Methodical recommendations for the special workshop "Cell culture and histogenesis"

Educational and Research Center "Institute of Biology" of Taras Shevchenko National University of Kyiv Liudmyla GARMANCHUK METHODOLOGICAL RECOMMENDATIONS FOR THE SPECIAL WORKSHOP "CELL CULTURE AND HISTOGENESIS" Kyiv - 2023 2 Methodical recommendations for the special workshop "Cell culture and histogenesis" / compiled by: Doctor of Biological Sciences, Professor of the Department of Ecology and Zoologie L.V.Garmanchuk - Kyiv: 2023 – 75 p. For students of medicine, laboratory diagnostic, biochemistry, scientists who want to acquire practical and theoretical skills in culturing cells of higher eukaryotes. Reviewers: I.V., Bielinska, Doctor of Biological Sciences, Docent L.D., Lyubich Doctor of Biological Sciences, Research Science Approved for publication at the meeting of the Academic Council of the Educational and Scientific Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv (Protocol №5 from 20.04.2023 ) Garmanchuk L.V., 2023 3 The main purpose of the methodological recommendations is to provide students with the opportunity to get acquainted with the methods of cell culture of higher eukaryotes, which are used at the present stage to acquire practical skills and gain new knowledge in conducting research in the field of Laboratory diagnostic, biochemistry, molecular and cell biology. INTRODUCTIO N The method of cell culture (method of "explantation" of cells) is one of the key methods of cell biology, which studies the features of proliferation, differentiation, migration, metabolic activity, and biochemical and molecular biological mechanisms of aging and death of a single cell and a population of genetically identical cells, for which all their properties are maintained in artificially created conditions in compliance with all the necessary rules of asepsis. The technique of tissue culture was first proposed at the beginning of the last century [Harrison, 1907; Carel, 1912] to study the properties of animal cells free from the influence of the systemic microenvironment, characteristic of multicellular organisms both in normal and pathological conditions. In the initial stages, this technology was based on working with undispersed tissues. The first cloned strain was L929, which was obtained from mouse L-cells by capillary method [Sanford et al., 1948]. In the 1950s, for the analysis of viral plaques, Dulbecco [Dulbecco, 1952] described a method of obtaining monolayer cultures using trypsin, which is now widely used in the practice of cell cultivation. The first permanent human cell line HeLa was obtained and cloned by a group of authors [Gey et al., 1952, Puck and Marcus, 1955] using an irradiated cell feeder layer. In the 50s of the last century, there was a boom in the development of cell technologies: the development of rules for working with cell cultures, the establishment of technologies for the production of the necessary equipment (laminar boxes, carbon dioxide incubators, inverted microscopes, devices for cryopreservation of cells), as well as artificial media with growth-stimulating factors and sterile plastic dishes for cell incubation. 5 The most significant achievements obtained based on cultured cells are hybridoma technology - the production of monoclonal antibodies and their use for the diagnosis and treatment of the most common diseases of various etiologies, artificial insemination and reproductive medicine technologies, technologies related to the production and use of stem cells. Thus, cell cultures can be used in the following studies: a study of intracellular processes - DNA replication and transcription, protein synthesis and energy metabolism, drug metabolism; study of intracellular and membrane transport of substances; interaction with trophic substrates. Important aspects are the study of mechanisms of cell infection, cytotoxic effects, and ligand-receptor interaction. Also, using cultured cells, it is relevant to study intercellular interaction, morphogenesis, paracrine and autocrine effects, and kinetics of cell proliferation, for which both single-type cells and models of co-cultivation of different cell types are used. Organotypic models of cultured cells find their application in regenerative medicine and transplantation, including pathological conditions. Formation and secretion of cell products, creation of bioreactors and obtaining final products, production technologies, and obtaining targeted biologically active products are integral parts of modern bioproduction. The advantages of cell culture are the ability to accurately control the physicochemical environment (pH, temperature, osmotic and partial pressure, oxygen and carbon dioxide content), as well as biochemical and physiological conditions, which contributes to the repeatability and reproducibility of results, mechanization, and certification of research, saving reagents and time of certain works. Cell culture very often replaces or complements existing models with 6 the use of animals. Often the use of cell culture is more convenient and cheaper compared to traditional experimental models using animals. Cell culture can represent with sufficient reliability the processes occurring in a certain type of cells in the body. In some cases, cell culture is indispensable, an example, in the case of the synthesis of chimeric proteins that require certain post-translational, such as glycosylation (R. Gouveia). The increasing use of recombinant therapeutic proteins in clinical practice, the transformation of antibodies into a powerful research tool, the gradual abandonment of animal models, and modern advances in stem cell research make cell culture a routine practice in many laboratories and encourages scientists to a more detailed understanding of the cellular processes that affect the cultivation of cells in artificial conditions. At the present stage of cell culture development, the question arises not only about the isolation and maintenance of certain cells, but also about the possibility of manipulating and modifying cells in culture for scientific or practical needs, such as the creation of recombinant or transgenic cell lines, modification of cells in the lines for artificial control of cell proliferation, increasing cell survival, reducing the associated metabolic products (lactate, ammonia, etc.), increasing productivity, regulation of post-translational modifications, etc. (M. Matascia, 2008,). Optimization of the cultivation process is due to several interrelated factors such as improvement of media and cultivation parameters (concentration of CO2, glucose, growth factors, etc.) as well as modification of cells by inclusion or exclusion of certain genes. In addition, the study of morphological and kinetic markers of cell lines is necessary for the correct maintenance of culture, and timely response to any changes in the state of cell culture. It is known that under certain circumstances 7 cell culture can lose its inherent properties or die and, thus, lead to incorrect interpretation of the results of the experiment, and loss of valuable achievements and time. Recently, very high hopes are placed on stem cells, which have a high potential for use in medical, genetic, applied, and fundamental research. The high proliferative and differentiation ability of stem cells makes them an extremely attractive object for development in the field of pharmacology, and personal and regenerative medicine (Stem cells and the future of regenerative medicine. National Academy Press Washington, D.C. 2003). This manual contains guidelines for working with cell cultures of higher eukaryotes, including humans. 8 I. PREPARATION OF THE BOX ROOM, REAGENTS, AND DISHES FOR WORK WITH CELL CULTURES. Lesson 1. Structure of the cell culture room, equipment, and safety. The purpose of the lesson: familiarization with biosafety techniques and basic rules of work in the cell culture laboratory. The basic rules of work in the laboratory for cell culture include: 1) safe handling of electrical, and gas equipment, as well as drainage; 2) all devices used for cell cultivation should be grounded, sockets and switches should be in a well-accessible and safe area; 3) the work of students and graduate students should take place only in the presence of responsible personnel; 4) the devices should be switched on and off only in agreement with the responsible personnel; 5) work in the culture laboratory should take place in the maximum permissible silence, without extraneous noise and distractions; 6) before starting to work under the laminar flow, the workplace should be equipped as conveniently as possible (chair height, access to utensils, automatic pipettes, gas or alcohol burner, etc;) 7) if questions arise during manipulations under the laminar flow, solve them only with the responsible personnel; 9 8) before starting work, you should find out what degree of biological safety is characteristic of a particular cell line or primary culture; 9) adhere to the rules and skills of sterile work as much as possible; 10) after finishing work, put the workplace in order and report on the work done. When establishing a laboratory for tissue culture, it is necessary to take into account, first of all, the volume of work, the type of cultured cells or tissues, a n d the class of biological safety. In accordance with this, the laboratory is created. Requirements for the premises of the cell culture laboratory: 1) sterile area, cleanliness, minimum movement of people, no through passage; 2) the location of the laboratory should be separate from the rooms where microbiological work and work with experimental animals are carried out; 3) the preparatory zone includes a sink, preferably divided into two blocks - for the primary processing of dishes, in which the cell cultivation was carried out, and a zone for successive washing (flowing, distilled, and deionized water) of dishes; 4) space for incubators; 5) refrigerators and freezers chambers for storage of reagents and cell culture media; 6) cabinets for storing sterile plastic and glassware; 7) freezers and equipment for cryopreservation and storage of biological material in liquid nitrogen. 16 Fig.2.Reducer to carbon dioxide cylinder It is also known that the cultivation of certain types of cells is more efficient with constant stirring. Structurally, these devices consist of a platform on which containers with cultivated cells are fixed and a drive mechanism that provides circular or reciprocating movement of the platform. When equipping CO2 -incubators with such platforms, the possibility of changing gas flows in the chamber environment should be taken into account. The next equally important device is an inverted microscope (Fig. 1, B), which is based on the principle of "invertedness", as a result of which cells attached to the bottom of a plate, vial, or Petri dish can be observed in the field of view. Using an inverted microscope, you can also observe cells growing in suspension. Most of the modern inverted microscopes are equipped with video cameras and digital cameras, which allow to carry out live imaging of cells at all stages of their cultivation, and microscopes have special software (e.g. AxioVision, Carl Zeis), which allow calculainge the area, staining intensity, size, and other parameters 17 of cultured cells, as well as constantly monitor the intensity of cell spreading on the substrate in a specific period of time. The fourth group of basic equipment is devices for cryopreservation and storage of cells. These are freezers (kelvinators) with temperatures down to -70о C, as well as Dewar tanks filled with liquid nitrogen. Usually, only personnel after special training is allowed to work with liquid nitrogen, taking into account all safety rules when handling reagents that can cause frostbite. To create cell banks equipped with cryo equipment, it is necessary to have a separate room where no other devices, especially heating devices, should be stored. Among the auxiliary necessary equipment, first of all, there are autoclaves and dry heat ovens, bidistillers and deionizers for water purification, devices for ultrafiltration of media and buffers used for cell cultivation, tabletop centrifuges, multi-well spectrophotometers, automatic liquid aspirators, water baths, pH meters, laboratory magnetic stirrers, automatic varipipipettes for different volumes, hemocytometers, as well as a wide range of biochemical, molecular biological, and chromatographic instruments necessary for the equipment of the culture laboratory for a particular research area (Fig.3). According to the rules of sterile work, the culture unit should be divided into several zones - a box in which there is a laminar flow cabinet, CO2 - incubator, inverted microscope, as well as a refrigerator for storing the necessary media for cell cultivation and a pre-box room with additional equipment. 18 Dry oven and autoclave (20 l) Semi-industrial autoclave (50 l) device for preparation of deionized water Tabletop centrifuge Multi-well spectrophotometer pH meter Fig.3.Auxiliary equipment when working with cultured cells 19 Outside the box with laminar flow and CO2-incubator in two separate rooms should be placed an autoclave and a dry heat cabinet for sterilization of solutions and dishes, as well as cryo equipment, which includes automated systems for cryopreservation of cells, as well as dewars for filling and storage of liquid nitrogen and dewars equipped with special numbered cassettes for longterm storage of cell lines, primary cultures, re-seeding strains, stem cells, clones, etc. Rules of sterile work: Remember: entrance to the cell culture room is restricted to specially trained personnel only. First of all, you need to have a change of protective clothing (gowns, caps, masks) and shoes (Fig.4). It is desirable to have overalls made of natural fabrics. Fig.4.Training of personnel to work in sterile conditions Before starting work, turn on the ultraviolet radiation in the cabinetlaminar flow (15-20 minutes), then the laminar air flow (REMEMBER - during direct work ultraviolet 20 is disconnected). Before working in the laminar flow it is necessary to treat hands with cotton wool dipped in alcohol. After this procedure, you should not put your hands outside the laminar flow unit. If this happens, the procedure should be repeated. The use of sterile gloves is not always safe, as the use of ethanol and a burner in the laminar flow can lead to a fire. The room in which sterile work with cells is carried out is not visited by strangers; it is not ventilated, but equipped with air conditioning. The walls and floors should be covered with a material that is well-washed with disinfectant solutions. It is desirable that the room is divided into two halves, the smaller one with the laminar, microscope, and CO2 - incubator, and the larger part with other necessary equipment. The room with the laminar flow should be entered with a change of clothes and shoes. Since the work with cells has a rather high cost of basic and auxiliary means, to admit a specialist to work, it is necessary to instruct him/her, as well as to go through all the stages of preparatory work, and especially work with gas burners in the laminar flow cabinet. STERILE WORK is not a set of methods, but rather a certain style of work. Under "style" in these circumstances it is understood that the quality of work, in this case, is determined not by "maximum", but by "minimum achievements". It is not difficult to work in such a way that there is no pollution, the only problem is that you should always work this way (regardless of your mood and condition). The rules of sterile work are almost identical for bacteria and eukaryotic cells. The only difference is that bacteria are usually more tolerant to their violation; purification of a valuable bacterial culture is a much simpler task than purification of a eukaryotic culture; the cost of a dead bacterial culture is much less than a lost eukaryotic culture. 21 The basic rule is to try to organize the work so that "clean" and "dirty" solutions and instruments are as far away from each other in time and space as possible. First, you should prepare sterile media, pour them into tubes and cups, close the stock solutions and take them out from under the laminar flow, and only then introduce the cell cultures under the laminar flow and manipulate them. It should be remembered that the main source of contamination during manipulations in the laminar flow cabinet is the hands of the experimenters, which are wiped with an alcohol solution before work. A container with germination of cantankerous microorganisms is a gap in sterile work. It is extremely important in this case to localize the problem. It is IMPOSSIBLE to work with bacteria, yeast, and cells of higher eukaryotes in one laminar flow tank. As a rule, separate rooms are used. If there is a need for one employee to work with these objects, the sequence of work is as follows - cells, bacteria, yeast. Stationary in the laminar flow box can be, cases with sterile pipettes, boxes with autoclaved plastic tips, the necessary amount of plastic dishes, sterile water, saline, and phosphate-salt buffer. It is unacceptable that culture media, serum, and protein solutions are in the laminar flow box. A separate refrigerator must be allocated for these components. Before use, ready-made media are heated in a thermostat. The sequence of work with cultured cells: 1. Preparation of media and reagents for cultivation. 2. Selection of the necessary amount of media required for a certain stage of work. 3. Cell transplantation and cryopreservation. 22 4. Seeding of cells in plates of different grades for short-term experiments. 5. Manipulation of cells in tablets. If several specialists work in the box laminar, you should follow the work schedule. Each of the workers must leave a perfectly clean workplace. Control questions: Basic safety rules in the laboratory for cell culture. Groups of basic equipment for working with cell cultures. How is a local sterile space created? Technical characteristics of laminar flow cabinets. What is a CO2 -incubator? Structure, temperature, and gas conditions of the CO2 -incubator. Principle of construction of inverted microscope. Auxiliary equipment for cultural boxing. What devices ensure the maintenance and long-term storage of cultured cells? What are the basic principles of sterile work? 23 Class 2.Preparation of solutions for cell culture. Most cell culture media are supplied by companies specializing in the production of sterile media and growth-stimulating solutions. In early studies, natural media based on tissue extracts and natural body fluids such as chicken embryo extract, serum, lymph, etc. were used for cell cultivation. With the proliferation of cell lines, the need for a large number of media has led to the introduction of media with chemically precisely defined composition based on biochemical analysis of natural body fluids. Eagle's basic Eagle's medium [Eagle, 1955] and minimal Eagle's medium (MEM) [Eagle, 1959] were widely used with various additions of calf sera, human sera, protein hydrolysates, and embryonic extracts. By that time many permanent cell lines (L929, HeLa, and others) had already been obtained and it became clear that these media are suitable for most cell lines. Nowadays, a number of modified media have been developed (for example, RPMI 1640 medium was developed for lymphoblastic cell lines), or modification of media for specific cultivation conditions (Leibovitz's L15 medium was developed for cell cultivation in the absence of C02 and NaHC03, [Leibovitz, 1963]). In the late 40s and early 50s, most cells were grown in plasma or fibrinogen clots in the presence of tissue extracts. In his classic work, Eagle investigated the nutrient requirements of the obtained cell lines, as a result of which he was able to achieve cell multiplication in a medium of a certain composition containing a mixture of amino acids, vitamins, salts, and carbohydrates, as well as small amounts of bovine and human serum, with 27 environmental factors identified as essential for cell growth. They form the basis of the nutrient medium, 24 known as Igla basic medium. Of the 20 amino acids that were part of the medium, 13 were essential, and the rest could be synthesized from other carbon sources. Removal of any of the 7 vitamins led to the development of symptoms of vitamin deficiency. When cells were cultivated in BSI, the medium had to be frequently renewed, so it was replaced by Eagle's minimal medium (EMI), in which the concentration of nutrients was increased, which ensured continuous cell growth in culture without replacing the medium. Today, the most popular media are Dulbecco's DMEM [Dulbecco & Freeman, 1959] or RPMI 1640 [Moore et al., 1967], in which serum is added. Serum-free media are also now used in industrial technology in order to simplify the process and reduce the risk of contamination of cell cultures by accidental infections. A popular compromise for many laboratories is a mixture of complex media such as Ham's F12 medium [Ham, 1965] with other media containing increased concentrations of amino acids and vitamins such as DMEM (Table 1). In most cases, commercial liquid culture media are used for cell cultivation (Fig.5). Fig.5 Liquid media for cell culture (Sigma, USA). 25 Table 1. Content of components (mM) in culture media MEM, DMEM, RPMI1640, F-12 Components (1) Amino acids MEM DMEM RPMI-1640 F-12 L-arginine L-asparagine 6,0х10-4 4,0 х10-4 1,1 х10-3 1,0 х10-4 1,0 х10-3 L-aspartic acid1, 5 x10-4 1,0 х10-4 L-cysteine2, 0 x10-4 L-cystine1. 0 x10-4 2.0 x10-4 1.1 x10-4 L-glutamic acid1, 4 x10-4 1,0 х10-4 L-glutamine 2,0 х10-3 4,0 х10-3 2,1 х10-3 1,0 х10-3 Glycine 4,0 х10-4 1,3 х10-4 1,0 х10-4 L-histidine 2,0 х10-4 2,0х10-4 9,7 х10-5 1,0 х10-4 L-oxyproline 1,5 х10-4 L-isoleucine 4,0 х10-4 8,0 х10-4 3,8 х10-4 1,0 х10-5 L-leucine 4,0 х10-4 8,0 х10-4 3,8 х10-4 1,0 х10-4 L-lysine HC1 4,0 х10-4 8,0 х10-4 2,2 х10-4 2,0 х10-4 L-methionine 1,0 х10-4 2,0 х10-4 1,0 х10-4 3,0 х10-5 L-phenylalanine 2,0 х10-4 4,0 х10-4 9,1 х10-5 3,0 х10-5 L-proline 1,7 х10-4 3,0 х10-4 L-serine 4,0 х10-4 2,9 х10-4 1,0 х10-4 L-threonine 4,0 х10-4 8,0 х10-4 1,7 х10-4 1,0 х10-5 L-tryptophan 4,9 х10-5 7,8 х10-5 2,5 х10-5 1,0 х10-5 L-tyrosine 2,0 х10-4 4,0 х10-4 1,1 х10-4 3,0 х10-5 L-valine Vitamins 4,0 х10-4 8,0 х10-4 1,7 х10-4 1,0 х10-5 P-aminobenzoic acid7, 7,3 x10-6 32 Task for topic 1: make calculations for the preparation of buffers for culturing and washing cells. Option 1. Perform calculations for the preparation of Hanks's BBS: volume - 15 ml, 2-fold solution; CaС12 in a 10% solution; glucose in a 20% solution Option 2. Perform calculations for the preparation of RBS with Ca2+ and Mg2+: volume -17ml; salt Na2HP04•7Н20 is replaced by NaH2PO4•12Н20; salt KH2P04 is replaced by KH2P04•7H20 Option 3. Perform calculations for the preparation of Hanks' BBS: volume 25 ml, 3x, Ca2+ in a 20% solution, glucose in a 10% solution; Na2HP047Н20 in 1M solution. Option 4. Make calculations for preparing VBS Earl: volume 185 ml; 4x, salt MgS04•7H20 in 0.5 M solution, NaCl in 3M solution, salt NaH2P04•H20 replaced by NaH2P04•12H20. Option 5. Perform calculations for the preparation of RBS without Ca2+ and Mg2+: volume - 12 ml; salt Na2HP04•7Н20 is replaced by Na2HP04•5Н20; KH2P04 salt in 1.5 M solution; NaCl is contained in a 20% solution. Option 6. Make calculations for the preparation of BBS Hanks: volume - 132 ml; 0.7x buffer, KH2P04 salt replaced by KH2P04•3H20; glucose in a 30% solution. Option 7. 33 Perform calculations for the preparation of RBS with Ca2+ and Mg2+: volume - 11 ml, 3x; salt MgS04•7H20 is replaced by MgS04•2H20; salt KH2P04 in a 1.5 M solution Option 8. Perform calculations for the preparation of RBS without Ca2+ and Mg2+: volume -110 ml; 3 times; KH2P04 salt is replaced by KH2P04•7H20; NaCl salt in a 2.5 M solution Option 9. Make calculations for the preparation of Earl's BBS: volume 630 ml; NaСl in a 4.5 M solution, salt NaH2P04•Н20 in a 2 M solution, glucose in a 5% solution Option 10. Make calculations for the preparation of BBS Hanks: volume -38 ml; 0.7x glucose in a 10% solution, CaC12 in a 2% solution for injections. Option 11 Perform calculations for the preparation of RBS with Ca2+ and Mg2+: a volume of 220 ml of CaС12 in a 5% injection solution, NaHCO3 and NaСl in 1.5 M solutions. Option 12 Perform calculations for the preparation of Hanks's BBS: volume 340 ml, 2.5x, CaС12 in 1% solution; Na2HP04х7Н20 was replaced by anhydrous NaCl in a 10% solution. Option 13 Perform calculations for the preparation of RBS with Ca2+ and Mg2+: a volume of 1.45 l CaС12 in a 2% injection solution, NaСl in a 16% solution. 34 Anhydrous KN2P04 salt is replaced by KN2P04 x3H20 salt. Option 14 Make calculations for preparing BBS Earl: volume 45.5 ml; 3.5 times solution, NaСl in 20% solution, NaH2P04•Н20 salt in 2 M solution, MgS04•7Н20 salt replaced by MgS04•2Н20 salt, glucose – in 2.7 M solution. Option 15. Make calculations for the preparation of RBS with Ca2+ and Mg2+: volume - 140 ml; 1.5x solution, salt Na2HP04•7Н20 in 1.2 M solution; NaСl in a 3.5 M solution, CaС12 in a 1.4 M solution. An example of solving a typical problem for the preparation of the necessary dosages of certain phosphate-salt solutions for a given volume, the corresponding multiplicity and modification of the components Perform calculations for the preparation of RBS without Ca2+ and Mg2+: volume - 530 ml; salt Na2HP04•7Н20 is replaced by Na2HP04•2Н20; salt KH2P04 is replaced by KH2P04•5H20. Progress of problem solving 1) We calculate the required weight of Na2HP04•2Н20 (g/l) instead of Na2HP04•7Н20 M.m. Na2HP04•2Н20 is 178. To prepare an 8.06 mM solution of Na2HP04•2Н20, it is necessary to prepare a weight of 178x0.00806=1.43 g/l; 2) We calculate the required amount of KH2P04•5H20 (g/l) instead of KH2P04. M.m. КH2P04•5Н20 is 226.1. To prepare a 1.47 mm solution of KH2P04•5H20, it is necessary to prepare a weight of 226.1x0.00147=0.33 g/l. 3) Taking into account the volume (530 ml), the content of all components of the solution, respectively, is: 35 CS1: 0.2x0.53=0.106 g/given volume КH2P04•5Н20: 0.33х0.53=0.175 g/given volume NaCl: 8x0.53=4.24 g/given volume Na2HP04 • 2Н20: 2.2x0.53=1.17 g/given volume. Note: The progress of problem solving must be presented in writing, with a full description of all the components of the given solution and the personal signature of the performing student. Task conditions can be changed. Question. 1. Why is it necessary to prepare phosphate-salt solutions from modified components (for example, instead of dry weight, concentrated solutions of certain components with a known molarity or percentage content are used)? 2. Why is it necessary to use multiple buffers (most often 2x buffers) when cultivating cells? 3. Describe the methods of sterilizing phosphate-salt buffers used for cell culture. Preparation of media for filtration. Nutrient media used for cell cultivation are sterilized by filtering through nitrocellulose sterile filters with a pore diameter of 0.22-0.45 microns. The filtration medium consists of a plastic or metal filter holder (Fig. 6) and the filter itself. The filtration sequence is as follows: the prepared culture medium is applied to the sterile membrane and pressed through syringe filter. When sterilizing solutions by autoclaving, it is necessary to prepare cotton gauze swabs, which are used to close the containers with solutions (Fig.) It is advisable to close the container with foil or lacquered paper on top of the cotton gauze plug. The autoclaving container is filled with liquid to 2/3 of the volume. The autoclaving mode for each solution is set according to its characteristics. Most often, it is 1.5 atmospheres for 30-40 minutes. 36 Figure 6. Cotton gauze swabs for autoclaving liquids. Figure 7. Sequence of ultrafiltration with using 0,22 micron nitrocellulose filters After autoclaving, the containers with solutions are closed with a rubber corks that are sterilized by autoclaving or boiling. In addition, it is advisable to burn the corks, moistened with 70% alcohol solution. Storage of cultivation media and phosphate-salt buffers is carried out at a temperature of +40 C. Storage of trypsin solution and fetal calf serum is carried out at -200 C, preferably in small aliquots necessary for cultivation. 37 Glutamine is added separately to the culture media, as this component can degrade in the complete nutrient medium. Therefore, glutamine can also be stored in small aliquots in frozen form. Often a mixture of antibiotics is used to cultivate certain cell lines or primary cultures. For each specific In this case, this mixture is specified in the culture conditions given in the certificate for cultured cells. Control questions: History of creation of cultivation media. What components are used for cultivation media? What components are used in cell culture to stimulate the growth of cell populations? How to prepare media from dry suspensions? Methods of sterilization of solutions for cultivation. Which solutions were recommended to be prepared by ultrafiltration only? Prepare an individual phosphate-salt solution (for example, calculate the necessary weights for the preparation of 2x Hanks' solution in a volume of 35 ml). Practical task: Calculate and prepare cotton swabs and autoclave individually prepared solution. 38 Class 3. Types of dishes for cell culture, preparation and sterilization of glassware. Cell culture dishes are divided into glass and plastic (Fig.8) Figure 8. Dishes for cell culture: plates, Petri dishes (glass and plastic), glass dishes for media, vials; pipettes. Plastic tableware is disposable and therefore cannot be sterilized. Depending on the tasks and main objectives of the scientific or production process, different types of dishes are used. The most popular are vials with different working surfaces (from 25 cm2 to 500 cm2 ) and Petri dishes with different diameters. Depending on the conditions of the experiment and the type of cultured cells (suspension and adherent), vials or cups with low and high adhesive properties are selected. The most commonly used tablets are 624 and 96 39 wells. 96-well dishes are of three types (flat-bottomed, V-shaped, and roundbottomed). For certain dishes, there are optimal rates of planting cells and the volume of culture medium used (Table 7). Table 7. Characteristics of plastic dishes used for cell cultivation. Vial type S (see2 ) V (ml) Wednesday C (cells) Vial 24 50 7 4,8х105 vial 25 70 7 5х105 vial 75 250 25 1,5х106 vial 83 260 30 1,6х106 vial 175 800 68 3,5х106 Cup 35x10 9,6 2,5 1,6х105 Cup 65x15 21 6,0 4,2х105 Cup 100x20 58 16,0 1,1х106 96 well plate 0,32 0,2 6х103 24 hole tablet 1,88 1,5 3,7х104 12 hole tablet 3,83 2,0 7,6х104 6 hole tablet 9,4 3,0 1,8х105 Among another plastic crockery should be noted – tubes for cryopreservation of cells, centrifuge tubes for different volumes, disposable pipettes, etc. Especially necessary are nozzles for reusable filtration of solutions with replaceable filters (nitrocellulose with a pore diameter of 0.22 microns) and disposable filters, so-called syringe filters designed to filter a small number of solutions and buffers (fig., given in the previous section). In addition, prefilters with pore diameters from 0.45 µm are used for filtration The most famous companies that are suppliers of plastic tableware are Nunc, Gibco, Falcon, Sarstadt, TPP, Cellstar. 40 Glassware - the main part of which belongs to the auxiliary dishes. These are dishes of various capacities, the main purpose of which is the preparation of solutions for cell cultivation. These dishes are specially prepared. Washing of dishes necessarily includes the procedure of soaking in special detergents or in a solution of potassium bichromate "chromovka" (93 g of potassium bichromate in rubber gloves and a respirator or cotton gauze bandage is well ground into a powdered mass in a mortar, the minimum required volume of water is added and 1 liter of sulfuric acid is added in portions). The dishes are washed clean in a certain detergent, preferably one that does not contain biological additives, rinsed in running water through a solution of "chrome", then washed well in running water, then rinsed in two or three portions of distilled and undistilled water. Sterilization of tableware is carried out in several ways: - hot steam sterilization using autoclaving; for this purpose, glassware is covered with two or three layers of foil, and carefully wrapped in autoclaving paper. A special strip should be attached to the top of one of the bottles or Petri dishes to certify the sterility of the dishes. If the mode in the autoclave is observed (40 minutes, 1.5 atmospheres), then black transverse stripes appear on this strip, and this indicates the sterility of the dishes. Also subject to autoclaving are tips for automatic varipipettes, which are placed in special closed racks (Fig. - sterilization with dry air using special "dry heat" cabinets (the mode is sufficient for sterilization at 150о C for 2-3 hours or 180о C for 40 minutes. For this purpose, the holes of glassware are again well closed with foil. Glass pipettes of different volumes are often used for cell cultivation. Pipette preparation also includes the procedure of 41 thorough washing, for which cylinders are used for soaking pipettes and their treatment in detergents, followed by thorough rinsing in many changes of running and distilled water. Before sterilization, the blunt ends of pipettes are closed with cotton swabs, and placed in special cases, or each pipette is wrapped separately in the paper that can withstand sterilization at 180о C. Sterilization modes are indicated above. Remember: the quality of work with cell cultures depends on the degree of absolute sterility of the dishes. Note: the dishes used for cell culture are never used for biochemical and molecular biological research. Immediately after sterilization, the dishes are also not used. It should be cooled to room temperature. Among the auxiliary dishes for which you can not follow strict sterilization rules are tubes in which biochemical manipulations with cells after their cultivation are carried out. Since all manipulations with cells and culture media are carried out using sterile pipettes, you should practice this procedure very well. Rules for working with sterile pipettes: 1) wipe the inner surfaces of the laminar box with an alcohol solution; 2) turn on ultraviolet irradiation and injection of sterile air in the box-laminar flow chamber for 30-40 minutes; 3) before start work it is necessary turn off the ultraviolet irradiation; 4) all manipulations in the box are carried out over a burner flame (alcohol or natural gas); 5) to ensure proper and safe operation in the laminar flow 48 For standardization determination concentration cells в within the framework of standard operating procedures create a protocol (SOP). SOP 1 STUDY OF CELL CONCENTRATION BY INCLUSION OF THE VITAL DYE TRYPAN BLUE 1. Purpose Determination of cell concentration and viability in suspension. 2. Distribution In laboratory diagnostics, clinical practice. 3. Purpose By incorporating trypan blue dye into dead cells, determine the cell concentration, a n d t h e ratio of live to dead cells in the suspension. 4. Object Cells 5. Materials and equipment Reagents Incubation environment: RPMI solution (10.2 g/l) + ETS - fetal calf serum (10%) + 0.4% trypan blue dye solution Laboratory glassware 96-well plates; Penicillin vials for media. Equipment Centrifuge. Pipettes. Microscope. 49 Goryaev camera. Thermostat. Materials. Tips for pipette dispenser (50, 100, 200, 300 µl), sterile. Filter paper cotton No. 1. 6. Preparation of solutions Preparation of trypan blue dye: dilute 400 mg of the dye in 100 ml of phosphatebuffered saline, pH 7.2 (or by increasing or decreasing the weight and volume of phosphate-buffered saline, respectively) 7. Preparation of samples for analysis Pellet the cell suspension by centrifugation at 1000 g for 5 min. Dilute the cell pellet in a certain volume (1 ml). 8. Research protocol А). The effluent concentration cells dilute by a factor of two, (3-4 dilution steps, Table 8) . Б). Take an aliquot of cells (50 µl) from each dilution and add an equal volume of trypan blue dye. В). Paint for 5 minutes. Г). After that, add the cells to a pre-prepared Goryaev chamber (each concentration under study in at least 2 parallels). Table8 Cell dilution scheme for counting in Goryaev chamber A) Theoretical: concentration/ml 1:2 1:-4 1:8 1:16 200 000 100 000 50 000 25 000 12 500 B) practical 50 12000 cel l 10000 nu m R 2 = 0.9993 be r 8000 6000 4000 2000 0 0 1 2 3 4 5 dilution of suspension Hole 1 2 3 4 А 100 000 50 000 28 000 13 000 В 100 500 52 000 25 000 12 000 M 100 250 51 000 26 500 12 500 m 1666,7 666,7 1000,0 333,3 9. Presentation of the results Fig.12. The curve of counting cells in the Goryaev chamber in the conditions of their successive 2-fold dilution. 10. Related documents and files Provide a protocol for cell counting and present the counting curve as a graph. 11. List of references 1. Molecular Biology of the Cell, B. Alberts, D. Bray, J. Lewis, et al. "The World", 1987 2. Adams R. Cell Culture Methods for Biochemists, M. "Mir", 1983, 263 12. Terms and abbreviations SOP - Standard Operating Procedure. A detailed, written instruction 51 to achieve reproducibility of each specific action. ETS - embryonic calf serum 13. Developer X To determine the correlation coefficient, compare the results of cell counting conducted by two researchers by Student's criterion (Table 9). Confidence limits for t with f - degrees of freedom (Petunin Y.I. Application of the theory of random processes in biology and medicine, Kyiv 1981, p.59) Table 9 Student's criterion, confidence limits for t with f - degrees of freedom f Bilateral limits f Bilateral limits 5% 1% 5% 1% 1 12,71 63,66 20 2,086 2,845 2 4,303 9,925 21 2,08 2,831 3 3,182 5,841 22 2,074 2,819 4 2,776 4,604 23 2,069 2,807 5 2,571 4,032 24 2,064 2,797 6 2,447 3,707 25 2,06 2,787 7 2,365 3,499 26 2,056 2,799 8 2,306 3,355 27 2,053 2,771 9 2,262 3,25 28 2,048 2,783 10 2,228 3,169 29 2,045 2,756 11 2,201 3,106 30 2,042 2,75 12 2,179 3,055 40 2,021 2,704 13 2,16 3,012 50 2,009 2,768 14 2,145 2,977 60 2 2,66 15 2,131 2,947 80 1,99 2,639 16 2,12 2,921 100 1,984 2,622 52 2 17 2,11 2,898 200 1,972 2,601 18 2,101 2,878 500 1,985 2,586 19 2,093 2,861 ~ 1,98 2,578 f 2,5% 0,5% f 2,5% 0,5% Unilateral borders Unilateral borders f= g+h-2; t=(M1 -M2 )/ m12+ m 2 Tasks Option 1. The concentration of cells counted in the Goryaev chamber is 5.5x103/ml, the percentage of dead cells is 7.4%, and the total volume of cells is 7.4 ml. Cells must be planted for cultivation at a concentration of 3.5x104/ml of living cells. Describe the sequence of preparation of cell suspension for planting. Option 2. In 43 small squares of Goryaev's chamber, the number of cells is 32, of which 7 are stained with trypan blue. Calculate the concentration of cells and the percentage of dead cells (10 μl of 1% trypan blue dye was added to 40 μl of the cell suspension). Option 3. There are 8 cells in 5 large squares of Goryaev's chamber. When planting cells for cloning by the limiting dilution method, it is necessary to plant 1 cell in a volume of 25 μl, 50 μl, 100 μl, and 150 μl. Calculate the initial concentration of cells in the suspension and the dilution in which the cells should be seeded. 53 Option 4. There are 2 cells in 13 small squares. Dead cells in the suspension were not recorded. What is the total number of cells in the suspension, the volume of which is 18.4 ml? Option 5. The concentration of cells in the suspension is 2.2x104 cells. What is the average number of cells contained in 10 small squares, 3 large squares when diluting the suspension with trypan blue dye in 1.25; 1.5 to 2 and 2.5 times, respectively? Option 6. Macrophage suspension was diluted 2, 5, and 10 times. When diluted 5 times, the number of cells in 40 small squares was 12 cells. How many cells are contained in 3 large squares when diluting the suspension by 2 times; in 56 squares at a 10-fold dilution and what is the initial concentration of macrophages. Option 7. When cloning cells in a volume of 122 ml, the cell content was 1.3x102. What manipulations should be carried out in order to sow cells in a 12-well plate in 4 wells (the volume of each is 1.25 ml) at a concentration of 1.75x103 cells/ml? When counting in Goryaev's chamber, how many cells will be contained in 72 small squares in 10 large squares and in 2 large and 5 small squares? Option 8. Solving a typical problem of counting cells in a Goryaev chamber. There are 33 cells in 80 small squares. Of them, 3 cells are dead. What is the 54 concentration of the initial cell suspension if 50 μl of the suspension with the addition of 25 μl of trypan blue dye are used for counting? According to the cell counting formula X=(a*4000*c)/b, a=33; b=80; c=1.5 times Therefore, X=33x4000x1.5/80=2475 cells/in 1 mm3 (1 μl). Accordingly, the total concentration in 1 ml = 2.475x106 cells. The percentage of dead cells can be calculated in 2 ways: A) 3 cells out of the total number of 33 are 9.1% dead, i.e. the cell suspension contains 90.9% live cells and 9.1% dead cells Control questions: Tell about the main purpose of the hemocytometer. Technical characteristics of the Goryaev chamber. How is the Goryaev chamber prepared for cell counting? How is the cell suspension prepared for counting? Determination of the percentage of viable cells. What is the standard operating procedure? Prepare a protocol for determining cell concentration using the Goryaev chamber after staining with trypan blue. Practical task. Calculate the concentration of cells in several parallels (at least 2), and determine the average value and the standard deviation. Present the results in the form of a graph. Determine the correlation coefficient by Student's criterion in pairs between researchers who counted cells according to Table 9. 55 Activity 4. MTT-colorimetric method of estimating the number of living cells by the activity of mitochondrial dehydrogenases of cells The MTT test is used to evaluate the proliferative parameters of cultured cells, the activity of their mitochondrial dehydrogenases, as well as to determine the cytotoxic/cytostatic effect [Alley M.C. et al., 1988; Mosmann T., 1983]. The basis of the method is the ability of mitochondrial enzymes of a living cell to transform 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) - a yellow salt into a purple crystalline MTT-formazan. In order to use this method of assessing the proliferative parameters of each studied cell line, control determinations of dehydrogenase activity are carried out. To do this, cells are planted in successively decreasing concentrations determined by counting cells with trypan blue and cultivated for a certain period of time. At all stages of cultivation, a parallel routine cell count is performed using trypan blue, and optical indicators are determined when water-soluble (almost colorless, but yellowish in stock concentrations) 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl -2-tetrazolium bromide, which is added to cultured cells and is transformed by dehydrogenases of living cells into purple formazan, which crystallizes inside the cell (Fig. 13). As shown in the photo, the color can be from light purple to pink. 56 Fig. 13. Incorporation of water-soluble 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2-tetrazolium bromide into cells and its transformation into formazan crystals. The transfer of formazan into a solution using suitable organic solvents, such as dimethylsulfoxide (DMSO) or isopropanol, and subsequent photometry 57 allow to accurately compare the change in optical density of the solution relative to the control with the change in the number of viable cells, and in cytotoxic studies to assess specific cell death that induced by one or another cytotoxic agent. The recovery of formazan is affected by the concentration of the dye in the incubation medium, and the optimal concentration varies widely for different cell lines, and the absorption spectrum of formazan depends on the pH and density of the cell suspension. Thus, in a suspension of higher density and with a critically low concentration of cells in the well, errors occur that lead to an overestimation of the results of chemosensitivity [Cherepovych et al., 2006]. Therefore, when using the MTT test on a large scale, for example, for screening potential therapeutic agents, or determining the cytotoxic effect, it is advisable, working with a certain culture, to determine the optimal concentration of cells in the control, at which there is no change in spectrophotometric indicators associated with large errors in parallel studies and, accordingly, not the reliability of the obtained results. In addition, it is known that dehydrogenase activity in living cells depends on the phase of the cell cycle. Thus, in the state of "proliferative rest" (G0), activation of catabolism enzymes was noted: phenylalanine hydroxylase, tyrosine, pyridine nucleosidase, lactic acid dehydrogenase, tricarboxylic acid cycle dehydrogenase, acid phosphatase, ATPase, and serine protease. Cells at rest are characterized by a significant decrease in respiratory activity. The rate of protein turnover in resting cells is generally more intense than in proliferating cells, although there is specificity for individual proteins and cell type. The main reason for accelerated protein renewal against the background of reduced synthesis is accelerated protein degradation. Therefore, in order to determine the number of living cells associated with the activity of mitochondrial dehydrogenases, 3-4 hours before the end of the cell incubation period, a pre-prepared 10-fold solution of MTT (Sigma, USA) is added to the wells of a 96-well plate (20 μl per 200 μl of incubation medium at the rate of 5 mg/ml phosphate-salt buffer) and incubated under the 64 11.Literature. - Colangelo D., Guo H.Y., Connors K.M., Silvestro L., Hoffman R.M. Noncolorimetric measurement of cell activity in three-dimensional histoculture using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide: the pixel image analysis of formazan crystals // Anal Biochem. – 1992. – Vol. 205(1). - P. 8-13 - Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assayas. J Immunol Methods 1983: 65: 55-63. Conclusion: So, the MTT test is an indirect method of determining the proliferative indicators associated with the activity of dehydrogenases of living cells. In some cases, this method may raise questions about the artifactuality of the obtained data. However, it has a number of advantages that are not available for the traditional method of direct counting in the Goryaev chamber. In particular, the MTT test detects cells at dilutions that are unattainable by direct counting, although the results obtained are also somewhat questionable. The next advantage of the MTT test is the speed and volume of the measurement, because the direct method requires careful counting under a microscope, and the MTT test uses only optical indicators to determine the amount, which can be quickly calculated using special screening equipment. Control questions: 1. What is the essence of the MTT-colorimetric method of research? 2. In which phase of the cell cycle is the activity of dehydrogenases the highest? 3. What parameters affect the recovery of formazan? 4. What color does 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-tetrazolium bromide have in aqueous solution and in formazan crystals? 5. What solvents are used to dissolve formazan crystals? 65 6. At what wavelength is extinction measured in the MTT test? 7. We recall what the standard operating procedure is. 8. List the equipment needed for colorimetric determination of cell viability in the MTT test. 9. How is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-tetrazolium bromide prepared, stored and used? 10. Perform calculations for 50 ml of single, 10-fold and 3.5-fold phosphate-salt buffer. 11. Present the sequence of research according to the protocol. 12. Draw a diagram of the experiment. 13. What conclusions can be drawn regarding the determination of proliferative indicators in the MTT test? 14. Carry out processing of the received data, statistical analysis by group and compare the data of routine counting in the Goryaev chamber and in the MTT test 66 Activity 5 Construction of a growth curve for cells of a permanent cell line . Cell culture is primarily influenced by the microenvironment. The acceptability and reliability of using cultured cells as a model for studying physiological functions in vivo is constantly subject to criticism. Often, due to the adaptation of cultured cells to artificially created culture conditions, the latter do not show the phenotype they have in vivo. As a result of the lack of cellular heterogeneity and three-dimensional architecture of tissues in cells in culture, intercellular and cell-matrix interactions are reduced. The culture lacks most of the humoral factors, and as a result, in the artificial microenvironment, non-specialized progenitor cells that spread, migrate and proliferate are favored. The influence of the microenvironment on culture occurs in the following directions: 1) the nature of the substrate on which the culture grows (dense such as plastic or other semi-liquid – collagen, gel, suspension); 2) degree of contact with other cells; 3) physico-chemical and physiological composition of the environment; 4) composition of the gas phase; 5) incubation temperature. Provision of an appropriate microenvironment, including adhesion to the substrate, concentration of nutrients, hormones or growth factors, as well as intercellular interaction are the main conditions for the manifestation of specialized cell functions (Alberts et al., 2002). Most cells from dense tissues grow as an attached monolayer unless transformed to become substrate-independent. Therefore, after tissue disaggregation or subculture, cells need to attach to the substrate before division begins. Cell adhesion is mediated by specific cell surface receptors for extracellular matrix molecules. Therefore, often during the cultivation of cells, plastic is covered with molecules of the extracellular matrix. It was shown 67 that the following main classes of transmembrane proteins are involved in the processes of intercellular and cell-matrix adhesion: Ca2+-independent (cell adhesion molecules, CAM) and Ca2+-dependent cadherins are mainly involved between homologous cells. Two identical molecules of such a protein interact with each other [Rosenman & Galatin, 1991; Alberts et al., 2002, Cavallaro & Christofori, 2004]. Cell entry into the cell cycle is regulated by signals from the microenvironment. The low density of cells leads to the fact that, without close contacts, the cell enters an active state and divides. Conversely, at a high cell density, the proliferation of normal cells is inhibited and the division of transformed cells is not inhibited. Inhibition of proliferation is initiated by cell contacts and enhanced by cell crowding as a result of cell shape changes. as well as limitation of distribution on the substrate. Intracellular control is mediated by cyclins and cyclin-dependent kinases [Planas-Sliva & Weinberg, 1997; Reed, 2003], which are affected by signaling cascades activated by phosphorylation of the intracellular domains of receptors upon their binding to growth factors. Negatively acting factors, such as p53 proteins [Sager, 1992; Mcllwrath et al., 1994], pl6 [Russo et al., 1998] or Rbgene expression products [Sager, 1992], stop the passage of the cycle at restriction points, or control points of the cycle. Communication between extracellular elements of cell cycle control (both positive, for example PDGF, and negative, for example TGF-β) and intracellular effectors is mediated by cell membrane receptors and biochemical signaling pathways, primarily those involving protein phosphorylation and the participation of secondary messengers such as cAMP, Ca2+ and diacylglycerol [Alberts et al., 2002]. That is why an important stage in the study of any population of cells disintegrated from the tissue, or already transferred into culture, is the determination of optimal conditions for their cultivation. The first step in studying any cell population (a line of genetically identical cells provided by the Cell Culture Bank or a primary culture explanted from a multicellular organism) is the construction of a growth curve. 68 Cell population growth curve It is known from the literature that the development of any cell population has a phase character (Fig. 15). The growth of cell populations in culture is described by a curve [Adams R, 1983, Freshny, 2009], on which a number of typical areas can be distinguished: at the initial stage, cells do not grow, their number and biomass do not change - this is the so-called adaptive period, or lag phase (Fig. 1, I). During this period, the incubation medium is adapted to a specific cell population (conditioning). The duration of the lag phase is determined by the optimal concentration of planted cells, cultivation conditions and the composition of the cell incubation medium. After that, the cells enter the phase of exponential growth (II), when the majority of the cell subpopulation is involved in intensive proliferation, and doubles after a certain period of time characteristic of this population of cells. In the logarithmic phase of cell population growth in the case of using a genetically identical cell line, when most cells synchronously enter the cycle phases, conditions are selected to determine the impact on the metabolism of proliferating cells. In particular, the cytotoxic/cytostatic effect of potential biologically active substances is investigated. The exponential growth phase of the cell population is followed by the slow growth phase (III). During this period, the number of cells participating in mitosis gradually decreases, and when the resources of the microenvironment and substrate area are exhausted, as a result of contact inhibition, the growth of the cell population enters the plateau or stationary phase (IY). 69 Fig. 15. Characteristic growth curve of the cell population in culture: I-lag phase; II-logarithmic phase; III-phase of slow growth; IY-stationary phase of growth; Yphase of degradation In this phase, the number of cells and their biomass almost does not change. If we extrapolate the phase of stationary growth to the phase of the cycle, we can assert the beginning of the period of "proliferative rest" for this population, which for most cells in the subpopulation is expressed in the G0/G1 transition. Transition of cells from this phase to proliferation is possible only with exogenous stimulation. Then, when the resources of the environment are completely exhausted, there is a phase of cell population degradation (Y), during which most of the cells die. However, if the population of transformed cells is considered, then in this phase, under conditions of nutrient substrate deficiency, a subpopulation of cells with high clonogenic potential can be selected. Therefore, to study the influence of biologically active agents, it is reasonable to model the growth phases of cell lines, which are represented by genetically identical cells and, accordingly, under the 70 same conditions, the microenvironment can be synchronized in a certain phase of growth, which is the basis for determining cytotoxic/pro-proliferative, about -/antiapoptotic effect on the cell subpopulation, as well as the ability of the cell population to differentiate in different directions (especially when it comes to stem cells) and their clonogenic potential. Thus, to construct a growth curve, cell cultivation is carried out for at least 5-10 days (sometimes up to 2 months of cultivation is required) and growth parameters for this population are determined at all stages of cultivation. 1. The most widely used method is cell counting using the vital dye trypan blue, which is incorporated into dead cells. 2. Today, methods of colorimetric visualization of cells based on the inclusion of vital dyes by mitochondrial dehydrogenases are also widely used. 3. Cytofluorimetric analysis is also an important step, which allows, by including intercalating dyes in DNA, to determine both the level of apoptotic cells and the distribution of cells by cell cycle phases. 4. The radioisotope method, which is based on the inclusion of H3-thymidine in DNA during its replication. Today, these methods are given a secondary role due to various considerations, first of all, it is related to occupational safety and admission according to the first list of experimental works dangerous to human health. The most widely used today is the MTT colorimetric method, which is based on the ability of mitochondrial enzymes of a living cell to reduce 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT, Sigma) is a yellow salt in purple crystalline MTT formazan. However, the activity of dehydrogenases at different phases of the cell cycle is different, so this method also has certain limitations. Actually, the complex use of three of the four methods proposed above can be used to construct a growth curve at different stages of cell population development. 71 Construction of growth curve for primary culture and cell line Thus, if a growth curve is required, the first step to be taken is to perform a routine cell count. using hemocytometers. The most common routine hemocytometer is the Goryaev chamber. For this, cells were seeded in 24-well (in a volume of 1 ml) or 12-well (in a volume of 2 ml) plates with a concentration of 3.5-5.0x104 cells/ml and cultivated for different periods of time. At each stage, cells are counted, viability and concentration are determined (Table 11). For example, the table shows the growth of the population of endothelial cells (a mouse endothelial cell line) and the primary culture of rat intestinal cells explanted by the trypsinization method. Both cultures were incubated for 8 days without changing the medium, in order to determine all stages of population growth. Table 11 The concentration of endothelial cells during long-term cultivation: counting using a Goryaev camera after staining the cells with trypan blue Time incubation cells «0» 12 hours 24 hours 36 hours 48 год 72 hours МАЕС (alive) 3,5 х104 4,2 х104 7,3х104 9,8 х104 14,1 х104 18,2 х104 МАЕС (deаd) 1,7 х103 2,2 х103 2,8 х103 5,5 х103 6,7 х103 1,1 х104 % deаd 4,6 % 4,9% 3,7% 5,3% 4,5% 5,7% Time incubation cells 96 hours 120 hours 144 hours 168 hours 192 hours 72 МАЕС (alive) 17,4 х104 16,4 х104 14,2 х104 11,1 х104 7,5 х104 МАЕС (dead) 1,3 х104 1,4 х104 2,2 х104 5,8 х104 8,4 х104 % alive 7,2% 7,9% 13,4% 34,3% 52,8% Fig. 16. Growth curve of endothelial cells during long-term cultivation without changing the medium. As you can see, from the given data, the growth of endothelial cells is described by a characteristic curve, from which it can be seen that the doubling time is approximately 22±2 hours, which is indicated in the certificate of these 73 cells. However, without changing the medium, there is a gradual death of the cell population and an increase in the subpopulation of necrotic cells, and on the 8th day of cultivation, the concentration of the dead reaches about 50%. Practical task: Sow cells in successively decreasing concentrations with "step" 2 according to Table 12. A) suspension culture (initial concentration 1x106 cells/ml) B) adhesive culture (initial concentration 2.5x105 cells/ml) Table 12 Cultivation scheme Dilution (cells/ml) 1 1:2 1:4 1:8 1:16 1:32 Suspension culture (theoretical dilution) 1х106 5х105 2,5х105 1,25х105 6,2х104 3,1х104 М1 Х1 Х1:2 Х1:4 Х1:8 Х1:16 Х1:32 М2 Х2 Х2:2 Х2:4 Х2:8 Х2:16 Х2:32 М3 Х3 Х3:2 Х3:4 Х3:8 Х3:16 Х3:32 М±m Σ (Xn/n) ±m Adhesive culture (theoretical dilution) 2,5х105 1,25х105 6,2х104 3,1х104 1,6х104 8х103 М1 Х1 Х1:2 Х1:4 Х1:8 Х1:16 Х1:32 М2 Х2 Х2:2 Х2:4 Х2:8 Х2:16 Х2:32 М3 Х3 Х3:2 Х3:4 Х3:8 Х3:16 Х3:32 М±m Σ (Xn/n) ±m