scholarly journals Long-term culture and characterization of patient-derived primary hepatocytes using conditional reprogramming

2019 ◽  
Vol 244 (11) ◽  
pp. 857-864 ◽  
Author(s):  
Shan Su ◽  
Cristina Di Poto ◽  
Rabindra Roy ◽  
Xuefeng Liu ◽  
Wanxing Cui ◽  
...  
Keyword(s):  
Liver Disease ◽  
Liver Diseases ◽  
Human Hepatocytes ◽  
In Vitro Cultivation ◽  
Primary Hepatocytes ◽  
Primary Human Hepatocytes ◽  
First Time ◽  
Liver Tissues

Cultivation of primary human hepatocytes (PHHs) often faces obstacles including failure of long-term in vitro culture, weak proliferation ability, rapid loss of liver-specific function and morphology, and tendency of fibrosis. Previous research focused on immortalization methods, such as telomerase and viral, to culture immortalized primary human hepatocytes, which may lose some of the normal properties. However, non-immortalized PHHs often fail to maintain long-term viability and functionality. These highlight the urgent need for developing new culture strategy for PHHs. In the present study, we isolated PHHs from fresh human liver tissues representing different liver diseases and age groups. We used conditional reprogramming, without permanent immortalization, for long-term in vitro primary human hepatocytes cultivation and characterization. For functional characterization, we assessed CYP3A4, 1A1 and 2C9 activities and measured the mRNA expression of albumin, s100a4, krt8, krt18, cyp1a1, cyp3a4, cyp2b6, cyp2c8, cyp2c9, and cyp2d6. Additionally, we compared the DNA fingerprint of the cells against their original liver tissues using short tandem repeat (STR) analysis. We found that PHHs-derived from young patients can survive for more than three months, while the lifespan of primary human hepatocytes derived from adult patients ranges from two to three months, which is longer than most commercial primary hepatocytes. Importantly, the cells at early passages retain strong CYP3A4, 1A1 and 2C9 activities and the DNA fingerprints are identical with their original tissues. Through conditional programming, we achieved, for the first time, a high level of success rate in the long-term in vitro cultivation of primary human hepatocytes-derived patients representing diverse liver disease. Moreover, the conditional programming cell culture technology reported in this paper requires neither co-culture with additive cells, nor complex and expensive components, such as collagen sandwich or spheroid culture. We thus believe that the patient-derived PHHs cultivation using conditional programming may provide a viable and valuable cell model to study liver disease-related mechanisms. Impact statement Commercially available primary human hepatocytes rapidly lose their proliferative ability and liver-specific functions over a few cultivation days. The demand for pharmaceutical toxicity screening and liver disease research requires the development of long-term primary hepatocyte culture methods. This manuscript addresses this challenge by introducing for the first time successful long-term in vitro cultivation of primary human hepatocytes from a range of liver transplantation patients using conditional reprogramming technique. The beauty of this technique is that it is not a permanent immortalization and does not require co-culture with additive cells. The primary human hepatocytes retain proliferative capacity, genetic stability, and hepatocyte-specific functions at early passages. In view of these, we believe that scientists and researchers will benefit from using these highly valuable cell models to study diverse liver diseases.

Long-term functional maintenance of primary human hepatocytes in vitro

Science ◽  
2019 ◽  
Vol 364 (6438) ◽  
pp. 399-402 ◽  
Author(s):  
Chengang Xiang ◽  
Yuanyuan Du ◽  
Gaofan Meng ◽  
Liew Soon Yi ◽  
Shicheng Sun ◽  
...  
Keyword(s):  
Cell Biology ◽  
Expression Profiles ◽  
Antiviral Drug ◽  
Gene Expression Profiles ◽  
Global Gene Expression ◽  
Human Hepatocytes ◽  
Hbv Infection ◽  
Primary Human Hepatocytes

The maintenance of terminally differentiated cells, especially hepatocytes, in vitro has proven challenging. Here we demonstrated the long-term in vitro maintenance of primary human hepatocytes (PHHs) by modulating cell signaling pathways with a combination of five chemicals (5C). 5C-cultured PHHs showed global gene expression profiles and hepatocyte-specific functions resembling those of freshly isolated counterparts. Furthermore, these cells efficiently recapitulated the entire course of hepatitis B virus (HBV) infection over 4 weeks with the production of infectious viral particles and formation of HBV covalently closed circular DNA. Our study demonstrates that, with a chemical approach, functional maintenance of PHHs supports long-term HBV infection in vitro, providing an efficient platform for investigating HBV cell biology and antiviral drug screening.

Long-term production of major coagulation factors and inhibitors by primary human hepatocytes in vitro: perspectives for clinical application

2007 ◽  
Vol 27 (6) ◽  
pp. 832-844 ◽  
Author(s):  
Kim A. Boost ◽  
Marcus K. H. Auth ◽  
Dirk Woitaschek ◽  
Hyun-Soo Kim ◽  
Philip Hilgard ◽  
...  
Keyword(s):  
Clinical Application ◽  
Coagulation Factors ◽  
Human Hepatocytes ◽  
Primary Human Hepatocytes

scholarly journals Primary Human Hepatocytes Maintain Long-term Functions in Porous Silk Scaffolds Containing Extracellular Matrix Proteins

2020 ◽  
Author(s):  
David A. Kukla ◽  
Whitney L. Stoppel ◽  
David L. Kaplan ◽  
Salman R. Khetani
Keyword(s):  
Human Liver ◽  
Type I Collagen ◽  
Collagen Scaffold ◽  
Human Hepatocytes ◽  
Type I ◽  
Primary Human Hepatocytes ◽  
Degradation Rates ◽  
Silk Scaffolds ◽  
Liver Tissues

ABSTRACTThe shortage of donor organs for transplantation has prompted the development of alternative implantable human liver tissues; however, the need for a clinically viable liver tissue that can be fabricated using physiologically-relevant primary human hepatocytes (PHHs) is unmet. Purified silk proteins provide desirable features for generating implantable tissues, such as sustainable sourcing from insects/arachnids, biocompatibility, tunable mechanical properties and degradation rates, and low immunogenicity upon implantation; however, the utility of such scaffolds to generate human liver tissues using PHHs remains unclear. Here, we show that the incorporation of type I collagen during the fabrication and/or autoclaving of silk scaffolds was necessary to enable robust PHH attachment/function. Scaffolds with small pores (73 +/- 25 µm) promoted higher PHH functions than large pores (235 +/- 84 µm). Further incorporation of growth-arrested 3T3-J2 fibroblasts into scaffolds enhanced PHH functions up to 5-fold for 5 months in culture, an unprecedented longevity, and functions were better retained than 2D configurations. Lastly, encapsulating PHHs within Matrigel™ while housed in the silk/collagen scaffold led to higher functions than Matrigel or silk/collagen alone. In conclusion, porous silk scaffolds are useful for generating long-term PHH +/- fibroblast tissues which may ultimately find applications in regenerative medicine and drug development.

Long-term in vitro cultivation of Histomonas meleagridis coincides with the dominance of a very distinct phenotype of the parasite exhibiting increased tenacity and improved cell yields

Parasitology ◽  
2017 ◽  
Vol 144 (9) ◽  
pp. 1253-1263 ◽  
Author(s):  
JANINE GRUBER ◽  
PETRA GANAS ◽  
MICHAEL HESS
Keyword(s):  
Cell Morphology ◽  
In Vitro Cultivation ◽  
Adverse Conditions ◽  
Distinct Phenotype ◽  
Morphological Aspects ◽  
Low Passage ◽  
First Time

SUMMARYThe majority of research on Histomonas meleagridis was performed in the first half of the last century, especially those on morphological aspects. In the present study identical monoxenic settings for cultures of the same H. meleagridis clonal strain in its virulent low passage and attenuated high passage form enabled a comparative analysis of parasite characteristics. For the first time, it could be shown that long-term in vitro cultivation led to a severe shift in cell morphology, with the occurrence of a very distinct phenotype expressing a flagellated and highly amoebic cell morphology. Furthermore, the attenuated parasites showed better growth rates and a higher tenacity when confronted with adverse conditions. During these experiments up to 100% of the parasites, both virulent and attenuated, assumed a completely rounded morphology elucidated by electron microscopy. The findings indicate that such previously reported cyst-like stages are a defence strategy of H. meleagridis, independent of the passage level in vitro and pathogenicity in vivo. In conclusion, long-term in vitro passaging of H. meleagridis led not only to an attenuation of the parasite, as previously demonstrated, but also to a shift in the parasite's phenotype regarding morphology, growth behaviour and a higher level of tenacity.

scholarly journals In-Depth Proteome Analysis Highlights HepaRG Cells as a Versatile Cell System Surrogate for Primary Human Hepatocytes

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 192 ◽  
Author(s):  
Georg Tascher ◽  
Audrey Burban ◽  
Sandrine Camus ◽  
Marine Plumel ◽  
Stéphanie Chanon ◽  
...  
Keyword(s):  
Proteome Analysis ◽  
Cell System ◽  
Human Hepatocytes ◽  
Primary Hepatocytes ◽  
Heparg Cell ◽  
Xenobiotic Detoxification ◽  
Primary Human Hepatocytes ◽  
Hepatic Energy Metabolism ◽  
Heparg Cells

Of the hepatic cell lines developed for in vitro studies of hepatic functions as alternatives to primary human hepatocytes, many have lost major liver-like functions, but not HepaRG cells. The increasing use of the latter worldwide raises the need for establishing the reference functional status of early biobanked HepaRG cells. Using deep proteome and secretome analyses, the levels of master regulators of the hepatic phenotype and of the structural elements ensuring biliary polarity were found to be close to those in primary hepatocytes. HepaRG cells proved to be highly differentiated, with functional mitochondria, hepatokine secretion abilities, and an adequate response to insulin. Among differences between primary human hepatocytes and HepaRG cells, the factors that possibly support HepaRG transdifferentiation properties are discussed. The HepaRG cell system thus appears as a robust surrogate for primary hepatocytes, which is versatile enough to study not only xenobiotic detoxification, but also the control of hepatic energy metabolism, secretory function and disease-related mechanisms.

Somatic Embryogenesis and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

1970 ◽  
Vol 19 (1) ◽  
pp. 89-99
Author(s):  
K. Choudhary ◽  
M. Singh ◽  
M. S. Rathore ◽  
N. S. Shekhawat
Keyword(s):  
Somatic Embryogenesis ◽  
Growth Regulators ◽  
Somatic Embryos ◽  
Basal Medium ◽  
Long Term Study ◽  
Continuous Application ◽  
First Time ◽  
Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l  of 2,4-D and 0.5 mg/l  of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture  legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

scholarly journals Characterization of Postharvest Fungicide-Resistant Botrytis cinerea Isolates From Commercially Stored Apple Fruit

2017 ◽  
Vol 107 (3) ◽  
pp. 362-368 ◽  
Author(s):  
Wayne M. Jurick ◽  
Otilia Macarisin ◽  
Verneta L. Gaskins ◽  
Eunhee Park ◽  
Jiujiang Yu ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Gray Mold ◽  
Apple Fruit ◽  
Sublethal Dose ◽  
Long Term Storage ◽  
Pose Control ◽  
First Time

Botrytis cinerea causes gray mold and is an economically important postharvest pathogen of fruit, vegetables, and ornamentals. Fludioxonil-sensitive B. cinerea isolates were collected in 2011 and 2013 from commercial storage in Pennsylvania. Eight isolates had values for effective concentrations for inhibiting 50% of mycelial growth of 0.0004 to 0.0038 μg/ml for fludioxonil and were dual resistant to pyrimethanil and thiabendazole. Resistance was generated in vitro, following exposure to a sublethal dose of fludioxonil, in seven of eight dual-resistant B. cinerea isolates. Three vigorously growing B. cinerea isolates with multiresistance to postharvest fungicides were further characterized and found to be osmosensitive and retained resistance in the absence of selection pressure. A representative multiresistant B. cinerea strain caused decay on apple fruit treated with postharvest fungicides, which confirmed the in vitro results. The R632I mutation in the Mrr1 gene, associated with fludioxonil resistance in B. cinerea, was not detected in multipostharvest fungicide-resistant B. cinerea isolates, suggesting that the fungus may be using additional mechanisms to mediate resistance. Results from this study show for the first time that B. cinerea with dual resistance to pyrimethanil and thiabendazole can also rapidly develop resistance to fludioxonil, which may pose control challenges in the packinghouse environment and during long-term storage.

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