Explants In Organ Culture Research Articles

Spontaneous development of gluconeogenesis in fetal rat livers during incubation in organ culture.

Freshly isolated fetal liver explants in organ culture did not convert L-[14C]alanine or L-[14C]lactate to carbohydrate, but L-[14C]serine and D-[14C]glycerol were both transformed. When explants were subjected to 42 hr of preliminary incubation without supplements, followed by transfer to fresh medium with added precursor, all four substrates underwent gluconeogenic transformation. It was concluded that the ability of fetal rat liver in organ culture to convert alanine and lactate to carbohydrate evolves slowly, but the conversion of glycerol, and to a lesser extent serine, to glucose and glycogen is initiated immediately.

Hormone induction of specific protein synthesis in midpregnant mouse mammary cell culture

Monolayer primary cell cultures of midpregnant mouse mammary cells were subjected to hormone stimulation under strictly defined conditions. Hormonal response was measured in terms of increase in rate of synthesis of mouse casein, using a double antibody precipitation technique. Cells stimulated by insulin plus prolactin plus cortisol plus estradiol plus progesterone showed a marked increase in rate of mouse casein synthesis over the controls. This specific product synthesis, which remains inducible in these cells for at least ten days, was detected either by labelled phosphoric acid or labelled amino acid incorporation. The mouse casein synthesized was identical, as judged by the identification techniques used, to that of mouse milk. Mouse midpregnant mammary explants in organ culture require insulin plus prolactin plus cortisol to express their full lactogenic capabilities. Nevertheless, when the same cells are dispersed and grown in monolayers they require ovarian steroids to elicit a lactogenic response as shown in this study. Ovarian steroids, therefore, are necessary in lactogenesis, although the fundamental nature of their action remains to be established.

The use of gelatin capsules in mounting organ culture explants for cryostat sectioning

The use of gelatin capsules in mounting organ culture explants for cryostat sectioning

Response of mammary tissue from pregnant goats to prolactin and growth hormone in organ culture.

Institute of Animal Physiology and Genetics, Czechoslovak Academy of Sciences, Uhrineves, Prague, Czechoslovakia (Received 19 March 1975) Nowadays organ explant cultures of mammary tissue are used to study the hormonal control of mammary development in non-ruminants. We have extended this technique to the culture of goat mammary gland. Nulliparous pure-bred Czech-white female goats with no obvious udder growth before pregnancy were used. They were killed by exsanguination and both mammary glands were removed and put in cold, sterile 0·85% NaCl. Batches of 20 expiants, selected at random, were put into Petri dishes containing 4 ml medium (Waymouth, 1959) and the required hormones. The Petri dishes were incubated at 37 °C under 95% O2 : 5% CO2. The secretory status of the expiants was studied histologically and graded according to Barnawell (1965). The mammary tissue of our goats at weeks 9 and 10 of pregnancy contained collections of

Induction of cystathionase in foetal rat liver explants effects of dexamethasone, N6, O2′-dibutyryladenosine 3′,5′-monophosphate and glucagon in vitro

At the 18th day of gestation and thereafter foetal rat liver explants in organ culture showed the competence to respond to dexamethasone by increased cystathionase activity, whereas the ability to respond to dibutyryl cyclic AMP or glucagon became evident at a later developmental stage (during the last 2 days prior to term). Simultaneous incubation with cycloheximide inhibited the stimulatory effect of these agents on foetal rat liver cystathionase activity in vitro. Dexamethasone and glucagon were both capable of increasing liver cystathionase activity both in newborn and 3-day-old animals in vivo.

Hormonal regulation of amino acid accumulation in human fetal liver explants Effects of dibutyryl cyclic AMP, glucagon and insulin

α-Aminoisobutyrate accumulation by human fetal liver explants in organ culture is stimulated by dibutyryl cyclic AMP ( N 6, 2′O- dibutyryl adenosine 3′–5′: cyclic monophosphate), glucagon or insulin. Theophylline increased the effect of submaximal concentrations of dibutyryl cyclic AMP or glucagon. Maximal concentrations of glucagon and dibutyryl cyclic AMP yielded the same results as either agent alone. A period of about 4–6 h was required to observe the stimulatory effect of dibutyryl cyclic AMP or insulin, which could be completely prevented by simultaneous incubation with cycloheximide. Maximal effects of either dibutyryl cyclic AMP or glucagon plus insulin produced additive results. These data support the hypothesis that insulin acts via a mechanism independent of the glucagon—cyclic AMP pathway in liver tissue. In addition, the pharmacologic receptor for glucagon was detected in liver explants from a 30-mm (crown - rump) specimen (6 weeks gestation). The liver had the competence to respond to dibutyryl cyclic AMP by the 36-mm stage. Tissue from a 36-mm specimen did not respond to insulin, but a clear response was elicited from a specimen at the 48-mm stage. These data demonstrate the ability of human fetal liver to respond to hormones at a very early stage in gestation.

Metabolism of cortisol by fetal rat liver explants.

Cortisol stimulated tyrosine aminotransferase activity (TAT) in fetal rat liver explants in organ culture only if the explants were subjected to a preliminary incubation. The effect was not due to loss of diffusible inhibitors or gain of a diffusible stimulator. In freshly isolated explants, 75% of added cortisol was accounted for after 5 hr as unconjugated tetrahydrocortisol and allo-tetrahydrocortisol. Other metabolites were also formed in small amounts. In explants subjected to preliminary incubation, 80% of the cortisol was recovered unaltered; the remainder appeared as tetrahydro metabolites. These observations are consistent with the hypothesis that maintenance of unaltered intracellular cortisol is essential for TAT induction, and that rapid inactivation of cortisol by the fetal liver retards the response of the tissue to the hormone. (Endocrinology 95: 466, 1974)

Scanning Microscopy of Human Intestinal Explants in Organ Culture

It has been reported that explants of human fetal intestine can be maintained in culture for up to 21 days in a viable condition and that these organ cultures support the growth of a variety of known viral agents responsible for enteric disease. Scanning electron microscopy (SEM) has been undertaken on several series of these explants to determine their appearance under routine culture conditions.Fresh specimens of jejunum obtained from normal human fetuses were washed, dissected into l-4mm pieces, and cultured in modified Leibowitz L-15 medium at 34° C as previously described. Serial specimens were fixed each day in 3% glutaraldehyde for 90 minutes at room temperature, rinsed, dehydrated, and dried by the CO2 critical point method in a Denton DCP-1 device. Specimens were attached to aluminum stubs with 3M transfer tape No. 465, and one sample on each stub was carefully rolled along the adhesive such that villi were broken off to expose their interiors.

The effect of prolactin and growth hormone on fatty acid synthesis by pregnant mouse mammary gland in organ culture.

SUMMARY The effect of various hormones on the incorporation of [14C]acetate into the fatty acids of pregnant mouse mammary gland explants in organ culture was studied. Of the hormones insulin (I), ovine prolactin (P), bovine growth hormone (GH) and cortisol (F) tested singly, only insulin stimulated fatty acid synthesis. There was synergism between cortisol or prolactin with insulin. The greatest stimulation in fatty acid synthesis occurred when explants were incubated in a medium containing either I + F + P or I + F + GH. Analysis by radio-gas-liquid chromatography of the fatty acids synthesized by explants after 14C labelling, showed that the pattern of fatty acids formed in the presence of I + F was distinctly different from that produced in the presence of I + F + P or I + F + GH. In the presence of I + F, the pattern of fatty acids resembled that found in mouse adipose tissue, whilst with I + F + P or I + F + GH the pattern resembled that of mouse milk fat. Synthesis of RNA was essential for the stimulation of fatty acid synthesis in explants incubated in medium containing I + F + P or I + F + GH. Results obtained when DNA synthesis was blocked with mitomycin C suggest that mitosis is important for the induction of milk-fatty acid synthesis. Puromycin had no effect for up to 8 h on explants which had been previously cultured in medium containing I + F, I + F + P or I + F + GH. This suggests a slow turnover rate of the enzymes involved in the synthesis of milk fatty acids.

A study on the effect of insulin on DNA, RNA and protein synthesis in mouse mammary gland tissue in organ culture.

SUMMARY The rates of synthesis of DNA, RNA and protein of mouse mammary gland explants in organ culture have been determined. Stimulation with insulin resulted in maximal rates of synthesis of these components, all occurring between 18 and 22 h of culture. The use of metabolic inhibitors of DNA, RNA or protein synthesis showed that after insulin stimulation, inhibition of any one of these processes was associated with a reduction in the synthesis of the other two components. Also the maximal rate of protein synthesis is governed by the net amount of RNA formed throughout the period of culture. Evidence is presented that the stimulation of DNA, RNA or protein synthesis by insulin is not due to increased transport of amino acids and that insulin appears to act rapidly on processes which subsequently lead to enhanced synthetic activity.

Use of silicone membranes for study of cells released by hemopoietic tissues in organ culture

Summary The use of silicone membranes protecting spleen explants in organ culture is described in detail. The method is compatible with long-term maintenance of hemic cell differentiation in vitro, and allows recovery of hemic cells released by the cultures. Several implications of this technique are briefly discussed with respect to long-term hemopoiesis in vitro and cell adhesiveness.

Effect of oral contraceptives on human endometrium in culture

Addition of oral contraceptives to the medium of human endometrial explants in organ culture caused histologically, histochemically, and biochemically detectable changes in the tissue. These alterations were similar to the response obtained in vivo and indicate that the synthetic hormones have a direct effect on the endometrium in addition to any other mode of action. The effects of the contraceptives in organ culture included proliferative and secretory changes similar to those cause by the natural ovarian hormones, increased deposition of glycogen in the glandular epithelium and in the stromal cells, and histologic changes in the stroma around the glands and the blood vessels.

Further differentiation of early neural tube explants in organ culture as studied by electron microscopy.

Chick embryo spinal cord has been explanted at 2-day stages, when few or no cells have formed axons, and cultured organotypically with adjacent tissues or isolated from all other tissues.Relatively mature nerve cells and glial cells, neurites and synapses could be seen in electron micrographs of cultures maintained for three to four weeks. Histological organization and some aspects of cell differentiation differed in the two types of cultures. Ependymal cells and randomly arranged cells, possibly modified glia, were seen only in cultures of neural tube with adjacent tissue; neurons and macroglia seemed to be more numerous in cultures of isolated neural tube.The development of characteristic cells, with variations according to culture conditions, provides the opportunity for further study of factors controlling patterns of proliferation and differentiation in the central nervous system from very early to advanced stages.

Synthesis of ovulating hormone in organ culture.

Adult female rat medial basal hypothalami (MBH) from the first and second days of diestrus, early and late proestrus and estrus of the estrous cycle were incubated with 25-day-old female rat pituitary explants in organ culture. At the end of 5 days the pituitary tissue was homogenized in the culture medium and the supernatant bioassayed for ovulating hormone (OH) activity in the PMS-primed immature female rat. The number of ova released was considered an index of OH potency. The pituitarytissue plus media from cultures containing MBH of adult female rats in the second day of diestrus and early and late proestrus induced the release of significantly more ova per rat than media from 25-day pituitary cultured alone or noncultured pituitary. It is concluded that the MBH of the adult female rat in the second day of diestrus and early and late proestrus of the estrous cycle is capable of stimulating synthesis of OH by the 25-day female rat anterior pituitary in organ culture. (Endocrinology 84: 778, 1969)

Conversion of phenylalanine to tyrosine in the bovine mammary secretory cell.

Abstract Studies with both dispersed cell and explant organ cultures prepared from a variety of both rat and cow tissues indicated that the liver of both species and the lactating mammary gland of the cow are able to effect a significant conversion of [14C]phenylalanine to [14C]tyrosine. This demonstration of phenylalanine hydroxylase activity in the secretory cell of the lactating bovine mammary gland explains the apparent lack of requirement for tyrosine as an essential amino acid by the cell in the synthesis of milk proteins. Heart, kidney, and muscle tissue and mammary tissue from the rat did not possess this enzymatic ability confirming the studies of previous investigators with tissue derived from a wide variety of mammalian species. Thus, lactating bovine mammary tissue apparently forms an exception to the common belief that significant synthesis of tyrosine takes place only in the liver of mammals.

An organ culture technique for maintaining the pulp tissue of intact human teeth

The classic organ culture method of Strangeways and Fell with all its subsequent modifications, which in fact comprise all existing organ culture techniques, strictly limits the size of explants which can be grown. Utilizing a new procedure which consists of perfusing the tissue with nutrient through small needles placed within the tissue mass, this limitation in explant size might be overcome. The test organ used in this study was the human tooth. Perfusion periods were up to 14 days. Either of two criteria was used to evaluate the success of the perfusion procedure. First, comparison of pulp tissue ATP levels in perfused, non-perfused, and immediately extracted teeth, showed immediately extracted pulp to contain 0.15 μg ATP, non-perfused saline or gauze control pulp to contain 0.02 μg ATP and pulps of all perfused teeth to contain 0.11 μg/mg ATP tissue. Or second, histologic observations on perfused, non-perfused and immediately fixed teeth revealed marked differences. In some of the perfused teeth, the connective tissue and odontoblast cells appeared intact, and blood vessels were both plentiful and clearly marked. In some areas numerous mononuclear phagocytes could be seen, indicating a chronic inflammatory response. In all non-perfused teeth frank necrosis, with little evidence of any cellular or blood vessel integrity except at the apices, was seen. These results suggest that the placement of small needles may allow for the growth or maintainance of explants in organ culture of much larger size than previously thought possible.

Hormones thyroïdiennes et enzymes de désulfuration de la l-cysteine du foie du rat

At the 18th day of gestation and thereafter foetal rat liver explants in organ culture showed the competence to respond to dexamethasone by increased cystathionase activity, whereas the ability to respond to dibutyryl cyclic AMP or glucagon became evident at a later developmental stage (during the last 2 days prior to term). Simultaneous incubation with cycloheximide inhibited the stimulatory effect of these agents on foetal rat liver cystathionase activity in vitro. Dexamethasone and glucagon were both capable of increasing liver cystathionase activity both in newborn and 3-day-old animals in vivo.