Animal Experiments in Medicine. Alternative methods In vitro techniques: Cell and tissue cultures. Gabriella Varga - PDF

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Animal Experiments in Medicine Alternative methods In vitro techniques: Cell and tissue cultures Gabriella Varga HURO/0901/069/ th October General background The general public by and
Animal Experiments in Medicine Alternative methods In vitro techniques: Cell and tissue cultures Gabriella Varga HURO/0901/069/ th October 1. General background The general public by and large accepts the experimental use of animals as necessary, but would like to eliminate any pain or distress in experiments involving living animals. Besides, there is a steady demand to replace animals in research by in vitro tests. Scientists too emphasize the need to reduce the pain and discomfort associated with experimental procedures, but understand that the replacement of in vivo experiments by in vitro methods is not an adequate solution. Some animal rights activists view the term alternative in the context of replacing all animal use with non-animal alternatives and fight to stop the use of animals in scientific experiments. Many of them are not experts in this field and do not know whether the alternatives are adequate or not to give answers to the questions arising in scientific research. There are questions that can be answered by in vitro experiments (for example, by using cell cultures), but these can not cover the whole area of scientific research. Working with living animals means the examination of questions in their complexity; in vitro experiments are able to answer only one special aspect of a question. Clearly, the method of cell cultivation is very useful in scientific research as certain questions can not be examined via in vivo models. Additionally, cell cultivation is emerging technology in many fields ( e.g. gene technology) and thus it is worthwhile to become acquainted with it, at least at a basic level. 2. In vitro procedures 2.1. Procedures that may be used for the replacement of live animals in research Biochemical tests and immunochemical techniques ( e.g. for the identification of bacterial toxins) Organ, tissue or cell cultures (e.g. for biochemical research) Microorganisms (to screen compounds for carcinogenicity and /or mutagenicity) Computer simulation and computer-based relationship models 2.2. Procedures that have been used to replace animals Monoclonal antibody production (see below) Pregnancy testing Vaccine potency test Virus vaccine production A hybridoma is a cell hybrid resulting from the fusion of a cancer cell (usually a myeloma or lymphoma) and a normal cell (lymphocyte) in order to combine de sired features of each, such as the ability of the cancer cell to multiply rapidly with the ability of a normal cell to dictate the production of a specific antibody. The hybridoma is immortal in the laboratory and makes the same products as its parent cells forever. The demand for hybridoma cell lines expressing highly specific monoclonal antibodies (MAbs) has increased dramatically in recent years due to the increased needs for MAbs used in diagnostic assays and. as novel therapeutic agents. These hybridoma cell lines are replacement alternatives for the production of most MAbs. 2 3. A brief history of tissue culture 1907 Ross Harrison described the technique of tissue culture, Alexis Carrel and Montrose Burrows later modified Harrison's technique The American Tissue Culture Association was founded at a conference in Hershey, Pennsylvania 1949 G.W. Hyatt created the US Navy Tissue Bank to store bone tissue collected during orthopedic surgery The first human tumor cell line, HeLa, was established from the cancerous cervical cells of Henrietta Lacks Leonard Hayflick created the first normal human diploid cell line Ananda Chakrabarty genetically engineered a strain of bacteria that could digest crude oil. 4. Tissue and cell cultures Cell and tissue culture is a technique by which cells are removed from a plant or animal organism and grown under controlled conditions in a sterile medium containing all the necessary nutrients. Cell culture is also an established global manufacturing technique in the biotechnology industry. 1-3 Cell cultures are usually used to screen compounds for carcinogenicity and/or mutagenicity; for the analysis of the cells themselves; to examine cell to cell communication; for an assessment of the cell's response to chemicals; and as a tool to produce cellular-derived protein products (biotechnology industry) 4.1. Cell/tissue culture terms Cell culture: The maintenance of dispersed cells, derived from primary tissue explants or cell suspensions. Tissue culture: The maintenance of a tissue in a way that may allow differentiation and preservation of the architecture and/or function. Monolayer: A single layer of cells growing on a surface. Subculture: The passaging of cells from one culture to another. Primary culture: A culture started from cells, tissues or organs taken directly from an animal. Organ culture: The maintenance of tissues, whole organs or parts of organs in a manner that may allow differentiation and preservation of the architecture and/or function. Explant culture: An excised fragment of an organ which usually retains some degree of tissue architecture. Cell line: This arises from the primary culture at the time of the first subculture it has a finite lifespan. Continuous cell line: A cell line which has been transformed It has an infinite lifespan Types of cell cultures Primary cell cultures can be generated from embryonic or adult tissue, typically have a finite lifespan in culture. The advantage of primary cultures is that the cells have not 3 been modified in any way (other than enzymatic or physical dissociation). The disadvantages of primary cultures are the mixed nature of each preparation, and the limited lifespan of the culture. Continuous cell lines are abnormal and are often transformed cell lines 5. Methods and conditions of cell cultivation Laminar flow hoods All media preparation and other cell culture work must be performed in a laminar flow hood. A vertical hood (biology safety cabinet) is best for working with hazardo us organisms. The filtered air blows vertically down from the top of the cabinet. In horizontal hoods, the filtered air blows out at the operator in a horizontal fashion. These are not useful for working with hazardous organisms, but offer the best protection for cultures. Both types of hoods involve the continuous displacement of air that passes through a HEPA (high-efficiency particle) filter that removes particulate matter from the air. The hoods are equipped with a short-wave UV light. CO 2 incubators Cells are grown in an atmosphere of 5-10% CO 2 because the medium used is buffered with sodium bicarbonate/carbonic acid and the ph must be strictly maintained in the physiological range, ph= The humidity must be maintained at about 100 % for cells growing in tissue culture dishes. Culture flasks should have loosened caps to allow for sufficient gas exchange. 1,2 Microscopes Inverted phase contrast microscopes are used to visualize cells Methods to prepare cell cultures 1. Preparation of solutions used for cell culture procedures. 2. Preparation of tissue for cell dissociation. 3. Dissociation of cells: Mechanical dissociation: disaggregation of cells by aspirating tissue through a 10 ml syringe equipped with a needle of appropriate width. Gentle forcing of cell clumps through the needle into the syringe (trituration) without any enzymatic treatment. Enzymatic dissociation: enzymatic digestion by collagenase, trypsin, trypsin- EDTA, dispase or protease treatment. 4. Filtration of cell suspension through a sterile nylon mesh to separate dispersed cells from the larger tissue pieces. 5. Washing of cells and centrifugation. 6. Resuspension and plating of cells. 7. Viable cell counts: a hemocytometer or a common microscope is used to determine total cell counts and viable cell numbers. Trypan blue is one of several stains recommended for use for viable cell counting in dye exclusion procedures. This method is based on the principle that live cells do not take up certain dyes, whereas dead cells do. Cells should be monitored daily for morphology and growth characteristics, fed every 2 to 3 days, and subcultured when necessary. 4 5.3. Cell attachment factors, cell adhesion molecules These compounds are used to promote cell adhesion: Collagen Fibronectins (cell surface and plasma proteins) Laminin (heteromeric glycoprotein) Poly L-lysine (polycationic form of the polyamino acid in the range 70, ,000 kda). Poly-L-Ornithin (polycationic form of the polyamino acid with MW: 30,000-70,000) Cell culture supplements Certain compounds are used for media supplementation: Fetal calf serum (FCS) is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth. FCS is the best supplementation for a basal medium, that is most frequently used for all types of cell cultures. Growth factors are naturally-occurring proteins, members of larger families of structurally and evolutionarily related proteins, that promote cell proliferation and cell differentiation The individual growth factor proteins are important for the regulation of a variety of cellular processes, acting as signaling molecules between cells ( e.g. epidermal growth factor (EGF), basic fibroblast growth factor (bfgf or FGF2), nerve growth factor (NGF), neurotrophins, erythropoietin (EPO), cytokines and hormones). Insulin Transferrin Serum albumin 6. Cell culture media types and their uses 6.1. Basic constituents of media are inorganic salts, carbohydrates, amino acids, vitamins, fatty acids and lipids, proteins and peptides and serum. Each type of constituent performs a specific function Cell culture media types and their uses (Table 1.) Media Examples Uses type Balanced salt solutions PBS DPBS Hanks BSS They form the basis of many complex media Basal media Earles BSS MEM DMEM Primary and diploid cultures Modification of MEM containing increased levels of amino acids and vitamins. Supports a wide range of cell types, including hybridomas 5 Complex media Serum Free Media Insect cells GMEM Glasgow s modified MEM was defined for BHK-21 cells. The medium was developed by modifying Eagle's BME by adding 10% tryptase phosphate and twice the normal concentrations of amino acids and vitamins. RPMI 1640 Originally derived for human leukemic cells. It supports a wide range of mammalian cells, including hybridomas Iscoves DMEM A further enriched modification of DMEM which supports high-density growth Leibovitz L-15 Designed for CO 2 -free environments TC 100 Designed for culturing insect cells Grace's Insect Medium Schneider's Insect Medium CHO For use in serum-free applications Ham F10 and derivatives Ham F12 DMEM/F12 Sf-900 II SFM, SF Insect- Medium-2 NOTE: These media must be supplemented with other factors such as insulin, transferrin and EGF. These media are usually HEPES buffered Specifically designed for use with Sf9 insect cells The Minimum Essential Medium (MEM), developed by Harry Eagle, is one of the m ost widely used of all synthetic cell culture media. MEM has been used for the cultivation of a wide variety of mammalian cells grown in monolayers. Figure 1 shows the components of the most frequently used cell culture media Dulbecco s Modified Eagle Medium (DMEM). Dulbecco s Modified Eagle Medium (DMEM component mg/l inorganic salts CaCl 2 *2H 2 O 264,00 Fe(NO 3 )3*9H 2 O 0,10 KCl 400,00 MgSO 4 *7H 2 O 200,00 NaCl 6400,00 6 NaHCO ,00 NaH 2 PO 4 *2H 2 O 141,00 other components D-Glucose 1000,00 Sodium-Pyruvate 110,00 amino acids L-Arginine*HCl 84,00 L-Cystine 48,00 Glycine 30,00 L-Histidine-HCl*H 2 O 42,00 L-Isoleucine 105,00 L-Leucine 105,00 L-Lysine-HCl 146,00 L-Methionine 30,00 L-Phenylalanine 66,00 L-Serine 42,00 L-Threonine 95,00 L-Tryptophane 16,00 L-Tyrosine 72,00 L-Valine 94,00 vitamins D-Ca-Pantothenate 4,00 Choline Chloride 4,00 Folic Acid 4,00 Inositol 7,20 Niacinamide 4,00 Pyridoxine-HCl 4,00 Riboflavin 0,40 Thiamine-HCl 4,00 Figure 1. The components of Dulbecco s Modified Eagle Medium (DMEM). 7 7. Culture dishes Figure 2. Cell culture flasks 8 Figure 3. Petri dishes 9 Figure 4. Multiwell dishes 10 8. Subculture method Proteolytic enzymes, trypsin, collagenase or pronase, usually in combination with EDTA, cause cells to detach from the growth surface. The enzymatic digestion is fast and reliable, but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum. 1,3 The steps of the subculture method are as follows: 1. Preparation of a trypsin - EDTA solution in a balanced salt solution (e. g. PBS without Ca ++ or Mg ++ ). 2. Removal of the medium from the culture dish by aspiration, washing of the cells in a monolayer in a balanced salt solution (without Ca ++ or Mg ++ ) to remove all traces of serum, and removal of the wash solution. 3. Addition of sufficient trypsin-edta solution in appropriate concentration to completely cover the cell monolayer. 4. Transfer of the culture to a 37 o C incubator for 2 min. 5. The coated cells are allowed to incubate until cells detach from the surface. 6. Monitoring of the cells under a microscope. 7. Progress can be checked by examination with an inverted microscope 8. The cells begin to detach when they appear rounded. 9. Dilution of the cells with serum, or with serum containing fresh medium and transfer to a sterile centrifuge tube. 10. Spinning of the cells, removal of the supernatant, and resuspension in culture medium (or freezing medium if the cells are to be frozen). 11. Addition of culture medium containing serum, and dilution into culture flasks or other culture vessels. Typically, 1:4 to 1:20 dilutions are appropriate for most cell lines. 9. Standard procedure for detaching adherent cells 1. Washing once with a buffer solution. 2. Release of cells from monolayer, surface treatment with dissociating agent, and observation of the cells under a microscope. 3. Incubation until the cells become rounded and loosen. 4. Transfer of the cells to a centrifuge tube and dilution with medium containing serum. 5. Spinning down of the cells, removal of the supernatant and replacement with fresh medium. 6. Counting of the cells in a hemocytometer, and dilution as appropriate into fresh medium. 10. Preservation and storage Freezing cells Harvesting of the cells as usual and washing once with complete medium. 2. Resuspension of the cells in complete medium and determination of the cell count/viability. 3. Centrifugation and resuspension in ice-cold freezing medium: 90% calf serum/10% DMSO, at cells/ml. maintenance of the cells on ice. Note: A cryoprotective agent such as glycerol or DMSO lowers the freezing point. It is best to use healthy cells that are growing in the log phase. 11 4. Transfer of 1 ml aliquots to freezer vials on ice. 5. Transfer to the -80 o C freezer overnight. Note: the cells are slowly cooled from room temperature to -80 o C to allow the water to move out of the cells before it freezes. The optimal rate of cooling is 1-3 o C per min. 6. Next day, they are transferred to liquid nitrogen, either in the liquid phase (-196 o C) or in the vapor phase (-156 o C) Thawing of frozen cells 1. Removal of the cells from frozen storage and quick thawing in a 37 o C water bath by gentle agitation of the vial. 2. As soon as the ice crystals have melted, gently pipetting into a centrifuge tube containing prewarmed growth medium ( ml complete growth medium per 1 ml frozen cells). 3. Pelleting of the cells by gentle centrifugation and discarding of the supernatant to remove cryopreservative (cryopreserved cells are fragile). 4. Careful resuspension of the cells in complete growth medium, followed by a viable cell count. 5. Plating of the cells. The cell inoculum should contain at least 3 x 10 5 viable cells/ml. 11. Production of artificial tissue ( tissue engineering ) There has been an enormous revolution in the biological sciences in the past twenty years, in the course of which a new area has emerged in biotechnology: namely human tissue engineering, a multidisciplinary field. Tissue engineering combines various aspects of medicine, cell and molecular biology, material sciences and engineering, for the purpose of developing tissue substitutes to regenerate, maintain or improve the function of damaged human tissues. Biotechnology engineering involves a uniquely interdisciplinary melding of engineering and medicine The history of tissue engineering The first definition of tissue engineering was given by Langer and Vacanti 4, who stated it to be an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ . MacArthur and Oreff6 7 defined it as understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use. 1. Tissue engineering (regenerative medicine) involves the repair or replacement of structural tissues (e.g. bone, cartilage, blood vessels, bladder, etc.). Tissue engineering uses living cells as engineering materials; it could be artificial skin which includes living fibroblasts, cartilage repaired with living chondrocytes, or other types of cells used in other ways. 2. Tissue transplantation (stem cells) is the transplantation of cells that perform a specific biochemical function (e.g. an artificial pancreas, or an artificial liver). 3. Biological engineering is a broader field that generally encompasses tissue engineering and related fields (e.g. biomaterials). 12 Cells became available as engineering materials when it was discovered in 1998 how to extend telomeres to produce an immortalized cell line. Before this, laboratory cultures of healthy, noncancerous mammalian cells would only divide a fixed number of times, up to the Hayflick limit. Leonard Hayflick observed in 1965 that cultured cells divide about 50 times before dying. Near to this limit cells show signs of old age (exceptions: stem cells and cancerous cells). The limit of the cell division number varies from cell type to cell type and from organism to organism. The human limit is about 52 and has been linked to the shortening of telomeres, a region of DNA at the end of the chromosomes. The production of engineered tissues is an emerging field which holds promise for the improvement of current medical therapies. Tissue engineering involves producing a 3D biocompatible scaffold with the proper amount of cells to implant, and then implanting the engineered tissue material in vivo. 6, Cell sources for tissue engineering Autologous cells are obtained from the same individual into which they will be reimplanted. Autologous cells give the fewest problems with rejection and pathogen transmission. Allogenic cells originate from a donor of the same species. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models. Primary cells are from an organism. Secondary cells are from a cell bank or from multipassaged primary cells. Xenogenic cells are those isolated from individuals of another species. In experiments aimed at the construction of cardiovascular implants, animal (pig) cells have extensively been used. Stem cells are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. Depending on their source, stem cells are divided into adult and embryonic stem cells. The first class being multipotent and the latter mostly pluripotent; some cells are totipotent, in the earliest stages of the embryo. Stem cells may be a promising tool for the repair of diseased or damaged tissues, or may be used to grow new organs. 6, The scaffolding technique Cells can generally be implanted or seeded into an artificial structure (usually referred to a scaffold) capable of supporting 3D tissue formation and serving at least one of the following purposes: cell at
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