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Mesenchymal stem cells (MSCs), first described in human bone marrow, are emerging as promising cell-based therapeutics for a wide range of diseases (Caplan and Correa, 2011). MSCs have been isolated from various organs in the body, and synovial MSCs were first reported by De Bari et al. (2001). We previously reported that synovial MSCs have superior proliferation and chondrogenic potentials as compared to bone marrow-, muscle-, and adipose- derived MSCs in humans (Sakaguchi et al., 2005) and rats (Yoshimura et al., 2007). In addition, administration of synovial MSCs for osteochondral defect promoted cartilage regeneration in a rabbit (Koga et al., 2008) and a pig model (Nakamura et al., 2012). In 2008, we started a clinical trial in human and obtained satisfactory results of symptoms and regenerated cartilage by Magnetic Resonance Imaging (Sekiya et al., 2015). We have also engaged in multiple research lines using synovial MSCs for meniscus regeneration in rats (Horie et al., 2009; Horie et al., 2012; Katagiri et al., 2013; Okuno et al., 2014; Ozeki et al., 2015). In this article, we demonstrated how to harvest the synovium including infrapatellar fat pad from a rat knee joint, and to describe the technique of isolation and culture of rat synovial MSCs.

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Preparation of Synovial Mesenchymal Stem Cells from a Rat Knee Joint
从大鼠膝关节制备滑膜间充质干细胞

干细胞 > 成体干细胞 > 间充质干细胞
作者: Nobutake Ozeki
Nobutake OzekiAffiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Bio-protocol author page: a3103
Takeshi Muneta
Takeshi MunetaAffiliation: Department of Joint Surgery and Sports Medicine, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
Bio-protocol author page: a3104
Mitsuru Mizuno
Mitsuru MizunoAffiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Bio-protocol author page: a3105
 and Ichiro Sekiya
Ichiro SekiyaAffiliation: Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan
For correspondence: sekiya.arm@tmd.ac.jp
Bio-protocol author page: a3106
Vol 6, Iss 9, 5/5/2016, 583 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1799

[Abstract] Mesenchymal stem cells (MSCs), first described in human bone marrow, are emerging as promising cell-based therapeutics for a wide range of diseases (Caplan and Correa, 2011). MSCs have been isolated from various organs in the body, and synovial MSCs were first reported by De Bari et al. (2001). We previously reported that synovial MSCs have superior proliferation and chondrogenic potentials as compared to bone marrow-, muscle-, and adipose- derived MSCs in humans (Sakaguchi et al., 2005) and rats (Yoshimura et al., 2007). In addition, administration of synovial MSCs for osteochondral defect promoted cartilage regeneration in a rabbit (Koga et al., 2008) and a pig model (Nakamura et al., 2012). In 2008, we started a clinical trial in human and obtained satisfactory results of symptoms and regenerated cartilage by Magnetic Resonance Imaging (Sekiya et al., 2015). We have also engaged in multiple research lines using synovial MSCs for meniscus regeneration in rats (Horie et al., 2009; Horie et al., 2012; Katagiri et al., 2013; Okuno et al., 2014; Ozeki et al., 2015). In this article, we demonstrated how to harvest the synovium including infrapatellar fat pad from a rat knee joint, and to describe the technique of isolation and culture of rat synovial MSCs.

Keywords: Synovium(滑膜), Mesenchymal stem cells(间充质干细胞), Rat(老鼠)

Materials and Reagents

  1. Culture dish (culture area: 56.7 cm2, diameter: 100 mm) (Thermo Fisher Scientific, catalog number: 150350)
  2. Cell strainer (70 µm) (VWR International, Greiner Bio-One GmbH, catalog number: 89508-344)
  3. Clip (Mitsuya, catalog number: GM-590) cut by pliers in a half size (Figure 1B)
  4. Styrene foam (RIKAKEN) (Figure 1C)
  5. 50 ml Falcon tube (Corning, catalog number: 352070)
  6. 8-12 weeks old Lewis rat (Charles River Laboratories International)
  7. Collagenase V (Wako Pure Chemical Industries, catalog number: 038-17851)
  8. α-minimal essential medium (α-MEM) (Thermo Fisher Scientific, catalog number: 12561-056)
  9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: 12483-020)
  10. Streptomycin, Penicilin, Amphotericin B (Antibiotic-Antimycotic, 100x) (Thermo Fisher Scientific, Gibco™, catalog number: 15240-062)
  11. Phosphate buffer saline (PBS) (Thermo Fisher Scientific, Gibco™, catalog number: 14190-235)
  12. α-MEM containing 10% FBS with antibiotics (see Recipes)
  13. 3 mg/ml Collagenase solution (for synovium from both knee joints) (see Recipes)

Equipment

  1. 37 degree, 5% CO2 forced-air incubator (ASTEC, catalog number: SCA-165DS)
  2. Centrifuge machine (KUBOTA Corporation, model: Model 8730)
  3. Scalpel holder (Natsume, catalog number: No.3 D-11) with blade (Natsume, catalog number: No.11 D-13), scissors (Natsume, catalog number: B-12), and tweezers (Natsume, catalog number: A-6) (Figure 1A)

Procedure

  1. Preparation of the surgery (10 min)
    1. Sacrifice a rat by delivering CO2 in a vinyl bag.
    2. Remove the hair around the knee joints, and sterile clean the legs with 70% ethanol (Figure 1D).
    3. Put the rat on the styrene foam, and cover the sterile sheet on the rat, keeping the right leg out of the hole. Stabilize the ankle with clip on the styrene foam (Figure 1E).


      Figure 1. Preparation of the surgery. A. Surgical instruments; B. Clip for stabilization of legs; C. Styrene form; D. Removal of the hair; E. Settings of the legs.

  2. Harvesting the synovium from the knee joint (Video 1) (10 min for both knee joints)
    1. Expose the patellar tendon with straight skin incision.
    2. Cut the patellar tendon transversely, and peel the tendon upward and downward to expose the infrapatellar fat pad.
    3. Separate the infrapatellar fat pad from the femur and tibia.
    4. Put the synovium into PBS in a Falcon tube.

      Video 1. Harvesting the synovium including infrapatellar fat pad from the right knee

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


  3. Isolation and culture of synovial MSCs
    1. Mince the synovium with a scalpel into 2-3 mm pieces.
    2. Put the minced synovium in collagenase solution.
    3. Place the tube in the incubator for 2-3 h. Shake it 2-3 times per hour.
    4. Filter the digested solution through a cell strainer.
    5. Centrifuge the tube at 580 x g for 5 min.
    6. Remove the supernatant, and plate the cells on the 56.7 cm2 dish (about 1,000 cells/ cm2).
    7. Change the medium twice a week and culture for 2 weeks with α-MEM containing 10% FBS with antibiotics.

Representative data

We can usually harvest 1 x 104 cells from one synovium (Figure 2). After 2 weeks, we can obtain 5 x 105 - 1 x 106 MSCs from one dish. The cells are heterogenous, and we usually use these cells of passage 2-4 in the experiments. We reported these data in our previous reports (Yoshimura et al., 2007).


Figure 2. Representative morphology of synovial MSCs (passage 1, day 7). Scale bar = 50 µm

Recipes

  1. α-MEM containing 10% FBS with antibiotics
    α-MEM 445 ml
    FBS 50 ml
    100x Antibiotic-Antimycotic 5 ml
  2. Collagenase solution 3 mg/ml (for synovium from both knee joints)
    α-MEM 3 ml
    Collagenase V 9 mg

Acknowledgments

We have no conflict of interest to prepare this article. This study was supported by the Highway Program for Realization of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) to IS.

References

  1. Caplan, A. I. and Correa, D. (2011). The MSC: an injury drugstore. Cell Stem Cell 9(1): 11-15.
  2. De Bari, C., Dell'Accio, F., Tylzanowski, P. and Luyten, F. P. (2001). Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 44(8): 1928-1942.
  3. Horie, M., Sekiya, I., Muneta, T., Ichinose, S., Matsumoto, K., Saito, H., Murakami, T. and Kobayashi, E. (2009). Intra-articular Injected synovial stem cells differentiate into meniscal cells directly and promote meniscal regeneration without mobilization to distant organs in rat massive meniscal defect. Stem Cells 27(4): 878-887.
  4. Horie, M., Choi, H., Lee, R. H., Reger, R. L., Ylostalo, J., Muneta, T., Sekiya, I. and Prockop, D. J. (2012). Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that enhances expression of type II collagen. Osteoarthritis Cartilage 20(10): 1197-1207.
  5. Koga, H., Shimaya, M., Muneta, T., Nimura, A., Morito, T., Hayashi, M., Suzuki, S., Ju, Y. J., Mochizuki, T. and Sekiya, I. (2008). Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect. Arthritis Res Ther 10(4): R84.
  6. Katagiri, H., Muneta, T., Tsuji, K., Horie, M., Koga, H., Ozeki, N., Kobayashi, E. and Sekiya, I. (2013). Transplantation of aggregates of synovial mesenchymal stem cells regenerates meniscus more effectively in a rat massive meniscal defect. Biochem Biophys Res Commun 435(4): 603-609.
  7. Nakamura, T., Sekiya, I., Muneta, T., Hatsushika, D., Horie, M., Tsuji, K., Kawarasaki, T., Watanabe, A., Hishikawa, S., Fujimoto, Y., Tanaka, H. and Kobayashi, E. (2012). Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs. Cytotherapy 14(3): 327-338.
  8. Okuno, M., Muneta, T., Koga, H., Ozeki, N., Nakagawa, Y., Tsuji, K., Yoshiya, S. and Sekiya, I. (2014). Meniscus regeneration by syngeneic, minor mismatched, and major mismatched transplantation of synovial mesenchymal stem cells in a rat model. J Orthop Res 32(7): 928-936.
  9. Ozeki, N., Muneta, T., Matsuta, S., Koga, H., Nakagawa, Y., Mizuno, M., Tsuji, K., Mabuchi, Y., Akazawa, C., Kobayashi, E., Saito, T. and Sekiya, I. (2015). Synovial mesenchymal stem cells promote meniscus regeneration augmented by an autologous Achilles tendon graft in a rat partial meniscus defect model. Stem Cells 33(6): 1927-1938.
  10. Sakaguchi, Y., Sekiya, I., Yagishita, K. and Muneta, T. (2005). Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum 52(8): 2521-2529.
  11. Sekiya, I., Muneta, T., Horie, M. and Koga, H. (2015). Arthroscopic transplantation of synovial stem cells improves clinical outcomes in knees with cartilage defects. Clin Orthop Relat Res 473(7): 2316-2326.
  12. Yoshimura, H., Muneta, T., Nimura, A., Yokoyama, A., Koga, H. and Sekiya, I. (2007). Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res 327(3): 449-462.


How to cite this protocol: Ozeki, N., Muneta, T., Mizuno, M. and Sekiya, I. (2016). Preparation of Synovial Mesenchymal Stem Cells from a Rat Knee Joint. Bio-protocol 6(9): e1799. DOI: 10.21769/BioProtoc.1799; Full Text



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