Environmental conditions of animal cell culture

part 5By : Saib H Al Owini

PH

• Most of human cells at 7,4 • Ph indicator : phenol red • Buffers - In organic NaHCO3 ,CO2

- Organic HEPES (N-2 hydroxyethyl peprazin N2 ethanosulfonic acid)

- Each has advantages and disadvantages

Co2 is needed !!!

• Pyruvate • Sodium B- glycerophosphate ( may without

co2 ) • Large # may be satisfied• The favoret HEPES + CO2 + HCO3

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%

• May be due to thickness or differentiation

• Selenium versus O2 WHY !!!

• Serum media don’t need

3- Osmolality

• Human 290 mosm/ kg• Mice 310 mosm /kg• Animal ( 260 -320) mosm /kg

• Open plates ( hypertonic)

• ( metabolic , evaporation , HEPES) make variations .

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

5- viscosity

• In agitated media

Prevention • Carboxymethylecellulose• polyvinylpyrolidone

BSS

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

Culture Vessels and Substrates

• Glass • Plastic ( polystyrene )- Gama irradiation - Chemically ( ionization)- Protein coat ( poly-lysin, poly-ornithin) - Matrix protein ( fibronectin, laminin, collagen)

Culture Vessels and Substrates

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).

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).

Multisurface Flask

Choice of vessel

• According to ; - cell mass -adherent or suspension - Vented or sealed - Frequency- Analysis- The cost

Small Stirrer Flasks

Treated surfaces

• Used glass or new • Used medium • Denaturated collagen + carbodiimide • Collagen : media = 1:10 • Fibronectin or laminin

Commercially available matrices

• From sarcoma EHS( laminin , fibronectin , proteoglycans , ..) Laminin predominant

ECM preparation

Feeder layer

• Benefits : • - metabolites • - Gf• Matrices • But it lead to changes ( !!!!) • Mechanism of changes

• 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).

Three dimensional matrices

• Collagen gel• Cellulose sponge • Fibrin clots / with thrombin • For tissue engineering