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

Podsumowanie

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.

Streszczenie

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

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

Wprowadzenie

Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic stem cells derived from bone marrow that manifest strong proliferation capability and multi-lineage differentiation potential^1,2,3,4. Indeed, BMSCs have been considered as an ideal candidate for bone tissue engineering and regeneration ever since they were discovered. For years, the iliac crest or long bones such as the tibia and femur have been the most common source of BMSCs for craniofacial regeneration. However, orofacial BMSCs, such as mandibular BMSCs (mBMSCs), display 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 BMSCs^5,6,7,8. 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 defect^10,11,12. 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 cells^13,14. 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.

Protokół

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

1. Preparation

1. Use two 5-week-old male Sprague Dawley rats for the experiment.
2. Sterilize all the instruments, including needle holders, tweezers and scissors at high temperature or immersed in 75% ethanol for 10 min.
   NOTE: Ethanol immersion should not be too long to avoid cell damage.
3. Prepare culture media beforehand, the composition of which is provided in Table 1. Supplement each medium as described below.
   1. Preparation of α-MEM culture medium (with 10% FBS): Supplement minimum essential medium alpha (α-MEM) with 10% fetal bovine serum and 1% penicillin and streptomycin.
   2. 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% β-glycerophosphate and 0.01% dexamethasone.
   3. Preparation of osteogenic induction medium: Mix 70% α-MEM culture medium (with 10% FBS) and 30% osteogenic differentiation medium.
   4. 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.
   5. 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.
   6. 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-β3.
      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.

2. Isolation and cultivation of rat mBMSCs

NOTE: All experimental operations should be performed on ice as much as possible to maintain cell viability.

1. Harvesting rat mandibles
   1. Euthanize two 5-week-old male Sprague Dawley rats by CO₂ asphyxiation. Ensure that the animal’s breathing is stopped before proceeding with the experiment.
   2. Rinse these rats in a beaker containing 75% ethanol for 3 min.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. 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.
   8. Separate the mandible body from the skull.
   9. Clean the adherent soft tissue on the bone surface using a wet gauze.
   10. Place the bone in a 10 cm sterile glass dish filled with precooled minimum essential medium-α (α-MEM) or phosphate buffer saline (PBS) on ice to preserve the viability.
2. Isolation and cultivation of the mBMSCs
   1. 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.
   2. Fill a 10 cm sterile glass dish with 10 mL of α-MEM (with 10% FBS).
   3. Use 1 mL syringe to aspirate α-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.
   4. Transfer the media containing the flushed cells into a 15 mL centrifuge tube and centrifuge at 800 x g in 4 °C for 5 min. Discard the supernatant.
   5. Resuspend the cells with 3 mL of α-MEM (with 10% FBS). Plate the cells in a new 10 cm culture dish and incubate at 37 °C in a 5% CO₂ incubator.
   6. 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 α-MEM (with 10% FBS).
   7. After 7 days of culture, P0 mBMSCs reached 70% to 80% confluence.
3. Flow cell sorting
   NOTE: P0 mBMSCs were phenotypically identified and primarily purified through flow cell sorting.
   1. Aspirate and discard the culture medium and then wash the dish with PBS. Discard the PBS.
   2. Add 1 mL of 0.25% trypsin with 0.02% EDTA in the dish. Digest the cells at 37 °C for 5 min, then add 2 mL of α-MEM (with 10% FBS) to stop the reaction.
   3. Transfer the cells suspension into a 15 mL centrifuge tube and centrifuge at 800 x g for 5 min.
   4. Resuspend the cells in 120 μL of PBS (with 10% FBS) after centrifugation.
   5. Block these cell suspensions with 1 μL of antibody against CD16/CD32 at 4 °C for 15 min.
   6. Transfer 100 µL 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 °C for 1 h in the dark^13,15. The concentration of antibodies used in this experiment is shown in Table 2. Use the other 20 µL of cell suspension as unstained negative control.
   7. 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).
   8. Add 10 μL of 0.01 mg/mL DAPI for 10 min before analysis.
   9. Use 40 nm filters placed on centrifuge tubes to filter the cells.
   10. 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.
   11. Collect the sorted CD29⁺CD90⁺CD45^- cells into a 15 mL centrifuge tube with 3 mL of α-MEM (with 10% FBS) pre-prepared.
   12. Centrifuge the tubes at 800 x g for 5 min. Remove the collection buffer and add 1 mL of fresh α-MEM (with 10% FBS) to resuspend the cells. Then plate them in a 6 cm culture dish.

3. Colony formation capability

NOTE: This step was performed to check for the division ability of mBMSCs.¹⁵

1. 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.
2. Culture the cells in α-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.
3. 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.
4. Remove the paraformaldehyde and rinse the wells twice with PBS.
5. Stain the cells with crystal violet staining solution and incubate them in 37 °C for about 10 min.
   NOTE: The staining time adjusted appropriately until the desired shade is achieved.
6. Remove the staining solution and wash the samples with distilled water to stop the reaction.
7. Image under a stereomicroscope and count scattered cell colonies which consisted of at least 50 cells.

4. Multilineage differentiation of mBMSCs

NOTE: The P2 mBMSCs were used for subsequent experiments unless otherwise described.

1. Osteogenic induction of mBMSCs
   1. 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 α-MEM (with 10% FBS).
   2. When the cell confluence reached 60-70%, change the medium into 30% osteogenic induction media. Culture the cells with α-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.
   3. After culturing for seven days, assess the calcification of these cells by alkaline phosphatase staining^16,17.
   4. 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.
   5. Then stain the cells with alkaline phosphatase staining solution and incubate at 37 °C for 10-30 min.
      NOTE: Incubation can be performed overnight at room temperature to obtain the required color.
   6. Wash the wells with distilled water to stop the reaction. Take photographs under light microscope. Red pigmented granules represent alkaline phosphatase (ALP) activity.
   7. After culturing the remaining cells for another 7 days, perform alizarin red staining to evaluate the mineralization capacity of the cells.
   8. Stain the cells with 0.5% alizarin red staining solution after cell fixation for 10 min^16,18. Stop the chromogenic reaction with distilled water after 3-5 min.
      NOTE: The reaction time can be extended depending on the color developed.
   9. Finally, place the culture plate under the light microscope to observe the effect of osteogenic staining; red nodules indicate calcium deposits of these cells.
2. Adipogenic induction of mBMSCs
   1. Seed P2 mBMSCs in a 12 well plate as described above.
   2. After the well become 90% confluent, culture the cells with adipogenic differentiation medium A. Use cells cultured in α-MEM culture medium (with 10% FBS) as negative control.
   3. After 2 days of induction, aspirate the medium A from the well and add 1 mL of adipogenic differentiation medium B in each well.
   4. 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.
   5. Apply the differentiation medium A and B alternately for three cycles and detect the adipogenesis of these cells by oil red O staining^16,18.
      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.
   6. Remove the medium and fix the cells with 4% paraformaldehyde for 10 min. Wash out the cells with PBS.
   7. Stain the cells with oil red O staining solution and incubate at 37 °C for approximately 3 min.
   8. Remove the staining solution and wash the wells with distilled water.
   9. Observe adipogenesis under light microscope.
3. Chondrogenesis induction of mBMSCs
   1. Seed P2 mBMSCs in 15 mL centrifuge tube at a density of 5 x 10⁵/cm².
   2. Centrifuge the tubes at 300 x g for 5 min. Discard the supernatant.
   3. Resuspend the cells with 0.5 mL of chondrogenic differentiation medium. Centrifuge the cells at 300 x g for 5 min.
   4. Loosen the cap of the centrifuge tube and incubate it at 37 °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.
   5. After 21 days of induction, fix the cell pellets with 4% paraformaldehyde, followed by dehydration, paraffin embedding and sectioning.
   6. After deparaffinization and hydration, stain the slides in Alcian blue solution for at least 15 min^18,19,20,21, then wash with distilled water to stop the reaction. Capture the photographs under light microscope.
   7. For immunofluorescent staining of collagen type II, perform the steps below.
      1. Incubate the slides with 5 µg/mL proteinase K for 15 min at 37 °C after the deparaffinization and hydration step.
      2. Incubate the slides in blocking buffer for 1 h at 37 °C.
      3. Apply 1 µL of collagen type II antibody in 200 µL of blocking buffer to the slides and incubate overnight at 4 °C.
      4. The next day, wash the slides with PBS twice and incubate with secondary antibodies at 37 °C for 1 h.
      5. Incubate the slices with 40,6-diamidino-2-phenylindole (DAPI) for 10 min.
   8. Take photographs of each slides under fluorescence microscopy.

5. Real-time PCR

1. Extract total RNA from mBMSCs using a guanidium isothiocyanate based commercially available reagent.
2. Perform reverse transcription of RNA into complementary DNA using a commercially available kit. The reaction conditions of reverse transcription were as follows: 65 °C for 5 min, 37 °C for 15 min, 85 °C for 15 s.
3. 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 °C for 5 min. 95 °C for 5 s, 60 °C for 30 s for 40 cycles.

Wyniki

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

Dyskusje

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,...

Materiały

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