Procedure For Cell Culture

Procedure For Cell Culture: A piece of tissue from the organism is usually quite complex and it contains connective tissue cells, a variety of blood cells, and reticuloendothelial cells. The first cell suspension is isolated and then inoculated into a new culture vessel along with a fresh medium, such culture is called primary cell culture or primary cell line. Three stages are used for the isolation of primary cell culture such as (i) isolation of the tissue (ii) disaggregation of tissue and (iii) seeding the culture into the culture vessel. All stages are performed in the laminar bench to avoid contamination (Fig.1). 

Laminar bench used for isolation and disaggregation of tissue
Fig.1: Laminar bench used for isolation and disaggregation of tissue 

(i) Isolation of the tissue: The explant from an excised portion of the body of an animal is used for the culture of animal cells in a suitable nutrient medium. The major explant tissues are collected (Fig.2) from laboratory animals like mice, rabbits, guinea pigs, etc.  Mouse embryos are a convenient source of cells for undifferentiated fibroblastic cultures. The explants from humans such as smooth muscle cells, alveolar cells, macrophages, leukocytes, etc are isolated and cultured in simulated media. Organs from which cells are to be isolated are surface sterilized with 70% alcohol and then removed aseptically. Excised tissues from the explant source are immediately transferred to a sterile nutrient medium or a well-balanced salt solution (BSS) containing antibiotics. The tissues isolated from the explant are either stored in freeze or used immediately.

Collection of explant tissues for cell lines
Fig.2: Collection of explant tissues for cell lines 

(ii) Disaggregation of tissue: A primary cell culture is obtained by disaggregating the tissue mechanically, enzymatically, or by use of a chelating agent (Fig.3)

Techniques for tissue disaggregation
Fig.3: Techniques for tissue disaggregation 

Mechanical or physical disaggregation: Physical disaggregation is a force-based method in which the tissue is carefully sliced and then exposed for sieving. The tissues may be forced through a syringe and needle or repeatedly pipetted. Sieving is a most important method where tissues are forced through gradually reduced mesh sieves. The cells are counted by hemocytometer and transferred into a medium to get the suspension of cells (104 cells/ml). This method gives a cell suspension more quickly than enzymatic disaggregation but may cause mechanical damage.

Enzymatic disaggregation: The enzymes used in enzymatic disaggregation are trypsin, mucase, pronase, collagenase, papain, pancreatin elastase, etc. This method is labor intensive and involves damage to cells. Code trypsin and collagenase enzymes are commonly used in tissue disaggregation. 

The process of treatment of the tissues with trypsin is called trypsinization. It is classified as warm trypsinization and cold trypsinization. In warm trypsinization, the tissue sample is chopped into 2 to 3 pieces and then transferred into a conical flask containing 100 ml warm trypsin (36.5 oC). The contents are mixed for 4 hours and then dissociated cells are collected every 30 minutes. The process may be repeated by adding fresh trypsin and incubating the contents. The dissociated cells are counted by using a hemocytometer and pooled in the medium containing serum for growth. In cold trypsinization, the tissue sample is chopped similarly to warm trypsinization. The whole contents are washed by using BSS and then transferred in a glass vial which is placed on ice for soaking with trypsin for 4 to 5 hours.  Trypsin is removed and tissues are incubated at 36.5 oC for 20 to 30 minutes. Serum (10 ml) is added to vials and cells are dispersed by repeated pipetting. The cell is counted, adjusted the cell density (104 cell/ml), and incubated for growth. 

The disaggregation by trypsin may damage some epithelial cells or it becomes ineffective for fibrous connective tissue. Collagenase has a more effective and simple method for the disaggregation of normal and malignant tissues. Extracellular matrix often contains collagen mainly in connective tissues and muscles. Hence, collagenase has been the first choice for the treatment of tissues. Biopsy tissues are mainly disaggregated in BSS-containing antibiotics. All chopped tissues are washed and then transferred in complete medium containing collagenase. The cells are separated by centrifugation and cultured in the medium for growth. 

Chelating agents: Chelating agents are mainly used for the preparation of cell suspensions from established cultures. Calcium and magnesium ions are treated with chelating agents (e.g. EDTA) which are required for epithelium tissues for their integrity. 

(iii) Seeding the culture into the culture vessel: The dissociated (primary) cells grow well when seeded on culture plates (Fig.4) at high density.

Culture vessel (T-25 culture flask)
Fig.4: Culture vessel (T-25 culture flask)

Most of the cells require the support of a substrate for growth. These cells are called anchorage-dependent (adherent) cells and it grows as a monolayer. Some cells do not require substrate called anchorage-independent (suspension culture) cells. The adherent cells can be obtained from organs (e.g. liver, kidney, etc) that are fixed in a place. These cells have positive surface charge except neuron and muscle cells. Anchorage-dependent cells may grow well by adhering to the negatively charged surface. Substrates used for the growth of such cells are glass (coverslip, slide, test tubes, plates, or flasks), plastic, and metals (stainless steel,  titanium, etc). The anchorage-independent cells or suspension cells are generally cultivated in a liquid medium and they do not attach to the surface of the vessel. It is classified into batch culture, feed batch culture, perfusion culture, semi-continuous culture, and continuous flow culture.

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