RESUMO
Rat hepatocytes were cultured on type I collagen-coated porous membranes, in Waxman's modified medium supplemented with very low concentrations of insulin (10(-9)m), glucagon (10(-10)m) and dexamethasone (10(-8)m). Under these experimental conditions, specific differentiated functions were well preserved for at least 15 days, as shown by measures of albumin secretion, EROD and PROD activities, by phase contrast microscopy and PAS and ORO staining for intracellular glycogen and lipid contents. These results suggest that this experimental system may be very useful for long-term in vitro pharmacotoxicological studies.
RESUMO
The aim of this study was to better characterize rabbit proximal kidney tubule cells cultured on collagen IV-coated porous inserts, as compared to the same cells seeded in standard plastic wells. Total protein contents in confluent monolayers on permeable membranes were about twofold higher than those measured in confluent cultures in plastic wells. Microscopy examinations suggested that such a difference was probably due to a higher cell density and to an impressive development of the apical brush-border membrane. Moreover, measurement of unidirectional transport of p-aminohippuric acid and tetraethylammonium bromide confirmed the high polarization level of cultures on porous inserts. Results of methyl(alpha-D-[U-14C]glyco)pyranoside uptake suggested that cell phenotype was probably influenced by culture conditions. Analysis of different markers as a function of time in culture showed decreases of alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), and Na(+)-K(+)-ATPase activities as well as increases in LDH, ATP, and glutathione levels, similar to those formerly reported for cells cultured in standard plastic plates. However, comparative data from 6-d-old monolayers have shown that AP, GGT, Na(+)-K(+)-ATPase, glutathione reductase (GRED), and selenium-dependent glutathione peroxidase (Se-GPX) activities were 2.8-, 2.6-, 1.6-, 1.2-, and 2.1-fold, respectively, better preserved on precoated permeable membranes. On the other hand, this paper reports for the first time in the literature that GRED and SE-GPX, two phase II detoxification enzymes, were well maintained in cultures of rabbit proximal kidney tubule cells. Our results show that culturing rabbit proximal kidney tubule cells on collagen IV-coated porous membranes was accompanied by an improvement of both morphological and biochemical properties of the cells.
Assuntos
Colágeno/farmacologia , Túbulos Renais Proximais/citologia , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Feminino , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/ultraestrutura , CoelhosRESUMO
Three recent progresses allow to perform plastic embedding of moderate or large samples of tissue in limits of time close to those of the paraffin embedding and to obtain slices between 1 and 2,5 mu on which all current histochemical or even histo-immunological reactions are possible. These technics prevent the retraction of the tissues, give a good preservation of the structure of the cells and permit a good analysis of the tiny lesions of all kinds of tissues. From our experience about 1362 varied biopsies, the best indications seems to be in order : kidney, hepatic, lung, medullary and nodes biopsies. It seems now justified to introduce these technics as a routine one, inside laboratories which have to detect tiny lesions on particular tissues or on experimental samples.
Assuntos
Técnicas Histológicas , Resinas Sintéticas , Animais , Medula Óssea/patologia , Humanos , Rim/patologia , Fígado/patologia , Pulmão/patologia , Linfonodos/patologia , Microtomia/instrumentaçãoRESUMO
Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days.