Growth of fish cell lines in glutamine-free media

The glutamine requirement for thein vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and raibow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitz's L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for thein vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement.Abbreviations BSA bovine serum albumin - CHSE-214 Chinook samon embryo cell line - dFBS dialyzed fetal bovine serum - FBS fetal bovine serum - GFSk-S1 goldfish skin cell line - GS glutamine synthetase - L-15 Leibovitz's L-15 media - L929 mouse fibroblast cell line - MEM minimum essential medium Eagle - PBS phosphate buffered saline - RTL-W1 rainbow trout liver cell line - RTSp-W1 rainbow trout spleen cell line     

Development of chick embryo liver during organ culture: requirement for zinc-insulin

Livers from five-day chick embryos maintained as organ cultures on Eagle's minimum essential medium (MEM) develop an ultrastructure similar to more mature liver cells, except for glycogen deposits and the smooth endoplasmic reticulum (SER) normally associated with such deposits. The enzymes, glycogen synthetase and glycogen phosphorylase, failed to develop in these cells. The addition of zinc-free insulin (insulin-HCl) to MEM promoted the development of small amounts of SER in the cultured cells, as well as an increase in both glycogen synthetase and phosphorylase activities. The addition of zinc-insulin also stimulated an increase in the activities of both enzymes, and promoted the development of greater amounts of SER and the deposition of glycogen, as well. In addition, both forms of insulin not only prevented the fall of total tissue protein which occurs during organ culture on MEM, but also stimulated net protein synthesis in the explanted liver (Benzo and de la Haba, '71).     

Isolation and primary culture of Necturus maculosus (Amphibia: urodela) hepatocytes

Summary In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7×10⁵ viable hepatocytes per gram body weight with an average viability of 86±5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8°C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.     

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B₁ (AFB₁) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR₅₀ was 20 mM for DMN and 0.072 µM for AFB₁ in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 µ uM for AFB₁ with 48 fr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR₅₀ values were 100 mM DMN and 1.8 µM AFB₁ respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.Abbreviations NR Neutral Red - MEM Eagle's Minimum Essential Medium - DMN dimethylnitrosamine - AFB₁ aflatoxin B₁ - LDH lactate dehydrogenase - HBSS Hanks balanced salt solution; - EDTA ethylene bis (oxyethylenenitrilo)-tetraacetic acid - L-15 Leibovitz's 15 - NADH B-nicotinamide adenine dinu - FBS fetal bovine serum - IA immediate autopsy Contribution No. 2816 from Laboratory of Genotoxicology.     

COMPARISON OF NEURONAL AND LENS PHENOTYPE EXPRESSION IN THE TRANSDIFFERENTIATING CULTURES OF NUERAL RETINA WITH DIFFERENT CULTURE MEDIA*

The effects of three different culture media (Eagle's MEM, F-12 and L-15) on the transdifferentiation of 8-day chick embryonic neural retina into lens cells, were examined with respect to the expression of two phenotypes. One type referred to neuronal specificity (as represented by the level of cholineacetyl-transferase, CAT, activity) and the other to lens specificity (as represented by content of α-and δ-crystallin). In 7-day cell cultures before the visible differentiation of lentoid bodies, CAT activity was detected in all media. But, its level was about 9 times higher in cultures with L-15 than in those with MEM and 3 times higher than in F-12. In 26-day cultures, CAT activity was practically undetectable. The production of α-and δ-crystallin was detected in cultures at 26 days. There were quantitative differences in the crystallin content with different media, and it was highest in cultures with L-15. The results indicate that conditions most favourable to the maintenance of the neuronal specificity in cell cultures of neural retina, can also support the most extensive transdifferentiation. The possibility of direct transdifferentiation of once neuronally specified cells into lens cells in cultures with L-15 has been suggested to explain the present results.     

In vitro differentiation of myoblasts from skeletal muscle of rainbow trout

Substrata, plating densities and tissue culture media were compared for their effects on the proliferation and differentiation of myoblasts from skeletal muscle of rainbow trout. Mononuclear cells were isolated from the lateralis muscle of 4–11-month-old trout and plated on to glass coverslips coated with fibronectin, laminin or Matrigel. Cell proliferation was estimated by determining the density of nuclei on successive days in culture, and myoblast differentiation was detected by immunostaining cultures with the myosin-specific monoclonal antibody MF20. Mononuclear cell proliferation was highest for cells cultured on fibronectin or laminin and lowest for cells cultured on Matrigel, but the total number of nuclei in myosin-positive cells did not differ between substrata. The percentage of nuclei in myosin-positive myocytes and myotubes was significantly higher for cells cultured on Matrigel. The proportion of cells adhering to Matrigel and undergoing differentiation increased with plating density. Of three media tested, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 (RPMI), Leibovitz's L-15 (L-15) supplemented with 1 or 10% fetal bovine serum (FBS), a significantly greater proportion of the myoblasts differentiated when cells were cultured in L-15+ 10% FBS. These results suggest that culturing trout muscle-derived cells on a substratum of Matrigel at a high density and maintaining cells in L-15+ 10% FBS provide the conditions that maximize the proportion of cells that actively synthesize muscle myosin and facilitate trout myoblast differentiation in vitro .     

Culture conditions affect induction of vitellogenin synthesis by estradiol-17 beta in primary cultures of tilapia hepatocytes

In vitro synthesis of vitellogenin (VTG), a female-specific protein, after estradiol-17 beta (E(2)) treatment was compared among different culture conditions using the hepatocytes of tilapia, Oreochromis mossambicus. VTG was measured by enzyme-linked immunosorbent assay. Comparison of Leibovitz's L-15 medium (L-15), Williams' medium E (WE) and Medium 199 (M199), which have been used for hepatocyte cultures in certain teleost fishes, showed that monolayer formation of the hepatocytes on the plate in WE and M199 was faster than in L-15 at the beginning of the culture. Morphological differences in the hepatocytes among the culture media were not evident by 96 h after culture. VTG synthesis in L-15 after E(2) treatment was higher than in WE and M199. A concentration of NaHCO(3) at 5 mM in L-15 resulted in faster monolayer formation of the cells and higher VTG synthesis than at 0 and 23 mM. Primary culture of the tilapia hepatocytes at 28 degrees C showed higher synthesis of VTG than at 23 and 33 degrees C. These results suggest that nutritional requirements are vitally different among species, and there are optimal ranges in the pH and the temperature in cultured hepatocytes.     

The growth of L-cells and Vero cells on an autoclavable MEM-peptone medium.

The growth of L-60TM cells (a suspension culture adapted L-cell) on media composed of MEM (minimum essential medium (Eagle)) and bactopeptone autoclaved together or separately under a variety of conditions has veen determined. It has been found that MEM autoclaved with 0.5% bactopeptone at 15 psi for 20 min, cooled and then neutralized with NaHCO3, consistently supported good cell growth of L-60TM and L-929 cells. Similar results were obtained when the MEM and bactopeptone were autoclaved separately. The cells grew initially as a monolayer, subsequently becoming a stationary suspension. Some experiments were carried out with agitated suspension culture of L-60TM cells in the autoclaved MEM-bactopeptone combination with and without added methylcellulose and results were obtained which indicate that large scale suspension culture is possible in this system. Other peptones were also found to support cell growth. The autoclaved MEM-bactopeptone combination also supported the growth of Chang liver and Vero cells. The Chang liver cells rapidly dissociated from the plastic surface but the Vero cells remained sufficiently securely attached so that it was possible to grow them near to confluency in roller bottles.     

Interactions of indole acetic acid with EGF and FSH in the culture of ovine preantral follicles

The mechanisms that regulate the gradual exit of ovarian follicles from the non-growing, primordial pool are very poorly understood. The objective of this study was to evaluate the effects of adding indole acetic acid (IAA), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) to the media for in vitro culture of ovine ovarian fragments and determine their effects on growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed in Bouin (control). The other fragments were cultured for 2 or 6 days in culture plates with: minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, bovine serum albumine and antibiotics (MEM+); MEM+ plus IAA (40 ng/mL); MEM+ plus EGF (100 ng/mL); MEM+ plus FSH (100 ng/mL); MEM+ plus IAA+EGF; MEM+ plus IAA+FSH; MEM+ plus EGF+FSH; or MEM+ plus IAA+EGF+FSH. After 2 or 6 days of culture in each treatment, the pieces of ovarian cortex were fixed in Bouin for histological examination. Follicles were classified as primordial or developing (primary and secondary) follicles. Compared to the control, in all media tested, the percentages of primordial follicles were reduced (P<0.05) and the percentages of developing follicles were increased (P<0.05) after 2 or 6 days of culture. Furthermore, culture of ovarian cortex for 6 days reduced the percentages of healthy, viable follicles when compared with the control (P<0.05), except for cultures supplemented with IAA+EGF and EGF+FSH. In conclusion, the addition of IAA and EGF or EGF and FSH to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture.     

Hepatocyte function in long-term organ culture of Amphimuma means liver.

Fragments of liver from the adult urodele Amphiuma means, the Congo eel, were maintained in organ culture for up to 70 days. The normal electrophoretic patterns of several enzymes were retained. The activities of ornithine transcarbamylase, arginase, glutamate oxalacetate transaminase and glutamate pyruvate transaminase, and urea production, glucose uptake and tissue glycogen content remained relatively constant throughout the culture period. Histological organization and hepatocyte ultrastructure were also retained. Liver fragments survived better in media based on MEM or BME than in medium based on Leibovitz L15. Since many aspects of tissue-specific structure and function are retained, long-term amphibian organ culture is well suited to studies on the control of hepatocyte function and on the effects of metabolites, hormones, drugs and toxins.     

Effects of ascorbic acid on in vitro culture of bovine preantral follicles

Summary The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles.     

Immunogenetic analysis of microsomal and non-microsomal lipoproteins from normal and malignannt mouse tissues for histocompatibility-2 (H-2) antigens

The H-2 antigenic properties of lipoprotein fractions from malignant (L-5178Y leukemia) and normal (spleen, thymus, liver, kidney) mouse tissues have been studied by serological and immunological tests, and the results compared to the previously described activities of these fractions in homograft-sensitization tests. Although, in general, the relative activities in the different assays parallel each other some notable exceptions were found. The non-microsomal lipoproteins from leukemic tissue, inactive in homograft-sensitization tests, did elicit H-2 antibody. Also, the liver microsomal lipoproteins, which are inactive in homograft-sensitization tests in amounts 400 × the minimal effective doses of spleen preparations, exhibited, in in vitro agglutinin-inhibition tests, approximately one-fourth the H-2 activity of the latter. Other findings of note include the high antibody-eliciting potency of the spleen and leukemia microsomal lipoproteins (15 μg protein was sufficient to initiate primary immunization and 1 μg protein to cause an anamnestic response); and the quantitive identity of H-2 antigen activity of the microsomal lipoproteins from spleen and thymus.     

Impact of monocrotophos on protein and carbohydrate metabolism in different tissues of albino rats.

The impact of monocrotophos on protein and carbohydrate metabolism in different tissues of albino rats was investigated. The monocrotophos (0.25 mg/ml) was orally intubated into an experimental group of rats. In another group, the same amount of water was orally intubated (control group) for 29 days. The protein content was increased in liver, serum and spleen of albino rats after treatment with monocrotophos. The protein content decreased in muscle and kidney, and overall the free sugar level decreased in all tissues. The glycogen content increased in muscle, serum and kidney after treatment with monocrotophos, and the glycogen content and reducing sugar level decreased in liver and spleen. The significance of these results is discussed.     

Ascorbic acid supplementation to primary culture of chicken hepatocytes with non-serum medium

Chicken liver is lack of ascorbic acid biosynthesis system, different from mammals and highly evoluted birds. Chicken hepatocytes cultured without ascorbate was expected to have lower ascorbate amounts than physiological levels. Intracellular was decreased as compared with intact liver by cell preparation performed with in situ collagenase perfusion. We added ascorbate to a primary culture of chicken hepatocytes in order to restore the amount of ascorbate. Serum-free Leivobitz's L-15 medium which do not contain ascorbate was used for control medium. Cells were cultured with several concentrations of ascorbate for 24 or 48 h. After ascorbate supplementation for 24 to 48 h, cellular ascorbate concentration increased depending on the dose of medium ascorbate. Medium lactate dehydrogenase activity derived from hepatocytes, an index of cell injury, decreased upon 5-100 mg/l of ascorbate supplementation for 48 h. Tyrosine aminotransferase activity, an index of liver function, increased following culture with 50 and 100 mg/l ascorbate for 48 h. The activities, however, decreased by supplementation with 1000 mg/l of ascorbate. In conclusion hepatocytes lost intracellular ascorbate during preparation by in situ collagenase perfusion. Supplementation of ascorbate restored cellular ascorbate concentration, lowered cell injury and raised tyrosine aminotransferase activitv in primary cultured chicken hepatocytes. Ascorbate treatment for 48 h at 50 mg/l was the best combination in this study for primary culture of chicken hepatpcyte with non-serum L-15 medium     

Initiation of Cartilage Cell Culture from Skate (<Emphasis Type="Italic">Raja porasa</Emphasis> Günther)

Abstract Cartilage tissues from the proboscis of skate (Raja porasa Günther) were used to initiate primary cultures of cartilage cells. Aseptically dissected cartilage tissues were immersed in MEM medium free of fetal bovine serum (FBS), pH 7.6, and minced into small pieces (1 mm³ on average). After hydrolysis with collagenase II, hyaluronidase, and trypsin for 2 hours at room temperature, the acquired cartilage cells were rinsed twice with 20% FBS-supplemented MEM medium and then inoculated into 25-cm³ cell culture flasks, and incubated at 24°C. The primary cultures were initiated successfully, and the cartilage cells grew gradually into a confluent monolayer at day 10. Effects of growth factors were also tested in this study, and it was found that 20 ng/ml of basic fibroblast growth factor and 100 ng/ml of insulin-like growth factor II together had the most prominent stimulating effect on the growth and division of cartilage cells in the series of concentration combinations employed. The induced cartilage cells cultured formed a confluent monolayer at day 7.     

Co-culture of rabbit one-cell embryos with rabbit oviduct epithelial cells

Summary Rabbit 1-cell embryos were co-cultured with rabbit oviduct epithelial cells (ROEC) to determine if ROEC can enhance embryo development in vitro. Primary ROEC were cultured in serum-free media at 39°C in a 5% CO₂:95% air environment. In experiment 1, 1-cell embryos were co-cultured in Ham's F10 with freshly collected or 4-d-old cultures of ROEC seeded in plastic culture wells or on collagen membranes. One-cell embryos cultured without ROEC served as controls. After 65 h in culture, embryos were stained with Hoechst 33342 to determine the number of cells per embryo. Cell numbers were higher (P<0.035) in all co-culture treatments when compared to controls. Optimal development was obtained by co-culture with 4-d-old ROEC grown on plastic (P<0.003). In experiment 2, Ham's F10, Medium 199, and CZB with glucose medium were compared for their ability to support embryo development in the presence or absence of 4-d-old ROEC growth on plastic. Cell number and the percentage of embryos becoming blastocysts were significantly (P<0.002) higher for embryos cultured in Medium 199 compared to the other media tested. In Medium 199, co-culture with ROEC resulted in only a slight, nonsignificant increase in cell number over culture in Medium 199 alone (110 vs. 96 cells). However, the percentage of embryos reaching the blastocyst stage when co-cultured in Medium 199 with ROEC (49%) was nearly twice (P=0.01) that of embryos in Medium 199 without ROEC (26%). In experiment 3, transfer of embryos cultured in Medium 199 with or without ROEC of 24 or 48 h resulted in no significant differences in posttransfer development. These data indicate a beneficial effect of ROEC on blastogenesis and a salvage effect of ROEC on cell proliferation in embryos grown in a less supportive medium such as Ham's F10. This work was supported by a Multicenter Cooperative Program on Non-HumanIn Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development, NIH, Bethesda, MD, through Cooperative Agreement HD 21939.     

Differentiation of Striated Muscle Fibers in Monolayer Cultures of Rat Anterior Pituitary

Striated muscle fibers appeared in monolayer cultures of rat anterior pituitary cells maintained in αMinimum Essential Medium (αMEM). As muscle differentiation in cultures of pituitary cells under ordinary conditions has not hitherto been reported, an in vitro study was undertaken to determine what factor(s) is responsible for this myogenesis. When dispersed anterior pituitary cells were culrured in three different media, αMEM, Medium 199 and Dulbecco's Modified Eagle Medium (DMEM), only αMEM induced a high incidence of striated muscles. The nature of the serum (fetal calf, calf and horse) and its concentration (1–10%) did not affect myogenesis.
In monolayers in αMEM, the sequence of differentiation of striated muscle was as follows: 1) Elongated cells, resembling myoblasts appeared; 2) these cells fused; and finally 3) cross striations appeared. Rhythmic contraction was most intense in striated muscle fibers, but it was also obsrved in myotubes without cross striations and even in myogenic cells before fusion. The possible origin of muscles in these pituitary cultures is discussed.     

Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro

Abstract. Three different culture media, Ham's F-12, medium 199, and Eagle's minimal essential medium (MEM), were compared with respect to the expression of neuronal (choline acetyl transferase activity: CAT) and glial (hydrocortisone-induced glutamine synthetase activity; GSase) markers of normal differentiation in cultures of 9-day chick embryo neuroretinal cells, and also with respect to the accumulation of a lens marker (δ crystallin) during so-called 'transdifferentiation' in these cultures.
MEM allows transient expression of both CAT and GSase activities in early cultures, but also permits extensive δ crystallin accumulation at later stages. F-12 medium gives somewhat higher levels of CAT and GSase activities, the former being noticeably prolonged as compared with parallel MEM cultures; δ crystallin accumulation, however, is largely inhibited in F-12 cultures. By contrast, medium 199 permits only low levels of CAT and GSase activities, perhaps because the neuronal cells are distributed individually over the glial cell sheet in 199 cultures, rather than forming aggregates as in MEM or F–12 cultures. Medium 199 also blocks δ crystallin accumulation.
The results of medium changeover between 'transdifferentiation'-permissive (MEM) and non-permissive (199, F-12) conditions suggest: (a) that potential lens precursor cells (whatever their nature) survive in F-12 medium for prolonged periods without extensive expression of the lens phenotype; (b) that such precursor cells become committed to subsequent differentiation as lens cells between 10 and 20 days of culture in permissive MEM medium (as judged by the accumulation of δ crystallin following transfer into F-12); and (c) that medium 199 can block expression of the lens phenotype even in cells already committed (by the above criteria) to lens differentiation, as for instance after 30 days of preculture in MEM.     

Growth- and development-dependent expression of gangliosides in rat hepatocytes and liver tissues.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.     

Differential effects of culture media on normal and foreign differentiation pathways followed by chick embryo neuroretinal cells in vitro

Three different culture media, Ham's F-12, medium 199, and Eagle's minimal essential medium (MEM), were compared with respect to the expression of neuronal (choline acetyl transferase activity: CAT) and glial (hydrocortisone-induced glutamine synthetase activity; GSase) markers of normal differentiation in cultures of 9-day chick embryo neuroretinal cells, and also with respect to the accumulation of a lens marker (delta crystallin) during so-called 'transdifferentiation' in these cultures. MEM allows transient expression of both CAT and GSase activities in early cultures, but also permits extensive delta crystallin accumulation at later stages. F-12 medium gives somewhat higher levels of CAT and GSase activities, the former being noticeably prolonged as compared with parallel MEM cultures; delta crystallin accumulation, however, is largely inhibited in F-12 cultures. By contrast, medium 199 permits only low levels of CAT and GSase activities, perhaps because the neuronal cells are distributed individually over the glial cell sheet in 199 cultures, rather than forming aggregates as in MEM or F-12 cultures. Medium 199 also blocks delta crystallin accumulation. The results of medium changeover between 'transdifferentiation'-permissive (MEM) and non-permissive (199, F-12) conditions suggest: (a) that potential lens precursor cells (whatever their nature) survive in F-12 medium for prolonged periods without extensive expression of the lens phenotype; (b) that such precursor cells become committed to subsequent differentiation as lens cells between 10 and 20 days of culture in permissive MEM medium (as judged by the accumulation of delta crystallin following transfer into F-12); and (c) that medium 199 can block expression of the lens phenotype even in cells already committed (by the above criteria) to lens differentiation, as for instance after 30 days of preculture in MEM.