1. Environmental conditions of animal cell culture part 5 By : Saib H Al Owini

2. PH Most of human cells at 7,4 Ph indicator : phenol red Buffers -In organic NaHCO 3, CO 2 -Organic HEPES (N-2 hydroxyethyl peprazin N2 ethanosulfonic acid) -Each has advantages and disadvantages

4. Co2 is needed !!!

5. Pyruvate Sodium B- glycerophosphate ( may without co2 ) Large # may be satisfied The favoret HEPES + CO2 + HCO3

6. 2- Oxygen Most cells use anaerobic ( O2 diffusion slow ) No Hb Large amount lead to free radical Small not adequate Scavengers ( glutathion,2- mercaptoethanol) Most cell culture use ATMO O2 only Ogran need mor about 95%

7. May be due to thickness or differentiation Selenium versus O2 WHY !!! Serum media don’t need

8. 3- Osmolality Human 290 mosm/ kg Mice 310 mosm /kg Animal ( 260 -320) mosm /kg Open plates ( hypertonic) ( metabolic, evaporation, HEPES) make variations.

9. 4- temprature Optimal according to animal body Or organ type Most at 37 Overheating more serious than under heating 4c or 196- But not 40

10. 5- viscosity In agitated media Prevention Carboxymethylecellulose polyvinylpyrolidone

11. BSS Inorganic May has ( glucose, Bicarbonate, HEPES) Function : -Dilution -Washing -Dissecting -Hours incubation ( passing media)

13. Culture Vessels and Substrates Glass Plastic ( polystyrene ) -Gama irradiation -Chemically ( ionization) -Protein coat ( poly-lysin, poly-ornithin) -Matrix protein ( fibronectin, laminin, collagen)

14. Culture Vessels and Substrates

16. Fig. 8.2. microtitration) plates. Plates are availableMultiwell Plates. Six-well, 24-well, and 96-well (with a wide range in the number of wells, from 4 to 144 (see Table 8.1 for sizes and capacities).

17. Fig. 8.4. Plastic Flasks. Sizes illustrated are 10 and 25 cm2 (Falcon, B-D Biosciences), 75 cm2(Corning), and 185 cm2 (Nalge Nunc) (see Table 8.1 for representative sizes and capacities).

18. Multisurface Flask

19. Choice of vessel According to ; - cell mass -adherent or suspension -Vented or sealed -Frequency -Analysis -The cost

20. Small Stirrer Flasks

21. Treated surfaces Used glass or new Used medium Denaturated collagen + carbodiimide Collagen : media = 1:10 Fibronectin or laminin

22. Commercially available matrices From sarcoma EHS ( laminin, fibronectin, proteoglycans,..) Laminin predominant

23. ECM preparation

24. Feeder layer Benefits : - metabolites - Gf Matrices But it lead to changes ( !!!!) Mechanism of changes

26. Morphology on Feeder Layers. (a) Fibroblasts from human breast carcinoma growing on plastic, in (b) growing on a confluent feeder layer of fetal human intestinal cells (FHI). Epithelial cells from human breast carcinoma growing (c) on plastic and (d) on same confluent feeder layer as in (b).

27. Three dimensional matrices Collagen gel Cellulose sponge Fibrin clots / with thrombin For tissue engineering