The use of cell cultures. Biotechnology technologies: cell cultures

The rudiments of organs grown outside the body (in vitro). The cultivation of cells and tissues is based on strict observance of sterility and the use of special nutrient media that ensure the maintenance of the vital activity of cultivated cells and are as similar as possible to the environment with which cells interact in the body. The method of obtaining cell and tissue culture is one of the most important in experimental biology. Cell and tissue cultures can be frozen and stored for a long time at liquid nitrogen temperature (-196°C). The fundamental experiment on the cultivation of animal cells was carried out by the American scientist R. Harrison in 1907, placing a piece of the rudiment of the nervous system of a frog embryo into a lymph clot. The germ cells remained alive for several weeks, nerve fibers grew out of them. Over time, the method was improved by A. Carrel (France), M. Burroughs (USA), A. A. Maksimov (Russia) and other scientists who used blood plasma and an extract from the tissues of the embryo as a nutrient medium. Later progress in obtaining cell and tissue cultures was associated with the development of media of a certain chemical composition for culturing various types of cells. Usually they contain salts, amino acids, vitamins, glucose, growth factors, antibiotics, which prevent infection of the culture with bacteria and microscopic fungi. F. Steward (USA) initiated the creation of a method for cell and tissue culture in plants (on a piece of carrot phloem) in 1958.

For the cultivation of animal and human cells, cells of different origin can be used: epithelial (liver, lungs, mammary gland, skin, bladder, kidney), connective tissue (fibroblasts), skeletal (bone and cartilage), muscle (skeletal, cardiac and smooth muscles ), the nervous system (glial cells and neurons), glandular cells that secrete hormones (adrenals, pituitary, cells of the islets of Langerhans), melanocytes, and various types of tumor cells. There are 2 directions of their cultivation: cell culture and organ culture (organ and tissue culture). To obtain a cell culture - a genetically homogeneous rapidly proliferating population - pieces of tissue (usually about 1 mm 3) are removed from the body, treated with appropriate enzymes (to destroy intercellular contacts), and the resulting suspension is placed in a nutrient medium. Cultures derived from embryonic tissues are characterized by better survival and more active growth (due to the low level of differentiation and the presence of progenitor stem cells in embryos) compared to the corresponding tissues taken from an adult organism. Normal tissues give rise to cultures with a limited lifetime (the so-called Hayflick limit), while cultures derived from tumors can proliferate indefinitely. However, even in a culture of normal cells, some cells spontaneously immortalize, that is, become immortal. They survive and give rise to cell lines with an unlimited lifespan. The original cell line can be obtained from a population of cells or from a single cell. In the latter case, the line is called clone, or clone. With prolonged cultivation under the influence of various factors, the properties of normal cells change, a transformation occurs, the main features of which are violations of cell morphology, a change in the number of chromosomes (aneuploidy). At a high degree of transformation, the introduction of such cells into an animal can cause the formation of a tumor. In organ culture, the structural organization of tissue, intercellular interactions are preserved, and histological and biochemical differentiation is maintained. Tissues dependent on hormones retain their sensitivity and characteristic responses, glandular cells continue to secrete specific hormones, and so on. Such cultures are grown in a culture vessel on rafts (paper, millipore) or on a metal mesh floating on the surface of the nutrient medium.

In plants, cell culture is based, in general, on the same principles as in animals. The differences in cultivation methods are determined by the structural and biological characteristics of plant cells. Most cells of plant tissues are totipotent: from one such cell, under certain conditions, a full-fledged plant can develop. To obtain a plant cell culture, a piece of any tissue (for example, callus) or organ (root, stem, leaf) in which living cells are present is used. It is placed on a nutrient medium containing mineral salts, vitamins, carbohydrates and phytohormones (most often cytokines and auxins). Plant cultures support at temperatures from 22 to 27°C, in the dark or under light.

Cell and tissue cultures are widely used in various fields of biology and medicine. The cultivation of somatic cells (all cells of organs and tissues with the exception of sex cells) outside the body has determined the possibility of developing new methods for studying the genetics of higher organisms using, along with the methods of classical genetics, methods of molecular biology. The molecular genetics of mammalian somatic cells has received the greatest development, which is associated with the possibility of direct experiments with human cells. Cell and tissue culture is used in solving such general biological problems as elucidating the mechanisms of gene expression, early embryonic development, differentiation and proliferation, interaction of the nucleus and cytoplasm, cells with the environment, adaptation to various chemical and physical influences, aging, malignant transformation, etc., it is used to diagnose and treat hereditary diseases. As test objects, cell cultures are an alternative to the use of animals in testing new pharmacological agents. They are necessary for obtaining transgenic plants, clonal propagation. Cell cultures play an important role in biotechnology in the creation of hybrids, the production of vaccines and biologically active substances.

See also cell engineering.

Lit.: Methods of cell cultivation. L., 1988; Culture of animal cells. Methods / Edited by R. Freshni. M., 1989; Biology of cultured cells and plant biotechnology. M., 1991; Freshney R. I. Culture of animal cells: a manual of basic technique. 5th ed. Hoboken, 2005.

O. P. Kisurina-Evgeniev.

I. Cell cultures

The most common are single-layer cell cultures, which can be divided into 1) primary (primarily trypsinized), 2) semi-transplantable (diploid) and 3) transplantable.

Origin they are classified into embryonic, neoplastic and from adult organisms; by morphogenesis- on fibroblastic, epithelial, etc.

Primary cell cultures are cells of any human or animal tissue that have the ability to grow as a monolayer on a plastic or glass surface coated with a special nutrient medium. The life span of such crops is limited. In each case, they are obtained from the tissue after mechanical grinding, treatment with proteolytic enzymes and standardization of the number of cells. Primary cultures derived from monkey kidneys, human embryonic kidneys, human amnion, chick embryos are widely used for virus isolation and accumulation, as well as for the production of viral vaccines.

semi-transplantable(or diploid ) cell cultures - cells of the same type, capable of withstanding up to 50-100 passages in vitro, while maintaining their original diploid set of chromosomes. Diploid strains of human embryonic fibroblasts are used both for the diagnosis of viral infections and in the production of viral vaccines.

transplanted cell lines are characterized by potential immortality and heteroploid karyotype.

The source of transplanted lines are primary cell cultures (for example, SOC, PES, VNK-21 - from the kidneys of day-old Syrian hamsters; PMS - from the kidney of a guinea pig, etc.), individual cells of which show a tendency to endless reproduction in vitro. The set of changes leading to the appearance of such features from cells is called transformation, and the cells of transplanted tissue cultures are called transformed.

Another source of transplanted cell lines are malignant neoplasms. In this case, cell transformation occurs in vivo. The following lines of transplanted cells are most often used in virological practice: HeLa - obtained from cervical carcinoma; Ner-2 - from carcinoma of the larynx; Detroit-6 - from lung cancer metastasis to the bone marrow; RH - from the human kidney.

For cell cultivation, nutrient media are needed, which, according to their purpose, are divided into growth and supporting ones. The composition of growth media should contain more nutrients in order to ensure active reproduction of cells to form a monolayer. Supporting media should only ensure the survival of cells in an already formed monolayer during reproduction of viruses in the cell.

Standard synthetic media, such as Synthetic 199 media and Needle media, are widely used. Regardless of the purpose, all nutrient media for cell cultures are designed on the basis of a balanced salt solution. Most often it is Hank's solution. An integral component of most growth media is the blood serum of animals (calf, bull, horse), without the presence of 5-10% of which, cell reproduction and the formation of a monolayer do not occur. Serum is not included in maintenance media.

I. Cell cultures - concept and types. Classification and features of the category "I. Cell cultures" 2017, 2018.

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K.K. - These are cells of a multicellular organism that live and multiply in artificial conditions outside the body.

Cells or tissues living outside the body are characterized by a whole complex of metabolic, morphological and genetic features that are sharply different from the properties of cells of organs and tissues in vivo.

There are two main types of single-layer cell cultures: primary and transplanted.

Primarily trypsinized. The term "primary" refers to a cell culture obtained directly from human or animal tissues in the embryonic or postnatal period. The life span of such crops is limited. After a certain time, phenomena of nonspecific degeneration appear in them, which is expressed in granulation and vacuolization of the cytoplasm, rounding of cells, loss of communication between the cells and the solid substrate on which they were grown. Periodic change of the medium, changes in the composition of the latter, and other procedures can only slightly increase the lifetime of the primary cell culture, but cannot prevent its final destruction and death. In all likelihood, this process is associated with the natural extinction of the metabolic activity of cells that are out of control of neurohumoral factors acting in the whole organism.

Only individual cells or groups of cells in the population against the background of degeneration of most of the cell layer can retain the ability to grow and reproduce. These cells, having found the potency of endless reproduction in vitro, give rise to transplanted cell cultures.

The main advantage of transplanted cell lines, in comparison with any primary culture, is the potential for unlimited reproduction outside the body and the relative autonomy that brings them closer to bacteria and unicellular protozoa.

Suspension cultures- individual cells or groups of cells grown in suspension in a liquid medium. They are a relatively homogeneous population of cells that are easily exposed to chemicals.

Suspension cultures are widely used as model systems for studying secondary metabolism pathways, enzyme induction and gene expression, degradation of foreign compounds, cytological studies, etc.

A sign of a "good" line is the ability of cells to rearrange metabolism and a high rate of reproduction under specific cultivation conditions. Morphological characteristics of such a line:

high degree of disaggregation (5-10 cells per group);

morphological uniformity of cells (small size, spherical or oval shape, dense cytoplasm);

Absence of tracheid-like elements.

Diploid cell strains. These are cells of the same type that are capable of undergoing up to 100 divisions in vitro, while retaining the failure of the original diploid set of chromosomes (Hayflick, 1965). Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production, as well as in experimental studies. It should be borne in mind that some features of the viral genome are realized only in cells that retain a normal level of differentiation.

130. Bacteriophages. Morphology and chemical composition

Bacteriophages (phages) (from other Greek φᾰγω - “I devour”) are viruses that selectively infect bacterial cells. Most often, bacteriophages multiply inside bacteria and cause their lysis. As a rule, a bacteriophage consists of a protein shell and the genetic material of a single-stranded or double-stranded nucleic acid (DNA or, less commonly, RNA). The particle size is approximately 20 to 200 nm.

The structure of particles - virions - of different bacteriophages is different. Unlike eukaryotic viruses, bacteriophages often have a specialized attachment organ to the surface of a bacterial cell, or a tail process, arranged with varying degrees of complexity, but some phages do not have a tail process. The capsid contains the genetic material of the phage, its genome. The genetic material of different phages can be represented by different nucleic acids. Some phages contain DNA as their genetic material, others contain RNA. The genome of most phages is double-stranded DNA, and the genome of some relatively rare phages is single-stranded DNA. At the ends of the DNA molecules of some phages there are "sticky areas" (single-stranded complementary nucleotide sequences), in other phages there are no sticky areas. Some phages have unique gene sequences in DNA molecules, while other phages have gene permutations. In some phages, DNA is linear, in others it is closed in a ring. Some phages have terminal repeats of several genes at the ends of the DNA molecule, while in other phages this terminal redundancy is ensured by the presence of relatively short repeats. Finally, in some phages, the genome is represented by a set of several nucleic acid fragments.

From an evolutionary point of view, bacteriophages that use such different types of genetic material differ from each other to a much greater extent than any other representatives of eukaryotic organisms. At the same time, despite such fundamental differences in the structure and properties of carriers of genetic information - nucleic acids, different bacteriophages show commonality in many respects, primarily in the nature of their intervention in cellular metabolism after infection of susceptible bacteria.

Bacteriophages capable of causing a productive infection of cells, i.e. an infection resulting in viable offspring is defined as non-defective. All non-defective phages have two states: the state of an extracellular, or free, phage (sometimes also called a mature phage) and the state of a vegetative phage. For some so-called temperate phages, the state of a prophage is also possible.

Extracellular phage are particles that have a structure characteristic of this type of phage, which ensures the preservation of the phage genome between infections and its introduction into the next sensitive cell. The extracellular phage is biochemically inert, while the vegetative phage, the active (“live”) state of the phage, occurs after infection of sensitive bacteria or after induction of a prophage.

Sometimes infection of sensitive cells with a non-defective phage does not result in the formation of viable progeny. This can be in two cases: during an abortive infection or due to the lysogenic state of the cell during infection with a temperate phage.

The reason for the abortive nature of the infection may be the active interference of certain cell systems in the course of infection, for example, the destruction of the phage genome introduced into the bacterium, or the absence in the cell of some product necessary for the development of the phage, etc.

Phages are usually classified into three types. The type is determined by the nature of the influence of a productive phage infection on the fate of the infected cell.

First type are truly virulent phages. Infection of a cell with a virulent phage inevitably leads to the death of the infected cell, its destruction, and the release of the progeny phage (excluding cases of abortive infection). Such phages are called truly virulent to distinguish them from virulent temperate phage mutants.

Second type- temperate phages. In the course of a productive infection of a cell with a temperate phage, two fundamentally different ways of its development are possible: lytic, in general (in its outcome) similar to the lytic cycle of virulent phages, and lysogenic, when the genome of a moderate phage passes into a special state - a prophage. A cell carrying a prophage is called a lysogenic or simply a lysogen (because it can undergo phage lytic development under certain conditions). Temperate phages that respond in the prophage state to the application of an inducing factor by the onset of lytic development are called inducible, and phages that do not react in this way are called non-inducible. Virulent mutants can occur in temperate phages. Virulence mutations lead to such a change in the sequence of nucleotides in the operator regions, which is reflected in the loss of affinity for the repressor.

The third type of phages are phages, the productive infection of which does not lead to the death of bacteria. These phages are able to leave the infected bacterium without causing its physical destruction. A cell infected with such a phage is in a state of constant (permanent) productive infection. The development of the phage results in some slowing down of the rate of bacterial divisions.

Bacteriophages differ in chemical structure, type of nucleic acid, morphology, and interaction with bacteria. Bacterial viruses are hundreds and thousands of times smaller than microbial cells.

A typical phage particle (virion) consists of a head and a tail. The length of the tail is usually 2-4 times the diameter of the head. The head contains genetic material - single-stranded or double-stranded RNA or DNA with the transcriptase enzyme in an inactive state, surrounded by a protein or lipoprotein shell - a capsid that preserves the genome outside the cell.

Nucleic acid and capsid together make up the nucleocapsid. Bacteriophages may have an icosahedral capsid assembled from multiple copies of one or two specific proteins. Usually the corners are made up of pentamers of the protein, and the support of each side is made up of hexamers of the same or a similar protein. Moreover, phages can be spherical, lemon-shaped, or pleomorphic in shape. The tail is a protein tube - a continuation of the protein shell of the head, at the base of the tail there is an ATPase that regenerates energy for the injection of genetic material. There are also bacteriophages with a short process, without a process, and filamentous.

The main components of phages are proteins and nucleic acids. It is important to note that phages, like other viruses, contain only one type of nucleic acid, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In this property, viruses differ from microorganisms that contain both types of nucleic acids in their cells.

The nucleic acid is located in the head. A small amount of protein (about 3%) was also found inside the phage head.

Thus, according to the chemical composition, phages are nucleoproteins. Depending on the type of their nucleic acid, phages are divided into DNA and RNA. The amount of protein and nucleic acid in different phages is different. In some phages, their content is almost the same, and each of these components is about 50%. In other phages, the ratio between these main components may be different.

In addition to these main components, phages contain small amounts of carbohydrates and some predominantly neutral fats.

Figure 1: Diagram of the structure of a phage particle.

All known phages of the second morphological type are RNA. Among phages of the third morphological type, both RNA and DNA forms are found. Phages of other morphological types are DNA-type.

131. Interferon. What it is?

Interfer O n(from lat. inter - mutually, among themselves and ferio - hit, hit), a protective protein produced by cells in the body of mammals and birds, as well as cell cultures in response to their infection with viruses; inhibits the reproduction (replication) of viruses in the cell. I. was discovered in 1957 by the English scientists A. Isaacs and J. Lindenman in the cells of infected chickens; later it turned out that bacteria, rickettsia, toxins, nucleic acids, synthetic polynucleotides also cause the formation of I.. I. is not an individual substance, but a group of low molecular weight proteins (molecular weight 25,000–110,000) that are stable in a wide pH zone, resistant to nucleases, and degraded by proteolytic enzymes. Formation in I.'s cells is associated with the development of a virus in them, that is, it is a reaction of the cell to the penetration of a foreign nucleic acid. After disappearance from a cell of the infecting virus and in normal cells And. it is not found. According to the mechanism of action, I. is fundamentally different from antibodies: it is not specific to viral infections (it acts against different viruses), does not neutralize the infectivity of the virus, but inhibits its reproduction in the body, suppressing the synthesis of viral nucleic acids. When it enters the cells after the development of a viral infection in them, I. is not effective. Besides, And., as a rule, is specific to the cells forming it; for example, I. of chicken cells is active only in these cells, but does not suppress the reproduction of the virus in rabbit or human cells. It is believed that not I. itself acts on viruses, but another protein produced under its influence. Encouraging results have been obtained in testing I. for the prevention and treatment of viral diseases (herpetic eye infection, influenza, cytomegaly). However, the widespread clinical use of I. is limited by the difficulty of obtaining the drug, the need for repeated administration to the body, and its species specificity.

132. Disjunctive way. What it is?

1.A productive viral infection occurs in 3 periods:

· initial period includes the stages of adsorption of the virus on the cell, penetration into the cell, disintegration (deproteinization) or "undressing" of the virus. The viral nucleic acid was delivered to the appropriate cell structures and, under the action of lysosomal cell enzymes, is released from protective protein coats. As a result, a unique biological structure is formed: an infected cell contains 2 genomes (own and viral) and 1 synthetic apparatus (cellular);

After that it starts second group virus reproduction processes, including average And final periods, during which repression of the cellular and expression of the viral genome occur. Repression of the cellular genome is provided by low molecular weight regulatory proteins such as histones, which are synthesized in any cell. With a viral infection, this process is enhanced, now the cell is a structure in which the genetic apparatus is represented by the viral genome, and the synthetic apparatus is represented by the synthetic systems of the cell.

2. The further course of events in the cell is directedfor viral nucleic acid replication(synthesis of genetic material for new virions) and implementation of the genetic information contained in it(synthesis of protein components for new virions). In DNA-containing viruses, both in prokaryotic and eukaryotic cells, viral DNA replication occurs with the participation of the cellular DNA-dependent DNA polymerase. In this case, single-stranded DNA-containing viruses first form complementary strand - the so-called replicative form, which serves as a template for daughter DNA molecules.

3. The implementation of the genetic information of the virus contained in the DNA occurs as follows: with the participation of DNA-dependent RNA polymerase, mRNAs are synthesized, which enter the ribosomes of the cell, where virus-specific proteins are synthesized. In double-stranded DNA-containing viruses, the genome of which is transcribed in the cytoplasm of the host cell, this is its own genomic protein. Viruses whose genomes are transcribed in the cell nucleus use the cellular DNA-dependent RNA polymerase contained there.

At RNA viruses processes replication their genome, transcription and translation of genetic information are carried out in other ways. Replication of viral RNA, both minus and plus strands, is carried out through the replicative form of RNA (complementary to the original), the synthesis of which is provided by RNA-dependent RNA polymerase, a genomic protein that all RNA-containing viruses have. The replicative form of RNA of minus-strand viruses (plus-strand) serves not only as a template for the synthesis of daughter viral RNA molecules (minus-strands), but also performs the functions of mRNA, i.e. goes to ribosomes and ensures the synthesis of viral proteins (broadcast).

At plus-filament RNA-containing viruses perform the translation function of its copies, the synthesis of which is carried out through the replicative form (negative strand) with the participation of viral RNA-dependent RNA polymerases.

Some RNA viruses (reoviruses) have a completely unique transcription mechanism. It is provided by a specific viral enzyme - reverse transcriptase (reverse transcriptase) and is called reverse transcription. Its essence lies in the fact that at first a transcript is formed on the viral RNA matrix with the participation of reverse transcription, which is a single strand of DNA. On it, with the help of cellular DNA-dependent DNA polymerase, the second strand is synthesized and a double-stranded DNA transcript is formed. From it, in the usual way, through the formation of i-RNA, the information of the viral genome is realized.

The result of the described processes of replication, transcription and translation is the formation daughter molecules viral nucleic acid and viral proteins encoded in the virus genome.

After that comes third, final period interaction between virus and cell. New virions are assembled from the structural components (nucleic acids and proteins) on the membranes of the cytoplasmic reticulum of the cell. A cell whose genome has been repressed (suppressed) usually dies. newly formed virions passively(due to cell death) or actively(by budding) leave the cell and find themselves in its environment.

Thus, synthesis of viral nucleic acids and proteins and assembly of new virions occur in a certain sequence (separated in time) and in different cell structures (separated in space), in connection with which the method of reproduction of viruses was named disjunctive(disjointed). With an abortive viral infection, the process of interaction of the virus with the cell is interrupted for one reason or another before the suppression of the cellular genome has occurred. Obviously, in this case, the genetic information of the virus will not be realized and the reproduction of the virus does not occur, and the cell retains its functions unchanged.

During a latent viral infection, both genomes function simultaneously in the cell, while during virus-induced transformations, the viral genome becomes part of the cellular one, functions and is inherited along with it.

133. Camelpox virus

Smallpox (Variola)- an infectious contagious disease characterized by fever and a papular-pustular rash on the skin and mucous membranes.
The causative agents of the disease belong to various genera and types of viruses of the smallpox family (Poxviridae). Independent species are viruses: natural cow yuspa, vaccinia (genus Orthopoxvirus), natural sheep pox, goats (genus Carpipoxvirus), pigs (genus Suipoxvirus), birds (genus Avipoxvirus) with three main species (causative agents of smallpox of chickens, pigeons and canaries).
Smallpox pathogens different animal species are morphologically similar. These are DNA-containing viruses characterized by relatively large sizes (170 - 350 nm), epitheliotropy and the ability to form elementary rounded inclusions in cells (Paschen, Guarnieli, Bollinger bodies), visible under a light microscope after Morozov staining. Although there is a phylogenetic There is a strong relationship between the causative agents of smallpox in different animal species, the spectrum of pathogenicity is not the same, and immunogenic relationships are not preserved in all cases. Variola viruses of sheep, goats, pigs and birds are pathogenic only for the corresponding species, and under natural conditions each of them causes an independent (original) smallpox. Variola cowpox and vaccinia viruses have a wide spectrum of pathogenicity, including cattle, buffalo, lo-boats, donkeys, mules, camels, rabbits, monkeys and humans.

Camel pox VARIOLA CAMELINA a contagious disease that occurs with the formation of a characteristic nodular-pustular smallpox rash on the skin and mucous membranes. The name of smallpox Variola comes from the Latin word Varus, which means crooked (pockmarked).

Epizootology of the disease. Camels of all ages are susceptible to smallpox, but young animals are more often and more severely ill. In stationary areas with smallpox problems, adult camels rarely get sick due to the fact that almost all of them get smallpox at a young age. In pregnant camels, smallpox can cause abortions.

Animals of other species are not susceptible to the original camelpox virus in natural conditions. In addition to cows and camels, buffaloes, horses, donkeys, pigs, rabbits and people who are not immune to smallpox are susceptible to the cowpox virus and vaccinia. Of the laboratory animals, guinea pigs are sensitive to cowpox and vaccinia viruses after the virus has been applied to the scarified cornea of ​​​​the eyes (FA Petunii, 1958).

The main sources of smallpox viruses are smallpox animals and people with vaccinia and recovering from hypersensitivity after immunization with vaccinia virus in smallpox calf detritus. Sick animals and people disseminate the virus in the external environment, mainly with the rejected epithelium of the skin and mucous membrane containing the virus. The virus is also released into the external environment with aborted fetuses (K. N. Buchnev and R. G. Sadykov, 1967). The causative agent of smallpox can be mechanically carried by domestic and wild animals immune to smallpox, including birds, as well as people immune to smallpox from children vaccinated with vaccinia.

Under natural conditions, healthy camels become infected through contact with sick animals in a virus-contaminated area through infected water, feed, premises and care items, as well as aerogenically by spraying virus-containing outflows by sick animals. More often, camels become infected when the virus enters the body through the skin and mucous membranes, especially when their integrity is violated or when vitamin A deficiency occurs.

In the form of an epizootic, smallpox in camels occurs approximately every 20-25 years. At this time, young animals are especially seriously ill. In the period between epizootics in zones that are stationary in terms of smallpox, among camels, smallpox occurs in the form of enzootic and sporadic cases that occur more or less regularly every 3-6 years, mainly among animals aged 2-4 years. In such cases, animals get sick relatively easily, especially in the warm season. In cold weather, smallpox is more severe, longer and is accompanied by complications, especially in young animals. In small farms, almost all susceptible camels fall ill within 2-4 weeks. It should be borne in mind that smallpox outbreaks among camels can be caused by both the original camelpox virus and cowpox virus, which do not create immunity against each other. Therefore, outbreaks caused by different smallpox viruses can follow one another or occur simultaneously.

Pathogenesis determined by the pronounced epitheliotropism of the pathogen. Once in the body of an animal, the virus multiplies and penetrates into the blood (viremia), lymph nodes, internal organs, into the epithelial layer of the skin and mucous membranes and causes the formation of specific exanthemas and enanthems in them, the severity of which depends on the reactivity of the organism and the virulence of the virus, pathways its penetration into the body and the state of the epithelial layer. Pocks develop sequentially in stages: from roseola with a nodule to a pustule with a crust and scar formation.

Symptoms. The incubation period, depending on the age of the camels, the properties of the virus and how it enters the body, ranges from 3 to 15 days: in young animals 4-7, in adults 6-15 days. Camels from non-immune camels may become ill 2-5 days after birth. The shortest incubation period (2-3 days) occurs in camels after they are infected with the vaccinia virus.

In the prodromal period, in sick camels, the body temperature rises to 40-41 ° C, lethargy and refusal to feed appear, the conjunctiva and mucous membranes of the mouth and nose are hyperemic. However, these signs are often seen, especially at the beginning of the onset of the disease on the farm.

The course of smallpox in camels, depending on their age, is also different: in young animals, especially in a newborn, it is more often acute (up to 9 days); in adults - subacute and chronic, sometimes latent, more often in pregnant camels. The most characteristic form of smallpox in camels is cutaneous with a subacute course of the disease (Fig. 1).

In the subacute course of the disease, clear, later cloudy, grayish-dirty mucus is released from the mouth and nose. Animals shake their heads, sniff and snort, throwing out the epithelium affected by the virus along with the virus-containing mucus. Soon, puffiness forms in the area of ​​the lips, nostrils and eyelids, sometimes spreading to the intermaxillary region, neck, and even to the dewlap area. Submandibular and lower cervical lymph nodes are enlarged. Animals have reduced appetite, they lie more often and longer than usual and get up with great difficulty. By this time, reddish-gray spots appear on the skin of the lips, nose and eyelids, on the mucous membrane of the mouth and nose; under them dense nodules are formed, which, increasing, turn into gray papules, and then into pustules the size of a pea and a bean with a sinking center and a roller-like thickening along the edges.

The pustules soften, burst, and a sticky liquid of a light gray color is released from them. The swelling of the head by this time disappears. After 3-5 days, the opened pustules become covered with crusts. If they are not injured by roughage, then the disease ends there. Removed or fallen off primary crusts have a reverse crater-like form of pustules. Scars remain in place of pockmarks. All of these lesions on the skin are formed within 8-15 days.

Pocks in sick camels often appear first on the head. At the age of one to four years, camels get sick, as a rule, easily. Lesions are localized on the scalp, mainly in the lips and nose. In camels, the udder is often affected. A few days after the opening of the primary pustules in the head area, smallpox lesions form on the skin and other low-haired areas of the body (in the areas of the breast, armpits, perineum and scrotum, around the anus, the inside of the forearm and thigh), and in camels also on the mucosa lining of the vagina. At this time, the body temperature of the camels usually rises again, sometimes up to 41.5 °, and the camels in the last month of pregnancy bring premature and underdeveloped camels, who, as a rule, soon die.

In some animals, the cornea of ​​​​the eyes (thorn) becomes cloudy, which causes temporary blindness in one eye for 5-10 days, and in camels more often in both eyes. Camel calves who fall ill shortly after birth develop diarrhea. In this case, within 3-9 days after the disease, they die.

With a relatively benign subacute course of smallpox and usually after infection with the vaccinia virus, animals recover in 17-22 days.

In adult camels, opening pustules on the oral mucosa often merge and bleed, especially when injured by roughage. This makes it difficult to feed, the animals lose weight, the healing process is delayed up to 30-40 days, and the disease becomes chronic.

With the generalization of the smallpox process, pyemia and complications (pneumonia, gastroenteritis, necrobacteriosis, etc.) sometimes develop. In such cases, the disease drags on for up to 45 days or longer. There are cases of disorders of the functions of the stomach and intestines, accompanied by atony and constipation. In some sick animals, swelling of the extremities is noted.

In camels with a latent course of smallpox (without characteristic clinical signs of the disease, only in the presence of fever), abortions occur 1-2 months before foaling (up to 17-20%).

The prognosis of the disease in adult camels is favorable, in camels with an acute course, especially at the age of 15-20 days and in camels born from non-immune to smallpox, unfavorable. Camels are seriously ill and up to 30-90% of them die. Camels at the age of 1-3 years are ill with smallpox more easily, and at an older age, although they are seriously ill, with signs of a pronounced generalized process, the mortality rate is low (4-7%).

Pathological changes are characterized by the lesions of the skin, mucous membrane and cornea of ​​the eyes described above. Pinpoint hemorrhages are noted on the epicardium and intestinal mucosa. In the chest cavity on the costal pleura, small hemorrhages and nodules ranging in size from millet grain to lentils of gray and gray-red color with curdled contents are sometimes also visible. The mucous membrane of the esophagus is covered with nodules the size of millet, surrounded by ridge-like elevations. The mucous membrane of the scar (sometimes the bladder) has similar hemorrhages and nodules with jagged edges, as well as small ulcers with a sunken pinkish center. In papules, elementary bodies such as Paschen bodies can be detected, which are of diagnostic value when microscopy of a smear preparation under immersion through a conventional light microscope.

The diagnosis is based on the analysis of clinical and epizootic data (taking into account the possibility of infection of camels from humans), pathological changes, positive results of microscopy (when processing smears from fresh papules using the Morozov silvering method) or electronoscopy, as well as bioassays on those susceptible to smallpox animals. It is possible to isolate the virus from the organs of aborted fetuses of camels with smallpox. When diagnosing smallpox, it is also recommended to use the diffusion precipitation reaction in agar gel and the neutralization reaction in the presence of active specific sera or globulins.

Differential diagnosis is carried out in doubtful cases (taking into account clinical and epizootic features). Smallpox must be differentiated from necrobacteriosis by microscopy of smears from pathological material and infection of white mice susceptible to it; from foot-and-mouth disease - infection of guinea pigs with a suspension of pathological material in the plantar surface of the skin of the hind legs; from fungal infections and scabies - by finding the corresponding pathogens in the examined scrapings taken from the affected areas of the skin; from brucellosis during abortions, miscarriages and premature foals - by examining the blood serum of camels RA and RSK and bacteriological examination of fetuses with the isolation of a microbial culture on nutrient media and microscopy (if necessary, use a bioassay on guinea pigs followed by bacteriological and serological tests of blood and sera).

When diagnosing smallpox in camels, it is also necessary to exclude a non-contagious, but sometimes widespread disease that occurs with skin lesions in the lips and nose - yantak-bash (Turkm.), Jantak-bas (Kazakh), which occurs from injuring them when eating shrubs called camel thorn (yantak, jantak, Alhagi). This disease can usually be observed in autumn in young camels, mainly under the age of one year. Adult camels are only slightly affected by camel thorn. With yantak-bash, there are usually no nodules or papular lesions, unlike smallpox, on the oral mucosa. The grayish coating that appears with yantak-bash is relatively easy to remove. However, it should be taken into account that yantak-bash contributes to the disease of smallpox in camels, and often proceeds simultaneously with it.

When isolating the smallpox virus, it is necessary to determine its type (original, cowpox or vaccinia), using the methods specified in the instructions of the Ministry of Health of the USSR of 1968. On the prevention of cowpox in humans, data obtained after infection (in isolated conditions) of camels who had had smallpox vaccinia virus and isolated pathogens.

Treatment of sick camels is mainly symptomatic. The affected areas are treated with a solution of potassium permanganate (1:3000), and after drying, they are lubricated with a mixture of 10% tincture of iodine with glycerin (1:2 or 1:3). After opening the smallpox, it is treated with a 5% emulsion of synthomycin on fortified fish oil, to which tincture of iodine is added in a ratio of 1:15-1:20; ointments - zinc, ichthyol, penicillin, etc. You can use 2% salicylic or boric ointment and 20-30% propolis ointment on petroleum jelly. In hot weather, 3% creolin ointment, tar and hexachlorane dust are indicated. The affected areas are lubricated with swabs soaked in emulsions and ointments 2-3 times a day.

The affected mucous membrane of the oral cavity is washed 2-3 times a day with a 10% solution of potassium permanganate or a 3% solution of hydrogen peroxide or decoctions of sage, chamomile and other disinfectants and astringents. With conjunctivitis, the eyes are washed with a 0.1% solution of zinc sulfate.

To prevent the development of a secondary microbial infection and possible complications, it is recommended to inject penicillin and streptomycin intramuscularly. With general weakness and complications, cardiac remedies are indicated.

From specific means of treatment in severe cases of the disease, you can use the serum or blood of camels who have had smallpox (subcutaneously at the rate of 1-2 ml per 1 kg of animal weight). The injection sites are carefully cut out beforehand and wiped with tincture of iodine.

Sick and convalescent camels are often given pure water, a mash of bran or barley flour, soft bluegrass or fine alfalfa hay, or cotton husks flavored with barley flour. In cold weather, sick animals, especially camels, are kept in a clean, dry and warm room or covered with blankets.

Immunity in naturally ill smallpox camels lasts up to 20-25 years, i.e., almost for life. The nature of immunity is skin-humoral, as evidenced by the presence of neutralizing antibodies in the blood serum of recovered animals and the resistance of camels to re-infection with the homologous smallpox virus. Camels born from camels who have had smallpox are not susceptible to the type of smallpox that the camel has had, especially in the first three years, that is, until puberty. Camel calves, who are under the uterus during the epizootic period, as a rule, do not get smallpox or get sick relatively easily and for a short time.

Prevention and control measures are in strict observance of all veterinary, sanitary and quarantine measures, timely diagnosis of the disease and determination of the type of virus. Persons should not be allowed to care for camels during vaccination and in the post-vaccination period until they (or their children) have completely completed their clinically pronounced reaction to vaccination smallpox. All camels, cows and horses entering the farm must be kept in an isolation cell for 30 days.

When smallpox appears among camels, cows and horses, by a special decision of the district executive committee, the area, settlement or district, pasture where this disease is found is declared unfavorable for smallpox and quarantine, restrictive and health measures are taken.

The appearance of smallpox is immediately reported to higher veterinary organizations, neighboring farms and districts for taking appropriate measures to prevent further spread of the disease.

In order to prevent infection of camels with cowpox, it is recommended to use a medical preparation - smallpox detritus, which is used to immunize all clinically healthy camels, regardless of their age, sex and physiological state (pregnancy and lactating camels) in disadvantaged and threatened cowpox farms. To do this, wool is cut off in the lower third of the camel's neck, treated with alcohol-ether or a 0.5% solution of carbolic acid, wiped dry with cotton wool or dried, the skin is scarified and applied with a thick needle, the end of a scalpel or a scarifier 2-3 shallow parallel scratches 2 in length -4 cm. 3-4 drops of the dissolved vaccine are applied to the freshly scarified skin surface and lightly rubbed with a spatula. Dissolve the vaccine as indicated on the labels of ampoules and ampoules boxes. Diluted and unused vaccine and vaccine ampoules are disinfected by boiling and destroyed. The tools used for vaccinations are washed with a 3% solution of carbolic acid or a 1% solution of formaldehyde and sterilized by boiling.

If the camel was not immune to cowpox, then on the 5-7th day after vaccination, papules should appear at the site of scarification. If they are not present, the vaccination is repeated, but on the opposite side of the neck and with a vaccine of a different series. Persons immune against smallpox and familiar with the rules of personal hygiene are allowed to care for immunized and sick camels. Young animals, especially from the weak group, can sometimes react strongly to vaccination and get sick with pronounced signs of smallpox.

Sick and highly responsive camels are isolated and treated (see above). Livestock premises and places contaminated with the smallpox virus are recommended to be disinfected with hot 2-4% solutions of caustic soda and caustic potash, a 3% solution of a sulfur-carbolic mixture or 2-3% solutions of sulfuric acid or clarified solutions of bleach, containing 2-6% active chlorine, which inactivate the smallpox virus within 2-3 hours (O. Trabaev, 1970). You can also use 3-5% solutions of chloramine and 2% formaldehyde solution. Manure must be burned or biothermally disinfected. The corpses of camels that have fallen with clinical signs of smallpox must be burned. Milk from camels sick and suspected of having smallpox, if it does not contain impurities of pus and is not contraindicated for any other reason, can be eaten only after boiling for 5 minutes or pasteurization at 85 ° -30 minutes. Wool and skin from camels killed during the period of trouble for smallpox farms are processed according to the instructions for disinfection of raw materials of animal origin.

It is recommended to remove restrictions from households and settlements that are unfavorable for smallpox no earlier than 20 days after the recovery of all animals and people with smallpox and after a thorough final disinfection.

134. Chemical composition and biochemical properties of viruses

1.1 Structure and chemical composition of virions.

The largest viruses (variola viruses) are close in size to small bacteria, the smallest (causative agents of encephalitis, poliomyelitis, foot-and-mouth disease) to large protein molecules directed to blood hemoglobin molecules. In other words, among viruses there are giants and dwarfs. To measure viruses, a conditional value called a nanometer (nm) is used. One nm is one millionth of a millimeter. The sizes of different viruses vary from 20 to several hundreds of 1 nm.

Simple viruses are composed of protein and nucleic acid. The most important part of a virus particle, the nucleic acid, is the carrier of genetic information. If the cells of humans, animals, plants and bacteria always contain two types of nucleic acids - deoxyribonucleic acid DNA and ribonucleic RNA, then only one type of either DNA or RNA was found in different viruses, which is the basis for their classification. The second mandatory component of the virion, proteins differ in different viruses, which allows them to be recognized using immunological reactions.

More complex in structure, viruses, in addition to proteins and nucleic acids, contain carbohydrates and lipids. Each group of viruses has its own set of proteins, fats, carbohydrates and nucleic acids. Some viruses contain enzymes. Each component of virions has certain functions: the protein shell protects them from adverse effects, the nucleic acid is responsible for hereditary and infectious properties and plays a leading role in the variability of viruses, and enzymes are involved in their reproduction. Usually, the nucleic acid is located in the center of the virion and is surrounded by a protein shell (capsid), as if dressed in it.

The capsid consists of similar protein molecules (capsomeres) arranged in a certain way, which form symmetrical geometric shapes in place with the nucleic acid of the virus (nucleocapsid). In the case of cubic symmetry of the nucleocapsid, the nucleic acid strand is coiled into a ball, and the capsomeres are tightly packed around it. This is how the viruses of polio, foot-and-mouth disease, etc.

With helical (rod-shaped) symmetry of the nucleocapsid, the virus thread is twisted in the form of a spiral, each of its coils is covered with capsomeres that are darkly adjacent to each other. The structure of capsomeres and the appearance of virions can be observed using electron microscopy.

Most of the viruses that cause infections in humans and animals have a cubic symmetry type. The capsid almost always has the form of an icosahedral regular twenty-sided hexahedron with twelve vertices and with faces of equilateral triangles.

Many viruses have an outer shell in addition to the protein capsid. In addition to viral proteins and glycoproteins, it also contains lipids borrowed from the plasma membrane of the host cell. The influenza virus is an example of a helical enveloped virion with a cubic symmetry type.

The modern classification of viruses is based on the type and shape of their nucleic acid, the type of symmetry, and the presence or absence of an outer shell.

Biochemical properties - see. manual!!!

135. Pieces of organs that retain functional and proliferating activity in vitro

Cell culture

cells of any animal tissue capable of growing in the form of a monolayer under artificial conditions on a glass or plastic surface filled with a special nutrient medium. The source of cells is freshly obtained animal tissue - primary cells, laboratory strains of cells - transplanted to-ry. cells. Embryonic and tumor cells have the best ability to grow under artificial conditions. The diploid to-ra of human and monkey cells is passaged a limited number of times, therefore it is sometimes called semi-transplantable to-swarm of cells. Stages of receiving to-ry of cells: crushing of a source; trypsin treatment; release from detritus; standardization of the number of cells suspended in a nutrient medium with antibiotics; pouring into test tubes or vials, in which the cells settle on the walls or bottom, and begin to multiply; control over the formation of a monolayer. To-ry cells are used to isolate the virus from the study. material, for the accumulation of viral suspension, the study of St. in. Recently, it has been used in bacteriology.

136. Parasthesias. What it is?

PARESTHESIA(from Greek para-near, in spite of and aisthesis-feeling), sometimes also called dysesthesias, sensations of numbness, tingling, goosebumps (myrmeciasis, myrmecismus, formicatio), burning, itching, painful cold (i.e., not caused by external irritation) n. psychroesthesia), movements, etc., sensations in apparently preserved limbs in amputees (pseudomelia paraesthetica). The causes of P. may be different. P. can occur as a result of local changes in blood circulation, with Renaud's disease, with erythromelalgia, with acroparesthesia, with endarteritis, as an initial symptom of spontaneous gangrene. Sometimes they occur with damage to the nervous system, with traumatic neuritis (cf. typical. P. with a bruise of the n. ulnaris in the sulcus olecrani area), with toxic and infectious neuritis, with radiculitis, with spinal pachymeningitis (compression of the roots), with acute and hron. myelitis, especially with compression of the spinal cord (tumors of the spinal cord) and with tabes dorsalis. Their diagnostic value in all these cases is the same as the diagnostic value of pain, anesthesia and hyperesthesia: appearing in certain areas, along the tract of one or another peripheral nerve or in the area of ​​one or another radicular innervation, they can give valuable indications of the location of the pathology. . process. Items are also possible as manifestations of cerebral damage. So, with cortical epilepsy, seizures often begin with P., localized in the limb from which convulsions then begin. Often they are also observed in cerebral arteriosclerosis or in cerebral syphilis and are sometimes harbingers of apoplectic stroke. - A separate position is occupied by the so-called. mental P., i.e. P. of psychogenic, hypochondriacal origin, for which it is especially characteristic that they have not an elementary, like organic, but a complex character - “crawling of worms under the scalp”, “raising a ball from the abdomen to the neck” (Oppenheim), etc. Their diagnostic value is, of course, completely different from that of organic P

137. Rules for working and safety precautions with virus-containing material

138. Infectious bovine rhinotracheitis virus

Infectious rhinotracheitis(lat. - Rhinotracheitis infectiosa bovum; English - Infectious bovine rhinotracheites; IRT, blistering rash, infectious vulvovaginitis, infectious rhinitis, "red nose", infectious catarrh of the upper respiratory tract) is an acute contagious disease of cattle, characterized mainly by catarrhal necrotic lesions of the respiratory tract, fever, general depression and conjunctivitis, as well as pustular vulvovaginitis and abortion.

The causative agent of IRT - Herpesvirus bovis 1, belongs to the family of herpesviruses, DNA-containing, the diameter of the virion is 120 ... 140 nm. 9 structural proteins of this virus have been isolated and characterized.

RTI virus is easily cultivated in a number of cell cultures, causing CPE. The reproduction of the virus is accompanied by the suppression of mitotic cell division and the formation of intranuclear inclusions. It also has hemagglutinating properties and tropism for the cells of the respiratory and reproductive organs and can migrate from the mucous membranes to the central nervous system, is able to infect the fetus at the end of the first and second half of pregnancy.

At - 60 ... -70 "C, the virus survives 7 ... 9 months, at 56 ° C it is inactivated after 20 minutes, at 37 ° C - after 4 ... 10 days, at 22 ° C - after 50 days. At 4 " With the activity of the virus decreases slightly. Freezing and thawing reduces its virulence and immunogenic activity.

Formalin solutions 1: 500 inactivate the virus after 24 hours, 1: 4000 - after 46 hours, 1: 5000 - after 96 hours. In an acidic environment, the virus quickly loses its activity, it remains for a long time (up to 9 months) at pH 6.0 ... 9.0 and a temperature of 4 °C. There is information about the survival of the virus in bull semen stored at dry ice temperature for 4 ... 12 months, and in liquid nitrogen - for 1 year. The possibility of virus inactivation in bull semen was shown when it was treated with a 0.3% trypsin solution.

Sources of the causative agent of infection are sick animals and latent virus carriers. After infection with a virulent strain, all animals become latent carriers of the virus. Breeding bulls are very dangerous, because after getting sick they secrete the virus for 6 months and can infect cows during mating. The virus is released into the external environment with nasal secretions, discharge from the eyes and genitals, with milk, urine, feces, and semen. Wildebeest are believed to be the reservoir of the RTI virus in African countries. In addition, the virus can replicate in ticks, which play an important role in causing the disease in cattle.

The factors of transmission of the virus are air, feed, semen, vehicles, care items, birds, insects, as well as humans (farm workers). Ways of transmission - contact, airborne, transmissible, alimentary.

Susceptible animals are cattle regardless of sex and age. The disease is most severe in beef cattle. In the experiment, it was possible to infect sheep, goats, pigs, and deer. Animals usually fall ill 10...15 days after entering a dysfunctional farm.

The incidence of RTI is 30...100%, mortality - 1...15%, may be higher if the disease is complicated by other respiratory infections.

In the primary foci, the disease affects almost the entire livestock, while mortality reaches 18%. IRT often occurs in industrial-type farms when completing groups of animals brought from different farms.

When it enters the mucous membranes of the respiratory or genital tract, the virus invades the epithelial cells, where it multiplies, causing their death and desquamation. Then ulcers form on the surface of the mucous membrane of the respiratory tract, and nodules and pustules form in the genital tract. From the primary lesions, the virus enters the bronchi with air, and from the upper respiratory tract it can enter the conjunctiva, where it causes degenerative changes in the affected cells, which provokes an inflammatory response of the body. Then the virus is adsorbed on leukocytes and spreads through the lymph nodes, and from there it enters the blood. Viremia is accompanied by general depression of the animal, fever. In calves, the virus can be carried by blood into the parenchymal organs, where it multiplies, causing degenerative changes. When the virus passes through the blood-brain and placental barriers, pathological changes appear in the brain, placenta, uterus and fetus. The pathological process also largely depends on the complications caused by the microflora.

The incubation period averages 2-4 days, very rarely more. Basically, the disease is acute. There are five forms of IRT: upper respiratory tract infections, vaginitis, encephalitis, conjunctivitis, and arthritis.

With the defeat of the respiratory organs, chronic serous-purulent pneumonia is possible, in which about 20% of calves die. In the genital form, the external genital organs are affected, endometritis sometimes develops in cows, and orchitis in sires, which can cause infertility. In bulls used for artificial insemination, IRT is manifested by recurrent dermatitis in the perineum, buttocks, around the anus, sometimes on the tail, scrotum. Virus-infected semen can cause endometritis and infertility in cows.

Abortions and death of the fetus in the womb are noted 3 weeks after infection, which coincides with an increase in the titer of antibodies in the blood of pregnant convalescent cows, the presence of which does not prevent abortions and fetal death in the womb.

A tendency of IRT to a latent course was noted with genital form. In the epithelium of the mucous membrane of the vagina, its vestibule and vulva, numerous pustules of different sizes are formed (pustular vulvovaginitis). Erosions and sores appear in their place. After healing of ulcerative lesions, hyperemic nodules remain on the mucous membrane for a long time. In sick bulls, the process is localized on the prepuce and penis. The formation of pustules and vesicles is characteristic. In a small proportion of pregnant cows, abortions, resorption of the fetus or premature calving are possible. Aborted animals, as a rule, had previously had rhinotracheitis or conjunctivitis. Among aborted cows, lethal outcomes due to metritis and fetal decomposition are not excluded. However, cases of abortions are not uncommon in the absence of inflammatory processes on the mucous membrane of the cow's uterus. With IRT, there are cases of acute mastitis. The udder is sharply inflamed and enlarged, painful on palpation. The milk yield is sharply reduced.

At meningoencephalitis along with oppression, a disorder of motor functions and an imbalance are noted. The disease is accompanied by muscle tremor, lowing, gnashing of teeth, convulsions, salivation. This form of the disease mainly affects calves 2-6 months of age.

Respiratory form infection is characterized by a sudden increase in body temperature up to 41 ... 42 "C, hyperemia of the nasal mucosa, nasopharynx and trachea, depression, dry painful cough, profuse serous-mucous discharge from the nose (rhinitis) and foamy salivation. As the disease develops, mucus becomes thick, mucous plugs and foci of necrosis are formed in the respiratory tract.In severe disease, signs of asphyxia are noted.Hyperemia extends to the nasal mirror ("red nose").The etiological role of the IRT virus in mass keratoconjunctivitis of young cattle has been proven.In young cattle, the disease sometimes manifests itself as encephalitis. It begins with sudden excitement, riot and aggression, impaired coordination of movements. Body temperature is normal. In young calves, some strains of RTI virus cause acute gastrointestinal disease.

In general, in sick animals, the respiratory form is clinically clearly expressed, the genital form often goes unnoticed.

An autopsy of animals killed or dead in acute respiratory form usually reveals signs of serous conjunctivitis, catarrhal-purulent rhinitis, laryngitis and tracheitis, as well as damage to the mucous membranes of the adnexal cavities. The mucous membrane of the turbinates is edematous and hyperemic, covered with mucopurulent overlays. In places, erosive lesions of various shapes and sizes are revealed. Purulent exudate accumulates in the nasal and adnexal cavities. On the mucous membranes of the larynx and trachea, petechial hemorrhages and erosions. In severe cases, the mucosa of the trachea undergoes focal necrosis; in dead animals, bronchopneumonia is possible. In the lungs there are focal areas of atelectasis. The lumen of the alveoli and bronchi in the affected areas are filled with serous-purulent exudate. Severe swelling of the interstitial tissue. When the eyes are affected, the conjunctiva of the eyelid is hyperemic, with edema, which also extends to the conjunctiva of the eyeball. The conjunctiva is covered with sebaceous plaque. Often, papillary tubercles about 2 mm in size, small erosions and sores are formed on it.

In the genital form, pustules, erosions and sores are visible on the highly inflamed mucous membrane of the vagina and vulva at different stages of development. In addition to vulvovaginitis, sero-catarrhal or purulent cervicitis, endometritis, and much less often proctitis can be detected. In sires, in severe cases, phimosis and paraphimosis join pustular balanoposthitis.

Fresh aborted fetuses are usually edematous, with minor autolytic phenomena. Small hemorrhages on the mucous membranes and serous membranes. After a longer period after the death of the fetus, the changes are more severe; in the intermuscular connective tissue and in the body cavities, a dark red liquid accumulates, in the parenchymal organs - foci of necrosis.

When the udder is affected, serous-purulent diffuse mastitis is detected. The cut surface is edematous, distinctly granulated due to an increase in the affected lobules. When pressed, a cloudy, pus-like secret flows from it. The mucous membrane of the cistern is hyperemic, swollen, with hemorrhages. With encephalitis in the brain, hyperemia of blood vessels, swelling of tissues and small hemorrhages are detected.

IRT is diagnosed on the basis of clinical and epizootological data, pathological changes in organs and tissues with mandatory confirmation by laboratory methods. Latent infection is established only by laboratory tests.

Laboratory diagnostics includes: 1) virus isolation from pathological material in cell culture and its identification in RN or RIF; 2) detection of RTI virus antigens in pathological material using RIF; 3) detection of antigens in the blood serum of sick and recovered animals (retrospective diagnosis) in RN or RIGA.

For virological examination, mucus is taken from sick animals from the nasal cavity, eyes, vagina, prepuce; from the forcedly killed and fallen - pieces of the nasal septum, trachea, lung, liver, spleen, brain, regional lymph nodes, taken no later than 2 hours after death. Blood serum is also taken for retrospective serological diagnosis. For laboratory diagnostics IRT use a set of bovine IRT diagnosticums and a set of erythrocyte diagnosticum for serodiagnosis of infection in RIGA.

Diagnosis of IRT is carried out in parallel with the study of the material for parainfluenza-3, adenovirus infection, respiratory syncytial infection and viral diarrhea.

Preliminary diagnosis for IRT in cattle is made on the basis of positive results of antigen detection in pathological material using REEF taking into account epizootological and clinical data, as well as pathological changes. The final diagnosis is established on the basis of the coincidence of the results of the RIF with the isolation and identification of the virus.

In the differential diagnosis of infectious rhinotracheitis, it is necessary to exclude foot and mouth disease, malignant catarrhal fever, parainfluenza-3, adenovirus and chlamydial infections, viral diarrhea, respiratory syncytial infection, pasteurellosis.

The disease is accompanied by persistent and long-term immunity, which can be transmitted to offspring with colostrum antibodies. The immunity of recovered animals lasts at least 1.5...2 years, however, even pronounced humoral immunity does not prevent the persistence of the virus in convalescent animals, and they should be considered as a potential source of infection for other animals. Therefore, all animals with antibodies to RTI should be considered as carriers of the latent virus.

139. The reservoir of nutrients in developing bird embryos is

Given the complex and rather lengthy process of embryogenesis in birds, it is necessary to form special temporary extra-embryonic - provisional organs. The first of these forms the yolk sac, and subsequently the rest of the provisional organs: the amniotic membrane (amnion), serous membrane, allantois. In evolution before, the yolk sac was found only in sturgeons, which have a sharply telolecithal cell and the process of embryogenesis is complex and lengthy. During the formation of the yolk sac, the yolk is overgrown with parts of the leaves, which we call extra-embryonic leaves or extra-embryonic material. But the extraembryonic endoderm begins to grow on the edge of the yolk. The extra-embryonic mesoderm is stratified into 2 sheets: visceral and parietal, while the visceral sheet is adjacent to the extra-embryonic endoderm, and the parietal - to the extra-embryonic ectoderm.

The extra-embryonic ectoderm pushes the protein aside and also overgrows the yolk. Gradually, the yolk masses are completely surrounded by a wall consisting of the extra-embryonic endoderm and the visceral sheet of the extra-embryonic mesoderm - the first provisional organ, the yolk sac, is formed.

Functions of the yolk sac. The endoderm cells of the yolk sac begin to secrete hydrolytic enzymes that break down the yolk masses. The cleavage products are absorbed and transported through the blood vessels to the embryo. So the yolk sac provides trophic function. From the visceral mesoderm, the first blood vessels and the first blood cells are formed and, therefore, the yolk sac also performs a hematopoietic function. In birds and mammals, among the cells of the yolk sac, cells of the genital bud, the gonoblast, are found early.

140. Reactivation. What it is?

By changing the genotype, mutations are divided into point (localized in individual genes) and gene (affecting larger parts of the genome).
Virus infection of sensitive cells is multiple in nature, i.e. several virions enter the cell at once. In this case, viral genomes in the process of replication can cooperate or interfere. Cooperative interactions between viruses are represented by genetic recombination, genetic reactivation, complementation, and phenotypic mixing.
Genetic recombination is more common in DNA-containing viruses or RNA-containing viruses with a fragmented genome (influenza virus). During genetic recombination, an exchange occurs between homologous regions of viral genomes.
Genetic reactivation is observed between the genomes of related viruses with mutations in different genes. When the genetic material is redistributed, a full-fledged genome is formed.
Complementation occurs when one of the viruses infecting a cell synthesizes a nonfunctional protein as a result of a mutation. The wild-type virus, synthesizing a complete protein, makes up for the absence of it in the mutant virus.

It is possible to maintain the life of tissues and organs outside the body by growing them in culture. For the first time, attempts to maintain the vital activity of human and animal cells under laboratory conditions were made in 1907 by Harrion and in 1912 by Carrel. However, it was only in 1942 that J. Monod proposed modern in vitro cultivation methods.

Cell culture is a population of genotypically the same type of cells that function and divide in vitro. Cell cultures obtained by targeted or random mutations are called cell lines .

The growth of cell cultures in vitro is complex. In general, the following phases are distinguished:

1. Induction period (lag phase). During the lag phase, there is no noticeable increase in the number of cells or the formation of products. This phase is usually observed after passage of the cell culture. In it, the cells adapt to the new culture medium, the cell metabolism is rebuilt.

2. Phase of exponential growth. It is characterized by rapid accumulation of biomass and waste products of cell cultures. In this phase, mitoses are most common compared to other growth phases. But in this phase, exponential growth cannot continue indefinitely. She moves on to the next phase.

Rice. 4.2. Cell culture Hep-2, 48 hours of cultivation, mitoses are visible.

3. Phase of linear growth. characterized by a decrease in the number of mitoses

4. slow growth phase. In this phase, the growth of the cell culture decreases due to a decrease in the number of mitoses.

5. Stationary phase . It is observed after the growth retardation phase, while the number of cells practically does not change. In this phase, either mitotic cell division ceases, or the number of dividing cells is equal to the number of dying cells.

6. The phase of dying culture, in which the processes of cell death predominate and mitotic divisions are practically not observed.

Successive transitions from phase 1 to phase 6 are observed to a large extent due to the depletion of the substrates necessary for the growth of the cell population, or due to the accumulation of toxic products of their vital activity. Substrates that limit the growth of cell cultures are called limiting .

Under conditions where the concentration of substrates and other components necessary for cell growth is constant, the process of increasing the number of cells is autocatalytic. This process is described by the following differential equation:

where N is the number of cells, μ is the specific growth rate.

Rice. 4.3. Cell culture RD, human rhabdomyosarcoma. Monolayer, living unstained cells.

Successive transitions from phase 1 to phase 6 are observed to a large extent due to the depletion of the substrates necessary for the growth of the cell population, or due to the accumulation of toxic products of their vital activity.

To maintain the life of cells in culture, a number of mandatory conditions must be observed:

A balanced nutrient medium is needed;

The strictest sterility;

Optimal temperature;

Timely passage, i.e. transfer to a new nutrient medium.

For the first time, J. Monod drew attention to the limitation of cell culture growth processes by substrates of enzymatic reactions. Substrates that limit the growth of cell populations are called limiting.

Almost all cell populations are characterized by a change in the growth rate under the influence of inhibitors and activators. There are inhibitors acting on DNA (nalidixonic acid), inhibitors acting on RNA (actinomycin D), inhibitors of protein synthesis (levomycetin, erythromycin, tetracycline), inhibitors of cell wall synthesis (penicillin), membrane-active substances (toluene, chloroform), inhibitors of energy processes (2,4 - dinitrophenol), inhibitors of the limiting enzyme.

One of the most important factors determining the kinetics of cell growth are hydrogen ions. Many cell cultures grow in a narrow pH range; a change in pH leads to a slowdown in their growth rate or to a complete cessation of growth

One of the first attempts to describe the phenomenon of population growth restriction was made by P. Ferhgulst in 1838. He suggested that in addition to the process of reproduction of organisms, there is a process of death of organisms observed due to "crowding", i.e. This process occurs when two individuals meet.

In the development of any cell population, there comes a period of cessation of cell growth and cell death. Obviously, growth arrest and cell death are no less important than their reproduction and growth. These processes are especially important for multicellular organisms. The uncontrollable and uncontrolled growth of individual cells is the cause of oncological diseases, stunting, aging and cell death is the cause of aging and death of the body as a whole.

Different populations and different cells behave quite differently. Bacterial cells and cells of unicellular organisms outwardly appear "immortal". When exposed to a suitable comfortable environment with an excess of a limiting substrate, the cells begin to multiply actively. The limitation of their growth is determined by the consumption of the substrate, the accumulation of inhibitor products, as well as a specific mechanism of growth limitation, which is called "progressive incompetence".

The cells of multicellular organisms behave quite differently. Differentiated cells make up organs and tissues, and their growth and reproduction are fundamentally limited. If the growth control mechanism breaks down, individual cells are created that grow indefinitely. These cells make up the population of cancer cells, their growth leads to the death of the organism as a whole.

Research into the problem of aging of "normal" cells in multicellular organisms has a very interesting history. For the first time, the idea that normal somatic cells of animals and humans should deterministically lose their ability to divide and die was expressed by the great German biologist August Weismann in 1881. Around the same time, scientists learned how to transfer animal and human cells into culture. At the beginning of the century, the famous surgeon, one of the founders of the in vitro cell culture technique, Nobel Prize winner Alexis Carrel set up an experiment that lasted 34 years. During this period, he cultivated fibroblast cells obtained from the heart of a chicken. The experiment was stopped because the author was sure that the cells could be cultured forever. These results convincingly demonstrated that aging is not a reflection of processes occurring at the cellular level.

However, this conclusion turned out to be erroneous. "Immortal" are reborn (transformed) cells that have lost control of growth and turned into cancer cells. Only in 1961 L. Hayflick, returning to the experiments of A. Carrel, showed that normal "not transformed" human fibroblasts are able to carry out about 50 divisions and completely stop reproduction. At present, there is no doubt that normal somatic cells have a limited replication potential.

To define the totality of the processes of "programmed" aging and cell death, the term "apoptosis". Apoptosis should be distinguished from necrosis - cell death due to random events or under the influence of external toxins. Necrosis leads to the release of cell contents into the environment and normally causes an inflammatory reaction. Apoptosis is a fragmentation of the contents of the cell from the inside, carried out by special intracellular enzymes, the induction and activation of which occurs when the cell receives an external signal or when the cell is forcibly injected with enzymes - activators of the apoptotic "machine", or when the cell is damaged by external factors that do not lead to necrosis, but capable of initiating apoptosis (ionizing radiation, reversible overheating, etc.).

The current interest of researchers in apoptosis is very high, it is determined by the awareness of the important role of apoptosis in the behavior of cell populations, since its role is no less than the role of the processes of growth and reproduction of cells.

The concept of “programmed” cell death existed for a very long time, but only in 1972, after the work of Kerr, Willy, and Currier, in which it was shown that many processes of “programmed” and “non-programmed” cell death are quite close, interest in apoptosis greatly increased. After the role of DNA degradation processes in apoptosis and, in many cases, the necessary de novo synthesis of RNA and specific proteins, was shown, apoptosis became the subject of biochemistry and molecular biology.

The molecular biology of apoptosis is very diverse. Apoptosis is studied by morphological changes in cells, by the induction, activity and appearance of transglutaminase products that "cross-link" proteins, by DNA fragmentation, by changes in calcium fluxes, by the appearance of phosphatidylserine on the membrane.

In 1982 S.R. Umansky suggested that one of the functions of the eukaryotic cell death program is the elimination of constantly emerging cells with oncogenic properties. This hypothesis is confirmed by the discovery of the p53 protein, an apoptosis inducer and tumor suppressor. The p53 protein is a transcriptional regulator capable of recognizing specific DNA sequences. The p53 gene activates several genes that delay cell division in the G 1 phase. After the action of factors that damage DNA (radiation, ultraviolet radiation), the expression of the p53 gene in cells is significantly enhanced. Under the influence of p53, cells with multiple DNA breaks are delayed in the G 1 phase, and if they enter the S phase (for example, in the case of tumor transformation), they undergo apoptosis.

Mutation of the p53 gene allows cells with damaged DNA to complete mitosis, preserves cells that have undergone tumor transformation, while they are resistant to radiation and chemotherapy. The mutant form of the p53 protein does not have the ability to stop the cell cycle.

The most common concept of "programmed" aging is currently based on the concept of the telomere. The fact is that DNA polymerase is not able to replicate the “tails” of the 3 / - end of the DNA template - several nucleotides at the 3 / - end. Multiple DNA replication during cell reproduction in this case should lead to a shortening of the read region. This shortening can be the cause of aging and a drop in the replication potential, a deterioration in the functioning of chromosomes. To prevent this process, the specific enzyme telomerase synthesizes the repeatedly repeated TTAGGG hexanucleotide at the ends of nuclear DNA, which forms an extended stretch of DNA called the telomere. The telomerase enzyme was predicted in 1971 by A. Olovnikov and discovered in 1985 by Greider and Blackburn.

In most cells of normal human tissues, telomerase is inactive, and therefore cells undergo apoptosis after 50–100 divisions, counting from their formation from the progenitor cell. In malignant tumor cells, the telomerase gene is active. Therefore, despite their "old age" in terms of the number of cell cycles passed and the accumulation of a large number of mutational changes in the DNA structure, the life span of malignant cells is almost unlimited. To overcome genome shortening and aging, according to these concepts, a cell must activate the telomerase gene and express more telomerase.

The growth of cell populations is limited by a number of factors leading to the existence of a limit in the accumulation of cell biomass. For animal and plant cells, growth restriction is a vital necessity; the growth of multicellular organisms is limited. The most important factors limiting the growth of cell populations include:

1. Depletion of the system by the limiting substrate;

2. The appearance in the population of cells that have lost the ability to divide.

3. Accumulation of products that are strong growth inhibitors.

Limiting the growth of a cell population may have the specific nature of a programmed failure. The biochemical mechanisms that stop cell proliferation seem to be of a different nature. It is now clear that in a number of cases growth arrest is associated with a loss of cell sensitivity to environmental growth factors. As an example, one can cite the features of the growth of the lymphocyte population induced by the action of growth factors. For example, the dynamics of the appearance and disappearance of the growth factor receptor on the cell membrane of T-lymphocytes is characterized by the fact that the rapid expression of the receptor is replaced by the stage of its loss. It is possible that the “desensitization” of the growth factor receptor is associated with the mechanism of its inactivation during the reaction.

To obtain a culture, it is best to use fresh cells taken from the tissues of an adult, an embryo, and even from malignant tumors. At present, cell cultures of the lung, skin, kidney, heart, liver and thyroid gland have been obtained. Cells are grown on solid or liquid nutrient media in the form of a monolayer culture, for example on glass, or as a suspension in vials or special devices - fermenters.

At present, to study the mechanisms underlying the growth and development of cell populations, methods of mathematical modeling using computer technology are increasingly being used. On the one hand, these approaches make it possible to fundamentally study the dynamics of processes, taking into account the totality of effects complicating population growth, on the other hand, they allow a reasonable search for technological regimes and fine control of the cell growth process.

The lifespan of some cell strains in culture can reach more than 25 years. However, according to Hayflick (1965), the lifespan of cells in culture does not exceed the lifespan of the type of organism from which they are taken. With a long duration of cell content in culture, they can degenerate into cancer cells. For example, the aging of diploid human fibroblasts in tissue culture corresponds to the aging of the whole organism. It is easier to maintain cell culture of poorly differentiated or undifferentiated tissues - cells of lymphocytes, fibroblasts, some epithelial cells. Highly differentiated and highly specialized cells of internal organs (liver, myocardium, etc.) grow poorly on nutrient media.

The method of tissue culture is of great importance for the study of malignant tumors and their diagnosis, the study of the patterns of regeneration (proliferation, regeneration factors, etc.), for obtaining a pure product of cell activity (enzymes, hormones, drugs), for the diagnosis of hereditary diseases. Cell culture is widely used in genetic engineering (isolation and transfer of genes, gene mapping, production of monoclonal antibodies, etc.). Cell cultures are used to study the mutagenicity and carcinogenicity of various chemical and biological compounds, drugs, etc.

At present, it is impossible to imagine the isolation and study of viruses without the use of cell cultures. The first report on the reproduction of the polio virus in cell cultures appeared in 1949 (Enders J.F. et al.). Cell cultures in virology are used for the following purposes: 1) isolation and identification of viruses; 2) detection of a viral infection by a significant increase in the number of antibodies in paired sera; 3) preparation of antigens and antibodies for use in serological tests. The main sources of tissues for obtaining monolayer cultures are animal tissues, for example, monkey kidneys, human malignant tumors, human embryonic tissues.

An important role in the study of the macrophage system is also played by the method of artificial cultivation. The role of this system in the infectious process, in the formation of antibodies, in the metabolism of blood pigments, in lipid metabolism disorders, in the metabolism of chemotherapeutic drugs, biochemical and biophysical properties, as well as the neoplastic potency of these cells, is being studied. Most of these studies are summarized in Nelson's monograph (Nelson D.S., 1969). In pure culture, macrophages were first isolated in 1921 by Carrel and Ebeling from chicken blood. Since many of the studies performed on macrophages are related to problems of human physiology and pathology, it is desirable to carry out such studies on cultures of human or mammalian macrophages, although mammalian macrophages do not reproduce on an artificial nutrient medium. Blood can serve as an available source of macrophages, but the yield of macrophages is low. The most widely used source of macrophages is the peritoneal fluid. It contains many macrophages and is usually free of other cells. Many free macrophages are present in the lungs (alveolar macrophages). They are obtained by washings from the alveoli and airways of the rabbit.

Analysis of the human karyotype is impossible without the use of cell culture. For this purpose, blood lymphocytes, spleen, lymph nodes, bone marrow cells, human fibroblasts and amniotic fluid cells are examined. To stimulate mitosis of lymphocytes, phytohemagglutinin is added to the culture medium. Cell growth lasts 48 - 72 hours. 4-6 hours before the end of cultivation, colchicine is added to the medium, which stops dividing cells in the metaphase, because inhibits spindle formation. In order to obtain a good spread of chromosomes on the metaphase plates, the cells are treated with a hypotonic solution (0.17%) of sodium chloride or other solutions.

In recent years, embryonic cell culture obtained by transabdominal amniocentesis has been widely used to diagnose many biochemical and cytogenetic defects of the embryo. Amniocentesis is performed between 15 - 18 weeks. pregnancy. The cell population of the amniotic fluid during this period consists mainly of desquamated cells of ectodermal origin: from amnion cells, skin epidermis, as well as the epithelium of the sweat and sebaceous glands, the oral cavity and partly the digestive tract and urinary tract and other parts of the embryo. In 1956, reports appeared on the determination of the chromosomal sex of the fetus based on the study of sex chromatin in the cells of the amniotic fluid. In 1963, Fuchs and Philip obtained a culture of amniotic fluid cells. Currently, several methods are used to obtain amniotic fluid cell cultures. Usually, 10 ml of a liquid sample is taken for analysis, centrifuged, the cell sediment is resuspended and seeded in plastic vials or Petri dishes in a special medium. Growth becomes noticeable after a few days. After reseeding, the cell suspension on days 14–21 is used to obtain metaphase plates.

Most of the modern knowledge in molecular biology, molecular genetics, and genetic engineering has been obtained from the study of cell cultures of microorganisms. This is determined by the fact that microorganisms and cell lines are relatively easy to cultivate, the process of generating a new generation takes from tens of minutes to several hours compared to macroorganisms, the growth of which takes years and decades. At the same time, development scenarios are similar for all populations developing in closed systems.

cell cultures

Cell culture technology consists in growing cells outside living organisms.

Plant cell cultures

Plant cell cultures are not only an important step in the creation of transgenic plants, but also environmentally acceptable and economically viable a source of natural products with therapeutic properties, such as paclitaxel (paclitaxel), contained in yew wood and produced as a chemotherapy drug called Taxol (Taxol). Plant cell cultures are also used to produce substances used by the food industry as flavors and colors.

Insect cell cultures

The study and application of insect cell cultures expands the possibilities for the development and use by humans of biological agents that destroy insect pests, but do not affect the viability of beneficial insects, and also do not accumulate in the environment. Although the merits of biological methods of pest control have been known for a long time, the production of such biologically active substances and pathogens for insects and microorganisms in industrial quantities is very difficult. The use of insect cell cultures can completely solve this problem. In addition, just like plant cells, insect cells can be used to synthesize drugs. This perspective is currently being actively explored. In addition, the possibility of using insect cells to produce VLP vaccines (VLP - virus-like particle - virus-like particles) for the treatment of infectious diseases such as SARS and influenza is being studied. This technique could greatly reduce costs and eliminate the safety concerns associated with the traditional chicken egg method.

Mammalian cell cultures

Mammalian cell cultures have been one of the main tools used by livestock breeding specialists for more than a decade. Under laboratory conditions, eggs obtained from cows of outstanding quality are fertilized with the spermatozoa of the respective bulls. The resulting embryos are grown in a test tube for several days, after which they are implanted in the uterus of surrogate mother cows. The same technique is the basis of human in vitro fertilization.

At present, the use of mammalian cell cultures goes far beyond artificial insemination. Mammalian cells may complement, and perhaps someday replace, the use of animals to test the safety and efficacy of new drugs. In addition, just like plant and insect cells, mammalian cells can be used to synthesize drugs, especially some animal proteins that are too complex to be synthesized by genetically modified microorganisms. For example, monoclonal antibodies are synthesized by mammalian cell cultures.

Scientists are also considering using mammalian cells to produce vaccines. In 2005, the US Department of Health and Human Services awarded Sanofi Pasteur a $97 million contract. The task of the company's specialists is to develop methods for cultivating mammalian cells in order to accelerate the development of influenza vaccines and, accordingly, increase humanity's preparedness for a pandemic.

Culture Based Therapies adult stem cells found in some body tissues (bone marrow, adipose tissue, brain, etc.) will also soon take their rightful place in clinical practice. Researchers have found that stem cells can be used by the body to repair damaged tissue. Adult hematopoietic stem cells have long been used as bone marrow transplants. They are necessary to restore the processes of maturation and formation of all types of blood cells. Such cells can be obtained in large quantities from cord blood, but their isolation is a rather complicated process.

Researchers are currently working on methods for isolating stem cells from the placenta and adipose tissue. A number of specialists are engaged in the development of methods for cellular reprogramming - returning mature cells of the body, for example, skin cells, to an undifferentiated state, and subsequent stimulation of their differentiation into cells of the required type of tissue.

Embryonic stem cells

Usage embryonic stem cells is also considered as a potential method of therapy for many diseases. As the name implies, fetal cells are obtained from embryos, in particular those that develop from eggs, fertilized in vitro (in vitro fertilization clinics) and, with the consent of donors, donated to researchers for scientific use. Blastocysts are usually used - 4-5-day-old embryos that look like balls under a microscope, consisting of several hundred cells.

For the isolation of human embryonic stem cells, the inner cell mass of the blastocyst is transferred to a nutrient-rich culture medium, where the cells begin to actively divide. Within a few days, the cells cover the entire surface of the culture plate. After that, the researchers collect dividing cells, divide them into parts and place them in new plates. The process of moving cells into new plates is called reseeding and can be repeated many times over many months. The cell passage cycle is called passage. Embryonic stem cells that have existed in culture for six or more months without differentiation (i.e., remaining pluripotent - capable of differentiating into cells of any tissue of the body) and retaining a normal set of genes are called embryonic stem cell line.

The inner surface of the culture plate is often covered with skin cells from mouse embryos genetically modified to fail to divide. These cells form a feeder layer - a "nutrient substrate", thanks to which the embryonic cells are attached to the surface. Scientists are trying to improve the existing method and eliminate the need to use mouse cells, since their presence always introduces the risk of viral particles and mouse proteins entering the culture of human cells that can cause an allergic reaction.

The maximum value of stem cell therapy and tissue engineering can be achieved if the therapeutic stem cells and tissues grown from them are genetically identical to the recipient's cells. Therefore, if the patient himself is not their source, stem cells must be modified by replacing their genetic material with the genes of the recipient and only then differentiated into cells of a specific type. At present, the procedure for replacing genetic material and reprogramming can only be successfully performed with embryonic stem cells.