1/5

Icahn School of Medicine at Mount Sinai LINCS Center for Drug

Toxicity Signatures

Standard Operating Procedure: PromoCell Primary Cardiomyocyte Plating for Drug Testing

DToxS SOP Index: CE-2.0 Last Revision: May 8, 2015 Written By: Evren U. Azeloglu and Bin Hu Approvals (Date): Joseph Goldfarb (5/8/2015) Marc Birtwistle (5/7/2015) Eric Sobie (5/7/2015) Ravi Iyengar (5/7/2015) Quality Assurance/Control (QA/QC) steps are indicated with green highlight. Metadata recording is highlighted with yellow highlight and superscript indices. ---------------------------------------------------------------------------------------------------- 1. 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 2. Thaw one vial of PromoCell cardiomyocytes (PCM)3 subcultured as described in DToxS

SOP CE-1.0 a. Warm one 50 mL aliquot of growth media (step 1) for 15 minutes in a 37ºC

waterbath b. Add 20 mL of growth media (step 1) into each of four 75 cm2 filter-capped flasks

(Corning, Cat: 430641) and put in 37ºC incubator. Label each flask with ID of frozen cells and date of transfer.

c. Place frozen cell stock in 37ºC water bath for 60 seconds (or until fully thawed) d. Slowly mix the thawed cell mixture into 11 mL of growth media (prepared in step

1) using a sterile 1000 µL micropipette to create a uniform cell suspension of 12 mL

e. Slowly add 3 mL of the cell suspension from step d into each flask making sure cells are plated evenly throughout the surface

f. Place flasks in a humidified 37ºC incubator with 5% CO2 3. Culture cells at 37ºC / 5% CO2 overnight and record confluence the next morning (QA/QC1)

2/5

a. Image a representative area from one of the flasks under low power phase contrast. We use a Leica DM-IL with 5X magnification and ThorLabs DCC3240 camera and the ThorCam software4

4. Culture cells until they reach 100% confluence changing media completely every other day with growth media (step 1) (QA/QC2).

a. Process takes 5-7 days depending on doubling time of the specific lot 5. Trypsinize and count cells

a. Warm 100 mL (two 50 mL aliquots) of growth media (step 1) at 37ºC b. Pipette 2 mL of warm growth media (step 1) into 24 60 mm cell culture dishes

(Corning, Cat: 430166) i. Label each dish with cell name (PCM Line #) and date

c. Aspirate media from each flask, rinse with 20 mL of 37ºC sterile PBS and aspirate thoroughly

d. Add 3 mL of 37ºC 0.05% Trypsin-EDTA, ensure even coverage of trypsin on flask bottom, and place in incubator for 2 minutes

e. Place 4 mL of fetal bovine serum (FBS)5 in 50 mL conical tube (this will be the collection tube for all cell suspensions)

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

g. Collect cell suspension with 10 mL of room temperature (RT) sterile PBS washes (5 mL wash, twice)

i. Collect trypsinized cell suspension into tube with 4 mL FBS ii. Rinse the flask with 5 mL of RT PBS, collect into same tube iii. Rinse the flask with 5 mL of RT PBS, collect into same tube

h. Count cells i. After slowly mixing cell suspension with a 10 mL pipette tip, pipette 10 µL

into each side of a clean hemacytometer (Hausser Scientific, Cat: 3200) with No.2 coverglass

ii. Count all the cells within the 5 large boxes of each side (10 boxes total) at 10X magnification under phase contrast, including all the cells on the upper-left sidelines and excluding all the cells on the lower-right sidelines

iii. Multiply cell count by 1,000 to obtain cell concentration per mL iv. If the count on one side is more than 25% different than the other side,

repeat the entire process from the beginning v. For 100% confluence, cell number per flask, should be 5-7.5 million; if

lower numbers are obtained, culture time should be increased for that given cell line.

i. Centrifuge suspension at 4ºC 250g for 5 minutes j. Resuspend pellet

i. Calculate the appropriate volume of growth media (step 1) to produce a final cell concentration of 400,000 cells/mL

ii. Aspirate supernatant from centrifuged cells iii. Slowly resuspend pellet by pipetting the calculated volume of growth

media (step 1) using a 10 mL pipette tip until no clumps are visible iv. Process takes 20-30 full-volume strokes

k. Plate 8x105 cells (2 mL of suspension) on each of the 24 60 mm dishes (final plating density of approximately 40,000 cells/cm2 with 4 mL growth media)

6. Culture cells at 37ºC / 5 % CO2, changing growth media (step 1) every other day until cells reach 100% confluence

a. Process takes 5-7 days depending on the doubling time of the line b. Record images of confluence at 100%6 (QA/QC3)

3/5

7. Aspirate media and replace with 4 mL PromoCell Myocyte Basal Media7 to start redifferentiation. Record date8.

8. Change half of the media (remove 2 mL and add 2 mL of new basal media) on Monday, Wednesday, and Friday for four weeks

4/5

Metadata

1. PromoCell Supplement Mix (Cat: C-39275): lot number 2. PromoCell Myocyte Growth Media (Cat: C-22270): 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. Phase contrast images of differentiating cardiomyocytes: magnification, pixel size,

image size, exposure time, software version, date, cell passage number, dish ID 7. PromoCell Myocyte Basal Media (Cat: C-22170): lot number 8. Dates for plating, start of redifferentiation, and drug treatment should be linked to each

experiment

5/5

Quality Assurance/Control Steps (QA/QC) QA/QC1-3: At each plating step, the confluence of the cells needs to be recorded. PromoCell recommends a minimum of 1-1.5x104 cells/cm2 at initial plating. In our preliminary experiments we found this density to be inadequate especially for extended differentiation periods where cells are deprived of serum. Therefore, cells are plated at an additional higher density for the final experimental plating. Frequent imaging ensures that cells have always achieved confluence at each passage point. If cell confluence at QC1 or QC2 is low, the flasks should be cultured for an additional week. QC3 is a final check prior to start of differentiation; if cell density is inadequate on the day of media switch, the experiment should be delayed until confluence is reached (see sample images below). If there is uneven growth between samples, the next step should be delayed until the least-dense dish reaches adequate confluence.

Inadequate cell density Inadequate cell density,

uneven plating Proper cell density