Culture of IMR90 Cells in Advanced MEM with Reduced Serum


 NOTE: Methods document and worksheet versions of this method are attached as PDFs.SCOPE OF APPLICATION (LIMITATIONS)This method describes the thawing, culturing, and cryopreservation of the human lung fibroblast cell line IMR90 in Advanced MEM-based growth medium. Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.


Introduction
CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS IMR90 cells are not known to be hazardous; however, they were derived from human samples and should be handled under BSL-2 precautions.
Researchers should wear appropriate personal protective equipment during the completion of this protocol. A face shield should be worn while handling liquid nitrogen and cell vials that have been recently removed from liquid nitrogen storage as they may explode unexpectedly. Parental stocks of IMR90-ATCC cells were obtained from ATCC at passage 10 and frozen at both passage 13 (on 8/28/2017) and passage 14 (on 8/31/2017). Since that time, we have started tracking cell growth in population doublings to obtain a more accurate representation of relative cell age. Thus, upon thawing of either passage 13/14 cells they are labeled with an "adjusted population doubling" (APD) of 0. Thereafter, their growth will be recorded by the addition of the calculated number of population doublings per passage. APD values for IMR90s will often increase by 2.0-2.5 per passage early in their lifespan and decrease to between 1.5-2.0 over time in culture. When the APD is regularly below 1.3, which typically takes about 12 passages according to the method described below, a new set of cells should be thawed.
Bicarbonate buffered solutions used for cell culture, such as growth medium and DPBS, should be "regassed" after each usage to facilitate the maintenance of proper pH. This can be accomplished by placing bottles in a tissue culture incubator for 15-30 minutes with the cap loosened, but still on the neck of the bottle, prior to closing/tightening the cap and storing at 4 °C until the next usage. Each user should have their own bottles of DPBS and growth medium to avoid cross contamination of cultures. Further, speci c sets of growth medium, DPBS, and trypsin should be used for each cell line/type being maintained by each user. Thawing Cryopreserved Cells 1. Warm growth medium to 37 °C and prepare materials prior to obtaining a vial of cells from cryostorage.
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. b. NOTE: It is preferable to subculture thawed cells for at least three passages before using in experiments.

Sub-Culturing Cells
Cells should be split three days after being plated at either 4.0 x 10 4 cells/mL NOTE: Lower APD IMR90 cells grow very quickly and should be plated at 3.5 x 10 4 cells/mL to start and then adjusted up to 4.0 x 10 4 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. 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. 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: