1. Primary Culture of Chick Myocyte

2. Establishment of primary cultures from various sources Consider:  Whether to use normal or tissue derivative tissue  Whether to obtain the tissue from an adult or embryo  Which species to use Isolation of cells e.g. mouse embryo hen’s egg human biopsy material

3. Requirements for primary culture  Remove fat and necrotic tissue  Chop tissue finely  Remove enzyme after digestion  Select proper medium for specific cell type  Plate more cells than the routine maintenance  Use embryo tissue to yield more viable cells

4. Isolation of tissue Dissection of tissue Fine dissection Primary explant Transfer 2nd explant Mechanical disaggregation Dispersed primary culture subculture Cell line outgrowth Cold trypsinwarm trypsin collagenase Long incubation O/N incubation centrifuge Resuspend and seed Fine dissection Primary explant Transfer 2nd explant Dispersed primary culture subculture outgrowth

5. Enzyme digestion: Incubation or tissue in proteolytic solution Enzymes:  Trypsin, collagenase for fibrous tissue  Elastase  Hyaluronidase  Pronase  Dispase

6.  Temperature cold trypsin 4 o C  Advantages: a. more cell types available i.e. epithelial cells b. may be treated over night c. may be used in small amount of tissue  Perfusion pumping of photolytic enzyme through the tissue and collecting cells in enzyme solution

7. Primary culture of chick myocyte 10 days chick embryo ( 6-8 embryos) ↓ Hearts taken (place in Hank’s balanced salt solution) ↓ Aortic vessel removed ↓ Heart tissue cut into small pieces ↓ Cells trypsinize with 3 ml trypsin ( 0.025%) ↓ Discard the supernatant from the first trypsinization ↓ Add 3 ml trypsin (0.025%) Incubate at 37 o C water bath, shake for 5 min ↓ Carefully collect supernatant from 2 d trypsinization and add to the culture medium in a fresh tube Repeat trypsinization for 2 more times ↓ Collect ad combine supernatant of all three trypsinization centrifuge 1200rpm, 5 min ↓ ↓ remove supernatant ↓ Resuspend cell in culture medium Cell culture in 37 o C CO 2 incubator

8. Culture medium for chick myocyte culture M 199 40% Penicilim /streptomycin 0.1% FCS 6% Low salt solution 54% Hank’s balance salt solution Trypsin 0.025%

9. Low salt solution mM g/l g/500ml NaCl 140 8.12 4.06 NaH 2 PO 4 1 0.156 0.078 MgSO 4 1 0.12 0.06 KCl 4 0.29 0.145 NaHCO 3 26 2.18 1.09 CaCl 2 1.1 0.121 0.06 Glucose 10 1.98 0.99 HEPEs 10 pH 7.4