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C Chesne

Publications and source records attributed to C Chesne.

17 recordsLinked to original sources
PubMed1999

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Adenosine Triphosphate↗
PubMed1999

Primary cultures of heart cells from the scallop Pecten maximus (mollusca-bivalvia).

Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.

Animals↗
PubMed1996

Comparison of the induction of hepatic peroxisome proliferation by the herbicide oxadiazon in vivo in rats, mice, and dogs and in vitro in rat and human hepatocytes.

Oxadiazon [5-ter-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)- 1,3,4-oxadiazol-2(3H)-one] was administered orally at 20-500 mg/ kg body wt/day to male Sprague-Dawley CD rats for 14 days, at 20-200 mg/kg body wt/day to male CD1 [CR1/CD-1(1GR)BR] mice for 28 days, and at 500 mg/kg body wt/day to male beagle dogs for 28 days. Although liver enlargement was observed in the three species, morphological studies indicated that peroxisome proliferation only occurred in rats and mice. Parallel biochemical investigations showed that there was a dose-dependent increase in the peroxisomal cyanide-insensitive palmitoyl CoA oxidase and acetyl carnitine transferase activities in treated rats and mice. Acetyl carnitine activity appeared to correlate well with the number and volume of peroxisomes as determined histologically. The increases in enzyme activities at 200 mg/kg body wt/day oxadiazon were comparable in rats and mice indicating that both rodent species were equally sensitive to oxadiazon-induced peroxisome proliferation. When added in vitro to cultured rat hepatocytes at concentrations ranging from 2.5 to 10 x 10(-5) M, oxadiazon induced a dose-dependent increase in the activities of palmitoyl CoA oxidase and acetyl carnitine transferase. The ratio obtained by comparing oxadiazon and clofibric acid on acetyl carnitine transferase activity at 5 x 10(-5) M in the present in vitro study on rat hepatocytes are equivalent to those that can be calculated from the results on this enzyme activity obtained in vivo in the rat with 500 mg/ kg body wt/day oxadiazon (this study) and clofibric acid (literature values), indicating that the rat hepatocyte cultures gave satisfactory results regarding peroxisome proliferation. Neither oxadiazon nor clofibric acid modified the activities of palmitoyl CoA oxidase and acetyl carnitine transferase in cultured human hepatocytes. The results presented here demonstrate clearly that oxadiazon induces peroxisome proliferation in rodents in vivo and in vitro, as determined both biochemically and morphologically, whereas dogs in vivo and human hepatocytes in vitro were refractory to peroxisome proliferation. This observation extends to the herbicide oxadiazon, which is structurally unrelated to other known peroxisome proliferators, the generally observed marked species difference in sensitivity to peroxisome proliferation, and has important implications in the human safety evaluation of this herbicide.

Animals↗
PubMed1996

Influence of alginate gel entrapment and cryopreservation on survival and xenobiotic metabolism capacity of rat hepatocytes.

Rat hepatocytes immobilized in calcium-alginate beads were tested for their ability to survive and function in vitro by comparison with hepatocyte monolayer cultures before and after cryopreservation. The freeze-thaw protocol previously designed for suspended hepatocytes was used; the freezing medium was the Leibovitz medium containing 10% fetal calf serum and 16% dimethylsulfoxide. The functions investigated included protein, lipid, and urea synthesis, albumin secretion, glycogen content, and various phase I and phase II enzyme activities. Cell viability was not altered by immobilization and the freeze-thaw procedure. Most functions tested were expressed at similar levels in unfrozen immobilized hepatocytes and corresponding hepatocyte monolayers. Only protein neosynthesis and some phase II drug metabolizing enzyme activities were decreased. The freeze-thaw process had no detrimental effect, whatever the function investigated. In addition, cryopreserved immobilized hepatocytes remained responsive to inducers: ethoxyresorufin O-deethylase activity was increased 11-12 times by 3-methylcholanthrene treatment in both fresh and cryopreserved alginate-entrapped hepatocytes. These results clearly show that hepatocytes immobilized in calcium-alginate beads remain functional even after cryopreservation indicating that they represent a promising approach for xenobiotic metabolism and toxicity studies.

Alginates↗
PubMed1996

Culture and drug biotransformation capacity of adult human keratinocytes from post-mortem skin.

The aim of this study was to analyse viability, growth, differentiation and drug metabolic capacity of cultured human keratinocytes obtained from post-mortem skin. Epidermal cells were prepared from 1-day post-mortem paired sun-exposed (outer) and sun-protected (inner) sites of the upper arm, of donors aged 47-80 years. The percentage of viable cells obtained from post-mortem skin was only slightly lower than that usually obtained for keratinocytes isolated from fresh skin, and no alterations of epidermal markers were noted. Keratinocytes isolated post-mortem from non-exposed skin had a higher viability (78 versus 73%), and a more active proliferation, while their attachment rate, keratin composition, lipid synthesis capacity and transglutaminase activity levels were similar to those of epidermal cells obtained from the sun-exposed skin. Keratinocytes isolated from post-mortem skin expressed various phase I and II activities at levels similar to those obtained with keratinocytes isolated from fresh skin while drug metabolizing enzyme activities were consistently higher in sun-exposed compared to sun-protected cells. The results support the conclusion that skin collected post-mortem can represent an alternative source of viable and functional epidermal cells, and that the functional changes that occur in adult keratinocytes habitually exposed to the sun, affect much more strongly the drug metabolism capacity than the expression of differentiation markers.

Aged↗
PubMed1994

Urokinase and type I plasminogen activator inhibitor production by normal human hepatocytes: modulation by inflammatory agents.

We examined the effects of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta) on the plasminogen activator system (urokinase, tissue-type plasminogen activator, type 1 plasminogen activator inhibitor) in primary cultures of human hepatocytes. We show that interleukin-1 beta and tumor necrosis factor-alpha increase urokinase-type plasminogen activator production, reinforcing the concept that increased urokinase production is associated with inflammatory processes. By contrast, the same agents (i.e., interleukin-1 beta and tumor necrosis factor-alpha) do not stimulate plasminogen activator inhibitor type 1 production. This latter observation rules out hepatocytes as a major cellular source of plasmatic plasminogen activator inhibitor type 1 during acute-phase-related responses. Among the inflammatory agents used, transforming growth factor-beta was found to be the most effective modulator of both urokinase-type plasminogen activator and plasminogen activator inhibitor type 1, inducing severalfold increases of activity of urokinase-type plasminogen activator, antigen and the corresponding mRNA and increasing plasminogen activator inhibitor type 1 antigen and mRNA levels. Urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 modulation by transforming growth factor-beta may play a critical role in hepatic pathophysiology.

Cells, Cultured↗
PubMed1992

Viability and primary culture of rat hepatocytes after hypothermic preservation: the superiority of the Leibovitz medium over the University of Wisconsin solution for cold storage.

Hepatocytes isolated from adult rat livers were hypothermically preserved for 24 or 48 hr before being plated under conventional culture conditions. They were stored either in the Leibovitz medium, a cell culture medium with and without polyethylene glycol (PEG), a compound known to suppress ischemia-induced cell swelling, or in the University of Wisconsin solution, the most effective solution for cold organ preservation. After 24 or 48 hr of storage at 4.5 degrees C in Leibovitz medium, cell viability and adherence efficiency to plastic dish, were only slightly reduced, whereas University of Wisconsin hepatocytes had a decreased viability and (especially after 48-hr storage) lost their adhesion ability; they did not survive in vitro. The metabolic competence of hepatocytes maintained in Leibovitz medium was retained over the 3 days of culture, as shown by low extracellular levels of the membrane-bound and cytosolic hepatic enzymes, as well as by intracellular glutathione content, albumin secretion rate and several phase I and phase II drug metabolic reactions very close to those found with fresh hepatocytes maintained under similar culture conditions. Addition of polyethylene glycol to the Leibovitz medium resulted in slightly higher viability and function of hepatocytes after cold storage. These results clearly demonstrate that viability of a transplanted liver does not correlate with long-term in vitro viability of isolated hepatocytes after hypothermic preservation in University of Wisconsin solution. They also suggest that nutritional and energy substrates as found in the Leibovitz medium are probably required to define a suitable solution for cold preservation of isolated parenchymal cells. The findings with Leibovitz medium favor the conclusion that hypothermically preserved hepatocytes could be used for various metabolic studies and for the treatment of liver insufficiency.

Adenosine↗
PubMed1992

[Extracorporeal bio-artificial liver using isolated hepatocytes. An experimental study in the rat].

A new type of bioartificial liver using isolated hepatocytes immobilized in alginate beads was developed and its capacity of correcting the metabolic deficiency of bilirubin conjugation in Gunn rats was assessed. Hepatocytes were isolated from Sprague-Dawley rats using the in situ collagenase perfusion technique, and they were then immobilized in Calcium alginate beads. The capacity of these immobilized hepatocytes to conjugate in vitro bilirubin was checked as compared to monolayer hepatocyte culture. The bioartificial liver consisted in a cylindrical bioreactor containing either alginate beads and hepatocytes (test group), or only alginate beads (control group). Gunn rats were connected to this bioreactor through and extracorporeal circulation system, and "bile samples were collected" every hour. Bilirubin mono and biconjugates were dosed in the bile using the high-performance liquid chromatography technique. The viability of alginate immobilized hepatocytes, determined before and after each experiment, was stable at 75%. In the test group, the total conjugate concentration rapidly increased to reach a maximum value of 204 +/- 16 microns after 3 hours, while in the control group, there were only conjugate traces (1 micron). These results show that the bioartificial liver is an efficient means to temporarily correct genetic deficiency in Gunn rats. Such a system could be of therapeutic interest in case of acute hepatic insufficiency.

Animals↗
PubMed1990

Transforming growth-factor-beta (TGF-beta) inhibits albumin synthesis in normal human hepatocytes and in hepatoma HepG2 cells.

We explored the effect of transforming growth factor beta (TGF-beta), a cytokine that appears to play a central role in inflammatory events, on albumin expression by normal adult human hepatocytes and hepatoma cells. Addition of TGF-beta to primary human hepatocyte cultures resulted in a dramatic decrease in albumin accumulation and synthesis. This effect was dose-dependent, took place after a 48h incubation period and was maintained over 96h. TGF-beta-decreased albumin protein levels were associated with reduced albumin mRNA content. Actin mRNA levels were concomittantly increased. Comparable qualitative effects of TGF-beta were observed on human hepatoma HepG2 cells.

Adult↗
PubMed1990

Primary culture of adult rat hepatocytes after 48-hour preservation of the liver with cold UW solution.

Rat livers were perfused and stored for 48 hr in cold University of Wisconsin solution before dissociation by the two-step collagenase method. At that time, glycogen content was significantly reduced, but no obvious changes in albumin, beta-actin and aldolase B mRNAs and in glutathione levels were observed. Enzymatic perfusion yielded 280 +/- 30 x 10(6) viable hepatocytes vs. 520 +/- 40 x 10(6) viable hepatocytes from unstored organs. Cell viability determined by trypan blue exclusion was 74% and 90%, respectively. Hepatocytes from University of Wisconsin-preserved livers had a 29% reduced adenosine triphosphate content, but glutathione levels did not significantly differ from those found in unstored cells. When put into culture, hepatocytes formed typical monolayers of granular epithelial cells and did not exhibit alteration of their fine structure when compared with cells from unstored organs. After 24 and 48 hr, they showed variations in cytochrome P-450 content and ethoxyresorufin O-deethylase activity similar to those observed with unstored cells. By contrast, overall protein synthesis and albumin secretion rate were 40% and 30% lower, respectively. Hepatocytes from University of Wisconsin-preserved organs could be cryopreserved and further cultured as unstored cells. The University of Wisconsin solution was also used to preserve isolated hepatocytes. Viability of freshly isolated hepatocytes was decreased by only 10% after 48 hr of hypothermic liver storage when assayed by intracellular lactate dehydrogenase content. However, after 4 hr of storage, in contrast with hepatocytes preserved in L15 Leibovitz medium, the cells attached poorly to plastic and exhibited morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMedDate not supplied

Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver.

A new extracorporeal bioartificial liver using alginate-entrapped hepatocytes was developed and evaluated for its ability to correct the lack of bilirubin conjugation in the Gunn rat. Hepatocytes were harvested from Sprague-Dawley rats by the two-step collagenase perfusion method and then immobilized in Ca(++)-alginate beads. The ability of immobilized hepatocytes to conjugate bilirubin was investigated in vitro by comparison with hepatocyte monolayer cultures. The bioartificial liver consisted of a cylindric bioreactor containing either alginate beads with hepatocytes (test group) or alginate beads alone (control group). Gunn rats were connected to this bioreactor via an extracorporeal circulation and bile fractions were collected at hourly intervals. Both bilirubin monoconjugates and bilirubin diconjugates were measured in the bile by high pressure liquid chromatography. Hepatocyte viability in alginate beads was determined prior to and at the end of each experiment and found to be unchanged (75%). In the test group, the concentration of bilirubin conjugates increase rapidly, attaining median values of 72.26 microM and 92.59 microM for mono and diconjugated bilirubin respectively, during a 3 h period of extracorporeal circulation. In the control group, the levels of either conjugate did not exceed 0.87 microM throughout the experiments. Statistical analysis showed a significant difference between the two groups (p < 0.0023). These results suggest that the bioartificial liver used in this study represents an effective method for the temporary correction of the Gunn rat's genetic defect. Such a system might be of therapeutic interest in acute liver failure.

Alginates↗
PubMedDate not supplied

An in vitro, ex vivo, and in vivo demonstration of the lipolytic effect of slimming liposomes: An unexpected alpha(2)-adrenergic antagonism.

Most of the slimming products already developed for cosmetic applications did not result from strategies that integrate whole lipolysis-regulating mechanisms. We thus focused our attention on a more complete integration of these mechanisms and we developed slimming liposomes (SLC) containing two micro-circulation activators, i.e., esculoside and Centella asiatica extracts, one phosphodiesterase inhibitor, i.e., caffeine, and one fatty acid-beta oxidation activator, i.e., L-carnitine. The validity of our approach was assessed through (a) in vitro tests demonstrating that SLC induced a dramatic increase in the cyclic adenosine monophosphate (cAMP) content in human adipocytes, with a subsequent rise in the nonesterified fatty acids (NEFA) content of human adipocyte incubation medium, and (b) in vivo studies showing that SLC could provide an actual potent slimming effect on human volunteers. Moreover, we give here, through binding experiments, the unambiguous demonstration that SLC is able to antagonize the alpha(2)-adrenergic receptor that is known to reduce intracellular AMPc content and, subsequently, to down-regulate lipolysis. This alpha(2)-adrenergic antagonism has never been reported for any component of SLC, and this work is the first demonstration of the alpha(2)-adrenergic antagonism of such a combination of active liposome compounds.

Adipocytes↗