Introduction:
Animal cell culture is a microbiological technique in which animal cells and tissues are obtained and maintained in a suitable environment.
It is further divided into,
Organotypic: Animal Organ Tissue Culture.
Histotypic: Animal Cell Culture.
Animal cells can be grown only upto certain generations.
Their growth requires typical conditions and a physicochemical environment.
Animal cell culture prepared from collecting tissue or cells from an organism is called a “Primary culture.”
The sequence of subcultures prepared from primary culture is called “Cell lines”.
History:
It was Ross Harisson from America who first cultured animal cells successfully in the laboratory.
Campbell, Wilmut and colleagues at Roslin Institute in Scotland successfully cloned the first animal cell using nuclear transfer technique and gave birth to a sheep named “Dolly” in 1996.
General procedure for cell Culture.
A piece of tissue of an organism is quite a complex substance as it is associated with cells of other tissues also like, cells from connective tissue, blood cells etc.
In the beginning the cell suspension is isolated and inoculated in a new culture vessel with fresh culture medium, such culture is called “Primary Cell Culture or Primary Cell Line”.
Following three stages are performed while isolating a primary culture,
Isolation of the tissue.
Disaggregation of the tissue.
Seeding of culture into the culture vessel.
All these stages are performed under a Laminar AirFlow hood in order to avoid any chance of contamination.
Isolation of the tissue:
A piece from a surgically removed portion of the body of an animal is used for the culture of animal cells in a suitable nutrient medium.
The major explant tissues for animal cell culture are collected from lab animals like rabbits, mice, guinea pigs etc.
The human cells like muscle cells, erythrocytes, leukocytes are also collected and cultured in suitable media.
Organs from which cells to be collected are surface sterilized with 70% alcohol and then aseptically removed.
The collected tissues are immediately transferred to a sterile nutrient medium or balanced salt solution (BSS) containing antibiotics.
The tissue is usually used immediately or stored in the freezer.
Disaggregation of the Tissue:
A tissue collected is disaggregated (Separated) from surrounding tissues by mechanical, enzymatic means or using chelating agents.
Mechanical disaggregation:
Also called “Physical Disaggregation”.
The mechanical approach involves slicing or harvesting tissue and subsequent harvesting of spill out cells.
This can be achieved by sieving, syringing and pipetting.
This procedure is inexpensive, rapid and simple, however, all these approaches involve the risk of cell damage, thus mechanical disaggregation is only used when the viability of the cells in the final yield is not very important.
Enzymatic Disaggregation:
This approach involves efficient disaggregation of cells with high yield by using enzymes such as trypsin, collagenase and others.
Enzyme based disaggregation allows hydrolysis of fibrous connective tissue and the extracellular matrix.
Currently, the enzymatic method is extensively used as it offers high recovery of cells without affecting the viability of cells.
Trypsin based disaggregation or trypsinization:
The process of enzymatic disaggregation of tissues using trypsin is called trypsinization.
Trypsinization is of two types,
Cold trypsinization.
Warm trypsinization.
This allows disaggregation of tissue using trypsin, usually crude trypsin because this trypsin contains other proteases.
In addition, cells can tolerate crude trypsin well and the ultimate effect of crude trypsin can easily be neutralized by serum or trypsin inhibitor (supplementation of trypsin inhibitor is required in the case of serum-free media).
Warm trypsinization:
This approach is extensively utilized for the disaggregation of cells.
During the initial step, sliced tissue is washed with dissection basal salt solution and is subsequently transferred to a container of warm trypsin (37 °C).
At regular intervals of 30 min the contents are stirred properly.
Then, the supernatant having dissociated, the cells are separated to disperse in a suitable medium.
Efficient dispersion of cells can be achieved by placing the container over ice.
Cold trypsinization:
This method is also called trypsinization with cold pre-exposure.
In this process the chance of cellular damage due to constant exposure to trypsin is reduced, which results in a high yield of viable cells with an improved survival rate for the cells (after 24 h of incubation).
Since this method does not involve frequent stirring or centrifugation, it can be conveniently adopted in the research laboratory.
During this process, after washing and chopping, tissue pieces are kept over ice in a vial and then subjected to treatment with cold trypsin for 6–24 h.
Then, after the cold trypsin treatment the trypsin is removed and discarded.
However, the tissue fragments still contain residual trypsin. These fragments are incubated at 37 °C (for 20–30 min) followed by repeated pipetting.
This will encourage the dispersion of cells. The fully dispersed cells can be counted using a cell counter and properly diluted, and then further utilized.
Drawbacks of trypsin disaggregation:
Trypsinization of cells can damage some cells, such as epithelial cells, and sometimes it is not effective for certain tissues, such as fibrous connective tissue, thus other enzymes are also recommended for dissociation of cells.
Collagenase based disaggregation:
Collagenase is an enzyme which is responsible for the cleavage of peptide bonds in collagen.
Collagen is a structural protein which is abundantly found in higher animals, mainly in the extracellular matrix of connective tissue and muscle.
Collagenase, mainly crude collagenase, can be successfully used for the disaggregation of several tissues that may or may not be sensitive to trypsin.
This process involves an initial transfer of the desired tissue into a basal salt solution which contains antibiotics.
This is followed by washing with settling and then transfer into a medium containing collagenase.
The solution is incubated for 1–5 days, followed by repeated pipetting for uniform dispersal of cells.
Separation of these dispersed cells is encouraged by keeping the solution in a stationary phase to further encourage the settling of cells.
c) Chelating Agents:
These are mainly used for preparation of cell suspensions.
These agents form chelats along with ions like Calcium, Magnesium etc.which are essential for maintaining integrity of epithelial cells.
3) Seeding of Culture:
The primary culture cell grows well when seeded into culture plates at high density.
Most Of the cell require support of substrate for growth, these cells are called “Anchorage Dependent Cells” e.g. liver, kidney etc.
Some cells don't require support, these cells are called “Anchorage independent Cells”.
The support materials used for Anchorage Dependent Cell are glass, plastic metals like stainless steel, titanium etc.
The Anchorage independent cells are cultivated in liquid medium.
Commonly Asked Question
Explain in detail different methods used for disaggregation of animal tissues.