A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells

Abstract

To explore a simple and easy-to-learn procedure for the isolation of human quiescent hepatic stellate cells (HSCs) that requires no advanced training. Thus reducing costs and increasing efficiency. This protocol will provide sufficient primary cells with minimal contaminants for future basic research on diseases associated with human HSCs. Normal liver tissues were isolated from patients undergoing hepatic hemangioma resection, and a single cell suspension of these tissues was prepared using the Gentle MACS tissue processor. By using this method, the difficulty of the procedure was reduced, fewer cells were lost during the preparation treatments, and the maximal activity of single cells was maintained. Following preparation of the cell suspension, the HSCs were further isolated using a Nycodenz density gradient. Cell viability was examined by trypan blue staining, and the purity of the quiescent human HSCs was determined by autofluorescence and oil red O staining. Activated and quiescent human HSCs were identified using immunofluorescence and Western blotting. The cell cycle distribution in activated and quiescent human HSCs was analyzed by flow cytometry.The recovery rate of the HSCs was approximately (2.1 ± 0.23) × 10 6 of tissue, with 94.43 ± 1.89% cell viability and 93.8 ± 1.52% purity. The technique used in this study is a simple, high-yield, and repeatable method for HSC isolation that is worthy of recommendation.

Mitochondria-specific delivery system for targeted regulation of mitochondrial gene expression

“…The detailed isolation of primary liver fibroblasts is as follows ( Figure 4 ). Collect surgically removed liver tissue during hepatic hemangioma resection or liver transplantation ( Friedman et al., 1992 ; Zhai et al., 2019 ). Place the liver tissue (∼20 g) into a 50 mL of sterilized glass vial on ice and add 20 mL of DMEM containing 10% FBS and 1% penicillin-streptomycin.…”

“…Collect surgically removed liver tissue during hepatic hemangioma resection or liver transplantation ( Friedman et al., 1992 ; Zhai et al., 2019 ).…”

Therapeutic targeting of STAT3 with small interference RNAs and antisense oligonucleotides embedded exosomes in liver fibrosis

“…Primary hepatic stellate cells (HSCs) isolated from wild-type (WT) mouse were cultured for 7 days, which led to their spontaneous activation (Figure S1A). [32][33][34] The activation of HSCs is characterized by gain of expression of alpha-smooth muscle actin (α-SMA, Figure S1B). Vesicles isolated from MSCs that were used to generate iExo containing siRNA and ASOs displayed the size distribution and expression of tetraspanin markers indicative of exosomes (Figure S1C,D).…”

Characterisation of a subpopulation of CD133+ cancer stem cells from Chinese patients with oral squamous cell carcinoma

“…www.nature.com/scientificreports www.nature.com/scientificreports/ Immunofluorescence staining. Immunofluorescence staining was performed as described previously 42 .…”