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.</li>
	<li>Prepare culture media beforehand, the composition of which is provided in Table 1. Supplement each medium as described below.
	<ol>
		<li>Preparation of &#945;-MEM culture medium (with 10% FBS): Supplement minimum essential medium alpha (&#945;-MEM) with 10% fetal bovine serum and 1% penicillin and streptomycin.</li>
		<li>Preparation of osteogenic differentiation medium: Supplement osteogenic differentiation basal medium with 10% fetal bovine serum, 1% glutamine, 1% penicillin-streptomycin, 0.20% ascorbic acid, 1% &#946;-glycerophosphate and 0.01% dexamethasone.</li>
		<li>Preparation of osteogenic induction medium: Mix 70% &#945;-MEM culture medium (with 10% FBS) and 30% osteogenic differentiation medium.</li>
		<li>Preparation of adipogenic differentiation medium A: Supplement adipogenic differentiation basal medium with 10% fetal bovine serum, 1% glutamine, 1% penicillin-streptomycin, 0.20% insulin, 0.10% IBMX, 0.10% Rosiglitazone and 0.01% Dexamethasone.</li>
		<li>Preparation of adipogenic differentiation medium B: supplement adipogenic differentiation basal medium with 10% fetal bovine serum, 1% glutamine, 1% penicillin-streptomycin and 0.20% insulin.</li>
		<li>Preparation of chondrogenic differentiation medium: Supplement chondrogenic differentiation basal medium with 0.01% Dexamethasone, 0.30% Ascorbic acid, 1% ITS, 0.10% Sodium pyruvate, 0.10% Proline and 1% TGF-&#946;3. 
		NOTE: The osteogenic differentiation medium must be used up within a month after configuration. The effective period of the configured chondrogenic differentiation medium is 12 h.</li>
	</ol>
	</li>
</ol>
2. Isolation and cultivation of rat mBMSCs
NOTE: All experimental operations should be performed on ice as much as possible to maintain cell viability.
<ol>
	<li>Harvesting rat mandibles
	<ol>
		<li>Euthanize two 5-week-old male Sprague Dawley rats by CO2 asphyxiation. Ensure that the animal&#8217;s breathing is stopped before proceeding with the experiment.</li>
		<li>Rinse these rats in a beaker containing 75% ethanol for 3 min.</li>
		<li>Place the rat inside a clean fume hood to harvest mandibles. 
		NOTE: Sterilize the fume hood by ultraviolet light radiation for 30 min before use.</li>
		<li>Place the rat in a supine position. Incise the skins and buccinator muscles from bilateral angulus oris to the posterior region of the mandible, which is the only movable bone with lower incisor. 
		NOTE: It is recommended to make a 2 cm incision on each side of the angulus oris to get a clear operation field.</li>
		<li>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.</li>
		<li>Completely disconnect the buccal muscles and the tendons attached to the coracoids and the inferior border of mandible. 
		NOTE: An increased mobility of the mandible is observed during this step because of the lack of muscle tension.</li>
		<li>Press the mandibular posterior teeth and rotate back and downwards until the condyles on both sides are clearly exposed. 
		NOTE: This step is performed to artificially open the mouth of the rat to the maximum extent possible to dislocate the condyle. The mandible is connected to skull through bilateral condyles, so the exposure of the condyles leads to the disconnection between the skull and mandible.</li>
		<li>Separate the mandible body from the skull.</li>
		<li>Clean the adherent soft tissue on the bone surface using a wet gauze.</li>
		<li>Place the bone in a 10 cm sterile glass dish filled with precooled minimum essential medium-&#945; (&#945;-MEM) or phosphate buffer saline (PBS) on ice to preserve the viability.</li>
	</ol>
	</li>
	<li>Isolation and cultivation of the mBMSCs
	<ol>
		<li>Cut off the anterior bone along the mesial edge of the first molar from the mandibular body. Remove the mandibular ramus including coracoid and condyle along the distal edge of the third molar to expose the marrow cavity.</li>
		<li>Fill a 10 cm sterile glass dish with 10 mL of &#945;-MEM (with 10% FBS).</li>
		<li>Use 1 mL syringe to aspirate &#945;-MEM culture medium. With the syringe needle inserted into the bone marrow cavity, repeatedly flush the bone marrow into the dish. Flush the bone cavity at least 3 times from both mesial and distal sides of the bone, respectively, until the bone turned white. 
		NOTE: This is the most critical step, as the bone marrow cavity of a rat mandible is not as obvious as in long bone and the proper point to insert the needle needs to be determined empirically. All experimental samples above should be stored on ice to maintain cell viability and so the operation time must be no longer than 2 h.</li>
		<li>Transfer the media containing the flushed cells into a 15 mL centrifuge tube and centrifuge at 800 x g in 4 &#176;C for 5 min. Discard the supernatant.</li>
		<li>Resuspend the cells with 3 mL of &#945;-MEM (with 10% FBS). Plate the cells in a new 10 cm culture dish and incubate at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>Check the morphological changes and growth of these cells on the 3rd day of culture. Remove the culture medium with nonadherent cells and tissue fragments. Gently add 10 mL of fresh &#945;-MEM (with 10% FBS).</li>
		<li>After 7 days of culture, P0 mBMSCs reached 70% to 80% confluence.</li>
	</ol>
	</li>
	<li>Flow cell sorting 
	NOTE: P0 mBMSCs were phenotypically identified and primarily purified through flow cell sorting.
	<ol>
		<li>Aspirate and discard the culture medium and then wash the dish with PBS. Discard the PBS.</li>
		<li>Add 1 mL of 0.25% trypsin with 0.02% EDTA in the dish. Digest the cells at 37 &#176;C for 5 min, then add 2 mL of &#945;-MEM (with 10% FBS) to stop the reaction.</li>
		<li>Transfer the cells suspension into a 15 mL centrifuge tube and centrifuge at 800 x g for 5 min.</li>
		<li>Resuspend the cells in 120 &#956;L of PBS (with 10% FBS) after centrifugation.</li>
		<li>Block these cell suspensions with 1 &#956;L of antibody against CD16/CD32 at 4 &#176;C for 15 min.</li>
		<li>Transfer 100 &#181;L of the cell suspension into a new microcentrifuge tube, stain the cells with Phycoerythrin (PE)-conjugated antibody against CD45, fluorescein-isothiocyanate (FITC)-conjugated antibody against CD90 and Allophycocyanin (APC)-antibody against CD29 at 4 &#176;C for 1 h in the dark13,15. The concentration of antibodies used in this experiment is shown in Table 2. Use the other 20 &#181;L of cell suspension as unstained negative control.</li>
		<li>Then centrifuge the tubes at 800 x g for 5 min, discard the suspension, and resuspend the cells in 0.5 mL of PBS (with 10% FBS).</li>
		<li>Add 10 &#956;L of 0.01 mg/mL DAPI for 10 min before analysis.</li>
		<li>Use 40 nm filters placed on centrifuge tubes to filter the cells.</li>
		<li>Analyze the cells on Fluorescence-activated cell Sorter. Set the panels as follows. Firstly, remove dead cells from total cell count by gating DAPI- cells, then gate CD29+CD90+CD45- in the selected cells as targeted mBMSCs.</li>
		<li>Collect the sorted CD29+CD90+CD45- cells into a 15 mL centrifuge tube with 3 mL of &#945;-MEM (with 10% FBS) pre-prepared.</li>
		<li>Centrifuge the tubes at 800 x g for 5 min. Remove the collection buffer and add 1 mL of fresh &#945;-MEM (with 10% FBS) to resuspend the cells. Then plate them in a 6 cm culture dish.</li>
	</ol>
	</li>
</ol>
3. Colony formation capability
NOTE: This step was performed to check for the division ability of mBMSCs.15
<ol>
	<li>Select second passage mBMSCs (P2) for this experiment. When the cell confluence reached 80% to 90%, reseed the cells in a serial gradient in a 6 well plate to evaluate their clonal proliferation ability. 
	NOTE: It is recommended to set the gradient from 1 x 102 to 1 x 103 cells per well, but the final dilutions of different cell lines of human or animal origin were empirically determined.</li>
	<li>Culture the cells in &#945;-MEM (with 10% FBS) for approximately two weeks until a good number of the colony forming units could be seen under light microscope. Refresh the culture medium every 3 days.</li>
	<li>Aspirate and discard the culture medium, wash the wells with PBS twice for 5 min at a time. Next add 4% paraformaldehyde solution to each well and fix for at least 10 min.</li>
	<li>Remove the paraformaldehyde and rinse the wells twice with PBS.</li>
	<li>Stain the cells with crystal violet staining solution and incubate them in 37 &#176;C for about 10 min. 
	NOTE: The staining time adjusted appropriately until the desired shade is achieved.</li>
	<li>Remove the staining solution and wash the samples with distilled water to stop the reaction.</li>
	<li>Image under a stereomicroscope and count scattered cell colonies which consisted of at least 50 cells.</li>
</ol>
4. Multilineage differentiation of mBMSCs
NOTE: The P2 mBMSCs were used for subsequent experiments unless otherwise described.
<ol>
	<li>Osteogenic induction of mBMSCs
	<ol>
		<li>Digest the mBMSCs as described above. Seed cells at a density of 2.5 x 105/cm2 in a 12 well plate supplemented with 1 mL &#945;-MEM (with 10% FBS).</li>
		<li>When the cell confluence reached 60-70%, change the medium into 30% osteogenic induction media. Culture the cells with &#945;-MEM (with 10% FBS) as a negative control and change the medium every 2 days. 
		NOTE: After a great good number of calcium nodules appear during osteogenesis, it is recommended to change the medium exchange pattern to a half volume medium exchange every 2 days, to prevent osteoblasts from floating.</li>
		<li>After culturing for seven days, assess the calcification of these cells by alkaline phosphatase staining16,17.</li>
		<li>Remove the culture medium and fix the cells with 4% paraformaldehyde for 10 min. Rinse the cells using 1 mL of PBS per well for 3 min twice.</li>
		<li>Then stain the cells with alkaline phosphatase staining solution and incubate at 37 &#176;C for 10-30 min. 
		NOTE: Incubation can be performed overnight at room temperature to obtain the required color.</li>
		<li>Wash the wells with distilled water to stop the reaction. Take photographs under light microscope. Red pigmented granules represent alkaline phosphatase (ALP) activity.</li>
		<li>After culturing the remaining cells for another 7 days, perform alizarin red staining to evaluate the mineralization capacity of the cells.</li>
		<li>Stain the cells with 0.5% alizarin red staining solution after cell fixation for 10 min16,18. Stop the chromogenic reaction with distilled water after 3-5 min. 
		NOTE: The reaction time can be extended depending on the color developed.</li>
		<li>Finally, place the culture plate under the light microscope to observe the effect of osteogenic staining; red nodules indicate calcium deposits of these cells.</li>
	</ol>
	</li>
	<li>Adipogenic induction of mBMSCs
	<ol>
		<li>Seed P2 mBMSCs in a 12 well plate as described above.</li>
		<li>After the well become 90% confluent, culture the cells with adipogenic differentiation medium A. Use cells cultured in &#945;-MEM culture medium (with 10% FBS) as negative control.</li>
		<li>After 2 days of induction, aspirate the medium A from the well and add 1 mL of adipogenic differentiation medium B in each well.</li>
		<li>After one day, remove medium B and start the cycle again with medium A added back into the well. Culture the cells alternately using medium A and B.</li>
		<li>Apply the differentiation medium A and B alternately for three cycles and detect the adipogenesis of these cells by oil red O staining16,18. 
		NOTE: Lots of round and big lipid droplets can be observed by culturing these cells with differentiation medium B for an additional 2 to 3 days.</li>
		<li>Remove the medium and fix the cells with 4% paraformaldehyde for 10 min. Wash out the cells with PBS.</li>
		<li>Stain the cells with oil red O staining solution and incubate at 37 &#176;C for approximately 3&#160;min.</li>
		<li>Remove the staining solution and wash the wells with distilled water.</li>
		<li>Observe adipogenesis under light microscope.</li>
	</ol>
	</li>
	<li>Chondrogenesis induction of mBMSCs
	<ol>
		<li>Seed P2 mBMSCs in 15 mL centrifuge tube at a density of 5 x 105/cm2.</li>
		<li>Centrifuge the tubes at 300 x g for 5 min. Discard the supernatant.</li>
		<li>Resuspend the cells with 0.5 mL of chondrogenic differentiation medium. Centrifuge the cells at 300 x g for 5 min.</li>
		<li>Loosen the cap of the centrifuge tube and incubate it at 37 &#176;C in a 5% CO2 incubator. Renew the medium every 2 days. 
		NOTE: Cells start pelleting after 24 h. It is recommended not to move the centrifuge tube for 48 h. Be careful not to aspirate the cell pellet when changing the medium.</li>
		<li>After 21 days of induction, fix the cell pellets with 4% paraformaldehyde, followed by dehydration, paraffin embedding and sectioning.</li>
		<li>After deparaffinization and hydration, stain the slides in Alcian blue solution for at least 15 min18,19,20,21, then wash with distilled water to stop the reaction. Capture the photographs under light microscope.</li>
		<li>For immunofluorescent staining of collagen type II, perform the steps below.
		<ol>
			<li>Incubate the slides with 5 &#181;g/mL proteinase K for 15 min at 37 &#176;C after the deparaffinization and hydration step.</li>
			<li>Incubate the slides in blocking buffer for 1 h at 37 &#176;C.</li>
			<li>Apply 1 &#181;L of collagen type II antibody in 200 &#181;L of blocking buffer to the slides and incubate overnight at 4 &#176;C.</li>
			<li>The next day, wash the slides with PBS twice and incubate with secondary antibodies at 37 &#176;C for 1 h.</li>
			<li>Incubate the slices with 40,6-diamidino-2-phenylindole (DAPI) for 10 min.</li>
		</ol>
		</li>
		<li>Take photographs of each slides under fluorescence microscopy.</li>
	</ol>
	</li>
</ol>
5. Real-time PCR
<ol>
	<li>Extract total RNA from mBMSCs using a guanidium isothiocyanate based commercially available reagent.</li>
	<li>Perform reverse transcription of RNA into complementary DNA using a commercially available kit. The reaction conditions of reverse transcription were as follows: 65 &#176;C for 5 min, 37 &#176;C for 15 min, 85 &#176;C for 15 s.</li>
	<li>Perform real-time PCR to detect osteogenesis and adipogenesis specific genes using primers listed in Table 3. The PCR amplification procedure was as follows: 95 &#176;C for 5 min. 95 &#176;C for 5 s, 60 &#176;C for 30 s for 40 cycles.</li>
</ol>

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M., Alzahrani, A. M., Abdel-Moneim, A. M., Ditzel, N., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reliable+protocol+for+long-term+culture+of+murine+bone+marrow+stromal(mesenchymal)+stem+cells+that+retained+their+in+vitro+and+in+vivo+stemness+in+long-term+culture.">A simple and reliable protocol for long-term culture of murine bone marrow stromal(mesenchymal) stem cells that retained their in vitro and in vivo stemness in long-term culture.</a> Biology Proceedings Online. 21, 3 (2019).</li></ol>

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(1X)</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&amp;amp;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(1X)</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...

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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 ...

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

Research

JoVE Journal

Medicine

5.2K Views.  National Clinical Research center of Stomatology. Dit artikel presenteert een methode die volledige beenmergaanhechting en flowcytometriesortering combineert voor het isoleren, cultiveren, sorteren en identificeren van beenmerg mesenchymale stamcellen uit rattenkaken.

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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Biology

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

Bioengineering

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

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

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