Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens

March, Sandra; Ramanan, Vyas; Trehan, Kartik; Ng, Shengyong; Galstian, Ani; Gural, Nil; Scull, Margaret A.; Shlomai, Amir; Mota, Maria M.; Fleming, Heather E.; Khetani, Salman R.; Rice, Charles M.; Bhatia, Sangeeta N.

doi:10.1038/nprot.2015.128

Cited by 130 publications

(144 citation statements)

References 96 publications

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“…falciparum [50] [5], improved development of liver form P . falciparum in cryopreserved primary human hepatocytes has recently been reported [51] [52] Based on or results, and other published reports, we infer that primary human hepatocytes may be a better system than the HCO-4 cell line to study the development and growth of liver form P . falciparum parasites [53].…”

Section: Discussionsupporting

confidence: 56%

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites

et al. 2016

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Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.

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Section: Discussionsupporting

confidence: 56%

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites

et al. 2016

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“…fibroblasts. 49,50 Using a number of clinically relevant drugs Khetani et al could show a higher sensitivity of the MPCC as compared to conventional short-term cultures for prediction of human hepatic drug toxicity. 51 More sophisticated tissue engineering strategies, considering the specific requirements of individual cell types, have to be conceived for the co-cultivation of more than two cell populations.…”

Section: Co-culture Of Hepatocytes With Npcmentioning

confidence: 99%

Cell sources forin vitrohuman liver cell culture models

Zeilinger

Freyer

Damm

et al. 2016

Exp Biol Med (Maywood)

186

160

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In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

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“…As noted earlier in this paper, methods for enhancing function of hepatocytes in vitro include co-cultivation with stromal cells such as fibroblasts 24, 25 . To demonstrate flexibility of our technology, we wanted to implement hepatocyte-fibroblast co-cultures with the inserts being developed in this study.…”

Section: Resultsmentioning

confidence: 94%

Harnessing endogenous signals from hepatocytes using a low volume multi-well plate

Gheibi

Son

Stybayeva

et al. 2017

Integrative Biology

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Hepatocytes are highly differentiated epithelial cells that lose their phenotype and function when removed from the in vivo environment. Given the importance of hepatic cultures for drug toxicity, bioartificial liver assist devices and basic biology studies, considerable efforts have been focused on maintenance of hepatic function in vitro. The methods used to date include co-cultivation of hepatocytes with stromal cells, organizing these cells into spheroids and imbedding them into bioactive gels. Our team has recently demonstrated that primary rat hepatocytes confined to microfluidic channels in the absence of convection maintained epithelial phenotype through upregulation of endogenous signals including hepatocyte growth factor (HGF). The objective of the present study was to transition from microfluidic devices, which are somewhat specialized and challenging to use and towards low volume multiwell plates ubiquitous in the biology laboratories. Using a combination of 3D printing and micromolding we have constructed inserts that could be placed into standard 12-well plates and could be used to create low volume culture conditions under where primary hepatocytes maintained differentiated phenotype. This phenotype enhancement was confirmed by assays including albumin synthesis and expression. Importantly we confirmed upregulation of HGF inside the low volume culture plates and demonstrated that inhibition of HGF signaling degraded hepatic phenotype in our cell culture platform. Overall, this study outlines a new cell culture system that leverages low volume effects of microfluidic channels in a multiwell plate format. Beyond hepatocytes, such system may be of use in maintenance of other difficult-to-culture cells including stem cells and primary cancer cells.

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Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens

Cited by 130 publications

References 96 publications

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites

Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites

Cell sources forin vitrohuman liver cell culture models

Harnessing endogenous signals from hepatocytes using a low volume multi-well plate

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