Serum-free Waymouth Research Articles

Hormonal effects on the regulation of hepatic heme biosynthesis.

Drug-induced porphyrin accumulation occurs in chick embryo liver cells maintained in serum-free Waymouth MD 705/1 medium. Addition of insulin and thyroxine to the medium results in a marked enhancement of porphyrin accumulation. The addition of hydrocortisone results in a further enhancement of porphyrine accumulation. Several agents which are reported to increase intracellular adenosine 3':5'-monophosphate (cAMP) levels, viz. glucagon, sodium fluoride, cAMP or its dibutyryl derivative, 3-isobutyl-1-methylxanthine and papaverine enhanced drug-induced porphyrin biosynthesis. On the other have, agents which are reported to decrease intra-cellular cAMP levels, viz. alloxan and imidazole, diminished drug-induced porphyrin accumulation. cAMP appears to enhance, but not to function as a "second messenger" in drug-induced porphyrin biosynthesis. Drug-induced porphyrin accumulation in chick embryo liver cells depend upon the insulin to glucagon ratio. A low level of porphyrin accumulation occurs at insulin to glucagon ratios similar to those found following glucose administration in vivo, suggesting a possible explanation for the therapeutic effect of glucose in hepatic porphyria. The 5 alpha A(A:B trans) and 5 beta H(A:Bcis) steroids are equipotent in inducing delta-aminolevulinic acid synthetase and porphyrin accumulation in chick embryo liver cells maintained in serum-free culture medium. Thus, there is no specific steric requirement for porphyrin-inducing activity in steroids.

Drug-induced porphyrin biosynthesis—XVIII. Effect of modulation of intracellular cyclic amp levels on drug-induced porphyrin biosynthesis in chick embryo liver cells maintained in serum-free medium

Chick embryo liver cells were maintained in serum-free Waymouth medium containing insulin and thyroxine. Several agents which are reported to increase intracellular adenosine 3': 5'-monophosphate (cyclic AMP) levels, viz. glucagon, sodium fluoride, cyclic AMP or its dibutyryl derivative, 3-isobutyl-1-methylxanthine and papaverine, enhanced allylisopropylacetanide (AIA)-induced porphyrin biosynthesis. The enhancement is particularly apparent at low doses of AIA. On the other hand, agents which are reported to decrease intracellular cyclic AMP levels, viz. alloxan and imidazole, diminished the ability of AIA to induce porphyrin accumulation. Adenosine, which is reported to inhibit cyclic AMP-dependent protein kinases, diminished the ability of AIA to induce porphyrin accumulation.

Drug-induced porphyrin biosynthesis—XVI. Effects of hydrocortisone, adenosine 3′:5′-cyclic monophosphoric acid, its dibutyryl derivative and a phosphodiesterase inhibitor on allylisopropylacetamide-induced porphyrin biosynthesis in chick embryo liver cells maintained in serum-free waymouth medium

Chick embryo liver cells were maintained in serum-free Waymouth medium containing insulin and thyroxine. Hydrocortisone (20.6 μM) enhanced allylisopropylacetamide (AIA)-induced δ-aminolevulinic acid (ALA) synthetase activity, as it was previously demonstrated to do in rat liver. It is likely that previous failures to observe a hydrocortisone effect in hepatic cell culture were due to the presence of hydrocortisone in serum added to the medium. Adenosine 3′: 5′-cyclic monophosphoric acid (cAMP; 0.3 mM) and its dibutyryl derivative (61 μM), although not essential for AIA induction of porphyrin biosynthesis, were observed to enhance the accumulation of porphyrins. In contrast, cAMP has been reported to be essential for AIA induction of ALA-synthetase activity in rat liver cell suspensions. The cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (135 μM), was shown to exert a similar effect on AIA-induced porphyrin biosynthesis as cAMP. It was concluded that cAMP enhances but does not function as a “second messenger” in AIA-induced porphyrin biosynthesis.

Chemically-defined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture

The effect on tissue differentiation and growth in vitro of certain of the factors implicated in collagen synthesis (ascorbic acid, alpha-ketoglutarate and oxygen) and the influence of hydrocortisone was studied using organ cultures of fetal mouse mandible as a mixed epithelial and connective tissue system. Using serum-free Waymouth's MB 752/1 chemically-defined medium, addition of high levels of ascorbic acid (300mug per ml), hydrocortisone (1mug per ml) and oxygen (95%) enhanced differentiation in a number of tissues, in particular skin and appendages, tooth germs and bone, while osteoid and dentine production were noticeable promoted. It is suggested that an essential aspect of media design for organ culture involves the incorporaation of collagen-promoting factors to the in vitro enviornment particularly with regard to the controlling role implicated for collagen in a variety of biological processess.

Drug-induced porphyrin biosynthesis—XV: Induction of porphyrin biosynthesis in chick embryo liver cells maintained in serum-free waymouth medium

Several drugs were shown to induce porphyrin accumulation in chick embryo liver cells maintained in serum-free Waymouth MD 705/1 medium. In this system, allylisopropylacetamide (AIA) was shown to increase δ-aminolevulinic acid (ALA)-synthetase activity 5-fold over control values. Addition of insulin to the medium resulted in enhanced porphyrin accumulation. Porphyrin accumulation in response to increasing doses of AIA, propylisopropylacetamide (PIA) and 3,5-diethoxycarbonyl-2,4,6-trimethylpyridine (Ox-DDC) was 3- to 4-fold greater in serum-containing than in serum-free medium. With 3,5-diethoxycarbonyl-1,4-dihydro-2.4.6-trimethylpyridine (DDC), however, porphyrin accumulation in response to increasing doses was slightly greater in serum-free than in serum-containing medium. In serum-free medium coproporphyrin accumulated in response to AIA and PIA, uroporphyrin in response to Ox-DDC. and a mixture of uro-, copro-, and protoporphyrin in response to DDC. The pattern of porphyrin accumulation in response to Ox-DDC and DDC was similar in serum-free and serum-containing medium. With AIA and PIA a mixture of uro-, copro- and protoporphyrin accumulated in serum-containing medium which contrasts with the accumulation of coproporphyrin alone in serum-free medium. It appears that porphyrin-inducing drugs might inhibit a variety of steps in the heme biosynthetic pathway.

Chick embryo liver cells maintained in serum-free waymouth MD 705/1 medium

Chick embryo liver cells maintained in serum-free waymouth MD 705/1 medium

The serial cultivation of suspended BHK-21/13 cells in serum-free Waymouth medium.

A simple medium system was developed to obtain growth of BHK-21 cells in shaker cultures in the absence of serum. These cells have now undergone over 80 serial passages in serum-free Waymouth medium and have been recovered from the frozen state after storage for over 1 month in medium containing 10% dimethyl sulfoxide (DMSO) and 1% bovine serum albumin (BSA). Various amounts of exogenous lipid in the form of sodium oleate were added to cultures of cells growing in serum-free Waymouth medium. Concentrations of 10-50 mug of sodium oleate/ml had no detrimental effects on the cells as measured by trypan blue uptake. Furthermore, the cells were serially passed ten times in the presence of 10 mug sodium oleate/ml. Depletion of calf serum from the growth medium and addition of known quantities of lipids to the system provides a means of revealing subtle changes in lipid synthesis and lipid turnover during cellular growth.