293细胞培养.doc

Protocol for Growth of HEK293 Cells in Suspension*Always wear lab coat and glovesBuffers and Media (see also file called media_buffers.doc)Detergents: Dodecyl-b-1-maltoside (DM) and n-octyl-b-D-glucoside (OG) can be obtained from Anatrace (Maumee, OH). Protein A-Sepharose was from RepliGen (Cambridge, MA), CNBr-activated Sepharose 4B was from Pharmacia. DMEM (Dulbeccos modified Eagles medium): Standard DMEM:Standard DMEM medium HB-Gro was purchased from Irvine Scientific (Santa Ana, CA). Powder for 10 L will be dissolved in 9 L Millipore water (distilled water). Add sodium bicarbonate if required (check manufacturers manual and/or powder container). Stir until dissolved, but avoid air bubbles. Fill up to 10 L and sterile filtrate into 20 autoclaved 0.5 L bottles. Before first use of a bottle add 10 % (v/v) heat inactivated (30 min, 56C) Fetal Bovine Serum (FBS) and penicillin (100 units/ml), streptomycin (100 mg/ml). You may have to add glutamine as well (check manufacturers manual and/or powder container). Store at 4C. Custom made DMEM:Custom-made DMEM lacking the amino acids of choice, sodium pyruvate, calcium and sodium bicarbonate was obtained from Atlanta Biologicals (Norcross, GA). Store at 4C.ORDMEM was made out of its components (e.g. Sigma cell culture tested). Store at 4C.*There are three types of DMEM used in this procedure. The DMEM with 1.4 mM Ca+ is the DMEM used for the steps up until the splitting into 20 plates. The subsequent steps use DMEM with 680 uM Ca+ and 340 uM Ca+. DMEM is usually prepared 2-3 L at a time with 1.4 mM and 340 uM Ca+ and stored in sterile bottles at 4C. The 680 uM can be obtained by mixing the two DMEM medias.Antibiotics and protease inhibitors: Geneticin (G418), trypsin, EDTA, penicillin, and streptomycin were obtained from Gibco/BRL (Gaithersburg, MD). Aprotinin, benzamidine, leupeptin, and pepstatin A were purchased from Boehringer Mannheim (Indianapolis, IN). Serum: Fetal bovine serum (FBS), cell culture grade calcium chloride, Pluronic F-68, PMSF and amino acids were from Sigma (St. Louis, MO). FBS (500 mL) was heat inactivated (56C, 30 min) and dialyzed (1 kDa cutoff) three times against 10 L PBS buffer (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 7.2.) over 3 days at 4C.Growing Cells on One PlateBefore retrieving cell lines, warm DMEM solution in a 37C water bath to prevent heat shock (for 1-2 h, dependent on the volume). Remove prepared geneticin G418 stock from -20C freezer and let it thaw in 4C refrigerator. The geneticin is usually purchased as a powder and made into stock solution. Geneticin is dissolved in DMEM media at a concentration of 50 mg of geneticin/mL of media). DMEM can be purchased (see above for vendors) or made with Millipore water (see media_buffers.doc) and is stored at 4C. (We currently are using commercial DMEM for the plates and making our own DMEM from individual components for the bioreactor since this DMEM contains 13C - labeled amino acids). Gather and prepare all materials needed for the growing of cells in tissue culture hood: pipettes, 15 mL conical tube, 10-cm culture dish, DMEM solution, and geneticin. This step is to minimize the amount of movement and possible contamination.*All glass bottles should be sprayed with 70% ethanol solution and wiped after handling.Retrieve cell lines from liquid nitrogen storage. Cells should be stored in 2 mL cryo-tubes in 1 mL DMEM containing 10% (v/v) DMSO solution. Use one cryo-tube of frozen cells for each 10-cm plate. Cells are to be thawed in a 37C water bath as fast as possible (i.e. as soon as the solution thaws, remove from the water bath and immediately go to the next step). *All subsequent steps should be done as quickly as possible. Transfer the cells from the cryotube to a sterile 15 mL conical tube with a pipette. * Always flame the DMEM solution bottle before and after usage. Pipette 10 mL of DMEM solution and transfer DMEM slowly into the conical tube. It is best to add a couple of drops at a time and then swirl to reduce osmotic shock. This step is to dilute the antifreeze in the cells. Centrifuge the tube with DMEM and cells at 1000 x g (rpm depends on machine) for three minutes. Remember to put a counterweight at the opposite end of the rotor. Remove the supernatant via vacuum or pipette in the hood. (There is a lot of pipetting in cell culture work, so connecting the pipette to a vacuum is useful.) Resuspend the pellet with the appropriate amount of DMEM (e.g. 9.6 mL). The total amount of DMEM and geneticin G418 together should equal 10 mL. Calculate the amount of geneticin G418 to add (2 mg/mL for our WT rhodopsin cell line, i.e. different cell lines will be selected with different amounts of geneticin) from stock solution. The rest of the solution is DMEM. (For example, 0.4 mL G418 stock solution + 9.6 mL DMEM solution). Pipette the solution (DMEM + cells) up and down in the conical tube to resuspend and break up the visible pellet. Pipette the solution onto a 10-cm culture dish.Add the calculated amount of geneticin onto the culture dish (e.g. 0.4 mL) and swirl to mix the solutions. Geneticin forces the cells to retain the extra DNA. Place the solution on an inverted microscope and check for single cells. These cells should move when the dish is swirled. i.e. they should not be floating clumps of cells or attached to the bottom of the plate at this time point.Place the covered culture dish in a 37C incubator at 5% CO2. (Incubator needs to be calibrated at the start to make sure it equilibrates at 5% CO2.) Check if the cells sink to the bottom of the dish in one or two hours. Cells that have sunk to the bottom should not move when the dish is swirled. It is normal that some cells may not have attached yet. Dispose used tubes/pipettes in bio-waste and return DMEM to 4C and geneticin to -20C or -80C freezers. Cells should be grown for approximately four days and fed once during the middle of this time period.Feeding the cells involves removing the old medium and adding new DMEM and geneticin (selection marker). Prepare all necessary materials ahead of time in the hood: pipettes, geneticin, DMEM.DMEM should be warmed to 37C in a water bath and geneticin thawed in a 4C refrigerator or at room temperature. Remove the culture from the incubator. Tilt the dish and vacuum the media (or use sterile pipettes) from the side of the dish. The cells should adhere to the bottom of the plate while the liquid media is removed.Add the same amount of DMEM and geneticin as the previous medium had (final volume 10 mL). *Remember to flame and practice sterile techniques. Place in incubator for an additional two days. After four days of growing the cells, there should be approximately 107 cells in the culture dish. The media in the dish should appear darker than the original media. Splitting the Cells onto 2 - 4 Plates of 15-cm in diameter. Prepare all the necessary materials needed to split the cells: 2 - 4 15-cm culture dishes (depends on how many cells needed), pipettes, conical tube, PBS, trypsin, DMEM, geneticin. The DMEM and PBS bottle should be sprayed with ethanol and wiped after handling. DMEM and PBS should also be at 37C and geneticin at 4C. The media from the culture dish is to be removed by vacuum or pipette and the cells subsequently washed with 1x PBS (10 mL). Add PBS and swirl. (see media_buffers.doc file for PBS composition).Remove PBS by vacuum or pipette. Add 1 mL of trypsin/EDTA solution (stored at -20C). (You can buy this solution commercially from Invitrogen.) Trypsin will detach the cells from the dish. Swirl the solution until all the cells have been removed form the bottom of the dish (you can also bang the plate against the hood wall). The cells appear as small whitish clumps that can be seen with the naked eye. Add 10 mL of DMEM to the culture dish. *Flame bottle. Pipette the solution up and down to mix and further break up the cells. OPTIONAL: pipette the solution and place it in a sterile 15 mL conical tube. Centrifuge the solution to remove the trypsin solution (supernatant). This is optional because the serum is a trypsin inhibitor. Remove the supernatant and resuspend the pellet in 12 mL of DMEM. Fill the 15-cm plates with DMEM solution. As with the preparation for the first culture dish, calculate the amount of geneticin (2 mg/mL), cells, DMEM needed to have a final volume of 30 mL. If the stock solution of geneticin is 50 mg/mL, there should be 26 mL DMEM, 3 mL cells, and 1.2 mL geneticin. Pipette 3 mL of the cells into each plate. Rotate while ejecting the cells so that they are spread out on the 15-cm culture dish. Be gentle. Add the appropriate amount of geneticin to each plate. Place the cells in 37C incubator at 5% CO2.Check the cells in one to two hours to see if they have attached to the bottom of the plate. Splitting the Cells onto 20 Plates of 15-cm in diameter. Approximately 72 hours later (3 days), split the cells onto 20 15-cm culture dishes containing the DMEM with the lower Ca+ concentration (680 M). The procedure is exactly the same as the cell split from the 10-cm plate to the (2-4) 15-cm plates. Cells are washed with 20 mL PBS (then drained). 2 mL trypsin/EDTA is added to detach the cells. There is no centrifugation in this step. Add DMEM directly to the old plates and then split onto the 20 15-cm plates. Add geneticin afterwards and place into 37C incubator. Check the cells in one to two hours to see if they have attached to the bottom of the plate. At this time point, the cells should appear scattered and circular and should be at approximately 10% confluence. 48 hours later (2 days), check on the cells again. The cells should now have expanded into a triangular shape and inhabit most of the plate (i.e. they should be at approximately 50-60% confluence). Feed the cells by removing the old medium and adding new DMEM (340 M Ca+). The procedure is detailed on page 2. Prepared all the materials ahead of time and warm the DMEM and thaw the geneticin. Add the same amount of DMEM and geneticin as in the previous medium (final volume 30 mL). *Remember to flame and practice sterile techniques.Replace the cells in the incubator and leave overnight for transfer into the bioreactor the next day. Transferring Cells and Growing in the BioreactorThe bioreactor should be prepared the day before the transfer of cells. It should be washed with Liquid-Nox detergent and scrubbed clean. It is then rinsed with Millipore water and autoclaved according to the manufacturers manual with PBS buffer (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 7.2). The volume of PBS buffer depends on the volume of the bioreactor growth. If 3 L of cells are grown (which is standard), then the bioreactor should be autoclaved with approximately 5 L PBS. Fill the jacket with water according to the manufacturers manual and autoclave the bioreactor with the jacket filled (we autoclave at 124C for 2h for 5L, check bioreactor manual). The tubing for the bioreactor should be washed in the dishwasher and then rinsed for 10 minutes with Millipore water. The tubing should be attached to the bioreactor ports with one clamp per tube 10 cm after the metal port. The glass adaptors should be greased (use autoclavable grease) and wrapped with aluminum foil to prevent contamination. Any other open ends should be stuffed with filters (the filters we use are housed in plastic containers that came with the starter kit for the bioreactor). The tubing is connected and autoclaved with the bioreactor. There should be no open ports. The pH electrode should be calibrated according to manufacturers manual before the bioreactor is autoclaved. (It has a long calibration time. Markus usually calibrates at pH = 7.0 overnight.) Switch the thermobath on BEFORE the bioreactor. The O2- electrode should be filled with alkaline solution every 2-3 bioreactor runs according to the manufacturers instructions. The autoclaved bioreactor and tubes are then connected to the controller. Connect the air filter (make sure there is no water in the filter) and attach the motor, temperature sensor, pH electrode, and O2 sensor. The water flow and heat jacket pipes should be connected with the outflow connected first and then the inflow. Turn the motor and air flow on. The air pressure should be 10 psi (check manual). On the screen, change the air to manual. The air should bubble out from the bottom of the bioreactor (bubbles should come out of the first three holes). The motor and air filter is to be left on until the end of cell growth. The only time it is off is when the PBS is exchanged with DMEM media. The bioreactor is left running with PBS to allow it to equilibrate for O2- electrode calibration. Check on the cells the next day. If they grew well overnight, they can be transferred into the bioreactor. Cells should be at 90% confluence (5 x 106 - 5 x 107 cells). They should appear very closely packed with approximately 5-10% free space. If the cells have not grown very well, they can be left in the incubator for an additional 24 hours. When the cells are ready to be transferred into the bioreactor, 3 L of DMEM needs to be ready, either commercial DMEM or DMEM made in the lab using labeled amino acids. (Note, when making the DMEM yourself, you may want to make stock solutions such that you can add the labeled amino acids separately depending on the exact labeling strategy you plan to use.)Prepare the hood by gathering all the required materials: several sterile bottles including feeding (5 L) and inoculation (0.5 L) bottle, pipettes, antibiotics, FBS + additional trace elements (for dialyzed FBS), and a large filter for sterile filtration. *All glass bottles should be flamed before and after opening. Add 10% dialyzed FBS volume (300 mL for 3L) into the DMEM solution and any additional trace elements according to recipe of manufacturer of the DMEM. Add to this solution 0.1% Pluronic F-68 (wt/vol) and 50 mg/L heparin (or 300 mg/L dextran sulfate). (Note, if you are using commericial DMEM for the first unlabeled bioreactor runs, then you do not need to dialyze the FBS. If you do not dialyze, then you do not need to add trace elements.)Swirl the bottle to mix the solutions. The medium is sterile filtered with a low protein binding cellulose acetate filter and usually with the use of a vacuum. The medium should be filtered into a sterile 5 L or large enough bottle. Add 100 units/mL of penicillin and 100 g/mL of streptomycin into the filtered solution. Approximately 200 mL of this solution should be removed and placed in a bottle where it will later be used to remove the cells from the plate. This part should be kept in the 37C water bath. The bottle is then capped with a special bioreactor cap that has tubes going in and coming out of the cap. The outside tube should be clamped and the cap flamed while placed on the bottle. The top part of the bottle should be covered with aluminum foil to prevent contamination. Calibrate the O2 sensor (100 % the equilibrated PBS, 0 % disconnected, check manual). Remove the aluminum foil and filter from the draining tube. Place the draining tube in a feed and begin draining the PBS from the bioreactor. The feed is to prevent air from flowing into the bioreactor. When there is a steady flow of PBS out, the tube can be taken out of the feed for faster outflow (gravity). PBS can be collected in bottles or buckets and drained into a sink. When almost all the PBS is drained, switch off the motor and the heat. Reattach the tube to the feed before all the PBS is drained. After the PBS is drained, reattach the clamps, filter, and place aluminum foil on the end of the draining tube. Reattach the O2 sensor, but leave the motor and heat off. Attach the container with the 3 L of DMEM solution to the bioreactor by attaching the two tubes glass adaptors (one on a bioreactor port and one from bottle head) with a clamp over a flame. Aluminum foil should cover the connection. Once attached, place the tube in a feed to prevent air inflow. Remove the clamps and allow the media to flow to the bioreactor, remove the tube from the feed to allow the media to drain faster by gravity. Allow the media to fill the bioreactor. When the media reaches the temperature sensor and is above the impeller, turn on the heat and motor. Reattach the tube to a feed and allow it to finish draining. When it is finished, place the clamps back on. The tube should be left in the feed and the connection can be left until the end of the experiment. Turn on the N2 and CO2 gas. The pressure for both should each be 10 psi. Connect the O2 gas; should be 10 psi. (Note, check the bioreactor instructions to make sure these are the recommended pressures for our model of reactor.) However, the oxygen gas can be added in 24 hours since air should be sufficient enough for the cells right now. The amount of oxygen consumption in the bioreactor can be used to determine the amount of cells growing. Allow the bioreactor to adjust to the media and stabilize the pH and temperature. This step takes approximately 3 hours depending on the volume of media. *Gather all materials into the hood: pipettes, trypsin, media, and sterile bottles. All glass bottles need to be flamed. After the media has been stabilized in the bioreactor, the cells can be removed from the incubator and the media from the plates vacuumed out. The cells are washed with 20 mL of PBS and the PBS is then drained. 2 mL trypsin/ is added to the plate and the cells are removed by swirling/shaking. Add 10 mL of the