THAWING MUSCLE CELLS AND ANTIOXIDANT TREATMENTS

General Notes

df = dilution factor = final volume / volume of stock transferredwhere final volume = volume of diluent + volume of stock
where final volume = volume of diluent + volume of stock
Food-grade materials: Carrot juice, V8, or turmeric
Lab-grade materials: Beta carotene, lycopene, or curcumin

Materials

Bovine satellite cell growth media (BSC-GM)
DMSO
Acetone
Beta carotene
Carrot juice
Lycopene
V8 juice
Curcumin
Turmeric
Filters150 mL bottle top filters for the juices Syringe filters and syringes for the powders
150 mL bottle top filters for the juices
Syringe filters and syringes for the powders
48-well plate
15 mL conical tubes
BSC cell suspension (100,000 cells/mL), prepared by instructors

Method

1. Prepare the antioxidant powders/juices
   1. NOTE: You will be assigned to one of three conditions
      1. Carrot juice & beta carotene
      2. V8 & lycopene
      3. Turmeric & curcumin
   2. Food-grade preparations (Carrot juice, V8, or turmeric)
      1. IF you are assigned [group i or ii], sterile filter the juice
         1. Add 20 mL juice to a 50 mL conical tube
         2. Spin at max speed on the centrifuge for 10 minutes
         3. Sterile filter through a 150 mL bottle top filter
      2. IF you are assigned [group iii], prepare turmeric “solution”
         1. In 15 mL tube, add 100 mg then add 9.9 mL water. In the hood, sterile filter into a labeled 15 mL tube.
   3. Lab-grade preparations: Purified carotenoids (10 mg/mL solution).
      1. [Group i] Beta carotene: In a 15 mL tube, add 15 mg then add 1.5 mL DMSO. In the hood, sterile filter into a labeled 15 mL tube.
      2. [Group ii] Lycopene: Already in solution at 10 mg/mL in acetone. Already sterile.
      3. [Group iii] Curcumin: In a 15 mL tube, add 10 mg then add 1 mL DMSO. In the hood, sterile filter into a labeled 15 mL tube.
2. Prepare media with added antioxidants.
   1. First, prepare solvent (DMSO or acetone) control at 0.1%
      1. To 15 mL of BSC-GM, add 15 µL of the appropriate solvent
   2. Prepare most concentrated lab-grade sample (10,000 ng/mL)
      1. To 10 mL of BSC-GM, add 10 µL of carotenoid powder solution
   3. Prepare most concentrated food-grade sample (“df 1”)
      1. Turmeric: To 9.5 mL BSC-GM, add 500 µL of filtered turmeric solution
      2. Carrot: To 9.3 mL BSC-GM, add 735 µL of filtered carrot juice
      3. V8 juice: To 9.6 mL BSC-GM, add 575 µL of filtered V8 juice
   4. Prepare serial dilutions with a dilution factor of 10 for each dilution
      1. For lab-grade solutions, to 4x 15 mL conical tubes, add 2.7 mL of BSC-GM with 0.1% of appropriate solvent. Label the tubes with 1,000 ng/mL, 100 ng/mL, 10 ng/mL, and 1 ng/mL
      2. For food-grade solutions, repeat as above but with just BSC-GM. Label tubes with dilution factor: df 10, df 100, df 1000, and df 10,000.
      3. To the first tube lab-grade solution tube (1,000 ng/mL), add 300 µL of the 10,000 ng/mL lab-grade carotenoid solution. Mix by pipetting up and down.
      4. Repeat the serial dilutions three times – 100 ng/mL, 10 ng/mL, 1 ng/mL – by adding 300 µL of the previous solution to the appropriate tube. Be sure to mix by pipetting thoroughly.
      5. Repeat the previous two steps but starting with the df1 food-grade solution. You will thus prepare df10, df100, df1000, and df10000 food-grade solutions.
3. With all your media prepared, add 200 µL media to your plates in triplicate, as in the layout below.

	1	2	3	4	5	6	7	8
A		BSC-GM	Food-grade df1	Food-grade df10	Food-grade df100	Food-grade df1000	Food-grade df10000
B		BSC-GM	Food-grade df1	Food-grade df10	Food-grade df100	Food-grade df1000	Food-grade df10000
C		BSC-GM	Food-grade df1	Food-grade df10	Food-grade df100	Food-grade df1000	Food-grade df10000
D		BSC-GM with 0.1% solvent	Lab-grade 10000 ng/mL	Lab-grade 1000 ng/mL	Lab-grade 100 ng/mL	Lab-grade 10 ng/mL	Lab-grade 1 ng/mL
E		BSC-GM with 0.1% solvent	Lab-grade 10000 ng/mL	Lab-grade 1000 ng/mL	Lab-grade 100 ng/mL	Lab-grade 10 ng/mL	Lab-grade 1 ng/mL
F		BSC-GM with 0.1% solvent	Lab-grade 10000 ng/mL	Lab-grade 1000 ng/mL	Lab-grade 100 ng/mL	Lab-grade 10 ng/mL	Lab-grade 1 ng/mL

1. To each well in the plate with media, add 10 µL of 100,000 cell/mL cell suspension to seed 1,000 cells per well
2. Place 48-well plate in incubator.
3. Feed with 200 µL of the appropriate media on Sunday and Tuesday.

References

1. Stout, A. J., Mirliani, A. B., Soule-albridge, E. L., Cohen, J. M. & Kaplan, D.L. Engineering carotenoid production in mammalian cells for nutritionally enhanced cell-cultured foods. Metab. Eng. 62, 126–137 (2020).

Reference volumes

Vessel	Surface (cm2)	~PBS volume	~Trypsin volume	~Media volume
6-well plate	9.6	1 mL	500 uL	2 mL
12-well plate	3.5	500 uL	250 uL	1 mL
24-well plate	1.9	500 uL	250 uL	1 mL
48-well plate	1.1	200 uL	100 uL	500 uL
96-well plate	0.32	100 uL	50 uL	200 uL
T-25	25	3 mL	1 mL	5 mL
T-75	75	5 mL	2 mL	12 mL
T-175	175	10 mL	3 mL	30 mL

This protocol was developed for BME 174: cultivated meat laboratory course at Tufts University. The course is administered by Professor David Kaplan's laboratory.

PROTOCOL DEX