Animal cell lines like HeLa cells and L-cells, and also normal cells which are transformed by viruses can grow and proliferate to form colonies in soft agar-based media. Most of the normal cells cannot grow in this media except some type of cells like cartilage cells. Isolation of clones is very easy, if they are cultured in semisolid media. Using a finely drawn pipette clones can be isolated from suspended state and easily be subcultured. However many types of cells require further treatments before isolating them from the semisolid media.
Normal baby hamster kidney line known as BHK-21 cannot grow in agar, but after transformation by polyoma virus these cells will grow in this media. But these cells will grow in suspension culture if pure agar devoid of sulfated polysaccharides is used. All these characteristics are used to measure the transformation of these cells.
a. 20ml of 2.5% Bacto-Agar in distilled water, the solution must be autoclaved before use.
b. 4.50ml of sterile complete growth medium
c. 3.20ml 2X base medium, can be prepared from 10X- powdered standard medium
d. 4.50ml of sterile growth medium prepared using 10% fetal bovine serum for dilution.
e. 10ml Fetal bovine serum
f. 6.80ml Nutrient mix, this is prepared by mixing 2X base medium (20ml), Fetal bovine serum (10ml), 1X base medium (50ml)
g. 60mm plastic dishes
h. 15ml plastic centrifuge tube
i. 10ml pipettes
j. 1ml pipettes
k. Water bath
l. Required cell suspension culture for plating
All required plates and dilution tubes are labelled, sterilized and prepared in advance.
1. Nutrient agar medium (1X) is prepared by following steps
a. 2.5% agar is melted using either autoclave or microwave oven or boiling water bath, then it is placed in 45 degree Celsius water bath. The temperature of the agar must be allowed to cool to the temperature of the water bath before proceeding further.
b. Nutrient mix is also prepared and kept in 45 degree Celsius water bath. The temperature of the nutrient mix must be allowed to reach to the temperature of the water bath before proceeding further.
c. 2.5% agar is poured into nutrient mix to create 1X nutrient agar medium. This medium is osmotically balanced, gently mixed avoiding the air bubbles. Until use this medium is kept in 45 degree Celsius water bath.
2. 7ml of the 1X nutrient agar media is pipette out into 60mm animal culture dish. Agar is allowed to harden. Once agar is hardened plates are kept in incubator. This medium will act as base for the growth of the animal cells and also it will keep the animal cells from attaching to the base of the animal culture dish.
3. 1ml of 1X nutrient agar medium is pipette into eight 15ml of centrifuge tubes and keep them in 45 degree Celsius water bath.
4. Animal cell suspension is prepared using complete growth medium. This suspension of animal cells is plated with the following cell concentrations: 1x105, 1x104, 1x103, and 1x102. Cell suspension 0.5ml is added to 4.5ml complete growth medium without agar to make the 1:10 dilutions.
a. 0.5ml of each dilution is added to tubes of nutrient agar mixture, gently mix the content and poured into the agar dishes. Care should be taken to maintain the temperature of the nutrient agar media. If the media is lower than the 45 degree celcius, then the cell suspension culture may form clumps. If the temperature is more than 45 degree Celsius, cells will die due to heat shock.
b. Same process is repeated for the remaining tubes and nutrient agar cell culture dishes. Each cell concentration is plated in duplicate.
5. Agar plates are allowed to harden for around 15 to 30 minutes on the bench top. After the agar gets hardened all the animal cell culture plates are kept in a carbon dioxide incubator. This carbon dioxide incubator provided all the necessary conditions which facilitates the growth and proliferation of the animal cells.
6. Every two or three days animal cell culture plates are observed for cell growth. This is continued until colonies are formed large enough to see with the naked eye or without any aid.
By following this procedure clonal growth of required animal cells can be achieved in a semisolid media.
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