The Complete CHO Culturing Guide
Figure 1: Cricetulus griseus
Deciding when to use mammalian cells:
In synthetic biology, cells can be one of the most valuable tools at a researcher's disposal. Bacterial and mammalian cells are of particular interest as they can be used for an infinite amount of applications including drug delivery, protein expression, DNA amplification, and of course vaccine production. When it comes time to choose the right cell type that best suits the project at hand, several design constraints must be taken into account. The biggest constraint is the actual function needed to be carried out. If the cell in question is going to be used to mimic the native environment of the human body, then mammalian cells are preferred. This should be obvious as human cells are mammalian cells or eukaryotic. Bacterial cells on the other hand are prokaryotes. There are several key differences between Prokaryotes and eukaryotes, chief among those is the fact that Eukaryotes are compartmentalized through organelles and Prokaryotes have no organelles. The lack of organelles means that molecules will interact completely differently inside bacterial cells compared to mammalian cells. Thus, when trying to understand how a certain drug, enzyme, or other molecule interacts within the human body, mammalian cells are the gold standard.
CHO DG-44
In the context of iGEM, immortalized cell lines are of particular interest as they are the perfect cell line for undergraduate researchers who aren’t too familiar with mammalian cell culture. Immortalized cell lines are cells that developed or were engineered to have mutations that allow them to divide an indefinite amount of times. They are robust and much hardier than primary cells that are harvested directly from tissue sources. CHO-DG44 cells (Chinese Hamster Ovarian cells) are an immortalized cell line that was derived from CHO-K1 cells. CHO-DG44 cells are distinguished by their lack of a dihydrofolate reductase gene. Without this gene, DG44 cells require glycine, hypoxanthine, and thymidine (GHT) to proliferate. There are several CHO variants on the market and it is important to understand what mutations are useful for specific applications. In the protocol below, culture of CHO-DG44 cells (and any CHO cell line) will be explained so that any and all future iGEM teams can start their own CHO cell culture.
All Reagents and Devices Needed:
CHO-DG44 cells CHO Complete Media 10 % Fetal Bovine Serum FBS Dulbecco’s Modified Eagle Medium DMEM 1% Penicillin Streptomycin Hypoxanthine Thymidine HT 100X PBS Phosphate Buffered Saline Store at room temperature DI Water NaCl KCl NA2HPO4 KH2PO4 0.5% Trypsin-EDTA DMSO 70% EtOH Serological Pipette 50 and 15mL centrifuge tubes 10,25, 2, and 1mL serological pipette tips
Hemocytometer With Disposable Glass Coverslips Counter Trypan Blue T75 Culture Flasks Kimtech Kimwipes 37℃ Water Bath Inverted Microscope Benchtop Centrifuge 37℃ CO2 Incubator Set at 5% CO2 -20℃ and 4℃ Fridges Two-Step Vacuum Filtration System For Aspiration Biological Safety Hood A Room With Negative Airflow
Reagent Guide and Storage
CHO Complete Media 500mL: Work in culture hood to keep reagents sterile 440mL Dulbecco’s Modified Eagle Medium DMEM Store at 4℃ 50mL 10 % Fetal Bovine Serum FBS Aliquot into 50mL tubes and store at -20℃ Thaw in 4℃ overnight 5mL 1% Penicillin Streptomycin Store at -20℃ Thaw in 37℃ water bath 5mL Hypoxanthine Thymidine HT100X If making from scratch: Dissolve 136 mg of hypoxanthine and 38 mg of thymidine in 100mL of molecular water Filter sterilize using a 0.22 micron filter Aliquot into 5mL and store at -20℃ Filter sterilize using a disposable filter sterilization kit with a 0.22 micron filter
Figure 2: Filter steralization
Remove the top clear lid and transfer all reagents into the top container Attach the vacuum hose nozzle and replace the clear lid Turn on the vacuum to sterilize Unscrew the now empty top container and crew on the colored cap to seal the now full container Make at least four 50mL aliquots and store the stock and aliquots at 4℃ When you have used up two of the four aliquots make another two to always have small batches of media at the ready
PBS 1L:
Obtain an autoclave safe 1L media bottle Fill bottle with 800mL of DI water 8.0g NaCL 0.2g KCl 1.44g Na2HPO4 0.24g KH2PO4 When weighing salts, be exact as possible to make pH balancing easier Dissolve salts in DI water Measure pH of water Add HCl or NaOH in small 1mL increments until the pH reaches 7.4 Try and be as exact as possible Autoclave for 20 minutes at 15psi and at 121℃ If you don’t have access to an autoclave, filter sterilize Store at room temperature
Cryofreezing Media: (Make the same day you are going to use it) This recipe can be scaled up or down depending on how many cryovials you want to fill 9.5mL of your Complete CHO Media 0.5mL DMSO Thaw at room temperature is frozen Store in -20℃ Filter sterilize and store at 4℃ or use immediately
Storage of other reagents: 0.5% Trypsin-EDTA (Store at -20℃ ) Thaw in 37℃ water bath 70%EtOH (Store at room temperature) Trypan Blue (Store at room temperature)
Setting Up Your Two Step Aspiration System:
Figure 3: Two step vaccum filter
#Good Practices to Follow:
Before beginning work in the culture hood, ensure you are working in a sterile environment by spraying the hood surface down with 70% EtOH Treat the culture hood’s air as sterile, but don’t treat the surface as sterile If media, pipettes, or cells are exposed to air outside the culture hood, they are no longer considered sterile and should not be used for culturing Minimize movement in and out of the culture hood as much as possible to keep the air entering the hood as sterile as possible. Before ANYTHING is brought into the culture hood or CO2 incubator, sterilize it by wiping it down with EtOH and a kimwipe. To avoid getting EtOH into the culture flasks or any reagents, spray a paper towel and whip down reagents When using a serological pipette, ensure that the tip doesn’t touch the outside of its packaging or anything that isn’t the liquid being pipetting. If it does, it is no longer considered sterile If anything ever spills in the hood, moisten a kim wipe with EtOH and wipe up the spill in a circular motion When warming reagents in water bath keep the lids from touching the water to preserve sterility Keep a bucket for non hazardous waste in the hood and one to hold unused pipettes
If your hood does not have a holder for your aspirating tube, put a command strip hook inside the hood. Then take a zip-tie and make a loop around the tube so that it can hang from the hook without touching the surface of the hood When handling cells, avoid any violent or jerking motions to put them under as little stress as possible Keep cell culture flasks caps loose to achieve optimal gas exchange if not perforated Make sure all movements are deliberate and smooth, spilling $500 of complete media is not worth an extra ten seconds. If you run out of 2mL aspiration tubes, take a 1mL tube and snap off the filter to create a makeshift aspirating tube. If these makeshift aspiration tubes fit loosely in your aspiration tube take a 1000µL tip and wrap it in parafilm. Shove the pipette into the aspirating tube so that the pointy side of the tip faces inward. You now have an aspirating tube that tapers inward and fits any serological tube smaller than a 2mL pipette.
If there are any signs of contamination, immediately bleach and get rid of the culture to prevent any chance of it spreading to your other cultures Your culture room must have negative air pressure When aspirating, tilt the flask so that all liquid moves to one corner of the flask. Place your aspirating tip in this corner to minimize the amount of cell loss
Initial Seeding of Your Culture:
Materials: Cryofrozen CHO-DG44 cells Complete CHO Media Ice T75 Culture Flask Warm up of 50mL aliquot of complete media in the 37℃ water bath Transfer 9mL of complete media to a 15mL centrifuge tube
Remove cells from dewar and place on ice, don’t take them out until right before you need them Whenever handling liquid nitrogen, wear appropriate PPE as designated by safety personnel in your lab. This includes liquid nitrogen resistant gloves and apron If liquid nitrogen gets on your skin immediately rinse with tepid water that is not hotter than 44℃ Do not apply hot water or try to warm up rapidly
Quickly thaw cells by agitating in 37℃ water bath (40-60sec) Keep the cap above water level Sterilize cell tube and media bottle with EtOH and bring into hood Gradually add 1mL of prewarmed complete media using a 1mL serological pipette Resuspend cells using the same pipette and transfer to the sterile 15 ml conical tube containing warm 9 ml complete media. Centrifuge the 15mL tube at 125xg for 7 min to pellet your cells Move back to the culture hood
Aspirate the medium and resuspend the pellet in 14 ml of fresh a-MEM and transfer to a T75 flask. Loosen the lid of the T-flask to allow CO2 exchange if not perforated. Be very careful not to aspirate your cells Leave ~100µL of media to avoid this Move flask to CO2 incubator
Maintaining the culture: (Done Every Other Day for Each Culture)
Materials: Complete CHO Media PBS Remove cells from the CO2 incubator and bring them to the microscope for analysis. Look for any signs of contamination, discoloration of media, morphology of cells, and the confluency (density of cells on flask surface). Morphology: Epithelial
Figure 4: CHO Cells Epithelial Morphology
Media color should be a hot pink color If it is yellowish orange it is too acidic and needs to be changed immediately
Figure 5: Happy culture left, sad culture right
Max confluency should be 85%
You should try to passage cells anywhere from 70-85% confluency
Figure 6: 100% confluency
Figure 7: 90% confluency
Figure 8: 80% confluency
70% Confluency
Wipe the flasks with EtOH and move into the fume hood to change out the spent media. Aspirate out the spent media from the flask Wash the cells with PBS Add 10mL of PBS per flask Swirl the flask using North to South, West to East motions for ~20 seconds Aspirate out the PBS Add 14mL of complete media to the flask and return to CO2 incubator for 48 hrs of incubation Passaging of cells: Materials: Complete CHO Media PBS 0.5% Trypsin-EDTA Trypan Blue Hemocytometer Glass Coverslips
Figure 9: Culture Hood Layout
When the culture reaches ~75-80% confluency, we can passage our cells Prep by putting your bottle of trypsin-EDTA, complete media aliquot, and PBS in a 37℃ water bath for ~20mins. Sterilize with EtOH before bringing into fume hood Begin by aspirating the spent media out of the flask.
Complete the next two steps as quickly as possible to minimize cell death Perform a wash on the cells with PBS to remove debris from the flask.
Add 5mL of PBS per flask to the face opposite of the attached cells to minimize any cell detachment Swirl the flask using North to South, West to East motions Aspirate out the PBS Add Trypsin to detach cells from flask surface Add 5mL of Trypsin to each flask and swirl in North to South, West to East motions. Tap the each side of the flask firmly to encourage detachment. Move flasks back into CO2 incubator for 5mins to allow cells to fully detach Remove flask from incubator and tap again to ensure complete detachment of cells from flask and each other Image under microscope to ensure morphological change
Add 5mL of complete media to the flask to neutralize the trypsin with a 10mL serological pipette Do not discard pipette after this step Use the same 10mL serological pipette to transfer the cell suspension to a 15mL STERILE conical tube Centrifuge the conical tubes at 125xG for 7 mins to pellet cells
Figure 10: Suspended cells
Figure 11: Pelleted Cells
Wipe the outside of the tube with EtOH and return inside the hood to aspirate out the supernatant. Be careful not to disturb the pellet by leaving ~100µL of supernatant in the tube Resuspend cells in the tube with 5mL complete media and gently pipetting cells 10-20 times It will not fully resuspend but try to break up big chunks as much as possible. Once media is cloudy your cells are resuspended enough to continue Remove 10µL of cell solution for counting. Move 10µL of the cell suspension to a 1.5mL microcentrifuge tube and add 10uL of trypan blue Pipette up and down to fully incorporate and place the tube in the CO2 incubator for ~3min to allow the dye to penetrate dead cells. Prepare the Hemocytometer and glass cover slip by spraying with EtOH and lightly dabbing with a kim wipe.
Microscope setup for Cell Counting
Leave a little EtOH on the edges of the cover slip so that it can better adhere to the surface of the hemocytometer. Place the cover slip on top of the surface of the hemocytometer so that half of the insertion notch is covered by glass. Take the trypan blue cell suspension out of the incubator, and move back into the culture hood. Take a 10µL pipette and pipette out 10µL of the suspension and carefully pipette it into the the notch of the hemocytometer covered by the cover slip The result should be a small blue rectangle as shown below
Figure 12: hemocytometer
Figure 13: Placement of Cell Suspension into Hemocytometer
Move the hemocytometer to a microscope and change the magnification until you can clearly make out grid lines and cells. For our purposes, we are going to be focusing on the corner squares consisting of 16 individual squares each.
Figure 14: hemocytometer grid
Move the top left set of 16 squares into focus and start counting the total number of alive and dead cells. To get a more accurate count, disregard any cells touching the right and top lines of any sub square. Do this for all four large squares. If one of the squares has a radically different number of cells compared to the other three, count the cells in the middle square and use those numbers instead. Once you have a cell count for four total squares, take the average of them. We will refer to this value as “α” Apply the following equation to calculate the total cell density, [C], of your cell suspension [C] = α * H V D Where: H = Hemocytometer factor (10,000 because each square being 0.1mm2 and 0.1mm in depth V = Total volume cells are resuspended in (5mL) D = Dilution Factor (2 because we added 10µL of trypan blue to 10µL of cell suspension Throw out the glass cover slip, and clean the hemocytometer with EtoH In our lab, we seed our cells at a density of 1.5x106 cells in 14mL of complete media for a T75 flask Calculate how much of your cell suspension is needed and how much fresh media is needed. Add the fresh media to a NEW T75 flask before adding the cell suspension as the cell suspension volume will be significantly less. When pipetting your cell suspension, try not to pipette out any large chunks of cells that were not fully resuspended. Label flask as follows: Culture Name: Culture A
Team Name: UC Davis iGEM Date of Passage: 10/20/21 Passage Number: P1
Leftover suspension should be bleached until solution is clear and aspirated Loosely close the cap on the flask to allow gas exchange if the cap is not perforated, and put the flask back in the CO2 incubator at 37℃
Cryo Freezing of Cells:
Materials:
10mL Cryofreezing Media Cryovials PBS 0.5% Trypsin-EDTA Dewar -80℃ Fridge
Prep:
Place a styrofoam container in the -80℃ fridge to cool overnight
Procedure:
This should be done at an early passage number Warm cryofreezing media, PBS, and 0.5% Trypsin-EDTA in 37℃ water bath Follow the passaging protocol all the way through Each cryovial can hold 1mL of cell suspension at a cell density of 1x106 cells/mL Transfer the desired amount of cells into a new 15mL conical tube If you want to fill five cryovials, transfer 5x106 cells to the tube Pellet your cells at 125xg for 7 mins in the tabletop centrifuge Aspirate the media being careful not to aspirate out any cells Resuspend the cells in cryofreezing media If you have 5x106 cell, pipette in 5mL of cryofreezing media Allow the cells to sit at room temperature for 15 minutes Take out your precooled styrofoam container from the -80℃ fridge and place your cryovials inside it Place the styrofoam container with your cells back into the -80℃ fridge and let it sit for 24hrs. After 24hrs move the cryovials into the dewar