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1.
Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers 总被引:4,自引:0,他引:4
Geraldine A. Hamilton Carl Westmoreland Elisabeth George 《In vitro cellular & developmental biology. Animal》2001,37(10):656-667
Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial. 相似文献
2.
Doo-Hoon Lee Hee-Hoon Yoon Ji-Hyun Lee Kwang-Woong Lee Suk-Koo Lee Sung-Koo Kim Jung-Eun Choi Young-Jin Kim Jung-Keug Park 《Biochemical Engineering Journal》2004,20(2-3):181-187
The performance of an extracorporeal bioartificial liver (BAL) support system depends on the functional activities of the hepatocytes immobilized in the system. One of the most promising techniques in retaining liver-specific functions is co-culturing hepatocytes with other cell types, such as epithelial cells, endothelial cells and dermal fibroblasts. Primary rat hepatocytes were suspension co-cultured with rat prostate endothelial cell line (RPEn) for 20 h in a spinner vessel to form hetero-spheroids, which contain the two types of the cells, i.e., hepatocytes and endothelial cells in the same spheroid. For the subsequent culture, the hetero-spheroids were entrapped in a Ca-alginate gel bead. From the results of incorporation efficiency test, it was found that RPEn cells have a significantly higher attachment affinity to hepatocytes than human dermal fibroblast and rat liver epithelial cells. We clearly found out that RPEn cells located on the surface of the hepatocyte spheroids from immunostained paraffin sections of the hetero-spheroids. Identical with in vivo liver tissue, laminin was stained at the surface of the hetero-spheroids. Ultrastructures of liver tissue, such as bile canaliculus-like and Disse’s space-like structures, were also found at the surface of the hetero-spheroids. In vivo liver tissue, in which hepatocytes were covered with sinusoidal endothelial cells, was partly mimicked by the endothelial cell-covered hepatocyte spheroids. And the hetero-spheroids showed significantly higher and stable albumin secretion and ammonia removal activities than pure spheroids for 12 days of observations.
Therefore, the endothelial cell-covered hepatocyte hetero-spheroids may offer a useful study model of epithelial–mesenchymal interactions and information about liver tissue engineering research as well as a substitute of a cell source of a BAL system. 相似文献
3.
The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form). A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells. In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc). Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device. 相似文献
4.
Ayse Karababa Katerina Groos-Sahr Ute Albrecht Verena Keitel Aygul Shafigullina Boris Görg Dieter Häussinger 《Neurochemical research》2017,42(3):737-749
Hepatic encephalopathy (HE) is associated with cerebral microglia activation. Ammonia, a major toxin of HE, activates microglia in vitro but does not trigger pro-inflammatory cytokine synthesis. In the present study we analysed effects of ammonia on lipopolysaccharide (LPS)-induced upregulation of microglia activation and cytokine mRNA as well as on cytokine secretion in mono-cultured microglia and co-cultured astrocytes and microglia. In mono-cultured microglia LPS (100 ng/ml, 18 h) strongly elevated mRNA levels of the microglia activation marker CD14 and the pro-inflammatory cytokines IL-1α/β, IL-6 and TNF-α. NH4Cl (5 mmol/l) had no effect on LPS-induced upregulation of CD14, IL-1α/β and IL-6 mRNA but enhanced LPS-induced upregulation of TNF-α mRNA in mono-cultured microglia. In co-cultured astrocytes and microglia, however, LPS-induced upregulation of IL-1α/β, TNF-α, IL-6, CD14 but not of IL-10, IL-12A/B or TGFβ1?3 mRNA was attenuated by NH4Cl. LPS-induced upregulation of IL-1α/β, IL-6 and TNF-α was also diminished by the TGR5-ligands allopregnanolone and taurolithocholic acid in mono-cultured microglia. NH4Cl also attenuated LPS-induced release of MCP-1, IL-6 and IL-10 in mono-cultured microglia. mRNA level of surrogate marker for microglia activation (CD14) and for the anti-inflammatory M2-type microglia (CD163, CXCL1, CXCL2) were also elevated in post mortem brain tissue taken from the fusiforme gyrus of patients with liver cirrhosis and HE. The findings suggest that ammonia attenuates LPS-induced microglia reactivity in an astrocyte-dependent way. One may speculate that these anti-inflammatory effects of ammonia may be triggered by neurosteroids derived from astrocytes and may account for absence of microglia reactivity in cerebral cortex of cirrhotic patients with HE. 相似文献
5.
We have developed an in vitro hepatocyte-adipose tissue explant (ATE) co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal adipose tissues. Expressions of inflammation related genes (IL-6, TNF-α, COX-2) were higher in the inguinal than the epididymal ATE. Similarly, expressions of marker genes of macrophage and monocyte (MPEG-1, CD68, F4/80, CD64) were higher in the stromal vascular fraction (SVF) isolated from inguinal ATE than that from epididymal ATE. However, expressions of lipolysis related genes (ATGL, HSL, perilipin-1) were higher in the epididymal adipocytes than inguinal adipocytes. Moreover, secretion of IL-6 and PGE(2) was higher from inguinal ATEs than from epididymal ATEs. There was a trend that the total levels of IL-6, TNF-α and PGE(2) in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2). Lipolysis, measured as glycerol release, was similar in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited a stronger cytotoxic response and higher level of insulin resistance in the co-cultured hepatocytes. In conclusion, our results reveal depot-dependent effects of ATEs on co-cultured primary hepatocytes, which in part may be related to a more pronounced infiltration of stromal vascular cells (SVCs), particularly macrophages, in inguinal adipose tissue resulting in stronger responses in terms of hepatotoxicity and insulin-resistance. 相似文献
6.
Keun-Hong Park Ju Young Park You Han Bae 《Biotechnology and Bioprocess Engineering》2001,6(4):264-268
Aggregates of specific cells are often regarded as a better form in artificial organs and mammalian cell bioreactors in terms
of cell-specific functionality. In this study, the morphology and liver-specific functions of freshly harvested primary rat
hepatocytes, which were cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were
examined and compared to a control (hepatocytes in single cell form). A copolymer of N-isopropylacrylamide (98 mole % in feed)
and acrylic acid (poly(NiPAAm-co-AAc)), a thermo-reversible copolymer gel matrix, was used to entrap hepatocytes either in
spheroids or single cells. During a 7-day culture period, the spheroids maintained higher viability and produced albumin and
urea at a relatively constant rate, while the single cell culture showed a slight increase in cell numbers and a reduction
in albumin secretion. Hepatocytes cultured as spheroids present a potentially useful three-dimensional cell culture system
for application in a bioartificial liver device. 相似文献
7.
Hollow fiber bioartificial liver utilizing collagen-entrapped porcine hepatocyte spheroids 总被引:2,自引:0,他引:2
Wu FJ Friend JR Lazar A Mann HJ Remmel RP Cerra FB Hu WS 《Biotechnology and bioengineering》1996,52(1):34-44
A xenogeneic hollow fiber bioreactor utilizing collagen-entrapped dispersed hepatocytes has been developed as an extracorporeal bioartificial liver (BAL) for potential treatment of acute human fulminant hepatitis. Prolonged viability, enhanced liver-specific functions, and differentiated state have been observed in primary porcine hepatocytes cultivated as spheroids compared to dispersed hepatocytes plated on a monolayer. Entrapment of spheroids into the BAL can potentially improve performance over the existing device. Therefore, studies were conducted to evaluate the feasibility of utilizing spheroids as the functionally active component of our hybrid device. Confocal microscopy indicated high viability of spheroids entrapped into cylindrical collagen gel. Entrapment of spheroids alone into collagen gel showed reduced ability to contract collagen gel. By mixing spheroids with dispersed cells, the extent of collagen gel contraction was increased. Hepatocyte spheroids collagen-entrapped into BAL devices were maintained for over 9 days. Assessment of albumin synthesis and ureagenesis within a spheroid-entrapment BAL indicated higher or at least as high activity on a per-cell basis compared to a dispersed hepatocyte-entrapment BAL device. Clearance of 4-methylumbelliferone to its glucuronide was detected throughout the culture period as a marker of phase II conjugation activity. A spheroid-entrapment bioartificial liver warrants further studies for potential human therapy. (c) 1996 John Wiley & Sons, Inc. 相似文献
8.
Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm. 相似文献
9.
Kohji Nakazawa Hiroshi Mizumoto Mitsuru Kaneko Hiroyuki Ijima Tomonobu Gion Mitsuo Shimada Ken Shirabe Kenji Takenaka Keizo Sugimachi Kazumori Funatsu 《Cytotechnology》1999,31(1-2):61-68
Porcine hepatocytes are used in the hybrid artificial liver support system that we are developing because of their high level
of liver functions in vitro and because human hepatocytes can not be used in Japan for ethical reasons. Spherical multicellular
aggregates or spheroids have been found to be effective in vitro for long-term maintenance of liver functions. Therefore,
we formed spherical multicellular aggregates (spheroids) of primary porcine hepatocytes using a polyurethane foam (PUF) as
a culture substratum and analyzed their drug metabolic functions in vitro. Primary porcine hepatocytes inoculated into the
pores of a flat PUF plate (25 × 25 × 1 mm), spontaneously formed spheroids within the range of 100 to 150 μm in diameter 24
to 36 h after inoculation. The formed spheroids were attached to the bottom surface of the PUF pores, and their morphology
and viability were maintained for more than 12 days. The P-450 activity in the spheroids of porcine hepatocytes was demonstrated
by detecting production of monoethylglycinexylidide from lidocaine. In addition, the conjugation enzyme activity was demonstrated
by detecting glucuronidation and sulfation of acetaminophen. These activities were maintained for 12 days at a level twice
as high as in the monolayer culture. This result shows that the porcine hepatocyte spheroids formed by using PUF can maintain
the drug metabolic functions important in a hybrid artificial liver device. Consequently, culturing porcine hepatocyte spheroids
using PUF seems to be promising for development of a hybrid artificial liver.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
10.
Our previous study found that co-culture with human vascular endothelial cells (HMVECs) is beneficial for dorsal root ganglion cells (DRGCs). The goal of the present study is to investigate whether co-culture with HMVECs could promote the development of DRGCs, and whether this effect is induced by the secretion of BDNF by HMVECs. DRGCs were mono-cultured, co-cultured with HMVECs or co-cultured with HMVECs that pre-transfected with BDNF siRNA, the expression of neurite formation and branching factors were determined. The results showed that transfecting with BDNF siRNA inhibited BDNF expression and reduced BDNF secretion. Co-culture with HMVECs increased the expression of Etv4, Etv5, FN-L, FN-M, and GAP-43 in DRGCs that accompanied by the activation of ERK pathway. However, these changes were all reversed by the inhibition of BDNF in HMVECs. In conclusion, our data demonstrate that HMVECs potentiated DRGCs development at least partly by the secretion of BDNF in the co-culture system. 相似文献
11.
Hepatocyte spheroids and hepatocyte were immobilized in chitosan/alginate capsules formed by the electrostatic interactions between chitosan and alginate. After encapsulation, there was a 10% decrease in the viability of spheroids due to the exposure of the cells to a pH 6 during the encapsulation process. However, the encapsulated hepatocyte spheroids maintained over 50% viability and liver specific functions for 2 weeks while the encapsulated hepatocytes, free hepatocytes and free hepatocyte spheroids showed low viability and liver specific functions. Therefore, encapsulated hepatocyte spheroid might be applied to the development of a bioartificial liver. 相似文献
12.
Wolfgang Metzger Barbara Rösch Daniela Sossong Monika Bubel Tim Pohlemann 《Cell biology international》2021,45(8):1633-1643
Spheroids are a promising tool for many cell culture applications, but their microscopic analysis is limited. Flow cytometry on a single cell basis, which requires a gentle but also efficient dissociation of spheroids, could be an alternative analysis. Mono-culture and coculture spheroids consisting of human fibroblasts and human endothelial cells were generated by the liquid overlay technique and were dissociated using AccuMax as a dissociation agent combined with gentle mechanical forces. This study aimed to quantify the number of apoptotic and proliferative cells. We were able to dissociate spheroids of differing size, age, and cellular composition in a single-step dissociation protocol within 10 min. The number of single cells was higher than 95% and in most cases, the viability of the cells after dissociation was higher than 85%. Coculture spheroids exhibited a higher sensitivity as shown by lower viability, higher amount of cellular debris, and a higher amount of apoptotic cells. Considerable expression of the proliferation marker Ki67 could only be seen in 1-day-old spheroids but was already downregulated on Day 3. In summary, our dissociation protocol enabled a fast and gentle dissociation of spheroids for the subsequent flow cytometric analysis. The chosen cell type had a strong influence on cell viability and apoptosis. Initially high rates of proliferative cells decreased rapidly and reached values of healthy tissue 3 days after generation of the spheroids. In conclusion, the flow cytometry of dissociated spheroids could be a promising analytical tool, which could be ideally combined with microscopic techniques. 相似文献
13.
Taku Matsushita Kohji Nakano Yasufumi Nishikura Kenta Higuchi Akifumi Kiyota Ryuichi Ueoka 《Cytotechnology》2003,42(2):57-66
Primary human fetal hepatocytes proliferated in monolayer culture up to the 9th passage. During proliferation, the cells changed
their morphology from a fibroblast-like shape after inoculation to an epithelia-like polygonal shape after they reached confluence.
The proliferation was associated with the loss of ammonia detoxification capacity, which is essential for the function of
bioartificial liver. The cells formed spheroids on a poly-glutamic acid- or poly-aspartic acid-coated polystyrene dish that
had a negatively charged surface at neutral pH. However, the cells did not form spheroids on a poly-lysine- or poly-arginine-coated
dish that had a positively charged surface, which is reportedly suitable to form spheroids for adult hepatocytes. The activity
of cytochrome P450 (CYP 1A1, CYP1A2) of the cells in spheroid culture was about twice as high as that of the cells in monolayer
culture. The ammonia detoxification activity of the cells was restored in spheroid culture by treatment with 2% dimethylsulfoxide.
These results suggest that the conditions for human fetal hepatocytes to form spheroids are different from that for adult
hepatocytes, and the use of poly-glutamic acid or poly-aspartic acid coating may improve spheroid culture of proliferative
human fetal hepatocytes.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
14.
Spheroid formation and differentiation into hepatocyte-like cells of rat mesenchymal stem cell induced by co-culture with liver cells 总被引:9,自引:0,他引:9
Mesenchymal stem cells (MSCs) derived from bone marrow have been shown to differentiate into hepatocytes, which would be an ideal resource for transplantation or artificial liver devices. Here we investigated the efficiency of co-culture system consisting of rat MSCs and adult liver cells to induce differentiation of MSCs into hepatocyte-like cells. Marked MSCs were either co-cultured with freshly isolated liver cells or treated with hepatocyte growth factor (HGF) for 21 days. In co-culture systems, MSCs formed spheroids of round-shaped cells while keeping normal proliferation and viability, strongly expressed albumin, alpha-fetoprotein, and cytokeratin-18 in mRNA and protein level from day 3 to 21. As a control, MSCs treated with HGF showed weak gene expressions in day 14 and had a few cells of protein staining in day 21. These results indicate that the co-culture microenvironment plays a decisive role for the hepatic differentiation of MSCs, and it is more efficient than HGF treatment. Insights gained from this study will be helpful to design optimal culture systems for the hepatic differentiation of human MSCs and the hepatic function maintenance of hepatocytes in vitro. 相似文献
15.
Tanya Debnath Sutapa Ghosh Usha Shalini Potlapuvu Lakshmi Kona Suguna Ratnakar Kamaraju Suprabhat Sarkar Sumanlatha Gaddam Lakshmi Kiran Chelluri 《PloS one》2015,10(3)
Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate. 相似文献
16.
Hu WS Friend JR Wu FJ Sielaff T Peshwa MV Lazar A Nyberg SL Remmel RP Cerra FB 《Cytotechnology》1997,23(1-3):29-38
Liver failure is a major cause of mortality. A bioartificial liver (BAL) employing isolated hepatocytes can potentially provide temporary support for liver failure patients. We have developed a bioartificial liver by entrapping hepatocytes in collagen loaded in the luminal side of a hollow fiber bioreactor. In the first phase of development, liver-specific metabolic activities of biosynthesis, biotransformation and conjugation were demonstrated. Subsequently anhepatic rabbits were used to show that rat hepatocytes continued to function after the BAL was linked to the test animal. For scale-up studies, a canine liver failure model was developed using D-galactosamine overdose. In order to secure a sufficient number of hepatocytes for large animal treatment, a collagenase perfusion protocol was established for harvesting porcine hepatocytes at high yield and viability. An instrumented bioreactor system, which included dissolved oxygen measurement, pH control, flow rate control, an oxygenator and two hollow fiber bioreactors in series, was used for these studies. An improved survival of dogs treated with the BAL was shown over the controls. In anticipated clinical applications, it is desirable to have the liver-specific activities in the BAL as high as possible. To that end, the possibility of employing hepatocyte spheroids was explored. These self-assembled spheroids formed from monolayer culture exhibited higher liver-specific functions and remained viable longer than hepatocytes in a monolayer. To ease the surface requirement for large-scale preparation of hepatocyte spheroids, we succeeded in inducing spheroid formation in stirred tank bioreactors for both rat and porcine hepatocytes. These spheroids formed in stirred tanks were shown to be morphologically and functionally indistinguishable from those formed from a monolayer. Collagen entrapment of these spheroids resulted in sustaining their liver-specific functions at higher levels even longer than those of spheroids maintained in suspension. For use in the BAL, a mixture of spheroids and dispersed hepatocytes was used to ensure a proper degree of collagen gel contraction. This mixture of spheroids and dispersed cells entrapped in the BAL was shown to sustain the high level of liver-specific functions. The possibility of employing such a BAL for improved clinical performance warrants further investigations. 相似文献
17.
Primary hepatocytes of small animals such as rat and rabbit were often used for the study of extracorporeal liver support
systems. Freshly isolated rat hepatocytes form spheroids within two days when cultivated as suspension in spinner vessels.
These spheroids showed enhanced liver specific functions and more differentiated morphology compared to hepatocytes cultured
as monolayers. However, shear stress caused by continuous agitation deteriorated spheroids gradually. In this work we immobilized
spheroids to prolong liver specific activities. First, hepatocyte spheroids were suspended in collagen solution containing
calcium chloride and then dropped into alginate solution. A thin layer of calcium alginate was formed around the droplet and
then was removed after the inner collagen was gelled by treatment of sodium citrate buffer. Spheroids embedded in collagen-gel
bead maintained liver specific functions such as albumin secretion rate longer than hepatocyte spheroids exposed to shear
stress. Therefore, we suggest that this immobilization technique may offer an effective long-term hepatocyte cultivation and
facilitate the development of a bioartificial liver support device. 相似文献
18.
Sung Mun Yang Doo Hoon Lee Jung Keug Park 《Biotechnology and Bioprocess Engineering》2000,5(2):99-105
Cell-cell interaction and the extracellular matrix (ECM) are believed to play essential roles duringin vitro culturing of primary hepatocytes in the control of differentiation and in the maintenance of tissue specific functions. The
objective of this study was to examine the effects of degree of cell-cell contact (DCC) on liver specific function of rat
primary hepatocytes. Hepatocyte aggregates with various degrees of cell-cell contact,i.e., dispersed cells, longish aggregate, rugged aggregate, and smooth spheroid were obtained at 1, 5–6, 15–20, and 36–48 hrs,
respectively in suspension cultures grown in spinner flasks embedded in Caalginate bead and collagen gel in order. The smooth
spheroids displayed a decrease in viability and functional activities. This may result from mass transfer limitation and shear
damage caused by agitation during aggregation. The rugged aggregate showed a higher viability and albumin secretion rate than
the dispersed cells or the other aggregates. This result indicates the possible enhancement of a bioartificial liver's (BAL)
performance using primary hepatocytes and the reduction in time to prepare a BAL through optimization of the immobilization
time. 相似文献
19.
Xiaoling Guo Rongjie Yu Ying Xu Ruiling Lian Yankun Yu Zekai Cui Qingshan Ji Junhe Chen Zhijie Li Hongwei Liu Jiansu Chen 《Journal of cellular and molecular medicine》2016,20(5):874-890
Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. However, little is known about its presence or effects in human adipose‐derived stem cells (hADSCs). In this study, the expression of PACAP type I receptor (PAC1R) was first confirmed in hADSCs. Maxadilan, a specific agonist of PAC1R, could increase hADSC proliferation as determined by Cell Counting Kit‐8 and cell cycle analysis and promote migration as shown in wound‐healing assays. Maxadilan also showed anti‐apoptotic activity in hADSCs against serum withdrawal‐induced apoptosis based on Annexin V/propidium iodide analysis and mitochondrial membrane potential assays. The anti‐apoptotic effects of maxadilan correlated with the down‐regulation of Cleaved Caspase 3 and Caspase 9 as well as up‐regulation of Bcl‐2. The chemical neural differentiation potential could be enhanced by maxadilan as indicated through quantitative PCR, Western blot and cell morphology analysis. Moreover, cytokine neural redifferentiation of hADSCs treated with maxadilan acquired stronger neuron‐like functions with higher voltage‐dependent tetrodotoxin‐sensitive sodium currents, higher outward potassium currents and partial electrical impulses as determined using whole‐cell patch clamp recordings. Maxadilan up‐regulated the Wnt/β‐catenin signalling pathway associated with dimer‐dependent activity of PAC1R, promoting cell viability that was inhibited by XAV939, and it also activated the protein kinase A (PKA) signalling pathway associated with ligand‐dependent activity of PAC1R, enhancing cell viability and neural differentiation potential that was inhibited by H‐89. In summary, these results demonstrated that PAC1R is present in hADSCs, and maxadilan could enhance hADSC viability and neural differentiation potential in neural differentiation medium. 相似文献
20.
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