Abstract
The lack of robust methods to preserve, purify and in vitro maintain the phenotype of the human liver’s highly specialized parenchymal and non-parenchymal cell types importantly hampers their exploitation for the development of research and clinical applications. There is in this regard a growing interest in the use of tissue-specific extracellular matrix (ECM) to provide cells with an in vitro environment that more closely resembles that of the native tissue. In the present study, we have developed a method that allows for the isolation and downstream application of the human liver’s main cell types from cryopreserved material. We also isolated and solubilized human liver ECM (HL-ECM), analyzed its peptidomic and proteomic composition by mass spectrometry and evaluated its interest for the culture of distinct primary human liver cells. Our analysis of the HL-ECM revealed proteomic diversity, type 1 collagen abundance and partial loss of integrity following solubilization. Solubilized HL-ECM was evaluated either as a coating or as a medium supplement for the culture of human primary hepatocytes, hepatic stellate cells and liver sinusoidal endothelial cells. Whereas the solubilized HL-ECM was suitable for cell culture, its impact on the phenotype and/or functionality of the human liver cells was limited. Our study provides a first detailed characterization of solubilized HL-ECM and a first report of its influence on the culture of distinct human primary liver cells.
Highlights
The liver fulfills a myriad of vital functions due to its complex assembly of highly specialized parenchymal and non-parenchymal liver cells
To assess whether the different major liver cell populations can be isolated from the cryopreserved liver cell fractions, we adapted a protocol previously applied to fresh human liver material [22] and identified the purified cell populations by a comparative analysis of the expression of cell-type specific genes by qPCR: albumin (ALB), cytochrome P450 3A4 (CYP3A4), hepatocyte nuclear factor 4α (HNF4A) and glucose-6-phosphatase catalytic subunit (G6PC) for HEPs, cytoglobin (CYGB) for hepatic stellate cells (HSCs), NO synthase 3 (NOS3) as a pan endothelial cells (ECs) marker, stabilin 1/2 (STAB1/2), Fcγ receptor 2b (FCGR2B) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) for liver sinusoidal endothelial cells (LSECs) and CD163 for MPs [14,16,22,27,28]
We found by dosing the protein content retained on Tissue culture plastic (TCP) following coating with different concentrations of solubilized HL-extracellular matrix (ECM) that a plateau in protein adsorption started at 50 μg/mL
Summary
The liver fulfills a myriad of vital functions due to its complex assembly of highly specialized parenchymal and non-parenchymal liver cells. Whereas the cryopreservation of freshly isolated HEPs has been the subject of numerous studies [6], the cryopreservation of the NPF, prior to and in view of its further processing for the purification of the different non-parenchymal liver cell types, has been poorly studied Another important hurdle constitutes the rapid loss of the specific phenotype of human primary liver cells in vitro. It is well-known that, upon in vitro culture, HEPs rapidly lose their polarity and metabolic properties [7,8], HSCs undergo an activation process during which they rapidly lose their retinoid-containing lipid droplets and acquire a fibrogenic, myofibroblast-like phenotype [9,10], and LSECs lose their fenestrations and sinusoidal-specific transcriptomic signature [11,12]. The composition of human liver-specific ECM (HL-ECM) and its potential impact on the function and phenotype of the main human liver cell populations has not yet been investigated
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