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1.
An alternative culture system has been developed based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can easily be scaled up from one to several chambers. The shape and the size of the area of cell growth may be designed to individual experimental demands. The whole culture chamber is optically accessible, so cell growth and morphology can be evaluated by light microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. A protocol for vitality tests using calcein-AM is worked out for an adherent cell line and for a suspension cell line. The lower detection limits are 7 × 102 cells cm-2 for the adherent cells and 5 × 104 cells mL-1 for the suspension cells. The upper limits are 1–2 × 105 cells cm-2 respectively 8 × 106 cells mL-1. 相似文献
2.
The potential of a DNA content assay, PicoGreen, for use in 3D bioengineered constructs was examined. The assay was tested
on ATDC5 cells in situ during culture in typical tissue engineering 3D constructs. Comparisons of cell standards from cell
lines and primary cells to λDNA standards was also conducted. An effective working range of the assay within 3D constructs was shown up to 2.5 × 105 cells ml−1. From significant variation found in DNA content between cell lines and primary cells, it was concluded that the most accurate
standard to use for the assay was from the cell type being examined. 相似文献
3.
Brady MS Lee F Petrie H Eckels DD Lee JS 《Cancer immunology, immunotherapy : CII》2000,48(11):621-626
Purpose: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon γ (IFNγ).
We have previously demonstrated that peptide-specific CD4+ T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce
interleukin-10 (IL-10) and (IFNγ). We asked whether the responding T cells kill the tumor cells and, if so, whether direct
cell contact was required. Methods: Two HLA class II+ melanoma cell lines derived from metastases were co-cultured with a human CD4+ T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma
cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked
immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized
and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required
for tumor cell death. Results: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNγ and IL-10 by the responding T cells. Direct T cell contact with
tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact
with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking
antibody to IFNγ but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays.
Conclusions: Peptide-specific CD4+ T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class
II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed
CD4+ T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so
with IFNγ.
Received: 1 July 1999 / Accepted: 17 September 1999 相似文献
4.
Kh. S. Vishniakova I. I. Kireev A. Ya. Dunina-Barkovskaya 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2011,5(4):355-363
Cell culture density is shown to alter the parameters characterizing phagocytic activity of cells in vitro. Phagocytosis index
(PI, mean number of beads per cell in the bead-containing population) and phagocytosis percent (PP, percentage of bead-containing
cells in cell population under study) for IC-21 macrophages incubated in the presence of non-opsonized 2-μm fluorescent latex
beads were determined using fluorescent microscopy and ImageJ software specially adapted for the purpose. Under control conditions
(DMEM without serum), increase in cell culture density was accompanied with a decrease of both parameters of the phagocytic
activity. At a mean density of 4 cells/105 μm2 (9 cells per a viewfield) PI was 7.1 ± 0.2 beads/cell and at 20 cells/105 μm2 (40 cells per a viewfield) PI dropped to 4.6 ± 0.1 beads/cell. PP was less sensitive, varied in the range of 95–100% but
also decreased as the cell density grew. At any density, PI was 1.5–2 times higher than the expected value (number of beads
per μm2 × cell contour area); apparently this divergence can be accounted for by cell locomotion and capture of a larger number of
beads than could drop onto a motionless cell with a constant contour area. Increase in cell density was also accompanied by
a decrease of the cell contour area (S
c), which amounted to 750 ± 16 μm2 at a density of 4 cells/105 μm2 and 346 ± 4 μm2 at a density of 20 cells/105 μm2. As the bead concentration was the same in all experiments, density-dependent decrease in PI and PP may be related with the
observed decrease in cell contour area. Yet, the bead number per cell area unit (PI/S
c) was bigger at higher density and PI/S
c was higher in cells with smaller S
c. Thus, individual (specific) activity of the cells did not lessen with an increase of the cell culture density in the range
studied (4–20 cells/105 μm2). Reduction of the cell contour area may reflect alteration in cell adhesion to the substrate as well as competitive relations
between adhesion and phagocytic processes. The data obtained imply that cell culture density has to be controlled as a factor
notably altering the phagocytic activity parameters. The effects of serum, methyl-β-cyclodextrin, and carbenoxolon reported
earlier [Golovkina et al. 2009. Biol membrany. 26 (5), 379–386] are re-evaluated and confirmed here. 相似文献
5.
A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor 总被引:1,自引:0,他引:1
Samia Rourou Arno van der Ark Samy Majoul Khaled Trabelsi Tiny van der Velden Héla Kallel 《Applied microbiology and biotechnology》2009,85(1):53-63
Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work.
Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent
experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of
3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h−1 were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus
production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI),
regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the
cells were infected at an MOI of 0.1; this level was equal to 107 FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should
be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor
pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1
microcarriers. An acceptable level of cell density (cell density level of 1.5 × 106 cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode
is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch
from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance.
A cell density level of 5.5 × 106 cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation
culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 107 FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements. 相似文献
6.
Lily I. Huschtscha Frances C. Lucibello Walter F. Bodmer 《In vitro cellular & developmental biology. Plant》1989,25(1):105-108
Summary A new method has been developed to count cells “in situ”, based on a fluorogenic enzyme assay that measures the activity of
alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship
did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers
over a range from about 104 to 5×105 for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and
HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a
useful method for studying growth assays.
Editor's Statement This paper describes a quick method for quantitation of cell number in microcultures. Such procedures are
valuable for the many situations in which minimizing cells and medium volume is desirable, although somewhat specialized equipment
is required for the procedure. An alternative procedure for quantitation of cells in microtiter culture appeared previously
in this journal (McCaffrey, et al., 24∶247–252). 相似文献
7.
Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell
growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate
reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth
in 0, 10−7, and 10−6 M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were
used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal
expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10−7 M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10−6 M MTX was 20% lower than that of recombinant CHO cells at 10−7 M MTX. There was no effect of 10−5 M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression. 相似文献
8.
Brillet K da Conceição MM Pattus F Pereira CA 《Bioprocess and biosystems engineering》2006,28(5):291-293
The paper describes a recombinant Schneider 2 (rS2) cell culture and protein expression in a bioreactor. S2 cells were transfected with a plasmid containing a fusion protein (human μ opioid receptor, hMOR, and green fluorescent protein, EGFP) under the control of inducible metallothionein promoter. A bioprocess in a bioreactor with 5% dissolved oxygen, 27°C and 120 rpm enabled the cell culture to attain 5.3×107 viable cells/mL at 96 h. The induction decreased the cell multiplication (2.5×107 viable cells/mL at 72 h). Glutamine and glucose and low levels of lactate were consumed. A fast recombinant protein synthesis took place and, at 6 h of induction, 2×104 receptors/cell could be detected by a functional binding assay. Fluorescence measurements showed a progressive increase of recombinant protein expression with a maximal value of 1.26×105 fluo counts/s at 24 h of induction. The data shown in this paper indicate a practical and scaleable cell culture bioprocess procedure for the preparation of recombinant proteins expressed in S2 cells. 相似文献
9.
Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe
based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium
feed rates with a bioreactor cell concentration of ∼5 × 106 cells mL-1. Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell
specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell-1 day-1. Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion
rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen
activator concentration was maximized (∼40 mg L-1) at 0.2 nL cell-1 day-1. The volumetric protein productivity (∼60 mg L-1 day-1 was maximized above 0.3 nL cell-1 day-1. The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion
cultures.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
M A Jackson J S Frymier B J Wilkinson P Zorner S Evans 《Journal of industrial microbiology & biotechnology》1998,21(4-5):237-241
Xanthomonas campestris MB245, a specific pathogen of the weedy grass Poa annua (annual bluegrass), is being developed as a bioherbicide to control this pest in turf. Nutritional and environmental factors
were evaluated based on their ability to support rapid submerged culture growth and high cell yield. Temperature optima for
the growth of X. campestris cells in submerged culture were between 27 and 30°C. At 30°C, optimal nutritional conditions for X. campestris growth supported generation times of 150–175 min and cell yields after 24 h growth of 1–2 × 1010 cells ml−1. Media containing sucrose or glucose as the carbon source and various organic nitrogen sources supported optimal X. campestris growth and cell yield. The addition of vitamin mixtures to complex and defined media had no significant effect on growth
or cell yield. The age of X. campestris cultures had a significant impact on cell survival after freeze drying. Following freeze drying, log phase cell survival
(44%) was significantly lower than early and late stationary phase cell survival, 62% and 68%, respectively. Cells harvested
in stationary phase, freeze dried and stored under vacuum at 4°C, showed no significant loss in viability after 6 months.
Thus, high cell concentrations of the bioherbicide X. campestris can be rapidly produced in submerged culture and stabilized as freeze-dried preparations.
Received 14 August 1998/ Accepted in revised form 8 October 1998 相似文献
11.
Zalfa A. Abdel Malek Kristie L. Kreutzfeld Mac E. Hadley Marvin D. Bregman Victor J. Hruby Frank L. Meyskens jr. 《In vitro cellular & developmental biology. Plant》1986,22(2):75-81
Summary Cell density is a factor that affects the capacity of Cloudman S91 melanoma cells to respond to melanotropins in monolayer
culture. Continuous exposure of melanoma cells to α-melanotropin or its potent analog [Nle4,D-Phe7]-α-MSH, resulted in maximal stimulation of tyrosinase after 2 d of treatment, but the magnitude of stimulation decreased
thereafter despite the continued presence of the melanotropins. However, when melanoma cells continually exposed to melanotropins
were subcultured to an initial low cell density and maintained in contact with α-MSH or [Nle4,D-Phe7]-α-MSH (long-term culture), tyrosinase activity was rapidly restored and greatly enhanced. Also, when cells were seeded at
initial densities ranging from 0.2 to 3.2×106 cells/flask, and exposed for 24 h to 10−7
M α-MSH, only the cultures seeded at low densities (0.2 and 0.4×106 cells/flask) exhibited maximal tyrosinase activity during the 24 h exposure to the melanotropins. Therefore, tyrosinase activity
was primarily affected by cell density rather than by the duration of time the cells were in culture or by continuous exposure
to melanotropin. Other flasks of various cell densities were treated with 10−7 M α-MSH or [Nle4,D-Phe7]-α-MSH for 24 h, followed byremoval of the melanotropins from the culture medium. The magnitude and duration of theresidual stimulation of melanoma tyrosinase activity by melanotropins were also found to be dependent on the initial cell density.
These results reveal that there is a limited range of optimal cell densities at which melanoma cells can respond to melanotropins
and express increased tyrosinase activity. 相似文献
12.
VERO cells were cultivated in microcarriers (MCs) using a bioreactor with a working capacity of 3.7 l. We studied the effect of MCs concentration in the proportion of MC-bearing cells and on the kinetics of cell growth, as well as the cell growth with a batch, fed-batch and perfusion mode operation. The data show that, in a batch system, with an initial VERO cell inoculum of 3×104 cells/mg of MCs, 65%, 55% and 50% of the MCs were shown to be totally covered by cells after 7 days in cultures with respectively 2, 5 and 10 mg/ml of MCs. It was observed, that higher concentrations of MCs produced higher final yields of VERO cells. With 10 mg/ml of MCs, after 7 days of culture, a final yield of 1010 cells in the bioreactor, was obtained. The study of cell growth with a batch, fed-batch or perfusion system showed that the medium renewal allowed the continuous cell growth with the obtention of a final yield of 4×109 cells in a culture with 2 mg/ml of MCs. The number of cells that can be easily reached in these culture systems, which can be even further improved, indicates its usefulness for cell propagation and the preparation of cell products such as viral antigens.This work was supported in part by grants from the Fundação de Amparo à Pesquisa do Estado de S. Paulo (FAPESP), Fundação Instituto Butantan, and European Economic Community (EEC). C.A. Pereira is recipient of a Conselho Nacional de Pesquisa (CNPq) fellowship. We thank A.C. Barbosa for technical assistance. 相似文献
13.
Biopolymer membrane was prepared using two oppositely charged natural biopolymer. The biopolymer membrane was used for the
encapsulation of two hybridoma cell (ATCC CRL-1606, ATCC BH-8852) to produce monoclonal antibodies. In order to reduce the
down stream steps, the pore size of the membrane was controlled to retain the monoclonal antibodies in the capsules based
on the diffusion experiments with standard proteins. T-flask culture showed cell densities of 8×107 cells/mL and 3×107 cells/mL, and MAb concentrations of 506 μg/mL and 109 μg/mL for encapsulated ATCC CRL-1606 and HB-8852, respectively. Two
liter perfusion culture with encapsulated ATCC HB-8852 was performed to enhance the MAb production. The MAb production of
the encapsulated hybridoma increased considerably comparing to the culture using silicone tubing for oxygen transfer. 相似文献
14.
The isolation, identification and quantification of exometabolites from culture media of the cyanobacterium Nostoc insulare are described. Besides the known exometabolite 4,4′-dihydroxybiphenyl (I), two more compounds, the β-carboline 9H-pyrido(3,4-b)indole
(norharmane, II) and N,N′-(4,5-dimethyl-1,2-phenylene)bis-acetamide (III), were discovered. Concentrations of all three compounds
in media and biomass of five 250 L cultures of N. insulare were determined. Culture medium values for I ranged between 200 and 1,250 μg L−1 (1.1–6.7 μmol L−1), for III between 115 and 390 μg L−1 (0.5–1.8 μmol L−1), whereas concentrations of II were conspicuously lower (2–16 μg L−1 or 0.014–0.094 μmol L−1). Amounts of III per volume of culture medium were about tenfold higher than in the biomass contained in an equal culture
volume. This difference is an indication for an active excretion of III. Amounts of I and II in biomass and media were of
no significant difference. In the neutral red uptake assay, I and II were found to be toxic against eukaryotic cells as follows:
I was of considerable cytotoxicity in concentrations from 1,000 to 10 mg L−1 and of lower cytotoxicity (causing a 27 % decrease of cell viability) in a concentration of 1,000 μg L−1, whereas II was merely of considerable cytotoxicity in concentrations from 1,000 to 100 mg L−1. Because of the cytotoxicity of I and because of the many known pharmacological effects of II there is a possibility that
a certain amount of risk to humans and livestock comes from cultures or even from biomass- free culture media of N. insulare. The natural function of the examined exometabolites is discussed. 相似文献
15.
When protoplasts of Saccharomyces cerevisiae T7 and IFO 0309 are cultured in a static liquid culture at 2.5 × 106 protoplasts/ml, cell wall regeneration does not occur and cell wall components (CWC) are released into the culture broth.
By using a specialized fluorometer, the concentrations of CWC could be measured on the basis of the fluorescence intensity
of the CWC after staining with Fluostain I. The inoculum concentration, pH, and osmotic pressure of the medium were important
factors for the production of CWC in culture. Under optimal culture conditions, S. cerevisiae T7 protoplasts produced 0.91 mg/ml CWC after 24 h. The CWC induced the tumor necrosis factor-α production about 1.3 times
higher than that of the commercially available β-1,3/1,6-glucan from baker’s yeast cells. 相似文献
16.
Jie Chen Heming Chen Xiangchen Zhu Yinghua Lu Shang-Tian Yang Zhinan Xu Peilin Cen 《Applied microbiology and biotechnology》2009,82(2):241-248
The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel
with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the
FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 108 cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional
free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l−1) was achieved with a high productivity (23 μg l−1 h−1). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving
a productivity of 9∼10 μg l−1 h−1 in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL. 相似文献
17.
Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation. 相似文献
18.
Tamaki T Okada Y Uchiyama Y Tono K Masuda M Wada M Hoshi A Akatsuka A 《Histochemistry and cell biology》2007,128(4):349-360
In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution
capacity of CD34−/CD45− (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP
transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen
gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2–8 × 104); however, the number increased 10–20 fold (2–16 × 105) after 6 days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal
and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type)
resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery
of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20%
recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can
differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve
(Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule)
and fluorescence in situ hybridization (FISH) analysis. 相似文献
19.
Chitosan was used as a matrix to induce three-dimensional spheroids of HepG2 cells. Chitosan films were prepared and used
for culturing Hep G2 cells. Attachment kinetics of the cells was studied on the chitosan films. The optimum seeding density
of the Hep G2 cells, required for three-dimensional spheroid formation was determined and was found to be 5 × 104/ml. The growth kinetics of Hep G2 cells was studied using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)
(MTT) assay, and morphology of the cells was studied through optical photographs taken at various days of culture. The liver
cell functions of the spheroids were determined by measuring albumin and urea secretions. The results obtained from these
studies have shown that the culture of Hep G2 cells on chitosan matrix taking appropriate seeding density resulted in the
formation of three-dimensional spheroids and exhibited higher amount of albumin and urea synthesis compared to monolayer culture.
These miniature “liver tissue like” models can be used for in vitro tissue engineering applications like preliminary evaluation
of the toxicity of drugs and chemicals. 相似文献
20.
The presence of hexavalent chromium salt in culture medium negatively affected the growth dynamics and physiological parameters
of the benthic microalga Attheya ussurensis. After 1 day of exposure to toxicant at concentrations of 2, 4, 7, and 10 mg/l, the cell counts were 10, 7.9, 5.6, and 4.3
× 103 cells/ml, respectively (versus 13 × 103 cells/ml in the control). A tendency towards a decrease in cell number remained until the end of the experiments; after 7
days of exposure the cell counts were 133, 102, 11, and 7.5 × 103 cells/ml (versus 204 × 103 cells/ml in the control). With increase in potassium bichromate concentration in the culture medium, there was an increase
in the ratio of cell height to width and a change in the form of the cell to horseshoe shaped. The contents of chlorophyll
a in microalgal cells after 1 day of exposure to 2, 4, 7, and 10 mg/l were 40, 37, 34, and 30 μg/l, respectively (45 μg/l in
the control). After 7 days, at chromium salt concentrations of 2 and 4 mg/l, the chlorophyll a content was higher (670 and 647 μg/l) than in the control (605 μg/l); at 7 and 10 mg/l, it significantly decreased to 87
and 65 μg/l, respectively. The contents of carotinoids in microalgal cells after 7 days of exposure to 2 and 4 mg/l were comparable
to the control values, while at 7 and 10 mg/l they decreased sharply. The amount of phaeophytin (as a percentage of total
chlorophyll a content) increased with increasing potassium bichromate concentration. 相似文献