Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures

Summary

A routine culture of bone marrow stromal cells (BMSCs) leads to the isolation of heterogeneous cell populations, with many cells being of hematopoietic origins. Here, we describe a method that utilizes low oxygen tension to greatly reduce hematopoietic contaminants in murine BMSC cultures.

Abstract

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this reason, BMSCs, which support hematopoiesis and contribute to all the functions of the skeleton, continue to be widely used to study multipotent mesenchymal progenitors (MMPs) and to infer SSC function. Moreover, given the breadth of transgenic murine models used to study musculoskeletal diseases, the use of BMSCs also serves as a powerful tool to examine the molecular mechanisms regulating MMPs and SSCs. However, common isolation procedures for murine BMSCs result in over 50% of recovered cells being of hematopoietic origins, potentially hindering the interpretation of the data generated during these studies. Here, we describe a method using low oxygen tension or hypoxia for the selective elimination of CD45⁺ cells in BMSC cultures. Importantly, this method can be easily implemented to not only reduce hemopoietic contaminants but to also enhance the percentage of MMPs and putative SSCs in BMSC cultures.

Introduction

Similar to hematopoietic stem cells (HSCs), SSCs are housed within the bone microenvironment; however, unlike HSCs, currently there is a lack of universally accepted cell surface markers that can be used to prospectively identity SSCs^1,2,3. However, in vitro culture systems and in vivo reconstitution assays demonstrate that a proportion of BMSCs have the capacity to support hematopoiesis, as well as the ability to differentiate into all cells of the mesenchymal lineages^4,5. Thus, while BMSCs represen....

Protocol

All methods described utilizing murine models were performed in compliance with approved Institutional Animal Care and Use Committee (IACUC) protocols (A187-19-08).

1. Dissection of hindlimb bones

1. Euthanize either 10-12-week-old male or female C57BL/6 mice by CO₂ asphyxiation using a flow rate of 2-4 L/min followed by confirmation of death by decapitation. Either female or male mice can used in this assay with no expected differences in the results. Spra.......

Discussion

Based on our observations, BMSCs adhere to tissue culture plastic earlier than macrophages and other cells of hemopoietic origins¹¹. For this reason, rinsing plates 3 h post plating is a critical step as this removes floating hematopoietic cells that have the potential to attach later time points. Moreover, these rinses should be done with care, specifically by pipetting media onto the side of the dish to prevent the disruption of loosely attached, rounded BMSCs that have not yet laid down matrix .......

Materials

Name	Company	Catalog Number	Comments
100mm2 tissue culture dishes	Corning	353003
1x Phosphate Buffred Saline (PBS)	Gibco	100010-023
Bright-Line hemocytometer	Sigma-Aldrich	Z359629
C57BL/6J	The Jackson Laboratory	664
HyClone Fetal Bovine Serum (U.S.), Characterized	GE Healthcare Life Sciences	SH30071.03
InvivO2 400	Baker Ruskinn	https://bakerco.com
MEMα, nucleosides	Gibco	12571-063
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140-122
Red Blood Cell Lysing Buffer Hybri-Max	Sigma-Aldrich	R7757
T-25cm2  tissue culture flasks	Corning	430168
Trypan Blue Solution	Sigma-Aldrich	T8154
Trypsin-EDTA (0.25%), phenol red	Gibco	25200-056