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. 2022 Apr 19;11(9):1379.
doi: 10.3390/cells11091379.

Optimized Isolation and Characterization of C57BL/6 Mouse Hepatic Stellate Cells

Alexandre Balaphas  1   2 , Jeremy Meyer  1 , Cécile Gameiro  3 , Aurélien Frobert  4 , Marie-Noëlle Giraud  4 , Bernhard Egger  4 , Leo H Bühler  4 , Carmen Gonelle-Gispert  4
Affiliations

Optimized Isolation and Characterization of C57BL/6 Mouse Hepatic Stellate Cells

Alexandre Balaphas et al. Cells. .

Abstract

To obtain meaningful results of hepatic stellate cell (HSC) function, it is crucial to use highly pure HSC populations. Our aim was to optimize HSC isolation from mice livers without exploiting the characteristically transient vitamin A autofluorescence of HSC. HSCs were isolated from C57BL/6 mice using a two-step collagenase digestion and Nycodenz gradient separation followed by CD11b-negative sorting step in order to remove contaminating macrophages and dendritic cells. Isolated cells were analyzed for yield, viability, purity, and potential new markers using immunofluorescence and flow cytometry. We obtained a yield of 350,595 ± 100,773 HSC per mouse liver and a viability of isolated cells of 92.4 ± 3.1%. We observed a low macrophage/dendritic cell contamination of 1.22 ± 0.54%. Using flow cytometry, we demonstrated that CD38 was expressed at the surface of HSC subpopulations and that all expressed intracellular markers specific for HSC in the liver. This isolation method, avoiding fluorescent activated cell sorting (FACS), allowed isolation of HSCs with high purity. Further, flow cytometry analysis suggests that CD38 may be a reliable marker of HSCs and may include subpopulations of HSCs without retinoid droplets.

Keywords: CD11b; CD38; MACS; autofluorescence; hepatic stellate cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) After Nycodenz separation, high-lipid-content cells float (white ring) between the Nycodenz solution and the GBSS + NaCl solution (yellow arrow). (B) Cell viability assessed with fixable EF 780 viability dye back gated in side scatter (SSC) and forward scatter (FSC)-contour plot with outliers as dots. Green: viable cells; red: dead cells. Proportion expressed as a mean ± SD, n = 3. (C,D) Quantitative polymerase chain reaction assessing DCN (coding for Decorin) (C) and CLEC4F (coding for Clefc4f) (D) gene expression in cell populations after the two major steps of HSC isolation (Nycodenz and MACS sorting). Values obtained were expressed as fold-increase (FI) where the value obtained by non-parenchymal cell (NPC) population was set as 1. Decorin has been described to be specific to HSCs whereas CLEC4F is expressed by Kupffer cells. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.00, if not specified, non-significant. n = 2.
Figure 2
Figure 2
Cytological observation of isolated hepatic stellate cells of HSCs a few hours after plating: contrast phase (lipid droplets are white relative to dark cytoplasm), UV-elicited autofluorescence (blue) and fusion of the first two images. White bar: 200 µm.
Figure 3
Figure 3
Flow cytometry characterization of isolated hepatic stellate cells. (A) Cell morphology allowing to gate cell populations. SSC: side scatter; FSC: forward scatter. (B) Selected cells/viable cells (see Figure 1B) gates were analyzed for their expression of CD45 (blue) and the presence of violet-induced autofluorescence (AF) (violet). CD45-/AF- cells are represented in gray. Proportions are expressed as a mean ± SD, n = 3. (C) Backgating analysis of the cell population morphology: AF+ cells (violet), CD45+ cells (blue), CD45-/AF- cells (gray). SSC: side scatter,;FSC: forward scatter. (D) Expression of F4/80 (pink) among the CD45+ population. Proportions are expressed as a mean ± SD, n = 3.
Figure 4
Figure 4
Assessment of CD38 expression on the hepatic stellate cell population sorted by MACS after the density gradient. (A) Histogram showing PE CY7 conjugated isotype (upper panel) or PE CY7 conjugated anti-mouse CD38 (lower panel) over cell count. The proportion is expressed as a mean ± SD, n = 3. (B) Backgating analysis of cell population morphology. Blue: CD45+ cells; violet: violet-induced autofluorescence (AF)+ cells; yellow: CD38+; gray: all viable cells (gate cell/viable cells). SSC: side scatter; FSC: forward scatter. (C) CD38 expression of HSC directly sorted from non-parenchymal cells according to their autofluorescence and morphology. Gray: without antibody; orange: CD38-PECY7 staining. n = 1, NA: no antibody.
Figure 5
Figure 5
The CD38+ cell population expresses hepatic stellate cell specific intracellular markers GFAP and Nestin. CD38 expression of backgated GFAP+ Nestin+ and violet-induced (AF)+ populations after isotype control (upper panel) and antibody (lower panel) directed against CD38. It appeared that the majority of CD38+ cells were positive for Nestin and GFAP and autofluorescent. Proportions (of viable cells) are expressed as a mean ± SD, n = 2.
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