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&#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 ...

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

Research

JoVE Journal

Medicine

5.1K Views.  National Clinical Research center of Stomatology. 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.

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 ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...