Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes

1991 ◽  
Vol 99 (1) ◽  
pp. 121-130
Author(s):  
K.S. Koch ◽  
G.G. Brownlee ◽  
S.J. Goss ◽  
A. Martinez-Conde ◽  
H.L. Leffert
Keyword(s):  
Reporter Gene ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Developmental State ◽  
Retroviral Vectors ◽  
Time Interval ◽  
Gene Products ◽  
Fetal Rat ◽  
Human Factor Ix

Retroviral vectors were used to transduce recombinant DNA encoding firefly luciferase, Escherichia coli beta-galactosidase or human factor IX into fetal rat hepatocytes in primary culture. Hepatocytes were transduced optimally during a restricted time interval, 2–4 days post-plating. Although efficient and stable expression of reporter gene products was observed in vitro, it was affected differentially by culture conditions (plating density, media constituents) and chemical modulators of hepatocyte growth and differentiation (gelatin, hydrocortisone, isobutylmethylxanthine). Cultured cells, mock-infected or infected with a luciferase-expressing vector, were harvested non-enzymatically and injected subcutaneously into the dorsal neck fascia of neonatal syngeneic rats. Tissue isolated from injection sites one week later contained hepatocyte foci. In animals transplanted with infected cells, the preliminary results suggest that luciferase activity was present at these sites in proportion to the numbers of injected cells. These findings and previous observations made with hepatocytes from neonatal and adult primary cultures, indicate that from day 19 in utero through maturity the transient temporal ‘period of susceptibility’ to infection in vitro is independent of the developmental state of starting tissue. Transplantability of cultured fetal hepatocytes infected with retroviral vectors and stably expressing reporter gene products suggests that such cells might provide promising models for liver gene therapy.

scholarly journals GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE

1974 ◽  
Vol 62 (3) ◽  
pp. 792-801 ◽  
Author(s):  
H. L. Leffert
Keyword(s):  
Dna Synthesis ◽  
Environmental Control ◽  
Primary Cultures ◽  
Growth Control ◽  
Rat Hepatocytes ◽  
Defined Medium ◽  
Crystalline Insulin ◽  
Fetal Rat ◽  
Primary Monolayer

The initiation of DNA synthesis has been studied in quiescent primary cultures of fetal rat hepatocytes using defined hormones and chemically defined medium. Preparations of crystalline insulin (0.01–10 µg/ml) or 20,000-fold purified somatomedin (0.01–1 µg/ml) are stimulatory. Growth hormone (0.025 µg/ml) and hydroxycortisone (0.025 µg/ml), 3':5'-GMP! (10-5 M) fail by themselves to initiate DNA synthesis but added together with insulin, enhance the stimulatory response by 50–100%. Thyroid hormones (L-T3, L-T4, 7.5–30 ng/ml) are by themselves without effect, but when they are added to thyroid hormone-depleted serum, the reconstituted mixtures show an enhanced capacity to initiate DNA synthesis. In contrast, glucagon (0.01 µg/ml) inhibits the insulin-stimulated response by about 50% without reducing basal DNA synthesis rates. The results described here and in the accompanying two reports indicate that environmental control over the initiation of DNA synthesis is complex, and can involve the participation of many factors. The in vitro findings are consistent with the concept that liver regeneration is hormonally controlled and raise the possibility that alterations of the intrahepatic ratio of insulin to glucagon are growth regulatory.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Expression of Liver Functions in Imrnortalised Rot Hepotocyte Cell Lines

1994 ◽  
Vol 13 (6) ◽  
pp. 439-444 ◽  
Author(s):  
Caroline MacDonald ◽  
Maureen Vass ◽  
Brian Willett ◽  
Alexander Scott ◽  
Helen Grant
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Hepatic Function ◽  
Glutathione S Transferase ◽  
Early Passage ◽  
Toxicological Studies ◽  
Well Differentiated

The differentiated hepatic function of two rat liver cell lines, P9 and SV40RH1, immortalised by transfection with SV40 DNA has been investigated in terms of the glutathione synthesis, and the activities of γ-glutamyltransferase, glutathione-S-transferase and UDP-glucuronosyltransferase. SV40RH1 is a highly differentiated cell line at early passage, but the expression of some aspects of its differentiated phenotype is unstable and some functions are lost by passage 12-13. P9 is a less-well differentiated cell line, with relatively stable expression of functions between passages 4 and 13. In terms of differentiated function both cell lines represent a marked improvement over primary cultures of rat hepatocytes which de-differentiate rapidly within 24-48 h in culture. This retention of liver function in proliferating cell lines offers the opportunity to use such cells in in vitro toxicological studies.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445 ◽  
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Abstract Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Manipulation of the phenotype of immortalised rat hepatocytes by different culture configurations and by dimethyl sulphoxide

2000 ◽  
Vol 19 (5) ◽  
pp. 309-317 ◽  
Author(s):  
M I Grant ◽  
K Anderson ◽  
G McKay ◽  
M Wills ◽  
C Henderson ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Toxicity Testing ◽  
Normal Liver ◽  
Rat Hepatocytes ◽  
In Vitro Toxicity ◽  
Collagen Gels ◽  
Testosterone Metabolism ◽  
Glutathione S Transferases ◽  
Cells Cultured

The liver-specific phenotype of immortalised rat hepato-cytes is not irretrievably lost as they age in culture but can be manipulated by modifying the culture environment. Testosterone metabolism was used to investigate the profile of cytochrome P450 isoenzymes present in two immortalised cell lines, P9 and LQC, and in primary cultures of rat hepatocytes, cultured on collagen films, gels and double gel cultures (sandwich configuration). The extent of testosterone metabolism, and the range of metabolites produced, was increased in immortalised cells by the presence of collagen as a substratum film or gel but survival was poorer and the range of metabolites was reduced in sandwich culture. In contrast, testosterone metabolism was retained in primary hepatocytes in sandwich cultures at a higher level than in collagen film or gel cultures. Expression of alpha class glutathione-S-transferases (GSTs) increased and that of GSTP1 decreased (changes which indicate a recovery of normal liver GST phenotype) when the medium of immortalised cell cultures was supplemented with dimethyl sulph-oxide (DMSO). DMSO also improved ethoxyresorufin 0-deethylation (EROD) and testosterone metabolism in immortalised cells. It also markedly inhibited prolifera-tion, DNA, RNA and protein synthesis. Maximal testosterone metabolism was observed in immortalised cells cultured on collagen gels in the presence of l1o (v/v) DMSO. Development of a protocol for treating immortalised liver cells cultured on collagen gels with DMSO to switch between proliferation and differentiation may provide a convenient system expres-sing the xenobiotic metabolising enzymes required for in vitro toxicity testing.

scholarly journals Retinoic acid increases zif268 early gene expression in rat preosteoblastic cells.

1991 ◽  
Vol 11 (5) ◽  
pp. 2503-2510 ◽  
Author(s):  
L J Suva ◽  
M Ernst ◽  
G A Rodan
Keyword(s):  
Retinoic Acid ◽  
Primary Cultures ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Early Gene ◽  
Regulatory Sequences ◽  
Fetal Rat ◽  
In Vitro Data

In this study we demonstrate that retinoic acid (RA) increases the expression of transcription factor zif268 mRNA in primary cultures of fetal rat calvarial cells and in simian virus 40-immortalized clonal rat calvarial preosteoblastic cells (RCT-1), which differentiate in response to RA, but not in the more differentiated RCT-3 and ROS 17/2.8 cells. The increased expression of zif268 mRNA is rapid (maximal within 1 h), transient (returns to basal levels by 3 h), detectable at RA doses of 10(-12)M, and independent of protein synthesis. The relative stimulation of zif268 mRNA by RA was much larger than that of other early genes, including c-fos, c-jun, and junB. The rate of transcription of RA-stimulated RCT-1 cells, estimated by nuclear run-on assays, was elevated, suggesting that RA regulation of zif268 gene transcription was at least in part transcriptional. Moreover, RA stimulated the transcriptional activity of a Zif268CAT (chloramphenicol acetyltransferase) plasmid containing 632 bp of zif268 5' regulatory sequences in RCT-1 cells but not in the more differentiated RCT-3 cells. These in vitro data support the in vivo observations which localize zif268 and RA receptor-gamma transcripts to bone and cartilage during development, suggesting that both RA and zif268 may play a role in osteoblast differentiation.

Hypothermic Storage of Periportal and Perivenous Rat Hepatocytes

1998 ◽  
Vol 7 (4) ◽  
pp. 345-355 ◽  
Author(s):  
Edgardo Elvio Guibert ◽  
María Gabriela Mediavilla ◽  
María Eugenia Mamprin ◽  
Joaquín Valentín Rodríguez
Keyword(s):  
Cold Storage ◽  
Trypan Blue ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Liver Cells ◽  
Enzyme Release ◽  
Isolated Cells ◽  
Trypan Blue Exclusion ◽  
Hypothermic Storage

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96h-4°C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37°C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.

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