Mammalian Tissue Culture Cell Growth Medium

There are a tremendous variety of recipies for growth medium for tissue culture cells. Some cell lines require that specific growth factors, such as insulin, be added to promote either their growth or phenotypic maintenence, however, most cell lines will either grow well, or can be adapted to grow well, in a fairly generic type of medium with 10% final concentration of fetal bovine serum. Some recipes follow:

final FBS	10%	15%	20%		10%	15%
DME-HG	500 ml	500 ml	500 ml		1000 ml	1000 ml	DMEM media with high glucose
FBS	57.5 ml	91.5 ml	130 ml		115 ml	183 ml	fetal bovine serum -- contains growth factors -- do NOT heat inactivate
NEAA	5.75 ml	6.1 ml	6.5 ml		11.5 ml	12.2 ml	non-essential amino acids from 100x stock
P/S	5.75 ml	6.1 ml	6.5 ml		11.5 ml	12.2 ml	pennicilin/streptomycin from 100x stock -- prevents bacterial contamination
l-Gln	5.75 ml	6.1 ml	6.5 ml		11.5 ml	12.2 ml	l-glutamine from 100x stock
plasmocin	57.5 μl	61 μl	65 μl		115 μl	122 μl	prevents mycoplasma contamination (stock 25 mg/ml)

Coriell's Mix: Coriell says to not add extra amino acids or vitamins¹

final FBS	5%	10%	15%
MEM (eagle/earle)	500 ml	500 ml	500 ml	minimal essential medium with earle's salts not modified minimal essential medium
FBS	26.9 ml	56.8 ml	90 ml	fetal bovine serum, NOT heat inactivated
P/S	5.4 ml	5.7 ml	6 ml	pennicillin/streptomycin, 100x
l-Gln	5.4 ml	5.7 ml	6 ml	l-glutamine, 100x
plasmocin	53.8 μl	56.8 μl	60.2 μl	anti-mycotic

Shake well to mix and store at 4C.

Fetal bovine serum, l-glutamine, and penn/strep stocks should be kept frozen for long-term storage at -20C. Working bottles of these solutions can be acceptably stored refrigerated at 4C. The stock bottles of DMEM and non-essential amino acids can be stored long-term at 4C.

Converting 10% FBS medium to 15% FBS medium
Add ¹/₁₇^th volume of FBS to medium already prepared at 10% FBS to bring the final FBS concentration to 15%. Eg: Add 588 μl FBS to 10 mL of 10% FBS medium

• supplementing smaller volumes of 10% FBS medium to 15% FBS in this way can be more efficient than making up an entire bottle of 15% FBS medium if large volumes of medium are not going to be required
  

pH indicators
Very often, media comes with the pH indicator phenol red pre-added. phenol red is:

• purple at pH 7.8
  
• pink at pH 7.6
  
• red at pH 7.4
  
• orange at pH 7.0
  
• yellow at pH 6.5
  

Growing cultures are healthy at around pH 7.4. As nutrients and the buffering capacity of the medium becomes exhaused through consumption and the build-up of metabolic waste products, the pH of the culture will gradually drop. The medium should be changed and/or the culture split at orange-red or lower pH colors. The 5% CO₂ typcially present in tissue culture incubators maintains pH stability through a carbonate based buffering system:
H₂O + CO₂ ⇌ H₂CO₃ ⇌ H⁺ + HCO₃^- ⇌ 2H⁺ + CO₃^-2

¹ Really. I talked to them directly and despite confusing and contradictory instructions on their web site, this is what they actually say to do.

Comments

• "DMEM" is Dulbecco's Modified Eagle Medium. It contains salts, pyruvate, essential amino acids and vitamins. The extra glucose provided in the "high glucose" formulation is useful to some cell lines and generally non-harmful to others.
  
• the "qualified" grade of Fetal Bovine Serum is generally sufficient. FBS certified to be free of endotoxin does not require heat inactivation
  
• the 100x stock of L-glutamine will generally precipitate into needlelike crystals when stored at 4C. There is no need to heat the solution to redissolve the crystals; simply mix well to achieve a relatively homogeneous suspension and add the appropriate amount of the suspension to the DMEM. The glutamine crystals will dissolve immediately upon this dilution.
  
  • L-glutamine is the least chemically stable of the essential amino acids with a half-life of approximately 8 days at 37C and 40 days at 4C. For this reason, extra is deliberately added in the generic medium recipe.