NuSerum,a synthetic serum replacement,supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

Summary In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23–24 chick embryos and plated at various densities (1, 5, 10, or 20 × 10⁶ cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [³H]leucine and [³H]thymidine. Chondrogenesis was determined by the incorporation of [³⁵S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 × 10⁶ cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 × 10⁶ cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [³⁵S]sulfate (2.6-fold), [³H]leucine (1.4-fold), and [³H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 × 10⁶ cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV. The observed effects of NuSerum and NuSerum IV may be attributed to a combination of factors, including lower concentrations of serum and its associated proteins, as well as supplemented growth factors and hormones known to promote cell proliferation and differentiation. Therefore, NuSerum and NuSerum IV are excellent, low-cost replacements for FBS in maintaining cellular growth and promoting chondrogenesis in LB mesenchymal cell cultures in vitro.     

Duct,exocrine, and endocrine components of cultured fetal mouse pancreas

Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10% bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin. When 10⁻⁶ M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10⁻⁸ M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e., duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method. This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute.     

Effects of cytochalasin B on pancreatic acinar cell structure and secretion

Summary The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.Supported by NIH grant GM-19998 from the United States Public Health Service. — We are indebted to Dr. John Heuser for advice throughout this project and assistance in the preparation of rapidly frozen acini, and to M. Lee for technical assistance     

Primary cultures of rat pancreatic acinar cells in serum-free medium

Summary Rat pancreatic acinar cells were isolated and cultured in Ham's F12 medium with 15% bovine calf serum. Caerulein, insulin, somatostatin, and dexamethasone (DEX) had no effect on intracellular or secreted amylase in these cultured cells. A serum-free medium, using Waymouth's MB 752/1 supplemented with albumin, epidermal growth factor (EGF), DEX, and HEPES, was then developed to avoid serum factors that might mask hormonal effects. In this SF medium, pancreatic acinar, cells maintained the morphological and ultrastructural characteristics of freshly isolated cells and secreted amylase in response to the secretagogue, carbamyl choline. Insulin, at a concentration of 1 μg/ml, significantly increased intracellular and secreted amylase activity after 3 d. This model cell system can be used to study the regulation of the synthesis of amylase and other pancreatic enzymes in vitro.     

Induction of cytochrome P-450-dependent hydroxylases in primary monolayer cultures of rat hepatocytes

Hydroxylation of androstenedione was studied in rat hepatocytes in primary monolayer culture following induction with phenobarbital. Six days after addition of phenobarbital and seven days after isolation of cells from liver, a maximal induction of total androstenedione hydroxylation of 5–6 times was seen at a phenobarbital concentration of 1·10⁻⁴ M. The 6β-, 7α- and 16α-hydroxylase activities showed different responses towards phenobarbital in agreement with the contension that different forms of cytochrome P-450 with different sensitivity towards phenobarbital participate in hepatic steroid hydroxylation. These results were obtained with cells supplemented with 1% (v/v) rat serum. The present cell culture system should be suitable for in vitro studies on mechanisms of induction of cytochrome P-450-dependent enzymes in normal liver cells.     

Factors contributing to the in vitro development of Ascaris suum from second-stage larvae to mature adults

When a three-step roller culture system was used, second-stage larvae of Ascaris suum, artificially hatched from eggs, developed in high numbers to the fourth stage, and a few to young and mature adults. The culture system consisted of (1) Medium KW-2 supplemented with 10 mM L-cysteine for the first 4 days, and with 5 mM L-cysteine for the following 7 days; (2) followed by Medium API-18 for 7 days; and (3) thereafter, by Medium API-1 supplemented with hemin (bovine) at a concentration of 24 micrograms/m1. Cultures were gassed with 95% nitrogen-5% carbon dioxide for the first 4 days and 85% nitrogen-5% oxygen-10% carbon dioxide thereafter, and incubated at 39 C. Two mature females that produced unfertilized eggs and a mature male with spermatozoa were the most advanced stages attained. The mature females were obtained in 67 and 73 days; and the largest female measured 110 mm. The latter produced 1,356,000 unfertilized eggs, from days 67 to 125. The mature male was obtained in 80 days; it measured 77 mm long and had paired spicules that were 1.5 mm long. Development of A. suum in three other culture systems showed that deletion of Medium API-18 or its substitution by Medium KW-2 limited development to late fourth stage and early, young adults, respectively; and the use of Medium API-1 without hemin limited development to early fourth stage.     

Mouse pancreatic acinar/ductlar tissue gives rise to epithelial cultures that are morphologically,biochemically, and functionally indistinguishable from interlobular duct cell cultures

Summary Most of the pancreatic exocrine epithelium consists of acinar and intralobular duct (ductular) cells, with the balance consisting of interlobular and main duct cells. Fragments of mouse acinar/ductular epithelium can be isolated by partial digestion with collagenase and purified by Ficoll density gradient centrifugation. We investigated whether previously developed culture conditions used for duct epithelium would result in the selective survival and proliferation of ductular cells from the acinar/ductular fragments. The fragments were cultured on nitrocellulose filters coated with extracellular matrix. After 2 to 4 wk the filters were covered with proliferating cells resembling parallel cultures of duct epithelium by the following criteria: protein/DNA ratio, light and electron microscopic appearance, the presence of duct markers (carbonic anhydrase [CA] activity, CA II mRNA, the cystic fibrosis transmembrane conductance regulator), the near absence of acinar cell markers (amylase and chymotrypsin), a similar polypeptide profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the presence of spontaneous and secretin-stimulated electrogenic ion transport. Both duct and ductular epithelia formed fluid-filled cysts in collagen gels and both could be subcultured. We conclude that acinar/ductular tissue gives rise to ductular cells in culture by some combination of acinar cell death and/or transdifferentiation to a ductular phenotype, accompanied by proliferation of these cells and preexisting ductular cells. These cultures may be used to investigate the properties of this part of the pancreatic duct system, from which most of the pancreatic juice water and electrolytes probably originates.     

In vitro secretion of zymogens by bovine pancreatic acini and ultra-structural analysis of exocytosis

The aim of this study is to establish a bovine pancreatic acinar cell culture model with longer viability and functionality. The cells could be maintained in a functional state for upto 20 days with normal morphology. Cells were positive for amylase as observed by immunofluorescence staining. Acinar cells are spherical and range about 2–3 µm in diameter. The porosome formed by exocytosis and heterogenous enzyme granules of size ranging 100–300 nm were seen on the surface of cells by electron microscopy. The activity of the enzymes was high on day 15 and the activity profile of the enzymes is in the order: protease>lipase>amylase and the enzymes were identified by SDS-PAGE. Long-term culture of bovine pancreatic acini could be useful in studying the pathogenesis of pancreatitis. Since the bovine genome shares about 80% identity with the human genome, the cells derived from bovine pancreas can be engineered and used as a potential xenotransplant to treat conditions like pancreatitis as the tissue source is abundantly available.     

Lack of pancreatic [3H]estradiol-binding activity in cystic fibrosis

A major characteristic of cystic fibrosis (CF) is the progressive degeneration of acinar cells in the pancreas. It is now well established that the normal pancreas contains an abundant amount of an [3H]estradiol-binding protein. Although the physiological function of this protein is unknown, available evidence suggests that it modulates resting secretion from acinar cells. Analysis of pancreatic autopsy samples from 13 patients who had CF demonstrated a high degree of correlation between loss of acinar cells and loss of [3H]estradiol-binding activity. Autopsy samples taken from individuals unaffected by CF contained large amounts of the [3H]estradiol-binding protein that were significantly correlated with the tissue content of amylase. This biological model demonstrates that the [3H]estradiol-binding protein in pancreas is localized primarily within acinar cells. Based on the presumed regulatory role this protein has on pancreatic secretion, an hypothesis is offered to account for acinar cell degeneration in individuals suffering from cystic fibrosis.     

Growth of exocrine acinar cells on a reconstituted basement membrane gel

Summary Methods have been developed for culturing a dividing population of morphologically differentiated rat parotid, lacrimal, and pancreatic acinar cells in vitro. Isolated acinar cells were plated onto tissue culture dishes coated with a three-dimensional, reconstituted basement membrane gel. After attachment in Ham’s nutrient mixture F12, the cells were cultured at 35°C in F12 supplemented with 10% heat inactivated rat serum, epidermal growth factor, dexamethasone, insulin, transferrin, selenium, putrescine, reduced glutathione, ascorbate, penicillin, streptomycin, and the appropriate secretagogue. Under these conditions, the cells attached rapidly and DNA synthesis was initiated within 2 to 3 d. Although the cells flattened on the substratum, they continued to maintain their differentiated morphology. The cells contained secretory granules, and the secretory enzymes peroxidase and amylase could be detected. The use of a reconstituted basement membrane gel proved critical for the attachment and growth of exocrine acinar cells.     

A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype

The development of in vitro models able to support the long-term viability and function of acinar cells is critical for exploring pancreatic pathophysiology. Despite considerable efforts, no long-term culture models for non-transformed pancreatic acini exist. Our aim was to develop and validate culture conditions for this purpose. An explant outgrowth culture design was established in which mouse pancreatic explants were cultured at the gas-liquid interphase. An enriched culture medium, pH 7.8, was employed to promote the selective outgrowth of acinar cells and to support their differentiated phenotype. After 7 days, the outgrown primary acinar cells were subcultured and maintained up to an additional 7 days as secondary monolayers on tissue culture plastic. Measurements of basal and caerulein-induced amylase secretion, phase-contrast microscopy and immunohistochemical analyses were used to characterize the cultures. Explants retained their pancreatic cytoarchitecture for 2 days in vitro. A triphasic dose response to caerulein was detected in 7-day primary cultures. The maximal rate of secretion was 1.2-fold versus basal (p=0.009) and 1.7-fold versus 1 pM caerulein (p=0.014). In secondary cultures the response was biphasic with maximal rates of secretion being 1.9-fold in 3- to 4-day cultures at 0.01 nM (p=0.049) and 2-fold in 6- to 7-day cultures at 0.1 nM (p=0.003). The present culture model provides a means to obtain functionally competent normal mouse acinar cells for long-term in vitro experimentation.     

Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen

Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n = 14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 μg/ml bovine pituitary extract, 5 μg/ml insulin, 10 ng/ml cholera toxin, and 50 μg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 μg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p < 0.05, n = 24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p < 0.05, n = 24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p < 0.05, n = 16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p < 0.05, n = 16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.     

Enhancement of hatching and trophoblastic outgrowth by mouse embryos cultured in Whitten's medium containing plasmin and plasminogen

Mice were induced to superovulate and 2-cell embryos were cultured in Whitten's medium with 10 mg bovine serum albumin/ml (WM) as control, Medium WM with 2.3, 4.6, 23.1 or 46.2 micrograms plasmin/ml, Medium WM with 14.6, 29.1 or 145.7 micrograms plasminogen/ml, Medium WM with 0.1, 0.2, 1.1 or 2.2 micrograms trypsin/ml; Medium WM with 0.2, 0.3, 1.6 or 3.3 micrograms pronase/ml and Medium WM with 10% heat-treated bovine serum (HTBS). Proteolytic activities in the culture media were evaluated at the start of the culture period and 10 days later. Blastocyst formation was significantly reduced in cultures supplemented with pronase and in the two higher levels of trypsin when compared to that in Medium WM. More embryos developed to the blastocyst stage in Medium WM + 2.3 or 23.1 micrograms plasmin/ml and Medium WM + 14.6 micrograms plasminogen/ml than in Medium WM (P less than 0.05). The incidence of hatching was significantly greater in Medium WM than in all plasminogen- and plasmin-supplemented media except for Medium WM + 29.1 micrograms plasminogen/ml. Although not significantly different, hatching was lower in Medium WM and Medium WM + 0.1 microgram trypsin/ml when compared to Medium WM + HTBS. Similar numbers of embryos completed the hatching process in Media WM, WM + 0.1 or 0.2 micrograms trypsin/ml and WM + 0.3 micrograms pronase/ml. Since dissolution of the zona pellucida occurred within 96 h for embryos cultured in Media WM + 1.6 or 3.3 micrograms pronase/ml and WM + 1.1 or 2.2 micrograms trypsin/ml, hatching could not be evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and monolayer culture of human fallopian tube epithelial cells

Summary In the present study we have established a pure monolayer culture system of human fallopian tube epithelial cells. The cells were isolated using collagenase digestion, and were cultured in Medium 199 supplemented with 15% fetal bovine serum. The epithelial cells derived from primary and secondary culture were characterized using immunocytochemical staining and electron microscopy. The cells continued to grow for 2 to 3 wk once the monolayer culture of the cells was established. It is currently possible to maintain the cultures until the third generation. Proliferation of these cells was enhanced by epidermal growth factor but not by basic-fibroblast growth factor, insulin, transferrin, estradiol-17β, or progesterone. This culture system offers a good model for determining characteristics of the tubal epithelium and would permit effective study of co-culture with embryos.     

Impairment of pancreatic acinar function by reserpine in vivo and in vitro

Summary Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired pancreatic secretion, and decreased pancreatic content of amylase. The mechanisms of altered acinar function and decreased amylase content in both CF and the reserpine-treated rat are unknown. To examine this alteration, the rate of [³H]phenylalanine (phe) incorporation into cellular protein was determined in pancreatic acinar cells after reserpine treatment of rats in vivo (7 d) and of cells in vitro (1 to 24 h). Acinar cells isolated from control, chronic reserpine-treated, and pair-fed rats were incubated in vitro with 0, 30, 50, or 100 μM reserpine. Reserpine treatment in vitro for 24 h of acinar cells from control rats significantly decreased amylase activity (20 to 70%), an effect similar to that of reserpine treatment in vivo. In vivo, reserpine treatment decreased [³H]phe incorporation (disintegrations per minute per milligram protein) 56% in freshly isolated cells, but did not alter intracellular specific activity (disintegrations per minute per nanomole phe, SA) of [³H]phe. Reserpine treatment (30 and 50 μM) in vitro for 1 h also decreased [³H]phe incorporation by freshly isolated cells from control (53 to 85%) and pair-fed (40 to 68%) rats. Reserpine treatment for 24 h in vitro significantly decreased [³H]phe incorporation by cells from control (82 and 98%), pair-fed (80 and 95%), and chronic reserpine-treated (90 and 97%) rats as compared with cells from respective in vivo treatments cultured with no reserpine. In vitro reserpine treatment also decreased the intracellular SA of [³H]phe in freshly isolated cells from control (14 and 36%) and pair-fed (35 and 39%) rats and in cultured cells from control (11 and 86%), pair-fed (60 and 88%), and chronic reserpine-treated (49 and 76%) rats. However, these alterations of SA by reserpine did not account for the decreased incorporation of [³H]phe into acinar protein, which remained significantly lower (70 to 88%) when expressed as total phe incorporation. These results suggest (a) that reserpine acts directly on acinar cells to alter function and (b) that the ability of the pancreas to synthesize digestive enzymes may be impaired in this model of cystic fibrosis. This study was supported in part by the Cystic Fibrosis Foundation, Bethesda, MD.     

Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William’s E Medium followed by Hepatocyte Differentiation Inducer Treatment

BackgroundHepatocyte differentiation inducer (HDI) lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.ResultsExpression levels of α-feto protein (AFP) were higher in cells cultured in WE and in Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham (DF12). 201B7 cells expressed the highest AFP and albumin (ALB) when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation) and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition).Conclusion201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.     

Differentiation of embryonic mouse GH cells and ACTH cells in vitro

The ability of cells that produce growth hormone (GH) and cells that produce adrenocorticotropic hormone (ACTH) to differentiate in various culture media was analyzed by means of ultrastructural immunocytochemistry on 13-day embryonic mouse pituitaries that were maintained in organ culture for 3-11 days. At the time of culture, relatively undifferentiated nongranulated or poorly granulated cells that were unreactive with anti-growth-hormone serum (anti-GH) and anti-adrenocorticotropic-hormone serum (anti-ACTH) were present in the pituitary. After 10-11 days in culture, immunoreactive GH cells were obtained only in media that were supplemented with cortisol, whereas ACTH cells were obtained in all media tested, including Medium 199 alone. In cortisol-supplemented media, the GH cells showed ultrastructural features typical of those that occur in vivo, and anti-GH immunoreactivity was obtained after as little as 3 days in culture, i.e., at a stage comparable to that which occurs in vivo. The results indicate that mouse GH cells are capable of differentiating in Medium 199 supplemented only with cortisol, without the addition of fetal calf serum or insulin; cortisol therefore appears to be an essential component of the embryonic milieu for the production of GH-secretory granules.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Proliferation of chick primordial germ cells cultured on stroma cells from the germinal ridge.

Fluorescent reagent-labelled PGCs isolated from the blood of 2-day-old chick embryos were cultured on stroma cells derived from 5-day-old germinal ridge in Medium 199 supplemented with 10% FBS, human IGF-1, bovine FGF-b, and murine LIF. In 7 experiments, the number of MCs increased by an average of 4.8 fold in 4 days. Intrinsic PGCs in the 5-day embryonic germinal ridge were observed loosely attached to the stroma cells, and they also increased 3.8 fold during culture for 4 days. These results indicate the possibility of applying this culture method to the production of transgenic chickens.     

Maternal dietary n-3 fatty acids alter cardiac ventricle fatty acid composition, prostaglandin and thromboxane production in growing chicks

The effects of feeding n-6 and n-3 fatty acids to broiler hens on cardiac ventricle fatty acid composition, and prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) production of hatched chicks were investigated. Fertile eggs obtained from hens fed diets supplemented with 3.5% sunflower oil (Low n-3), 1.75% sunflower+1.75% fish oil (Medium n-3), or 3.5% fish oil (High n-3) were incubated. The hatched chicks were fed a diet containing 18:3 n-3, but devoid of longer chain n-6 and n-3 fatty acids for 42 days. Arachidonic acid content was lower in the cardiac ventricle of High n-3 and Medium n-3 compared to Low n-3 birds for up to 2 weeks (P<0.002). Long chain n-3 fatty acids were higher in the cardiac ventricle of chicks from hens fed High and Medium n-3 diets when compared to chicks from hens fed the Low n-3 diet. Differences in long chain n-3 fatty acids persisted up to four weeks of age (P<0.001). Peripheral blood mononuclear cells (PBMNC) of 7-day-old High n-3 broilers produced significantly lower PGE2 and TXA2 than PBMNC from Low n-3 and Medium n-3 birds. These results indicate that maternal dietary n-3 fatty acids increases cardiac ventricle n-3 fatty acids while reducing arachidonic acid and ex vivo PGE2 and TXA2 production during growth in broiler chickens.