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Cell Culture using Cell Line

Cell culture using cell line as shown above picture. RPMI 1640, also known as RPMI medium, is a growth medium used in cell culture. and it is a synthetic medium. RPMI 1640 was developed by George E. Moore, Robert E. Gerner, and H. Addison Franklin in 1966 at Roswell Park Memorial Institute, from where it derives its name. It is a  modification of McCoy′s 5A medium or RPMI 1630 and was originally formulated to support lymphoblastoid cells in suspension cultures, but can also support a wide variety of adherent cells.

Preparation of culture media (RPMI complete media)

•  RPMI incomplete media (+/- Glutamine):

FBS -10%

Antibiotics – 1.2 % (2mM working solution )

HEPES -25Mm working solution

Mix all the components to make complete media.

The complete composition of RPMI

One liter of RPMI 1640 contains-

• Glucose: 2 gm
• phenol red: 5 mg
  sodium chloride: 6 g
• sodium bicarbonate:2 g
• disodium phosphate: 1.512 gm
• potassium chloride: 400 mg
• magnesium sulfate: 100 mg,
• calcium nitrate:  100 mg
  glutamine: 300 mg
• arginine: 200 mg
• each asparagine, cystine, leucine, and isoleucine: 50 mg
• lysine hydrochloride: 40 mg
• serine: 30 mg
• each aspartic acid, glutamic acid, hydroxyproline, proline, threonine, tyrosine, and valine: 20 mg
• each histidine, methionine, and phenylalanine: 15 mg
• glycine: 10 mg
• tryptophan: 5 mg
• reduced glutathione:  1 mg
  I-inositol: 35 mg
• choline chloride: 3 mg
• each para-aminobenzoic acid folic acid, nicotinamide, pyridoxine hydrochloride, and thiamine hydrochloride: 1 mg
• calcium pantothenate: 0.25 mg
• each biotin and riboflavin: 0.2 mg
• cyanocobalamin: 0.005 mg

Thawing of frozen cells :

1. Take out the vial from the 80 °C freezers.
2. Dip the via in a water bath maintained at 37° C with slight shaking. (Do not dip the vial up to the cap ). When there is still a small crystal of ice in the vial,  stop thawing of cells and transfer the cells in 5 ml of previously prepared warm (37 °C ) Complete media.

Culture of cells

1. Transfer the cells to a 25 cm culture flask. ( keep the flasks horizontally with raised part of the cap facing upwards).
2. Do not fully tighten the cap. The facilities the passage of gases.
3. Observe the cells under an inverted microscope.
4. Incubate the culture cells in the CO2 incubator maintained at 37º C.

Keynotes:

• RPMI stands for  Roswell Park Memorial Institute.
• HEPES stands for 2-hydroxyethyl -1-piperazineethanesulfonic acid and it is a zwitterionic sulfonic acid buffering agent.