Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Comparison of colony-formation efficiency of bovine fetal fibroblast cell lines cultured with low oxygen, hydrocortisone, L-carnosine, bFGF, or different levels of FBS

A comparison of colony-formation efficiency (CFE) was made between six independent bovine fetal fibroblast (BFF) cell lines used in somatic cell nuclear transfer. Variation in CFE was assessed under different culture conditions. The conditions examined were ambient atmosphere (approximately 20% oxygen) culture versus 5% oxygen culture, three levels of fetal bovine serum (FBS) in the medium (5%, 10% or 20%), and the amendment of 10% FBS medium with basic fibroblast growth factor (1 ng/mL), L-carnosine (20 mM), or hydrocortisone (1 microM). The six BFF cell lines showed significant differences from one another in CFE. No significant difference in CFE was found with reduced oxygen culture. L-Carnosine also had no significant effect on CFE. A FBS concentration of 10% was found to produce the best overall CFE. Hydrocortisone treatment reduced the size of colonies although the number of colonies formed was not affected. Basic FGF increased the size of colonies but the number of colonies formed was not affected. The results showed that different BFF cell lines varied significantly in their CFE. Also, some medium supplements or culture conditions that have shown positive CFE effects on the fibroblasts of other species failed to show significant positive CFE effects on the BFF cell lines tested.     

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.     

The differentiation of L5/A10 myoblast cell line (a subclone of L5 line) is controlled by changes of culture conditions

We report here that it is possible to induce differentiation in a subline of L5 myoblast line (L5/A10) by manipulating the culture media. When L5/A10 myoblast are cultured in F14 supplemented with 10% fetal calf serum the cells grow with a division time of 12 h and reach confluency at a cell density of approximately 2.4 X 10(5) cells per cm2, without undergoing differentiation, characterized, morphologically, by formation of multinucleated fibers, and biochemically, by the synthesis of muscle specific proteins such as creatine phosphokinase or myokinase. However, cells, grown in F14 + 10% fetal calf serum, will undergo regular differentiation after a limited number of division when transferred to F14 medium supplemented with limiting concentrations (1-2%) of fetal calf serum. Investigations of the biochemistry of myoblast differentiation in cell culture will be facilitated by the availability of a cell line that can undergo differentiation under controlled conditions.     

Oncostatin M stimulates proliferation and functions of mouse fetal liver cells in three-dimensional cultures

In order to develop a tissue engineered bioartificial liver (BAL), long-term three-dimensional (3-D) culture of fetal liver cells (FLCs) utilizing porous polymer as a scaffold was performed for up to 1 month. The effects of the basal medium and supplementation with oncostatin M (OSM) on the proliferation and differentiation of mouse FLCs were examined in both 3-D culture and conventional monolayer dish culture. Compared with monolayer culture, cell numbers and hepatic function of FLCs were better maintained by 3-D culture. When two kinds of basal media were tested in this study, Williams' medium E (WE) was superior to minimum essential medium alpha (alphaMEM) in expressing hepatic function of FLCs in both 3-D and monolayer cultures, although higher cell densities were obtained with alphaMEM. OSM potently stimulated both cell growth and metabolic activity, especially in 3-D culture. When WE supplemented with OSM was used for 3-D culture, albumin secretion by FLCs increased dramatically after day 5, and a high level of secretion was maintained until the end of culture. During a period of over 1 month, no decrease of albumin secretion was observed. In conclusion, this 3-D culture method was expected to be one of the realistic attempts to develop a tissue engineered BAL.     

Adaptation of BHK-21 Cells to Growth in Shaker Culture and Subsequent Challenge by Japanese Encephalitis Virus

Baby hamster kidney (BHK-21) cells were adapted to grow in shaker culture using Waymouth medium 752/1 containing 20 mM N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid buffer and supplemented with 2.5% (vol/vol) calf serum, 0.002% (wt/vol) sodium oleate, and 0.2% fatty acid-free bovine serum albumin (WO2.5). Infectivity of Japanese encephalitis virus grown in the cells adapted to WO2.5 approached 2 x 10(8) plaque-forming units per ml. The culture volume of infected cells was reduced fivefold 12 h after infection. This step resulted in a 10-fold increase in infectivity over that obtained from infected cultures not subjected to volume reduction.     

Autocrine growth regulation of W12 and GCA cells in culture

Two rat kidney cell lines transformed by two strains of ASV virus were investigated. It was demonstrated that these two lines (1) showed density-independent growth, (2) had a decreased requirement for serum in the culture medium, (3) had the ability to grow in a chemically defined medium (without serum), and the rate of this growth had increased with the increase in starting density of cells, and (4) had the ability of anchrage-independent growth, even without serum. These results confirmed autostimulation of growth of W12 and GCA cells. It was also shown that the crude conditioned media contained autocrine growth factors, which could be extracted with 1M acetic acid. The extracts (AEs) stimulated the growth of the parental cells and NRK-49F cells almost as well as 5% calf serum and the extraction resulted in several-fold purification of mitogenic substances. These substances were not only specific to parental lines, but also stimulated growth of other transformed lines and normal NRK-49F cells. Extracts from the conditioned media of W12 and GCA cells intensified the rate of anchorage-independent growth in the concentration-dependent manner. In AE-W12, two peaks of mitogenic activity were detected (F1, F2) and similarly in AE-GCA (F3, F4). Fractions F2 (approximately 8 kDa), F3 (approximately 25 kDa) and F4 (approximately 12 kDa) were thermostable but F1 (approximately 45 kDa) was thermolabile. All four fractions were sensitive to trypsin and DTT treatment, and were acid-stable. Using ELISA kit it was shown that W12 and GCA cells released TGFbeta1 and GCA cells released very small quantities of bFGF. These results confirmed the autocrine regulation of growth in both cell lines.     

Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth medium supplemented with serum (3T3 system) or without feeder cells in a dedicated serum-free medium (EpiLife). During the culture, the cells were maintained either at ambient oxygen tension (20%) or at different levels of hypoxia (15, 10, 5, and 2% oxygen). The effect of oxygen on cell growth, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET).     

Calcitonin-responsive clonal cell line from porcine kidney (PK(15)) in serum-free medium

PK(15), a homogeneous epithelial cell line from porcine kidney which was originally established through single cell cloning from PK-2a, was found to respond to [Asu1,7]eel calcitonin with an increase in adenosine 3':5'-cyclic monophosphate (cAMP) content, but do not respond to parathyroid hormone or arginine vasopressin. These cells were able to grow in a synthetic medium (a 1:1 mixture of Dulbecco's modified Eagle's MEM and Ham's F12 medium) without any supplementary factor. The medium supplemented with selenous acid, transferrin, and insulin permitted a growth rate equivalent to those in serum containing medium. When grown in the serum-free defined medium, these cells showed an increase in cAMP content in response to [Asu1,7]eel calcitonin to approximately the same degree as in the serum containing medium (10% fetal calf serum). Our present study first indicates that PK(15) cells are capable of growing in the serum-free defined medium retaining the calcitonin responsiveness of the original cells.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Effects of Fetal Bovine Serum on Ferrous Ion-Induced Oxidative Stress in Pheochromocytoma (PC12) Cells

Ferrous ion (Fe²⁺) has been considered to be a cause of neuronal oxidative injury. Since body fluids contain protein and serum is an essential component of tissue culture medium, we have examined the role of serum protein on Fe²⁺-mediated oxidative stress using PC12 cells and rat cerebral cortices. Fe²⁺ or the combination of ascorbate and Fe²⁺ increased concentrations of thiobarbituric acid reactive substances (TBARS) in PC12 cells and cerebrocortical homogenates in medium (RPMI 1640), but did not increase TBARS when the medium was supplemented with 10% fetal bovine serum. Treatment with ascorbate/Fe²⁺ in serum-free medium reduced endogenous glutathione (GSH) concentration in PC12 cells. However, the medium supplemented with serum did not reduce GSH concentrations. PC12 cell death induced by ascorbate/Fe²⁺ was alleviated by increasing serum or bovine albumin concentrations in the medium. These observations indicated that oxidative injury caused by the transition metal ion could be lessened by adding fetal bovine serum to culture medium.     

Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Effects of low density lipoproteins and mevinolin on sympathetic responsiveness in cultured chick atrial cells. Regulation of beta-adrenergic receptors and alpha s

We have previously demonstrated that cultures of myocytes from embryonic chick atria grown in media supplemented with fetal calf serum from which lipoproteins have been removed demonstrate a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared with cells grown with control medium. This increased response to carbamylcholine was associated with a 1.4-fold increase in total cell cholesterol, a 2-fold increase in the number of muscarinic receptors which bind agonist with high affinity, and a 2-fold increase in the levels of the alpha subunits of Go and Gi (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In the studies reported here, we determined the responsiveness of cells grown in lipoprotein-depleted serum (LPDS) to beta-adrenergic stimulation. Isoproterenol stimulated a contractile response of 58% measured as an increase in amplitude of contraction with a half-maximal effect at 3 x 10(-7) M for cells grown in fetal calf serum, but had no significant effect on amplitude of contraction on cells grown in LPDS. In cells grown in media supplemented with fetal calf serum, isoproterenol (1 x 10(-3) M) stimulated adenylate cyclase activity 100% over basal with an EC50 of 7 x 10(-6) M compared with an increase of 32% in cells grown in media supplemented with LPDS. beta-Adrenergic receptor number as measured by the binding of 125I-pindolol decreased from 24 +/- 3 (+/- S.E., n = 6) fmol/mg protein in cells grown under control conditions to 12 +/- 2 (n = 6) fmol/mg protein in media supplemented with LPDS. The level of alpha s as measured both by ADP-ribosylation with cholera toxin in the presence of 32P-NAD and by immunoblotting with specific antibody to alpha s decreased by 3-fold in cells grown in media supplemented with LPDS compared with control. All of these effects of growth of cells in LPDS were reversed by incubating cells with LPDS plus 30 microM mevinolin, an inhibitor of endogenous cholesterol synthesis. These studies indicate that growth of cells in media supplemented with LPDS results in a coordinate decrease in the levels of beta-adrenergic receptors and alpha s. Taken together with our previous studies these data support the hypothesis that the receptors and guanine nucleotide-binding proteins which mediate sympathetic and parasympathetic responsiveness in the heart are reciprocally regulated.     

Factors influencing the release of proteins by cultured schwann cells

Cultured rat schwann cells grown in association with sensory neurons when labeled with [(3)H]leucinem, [(3)H]glucosamine, or [(35)S]methionine release labeled polypeptides into the culture medium. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of more than 20 polypeptides with molecular weights ranging from 15,000 to more than 250,000. Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for approximately 40 percent of the leucine or methionine radioactivity in medium polypeptide. Schwann cells grown in a serum-free defined medium, in which schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides tha cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides. Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE. This change in the pattern of polypeptides release by schwann cells is accompanied by changes in the association between schwann cells and axons. An early step in the establishment of normal axon-schwann cell relations appears to be an inward migration of schwann cells into axonal bundles and spreading of schwann cells along neurites. These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by schwann cells may be important for the development of normal axonal ensheathment.     

Establishment and characterization of a diploid cell line from the tick, Dermacentor parumapertus Neumann (Acarina: Ixodidae)

Establishment of a continuous cell line (RML-14) from embryonic tissues of the tick Dermacentor parumapertus Neumann is reported. The culture medium employed consisted of a combination (2:1) of Eagle's and L-15 (Leibovitz) media supplemented with 20% fetal bovine serum, 10% tryptose phosphate broth, and 0.1% bovine plasma albumin. At the 8th passage, 99% of dividing cells had the female chromosome complement, among which more than 70% had a diploid chromosome number of 22. At the 13th passage, cell population showed approximately a 3-fold increase during the first 8 days of culture. As of December 1976, had been subcultured 40 times.     

Rat pleural mesothelial cells in culture

Summary A culture system has been developed for long-term maintenance of rat pleural mesothelial cells. Mesothelial cells were isolated from the parietal pleura of rats and cultured in NCTC 109 medium supplemented with 10% fetal bovine serum. The cell explants attached to the dish and formed a confluent monolayer of polygonal cells within 10 to 15 days. Subcultures were made in the same medium. The mean population doubling time was approximately 30 hr. The ultrastructure of the mesothelial cells in culture was studied by light and electron microscopy and was compared with that of cells obtained from submesothelial components. This work was supported by CEE Grant No. 264 77 6 ENV F.     

Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment

In the present study in vitro expansion of human keratinocytes by supplementing dermal fibroblasts conditioned medium (DFCM) has been reported. Effect of two different DFCM acquired by culturing fibroblasts in keratinocyte-specific medium (defined keratinocytes serum free medium, DFCM-DKSFM) and fibroblast-specific serum free medium (F12: DMEM nutrient mix, DFCM-FD) have been compared. Growth kinetics of keratinocytes in terms of efficiency of cell attachment, expansion index, apparent specific growth rate and growth potential at the end of culture was evaluated in culture supplemented with DFCM-DKSFM and DFCM-FD in comparison with control i.e. DKSFM only. Results indicated that supplementation of DFCM caused significant increase in keratinocyte attachment. Efficiency of keratinocyte attachment in culture supplemented with bFCM-DKSFM was significantly higher compared to those cultured in DFCM-FD and DKSFM. In addition, the expansion index of keratinocytes in cultures supplemented with DFCM-DKSFM and DFCM-FD were 3.7 and 2.2 times higher than that of control condition even though the apparent growth rate and proliferative potential was found significantly lower. These results suggested that supplementation of DFCM enhanced expansion of keratinocyte by increasing efficiency of cell attachment, and DFCM-DKSFM provided suitable condition for in vitro expansion of keratinocytes compared to DFCM-FD and control condition.     

Application of hypoosmolar medium to fed-batch culture of hybridoma cells for improvement of culture longevity

Cell culture longevity in fed-batch culture of hybridomas is often limited by elevated medium osmolality caused by repeated nutrient feeding. Shotwise feeding of 10x Dulbecco's modified Eagle's medium (DMEM) concentrates elevated the osmolality of medium up to 540 mOsm/kg at the end of fed-batch culture of S3H5/gamma2bA2 hybridoma which is known to be lethal to most hybridomas. S3H5/gamma2bA2 hybridoma has been shown to grow without significant growth depression at 219 mOsm/kg in DMEM supplemented with 10% fetal bovine serum. To improve culture longevity in fed-batch cultures of S3H5/gamma2bA2 hybridoma, a hypoosmolar medium (223 mOsm/kg) was used as an initial basal medium. The use of hypoosmolar medium delayed the onset of severe cell death resulting from elevated osmolality and allowed one more addition of 10x DMEM concentrates to the culture. As a result, a final antibody concentration obtained was 121.5 microg/mL which is approximately 1.5-fold higher compared to fed-batch culture using a standard medium (335 mOsm/kg). When compared to batch culture, a more than 5-fold increase in the final antibody concentration was achieved. Taken together, the use of hypoosmolar medium as an initial medium in fed-batch culture improved culture longevity of S3H5/gamma2bA2 hybridoma, resulting in a substantial increase in the final antibody concentration.     

Prolactin augments luteinizing hormone binding to rat Leydig cells in serum-free culture

Effect of prolactin on the testicular luteinizing hormone binding was studied in a serum-free culture system. By the collagenase digestion of decapsulated testes taken out from 25-day-old rats, Leydig cells were isolated and cultured for 7 days in DME/F12 (1:1) medium supplemented with insulin, transferrin, epidermal growth factor, and gentamicin. The cultured cells exhibited the 3β-hydroxysteroid dehydrogenase activity. Hill plots constructed from the data of competition experiment showed that the dissociation constant (K_d) was 0.33 × 10^–10M. The K_d value was approximately the same as the known value for the rat testicular homogenates. When the Leydig cells were cultured with ovine prolactin for the last 3 days of 7-day culture period, the binding of luteinizing hormone increased to 1.7-fold ofthat in the control group. From these results it is concluded that prolactin acts to up-regulate the binding of luteinizing hormone to rat testicular Leydig cells in serum-free culture