CULTURES OF CELLS AND FABRICS

From Big Medical Encyclopedia

CULTURES OF CELLS AND FABRICS — the cells, pieces of fabric, rudiments of body growing or keeping viability out of an organism «in a test tube» (in vitro).

In cellular cultures of a cell are not organized in fabric whereas for fabric and organ cultures a necessary condition is preservation of a fabric and organotipichesky differentiation and (or) function.

History

the First attempts of cultivation of cells of in vitro were made in 19 century. In 1885 W. Roux was succeeded to keep within several days development of a medullary plate of a chicken embryo in salt solution, and in 1887 to J. Arnold — to observe migration of leukocytes of a frog. However at this time there was no real base for creation of the conditions capable to support life activity of cells and in vitro fabrics. Out of an organism experiences of R. G. Harrison who in 1907 eksplantirovat pieces of a medullary tube of an embryo of a frog in clots of a lymph of a frog are considered as the original beginning of cultivation of cells. In several weeks of cultivation at these cells axons grew. Method of cultivation in a clot of a lymph, is later in a clot of plasma, with addition of embryonal extracts became one of standard and it is used to a crust, time.

Development of methods of cultivation was promoted in many respects by researches of A. Karrel who showed a possibility of long cultivation of cells in the conditions of a strict asepsis. Karrel's bottles are a necessary element of the equipment of modern cultural laboratories. The big contribution to development of methods of cultivation of cells was made by domestic scientists A. A. Maximov, A. V. Rumyantsev, G. K. Hrushchov, N. G. Hlopin and A. D. Timofeevsky. The group of researchers from National cancer in-that (USA) under the leadership of W. Earle developed methods of cultivation of cells on glass, received cultures of single cells and cells in suspension. Result of works of Fischer (D. Fischer, 1948), Morton, Parker, Morgan (N. of J. Morton, R. Parker, J. F. Morgan, 1950), Eagle (H. Eagle, 1955) was creation of modern mediums for cultivation of cells of mammals of in vitro.

After in the 50th 20 century the possibility of reproduction of a virus of poliomyelitis on a number of cellular cultures was shown and there was a practical interest in development of methods K. to. and t., for receiving vaccines this area of researches began to develop promptly. Creation of special environments of cultivation allowed to receive cultures of cells from different bodies and fabrics of many species of mammals and the person (see the table). The special suspension cultures coming on the abilities to give big biomass to cultures of microorganisms were removed. Suspension cultures found application in virology for accumulation of viruses of encephalomyelitis of horses, a tick-borne encephalitis, vesicular stomatitis, a variolovaccine, flu, a disease of Newcastle, and also in production biol, active drugs: enzymes, antibodies, vaccines. Various Cultures of cells and fabrics are applied with the diagnostic purposes for cultivation of viruses (see).

Detection of the phenomenon of spontaneous malignant transformation of cells of in vitro, and also transformation by means of a number of viruses and chemical carcinogens was an experimental basis of studying of mechanisms of carcinogenesis (see. Carcinogenesis ). Methods of cell fusion are developed for formation of hybrids of somatic cells (see. Genetics of somatic cells ), which are used for mapping of genes of mammals. Improvement of methods of cultivation of cells was a basis of development of cytogenetics of the person, identification of the syndromes connected with disturbance of number and structure of chromosomes (syndromes of Down, Shereshevsky — Turner, Klaynfelter, Edwards, Patau, cat's shout etc.). Achievements in the field of cytogenetics and mapping of genes are connected with development of essentially new methods of differential coloring of chromosomes. Improvement of methods of cultivation of amniotic cells of a fruit and equipment amniocentesis (see) allowed to approach the solution of the main questions of medicogenetic consultation — prenatal diagnosis of chromosomal diseases and hereditary biochemical, disturbances of exchange.

The culture of cells is one of pilot models genetic engineering (see).

Besides, methods K. to. and t. are applied in histology to studying of a morphogenesis and funkts, anatomy of cells and fabrics, in immunology to cultivation immunocompetent cells (see) and studying of their functions in hematology for cultivation of cells of marrow and peripheral blood.

The main culture conditions of cells and fabrics

For production, receiving and storage of Cultures of cells and fabrics are necessary sterile rooms, the wide range of salt solutions, mediums and antibiotics. Besides, the following equipment is required: sterile cabinets with uv radiation and installations with a laminar flow of sterile air; microscopes — inverted for viewing of cultures and usual for the analysis of drugs; centrifuges; thermostats with water heating and with controlled giving of CO 2  ; refrigerators for storage of environments, antibiotics, serums, enzymes; installations for freezing and capacity with liquid nitrogen for storage of cellular lines; magnetic stirrers; rubber bungs, tubes, tips; ware from neutral glass like «Pyrex». Ware is boiled in special detergents, repeatedly rinsed a dist, water, sterilized dry heat or autoclaving. Used biol, liquids will sterilize filtering under pressure via membrane filters with a pore size of 220 nanometers after prefiltering via deep filters (from asbestos or glass fiber).

Except the traditional equipment, the modern cultural laboratory uses micropolycups (plastic panels with deepenings) with a set of automatic micropipets and microdosimeters, rolling and suspension installations. Use of plastic ware disposable is desirable.

At maintenance of life activity of cells of in vitro the osmotic pressure, hydrogen ion concentration (pH) and some other inorganic ions, structure of a gaseous fluid, temperature are important. Osmotic pressure in cells of mammals makes at t ° 38 ° apprx. 7,6 atm, and it needs to be supported in culture medium. Osmotic pressure in mediums is created by hl. obr. concentration of sodium chloride. A part is played by other inorganic ions and glucose. High-molecular compounds influence the size of osmotic pressure a little.

Optimum hydrogen ion concentration, i.e. pH of the environment, for growth of the majority of cells is close to neutral values. For growth of diploid fibroblasts it is necessary to support pH within 7,2 — 7,4. The majority of cells, especially constant cellular lines, i.e. the oversown lines, maintain pH from 6,8 to 7,6. The size pH in culture medium depends on a ratio Gidrokarbonat of sodium is a part of the balanced salt solution of a medium, CO 2 it is formed as a result of breath of cells. At cultivation in the closed vessels, in case of low initial concentration of cells, the pH level moves sharply in the alkaline party that affects viability of cells. It can be avoided, blowing into bottles of CO 2 right after crops. Cultivation in open vessels, in thermostats with controlled giving of CO is preferable 2 (5 — 10% of CO 2 and 95 — 90% of air). Wednesdays with a big buffer capacity on the basis of the phosphatic buffer, Tris or HEPES are made (oxymethyl-aminomethane and N-2-гидроксиэтилпиперазин-N-этансульфоновая to - that).

For constant control behind the pH level enter the pH indicator into structure of growth environments, in quality to-rogo often apply phenol - red or phenol-sulfontalein. These substances are not toxic for cells in concentration to 0,002 g/l.

Ions of sodium, calcium, magnesium, iron which not only participate in maintenance of constancy of osmotic pressure are necessary for maintenance of life activity of cells and in vitro fabrics, but are necessary for functioning of a number of cellular enzymes. It is essential as well sufficient concentration of oxygen, edge in the closed vessels is provided with certain relations of air volume and volume of the environment.

Mediums

Cultivation To. to. and t. it is provided first of all with the difficult balanced enrollment of a number of nutritious components of the environment. Wednesdays share on growth and supporting. Growth environments provide growth of cells and fabrics whereas supporting are intended to hl. obr. for preservation of viability and funkts, activities To. to. and t. for an appreciable length of time. In turn, it is possible to distinguish natural mediums from growth environments — products of partial processing of natural highly nourishing substances: the serum of cattle, horse, the person, embryos of calfs inactivated by heating; a hydrolyzate of lactalbumin — a product of enzymic hydrolysis of proteins of milk; Aminopeptidum — an enzymatic hydrolyzate of protein; a haemo hydrolyzate — a product of zymolysis of blood of cattle and the person; embryonal chicken extract. An essential lack of all natural (native) mediums is their non-standard and complexity of sterilization. Synthetic mediums are prepared from certain chemical substances without inclusion of natural products.

On synthetic environments without addition of serum or other natural nutrients not all cells grow. The environment 199, the environment of Eagle and its modification, the F-10 and F-12 circle (Ham), CMRL-1066, NCTC-109, the environment of IAC-which, etc. are most widely applied. Irreplaceable amino acids in a L-form (arginine, a histidine, a leucine, an isoleucine, a lysine, methionine, phenylalanine, threonine, tryptophane, valine) are a part of synthetic mediums, as a rule. Also cysteine and tyrosine are necessary. At the transformed cells the need for a glutamine is sharply increased. For improvement of growth of single cells it is necessary to add serine. The complex of vitamins includes vitamins of group B (paraaminobenzoic to - that, biotin, sincaline, folic to - that, nicotinic to - that, pantothenic to - that, piridoksal, Riboflavinum, thiamin, inositol). Some environments (environment 199, NCTC-109) contain coenzymes and fat-soluble vitamins. The important group of components is made by reducers (ascorbic to - that, L-cysteine, glutathione) and predecessors nucleinic to - t. Glucose, a 2-dezoksi-D-ribose, a D-ribose are a source of carbohydrates. The structure of environments is constantly improved. Addition of serum to synthetic environments improves growth of cells. Synthetic environments with addition 5 — 20% of serum have broad use. For cellular cultures use serum of various origin. H ai has the best growth properties embryonal serum. Completely the role of all components of serum is not found out. Peptides, lipids, amino acids are sources of nutrients. High-polymeric fractions of serum alpha and beta globulins perform protective function, protecting cells from damage. Serum promotes an attachment of cells to substrate and a rasplastyvaniye on it.

Supporting medium, as a rule, the growth environment, in to-ruyu does not enter serum. The special supporting mediums including to gelatin or skim milk are created.

For preparation of working solutions and Wednesdays use the water bidistilled or demineralized by means of ion exchangers. Many primary cultures well grow on the Wednesday containing a hydrolyzate of lactalbumin or a haemo hydrolyzate and serum. Good results are yielded by the F-10 circle (Ham) from 2 — 10% of serum. For cultivation of constant cellular lines use the main environment of Eagle or the environment 199 with addition of 10% of serum, often use the minimum environment of Eagle (MEM). During the work with cellular lines of the increased nutritious requirement environments of IAC-which, Dulbekko, etc. are recommended. Cells of mammals easily adapt to different environments, however cases of difficult adaptation and toxicity of environments are observed. As a rule, the specified cases are connected with disturbance of reference conditions in production of Wednesdays or serums.

Contamination of cultures of cells use of antibiotics

Despite observance of rules of sterility, in the laboratories working with cellular cultures cases of bacterial, mycoplasmal, fungal and virus contamination of cellular cultures happen microorganisms and. Researchers, serum and air can be a source of contamination by bacteria and mycoplasmas. Viruses can get to culture with cells if they are taken from the infected organism, and also with trypsin or serum. Basis of selection of the antibiotics having ability to kill microorganisms are results bacterial, the analysis of pollution and testing of efficiency of action of antibiotics for them. Most often for fight against bacterial pollution use penicillin, streptomycin, Neomycinum, lincomycin, gentamycin, polymyxin, for fight against mycoplasmas — Kanamycinum, miokrizin, Aureomycinum and tylosin, and with fungi — Fungizonum (Amphotericinum In).

Methods of preparation and the characteristic of cultures of cells and fabrics

Cultures of the worrying fabrics

As a rule, for short-term maintenance of life of cells and fabrics use the balanced salt solutions with a certain osmotic pressure and pH of the environment. As an energy source add glucose. For maintenance of the worrying cultures most widely use Hanks, Earl, Tirod, Dulbekko's solutions and Trouell's circle — T8.

Cultures of the growing fabrics

Obtaining cultures of fabrics is based on primary explantation of the growing fabrics isolated directly from an organism. In what slide plates with holes, Karrel's bottles, and also test tubes and perfused cameras are used belong to a basic group of methods.

Explantation of small pieces of fabric for morfol, researches in combination with microcinematography is carried out on a slide plate, a cut then covered with special cover glass and stuck. Modifications of this method are better known: explantation on unary cover glass with a clot of plasma, a cut place over a hole of a slide plate (Garrison); Maximov's method with double cover glass; a method of cultivation in a drop of Wednesday on cover glass [Lewis and Lewis (W. Lewis, M. Lewis)]. Methods of cultivation To. to. and t. on glasses are convenient for short-term researches since long maintaining is connected with need of difficult replacement of a medium. Cultures on cover glasses can be oversown. For this purpose explant is cut on pieces and transferred to new cover glasses. Each piece is processed then as new explant.

Longer cultivation of explants is possible in a clot of plasma in Karrel's bottles [Earl (W. R. Earle)]. The method is used during the formation of lines and strains of cells. For this purpose place a drop of mix of plasma and embryonal extract in Karrel's bottles, to-ruyu distribute on a bottom of a bottle, place pieces of fabric in plasma and allow to be formed to a clot, and then flow a medium. Fabric can be transferred from a vessel to a vessel, cutting pieces or tripsiniziruya fabric and a clot, as in case of obtaining initially tripsinizirovanny cultures.

Cultivation in a clot of plasma in the motionless or rotating test tube, a method of «the flying glasses» are close to cultivation in Karrel's bottles. Explants can be strengthened on walls of a test tube or on narrow cover glasses which place in test tubes with a medium. Test tubes can be placed in inclined situation in the rotating drum for the best contact of explant with Wednesday. The method of «the flying glasses» was widely used for a research of a large number of explants. Instead of a clot of plasma it is possible to use substrates: collagenic, mylar (for further electronic microscopic examinations), plastic, agar, from porous paper, an atsetattsellyuloza. It is possible to eksplantirovat fabric and directly on glass, in particular at the explantation of pieces of skin, macrophages and other cells capable to be attached to glass. A problem of a method of organ cultures is creation of the culture conditions closest to conditions of in vivo. The philosophy of a method consists that explant is placed on border of two phases — the nutritious environment / gas mixture. There are several options of maintenance of organ cultures: cultivation on a surface of a plasma clot, on clock glass in a Petri dish [Fell (N. Fell)] — the classical equipment which is a basis of modern methods; cultivation in the soft (semisolid) agar saturated with nutrient medium, embryonal extract, serum; cultivation on metal (tantalum or stainless steel) the grids covered with an agar [Trouell (O. Trowell)] which lower surface concerns a liquid medium; cultivation on «raft» from an atsetattsellyuloza or tissue paper [J. Chan, 1954; Richter, 1955]; cultivation on milliporovy filters [Grobstein (S. Grobstein), 1955]. For creation of optimal conditions of culture incubate in the gas mixture consisting of 95% of O 2 and 5% of CO 2 with daily shift of a medium. In the equipment of fabric and organ cultures the perfused cameras allowing have a wide spread occurance it is intravital to study developments.

Primary cultures, diploid strains and cellular lines

Irrespective of the relation to substrate it is necessary to distinguish primary cultures and cellular lines. Primary cultures receive directly from normal or tumoral tissues of the person or animals by method of explantation or a tripsinization of the fabrics possessing growth potentialities (embryonal: musculoskeletal fabric, amnion, lung etc.; adults: skin, renal parenchyma etc.). At the same time most often receive cultures of epithelial cells or fibroblasts.

The culture is considered primary until it underwent at least single browning. In the course of browning primary cultures turn into cellular lines.

Cellular lines can be or short-term, i.e. capable to pass limited number of passages, or the constants (oversown, intertwined, stable, transformed), i.e. in vitro, capable to unlimited browning.

Fig. 1. Single-layer (monolayer) culture of cells: on all field of vision the cells of the Chinese hamster growing on glass are visible; x 300.

The general for modern ways of obtaining primary tripsinizirovanny single-layer cultures is processing of fabrics trypsin, is more rare chymotrypsin, papain, Pancreatinum, hyaluronidase, a collagenase. In sterile conditions fabric is crushed, washed out and placed in solution of enzyme on the magnetic stirrer. The received suspension of cells is centrifuged, the deposit of cells is parted with the growth environment to optimum concentration (depending on type of cellular cultures from 100 000 to 500 000 cells on 1 ml of the environment). After crops in cultural bottles of a cell are attached to glass and adapt to conditions of in vitro (a phase of stabilization, a lag-phase), then they begin to proliferate actively (a phase of logarithmic growth, a log-phase), entering a stationary phase with formation of a monolayer (fig. 1).

Kind of monolayer cultures are rolling cultures. Rolling cultivation of cells allows to use all inner surface of a bottle, edges is washed Wednesday as a result of continuous rotation of a bottle with low speed (0,5 — 1 rpm). Alternation liquid and the air phases washing cells creates the most favorable conditions for growth of cells and increases their productivity. Rolling cultivation is applicable for primary cultures, diploid strains, cells, and also constant cellular lines with the expressed adhesive properties of a cellular surface. Success of cultivation on scooters depends also on structure of the environment. Most high performers with perfusion of the environment in the rotating scooters.

There are quantitative methods of assessment of proliferative activity of cells in culture. Mitotic activity is characterized by number of the cells which are in different phases of a mitosis on 1000 counted. Values express in per mille. Depending on type of cellular cultures and culture conditions this size varies. Mitotic activity reaches the greatest values in a phase of logarithmic growth. Thanks to existence at normal cells of a phenomenon contact inhibition of growth (see) formation of a continuous monolayer leads to the termination of cellular proliferation. For receiving new cellular generation the created monolayer is tripsinizirut before formation of single cells which part with the growth environment and pereseivat in new bottles. The number of passages at primary cultures is limited, and after certain time the phenomena of the nonspecific degeneration leading to death of cells develop in them. Change of the environment, creation of optimal conditions of cultivation do not prevent this process.

Fig. 2. The clone of cells of the Chinese hamster growing in a soft agar owing to smaller affinity to substrate; x 200.
Fig. 3. Microdrug of the cells received as a result of several mitotic divisions of one cell — a clone; x 250.

Separate cells in primary cultures can give rise to lines of the changed cells which have a potentiality of boundless growth in culture. Removal of lines of cells from primary cultures lasts for several months. Constant cellular lines (fig. 2) are characterized by the inherited changes of a cellular surface which in general define their big autonomy, smaller affinity to substrate in comparison with stem cells of primary cultures. In some cases the transformed cells lose property of contact inhibition of growth that in turn leads to formation of focuses of multilayer growth. Cells of cellular lines have, as a rule, an unstable heteroploid set of chromosomes. Set of all these features along with property of infinite reproduction in vitro is called transformation. To Mozhnoindutsirovat transformation of cells by means of some viruses and chemical connections. Malignant new growths are other source of receiving constant cellular lines (e.g., the HeLa-line is received from the woman with a planocellular carcinoma of a neck of uterus). However not from all malignant new growths it is possible to receive cellular lines. Use of a method of cloning of cells allows to receive genetically homogeneous line — the clone which is posterity of one cell (fig. 3).

From primary cultures of normal fabrics it is possible to allocate diploid strains of cells which are morphologically homogeneous cultures. Diploid strains shall keep the diploid set of chromosomes characteristic of an individual in the course of browning, from a cut the strain is received. Diploid strains pass three phases of development: stabilization, active growth and aging; they have the long, but limited term of life. The strain of WI-38 of diploid cells of an easy embryo of the person removed by Heyflik and Murkhed is widely known (L. Hayflick, P. S. Moorhead, 1961). Diploid strains receive by method of explantation of pieces of embryonal fabric and crops of a suspension of the embryonic cells shchadyashche processed by trypsin at t ° 4 °. Initial sowing concentration — 10 5 cells on 1 ml of the environment and pH 7,2 — 7,4 is important. Receiving homogeneous suspension since not completely tripsinizirovanny pieces are the reason of early aging of culture is very important. Diploid strains of cells of an embryo of the person maintain apprx. 50 passages. The strains received from fabrics of an adult organism have lower passazhny potential. Cultures of diploid cells of the person use for preparation of the following virus vaccines: poliomyelitic, adenoviral, clumsy, krasnushny, smallpox, rinovirusny, antirabic, against a tick-borne encephalitis. Criteria of suitability of strains of diploid cells for production of vaccines are kariologichesky stability, absence virus, bacterial, mycoplasmal and other contaminations, lack of tumorogenny activity at an inoculation an animal. For use in practice of receiving vaccines strains on the first passages of a phase of active growth freeze and preserve at a temperature of liquid nitrogen.

Except use in virology, diploid strains of cells widely apply to studying of action of various damaging agents on the genetic device of a cell for a research of fermental systems of a cell, in experiments on cellular hybridization, for studying of processes of the induced transformation.

Suspension cultivation

the Method of suspension cultivation gives the chance to receive a large number of cells, in particular for production of the enzymes and other biologically active compounds having scientific and practical value.

The method of suspension cultivation is based on changes of properties of a cellular surface of the oversown cells which have the lowered affinity to substrate. At suspension cultivation high uniformity of cells in culture medium, their long maintenance in a logarithmic growth phase is reached. The easiest way of cultivation — accumulative culture, in a cut of a cell constantly mix up (by means of the magnetic stirrer) in the irreplaceable environment. Speed of hashing is 200 — 300 rpm. Lymphocytes require more high speed — 600 rpm. More effective hashing is reached at circulation of cells in tubes. Without change of the environment in suspension cultures all growth phases of cellular cultures of in vitro are observed: lag-phase, log-phase, stationary phase and phase of logarithmic dying off.

Addition of fresh portions of the environment in devices of suspension cultivation in a logarithmic phase gives the chance to support cells in this phase beyond all bounds long. On the basis of this observation flowing suspension systems in which the speed of receipt of the environment is regulated by the speed of reproduction of cells were developed. Flowing suspension systems can be like hemostat in which giving of the environment is carried out according to the set program with a certain time slice (on the basis of the received parameters of cellular reproduction). The system of turbidostat in which giving of the environment is regulated on the basis of photometric measurements of density of cellular suspension is more perfect. For smooth operation chemo - and turbidostat it is necessary to support optimal conditions of cultivation: pH of the environment, concentration of O 2 and CO 2 , density of cells in suspensions, the corresponding composition of nutrient mediums, necessary temperature.

Suspension cultures successfully use for reproduction of influenza viruses of the person and birds, encephalomyelitis of horses, a tick-borne encephalitis etc. Most widely apply in suspension cultivation of the environment of Higuti, Bertsch, Neygl.

Cultures of cells of in vivo. Optimal conditions for life activity of cells are provided with an organism. Cultivation of cells and in vivo fabrics or browning through a live organism is carried out by their transplantation: a) to embryos; b) an animal with tolerance to cells of the donor; c) to genetically identical individuals; d) to the areas deprived of direct blood supply (an anterior chamber of an eye, a brain, a cornea); e) in diffusion chambers from milliporovy filters; e) using the means oppressing immune system of the recipient. Convenient model of cultivation of cells and fabrics is chorion-allantoisnaya a cover of a chicken embryo.

Many experimental tumors support by serial passages on genetically identical animals. Heterografting in an anterior chamber of an eye is applied to studying of a differentiation of various normal and tumor cells, and also to cultivation of organ cultures.

Fig. 4. Skanoelektronogramma of a cellular surface (it is specified by an arrow) of the cultivated mouse gepatoma; X 3000.

Cultivation of cells in diffusion chambers from milliporovy filters (the AA or HA type, with the time which is not passing a cell) is carried out in an abdominal cavity of animals. Cells in cameras are capable not only is long to exist, but also to be differentiated. Especially often this method is applied to maintaining organ cultures. Possibilities of a research of the cells growing in diffusion chambers, methods of a submicroscopy are perspective (fig. 4.).

Astsitny tumors — a number of the intertwined tumors of animals which are formative in the form of freely weighed cells in exudate of an abdominal cavity. Some of astsitny tumors it is possible to cultivate in vitro that creates unique model system for carrying out experiments, combining methods of the research in vivo and in vitro. Most widely in experimental practice the astsitny form of a carcinoma of Ehrlich is used; besides, the large number of astsitny forms of tumors at rats and mice who are viable in the conditions of in vitro is received.

Receiving gomo-, heterokarionums and cellular hybrids. Methods of receiving gomo-, heterokarionums and hybrids of somatic cells of in vitro are based on opening of G. Barski et al. (1960), found out that somatic cells can merge, forming viable hybrids. Hybridization of somatic cells takes place several stages. At first cells stick together, then cellular covers collapse and the cytoplasmatic bridge is formed. Full cell fusion comes to an end with formation of one general cover. Thus, two-nuclear or multinuclear gomokariona (if identical cells merge) or heterokarionums turn out (if cells of a different origin merge). Gomokariona are of interest to studying of behavior of multinucleate cells in culture. Heterokarionums found application at the solution of questions of regulation, repression and derepression of genes. The large number of researches is conducted on heterokarionums of chicken erythrocytes with HeLa-cells.

Hybrids of somatic cells arise spontaneously with a low frequency of 10 - 6 — 10 - 8 . Processing of cells a parainfluenza virus Sendai (previously inactivated uv radiation or beta propiolactone) increases the frequency of merge to 10 - 2 — 10 - 3 . With use of a virus Sendai is possible to receive hybrids of the somatic cells belonging to organisms of different types, classes (mammal X birds) and even different types (people the X mosquito). For formation of hybrids successfully use polyethyleneglycol (PEG) which allowed to receive cell fusion from representatives of plant kingdom and animals (a vegetable protoplast the X chicken).

For allocation of hybrid cells after their education use special selection environments in which initial, parent, cells perish, and only the hybrids different from parent cells a certain combination of markers, napr, the increased resistance to action of analogs of the bases of DNA or the lowered need for certain metabolites survive.

Fig. 5. A karyotype of a rat (a), mouse (b) and a hybrid cell (in); the Karyotype contains chromosomes of parent lines of cells in a hybrid cell.

For confirmation of a hybrid origin of the allocated cells investigate their Karyotype (fig. 5), define presence of marker parent chromosomes, investigate enzymes and define availability of antigens of parent cells.

Hybridization of somatic cells serves as the main experimental approach to mapping of genes of mammals, in particular the person. It was revealed that at interspecies hybrids at cultivation preferable loss of chromosomes of one of parents is observed. So, at hybrids the mouse of the X people is consistently eliminirut by chromosomes of the person that is used for mapping of chromosomes of the person. Development of methods of hybridization of somatic cells made possible the analysis of inheritance of a zlokachestvennost at the cellular level, studying of mechanisms of a differentiation of cells and a problem of regulation of genes.

Except the described hybridization of somatic cells, special methods of receiving hybrids are developed for the analysis of mechanisms of cytoplasmic inheritance. Also hybrids of cells of one type with separate chromosomes from cells of other type, with biochemical confirmation of functioning of these chromosomes in a hybrid cell are received.

Freezing and storage of cells at a low temperature

at the end of 19 century was shown that bacteria, seeds of plants and difficult biochemical, connections can is long to remain at low temperatures. In the 50th 20 century attempts to use during the freezing of cells of substance, increasing survival were made — cryoprotectors. So, Poldzh (S. of Polge, 1949) found out that the glycerin added to suspension of cells considerably increases their survival. Since 60th the dimethyl sulfoxide (DMSO) came under the spotlight. It is widely used as a cryoprotector for many types of cells. Consider that strengthening of electrolytes in a cell or about it is the main reason of damage of cells during the freezing. At a temperature below 0 ° in cells crystals of ice which increase in process of fall of temperature are formed, in the remained environment also concentration of electrolytes respectively increases. Crystals of ice are not formed at gradual decrease in temperature. Electronic and microscopic observations demonstrate that very slow freezing and bystry thawing cause the smallest damage of cytoplasmatic structures of a cell. For conservation the growing cells of culture are removed from glass trypsin, washed out the growth environment and place in the environment of freezing in concentration of cells 1 million/ml and more. The environment of freezing — the usual growth or supporting medium containing from 2 to 25% of serum and cryoprotectors: glycerin of 5 — 10% or DMSO of 5 — 15%. Cells place in special sterile plastic or glass ampoules, close or solder and begin to cool. To — 20 ° fall of temperature is conducted with a speed of 1 ° in 1 min. During the freezing of pieces of fabric they are crushed to the size of 1 mm3 and carry out similar procedures. Further cooling from — 20 ° and up to the temperature of storage is carried out quickly. Store the frozen cells in refrigerators with liquid nitrogen. Storage in a gas phase is preferable, over liquid nitrogen where temperature makes apprx. — 130 °.

Process of thawing shall be very bystry. Ampoules from liquid nitrogen transfer to the water bath t ° 40 °. As soon as suspension completely thaws, it is parted by 1: The 10th warm growth environment also disseminate. It is possible to ottsentrifugirovat previously cells and to fill in with the fresh growth environment. Pieces wash out and as soon as possible inoculate. After 6 months of storage of the cultures frozen and thawed in the described way 100% survival of cells are observed.

Transportation of cells on long distances is possible either in the form of suspension, or in the form of a monolayer in the cultural bottle which is completely filled with the environment. Transportation of cells in the frozen state at a temperature of boiling of liquid nitrogen in special containers is possible and on dry ice.

Cultures of cells and fabrics in radiobiology

Cultures of cells and fabrics are widely applied in radiobiology. Interest in them is caused by an opportunity to study the radiopathology of a cell isolated from influence of other cellular systems or neurohumoral factors which take place in an organism.

Cultures of cells use during the development of questions of the mechanism of action of ionizing radiation on a cell (see. «Targets» theory ), problems of cellular radio sensitivity, questions of defeat and recovery of cells at action of ionizing radiation, etc. Consistent patterns of change of frequency of aberation chromosomes depending on dosage rate of radiation are determined, e.g. At rather big dosage rates of radiation (more than 0,5 — 1 r / mines) the frequency of aberation chromosomes grows with increase in an ionizing radiation dose and makes about 0,15% for everyone is glad doses. In the conditions of use of low powers of doses of radiation (0,2 — 0,0002 r / mines) this dependence (a dose — effect) significantly changes; growth of effect (e.g., frequencies of aberation chromosomes) from an ionizing radiation dose is expressed to those more weakly, than dosage rate of radiation is less that demonstrates decrease biol, efficiency of ionizing radiation. At long action of low powers of ionizing radiation doses there is a stabilization of effect, at a cut, despite the continuing radiation of cultures, there is no further increase in frequency of chromosomal damages any more though the total doses received by cell population can make 5000 — 10 000 rubles. Level of stabilization of effect in these conditions is various and proportional to the size of dosage rate of radiation. Observed changes connect with the fact that in the conditions of long action of ionizing radiation radiation takes a lineage of cells. In the irradiated population balance between intensity of emergence of beam damages to cells and speed of their reparation is established.

Use of synchronous cultures allowed to obtain new data on action of radiation on cell life cycle: considerable fluctuations of radio sensitivity are revealed, the nature of emergence of aberation and chromatid chromosomes is in details tracked, the sites of a cellular cycle responsible for a delay of synthesis of DNA and a mitosis, etc. are found.

For example, the greatest radio sensitivity is, as a rule, characteristic of the cells which are at the time of radiation in the period of a mitosis (M) whereas in presinteticheskiya (G1) or premitotic (G2) the periods they by 2 — 5 times more radiorezistentna. During synthesis of DNK(S) radio sensitivity has intermediate value more often, but signs of the increased reparative ability of cells are noted.

In radiogenetics methods of cultivation provide not only a possibility of preparation of the high-quality drugs for the thin chromosomal analysis (metaphase plates) of different types of cells including and which are seldom sharing (cultivation with phytohemagglutinin), besides, cultures of various cells are an object for versatile researches of action of radiation on genetic structures of cells. They have paramount value in a research of gene transmutations and mutations. With their help are revealed biochemical, the mutants steady against medicines, with the changed radio sensitivity, etc.

fig. 6. Dependence of frequency of damages of chromosomes (emergence of dicentrics) on an exposure dose to culture of leukocytes of peripheral blood counting on one cell: the effect increases at increase in an exposure dose (curves are constructed according to various authors).

For diagnosis of radiation defeats the greatest value has determination of frequency and type of the aberation chromosomes arising in cells of marrow and peripheral blood (fig. 6) so far.

Fig. 7. The curves characterizing survival of the cells of the Chinese hamster irradiated in a presintetichesky stage (2) in combination with administration of drug Budr (1) and Cysteaminum (3): the protective effect of Cysteaminum and the radio sensibilizing effect of drug BUDR is noted.

Cultures of cells find application during the studying of operation of chemical radioprotectors and radio sensitizers. Administration of various chemical substances in a medium of the irradiated cultures with the subsequent analysis of survival, frequency of aberation chromosomes or other objective indicators of radiation injury of cells allows quickly and with an adequate accuracy to judge character and efficiency of their action on cells (fig. 7).

There are, however, also certain restrictions of use of cultures in radiobiol. and other researches. They are connected first of all with the fact, that in single-layer cultures it is possible to provide only reproduction of cells whereas the second extremely important process of cell activity — process of a differentiation — quickly fades. Therefore in radio biological researches use the intertwined cellular lines, but not primary cultures more often.

Cultures of cells and fabrics in oncology

Cultures of cells and fabrics as the method of scientific research is widely applied in modern oncology. The method allows to study at the cellular level etiol, factors and pathogenetic mechanisms of process of a malignancy, biol, features and patterns of behavior and interaction of normal and tumor cells, mechanisms of effect of carcinogenic and antineoplastic substances, and also others chemical, physical. and biol, agents. In system K. to. and t. cultivation is possible oncogenous viruses (see).

In conditions To. to. and t. naturally the phenomenon of carcinogenesis or a malignancy of normal cells is reproduced. A malignancy of cells in To. to. and t. can occur under the influence of oncogenous viruses, chemical carcinogenic substances, and also without special influences (spontaneously).

A. Karrel in 1924 reported about the first attempts to cause a malignancy of cells out of an organism. For this purpose the author used a virus of sarcoma of Raus. Developing of tumors on site of an inoculation of the chicken monocytes processed by a virus left open a question whether development of tumors is result of a malignancy of cells of in vitro or a consequence of action of the virus which bred in culture of cells. Further, in process of allocation and studying of other oncogenous viruses, their direct action on cells in was shown To. to. and t. Oncogenous viruses (DNA - and RNA-containing) cause in To. to. and t. development of process of transformation, in the course to-rogo a cell undergo inherited morfol., kariologichesky, fiziol., antigenic and other changes. In cases of malignant transformation of a cell gain ability to form at an inoculation an animal of a tumor. It is most studied in To. to. and t. action of such oncogenous viruses as virus of a polioma, SV40, adenovirus, virus of sarcoma of Raus, etc. Some Oncogenous viruses are capable to cause malignant transformation of cultures of cells of the person.

Since Fischer's experiences (A. Fischer, 1926) conducts researches with the purpose to cause a malignancy of cells chemical carcinogenic substances. M. A. Magat, A. D. Timofeevsky, L. F. Larionov, etc. after influence by multinuclear aromatic hydrocarbons (1, 2, 5, 6-dibenzanthracene, 3,4 benzpyrene) observed in cultures emergence morfol, the changes similar to transformation of a virus origin. In the subsequent experiences with carcinogenic substances, hl. obr. in the conditions of monolayer cultures, different authors managed to cause approach of malignant transformation. Nevertheless the possibility of presence at cells latent, including oncogenous, viruses leaves open a question of independent maligniziruyushchy action on cells of chemical carcinogenic substances.

In 1941 — 1943 in Guy's laboratories (G. The lake of Gey) and W. Earle was established that the malignancy of cells can result from long cultivation without special influences. The so-called spontaneous malignancy comes in monolayer cultures several months later after the beginning of cultivation. In cultures signs of transformation are observed: the morphology changes, chromosome number (heteroploidy) increases in cells, growth rate increases. The inoculation of such cultures an animal often causes development of tumors. The reasons of a spontaneous malignancy in To. to. and t. finally are not studied. Significance is attached to factors of a medium, lack of homeostatic influences of an organism, transfer of cells in specific conditions To. to. and t. etc. There are data on participation in a spontaneous malignancy of oncogenous viruses, mycoplasmas.

Cultures of cells and fabrics are convenient experimental system for cultivation and studying of tumors out of an organism.

The first attempts to cultivate (to eksplantirovat) tumors of the person were made in 1910 by A. Karrel and Burroughs (M. T. Burrows). In 1913 P. P. Avrorova and A. D. Timofeevsky for the first time managed to be grown up a blood cell and marrow of patients with leukoses, and in 1914 they eksplantirovat several sarcomas of the person. The subsequent numerous researches of explants of tumors showed that tumor cells in To. to. and t. could show signs of fabric accessory that allowed to investigate a histogenesis of a number of tumors of the person. The myogenetic nature of some sarcomas was shown, genesis of new growths of ovaries, a uterus, an enclavoma of a parotid sialaden, the low-differentiated tumors of a lung, new growths of c is specified. the N of page, proved the neurogenic nature of melanomas etc. Noted ability of anaplazirovanny tumor cells to a differentiation (maturing) according to their genesis, according to A. D. Timofeevsky, allows to raise a question of a possibility of the directed change them biol, properties.

Fig. 8. Native microdrugs of cultures of cells of tumors of the person (shooters specified epithelial cells of a tumor): and — a solid breast cancer, x 56; — a cystadenocarcinoma of an ovary, x 56; in — bronchogenic cancer of a lung, x 80; — an adenocarcinoma of a stomach, x 80

The habit view of some cultures of cells of tumors of the person is presented in the figure 8.

Fig. 9. Development of epithelial-like heteroploid cells of the cellular CaVe line from a carcinoma of a stomach of the person: and — microscopic section of an initial solid carcinoma of a stomach; coloring hematoxylin-eosine, x 80; — transformation of culture in 1,5 — 2 months after crops of an initial tumor (shooters specified emergence of islands in epithelial-like cells); coloring by hematoxylin, X 80; in — a monolayer of homogeneous epithelial-like cells (native drug, x 80)

Explantation of tumors of the person pursued the aim to receive long cultures of tumor cells. However at cultivation on a plasma layer the growing capacity of cultures weakened and cells perished. Only development of a technique of single-layer cultures using synthetic mediums allowed is long to support growth and reproduction of tumor cells of the person and animals. In the conditions of a monolayer, as well as on a plasma layer, life expectancy of tumor cells is limited if in culture there do not occur the changes which are expressed in emergence of epithelial-like heteroploid cells which force out other cellular forms (fig. 9). Cells of such transformed cultures of tumors are capable to unlimited growth out of an organism. In transformation the role of viruses, mutagen substances, radiation etc. is not excluded.

Cultures of the transformed tumor and normal cells (cellular lines) find similarity of characteristics. Along with similar epithelial-like morphology and a submicroscopic structure in cultures cells with heteroploid, more often okolotriploidny karyotypes prevail. The proliferative pool of such populations is high, and duration of a cellular cycle is small. The transformed cultures, losing the majority of antigens, keep specific specificity and buy cultural antigen. Tumor cells keep tumoral antigen, and normal at a malignancy — get it. In cultures against the background of the lowered breath glycolysis amplifies, affinity to glucose increases, the isoenzymatic range of some enzymes etc. changes. Standardization of characteristics of cultures in some cases raises a question of pollution (contamination) of one cellular cultures by others, including cells of HeLa that in each case shall decide individually.

Fig. 10. Microdrugs of cellular lines from tumors of the person (monolayer cultures): and — the HeLa line from a carcinoma of a neck of uterus; — the KV line from a carcinoma of an oral cavity; in — the CaPa line from a carcinoma of a pancreas; — the AS line from an angiosarcoma. Coloring by hematoxylin, x 200.

The first HeLa line of tumor cells of the person was received in 1951 from planocellular cancer of a neck of uterus (Guy et al., 1952). Are known and are used in various researches of the KV line from a carcinoma of an oral cavity [Eagle (N. by Eagle), 1955], H ER-2 of a carcinoma of a throat [Moore (And. Moore) et al., 1955], series of the cellular Detroit lines [Berman and Stalberg (L. Berman, S. Stulberg), 1957], etc. In our country are received and are supported the AS line from an angiosarcoma (A. M. Eroshkina, 1956), the DAPT line from the differentiated astrocytoma (M. S. Benyumovich with sotr., 1962), the lines CaPa, CaVe, CaOv, TuWi from carcinomas of a pancreas, stomach, ovary and Vilms's tumor (Ya. V. Dobrynin with sotr.). Dozens of lines of tumor cells of the person and animals which are grown up in single-layer culture (fig. 10), and also the line of blood cells and marrow of patients with leukoses, supported in the form of suspension cultures are described. Many lines of tumor cells of the person supported in single-layer culture are options of the HeLa line.

Fig. 11. Skanoelektronogramma of cells in monolayer culture (are specified by shooters): and — the normal fibroblasts spread and well attached to a substrate; — the malignizirovanny fibroblasts of the line L (rounded and poorly attached to a substrate of a cell); X 2000.

V K. to. and t. features of behavior and interaction of tumor cells are studied. According to Aberkrombi and Ambrose (M. of Abercrombie, E. Ambrose, 1958), the behavior of tumor cells is defined by loss of ability to slow down the movement at contact with other cells (see. Contact inhibition of growth ), to detain the introduction in a mitosis at contact, and also to be guided by surfaces. According to Yu. M. Vasilyev (1968), in a basis patol, behavior inability of tumor cells to contact inhibition of the movement, how many the broken reaction of an attachment due to defect in the organization of lamellar cytoplasm — the structure forming at contact of a cellular surface with substrate (fig. 11) lies not so much.

V K. to. and t. decrease was for the first time noted and loss of opukholeobrazuyushchy properties it is long the cultivated tumor cells. These observations concerned as cells, malignizirovavshikhsya in culture [Earl and Sanford (K. K. Sanford), 1958], and received directly from tumors [G. Foley, 1965]. Phenomenon of loss of a zlokachestvennost in To. to. and t. it is a little studied. It is supposed that decrease and loss of opukholeobrazuyushchy ability can be caused by selection of more mature and less malignant cells under the influence of factors of the environment.

It is assumed that the perversion of an antigenic complex owing to long cultivation of tumor cells can lead at transplantation to development of a tissue incompatibility.

Cultures of cells and fabrics are used for studying of effects and mechanisms of action various biol., physical. and chemical agents. Use is important To. to. and t. for studying of the chemotherapeutic drugs used in onkol. to clinic, and selection of new antineoplastic substances. Total quantity and death toll and viable cells, contents in cultures of protein and nucleinic to - t, extent of oppression of enzymes, level of inclusion by cells of radioactive precursors of synthesis nucleinic to - t, a squirrel etc. can be criteria of biological (cytotoxic) effect of such substances. Cultures of cells and fabrics allow to select potentially antineoplastic cytotoxic substances while the research of their specific antineoplastic activity is possible only in animal experiments.

V K. to. and t. define individual sensitivity of tumors of the person to antineoplastic drugs for the purpose of the subsequent medicinal treatment. For this purpose preferential use short-term cultures of tumors. Criteria for evaluation of effect of drugs the same that at selection of tsitostatik. Definition of a range of sensitivity of tumors to a set of antineoplastic drugs (onko-biogramm) allows to choose more effective for this patient antineoplastic drug On correlation yielded onkobiogramma and there are no results of the subsequent treatment a consensus.

Development and improvement of a method K. to. and t. promoted its wide and successful use in various onkol, researches. Many are studied biol, properties and features of tumor cells, some reasons and mechanisms of carcinogenesis etc. However a number of key problems of oncology, such as spontaneous malignancy, the differentiation of tumor cells, loss of opukholeobrazuyushchy and other properties by them, still expects the decision.

Cultivation of marrow

Marrow is an irreplaceable object for studying of processes of proliferation and a differentiation of cells in general and the hemopoietic cells in particular. It represents the heterogeneous cell population consisting from the proliferating and differentiated cells. The differentiation proceeds in the form of a number of consecutive stages, easily distinguishable functionally or morphologically. Unlike the majority of other fabrics, marrow can be easily received from the living person.

History of studying of marrow in culture begins with A. Karrel and Burroughs's work (M. T. Burrows) cultivating marrow and a spleen in plasma a clot in 1910.

In the next years methods of cultivation of the hemopoietic fabrics developed and used by A. A. Krontovsky, A. A. Maximov, A. Fischer, Efrussi (V. Ephrussi), etc. In spite of the fact that in these works the main features of behavior of the hemopoietic cells in cultures were established, methodical approaches of cultivation of marrow are developed, schemes of a hemopoiesis are specified (first of all A. A. Maximov), researchers did not manage to receive true cultures of marrow, i.e. cultures in which there is an increase of mass of eksplantirovanny cells. This task is not solved also by the end of the 70th of 20 century. At all used methods of cultivation the hemopoietic cells it is long worry and samopodderzhivatsya without essential increase in their number.

Methods of cultivation of marrow can be divided into two groups — clonal and not clonal.

Clonal methods

the Basic purpose of such cultures consists in identification of various categories of progenitors the, i.e. cells capable to do sufficient number of divisions for formation of a clone from several tens — thousands of cells (see. Hemopoiesis ). Necessary conditions for obtaining clonal growth — the Wednesday providing a relative immobilization of cells (that cells descendants did not migrate far from a zone of proliferation), the low density of crops (that clones did not block each other) and presence at the environment of the factors promoting proliferation and a differentiation of klonoobrazuyushy cells. These conditions managed to be satisfied for progenitors of all three sprouts of a hemopoiesis.

Cultivation of progenitors of granulocytes and macrophages. As the immobilizing environment use an agar (final concentration of 0,3%) or methyl cellulose (final concentration of 0,8%). For growth use the chemical environments differing for different types of animals. Most often use the environment of IAC-which 5A, the environment 199, RPMI 1640, the environment of Eagle, etc. In serum-free environments clones are not formed therefore addition 10 — 30% of serum is obligatory (most often embryonal veal, placental human, etc.). Without addition of an exogenous factor — colony stimulating activity (KSA) of colony are not formed at the usual densities of plating (1 — 3 X 10 5 cells on 1 ml of the environment); the insignificant number of colonies develops at the high density of cells, an order 10^6 cells on 1 ml of the environment above. As a rule, cultures conduct in the presence of an exogenous factor — colony stimulating activity. As the last use the conditioned environments from cultures of various cells and fabrics (fibroblasts, monocytes, malignant cells, kidneys of newborn animals, lungs, a placenta, etc.), serums from normal animals or animals with a leukosis or receiving endotoxin etc. Apply as well a feeder (the cellular underlayer conditioning the environment). In this case the feeder is usually cultivated in a sublayer of more dense agar (0,5%) on which layer cells of marrow in a soft agar (0,3%). Such option of a method is most often used at cultivation of marrow of the person. In this case as a feeder use leukocytes of peripheral blood in concentration of 106 cells on 1 ml of the environment.

Fig. 12. Types of colonies of cells of marrow in agar culture: and — compact (granulotsitarny); — diffusion (Macrophagic); in — mixed; x 36.

Density of cells of marrow at crops usually 1 — 3 X 105 cells on 1 ml of the environment. Colonies are recorded under the inverted microscope after an incubation within 7 — 14 days at t ° 37 ° in the atmosphere representing mix 5 — 10% of CO 2 with air (gas phase). Take the cellular accumulation consisting usually from more than 50 cells for colony; all cellular accumulations of smaller size are called clusters. It is supposed that clusters are formed by more mature cells capable to do smaller number of mitoses in culture. Three types of colonies — compact (granulotsitarny), diffusion are observed (macrophagic) and mixed (fig. 12).

Cultivation of progenitors of erythrocytes. The principles of such cultures the same, as for predecessors of granulocytes. As the supporting substrate use a plasma clot (10% citrate plasma of cattle) or methyl cellulose (the agar is not suitable for these cultures). Emergence of clones requires presence at the environment of erythropoetin (apprx. 0,25 pieces/ml). Distinguish two types of colonies. One of them — the small colony consisting of 8 — 32 cells. The account of such colonies is carried out in 2 — 3 days after explantation. In later terms of a cell in colonies ripen to erythrocytes and lyse. In 5 — 10 days of cultivation in the presence of higher concentration of erythropoetin (to 10 pieces/ml) there are big colonies (over 32 cells, usually — several hundred cells) or the centers from several small colonies. Such center received the name of a burst (English burst-explosion). Assume that these types of colonies are formed by different predecessors. Less mature predecessors produce bursta, more mature — small colonies.

Cultivation of progenitors of megacaryocytes. These predecessors form colonies of magakariotsit in agar cultures in the presence of mercaptoethanol (final concentration 5 X 10 - 5 М). As a source of colony stimulating activity use a conditioned medium of cultures of splenic lymphocytes. Duration of cultivation is 7 — 10 days.

Fig. 13. Colonies of fibroblasts in monolayer cultures of marrow of a Guinea pig (are specified by shooters): and — a habit view of colonies (it is reduced twice); — a fragment of colony; x 400.

Cultivation of progenitors of stromal mekhanotsit. At cultivation of marrow in culture proliferate not only the hemopoietic, but also stromal cells. The last are strongly attached to glass and are rather slow-moving in this connection formation of clones can be observed not only in semi-fluid, but also in liquid cultures where clones are formed in the form of the cellular accumulations (fig. 13) attached to a bottom of a vessel. Such cultures are conducted in complete mediums (chemically certain environment with additive of 10 — 30% of serum, most often embryonal veal). Cellular concentration at explantation 1 — 3 X 105 cells on 1 ml of the environment. The quantity of the environment decides by the area of a bottom of the culture vessel (about 1 ml of the environment on cells on 4 cm2). Colonies consider in 10 — 14 days of cultivation a naked eye after coloring (e.g., hematoxylin).

Clonal methods of cultivation of marrow use very widely; they became already usual methods of a research of patients in many gematol, clinics of the world. With their help extremely important data on the hemopoietic system were obtained. In particular, it was succeeded to show that marrow and other hemopoietic bodies contain progenitors, intermediate between a stem hemopoietic cell and morfol, recognizable cells of the corresponding ranks of a hemopoiesis. In this regard all categories of the hemopoietic predecessors combine in the department of the committed predecessors which received the name of buffer department. The following categories of predecessors are revealed: a progenitor of granulocytes and macrophages, colony-forming unit in culture (BEDS); early erythroidal predecessor, burstoobrazuyushchy unit (BOEe); late erythroidal predecessor, colony-forming unit erythroidal (KOEe); predecessor of megacaryocytes, colony-forming unit megakariotsitarny, (WHICH); stromal predecessor, colony-forming unit fibroblastny (KOEf). All categories of the hemopoietic predecessors represent morphologically a cell, intermediate between a lymphocyte and a blast. At a normal hemopoiesis these cells proliferate and rate of their proliferation is regulated by hormones. Only one of such hormones — erythropoetin is in details characterized; whether leykopoetina are and trombopoetina regulators, experimentally it is not proved. Cellular cycles of predecessors and their descendants in culture are established. The factors stimulating proliferation of colony-forming units in culture are found and some marrowy populations capable to products of these factors are revealed. It is proved that at leukoses can suffer both progenitors, and producing colony stimulating activity of a cell. Correlations between the course of a leukosis and the characteristic of colony-forming units in culture etc. are found.

It turned out that cells of stromal predecessors at a stable hemopoiesis in an organism do not proliferate. They are capable to construct a stroma of marrow at transplantation, on to-ruyu there is a repopulyation of the hemopoietic cells, and there is a center of an ectopic hemopoiesis.

In general use of clonal methods of cultivation of marrow promoted detection of new data on a structure of the hemopoietic system, about existence and properties of a number of categories of progenitors, about regulation of proliferation and a differentiation of these cells etc. Use of these methods continues to grow intensively.

Not clonal methods of cultivation of marrow

the Main objective of these methods of cultivation — obtaining stable long cultures of the hemopoietic fabric, with constant products of the hemopoietic cells, including and stem hemopoietic cells — is still not solved. At all existing methods of culture more or less quickly are exhausted or in them growth, but not the hemopoietic, but stromal cells continues. However and in such look not clonal cultures widely use for studying of a number of fundamental issues of functioning of marrow.

Organ cultures of marrow. In organ cultures the organotipichesky growth of fabric, without increase in cellular mass of explant is supported. In such cultures marrow a fragment or a dense suspension is eksplantirut on the substrate (milliporovy filters, various membranes, etc.) which is on a surface of the environment. Nutrients diffuse from the environment in explant, and waste products of the last diffuse on Wednesday. Growth on border of liquid and gas phases promotes preservation of organ structures that extends functioning of culture. Such hemopoietic fabric as an embryonal liver, can be supported in culture within several weeks. Composition of nutrient medium and a gas phase same, as well as at clonal methods of cultivation.

Cultivation of marrow in fluid mediums. Unlike organ cultures, at cultivation of marrow in fluid mediums use cellular suspensions, but not fragments of the hemopoietic fabrics. Density of explantation — usually from 0,5 to 2 million cells in 1 ml. A medium is changed in process of acidulation, depending on intensity of growth of cells. Option of cultures of this sort is Marbruk's system, at a cut marrowy cells with a small amount of the environment (1 ml) place in a vessel with a bottom from a cellophane membrane. This vessel is placed in a flask from 50 ml of a medium. Such system does not demand frequent change of the environment and at the same time provides sufficient cellular density.

Not clonal methods of cultivation of the hemopoietic cells widely use for studying of kinetics of various cell populations, for studying of various biochemical processes in cells and mechanisms of their induction (e.g., synthesis of hemoglobin in erythroidal cells), for a research of cellular differentiations and morfofunktsionalny characteristics of various populations of the hemopoietic cells. The important place is taken by studying of cooperative interactions of cells in culture, humoral factors of regulation of a hemopoiesis etc. Attempts of obtaining true culture of marrow with increase in mass of the hemopoietic cells continue.

By means of not clonal cultures cellular cycles of many categories of the hemopoietic cells are studied, the factors inducing a differentiation in normal and some leukemic cells are investigated, the most sensitive system of definition of erythropoetin is created, many factors stimulating a hemopoiesis, etc. are characterized. The first data on a possibility of long maintenance of a hemopoiesis in culture are obtained.

Cultivation of marrow in diffusion chambers. At such method of cultivation marrow in the form of a cellular suspension in a full medium is placed in the cell (volume of 0,1 — 0,2 ml) with walls from milliporny or, better, nukleoporny filters which size of a time (0,22 microns) does not interfere with penetration into the camera of nutrients and removal of products of exchange. At the same time cameras are impenetrable for cells. Diffusion chambers place intraperitoneally to animals, usually mice. Since cameras are impenetrable for cells, including and for lymphocytes, expressed immunol, reaction against cells in the camera does not happen, and in an abdominal cavity of mice it is possible to cultivate cells of any kinds of animals and the person. Growth of cells is better if the camera is placed in an organism of the irradiated recipient. Usually cultures investigate during the first 7 — 9 days. If longer cultivation is required, cameras clear of the connecting fabric which accrued outside once a week and transfer to the following recipient. At renting of cultures the camera is processed enzyme pronazy, edges releases the hemopoietic cells and allows to transfer them to a suspension for studying of number, morfol, and funkts, features etc.

Such option of cultures along with obvious shortcomings (impossibility of studying of humoral factors, feeding habits, the nature of regulators etc.) has a number of advantages — simplicity, good reproducibility, simplicity of environments, the best conditions for cells that does a method more and more popular. With its help kinetics of transformation of stock cultures in cameras, the direction of a differentiation of the hemopoietic cells, intercellular interactions (in the double cameras divided by the filter), etc. are studied.

The problem of cultivation of marrow is in a stage of active development. In the long term these researches — creation of a method of definition of stem hemopoietic cells in culture, creation of stable cultures of the hemopoietic cells, receiving a gain of cellular weight in cultures of marrow. The solution of these problems will be one of the major steps in progress of knowledge in the field of a normal hemopoiesis and mechanisms of its malignancy.


Table. The short characteristic of the main cultures of cells (according to domestic and foreign literature)

— Means lack of data on this question.



Bibliography: Golubev D. B., Sominina A. A. and Medvedev M. N. The application guide of cellular cultures in virology, L., 1976, bibliogr.; Iranov S. D. and Hesin Ya. E. Organ cultures in virologic researches, M., 1977, bibliogr.: Culture of nervous tissue, under the editorship of Yu. M. Zhabotinsky, M., 1977, bibliogr.; New methods of culture of animal fabrics, the lane with English, under the editorship of Yu. M. Olenov, M., 1976; P about D. Kultur's l of cells and fabrics, the lane with English, M., 1963; The Guide to cultivation of nervous tissue, under the editorship of B. N. Veprintse-va, M., 1976, bibliogr.; The X l about p and N. G. Kultur's N of fabrics, L., 1940; about N e, All-biological and experimental fundamentals of histology, L., 1946, bibliogr.; E f r at with with and B. Hybridization of somatic cells, the lane with English, M., 1976, bibliogr.; Cells and tissues in culture, ed. by E. N. Willmer, v. 1—2, L. — N. Y., 1965; D u 1 b e with with o R. Topoinhibition and serum requirment of transformed and untra-nsformed cells, Nature (Lond.), v. 227, p. 802, 1970; HayflickL. Moorhead P. The limited in vitro lifetime of human diploid cell strains, Symp. Int. Soc. Cell Biol., v. 3, p. 155, N. Y. — L., 1964; L a-v a p p a K. S. Survey of ATCC of human cell lines for HeLa contamination, In vitro, v. 14, p. 465, 1978; Readings in mammalian cell cultures, ed. by R. Pollack, N. Y., 1975; Tissue cultures, ed. by P. F. Kruse and. M of K. Patterson, N. Y., 1973. To. to. u of t. in radiobiology — Bochkov H. P. Chromosomes of the person and radiation, M., 1971, bibliogr.; To about r about of odes and V. I N. Problems of post-radiation recovery, M., 1966, bibliogr.; About to and d and Highway. Radiation biochemistry of a cell, the lane with English, „M., 1974.

To. to. and t. in oncology — Vasilyev Yu. M. and Malenkov A. G. Cellular surface and reactions of a cell, L., 1968, bibliogr.; Timofeevskaya E. A. Assessment of effect of antineoplastic drugs in culture of fabrics, Vestn. USSR Academy of Medical Sciences, No. 9, page 70, 1966; Timofe-e in with to and y A. D. Eksplantation of tumors of the person, M., 1947, bibliogr.; it, Long cultures of fabric and malignancy of cells, * Vestn. USSR Academy of Medical Sciences, No. 11, page 3, 1964; Frankfurt O. S. Cellular mechanisms of chemotherapy of tumors, M., 1976, bibliogr.; III and and d L. M., Kolesnichenko T. S. and Sorokin Yu. D. Transplacental blastomogenesis and organ cultures, M., 1975; Advances in tissue culture, ed. by C. Waymouth, Baltimore, 1970; Aktuelle Probleme der Zellzuchtung, hrsg. v. B. Mauersberger, Jena, 1971; Harris M. Cell culture and somatic variation, N. Y., 1964; Human tumors in short term culture, ed. by P. P. Dendy, L. — N. Y., 1976.

Cultivation of marrow — Luriya E. A. The hemopoietic and adenoid tissue in cultures, M., 1972, bibliogr.; Chertkov I. L. and F r and d e of N matte of A. Ya. Cellular bases of a hemopoiesis (Hemopoietic progenitors), M., 1977; Metcalf D. Hemopoietic colonies, in vitro cloning of normal and leukemic cells, B., 1977, bibliogr.; Metcalf D. a. The M o o r e M. of A. S. Haemopoie-tic cells, Amsterdam — L., 1971; Murray M. R. a. K o p e with h G. A bibliography of the research in tissue culture, 1884 to 1950, v. 1—2, L. — N. Y., 1953.


T. H. Ignatova, T. V. Pospelov; V. A. Gubin (I am glad.), Ya. V. Dobrynin (PMC.), I. L. Chertkov, (gems.), authors of the tab. T. N. Ignatova, T. V. Pospelov.

Яндекс.Метрика