| Background and Aim:The combination of in situ collagenase digestion by liver perfusion and density gradient centrifugation are the most widely used technique for isolating liver non-parenchymal cells (NPCs). However, cell sample prepared by commonly-used two-layer density gradient centrifugation may still contain non-viable cell components, which could lead to non-specific staining during flow cytometry analysis. Thus, the aim of this study was to develop a modified four-layer density gradient centrifugation method to get higher Kupffer cell yield and less non-viable components contamination.Methods:After in situ collagenase digestion by portal perfusion, a four layer density gradient centrifugation was performed with7.2%,11.2%,13.2%and18%w/v OptiPrepTm solution. Flow cytometry was used to analyze cell components of each cell band at different density gradient interface. Cell counts of Kupffer cell in each cell groups were measured. And7-AAD staining was used to detect non-viable Kupffer cells. Then the new four-layer method and the traditional two-layer method (with11.5%and17.6%w/v OptiPrepTM solution) were compared upon the percentage of F4/80positive cells, F4/80mean fluorescence intensity (MFI) and the percentage of F4/80CD146double positive cells.Results:Four cell groups were formed after centrifugation, and the cell band at7.2/11.2%iodixanol interface contained the highest percentage of Kupffer cells with the lowest7-AAD positivity (P<0.05). Compared to the two-layer density gradient centrifugation, the modified four-layer method yielded more Kupffer cells and provided better F4/80preservation with lower percentage of F4/80CD146double positive (P<0.05).Conclusion:The four-layer density gradient centrifugation yielded more Kupffer cells with higher purity, less disruption of F4/80antigen and less non-specific staining with non-viable components during flow cytometry analysis. Background and Aim:The classic view believed that macrophages originate and constantly renew from hematopoietic stem cells (HSC) in the bone marrow via monocyte intermediates. However, new data showed that tissue macrophage developed from embryonic yolk-sac progenitors independent of HSCs and could self-renew through adulthood. But neither of the two theories can explain the coexistence of bone-marrow-derived Kupffer cells and liver sessile Kupffer cells in green fluorescent protein (GFP) chimeras. Thus, this study was aim to identify the role of bone marrow progenitors, especially HSCs in Kupffer cell recovery in mice.Methods:Bone marrow chimeric mice undergone liver-shield irradiation and GFP+bone marrow transplantation (LSI-BMT) was established. Flow cytometric analysis, fluorescence-activated cell sorting (FACS) and immunofluorescent staining were used to detect GFP expression on KCs, HSCs and circulating monocytes at steady state and after different liver injury. High-throughput cell surface marker screening was applied to identify different types of bone-marrow-derived macrophage. Results were retested by adoptive cell transfer.Results:Until the40th week after bone marrow transplantation, there were few GFP+KCs detected in LSI-BMT mice. And liver sessile KCs population remained no change after irradiation and all KCs remained of GFP-host origin. But after irradiation injury, the number of GFP-liver sessile KCs dramatically decreased and remained unchanged throughout the40-week observational period without any signs of cell proliferation. At the meanwhile, GFP+BM-derived macrophages appeared at the second week, continuously increased to reach a plateau at the fourth week and finally settled down. Similar results were observed after clodronate liposomes treatment and repeated carbon-tetrachloride (CCl4) injections, showing that bone-marrow-derived settled macrophages contributed to Kupffer cell recovery. However, bone-marrow-derived macrophage only stayed in liver for a short time after single dose of CCl4. The above two types of bone-marrow-derived macrophages had distinct gene expression and cell surface marker. Compare to monocytic migratory (short stay) macrophages, settled macrophages showed HSC-specific markers. And the following adoptive cell transfer confirmed that only when GFP+HSCs (rather than monocytes) were transplanted into clodronate liposomes treated wild-type mice did GFP+KCs appear in the liver of recipients.Conclusion:At stead state, liver sessile KCs can long-term self-maintain. But after radiation, clodronate-liposomes clearance or repeated CCl4treatment, they are damaged and replaced by settled macrophages derived from HSCs independent of monocyte intermediate. |
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