1. Islamic University _Gaza Faculty of science Department of Biotechnology By: Mahmoud W. El-Hindi 2013_2014 1

2. Mammalian Cell Culture Media Initial attempts to culture cells were performed in natural media based on tissue extracts and body fluids, such as chick embryo extract, serum and lymph. Eagle’s Minimal Essential Medium (MEM) became widely adopted, variously. Supplemented with calf, human, or horse serum, protein hydrolysates, and embryo extract. 2

3. Cont. As more continuous cell lines became available (HeLa, etc. Most cell lines grow well at pH 7.4. Although the optimum pH for cell growth varies relatively little among different cell strains. 3

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5. Cont. Some normal fibroblast lines perform best at pH 7.4– 7.7, and transformed cells may do better at pH 7.0–7.4. Epidermal cells could be maintained at pH 5.5 but this level has not been universally adopted. 5

6. What is in the media? Dulbecco’ Modified Eagle’s Media (DMEM) Contains glucose, some proteins, and essential salts. Contains a pH indicator (phenol red) Media looks pink/red at pH 7.2 Acidic -yellow or orange (cell growth, bacterial growth). Basic -purple (no cell growth, not enough CO 2 ) 6

7. More media components Antibiotics (penicillin and streptomycin) Prevent bacterial contamination. Salts and buffers To stimulate in vivo environment. Serum Portion of blood after the cells and fibers have clotted From cow, horse, sheep added to media as a nutrient source for growing cells. Lipids, proteins 7

8. Mammalian Cell Culture Media Preparation 1. To a mixing container that is as close to the final volume as possible, add 10% less distilled water than the desired total volume of medium. 2.Add powdered medium to room temperature water with gentle stirring. Do not heat water. 3.Rinse inside of package to remove all trace of powder. 4.Add Sodium Bicarbonate as required. reassociate 8

9. Cont. 5.Dilute the medium to the desired volume with distilled water and stir until dissolved. Do not over mix. 6.Adjust the pH to between 0.2 – 0.3 below the desired final working pH by slowly adding, with stirring, 1NaOH/ 1N HCI. The pH usually will rise 0.2 – 0.3 units upon filtration. Keep the container closed until the medium is filtered. 7.Process the medium immediately into sterile containers by membrane filtration using 0.2 micron filter. 9

10. Dulbecco’ Modified Eagle’s Media (DMEM) Prepare 900ml of distilled water in clean graduated cylinder. Water temperature should be 15-30ºC. Put the beaker on magnetic stirrer. Add 15.4 g/L DMEM media powder to the water and stir gently. Stir till completely dissolved. Add 1.2 gram Sodium Bicarbonate (or 49.4 ml of 7.5% Sodium Bicarbonate Solution). 10

11. Cont. Add 10% (100ml) of Fetal Bovine Serum. Place the FBS in water bath to don’t prevent cell growth. Add 10ml from Antibiotics (penicillin, streptomycin). Adjust pH to 0.2-0.3 units below the required pH using 1N HCl or 1N NaOH. The pH will rise by 0.1-0.3 units after filtration. 11

12. Cont. 7.4 The required media pH is 7.4. 1 Add distilled water up to 1liter. Filter for sterility with 0.2µ filter into sterile bottles. 12

13. RPMI 1640 culture media preparation Prepare 900ml of distilled water in clean graduated cylinder. Water temperature should be 15-30ºC. Put the beaker on magnetic stirrer. Add equivalent powder to the water and stir gently. Stir till completely dissolved. Add 2.0 gram Sodium Bicarbonate (or 26.67 ml of 7.5% Sodium Bicarbonate Solution). 13

14. Cont. Adjust pH to 0.2-0.3 units below the required pH using 1N HCl or 1N NaOH. The pH will rise by 0.1-0.3 units after filtration. Add distilled water up to 1 liter. Filter for sterility with 0.2µ filter into sterile bottles. 14

15. Trypsin EDTA (TRED) An enzyme used to detach the cells from a culture dish. EDTA binds calcium ions in the media that would normally inhibit trypsin. 15

16. Phosphate Buffered Saline Used to wash/remove excess serum that inhibits the function of TRED. Must be warmed in the water bath before use so cells are not shocked by cold liquid. 16

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18. Culture Medium Sterilization 18

19. Culture flask Culture Plate 19

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