Biology of Cultured Cells PDF

Biology of Cultured cells  Extracellular Matrix Tissues are not made up solely of cells. A substantial part of their volume is extracellular space, which is largely filled by an intricate network of macromolecules constituting the extracellular matrix This matrix is compos...

Biology of Cultured cells  Extracellular Matrix Tissues are not made up solely of cells. A substantial part of their volume is extracellular space, which is largely filled by an intricate network of macromolecules constituting the extracellular matrix This matrix is composed of a variety of proteins and polysaccharides that are secreted locally and assembled into an organized meshwork in close association with the surface of the cell that produced them. The vertebrate extracellular matrix serves as a relatively inert scaffold to stabilize the physical structure of tissues and has a far more active and complex role in regulating the behavior of the cells that contact it, influencing their survival, development, migration, proliferation, shape, and function. The extracellular matrix has a correspondingly complex molecular composition. Cells surrounded by spaces filled with extracellular matrix Schematic representation of cell adhesion  Cellular Adhesion Cellular adhesion is the binding of a cell to another cell or to a surface or matrix. Cellular adhesion is regulated by specific adhesion molecules that interact with molecules on the opposing cell or surface. Adhesion molecules are also termed "receptors" and the molecules they recognize are termed "ligands" (and sometimes "counterreceptors"). Since cells are not often found in isolation, rather they tend to stick to other cells or non- cellular components of their environment, a fundamental question is: what makes cells sticky? Cell adhesion generally involves protein molecules at the surface of cells, so the study of cell adhesion involves cell adhesion proteins and the molecules that they bind to.  Cell Adhesion Molecules (CAM) Three major classes of transmembrane proteins are involved in cell-cell and cell-substrate adhesion. Cell-Cell Adhesion Molecules: CAMs (Ca2+- independent), and cadherins (Ca2+ dependent) are involved primarily in interactions between homologous cells. These proteins are self interactive. Cell substrate interactions: Mediated by integrins, receptors for matrix molecules such as fibronectin, entactin, laminin, and collagen. Disaggregation of the tissue, or an attached monolayer culture, with protease will diggest some of the extracellular domains of transmembrane proteins, allowing cells to become dissociated from each other. The cells must resynthetize matrix proteins before they attach or must be provided with matrix- coated substrate.  Cell Proliferation: Cell Cycle Cell cycle made up of 4 phases: M phase: Chromosomes condenses into chromosomes, and the 2 individual chromatids segregates to each daughter cell. G1 (Gap 1) phase: Cell progress toward DNA synthesis and another division cycle or exits the cell cycle reversibly (G0) or irreversibly to commit to differentiation. S phase (DNA synthesis): DNA replicates G2 (Gap 2) phase: Cell prepares for reentry into mitosis. Determine the integrity of the DNA and will halt the cell cycle to allow DNA repair or entry into apoptosis if repair is impossible. Apoptosis, or programmed cell death, is a regulated physiological process whereby a cell can be removed from a population. Apoptosis is marked by DNA fragmentation, nuclear blebbing, and cell shrinkage.  Cell Proliferation: Control of Cell Proliferation Entry into the cell cycle is regulated by signals from the environment: Low cell density: Renders cells capable of spreading, which permits their entry into the cycle in the presence of mitogenic growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), or plateled-derived growth factor (PDGF). The growth factors interacts with cell surface receptors. High cell density: Inhibits the proliferation of normal cells. Inhibition of proliferation is initiated by cell contact and is accentuated by crowding and the resultant change in the shape of the cell and reduced spreading.  Synchronization of cell cultures Several methods can be used to synchronize cell cultures by halting the cell cycle at a particular phase. Serum starvation and treatment with Thymidine or Aphidicolin halt the cell in the G1 phase, Mitotic shake-off, treatment with colchicine Treatment with Nocodazole halt the cell in M phase Treatment with 5-fluorodeoxyuridine halts the cell in S phase.  Differentiation The expression of differentiated properties in cell culture is often limited by the promotion of cell proliferation, which is necessary for the propagation of the cell line. The conditions required for the induction of differentiation may often be antagonistic to cell proliferation and vice versa. If differentiation is required, it may be necessary to define distinct sets of conditions: Optimize cell proliferation Optimize cell differentiation. Conditions required for cell differentiation: - High cell density, - Enhanced cell-cell and cell-matrix interaction, - Presence of various differentiation factors. Maintenance of differentiation requires culturing cells on or in special matrices, such as collagen, or gelatine sponge, or matrices from other natural tissue matrix such as fibronectin, chondronectin, and laminin.  Dedifferentiation Dedifferentiation: The inability of cell lines to express the in vivo phenotype of the cells from which they were derived. Differentiated cells lose their specialized properties in vitro but it is unclear whether, Undifferentiated cell of the same lineage overgrow terminally differentiated cells of reduced proliferative capacity, The absence of the appropriate inducers (hormones: cell or matrix interaction) causes deadaptation. Continuous proliferation may select undifferentiated precursors, which in the absence of the correct inductive environment, do not differentiate.  Energy Metabolism Most culture media contain 4-20 mM glucose, which is used as a carbon source for glycolysis, generating lactic acid as an end product. Under normal culture conditions (atmospheric oxygen) oxygen is low. In the absence of an appropriate carrier, such as hemoglobin, raising the O2 will generate free radicals that are toxic to the cell, so O2 is usually maintained at atmospheric level (low). This results in anaerobic conditions and the use of glycolysis for energy metabolism. However, the citric acid cycle remains active, amino acids (glutamin) can be utilized as a carbon source by oxidation to glutamate by glutaminase and entry into the citric acid cycle by transamination to 2-Oxoglutarate. Deamination of the glutamine tends to produce ammonia, which is toxic to the cells and can limit cell growth. The use of dipeptide such as glutamyl-alanine or glutamyl-glycine minimize the production of ammonia.  Initiation of the culture A culture is derived either by the outgrowth of migrating cells from a fragment of tissue or by enzymatic or by mechanical dispersal of the tissue. Primary culture is the first in a series of selective processes that may give rise to a relatively uniform cell line. In primary explantation, selection occurs by virtue of the cell’s capacity to migrate from the explant, while with dispersed cells, only those cells that both survive the disaggregation technique and adhere to the substrate or survive in suspension will form the basis of a primary culture.  Initiation of the culture: Selection A further selection occurs if primary culture is kept for more than few hours: - Cells capable of proliferation will increase. - Some cell types will survive but not increase. - Others will be unable to survive under the particular conditions of the culture. In case of monolayer culture, the relative proportion of each cell type will change and will continue to do so until all available culture substrate is occupied. After confluence is reached: - Cells whose growth is sensitive to density limitation will stop dividing, - Transformed cells, which are insensitive to density limitation will tend to overgrow. Keeping the cell density low (by frequent subculture) helps to preserve the normal phenotype in culture. Selection in Cell Line Development  Evolution of cell lines - After the first subculture, or passage, the primary culture becomes known as cell line and may be propagated and subcultured several times. - After each successive subculture, the component of the population with the ability to proliferate most rapidly will gradually predominate. - Nonproliferating cells will be diluted out.  Senescence Normal cell can divide only for a limited number of times. Cell lines derived from normal tissue will die out after a fixed number of population doublings. This genetically determined event is known as senescence. Senescence is determined by the inability of terminal sequences of DNA in the telomeres to replicate at each cell division. Progressive shortening of the telomeres is observed and the cell is unable to divide further. Exceptions: Germ cells, stem cells, and transformed cells, which often express the enzyme telomerase, which is capable of replicating the terminal sequences of DNA in the telomeres and extending life span of the cells, infinitely in the case of germ cells and some tumor cells. The Development of Continuous Cell Lines Some cell lines may give rise to continuous cell lines. The ability of a cell line to grow continuously probably reflects its capacity for genetic variation, allowing subsequent selection. Genetic variation often involves the deletion or mutation of the p53 gene, which would normally arrest cell cycle, if DNA were to become mutated, and overexpression of the telomerase. Human fibroblasts remain euploid throughout their life span in culture and never give rise to continuous cell line. Mouse fibroblasts and cell culture from a variety of human and animal tumors often give rise to continuous cultures. A common feature of many human continuous cell lines is the development of a subtetraploid chromosome number. The alteration in a culture that give rise to a continuous cell line is called in vitro transformation and may occur spontaneously, or be chemically or virally induced.  The Development of Continuous Cell Lines Continuous cell lines are usually aneuploid and often have a chromosome number between the diploid and tetraploid value. There is considerable variation in chromosome number and constitution among cells in the population (heteroploidiy)