Partial differentiation of rat egg cylinders in serum-free and protein-free medium

Modified organ cultures of rat egg cylinders were grown for 2 weeks in Eagle's MEM without serum or with serum added at different times. Explant survival was decreased only in cultures grown for the entire 2 weeks in serum-free medium, whereas the explant growth was impeded in all but the cultures grown for 2 weeks in 50% MEM plus 50% serum. Differentiation of epidermis and cartilage in the cultures deprived of serum for the entire 2-week period was comparable to that in fully serum-supplemented medium, whereas other differentiated tissues were rare or absent. In explants cultivated without serum for only the first week, neuroblasts were scarce.     

Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [14C]glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.     

Growth and hemolysin production by Haemophilus pleuropneumoniae cultivated in a chemically defined medium

A chemically defined medium (CDM) has been developed which supports both growth and hemolysin production by Haemophilus pleuropneumoniae. Although the growth rate in stationary cultures was substantially slower in CDM than in trypticase soy broth plus 0.6% yeast extract (TSBYE) and slightly slower than in heart infusion broth (HIB), extracellular hemolysin activity in CDM was slightly higher than in HIB and 16-fold greater than in TSBYE. Maximum hemolytic activity was produced in CDM in early to mid log phase of growth. Hemolytic activity in sterile, cell-free culture supernatant fluids persisted for over 10 days at 4 degrees C and 3-5 days at 37 degrees C, but was completely destroyed at 56 degrees C after 30 min. Total hemolysin inactivation was also achieved in the presence of trypsin or pronase (10 units/mL), but no decrease in hemolytic activity was noted in the presence of DNase or RNase. Iron had little effect on the hemolytic activity in the early stages of growth. However, in the later stages of growth, iron had a pronounced effect with hemolytic activity decreasing as the iron concentration increased from 1 to 500 microM. None of these iron concentrations had any effect on the hemolytic activity when added directly to prepared cell-free culture supernatant fluids. The extracellular hemolysin produced by H. pleuropneumoniae in CDM appears to be a heat-labile protein the activity of which is influenced by iron at certain phases of growth.     

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.     

Endocytosis in guinea pig seminal vesicle epithelial cells cultivated in chemically defined medium

We have developed a chemically defined monolayer culture system for guinea pig seminal vesicle epithelial cells (SVEP). The cells appeared as a polarized monolayer with apical microvilli, tight junctions and desmosome-like junctions, and often dilated intercellular spaces. SVEP expressed epithelial-specific cytokeratins as detected immunocytochemically. Growth was obtained during the first week of culture. In this period, the cells were exposed to unconjugated horseradish peroxidase (HRP), a ricin-peroxidase conjugate (Ri-HRP), or cationized ferritin (CF). HRP was endocytosed without binding to the SVEP surface (fluid-phase endocytosis) and was found mainly in multivesicular endosomes and lysosomes. Ri-HRP and CF, however, were endocytosed following binding to the cell surface. Initially these markers were present in multivesicular endosomes, but later also in smaller tubular and vesicular endosomes, some Golgi-associated elements (but not Golgi stacks), and lysosomes. We conclude that our SVEP culture system may be useful in further studies, on e.g. hormonal regulation of endocytosis and other processes of importance for SVEP maintenance and modulation of the seminal fluid in vivo.     

Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium

By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum. The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi.     

Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Embryonic stem cell development in a chemically defined medium

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cell in vitro differentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported in Xenopus, due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possibly in vivo.     

Morphological and biochemical differentiation of limb bud cells cultured in chemically defined medium

When chick limb buds were isolated from stage 22–23 embryos and cultured in chemically defined medium “BGJb,” the explants grew and, after about 9 days, showed good chondrogenesis of recognizable cartilage segments. Cartilage-type proteoglycan (termed PCS-H) was not synthesized during early days of culture, but by Day 9, it became a major proteoglycan constituent of the tissue. Freshly dissociated limb bud cells, when plated as monodispersed cultures at a density of 7 × 10⁶ cells/ml of BGJb, did not undergo chondrogenic differentiation and, instead, assumed the appearance of unhealthy or degenerated cells. During 9 days of culture, even though proteoglycans were synthesized, they were nevertheless of much smaller molecular size than PCS-H. When limb bud cells were cultured as a pellet containing 7 × 10⁶ cells in 1 ml of BGJb, a more tightly packed aggregate of about 2 × 10⁶ cells appeared in an inner region of the pullet during the first 24 hr of culture, and by Day 12 the aggregate had differentiated into a cartilage nodule surrounded by a thin layer of what appear to be ectodermal cells. As the conversion of aggregate into cartilage nodule progressed, newly formed proteoglycans gradually became more like cartilage-type proteoglycans, and by Day 12 they had many chemical and physical characteristics similar to those of the proteoglycans isolated from fully differentiated cartilages. The results indicate that the initial association of limb bud cells is an important factor for the chondrogenesis in BGJb and further suggest that the tight binding of the cell surfaces to one another may directly or indirectly stimulate the mechanism of synthesis of cartilage-type proteoglycans.     

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.     

Functional integrity of fetal rat liver explants cultured in a chemically defined medium.

A technique is described for the rapid preparation of large numbers of fetal rat liver explants for the study of developmental changes. Uniform pieces (1 mm³) of liver at 16 and 20 days of gestation were cultured for 3 days in a chemically defined medium in the absence of serum or exogenous proteins. Explants of both gestational ages appeared morphologically normal at the end of 3 days. Net uptakes of glucose remained relatively constant over the culture period. There was an initial loss of protein and DNA which could be largely accounted for by loss of erythropoietic cells. After the first or second day in culture, the DNA content of the explants remained constant. The amount of nonsedimentable protein (nonerythrocyte) released into the medium was 16 to 20 μ per explant per day. Between 40 and 50% of this protein was identified as albumin and α-fetoprotein by immunologic and electrophoretic techniques. A minor protein component was identified as transferrin. These proteins, normally secreted into amniotic fluid, were released in amounts that markedly exceeded their concentrations in the explant. This release was blocked by the presence of cycloheximide in the culture medium. This technique provides a simple method for maintaining viable fetal liver explants in a chemically defined medium, and should be useful for studying metabolic development in antenatal liver.     

Lovastatin biosynthesis by Aspergillus terreus in a chemically defined medium

Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract.     

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...     

Morphological and functional properties of rat dorsal root ganglion cells cultured in a chemically defined medium

A culture procedure for dorsal root ganglion (DRG) cells is presented using a completely defined culture medium without antibiotics, in combination with mechanical dissociation procedures. This culture procedure allows all dorsal root ganglion cell types to be cocultured for periods of at least 106 days. Some of the dorsal root ganglion neurons, which could be identified by their neurofilaments and the presence of fluoride resistant acid phosphatase, regained their original T-cell appearance within two weeks. After one month in culture ganglion-like reaggregates appeared. Schwann cells, satellite cells and fibroblasts were identified using morphological criteria. All neurons tested maintained excitability during, at least, the first 35 days in culture, since in all cases action potentials could be evoked by current pulses. The method has proved to be useful in the study of morphological, cytochemical and electrophysiological aspects of dorsal root ganglion cell differentiation in vitro.     

Improved cryopreservation of domestic cat sperm in a chemically defined medium

The objective was to compare a proprietary egg yolk-based cryopreservation medium with a chemically defined soy-based medium, as well as to examine effects of temperature of glycerol addition on sperm parameters and IVF after freezing and thawing of domestic cat sperm. Semen was collected from adult cats (four males and three ejaculates per male), divided in four equal aliquots, and extended in either egg yolk with 4% glycerol added before (EYG) or after (EY) cooling to 5 °C, or soy-lecithin with 4% glycerol added before (SLG) or after (SL) cooling to 5 °C. Extended sperm were frozen in straws over liquid nitrogen vapor. Sperm progressive motility (%) and rate of progressive movement (scale of 0–5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation, acrosome integrity, and DNA integrity were assessed at 15 min post-thaw. Effects of media (EY or SL) on IVF success was also examined (three males and three ejaculates per male). Sperm motility was greater (P < 0.05) in soy-based compared with egg yolk-based media at 3, 6, and 24 h post-thaw. A higher (P < 0.05) percentage of noncapacitated sperm (pattern F) were present in soy-based (SLG, 63.7 ± 9.2%; and SL, 64.1 ± 9.2%) compared with egg yolk-based (EYG, 49.9 ± 7.9%; and EY, 52.4 ± 18.6%) cryopreservation media, regardless of temperature of glycerol addition. Addition of glycerol at 5 °C increased (P < 0.05) percentage of sperm motility at 6 h (EYG 16.3 ± 8.3% vs. EY, 24.0 ± 11.7%; SLG, 36.7 ± 6.5% vs. SL, 42.9 ± 10.1%) and 24 h (EYG, 2.1 ± 3.3% vs. EY, 8.3 ± 3.9%; SLG, 11.3 ± 8.3% vs. SL, 18.8 ± 7.4%) post-thaw in both media. There were no differences (P > 0.05) between cryodiluents in embryo cleavage, percentage of embryos reaching blastocyst, or cell number per blastocyst. The chemically defined, soy-based medium resulted in better preservation of long-term motility and capacitation status of frozen-thawed domestic cat sperm compared with a commercial egg yolk-based extender, without compromising fertilizing ability.     

Leakage and delivery of liposome-encapsulated methotrexate-gamma-aspartate in a chemically defined medium

A chemically defined medium was developed to study liposome-mediated delivery of methotrexate-gamma-aspartate to cells under conditions where dilute suspensions of negatively charged liposomes to not leak extensively. The defined medium induced 14% leakage of methotrexate-gamma-aspartate from egg phosphatidylglycerol/cholesterol (67:33) liposomes diluted to 53 nM lipid. In contrast, commercially available serum replacements induced up to 91% leakage from the same liposomes. The growth inhibitory properties of non-loaded phosphatidylglycerol liposomes were greater in the chemically defined medium that they were in medium supplemented with 10% serum. Egg phosphatidylglycerol, dioleoylphosphatidylglycerol and dilaurylphosphatidylglycerol liposomes inhibited cell growth more than dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol liposomes. In 10% serum, phosphatidylglycerol liposomes with widely varying phase-transition temperatures were nearly equally effective to deliver drug to CV1-P and L929 cells, despite great differences in liposome stability. Liposome encapsulated methotrexate-gamma-aspartate was more potent when the cells were grown in the defined medium, and the increase in drug delivery was observed from phosphatidylglycerol liposomes of different phase-transition temperatures. The minimum fraction of negatively charged phospholipid required for optimal liposome-mediated drug delivery varied between cell types and among growth media. The growth inhibitory effects of liposome-encapsulated methotrexate-gamma-aspartate was also determined under conditions where the cells were exposed to drug for periods shorter than the entire growth assay. Reduction of the exposure time decreased the potency of both encapsulated and free drug in medium containing 10% serum, and decreased the potency of free drug in the defined medium. However, the potency of encapsulated drug in the defined medium was similar for all exposure lengths between 1 and 48 hours.     

Antibacterial testing of metal ions using a chemically defined medium

Data from in-vitro tests on potential germicides can be greatly influenced by the culture medium. The bioavailability and biochemical reactivity of the biocides can be influenced by chemical interference with media components (Spooner & Sykes 1972). Bird et al . (1985) showed that metal ions are particularly prone to chemical interferences. A chemically defined solid medium has been developed to monitor the antibacterial activity of metal ions. The minimum inhibitory concentrations of zinc and silver have been determined against a range of bacteria using this medium.