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1.
Human epidermal keratinocytes (HEK) are skin cells of primary importance in maintaining the body’s defensive barrier and are
used in vitro to assess the irritation potential and toxicity of chemical compounds. Microfluidic systems hold promise for
high throughput irritant and toxicity assays, but HEK growth kinetics have yet to be characterized within microscale culture
chambers. This research demonstrates HEK patterning on microscale patches of Type I collagen within microfluidic channels
and maintenance of these cells under constant medium perfusion for 72 h. HEK were shown to maintain 93.0%–99.6% viability
at 72 h under medium perfusion ranging from 0.025–0.4 μl min−1. HEK maintained this viability while ∼100% confluent—a level not possible in 96 well plates. Microscale HEK cultures offer
the ability to precisely examine the morphology, behavior and viability of individual cells which may open the door to new
discoveries in toxicological screening methods and wound healing techniques. 相似文献
2.
Protocorm cultures of Dendrobium candidum were established in balloon type bubble bioreactors using Murashige and Skoog (MS) medium with 0.5 mg l−1 α-naphthaleneacetic acid (NAA), 2.5% (w/v) sucrose, 5:25 mM NH4:NO3 and 1% (v/v) banana homogenate for the production of biomass and bioactive compounds. In 3 l bioreactor containing 2 l medium, a maximum protocorm biomass (21.0 g l−1 dry biomass) and also optimum quantities of total polysaccharides (389.3 mg g−1 DW), coumarins (18.0 mg g−1 DW), polyphenolics (11.9 mg g−1 DW), and flavonoids (4.5 mg g−1 DW) were achieved after 7 weeks of culture. Based on these studies, 5 and 10 l bioreactor cultures were established to harvest 80 g and 160 g dry biomass. In 10 l bioreactors, the protocorms grown were accumulated with optimal levels of polysaccharides (424.1 mg g−1 DW), coumarins (15.8 mg g−1 DW), polyphenols (9.03 mg g−1 DW) and flavonoids (4.7 mg g−1 DW). The bioreactor technology developed here will be useful for the production of important bioactive compounds from D. candidum. 相似文献
3.
4.
I. C. Hsu M. M. Lipsky K. E. Cole C. H. Su B. F. Trump 《In vitro cellular & developmental biology. Plant》1985,21(3):154-160
Summary Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function.
Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intack lobe with collagenase or by digestion
of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 106 cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes,
632 pmol/mg protein of cytochromep
450 and 536. pmol/mg protein cytochromeb
5 were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells
maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes
in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired
DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture.
This work was supported by EPA Grants R-809835-01-1, R-809599010 and DOE Contract DE-A505-83ER60158. Cobtribution no. 1762
from the Cellular Pathobiology Laboratory, University of Maryland School of Medicine. 相似文献
5.
We present a simple technique for cell loading, culturing, and phenotypic study in a multi-chamber microfluidic device made of polydimethylsiloxane (PDMS). This technique is based on the use of degassing induced aspiration of PDMS which allows loading cells into micro-cavities within 1 min. A large number of triangle cavities are patterned aside main flow channels with narrow connections so that cells can be loaded by aspirating into each cavity. In our device, high throughput and long-term monitoring can be done with minimum shear force of the flow. As a demonstration, we show a controlled loading at single cell level and the phenotypic variation of gene expression of the yeast strain w303 as a function of copper ion concentration of the medium. 相似文献
6.
Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro 总被引:1,自引:0,他引:1
George E. Milo William B. Malarkey John E. Powell James R. Blakeslee David S. Yohn 《In vitro cellular & developmental biology. Plant》1976,12(1):23-30
Summary Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human
fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did
not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and
growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from
company C supported cell growth in media without serum.
Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported
cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting
sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per
ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth.
Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory
effect, implies that these hormones competitively regulate growth of responsive cells in vitro.
Supported in part by NIH-NCI-EC2074. 相似文献
7.
The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell culture 总被引:2,自引:0,他引:2
Fred H. Schindler Marsha A. Ose Michael Solursh 《In vitro cellular & developmental biology. Plant》1976,12(1):44-47
Summary This report describes a method for preparing primary cell cultures of differentiated rabbit sternal and human vertebral cartilage
cells. These cell cultures were shown to synthesize primarily α1 chains, which is taken to mean that at least 82% of the collagen
produced is cartilage specific collagen (type II).
This work was supported in part by grant HD-05505 from NIH. 相似文献
8.
An automated laboratory‐scale methodology for the generation of sheared mammalian cell culture samples
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Adrian Joseph Stephen Goldrick Michael Mollet Richard Turner Jean Bender David Gruber Suzanne S. Farid Nigel Titchener‐Hooker 《Biotechnology journal》2017,12(5)
Continuous disk‐stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing‐scale. The use of scale‐down methods to characterise disk‐stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot‐scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines. This paper describes an alternative and easy‐to‐assemble automated capillary‐based methodology to generate levels of EDRs consistent with those found in a continuous disk‐stack centrifuge. Variations in EDR were achieved through changes in capillary internal diameter and the flow rate of operation through the capillary. The EDRs found to match the levels of shear in the feed zone of a pilot‐scale centrifuge using the experimental method developed in this paper (2.4×105 W/Kg) are consistent with those obtained through previously published computational fluid dynamic (CFD) studies (2.0×105 W/Kg). Furthermore, this methodology can be incorporated into existing scale‐down methods to model the process performance of continuous disk‐stack centrifuges. This was demonstrated through the characterisation of culture hold time, culture temperature and EDRs on centrate quality. 相似文献
9.
Organ decellularization is emerging as a promising regenerative medicine approach as it is able to provide an acellular, three-dimensional biological scaffold material that can be seeded with living cells for organ reengineering. However this application is currently limited to donor-derived decellularized organs for reengineering in vitro and no study has been conducted for re-engineering the decellularized organ in vivo. We developed a novel technique of a single liver lobe decellularization in vivo in live animals. Using a surgical method to generate a by-pass circulation through the portal vein and infra-hepatic vena cava with a perfusion chamber system, we decellularized the single liver lobe and recellularized it with allogenic primary hepatocytes. Our results showed that the decellularization process in vivo can preserve the vascular structural network and functional characteristics of the native liver lobe. It allows for efficient recellularization of the decellularized liver lobe matrix with allogenic primary hepatocytes. Upon the re-establishment of blood circulation, the recellularized liver lobe is able to gain the function and the allogenic hepatocytes are able to secret albumin. Our findings provide a proof of principle for the in vivo reengineering of liver. 相似文献
10.
Julio C. Davila Chandrasekara G. Reddy Patrick J. Davis Daniel Acosta 《In vitro cellular & developmental biology. Plant》1990,26(5):515-524
Summary The present study was undertaken to assess and compare the toxic effects of papaverine hydrochloride and its metabolites.
Primary cell cultures of rat hepatocytes were treated with papavarine (papaver), 3′-O-desmethyl (3′-OH), 4′-O-desmethyl (4′-OH),
and 6-O-desmethyl (6-OH) papaverine at 1×10−5, 1×10−4, and 1×10−3
M for 4,8, 12, and 24-h periods. Cell injury was determined by: a) cell viability using the trypan blue exclusion test; b)
cytosolic enzyme leakage of lactate dehydrogenase and aspartate aminotransferase; c) morphologic alterations; and d) lactate:
pyruvate (L:P) ratios. Cell cultures showed concentration-and time-dependent responses. For example, a decrease in cell viability
and an increase in enzyme leakage were observed after cell treatment with 1×10−4 and 1×10−3
M papaver for 8 h; 1×10−3
M 6-OH papaverine for 8 h and 1×10−4
M for 24 h; and 1×10−3
M 4′-OH papaverine for 24 h (P<0.05). Furthermore, changes in morphology correlated to cell viability and enzyme release in those cultures treated with
papaver, 4′-OH and 6-OH papaverine. Some of these changes included size deformation, cell detachment from the dishes, and
cell necrosis. On the other hand, an increase in L:P ratios (P<0.05) was detected with papaver as early as 8 h with 1×10−4 and 1×10−3
M and 12 h with 1×10−5
M; 6-OH showed an increase, in L:P ratios at 8 h with 1×10−3
M and 12 h with 1×10−4
M; these changes were evident with 4′-OH at 12 h with 1×10−3
M. In contrast, cells treated with 3′-OH papaverine did not show significant damage with any time period and concentration
used in this study. The results of this study indicate that papaverine-derived metabolites are less cytotoxic than its parent
compound, papaver. The toxicity was ranked as follows: papaver>6-OH>4′-OH>−3′-OH.
This work was supported in part by grant ES04200-02 from the National Institute of Environmental Health Sciences, Bethesda,
MD.
Presented in part at the fall ASPET meeting in Salt Lake City, August, 1989. Daniel Acosta is a Burroughs Wellcome Scholar
in Toxicology. 相似文献
11.
Kazunori Furukawa Yohichi Mochizuki Norimasa Sawada 《In vitro cellular & developmental biology. Plant》1984,20(7):573-584
Summary Alterations in peroxisomes and catalase activity and their responsiveness to clofibrate in adult rat hepatocytes in primary
culture were investigated.
The numbers of peroxisomes with and without crystalloid nucleoids per unit cytoplasmic area were preserved in cultured hepatocytes
for 2 d after seeding at a level comparable to that of freshly isolated hepatocytes. At Day 3 in culture, the number of anucleoid
peroxisomes was reduced in untreated hepatocytes, accompanied by more significant reduction in the number of nucleoid-containing
peroxisomes, which decreased until Day 5. Peroxisome diameters were reduced in untreated hepatocytes at Day 2 and this decrease
in the diameter was continued until Day 7. Catalase activity in untreated hepatocytes decreased markedly with culture age.
The number of anucleoid peroxisomes was significantly greater in hepatocytes treated with 2mM clofibrate in culture than in freshly isolated hepatocytes for 2 d or in untreated hepatocytes of the same culture age through
7 d. The number of nucleoid-containing peroxisomes in the treated cells began to decrease in 3 d, but was greater than that
of untreated cells at Days 3 and 5. Peroxisomes with well-developed nucleoids were observed frequently in the treated cells
even at Day 7. Peroxisome diameters were greater in the treated cells than in untreated cells at Days 3, 5, and 7. Catalase
activity was always higher in the treated cells than in untreated cells. These results suggest that clofibrate is effective
in inducing peroxisome proliferation as well as in maintaining the organelles in cultured hepatocytes.
This work was supported in part by Grants-in-Aid for Scientific Research from Ministry of Education, Science and Culture,
Japan, 448143, 50168, 501069, and 577196, and by a Grant-in-Aid from Hokkaido Geriatrics Research Institute. 相似文献
12.
Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor
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Qasim A. Rafiq Mariana P. Hanga Thomas R. J. Heathman Karen Coopman Alvin W. Nienow David J. Williams Christopher J. Hewitt 《Biotechnology and bioengineering》2017,114(10):2253-2266
Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum‐free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253–2266. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. 相似文献
13.
The ability of polycyclic aromatic hydrocarbons and glucocorticoids to regulate monooxygenase activity of human fetal liver has been studied using hepatocytes prepared by collagenase digestion of liver samples from human abortuses of 13 to 19 weeks of gestational age, and maintained in primary monolayer culture for periods up to 5 days. Addition of 1,2-benzanthracene to the cells caused an increase in monooxygenase activity (3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin) in a time-and concentration-dependent fashion. The concentration of 1,2-benzanthracene required to achieve half-maximal induction was 5 microM. The inductive effect of the polycyclic hydrocarbon was potentiated approximately 2.5-fold when dexamethasone (250 nM) or other glucocorticoids were included in the culture medium. Dexamethasone alone had little or no effect on the induction of monooxygenase activity. The concentration of dexamethasone required for half-maximal stimulation of monooxygenase activity in the presence of 1,2-benzanthracene was 5-10 nM, and the action of dexamethasone was reversed by the addition of cortisol 21-mesylate, consistent with the concept that the action of dexamethasone was mediated by binding to a glucocorticoid receptor. These results are suggestive that glucocorticoids, which are produced by the fetal adrenal and have an important role in the regulation of fetal development, act synergistically with polycyclic aromatic hydrocarbons to induce the activity of liver monooxygenases in the human fetus. 相似文献
14.
15.
Ying Luo Cheng Lou Sui Zhang Zhengyan Zhu Qianzhe Xing Peng Wang Tong Liu Hui Liu Chenglong Li Wenxia Shi Zhi Du Yingtang Gao 《Cytotherapy》2018,20(1):95-107
Background aims
Human induced pluripotent stem cells (hiPSCs) are becoming increasingly popular in research endeavors due to their potential for clinical application; however, such application is challenging due to limitations such as inferior function and low induction efficiency. In this study, we aimed to establish a three-dimensional (3D) culture condition to mimic the environment in which hepatogenesis occurs in vivo to enhance the differentiation of hiPSCs for large-scale culture and high throughput BAL application.Methods
We used hydrogel to create hepatocyte-like cell (HLC) spheroids in a 3D culture condition and analyzed the cell-behavior and differentiation properties of hiPSCs in a synthetic nanofiber scaffold.Results
We found that treating cells with Y-27632 promoted the formation of spheroids, and the cells aggregated more rapidly in a 3D culture condition. The ALB secretion, urea production and glycogen synthesis by HLCs in 3D were significantly higher than those grown in a 2-dimensional culture condition. In addition, the metabolic activities of the CYP450 enzymes were also higher in cells differentiated in the 3D culture condition.Conclusions
3D hydrogel culture condition can promote differentiation of hiPSCs into hepatocytes. The 3D culture approach could be applied to the differentiation of hiPSCs into hepatocytes for bioartificial liver. 相似文献16.
M. E. Bouma M. Pessah G. Renaud N. Amit D. Catala R. Infante 《In vitro cellular & developmental biology. Plant》1988,24(2):85-90
Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a
serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of
the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies
directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system
revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein
fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density
lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining.
VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles
isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein.
The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation
of [3H]glycerol, more than 92% of the [3H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures
of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein
metabolism in human liver. 相似文献
17.
M. Sensenbrenner J. Treska-Ciesielski Z. Lodin P. Mandel 《Cell and tissue research》1970,106(4):615-626
Summary Dissociated cells from 9, 12 and 15 day-old chick embryo spinal ganglia were cultivated in presence of total embryo-extract, brain embryo extract, or total embryo extract supplemented with purified nerve growth factor (NGF). The cells were maintained during 4 days in Maximow assembly and during 1 month in Rose chamber. Neurons showed growth of nerve fibres. The non-neural cells evolved to spindle cells, Schwann cells, or fibroblasts.Ribonucleic acid (RNA) synthesis was followed with tritiated uridine by autoradiography. Some nerve cells showed tritiated uridine incorporation. The highest incorporations for short-term cultures were at 15 hours in presence of NGF, at 48 hours in presence of total or brain extract, and for long-term cultures at 8 days. These periods corresponded to the highest growing activity of the nerve fibres. After 4 days all the non-neural cells incorporated tritiated uridine.The tritiated uridine was first incorporated into the RNA of the nucleus and, afterwards was found also in the cytoplasm. The presence of brain extract or of NGF stimulates the incorporation of labelled uridine into RNA. No labelling was found in the nerve fibres, even after 4 hours incubation.Chargée de Recherche au C.N.R.S.This communication is a part of the Doctorat és-Sciences thesis, presented by Mrs. J. Treska-Ciesielski.With the technical assistance of Mrs. M. F. Knoetgen and A. Bieth. 相似文献
18.
Matsunaga T Maruyama M Harada E Katsuyama Y Sugihara N Ise H Negishi N Ikeda U Ohmori S 《Biochemical and biophysical research communications》2004,318(2):428-434
CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR. 相似文献
19.
Incidents with single cells and their genesis have not been the major focus of science up to now. This fact is supported by
the difficulties one faces when wanting to monitor and cultivate small populations of cells in a defined compartment under
controlled conditions, in vitro. Several approaches of up- and down-scaling have often led to poorly understood results which
might be better elucidated by understanding the cellular genesis as a function of its microenvironment. This review of the
approaches of scale-up and scale-down processes illustrates technical possibilities and shows up their limitations with regard
to obtainable data for the characterisation of cellular genesis and impact of the cellular microenvironment. For example,
stem cell research advances underline the lack of information about the impact of the microenvironment on cellular development.
Finally, a proposal of future research efforts is given on how to overcome this lack of data via a novel bioreactor setup. 相似文献
20.
The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified
and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free
medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics
of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed
in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media
to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell
specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension
culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free
condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII
in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in
a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture
system with a spin filter is employed.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献