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Modern Principles of Classification and Development of Nutrient Media for Culturing of Human and Animal Cells V. Yu. Tabakov1,2, Yu. V. Schepkina1,2, and V. V. Chestkov1,2

Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 47-56, January, 2013 Original article submitted June 20, 2012 Analysis of the main principles of classification and development of nutrient media used for culturing of human and animal cells in biology and medicine is presented. The key moments of induction and regulation of mitogenic cascades and their differences in cells of continuous lines, diploid cells, and primary cultures are discussed. Some variants of classification of nutrient media for various cell culture types are described. Peculiarities of composition and the prospects of using serum-free media are discussed. Practical results obtained by the authors in the development of diverse purpose nutrient media are presented. Key Words: cell cycle; nutrient media; growth factors; cell culture; cell line Introduction of cell culture technologies into biology, medicine, and biotechnology implies solution of the problem of cell cycle (CC) control for ex vivo cell culturing. Practical solution of this problem is closely related to the composition of the used nutrient media. The present study focuses on the dependence of rational approach to the construction and selection of culture media on genetic characteristics of the cultured cells.

Types of cell cultures All variants of cell cultures can be divided (differentiated) into 3 types by their genetically determined potencies to in vitro development [26]. Primary tissue/cell cultures are obtained from live tissues of the multicellular organisms. The tissue sources are explants, i.e. biopsy specimens, operation and autopsy materials, and cell isolates. The primary culture is formed by the explant cells during in vitro expansion of mobile and/or proliferating subpopulations. The primary culture cells retain genome structure and cell homeostasis of the source. Their vital activity depends on the presence of signals regulat1 Medical Genetic Research Center, Russian Academy of Medical Sciences; 2PanEco Company, Moscow, Russia. Address for correspondence: [email protected]. V. V. Chestkov

ing the main metabolic pathways and initiation of the cell cycle and differentiation programs in the nutrient medium or matrix. These signals (e.g. growth factors, hormones, bioactive metabolites) are present in the explant or are added to the culture. By its population composition, the primary culture is characterized by high heterogeneity due to the presence of cell of different specificity at the initial stage of in vitro culturing. The maintenance of proliferating cell populations of common differentiation lineages is often related to gradually exhausting pool of stem/progenitor cells residing in reserve niches of the explants [1,26] The working potential of the primary cell culture is limited by the first (or the so-called zero passage). After the first subculturing procedure, the cell material cannot be considered primary culture. Primary cultures are required for cellular, biochemical, and molecular cytogenetic diagnosis and selection of new promising cell lines. Semi-transformed cultures (pseudo lines) of diploid eukaryotic cells are usually prepared by repeated passaging of the primary cultures; this process is associated with partial selection of subclones capable of limited self-renewal during ex vivo culturing. Gradual deceleration of cell proliferation in semi-transformed cultures eventuating in cell proliferation arrest is a result of cell aging (replicative aging) [29,49].

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V. Yu. Tabakov, Yu. V. Schepkina, and V. V. Chestkov

The obtained pseudolines are characterized by different proliferative potential, differentiation lineages, and rate of differentiation depending on the tissue specificity of the primary material. These cultures are characterized by unsteady cell composition, morphology, and ploidy. Similarly to the primary cultures, the majority of pseudo lines retain diploid karyotype. Changes in cytomorphology and cell biochemistry increasing during culturing are determined by genetic mutations and epigenetic modifications aimed at adaptation to in vitro culturing conditions [2,26]. In light of this, the number of passages is an important characteristic of pseudo line culture. It was demonstrated that experiments on pseudo lines are correct within 4-8 passages. Cultures of this type can be used in various model studies on cells of different tissue specificity. Cells of continuous (transformed, immortalized) cell lines are capable of unlimited in vitro proliferation. Mitotic activity of continuous cell lines far surpasses the corresponding parameter of primary culture cells and diploid cells. This is attained due to greater number of actively proliferating cells at the state of realization of the cell cycle program. Population composition of continuous line cultures under standard culturing conditions can be maintained from passage to passage for a long time. The stable line is usually presented by a cell strain of certain cytomorphological specificity. Continuous cell lines have aneuploid karyotype; modal number of chromosomes for each line is specified in culture certificate [6]. These characteristics reflect selection of mutations related to phenotypic manifestations of constitutively high activity of mitogenic cascade components and disturbances in extra- and intracellular elements of cell cycle negative control. Selection of these mutations is related to the so-called spontaneous or induced cell transformation. Active proliferation of transformed cells does not require initiating signals for cell cycle activation cascades. However, tumor origin of the tissue usually does not guarantee in vitro proliferation capacity and transformation of the primary culture of tumor cells into continuous cell line. Hence, in vivo or in vitro transformation should involve genes responsible for autonomic metabolism and ex vivo proliferation of cells. Despite considerable differences from native tissue cells, continuous cell lines are a convenient tool for biotechnology, vaccinology, and experimental studies not requiring strict extrapolation for the organization of body cell systems.

Cell cycles: mechanisms of induction and possible regulation All the cells of multicellular eukaryotes originate from a fertilized egg (zygote) and pass through successive

165 stages of precursor cells with different proliferation and differentiation potential (potency) [41,49]. Starting from the late embryonic stage, the multicellular organism represent a hierarchical system of cell differons that have common precursors, SC [1,49]. The ontogenesis program at the cellular level unfolds from totipotent embryonic blastomeres through multipotent SC of the germ layers and tissue precursors to oligopotent and unipotent progenitor elements of the tissue reserve (Fig. 1). During cell maturation and differentiation, the loss of differentiation potential is accompanied by the loss of cell capacity to selfrenewal at successive stages of maturation and their proliferation more and more depends on specific factors regulating growth and development. Thus, the level of SC of regional tissue reserve requires specific growth factors for triggering CC program and entry into bidirectional development. At the level of maturing elements of differons, active progression through CC stages is completely controlled by growth factors and neuroendocrine stimulators. An important factor during in vitro culturing of various cell types is the maintenance of active cell cycle: G1SG2M. Primary cell cultures derived primarily from regional tissue explants contain SC of the tissue reserve, progenitor, maturing, and differentiated elements [26]. For reproduction of these cell subpopulations in vitro, the presence of initiating mitogenic growth factors in the medium is required. Semi-transformed cultures of pseudo lines of diploid cell, as products of partial selection of in vitro adapted primary cultures, rapidly lose the pool of reserve tissue cells and progenitors during passaging and therefore are more dependent on the presence of factors triggering the CC program. These factors are used for mobilization of progenitor cells from “inactive” G0 state to G1 stage of CC, initiation of proliferation of maturing cells, and their G1S transition [25]. The best-known factors of mitogenic cell activation are platelet-derived growth factor (PDGF) and FGF [33]. EGF and insulin-like growth factors (IGF) maintain proliferative stimulation of cells during their progression through G1 checkpoint [32,33]. Binding of inducing growth factors with membrane receptors on the target cells leads to receptor dimerization followed by their activation and autophosphorylation of cytoplasmic tyrosine kinase domains (Fig. 2). Phosphorylated tyrosine kinase sites interact with signaling proteins activating signal transduction cascade through the system of mitogen-activated protein kinases (MAPK). The best-studied signaling pathways are coupled with MAPK cascades: ERK, p38, JNK [50,54]. The main result of the primary mitogenic stimulation is cyclin D1 production and its binding to cy-

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Fig. 1. Dependence of cell cycle characteristics on the potency level of animal cells and their relationship with the main stages of ontogeny. ESC: embryonic SC; GSC: SC of germ layers and tissues; TSC: tissue SC.

clin-dependent kinase CDK4. Activated cyclin-kinase complexes can step-by-step phosphorylate retinoblastoma protein pRb, an inhibitor of positive transcription regulators of the E2F family stimulating transcription of cyclins, kinases, and factors of DNA replication essential for the transition from G0-G1 to S, G2, and M phases of CC [22,30,44]. Successive activation of all components of the mitogenic cascades is a necessary condition for the maintenance of diploid cell in primary and semi-transformed cultures during realization of the CC program. Thus, an important aspect in construction of effective growth and proliferation media for culturing of non-transformed cells is determination of the qualitative and quantitative composition of ingredients initiating or reinforcing the work of mitogenic cascade components. Figure 2 demonstrates three conceivable contours of regulation of mitogenic cascades in the cell. The first (signal) level is related to optimal choice of qualitative and quantitative parameters of mitogenic growth factors (common or specific set) [26,43]. The second (membrane) level implies targeted modification of the structure and properties of the cell membrane facilitating perception of the mitogenic signal. This can be achieved via the use of additional signal factors or factors modifying the membrane and/or receptors and extracellular matrix components [27,31,37].

The third (intracellular) level consists in the maintenance of mitogenic cascade components in the active state by using substances maintaining phosphorylated status of proteins involved in the transmission of mitogenic signals [47]. Modification of the mitogenic response at the level of mitogenic cascade components or at the genetic level can be achieved by using hormonal compounds, but the mechanisms of hormonal stimulation are often unclear and ambiguous, because they are realized against the background of other mitogenic influences [46,51]. The maintenance of active cell proliferation in the majority of continuous lines seems to be a simpler task, because transformed cells constitutively express high activity of the key mitogenic cascade components and therefore do not require the presence of additional specific initiating factors in the culture medium.

Principles of classification, usage, and development of nutrient media Specific biological characteristics of types and specific variants of cell cultures necessitate the use of nutrient media for cell culturing ensuring the maintenance of the basal and studied physiological functions. All nutrient media have been grouped on the basis of their historical origins, composition, and application purposes [4,26]. However, widening of the spectrum of nutrient media and applications of the cell technolo-

V. Yu. Tabakov, Yu. V. Schepkina, and V. V. Chestkov

gies in biology, biotechnology, and medicine require creation of target classification systems simplifying the choice of the medium with desired properties. Spontaneously developed and practically used system of culture media classification is based on the three principles: maintenance of proliferative activity, specificity of the media for the objects and experimental tasks, and medium composition (Table 1). Classification of the culture media based on their specificity and taking into account branching application fields and trends in culturing technologies is the most controversial, because many culture media can be used for solving different problems and for different cell cultures. It can be assumed that the media intended for primary cultures of cells and long-term passaging of semi-transformed pseudo lines have most complex composition, because these cultures require additional regulatory signals for their ex vivo survival. Traditionally, complex substances (fluids and tissue extracts) of the multicellular organisms that were used in the first experiments on ex vivo culturing of tissue and organ cells served as the sources of these signals. The development of cell physiology and biochemistry led to creation of the basic recipes of the so-called synthetic classical media and minimization of the number of additives of undefined composition necessary for cell growth and proliferation [23,26]. Classical media enriched with complex additives of plant and animal origin are most often used as the growth media during collection passaging, experimen-

167 tal studies, biotechnological and biomedical processes with cells of continuous lines and pseudo lines, and for preparing primary tissue cultures and cell isolates from animals. Blood serum (usually embryonic calf serum, ECS, 1-2 to 50% v/v) is most widely used as the complex component for optimization (enrichment) of synthetic basic classical media [4,26]. The use of blood serum remains an essential factor in the construction of proliferative media providing accelerated cell proliferation and rapid accumulation of the biomass in the culture. Additional factors and cell growth stimulators are also introduced into proliferative media. These specific supplements maintain proliferation-active cells in the state of CC program realization, promote rapid progression through stage G1, and prevent terminal differentiation and apoptosis. Figure 3 (a, b) shows cultures of adult human skin fibroblasts in a classical growth medium (DMEM+10% ECS) and proliferative medium (Amniokar). The cell yield in the culture grown in Amniokar medium more than 3.8-fold surpassed that in DMEM medium [9]. Some primary cultures and strains of diploid cells (e.g., amniotic fluid cells, mesenchymal SC, and human umbilical vein endothelial cells HUVEC) can be effectively cultured and passaged only in the presence of a sufficient amount of cell growth stimulators in proliferative media [3,9,26]. Moreover, proliferative media can be successfully used for the diagnosis and analysis of the prop-

Fig. 2. Scheme of induction and three possible mitogenic cascade regulation contours in animal cells. GF: growth factors; P: phosphorylated state of proteins; MAPKs: mitogen-activated protein kinases; CKC: cyclin-kinase complexes; CDK 4/6:cyclin-dependent kinases, E2F: positive transcription regulators for CC proteins; G1, S: initial stages of CC.

Cell Technologies in Biology and Medicine, No. 1, May, 2013

168 TABLE 1. Modern Classification of Nutrient Media Principle Maintenance of proliferative activity

Specificity

Composition

Category

Purpose

Maintaining

Short-term maintenance of culture viability in experiments and processes

Growth

Routine culturing, passaging, and experimental studies

Proliferative

Rapid production of the biomass, accumulation of proliferating cells, and maintenance of undifferentiated phenotype

Low-specific (classical)

Routine culturing, optimization of the medium certain types of cell cultures, experimental studies

Special:

Experiments on cultures with specific needs and/or for special experimental purposes

•

by the cultured objects

•

by experimental tasks

•

experimental

•

diagnostic

•

for medical cell technologies

•

IVF

•

for biotechnology

Serum-free media of specific composition: •

protein-free

•

protein-containing

•

enriched

Enriched media: •

low-serum

•

serum-containing

•

containing organ and tissue extracts

•

containing peptones and hydrolysates

•

conditioned

erties of tumor tissue; the possibility of culturing and the type of in vitro culture in this case is an additional measure of malignancy of the analyzed neoplasm. Thus, proliferation medium Clonogen specially designed for culturing of tumor tissue cells was used for cytomorphological diagnosis and metaphase FISH-analysis of the status of protooncogenes Her2/ neu and N-myc in breast cancer and neuroblastoma samples, respectively [10]. Despite the effective use of complex biological substances as the unspecific stimulators of cell growth and proliferation, there are significant potential risks of working with enriched media: high cost, uncertainty of the qualitative and quantitative composition affecting the results of cell product isolation and purification, and the risk of contamination of cells and their products with pathogenic agents [24,26].

Avoiding contamination of cells and cell synthesis products, reduction of substrate immunogenicity, ensuring targeted effects of the studied factors, reproducibility of the results of experiments and processes, and facilitation of cell product purification Attaining of high growth rates in cell cultures in the absence of strict control of nutrient medium composition

This substantiates the need in practical studies and development of serum-free media [11,12,26]. Growth potential of these media is restricted by continuous lines not requiring addition of CC initiation factors and regulation of the background metabolism. However, serum-free medium Gibris developed in Russia allows unlimited culturing of some cell lines with population doubling time T2 [26] slightly exceeding the corresponding parameter for serum-containing media (Table 2). At the same time, considerable inhibition of the growth rate has been demonstrated for some cell lines in comparison with that in serum-containing medium. This attests to the contribution of some serum factors into proliferative activity of these lines. Thus, mouse embryonic fibroblasts adapted to serum-free culturing conditions, NIH/3T3-SF subline, proliferate after ad-

V. Yu. Tabakov, Yu. V. Schepkina, and V. V. Chestkov

dition of mitogenic growth factors (e.g., bFGF) to the serum-free medium [38]. Significant differences in the effectiveness and properties of continuous cell lines cultured without serum substantiate the need in large number of individual formulations of serum-free media for different objects and processes [12]. Transition to serum-free culturing conditions modifies biochemical and morphological characteristics of the cell lines, which manifests in changes in cell morphology and impairment of adhesion properties of cells in adhesion cultures. However, different cell lines demonstrate different responses to transition to serum-free culture media. For instance, HeLa cells cultured in Gibris medium get spherical shape and

169 transit into a suspension. CHO-K1 cells partially retain adhesion capacity, but do not spread. MDCK cells remain a monolayer culture in serum and serum-free media and can be used for virus particle titering and routine production of viral antigens for vaccines [7]. Serum-free Gibris medium was effective in in vitro virus reproduction systems for the virus of spring viremia of carp on monolayer cultures of continuous cell lines of Mozambican telapia (TmB) and carp (CTF/T) river fish [13]. Special conditions of mitogenic stimulation in a serum-free medium provide prerequisites of creation of effective technologies for culturing of human peripheral blood lymphocytes. We have shown that lymphocytes cultured in serum-free media responded

Fig. 3. Cytomorphology and adult human skin fibroblasts (HSFW58 strain) in different culture media. a) serum-containing growth medium DMEM+10% ECS; b) Aminokar proliferative medium; c) Gibris-F serum-free enriched medium (SF). Phase contrast, 150.

Cell Technologies in Biology and Medicine, No. 1, May, 2013

170 TABLE 2. Comparison of Doubling Time (T2) in Continuous Cell Cultures in Classic Serum-Containing Medium (DMEM+10% ECS) and Sublines Adapted to Serum-Free Conditions in Serum-Free Gibris Medium T2, h Cell line (DMEM+10% ECS)

Gibris

Sp2/0

12-16

13-18

HeLa

20-25

22-26

CHO-K1

16-21

19-23

Vero

22-26

40-48

NIH/3T3

18-22

33-38 after introduction of additional growth factors

MDCK

23-28

25-31

to 2-10-fold lower doses of bioactive substances than cells in RPMI-1640 medium containing 5-10% ECS [8]. Our study eventuated in introduction of serumfree media Limfokar-2 into medical cytogenetic diagnostics. In contrast to lymphocytes characterized by high natural potential of mitogenic induction, culturing of diploid cell pseudo lines (fibroblasts, keratinocytes, myocytes, SC, etc.) requires enriched serum-free media containing specific stimulators of cell growth and differentiation. Cell growth in these media is limited by few passages due to rapid exhausting of cell proliferative potential in the absence of serum factors regulating CC in non-transformed cell [39,40]. Russianmade serum-free medium for culturing of human skin fibroblasts over 4-6 passages is presented by Gibris-F (BS) (Fig. 3, c) [55]. This category of culture media provides solution of experimental tasks requiring controlled culturing conditions. Moreover, enriched serum-free media are used for cell biomass expansion in the absence of immunogenic substances and xenogenic contaminants for the purposes of cell replacement therapy and tissue transplantation [21,35,36]. Low-serum media occupy an intermediate position between serum-containing and serum-free media. This trend in creation of enriched media is based on practical experience of using sera in minimal amounts (1-2% v/v). The growth potential of low-serum media can be strengthened via introduction of cell growth stimulators, their qualitative and quantitative parameters varying depending on the nature of cultured cells and/or experimental tasks [20,52]. This optimization can provide long-term maintenance pseudo lines of diploid cells in low-serum media or improve the pa-

rameters of serum-free media to the level of growth and proliferative media [20,54]. The low-serum media containing allogeneic or autologous serum can be very promising in the development of technologies for cell, tissue, and organ transplantation [42]. A very promising trend from the point of view of modern cellular technology is manipulations with SC (embryonic, mesenchymal, hemopoietic, and artificially induced), which can help to solve many problems of replacement therapy at the cellular, tissue, and organ levels [15]. At the same time, the problems of long-term maintenance of SC cultures and tissue-specific differentiation require special attention. The choice between SC culture maintenance in undifferentiated self-renewing state or differentiation depends on the set and proportion between growth factors, composition of the nutrient medium, and the presence of specific and nonspecific differentiation signals [28,45,48]. In light of this, the use of serum-free media for SC culturing is limited despite their high proliferative potential. Nevertheless, some serum-free culture media formulations were proposed for the maintenance and targeted differentiation in vitro [18,19,34]. Products for in vitro fertilization technologies (IVF) occupy a special place in the application-based classification of nutrient media [14]. In contrast to classical nutrient media for culturing of isolated cells and tissues, IVF media are used for manipulations with gametes, fertilization, culturing of the organism at the early stages of embryonic development, and introduction of the multicellular product into mother’s body for completion of the prenatal development under natural condition in vivo. The zygote has considerable stores of plastic and energy substrates fully ensuring the first stages of embryogenesis. Since the natural cell-cell interactions in the cultured embryo are sufficient to ensure proper development at the early stages, there is no need to introduce cell growth factors to IVF media. These peculiarities together with high sensitivity of the developing embryos to the presence of endotoxins and protein factors in the medium substantiate preferential use of serum-free media for IVF technologies. The principal criterion during the development of culture media for IVF is their adequacy to physiological conditions surrounding the embryo in woman’s body during its passage through the Fallopian tubes and implantation into the uterine wall. Changes in the content of normal metabolites (e.g. glucose and lactate) in the medium are a regulatory factor ensuring successful division of the zygote until the morula stage [5]. All this explains great variety of nutrient media used for experimental and practical work in the bio-

V. Yu. Tabakov, Yu. V. Schepkina, and V. V. Chestkov

logy, biotechnology, and medicine. Thus, the development and optimization of nutrient media is a necessary condition for the solution of objective tasks of ex vivo cell culturing.

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