Primary Cell Culture
Primary cell culture refers to the culturing techniques that occur after the isolation of cells, but before the first subculture.
Primary cultures come from large tissue masses.
These cultures may have different types of cells, like fibroblasts, lymphocytes, macrophages, and epithelial cells.
Factors for Efficient Primary Cell Culture
Embryonic tissues are preferred over adult tissues for primary cultures because:
Embryonic cells are easier to disaggregate (break apart).
They give more viable cells that grow faster in laboratory conditions (in vitro).
The amount of cells in primary cultures should be high, because they survive less than cells in subcultures.
Tissues need to be processed carefully to minimise damage to the cells.
Dead cells must be removed.
An appropriate nutrient-rich medium is important for growth.
If using serum, it is best to use fetal bovine serum instead of serum from calves or horses.
Enzymes used for breaking up tissues should be removed by centrifugation.
Techniques for Primary Culture
There are three common techniques to obtain primary cultures from tissues:
Mechanical disaggregation – physically breaking the tissue apart.
Enzymatic disaggregation – using enzymes to break down the tissue.
Primary explant technique – placing small pieces of tissue directly in a culture medium.
These methods help ensure that cells are properly cultured and ready for further experiments or growth in the lab.
Mechanical Disaggregation
Mechanical disaggregation is used for breaking up soft tissues like the spleen, brain, embryonic liver, and soft tumours. This method involves chopping or slicing the tissue to release cells, which are then collected. Cells can be collected in two ways:
Pressing the tissue through a series of sieves with gradually smaller mesh sizes.
Forcing the tissue fragments through a syringe and needle.
Although mechanical disaggregation can damage cells, the process is less expensive, quick, and simple. It works best when there's plenty of tissue, and precision is not crucial. However, the cell viability (how many cells stay alive) is lower compared to enzymatic techniques.
Enzymatic Disaggregation
Enzymatic disaggregation is mostly used when you need high recovery of cells from tissue. It works especially well with embryonic tissues because they contain less fibrous connective tissue and extracellular matrix.
This method involves using enzymes like trypsin, collagenase, or others to break down the tissue.
Disaggregation by Trypsin
Trypsinization refers to the use of the enzyme trypsin to break up tissues.
Many people use crude trypsin rather than pure trypsin because:
Crude trypsin contains other proteases (enzymes that break down proteins), making it more effective.
Cells can tolerate crude trypsin better.
The remaining crude trypsin activity can easily be neutralised by the serum in the culture medium. This ensures the enzyme doesn’t keep breaking down the cells.
Enzymatic disaggregation is more effective when you need to ensure a higher yield of viable cells compared to mechanical methods. Disaggregation of cells can be done using pure trypsin, which is less toxic and more specific in its action. The tissue is cut into small pieces (2-3 mm) and then treated with trypsin to break it down into cells. There are two main techniques of trypsinization:
Warm Trypsinization
This method is commonly used to separate cells.
The tissue is chopped and washed with DBSS (Dissection Basal Salt Solution).
It is then transferred to a flask with warm trypsin (37°C).
The mixture is stirred, and every 30 minutes, the separated cells (in the liquid) are collected.
After removing the trypsin, the cells are preserved (kept on ice).
Fresh trypsin is added at 30-minute intervals, and the process continues for about 4 hours.
The separated cells are then counted, diluted, and used.
Cold Trypsinization
This is sometimes called trypsinization with cold preexposure.
It reduces the risk of cell damage, which can happen if cells are exposed to trypsin at 37°C for too long (as in warm trypsinization).
The chopped tissue is kept in a vial on ice and soaked in cold trypsin for 6-24 hours.
After this, the trypsin is removed.
The tissue (with remaining trypsin) is incubated at 37°C for 20-30 minutes.
The cells are separated by pipetting, counted, diluted, and used.
Advantages and Limitations
Cold trypsinization results in higher cell yields and better survival after 24 hours of incubation.
This method is easier because it doesn’t need stirring or centrifugation, making it more convenient for labs.
However, the major limitation is that cold trypsinization cannot be used for large quantities of tissue.
Limitations of trypsin disaggregation:
Using trypsin for disaggregation of cells may cause damage to some types of cells, like epithelial cells.
It may also be almost ineffective for certain tissues, such as fibrous connective tissue.
Therefore, other enzymes are sometimes used to dissociate cells more effectively.
Disaggregation by collagenase:
Collagen is the most abundant structural protein in higher animals and is mainly found in the connective tissue and muscle matrix.
Collagenase is an enzyme used for disaggregation of tissues that may be too sensitive for trypsin.
The enzyme is often a crude one, contaminated with other proteases, which may lead to less effective results.
However, highly purified collagenase has been tried with more success, but it is still less effective compared to crude collagenase in certain cases.
Important Stages in Collagenase Disaggregation:
Tissue is suspended in a basal salt solution with antibiotics.
It is chopped into smaller pieces and washed by settling.
The pieces are incubated in a medium containing collagenase for 1-5 days.
After this, the tissue is pipetted to disperse the cells.
The fibroblastic cells and epithelial cells are separated by settling.
Collagenase disaggregation uses:
Collagenase has been successfully used for disaggregation of tissues from the human brain, lungs, and other tissues, like tumours.
This technique is also effective for various animal tissues.
Hyaluronidase is another enzyme that helps break down carbohydrates on the surface of cells, promoting disaggregation.
Combining collagenase with hyaluronidase is very effective, especially for tissues like rat or rabbit liver.
Use of other enzymes in disaggregation:
Trypsin and collagenase are the most commonly used enzymes for tissue disaggregation.
Certain bacterial proteases (such as pronase and dispase) have been tried but with limited success.
Besides hyaluronidase, neuraminidase is sometimes used along with collagenase to help degrade carbohydrates on cell surfaces.
I have explained all the concepts step-by-step, and here is the simplified diagram showing the disaggregation of tissue using collagenase. It follows the same steps as in the text:
The tissue is placed in a basal salt solution (BSS) with antibiotics.
The tissue is chopped into small pieces and washed by settling.
It is incubated in a medium containing collagenase for 1-5 days.
The tissue is pipetted to disperse the cells.
Fibroblastic cells and epithelial cells are separated.
Primary Explant Technique
The primary explant technique was first developed by Harrison in 1907. Although it has undergone several modifications, this technique is still in use. Below is a simplified description of the procedure:
The tissue is placed in basal salt solution, where it is finely chopped and then washed.
The basal salt solution is then removed.
Tissue pieces are evenly spread over the growth surface.
After adding the appropriate medium, the tissue is incubated for 3–5 days.
The medium is changed weekly until a substantial outgrowth of cells is observed.
The explants are then removed and transferred to a fresh culture vessel.
When is Primary Explant Technique Useful?
This technique is particularly useful for disaggregating small quantities of tissues, like skin biopsies. Unlike mechanical or enzymatic disaggregation, which are better suited for large tissue amounts, the primary explant technique works well for small samples, reducing the risk of losing cells.
However, a drawback of this method is that some tissues do not adhere well to the growth surface, making it difficult to select cells from the outgrowth. But overall, the primary explant technique can be applied to a wide variety of embryonic cells, such as:
Fibroblasts
Myoblasts
Epithelial cells
Glial cells
It remains a reliable method for isolating and growing cells in tissue culture, especially when handling small samples.
