In this primary culture in animal tissue culture post we have briefly explained about primary cell line culture initiation, techniques, what is viable and nonviable cells.
Primary Cell Culture
Primary cell line culture is cells derived directly from tissues following enzymatic dissociation or from tissue fragments referred to as explants. These are usually the cells of preference, since it is argued that primary cell line culture retain their characteristics and reflect the true activity of the cell type in vivo. The disadvantage in using primary cell line culture, however, is that their isolation can be labour-intensive and may produce a heterogeneous population of cells. Moreover, primary cell line culture have a relatively limited lifespan and can be used over only a limited period of time in culture.
Primary cell line culture (figure 2) can be obtained from many different tissues and the source of tissue used generally defines the cell type isolated. For instance, cells isolated from the endothelium of blood vessels are referred to as endothelial cells whilst those isolated from the medial layer of the blood vessels and other similar tissues are smooth muscle cells. Although both can be obtained from the same vessels, endothelial cells are different in morphology and function, generally growing as a single monolayer characterised by a cobble-stoned morphology. Smooth muscle cells on the other hand are elongated, with spindle-like projections at either end, and grow in layers even when maintained in culture.
In addition to these cell types there are several other widely used primary cell line culture derived from a diverse range of tissues, including fibroblasts from connective tissue, lymphocytes from blood, and neurons from nervous tissues and hepatocytes from liver tissue.
Culture Initiation Process
After isolation, a primary cell line culture may be obtained either by allowing cells to migrate out from fragments of tissue adhering to a suitable substrate or by disaggregating the tissue mechanically or enzymatically to produce a suspension of cells, some of which will ultimately attach to the substrate. It appears to be essential for most normal untransformed cells, with the exception of hematopoietic cells and stem cells, to attach to a flat surface in order to survive and proliferate with maximum efficiency.
Primary Culture Methods
Primary cell line culture broadly involves the culturing techniques carried following the isolation of the cells, but before the first subculture. Primary cell line culture are usually prepared from large tissue masses. Thus, these cultures may contain a variety of differentiated cells e.g. fibroblasts, lymphocytes, macrophages, epithelial cells. Among the various techniques devised for the primary cell line culture of isolated tissues, three techniques are most commonly used:
As there is a risk of proteolytic damage to cells during enzymatic digestion, many people have chosen to use mechanical disaggregation (Figure 1). This procedure gives a cell suspension more quickly than enzymatic digestion but may cause mechanical damage. Spillage and sieving are probably the gentlest mechanical methods, while pipetting and, particularly, syringing, are most likely to generate shear stress. Only soft tissues, such as spleen, embryonic liver, embryonic and adult brain, and some human and animal soft tumors, respond well to this technique. Even with brain, for which fairly complete disaggregation can be obtained easily, the viability of the resulting suspension is lower than that achieved with enzymatic digestion, although the time taken may be very much less.
Figure 1: Mechanical Disaggregation Process
Enzymatic disaggregation is mostly used when high recovery of cells is required from a tissue. Disaggregation of embryonic tissues is more efficient with higher yield of cells by use of enzymes. This is due to the presence of less fibrous connective tissue and extracellular matrix. Enzymatic disaggregation can be carried out by using trypsin, collagenase or some other enzymes.
Disaggregation: The term trypsinization is commonly used for disaggregation of tissues by the enzyme, trypsin. The choice of which trypsin grade to use has always been difficult, as there are two opposing trends: (1) the purer the trypsin, the less toxic it becomes, and the more predictable its action; (2) the cruder the trypsin, the more effective it may be, because of the presence of other proteases.
Warm trypsinization: It is important to minimize the exposure of cells to active trypsin in order to preserve maximum viability. Hence, when whole tissue is being trypsinized at 37◦C, dissociated cells should be collected every half hour, and the trypsin should be removed by centrifugation and neutralized with serum in medium. The warm trypsin technique is useful for the disaggregation of large amounts of tissue in a relatively short time, particularly for chopped whole mouse embryos or chick embryos.
Figure 2: Primary Cell Culture Protocol
Cold trypsinization: One of the disadvantages of using trypsin to disaggregate tissue is the damage that may result from prolonged exposure to the tissue to trypsin at 37⁰C hence the need to harvest cells after 30 min incubations in the warm trypsin method rather than have them exposed for the full time as required to disaggregate the whole tissue (Figure 3). A simple method of minimizing damage to the cells during exposure is to soak the tissue in trypsin at 4⁰C for 6 to 18 h to allow penetration of the enzyme with little tryptic activity. The cold trypsin method usually gives a higher yield of viable cells, with improved survival after 24-h culture, and preserves more different cell types than the warm method.
Figure 3: Cold trypsinization Process
Collagenase: This technique is very simple and effective for many tissues, embryonic, adult, normal, and malignant (Figure 4). It is of greatest benefit when the tissue is either too fibrous or too sensitive to allow the successful use of trypsin. Crude collagenase is often used and may depend, for some of its action, on contamination with other nonspecific proteases. More highly purified grades are available if nonspecific proteolytic activity is undesirable, but they may not be as effective as crude collagenase. Disaggregation in collagenase has proved particularly suitable for the culture of human tumors, mouse kidney, human adult and fetal brain, liver, lung, and many other tissues, particularly epithelium.
Figure 4: Collagenase Process
c. Primary Explant
Primary cell line culture explant technique was, in fact the original method, developed by Harrison in 1907. This technique has undergone several modifications, and is still in use. The tissue in basal salt solution is finely chopped, and washed by settlings. The basal salt solution is then removed. The tissue pieces are spread evenly over the growth surface. After addition of appropriate medium, incubation is carried out for 3-5 days. Then the medium is changed at weekly intervals until a substantial outgrowth of cells is observed. Now, the explants are removed and transferred to a fresh culture vessel.
Primary cell line culture explant technique is particularly useful for disaggregation of small quantities of tissues (e.g. skin biopsies). The other two techniques mechanical or enzymatic disaggregation however, are not suitable for small amounts of tissues, as there is a risk of losing the cells. The limitation of explant technique is the poor adhesiveness of certain tissues to the growth surface, and the selection of cells in the outgrowth. It is however, observed that the primary cell line culture explant technique can be used for a majority of embryonic cells e.g. fibroblasts, myoblasts, epithelial cells, glial cells.
When tissue is disaggregated and seeded into primary cell line culture, only a proportion of the cells are capable of surviving and generating a primary cell line culture. Some cells may not be capable of attachment but yet viable; others are nonviable, necrotic or apoptotic. If it is important to do so, the proportion of necrotic and apoptotic cells may be determined by viability staining and flow cytometry. Normally nonviable cells are removed at the firs change of medium. With primary cell line culture maintained in suspension, nonviable cells are gradually diluted out when cell proliferation starts.
If necessary, however, nonviable cells may be removed from the primary cell line culture disaggregate by centrifuging the cells on a mixture of Ficoll and sodium metrizoate. This technique is similar to the preparation of lymphocytes from peripheral blood. The viable cells collect at the interface between the medium and the Ficoll/metrizoate, and the dead cells form a pellet at the bottom of the tube.