Human primary cultured hepatic stellate cells can be cryopreserved

Abstract

We compared the morphological and functional characteristics of cultured unfrozen hepatic stellate cells (HSCs) and cryopreserved HSCs obtained from human livers. We used liver tissues obtained by surgical resection from patients with metastatic liver cancer or with hepatocellular carcinoma. HSCs were isolated and allowed to spread in culture. Comparison of morphological and functional features between the unfrozen HSCs and cryopreserved HSCs was performed at each passage using the following techniques: light microscopy, immunohistochemistry, cell growth curve, metallothionein (MTT) assay, and PI staining, Western blot, real-time polymerase chain reaction (PCR), and gene expression analysis using microarrays. The purity of HSCs was more than 90% in all passages. alpha-Smooth muscle actin (SMA-)positive HSCs gradually increased in successive passages, and the positive cell rate and rate of increase in cell number were similar in both groups. Expression of platelet-derived growth factor (PDGF) receptor, transforming growth factor (TGF)-beta receptor, and alpha-SMA mRNAs and protein was similar during each passage in the two groups. Gene expression was nearly identical at each passage in unfrozen and frozen/thawed samples obtained from the same patient. In conclusion, an adequate protocol for the cryopreservation of human primary cultured HSCs could be established.

Rebaudioside A administration prevents experimental liver fibrosis: an in vivo and in vitro study of the mechanisms of action involved

“…Human HSCs (hHSCs) were obtained from hepatic biopsy patients with obesity as described previously in detail (Nakamura et al, ; Reyes‐Gordillo et al, ). VL‐17A cells were kindly provided by Dr. Dahn L. Clemens.…”

Stevioside inhibits experimental fibrosis by down‐regulating profibrotic Smad pathways and blocking hepatic stellate cell activation

“…Human HSCs (hHSCs) were isolated, as described elsewhere, using collagenase and pronase digestion and fractionation on an OptiPrep (Sigma, St Louis, MO, USA) gradient, from human liver biopsies from patients with morbid obesity who were subjected to bypass surgery by an approved protocol (IRB 070701). Written informed consent was obtained from the study participants, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as was reflected in prior approval by the Institutional Review Committee.…”

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells

“…Primary HSCs (Nakamura et al 2010) and LX-2 cells (Xu et al 2005) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nippon Suisan Kaisha, Ltd., Tokyo, Japan) supplemented with 20 and 2 % foetal bovine serum (Biowest, Inc., Nuaillé, France), respectively, with 100 U/mL penicillin and 100 lg/ mL streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere of 5 % CO 2 . When cytokine stimulation was carried out, primary HSCs and LX-2 cells were exposed to cytokine concentrations of 1 ng/mL for each following serum starvation for 24 h using DMEM/0.5 % serum.…”

“…For immunofluorescence labelling, cells were fixed in 4 % paraformaldehyde, and incubated with a primary antibody for 1 h at 4°C (Nakamura et al 2010). The primary antibody was monoclonal mouse anti-human SMA (Clone 1A4) (Agilent Technologies, Inc., Santa Clara, CA, USA), with a secondary Cy3-conjugated AffiniPure anti-mouse IgG (Jackson ImmunoResearch, Inc., West Grove, PA, USA).…”

“…To investigate the effect of these inflammatory cytokines on HSCs, we prepared primary human HSCs from excised human livers (Nakamura et al 2010) prior to assaying.…”