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ORIGINAL RESEARCH article

Front. Immunol.

Sec. Molecular Innate Immunity

Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1617203

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

Provisionally accepted
Xoan Thi HoangXoan Thi Hoang1*Phu Chi VuPhu Chi Vu2Nhat Minh DangNhat Minh Dang2Jonghyeok JungJonghyeok Jung2Min Hyek KimMin Hyek Kim2Min Guk LeeMin Guk Lee2Joohyun ShimJoohyun Shim3Jae Young KimJae Young Kim2*
  • 1NTT Hi-Tech Institute, Nguyen Tat Thanh University, Hồ Chí Minh, Vietnam
  • 2Department of Life Science, Gachon University, Seongnam, Kyeonggi-Do 13120, Korea, Seongnam, Republic of Korea
  • 3Department of Transgenic Animal Research, Optipharm Inc., Cheongju, Korea, Cheongju, Republic of Korea

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In this study, we established a comparative approach to isolate and characterize red pulp macrophages (RPMs) from pig splenocytes using two methods: CD163 antibody-based sorting and magneticactivated cell sorting (MACS), leveraging the natural iron content and autofluorescence of RPMs. Flow cytometry identified an autofluorescent population, a hallmark of RPMs, within pig splenocytes. The 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 showed that isolated RPMs expressed high levels of CD16 and CD163, moderate levels of CD11b, and low or undetectable levels of CD14, CD32, and CD169. Functional assays confirmed the phagocytic activity of RPMs against senescent red blood cells, along with upregulation of genes related to heme and iron metabolism. This study provides an optimized protocol for RPM isolation and characterization, offering an efficient strategy applicable to immunological research and splenic function studies.

Keywords: Pig spleen; red pulp macrophages, autofluorescence, CD163, RBC clearance, Iron retention

Received: 24 Apr 2025; Accepted: 14 Aug 2025.

Copyright: © 2025 Hoang, Vu, Dang, Jung, Kim, Lee, Shim and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Xoan Thi Hoang, NTT Hi-Tech Institute, Nguyen Tat Thanh University, Hồ Chí Minh, Vietnam
Jae Young Kim, Department of Life Science, Gachon University, Seongnam, Kyeonggi-Do 13120, Korea, Seongnam, Republic of Korea

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Supplementary Material