Cellular biotechnology in crop production. Technologies using GMOs and biotechnology

A special area of ​​application of cell cultures and cell technologies is tissue engineering, associated with the development of bioartificial organs and tissues. Currently, on the basis of accumulated fundamental knowledge, technologies for maintaining cell cultures of various origins (plant cells, insect and mammalian cells) have been mastered, including on an industrial scale.

Plant cells and plant tissue culture make it possible to regenerate an entire plant from protoplasts and cells. A feature of plant cell cultures is their ability to totipotency, i.e. in a certain environment and certain conditions, it is possible to regenerate a whole plant from a single cell. This technique provides relatively short term obtaining numerous cell populations under controlled conditions and makes it possible to identify plant lines with increased biological productivity.

Plant cell engineering is based on the use of isolated cell cultures, tissues, and protoplasts. There are several areas for using these technologies in crop production.

The first is associated with the ability of isolated plant cells to produce valuable biologically active compounds in culture, including ginseng or idiolites, essential oils, alkaloids, glucosides, etc.

The second direction is the use of isolated tissue culture for clonal propagation of plants and improvement of planting material.

The third direction is the use of isolated cells in plant breeding. Plant cells cultured on artificial media are characterized by great heterogeneity; In this case, it is possible to select cells that are resistant to certain unfavorable factors - drought, low temperature, phytopathogens, etc.

Plant cell cultures are used for the biotransformation of chemical compounds and for the efficient de novo synthesis of biologically active compounds. In cell culture, the ability to produce biologically active compounds characteristic of the original whole plant is retained, which makes it possible to organize conditions that ensure the synthesis of valuable products not previously found in the original intact plants. For example, in plant cell cultures it has become possible to obtain such valuable compounds as pericin, pericalin, hinokiol, ferriginol, aquammalin, etc.

Large-scale plant cell culture systems have been implemented to obtain various valuable substances- menthol, ginseng, ubiquinone-10, betanin, camptothecin (anti-carcinogen), polypeptides - phytovirus inhibitors, agar-agar, etc. For example, an effective and expensive cytostatic drug paclitaxel, traditionally obtained from the bark of the Mediterranean Yew (8 tons of raw materials provide 1 kg of the drug), is currently obtained with greater efficiency in cell culture.

Processes using immobilized plant cells turned out to be even more effective. Such biological systems are more resistant to mechanical damage, and the phase of cell growth coincides with the phase of product formation; cells are easily transferred to a new environment or other culture conditions. Once the ability of the apical meristem (the small area of ​​undifferentiated cells at the tip of a stem) to grow to form a whole plant was established, this technique was used to clone plant lines.

Plant tissue culture, similar to cell culture, allows one to quickly obtain healthy plant clones and, on this basis, promising planting material on an almost unlimited scale.

Insect cell cultures make it possible to obtain new types of biological agents to combat insect pests without negative influence for viability useful species insects, as well as those that do not accumulate in the environment. The advantages of biological methods of pest control have been known for a long time; these are bacterial, fungal and viral preparations, the production of which requires specialized equipment and conditions. This is especially true for drugs of the viral group, the production of which is based on the mass reproduction of the host insect on artificial media. Due to the rather labor-intensive nature of production, these drugs have not found widespread use until recently.

The use of insect cell cultures can completely solve this problem. The technique of insect cell cultures for propagating viruses is very promising. This requires obtaining highly productive cell lines, optimizing culture media, and choosing effective virus-cell systems. Using this technology, the drug “Elkar” is produced in the USA. Developments in recombinant baculoviruses with genes encoding water exchange insects After using this drug, insects die within 5 days from dehydration or oversaturation with water.

New biotechnology methods may affect the price of viral drugs. In addition, just like plant cells, insect cells can be used to synthesize medicines. The implementation of the potential of insect cells for the production of VLP vaccines (VLP - virus-like particles) has begun, intended for the treatment of infectious diseases such as atypical pneumonia and flu. This technique could greatly reduce costs and eliminate the safety concerns associated with the traditional method using chicken eggs.

Animal cell cultures are widely used as test objects to evaluate the safety and effectiveness of new drugs. In addition, mammalian cells are suitable for the synthesis of drugs, especially some animal proteins that are too complex to be synthesized using genetically modified microorganisms, as well as monoclonal antibodies and vaccines.

For example, Sanofi Pasteur (USA), commissioned by the US Department of Health and Human Services, is developing methods for culturing mammalian cells for the purpose of effectively synthesizing influenza vaccines. A special area of ​​application of cell cultures, especially stem cells, and technologies is therapy and reconstructive surgery damaged organs and tissues.

The list of diseases the treatment of which becomes possible through the use of cell therapy and transplantation is rapidly growing. The most advanced currently is the use of cellular technologies in cardiology for the treatment of myocardial infarction, restoration of blood flow in ischemic organs and tissues, increasing the pumping function of the heart, as well as the treatment of dyslipidemia and atherosclerosis. In neurology, cell transplantation technologies have begun to be used to treat Parkinson's disease and Haginton's disease.

There are examples of the positive use of bone marrow stem cells for the healing of burns and deep skin wounds, and the treatment of systemic and local bone defects. In connection with the identified antitumor activity of poorly differentiated hematopoietic cells and their ability to directly suppress tumor growth, research is being conducted aimed at the clinical use of stem cells in oncology.

The search for new technologies to restore the lost function of an organ or system has led to the emergence of tissue engineering (regenerative medicine and organogenesis) at the intersection of biotechnology and medicine. The future of medicine is directly linked to the development of cellular technologies, which make it possible, without changing a damaged organ, to “renew” its cellular composition. This “renewal” of the structural and functional elements of an organ makes it possible to solve the same problems as organ transplantation. At the same time, this technology greatly expands the possibilities of transplantation treatment, making it accessible to a wide range of different categories of patients.

The basis for the development of the latest reconstructive technologies are functioning cells capable of forming tissues of different types, depending on the microenvironment.

The list of diseases for which cellular technologies are already being used or are planned to be used in the near future is growing rapidly. This list will apparently include all the diseases drug treatment which are ineffective. The interdisciplinary approach used in tissue engineering is aimed primarily at creating new biocomposite materials to restore the lost functions of individual tissues or organs as a whole.

The basic principles of this approach are the development and use of carriers made of biodegradable materials, which are used in combination with donor cells and/or bioactive substances, when implanted into a damaged organ or tissue.

Revolutionary transformations of traditional biotechnological processes are associated with the use of methods genetic engineering. The recombinant DNA method is the cornerstone of modern biotechnology. Making recombinant DNA means combining (recombining) two pieces of DNA from different species.

With the help of genetic engineering, various socially significant technologies and processes have been developed and used:
- production of new medicines and safe vaccines;
- treatment of certain genetic diseases;
- creation of biocontrol agents for agriculture;
- increasing productivity and reducing production costs;
- reducing the allergenicity of some products;
- improvement nutritional properties products;
- development of biodegradable plastics;
- reducing the level of water and air pollution;
- slowing down the rate of food spoilage;
- control of viral diseases.

Molecular or genetic cloning - the process of creating genetically identical DNA molecules - is the basis of molecular biology, a fundamental method of biotechnology research, and the basis for the development and commercialization of biotechnology. The vast majority of practical applications of biotechnology, from drug development to the creation of transgenic crops, are based on genetic cloning methods.

With the help of molecular cloning it became possible: identification, localization and description of genes; creating genetic maps and sequencing entire genomes; drawing parallels between genes and their associated traits; establishing the molecular basis for the manifestation of symptoms. The scope of cloning is extremely wide.

To the number promising directions biotechnology refers to the modification of the genome of cultivated plants in order to increase productivity and improve their resistance to pests and unfavorable conditions environment. Naturally, the greatest interest is generated by the possibility of transformation of monocotyledonous plants (primarily cereals).

Transformation of the genome of higher plants is carried out by two methods: ballistic and using a natural system for transferring genetic material - part of the Ti-plasmid (T-DNA) - Agrobacterium tumefaciens, the causative agent of bacterial cancer or crown gall in dicotyledonous plants. Antibiotic resistance genes are used as a marker system, as well as new systems - Lux genes or the bacterial P-glucuronidase gene, which causes staining of the selective medium. Ti plasmids modified with different genes are transferred into plants. The transfer of genetic information using a Ti plasmid was also carried out using the example of monocotyledonous plants: gladiolus bulb tissue with the side parts removed was used.

Cylindrical explants were infected with virulent strains of Agrobacterium carrying a Ti plasmid, which contains genes encoding the synthesis of opines; after 24 hours, tumors appeared on the surface of the infected plant. Experts from Carnell University (USA) have proposed a new method - shooting tungsten bullets coated with genetic material at plant cells. “Shelling” the host cells occurs at a speed of over 1,000 km/h with a million metal pellets coated with a layer of DNA fragments; the method turned out to be universal.

With the help of genetic engineering, it has become possible to obtain salt-, herbicide- and frost-resistant plants. According to experts, in the United States, annual damage from frost is estimated at $6 billion. It turned out that the most active centers of crystallization are individual bacteria (representatives of Pseudomonas, Erwinia), capable of causing ice formation at 0 °C; They were called INA bacteria - active water crystallizers (Ice-nucleation Active). In these bacteria, a specific membrane protein has been identified that causes crystallization; ice genes have been cloned and modified.

Removal of the middle portion resulted in loss of the ability to excrete protein crystallization. As a result, bacteria with a modified ice gene lost the ability to crystallize water at -1-5 oC. Inoculating plants with these genetically modified bacteria at bud break prevents colonization by other bacteria, so the plants are effectively immune to infection by wild-type INA bacteria and are immune to short-term frosts.

The second wave of the “green revolution” is focused on obtaining genetically modified “self-fertilizing” plants. It has been established that the genome of nitrogen-fixing symbiotic bacteria contains a group of genes responsible for symbiosis with plants and localized in large symbiotic plasmids pSym; the process of nodule formation is controlled by nod genes and, finally, nif genes are responsible for the synthesis of nitrogenase, i.e. nitrogen fixation.

Currently, large amounts of money in the United States and other countries are being invested in a program for producing transgenic cereals with nitrogen fixation genes. However, when nitrogen fixation genes are transferred to higher plants, in addition to difficulties genetic nature, there are others. The regulation of the relationship between nitrogen fixation genes and the genes responsible for the synthesis of electron carriers and cofactors necessary for the functioning of the nitrogenase enzyme has not yet been adequately studied.

The latter must be protected from the inhibitory effects of oxygen. Intensive research is also being carried out on plant genetics to select effective host plants, as well as research aimed at modifying the genome of microorganisms to produce organisms that can exist in symbiosis not only with leguminous plants (for example, cereals). Basic Research on the transfer of nitrogen fixation genes into higher plants will apparently lead to promising discoveries and a radical revolution in the practice of nitrogen nutrition of plants.

The second, very important area of ​​application of genetic engineering is to give crop plants resistance to infection by leaf-eating insects. Natural plant pathogens, such as the bacteria Bacillus thuringiensis (Bt), which produce toxins effective against leaf-eating insects, have become the source of genes for making plants resistant to these pests. The synthesis of Bt toxins is controlled by a single gene; available methods allow work aimed at improving existing Bt producers and products.

It is known that the genes controlling the synthesis of Bt crystals are localized on a small number of plasmids of significant molecular weight. The toxic protein produced by Bt has been cloned into E. coli and B. subtilis and has been expressed even during the vegetative growth phase. There is information about cloning a protein toxic to butterflies in tobacco cells.

In a fully grown tobacco plant, every cell produced a toxin. Thus, a plant that has acquired the toxin itself becomes resistant to insects: by eating the leaves, the caterpillar dies without causing significant harm to the plant.

The American companies Monsanto and Agrocetus have conducted field tests and released varieties of cotton, soybeans and a number of other crops with the Bt genome embedded in the chromosome. Resistance to caterpillars is transmitted to seeds and subsequent generations of plants. The production of transgenic potato and tomato seedlings with the introduced Bt gene, which is toxic to lepidopterans, has begun. A transgenic insect-resistant poplar with an antitrypsinase gene introduced into tissue cells was created. The enzyme reduces protein absorption by insects, which leads to population decline.

Herbicide resistance genes have been cloned; their cloning into plants is intended to ensure the safety of the use of pesticides in agriculture. However, cloning genes into crop plants is associated with certain risks. Concerns are associated with the possibility of genetic vectors and transgenic plants leaving the control of biotechnologists. Therefore, there are concerns about the transformation of genetically engineered plants into weeds, although the “weediness” complex (a set of traits that ensures rapid spread to the detriment of cultivated plants, resistance to adverse factors, effective seed dispersal mechanisms, etc.) can hardly be formed as a result of transplantation of one or a few genes.

At the same time, herbicide resistance encoded by a single gene can cause significant problems in crop rotation practices. Thus, a plant resistant to a certain drug, cultivated in a certain area, will act in relation to it as a weed resistant to this herbicide the next year when changing crops in this field. This requires careful testing of all genetically engineered plants before they are released into the field.

A new way of genome modification is the use of antisense RNAs, i.e. suppression of the synthesis of a certain protein. The introduction of a complementary mRNA oligonucleotide into the cell prevents the reading of information. The use of this technology made it possible to obtain a tomato variety that is preserved long time by blocking the function of the polygalacturonase gene (breaks down carbohydrates in the cell, stimulating ripening), or coffee with low caffeine levels as a result of introducing a gene that suppresses production; those. This is a way to remove unpleasant bitter and other substances from agricultural products, suppress viral infections, etc.

Genetic engineering of animals is aimed at breeding animals with high performance properties.

Genetic engineering methods, together with animal cloning, also make it possible to obtain models for studying human diseases, aging processes and the formation of malignant neoplasms. In the future, these techniques could be used to develop new drugs and evaluate the effectiveness of treatments such as gene and cell therapy. Animal cloning also provides an opportunity to save endangered species.

Biotechnological methods of animal reproduction include artificial insemination, induction of labor, transplantation, regulation of the ratio of male and female female in the population, this also includes early diagnosis of pregnancy, the use of hormones to regulate reproductive functions and growth, etc.

Artificial embryo division is a routine cloning method. The method makes it possible to obtain large number replica animals from high-value producers. It is possible to obtain a large number of copies of identical twins by dividing embryos at the blastula or morula stage into parts. These parts of the embryo are introduced into the empty shells of pig eggs, placed in agar cylinders for several days, then introduced into the oviducts of sheep.

Normally developed embryos are surgically transplanted into recipient cows on days 6-7 of the reproductive cycle. The survival rate for halves is up to 75%, for quarters it is lower, about 40%. The resulting embryos are implanted into the uterus of the surrogate mother, who ensures their gestation and birth. Since the embryos come from the same zygote, they are genetically identical.

Embryo manipulation is used to produce embryos of various animals. The approach makes it possible to overcome the interspecies barrier and create chimeric animals. In this way, for example, sheep-goat chimeras were obtained.

The first experiments showed the possibility of transforming the genome of animals with human genes; in the USA it was possible to obtain pigs carrying the gene for human growth hormone. At the Edinburgh Biotechnology Center, sheep were obtained with transferred human factor 9, which is secreted in the milk.

The new method - somatic cell nuclear transfer (or reprogramming of eggs) begins with the isolation of a somatic cell from the body - any cell that is not involved in the reproduction process (i.e., any other than germ cells - sperm and eggs). In mammals, any somatic cell contains a complete double set of chromosomes (in each pair, one chromosome is obtained from the mother's egg, the second from the father's sperm). The genome of any germ cell consists of only one set of chromosomes.

To create Dolly the sheep, the researchers transferred the nucleus of a somatic cell obtained from an adult sheep into an egg whose nucleus had previously been removed. After certain chemical manipulations, the egg with the replaced nucleus began to behave like a freshly fertilized egg. As a result of its division, an embryo was formed, which was implanted into a surrogate mother and carried throughout the entire pregnancy. The appearance of Dolly demonstrated the possibility of removing the genetic program of the nucleus of a specialized somatic cell and reprogramming it in the laboratory by placing it in an egg.

Within 5-6 days, the egg develops into an embryo that is genetically identical to the donor animal. The cells of this embryo can be used to obtain any type of tissue that, when transplanted, will not be rejected by the body of the nucleus donor. This method could well be used to grow cells and tissues for replacement therapy. This method of animal cloning is very actively used at present.

Improving biotechnological methods and means of diagnosis and treatment, the ability to create effective breeds of farm animals and varieties of cultivated plants using accelerated methods - all this helps to improve the quality of human life.

Let us feel the contribution of biotechnology to increasing mineral and energy resources, as well as to protecting the environment. In connection with the latter, the direction focused on the development of environmentally friendly new materials becomes especially significant. Creation of environmentally friendly materials with beneficial properties remains one of the key problems of our time. To date, production volumes of synthetic plastics that are not degradable in the natural environment have reached 180 million tons/year, which has created a global environmental problem.

A radical solution to this problem is the development of polymers capable of biodegrading into components that are harmless to living and nonliving nature. In this regard, in recent years there has been significant progress in the synthesis of degradable biopolymers - high-molecular polymer materials capable of degrading in the natural environment to harmless products (carbon dioxide and water).

ON THE. Voinov, T.G. Volova

Microorganisms differ significantly from each other in morphology, cell size, relation to oxygen, requirements for growth factors, ability to assimilate different components of the substrate, etc.
Of the more than 100,000 known species of microorganisms, relatively few are used in industry - about 100 species. They must meet the following requirements:
1. Grow on cheap and accessible substrates;
2. Possess high speed biomass growth and provide high productivity of the target product with economical consumption of nutrient substrate;
3. Exhibit targeted biosynthetic activity with minimal formation of by-products (Fig. 2.1);

Rice. 2.1. Microorganisms used in industrial synthesis various connections
A – A cetobacter aceti; B – Aspergillus niger; B – Penicillium chrysogenum; D – Lactobacillus delbruecki; E – Production of levan during fermentation of a medium containing Zymomonas mobilis

4. Be genetically homogeneous, stable in terms of productivity and requirements for nutrient substrate, as well as cultivation conditions;
5. Be resistant to phages and other foreign microflora;
6. Be harmless (not have pathogenic properties) for people and the environment;
7. It is desirable that the producers be thermophilic and acidophilic, since in this case it is easier to protect the fermentable substrate from invasion of foreign microflora;
8. The target biosynthesis product must have economic and national economic value and be easily isolated from the fermented substrate.
Anaerobic microorganisms are of increasing interest because they do not require energy-intensive aerating devices during cultivation.
Supersynthesis, that is, the ability of a microorganism to synthesize a certain product in quantities exceeding its physiological needs is quite common in nature. Often, one or another metabolic product (organic acids, alcohols, antibacterial substances) released by a microorganism into the environment is toxic to other species and serves the producer as a means of protecting the inhabited space or as a nutrient reserve. Microorganisms with such properties were the first to be attracted to economic activity humans thousands of years ago and a spontaneous selection of the most productive forms was carried out. Now such natural strains of microorganisms, sometimes after deliberate selection, are used to produce microbial biomass (microbial protein) as bacterial nitrogen fertilizers, biopesticides, in food production and other sectors of the national economy. However, the main contingent of industrial microorganisms is represented by artificially selected strains.
Currently, three types of strains are used in industry:
1. Natural strains, often improved by natural or artificial selection;
2. Strains changed as a result of induced mutations;
3. Culture strains obtained by genetic or cell engineering methods.
Principles of selection of organisms. Starting the conscious selection of microorganisms, man set the goal of creating industrial organisms with properties unusual for wild microbes. Methodologically, a person solves this goal in two ways:
1. Correction of the genetic information of the microbial cell, excluding properties that are not desirable for industrial synthesis and enhancing the desired characteristics.
2. Induction of completely new information in the genetic program of the cell.
To do this, it is also necessary to solve the following problems:
1. Significantly increase the productivity characteristic of a given type of microbe and its metabolic product;
2. Genetically program the biosynthesis of substances that are unusual for a given species or even unusual for the microbial world.
Unlike the selection of cultivated plants and domestic animals, which has a thousand-year history and rich experience, targeted selection and selection of microorganisms began only after recognition of the microcosm and developed in parallel with the achievements of genetics as a scientific discipline.
When selecting any living organism, a person relies on the natural driving forces of evolution - hereditary changes and selection of positive specimens. However, microorganisms, as objects of selection, have a number of features:
1. Growing a microbial culture from one cell - a common technique for microbial selection - leads to the fact that an individual clone (having genetic uniformity) always ends up in the hands of the breeder as the starting material for selection. On the other hand, a clone of microorganisms quickly reaches such a number that, due to natural mutations, it turns into a population - a collection of cells with different genotypes;
2. Most microorganisms are haploid, with one copy of chromosomes, so they do not have hidden variability, which is the basis for selection of higher organisms;
3. The ability for hybridization (sexual reproduction) is still unknown in most microorganisms of industrial importance. This means that cell selection can only be carried out vegetatively;
4. Microorganisms are characterized by an exceptionally rapid change of generations, therefore the microbiologist has much more opportunities for selecting positive specimens. The assessment of the selected microorganism can be carried out in a matter of days of cultivation, in contrast to macroorganisms, where the results of the work are visible after several years;
5. The microbial breeder has a huge number of individuals to select, which fundamentally expands his capabilities, but assessing the productivity of each clone requires labor-intensive work.
The culture to be selected can be selected from the cultures collected in the collection (museum cultures); well-known industrial producers can be used; microbes can be isolated from natural substrates.

Ideas about the biochemistry and physiology of microorganisms guide the breeder towards certain groups of microorganisms with the most likely potential supersynthesis substance of interest. Antibiotic producers are mainly found among ascomycete and actinomycete fungi; supersynthesis of amino acids is easier to obtain in corynebacteria; extracellular enzymes are often synthesized by yeast. The main stages of microorganism selection are shown in Figure 2.2.

Rice. 2.2. Selection scheme for microorganisms

Table 2.1. Groups of cell technology methods

Methods for creating distantly related hybrids

Cell technologies in plant breeding . Enrichment of the gene pool of grain crops can be achieved using huge genetic resources their wild relatives. Types of cereals with high resistance to:

• diseases and pests,
• temperature and water stress,
• salinity and high acidity of soils.

In breeding work, to overcome interspecific (or even intergeneric) incompatibility, the following methods are used:

• in vitro fertilization,
• embryo culture,
• backcrossing and other modern techniques (Feldman and Sears, 1984).

Genetic engineering of plants creates a completely new mechanism of genetic variability - transgenosis, which, unlike previously existing ones (recombiogenesis, mutagenesis), is characterized by the possibility of transferring individual genes. True, this feature makes it difficult to use methods genetic engineering to improve a number of economically valuable traits inherited polygenically. But chimeric plants have already been obtained that carry genes for resistance to diseases, insects, herbicides, etc. (Kilchevsky, Khotyleva, 1997).

Distant hybridization of cultivated cereals with wild relatives, the goal is to transfer single genes or small fragments of chromosomes from wild ones to the genome of cultivated species. But for this it is necessary to overcome incompatibility barrier- lack of chromosome conjugation in meiosis. In wheat, genes affecting chromosome conjugation were discovered on chromosome 5B, and thus the ability to control this process to a certain extent was revealed.

At the University of Madrid (Spain) and the Max Planck Institute for Breeding (Cologne, Germany), a direct gene transfer method, which makes it possible to obtain transgenic plants in a relatively simple way in vivo, eliminating the need to regenerate plants from cells. The most extensive work is being done with Azospirillum bacteria (Postgate, 1989). In a series of experiments conducted in various states of India, inoculation of seeds of wheat, rice, sorghum, and millet with rhizosphere nitrogen fixers provided an increase in grain yield of up to 30% (Subba Rao, 1982.

Another important area of ​​modern biotechnology has now received wider practical application - cell selection as a method of creating new forms of plants by isolating mutant cells and somaclonal variations under selective conditions. Cellular selection is, as it were, a development of mutation selection, but is implemented at the level of single cells using in vitro technology, which gives it, on the one hand, greater opportunities, and on the other hand, creates significant difficulties due to the need to regenerate full-fledged cells from individual cells. plants.

Advantage cell selection before traditional methods consists of:

• lack of seasonality in work,
• the possibility of using millions of cells during selection,
• direction of selection through the use of selective media,
• performing work in laboratory conditions

With the development of in vitro culture, there is a real possibility of wider use of haploidy in crop breeding. The use of the cell culture method made it possible to regenerate plants from generative cells containing haploid set chromosomes. Mass production of haploids became possible. Practical significance in selection currently obtained culture anthers (androgenesis), ovaries and ovules (gynogenesis) and the haploproducer method, which is a type of gynogenesis

Lecture outline

Topic 5.6. Preparation for agriculture. Protein of unicellular microorganisms

OBTAINING MICROBIAL BIOMASS

MODULE 2.

Lecture format: brain attack

1 Biotechnology and crop production.

2 Biotechnology and animal husbandry.

3 Technological bioenergy.

4 Production of feed protein.

5 Use of yeast and bacteria.

6 Use of algae and microscopic fungi.

Problem to solve: areas of application of biotechnology in agriculture.

Students express ideas for solving this problem. The ideas are then analyzed by a group of experiments with the help and advice of the teacher. The rule of brainstorming is that any ideas, even the most absurd, are expressed; criticism of ideas at the time of attack is prohibited. All ideas are recorded by the facilitator and ensured that they are reviewed by the participants. Such a lecture activates students’ mental activity and develops heuristic abilities.

Cultivated plants suffer from weeds, rodents, insect pests, nematodes, phytopathogenic fungi, bacteria, viruses, unfavorable weather and climatic conditions. These factors, along with soil erosion and hail, significantly reduce the yield of agricultural plants. The Colorado potato beetle and the fungus cause enormous damage to potato growing. Phytophthora- causative agent of early rot (late blight) of potatoes.

Corn is susceptible to devastating outbreaks of southern leaf rot, the damage from which in the United States in 1970 was estimated at $1 billion.

In recent years great attention are given to viral plant diseases. Along with diseases that leave visible marks on cultivated plants (mosaic disease of tobacco and cotton, winter disease of tomatoes), viruses cause hidden infectious processes that significantly reduce the yield of agricultural crops and lead to their degeneration.

Biotechnological ways to protect plants from the considered harmful agents include:

Breeding plant varieties resistant to adverse factors;

Chemicals control (pesticides), weeds (herbicides), rodents (raticides), insects (insecticides), nematodes (nematicides), phytopathogenic fungi (fungicides), bacteria, viruses.;

Along with plant protection, the task is to increase the productivity of agricultural crops, their nutritional (feed) value, and the creation of plant varieties growing on saline soils, in arid and swampy areas. Developments are aimed at increasing the energy efficiency of various processes in plant tissues, from the absorption of light quantum to the assimilation of CO 2 and water-salt metabolism.

Most biomedical research is carried out on cells in vitro(that is, not on a living organism, but on cells “in vitro”). Cells are used as a model biological object in scientific research, testing and drug production. In addition, scientists have learned to correct genetic errors in cells and give them the ability to resist certain diseases, which serves as the basis for future medical technologies - gene and cell therapies. This article will discuss methods for working with cells, as well as the possibilities and limitations associated with their use.

The general partner of the cycle is the company: the largest supplier of equipment, reagents and Supplies for biological research and production.

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A living object consists of smaller components: organs, tissues, cells, organelles, biomolecules and at the same time is part of larger systems - such as food chains, ecosystems, communities. Even Hippocrates and Aristotle wondered about the relationship between the whole and its parts and agreed on the understanding that the whole is more than the sum of the parts. However modern science relies almost entirely on an alternative principle - reductionism - according to which knowledge of the whole is carried out through knowledge of its component parts. With this approach, connections between parts are lost, in our case between cells. Although each cell is also, in essence, a whole, outside the body it is no longer exactly the same cell that was in its composition. What changes with this transition?

A clear illustration is provided by slime molds. These single-celled organisms have the ability to combine tens of thousands of cells into plasmodium, which, in search of food, can move quickly and throw out spores over a long distance, and then again disintegrate into individual cells (video 1). Many bacteria also have the ability to form dense conglomerates - biofilms, or biofilms, in which internal cells are protected by external ones from the environment. That is, association into multicellular structures leads to the emergence of new functions that are absent in individual cells. Competitive advantages through the acquisition of new abilities caused the unification of unicellular organisms into multicellular ones about 1 billion years ago.

Video 1. Dictyostelium discoideum- single-celled slime mold. When slime molds search for food, individual cells unite and form a plasmodium up to 2 mm long.

In multicellular organisms, cells exist in inextricable connection with each other over hundreds of millions of years of evolution and during this time they have been completely reorganized to perform specific functions. The spectrum of genes expressed by a cell, its functions and activity, shape and size, rate of division and death are regulated by connections with other cells of the body. In mammals, this connection is based on all kinds of signals: chemical (hormones, cytokines, growth factors, nutrients, oxygen derivatives, metal ions, etc.), mechanical and electrical (transmitted by neurons) (Fig. 4).

Figure 4. Main types of cell-cell interactions. Within the body, cells are closely interconnected, and their activities are strictly coordinated. For this purpose the following are used: electrical signals ( above), transmitted by neurons, chemical signals ( in the middle), transmitted through blood (endocrine) or intercellular fluid(paracrine), as well as mechanical forces ( from below). These connections exist both between neighboring cells and between even the most distant cells of the body. The cell converts incoming signals into instructions to change the spectrum of active genes, and therefore the properties of the cell itself.

Intercellular communication underlies processes studied by almost all medical disciplines, in particular endocrinology, neurology, cardiology, hematology, etc., but there is no talk yet of a complete deciphering of these interactions. If a cell in the body is disrupted in its normal connections with its environment, it either dies (for this there are special mechanisms of self-destruction - apoptosis and anoikis), or becomes “asocial”, that is, cancerous. When isolated from the body and transferred to a Petri dish, the cells also lose almost all external connections, but under favorable conditions they live and divide for some time (however, they can also become cancerous).

In new ones, artificial conditions cells retain only the genome, and all other properties change - size, growth rate, gene expression, functional activity, sensitivity to drugs, metabolism, membrane composition and others. In this regard, today there is an active search for conditions in vitro, which maximally reproduce the conditions in vivo. Most body tissues contain several types of cells, have a complex structure of the extracellular matrix, and are penetrated by a network of vessels and nerves (Fig. 5). Completely recreate such an architecture in vitro is not yet possible, which means that cells in culture still remain only an approximation to the cells in the body.

Figure 5. Complex organization of connective tissue in vivo consisting of: several types of cells (fibroblasts, fat cells, macrophages and other leukocytes), several types of fibers (1 - elastic, 2 - reticular and 3 - collagen), nerves and vessels enclosed in a hydrogel made of polysaccharides.

Cell isolation and cultivation

The source of cells can be any tissue in the body. In most tissues, cells are located within what is called extracellular matrix, which divides the tissue into regions and shapes its architecture (Fig. 5 and 6). The extracellular matrix consists of proteins that form fibrils: collagen, fibronectin, elastin and proteoglycans with long polysaccharide branches (glycosaminoglycans). The latter perfectly retain liquid and form a so-called hydrogel in most of the extracellular space. In the case of bone, the matrix is ​​formed from crystallized calcium phosphate. The most common human protein is collagen: it accounts for up to 30% of the mass of all proteins in the body. Matrix components are synthesized by many cells, but fibroblasts specialize in this.

Figure 6. What tissues look like under a microscope: intravital multiphoton microscopy various areas skin containing loose ( A ) and dense ( b ) connective tissues, muscular ( V ) And nerve fibers (G ), fat cells ( d ) and microvesicles ( e ). Collagen fibers are shown in red (except V ), blood vessels - green, cells and microvesicles - blue (on b - green). Size scale: 50 µm. To see the picture in full size, click on it.

To isolate cells, it is necessary to destroy the extracellular matrix and break the bonds between cells, after which the cells “scatter” (Fig. 7). A cell culture can also be obtained in another way: by placing an entire piece of tissue in a culture medium, and then some of the cells will gradually crawl out and seed the surrounding space. This type of culture is called explant. Isolation and manipulation of cells are usually carried out in a special sterile box - a laminar flow hood (Fig. 8 A ).

Figure 7. Scheme of cell isolation from tissue. A tissue fragment obtained intravitally (by biopsy) or after death is crushed mechanically and treated with proteolytic enzymes that break down the extracellular matrix (trypsin, collagenase, hyaluronidase, papain or combinations thereof). In addition to enzymes, calcium-binding compounds (chelators) are used, which take calcium from cell adhesion molecules and weaken their connection with each other and with the extracellular matrix. The addition of DNase prevents cells from sticking together into sticky clots due to electrostatic interactions with DNA released from damaged cells. After this, the cells are separated from matrix fragments (debris) by centrifugation and/or filtration and planted in culture flasks or Petri dishes.

Some tissues do not have an extracellular matrix or the cells are not associated with it, which greatly simplifies the isolation procedure. This applies mainly to blood cells and their precursors found in the bone marrow. Due to the ease of isolation and preservation of surface molecules for these cells, the variety of types and the routes of their formation from blood stem cells (hematopoietic SC) are the most well studied.

After isolation, the cells are placed in a nutrient medium and grown in Petri dishes or flasks in an atmosphere of carbon dioxide (5% CO 2) and close to 100% humidity in CO 2 incubators (Fig. 8 b ). The nutrient medium consists of physiological salts, pH buffer, amino acids, vitamins and glucose, and protein growth and nutritional factors (see box below). The phenol red indicator added to the medium allows you to visually monitor the pH and gives the medium a red color.

Figure 8a. The main equipment for cultural work is a laminar flow cabinet. The laminar flow cabinet provides a uniform vertical flow of sterile air, protecting the cell culture from microbial contamination.

Figure 8b. The main equipment for cultural work is a CO 2 incubator. Carbon dioxide in a CO 2 incubator, firstly, it allows you to better reproduce the conditions in vivo, and, secondly, it is necessary to maintain the pH of the environment, in which, as a rule, a bicarbonate CO 2-dependent buffer system is used. The incubator maintains humidity close to 100%, which prevents the evaporation of the liquid and the concentration of its components.

Protein growth and nutritional factors

Protein factors are the most delicate area when culturing cells. The most common source of these factors is animal serum, usually fetal calf serum, less often horse serum, or from the same animal as the cells themselves. Cells are supplemented with filtered whole serum diluted to 1–10%. When studying the effect of any substances on cells (growth factors, protein hormones, cytokines), serum creates a strong background, which must be reduced by growing cells without serum (deprivation) for 2–72 hours before the experiment. The use of serum is almost impossible to standardize and is a potential source of infection, which significantly limits its use in the production of biomedical products. An alternative to whey is protein supplements of a standard composition made from purified and recombinant proteins.

Problems of cultivation and reproducibility in experiments with living cells (solutions from Diaem)

Some crops are very sensitive to external influences or fluctuations in the chemical composition of the environment, which raises a number of questions:

• How to bring cultivation conditions in vitro to conditions comparable to those in vivo? Some processes occurring in living organisms require the creation of special conditions (microenvironment) for cells.
• How to organize a long-term experiment and observe over time? Cell cultures are an ideal object for long-term experiments. But the problem of environmental homeostasis arises: cells constantly consume nutrients and release metabolites. If the issue is solved by simply changing the culture medium, then the concentration of substances in the medium between replacements will not be constant. In addition, the cell culture will have to be regularly removed from the incubator in order to change the medium or observe under a microscope.
• How to protect a cell culture from contamination by bacteria, mycoplasmas and fungi, which can ruin all efforts to isolate cells from tissues?
• How to achieve reproducibility of an experiment? Cells are living objects whose behavior can be influenced by even minor factors: frequent opening of the incubator door, different time the presence of the culture outside the incubator, lighting when observing cultures under a microscope - all this can have a significant impact on the appearance of artifacts in the experiment. So the second task is to achieve stability of the experimental parameters.

Solution:

Cancer cells, even in the body, are very different from normal ones, and in culture this difference only increases. Thus, many cancer lines have an increased number of chromosomes (aneuploidy), which makes them more resistant to DNA damage. In addition, many types of cancer are caused by viral infections that are transmitted to cell cultures, which does not allow the use of these cells in the production of vaccines (see below). In this regard, lines of “normal” - non-cancerous - cells are in widespread demand in pharmacology and scientific research.

It was the production of vaccines that served as the main motive for obtaining a culture of “normal” cells. To this end, Leonard Hayflick (Fig. 10 b ) isolated cells from abortive material and discovered the presence of a limit on the number of cell divisions in culture, called the Hayflick limit. For human cells, this limit is 50–70 divisions and is due to telomere shortening, a fundamental mechanism of cell aging.

In 1965, Hayflick obtained the lung fibroblast line WI-38 (Fig. 10 A ), which is mortal, but was produced and frozen in sufficient quantities to support research around the world to this day. Due to its human origin, the absence of viral infections and cancer transformation, this line has found wide application in the production of vaccines. Now, however, pharmaceutical companies are afraid that the finite resource of this line will not allow them to complete the research they have begun, and are moving to other lines. Currently, this line is widely used to study the molecular mechanisms of aging.

Some compromise between cancerous and normal is immortalized cells: derived from normal tissue, they have acquired the ability to undergo an unlimited number of divisions. Such a transition to immortality can occur either spontaneously or as a result of the artificial introduction of certain genes or fusion with cancer cells, as in the case hybrid. Oncogenes can give “immortality” to cells: large T-antigen of the SV40 virus, H-Ras, c-myc, E1A. These genes essentially cause tumor transformation of cells with all the attendant disadvantages (genomic instability, loss of physiological functions, etc.). The most delicate and increasingly widespread method of immortalization is the introduction of the telomerase reverse transcriptase (TERT) gene into the cell, which completes telomeres and prevents their shortening during division (Fig. 11). The Nobel Prize in Physiology or Medicine was awarded in 2009 for the discovery of telomerase.

Although such transduction is also essentially oncogenic, it allows for better preservation of physiological functions and less destabilization of the genome compared to transduction with other oncogenes. It's also worth noting that this method does not work for all cells: for example, it does not work for T and B cells.

Figure 11. The ends of chromosomes - telomeres - are shortened with each cell division, and when they are depleted, a self-destruction mechanism is triggered in the cell. Telomeres in vertebrates consist of repeated nucleotide sequences TTAGGG. With each division, these sections are shortened, but telomerase TERT provides its own single-stranded DNA as a template, with which it synthesizes first one chain, and then DNA polymerase completes the second strand of telomere DNA. Thus, telomerase is able to maintain cell immortality.

Differentiated cells(which have acquired their final specialization) make up the majority of the body’s cells and practically do not divide. Being the main components and “workhorses” in all organs and tissues, they serve as the target of action of most drugs, which makes them very popular for research. They can be obtained in culture by directed differentiation pluripotent stem cells into the desired cell type (see below), however, this path has significant technical and, in the case of human cells, also ethical difficulties.

The most common differentiated cell cultures are primary cell cultures- cells isolated from mature tissue and not passaged in vitro(Fig. 12). The number of divisions of such cells critically depends on the culture conditions, but is rarely more than 5–10 times. The exceptions are stem, progenitor and some specialized cells, for example, activated T and B lymphocytes. In addition, with prolonged cultivation, the primary culture is prone to replacement by fibroblasts.

Many studies are carried out specifically on primary cell cultures, which have a number of advantages compared to cancer and immortalized cells:

1. They fit the cells much better in vivo: neurons conduct electrical impulses, hepatocytes secrete albumin, macrophages phagocytose bacteria, etc.
2. If we talk about animal cells, they are easily available if you have a vivarium - all that remains is to obtain them (which, however, is quite labor-intensive). The availability of human cells is determined by the type of tissue: cultures of endothelial cells from the umbilical vein (HUVEC, Fig. 12d) and mesenchymal stromal cells from adipose tissue, the sources of which are by-products obstetrics and surgery respectively.
3. They carry the donor's genotype and can therefore be used to study the causes of pathologies in a particular patient at the molecular level.

Cell cultures of humans, mice, rats, rabbits and other animals are collected in collections stored in liquid nitrogen at a temperature of −196 °C (Fig. 13 A ). The most complete collection - ATCC in the USA - contains more than 4600 eukaryotic cell cultures. At Moscow State University they are now creating a depository of living systems, unique in its scale, “Noah’s Ark”, in which, among all kinds of samples of living objects, there are also animal and human cells (Fig. 13 b ). Some institutes also have their own collections: for example, at the Institute of Bioorganic Chemistry of the Russian Academy of Sciences, the collection is organized according to the principle of a cloud storage with a common journal and cells distributed among different laboratories.

Cell lines from the collections can be purchased; they exist for almost every type of tumor and healthy tissue and have been characterized in detail. This allows you to select the most suitable lines for a particular study and compare the results with those previously obtained in your own or in other laboratories.

Diaem: everything you need to work with cell cultures

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Culture plastic, media, serum, substitutes, additives:

Material provided by our partner - Diaem company

Use of cells in scientific research

Cell cultures are primarily a tool for scientific research. What capabilities does this tool provide and what can it be used for? For cells in culture there is a rich arsenal of manipulation and analysis methods: some are included in the 12 methods of this special project, and many others are described in separate articles on Biomolecule. Here we will briefly discuss the main ones, but before that we will discuss the unique advantages of cell cultures as model objects.

Unlike cells in the body, for cells in culture the researcher completely determines the external conditions. Although these conditions often do not correspond to the conditions in vivo, reproducibility and control allow precise experiments to be carried out and the response of cells to specific stimuli to be revealed. This opportunity is unique for studying intracellular processes, but we must remember that, rather than stronger cells cells in culture differ from cells in the body, the more likely it is that the mechanisms of these processes will also differ.

The cell contains not a dilute solution used in biochemical and structural studies, but a very dense and rich microenvironment ( crowded environment) (Fig. 14 and video 2). As a result, the activities of molecules in an artificial solution and “in real life” sometimes differ by several orders of magnitude. That is why the cell is a much more adequate model when studying the activities and interactions of biomolecules and when analyzing the effect of potential drugs on target molecules in pharmacology.

Video 2. Three-dimensional reconstruction of various intracellular structures in a slice from Figure 14. signature

Yellow - endoplasmic reticulum, blue - membrane-bound ribosomes, orange - free ribosomes, light green threads- microtubules, blue - dense core vesicles, white - clathrin-negative vesicles, light red - clathrin-containing vesicles, purple - clathrin-negative compartments, cavities with light and dark green internal and external surfaces - mitochondria. Individual molecules and small complexes are not shown.

How to deliver a gene into a cell?

If the molecule we are interested in is a protein, then the easiest way to put it into a cell and study it is to force it to be synthesized directly “on the spot” by introducing the gene for this protein into the cell. There are many methods for delivering genes into cells (Fig. 15), both based on purely physicochemical principles ( transfection), and using viruses as a carrier ( transduction).

Efficiency transfections depends significantly on the cell type and technique: for cancerous and immortalized cells it is usually 30–80%, however for primary cells it is rarely more than 10%. An exception is the electroporation method, which sometimes allows for highly efficient gene delivery to primary cultures. Chemical methods transfections are not designed to integrate the introduced gene into the cell's genome, and over time the introduced DNA is lost.

A small fraction of DNA can still become fixed in the genome during transfection and be inherited, which makes it possible to obtain a line with stable expression of the introduced gene. Selection is carried out by selecting such cells for antibiotic resistance (along with the gene under study, a gene that provides such resistance is usually inserted) or using several cycles of cell sorting.

Figure 15. Transfection and transduction methods. To introduce a gene into a cell, it is necessary to overcome the outer membrane. Most often, nano-sized complexes of DNA with lipid vesicles (lipofection), polymer carriers or calcium crystals, which themselves are absorbed by the cell, are used for this purpose. You can also temporarily perforate the membrane using an electrical discharge - electroporation. For cases where the gene needs to be introduced into only a few cells (for example, when studying single neurons), the microinjection method is used. In the case of transduction, viral particles are used, in which the necessary gene is placed instead of part of their own genome. At the same time, viruses retain the ability to penetrate cells, effectively deliver the gene to the nucleus and, in the case of some viruses, integrate it into the DNA of the cell.

Viral methods transduction have a number of advantages over transfection: high efficiency in relation to primary cell lines, the possibility of inserting the gene into the genome, applicability in vivo and selectivity of the action of viruses on certain types of cells and tissues.

For transfection, artificial viral particles are created that are externally similar to natural viruses and penetrate the cell using the same mechanisms. But inside, instead of part of their own DNA, they carry the genes the researcher needs and are deprived of a self-replication program (that is, such particles are not infectious). Today, about a dozen genetic engineering constructs based on various viruses are widely used. To change the selectivity of viral particles, they can be further modified with specified surface molecules.

Edit this

The capabilities of cellular technologies are now reaching a new level after the recent breakthrough in genome editing methods based on the CRISPR/Cas-9 system. The technique is already actively used, and kits for selectively removing certain genes from cells (genetic knockout) are commercially available. The advantage of CRISPR/Cas-9 is its simplicity compared to previously available genome editing methods (zinc fingers, TALEN). Using CRISPR/Cas-9, you can not only delete and turn off genes, but also change and restore them. Today, great hopes are placed on CRISPR/Cas-9 in the treatment of monogenic hereditary diseases.

Let there be light

The relative ease of introducing the desired genes into cells opens up access to the rich possibilities of perhaps the most powerful tool of molecular biology today - genetically encoded fluorescent proteins. They allow you to track individual cells in the body and even molecules in cells, stain certain organelles, study intermolecular interactions and protein conformation, measure the concentrations of secondary messengers (Ca 2+, H 2 O 2, cAMP, H +, ATP/ADP, NADH, etc. .), visualize and determine the activity of various proteins.

In addition, in the last five years, optogenetics has become widespread - a method in which, using genetically encoded light-sensitive proteins, you can control cellular processes with high temporal and spatial resolution, literally by shining a laser into the desired area (Fig. 16).

Figure 16. Optogenetic constructs allow using a laser to given point cells and launch various processes at a given time: 1 - ion current through the membrane and electrical impulses, 2 - gene activity, 3 - protein activity, 4 - receptor activity and signal transmission into cells, 5 - absorption of substances from the outside, 6 - interactions between proteins, 7 - movement of intracellular vesicles, 8 - synthesis and degradation of proteins, 9 - mitochondrial respiratory chain and cell death, 10 - signal transmission by secondary intermediaries.

Fluorescent proteins and optogenetics are showing their full potential when coupled with modern fluorescence microscopy techniques. These methods have achieved sensitivity of single molecules with a spatial resolution of several tens of nanometers, and with a temporal resolution of up to several tens of microseconds (Fig. 17). Although this spatial resolution is lower than that of electron microscopy, the availability of temporal resolution (i.e., applicability to living cells) and the ability to analyze multiple molecules simultaneously make optical microscopy methods unique for a wide range of applications.

Figure 17. Spatial and temporal resolution of modern high-resolution optical microscopy techniques. Designations: FRET - Forster resonant energy transfer (Forster resonant energy transfer); SPT - single particle tracking (microscopy of single particle trajectories); PALM/STORM - photoactivation localization microscopy/stochastic optical reconstruction microscopy (photoactivated localization microscopy/stochastic optical reconstruction microscopy); STED - stimulated emission depletion (stimulated emission suppression microscopy); FCS - fluorescence correlation spectroscopy (fluorescence correlation spectroscopy); TIRF - total internal reflection (microscopy of total internal reflection); SIM - structured illumination microscopy; - confocal microscopy (confocal microscopy).

Typing and sorting

Another powerful method that only applies to cells in vitro And ex vivo(immediately after selection), is flow cytometry- individual study of cells in a fluid flow: cells one by one pass through the laser beam, and special detectors catch the fluorescence and light scattering signal from the cell illuminated by the laser. Unlike microscopy, it is an initially quantitative method, that is, it allows you to accurately measure fluorescence intensity and has high productivity: the processing speed reaches a million cells per minute. This allows for a “census” of heterogeneous cell populations, which are all primary cell cultures. In particular, there are no alternatives to it when identifying, counting and sorting rare cells in a population, such as stem cells or antigen-specific lymphocytes, of which there may be only a few cells per million. Cytometry is most widespread in immunology for the analysis of subpopulations of leukocytes without transferring them into culture. An important type of cytometry is flow cell sorting, which allows not only to analyze, but also to isolate individual subpopulations from heterogeneous cell mixtures with a purity of more than 99%.

From cells to organisms using stem cells

Previously, we looked at traditional methods of working with cells, many of which were proposed in the second half of the last century. At the time, these were cutting-edge technologies that subsequently made significant contributions to the development of biomedical science and molecular biology, and are now part of routine laboratory practice. Today, advanced cellular technologies allow achieving impressive results. However, in scientific and, especially, popular science articles, authors often tend to exaggerate the scale of the discovery and gloss over the difficulties and limitations. This creates a somewhat distorted picture, in which already today there are doctors somewhere who print organs on a 3D printer, save people from HIV and blindness, and are able to cure almost any cancer. Indeed, the success of cellular technologies allows us to hope that this will indeed become possible in the future, but for now leading experts urge not to rush into predictions, since the body and tissue are much more complex than just the sum of their component cells.

Embryonic stem cells

Figure 20a. Stimulation of the formation of embryoid bodies in suspended drops.

Figure 20b. Cultivation of cells in a Maximov chamber. In it, a drop with a piece of tissue or cells is placed on a small cover glass. With the help of a drop of water and capillary forces, the small cover glass adheres to the large one, which, in turn, is placed on the base and sealed with paraffin.

Background information on working with cell cultures from Diaem

Would you like to learn more about the intricacies of working with cell cultures or see how a cell laboratory is organized?

Additional information on the topic “Cell Technologies”.