Calf articular cartilage in organ culture in a chemically defined medium

In Vitro Cellular &; Developmental Biology - Plant - Articular cartilage from 6-month-old calves was maintained in organ culture in Eagle's minimum essential medium at different oxygen...     

Altered cartilage proteoglycans synthesized by chick limb bud chondrocytes cultured in serum-free defined medium

Chick high-density culture chondrocytes synthesize cartilage-specific proteoglycans with much structural similarity to the proteoglycans made by cartilage in vivo. Such cultures can be maintained in a defined medium formulated in this laboratory in which chondrogenesis occurs without the addition of serum. The proteoglycans synthesized by the chondrocytes in the presence of defined medium are of a cartilage-specific structure but differ in some aspects from the proteoglycans made in serum-containing medium. While their buoyant density, ability to aggregate with hyaluronic acid, and keratan sulfate chain size are unchanged, the proteoglycans synthesized in defined medium have altered chondroitin sulfate chains. This chondroitin sulfate is of significantly larger size and has a different sulfation pattern relative to that produced in serum-containing medium. The larger size of the chondroitin sulfate results in a larger monomer size of the defined medium proteoglycans. These differences have implications about the regulation of the structure of chondroitin sulfate proteoglycans.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Nitric oxide regulates steroid synthesis by bovine antral granulosa cells in a chemically defined medium

Nitric oxide (NO) in bovine ovary has been characterized as one of the controllers of granulosa cells’ (GC) steroidogenesis and apoptosis. One of the pathways used by NO to have these effects is cGMP. The objectives of the present study were to verify the effect of sodium nitroprusside (SNP), a NO donor, on steroidogenesis, cell viability (mitochondrial activity) and GC cell cycle distribution and if this effect occurs by the NO-cGMP signaling pathway with the addition of SNP with or without 1H-[1,2,3] oxadiaziolo[4,3a]quinoxaline-1-one (ODQ), a selective soluble guanylate cyclase inhibitor. The antral GC from 3 to 5 mm diameter cattle follicles was cultured without treatment (control), with ODQ (10⁻⁴ M) and 10⁻⁵, 10⁻³ and 10⁻¹ M SNP with or without ODQ for 24 h. Nitrate/nitrite (NO₃⁻/N0₂⁻) concentrations were evaluated by Griess method, progesterone (P₄) and 17β-estradiol (E₂) concentrations by chemiluminescence, viability and cell cycle stage by MTT method (3-[4,5-dimethylthiazol-2yl]-2,3 dipheniltetrazolium bromide) and flow cytometry, respectively. Nitrate/nitrite concentration in culture medium increased (P < 0.05) in a dose-dependent manner according to SNP concentration added to the culture medium. The GC cultured without treatment, with ODQ and with 10⁻⁵ M SNP in the presence or absence of ODQ developed into cell aggregates and did not vary in cell viability (P > 0.05), while GC cultured with 10⁻³ and 10⁻¹ M SNP with or without ODQ presented disorganized GC aggregates or did not develop into cell aggregates and also had substantially decreased cell viability (mitochondrial activity inhibition) and steroids synthesis (P < 0.05), and effects were not reversed with us of ODQ. Most GC cultured without treatment (control) or with ODQ, 10⁻⁵ and 10⁻³ M SNP with or without ODQ were in the G0/G1 (80–75%) stage and in a lesser proportion (20–25%) in the S + G2/M stage of the cell cycle, while the 10⁻¹ M SNP treatment resulted in GC in G1 phase arrest. The treatment with 10⁻⁵ M SNP increased (P < 0.05) E₂ synthesis and inhibited (P < 0.05) progesterone synthesis. The addition of ODQ reversed (P < 0.05) the stimulatory effect of 10⁻⁵ M SNP treatment on E₂, but not on P₄ synthesis (P > 0.05). These results demonstrated that E₂ synthesis by antral GC from small follicles is modulated by lesser NO concentrations via the cGMP pathway, but not P₄ while steroids inhibition cGMP pathway independent, mitochondrial damage and the interference on cell cycle progression caused by greater NO concentration can lead to cell death.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium.

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Fibroblast growth factor 10 regulates Meckel's cartilage formation during early mandibular morphogenesis in rats

Fibroblast growth factors (FGF) are pluripotent growth factors that play pivotal roles in the development of various organs. During mandibular organogenesis, Meckel's cartilage, teeth, and mandibular bone differentiate under the control of various FGF. In the present study, we evaluated the role of FGF10 in rat mandibular chondrogenesis and morphogenesis using mandibular organ culture and mandibular cell micromass culture systems. The overexpression of Fgf10 induced by the electroporation of an FGF10 expression vector not only altered the size and shape of Meckel's cartilage, but also upregulated the expression of the cartilage characteristic genes Col2a1 and Sox9 in a mandibular organ culture system. Meckel's cartilage was deformed, and its size was increased when Fgf10 was overexpressed in the lateral area of the mandible. Meanwhile, no effect was found when Fgf10 was overexpressed in the medial portion. In the mandibular cell micromass culture, recombinant FGF10 treatment enhanced chondrogenic differentiation and endogenous ERK (extracellular signal-regulated kinase) phosphorylation in cells derived from the lateral area of the mandible. On the other hand, FGF10 did not have significant effects on mandibular cell proliferation. These results indicate that FGF10 regulates Meckel's cartilage formation during early mandibular morphogenesis by controlling the cell differentiation in the lateral area of the mandibular process in rats.     

Cultivation of Pasteurella haemolytica in a chemically defined medium

A chemically defined medium was developed for the aerobic cultivation of Pasteurella haemolytica. Studies on the growth of strain H44L were conducted in a medium consisting of 15 amino acids, inorganic salts, citrate, nicotinamide, pantothenate, thiamine or thiamine monophosphate, and carbon sources. The amino acids were provided as l isomers, because racemic mixtures of some amino acids inhibited growth. The carbon source consisted of a mixture of 1.0% d-galactose and 0.1% d-glucose. Culture populations of strain H44L reached 2 x 10(10) cells per milliliter after 16 hr of incubation at 37.5 C. Other strains of P. haemolytica, from a wide variety of sources, were tested for growth in the medium, and 23 of 24 strains grew well. Five strains of P. haemolytica var. ureae failed to grow in the medium.     

Embryonic mouse lung morphogenesis and type II cytodifferentiation in serumless, chemically defined medium using prolonged in vitro cultures

The timing, position and mechanism(s) for determining type II cytodifferentiation during mammalian lung development are not known. To approach this problem, we have cultured Theiler stage 16 embryonic B10.A strain mouse lung primordia (12-days gestation, E12) in serumless, chemically defined medium in the presence or absence of dexamethasone (DEX) for periods up to 27 days in vitro. Morphogenesis and cytodifferentiation were evaluated by light and transmission electron microscopy and immunochemical techniques. Pulmonary surfactant-associated apoproteins (PSAP) were initially expressed by type II cells at 16.5-day gestation in vivo. DEX-supplementation to the culture medium resulted in the accelerated expression of PSAP; the apoprotein isoforms (A1, A2, and A3) produced in vitro were comparable to those synthesized during fetal and postnatal in vivo development by high resolution, two-dimensional gel electrophoresis coupled with immunoblot staining. Cultures without DEX produced PSAP A2 and A3 isoforms, but did not produce A1 (26-31 kDa, pI 5.2-5.3). DEX-treated cultures produced more lamellar bodies within type II cells than non-treated controls. The results demonstrate that long-term cultures of embryonic lung primordia express morphogenesis, cytodifferentiation and the synthesis and secretion of PSAP in the absence of exogenous hormones or growth factors. The data set further supports the hypothesis that morphogenesis and type II cytodifferentiation are regulated by autocrine and paracrine factors intrinsic to the embryonic lung developmental program and independent of exogenous hormone controls.     

The uptake of amino acids from a chemically defined medium byFusobacterium species

The uptake of amino acids from a chemically defined medium was determined for various species ofFusobacterium. Reference strains utilized a wider range and higher levels of amino acids than clinical isolates. Among the acidic and basic amino acids, arginine, histidine, and glutamate were used by most species. In general, nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, proline, and methionine were poorly utilized. Apart from glycine and tyrosine, the neutral but polar amino acids such as serine, cysteine, and asparagine were incorporated at significantly high levels. ThusFusobacterium species, unlike several Gram-negative anaerobic bacteria, have the capacity to utilize both free amino acids and peptides as energy sources and may partly account for the wide distribution of these species in areas of tissue degradation.     

A new diploid nontumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium

Summary A new, nontumorigenic human breast epithelial cell line, HMT-3522, has been established from fibrocystic breast tissue. Cells were explanted and propagated in chemically defined medium including insulin, transferrin, epidermal growth factor, hydrocortisone, estradiol, prolactin, and Na-selenite. The epithelial nature of the cell line was established by immunocytochemical detection of cytokeratins. Moreover, electronmicroscopy revealed monolayers of polarized cells connected by desmosomes and provided with apical microvilli. Milk fat globule membrene antigen, specific for the apical membrane domain of normal, luminal breast epithelial cells, was expressed only in confluent cultures where some cells overlaid others, indicating “stem cell”-like properties. After 25 to 30 passages, the cells are diploid with a few marker chromosomes and loss of chromosomes in the D-group. The cells are nontumorigenic in athymic mice; they lack estrogen receptors, and estradiol does not stimulate growth. The HMT-3522 cell line may represent a useful model for the study of brest cell differentiation and carcinogenesis in vitro. This work was supported by a grant from the Danish Cancer Society.     

The subunits of chemically treated proteoglycan isolated from bovine nasal cartilage

1. The light fraction of the proteoglycan of bovine nasal cartilage was split by treatment with 0.1m-hydrochloric acid in acetone. The products were separated by gel filtration on 4% agarose and two retarded fractions were detected and isolated. These two fractions were found to have a Stokes radius of 134 and 47 A respectively, as determined by calibration of the column against proteins of known hydrodynamic volumes. 2. The 47 A fraction had a protein content of 4% and a glucosamine/galactosamine ratio 1:23. The 134 A fraction had a protein content of 20% and a glucosamine/galactosamine ratio 1:4.8. 3. The results of the viscometric studies on both fractions suggested that the 134 A fraction alone exhibited the property of undergoing reversible pH-dependent aggregation with a transition point at pH4.9. 4. It was concluded that these fractions could represent subunits of the native cartilage proteoglycan.     

Explant culture of rat esophagus in a chemically defined medium

Summary Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, β-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [³H]thymidine into DNA and [³H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [³H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30° C, and in either 50 or 20% O₂. Ninety-five percent O₂ was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.     

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud

In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.     

Birth of piglets derived from porcine zygotes cultured in a chemically defined medium.

We evaluated the in vitro development of porcine zygotes that were cultured in a novel culture medium, porcine zygote medium (PZM), under different conditions and compared to in vivo development. The viability of these zygotes to full term after culture was also evaluated by embryo transfer to recipients. Porcine single-cell zygotes were collected from gilts on Day 2 after hCG injection. Culture of zygotes in PZM containing 3 mg/ml of BSA (PZM-3) produced better results in terms of proportion of Day 6 blastocysts, Day 8 hatching rate, and numbers of inner cell mass (ICM) cells and total cells in Day 8 embryos than that in North Carolina State University (NCSU)-23 medium. In culture with PZM-3, embryo development was optimized in an atmosphere of 5% CO2:5% O2:90% N2 compared to 5% CO2 in air. The ICM and total cell numbers in Day 6 embryos cultured in PZM-3 or in PZM-3 in which BSA was replaced with 3 mg/ml of polyvinyl alcohol (PZM-4) were also greater than those of NCSU-23 but less than those developed in vivo. However, no difference was found in the ratio of ICM to total cells among embryos developed in PZM-3, PZM-4, or in vivo. When the Day 6 embryos that developed in PZM-4 (99 embryos) or in vivo (100 embryos) were each transferred into six recipients, no difference was found in the farrowing rate (83.3% for both treatments) and in the number of piglets born (33 and 42 piglets, respectively). Our results indicate that porcine zygotes can develop into blastocysts in a chemically defined medium and to full term by transfer to recipients after culture.     

A chemically defined fermentation medium for the growth of Micromonospora purpurea

A chemically defined medium for Micromonospora purpurea has been devised, consisting of glucose, a nitrogen source, calcium carbonate, magnesium sulfate, dibasic potassium phosphate, and the required trace quantities of iron, copper, zinc, and manganese. Using washed cell inocula, dry mycelial weights of more than 16 mg./ml. were obtained in 7-day shaken-flask fermentations. Nutrient requirements for M. purpurea are discussed and growth data presented. Sucrose, maltose, starches and dextrins could be substituted for glucose, and resulted in good growth of the organism. A number of amino acids and inorganic nitrogen-containing compounds were capable of utilization as sole nitrogen sources. Weekly serial transfers of the culture in defined medium have shown no diminution in mycelial weights over a four-month period.