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1.
Neil C. Talbot Caird E. Rexroad Jr. Anne M. Powell Vernon G. Pursel Thomas J. Caperna Sherry L. Ogg Neil D. Nel 《In vitro cellular & developmental biology. Animal》1994,30(12):843-850
Summary Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The
cells were polygonal and had phase-contrast dark, granular cytoplasm with prominent nuclei and nucleoli. These feeder-dependent
cell cultures differentiated into large, multicellular, secretory, duct-like structures or formed small canaliculi between
individual cells. Alternatively, the cells accumulated droplets that stained intensely with Oil Red O, a lipid-specific stain.
Alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNAs were expressed as the cells differentiated in culture. Serum-free
medium that was conditioned by the cells contained transferrin, AFP, and albumin. The growth and viability of the cells were
inhibited by transforming growth factor β1 (TGFβ1) at concentrations ≥1 ng/ml. The cell cultures grew slowly with doubling
times of 2 to 3 d. One of the cultures, pig inner cell mass-19 (PICM-19), was passaged continuously for over 2 yr [>100 population
doublings (PD)] and appears to be an infinitely self-renewing cell population. The stem cell characteristics of the epiblast-derived
fetal hepatocytes indicate that the cells may be unique for investigations of liver differentiation and organogenesis. 相似文献
2.
Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal
human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to
22 h after death) was not viable. When mucosal specimens (1.5 mm2) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48
h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman
sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth
kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased
viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8
and no growth at 6.2.
Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould
increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate
compared to platic.
The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation
and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium.
Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared
in Clin. Res. 30: 93A; 1982. 相似文献
3.
T Matsuda K Okamura Y Sato A Morimoto M Ono K Kohno M Kuwano 《Journal of cellular physiology》1992,150(3):510-516
We have previously demonstrated that epidermal growth factor (EGF) induces cell migration, tissue-type plasminogen activator synthesis, as well as tubular formation in microvascular endothelial cells from human omental tissue. In this study, we compared the responsiveness to EGF of late passaged (senescent) human omental microvascular endothelial (HOME) cells with that of early passaged (young) HOME cells. We have employed HOME cells derived from surgically resected omental samples from 14 patients. EGF-stimulated cell migration significantly more in the young cells than in the senescent cells during serial cultivation (aging) in vitro. Scatchard analysis demonstrated that the number for both high and low affinity receptors for EGF in HOME cells was decreased dramatically during serial cultivation. The expression of EGF receptor mRNA was also decreased in the senescent HOME cells. Treatment of HOME cells with EGF significantly increased cellular mRNA levels of tissue-type plasminogen activator, and two protooncogenes, c-fos and c-myc, in young HOME cells, but not in senescent HOME cells. Thus HOME cells aged in vitro show a decreased responsiveness to EGF, resulting in decreased migration of human endothelial cells. The serial cultivation of human endothelial cells in vitro may downregulate EGF receptor and decrease responsiveness to exogenous EGF, a potent angiogenic factor. 相似文献
4.
Heng BC Liu H Rufaihah AJ Cao T 《In vitro cellular & developmental biology. Animal》2006,42(3-4):54-57
Summary Human embryonic stem (hES) cells require cooperative interactions with each other for their survival. Previously, the size
of hES cell clumps has been reported to be an important factor in determining their viability during routine serial passage.
However, the effects of seeding density of the hES cell clumps per se have not yet been investigated. Therefore, this study
attempted to compare the level of spontaneous differentiation of hES colonies passaged at two different split ratios (1∶3
and 1∶8) of a single confluent well of a six-well dish. After 7 d of in vitro culture following serial passage, hES colonies
were assigned into three grades according to their degree of spontaneous differentiation: (1) Grade A, which was completely
or mostly undifferentiated; (2) grade B, which was partially differentiated; and (3) grade C, which was mostly differentiated.
Assessment of the degree of spontaneous differentiation was based on morphological observations under bright-field and phase-contrast
microscopy, as well as on immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. We observed that,
at a split ratio of 1∶3, the percentages of grade A, B, and C colonies were 89.5, 8.8, and 1.7%, respectively. This was significantly
different from the corresponding values of 52.7, 31.3, and 16.0%, respectively, obtained at a split ratio of 1∶8. Hence, our
results indicated that a lower passage density led to a higher degree of spontaneous differentiation of hES colonies. 相似文献
5.
Continuous multiplication of rabbit tracheal epithelial cells in a defined,hormone-supplemented medium 总被引:16,自引:0,他引:16
Summary An improved Ham’s F12 nutrient medium supplemented with epidermal growth factor (EGF), insulin (INS), and transferrin (TF)
was developed for continuous proliferation and clonal growth of primary rabbit tracheal epithelial (TE) cells in culture.
The addition of small quantities of fetal bovine serum (FBS) (0.01 to 0.1%) to cultures had little measurable stimulation
on TE cell growth and plating efficiency. However, serum levels higher than 0.1% inhibited cell growth and also masked the
growth stimulating activities of EGF and INS despite an increase in cell attachment. Under this defined, hormone-supplemented
medium, and in the presence of a trace amount of serum (0.01%), 10 to 20% of the protease-dissociated TE cells attached to
the culture dish followed by at least four population doublings during 7 to 10 d of culture. Clonal growth occurred at a seeding
density of 17 cells/cm2 with a plating efficiency of 6 to 8%. Confluent primary cultures could be passaged two to four times by treatment with a
0.1% trypsin-1 mM EDTA solution and a total of 10 to 30 population doublings of in vitro life span were obtained. The epithelial nature of
cultured cells was confirmed by indirect immunofluorescent staining with antikeratin antibody as well as by transmission electron
microscopy. This study shows that using this improved hormone-supplemented medium, rabbit TE cells can be maintained in culture
for extended periods of time without the aid of a fibroblast feeder layer or explant tissue. This system could be useful for
the study of cell differentiation of tracheal epithelium. 相似文献
6.
Propagation and morphologic phenotypes of human umbilical cord artery endothelial cells 总被引:1,自引:0,他引:1
V W van Hinsbergh A M Mommaas-Kienhuis R Weinstein T Maciag 《European journal of cell biology》1986,42(1):101-110
Human umbilical cord artery endothelial cells can be propagated on fibronectin-coated dishes for approximately 50 cumulative population doublings in the presence of crude preparations of endothelial cell growth factor (ECGF) and serum. The cells were characterized by immunofluorescent staining and their ultrastructure. Different morphologic phenotypes could be demonstrated: closely attached cell monolayers, atypical cells, giant cells, tube-like structures. The formation of tube-like structures can be induced by proteolytic modification of fibronectin. Our data demonstrate that umbilical arteries may provide an excellent source for the routine serial cultivation of human arterial endothelial cells. 相似文献
7.
Norbert E. Fusenig Wolfgang Thon Walter Samsel 《In vitro cellular & developmental biology. Plant》1979,15(5):315-325
Summary A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well
as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures
on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was
applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly
transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture
plastic petri dishes (by heated stamps), 0.2 to 4×105 cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling, with radioactive
precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures,
the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation
counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples
compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric
methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 μg DNA or
5 μg protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied
to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small
Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this
method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a
factor of 10, test substances and time), it might also be advantageous, for other purposes as well where the availability
of cells or test substances are limiting factors for large test series. 相似文献
8.
A β-galactosidase activity has recently been used as a histochemical marker of replicative senescence in human fibroblasts and keratinocytes. To establish whether this marker could be used to detect senescence of vascular cells, we have investigated its presence in cultures of serially passaged human umbilical vein endothelial cells and rabbit aortic smooth muscle cells. β-Galactosidase activity was detected by light microscopy using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β-
-galactopyranoside. In endothelial cell cultures, lysosomal β-galactosidase activity, which is detected at pH 4.0, was present in all cells regardless of their replicative age. In contrast, senescence-associated β-galactosidase activity, which is detected at pH 6.0, was absent in the majority of cells in early passage cultures (<15 cumulative population doublings), but was present in a large proportion of cells (up to 62%) in late passage cultures (>30 cumulative population doublings); in intermediate passage cultures (15–30 cumulative population doublings) it was found in fewer than 15% of the cells. The increase in the percentage of senescence-associated β-galactosidase-positive cells correlated with a decrease in the cell density at confluence and with a marked increase in cell size. Counterstaining with an antibody directed against the endothelial cell marker CD31 showed that senescent cells retained the expression of this antigen. Senescence-associated β-galactosidase was also detected in serially passaged, but not in primary explant cultures of rabbit aortic vascular smooth muscle cells. The presence of senescence-associated β-galactosidase in cultured vascular smooth muscle cells and endothelial cells suggests that this marker could be used to study the role of cellular senescence in vascular disease. 相似文献
9.
A serum-free medium formulation – TUD-1 – was developed supporting growth of HUVEC in tissue culture. Special features of
the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine
and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone
supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free
long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences
in final cell density compared to controls cultivated with serum.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
Panagiota Miltiadous Antonios Stamatakis Fotini Stylianopoulou 《Cellular and molecular neurobiology》2010,30(3):347-360
Insulin-like growth factor I (IGF-I) has been shown to act as a neuroprotectant both in in vitro studies and in in vivo animal
models of ischemia, hypoxia, trauma in the brain or the spinal cord, multiple and amyotrophic lateral sclerosis, Alzheimer’s
and Parkinson’s disease. In the present study, we investigated the neuroprotective potential of IGF-I in the “kainic acid-induced
degeneration of the hippocampus” model of temporal lobe epilepsy. Increased cell death—as detected by FluoroJade B staining—and
extensive cell loss—as determined by cresyl violet staining—were observed mainly in the CA3 and CA4 areas of the ipsilateral
and contralateral hippocampus, 7 days following intrahippocampal administration of kainic acid. Kainic acid injection also
resulted in intense astrogliosis—as assessed by the number of glial fibrillary acidic protein (GFAP) immunopositive cells—in
both hemispheres, forming a clear astroglial scar ipsilaterally to the injection site. Heat-shock protein 70 (Hsp70) immunopositive
cells were also observed in the ipsilateral dentate gyrus (DG) following kainic acid injection. When IGF-I was administered
together with kainic acid, practically no signs of degeneration were detected in the contralateral hemisphere, while in the
ipsilateral, there was a smaller degree of cell loss, reduced number of FluoroJade B-stained cells, decreased reactive gliosis
and fewer Hsp70-positive cells. Our present results extend further the cases in which IGF-I is shown to exhibit neuroprotective
properties in neurodegenerative processes in the CNS. 相似文献
11.
There is a wide range of reported values for prostacyclin (PGI2) synthesis by cultured endothelial cells from human umbilical veins (HUVE). Part of this variation may be due to differences in isolation and culture conditions, but part may be due to previously unstudied variation in the number of population doublings (PDs) which the cells have undergone in vitro. Attention is now shifting to arachidonic acid (AA) metabolism by cells from adult human vessels and these cells may require increased PDs to obtain confluent cultures for testing. Therefore, we have examined the effect of number of cell population doublings as well as number of subcultivations on PGI2 synthesis using HUVE as a model system. Primary and first subcultivation cultures inoculated at high density, so that PDs at confluence were less than 4, synthesized 10 times as much PGI2 as the same isolates inoculated at low density with PDs greater than 4. Isolates inoculated and subcultivated so that the PDs at confluence after the fourth subcultivation were less than 6, showed 50% less PGI2 synthesis between the primary and first subcultivation and between the first and second subcultivations. Isolates with less than 4 PDs after the fourth subcultivation were carried further to determine the effect of extensive subcultivation. Four of six isolates showed a sudden increase in PGI2 synthesis which occurred between subcultivations 5 and 12 (PDs 4-6). These results demonstrate that AA metabolism is markedly affected by growth in culture and serial subcultivation. 相似文献
12.
Toshihiro Mitaka Takashi Kojima Toru Mizuguchi Yohichi Mochizuki 《In vitro cellular & developmental biology. Animal》1996,32(8):469-477
Summary To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The
hepatocytes were cultured in serum-free modified Dulbecco’s modified Eagle’s medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6×105 cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to
Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered
and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near
confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the
second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could
attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo. but they never overgrew.
Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells
possessed hepatic characteristics such as peroxisomes with a crystalline nucleoid and bile-canaliculus structures. When 10%
fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate
very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could
differentiate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells.
In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided
evidence that the subcultured cells still have the potential to proliferate and to differentiate. 相似文献
13.
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
14.
Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor 总被引:2,自引:0,他引:2
Summary BC3H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified
Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin,
and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a
rate equal to that of serum-supplemented medium, and at low cell input (103 cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell
survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency
of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF. 相似文献
15.
Isolation,growth requirements,cloning, prostacyclin production and life-span of human adult endothelial cells in low serum culture medium 总被引:3,自引:0,他引:3
Hiroyoshi Hoshi Wallace L. McKeehan 《In vitro cellular & developmental biology. Plant》1986,22(1):51-56
Summary Endothelial cells from autopsy and biopsy specimens from a variety of adult human vascular tissue were harvested by collagenase
treatment and gentle swabbing of the lumenal surface. Nutrient medium MCDB 107 containing a partially purified brain-derived
growth factor (5 μg/ml), epidermal growth factor (10 ng/ml) and only 2% (v/v) fetal bovine serum supported clonal and long-term
serial culture (17.6 to 26.1 cumulative population doublings) of endothelial cells from vena cava, thoracic aorta and tibial
arteries at a 70% rate of success. Cumulative doublings of the cell population from eight cultures were inversely proportional
to age of donor of the vascular tissue from which cells were isolated. Heparin had an enhancing effect on cell growth that
varied with cell strain. Prostacyclin production of human adult endothelial cell cultures was stimulated by aracidonate and
thrombin by 17 to 20 and 2 to 3-fold respectively. Endogenous and stimulated rates of prostacyclin production by human adult
endothelial cells were 2 to 3 times that of human adult smooth muscle cells and 20 to 30 times that of human fibroblasts.
The work was supported by Public Health Service Grant AGO3275 and Grant No. 1718 from the Council for Tobacco Research.
Editor's statement This paper provides an opportunity for relatively rapid, easy growth and cloning of endothelial cells from
various human specimens which are more difficult to deal with than those obtained from an intact artery or intact umbilical
vein. Russel Ross 相似文献
16.
Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes 总被引:11,自引:0,他引:11
Norimasa Sawada Akito Tomomura Carol A. Sattler Gerald L. Sattler Hynda K. Kleinman Henry C. Pitot 《In vitro cellular & developmental biology. Plant》1987,23(4):267-273
Summary Some effects of culturing adult rat hepatocytes on each of four different substrates—laminin (LN), collagen type I (C-I),
collagen type IV (C-IV), and fibronectin (FN)—have been investigated under defined conditions. No differential effect on the
attachment of the cells to the various substrates was noted; however, the spreading of hepatocytes shortly after initial plating
was most strikingly enhanced by FN, whereas LN exhibited little or no such enhancement. The two collagen substrates enhanced
the spreading of hepatocytes more than did LN, but less than FN. The different substrates had no differential effect on the
induction of tyrosine aminotransferase by dexamethasone and glucagon for at least the first 10 d in culture. The longevity
of the hepatocytes was not changed significantly by any of the substrates, at least through the 14th d of culture. During
the culture periods the hepatocytes at high cell density were maintained as confluent monolayers, regardless of the substrate
on which they had been cultured. After 14 d of culture, γ-glutamyltranspeptidase activity was highest in cells cultured on
C-IV, and lowest in those on FN. DNA synthesis in cultured hepatocytes at a low cell density was highest in cells cultured
on FN, with decreasing levels of this parameter in cells cultured on C-IV, C-I, and LN, respectively. These results demonstrate
that specific components of the extracellular matrix modulate both differentiated functions and the replication of hepatocytes
cultured in serum-free medium.
This work was supported in part by grants (CA-07175, CA-09135, CA-22484) from the National Cancer Institute, Bethesda MD.
N. Sawada was supported by a Cancer Research Campaign Grant D (U.K.) from the International Union Against Cancer. 相似文献
17.
Per E. Schwarze Anne E. Solheim Per O. Seglen 《In vitro cellular & developmental biology. Plant》1982,18(1):43-54
Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number
of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both
as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein
degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations,
and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at
least a week without any significant net loss of cells or cellular protein.
The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities.
An erratum to this article is available at . 相似文献
18.
Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model 总被引:6,自引:0,他引:6
Ries K Krause P Solsbacher M Schwartz P Unthan-Fechner K Christ B Markus PM Probst I 《In vitro cellular & developmental biology. Animal》2000,36(8):502-512
Summary The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal
bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal
(stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal
growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the
2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved
sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells
declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto
stromal cells precultured for 4–14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells.
Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes
and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte
differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in
cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate
carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated
urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30%
and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in
the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from
stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for
the investigation of stroma-derived differentiation factors. 相似文献
19.
Hiroyoshi Hoshi Mikio Kan Wallace L. McKeehan 《In vitro cellular & developmental biology. Plant》1987,23(10):723-732
Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on
human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase
mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented
medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth
factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed
that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported
attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined
as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically
stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte
number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could
be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned
medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein
(1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The
results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification
and characterization of additional normal hepatocyte growth factors.
This work was supported by NIH grant DK35310.
Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes.
In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that
factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional
growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate
that the malignant transformation of these cells may involve the production of autocrine growth stimulators. 相似文献
20.
Hepatocyte transplantation is considered an alternative to whole organ transplantation. However, the availability of human
cadaveric livers for the isolation of transplantation-quality hepatocytes is increasingly restricted. Xenogeneic porcine hepatocytes
may therefore serve as an alternate cell ressource. The propagation of hepatocytes is often necessary to yield a sufficient
cell number for downstream applications in xenotransplantation and in, for example, bioartificial liver support or pharmacological
and toxicological studies. Our goal has been to propagate primary porcine hepatocytes in vitro and to determine the functional
maintenance of the propagated cells. Porcine hepatocytes were cultured under serum-free conditions in the presence of hepatocyte
growth factor and epidermal growth factor and passaged several times. The viability, proliferation and maintenance of liver-specific
functions were determined as culture proceeded. Total cell number increased by 12-fold during four sequential passages, although
the proliferative capacity was higher in primary cells and early passages as compared with late passages. Xenobiotics metabolism
and urea synthesis gradually decreased with ongoing culture but could be restored by treatment with appropriate stimuli such,
as β-naphthoflavone and cAMP. The expression of hepatocyte-specific genes was generally lower at the beginning than at later
time-points of culture of individual passages. Porcine hepatocytes can thus be propagated in vitro. The partial loss of hepatocyte
function may be restored in vitro by appropriate stimuli. This may also be achieved in a recipient liver after hepatocyte
transplantation provided that the proper physiological environment for the maintenance of the differentiated hepatocyte phenotype
is present.
This study was supported by grants to B. Christ from the German Ministry of Education and Research (01 ZZ 0109 and NBL3-NG4)
as well as by grants from the Federal State of Saxonia-Anhalt through the Wilhelm-Roux-Program at the Medical Faculty of the
Martin-Luther-University of Halle-Wittenberg to B. Christ (09/07 and 04/03). 相似文献