Prabhakar Singh- IV_SEM-Basic Techniques of Mammalian cell culture in vitro
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This document discusses techniques for primary cell culture, including mechanical disaggregation, enzymatic disaggregation using trypsin or collagenase, and the primary explant technique. It provides details on each technique, such as how tissues are chopped or sliced, the use of enzymes at different temperatures, and factors that promote efficient development of primary cultures. The document also covers criteria for cell separation methods, cell differentiation, methods of cell transformation, and characteristics of transformed cells.
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Prabhakar Singh- IV_SEM-Basic Techniques of Mammalian cell culture in vitro
- 1. TEJASVI NAVADHITAMASTU “Let our (the teacher and the taught) learning be radiant” Let our efforts at learning be luminous and filled with joy, and endowed with the force of purpose Paper XII: CLINICAL AND DIAGNOSTIC BIOCHEMISTRY Dr. Prabhakar Singh. D.Phil. Biochemistry Department of Biochemistry, VBSPU, Jaunpur Basic techniques of mammalian cell culture in vitro; disaggregation of tissue, primary culture, cell separation. Methods of generation of transformed cells. Biology and characterization of cultured cells.
- 2. TECHNIQUE USED FOR PRIMARY CELL CULTURE The three types of technique are: (1)Mechanical Disaggregation (2) Enzymatic Disaggregation (3) Primary Explant Technique. Primary culture broadly involves the culturing techniques carried following the isolation of the cells, but before the first subculture. Primary cultures are usually prepared from large tissue masses. Thus, these cultures may contain a variety of differentiated cells e.g. fibroblasts, lymphocytes, macrophages, epithelial cells.
- 4. TECHNIQUE # 1. MECHANICAL DISAGGREGATION: For the disaggregation of soft tissues (e.g. spleen, brain, embryonic liver, soft tumors), mechanical technique is usually employed. This technique basically involves careful chopping or slicing of tissue into pieces and collection of spill out cells. The cells can be collected by two ways: i. Pressing the tissue pieces through a series of sieves with a gradual reduction in the mesh size. ii. Forcing the tissue fragments through a syringe and needle. Although mechanical disaggregation involves the risk of cell damage, the procedure is less expensive, quick and simple. This technique is particularly useful when the availability of the tissue is in plenty, and the efficiency of the yield is not very crucial. It must however, be noted that the viability of cells obtained from mechanical techniques is much lower than the enzymatic technique.
- 5. TECHNIQUE # 2. ENZYMATIC DISAGGREGATION 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 by trypsin: The term trypsinization is commonly used for disaggregation of tissues by the enzyme, trypsin. Many workers prefer to use crude trypsin rather than pure trypsin for the following reasons: i. The crude trypsin is more effective due to the presence of other proteases ii. Cells can tolerate crude trypsin better. iii. The residual activity of crude trypsin can be easily neutralized by the serum of the culture media (when serum-free media are used, a trypsin inhibitor can be used for neutralization). Disaggregation of cells can also be carried out by using pure trypsin which is less toxic and more specific in its action. The desired tissue is chopped to 2-3 mm pieces and then subjected to disaggregation by trypsin. There are two techniques of trypsinization-warm trypsinization and cold trypsinization
- 7. WARM TRYPSINIZATION This method is widely used for disaggregation of cells. The chopped tissue is washed with dissection basal salt solution (DBSS), and then transferred to a flask containing warm trypsin (37° C). The contents are stirred, and at an interval of every thirty minutes, the supernatant containing the dissociated cells can be collected. After removal of trypsin, the cells are dispersed in a suitable medium and preserved (by keeping the vial on ice). The process of addition of fresh trypsin (to the tissue pieces), incubation and collection of dissociated cells (at 30 minutes intervals) is carried out for about 4 hours. The disaggregated cells are pooled, counted, appropriately diluted and then incubated.
- 8. COLD TRYPSINIZATION This technique is more appropriately referred to as trypsinization with cold pre- exposure. The risk of damage to the cells by prolonged exposure to trypsin at 37°C (in warm trypsinization) can be minimized in this technique. After chopping and washing, the tissue pieces are kept in a vial (on ice) and soaked with cold trypsin for about 6-24 hours. The trypsin is removed and discarded. However, the tissue pieces contain residual trypsin. These tissue pieces in a medium are incubated at 37°C for 20-30 minutes. The cells get dispersed by repeated pi- pettings. The dissociated cells can be counted, appropriately diluted and then used. The cold trypsinization method usually results in a higher yield of viable cells with an improved survival of cells after 24 hours of incubation. This method does not involve stirring or centrifugation, and can be conveniently adopted in a laboratory. The major limitation of cold trypsinization is that it is not suitable for disaggregation of cells from large quantities of tissues. LIMITATIONS OF TRYPSIN DISAGGREGATION Disaggregation by trypsin may damage some cells (e.g. epithelial cells) or it may be almost ineffective for certain tissues (e.g. fibrous connective tissue). Hence other enzymes are also in use for dissociation of cells.
- 9. DISAGGREGATION BY COLLAGENASE Collagen is the most abundant structural protein in higher animals. It is mainly present in the extracellular matrix of connective tissue and muscle. The enzyme collagenase (usually a crude one contaminated with non-specific proteases) can be effectively used for the disaggregation of several tissues (normal or malignant) that may be sensitive to trypsin. Highly purified grades of collagenase have been tried, but they are less effective when compared to crude collagenase. The important stages in collagenase disaggregation, depicted in Fig. 36.3, are briefly described hereunder. The desired tissue suspended in basal salt solution, containing antibiotics is chopped into pieces. These pieces are washed by settling, and then suspended in a complete medium containing collagenase. After incubating for 1-5 days, the tissue pieces are dispersed by pipetting. The clusters of cells are separated by settling. The epithelial cells and fibroblastic cells can be separated. Collagenase disaggregation has been successfully used for human brain, lung and several other epithelial tissues, besides various human tumors, and other animal tissues. Addition of another enzyme hyaluronidase (acts on carbohydrate residues on cell surfaces) promotes disaggregation. Collagenase in combination with hyaluronidase is found to be very effective for dissociating rat or rabbit liver. This can be done by per-fusing the whole organ in situ. Some workers use collagenase in conjunction with trypsin, a formulation developed in chick serum, for disaggregation of certain tissues.
- 10. USE OF OTHER ENZYMES IN DISAGGREGATION Trypsin and collagenase are the most widely used enzymes for disaggregation. Certain bacterial proteases (e.g. pronase, dispase) have been used with limited success. Besides hyaluronidase, neuraminidase is also used in conjunction with collagenase for effective degradation of cell surface carbohydrates.
- 11. Technique # 3. PRIMARY EXPLANT TECHNIQUE The primary 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 simplified procedure adopted for primary explant culture is depicted in Fig. 36.4, and briefly described below.
- 12. 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. The primary 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 explant technique can be used for a majority of embryonic cells e.g. fibroblasts, myoblasts, epithelial cells, glial cells. SEPARATION OF VIABLE AND NON-VIABLE CELLS: It is a common practice to remove the nonviable cells while the primary culture is prepared from the disaggregated cells. This is usually done when the first change of the medium is carried out. The very few left over non-viable cells get diluted and gradually disappear as the proliferation of viable cells commences. Sometimes, the non-viable cells from the primary cultures may be removed by centrifugation. The cells are mixed with ficoll and sodium metrizoate, and centrifuged. The dead cells form a pellet at the bottom of the tube.
- 15. CRITERIA/ CHARACTERISTICS FOR EFFICIENT DEVELOPMENT OF PRIMARY CULTURES: 1. Embryonic tissues rather than adult tissues are preferred for primary cultures. This is due to the fact that the embryonic cells can be disaggregated easily and yield more viable cells, besides rapidly proliferating in vitro. 2. The quantity of cells used in the primary culture should be higher since their survival rate is substantially lower (when compared to subcultures). 3. The tissues should be processed with minimum damage to cells for use in primary culture. Further, the dead cells should be removed. 4. Selection of an appropriate medium (preferably a nutrient rich one) is advisable. For the addition of serum, fetal bovine source is preferred rather than calf or horse serum. 5. It is necessary to remove the enzymes used for disaggregation of cells by centrifugation.
- 16. They are broadly categorized into two groups: 1. Physical fractionation for cell separation. 2. Chemical blockade for cell separation. CELL SEPARATION Cell Separation by Physical Means: Physical fractionation or cell separation techniques, based on the following characteristics are in use: a. Cell density. b. Cell size. c. Affinity of antibodies on cell surface epitopes. d. Light scatter or fluorescent emission by labeled cells. The two commonly used techniques namely centrifugal elutriation and fluorescence-activated cell separation are briefly described hereunder.
- 17. Cell Differentiation: The various cell culture conditions favour maximum cell proliferation and propagation of cell lines. Among the factors that promote cell proliferation, the following are important: i. Low cell density ii. Low Ca2+ concentration iii. Presence of growth factors For the process of cell differentiation to occur, the proliferation of cells has to be severely limited or completely abolished. Cell differentiation can be promoted (or induced) by the following factors: i. High cell density. ii. High Ca2+ concentration. iii. Presence of differentiation inducers (e.g. hydrocortisone, nerve growth factor). As is evident from the above, different and almost opposing conditions are required for cell proliferation, and for cell differentiation. Therefore if cell differentiation is required two distinct sets of conditions are necessary. 1. To optimize cell proliferation. 2. To optimize cell differentiation.
- 18. METHODS OF CELL TRANSFORMATION AND CHARACTERISTICS OF TRANSFORMED CELLS Cell transformation due to changes in the genetic material, and cell cloning involving the production of a population single cell are described here. The four aspects of Cell Transformation are: (1) Genetic Instability (2) Immortalization (3) Aberrant Growth Control and (4) Tumorigenicity. Transformation of Cells: Transformation broadly refers to the change in phenotype of a cell due to a new genetic material. As regards the cultured cells, transformation involves spontaneous or induced permanent phenotypic alterations as a result of heritable changes in DNA, and consequently gene expression. Transformed Cells: Transformation is the phenomenon of the change in phenotype due to the acquirement of new genetic material. Transformation is associated with promotion of genetic instability.
- 19. 1. Growth rate 2. Mode of growth 3. Longevity 4. Tumorigenicity 5. Specialized product formation. While characterizing the cell lines, it is necessary to consider the above characters to determine whether the cell line has originated from tumor cells or has undergone transformation in culture The transformed and cultured cells exhibit alterations in many characters with reference to
- 20. Transformation of cells may occur due to any one of the following causes that ultimately result in a changed genetic material: i. Spontaneous. ii. Infection with transforming virus. iii. From gene transfection. iv. Exposure to chemical carcinogens. v. Exposure to ionizing radiations. The normally occurring genetic variations in the cultured cells are due to the following causes: 1. High rate of spontaneous mutations in the in vitro conditions, possibly due to high rate of cell proliferation. 2. The continued presence of mutant cells in the culture, as they are not normally eliminated.
- 21. CHARACTERISTICS OF TRANSFORMED CELLS The general characters of transformed cells are given in Table.They are grouped as genetic, structural, growth and neoplastic, and listed. Transformation is associated with genetic instability, immortalization, aberrant growth control and malignancy. These aspects are briefly described. Genetic Instability: In general, the cell lines in culture are prone to genetic instability. A majority of normal finite cell lines are usually genetically stable while cell lines from other species (e.g. mouse) are genetically unstable, and can get easily transformed. The continuous cell lines derived from tumors of all species are unstable.
- 22. Immortalization The acquisition of an infinite life span by a cell is referred to as immortalization. Most of the normal cells (from different species) have a finite life span of 20-100 generations. But some cells from mouse, most of the tumor cells have infinite life span, as they go on producing continuous cell lines. Control of finite life span of cells The finite life span of cultured cells is regulated by about 10 senescence genes. These dominantly acting genes synthesize products which inhibit the cell cycle progression. It is strongly believed that immortalization occurs due to inactivation of some of the cell cycle regulatory genes e.g. Rb, p53 genes.
- 23. Among the above viral genes, SV40LT is most commonly used to induce immortalization. The product of this gene (T antigen) binds to senescence genes such as Rb and p53. This binding restricts surveillance activity of senescence genes. The result is an increased genomic instability and activity, leading to further mutations favouring immortalization. For the process of immortalization, the cells are infected with retroviruses containing immortalizing gene before they enter senescence. By this way, the life span of the cells can be extended by 20-30 population doublings. Thereafter, the cells cease to proliferate, and enter a crisis phase that may last for several months. At the end of the crisis phase, a small portion of cells can grow, and eventually become immortalized. Immortalization of cells by viral genes: Several viral genes can be used to immortalize cells. Some of these genes are listed below. 1. SV40LT 2. HPV16E6/E7 3. hTRT 4. Ad5E1a 5. EBV.
- 24. Immortalization of human fibroblasts: The human fibroblasts are most successfully immortalized by the viral gene namely SV40LT. The process of fibroblast immortalization is complex and indirect with a very low probability i.e. about 1 in 107cells. Immortalization of cells by telomerase-induction: The most important cause of finite life span of cells (i.e. senescence) is due to telomeric shortening, followed by cell death (apoptosis). If the cells are transfected with telomerase gene htrt, the life span of the cells can be extended. And a small proportion of these cells become immortal.
- 25. CELL CULTURE The cell culture can be initiated by the cells derived from a tissue through enzymatic or mechanical treatments. Primary culture is a selective process that finally results in a relatively uniform cell line. The selection occurs by virtue of the capacity of the cells to survive as monolayer cultures (by adhering to substrates) or as suspension cultures. Among the cultured cells, some cells can grow and proliferate while some are unable to survive under the culture environment. The cells continue to grow in monolayer cultures, till the availability of the substrate is occupied. The term confluence is used when the cultured cells make close contact with one another by fully utilizing the available growth area. For certain cells, which are sensitive to growth limitation due to density, the cells stop growing once confluence is reached. However, the transformed cells are insensitive to confluence and continue to overgrow. When the culture becomes confluent, the cells possess the following characters: 1. The closest morphological resemblance to the tissue of origin (i.e. parent tissue). 2. The expression of specialized functions of the cells comparable to that of the native cells.
- 26. BIOLOGY OF CULTURED CELLS. Development of continuous cell lines: Certain alterations in the culture collectively referred to as transformation, can give rise to continuous cell lines. Transformation may be spontaneously occurring, chemically or virally- induced. Transformation basically involves an alteration in growth characteristics such as loss of contact inhibition, density limitation of growth and anchorage independence. The term immortalization is frequently used for the acquisition of infinite life span to cultured cells. Genetic variations: The ability of the cells to grow continuously in cell lines represents genetic variation in the cells. Most often, the deletion or mutation of the p53 gene is responsible for continuous proliferation of cells. In the normal cells, the normal p53 gene is responsible for the arrest of cell cycle. Most of the continuous cell lines are aneuploid, possessing chromosome number between diploid and tetraploid value. Normal cells and continuous cell lines: A great majority of normal cells are not capable of giving rise to continuous cell lines. For instance, normal human fibroblasts go on proliferating for about 50 generations, and then stop dividing. However, they remain viable for about 18 months. And throughout their life span, fibroblasts remain euploid. Chick fibroblasts also behave in a similar fashion. Epidermal cells and lymphoblastic cells are capable of forming continuous cell lines.
- 29. Evolution and Development of Cell Lines: The primary culture grown after the first subculture is referred to as cell line. A given cell line may be propagated by further sub culturing. As the subcultures are repeated, the most rapidly proliferating cells dominate while the non- proliferating or slowly proliferating cells will get diluted, and consequently disappear. Senescence: The genetically determined event of cell divisions for a limited number of times (i.e. population doublings), followed by their death in a normal tissue is referred to as senescence. However, germ cells and transformed cells are capable of continuously proliferating. In the in vitro culture, transformed cells can give rise to continuous cell lines. The evolution of a continuous cell line is depicted in Fig. 35.4. The cumulative cell number in a culture is represented on Y-axis on a log scale, while the X-axis represents the time in weeks. The time for development of a continuous cell line is variable. For instance, for human diploid fibroblasts, the continuous cell line arises at about 14 weeks while the senescence may occur between 10 to 20 weeks; usually after 30 and 60 cell doublings. Dedifferentiation: Dedifferentiation refers to the irreversible loss of specialized properties of cells when they are cultured in vitro. This happens when the differentiated in vitro cells lose their properties In the in vivo situation, a small group of stem cells give rise to progenitor cells that are capable of producing differentiated cell pool. On the other hand, in the in vitro culture system, progenitor cells are predominantly produced which go on proliferating. Very few of the newly formed cells can form differentiated cells. The net result is a blocked differentiation. Dedifferentiation implies an irreversible loss of specialized properties of the cells. On the other hand, de-adaptation refers to the re-induction of specialized properties of the cells by creating appropriate conditions.
- 31. CHARACTERIZATION OF CULTURED CELLS Characterization of cultured cells or cell lines is important for dissemination of cell lines through cell banks, and to establish contacts between research laboratories and commercial companies. Characterization of cell lines with special reference to the following aspects is generally done: 1. Morphology of cells, 2. Species of origin, 3. Tissue of origin. 4. Whether cell line is transformed or not, 5. Identification of specific cell lines. Morphology of Cells: A simple and direct identification of the cultured cells can be done by observing their morphological characteristics. However, the morphology has to be viewed with caution since it is largely dependent on the culture environment. For instance, the epithelial cells growing at the center (of the culture) are regular polygonal with clearly defined edges, while those growing at the periphery are irregular and distended (swollen). The composition of the culture medium and the alterations in the substrate also influence the cellular morphology. In a tissue culture laboratory, the terms fibroblastic and epithelial are commonly used to describe the appearance of the cells rather than their origin. Fibroblastic cells: For these cells, the length is usually more than twice of their width. Fibroblastic cells are bipolar or multipolar in nature. Epithelial cells: These cells are polygonal in nature with regular dimensions and usually grow in monolayers. The terms fibroblastoid (fibroblast-like) and epitheloid (epithelial-like) are in use for the cells that do not possess specific characters to identify as fibroblastic or epithelial cells.
- 32. Measurement of Growth Parameters of Cultured Cells: Information on the growth state of a given culture is required to: a. Design culture experiments. b. Routine maintenance of culture. c. Measurement of cell proliferation. d. Know the time for subculture. e. Determine the culture response to a particular stimulus or toxin. Some of the commonly used terms in relation to the measurement of growth of cultured cells are explained. Population doubling time (PDT): The time interval for the cell population to double at the middle of the logarithmic (log) phase. Cell cycle time or generation time: The interval from one point in the cell division to the same point in the cycle, one division later. Thus cell cycle time is measured form one point in the cell cycle until the same point is reached again. Confluence: It denotes the culture stage wherein all the available substrate (growth area) is utilized, and the cells are in close contact with each other. Contact inhibition: Inhibition of cell motility and plasma membrane ruffling when the cells are in complete contact with other adjacent cells. This mostly occurs at confluence state, and results in the ceasation of the cell proliferation. Cell density: The number of cells per ml of the medium. Saturation density: The density of the cells (cells/ml2, surface area) in the plateau phase.