Add to 10 L use volumetric flask or graduated cylinder. Store at room temperature. Return solution to graduated cylinder and add appropriate volume of DI water to bring total volume to 1 liter. Add 100 mL 10X stock solution to graduated cylinder. Transfer solution to bottle.8 141 294 Making 10 L of 1X solution: A. 5.0 mM 1360 26.4 10X g/L 80 2. 13 .3 for 1 x solution. Do not adjust pH. Dissolve compounds in 700 mL distilled H2O B. Dispense 1X solution in bottles and autoclave at 250°F for 30 minutes (liquid mode). autoclave the PBS in the bottle with cap loosened and covered in tin foil. Making 1 L of 10X solution: A. Add about 800 mL DI water to graduated cylinder (will help absorb heat produced during dilution) 2. 3.7. Add to 1 L using volumetric flask or graduated cylinder C.0 0.2 .0 liters of double distilled water. C.0 20 4.20 2 0. B. Dissolve compounds in 7. pH the solution to 7. Transfer to beaker with stir bar.4 mM 136 2. Check and adjust pH to 7.68 14.2 4. D. liquid cycle.1 29.PREPARATION OF PHOSPHATE BUFFERED SALINE (WITHOUT MG OR CA) UPDATED 2/22/02 Materials NaC1 KC1 Na2HPO4 KH2PO4 1X g/L 8. Adjust pH and sterilize when diluted to working concentration.
Caps will be sterilized separately in baskets. Variable speed peristaltic pump (Randolph 610). 8. date. i. 4. Powdered medium. 6. After preparation. type of medium. i) Add half of the quantity of the water to the container. filtration and dispensing. Polypropylene mixing paddle. Label all cases clearly. the medium should be left at room temperature for 2 days and supplemented with serum if no contamination is detected. iii) Stir continuously to prevent clumping. 3. penicillin.90 can be used as a back-up system. 2. ii) Add the appropriate amount of powdered medium to the water. Add medium slowly. Preparation of Medium a. 15 cases are required. 7. 500 ml bottles. Bottles should be sterilized and covered with a piece of heavy duty tin foil. Dry cycle (30 minutes + 30 minutes dry). b. 20 per case. The water is collected in the 20 liter (or 50 liter) polypropylene tank in which the media is to be prepared. lot number and type of media. c. 293 mm Millipore filter holder set up or Millipore Twin . C. Each case will be clearly labeled as to date. Double distilled water collected in a 200-liter polypropylene tank.PREPARATION OF 1 X CELL CULTURE BASAL MEDIUM UPDATED 2/22/02 A. or HCL (for REMI). 2. GENERAL Medium will usually be prepared in 20 to 50 liter lots and dispensed in 500 ml bottles (450 ml/bottle). B. Supplemented media will be frozen at -20°C and will not be used until sterility testing is complete (this medium will be designated for the maintenance of the cells). 15 .e. 20 bottles per case. Steps in Preparation 1. Add sodium bicarbonate. Add the appropriate amount of powdered medium to the water slowly and with stirring to prevent clumping. Preparation of Equipment a. For the average 150-liter lot. Sodium bicarbonate. Water: Milli Q water is used as the source for the Bellco Still. etc.. Sterile filter should be obtained from a secure location where the sterile packaging has not been compromised. streptomycin. Bottles: Bottles are sterilized in large boxes (24 bottles per case) in autoclave ("A" or "B" autoclaves). Materials Required 1. Source of compressed CO2 for pH adjustment. Filters used in media preparation must be stored by the user in a secure location in order to ensure the sterile packaging has not been compromised. Pall #3001 ultrapore filter. 5.
8-6. The 293-mm Millipore Filtration Assembly should be used (non-sterilized) as a pre-filter when the Twin-90 is used for filtration of batches of media in excess of 125 liters. and caps should be placed on tightly. or supplement immediately and freeze. Fill bottles using a flow rate of 4 to 6 liters/minute. Information concerning the assembly of the 293-mm filter may be obtained from the files in the office or in the general information notebook in the media production room. Optional Use of 293 mm Millipore Filtration Assembly a. (incubation at 37°C for 14 days. i) Place hose from pump all the way to bottom of the tank.(we just store william E in cold room) 3. f. seal off the valve. and individual bottles checks substantiate sterility.2 by the addition of HC1. This will wash out any residual water in the hoses and filter from the autoclaving (this step hasn’t been seen in the lab). and last bottle of each lot and use for sterility tests. Once the media starts to flow out. Place lid on tank. ii) RPMI medium requires pH adjustment to 7.) i. d. This filter is package pre-sterilized and does not require a media bell for dispensing. g. the entire lot should be stored at -20°C until supplementation. 16 . Remove the first.i) Adjust pH of DMEM to 6. c. as this will cause cracking during freezing. leave the top screw valve loose to permit the release of any air which may be trapped in the unit. 4. A 500-ml bottle should be filled to the 450-ml level using a "guide" bottle filled to that volume. c. ii) When adding 1 N HC1 to RPMI. The 293-mm filter may be utilized as a pre-filtration unit with the Pall #3001 filters. All bottles should be flamed before and after filling. e. No bottle should be filled past the level where the bottle narrows. When initially pumping media through the tubing and filter unit. b. Allow the entire lot to remain at room temperature for two days.9 by adding 1 N HCl into the medium using a Pasteur pipette. Return the first four bottles of filtered media to the polypropylene tank and stir thoroughly. Attach tubing from CO2 tank and allow a slow but steady stream of CO2 to blanket the medium. middle. use small amounts and mix well. Attach media bell to the ring stand in the sterile room and carefully remove foil and wrapping. The Twin-90 should only be used for back-up purposes. h. b. If preliminary results of incubated samples indicate that the batch is sterile. b. Use of Millipore Twin-90 Filter a. The Pall #3001 filter is the primary media production filter.
In Media N/A ~68. Perform sterility test 10.0593 g 0.07212.252 g 4g 1.IMX MEDIA UPDATED 2/22/02 Cells: Chinese Hamster Ovary Cells (CHO) Component IMX 1. Add intralipid (50 ml for each 500 ml bottle) & gently mix 9.17 mM ~68. Add above components (except intralipid) into a clean 1L erlenmeyer flask 3.1 mM (w/ IMX 1. Store at 4C until use 30 .15 mM 6.3 mM N/A ~36.5 mM 200 mM N/A N/A 20% fat emulsion Conc.0500 Amino acid cabinet Sigma Cellgro Cellgro Cellgro Sigma Gibco BRL Sigma Sigma Sigma G5146 99-182-LI 99-175-Cl 99-176-LI M8407 25030-081 PEN-NA S6501 I141 Organic Cabinet 4C 4C 4C minus 20C minus 20C 4C 4C 4C Procedure: 1.2 NaCl Na Bicarbonate Soy Peptone Glucose Trace Elements 1000xA Trace Elements 1000xB Trace Elements 1000xC Methotrexate L-Glutamine Penicillin G Streptomycin Intralipid Vendor Cat # Storage Mixing 13. Gently invert and swirl so as to avoid bubbles 5.4 mM ~14.1 g 100 ml Stock Conc. Add < 1 L of DI/UF water 4. Adjust pH to 7. N/A N/A N/A N/A N/A N/A N/A N/A 1.0 6. Adjust to final volume (1L) 7. Sterilize 2 500 ml bottles 2.6 M N/A JRH Biosciences 571004-50L 4C Sigma S5886 Inorganic Cabinet Sigma S4019 Organic Cabinet EM Science 1. Filter sterilize into 500 ml bottles using a 0.2) N/A N/A N/A ~0.2 g 5g 2.4 mM ~0.22 mm filter 8.0 g 1 ml 1 ml 1 ml 100 ml 32 ml 0.
Add remaining 5 compounds 5. Store at -200 C 27 .5 or 5 mL sized aliquots. Add 43. weighing dish/paper Sterile pipets Magnetic Stir bar Magnetic Stir plate Syringe and syringe filter 20 or 40 Falcon Tubes Procedure: 1.POOLED SUPPLEMENTS UPDATED 2/22/02 Component Albumin Linoleic Acid EGF Sigma Cat # A-2058 L-5900 E-4127 Mixing 5g 50mg/25 mL H2O 0. Filter sterilize (use pre-filter) and aliquot into 2. Add Albumin to sterile 100 mL bottle 2.2 µg/mL 0.5 mL 0.210 mL Concentration Conc.1 ng/mL Dexamethasone D-1756 Transferrin Glucagon Na2SeO3 T-1147 G-3157 S-5261 Materials: 100 mL bottle Balance. Mix together and add 25 mL DI water (mix until Albumin dissolves) 4. Use sterile falcon tubes (which can withstand freezing) 7.6 mg/mL 0. Add 5 mL DI water to each of 5 bottles of Linoleic Acid 3.2 mL 0. In Media 50 mg/mL 500 µg/mL 15 µg/mL 150 ng/mL 0.392 mL 3 mL 0.4 µg/mL 0.5 µg/mL 39.1mg/5mL PBS 100mg/(8m PBS+2mL EtOH) 100mg/5mL H2O 2mg/10mL PBS 1mg/10mL H20 Vol in Pooled Supplements 5g 25 mL 2.3 mL DI water to bring final volume to 100 mL 6.21 µg/mL 5 ng/mL 392 ng/mL or 1µM 6 µg/mL 4 ng/mL 2.
Aliquot into approximately 5 mL size aliquots in sterile centrifuge tubes. CuSO4*5H20 will take a while to enter dissolve. Sterilize using a syringe filter. Stir solution. add Na2SeO3 and ZnSO4*7H20. Once dissolved. Date and label.065 mg/50mL water Concentration: Concentration in 10 mM 300 µg/mL 5 µM 0.015g/50 mL water .125g/50 mL water 0.TRACE ELEMENTS UPDATED 2/22/02 Component: Media CuSO4*5H20 Na2SeO3 ZnSO4*7H20 Sigma Cat #: Mixing: C-8027 S-5261 Z-0251 0.1 µM 3 ng/mL 50 pM Materials: Small Beaker Magnetic Stir Bar Magnetic Stir Plate Balance. store at 40C. weighing dish/paper Syringe and Syringe Filter 10 sterile 15 mL centrifuge tubes Procedure: Add the CuSO4*5H20 to a small beaker with 50 mL of water and a stir bar. 26 .
9. until the suspension is homogeneous. Invert to mix. media. 5. 3. Using a pipette remove suspension from vial. 17. If 60-80% of the cells are viable that reflects on a good freeze technique. loosen cap. Thaw IMMEDIATELY by holding cryotube in 37°C water bath in such a way that the juncture of cap and tube is not submerged in the water. sterile 3. Suck in and out as few times as possible to break up major lumps. 12. gently invert to mix. centrifuge tubes. 15. 15ml. room temperature. Suck off the supernatant media. *Steps 12 & 13 are applicable only when there is enough volume. 13. 16. trypan blue dye. Add enough media to again double the new volume (you have now diluted the cell suspension 8-10 times). Remove a measured amount of cell suspension from the T-flask. Thump. Count both the dead (dark blue) and the live (colorless). Remove cryotube from liquid nitrogen and record it as removed in Liquid Nitrogen Database. 8. Add l ml media for each l ml thawed cell suspension S-L-O-W-L-Y drop by drop.THAWING CELL FROM DEEP FREEZE STATE UPDATED 2/22/02 Materials: 1. Let centrifuge tube containing cells and media sit in 37°C water bath for 5 minutes. sterile 2. warmed to 37°C 4. Place T-flask in 37°C CO2 incubator. 4. stock solution Procedure: 1. Let sit in 37°C water bath for 5 minutes. Shoot the media in to break up cell clumps. Transfer the cell suspension into the T-flask (spinner). For anchorage dependent cells. 21 . without creating air bubbles. change medium to remove residual DMSO and dead cells on the second day. 7. SLOWLY but not dropwise add enough media to double the current volume. 11. Replace the 8-10 ml you just sucked off with fresh media. 14. and place in a centrifuge tube. 2. pipettes. dilute with the same amount of Trypan Blue dye (1:2 dilution). Incubate overnight. 10. in the case of non-anchorage dependent cells. Using the hemocytometer count the cells for a viable cell count. Centrifuge at 700-800 rpm. Thump to mix until homogeneous. same size T-flask from which cells originally came 5. Repeat viable counts. every 24 hours. You will see a nonhomogeneous layer of medium on top. 6. note volume withdrawn. 5 minutes.
*All unlabeled T-flasks will be discarded. 2. Slap the side of the flask--cells should slide off. 18 . then remove 2ml of the mixed cell suspension to each T-flask the same size as the original. initials. cell line name. media. Label flask with split ratio. Do not suck up all of the volume or release the entire volume at once or else you will get bubbles and subsequent protein degeneration. Remove appropriate amount from parent flask for each daughter flask. passage number. For example: Take 2ml out of the total of 8ml for each of 4 flasks for a 1:4 dilution as in example above. incubate longer d) add medium containing serum to stop reaction e) shear with a pipette to mix SPLITTING CELLS FROM T-FLASKS UPDATED 2/22/02 Procedure: 1. Shear gently for the purpose of dispersing cells in newly added media. Add enough medium to stop reaction (minimum is twice the volume of trypsin) plus enough volume to split comfortably. date. 4.b) place T-flask in incubator for 2-3 minutes. If cells are not coming off. return flask to incubator and loosen caps. cap loosened c) at the end of 2-3 minutes to check if cells are coming off. 3. The responsible person will be disciplined. For example: for a 1:4 split add 7ml to the already existing l ml of trypsin.
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