Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

Jauregui, Hugo O.; Hayner, Nancy Thompson; Driscoll, James L.; Williams-Holland, Rhonda; Lipsky, Milton H.; Galletti, Pierre M.

doi:10.1007/bf02618612

Cited by 281 publications

(116 citation statements)

References 31 publications

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“…This prompted us to investigate the degree of cell lysis in medium CHO-T1-SF after inoculation at higher cell densities (2·10 4 cells/cm 2 ). The extent of cell lysis during the cultivation of mammalian cells can be determined by measuring the activity of L-LDH in conditioned media (Bour et al, 1988;Goergen et al, Schröder, Matischak, and Friedl 14 1993;Jauregul et al, 1981;Legrand et al, 1992;Racher et al, 1990). However, we were not able to detect any L-LDH activity in media conditioned by high-density cultures of any one of the anchorage- (Schröder and Friedl, 1997a).…”

Section: Growth Characteristics In Serum-free Medium Cho-t1-sfmentioning

confidence: 62%

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Schröder

Matischak

Friedl

2004

Journal of Biotechnology

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Citation for published item:hr¤ oderD wF nd w tis h kD uF nd priedlD F @PHHRA 9 erumE nd proteinEfree medi formul tions for the ghinese h mster ov ry ell line h u fIIF9D tourn l of iote hnologyFD IHV @QAF ppF PUWEPWPF Further information on publisher's website: httpXGGdxFdoiForgGIHFIHITGjFj iote FPHHQFIPFHHS Publisher's copyright statement:Additional information: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. AbstractThe production of therapeutic proteins in mammalian cell lines is of outstanding importance. The maintenance of most mammalian cell lines in culture requires the addition of serum to the culture medium.The elimination of serum from mammalian cell culture is desirable since serum is expensive and a source of contaminants, e.g. viruses, mycoplasma or prions. Here we describe the composition of serum-and protein-free media for the Chinese hamster ovary (CHO) cell line DUKXB11. The serum-free formulation supports excellent growth of CHO DUKXB11 cells at low (23 cells/cm 2 ) and high (2·10 4 cells/cm 2 ) seeding densities characterized by a generation time of 10 -12 h, and, after addition of 0.2% pluronic F-68, the growth of a recombinant suspension cell line derived from DUKXB11. In addition, this formulation also allowed us to adapt recombinant cell lines expressing various amounts of human antithrombin ATIII (ATIII)to serum-free conditions. Secretion of ATIII was readily observed in the serum-free medium. Minor changes to the serum-free formulation resulted in a protein free formulation that supported growth of CHO DUKXB11 cells, growth of recombinant CHO cells expressing ATIII, and production of ATIII. Key wordsChinese hamster ovary cells, serum-free medium, protein-free medium, recombinant protein production,

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Section: Growth Characteristics In Serum-free Medium Cho-t1-sfmentioning

confidence: 62%

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Schröder

Matischak

Friedl

2004

Journal of Biotechnology

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“…In the first experiment, we determined the range of noncytotoxic concentrations of bilobalide by measuring LDH release, which is a marker of cytotoxicity (Jauregui et al, 1981). Bilobalide at 1 to 100 M did not increase LDH release in cultured rat hepatocytes or HepG2 cells (Fig.…”

Section: Ldh Release In Cultured Hepatocytes and Hepg2 Cellsmentioning

confidence: 99%

Species-Dependent and Receptor-Selective Action of Bilobalide on the Function of Constitutive Androstane Receptor and Pregnane X Receptor

Lau

Yang²,

Rajaraman³

et al. 2011

Drug Metab Dispos

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ABSTRACT:Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6␤-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16␣-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.

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“…20 The viability of GEPH was determined by a fluorescence vital staining technique with fluorescein diacetate:ethidium bromide (FDA: EB). 21 GEPH were incubated in working FDA:EB (5 g/mL:10 g/ mL) solution for 5 minutes at 37°C, and then washed twice in phosphate-buffer solution.…”

Section: Determination Of Cell Viabilitymentioning

confidence: 99%

Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes

et al. 2001

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Cryopreserved porcine hepatocytes are a ready source of metabolic function for use in a bioartificial liver (BAL). However, cryopreservation is associated with a loss of hepatocyte viability. The mechanism of cell death during cryopreservation is incompletely understood, but may involve apoptosis through caspase activation. This study evaluates the cytoprotective effect of a global caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (ZVADfmk) during cryopreservation of porcine hepatocytes. Freshly isolated porcine hepatocytes (viability, 97.4% ؎ 0.9%) were cryopreserved in 60 mol/L ZVAD-fmk (؉ZVAD group) or without ZVAD-fmk (؊ZVAD group) for 24 to 72 hours. Apoptotic and necrotic death were both observed after thawing and after 24 hours of culture. Caspase 3-like activity was significantly reduced by ZVADfmk, and was associated with improved viability and reduced apoptotic death of porcine hepatocytes after cryopreservation. Mitochondrial membrane potential (MMP) was increased in cultures of porcine hepatocytes that were cryopreserved in ZVAD-fmk. These results demonstrate the following: 1) Caspase 3-like protease activation and apoptosis occurs in porcine hepatocytes during cryopreservation; and 2) mitochondrial injury in this process is reduced by caspase inhibition. (HEPATOLOGY 2001;33:1432-1440.)A number of liver-assist devices have been developed, including the extracorporeal use of isolated mammalian hepatocytes to provide metabolic function in a bioartificial liver (BAL). 1,2 Criteria for successful application of a BAL include bridging patients to liver transplantation, reduction of intracranial hypertension and cerebral edema, and avoidance of liver transplantation in cases of reversible hepatic failure. 3-5 To achieve these goals, hepatocytes in the BAL provide metabolic functions to the patient in liver failure. The extent to which these goals can be achieved is dependent on several variables, including the number of viable and metabolically active hepatocytes in the device. 6 Unfortunately, hepatocyte viability declines over time following isolation, and this decline limits the effectiveness and duration of BAL therapy. Cryopreservation, which allows cells to be used weeks and months after isolation, may also contribute to premature cell death. 7,8 Several BAL systems have been designed to maintain hepatocyte viability ex vivo. One such BAL system involves the entrapment of hepatocytes in cylindrical collagen gels located in the fibers of a hollow fiber bioreactor. 9,10 An in vitro model of this BAL system using gel-entrapped hepatocytes has been developed for static culture experimentation, such as the study of mechanisms of hepatocyte death. 11 The in vitro model avoids the expenses of largescale testing, and multiple experiments can be performed simultaneously. Potential immune interactions, present with in vivo models of BAL testing, are also avoided with in vitro testing.Our recent work has shown that cell death of freshly isolated hepatocytes occurs, in part, by apoptosis. 12...

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Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells, cell suspensions, and primary monolayer cultures

Cited by 281 publications

References 31 publications

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11

Species-Dependent and Receptor-Selective Action of Bilobalide on the Function of Constitutive Androstane Receptor and Pregnane X Receptor

Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes

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