Marrow Stroma Cell Culture and Identification of SD Rat in Vitro_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LIU Kun ¹ , LUO Jun ¹ , LOU Fan ² , MA Yan ³

1.Center of Digestion and Vascular Surgery, The First Teaching Hospital of Xinjiang Medical University， Urumqi 830054， China;;
2.Center of Neoplastic Diagnose and Treat, The First Teaching Hospital of Xinjiang Medical University， Urumqi 830054， China;;
3.Center of Medical Research， The First Teaching Hospital of Xinjiang Medical University， Urumqi 830054， China;

Corresponding author：

Keywords：

Marrow stroma cell; Adult stem cells; Cell culture; Immunohistochemistry

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Objective To research the methods and techniques of SD rat bone marrow stromal cells （MSCs）culture in vitro and to provide a large number of MSCs for cell therapy. Methods Bone marrow from the femur and tibia of the early age SD rats was taken to culture to the passage 1-4 （P1-P4）, its growth was observed and P3 cells were evaluated by HE staining and immunohistochemisty. Results The growing speed of P1-P3 was faster than that of P0, cells fusion was 85%-90% after 3-4 d and the cells were arranged in groups liked whirlpool shape or parallel; （3.4-3.6）×104/cm2 cells were gained and the total cell number of P1-P3 was 4.08×106, 2.44×107 and 2.85×108 respectively, the rate of trypan blue rejecting stained was 95%-97%. P4’s growing speed was slower than before, 1.42×109 in 3.0×104/cm2 cells were gained, and the rate of rejecting stained was 95%. P4-cell output was amplified nearly 2 000-fold higher than P0-cell. P3 immunohistochemical analysis indicated CD105+ cells 61.9%， CD44+ 45.4%， CD29+ 16.8%， CD45+ 8.2%， CD31+ 13.7%， CD34+ 8.3% and CD11b+ 1.5%, respectively. Conclusion The culture of whole bone marrow is suitable for a large number of MSCs provision in vitro, and can meet the needs of the cell therapy research.

Citation： LIU Kun,LUO Jun,LOU Fan,MA Yan. Marrow Stroma Cell Culture and Identification of SD Rat in Vitro. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2009, 16(11): 875-879. doi: Copy

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10.	Gastens MH, Goltry K, Prohaska W, et al. Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy [J]. Cell Transplant, 2007; 16(7): 685-696.
11.	Burt RK, Testori A, Oyama Y, et al. Autologous peripheral blood CD133+ cell implantation for limb salvage in patients with critical limb ischemia [J]. Bone Marrow Transplant, 2009 May 18. [Epub ahead of print].
12.	Ishikane S, Ohnishi S, Yamahara K, et al. Allogeneic injection of fetal membrane-derived mesenchymal stem cells induces therapeutic angiogenesis in a rat model of hind limb ischemia [J]. Stem Cells, 2008; 26(10): 2625-2633.
13.	汪忠镐，李震. 我国血管外科的现状和发展 [J]. 中国普外基础与临床杂志, 2006; 13(6)： 625-628.
14.	Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science, 1999; 284(5411): 143-147.
15.	Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature, 2002; 418(6893): 41-49.
16.	Davani S, Marandin A, Mersin N, et al. Mesenchymal progenitor cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a rat cellular cardiomyoplasty model [J]. Circulation, 2003; 108 Suppl 1: II253-II258.
17.	罗蔚锋, 包仕尧, 张志琳, 等. 大鼠骨髓基质细胞的体外培养及鉴定 [J]. 苏州大学学报(医学版), 2005; 25(1)： 51-53.
18.	黄涛, 徐如祥, 姜晓丹, 等. 大鼠骨髓基质细胞的培养及其表面抗原的检测[J]. 中国临床解剖学杂志, 2008，26(2)：171-174.
19.	Sessarego N, Parodi A, Podestà M, et al. Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application [J]. Haematologica, 2008; 93(3): 339-346.
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22.	王君, 李海红, 付小兵, 等. 创伤条件下骨髓细胞向皮肤软组织细胞分化的研究进展 [J]. 中国危重病急救医学, 2006; 18（1）： 58-61.
23.	Bhattacharya V, McSweeney PA, Shi Q, et al. Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34(+) bone marrow cells [J]. Blood, 2000; 95(2): 581-585.
24.	Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization [J]. Circ Res, 1999; 85(3): 221-228.
25.	Kaiser S, Hackanson B, Follo M, et al. BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype [J]. Cytotherapy, 2007; 9(5): 439-450.
26.	Iba O, Matsubara H, Nozawa Y, et al. Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs [J]. Circulation, 2002; 106(15): 2019-2025.

1. Psaltis PJ, Zannettino AC, Worthley SG, et al. Concise review: mesenchymal stromal cells: potential for cardiovascular repair [J]. Stem Cells， 2008; 26(9): 2201-2210.
2. Haack-Sorensen M, Friis T, Bindslev L, et al. Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy [J]. Scand J Clin Lab Invest， 2008; 68(3): 192-203.
3. 缪健航，师天雄，胡锡祥，等. 自体外周血干细胞移植治疗下肢动脉缺血性疾病的临床应用 [J]. 中国普外基础与临床杂志, 2008； 15(2)： 106-108.
4. 张鸿坤，李鸣，封华, 等. 骨髓动员外周血干细胞移植治疗严重肢体缺血的临床应用 [J]. 浙江大学学报(医学版), 2007； 36(4): 360-363.
5. Saito S, Nishikawa K, Obata H, et al. Autologous bone marrow transplantation and hyperbaric oxygen therapy for patients with thromboangiitis obliterans [J]. Angiology， 2007; 58(4): 429-434.
6. Amann B, Luedemann C, Ratei R, et al. Autologous bone marrow cell transplantation increases leg perfusion and reduces amputations in patients with advanced critical limb ischemia due to peripheral artery disease [J]. Cell Transplant， 2009; 18(3): 371-380.
7. Procházka V, Gumulec J, Chmelová J, et al. Autologous bone marrow stem cell transplantation in patients with end-stage chronical critical limb ischemia and diabetic foot [J]. Vnitr Lek， 2009; 55(3): 173-178.
8. Nevskaya T, Ananieva L, Bykovskaia S, et al. Autologous progenitor cell implantation as a novel therapeutic intervention for ischaemic digits in systemic sclerosis [J]. Rheumatology (Oxford), 2009; 48(1): 61-64.
9. Sotiropoulou PA, Perez SA, Papamichail M. Clinical grade expansion of human bone marrow mesenchymal stem cells [J]. Methods Mol Biol, 2007; 407: 245-263.
10. Gastens MH, Goltry K, Prohaska W, et al. Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy [J]. Cell Transplant, 2007; 16(7): 685-696.
11. Burt RK, Testori A, Oyama Y, et al. Autologous peripheral blood CD133+ cell implantation for limb salvage in patients with critical limb ischemia [J]. Bone Marrow Transplant, 2009 May 18. [Epub ahead of print].
12. Ishikane S, Ohnishi S, Yamahara K, et al. Allogeneic injection of fetal membrane-derived mesenchymal stem cells induces therapeutic angiogenesis in a rat model of hind limb ischemia [J]. Stem Cells, 2008; 26(10): 2625-2633.
13. 汪忠镐，李震. 我国血管外科的现状和发展 [J]. 中国普外基础与临床杂志, 2006; 13(6)： 625-628.
14. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science, 1999; 284(5411): 143-147.
15. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature, 2002; 418(6893): 41-49.
16. Davani S, Marandin A, Mersin N, et al. Mesenchymal progenitor cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a rat cellular cardiomyoplasty model [J]. Circulation, 2003; 108 Suppl 1: II253-II258.
17. 罗蔚锋, 包仕尧, 张志琳, 等. 大鼠骨髓基质细胞的体外培养及鉴定 [J]. 苏州大学学报(医学版), 2005; 25(1)： 51-53.
18. 黄涛, 徐如祥, 姜晓丹, 等. 大鼠骨髓基质细胞的培养及其表面抗原的检测[J]. 中国临床解剖学杂志, 2008，26(2)：171-174.
19. Sessarego N, Parodi A, Podestà M, et al. Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application [J]. Haematologica, 2008; 93(3): 339-346.
20. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement [J]. Cytotherapy, 2006; 8(4): 315-317.
21. Harting M, Jimenez F, Pati S, et al. Immunophenotype cha-racterization of rat mesenchymal stromal cells [J]. Cytotherapy, 2008; 10(3): 243-253.
22. 王君, 李海红, 付小兵, 等. 创伤条件下骨髓细胞向皮肤软组织细胞分化的研究进展 [J]. 中国危重病急救医学, 2006; 18（1）： 58-61.
23. Bhattacharya V, McSweeney PA, Shi Q, et al. Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34(+) bone marrow cells [J]. Blood, 2000; 95(2): 581-585.
24. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization [J]. Circ Res, 1999; 85(3): 221-228.
25. Kaiser S, Hackanson B, Follo M, et al. BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype [J]. Cytotherapy, 2007; 9(5): 439-450.
26. Iba O, Matsubara H, Nozawa Y, et al. Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs [J]. Circulation, 2002; 106(15): 2019-2025.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Marrow Stroma Cell Culture and Identification of SD Rat in Vitro

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