Single-layer cell cultures. Cell culture. Application in biotechnology

S. Ringer developed a saline solution containing sodium, potassium, calcium and magnesium chlorides to maintain the heartbeat of animals outside the body. In 1885, Wilhelm Roux established the principle of tissue culture by extracting part of the bone marrow from a chicken embryo and keeping it in a warm saline solution for several days. Ross Granville Harrison, who worked at the Johns Hopkins School of Medicine and then at Yale University, published the results of his experiments in 1907–1910, creating a tissue culture methodology. In 1910, Peyton Routh, working with chicken sarcoma cell culture, induced the formation of tumors in healthy animals. This later led to the discovery of oncogenic viruses (Nobel Prize in Physiology or Medicine 1966).

Cell culture techniques developed significantly in the 1940s and 1950s in connection with research in the field of virology. Growing viruses in cell cultures has made it possible to obtain pure viral material for the production of vaccines. The polio vaccine was one of the first drugs mass produced using cell culture technology. In 1954, Enders, Weller and Robbins received the Nobel Prize "for their discovery of the ability of the polio virus to grow in tissue cultures." In 1952, the well-known human cancer cell line HeLa was obtained.

Basic principles of cultivation

Cell isolation

For cultivation outside the body, living cells can be obtained in several ways. Cells can be isolated from blood, but only leukocytes are capable of growing in culture. Mononuclear cells can be isolated from soft tissues using enzymes such as collagenase, trypsin, pronase, which destroy the extracellular matrix. In addition, pieces of tissue can be placed in the nutrient medium.

Cultures of cells taken directly from the object (ex vivo) are called primary. Most primary cells, with the exception of tumor cells, have a limited lifespan. After a certain number of divisions, these cells become old and stop dividing, although they may not lose viability.

There are immortalized (“immortal”) cell lines that can reproduce indefinitely. In most tumor cells, this ability is the result of a random mutation, but in some laboratory cell lines it is acquired artificially, by activating the telomerase gene.

Cell culture

Cells are grown in special nutrient media at a constant temperature, and mammalian cells usually also require a special gas environment maintained in a cell culture incubator. As a rule, the concentration of carbon dioxide and water vapor in the air is regulated, but sometimes also oxygen. Nutrient media for different cell cultures differ in composition, pH, glucose concentration, composition of growth factors, etc. Growth factors used in culture media are most often added along with blood serum. One of the risk factors in this case is the possibility of infection of the cell culture with prions or viruses. In cultivation, one important goal is to eliminate or minimize the use of contaminated ingredients. However, in practice this is not always achieved. The best, but also most expensive way is to add purified growth factors instead of whey.

Culturing human cells is somewhat contrary to the rules of bioethics, since cells grown in isolation can outlive the parent organism and then be used for experimentation or to develop new treatments and profit from it. The first ruling in this area came from the California Supreme Court in John Moore v. University of California, which held that patients have no property rights in cell lines obtained from organs removed with their consent.

Hybridoma

Use of cell cultures

Mass cell culture is the basis for the industrial production of viral vaccines and a variety of biotechnology products.

Biotechnology Products

Industrially, products such as enzymes, synthetic hormones, monoclonal antibodies, interleukins, lymphokines, and antitumor drugs are obtained from cell cultures. Although many simple proteins can be produced relatively easily using rDNA in bacterial cultures, more complex proteins such as glycoproteins can currently only be produced from animal cells. One of these important proteins is the hormone erythropoietin. The cost of growing mammalian cell cultures is quite high, so research is currently being conducted into the possibility of producing complex proteins in insect or higher plant cell cultures.

Tissue culture

Cell culture is an integral part of tissue culture and tissue engineering technology because it defines the basis for growing cells and maintaining them in a viable state ex vivo.

Vaccines

Vaccines against polio, measles, mumps, rubella, and chickenpox are currently being produced using cell culture techniques. Due to the threat of an influenza pandemic caused by the H5N1 virus strain, the United States government is currently funding research to obtain an avian influenza vaccine using cell cultures.

Non-mammalian cell cultures

Plant cell cultures

Plant cell cultures are usually grown either as a suspension in a liquid nutrient medium or as a callus culture on a solid nutrient base. Cultivation of undifferentiated cells and callus requires maintaining a certain balance of plant growth hormones, auxins and cytokinins.

Bacterial, yeast cultures

Main article: Bacterial culture

To cultivate small numbers of bacterial and yeast cells, the cells are plated on a solid nutrient medium based on gelatin or agar-agar. For mass production, cultivation in liquid nutrient media (broths) is used.

Viral cultures

I. Cell cultures

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

By origin they are classified into embryonic, tumor and from adult organisms; by morphogenesis- fibroblastic, epithelial, etc.

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

Semi-leathered(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.

Continuous cell lines are characterized by potential immortality and a heteroploid karyotype.

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

Another source of transplantable cell lines is 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 laryngeal carcinoma; Detroit-6 - from lung cancer metastasis to the bone marrow; RH - from human kidney.

To cultivate cells, nutrient media are required, which, according to their purpose, are divided into growth and supporting media. Growth media must contain more nutrients to ensure active cell proliferation to form a monolayer. Supporting media should only ensure that cells survive in an already formed monolayer during the multiplication of viruses in the cell.

Standard synthetic media, such as synthetic media 199 and Eagle's media, are widely used. Regardless of the purpose, all cell culture media are constructed using a balanced salt solution. Most often it is Hanks solution. An integral component of most growth media is animal blood serum (veal, bovine, horse), without the presence of 5-10% of which cell reproduction and monolayer formation do not occur. Serum is not included in the maintenance media.

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

Cell cultures

Cell culture technology involves growing cells outside of 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 justified a source of natural products with therapeutic properties, such as paclitaxel, found in yew wood and marketed as a chemotherapy drug called Taxol. Plant cell cultures are also used to produce substances used by the food industry as flavorings and colors.

Insect cell cultures

The study and use 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 do not accumulate in the environment. Despite the fact that the advantages 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 prospect is currently being actively studied. In addition, the possibility of using insect cells to produce VLP vaccines (virus-like particles) to treat 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 method using chicken eggs.

Mammalian cell cultures

Mammalian cell cultures have been one of the main tools used by livestock breeding specialists for decades. In laboratory conditions, eggs obtained from cows with outstanding qualities are fertilized with sperm from the corresponding bulls. The resulting embryos are grown in vitro for several days, after which they are implanted into the uterus of surrogate mother cows. The same technique is the basis of in vitro fertilization in humans.

Currently, the use of mammalian cell cultures goes far beyond artificial insemination. Mammalian cells can complement, and perhaps one day replace, the use of animals to test the safety and effectiveness of new drugs. In addition, 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 specifically by mammalian cell cultures.

Scientists are also considering the possibility of 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 culturing mammalian cells in order to speed up the development of influenza vaccines and, accordingly, increase humanity’s preparedness for a pandemic.

Culture-based therapies adult stem cells, found in some tissues of the body (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 umbilical cord blood, but their isolation is a rather complex process.

Researchers are currently working on methods to isolate stem cells from the placenta and adipose tissue. A number of specialists are busy developing methods of 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 tissue type.

Embryonic stem cells

Usage embryonic stem cells is also considered as a potential therapy for many diseases. As the name suggests, embryonic cells are obtained from embryos, specifically those that develop from eggs that have been fertilized in vitro (in vitro fertilization clinics) and, with the consent of the donor, given to researchers for scientific use. Typically, blastocysts are used - 4-5 day old embryos that look like balls under a microscope, consisting of several hundred cells.

To isolate 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. The researchers then collect the dividing cells, divide them into pieces, 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 subculture 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 that have been genetically modified to be unable to divide. These cells form a feeder layer - a “nutrient substrate”, thanks to which embryonic cells attach 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 and tissue engineering therapies can be achieved when the therapeutic stem cells and the tissues grown from them are genetically identical to the recipient cells. Therefore, if the patient himself is not their source, stem cells must be modified by replacing their genetic material with the recipient's genes and only then differentiated into cells of a specific type. Currently, the procedure for replacing genetic material and re-programming can only be successfully performed with embryonic stem cells.

1966).

Cell culture techniques developed significantly in the 1940s and 1950s in connection with research in the field of virology. Growing viruses in cell cultures has made it possible to obtain pure viral material for the production of vaccines. The polio vaccine was one of the first drugs mass produced using cell culture technology. In 1954, Enders, Weller and Robbins received the Nobel Prize "for their discovery of the ability of the polio virus to grow in tissue cultures." In 1952, the well-known human cancer cell line HeLa was obtained.

Basic principles of cultivation

Cell isolation

For cultivation outside the body, living cells can be obtained in several ways. Cells can be isolated from blood, but only leukocytes are capable of growing in culture. Mononuclear cells can be isolated from soft tissues using enzymes such as collagenase, trypsin, pronase, which destroy the extracellular matrix. In addition, pieces of tissue and materials can be placed in the nutrient medium.

Cultures of cells taken directly from the object (ex vivo) are called primary. Most primary cells, with the exception of tumor cells, have a limited lifespan. After a certain number of divisions, these cells become old and stop dividing, although they can still remain viable.

There are immortalized (“immortal”) cell lines that can reproduce indefinitely. In most tumor cells, this ability is the result of a random mutation, but in some laboratory cell lines it is acquired artificially, by activating the telomerase gene.

Cell culture

Cells are grown in special nutrient media at a constant temperature. Plant cell cultures use controlled lighting, and mammalian cells usually also require a special gas environment maintained in a cell culture incubator. As a rule, the concentration of carbon dioxide and water vapor in the air is regulated, but sometimes also oxygen. Nutrient media for different cell cultures differ in composition, glucose concentration, composition of growth factors, etc. Growth factors used in mammalian cell culture media are most often added along with blood serum. One of the risk factors in this case is the possibility of infection of the cell culture with prions or viruses. In cultivation, one of the important goals is to eliminate or minimize the use of contaminated ingredients. However, in practice this is not always achieved. The best, but also most expensive way is to add purified growth factors instead of whey.

Cross-contamination of cell lines

When working with cell cultures, scientists may encounter cross-contamination problems.

Features of growing cells

When growing cells, due to constant division, there may be an excess of them in the culture, and, as a result, the following problems arise:

• Accumulation of excretory products, including toxic ones, in the nutrient medium.
• Accumulation of dead cells in the culture that have ceased to function.
• The accumulation of a large number of cells has a negative effect on the cell cycle, growth and division slow down, and cells begin to age and die (contact growth inhibition).
• For the same reason, cellular differentiation may begin.

To maintain the normal functioning of cell cultures and also to prevent negative phenomena, the nutrient medium is periodically replaced, cell passaging and transfection are carried out. To avoid contamination of cultures with bacteria, yeast, or other cell lines, all manipulations are usually carried out aseptically in a sterile box. To suppress microflora, antibiotics (penicillin, streptomycin) and antifungal drugs (amphotericin B) can be added to the nutrient medium.

Culturing human cells is somewhat contrary to the rules of bioethics, since cells grown in isolation can outlive the parent organism and then be used for experimentation or to develop new treatments and profit from it. The first ruling in this area came from the California Supreme Court in John Moore v. University of California, which held that patients have no property rights in cell lines obtained from organs removed with their consent.

Hybridoma

Use of cell cultures

Mass cell culture is the basis for the industrial production of viral vaccines and a variety of biotechnology products.

Biotechnology Products

Industrially, products such as enzymes, synthetic hormones, monoclonal antibodies, interleukins, lymphokines, and antitumor drugs are obtained from cell cultures. Although many simple proteins can be produced relatively easily using rDNA in bacterial cultures, more complex proteins such as glycoproteins can currently only be produced from animal cells. One of these important proteins is the hormone erythropoietin. The cost of growing mammalian cell cultures is quite high, so research is currently being conducted into the possibility of producing complex proteins in insect or higher plant cell cultures.

Tissue culture

Cell culture is an integral part of tissue culture and tissue engineering technology because it defines the basis for growing cells and maintaining them in a viable state ex vivo.

Vaccines

Vaccines against polio, measles, mumps, rubella, and chickenpox are currently being produced using cell culture techniques. Due to the threat of an influenza pandemic caused by the H5N1 virus strain, the United States government is currently funding research to obtain an avian influenza vaccine using cell cultures.

Non-mammalian cell cultures

Plant cell cultures

Plant cell cultures are usually grown either as a suspension in a liquid nutrient medium or as a callus culture on a solid nutrient base. Cultivation of undifferentiated cells and callus requires maintaining a certain balance of plant growth hormones, auxins and cytokinins.

Bacterial, yeast cultures

Main article: Bacterial culture

To cultivate small numbers of bacterial and yeast cells, the cells are plated on a solid nutrient medium based on gelatin or agar-agar. For mass production, cultivation in liquid nutrient media (broths) is used.

Viral cultures

Cell cultures - These are genetically homogeneous populations of cells growing under constant environmental conditions. These can be strains of normal human, animal, plant, or malignant tumor cells.

Cultivation conditions

Cells are usually placed in glass vessels, hence the name in vitro studies (from the Latin In - in, vitro - glass), although cultures are now more often grown in plastic vessels. Cells isolated from tissues are incubated at a temperature of 38 ° C 39 ° C (for cells of animal and human organisms) and at 22 ° C 28 ° C (for plant cells) in a nutrient medium of the appropriate composition. The cells then grow as a suspension or monolayer. Suspension culture is the cultivation of individual cells or small groups of them in suspension in a liquid nutrient medium using equipment that ensures their aeration and mixing. A characteristic feature of suspension cultures is their morphological and biochemical heterogeneity. A cell population contains cells that vary in size and shape.

Application

Application in cytology

In cytology, this method is convenient because cells in culture are easily accessible for various biochemical manipulations. When working with them, radioactive substances, poisons, hormones, etc. can be introduced in the required concentration for the required time. The amount of these substances may be an order of magnitude less than in experiments on animals. There is no risk that the substance will be metabolized by the liver, excreted by the kidneys or deposited in the muscles. This provides real values ​​for the rate of action of the substance on the cell or its absorption by the cell.

To study living plant cells, a culture of isolated protoplasts is used. Isolated protoplasts can be defined as "naked" plant cells because the cell wall is removed mechanically or enzymatically. The system of isolated protoplasts makes it possible to conduct selection at the cellular level, to work in a small volume with a large number of individual cells, to obtain new forms of plants through direct gene transfer, and to obtain somatic hybrids between species that are systematically distant. Since regeneration of the cell membrane immediately begins in isolated protoplasts, they are a convenient object for studying the formation of cellulose microfibrils.

Application in virology

In virology, cell cultures are used very widely because they are relatively easy to work with in the laboratory, unlike other methods - growing viruses in chicken embryos or in living animals. In addition, on a monolayer of cell culture, the cytopathic effect of viruses can be well studied by the formation of intracellular inclusions, plaques, in hemadsorption and hemagglutination reactions, and by color breakdown. When working with cell cultures, significant results can be obtained by working with a small number of cultures. Experiments that require hundreds or thousands of laboratory animals to confirm a particular fact can be carried out with equal statistical reliability on the same number of cell cultures. Thus, there is no need to keep a vivarium in the laboratory and there are no ethical aspects of handling sick animals.

The transformation of cells by viruses is also being studied, the mechanism of which is similar to the mechanism of the occurrence of malignant tumors.

Application in pharmacology

Cell cultures are widely used to test the effects of substances that can be used as drugs. Despite the fact that the results obtained on cell cultures cannot be extrapolated to the entire body, there is no doubt that if a particular substance disrupts the activity of cells from several different culture lines, then a negative effect should be expected when this substance is introduced into the body.

Application in biotechnology

Specific cell cultures are a valuable source of hormones and other biologically active substances. They are already being used to produce the antiviral protein interferon.

Application in genetics

In genetics, the ability of cells to grow in culture is used in the following areas:

• Cloning
• Cell storage
• Obtaining mutant cells and working with them

Types of Cell Cultures

1. Primary trypsinization - obtained from crushed human and animal tissues by treating them with trypsin or other enzymes. They can withstand only 5-10 divisions (passages).

2. transplantable - cells that have acquired the ability to reproduce unlimitedly, since they are derivatives of human and animal tumors.

3. Napivresplyuvani (diploid) - can withstand up to 100 passages, while maintaining the original diploid set of chromosomes.

Most common cell lines