GROWTH AND CULTIVATION OF BIOOBJECTS - V. M. Samygin - 2016

CHAPTER 12. ANIMAL CELL CULTIVATION

The possibility of maintaining living Tissues outside the Organism was demonstrated in 1885 by W. Roux, who kept a chick embryo membrane viable in a warm physiological saline solution. Later, in 1887 (note: retaining original text meaning, though author wrote 1897), Loeb maintained Blood and Connective Tissue Cells viable in test tubes containing serum and Blood Plasma. Ljunggren (1898) showed that human Skin explants could be kept viable in an acidic environment while retaining their capacity for reimplantation. In 1913, A. Carrel used blood plasma enriched with an embryo extract. This work was sustained over a long period and became known as the cultivation of "immortal" cells. Thus, the recognition that cells from higher animal tissues can be isolated from the organism and subsequently provided with conditions for GROWTH AND REPRODUCTION in vitro dates back to the first decade of the 20th century.

Once it became known that such processes were feasible, researchers demonstrated the possibility of growing and reproducing filterable infectious agents—Viruses—in these cells, thereby yielding large quantities of viral material for vaccine production. Furthermore, it became possible to introduce specific exogenously obtained genes into cells and achieve their expression, as well as to grow whole populations from a single Cell in culture. When such populations began to be derived from a cell that secreted Antibodies in vitro, all the antibody molecules in the supernatant turned out to be identical.

The cultivation of animal Cells and Tissues has now become widespread across various fields of research—from cell and molecular biology to rapidly advancing applied areas of biotechnology. Animal tissue culture is used to study the MECHANISMS OF CELL growth and differentiation, intertissue and intercellular interactions, METABOLISM, and so forth. Animal Cell Cultures serve as important producers of many biologically vital substances. They are used to grow viruses for identification and vaccine production. Cell cultures are also frequently employed in testing and studying the Mechanisms of action of drugs, cosmetics, pesticides, and preservatives.

Cell culture Methods have found wide application in the reconstruction of various tissues and Organs. For instance, skin cell culture is used for replacement therapy in burn victims, and endothelial cell culture is utilized for blood vessel wall reconstruction. The capacity of cells to grow in culture has driven The Development of methods for cell cloning, storage, and fusion, which in turn has given rise to a new scientific field: somatic cell genetics. Organ cultures are used to study the regularities of organ development and ways to maintain the viability of isolated organs intended for transplantation.

Demonstrating that animal cells can grow and divide in culture required mastering a range of approaches and techniques. These include methods for obtaining cells free of exogenous prokaryotes and Fungi; developing media in which the growth of "tissue-cut" or isolated cells is not suppressed; monitoring cells throughout the dynamics of their development; and continuously cultivating animal cell cultures in vitro while keeping them free from other biological agents.

The scientific foundation for developing these methodologies rests on THE CONCEPT OF The Cell as the primary structural element of living organisms of both animal and plant origin. The idea that animal tissue cells can be isolated from the organism and then provided with conditions for growth and reproduction in vitro originated from a concept put forward by Claude Bernard. He hypothesized that Living organisms are not the only entities capable of maintaining internal environmental constancy despite external fluctuations; a cell outside an animal organism will also strive to maintain its internal conditions. If the differences between internal and external conditions are minor, the probability of Cell Growth and Division is high. This understanding led to the necessity of developing media capable of supporting and stimulating cell growth outside the organism. In the course of this work, several adjustments were made to the culture medium formulation, along with the Introduction of a supplementary and highly crucial approach in cell handling techniques—primarily The Use of Trypsin to release cells from the tissue matrix in which they reside.

Cell clones in culture derived from a single cell were first obtained by Earle and coworkers in 1948. Eagle (1955) systematically investigated the Nutritional Requirements of cells. Until Hayflick and Moorhead isolated the human diploid cell strain (HDCS) WI-38 in 1961, it was believed that an established cell line had an unlimited lifespan. However, its survival period in culture was found to be limited to approximately 50 population generations, and upon dying out, these cells remained diploid without showing signs of senescence or malignant transformation. Cells isolated from cancerous tumors or transformed during cultivation are characterized by "immortality" and correlate with heteroploidy. Moreover, the first obtained suspension cultures of animal cells were generally based on malignant tissue cells (HeLa cells).

The subsequent phase in the Selection/37.html">History of Human diploid cell cultivation is associated with the ESTABLISHMENT OF THE fact that they are genetically stable and free from all known latent and Oncogenic Viruses. Therefore, human diploid cell strains are authorized for use

in The production of biological products intended for humans. This dogma remains in effect today, even though recent discoveries have clearly revealed the presence of potential oncogenes in cells isolated from normal tissues. These oncogenes are identical to those found in known oncogenic viruses (Sarcoma viruses). It was later established that A number of viruses are capable of inducing tumor formation, and such viruses were termed oncogenic.

12.1. Characteristics of Cells Cultivated in Vitro

Cells of the same type within a tissue interact with one another and coordinate their division rates to maintain an appropriate population density. This type of control is clearly manifested during injury responses. For example, when the epithelium is damaged, cells at the wound edges are stimulated to divide and crawl over the exposed surface until it is covered again, after which cell proliferation and movement cease. A similar phenomenon can be observed in dissociated cells in culture. Epithelial cells or fibroblasts placed in a dish in the presence of serum will "attach" to the surface, flatten, and divide until a confluent monolayer is formed, in which adjacent cells are in contact with one another.

Normal cells stop dividing, a phenomenon known as density-dependent inhibition of proliferation. If such a monolayer is "wounded" with a needle in such a way as to create a cell-free strip across the dish, cells from the edges of this strip begin to migrate into the vacant space and divide.

The cell population density at which cells in a confluent monolayer cease dividing increases with higher concentrations of growth factors in the medium. In addition, it was found that if the culture fluid flows across The surface of a dish containing islands of

cells, those cells washed by medium that has just passed over other cells will divide more slowly than those washed by medium that has passed over cell-free areas. The medium that has flowed over cells is deficient in certain essential nutrients or growth factors.

Growth factors are typically present in the medium at a concentration of about 10^-10 M (approximately one molecule per the volume of a sphere 3 µm in diameter), and each cell possesses enough receptors to bind all the growth factor molecules. Competition for growth factors and nutrients is not the only factor influencing the division rate in cell culture. The shape of the cells during their flattening and movement across the substrate onto vacant spaces also strongly affects their capacity to divide. When normal cells are cultivated in suspension—meaning they are not attached to a solid surface and thus retain a rounded shape—they almost never divide.

The Effect of cell spreading on proliferation can be observed when growing cells on substrates with varying surface adhesiveness, or on substrates featuring only tiny adhesive patches where a cell can attach but cannot spread. In such cases, the Cell Division rate increases with the degree of cell spreading. It is possible that heavily spread cells can capture more growth factors and absorb more nutrients due to their increased surface area.

Changes in the growth properties of cultured cells are referred to as transformation. Transformed cells are capable of growing under conditions where the surface area-to-volume ratio is less favorable. Transformed cells lack a limited lifespan, which gives them a distinct advantage in BIOTECHNOLOGY AS A substrate for use.

Today, virtually any Human and Animal cells can be (or already have been) brought into culture, thereby serving as a tool and object in numerous biomedical studies. These include human connective tissue elements (fibroblasts), Skeletal Tissues (bone and Cartilage), cardiac and smooth Muscles, Epithelial Tissues (Liver, Lungs, Kidneys, etc.), Nervous system cells, endocrine cells, melanocytes, and various tumor cells.

Fibroblast cultures are the most widely used. This is due to the ease of their cultivation, as well as the fact that they constitute a significant portion of body mass and the stroma of many organs. The feasibility of extrapolating data obtained from cultured fibroblasts to in vivo conditions has been proven (Greenberg, 1978). The primary evidence is as follows. First, fibroblasts in vitro retain the essential features characteristic of cells in the organism, as well as the ontogenetic and individual genotypic Properties of the donor organism. Second, no other cell type can so fully represent The properties of the organism's cells. Third, the changes that occur when fibroblasts are introduced into culture can be easily controlled and minimized by establishing appropriate conditions.

Two main directions in animal cell cultivation can be distinguished: cell cultures and Organ and tissue cultures (organ cultures). Cells multiply in cultures, which ensures the yield of a large cell mass. They are then identified, divided into identical parallel replicates, and, if necessary, preserved. Since the dynamic properties of cultured cells can be difficult to control and certain in vivo cellular interactions are hard to reconstruct in vitro, preference is often given to cell systems that preserve the structural integrity of the original tissue.

A cell population is not always homogeneous and does not possess a fixed phenotype. Some cultures, such as epidermal cells, contain stem cells, progenitor cells, and squames (or squamous cells). Such cultures undergo continuous renewal driven by stem cells, proliferation and maturation of progenitor cells, and irreversible differentiation accompanied by the "shedding" of squamous cells into the culture medium.

12.2. Animal Cell Culture Systems

For introduction into culture, it is preferable to use cells derived from embryonic tissues. They exhibit better survival rates and more active growth compared to corresponding cells from mature tissues. The reason for this lies in the low level of specialization and the presence of replicating progenitor cells in embryos. The proliferative capacity of adult tissues is lower, as they contain a higher proportion of non-dividing specialized cells.

There are three main Types of Animal cell cultures: primary cultures, which can be derived from almost any organ and survive only until the first subculture; diploid cultures, typically obtained from embryonic tissues, which maintain a diploid set of Chromosomes for up to 50 passages before eventually transforming into continuous (established) heteroploid cultures capable of surviving outside the organism for decades. In addition, tissue cultures can be classified by the species of origin; the specific tissue type; the physiological state of the tissue at the time of extraction (normal versus tumorous); and the cultivation method (monolayer, suspension, microcarrier-based, etc.). The primary factor when selecting a tissue or cell line for further study is The Nature of the biological process under investigation.

12.2.1. Primary Cultures

Cells harvested from an animal and maintained in culture are designated as primary until they undergo their first passage (subculturing). Primary cell cultures are typically heterogeneous and exhibit low proliferation rates. They represent the cellular Water/126.html">Diversity of the source tissue most comprehensively. However, a primary culture lacks many of the cell types present in the original tissue, as not all cells are capable of attaching to a substrate and surviving under in vitro conditions. Subculturing extends the lifespan of the culture, enables cloning, and facilitates the study and preservation of cellular properties. This process yields more homogeneous populations, albeit at the cost of losing specialized cell types.

Primary cell cultures are established by the aseptic excision of a tissue fragment followed by its mechanical or enzymatic disaggregation. In mechanical disaggregation, the tissue fragment is minced into pieces roughly 1 mm in size, which then adhere to a substrate via their own adhesiveness, the surface of the culture vessel, or a plasma clot. Enzymatic disaggregation, using 0.01–0.25% trypsin or 200–2000 units/mL of collagenase, yields a higher cell recovery, although this method is selective since not all cells survive dissociation. The principal advantage of deriving cell lines from primary cultures is the generation of a large volume of stable material suitable for long-term use.

12.2.2. Continuous Cultures

After several passages, a cell line either dies out or undergoes transformation to become a continuous cell line. The Emergence of a continuous cell line is indicated by morphological changes (reduction in cell size, decreased adhesiveness, a rounded appearance, and an increased nucleus-to-Cytoplasm ratio); an elevated growth rate (with cell doubling time dropping from 36–48 to 12–36 hours); reduced dependence on serum and substrate, leading to an increased capacity for suspension growth; heightened heteroploid characteristics (chromosomal variations among cells); and increased tumorigenicity. However, normal cells that spontaneously transform into a continuous line do not become malignant, despite sharing certain features. Thus, the advantages of continuous cell lines include rapid growth rates, The ability to reach higher cell densities, suitability for suspension culture, and the use of less expensive media. Disadvantages include increased chromosomal instability, deviation from the donor phenotype, and the loss of specific markers.

12.3. Nutrient Media and Cultivation Conditions

Once cells are isolated from a tissue or organism, the culture medium used for their growth must replicate all the environmental conditions the cells experienced in vivo. This Supports cell survival, proliferation, and differentiation. The extracellular environment must supply nutrients and hormonal factors, thereby providing everything necessary for cell growth and viability.

Animal and human cell cultures impose specific requirements regarding the liquid phase (nutrient medium), gaseous phase (gas concentrations), and solid phase (substrate surface). Salt solutions form the backbone of nutrient media. The mineral components in these solutions are balanced so that the medium acts as a buffer, maintaining a constant acid-base balance throughout cultivation. Maintaining a stable pH is a fundamental requirement for successful cell culture.

Earle's and Hank's balanced salt solutions are commonly used to prepare culture media. These solutions, alongside Dulbecco's and Vogt's phosphate-buffered saline, are also employed for irrigating and washing cells during subculturing, isolating cell lines, and performing other culture manipulations. Osmotic pressure represents another critical cultivation parameter. It is determined by the number of moles of osmotically active particles (ions and unionized molecules) of dissolved substances per kilogram of solvent (osmolality) or per liter of solution (osmolarity). The pH and osmolality ranges compatible with cell proliferation are narrow and vary depending on the cell type. To maintain physiological pH, most media utilize a bicarbonate buffer system. Some solutions contain low levels of bicarbonate (Hank's solution) and are designed to maintain pH in tightly sealed vessels. Others (Earle's solution) contain higher bicarbonate concentrations and are intended for systems with an elevated partial pressure of CO₂. When cultures are grown outside a CO₂ incubator, where maintaining pH is more challenging, alternative buffer systems are employed. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) serves as an excellent buffer. HEPES is readily soluble in water, does not bind divalent cations, and is non-cytotoxic at concentrations up to 0.05 M.

The development of nutrient media for animal cell cultures has progressed along two main paths: media containing natural additives (such as sera and embryonic extracts) and media with a chemically defined composition. Currently, the most widely used media contain Energy Sources (CARBOHYDRATES) and nitrogen sources, Essential Amino Acids, Vitamins, inorganic salts (providing macro- and microelements), nucleosides, Lipids and fat-soluble components, Hormones, growth factors, as well as serum (up to 10%) and various other supplements.

Serum is a complex mixture of small and large molecules that can either promote or inhibit cell growth. Its primary Functions include supplying hormonal factors that stimulate cell growth and function; providing factors essential for cell attachment and spreading; and acting as a carrier protein for hormones, minerals, lipids, and other vital substances. Serum Proteins that directly and specifically participate in stimulating cell division are known as growth factors. The same cell type can often be stimulated by multiple different growth factors.

Carrier proteins are required to transport low-molecular-weight factors (such as vitamins, amino acids, and lipids). Albumin fulfills this carrier role, while Collagen acts as a cell attachment and spreading factor.

Culturing cells in the presence of serum presents several drawbacks. This is largely because, for most cells, serum is not a physiological fluid they encountered in their native tissue. Furthermore, sera may contain inadequate levels of species-specific growth factors, exhibit batch-to-batch biological Variability, and, in some instances, display cytotoxic effects.

In recent years, serum-free media containing a blend of hormones and growth factors have been developed. Serum-free media offer distinct advantages: they are non-toxic, provide superior experimental reproducibility due to compositional stability, and reduce the risk of contamination by viruses, fungi, and Mycoplasmas. Such media minimize the Interference of serum proteins with biological research outcomes and are easier to process for the removal of cellular metabolic waste. Nevertheless, serum-free media possess significant limitations. Notably, supplementing the medium with hormones and growth factors makes it just as costly as serum-supplemented media. Additionally, serum-free media are frequently suitable only for a restricted range of cell types.

Among the most prevalent standard media for animal cell cultures are Eagle's MEM (Minimal Essential Medium) and BME (Basal Medium, Eagle). MEM is more commonly used and contains minerals, amino acids, Water-Soluble Vitamins, and Choline and Inositol, which serve as carbohydrate substrates. MEM is used exclusively with serum supplementation since it lacks biotin, vitamin B₁₂, iron ions, and Trace Elements. It is formulated on The basis of Earle's salt solution. In addition to these, Dulbecco's medium, Iscove's medium, McCoy's 5A, and Medium 199 are used with serum for rapidly proliferating cells.

12.4. Cell Cultivation Systems

There are two primary systems for cell cultivation: batch cultures and continuous cultures.

Batch cultures refer to systems where cells are inoculated into a fixed volume of medium. These systems are suitable for cultivating cells in both monolayers and Suspensions. As cells proliferate, nutrients are consumed and metabolites accumulate; consequently, the medium must be periodically replaced. This induces shifts in cellular metabolism, also known as physiological differentiation. Over time, nutrient depletion leads to a cessation of cell proliferation. Several techniques are used to extend the lifespan of batch cultures: replacing a portion of the culture with an equal volume of fresh medium (intermittent feeding); increasing the culture volume at a constant low rate while periodically harvesting small cell aliquots (continuous-volume method); and continuously feeding fresh medium into the culture while simultaneously withdrawing an equal volume of spent cell-free medium (perfusion method). Perfusion can be open, where all spent medium is discarded, or closed, where the harvested medium passes through an auxiliary vessel to restore its pH and aeration before being returned to the culture vessel. All batch culture systems are characterized by some degree of waste accumulation and fluctuating environmental conditions.

Continuous cultures provide true homeostatic conditions without altering the concentrations of nutrients and metabolites or cell density. This is the sole system in which all cells are homogeneous and maintain that homogeneity over extended periods. Homeostasis is achieved by the continuous inflow of fresh medium into the culture coupled with the simultaneous removal of an equivalent volume of medium containing cells. Such systems are well-suited for suspension cultures and monolayer cultures grown on microcarriers.

12.5. Monolayer and Suspension Cultures

Animal cell cultivation encompasses two major approaches: monolayer cultures and suspension cultures.

12.5.1. Monolayer Cultures

The majority of non-transformed mammalian cells can proliferate only as a monolayer, attached to a solid substrate such as neighboring cells, Glass, plastic, or metal. Cells anchor themselves via Electrostatic Interactions, which requires culture vessel surfaces to be wettable and negatively charged. Cell attachment also necessitates cross-linking with Glycoproteins and the presence of divalent calcium and magnesium cations.

Primary cells actively dividing in culture may undergo what is known as contact inhibition of locomotion. Primary cells cannot overgrow one another, and in most cases, The formation of a confluent monolayer halts cell division. This phenomenon is characteristic not only of primary cells but is also observed in numerous cell lines. Non-transformed cells can remain viable in this quiescent state for some time, but if they are not subcultured, they ultimately perish. Several factors capable of overcoming contact inhibition have been isolated from serum.

Cells transformed by viruses are characterized by impaired contact inhibition, allowing them to reach a higher final density. Transformed cells are capable of growing until the medium is completely depleted; if the medium is not replenished thereafter, the cells rapidly die. Monolayer cultivation is carried out in tissue culture flasks or tubes that provide a growth surface of 5-200 cm².

Monolayer cultures offer several advantages:

- they allow for the replacement of the nutrient medium and washing of the cells prior to The addition of fresh medium, which is crucial when cell growth takes place under one set of conditions and product accumulation under another (for example, when transferring cells from a serum-containing medium to a serum-free one);

- they make it possible to achieve high cell densities using perfusion techniques;

- they ensure close intercellular contacts (which is required, for instance, for virus propagation);

- they provide a more pronounced expression of the target product because the cells are attached to a substrate;

- they are suitable for any cell type and allow the use of identical equipment with varying cell-to-medium ratios, which can be easily adjusted during the experiment.

However, monolayer cultures also have certain drawbacks:

- the requirement for large space;

- increased cost and labor intensity as the scale increases;

- less effective control due to sampling limitations;

- difficulties in determining and maintaining parameters such as pH and O₂ levels, as well as ensuring cell culture homogeneity.

12.5.2. Suspension Cultures

For scaling up cell mass, suspension cultures are generally more convenient than monolayer cultures. As a rule, cells that have detached from their growth substrate are incapable of growing in suspension and rapidly degrade. However, if certain cells are cultivated in a rotating flask (2 rpm) to prevent surface attachment in a medium containing methylcellulose (which inhibits cell aggregation), viable suspension cell lines can be obtained. Sometimes this is sufficient to establish a suspension culture, but specialized suspension culture vessels and media deficient in calcium and magnesium ions are usually required.

Suspension cultures can also be established by treating a monolayer cell culture harvested for subculturing with a 0.02% chymopsin solution in phosphate-buffered saline or with 0.1% trypsin and 0.01% EDTA. Suspension cultures grow best within a narrow range of cell concentrations and when the culture vessel is half-filled.

Suspension cultivation of primary and continuous cell lines is carried out in roller bottles, which provide favorable conditions for constant mixing of the liquid and gas phases, while reducing the total medium volume by half compared to stationary conditions.

Suspension cell cultivation can also be performed in fermenters designed for microbial suspension cultures, where continuous mixing of cells in the medium is achieved using magnetic or mechanical stirrers operating at high rotation speeds that prevent cells from adhering to the vessel walls.

Suspension cultivation ensures at least a 2-3-fold saving in nutrient media compared to conventional stationary monolayer cultivation, while completely eliminating expensive Proteolytic Enzymes and Buffer solutions. Furthermore, suspension cultures are preferable for obtaining large quantities of biomass.

12.5.3. Monolayer Cultivation on Microcarriers

Microcarrier systems combine the advantages of both monolayer and suspension cultivation. Microcarriers are small solid particles (kept in suspension through agitation) upon whose surfaces cells grow as a monolayer. Microcarriers must be non-toxic, non-sorptive toward nutrient media components and cell metabolic products, and possess a surface charge or exchange capacity sufficient for cell attachment. Commercial microcarriers have a diameter of 100-200 µm and are classified into dextran-based (in two variants); collagen- or gelatin-coated microcarriers; polystyrene microcarriers; and glass or Cellulose microcarriers.

12.6. Cell Cycle AND Growth Cycle

Following inoculation into a flask, cells enter a lag phase lasting 2-24 hours, which is followed by the exponential growth phase (logarithmic phase). At the end of this phase, the cells reach complete monolayer confluence and enter a period of slowed growth or quiescence (stationary phase). This is subsequently followed by a decline phase characterized by a decrease in cell number and cell death.

Growing cells divide approximately once every 24 hours. In the absence of any limitations, cells are evenly distributed throughout the cell cycle; if the cell number doubles at regular intervals, the culture is said to be in the exponential growth phase. Typically, after a brief period of exponential growth, one factor or another becomes limiting. This may occur As a result of the depletion of a nutrient medium component or because the cultured cells have completely covered the growth surface.

In suspension, cells can maintain exponential growth for a prolonged period—theoretically indefinitely—if cultivated in a chemostat where an increase in nutrient supply is balanced by an equivalent outflow of cell suspension. This results in constant environmental conditions and cell numbers. In practice, however, this is difficult to achieve due to bacterial contamination.

When it is necessary to obtain a synchronous population—that is, a population of cells at the exact same phase of cell growth—two methodological approaches are used. In the first approach, cells are arrested using chemical or physical treatments so that they remain at the same stage of the cell cycle. However, such treatments may cause cells to become abnormal in certain parameters. In the second approach, cells at a specific cell cycle stage are selected. Selection can be based on particular physical properties, such as weak cell attachment to the substrate or varying DNA content in cells at different cell cycle stages. Alternatively, cells can be selected based on chemical properties by selectively killing off the remaining cells.

When working with cell cultures, glass and plasticware, water purification systems, devices for dispensing, dilution, and sampling, equipment for preparing culture media, laboratory thermostats, CO₂ incubators, aerators, shakers, fermenters, and other equipment are used.