Selection of a chemically defined medium for culturing fetal mouse small intestine

Summary We evaluated six commercially available tissue culture media in their capacity to support villi morphogenesis and enterocyte differentiation during duodenal development of the fetal mouse in vitro: McCoy's 5A, Medium 199, Swim's S77, Trowell T8, Leibovitz L-15, and RPMI-1640. The duodenal segments were resected at 15 d gestation, before the formation of intestinal villi. In the segments cultured with the first four media, no villi differentiated even at 72 h culture. The number of epithelial cells per transverse section of the explants did not increase at 24 h and thereafter the number of epithelial cells decreased, except with McCoy's 5A. With the Leibovitz and RPMI media, rudimentary villi differentiated at 24 h of culture and they attained their longest length at 48 h. With the RPMI medium, the number of epithelial cells doubled at 24 h of culture and with Leibovitz medium it doubled at 48 h. At the fine structural level absorptive cells remained poorly differentiated with all the media studied. Goblet cells were easily identified after 24 h culture; they had a well developed rough endoplasmic reticulum and numerous mucous granules. Endocrine cells differentiated in culture and they were loaded with secretion granules. It was concluded that the small intestine of the fetal mouse can be kept in organ culture for at least 72 h. Full maturation of absorptive cells seemed to require some additional factor(s) as they remained poorly differentiated with all the media studied. Because well differentiated endocrine cells were present in all the explants, it appeared that gastrointestinal hormones do not affect villi morphogenesis and absorptive cells differentiation. This investigation was supported by Grant MA-6069 from the Medical Research Council of Canada. Mr. P. A. Micheletti was supported by a studentship from the FCAC.     

Permissive effect of glutamine on the differentiation of fetal mouse small intestine in organ culture

The proximal third of the small intestine of 15-day-old mouse embryo can be cultured for 72 h at 37 degrees C. When Trowell-T8 medium is used, the integrity of the explants is maintained, but villi do not form and absorptive cells are poorly differentiated. However, when Leibovitz L-15 or RPMI-1640 medium is used, one can observe the formation of medium-sized villi, and absorptive cells in the explants are more differentiated. Since the chemical composition of T8 medium is quite different from that of the other two media, we decided to test the importance of two major differences, i.e., three amino acids and five vitamins, in order to find out which element(s) is necessary to permit the formation of intestinal villi. Subsequent testing demonstrated that the three amino acids are responsible for the effect on differentiation, and that glutamine is the only critical difference between T8 and the two other media. The results show that the addition of L-glutamine to T8 medium permits the formation of villi, the initiation of absorptive cell differentiation, an increase in DNA synthesis, and finally, an increase in the number of epithelial cells. These findings indicate that undifferentiated fetal mouse small intestine is able to express its phenotype in organ culture, even without any extrinsic regulatory influences, provided that L-glutamine is present at a sufficient level in the culture medium. The use of inhibitors indicated that L-glutamine may be essential as an energetic substrate and/or a precursor for glucosamine.     

Influences of dexamethasone on the maturation of fetal mouse intestinal mucosa in organ culture

The effects of glucocorticoids on the maturation of the fetal small intestinal mucosa have been studied using duodenal explants resected at 17 days of gestation and cultured in a serum-free medium in the presence or absence of dexamethasone (30-300 ng/ml). Dexamethasone (a) increases specifically alkaline phosphatase, maltase, trehalase and sucrase activities and (b) allows an accumulation of goblet cells along the villi at a faster rate than that occurring in utero. These results indicate that glucocorticoids influence directly the differentiation of absorptive cells and goblet cells in the small intestine during the fetal period.     

Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17beta-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 microg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.     

Mitogenic response to epidermal growth factor (EGF) modulated by platelet-derived growth factor in cultured fibroblasts

The mitotic effects of epidermal growth factor (EGF) were investigated in two cultured fibroblast lines, BALB/c-3T3 and C3H 10T1/2 cells. EGF (30 ng/ml) added to quiescent 3T3 cells in medium containing either platelet-poor plasma or 10(-5) M insulin caused only minimal increases in the percentage of cells stimulated to initiate DNA synthesis. In contrast, EGF acted synergistically with either insulin or plasma to stimulate DNA synthesis in quiescent cultures of 10T1/2 cells, although the maximum effects of EGF were measured at concentrations several-fold greater than those found in either serum or plasma. In either 3T3 or 10T1/2 cells a transient preexposure to platelet-derived growth factor (PDGF) caused over a 10-fold increase in the sensitivity to the mitogenic effects of EGF. It is therefore possible that a primary action of PDGF is to increase the sensitivity of fibroblasts to EGF, independent of whether EGF alone is found to be mitogenic.     

Requirement of serum components for the preservation of primordial germ cells in testis cords during early stages of testicular differentiation in vitro in the mouse

Mouse gonadal primordia were isolated from embryos on the 11th day of gestation and cultured in vitro. They developed into either testes or ovaries after 7 days of culture in Eagle's minimum essential medium (MEM) supplemented with horse serum, whereas they did not differentiate in MEM alone. We studied how serum components are required for testicular development in vitro. When gonadal primordia were cultured in MEM alone for the first 1-3 days and subsequently in MEM supplemented with serum, testis cords developed while germ cells disappeared or only a few remained in the testis cords. In contrast, when serum was present in the medium during the first day of culture and omitted thereafter, germ cells were retained within testis cords. These results suggested that some serum component(s) is specifically required by germ cells independent of testis cord organization. Of more than 10 serum components tested, low and very low density lipoprotein fractions increased the number of germ cells in testicular explants.     

Serial propagation of human ovarian surface epithelium in tissue culture

Most human ovarian cancers are thought to arise in the ovarian surface epithelium (OSE). The precise role of OSE in carcinogenesis has not been defined because no appropriate animal models for the study of this tissue exist and culture of human OSE has been limited to primary outgrowths. In this report, we describe conditions for serial cultivation of normal human OSE. Premenopausal ovarian tissue was obtained at surgery. OSE growth was compared in media MCDB 202, 199 and Waymouth's 752/1 (WM) supplemented with 5, 15, or 25% fetal bovine serum (FBS), with/without 20 ng/ml epidermal growth factor (EGF) and 0.4 micrograms/ml hydrocortisone (HC). The rate and extent of OSE outgrowths from explants in primary culture were greatest in either WM or 199/202 (1:1), supplemented with 15% FBS/EGF/HC. In early passage cultures, cell proliferation was most rapid and extensive in 199/202 with 15% FBS, EGF, and HC. In this medium, OSE cells were subcultured up to 10 times and underwent 20-25 population doublings over 5 weeks. The population doubling time during rapid growth was approximately 48 h. Seeding efficiencies of up to 53% and cloning efficiencies of up to 13% were obtained. Early passage OSE cells reversibly modulated from a slow growing, epithelial, intensely keratin-positive form in 199/202 medium lacking EGF/HC, to a rapidly proliferating, elongate, less keratin-positive form in medium with EGF/HC. OSE cells grown in WM/5-15% FBS were epithelial and near-stationary. Thus, culture conditions have been defined for ovarian carcinogen assays requiring either proliferating or stationary cell populations, and for further studies of the role of OSE in ovarian biology.     

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

Density dependent proliferation of human glia cells stimulated by epidermal growth factor.

Epidermal growth factor (EGF) isolated from mouse salivary glands, enhanced the multiplication and [³H]TdR incorporation of human normal glia cells in serum-free medium supplemented with human serum albumin. Optimal dose was 2 ng/ml for both dense and sparse cultures but dense cultures were stimulated by EGF to a much less extent than sparse cultures. Data are presented that make the possibility unlikely that the density dependent inhibition of the EGF response is due to depletion of EGF in the medium or a local, juxtacellular starvation for the factor.     

Stimulation of sugar uptake in cultured fibroblasts by epidermal growth factor (EGF) and EGF-binding arginine esterase.

Mouse epidermal growth factor causes a rapid increase in 2-deoxyglucose uptake in stationary phase mouse (3T3) cells or human fibroblasts. Maximum effect is approximately two fold over control levels for 3T3 cells and about 50% over controls for human fibroblasts. Maximum effect on 3T3 cells is seen about two hours after addition of 10 ng/ml EGF to the culture medium. Stimulation is easily measureable within the first fifteen minutes after addition of the hormone and may be detected at hormone concentrations as low as 0.1 ng/ml. The EGF-binding arginine esterase found associated with EGF in the mouse submaxillary gland causes an enhancement of the EGF effect. In serum-free medium, the EGF effect is still readily observed, but no enhancement by the esterase is seen. SV40 virus-transformed 3T3 cells show no effect on deoxyglucose uptake after addition of 10 ng/ml EGF to the culture medium, but a response may be demonstrated after these cells are incubated for 12 hours or more in serumless medium. EFG stimulates transport of 3-O-methylglucose in stationary phase 3T3 and human fibroblasts but no EGF stimulation of alpha-amino-isobutyrate uptake in 3T3 cells is seen under conditions is reproted to inhibit intracellular degradation of human EGF by human fibroblasts, does not diminish the EGF effect on deoxyglucose uptake in human fibroblasts.     

Changes in the starch content during organogenesis in in vitro cultured Begonia rex stem explants

Stem explants, excised from greenhouse-grown Begonia rex plants, were cultured on basal medium (T. Murashige and F. Skoog, Physiol. Plant. 15: 473–497, 1962) contained in sterile Petri dishes. The medium was supplemented with benzyladenine (0.1 mg 1⁻¹) naphthaleneacetic acid (0.01 mg 1⁻¹) and, according to experimental requirements, with either sucrose (3%) or mannitol (3%). Histochemical and biochemical examination of the starch content of the explant was carried out over several days. There was no starch deposition or organogenesis in tissue cultured on mannitol and carbohydrate-free growth medium. The most dramatic finding was the heavy accumulation of starch in tissue cultured on sucrose medium. This copious accumulation preceded any organ formation and was mainly in regions which ultimately gave rise to shoot primordia. The heavy build-up of starch preceding organogenesis was also observed when explants previously cultured on mannitol medium were transferred to medium containing sucrose. During shoot primordia development there was a decrease in the starch content of the cultured tissue indicating the utilization of the polyglucan in the organogenic process.     

Effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fusion of embryonic palates in serum-free organ culture using wild-type, EGF knockout, and TGFalpha knockout mouse strains

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor-alpha (TGFalpha) in the palate and affects proliferation and differentiation of medial epithelial cells. EGF knockout embryos are less susceptible to the induction of CP by TCDD. This study used palate organ culture to examine the hypothesis that EGF enables a response to TCDD. METHODS: The midfacial tissues from wild-type (WT), EGF knockout, C57BL/6J, and TGFalpha knockout embryos were placed in organ culture on gestational day (GD) 12. Palatal explants were cultured for 4 days in serum-free Bigger's (BGJ) medium with 0.1% dimethyl sulfoxide (DMSO) or 1 x 10(-8) M TCDD with or without 2 ng of EGF/ml, 1 or 2 ng of TGFalpha/ml. Effects on palatal fusion were evaluated on day 4 of culture. EGF levels in explants and medium were determined using Luminex technology. RESULTS: In serum-free, control medium, palates from all of the strains fused. EGF knockout palates cultured with TCDD (no EGF) fused, but those cultured with TCDD + 2 ng of EGF/ml failed to fuse (p < 0.05 vs. control or TCDD without EGF). TGFalpha knockout palates failed to fuse when cultured with TCDD + 2 ng of TGFalpha/ml. EGF levels increased in tissue and accumulated in the medium after 24 hr of culture. CONCLUSIONS: This study demonstrated that providing EGF to the palates of EGF knockout mice restored the response to TCDD. These studies support the hypothesis that the mechanism for induction of CP by TCDD is mediated via the EGFR pathway.     

Plant regeneration in <Emphasis Type="Italic">Arachis stenosperma</Emphasis> Krapov. and W. C. Gregory from roots and calluses derived from leaflets of <Emphasis Type="Italic">in vitro</Emphasis> plants

Seed explants of A. stenosperma were cultured on MS medium supplemented with 6-benzylaminopurine with the aim of rescuing nonviable accessions stored in seed bank conditions. The regeneration potential of leaf explants from in vitro plants derived from embryonic axes was studied by using whole leaflets and leaflet segments. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine and naphthalene acetic acid. Indirect organogenesis was observed in response to 6-benzylaminopurine, either alone or in association with naphthalene acetic acid, in both explant types. Media supplemented with naphthalene acetic acid as the sole growth regulator induced rhizogenesis in whole leaflets and leaflet segments, with subsequent shoot production directly from the roots.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles

Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.     

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.     

Phenotypic transformation of normal rat kidney cells in a growth-factor-defined medium: induction by a neuroblastoma-derived transforming growth factor independently of the EGF receptor

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth-factor-inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth-factor-defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth-factor-defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage-independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.     

Induction of the synthesis of the pregnancy-specific protein p70 in the endometrium by intramuscular injection of recombinant bovine interferon-alpha I1 in nonpregnant ewes.

We examined the effect of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1) or recombinant bovine trophoblast protein-1 (rbTP-1) on protein synthesis by endometrial explants from Day-13 cyclic ewes and studied the ability of rboIFN-alpha I1 injected i.m. to influence subsequent protein secretion by endometrial tissue explants. In Expt 1, ewes were injected with either 2 mg rboIFN-alpha I1 or vehicle alone at 12 h intervals beginning on Day 11 of the oestrous cycle and ending on the morning of Day 13; 8 h after the last injection, ewes were hysterectomized and endometrial explant cultures were prepared. Explants were cultured for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. For Expt 2, additional explants were prepared from Expt 1 controls. Explants were cultured in the presence of 0, 20 or 200 ng/ml of either rboIFN-alpha I1 or rbTP-1 for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. Secreted proteins were analysed by two-dimensional electrophoresis and fluorography. There was a marked enhancement of a 70 kDa acidic protein, p70, in explants cultured in the presence of rboIFN-alpha I1 or rbTP-1. This polypeptide is a product of the gravid uterine horn from Day 14 to Day 20 of pregnancy and is a useful marker of the action of interferon-alpha (IFN-alpha) on endometrium. Enhanced production of p70 also occurred in ewes injected i.m. with rboIFN-alpha I1.(ABSTRACT TRUNCATED AT 250 WORDS)