Simple Isolation of Human Bone Marrow Adipose Tissue–Derived Mesenchymal Stem/Stromal Cells

Current protocols Pub Date : 2025-01-03 DOI:10.1002/cpz1.70081

Gülsena Tonyalı, Emine Kiliç, Bihter Muratoğlu, Esin Alpdündar-Bulut, Cansu Özdemir, Duygu Uçkan-Çetinkaya

{"title":"Simple Isolation of Human Bone Marrow Adipose Tissue–Derived Mesenchymal Stem/Stromal Cells","authors":"Gülsena Tonyalı, Emine Kiliç, Bihter Muratoğlu, Esin Alpdündar-Bulut, Cansu Özdemir, Duygu Uçkan-Çetinkaya","doi":"10.1002/cpz1.70081","DOIUrl":null,"url":null,"abstract":"Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM-MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non-enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell & Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC.Basic Protocol 1: Isolation of human BMAT-MSCs from the fatty layer of the bone marrowBasic Protocol 2: Culture expansion, trypsinization, and cryopreservation of BMAT-MSCsSupport Protocol 1: Immunophenoypic characterization of human BMAT-MSCs by flow cytometrySupport Protocol 2: In vitro characterization of multilineage differentiation potential of human BMAT-MSCsSupport Protocol 3: Further characterization of gene expression in human BMAT-MSCs using qRT-PCR","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.70081","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}

引用次数: 0

Abstract

Bone marrow adipose tissue (BMAT) has garnered significant attention due to its critical roles in leukemia pathogenesis, cancer metastasis, and bone marrow failure. BMAT is a metabolically active, distinct tissue that differs from other fat depots. Marrow adipocytes, closely interacting with hematopoietic stem/progenitor cells and osteoblasts, play a pivotal role in regulating their functions. However, standardized methods for isolating and defining human BMAT (hBMAT) remain unclear. In animal models, BMAT is commonly isolated directly from the bone marrow through flushing, enzymatic digestion, or mechanical disruption. In humans, BMAT isolation often involves the adipogenic induction of bone marrow mesenchymal stem/stromal cells (BM-MSCs) derived from bone marrow aspirates. However, this approach reflects cellular responses to chemical stimuli and may not accurately represent in vivo differentiation potential. Similarly, BMAT obtained from hip or knee replacement surgeries might not reflect basal physiological conditions due to inflammatory influences. Here, we describe a direct method for culturing BMAT from the fatty layer of bone marrow aspirates obtained from healthy transplant donors. This protocol employs centrifugation and washing steps using basic laboratory equipment, offering simple and non-enzymatic approach. For validation, isolated cells are characterized according to the International Society for Cell & Gene Therapy (ISCT) criteria. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of human BMAT-MSCs from the fatty layer of the bone marrow

Basic Protocol 2: Culture expansion, trypsinization, and cryopreservation of BMAT-MSCs

Support Protocol 1: Immunophenoypic characterization of human BMAT-MSCs by flow cytometry

Support Protocol 2: In vitro characterization of multilineage differentiation potential of human BMAT-MSCs

Support Protocol 3: Further characterization of gene expression in human BMAT-MSCs using qRT-PCR

查看原文本刊更多论文

人骨髓脂肪组织来源间充质干细胞/基质细胞的简单分离。

骨髓脂肪组织（Bone marrow aditissue， BMAT）因其在白血病发病、肿瘤转移和骨髓衰竭中的重要作用而受到广泛关注。BMAT是一种代谢活跃的独特组织，与其他脂肪库不同。骨髓脂肪细胞与造血干细胞/祖细胞和成骨细胞密切相互作用，在调节其功能中起着关键作用。然而，分离和定义人BMAT （hBMAT）的标准化方法仍不清楚。在动物模型中，BMAT通常通过冲洗、酶消化或机械破坏直接从骨髓中分离出来。在人类中，BMAT的分离通常涉及骨髓间充质干细胞/基质细胞（BM-MSCs）的成脂诱导。然而，这种方法反映了细胞对化学刺激的反应，可能不能准确地代表体内分化潜力。同样，由于炎症的影响，髋关节或膝关节置换手术获得的BMAT可能不能反映基础生理状况。在这里，我们描述了一种从健康移植供者获得的骨髓抽吸液的脂肪层培养BMAT的直接方法。该方案采用离心和洗涤步骤，使用基本的实验室设备，提供简单和非酶的方法。为了验证，分离的细胞根据国际细胞与基因治疗学会（ISCT）的标准进行表征。©2025威利期刊LLC。基本协议1:孤立的人类BMAT-MSCs骨髓脂肪层的基本协议2:文化扩张,胰蛋白酶化,和低温贮藏BMAT-MSCs支持协议1:Immunophenoypic表征人类BMAT-MSCs通过流式细胞术支持协议2:体外表征multilineage分化潜力的人类BMAT-MSCs支持协议3:进一步描述基因表达在人类BMAT-MSCs使用中存在。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Current protocols

CiteScore

4.00

自引率

0.00%

发文量