Serum-free culture conditions for analysis of secretory proteinases during myogenic differentiation of mouse C2C12 myoblasts.

We have been studying extracellular proteins such as proteinases and attachment factors under serum-free culture conditions. A number of studies on myogenesis using an in vitro culture system have reported that proteinases and ECM components play significant roles in muscle differentiation. However, most of the studies were performed in the presence of serum. Serum is abundant in the aforementioned proteins and its use in serum-free culture affects many cellular functions significantly. In this study, we tried to establish serum-free culture conditions for analyzing extracellular proteins involved in mouse myogenic differentiation. By evaluating media, supplements, and procedure of cell inoculation under serum-free conditions and by comparing the resultant conditions with conventional conditions on differentiated characteristics of the cells, it was revealed that serum-free Dulbecco's modified Eagle's medium/Ham's F-12 plus insulin more efficiently supported myogenesis morphologically and biochemically than conventional 2% horse serum-containing culture and that secretory proteinases obtained from our serum-free culture were different from those obtained utilizing conventional serum-free cultures in their activities and patterns. Since our serum-free medium consists of simple components, the medium is low cost and easy to prepare. Furthermore, the results suggest that our culture conditions are superior to conventional conditions biochemically and morphologically and will provide more precise and accurate information on extracellular proteins involved in myogenesis.     

Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.     

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt₂-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.     

Isolation and culture of primary equine tracheal epithelial cells

Culture of airway epithelial cells is a useful model to investigate physiology of airway epithelia and airway disease mechanisms. In vitro models of airway epithelial cells are established for various species. However, earlier published method for isolation and culture of equine tracheal epithelial cells requires significant improvements. In this report, the development of a procedure for efficient isolation, characterization, culture, and passage of primary equine tracheal epithelial cells are described. Epithelial cells were isolated from adult equine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue and smooth muscle. The tissue was minced and dissociated enzymatically using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution for 2 h at 37 degrees C. Cells were collected by sieving and centrifugation, and contaminating fibroblasts were removed by differential adhesion. This procedure resulted in a typical yield of 1 x 10(7) cytokeratin-positive epithelial cells per gram tracheal lining tissue. Viability was 95% by trypan blue exclusion and isolates contained approximately 94% cytokeratin-positive cells of epithelial origin. Cells seeded at a density of 6.9 x 10(4) cells/cm(2) in serum-free airway epithelial cell growth medium formed monolayers near confluency within a week. Confluent cells were dissociated using dispase II and first passages (P1) and second passages (P2) were successfully established in serum-free medium. Collagen coating of tissue culture flask was not required for cell adhesion, and cultures could be maintained at the level of P2 over 30 d. In the present study, we could establish a high-yield protocol for isolation and culture of equine tracheal epithelial cells that can serve for in vitro/ex vivo studies on the (patho-)physiology of equine airway disease as well as pharmacological and toxicological targets relevant to airway diseases.     

Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes.

Normal adult rat liver cells have been successfully cultured as monolayers without subjecting donor animals to a partial hepatectomy before cell isolation. Coating plastic tissue culture dishes with acid soluble calf skin collagen increases the efficiency of cell attachment. Hepatocytes form a monolayer in 24 hr in serum-free L-15 medium although 10% fetal calf serum for 24 hr increases efficiency of attachment. In serum-free medium the mono-layer remains viable for at least one week without added insulin. Cultured cells maintain tyrosine transaminase and four of the five urea cycle enzymes at levels above or equal to freshly isolated cells for 72 hr. Adenylyl cyclase activities are maintained for at least 72 hr, and are stimulated by epinephrine, glucagon and fluoride. Tyrosine transaminase activity is increased in cultured cells by glucagon and dexamethasone, but urea cycle enzymes are not.     

A serum-free defined culture system which maintains follicle-stimulating hormone responsiveness and differentiation of porcine granulosa cells

A serum-free defined culture system has been developed that maintains follicle-stimulating hormone (FSH)-dependent differentiation of porcine granulosa cells from small follicles for up to six days in culture. Confluent monolayers of epithelioid cells were established after culture on fibronectin-coated culture dishes (FBN, 2 micrograms/cm2) in nutrient medium supplemented with human low-density lipoprotein (LDL, 10 micrograms/ml), insulin (I, 1 microgram/ml), and thrombin (TH, 1 NIH U/ml). Each of these factors was necessary to maintain the epithelioid morphology of the monolayers that attained 70% of the protein content and 71% of the cell number of replicate cultures maintained in nutrient medium supplemented with 10% fetal calf serum and insulin. Addition of FSH to the FBN/LDL/I/TH-supplemented cultures resulted in dose-dependent increases in progesterone secretion and [125I]-iodo-human chorionic gonadotropin (hCG) binding comparable to those obtained in the cultures containing serum. These results indicate that the attachment, epithelioid morphology, and differentiated function of porcine granulosa cells (GCs) can be maintained in defined culture conditions. This culture system will facilitate study of the effects of growth promoters and differentiative agents on GC function in the absence of poorly defined serum supplements.     

Acute radiation effects on the content and release of plasminogen activator activity in cultured aortic endothelial cells

Confluent monolayers from three lines of bovine aortic endothelial cells were exposed to a single dose of 10 Gy of 60Co gamma rays. Seventy-two hours later, the morphology of the irradiated and sham-irradiated monolayers was examined, and cellular DNA and protein contents were determined. In addition, the release of plasminogen activator (PA) activity into the culture media and PA activity in the cell lysates were assayed. Irradiated monolayers maintained their cobblestone appearance, but individual endothelial cells were enlarged considerably compared to sham-irradiated cells. DNA and protein contents in the irradiated monolayers were reduced to 43-50% and 72-95% of the control levels, respectively. These data indicate that radiation induced cell loss (detachment and/or lysis) from the monolayer, with hypertrophy of surviving (attached) cells to preserve the continuity of the monolayer surface. Total PA activity (lysate plus medium) in the irradiated dishes was reduced to 50-75% of the control level. However, when endothelial PA activity was expressed on the basis of DNA content, the irradiated monolayers from two of the three cell lines contained significantly more PA activity than did sham-irradiated monolayers. Most importantly, the percentage of the total PA activity released into the culture medium by irradiated cells (5-22%) was significantly (P less than 0.001) lower than that released by sham-irradiated cells (23-68%). These data suggest that fibrinolytic defects observed in irradiated tissues in situ may be attributable at least in part to a radiation-induced inhibition of PA release by vascular endothelial cells.     

Rabbit chondrocytes maintained in serum-free medium : I. Synthesis and secretion of hydrodynamically-small proteoglycans

The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared. Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum. After one day, the major peak of incorporated 35SO4 in medium fraction dAl eluted as a retarded peak (Kav 0.28) on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions. The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures. The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions. Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium. Culture medium fraction dAlDl from serum-free culture medium aggregated poorly when incubated with human hyaluronic acid (HA) in the presence of bovine link protein or when dialysed against bovine nasal cartilage proteoglycan aggregate. Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions. No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types.     

The Polarity of Choroid Plexus Epithelial Cells In Vitro Is Improved in Serum-Free Medium

Abstract: The influence of culture conditions on the development of normal characteristics of the choroid plexus epithelium has been investigated in vitro with respect to polarity, barrier properties, transport, and secretory activity. Withdrawal of serum supplement in the culture medium of cells grown on filters caused morphologically visible changes by an increased trimming of microvilli at the apical membrane side, which is accompanied by an increased expression of the Na⁺,K⁺-ATPase. Moreover cells under serum-free conditions exhibit structural changes in tight junctional zonula occludens protein-1 (ZO-1) organization, a reduced permeability, and a drastically increased electrical resistance from 150 Ω· cm² in the presence of serum to 1,500 Ω· cm² after serum withdrawal. Under these conditions, cell monolayers are able to build up a transcellular proton gradient and to secrete fluid into the upper (apical) filter compartment, which is accompanied by a polarized secretion of proteins like transthyretin. Active transport of the dyes fluorescein and phenol red by the organic anion transporter is found to be driven by the Na⁺,K⁺-ATPase. We come to the conclusion that removal of serum favors the differentiation process of the plexus epithelium in vitro, which brings the cell culture model closer to the physiological situation in vivo. We present preliminary evidence that epidermal growth factor may be one component in serum preventing the proper in vitro differentiation.     

Defined medium for primary culture de novo of adult rat alveolar epithelial cells

Summary Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resemble type I cells. Concurrently, the cells express type I cell surface epitopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiation. To define specific soluble growth factors and matrix substances that may regulate these processes, it would be preferable to culture isolated pneumocytes de novo under completely defined, serum-free conditions. In this study, we developed a completely defined serum-free medium that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioelectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric properties, morphology, and binding of a type I cell-specific monoclonal antibody were determined as functions of time. Plating efficiency plateaus at about 14% by Day 3 in culture. Transepithelial resistance rises to high levels, peaking at 1.76±0.14 KΩ-cm² by Day 5 in culture. Short-circuit current peaks on Day 3 in culture at 2.71±0.35 μA/cm². With time, the cells gradually become flattened with protuberant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope. These observations indicate that it is feasible to culture alveolar epithelial cell monolayers under completely defined serum-free conditions de novo. This culture system should prove useful for identifying soluble growth factors and matrix substances that modulate alveolar epithelial cell biological properties.     

Alveolar pre-type II cells from the fetal rabbit lung: effect of confluence on the production of disaturated phosphatidylcholine

Pre-type II alveolar cells isolated from the fetal rabbit lung on the 24th gestational day have been maintained in vitro for 14 days in a chemically defined medium supplemented with hormone-stripped serum. These cells replicate in culture. Measurement of the incorporation of [14C]choline into cellular disaturated phospholipid indicated that those cells grown in vitro under standard conditions for 8 days (pre-confluent) incorporate the radioactive precursor at a similar rate to cells maintained for 14 days (post-confluent). Both dexamethasone and serum-free medium conditioned by monolayer cultures of fetal rabbit lung fibroblasts stimulated [14C]choline incorporation into disaturated phosphatidylcholine (PC) by the pre- and post-confluent cultures after 24 or 48 h of exposure: the conditioned medium was more effective than the steroid. These treatments had little effect on choline incorporation into disaturated phosphatidylcholine of preconfluent cells during the first 12 h. A marked response occurred by 24 h after which the labelling of disaturated phosphatidylcholine plateaued. In contrast, with post-confluent cells labelling of disaturated PC increased in a more linear fashion and only plateaued after 72 h. Determination of the ratio of incorporation of [14C]choline into disaturated versus unsaturated phospholipid indicated that serum-free medium conditioned by monolayer cultures of fetal lung fibroblasts specifically increased the level of radioactive precursor in the disaturated phospholipid in both the pre- and post-confluent cell monolayers.     

Co-culture,oviduct secretion and the function of oviduct-specific glycoproteins.

Co-cultures of embryos with somatic cells, usually in the form of monolayers, or conditioned medium from these somatic cells, results in development past the early stage blocks and the formation of hatched blastocysts. Optimum rates of development are not achieved, however, and the task is to investigate components of the oviduct that are obligatory or facilitative for embryo development. Glycine and alanine are amino acids present in much higher concentrations in oviduct fluid than in serum or culture media. Glycoproteins specifically produced by the oviduct around oestrus bind to embryos and aid development but are absent from most culture media. These glycoproteins are induced by oestrogen in vivo but not in vitro. It is our contention that co-cultures of mammalian embryos should include appropriate concentrations of amino acids and a source of embryotrophic glycoproteins as an additive or by including stromal cells in addition to epithelial cells.     

Establishment of endometrial glandular epithelial cell subculture in a serum-free,hormonally defined medium,on a basement membrane matrix

Summary— Epithelial glands were isolated from guinea-pig endometrium. In order to reduce the requirement for a serum supplement and the contamination by non epithelial cells in primary culture, various coatings of the culture dishes were tested using serumfree Ham's F12 containing defined chemicals including 17β-estradiol. while epithelial glands seeded on culture dishes coated with Matrigel, a basement membrane matrix-failed to spread, they formed on poly-d -lysine plus serum-coated dishes, a subconfluent monolayer (5–7 days) enriched in cytokeratin-immunostained cells (78%). Cells from subconfluent primary cultures, obtained on poly-d -lysine plus serum-coated dishes in serum-free hormonally defined medium, were passaged on Matrigel-coated dishes in serum-free hormonally defined medium. These subcultures contained, at confluence (4–5 days), a high percentage (> 95%) of cytokeratin-immunostained cells. These monolayers consisted of well-differentiated cells which exhibited ultrastructural features characteristics of endometrial epithelial cells. Moreover, these confluent cells contained 50% immunostained nuclei for progesterone receptors. Progesterone receptor amounts decreased in confluent subcultures treated with progesterone and became undectable after longterm treatment, suggesting responsiveness of these cells to progesterone. This culture system provides a well-defined model for the study of protein synthesis and secretion by endometrial glandular cells under hormonal control.     

Characterization of newly established clonal oviductal cell lines and differential hormonal reculation of gene expression

Summary Oviductal functions have been studied mainly in primary epithelial cell culture and organ culture. However, secretory cells and ciliated cells coexist in the epithelium, and the small size of the oviduct limits the sources of both epithelial and stromal cells. To circumvent the limits, we attempted to establish clonal cell lines from an oviduct of a p53-deficient mouse. An oviduct was enzymatically digested and cultured in medium containing 10% fetal calf serum supplemented with estradiol-17β. Morphologically distinct clones (10 epithelial and 4 fibroblastic clones) were established, and all clones expressed estrogen receptor α and progesterone receptor. Expression of a mouse oviduct-specific glycoprotein gene as a marker of secretory cells was limited in one clone and was stimulated by estrogens and suppressed by progesterone. Expression of helix factor hepatocyte nuclear factor/forkhead homologue-4 gene as a marker of ciliated cells was limited in two clones and was suppressed by estrogens. The two genes were never coexpressed in any clones. The results strongly suggest that the oviductal epithelium consists of two functionally determined populations. To our knowledge, this is the first establishment of functional clonal cell lines of the oviduct and makes it possible to study independently two oviductal functions, secretion and ciliogenesis.     

Confrontation of an invasive (MO4) and a noninvasive (MDCK) cell line with embryonic chick heart fragments in serum-free culture media

Summary Confronting cultures of precultured embryonic chick heart fragments (PHF) with aggregates of malignant cells in vitro have been shown to be relevant for a number of aspects of tumor invasion in vivo. Preculture of the heart fragments, formation of cell aggregates and subsequent culture of confronting pairs have so far been done only in serum-containing culture media. We describe here confronting cultures of PHF with invasive MO₄ mouse cell aggregates or noninvasive MDCK dog kidney cell aggregates in serum-free media. Heart fragments precultured in the absence of serum seemed to be necrotic after confronting culture in serum-free media. However, preculturing in media supplemented with 10% fetal bovine serum allowed us to do subsequent confronting cultures in absence of serum. Cell aggregates were also prepared in serum-containing medium. MO₄ cells occupied and replaced the heart tissue within 4 d, whereas MDCK cells remained at the periphery, of the PHF. This indicates that serum-free confronting cultures can discriminate between invasive and noninvasive cells. The viability of individual PHF and cell aggregates cultured in the same way as in confrontations was ascertained by histology and by explantation and postculturing on a solid tissue culture substrate. Growth of the cultures was smaller in serum-free media than in media supplemented with 10% fetal bovine serum. The main advantage of serum-free culture conditions in vitro is the elimination of the influence of serum components on invasion, and the ability to examine the effect on invasion of drugs that are, susceptible to inactivation by serum. This work was supported by the Fonds van de Sport Vereniging tegen de Kanker, Brussels, Belgium, and the Fonds voor Geneeskundig Wetenschappelijk Onderzoek Brussels, Belgium     

A mouse fibroblast line cycles between monolayer and spheroid forms, regulates Met and HGF expression, and releases an attachment and growth-promoting substance

A subline of mesoderm-derived mouse NIH3T3 fibroblasts was selected for its ability to proliferate in serum-free media. This cell line (SFDH) grows as a monolayer at low density and spontaneously forms dense, multicellular spheroids at high density. Spheroid formation can also be induced by the addition of dexamethasone, polybrene, or heparin. Spheroids eventually detach from the substrate, but will reattach and re-form monolayers when transferred to fresh culture vessels and media, repeating the cycle again upon reaching high density. Thin section analysis of spheroids shows morphologically-distinct regions of cells, including an attenuated outer surface and a cuboidal interior with occasional lumen-like areas. Over time in culture, spheroids express increasing levels of met, the Met ligand-SF/HGF and cytokeratin, an epithelial marker, in comparison to monolayers. Both monolayer and spheroid-derived cells are rapidly tumorigenic in nude mice. Media conditioned by SFDH cells contain factors that stimulate growth and attachment of a variety of tumorigenic and non-tumorigenic cell lines, inducing cells to divide in serum-free media for up to 14 days when plated on tissue culture-treated and nontreated plastic surfaces pre-coated with SFDH conditional media. The growth-stimulating activity fractionates as a single peak over a sepharose column in the presence of 6 m urea, and sediments as a high molecular weight complex. Growth-stimulating activity can be neutralized by several antisera specific for hepatocyte growth factor, and the same sera recognize a novel approximately 37 kD protein in active supernatants. The cyclic, continuous nature of alternating monolayer and spheroid forms makes this cell line appropriate for studying changing gene expression patterns in progressive cell-cell/cell-matrix interactions.     

Behavior of primary and serially transplanted estrogen-dependent renal carcinoma cells in monolayer and in collagen gel culture

Summary When estrogen is present in culture medium, enzyme-dissociated cells from estrogen-induced primary renal tumors in Syrian hamsters and from 1st to 4th serially transplanted carcinomas in monolayer culture contain progesterone receptor levels that are similar and comparable to those in tumors in vivo (i.e., 2 pmol/3×10⁶ cells). Despite the similarity of receptor levels in cultured cells isolated from primary and transplanted tumors, the ability of cells to be maintained in culture differs considerably from one tumor stage to another. When cultured as monolayers in plastic flasks, isolated cells from primary tumors exhibit a marked decline in cell number after 4 to 6 d in culture. On the other hand, monolayer-cultured cells from first and second transplantation tumors remain essentially constant in cell number over a 2 wk culture period and cells from third transplantation tumors undergo a two- to threefold increase in cell number during 2 wk in culture. When primary tumor cells are cultured in collagen gels, the decline in cell number over a 2 wk culture period is prevented and progesterone receptor levels remain elevated. Cells cultured from first transplantation tumors exhibit a delayed decline in cell number beginning after 2 wk in monolayer culture. The decline in cell number in monolayer culture, like that for cells from primary tumors, can be prevented by culturing cells from first transplantation tumors in collagen gels. Neither cells from primary nor first transplantation tumors exhibit significant increases in cell number in collagen gels. Increasing the serum concentration of growth medium to 30% does not stimulate growth of cells under these conditions. Cells isolated from fourth transplantation tumors undergo a fourfold increase in cell number over a 1 month culture period whether cells are cultured as monolayers or in collagen gels. This investigation was supported by Grant CA 22008 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by institutional research funds from Marquette University.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Establishment and optimization of epithelial cell cultures from human ectocervix,transformation zone,and endocervix optimization of epithelial cell cultures

Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum-free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step-by-step protocol for culturing cells from each region of human cervix.