Introduction to tissue culture-Chapter 1.pdf

Animal Tissue Culture What is cell/tissue culture? Propagation of cells outside the organism Tissue culture involves both plant and animal cells Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture) Cell culture is...

Animal Tissue Culture What is cell/tissue culture? Propagation of cells outside the organism Tissue culture involves both plant and animal cells Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture) Cell culture is the technique of growing and maintaining the cells of multicellular organisms outside of the organism in specially designed containers, which are located in conditions which attempt to mimic the precise environmental conditions such as temperature, humidity, nutrition, and contamination-free conditions that were present in that organism. “Tissue culture can be a powerful technique if conducted properly and a great waste of time and money when done sloppily” Bernice Martin, Ph.D.  History of Cell culture 1801 Bichat names the tissues of the body 1880 Roux maintains medullary plate of embryonic chicken in saline for a few days 1907 Harrison demonstrated the in vitro growth of living animal tissue – Explant of nerve cord from a tadpole in a frog lymph – Sprouting of axons 1911-1912 Burrows and Carrel grow explants from adult animals and malignant tissues – Aseptic techniques – Carrel Flask Carrel begins a cell line from a heart of chicken embryo – Grown later by Albert Ebeling for 34 years 1914 Thompson begins to experiment with organ culture, explanting toes, feather germs, optic lens from embryonic chicks Ebeling succeeds in culturing epithelial cells Parker and Nye inoculate fragments of rabbit testes with vaccinia virus and cultivate them in plasma 1940/1950’s polio epidemics Need for polio vaccine 1948 Enders, Weller and Robbins prove that poliomyelitis virus can be grown in vitro without nerve tissue – Primary monkey kidney cells – Human diploid lung fibroblast 1952 George Gey established HeLa cell line from human cervical carcinoma (the first human cell line) 1955 chemically defined media (Eagle, Earle) and attachment factors – Consistency – Easy sterilization – Reduced chance of contamination 1958 Harriss induces hybridization of cells on a large scale including different species and different types of cells from the same species 1961 Hayflick and Moorhead described the finite life span of normal human diploid cells 1970s – recombinant DNA technology Growing proteins in E-coli – No post-translational modifications Glycosylation Folding Excretion vs inclusion bodies – Large complex proteins require animal cells Recent Serum-free media Genetic modifications in cells Bioreactors Large-scale (20 m3) cultures for commercial applications  Terminology Organ Culture A three dimensional culture of undisaggregated tissue retaining some or all of the features of the tissue in vivo Cell Culture Single cells, no longer organised as tissues. Derived from dispersed cells taken from the original tissue Primary Cell Culture Derived from an explant, directly from the animal Usually only survive for a finite period of time Involves enzymatic and/or mechanical disruption of the tissue and some selection steps to isolate the cells of interest from a heterogeneous population  Terminology Clone A population derived from a single cell Sub-culture Transplantation of cells from one vessel to another Established or Continuous Cell Lines A primary culture that has become immortal due to some transformation Most commonly tumour derived, or transformed with a virus such as Epstein-Barr One of the most commonly used cells are Chinese Hamster Ovary cells (CHO) The SH-SY-5Y cells a human neuroblastoma derived cell line Passage Number Number of successive sub-cultures from primary culture  Why culture cells in vitro? Research – Understand function of cells – Toxicology Tissue engineering – Skin, Liver – Bone and Cartilage Production of biologicals (pharmaceutical proteins) – Monoclonal antibodies – Hormones (FSH) – Enzymes (α-glucosidase) – Vaccines (Polio) Tissue Culture Applications  Culture Environment Substrate: Can be: solid, semisolid or liquid. solid: glass, plastic (treated polystyrene); coat plastic with extra cellular matrix (ECM) components e.g. collagen, cells usually grown in monolayers, then passaged and expanded. semisolid agar/ECM based e.g. three dimensional matrices: collagen gel cellulose-sponge; aids e.g. tube formation. Liquid: simply suspension cultures. Feeder layers - layers of e.g. mouse embryo fibroblasts, which are treated (irradiation, or chemically) to stop them from proliferation to form the basis on which other cells such as ES cells (embryonic stem cells) can be cultured. Gas phase Air and added carbon dioxide (usually 5%, dependent on NaHCO3 level in the medium) Oxygen - usually atmospheric tension but: some organ cultures require increased levels i.e. 95% O2, others grow better with reduced oxygen levels (i.e. chondrocytes, some neurons). Carbon dioxide level: for bicarbonate buffered media, CO2 tension regulates pH. Media The choice of media is cell type specific and often empirical - no "all purpose" medium. Should provide: nutrients, buffering capacity, isotonic, and be sterile  Advantages of Tissue Culture ⚫ Study of cell behaviour without the variations that occur in animal ⚫ Control of the growth environment leads to uniformity of sample ⚫ Characteristics of cells can be maintained over several generations, leading to good reproducibility between experiments ⚫ Cultures can be exposed to reagents e.g. radio-chemicals or drugs at defined concentrations ⚫ Large quantities of cells can be obtained ⚫ Finally it avoids the legal, moral and ethical problems of animal experimentation  Advantages of Tissue Culture Category Advantages Physico-chemical Control of pH, temperature, osmolarity, dissolved Environment gases Physiological conditions Control of hormone and nutrient concentration Microenvironment Regulation of matrix, cell-cell interaction, gaseous diffusion Cell line homogeneity Availability of selective media, cloning Characterization Cytology and immunostaining easily performed Preservation Can be stored in liquid nitrogen Validation and Origin, history, purity can be recorded accreditation Replicates and variability Easy quantitation Reagents saving Reduced volumes, direct access, lower cost Control of C X T Ability to define dose, concentration, and time Mechanization Available with microtitration and robotics Reduction of animal use Cytotoxicity and screening of pharmaceutics, cosmetics, etc.  Shortcomings Not at all like an animal – In cell cultures cells are no longer organized into tissues – Monolayer is nothing like a 3D tissue Environment is not like in vivo – Extra cellular matrix (ECM) scaffolding Effects of serum, plasma, etc.  Disadvantages Have to develop standardised techniques in order to maintain healthy reproducible cells for experiments Takes time to learn aseptic technique Quantity of material is limited Dedifferentiation and selection can occur and many of the original cellular mechanisms can be lost  Limitations of Cell Culture - Cell culture is referred to as an ex vivo study of the cellular milieu. This is a problem because the cell is not in its normal physiological and original environment. - Cell culture is simply an attempt to provide a simulated environment. "The study of cells in cell culture is akin to studying polar bear habits at the local zoo." - Also a problem in cell culture is the usage of cells which are transformed. HepG2 cells for example are derived from a liver cancer cell line from humans, and thus are a hepatocarcinoma cell line. The disadvantage of this is these cells often differ markedly from normal cells in that they have altered or a loss of their specific cell function due to mutations. - Alternatives to using transformed cells is Primary cell culture, which is the culture of primary cells, which are untransformed, normal cells isolated recently from an animal by tissue culture.  Limitations of Cell Culture Category Examples Necessary expertise Handling Chemical contamination Microbial contamination Environmental control Workplace Incubation, pH control Containments and disposal of biohazards Quantity and cost Capital equipment Consumables Medium, serum, plastics Genetic instability Heterogeneity, variability Phenotypic instability Dedifferentiation Adaptation Selection Identification of cell type Expression of markers Histology, cytology Geometry and microenvironment  Missing Features in Cell Culture - Cell culture is missing the original blood circulation. - Cell culture often is missing the original tissue organization and structure. - Factors in the blood such as hormones are often missing. - Cells are often not always and entirely in contact with other cells, as cultures are never left to 100% confluency. This is a problem as normal cells (primary cells ) need cell to cell contacts. Transformed cells usually can grow and divide quite well in the absence of cell-cell contacts. - Also of note is hormones are usually added at very high concentrations to treat cells and are not usually physiological.  Safety in Cell Culture Substances Hazardous to Health Carcinogen A substance that can cause Cancer Teratogen A substance that can cause damage to the developing Foetus Mutagen A substance that can cause a mutation in the genetic material that can be passed to the next generation Gentamycin and Thapsigargin Possible Teratogens Hygromycin Possible Carcinogen Streptomycin Mutagen Types of Tissue Culture  Isolation of cells Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood, however only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture. Cells that are cultured directly from an animal or person are known as primary cells. With the exception of some derived from tumours, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence (Cellular senescence is the phenomenon where normal diploid differentiated cells lose the ability to divide) and stop dividing, while generally retaining viability. An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types.  Properties of Different Types of Culture Category Organ culture Explant Cell culture Source Embryonic organs, Tissue fragment Disaggregated tissue primary adult tissue fragments culture, propagated cell line Effort High Moderate Low Characterization Easy, Histology Cytology and Markers Biochemical, molecular, immunological, and cytological assays Histology Informative Difficult Not applicable Biochemical Possible Difficult Not applicable differentiation Propagation Not possible Possible Standard procedure Replicate sampling, High intersample High intersample variation Low level of variation reproducibility, variation homogeneity Quantitation Difficult Difficult Easy, many techniques available  Subculture Advantages Disatvantages Propagation Trauma of enzymatic or mechanical disaggregation More cells Selection of cells adapted to culture Increased Homogeneity Genetic instability Characterization of Loss of differentiated properties replicate samples (may be inducible) Frozen storage