Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Summary

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Abstract

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Introduction

Primary murine BMSCs are commonly used as an in vitro model of mesenchymal stem cells since their discovery in the early 1980s¹. Indeed, cultures of plastic-adherent cells flushed from the bone marrow cavity of long bones maintain the capacity to be differentiated into osteoblasts, osteoclasts, chondrocytes, or adipocytes in many studies^2,3,4,5. However, the bone marrow is a unique tissue composed of many different cell populations including, but not limited to, BMSCs, HSCs, endothelial, and immune cells. Thu....

Protocol

All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Maine Medical Center Research Institute.

1. Collection Tubes Preparation

1. Make the BMSC culture media by supplementing Minimum Essential Medium α (MEM α) with 10% Fetal Bovine Serum and 1% of Penicillin/Streptomycin.
2. Place 100 µL of BMSC culture media in 1.5 mL microcentrifuge tubes. 3 bones will usually fit in one single tube so prepare accordingly.
3. Cut the ends of 200 µL pipette tips enough so that they fit in the centrifuge tubes. Insert the tips inside the tubes and ensure that the lids c....

Results

Overview of the Cell Cultures
The two culture techniques allow the assessment of differentiation on a mixed population of BMCs comprised of BMSCs and HSCs (Total BMCs, Figure 1A) or a population of BMSCs split from HSCs (Adherent BMSCs, Figure 1B). 48 hr after the cells from the marrow are isolated and plated (Figure 1C), they rapidly attach to the bottom of the plastic culture p.......

Discussion

In this article, two methods of culture of BMSCs are presented with their advantages and limitations. Isolating cells coming from the bone marrow is a relatively effortless process. However, obtaining a cell population representative of the mesenchymal stem cells or osteoclastic progenitors can be challenging due to the diverse cellular environment of the marrow cavity.

Culturing the entirety of the bone marrow contents provides a close representation of the in vivo microenvironment. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
200μL pipet tips	Rainin	17014401
1.5 mL centrifuge tubes	USA Scientific	1615-5500
15 mL conical tube	VWR	89039-668
50 mL conical tube	VWR	89039-660
Phosphate Buffered Saline (PBS)	Sigma-Aldrich	21-040-CM
Ethanol	Fisher Science	04-355-451
Dissection tools
70 μm filters	BD Falcon	352350
0.25% trypsin	Gibco	25200
Kimwipe	VWR	82003-820
Paraformaldehyde (PFA)	Electron Microscopy Science	15710
Alkaline Phosphatase kit	Sigma-Aldrich	86R
Silver Nitrate	Sigma-Aldrich	S6506
Sodium Thiosulfate	Sigma-Aldrich	S7026
Oil Red O	Sigma-Aldrich	O0625
Isopropanol	Sigma-Aldrich	190764
10% Neutral Buffered Formalin	Sigma-Aldrich	F5554-4L
Whatman filter Grade 1	Sigma-Aldrich	Z274852
Tartrate-Resistant Acid Phosphatase kit	Sigma-Aldrich	387A
Glutaraldehyde	Electron	16220
MEMα	Gibco	12571
Fetal Bovine Serum	VWR	97068-085
Penicillin/Streptomycin	Invitrogen	15140-122
β-glycerol phosphate	Sigma-Aldrich	G9891
Ascorbic acid	Sigma-Aldrich	A4544
DMEM High Glucose	Sigma-Aldrich	D5796
Rosiglitazone	Cayman Chemical	717410
Insulin	Sigma-Aldrich	I6634
IBMX	Sigma-Aldrich	I5879
Dexamethasone	Sigma-Aldrich	D4902
RANKL	Peprotech	310-01
mCSF	Peprotech	315-02
Axio Observer inverter microscope	Zeiss