We thank for the assistance of Laboratory for Digitized Stomatology and Research Center for Craniofacial Anomalies of Shanghai Ninth People&#39;s Hospital. The work of this manuscript is supported by grants from the National Natural Science Foundation of China (NSFC) [81570950,81870740,81800949], Shanghai Summit &#38; Plateau Disciplines, the SHIPM-mu fund from Shanghai Institute of Precision Medicine, Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine [JC201809], the Incentive Project of High-level Innovation Team for Shanghai Jiao Tong University School of Medicine. And L.J. is a scholar of the Outstanding Youth Medical Talents, Shanghai &#34;Rising Stars of Medical Talent&#34; Youth Development Program and the &#8220;Chen Xing&#8221; project from Shanghai Jiaotong University.

All animal experimental procedures in this paper were approved by the Animal Care Committee of Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine.
1. Preparation
<ol>
	<li>Use two 5-week-old male Sprague Dawley rats for the experiment.</li>
	<li>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.<...

<ol>
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L., McClung, G., Mammone, R. M., Ortved, K. 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All authors state that they have no conflicts of interest.

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,...

Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic stem cells derived from bone marrow that manifest strong proliferation capability and multi-lineage differentiation potential1,2,3,4. 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 some differences from long bone BMSCs, such as different embryonic origin and development pattern. Mandibles arise from neural crest cells of the neuroectoderm germ layer and undergo intramembranous ossification, while axial and appendicular skeletons are from the mesoderm and undergo endochondral ossification. Furthermore, clinical observations and experimental animal studies have consistently indicated that there are functional differences between orofacial and iliac crest BMSCs5,6,7,8. Reports have shown that BMSCs derived from craniofacial bone such as mandible, maxillary bone, and alveolar bone exhibited superior proliferation, life span, and differentiation capability than those from axial and appendicular bones9. mBMSCs, therefore, are considered to be the preferred resources for future therapeutic applications of craniofacial diseases such as cherubism, jaw tumor, osteoporosis of jaw bone, and periodontal tissue defect10,11,12. To understand the treatment potential in preclinical experiments, it is essential to establish a method for rapidly isolating and culturing mBMSCs in vitro.
In this study, the aim was to obtain purified mBMSCs by whole bone marrow adherence and flow cytometry sorting. The anatomical morphology of rat mandible, clearly observed through micro computed tomography (Micro-CT) and histological sections, showed that the trabecular bone of the mandible was between the incisor medullary space and the alveolar bone. The bone marrow from trabecular bone was flushed to obtain mandibular marrow cells, but the cells cultured in this way were not pure mBMSCs and were likely to consist of multiple types of cells with uncertain potencies and diverse lineages such as cells from bone, fat and endothelial cells13,14. The next step of cell purification was particularly important. Flow cytometry filters cells by recognizing a combination of cell-surface proteins and has been widely adopted in the enrichment of mesenchymal stem cells. Cell homogeneity is the main advantage of flow cytometry, but the process does not determine cell viability and can result in a limited cell yield. In this study, the P0 mBMSCs obtained from whole bone marrow adherence were sorted by flow cytometry to obtain mBMSCs with high purity and strong proliferation capacity.
This study introduces a reproducible and reliable protocol for isolation, culture, and differentiation of rat mandibular BMSCs using a combination of whole bone marrow adherence and flow cytometry sorting. It is a reliable and convenient method for researchers in related fields to use.

<table>
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA&#65288;1X&#65289;</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>10cm culture dish</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>acutenaculum</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adipogenic differentiation medium</td>
			<td>Cyagen biosciences inc.</td>
			<td>MUBMX-90031</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue</td>
			<td>Beyotime Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Color Development Kit</td>
			<td>Beyotime Biotechnology</td>
			<td>C3206</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha-Minimum essential medium</td>
			<td>GE Healthcare HyClone Cell Culture</td>
			<td>SH30265.01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti -CollagenII Rabbit pAb</td>
			<td>Abcam</td>
			<td>ab34712</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies against CD16/CD32</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antifade Mounting Medium with DAPI</td>
			<td>Beyotime Biotechnology</td>
			<td>P0131</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse/rat CD29 Antibody</td>
			<td>biolegend inc</td>
			<td>102215</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Esco</td>
			<td>AC2-4S8-CN</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (OX1), PE, eBioscience</td>
			<td>Invitrogen</td>
			<td>12-0461-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), FITC, eBioscience</td>
			<td>Invitrogen</td>
			<td>11-0900-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>cence</td>
			<td>L500</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondrogenesis differentiation medium</td>
			<td>cyagen biosciences inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td>LSM880</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal Violet Staining Solution</td>
			<td>Beyotime Biotechnology</td>
			<td>C0121</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GE Healthcare HyClone Cell Culture</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Esco</td>
			<td>CCL-170B-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>Magzol reagent(Trizol reagent)</td>
			<td>Magen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>micropipettor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osteogenic differentiation medium</td>
			<td>cyagen biosciences inc.</td>
			<td>MUBMX-90021</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline&#65288;1X&#65289;</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Kit</td>
			<td>TakaRa Bio Inc</td>
			<td>RR036A</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P6556</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickBlock Blocking Buffer</td>
			<td>Beyotime Biotechnology</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR1 Premix</td>
			<td>TakaRa Bio Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Beyotime Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cultivation of mandibular bone marrow mesenchymal stem cells in rats

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&#8722; 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.

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&#8722; mBMSCs were purified18,22, which accounted for about 81.1% in the P0 cells (Figure 1C...

Watch this Scientific Journal Video about Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats at JoVE.com

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

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.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Studies has shown that the BMSCs derived from cranial facial bone exhibit superior capabilities than those from axial and appendicular bones. Therefore, mandibular BMSCs are considered to be the preferred resource for developing future therapeutics of craniofacial diseases. This protocol combines the advantages of whole bone marrow adherence and fluorescent cell sorting to obtain significant numbers of purified mandibular BMSCs in a short time.If you are trying this protocol for the first time I think it's very important to understand the anatomic structure and the functional movement of the rat mandible, which can be achieved by watching this video or reading related literature. After euthanizing the rat, place it in a clean fume hood in a supine position. Incise the skin and buccinator muscles from bilateral angulus oris to the posterior region of the mandible.Open the mouth of the rat by pushing the maxillary and mandibular incisors towards the opposite direction with both thumbs, to expose the mandibular teeth, which are attached to the mandible. Completely disconnect the buccal muscles and the tendons attached to the coracoids and the inferior border of the mandible. Then press the mandibular posterior teeth and rotate them back and downwards until the condyles on both sides are clearly exposed.Separate the mandible body from the skull, and clean the adherence soft tissue on the bone surface using a wet gauze. Place the bone in a 10 centimeter sterile glass dish filled with precooled minimum essential medium alpha, or PBS on ice, to preserve the viability. Cut off the anterior bone along the mesial edge of the first molar from the mandibular body, then remove the mandibular ramus including coracoid and condyle along the distal edge of the third molar to expose the marrow cavity.Fill a 10 centimeter sterile glass dish with 10 milliliters of alpha-MEM with 10%FBS. Aspirate the medium with a 10 milliliter syringe, then insert the needle into the bone marrow cavity and repeatedly flush the bone marrow into the dish. Flush bone cavity at least three times from both mesial and distal sides of the bone respectively, until the bone turns white.Transfer the media containing the flushed cells into a 15 milliliter centrifuge tube, and centrifuge it at 800 RPM and for five minutes. Discard the supernatant, and resuspend the cells with three milliliters of alpha-MEM with 10%FBS. Plate the cells in a new 10 centimeter culture dish and incubate them at 37 degrees Celsius in a 5%carbon dioxide incubator.Aspirate the culture medium and wash the dish with PBS. Add two milliliters of 0.25%trypsin with 0.02%EDTA in the dish and digest the cells at 37 degrees Celsius for five minutes. Then add four milliliters of alpha-MEM with 10%FBS to stop the reaction.Transfer the cell suspension into a 15 milliliter centrifuge tube and centrifuge it at 800 RPM for five minutes. Resuspend the cells in 120 microliters of PBS with 10%FBS after centrifugation. Transfer 100 microliters of the cell suspension into a new micro centrifuge tube.Block the cell suspensions with one microliter of antibody against CD16 through CD32 at four degrees Celsius for 15 minutes, and stain the cells with PE-conjugated antibody against CD45, FITC-conjugated antibody against CD90, and APC-antibody against CD29, at four degrees Celsius for one hour in the dark. Use the other 20 microliters of cell suspension as unstained negative control. Centrifuge the tubes at 800 RPM for five minutes.Discard the supernatant and resuspend the cells in 0.5 milliliters of PBS with 10%FBS. Add 10 microliters of 0.01 milligrams per milliliter DAPI 10 minutes before analysis. Use 40 nanometer filters placed on centrifuge tubes to filter the cells, then analyze the cells on a fluorescence activated cell sorter.First remove dead cells from total cell count by gating DAPI negative cells, then gate for CD29 positive, CD90 positive, CD45 negative, in the selected cells as targeted mBMSCs. Collect the sorted mBMSCs into a 15 milliliter centrifuge tube with five milliliters of alpha-MEM with 10%FBS. Centrifuge the tubes at 800 RPM for five minutes and remove the collection buffer.Add one milliliter of fresh medium to resuspend the cells, then plate them in a six centimeter culture dish. Using this protocol, a large proportion of cells adhered to the plate on the third day after the initial culture. After an additional three to four days of culture, the cell confluence reached 70 to 80%Fluorescent cell sorting was used to purify mBMSCs which accounted for about 81.1%in the P0 cells.After culturing P2 mBMSCs in a six well plate for a week, a significant amount of colony forming units were observed. To assess the multi-lineage differentiation ability of the mBMSCs they were induced into osteo-chondro-and adipo-lineages. Increased activity of ALP, red calcific nodules under alizarin red staining, and increased expression of osteogenic specific genes Runx2, Alp, Bsp and Ocn, indicated osteogenic induction.Oil-red-O staining was used to identify adipogenesis. Numerous lipid rich vacuoles were evident after nine days of induction. Furthermore expression of adipogenic specific genes Ppar-gamma-1 and Cebpa was increased.The samples showed positive staining for Alcian blue during microscopic observation of chondrogenic differentiation. In addition, immunostaining with anti-type II collagen antibody showed enhanced accumulation of cartilage matrix. Ensuring the viability of mBMSCs is very important in this protocol.This purpose can be achieved by choosing proper experimental animal, operating on ice, and shortening the experimental time. Utilizing this in vitro model, one can obtain a high number of proliferative mandibular BMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mandibular BMSCs.

isolation-cultivation-mandibular-bone-marrow-mesenchymal-stem-cells

Research

JoVE Journal

Medicine

5.0K Görüntüleme.  National Clinical Research center of Stomatology. Studies has shown that the BMSCs derived from cranial facial bone exhibit superior capabilities than those from axial and appendicular bones. Therefore, mandibular BMSCs are considered to be the preferred resource for developing future therapeutics of craniofacial diseases. This protocol combines the advantages of whole bone marrow adherence and fluorescent cell sorting to obtain significant numbers of purified mandibular BMSCs in a short time.If you are trying this protocol for the fi...