Prepare Growth Medium
1. Remove 20 mL a new bottle of Advanced MEM (AMEM) basal medium and discard.
2. Add the following to the remaining volume of AMEM in the bottle:
a. 10 mL of fetal bovine serum (FBS).
b. 10 mL of GlutaMAX.
c. Add 1 mL of 100X penicillin/streptomycin.
3. Mix thoroughly and label with the date and additives.
Collagen Coating of Tissue Culture Plates
IMR90 cells should be grown on dishes/plates/inserts that have been coated with 50 mg/mL bovine collagen I solution, as described in McNabb and McCullough (2019).
Thawing Cryopreserved Cells
1. Warm growth medium to 37 °C and prepare materials prior to obtaining a vial of cells from cryostorage.
2. Remove vial from liquid nitrogen (wearing eye protection) and thaw in 37 °C water bath (1-2 min).
a. It is important that cells are thawed quickly to reduce the duration of exposure to the high concentration of DMSO in the freezing medium, which will negatively impact cell viability.
3. Add 24 mL of growth medium to a 50 mL conical tube then transfer the thawed cells from the cryovial into the growth medium using a P1000 pipette and gently mix by inverting 10 times.
NOTE: do not vortex
4. Pellet cells via centrifugation at 1,000 x g for 4 minutes at room temperature in centrifuge with a swinging bucket rotor.
5. Carefully aspirate the supernatant.
6. Gently resuspend the cells in 12 mL of pre-warmed growth medium, add 13 mL of additional growth medium, and transfer into a collagen-coated 15 cm dish. Distribute cell suspension on the dish through a combination of gentle rocking and swirling of the dish for approximately 15 seconds.
7. Place in a tissue culture incubator overnight.
8. Check cells the following day for attachment to the dish. Aspirate and replace medium.
a. NOTE: Use 10 and 25 mL of medium for 10 and 15 cm plates, respectively.
b. NOTE: It is preferable to subculture thawed cells for at least three passages before using in experiments.
Cells should be split three days after being plated at either 4.0 x 104 cells/mL
NOTE: Lower APD IMR90 cells grow very quickly and should be plated at 3.5 x 104 cells/mL to start and then adjusted up to 4.0 x 104 cells/mL accordingly as their growth rate declines over time in culture.
1. Pre-warm growth medium and DPBS in a 37 °C water bath and a trypsin aliquot at room temperature for approximately 30 minutes.
2. Aspirate the growth medium from plates to be passaged.
3. Rinse cells with pre-warmed DPBS and swirl gently for approximately five seconds.
a. 20 mL for 15 cm dishes
b. 10 mL for 10 cm dishes
4. Aspirate the DPBS wash.
5. Add trypsin and rotate gently to distribute evenly across the plate.
a. 1.5 mL (5 mL pipet) for 15 cm dish
b. 750 μL (P1000 pipette) for 10 cm dish
6. Place in tissue culture incubator for 4 minutes, making sure not to stack dishes on top of each other. Following incubation, use the palm of your hand to gently tap the side of the dish to facilitate detachment. If cells are still attached, incubate for another 2 minutes. Incubate and tap dish until >95% of the cells are detached. Check detachment with microscope.
a. NOTE: the duration of trypsinization may need to be optimized for each lot of trypsin.
7. Add 12 mL growth medium, gently wash the surface of the dish with a pipette, and triturate once to break up any large cell clumps. Transfer the cell suspension to a 50 mL conical tube.
8. Wash the dish with an additional 12 mL of growth medium, collect, triturate, and add to the cell suspension from Step #7.
9. Pellet cells at 1,000 x g for 4 minutes at room temperature in centrifuge with a swinging bucket rotor.
10. Aspirate the supernatant and resuspend the cell pellet in 12 mL of growth medium. Triturate at least four times to thoroughly break up the majority of clumped cells. Add additional growth medium to dilute the cell suspension if desired. Typically, growth medium is added such that there is a total volume of 12 mL per 2-3 15 cm plates collected. Mix thoroughly by inverting the tube 10 times.
11. Prepare a 1:1 dilution of the cell suspension in trypan blue (50 μL trypan blue + 50 μL cell suspension) in an Eppendorf tube.
12. Count cells that exclude trypan blue (i.e., appear clear) on both sides of a hemocytometer (do not use an automated cell counter as these cells display variability in size and are not accurately quantified by automated methods).
a. NOTE: Blue staining of cells indicates a damaged membrane. Blue-stained cells are considered to be non-viable.
13. Calculate the cell concentration in the cell suspension.
14. Determine the new culture APD.
a. Calculate and record the number of cells obtained per plate during this passage.
b. Calculate the number of population doublings that occurred during the last passage (include the indicated values in the associated worksheet) and adding that number to the previous APD:
([(3.32(log(cells collected per plate)-log(cells plated per plate)) + previous APD])
15. Dilute cells to 4.0 x 104 cells/mL in growth medium in a 50 mL tube (or larger sterile bottle based on needed volume) to the total volume that will be used for plating (10 mL for each 10 cm plate and 25 mL for each 15 cm plate, making 2-5 mL extra).
16. Mix thoroughly by inverting 10 times and dispense the diluted cell suspension into each dish. Distribute cell suspension on the dish through a combination of gentle rocking and swirling of the dish for approximately 15 seconds.
17. Place in a tissue culture incubator. Cells will be ready to split three days after plating.
a. NOTE: Plating cells at a determined cell density will result in more predictable sub-culture and more reproducible performance in subsequent experimental assays.
18. Place bottles of growth medium and DPBS in 5% CO2 incubator with lids partially unscrewed for 15 minutes to adjust pH before returning to 4°C for storage.
Preparing cells for cryopreservation
1. Prepare freezing medium.
a. 50% FBS, 40% growth medium, 10% DMSO.
b. Filter through a 0.2 μm pore syringe filter into a 50 mL conical tube.
2. Follow sub-culturing protocol through Step #10.
3. Determine the number of cells present and pellet cells at 1,000 x g for 4 minutes at room temperature in centrifuge with a swinging bucket rotor.
4. Carefully aspirate the supernatant and resuspend the cell pellet in freezing medium at a density of 1.25 x 106 cells/mL.
5. Mix cell suspension by gently inverting 10 times and dispense 1 mL aliquots into cryovials with a P1000 pipette.
6. Place cryovials in a Mr. Frosty freezing container (filled to indicated line with isopropyl alcohol) and place in a -80 °C freezer overnight.
a. The chamber regulates cooling rate at approximately 1 °C per minute.
b. NOTE: Isopropanol should be changed after every three freezing cycles.
c. NOTE: Do not leave vials at -80 °C longer than overnight as it will impact viability after thawing.
7. Transfer the frozen vials in liquid nitrogen storage.