Long-term culture of human aortas. Development of atherosclerotic-like plaques in serum-supplemented medium

Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures.     

Expression of cathepsin B and aryl hydrocarbon hydroxylase activities,and of apolipoprotein B in human hepatoma cells maintained long-term in a serum-free medium

Summary We have established the human hepatoma cell line, HepG2, in a defined, serum-free medium. These cells were maintained and studied over a 100-generation period (i.e. 10 serial transfers). Cells maintained in serum-free medium exhibited growth parameters (i.e. saturation density, efficiency of plating, and population doubling time) similar to those obtained with HepG2 cells maintained in serum-supplemented medium. Serum-free cells were also similar to their serum-supplemented counterparts with respect to the expression of cathepsin B activity and the induction of aryl hydrocarbon hydroxylase by 2,3,7,8-tetra-chlorodibenzo-p-dioxin. Significantly, HepG2 cells maintained in serum-free conditions also retained the ability to synthesize and secrete proteins, including the liver plasma protein, apo-lipoprotein B. These results indicate that the serum-free medium used in this study supports the long-term growth and maintenance of human hepatoma, HepG2, cells in culture. Inasmuch as these cells retain phenotypes, including differentiated markers previously reported for their serum-supplemented counterparts, they may provide a more reliable, standardized culture system to study the expression, secretion, and regulation of proteins during biological and pathologic processes.     

A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.     

Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

Effects of B-retinyl acetate on human breast epithelium in explant culture

Explants of human breast (obtained aseptically from ten women ages 18-37) were examined and the vitamin A compound B-retinyl acetate was tested (at 3 X 10(-5) and 3 X 10(-6) M) for its effects on the fine structure and growth of the epithelium. In the absence of B-retinyl acetate, cells growing out from the explant (outgrowth cells) underwent squamous metaplasia, began to accumulate many intermediate filaments (tonofilaments), and revealed large desmosomes after 2 weeks in culture. In the presence of either concentration of B-retinyl acetate, the epithelial cells were largely prevented from undergoing squamous metaplasia. The glandular epithelium inside the explant maintained a typical secretory appearance for 2 weeks in control cultures (without the retinoid) and then began to show increased numbers of lysosomes and a loss of secretory granules. These glandular epithelial cells did not undergo squamous metaplasia, but they contained increased numbers of intermediate filaments. In contrast, glandular epithelium cultured in either concentrations of B-retinyl acetate appeared secretory for as long 6 weeks in culture, and intermediate filaments were not obvious. Autoradiographs demonstrated that both concentrations of B-retinyl acetate inhibited cell division in the outgrowth epithelium and in the (internal) glandular epithelium. The ability of B-retinyl acetate to reverse squamous metaplasia in the outgrowth cells was also tested. Outgrowth cells reversed to a more normal ductlike appearance after 6 weeks culture in standard medium followed by only 1 week in 20 micrograms/ml B-retinyl acetate. After 7 weeks in standard medium and 1 week in 10 micrograms/ml B-retinyl acetate, the cells showed a partial reversal of the squamous metaplasia.     

Three-dimensional culture of human tumor cells under standardized medium conditions

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution.The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase.The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.     

Control of enzyme activity levels by serum and hydrocortisone in neonatal rat heart cells cultured in serum-free medium

A serum-free, hormone-supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum-supplemented media. To quantitatively compare cell behavior in SFHM with serum-supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+-linked sn-glycerol-3-phosphate dehydrogenase, malate dehydrogenase, NAD+-linked isocitrate dehydrogenase, NADH-cytochrome c reductase, and succinic cytochrome c reductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum-supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: 1) many enzyme activities that may represent dedifferentiation are elevated by serum; 2) NAD-linked glycerol-3-phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; 3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; 4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 micrograms/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in cultured neonatal rat heart cells.     

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Establishment of cell strains from human urothelial carcinoma and their morphological characterization

We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.     

Establishment of cell strains from human urothelial carcinoma and their morphological characterization

Summary We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain. This work was supported in part by Grants 5319 and 5322 in aid for cancer research from the Ministry of Health and Welfare, Japan.     

Influence of retinoic acid on the ultrastructure and hyaluronic acid synthesis of adult human epidermis in whole skin organ culture

Normal human skin was maintained in organ culture under chemically defined conditions. All-trans retinoic acid was added to the culture medium at the final concentration of 5 mumol/l. After 5 days in culture samples were either harvested for electron microscopy or labeled with 3H-glucosamine for 24 h. After labeling, epidermis was separated from dermis and both tissue compartments were analyzed for the content of 3H-labeled glycosaminoglycans (GAGs) using CPC-precipitation and thin layer chromatography after enzymatic degradation into specific disaccharides. Retinoic acid caused a marked change in the epidermal tissue architecture. The epidermal cells were flattened and contained fewer desmosomes and tonofilaments than control explants. Retinoic acid induced accumulation of fine granular material in the intercellular spaces in the upper, and less dense, flocculent material in the lower epidermis. The analysis of 3H-glycosaminoglycans showed that in the epidermis retinoic acid elevated the amount of labeled hyaluronate by 70%, whereas sulfated GAGs were not significantly increased. In dermis the incorporation of 3H-glucosamine into neither hyaluronate nor sulfated GAGs was stimulated by the retinoic acid. It is concluded that retinoic acid significantly modifies the differentiation of normal adult human epidermis by decreasing cytoskeleton components and by inducing the synthesis of new intercellular material, at least a part of which is hyaluronic acid. As a consequence, the cohesion between the epidermal cells was apparently weakened.     

Effects of thyroliberin (TRH) on cell proliferation and prolactin secretion by GH3/B6 rat pituitary cells: a comparison between serum-free and serum-supplemented media

Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum-free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum-supplemented medium to serum-free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum-free medium, TRH stimulates prolactin production by as much as 5-fold instead of approximately 2-fold in serum-supplemented medium. Furthermore, this increased responsiveness to TRH in serum-free medium is accompanied by a 10-fold decrease in the ED50 for TRH (concentration needed for half-maximal response) and paradoxically by a 2-fold reduction in the number of high-affinity TRH binding sites without significant change of their association constant.     

Increased glucocorticoid responsiveness of CD4+ T-cell clonal lines grown in serum-free media

Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for 3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid hormone action may be pursued more precisely in a clearly defined culture medium. This work was conducted in conjunction with the Walls Medical Research Foundation.     

Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/somatomedin-C

Hepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8-16 weeks gestation obtained following prostaglandin-induced pregnancy terminations. They were maintained on collagen-coated tissue culture dishes in selective arginine-free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H-thymidine. The labeling index of hepatocytes was consistently enhanced by 25-250 ng/ml human placental lactogen (HPL), 25-250 ng/ml human growth hormone (HGH), 10-50 ng/ml insulin-like growth factor I/somatomedin-C (IGF I/Sm-C), and 10% dialyzed fetal calf serum, reaching a maximum of three- to four-fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF I/Sm-C. Although this proportion did not change following treatment with HGH and HPL, IGF I/Sm-C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF I/Sm-C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF I/Sm-C.     

Factors controlling embryonic heart cell proliferation in serum-free synthetic media

Summary Embryonic chick cardiac cell cultures, plated on collagen-coated dishes, containing serum-free synthetic media proliferate actively. The basic medium contained Ham's F12 nutrient mixture, fetuin, ascorbic acid, and bovine serum albumin. This medium was supplemented with various combinations of factors; endothelial cell growth supplement (ECGS), epidermal growth factor (EGF), insulin (I), transferrin (T), selenium (S), hydrocortisone, and thyroxine or supplemented alone. Basic medium supplemented with ECGS alone contributes to the highest final cell density among all other factors used in various combinations or alone. The final cell density of the control culture with 2% fetal bovine serum was higher than those of all experimental cultures and an additional control culture grown in the basic medium. Combinations of factors without ECGS do not promote significant cell proliferation. Thyroxine is required to induce optimal differentiation and contractility of cardiac myocytes in vitro. Fibronectin and laminin did not show any more influence than collagen did on the growth and maintenance of cardiac myocytes in serum-free media. The proportion of cardiac muscle cells in ECGS-containing media was higher than those in other experimental media and control media with the exception of ECGS and ITS-containing medium that showed lower proportion of cardiac myocytes than that of serum-containing medium on Days 3 and 5. The profiles of incorporation of [³H]thymidine into DNA of heart cells in experimental and control cultures showed a peak in incorporation values within the first week of culture and subsequently declined. Autoradiography studies revealed that cardiac myocytes in culture supplemented with ECGS alone attained a peak in labeling index on Day 1 with approximately 62% labeled cells. Subsequently, the labeling indices declined. Cardiac myocytes grown in media without ECGS showed significantly lower labeling indices than those in ECGS-containing media. This study has demonstrated the influence of ECGS, EGF and ITS in promoting the growth of cardiac myocytes and also in contributing to the maintenance of contractile cardiac myocytes in serum-free, long-term culture. The influence of ECGS on heart cell proliferation is considered to be superior to that of EGF and ITS. This study was supported in part by a grant HL-25482 from the National Heart Lung and Blood Institute and a grant from the American Heart Association of Michigan.     

Use of synthetic serum-free medium for culture of human dermal fibroblasts to establish an experimental system similar to living dermis

In this study, we sought to establish a defined experimental system for fibroblast growth similar to that of the living dermis. To this end, we evaluated the growth and biochemical characteristics of fibroblasts cultured with serum-free HFDM-1, a finely tuned synthetic medium for human fibroblast culture. Three culture conditions were used to grow fibroblasts obtained from primary culture: (1) culture with Dulbecco’s modified Eagle medium (DMEM) plus 10 % fetal bovine serum (serum-supplemented DMEM), (2) culture with DMEM (serum-free DMEM), and (3) culture with HFDM-1 (HFDM-1), and fibroblast morphology, growth, collagen type I production, and lipid composition were analyzed. Fibroblasts grown in HFDM-1 maintained cell numbers at nearly 100 % from days 14 to 21 and produced more collagen type I than cells grown in serum-supplemented and serum-free DMEM. Arachidonic acid (20:4) and total polyunsaturated fatty acids were lower in cells grown in serum-free DMEM and HFDM-1 than in serum-supplemented DMEM. These results suggested that HFDM-1 recapitulated growth conditions in the dermis better than traditional, serum-supplemented DMEM. In addition, the controlled chemical composition of HFDM-1 eliminated a potential source of variability in cell culture conditions.     

Effects of growth factors on proliferation of basal and luminal cells in human breast epithelial explants in serum-free culture

Summary A method of culturing human breast epithelium is described in which viable explants can be maintained in protein-free medium while retaining the capacity of responding to added hormones and growth factors for at least 7 days. Culture parameters were chosen to provide maximum sensitivity of detection of proliferative responses by autoradiography. Under basal conditions, the mean thymidine labeling index of the explants was 0.08%. After stimulation with insulin, hydrocortisone, and cholera toxin (I,H,CT), a combination known to stimulate proliferation in human breast epithelium in vitro, the mean labeling index was 15.7%. Stimulation of explants with epidermal growth factor (EGF) and transforming growth factor (TGF)-α resulted in mean labeling indices of 6.6 and 10.8%, respectively. Autoradiography at the ultrastructural level demonstrated that in I,H,CT-stimulated explants the majority of the labeled cells were luminal, with only 1.5% being basal cells. In contrast, after EGF and TGF-α basal cells accounted for 11.5 and 18.5% of the labeled population. These results indicate that this system provides an in vitro assay of proliferative activity in the normal human breast that enables comparisons to be made between both the luminal and the basal cells in the explants and their counterparts in monolayer culture prepared from flow sorted cells. Thus, growth responses dependent on cell-to-cell interactions or stromal modulation can be identified.     

Biomedical advances from tissue culture

The demonstration that the “dedifferentiation” of cells commonly observed in the early days of tissue culture was due to selective overgrowth of fibroblasts led to enrichment culture techniques (alternate animal and culture passage) designed to give a selective advantage to functionally differentiated tumor cells. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types. These results gave rise to the hypothesis that cells in culture accurately represent cells in vivo but without the complex in vivo environment. This concept has been strengthened with the development of hormonally defined culture media in combination with functionally differentiated clonal cell lines, which have augmented the potential of tissue culture studies. The use of hormonally defined media in place of serum-supplemented media demonstrates that hormonal responses and dependencies can be discovered in culture. Discoveries of hormonal dependencies of cancer cells has led to therapies targeting intracellular signaling pathways while discoveries of hormonal responses of pluripotent cells are helping to identify the potential application of stem cells. In these and other ways tissue culture technology will continue to contribute to solving problems of human health.     

Mechanical and biochemical characterization of cartilage explants in serum-free culture

Allografts of articular cartilage are both used clinically for tissue-transplantation procedures and experimentally as model systems to study the physiological behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Chemical mediators in poorly defined culture media can stimulate cells to quickly degrade their surrounding extracellular matrix. This is particularly true of juvenile cartilage which is generally more responsive to chemical stimuli than mature tissue. By carefully modulating the culture media, however, it may be possible to preserve allograft tissue over the long-term while maintaining its original mechanical and biochemical properties. In this study juvenile bovine cartilage explants (both chondral and osteochondral) were cultured in both chemically defined medium and serum-supplemented medium for up to 6 weeks. The mechanical properties and biochemical content of explants cultured in chemically defined medium were enhanced after 2 weeks in culture and thereafter remained stable with no loss of cell viability. In contrast, the mechanical properties of explants in serum-supplemented medium were degraded by ( approximately 70%) along with a concurrent loss of biochemical content (30-40% GAG). These results suggest that long-term maintenance of allografts can be extended significantly by the use of a chemically defined medium.