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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, 1221 views, 0 Q&A
DOI: https://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)

[Abstract]

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.

材料和试剂

  1. 培养皿(培养面积:56.7cm 2,直径:100mm)(Thermo Fisher Scientific,目录号:150350)
  2. 细胞过滤器(70μm)(VWR International,Greiner Bio-One GmbH,目录号:89508-344)
  3. (图1B)用剪钳切割的夹子(Mitsuya,目录号:GM-590)
  4. 苯乙烯泡沫(RIKAKEN)(图1C)
  5. 50ml Falcon管(Corning,目录号:352070)
  6. 8-12周龄Lewis大鼠(Charles River Laboratories International)
  7. 胶原酶V(Wako Pure Chemical Industries,目录号:038-17851)
  8. α-最小必需培养基(α-MEM)(Thermo Fisher Scientific,目录号:12561-056)
  9. 胎牛血清(FBS)(Thermo Fisher Scientific,目录号:12483-020)
  10. 链霉素,青霉素,两性霉素B(抗生素 - 抗真菌剂,100x)(Thermo Fisher Scientific,Gibco TM,目录号:15240-062)
  11. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190-235)
  12. 含有10%FBS和抗生素的α-MEM(参见配方)
  13. 3mg/ml胶原酶溶液(用于来自两膝关节的滑膜)(参见配方)

设备

  1. 37℃,5%CO 2强制空气培养箱(ASTEC,目录号:SCA-165DS)中。
  2. 离心机(KUBOTA Corporation,型号:8730型)
  3. 手术刀(Natsume,目录号:11D-13),剪刀(Natsume,目录号:B-12)和镊子(Natsume, :A-6)(图1A)

程序

  1. 手术准备(10分钟)
    1. 通过在乙烯袋中输送CO 2来牺牲老鼠。
    2. 取出膝关节周围的头发,用70%乙醇无菌清洁腿(图1D)
    3. 把大鼠放在苯乙烯泡沫,并覆盖在大鼠的无菌板,保持右腿出洞。用苯乙烯泡沫上的夹子稳定踝部(图1E)。


      图1.手术准备 A.外科器械; B.用于稳定腿的夹子; C.苯乙烯形式; D.去除头发; E.腿的设置。

  2. 从膝关节收获滑膜(视频1)(两个膝关节10分钟)
    1. 暴露髌腱与直皮肤切口。
    2. 切割髌腱横向,并向上和向下剥离腱暴露下髌骨脂肪垫。
    3. 从股骨和胫骨分离髌下脂肪垫。
    4. 将滑膜置于Falcon管中的PBS中。

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      视频1.从右膝收获包括髌骨下脂肪垫的滑膜
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  3. 滑膜MSC的分离和培养
    1. 用刮刀将滑膜切成2-3毫米的块
    2. 将切碎的滑膜置于胶原酶溶液中
    3. 将管在孵化器中2-3小时。每小时摇动2-3次。
    4. 通过细胞过滤器过滤消化的溶液。
    5. 以580×g离心管5分钟。
    6. 除去上清液,将细胞平铺在56.7cm 2培养皿(约1,000细胞/cm 2 )上。
    7. 每周更换培养基两次,用含有10%FBS和抗生素的α-MEM培养2周

代表数据

我们通常可以从一个滑膜收获1×10 4个细胞(图2)。 2周后,我们可以从一个培养皿中获得5×10 5个<1> 1×10 6个 MSCs。细胞是异质的,我们通常在实验中使用2-4代的这些细胞。我们在之前的报告中报告了这些数据(Yoshimura等人,2007年)。


图2.滑膜MSC的代表性形态(第1天,第7天)。比例尺=50μm

食谱

  1. 含有10%FBS和抗生素的α-MEM α-MEM 445ml
    FBS 50ml
    100x抗生素 - 抗真菌5ml ml
  2. 胶原酶溶液3mg/ml(用于来自两膝关节的滑膜)
    α-MEM 3ml
    胶原酶V 9mg

致谢

我们没有利益冲突准备这篇文章。这项研究得到了日本医学研究开发机构(AMED)到IS的再生医学实现高速公路项目的支持。

参考文献

  1. Caplan,A.I。和Correa,D。(2011)。 MSC:伤害药店 细胞干细胞 9(1):11-15。
  2. De Bari,C.,Dell'Accio,F.,Tylzanowski,P.and Luyten,F.P。(2001)。 来自成人人滑膜的多功能间充质干细胞。关节炎风湿 em> 44(8):1928-1942。
  3. Horie,M.,Sekiya,I.,Muneta,T.,Ichinose,S.,Matsumoto,K.,Saito,H.,Murakami,T.and Kobayashi,E。(2009)。 关节内注射的滑膜干细胞直接分化为半月板细胞,促进半月板再生而不移动到大鼠大规模半月板缺损中的远端器官。干细胞 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)。 人间充质干细胞(MSC)的关节内注射通过被激活表达来促进大鼠半月板再生印度刺猬增强II型胶原蛋白的表达。骨关节炎软骨 20(10):1197-1207。
  5. Koga,H.,Shimaya,M.,Muneta,T.,Nimura,A.,Morito,T.,Hayashi,M.,Suzuki,S.,Ju,YJ,Mochizuki,T。和Sekiya, )。 用于移植间充质干细胞作为软骨缺损的潜在治疗的局部粘附技术。 > Arthritis Res Ther 10(4):R84。
  6. Katagiri,H.,Muneta,T.,Tsuji,K.,Horie,M.,Koga,H.,Ozeki,N.,Kobayashi,E。和Sekiya, 滑膜间充质干细胞聚集体的移植在大鼠大量半月板缺损中更有效地再生半月板。 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, Tanaka,H。和Kobayashi,E。(2012)。 关节镜,组织学和MRI分析软骨修复后微创移植同种异体滑膜间质基质的方法细胞进入猪的软骨缺陷。 Cytotherapy 14(3):327-338。
  8. Okuno,M.,Muneta,T.,Koga,H.,Ozeki,N.,Nakagawa,Y.,Tsuji,K.,Yoshiya,S.and Sekiya, 在大鼠模型中滑膜间充质干细胞的同基因,轻微错配和主要错配移植的半月板再生。 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, Saito,T。和Sekiya,I.(2015)。 滑膜间质干细胞在大鼠部分半月板缺损模型中促进由自体跟腱移植增强的半月板再生。 Stem Cells 33(6):1927-1938。
  10. Sakaguchi,Y.,Sekiya,I.,Yagishita,K.and Muneta,T。(2005)。 源自各种间充质组织的人类干细胞的比较:作为细胞来源的滑膜的优势。 Arthritis Rheum 52(8):2521-2529。
  11. Sekiya,I.,Muneta,T.,Horie,M。和Koga,H。(2015)。 关节镜移植滑膜干细胞改善膝盖软骨缺损的临床结果 Clin Orthop Relat Res 473(7):2316-2326。
  12. Yoshimura,H.,Muneta,T.,Nimura,A.,Yokoyama,A.,Koga,H.and Sekiya,I。(2007)。 来自骨髓,滑膜,骨膜,脂肪组织和肌肉的大鼠间充质干细胞的比较。/a> Cell Tissue Res 327(3):449-462
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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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