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1.
Morphology and function of cultured hepatocytes isolated from rats with experimental toxic hepatitis
E. V. Baidyuk A. P. Shiryaeva N. N. Bezborodkina G. A. Sakuta 《Cell and Tissue Biology》2009,3(6):565-572
The goal of the study was to examine the morphology and function of primary hepatocytes isolated from rats with toxic hepatitis
induced by a combination of CCl4 and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed
actin cytoskeleton, cell-cell, and cell-matrix contacts. In this investigation, the morphology of mitochondria and their localization
in hepatocytes was assayed with Rhodamine 123 staining. Glycogen and DNA contents in cultured hepatocytes were determined
by fluorescent cytometry. It was found that the ploidy of hepatocytes isolated from normal and injured livers were different.
Cells were maintained in culture for 5 days and no changes in ploidy distribution were observed. The glycogen content was
50% higher in the experimental group than the control one; it was decreased in control and cirrhotic hepatocytes treated with
collagenase. Intact hepatocytes accumulated glycogen within 3 days; the glycogen level remained low in pathologic hepatocytes. 相似文献
2.
Yumi Kono Suyun Yang Eve A. Roberts 《In vitro cellular & developmental biology. Animal》1997,33(6):467-472
Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of
collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that
of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells.
Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period
of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin
secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion
could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer
of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture.
These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes
in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves
differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system
is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro. 相似文献
3.
Neil C. Talbot Vernon G. Pursel Caird E. Rexroad Jr. Thomas J. Caperna Anne M. Powell Roger T. Stone 《In vitro cellular & developmental biology. Animal》1994,30(12):851-858
Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary
cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver
cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small
biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing
gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and
β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder
cell co-culture may be useful for the sustainable culture of hepatocytes from other species. 相似文献
4.
Elena L. Sun Danielle G. Aspar Roger G. Ulrich George W. Melchior 《In vitro cellular & developmental biology. Plant》1990,26(2):147-150
Summary A method is described for the preservation and subsequent recovery of hepatocytes obtained by collagenase perfusion of cynomolgus
monkey (Macaca, fascicularis) livers. The fresh cells are suspended in fetal bovine serum containing 10% dimethylsulfoxide and, using a microprocessor-controlled,
liquid nitrogen freezing chamber and a specific cooling protocol, processed in such a way that they can be stored in liquid
nitrogen for several months and still restored to active culture. When the cryopreserved cells were established in culture
they were found to actively synthesize and secrete both albumin and apolipoprotein A-I. That, taken together with morphologic
evidence, was viewed as indication that the cells recovered in culture were in fact hepatocytes and not some other cell type
from the monkey liver. The availability of this procedure for storing hepatocytes should contribute significantly to the efficient
use of nonhuman primates as models with which to study hepatic metabolism. 相似文献
5.
Shu-Ren Wang Guy Renaud Jacqueline Infante Danièle Catala Recaredo Infante 《In vitro cellular & developmental biology. Plant》1985,21(9):526-530
Summary Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance,
viability (94.5%) and yield (1.76.107 cells/g liver) compare well with those of previously described methods using collagenase. Differentiated functions of the
hepatocytes in primary culture such as albumin secretion (10.9 μg/mg cell protein/d) and triglyceride synthesis and secretion
are maintained. Induction of triglyceride synthesis and secretion by oleic acid takes place to an extent similar to that observed
in vivo and liver perfusion. Particles with a lipid composition resembling circulating very low density lipoproteins are secreted
into the medium. These characteristics demonstrate the ability of hepatocytes isolated with EDTA and subsequently used in
primary culture to retain complex and highly differentiated functions of the intact liver. 相似文献
6.
Annettee Athari Kirsten Unthan-Fechner Peter Schwartz Irmelin Probst 《In vitro cellular & developmental biology. Plant》1988,24(11):1085-1091
Summary A technique has been devised to attach adult rat hepatocytes to collagen-coated dextran microcarriers. Cells were cultured
serum-free for 2 d and their viability, enzyme activities, glucose metabolism, and hormone responsiveness were compared to
data obtained from conventional dish cell culture. The two diffeent culture methods showed no difference in cell viability
and morphology. Microcarrier-cultured cells exhibited hormone responsiveness comparable to dish cultures; glycolysis could
be activated three-fold by the sole addition of insulin, and gluconeogenesis was increased by 40 to 50% by glucagon. During
the 48-h culture glucokinase and phosphoenolpyruvate carboxykinase activities declined at a similar rate in both culture systems.
Long-tem culture wih 0.1 μM insulin prevented the decrease of glucokinase activity. Insulin responsiveness (activation of glycolysis) was still pronounced
after 48 h in culture. The microcarrier technique establishes a new in vitro liver system in which acute and long-term hormonal
actions can be investigated using the technical advantages of a suspensions cultures.
This study had been supported by the Deutsche Forschungsgemeinschaft. 相似文献
7.
Joanne Zurlo Linda M. Arterburn 《In vitro cellular & developmental biology. Animal》1996,32(4):211-220
Summary An hepatocyte culture system was developed for potential use in toxicological studiesin vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox™ dishes in a modified
Chee’s medium containing 1μM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase
release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained
at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone
metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A.
Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the
conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and
UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These
results strongly support the use of this hepatocyte culture system forin vitro toxicological studies.
A patent has been filed for the use of the system described herein as anin vitro test kit. 相似文献
8.
Alexandre E Cahn M Abadie-Viollon C Meyer N Heyd B Mantion G Cinqualbre J David P Jaeck D Richert L 《Cell and tissue banking》2002,3(4):223-233
The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor
liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste
after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy,
alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes.
Pre-operative biological and anatomopathological data indicated that, while mild steatosis (≤10% steatotic hepatocytes) did
also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte
yield. Cholestasis, as assessed by γ-glutamyl transferase serum values, significantly negatively correlated with the percentage
of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min
was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage
of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage
of digested tissue and the yield of viable cells.
In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend,
for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and
to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing
warm ischaemia or clamping during resection due to the decrease in cell yield.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
Hugo O. Jauregui Paul N. McMillan Karen Hevey Sharda Naik 《In vitro cellular & developmental biology. Plant》1988,24(5):401-412
Summary A quantitative evaluation of lectin binding to adult rat hepatocyte cell surfaces was done using cells isolated by two different
collagenase perfusion methodologies and cultured as monolayers with two different tissue culture media formulations (protocol
I vs. protocol II). The presence of α-D-mannosyl and α-D-glucosyl groups was detected by the binding of Concanavalin A (Con
A), Lens culinaris agglutinin (LCA), and Pisum sativum agglutinin (PSA) to freshly isolated cells. Furthermore, β-D-galactose
[Ricinus communis agglutinin (RCA)] and sialic acid residues [wheat germ (WGA)] were also found. Protocols I and II served
as models for evaluation of: a) the stripping effect of collagenase separation procedures, b) the restoration in culture of
collagenase-stripped sugar residues, c) the effect of the culture environment on cell viability [as measured by lactic acid
dehydrogenase (LDH) leakage] and the protein content of hepatocytes, and d) the presence of cell surface sugar residues as
a function of culture duration. The ultrastructural morphology of freshly isolated and cultured hepatocytes was also evaluated.
These studies indicated that a decline in lectin binding invariably occurred earlier than a massive leakage of LDH and a decrease
in the protein content of the cells in culture. Ultrastructurally, autophagocytosis was an early phenomenon in cells isolated
and cultured by protocol I, which was also inferior to protocol II regarding the preservation of hepatocyte glycocalyces.
Sugar residues lost due to the collagenase-stripping effect were restored, as shown by lectin binding, within the first 24
h of culture. This stripping effect was confirmed by quantitative evaluations of lectin binding to hepatocytes in culture
after an incubation with collagenase. This study shows that the binding of peroxidase-labeled lectins is a useful tool for
quantitative evaluation of the sugar composition of hepatocyte cultures.
This study was supported by grant I-ROI-AM 26520 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney
Diseases, Bethesda, MD, and by W. R. Grace Corporation. 相似文献
10.
Summary Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of
0.25 × 106 cells/cm2 in plastic culture dishes precoated with trout skin extract (7.6 μg skin protein/cm2) to facilitate cell attachment were maintained at 16° C. Cells were treated with DEX (10−9 to 10−7
M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72
h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly
lower in DEX (10−8 to 10−7
M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual
decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10−8 to 10−7
M). DEX at 10−9
M was ineffective. Concomitant addition of 10−6
M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10−7
M DEX abolished the DEX effect. RU486 at 10−8
M was ineffective. Spironolactone (10−8 to 10−6
M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10−6
M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and
this regulation is mediated through the type II glucocorticoid receptor(s). 相似文献
11.
Gary K. Ostrander James B. Blair Beverly A. Stark Garry M. Marley Wesley D. Bales Robert W. Veltri David E. Hinton Mark Okihiro Lisa S. Ortego William E. Hawkins 《In vitro cellular & developmental biology. Animal》1995,31(5):367-378
Summary Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained
viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures
for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared
and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and
passaged.
The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell
types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis,
and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical
staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell
populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation
or pluripotent cells, were observed with increased time in primary culture. 相似文献
12.
13.
James E. Klaunig Randall J. Ruch Peter J. Goldblatt 《In vitro cellular & developmental biology. Plant》1985,21(4):221-228
Summary Rainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×106 liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes
in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations
of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the
incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep
450 activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d
of culture. Culture temperature also influenced thep
450 activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated
hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes
between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and
the appearance of bundles of intermediate filaments also were observed with increased time in culture.
This work was supported in part by an American Cancer Society Grant (Ohio Division, Inc.) and an NIH Biomedical Research Support
Grant 5507RR05700010. 相似文献
14.
M. Teresa Donato M. José Gómez-Lechón José V. Castell 《In vitro cellular & developmental biology. Plant》1990,26(11):1057-1062
Summary We have developed new co-cultures of continuous cell lines 3T3 (clone A31) and C3H/10T1/2 (colone 8) with hepatocytes as an
alternative to co-cultures with nonconinuous epithelial cells. In this biological system we studied in detail the expression
of the hepatic biotransformation system. After 7 d in culture, total cytochrome P-450 content and the monooxygenase activities
aryl hydrocarbon hydroxylase and 7-ethoxycoumarino-deethylase still maintained about 30% of their initial value, whereas in pure cultured hepatocytes these activities were
undetectable. A significant response to induction by methylcholanthrene and phenobarbital of monooxygenase activities was
observed in co-cultures for 7 d. NADPH-cytochrome c reductase activity remained unchanged for at least 7 d in co-cultured
hepatocytes, whereas in pure cultures this activity was reduced to about 75% of the initial value after only 24 h. Finally,
the activity of the conjugating enzymes UDP-Gt and GSH-t was maintained at nearly the initial levels during the complete period
of study. The easy handling of continuous cell lines and the maintenance of the biotransformation system of hepatocytes in
co-culture make this approach simpler and easier to standardize.
This investigation was supported by grants 86/1098 and 87/1022 from Fondo de Investigaciones Sanitarias der la Seguridad Social,
Ministerio de Sanidad y Consumo Espa?ol. 相似文献
15.
For long-term maintenance of functional hepatocytes in primary culture, a new culture system with chemically modified type-I collagen gel was developed. Isolated hepatocytes spread as flat cells and rapidly lost their viability and functions when cultured on native collagen gel. In contrast, they survived for several weeks when cultured on collagen gels that had been modified by treatment with sodium-borohydride (NaBH4) or by digestion with pepsin, which resulted in destruction of crosslinking of collagen fibers and marked decrease in meachanical strength of the gels. These long-lived cells were round and aggregated and maintained high levels of various differentiated liver functions including albumin secretion and activities of tyrosine aminotransferase and P450. Moreover on collagen gels modified by treatment with NaBH4 or pepsin, the cell showed less DNA synthesis in response to mitogenic stimulation than cells cultures on gel containing native collagen. Interestingly, crosslinking of these chemically modified gels with D-ribose resulted in changes in various phenotypes of hepatocytes cultures on them including shape, longevity, and functions expressed when the cells were cultured on native collagen gel, suggesting that the effect of modification of the collagen gel is reversible. Thus the structure of collagen gels, probably due to the degree of crosslinking, seems to affect the morphology, maintenance of differentiated functions, and growth of primary cultured hepatocytes. 相似文献
16.
Vanhaecke T Foriers A Geerts A Shephard EA Vercruysse A Rogiers V 《Alternatives to laboratory animals : ATLA》2001,29(3):335-346
The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST. 相似文献
17.
rES (rhesus monkey embryonic stem) cells have similar characteristics to human ES (embryonic stem) cells, and might be useful as a substitute model for preclinical research. Before their clinical application, it is critical to understand the roles of factors that control the differentiation of ES cells into hepatocytes. Here, we analysed the effect of collagen gels on rES cells differentiation into hepatocytes by stepwise protocols. About 80% of DE (definitive endoderm) cells were generated from rES cells after being treated with activin A. The DE cells were then plated on to collagen gels or type I collagen-coated wells with growth factors to induce hepatocyte differentiation. In type I collagen systems, characteristics of immature hepatocytes were observed, including the expression of immature hepatic genes and the generation of 15±3% AFP (alpha fetoprotein)/CK (cytokeratin)18 double-positive cells. In collagen gel culture, differentiated cells exhibited typical hepatocyte morphology and expressed adult liver-specific genes. The mRNA expression of AFP (immature hepatic gene) was detected at day 11 but decreased at day 18. In contrast, mRNA expression of albumin (mature hepatic gene) was detected at day 11 and increased at day 18. Compared with type I collagen systems, significantly higher AFP/CK18 double-positive cells (68±7%) were produced in collagen gel culture. Furthermore, some differentiated cells acquired the hepatocytic function of glycogen storage. However, only immature hepatic genes were observed in collagen gel systems if growth factors were absent. Thus, collagen gels combined with hepatocyte-inducing growth factors efficiently promoted differentiation of hepatocytes from rES. 相似文献
18.
Gary W. Long Simon Leath Richard Schuman Michael R. Hollingdale W. Ripley Ballou Betty Kim Lee Sim Stephen L. Hoffman 《In vitro cellular & developmental biology. Plant》1989,25(9):857-862
Summary
Plasmodium berghei exoerythrocytic (EE) stages have been cultured in vitro in human continuous cell lines and primary cultures of both human
and rat hepatocytes. Although the predominant experimental model of irradiated sporozoite-induced protective immunity is the
mouse,P. berghei has not been cultivated in primary mouse hepatocytes or in continuous mouse lines. Because of this, target cells are not
available for determining if these immunized mice produce cytotoxic T lymphocytes (CTLs) that recognizeP. berghei antigens expressed on hepatocytes in the context of class I major histocompatability (MHC) antigens. We report the development
of methods for cultivating the (EE) stage ofP. berghei in murine hepatocytes and in two cell lines derived from the livers of BALB/c mice; one line produced from a primary hepatocyte
culture and the other produced by fusion of mouse hepatocytes with a continuous rat liver line. Mature parasites were detected
by microscopy and by DNA probe in both cell lines, each of which supported complete development ofP. berghei liver stages and production of infectious merozoites. Since class I MHC antigens are present on the surface of primary hepatocytes
and the mouse X rat hybrid line, these cells can be used to detect cytotoxic T cells against liver stage parasites.
This work was supported by the Naval medical Research and Development Command, Bethesda, MD, work unit no. 3M161102B510AK111,
ONR contract N00014-83-C-0355, and by contract DPE-0453-C-00-3051-00 of the U.S. Agency for International Development, Washington,
D.C.
The opinions and assertions herein are not to be construed as official or as reflecting the views of the Navy Department or
the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current
edition of the “Guide for the Care and Use of Laboratory Animals”, Institute of Laboratory Animal Resources, National Research
Council, DHHS, Pub. no. (NIH)85-23 相似文献
19.
Serene M.L. Lee Celine Schelcher Maresa Demmel Maria Hauner Wolfgang E. Thasler 《Journal of visualized experiments : JoVE》2013,(79)
The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes. 相似文献
20.
Elsie M. B. Sorensen Daniel Acosta 《In vitro cellular & developmental biology. Plant》1984,20(10):763-770
Summary Freshly isolated hepatocytes from neonatal rats were cultured for approximately 24 h; incubated for 5, 30, or 60 min in solutions
containing 0, 50, 100, or 200 μM cadmium; embedded in plastic; and sectioned for optical microscopy. The exeent of cadmium-induced hepatotoxicity was evaluated
by double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of threedimensional
information) whereby hepatocytes were classified on the basis of the severity of morphologic damage at the optical level.
Both time and concentration effects were studied. Cultures exposed to 200 μM cadmium, for various intervals of time from 5 to 60 min, showed statistically significant reductions in the relative volume
percent of normal hepatocytes, elevations (then reductions) in the relative volume percent of slightly damaged hepatocytes,
increases in the relative volume percent of moderately damaged cells, and increases in the relative volume percent of severely
damaged liver cells. As the concentration of cadmium was increased from 50 to 200 μM cadmium (during both 30 and 60-min exposures), significant trends were observed in cellular distribution patterns based on
relative volume percent. Morphologically normal cells decreased, both slightly damaged and moderately damaged cells increased,
and severely damaged cells remained unchanged. These results indicated that morphometric analysis at the optical level provided
quantitative estimates for the evaluation of time- and concentration-effects of cadmium on cultured hepatocytes.
This work was supported by a grant from the Johns Hopkins University Center for Alternatives to Animal Testing. 相似文献