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Primary Culture of Chick Myocyte. Establishment of primary cultures from various sources Consider:  Whether to use normal or tissue derivative tissue.

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Presentation on theme: "Primary Culture of Chick Myocyte. Establishment of primary cultures from various sources Consider:  Whether to use normal or tissue derivative tissue."— Presentation transcript:

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 % 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 NaH 2 PO MgSO KCl NaHCO CaCl Glucose HEPEs 10 pH 7.4


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