Primary Culture of Chick Myocyte
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
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
Isolation of tissue
Dissection of tissue
Fine dissection
Mechanical
disaggregation
Cold trypsin
Primary explant
O/N incubation
Transfer
warm trypsin
collagenase
Long incubation
centrifuge
outgrowth
2nd explant
Dispersed primary
culture
subculture
Cell line
Resuspend and seed
Enzyme digestion:
Incubation or tissue in proteolytic solution
Enzymes:
 Trypsin , collagenase for fibrous tissue
 Elastase
 Hyaluronidase
 Pronase
 Dispase
 Temperature
cold trypsin 4oC
 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
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 37oC water bath, shake for 5 min
↓
Carefully collect supernatant from 2d 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 37oC CO2 incubator
Culture medium for chick myocyte culture
M 199
Penicilim /streptomycin
FCS
Low salt solution
40%
0.1%
6%
54%
Hank’s balance salt solution
Trypsin
0.025%
Low salt solution
NaCl
NaH2PO4
MgSO4
KCl
NaHCO3
CaCl2
Glucose
HEPEs
pH 7.4
mM
g/l
g/500ml
140
1
1
4
26
1.1
10
10
8.12
0.156
0.12
0.29
2.18
0.121
1.98
4.06
0.078
0.06
0.145
1.09
0.06
0.99