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Icahn School of Medicine at Mount Sinai LINCS Center for Drug

Toxicity Signatures

Standard Operating Procedure: PromoCell Primary Cardiomyocyte Subculture

DToxS SOP Index: CE-1.0 Last Revision: May 8, 2015 Written By: Evren U. Azeloglu and Bin Hu Approvals (Date): Joseph Goldfarb (5/8/2015) Marc Birtwistle (5/1/2015) Eric Sobie (5/7/2015) Ravi Iyengar (5/6/2015) Quality Assurance/Control (QA/QC) steps are indicated with green highlight. Metadata recording is highlighted with yellow highlight and superscript indices. ----------------------------------------------------------------------------------------------------

1. This subculture protocol has been modified from the aseptic culture protocol provided by PromoCell, the commercial supplier of cardiomyocytes

2. Growth media supplied by the manufacturer needs to be prepared by adding “Promocell Supplement Mix1” into Promocell “Myocyte Growth Media2” under sterile conditions

a. Remove 25 mL from a new 500 mL bottle of Promocell “Myocyte Growth Media” and add 25 mL of Promocell “Supplement Mix”

b. Prepare ten 50 mL sterile aliquots of this “Growth Media” in Falcon tubes (BD, Cat: 3562098)

c. Label tubes with “Growth Media”, media lot number, date and store at 4ºC for at most one month

3. Under sterile conditions, thaw a 1 mL vial of Promocell cardiomyocytes (PCM)3, which are delivered as frozen P2 stock on dry ice:

a. If not used immediately, frozen stock should be stored in liquid nitrogen b. Label a 75 cm2 filter-capped flask (Corning, Cat: 430641) with PCM-Line #,

passage # and date c. Add 20 mL of growth media (step2) into flask and warm it up by placing in a

humidified 37ºC / 5% CO2 incubator for 5 minutes d. Place frozen cell stock vial in a 37ºC water bath for 60 seconds (or until fully

thawed) e. Slowly mix the thawed 1 mL cell suspension into the warm media-containing

flask using a sterile 1000 µL micropipette f. Gently move the flask; making sure cells are plated evenly throughout the

surface. Be sure not to let the media contact the upper neck of the flask or the cap at any point.

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g. Place the flask in a humidified 37ºC / 5% CO2 incubator 4. Culture cells at 37ºC / 5% CO2 overnight and record confluence the next morning:

a. Image cells under low magnification. We use 5X phase contrast on the Leica DM-IL with ThorLabs DCC3240 camera and the ThorCam software4

5. Culture cells until they reach 80-90% confluence, changing the media completely every other day with 20 mL pre-warmed 37ºC growth media (step2) (QA/QC1)

a. Always pre-warm the media to 37ºC before media changes b. Culturing takes 10-12 days depending on doubling time of the specific lot

6. Split cells 1:4 into 75 cm2 filter-capped flasks a. Warm 100 mL (2 aliquots) of growth media (step 2) in a 37ºC water bath b. Aspirate media from cell culture, rinse with 20 mL of room temperature sterile

phosphate buffered saline (PBS); aspirate thoroughly c. Add 3 mL of room temperature 0.05% Trypsin-EDTA, ensure the trypsin solution

covers the bottom flask surface, and place the flask in the incubator for 2 minutes d. Place 1 mL of room temperature fetal bovine serum5 (FBS) in 15 mL conical tube

(BD, Cat: 3562097), which will be the collection tube for the cell suspension e. Tap gently at the side of the flask to dislodge any adherent cells

i. Check under 5X phase contrast to make sure all cells are detached f. Collect cell suspension with two PBS washes

i. Collect trypsinized cells into tube containing 1 mL FBS ii. Rinse the flask with 5 mL of room temperature PBS, add into same tube iii. Rinse the flask a second time with 5 mL of room temperature PBS, add into

same tube g. Spin down suspension at 4ºC 250g RCF for 5 minutes h. Carefully aspirate supernatant and gently resuspend pellet with 12 mL of growth

media (step 2) warmed to 37ºC, pipetting slowly using a 10 mL pipette (Corning, Cat: 4488) until no clumps are visible i. Process takes 20-30 full-volume strokes

i. Pipette 20 mL of 37ºC growth media (step 2) into each of the four 75 cm2 filter-capped flasks

j. Label each flask with cell name (PCM-Line #), date, and passage # at replating i. At each lifting with trypsin, the passage number increments by one; this

applies to cells that are being frozen k. Slowly add 3 mL of cell suspension into each flask and rock gently to ensure

homogeneous cell density throughout the growth media 7. Culture cells at 37ºC / 5% CO2 overnight and observe confluence the next morning 8. Culture cells until they reach >90% confluence changing media completely every other

day with 20 mL 37ºC growth media 9. Repeat items 5 through 8 for each flask, splitting cells 1:4 into 75 cm2 filter-capped flasks

one more time and labeling with cell name (PCM-Line #), passage number at replating, and date (QA/QC2)

a. At this point there should be 16 flasks with >90% confluence 10. Trypsinize and cryopreserve cells

a. Label seventeen 2 mL cryopreservation tubes (Nunc, Cat: 377267) with cell name (PCM-Line #), passage at the replating (P5), date, and tube number

b. Aspirate media, rinse with 20 mL of room temperature sterile PBS, aspirate thoroughly

c. Add 3 mL of room temperature 0.05% Trypsin-EDTA, place in incubator for 2 minutes

d. Place 5 mL of fetal bovine serum (FBS) in a 50 mL conical tube (this will be the collection tube for the cell suspension) per flask quartet (4 tubes for 16 flasks)

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e. Tap gently at the side of each flask to dislodge any remaining adherent cells i. Check under 5X phase contrast to make sure all cells are detached

f. Collect cell suspension with 10 mL of room temperature PBS with two washes, as in step 6f above

g. Spin down suspension at 4ºC 250g RCF for 5 minutes h. During spin, mix 15.3 mL of FBS with 1.7 mL of sterile dimethyl sulfoxide

(DMSO)6 at room temperature. This is the final suspension media for cryopreservation of cells.

i. Aspirate supernatant and gently resuspend pellet with FBS-DMSO mix i. Using 10 mL of freezing solution with a 10 mL pipette, resuspend one pellet

at a time pipetting slowly, moving from one conical tube to the next ii. At the end, this 10 mL of solution should have cells from 16 flasks and be in

the final 50 mL tube iii. Use the remaining 7 mL (with a 10 mL pipette tip) to slowly rinse the tubes

and collect the leftovers into the same final 50 mL conical tube j. Pipette 1 mL cell suspension (with 10 mL pipette tip) into individually labeled

cryotubes and place tubes in a CoolCell freezing container (Biocision, Cat: BCS-405) and place into -80ºC overnight

k. Move the tubes into liquid nitrogen for extended storage the following day

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Metadata

1. PromoCell Supplement Mix (Cat: C-39275): lot number 2. PromoCell Myocyte Growth Media (Cat: C-22170): lot number 3. PromoCell Cardiomyocytes (Cat: C-12810): LINCS ID, line ID, lot number, subject

age, sex, cell doubling time 4. Phase contrast images of growing cardiomyocytes: magnification, pixel size, image

size, exposure time, software version, date, cell passage number, dish ID 5. Fetal bovine serum (Sigma Aldrich, Cat: F2442-500ML): lot number 6. Dimethyl sulfoxide (Sigma Aldrich, Cat: D2650-100ML): lot number

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Quality Assurance/Control Steps (QA/QC) QA/QC1-2: At each plating step, the confluence of the cells needs to be checked. If cell confluence at QA/QC1 or QA/QC2 is below 90% (shown below as proper cell density) the flasks should be cultured for additional time. If there is uneven growth in between samples, the next step should be delayed until the least-dense dish reaches adequate confluence.

Inadequate cell density (70%) Proper cell density (90%)