Animal Cell Culture & Biology of Cultured Cells Lecture 1 PDF

Animal Cell Culture: Introduction Dr Mohd Nazmi Bin Abd Manap 29/03/2023 Dr Ismatul N.A. Ismail 1  Topics Outline ❑ What is Animal Cell Culture? ❑ Historical Background ❑ Ty...

Animal Cell Culture: Introduction Dr Mohd Nazmi Bin Abd Manap 29/03/2023 Dr Ismatul N.A. Ismail 1  Topics Outline ❑ What is Animal Cell Culture? ❑ Historical Background ❑ Types of Tissue Culture ❑ Advantages and Limitations of Animal Cell Culture ❑ Animal Cell Culture Applications 29/03/2023 Dr Ismatul N.A. Ismail 2  What is animal cell culture? ✓ The growth of tissues or cells separates from the organism. ✓ In vitro culture (maintain and/or proliferate) of cells, tissues or organs. ✓ This is typically facilitated via the use of a liquid, semi-solid, or solid growth medium (broth or agar). ❑ Animal cell culture is a process of growing animal cells in vitro, outside of their natural environment, under controlled conditions. 29/03/2023 Dr Ismatul N.A. Ismail 3 Historical Background 29/03/2023 Dr Ismatul N.A. Ismail 4  Three Types of Tissue Culture (A) Cell culture: The tissue, or outgrowth from the primary explant, is dispersed (mechanically or enzymatically) into a cell suspension. Then cultured as: 1. Adherent monolayer on a solid substrate (various cell types). 2. Suspension in the culture medium (few cell types). 29/03/2023 Dr Ismatul N.A. Ismail 5 (B) Primary explant* culture: Fragment of tissue is placed at a glass (or plastic)–liquid interface. After attachment, migration is promoted in the plane of the solid.substrate *Explant: living cells, tissues, or organs from animals or plants that transfer to a nutrient medium. 29/03/2023 Dr Ismatul N.A. Ismail 6 (C) Organ culture: The entire embryos or organs are excised from the body and culture. The architecture characteristic of the tissue in vivo is retained, at least in part, in the culture. The tissue is cultured at the liquid–gas interface (on a raft, grid, or gel), which favors the retention of a spherical or three-dimensional shape. 29/03/2023 Dr Ismatul N.A. Ismail 7  Cell Culture Morphology Morphologically cell cultures take one of two forms: 1. growing in suspension (as single cells or small free-floating clumps). cell lines derived from blood (leukaemia, lymphoma) 29/03/2023 Dr Ismatul N.A. Ismail 8 2. growing as a monolayer that is attached to the tissue culture flask. Cells from solid tissue (lungs, kidney, breast), endothelial, epithelial, neuronal, fibroblasts. Human breast cancer cells (MCF-7) HeLa-epithelial cells Human Fetal Lung Fibroblast Cells (MRC-5 Line) SHSY5Y-Neuronal Cells Endothelial cells 29/03/2023 Human kidney cells (HT-1080)Dr Ismatul N.A. Ismail 9  The immortal cells of Henrietta Lacks 29/03/2023 https://youtu.be/22lGbAVWhro Dr Ismatul N.A. Ismail 10  Advantages of Animal Cell Culture 1. Cell culture is a superior method in biotechnology. Allows for altering various physiological and physiobiological conditions like pH, temperature and osmotic pressure. 2. Enable studies related to cell metabolism and understand the biochemistry of cells. 3. Permits observation of the effects of various compounds like drugs and proteins on different cell types. 4. The results from animal cell cultures are consistent if a single cell type is used. 5. This technique facilitates the identification of various cell types on the basis of the appearance of markers like molecules or through karyotyping. 6. The usage of animal cells for testing and other purposes prevents the use of animals in experiments. 7. Can be used for the production of large quantities of proteins and antibodies, which would otherwise require a large investment. 29/03/2023 Dr Ismatul N.A. Ismail 11  Disadvantages of Animal Cell Culture 1. It is a specialized technique that requires trained personnel and aseptic conditions. 2. The technique is an expensive process as it requires costly equipment. 3. The subsequent subculture of the cell culture might result in differentiated properties as compared to the original strain. 4. The method produces a minuscule amount of recombinant proteins, which further increases the expenses of the process. 5. Contamination with mycoplasma and viral infection occur frequently and are difficult to detect and treat. 6. The cells produced by this technique lead to instability due to the occurrence of aneuploidy chromosomal constitution. 29/03/2023 Dr Ismatul N.A. Ismail 12  Cell Culture Application ❑ Excellent model systems for studying: 1. The normal physiology and biochemistry of cells. 2. The effects of drugs and toxic compounds on the cells. 3. Mutagenesis and carcinogenesis. ❑ Used in drug screening and development. ❑ Large-scale manufacturing of biological compounds (vaccines, insulin, interferon, other therapeutic proteins) ❑ Cancer Research: study the differences between cancer cells and normal cells ❑ The differences allow more detailed studies on carcinogenic substances’ potential causes and effects. ❑ Normal cells can be cultured to form cancer cells by the use of certain chemicals, viruses, and radiation. ❑ Cancer cells can also be used as test systems for studies related to the efficiencies of drugs and techniques used in cancer treatment. 29/03/2023 Dr Ismatul N.A. Ismail 13  An Introduction to Cell Culture https://youtu.be/RpDke-Sadzo 29/03/2023 Dr Ismatul N.A. Ismail 14  Is Lab-grown Meat is the Future of Meat? https://youtu.be/29GFYxI4tek 29/03/2023 Dr Ismatul N.A. Ismail 15  https://www.labxchange.org/library How To: Tissue Culture https://www.labxchange.org/library/items/lb:LabXchange:11840d21:video:1 29/03/2023 Dr Ismatul N.A. Ismail 16 Biology of Cultured Cells The culture environment Cell that is grown invitro favours spreading, migration and proliferation of unspecialised progenitor cells Cells growth will not favour expression of differentiated function Cell interaction are reduced as cell is lacking 3D scaffolding found in vivo and heterogeneity found in vivo Absent of normal stimuli and hormones found in vivo. Therefore cell does not express the correct phenotype as cells microenvironment has changed. The culture environment The influence of the environment on the culture is expressed through this route: Nature of substrate Degree of contact with other cells Medium’s physicochemical and physiological constitution Constitution of gas phase Incubation temperature Cell adhesion Most cells from solid tissues will form/grown as adherent monolayer Cells adhesion is mediated by specific cell surface receptors for molecular in extracellular matricx (ECM). Spreading of cells start with secretion of ECM matrix protein and proteoglycan. Matrix adheres to charged substrate and cells bind to matrix via receptors Glass material – slight negative charge Plastic is treated with an electric ion discharge Cell adhesion Glass or plastic that has been conditioned by previous cell growth provide a better surface for attachment. Substrates pretreated with matrix constituents, such as fibronectin or collagen, or derivatives such as gelatin, will help more fastidious cells attach and proliferate. For fibroblast-like cells, the main requirement is for substrate attachment and cell spreading; usually the cells migrate individually at low densities. Epithelial cells may also require cell–cell adhesion for optimum survival and growth, and consequently they tend to grow in patches.  Major classes of transmembrane proteins: Cadherin (Ca2+ dependent)- interactions between homologous cells via adherens junction or desmosomes Cell CAMs – (Ca2+independent) cell-cell adhesion molecules – interacts with integrins in immunological synapse adhesion Integrins – Mediate cell-matrix adhesion, interact with molecules such as fibronectin, molecules entactin, laminin, and collagen that bind to them via a specific motif usually containing the arginine-glycine-aspartic acid (RGD) Transmembrane proteoglycans- interact with matrix constituents such as other proteoglycan or collagen not via RGD motif.  Cell adhesion Diagrammatic representation of a layer of epithelial cells above connective tissue containing fibrocytes and separated from it by a basal lamina. CAMs and cadherins are depicted between like cells, and integrins and proteoglycans between the epithelial layer and the matrix of the basal lamina. Cytoskeleton Cell adhesion molecules basically attach to cytoskeleton Attachment of integrins to actin microfilament via linker protein is linked to reciprocal signalling between the cell surface and the nucleus Cadherin molecules also linked the actin cytoskeleton in adherens junctions – mediating changes in the cell shape Intercellular Junctions Adhesion molecules are arranged in plasma membrane of adjacent cells while others are organised in the intercellular junctions Desmosomes – hold epithelial cells together (anchoring junctions) Tight junctions – seal the space between cells and prevent leakage of extracellular fluid Gap junctions – allow ions, nutrient and small signalling molecules to pass (communicating junctions) Extracellular Matrix (ECM) Intercellular spaces are filled with ECM ECM constituents is determined by the type of cell. Example: fibrocytes secrete type I collagen and fibronectin into the matrix, whereas epithelial cells produce laminin. ECM is a significant component in the phenotypic expression of the cells. Mostly cultured cell lines are allowed to generate their own ECM, but primary culture and propagation of some specialized cells, and the induction of their differentiation, may require exogenous provision of ECM.  Cell motility Most cultured cell are capable of movement. Most motile are fibroblast at a low cell density when the cells are not in contact. The least motile are dense epithelial monolayers. Examples shown the ability of cells to stretch and subsequently ‘slingshot’ forward. Evolution of cell line Growth characteristic of the Primary cell culture: Lag phase – no significant growth. Log phase – cell grows exponentially. Plateau phase – cell growth reach plateau; limited by the medium, cells confluency and cell reach senecensce. Evolution of Cells lines Maximum cells can be passage approx. 10 passages. Senescence follows after: Due to shortening of telomeres Transformation of cells is required to continues cell division. What is senescense? Refers to a state of permanent cell growth arrest that occurs when cells undergo a finite number of divisions. This phenomenon was first described in the 1960s by Leonard Hayflick, who observed that normal human cells could only divide a limited number of times before entering a senescence state (Hayflick limit). At each DNA replication – the telomere region will be shortened Progressive shortening of telomeres region leading to cells unable to divide. Initiation of cell lines Primary Culture - culture derived from main tissue. Cells capable to proliferate will increase in number Some cells are only capable to survive but doesn’t proliferate Some cells will die Certain aspects of cells function are best study when it reach confluency as it mimic the morphological resemblance of its origin (in vivo) Initiation of cell lines Cell Line - culture derived from primary culture (1st subculture/passage). This cell line may be further propagated and subcultured for several time. In each subculture, population with the ability to proliferate most rapidly will gradually predominate, and non-proliferating or slowly proliferating cells will be diluted out. This give rise to many types of cell lines. Ex: MRC-5 human embryonic lung fibroblast Subculture - Continuous cultures or passage of cell cultures derived from cell line. Continuous cell lines Finite cell lines could change to continuous cell lines Often by p53 gene mutation or deletion Overexpression of telomerase Transformation vs Immortalization Transformation is additional changes in growth characteristics Immortalization – infinite lifespan Aneuploidy is a characteristic of continuous cells line Usually chromosomes number is in between diploid and tetraploid value Heteroploidy is also observed (considerable variation in chromosomes number) Most cells does not become continuous cell lines  Continuous cell lines Characteristics Reduced serum requirement. Reduced density limitation of growth. Ability to grow in semisolid media, and aneuploidy. All traits above are associated with malignant transformations. Similar morphological and behavioral changes can also be observed in cells that have undergone virally or chemically induced transformation. Understanding cell proliferation G1 phase (growth 1 phase)—cells induce growth by first making RNA and proteins, DNA content does not change S phase (synthesis phase)—DNA synthesis proceeds with DNA replication and cells have variable amounts of DNA G2 phase (growth 2 phase)—have completed DNA synthesis, and continue to grow and prepare for mitosis with DNA maintained at double the original amount M phase (mitosis)—nuclear division and cytoplasmic division giving rise to two daughter cells Checkpoint in cell cycle G1 checkpoint - cell is susceptible to control of cell cycle progression at a number of restriction points, cell either progress towards DNA synthesis & another division cycle or exits the cell cylcle G2 checkpoint - determines integrity of DNA. Cell cycle will be halted to allow DNA repair or entry into apoptosis (program cell death) if it is irreparable  Cell cycle progresses by Cdks (cell division How cell cycle kinases) with presence inhibitors regulate cell of mitogen proliferation Through Rb (retinoblastoma) gene product and p53 Cell cycle arrested in G1 Blocking cell cycle progression by Rb (no mitogens) at checkpoint Damage on these gene leads to cancerous cells Inactivation is possible by phosphorylation Cell cycle arrested by p53 if there is DNA damage. Intracellular control of cell proliferation Positive acting factors (Let cell cycle progress) Cyclins - cyclins activated by cell division cycle kinases (Cdks), Negative acting factors (block cycle progression at checkpoint) Rb gene product P53 Differentiation vs Proliferation Regulation of cell proliferation Growth environment regulates normal cells entry into the ‘cell cycle’ Growth factors added externally could promote cell proliferation: Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), Fibroblast Growth Factors (FGF) - (+ve) Transforming growth factor (TGF) - (-ve) High confluency (dense cell) also inhibits cells proliferation (inhibition by contact) Internal control of cell proliferation: (+ve) – cyclins, Growth Factor Receptor Activation (-ve) – p53, Rb, Checkpoints Cell Proliferation VS Cell Differentiation Normal culture conditions (low cell density, mitogens in the medium) will favor cell proliferation High cell density and addition of differentiation factors will induce differentiation. The position of the equilibrium will depend on culture conditions. Proliferation does not promote Differentiation Dedifferentiation Inability of cell lines to express the characteristic in vivo Factors leading to dedifferentiation: Inability of cell lines to differentiate Wrong lineage of cells is selected in vitro Undifferentiated cells of same lineage overgrow terminally differentiated cells of reduced proliferative capacity Absence of inducers (hormones, cell or matrix interaction) lead to reversible loss of differentiatd properties Dedifferentiation vs Deadaptation Dedifferentiation Specialized properties of the cell are lost by conversion to a more primitive phenotype. (irreversible) Example a hepatocyte would lose its characteristic enzymes (arginase, aminotransferases, etc.) and could not store glycogen or secrete serum proteins, because of reversion or conversion to a precursor cell. Deadaptation Environmental growth condition suppresses phenotype (reversible) Synthesis of specific product can be reinduced if the correct condition can be recreated. Tissue Retains function longer 3-Dimensional tissue could retains its properties longer but could not be propagated Ways to overcome this: Cells are cultured on matrices Matrigel is commercially available Not perfect but could offer great results Potential in generating heterotypic cultures Offer possibility to study pathological behaviour  Also known as cell communication or signal transduction, is the process by which cells communicate with each other to coordinate their activities and respond to changes in their environment Cell proliferation, migration, differentiation, and apoptosis in vivo are regulated by cell–cell Cell interaction, cell–matrix interaction, and nutritional and hormonal signals Signaling Some is contact mediated via cell adhesion molecule Invitro condition – only autocrine (cell signaling within the same cell) signaling works In vivo condition – all works including paracrine (signal arise from different cell type) and endocrine (signal arise from endocrine glands)