Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Summary

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Abstract

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.

In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29⁺CD90⁺CD45⁻ mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

Introduction

Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic stem cells derived from bone marrow that manifest strong proliferation capability and multi-lineage differentiation potential^1,2,3,4. Indeed, BMSCs have been considered as an ideal candidate for bone tissue engineering and regeneration ever since they were discovered. For years, the iliac crest or long bones such as the tibia and femur have been the most common source of BMSCs for craniofacial regeneration. However, orofacial BMSCs, such as mandibular BMSCs (mBMSCs), display so....

Protocol

All animal experimental procedures in this paper were approved by the Animal Care Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine.

1. Preparation

1. Use two 5-week-old male Sprague Dawley rats for the experiment.
2. Sterilize all the instruments, including needle holders, tweezers and scissors at high temperature or immersed in 75% ethanol for 10 min.
   NOTE: Ethanol immersion should not be too long to avoid cell damage.
3. Prepare culture media beforehand, the composition of which is provided in Table 1. Supplement each medium as described belo....

Results

Using this protocol, a large proportion of cells adhered to the plate on the third day after the initial culture. Typically, after an additional 3-4 days of culture, the cell confluence reached to 70 to 80% (Figure 1B). With fluorescent cell sorting, DAPI^-CD29⁺CD90⁺CD45⁻ mBMSCs were purified^18,22, which accounted for about 81.1% in the P0 cells (Figure 1C).

.......

Discussion

This protocol describes a method to isolate BMSCs from rat mandibles in vitro by combining whole bone marrow adherence and fluorescent cell sorting, which is a simple and reliable way to obtain proliferative mBMSCs with strong differentiation ability. This method could preliminarily purify mBMSCs by flow cell sorting, but if there are higher requirements for cell homogeneity, more precise purification methods may be required.

Currently, there are four main techniques used for isolating mBMSCs,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA(1X)	Gibco	25200072
10cm culture dish	Corning
acutenaculum
Adipogenic differentiation medium	Cyagen biosciences inc.	MUBMX-90031
Alcian Blue	Beyotime Biotechnology
Alizarin red	Sigma-Aldrich	A5533
Alkaline Phosphatase Color Development Kit	Beyotime Biotechnology	C3206
alpha-Minimum essential medium	GE Healthcare HyClone Cell Culture	SH30265.01B
Anti -CollagenII Rabbit pAb	Abcam	ab34712
Antibodies against CD16/CD32
Antifade Mounting Medium with DAPI	Beyotime Biotechnology	P0131
APC anti-mouse/rat CD29 Antibody	biolegend inc	102215
Biosafety cabinet	Esco	AC2-4S8-CN
CD45 Monoclonal Antibody (OX1), PE, eBioscience	Invitrogen	12-0461-82
CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), FITC, eBioscience	Invitrogen	11-0900-85
Centrifuge	cence	L500
Chondrogenesis differentiation medium	cyagen biosciences inc.
Confocal laser scanning microscope	Zeiss	LSM880
Countess II FL Automated Cell Counter	Invitrogen	AMQAF1000
Crystal Violet Staining Solution	Beyotime Biotechnology	C0121
Fetal Bovine Serum	GE Healthcare HyClone Cell Culture	SH30084.03
Goat Anti-Rabbit IgG H&L (Alexa Fluor 488)	abcam	ab150077
Incubator	Esco	CCL-170B-8
Inverted microscope	olympus	CKX53
Magzol reagent(Trizol reagent)	Magen
micropipettor	Eppendorf
Oil Red O
Osteogenic differentiation medium	cyagen biosciences inc.	MUBMX-90021
Penicillin-Streptomycin	Gibco	15070063
Phosphate-buffered saline(1X)	Gibco	20012027
PrimeScript RT Master Kit	TakaRa Bio Inc	RR036A
Proteinase K	Sigma-Aldrich	P6556
QuickBlock Blocking Buffer	Beyotime Biotechnology	P0260
scissor
SYBR1 Premix	TakaRa Bio Inc
Toluidine Blue	Beyotime Biotechnology
Trypan Blue Solution, 0.4%	Gibco	15250061