Cell Culture Characteristics - PDF

CHARCTERISTICS OF CELLS IN CULTURE 1 ▪ WHERE TO OBTAIN CELLS? ▪ CELLS FROM TISSUE: A PRIMARY CULTURE ▪ CELL TYPES ▪ HOW DO YOU SELECT A PARTICULAR CELL TYPE? ▪ WHAT IS A NORMAL CELL? ▪ ANCHORAGE DEPENDENCE ▪ THE CULTURE OF DIFFERENTIATED CELLS ▪ EMB...

CHARCTERISTICS OF CELLS IN CULTURE 1 ▪ WHERE TO OBTAIN CELLS? ▪ CELLS FROM TISSUE: A PRIMARY CULTURE ▪ CELL TYPES ▪ HOW DO YOU SELECT A PARTICULAR CELL TYPE? ▪ WHAT IS A NORMAL CELL? ▪ ANCHORAGE DEPENDENCE ▪ THE CULTURE OF DIFFERENTIATED CELLS ▪ EMBRYONIC STEM CELLS ▪ ADULT STEM CELLS ▪ TRANSFORMED CELLS ▪ CELLS FROM A CULTURE COLLECTION 10/21/20 Prof. Dr. Emad El-Zayat 22 2 1- Where to obtain cells A major choice has to be made when establishing a cell culture as to whether cells are obtained directly from animal tissue or from a culture collection (cell banks). The choice will depend upon the objective of the project and the nature of the experiments planned. Prof. Dr. Emad El-Zayat 10/21/2022  Isolation directly from tissue (a primary cell 3 culture) offers a means of culturing cells close to their state in vivo. However, the isolation process is far more demanding and troublesome compared with establishing a culture from a cell sample that could be obtained from a culture collection. 🠶 Culture collection: are banks in which cells have been well characterized in terms of growth, origin, and genetic traits. Prof. Dr. Emad El-Zayat 10/21/2022  2- Cells from tissue: a primary culture 4 A primary culture is established when the cells taken directly from animal tissue are added to growth medium. Primary cultures are often established from embryonic tissue because the cells are more easily dispersed and have a superior growth potential. The objective of establishing a primary culture is: to select a single cell type from a tissue of ordered Prof. Dr. Emad El-Zayat 10/21/2022 structure. 5 The original methods developed for tissue culture involve the maintenance of tissue fragments (or explant) on a solid surface and supplied with suitable nutrients. However, such cultures are of greater use if the individual cells are separated out before culture. Prof. Dr. Emad El-Zayat 10/21/2022 6 Prof. Dr. Emad El-Zayat 10/21/2022  This is done by fragmenting tissue with forceps and 7 scissors followed by treatment with a proteolytic enzyme such as trypsin or collagenase. The proteolytic action of the enzymes disaggregates the tissue into individual cells. Cells are then isolated by low-speed centrifugation. The time that the cells are in contact with the degradative enzymes should be minimized otherwise membrane damage may occur. Prof. Dr. Emad El-Zayat 10/21/2022 Cells can be bathed in trypsin for 10–20 minutes.  Longer exposure times are 8 undesirable because breakdown of the protein components of cell membranes could occur. Collagenase: degrades collagen and is more recommended because it is less harmful to cell membranes, but it is more expensive. Prof. Dr. Emad El-Zayat 10/21/2022  This technique works well for most tissues 9 although some modifications to the general procedure may be necessary to ensure the maximum yield of a particular cell type. One of the major difficulties and reasons for failure at this stage is that the cell population becomes contaminated with bacteria or fungi. To avoid this problem it is important to maintain aseptic techniques throughout the process of establishing the primary culture. Prof. Dr. Emad El-Zayat 10/21/2022 10 NOTE All the dissection instruments should be sterile and all working surfaces should be swabbed with 70% alcohol. Prof. Dr. Emad El-Zayat 10/21/2022  NB: 11 The entire procedure should be carried out in a laminar flow cabinet under sterile conditions. The process combines proteolytic digestion with mechanical agitation to cause individual cells to disperse into a liquid suspension. The addition of a serum-supplemented medium after trypsinization is important to inhibit further activity of the trypsin. Digestion of the tissue may be aided by the proteolytic enzyme collagenase and by the addition of chelating agents such as Versene or ethylenediaminetetra acetic acid (EDTA). Prof. Dr. Emad El-Zayat 10/21/2022 12 When the cells in a primary culture stop growing (confluence) checked by the inverted microscope a new culture may be established by inoculating some of the cells into fresh medium. This is called subculturing, splitting or passaging. A secondary culture is established after the first passage of the primary culture. Prof. Dr. Emad El-Zayat 10/21/2022  However it should be noted that: the greatest 13 chance of genetic alteration occurs in the first few passages following the primary culture as cells adapt to a new chemical environment. “The passage number”: is often recorded as the number of subcultures from the primary source or culture. The chick embryo fibroblasts for example may grow for around 30 passages before Prof. Dr. Emad El-Zayat 10/21/2022  3- Cell types and cell morphology 14 Animal cells are usually defined by the tissue from which they have been derived and have characteristic shapes that can be observed and recognized easily through a light microscope. Figure 2.2 illustrates the morphology of the cells commonly grown in culture. These are derived from five main types of animal tissue. Prof. Dr. Emad El-Zayat 10/21/2022 15 Prof. Dr. Emad El-Zayat 10/21/2022  Epithelial tissue 16 Consists of a layer of cells which cover organs and line cavities; examples include skin and the lining of the alimentary canal. The epithelial cells grow well in culture as a single cell monolayer and have a characteristic cobble-stone appearance. Prof. Dr. Emad El-Zayat 10/21/2022 17 🠶Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches. Prof. DR. Emad El-Zayat 10/21/ 2022  Connective tissue 18 Forms a major structural component of animals, consisting of a fibrous matrix and including bone, cartilage. The tissue contains fibroblasts which are amongst the most widely used cells in laboratory cultures. Fibroblasts are bound to the fibrous protein collagen in the connective tissue. The cells are spherical when first dissociated by trypsin from the tissue but elongate to a characteristic spindle-shape on Prof. Dr. Emad El-Zayat 10/21/2022 attachment to a solid surface. 19 Fibroblasts have excellent growth characteristics and have been the ‘favorite’ cells for establishing cultures. Fibroblast and epithelial cells adapt relatively easily to culture and have growth rates with a doubling time of 18–24 hours. 10/21/202 Prof. Dr. Emad El-Zayat 2  🠶 Fibroblastic (or fibroblast-like) cells are bipolar or 20 multipolar, have elongated shapes, and grow attached to a substrate. Prof. DR. Emad El-Zayat 10/21/20 22 21 Muscle tissue Consists of a series of contractile tubules formed from precursor cells (myoblasts) which fuse to form a multinucleate complex which also contain the structural proteins actin and myosin. The precursor cells are myoblasts which are capable of differentiation to form myotubes—a process that can be observed in culture. The following Fig. shows the myoblast alignment that occurs during the process. Prof. Dr. Emad El-Zayat 10/21/2022 22 Prof. Dr. Emad El-Zayat 10/21/2022 23 Nervous tissue Consists of characteristically spindle-shaped neurons which are responsible for the transmission of electrical impulses and supporting cells, called glial cells. Neurons are highly differentiated cells and have not been observed to divide in culture. However, the addition of nerve growth factor to cultures of neurons may cause the formation of cytoplasmic outgrowths called neurites. Prof. Dr. Emad El-Zayat 10/21/2022 24 Prof. Dr. Emad El-Zayat 10/21/2022 25 Some of the characteristics of nerve cells can be observed with neuroblastomas, which are tumor nerve cells that undergo cell growth (division) in culture. Prof. Dr. Emad El-Zayat 10/21/2022 26 Blood and lymph Belong to connective tissues which contain a range of cells in suspension. Some of these will continue growth in a culture suspension. These include the lymphoblasts which are white blood cells and are used extensively in culture because of their ability to secrete immuno- regulating compounds (cytokines). Prof. Dr. Emad El-Zayat 10/21/2022  Standard Blood Smear 27 Prof. Dr. Emad El-Zayat 21/10/2022  🠶 Lymphoblast-like cells are spherical in shape and 28 usually grown in suspension without attaching to a surface. Prof. DR. Emad El-Zayat 10/21/2 022  4- How to select a particular cell type 29 The primary culture will almost certainly contain a variety of different cell types with differing growth capacities. However, for most experimental work it is important to isolate a single cell type from the culture population. There are several ways this can be achieved: Prof. Dr. Emad El-Zayat 10/21/2022  (1) Allow the cells to grow. Fast-growing cell 30 types may assume dominance in a population. For example, fibroblasts have relatively short population doubling times and may outgrow other cells after a few generations (called ‘fibroblast overgrowth'). (2) Control the composition of the growth medium. The addition of specific growth factors or known growth inhibitors may allow selective growth of certain cell types. Prof. Dr. Emad El-Zayat 10/21/2022  (3) Separate cells by using gradient 31 centrifugation: The cells sediment to an equilibrium position equivalent to their own density—a process called isopycnic sedimentation. The gradients can be formed by nontoxic, high- molecular-weight material such as colloidal silica as developed by Pharmacia in their formulations ‘Ficoll’ and ‘Percoll’. This method is particularly effective for the isolation of certain cell types in sterile medium, for example Prof. Dr. Emad El-Zayat 10/21/2022 32 Prof. Dr. Emad El-Zayat 10/21/2022 33 Notes The upper layer following the initial centrifugation contains blood plasma and is a good source of platelets. Washing the lymphocytes in salt solution ensures removal of the platelets. Granulocytes and erythrocytes form the lower layer after centrifugation. Transferring a minimal volume of the lymphocyte band ensures minimal granulocyte contamination. Typically this procedure recovers up to 80% of the lymphocytes from the original blood sample Prof. Dr. Emad El-Zayat 10/21/2022 34 🠶 (4) Fluorescence Activated cell sorting (FACS): which is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and flouresecent characteristics of each cell. Prof. Dr. Emad El-Zayat 10/21/2022  5- What is a ‘normal’ cell? 35 In the 1960s, ‘normal’ mammalian cells were required as hosts for the production of human vaccines in order to ensure the safety of these products. In order to meet this requirement, a number of characteristics of ‘normal’ animal cells were defined by Hayflick and Moorhead following their work with human embryonic cells. Prof. Dr. Emad El-Zayat 10/21/2022  Characteristics of normal animal cells 36 (1) Diploid chromosome number: which indicates that no gross chromosomal damage has occurred. (2) Anchorage dependence: the cells require a solid substratum for attachment and growth. (3) Finite lifespan: this is a reflection of the intrinsic growth potential of the cells (cell biological clock) (4) Nonmalignant: the cells are not cancerous. This can be shown by the inability of the cells to form a tumor following injection into immuno-compromised mice. Prof. Dr. Emad El-Zayat 10/21/2022 37 6- Anchorage-dependence Anchorage-dependence: is the requirement of cells for a solid substratum for attachment before growth can occur. At the laboratory scale this substratum can be provided by the solid surface of Petri dishes, T- flasks, or Roux bottles which are made of -vely charged- treated glass or plastic. The interaction between the cell membrane and the growth surface is critical and involves a combination of electrostatic attraction and van der Waal’s forces. Prof. Dr. Emad El-Zayat 10/21/2022  Cell adhesion occurs by divalent cation 38 (usually Ca2+) and basic proteins (+vely charged) like fibronectin, forming a layer between the solid substratum and the cell surface. In most cases the cell-surface interaction is provided by a range of nonspecific proteins which form a 2.5 nm-thick layer on the substratum prior to cell attachment. Prof. Dr. Emad El-Zayat 10/21/2022 39 Serum-derived glycoproteins (e.g. fibronectin) can provide a surface coating conducive to cell attachment. Conditioning factors are released by cells into the medium and help in forming a bond between cell surface glycoproteins and the substratum Prof. Dr. Emad El-Zayat 10/21/2022  The density of the electrostatic charge on the solid 40 substratum is also critical in maximizing cell attachment. A negative charge is provided on glass surface containers by alkali treatment. Tissue culture-grade plastic ware consists of sulfonated polystyrene with a surface charge of 2–5 negatively charged groups per nm2. Culture systems are also available for the large-scale Prof. Dr. Emad El-Zayat 10/21/2022 production of anchorage-dependent cell. 41 Prof. Dr. Emad El-Zayat 10/21/2022  7- The culture of differentiated cells 42 Differentiation: is a process whereby cells slowly change their characteristics to become specialized cells with characteristic phenotypes. This process occurs in vivo during: (a) embryonic development or (b) wound healing and leads to the formation of cells with specialized function (differentiated) such as neurons or muscle cells or (c) normal cell replacement, as is necessary in the bloodstream. Prof. Dr. Emad El-Zayat 10/21/2022 The undifferentiated precursors of this process are 43 Prof. Dr. Emad El-Zayat 10/21/2022  Most stem cells or embryonic cells grow 44 well in culture. As cells become more specialized (differentiated) they tend to lose their growth capabilities and this is reflected by poor growth in culture. For most cell types proliferation is incompatible with the expression of differentiated properties. Prof. Dr. Emad El-Zayat 10/21/2022  When some cells derived from a tissue are 45 placed in culture there can be an apparent loss in differentiated properties in the surviving cell population (why??) Some explanation can be offered for this: a- Selective outgrowth of undifferentiated cell types. These include fibroblasts and epithelial cells that may be obtained from non growing animal tissue; b- Adaptive response of cells to the culture media. c- Loss of cell-cell interaction necessary to maintain Prof. Dr. Emad El-Zayat 10/21/2022  Tumor cells are, in most cases, undifferentiated 46 and have good growth characteristics. However, there are also some differentiated tumor cells which have proved extremely valuable. For example neuroblastomas (differentiated tumor cells) are fast-growing tumor cells which have been used to study response effects with nerve growth factor. Differentiated tumor cells: retain the phenotypic Prof. Dr. Emad El-Zayat 10/21/2022 characteristics of normal differentiated cells but  The following 4 factors may allow some 47 differentiated properties of normal cells to be maintained in culture: (1) Hormones and growth factors. There are a number of media formulations containing selective components that can maintain the differentiated state of specific cell types, for example keratinocytes, hepatocytes and nerve cells. (2) Chemical agents. Solvents such as dimethyl sulfoxide (DMSO) may allow the maintenance of a differentiated state by an effect on membrane fluidity. Prof. Dr. Emad El-Zayat 10/21/2022  (3) Cell-cell interactions. Contact between cells may allow the 48 formation of gap junctions and allow metabolites to synchronize the expression of differentiation within a cell population. This may also play a part in the arrest of growth when a cell population has covered an available growth surface (defined as ‘confluence'). (4) Interaction with the growth surface. Collagen has been found to be essential for maintaining the polarity of hepatocytes in relation to the attachment surface. Cell polarity is governed by an asymmetrical distribution of 10/21/2022 ion currents (particularly Ca2+) which allows one end of the cell Prof. Dr. Emad El-Zayat 49 Some culture systems have been extremely valuable in investigating the metabolic changes that are associated with differentiation. However, growth in these cultures is either nonexistent or can be prolonged only for a short period (weeks). Prof. Dr. Emad El-Zayat 10/21/2022  8- Embryonic stem cells 50 These cells are capable of apparently unlimited growth but have the capacity, given the appropriate stimuli, to differentiate into any other cell type. Human embryonic stem cells were first isolated in 1998 by J. Thomson at the University of Wisconsin, who derived several cell lines and showed their capacity for growth for up to at least 300 population doublings. The cells were derived from the inner cell mass (~30 cells) of a human blastocyte formed from several days (~ 4-5) growth of an embryo following in vitro fertilization. Prof. Dr. Emad El-Zayat 10/21/2022 51 Embryonic Stem Cells Prof. Dr. Emad El-Zayat 10/21/2022 Embryonic stem cells in the dish: What do cultured ES cells look like? Fluorescent imaging of embryonic stem cell colonies  These embryonic stem cells have been shown to 54 have these 7 important properties: (1) Pluripotent, which means they have the capacity for differentiation into the cells of the three major tissue types (ectoderm, mesoderm and endoderm). This means that they have the potential to act as precursors for all cells of the body. (2) propagated indefinitely (self-renewal) in a non- differentiated state. Prof. Dr. Emad El-Zayat 10/21/2022  (3) Directed differentiation. They can be 55 induced to follow a specific pathway of differentiation, given the appropriate chemicals, growth factors or cell contact. (4) Associated with specific cell markers, e.g. Oct-4 transcription factor and stage- specific embryonic antigen (SSEA). (5) Have a normal diploid karyotype Prof. Dr. Emad El-Zayat 10/21/2022  (6) Have a high activity of telomerase. 56 This tends to correlate with immortality in human cell lines. (7) Form a teratoma, if injected into immuno-compromised mice, which is a clump of cells in which some of the major differentiated cell types can be distinguished or Embyoid body if grown in vitro Prof. Dr. Emad El-Zayat 10/21/2022 57 8.1- Directed differentiation If embryonic stem cells are allowed to clump then they form an embryoid body in which the cells begin to differentiate spontaneously. However, through directed differentiation the addition of specific growth factors may direct the cells down a specific pathway of change. The ability to direct such events is extremely useful for both: studying developmental changes and cell used therapy (regenerative medicine). Prof. Dr. Emad El-Zayat 10/21/2022 58 Prof. Dr. Emad El-Zayat 10/21/2022 59 9- Adult stem cells These are undifferentiated cells found among differentiated cells in a tissue or organ. Normally these cells can differentiate along a more limited pathway than embryonic stem cells to produce cells associated with the tissue. The cells serve to replace cells or repair tissue damage. Prof. Dr. Emad El-Zayat 10/21/2022  Under certain conditions adult stem 60 cells may be induced to differentiate into cell types other than those associated with the tissue from which they were derived. This is known as transdifferentiation or plasticity and is presently an active area of research. Prof. Dr. Emad El-Zayat 10/21/2022  Hematopoietic Stem cells (HSC) found in 61 the bone marrow differentiate through the hematopoietic pathway to provide the extensive range of mature blood cell types. The hematopoietic stem cells are important for the continuous replacement of the cells found in the blood system. The hematopoietic pathway involves differentiation of cells through four stages: hematopoietic stem cells → early progenitor Prof. Dr. Emad El-Zayat 10/21/2022  The two distinct progenitor lineages are 62 lymphoid and myeloid lineage. The lymphoid progenitor cells produce the mature T-lymphocytes, B-lymphocytes and Natural killer cells when they are stimulated to differentiate by various interleukins : IL-2, IL-3, IL-6, IL-7 Prof. Dr. Emad El-Zayat 10/21/2022 63 Prof. Dr. Emad El-Zayat 10/21/2022 64 10- Transformed cells Transformation has two different meanings in cell biology: a- Expression of foreign genes in bacteria or animal cells b- Change of animal cells from normal to infinite growth capacity. Prof. Dr. Emad El-Zayat 10/21/2022 65 ‘Normal’ animal cells have a finite growth capacity. But some normal cells acquire a capacity for infinite growth and such a population can be called an “immortal”, “established”, “continuous” or “cancerous” cell line. This requires cells to go through a process called transformation, which causes the cells to lose their sensitivity to the stimuli associated with Prof. Dr. Emad El-Zayat 10/21/2022  Transformed cells may also lose their 66 anchorage-dependence and often show some chromosomal fragmentation. This genetic state is referred to as aneuploidy, which means that there is a slight alteration from the normal diploid state. The transformed cells have a high capacity for growth in relatively simple growth medium Prof. Dr. Emad El-Zayat 10/21/2022 (poor medium) and without the need for  Carcinogenesis in vivo is analogous, but not 67 identical to the transformation of cells in vitro. Not all transformed cells are malignant, a characteristic defined by the ability to form tumors in animals. But all tumor-derived cells grow continuously in culture. Examples of carcinogenesis in vivo include HeLa cells, which are derived from a cervical cancer and Namalwa cells, which are derived from a human lymphoma. Prof. Dr. Emad El-Zayat 10/21/2022  These cells are relatively easy to grow. They are 68 robust and show good growth characteristics, which include a short doubling time and a low requirement for growth factors. Cells can be transformed or ‘immortalized’ by a variety of techniques which include: treatment with mutagens, viruses or oncogenes Prof. Dr. Emad El-Zayat 10/21/2022 69 An oncogene is defined as a gene that induces the formation of tumorigenic cells. Infection by retroviruses is a particularly effective method of immortalizing cells. These retroviruses express activated oncogenes (e.g. myc and ras), which cause cell transformation. Prof. Dr. Emad El-Zayat 10/21/2022  11- Cells from a culture collection 70 For many applications, cell lines may be obtained from cell culture collections (′cell banks'), which have a large selection of well-characterized cell lines. This is far easier than having to rely on primary animal tissue for establishing cultures. Samples of cell suspensions (~107 cells/ml) are offered for sale by the cell collections in frozen vials that can be transported in dry ice. 10/21/2022 Prof. Dr. Emad El-Zayat  The largest and most well-known international animal 71 cell culture collections are given below: 1- The American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, USA: website: www.atcc.org. 2- The European Collection of Animal Cell Culture (ECACC), Public Health Laboratory Service (PHLS), Centre for Applied Microbiology Research (CAMR), Porton Down, Salisbury SP4 OJG, UK: website: www.ecacc.org. Prof. Dr. Emad El-Zayat 10/21/2022  On arrival the cells should be thawed and 72 inoculated immediately into culture media or stored in liquid nitrogen. Table 2.1 lists a few examples of commonly used cell lines which can be obtained from the major international culture collections. All these cells grow well in culture when provided with an appropriate growth medium. Prof. Dr. Emad El-Zayat 10/21/2022 73 Prof. Dr. Emad El-Zayat 10/21/2022  Other services offered by these establishments 74 are: 1- The safe storage of private cell lines. This is useful for the maintenance of a master stock of important cells 2- Tests for contamination in cell lines. 3- Characterization of cell lines. This includes isoenzyme analysis, karyotyping and Prof. Dr. Emad El-Zayat 10/21/2022 DNA fingerprinting, flow cytometry etc…