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猪脾细胞红髓巨噬细胞分离技术的比较评估与特性分析

Comparative evaluation of isolation techniques and characterization of red pulp macrophages from pig splenocytes.

作者信息

Vu Phu Chi, Dang Nhat Minh, Jung Jonghyeok, Kim Min Hyuk, Lee Min Guk, Shim Joohyun, Hoang Thi Xoan, Kim Jae Young

机构信息

Department of Life Science, Gachon University, Seongnam, Kyeonggi-Do, Republic of Korea.

Department of Transgenic Animal Research, Optipharm Inc., Cheongju, Republic of Korea.

出版信息

Front Immunol. 2025 Sep 1;16:1617203. doi: 10.3389/fimmu.2025.1617203. eCollection 2025.

DOI:10.3389/fimmu.2025.1617203
PMID:40959078
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12434077/
Abstract

INTRODUCTION

Red pulp macrophages (RPMs) play a central role in iron recycling and immune regulation within the spleen, yet optimized methods for the isolation and characterization of pig RPM remain limited.

METHODS

We compared two approaches for isolating RPMs from pig splenocytes: CD163 antibody- based sorting and magnetic-activated cell sorting (MACS), which leverages the natural iron content and autofluorescence of RPMs. Isolated cells were evaluated by flow cytometry for marker expression, and functional assays were performed to assess phagocytic activity and gene expression related to iron metabolism.

RESULTS

Flow cytometry identified an autofluorescent population, a hallmark of RMPs, within the pig splenocytes. CD163-based method enriched RPMs to 71.8% autofluorescent cells, while the MACS- based approach achieved a higher yield of 81% autofluorescent cells without using antibodies, demonstrating greater cost-effectiveness and efficiency. Marker analysis revealed high expression of CD16 and CD163, moderate expression of CD11b, and low or undetectable levels of CD14, CD32, and CD169. Functionally, isolated RPMs demonstrated robust phagocytosis of senescent red blood cells and upregulation of genes involved in heme and iron metabolism.

DISCUSSION

These findings establish an optimized, antibody-free protocol for efficient isolation of pig RPMs. The approach provides a reliable platform for studying splenic macrophage biology, iron homeostasis, and immunological research and splenic function studies.

摘要

引言

红髓巨噬细胞（RPMs）在脾脏内的铁循环和免疫调节中发挥着核心作用，但用于分离和鉴定猪RPMs的优化方法仍然有限。

方法

我们比较了两种从猪脾细胞中分离RPMs的方法：基于CD163抗体的分选和磁激活细胞分选（MACS），后者利用了RPMs的天然铁含量和自发荧光。通过流式细胞术评估分离细胞的标志物表达，并进行功能测定以评估吞噬活性和与铁代谢相关的基因表达。

结果

流式细胞术在猪脾细胞中鉴定出一个自发荧光群体，这是RMPs的一个标志。基于CD163的方法将RPMs富集到71.8%的自发荧光细胞，而基于MACS的方法在不使用抗体的情况下获得了更高的81%自发荧光细胞产量，显示出更高的成本效益和效率。标志物分析显示CD16和CD163高表达，CD11b中度表达，CD14、CD32和CD169低表达或未检测到。在功能上，分离的RPMs表现出对衰老红细胞的强大吞噬作用以及血红素和铁代谢相关基因的上调。

讨论

这些发现建立了一种优化的、无抗体的方案，用于高效分离猪RPMs。该方法为研究脾脏巨噬细胞生物学、铁稳态以及免疫学研究和脾脏功能研究提供了一个可靠的平台。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0633/12434077/9ac1cd127e43/fimmu-16-1617203-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0633/12434077/52dd376b7ab9/fimmu-16-1617203-g001.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0633/12434077/9ac1cd127e43/fimmu-16-1617203-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0633/12434077/52dd376b7ab9/fimmu-16-1617203-g001.jpg
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