低氧条件下骨髓间充质干细胞在中空纤维膜反应器中的扩增
|
摘要
骨髓间充质干细胞(Mesenchymal stem cells,MSCs)是一类存在于骨髓间质层的极少量细胞,不但具有强大的自我更新及多向分化潜能,还具有支持造血的重要功能,并且植入反应弱,易于被外源基因转染并稳定表达,是组织工程和细胞/基因治疗中较为理想的种子细胞,受到广泛关注。
但是骨髓中的MSCs的含量非常少,并且在正常培养条件下细胞在传代过程中容易失去增殖和分化活性,这极大影响了间充质干细胞的可用性。所以在扩增过程中,如何在保证增殖数量的同时,减少在细胞增殖过程中产生的损伤,保持干细胞的特性及分泌基质的能力,这是组织工程中首先应解决的问题,也是临床利用这些细胞治疗疾病的前提。
为了提高间充质干细胞的扩增倍数并保持干细胞的特性,本文在低氧条件下采用灌注式中空纤维膜生物反应器(Hollow-fiber Membrane Bioreactor)进行体外扩增MSCs。采用密度梯度离心法分离SD大鼠骨髓间充质干细胞,将第五代MSCs接种于醋酸纤维素中空纤维膜上预培养24小时后,转移至反应器中。根据骨髓中溶解氧对应的气相氧气浓度的范围是1%-7%,用三气培养箱制造低氧环境,低氧组设为5%O_2,对照组为20%O_2。连续培养7天后,收获细胞。通过计算细胞的扩增倍数、成纤维细胞-集落形成实验、多向诱导分化检测和流式细胞仪分析,考察低氧动态环境对骨髓间充质干细胞增殖的影响。
结果表明,低氧条件下在灌注式中空纤维膜生物反应器内,O_2含量基本保持恒定;MSCs扩增倍数明显增加,7天后扩增倍数达到11倍,明显高于常氧组8倍的扩增倍数。在低氧动态环境下扩增的细胞具有更强的成集落能力,低氧组集落数为112,而对照组为53,并且细胞的增殖潜能和代谢活性都有所增强。多向诱导分化检测和流式细胞仪分析结果显示,骨髓间充质干细胞在低氧动态条件下培养具有更强的诱导分化能力,保持了较好的干细胞特性。因此,本文所采用的培养方法是体外培养与扩增MSCs的一种有效的方法。
Rat mesenchymal stem cells (MSCs) represent a small portion of the cells in the stromal compartment of bone marrow, capable of differentiating in vitro into mature-like cells from all three germ layers. They have the potential to differentiate into cartilage cells, cardiac muscle cells, adipose cells, nerve cells and osteoblasts. They are the promising "seed cells" in tissue engineering and ideal target cells in gene therapy for their low implanting response, being easy to be transfected and expressed steadily and function of supporting hematopoiesis.
However, MSCs are in limited quantity and during the process of proliferation, MSCs may lose the activity of proliferation and differentiation gradually under the normal culture conditions, which can not meet the need of autologous repairation and clinical therapy. So how to reduce the damage during the cell proliferation, and retain the character and the ability of secreting matrix of the stem cells is very important.
In order to raise the expansion fold and retain the stemness, a perfusion hollow fiber membrane bioreactor was developed and used to culture and expand MSCs in vitro. SD rat mesenchymal stem cells (rMSCs) were isolated with density gradient centrifugation. The cells were seeded on cellulose acetate hollow-fiber membrane constructs and preincubated for 24 hours. Then the constructs were transferred to the bioreactor and cultured for up to 7 days. Because the partial pressure of the atmospheric oxygen (pO_2) in bone marrow is much lower than that in the air, rMSCs were grown under low-pO2 conditions (5% oxygen) and air (21% oxygen) for comparison. The cell expansion fold, colony formation, multi-differentiation were assayed and fluorescence-activated cell sorter analysis were carried out to examine the role of different pO_2 in dynamic condition on rMSC proliferation.
The results showed that in hollow-fiber membrane bioreactor, O_2 concentration kept almost constant; MSCs expansion fold in 5%O_2 is 11, while 8 in control oxygen and proliferation potential and metabolic activity were higher for cultures maintained in low oxygen than those in control oxygen. In 5%O_2 condition rMSCs on hollow-fiber membranes can maintain significantly higher colony-forming unit capabilities than normoxic controls, approximately 112 and 53 respectively.
Hypoxic rMSCs also expressed higher levels of CD44~+ and CD29~+ markers than normoxic controls. After being induced, the expanded cells still reserved the strong muti-differentiation potential. Therefore, it's safe and effective for MSCs culture and expansion in vitro with the method used here.
但是骨髓中的MSCs的含量非常少,并且在正常培养条件下细胞在传代过程中容易失去增殖和分化活性,这极大影响了间充质干细胞的可用性。所以在扩增过程中,如何在保证增殖数量的同时,减少在细胞增殖过程中产生的损伤,保持干细胞的特性及分泌基质的能力,这是组织工程中首先应解决的问题,也是临床利用这些细胞治疗疾病的前提。
为了提高间充质干细胞的扩增倍数并保持干细胞的特性,本文在低氧条件下采用灌注式中空纤维膜生物反应器(Hollow-fiber Membrane Bioreactor)进行体外扩增MSCs。采用密度梯度离心法分离SD大鼠骨髓间充质干细胞,将第五代MSCs接种于醋酸纤维素中空纤维膜上预培养24小时后,转移至反应器中。根据骨髓中溶解氧对应的气相氧气浓度的范围是1%-7%,用三气培养箱制造低氧环境,低氧组设为5%O_2,对照组为20%O_2。连续培养7天后,收获细胞。通过计算细胞的扩增倍数、成纤维细胞-集落形成实验、多向诱导分化检测和流式细胞仪分析,考察低氧动态环境对骨髓间充质干细胞增殖的影响。
结果表明,低氧条件下在灌注式中空纤维膜生物反应器内,O_2含量基本保持恒定;MSCs扩增倍数明显增加,7天后扩增倍数达到11倍,明显高于常氧组8倍的扩增倍数。在低氧动态环境下扩增的细胞具有更强的成集落能力,低氧组集落数为112,而对照组为53,并且细胞的增殖潜能和代谢活性都有所增强。多向诱导分化检测和流式细胞仪分析结果显示,骨髓间充质干细胞在低氧动态条件下培养具有更强的诱导分化能力,保持了较好的干细胞特性。因此,本文所采用的培养方法是体外培养与扩增MSCs的一种有效的方法。
Rat mesenchymal stem cells (MSCs) represent a small portion of the cells in the stromal compartment of bone marrow, capable of differentiating in vitro into mature-like cells from all three germ layers. They have the potential to differentiate into cartilage cells, cardiac muscle cells, adipose cells, nerve cells and osteoblasts. They are the promising "seed cells" in tissue engineering and ideal target cells in gene therapy for their low implanting response, being easy to be transfected and expressed steadily and function of supporting hematopoiesis.
However, MSCs are in limited quantity and during the process of proliferation, MSCs may lose the activity of proliferation and differentiation gradually under the normal culture conditions, which can not meet the need of autologous repairation and clinical therapy. So how to reduce the damage during the cell proliferation, and retain the character and the ability of secreting matrix of the stem cells is very important.
In order to raise the expansion fold and retain the stemness, a perfusion hollow fiber membrane bioreactor was developed and used to culture and expand MSCs in vitro. SD rat mesenchymal stem cells (rMSCs) were isolated with density gradient centrifugation. The cells were seeded on cellulose acetate hollow-fiber membrane constructs and preincubated for 24 hours. Then the constructs were transferred to the bioreactor and cultured for up to 7 days. Because the partial pressure of the atmospheric oxygen (pO_2) in bone marrow is much lower than that in the air, rMSCs were grown under low-pO2 conditions (5% oxygen) and air (21% oxygen) for comparison. The cell expansion fold, colony formation, multi-differentiation were assayed and fluorescence-activated cell sorter analysis were carried out to examine the role of different pO_2 in dynamic condition on rMSC proliferation.
The results showed that in hollow-fiber membrane bioreactor, O_2 concentration kept almost constant; MSCs expansion fold in 5%O_2 is 11, while 8 in control oxygen and proliferation potential and metabolic activity were higher for cultures maintained in low oxygen than those in control oxygen. In 5%O_2 condition rMSCs on hollow-fiber membranes can maintain significantly higher colony-forming unit capabilities than normoxic controls, approximately 112 and 53 respectively.
Hypoxic rMSCs also expressed higher levels of CD44~+ and CD29~+ markers than normoxic controls. After being induced, the expanded cells still reserved the strong muti-differentiation potential. Therefore, it's safe and effective for MSCs culture and expansion in vitro with the method used here.
引文
[1]何红媛,王萍,夏冬.干细胞调控机制及体外培养影响因素的研究.四川解剖学杂2002,10(4):217-221.
[2]高书言,邱庆祥.干细胞的基础研究及开发利用.江西中医学院学报,2002,14(2):57-60.
[3]Ogawa M.Differentiation and proliferation of hematopoietic stem cells.Blood,1993,81:2844-2851.
[4]Filip S,Morkr J,Hrusk A.Adult Stem Cells and Their Importance in Cell Therapy.Folia Biologica,2003,49:9-14.
[5]J.E.Till,E.A.McCulloch.Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells.Radiation Research,1961,14(2):213-222.
[6]戴育成.干细胞研究进展.江西医学检验,2002,20(4):234-235.
[7]James A.Thomson,Joseph Itskovitz Eldor,Sander S.Shapiro et al.Embryonic Stem Cell Lines Derived from Haman Blastocysts.Science,1998,282(6):1145-1147.
[8]周林.干细胞研究进展及意义.21世纪青年学者论坛.23(6):23.
[9]Hall F L,Han B,Kundu R K et al.Phenotypic differentiation of TGF betal responsive pluripotent premesenchymal prehematopoietic progenitor(P4 stem)cells from murine bone marrow.Hematother Stem Cell Res,2001,10(2):261-271.
[10]颉语欣,王旭,张进贵.间充质干细胞的研究进展.河北医药,2005,27(2):130-132.
[11]Friedenstein AJ,Shapiro-Piatetzky II,Petrakova K V.Osteogenesis in transplants of bone marrow cells.J Embryol Exp Morphol,1966,16:381-390.
[12]Friedenstein AJ,Chailakhyan RK,Gerasimov UV.Bone marrow osteogenic stem cells:in vivo cultivation and transplantation in diffusion chambers.Cell Tissue Kinet,1987.20:263-272.
[13]Bianco P,Riminucci M,Gronthos S et al.Bone marrow stromal stem cells:nature,biology,and potential application.Stem Cells,2001,19:180-192.
[14]余勤,连俊兰,王艳.大鼠骨髓间充质干细胞体外培养扩增及生物学特性研究.浙江中医药大学学报,2006,30(2):189-191.
[15]艾国平,粟永萍,闰国和.骨髓间充质干细胞的分离与培养.第三军医大学学报,2001,23(5):553-555.
[16]Conger P A,Minguell J.Phenotypica and functional properties of human bone marrowmesenchymal progenitor cells.J Cell Physiol,1999,181(1):67-73.
[17]Galmiche MC,Briere J.Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway.Blood,1993,82:66-76.
[18]胡静波,周燕,蒋丹丹等.体外扩增过程中人骨髓间充质干细胞的增殖与分化规律.细胞与分子免疫学杂志,2006,22(1):7-10.
[19]Alexandra Stolzing,Andrew Scutt.Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells.Free Radical Biology and Medicine,2006,41:326-338.
[20]Bianca P,RiminucciM.Bone marrow stromal stem cells:nature,biology,and potential applications.Stem Cells,2001,19:180-192.
[21]Gronthos S,Graves SL,Ohta S et al.The Stro- 1 fraction of adult human bone marrow contains the osteogenic precursors.Blood,1994,4:4164-4173.
[22]Simmons PJ,Gronthos S,Zannettino A et al.Isolation,characterization and functional activity of human marrow stromal progenitors in hemopoiesis.Prog Clin Biol Res,1994,389:271.
[23]Waller EK,Olwens J,Lund-Johansen F et al.The "common stem cell" hypothesis reevaluated:human fetal bone marrow contains separate populations of hematopoiefic and dtromal progenitors.Blood,1995,85:2422
[24]李秀森,郭子宽,杨靖清等.骨髓间充质干细胞的生物学特性.解放军医学杂志,2000,25:346-348.
[25]付文玉,路艳蒙,朴英杰.人骨髓间充质干细胞的分化与端粒酶活性.第一军医大学学报,2001,21(11):801-804.
[26]Sale G E,Storb R.The bone marrow transplant into blood.Exp Hematol,1983,10:961-966.
[27]Hanada K.Stimulatory effects of basic fibroblast growth factors and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow- derived mesenchymal stem cells.J Bone Miner Res,1997,12:1606-1641.
[28]Marrin I,Muraglia A,Campanile G et al.Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors form human bone marrow.Endocrinology,1997,138(10):4456-4462.
[29]Hanada K,Demis J E,Caplam A J,et al.Stimulatory effect of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-enroved emsenchymal stem cells[J].J Bone Miner Res,1997,35(12):1606-1614.
[30]Douncis I S,Groomer E R.Chondrogenic phenotype of perichondriunr derivered chondroprogenitor cells is influenced by transforming growth factorβ_1.J Orthop Res,1998,15:803.
[31]Worster AA,Nixon AJ,Brower-Toland BD et al.Effect of transforming growth factor betal on chondrogenic differentiation of cultured equine mesenchymal stem cells.Am J V et Res,2000,61(9):1003-1010.
[32]Reyes M,Lund T,Lenvik T et al.Purification and ex vivo expansion of postnatal human marrow mesenchymal progenitor cells.Blood,2001,98(9):2615-2625.
[33]郭丽,尹飞,王晓丽等.骨髓间充质干细胞向神经元的诱导分化.吉林大学学报医学版,2003,29(1):51-53.
[34]Cui Q,W ang GJ,Balian G.Pluripotential marrow cells produce adipocytes when transplanted into steroid-treated mice.Connect Tissue Res,2000,41(1):45-56.
[35]Ferrari G,Cusella-De Angelis G,Coletta H et al.Muscle regeneration by bone marrow-derived myogenic progenitors.Science,1998,279(5356):1528-1530.
[36]Orlic D J,Lcajstura S,Chimenti F et al.Mobilized bone marrow cells repair the infarcted heart,improving function and survival.Pro Natl Acad Sci USA,2001,98:10344-10349.
[37]Friedenstein A J,Chailakhyan R K,Gerasimov V V.Bone marrow osteogenic stem cells:invitro cultivation and transplantation in diffusion chambers.Cell Tissue Kinet,1987,20:263-272.
[38]路艳蒙,付文玉,朴英杰.大鼠间充质干细胞的培养.解剖学杂志,2000,23:160-163.
[39]Deryugina E I,Muller-sieburg C E.Stromal cells in longerm cultures:keys to theelucidation of hematopoietic development.Crit Rev Immunol,1993,13:115-150.
[40]Deryugina E I,Muller-Sieburg C E.Stromal cells in long-term cultures:keys to the elucidation of hematopoietic development.Crit Rev Immunol,1993,13(2):115-150.
[41]M.Q.Wickham,O.R.Erickson,J.M.Gimble et al.Multipotent Stromal Cells Derived From the Infrapatellar Fat Pad of the KneeClin.Orthop,2003,412.
[42]Kuznetsov S A,Friedenstein A J,Robey P G.Factors required for bone marrow stromal fibroblast colony formation in vitro.Br J Haematol,1997,97:561.
[43]Colter O C,Class R,Oigirolamo C M et al.Rap expansion of recycling stem cells in cultures plastic-adherent cells from human bone marrow.Proc Natl Acad Sci USA,2000,97(7):3213-3218.
[44]Glowacki J,Mizuno S,Greenberger J S.Perfusion enhances functions of bone marrow stromal cells in three-dimensional culture.Cell Transplant,1998,7(3):319-326.
[45]Bruder S P,Shea M.Bone regeneration by implantation of purified,culture expanded human mesenchymal stem cells.J Orthop Res,1998,16(2):155-162.
[46]Horwitz E M,Prockop O J,Fitzpatrick L A et al.Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis inperfecta.Nature Medicine,1999,5:309-313.
[47]Deans R J,Moseley A B.Mesenchymal stem cells:biology and potential clinical uses.ExpHematol,2000,28(8):875-884.
[48]Ahdjoudj S,Fromigue O,Marie PJ.Plasticity and regulation of human bone marrow stromal osteoprogenitor cells:potential implication in the treatment of agerelated bone loss.Histol Histopathol,2004,19(1):151-157.
[49]Koc O N,Gerson S L.Rapid hematopoietic recoveryafter co-infusion of autologous blood stem cells and culture expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high dose chemotherapy.J Clin Oncol,2002,18(2):307-316.
[50]Lu D,Li Y,Wang L et al.Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury.J Neurotrauma,2001,18(8):813-819.
[51]华松,武浩.骨髓间充质干细胞的研究进展.中国畜牧杂志,2004,40(10):38-41.
[52]Natalia Juncosa-Melvin,Gregory P.Boivin,Cynthia Gooch et al.The Effect of Autologous Mesenchymal Stem Cells on the Biomechanics and Histology of Gel- Collagen Sponge Constructs Used for Rabbit Patellar Tendon Repair.Tissue Engineering.2006,12(2):369-379.
[53]孙吉平,贾延劫,罗芳等.脂质体介导外源性基因在骨髓基质细胞表达.中南大学学报医学版,2005,30(2):140-144.
[54]孙吉平,贾延劫,王晓莉等.Superfect高效介导PDX-1基因在骨髓间质干细胞表达.中国病理生理杂志,2007,23(1):12-17.
[55]于美娟,张成,王淑辉等.逆转录病毒介导的人抗肌萎缩蛋白小基因在mdx鼠骨髓间充质干细胞的表达.中国医学科学院学报,2006,28(4):558-561.
[56]Schwarz E J,Alexander G M,Prockop D J.Multipotential marrow stromal cells transduced to produce L-DOPA:engraft in a rat model of Parkinson disease.Hum Gene Ther,1999,10(15):2539-2549.
[57]Baroffio A,Oabbiani G.Heterogeneity in the progeny of single human muscle satellite cells.Differentiation,1995,59(4):259-268.
[58]Nielsen L K.Bioreactors for Hematopoietic Cell Culture.Annu.Rev.Biomed.Eng,1999,01:129-152.
[59]Cabrita G J M,Ferreira B S,da silva C L et al.Hematopoietic stem cells:from the bone to the bioreactor.TRENDS in Biotechnology.2003,21(5):233-240.
[50]Natanel Korin,Avishay Bransky,Uri Dinnar et al.A parametric study of human fibroblasts culture in a microchannel bioreactor.Lab Chip,2007,7,611-617
[61]王玉琨,田浩,岳延盛.氧与活性氧.西安石油学院学报,1998,13(2):57-60.
[62]Frank L,Massaro D.Oxygen toxicity.Am J Med,1980,69(1):117-126.
[63]Halliwell B,Gutteridge JM.Oxygen toxicity,oxygen radicals,transition metals and disease.Biochem J,1984,219(1):1-14.
[64]Marie Csete.Oxygen in the Cultivation of Stem Cells.2005 New York Academy of Sciences.
[65] Gianluca D'Ippolito, Sylma Diabira. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone, 39(3): 513-522.
[66] TondevoldE, Eriksen J, JansenE. Observations on long bone medullary pressures in relation to arterial PO_2, PCO_2, and pH intheanaesthetized dog. ActaOrthop Scand, 1979, 50(6): 645-651.
[67] Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hematopoietic stem cells. Blood, 1993, 82:2031-7.
[68] Chow DC, Wenning LA, Miller WM et al. Modeling pO_2 distributions in the bone marrow hematopoietic compartment:I. Krogh's model. Biophys, 2001,81: 675-84.
[69] Donald P, Lennon et al .Cultivation of Rat Marrow-Derived Mesenchymal Stem Cells in Reduced Oxygen Tension: Effects on In Vitro and In Vivo Osteochondrogenesis. Journal of Cellular Physiology, 2001, 187: 345-355.
[70] Warren L, Grayson, Feng Zhao et al. Effects of Hypoxia on Human Mesenchymal Stem Cell Exoansion and Plasticity in 3D Constructs. Cellular Physiology, 2006, 207:331-339.
[71] Moussavi-Harami F, Duwayri Y, Martin JA, et al. Oxygen effects on senescence in chondrocytes and mesenchymal stem cells:consequences for tissue engineering. Iowa Orthop J, 2004, 24:15-20.
[72] Toshihiko Ezashi, Padmalaya Das, R. Michael Roberts. Low O_2 tensions and the prevention of differentiation of hES cells.PNAS,2005, 102(13): 4783-4788.
[73] M. Lash , H.G. Bohlen. Perivascular and tissue PO_2 in contracting rat spinotrapezius muscle. AJP - Heart and Circulatory Physiology, 252(6): 1192-1202.
[74] Packer, Fuehr. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature, 1977, 267:423-425.
[75] Minchenko A, Bauer T, Salceda S et al. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest, 1994, 71 (3):374-9.
[76] Yoko Horino, Shigeru Takahashi, Takashi Miura. Prolonged hypoxia accelerates the posttranscriptional process of collagen synthesis in cultured fibroblasts. Life Sciences, 2002, 71 (15) :3031-3034.
[77] Deren JA, Kaplan FS, Brighton CT. Alkaline phosphatase production by periosteal cells at various oxygen tension in vitro. Clin Orthop, 1990, (252):307-312.
[78] Zhu LL , Wu LY , Yew DT et al. Effects of hypoxia on the proliferation and differentiation of NSCs. Mol Neurobiol,2005,31(13):231-242.
[79] Lorenz S, Marie C, Lee S H et al. Enhanced proliferation , survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. Neurosc , 2000, 20(19) : 7377-7383.
[80] Zhao HQ, Zhu LL, Zhao T et al. Effects of hypoxia on the proliferation of embryonic stem cell. Chin J Appl Physiol, 2004,20(3):209-213.
[81]赵彤,张翠萍,朱玲玲.低氧促进神经干细胞向多巴胺能神经元分化.Acta Physiologica Sinica,2007.59(3):273-277.
[82]Morrison SJ,Csete M,Groves AK et al.Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.J Neurosci,2000,20:7370-7376.
[83]Eun Ji Gang,Seung Hyun Hong,Ju Ah Jeong et al.In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells.Biochemical and Biophysical Research Communications,2004,321:102-108.
[84]Dolores Baksha,John E.Daviesa,Peter W.Zandstra.Adult human bone marrow - derived mesenchymal progenitorcells are capable of adhesion-independent survival and expansion.Experimental Hematology,2003,31:723-732.
[85]Collins P C.,Nielsen L K.,Patel S D et al.Characterization of Hematopoietic Cell Expansion,Oxygen Uptake,and Glycolysis in a Controlled,Stirred-Tank Bioreactor System.Biotechnol.Prog,1998,14:466-472.
[86]D.Schop,F.W.Janssen,E.Borgartl et al.Expansion of mesenchymal stem cells using a microcarrier-based cultivation system:growth and metabolism.J Tissue Eng Regen Med,2008,2(2-3):126-35.
[87]Chert Zhaolie,Pirklutkemeyer,Kai Iding.High-density culture of recombinant chinese hamster ovary cells producing prothrom in protein-free medium.Biotechnology Letters,2001,23(1):767-770.
[88]Schwarz R P,Goodwin T J,Wolf D A.Cell culture for three-dimensional modeling in rotating-wall vessels:an application of simulated microgravity.J.Tissue Cult.Method,1992,14(2):51-58.
[89]Liu Y,Liu T,Fan X,Ma X,Cui Z:Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel.J Biotechnol 2006,124:592-601.
[90]王佃亮,韩梅胜.动物细胞培养用生物反应器及相关技术.中国生物工程杂志,2003,11:24-27.
[91]候旭,黄华,万昌秀等.组织工程生物反应器及其仿生力学环境探讨.航天医学与医学工程,2006,19(2):153-156.
[92]朱琳.气升式环流中空纤维膜生物反应器内骨髓间充质干细胞的培养与扩增:硕士学位论文.大连:大连理工大学,2007.
[93]Feng Zhao,Pragyansri Pathi.Effects of Oxygen Transport on 3-D Human Mesenchymal Stem Cell Metabolic Activity in Perfusion and Static Cultures:Experiments and Mathematical Model.Biotechnol.Prog,2005,21:1269-1280.
[94]Pankaj Godara,Clive D McFarland,Robert E Nordon.Mini-review Design of bioreactors for mesenchymal stem cell tissue engineering.Chem Technol Biotechnol,2008,83:408-420.
[95]张前程,张凤宝,姚康德.动物细胞培养生物反应器研究进展.化工发展,2002,21(8):560-563.
[96]安东莫泽尔.生物工艺技术学.曲音波等译.长沙:湖南省科学技术出版社,1993.
[97]Nyberg SL,Platt JL,Shirabe K et al.Immunoprotection of xenocytes in a hollow fiber bioartificial liver.ASAIO J,1992,38(3):463-7.
[98]Richard A.Knazekl,Pietro M et al.Cell Culture on Artificial Capillaries:An Approach to Tissue Growth in vitro.Science,1972,178(4056):65-67.
[99]Catapano G.Mass transfer limitations to the performance of membrane bioartificial liver support devices.The International Journal of Artificial Organs,1996,19(1):18-31.
[100]Randall E.McClelland,Robin N.Coger.Use of Micropathways to Improve Oxygen Transport in a Hepatic System.Biomechanical Engineering,2000,122(3):268-273.
[101]Stephen P.Wolfe,Edward Hsu,Lola M.Reid et al.A Novel Multi-Coaxial Hollow Fiber Bioreactor for Adherent Cell Types.Part 1:Hydrodynamic Studies.Biotechnology and Bioengineering,2002,77(1):83-90.
[102]Hironori Yamazoe,Hiroo Iwata.Efficient generation of dopaminergic neurons from mouse embryonic stem cells enclosed in hollow fibers.Biomaterials,2006,27:4871-4880.
[103]王常勇,王永红,郭希民等.应用旋转生物反应器和微载体技术体外大规模扩增人骨髓间充质干细胞.中华实验外科杂志,2002,19(4):378.
[104]吴清法,吴祖泽,董波.微载体悬浮培养成人骨髓间充质干细胞.中国实验血液学杂志,2003,1(1):15-21.
[105]王飚,王智明,伍锦华等.旋转生物反应器内微载体扩增骨髓间充质干细胞组织工程修复兔下颌骨缺损.中国美容整形外科杂志,2007,18(2):143-146.
[106]蒋丹丹,胡静波,周燕等.兔骨髓间充质干细胞在Cytodex 3微载体悬浮培养系统中的贴附条件优化.生物医学工程学杂志,,2007,24(4):884-888.
[107]邱垂源.人MSC灌注生物反应器培养与生物材料生物相容性研究:硕士学位论文.广州:暨南大学,2007.
[108]MeinelL,Karageorgiou,VFajardoR,etal.Bone tissue engineering using humnamesenehymal stem cells:effets of scaffold material and medium flow.Ann BiomedEng.2004,32(1):112-122.
[109]RdaisieM,EulothM,YangL,etal.High-density seeding of myoeyte cells for cardiac tissue engineering.Bioteehnol Bioeng,2003,82(4):403-414.
[110]Kuhn D M,Balkis M,Chandra J,et al.Uses and limitations of the XTT assay in studies of Candida growth and metaboliam[J].J Clin Microbiol,2003,41(1):506-508.
[111]Natanel Korin,Avishay Bransky,Uri Dinnar and Shulamit Levenberg.A parametric study of human fibroblasts culture in amicrochannel.Bioreactor.Lab Chip,2007,7:611-617.
[112]Lorenz Meinel,Vassilis Karageorgiou,Robert Fajardo,et al.Bone Tissue Engineering Using Human Mesenchymal Stem Cells:Effects of Scaffold Material and Medium Flow.Annals of Biomedical Engineering,Vol.32(1):112-122.
[113]Feng Zhao,Teng Ma.Perfusion Bioreactor System for Human Mesenchymal Stem Cell Tissue Engineering:Dynamic Cell Seeding and Construct Development.Biiotechnology and Bioengineering,2005,4(91):482-493.
[114]Michael Scott Kallos.The development of bioreactor protocols for the large scale expansion of mammalian neural stem cells(PhD).Canada,University of Calgary,1999
[115]赵虎.成骨细胞在新型生物反应器内的大规模扩增:硕士学位论文.大连:大连理工大学,2006.
[116]王艳芳.生物反应器内培养兔骨髓间充质干细胞:硕士学位论文.大连:大连理工大学,2006.
[117]李洪鹏,许建中,周强,等.流体剪切应力对人骨髓间充质干细胞增值及细胞周期的影响.第三军医大学学报,2005,27(16):1637-1639.
[118]Riddle RC,Taylor AF,Genetos DC,et al.MAP kinase and calciums ignaling mediate fluid flow-induced human mesenchymal s tem cell proliferation.Am J Phys iol Cell Physiol,2006,290(3):776-84.
[119]余勤、连俊兰、王艳.大鼠骨髓间充质干细胞体外培养扩增及生物学特性研究.浙江中医药大学学报,2006,30(2):189-191.
[120]艾国平,粟永萍,闫国和.骨髓间充质干细胞的分离与培养.第三军医大学学报.2001,23(5):553-555.
[121]Donaldp,Lennon.Cultivation of Rat Marrow-Derived Mesenchymal Stem Cells in Reduced Oxygen Tension:Effects on In Vitro and In Vivo Osteochondrogenesis.Journal of cellular physiology,2001,187:345-355.
[122]D'Ippolito G,Diabira S,Howard GA,et al.Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells.Bone,2006;39:513-522.
[123]Patel S D,Papoutsakis E T,Winter J N,et al.The lactate issue revisited:novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures.Biotechnol Prog.2000,16(5):885-892.
[124]王佃亮,韩梅胜.动物细胞培养用生物反应器及相关技术.中国生物工程杂志.2003,11:24-27.
[125]李胜富,黄定强,卢晓风等.兔骨髓间充质干细胞定向诱导分化为脂肪细胞及成骨细胞的研究.生物医学工程学杂志.J Biomed Eng,2003,20(2):209-213.
[126]李冬梅,金连弘,张宇等.骨髓间充质干细胞定向诱导成类软骨细胞的实验研究.解剖科学进展,2004,10(4):311-314.
[2]高书言,邱庆祥.干细胞的基础研究及开发利用.江西中医学院学报,2002,14(2):57-60.
[3]Ogawa M.Differentiation and proliferation of hematopoietic stem cells.Blood,1993,81:2844-2851.
[4]Filip S,Morkr J,Hrusk A.Adult Stem Cells and Their Importance in Cell Therapy.Folia Biologica,2003,49:9-14.
[5]J.E.Till,E.A.McCulloch.Measurement of the Radiation Sensitivity of Normal Mouse Bone Marrow Cells.Radiation Research,1961,14(2):213-222.
[6]戴育成.干细胞研究进展.江西医学检验,2002,20(4):234-235.
[7]James A.Thomson,Joseph Itskovitz Eldor,Sander S.Shapiro et al.Embryonic Stem Cell Lines Derived from Haman Blastocysts.Science,1998,282(6):1145-1147.
[8]周林.干细胞研究进展及意义.21世纪青年学者论坛.23(6):23.
[9]Hall F L,Han B,Kundu R K et al.Phenotypic differentiation of TGF betal responsive pluripotent premesenchymal prehematopoietic progenitor(P4 stem)cells from murine bone marrow.Hematother Stem Cell Res,2001,10(2):261-271.
[10]颉语欣,王旭,张进贵.间充质干细胞的研究进展.河北医药,2005,27(2):130-132.
[11]Friedenstein AJ,Shapiro-Piatetzky II,Petrakova K V.Osteogenesis in transplants of bone marrow cells.J Embryol Exp Morphol,1966,16:381-390.
[12]Friedenstein AJ,Chailakhyan RK,Gerasimov UV.Bone marrow osteogenic stem cells:in vivo cultivation and transplantation in diffusion chambers.Cell Tissue Kinet,1987.20:263-272.
[13]Bianco P,Riminucci M,Gronthos S et al.Bone marrow stromal stem cells:nature,biology,and potential application.Stem Cells,2001,19:180-192.
[14]余勤,连俊兰,王艳.大鼠骨髓间充质干细胞体外培养扩增及生物学特性研究.浙江中医药大学学报,2006,30(2):189-191.
[15]艾国平,粟永萍,闰国和.骨髓间充质干细胞的分离与培养.第三军医大学学报,2001,23(5):553-555.
[16]Conger P A,Minguell J.Phenotypica and functional properties of human bone marrowmesenchymal progenitor cells.J Cell Physiol,1999,181(1):67-73.
[17]Galmiche MC,Briere J.Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway.Blood,1993,82:66-76.
[18]胡静波,周燕,蒋丹丹等.体外扩增过程中人骨髓间充质干细胞的增殖与分化规律.细胞与分子免疫学杂志,2006,22(1):7-10.
[19]Alexandra Stolzing,Andrew Scutt.Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells.Free Radical Biology and Medicine,2006,41:326-338.
[20]Bianca P,RiminucciM.Bone marrow stromal stem cells:nature,biology,and potential applications.Stem Cells,2001,19:180-192.
[21]Gronthos S,Graves SL,Ohta S et al.The Stro- 1 fraction of adult human bone marrow contains the osteogenic precursors.Blood,1994,4:4164-4173.
[22]Simmons PJ,Gronthos S,Zannettino A et al.Isolation,characterization and functional activity of human marrow stromal progenitors in hemopoiesis.Prog Clin Biol Res,1994,389:271.
[23]Waller EK,Olwens J,Lund-Johansen F et al.The "common stem cell" hypothesis reevaluated:human fetal bone marrow contains separate populations of hematopoiefic and dtromal progenitors.Blood,1995,85:2422
[24]李秀森,郭子宽,杨靖清等.骨髓间充质干细胞的生物学特性.解放军医学杂志,2000,25:346-348.
[25]付文玉,路艳蒙,朴英杰.人骨髓间充质干细胞的分化与端粒酶活性.第一军医大学学报,2001,21(11):801-804.
[26]Sale G E,Storb R.The bone marrow transplant into blood.Exp Hematol,1983,10:961-966.
[27]Hanada K.Stimulatory effects of basic fibroblast growth factors and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow- derived mesenchymal stem cells.J Bone Miner Res,1997,12:1606-1641.
[28]Marrin I,Muraglia A,Campanile G et al.Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors form human bone marrow.Endocrinology,1997,138(10):4456-4462.
[29]Hanada K,Demis J E,Caplam A J,et al.Stimulatory effect of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-enroved emsenchymal stem cells[J].J Bone Miner Res,1997,35(12):1606-1614.
[30]Douncis I S,Groomer E R.Chondrogenic phenotype of perichondriunr derivered chondroprogenitor cells is influenced by transforming growth factorβ_1.J Orthop Res,1998,15:803.
[31]Worster AA,Nixon AJ,Brower-Toland BD et al.Effect of transforming growth factor betal on chondrogenic differentiation of cultured equine mesenchymal stem cells.Am J V et Res,2000,61(9):1003-1010.
[32]Reyes M,Lund T,Lenvik T et al.Purification and ex vivo expansion of postnatal human marrow mesenchymal progenitor cells.Blood,2001,98(9):2615-2625.
[33]郭丽,尹飞,王晓丽等.骨髓间充质干细胞向神经元的诱导分化.吉林大学学报医学版,2003,29(1):51-53.
[34]Cui Q,W ang GJ,Balian G.Pluripotential marrow cells produce adipocytes when transplanted into steroid-treated mice.Connect Tissue Res,2000,41(1):45-56.
[35]Ferrari G,Cusella-De Angelis G,Coletta H et al.Muscle regeneration by bone marrow-derived myogenic progenitors.Science,1998,279(5356):1528-1530.
[36]Orlic D J,Lcajstura S,Chimenti F et al.Mobilized bone marrow cells repair the infarcted heart,improving function and survival.Pro Natl Acad Sci USA,2001,98:10344-10349.
[37]Friedenstein A J,Chailakhyan R K,Gerasimov V V.Bone marrow osteogenic stem cells:invitro cultivation and transplantation in diffusion chambers.Cell Tissue Kinet,1987,20:263-272.
[38]路艳蒙,付文玉,朴英杰.大鼠间充质干细胞的培养.解剖学杂志,2000,23:160-163.
[39]Deryugina E I,Muller-sieburg C E.Stromal cells in longerm cultures:keys to theelucidation of hematopoietic development.Crit Rev Immunol,1993,13:115-150.
[40]Deryugina E I,Muller-Sieburg C E.Stromal cells in long-term cultures:keys to the elucidation of hematopoietic development.Crit Rev Immunol,1993,13(2):115-150.
[41]M.Q.Wickham,O.R.Erickson,J.M.Gimble et al.Multipotent Stromal Cells Derived From the Infrapatellar Fat Pad of the KneeClin.Orthop,2003,412.
[42]Kuznetsov S A,Friedenstein A J,Robey P G.Factors required for bone marrow stromal fibroblast colony formation in vitro.Br J Haematol,1997,97:561.
[43]Colter O C,Class R,Oigirolamo C M et al.Rap expansion of recycling stem cells in cultures plastic-adherent cells from human bone marrow.Proc Natl Acad Sci USA,2000,97(7):3213-3218.
[44]Glowacki J,Mizuno S,Greenberger J S.Perfusion enhances functions of bone marrow stromal cells in three-dimensional culture.Cell Transplant,1998,7(3):319-326.
[45]Bruder S P,Shea M.Bone regeneration by implantation of purified,culture expanded human mesenchymal stem cells.J Orthop Res,1998,16(2):155-162.
[46]Horwitz E M,Prockop O J,Fitzpatrick L A et al.Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis inperfecta.Nature Medicine,1999,5:309-313.
[47]Deans R J,Moseley A B.Mesenchymal stem cells:biology and potential clinical uses.ExpHematol,2000,28(8):875-884.
[48]Ahdjoudj S,Fromigue O,Marie PJ.Plasticity and regulation of human bone marrow stromal osteoprogenitor cells:potential implication in the treatment of agerelated bone loss.Histol Histopathol,2004,19(1):151-157.
[49]Koc O N,Gerson S L.Rapid hematopoietic recoveryafter co-infusion of autologous blood stem cells and culture expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high dose chemotherapy.J Clin Oncol,2002,18(2):307-316.
[50]Lu D,Li Y,Wang L et al.Intraarterial administration of marrow stromal cells in a rat model of traumatic brain injury.J Neurotrauma,2001,18(8):813-819.
[51]华松,武浩.骨髓间充质干细胞的研究进展.中国畜牧杂志,2004,40(10):38-41.
[52]Natalia Juncosa-Melvin,Gregory P.Boivin,Cynthia Gooch et al.The Effect of Autologous Mesenchymal Stem Cells on the Biomechanics and Histology of Gel- Collagen Sponge Constructs Used for Rabbit Patellar Tendon Repair.Tissue Engineering.2006,12(2):369-379.
[53]孙吉平,贾延劫,罗芳等.脂质体介导外源性基因在骨髓基质细胞表达.中南大学学报医学版,2005,30(2):140-144.
[54]孙吉平,贾延劫,王晓莉等.Superfect高效介导PDX-1基因在骨髓间质干细胞表达.中国病理生理杂志,2007,23(1):12-17.
[55]于美娟,张成,王淑辉等.逆转录病毒介导的人抗肌萎缩蛋白小基因在mdx鼠骨髓间充质干细胞的表达.中国医学科学院学报,2006,28(4):558-561.
[56]Schwarz E J,Alexander G M,Prockop D J.Multipotential marrow stromal cells transduced to produce L-DOPA:engraft in a rat model of Parkinson disease.Hum Gene Ther,1999,10(15):2539-2549.
[57]Baroffio A,Oabbiani G.Heterogeneity in the progeny of single human muscle satellite cells.Differentiation,1995,59(4):259-268.
[58]Nielsen L K.Bioreactors for Hematopoietic Cell Culture.Annu.Rev.Biomed.Eng,1999,01:129-152.
[59]Cabrita G J M,Ferreira B S,da silva C L et al.Hematopoietic stem cells:from the bone to the bioreactor.TRENDS in Biotechnology.2003,21(5):233-240.
[50]Natanel Korin,Avishay Bransky,Uri Dinnar et al.A parametric study of human fibroblasts culture in a microchannel bioreactor.Lab Chip,2007,7,611-617
[61]王玉琨,田浩,岳延盛.氧与活性氧.西安石油学院学报,1998,13(2):57-60.
[62]Frank L,Massaro D.Oxygen toxicity.Am J Med,1980,69(1):117-126.
[63]Halliwell B,Gutteridge JM.Oxygen toxicity,oxygen radicals,transition metals and disease.Biochem J,1984,219(1):1-14.
[64]Marie Csete.Oxygen in the Cultivation of Stem Cells.2005 New York Academy of Sciences.
[65] Gianluca D'Ippolito, Sylma Diabira. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone, 39(3): 513-522.
[66] TondevoldE, Eriksen J, JansenE. Observations on long bone medullary pressures in relation to arterial PO_2, PCO_2, and pH intheanaesthetized dog. ActaOrthop Scand, 1979, 50(6): 645-651.
[67] Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hematopoietic stem cells. Blood, 1993, 82:2031-7.
[68] Chow DC, Wenning LA, Miller WM et al. Modeling pO_2 distributions in the bone marrow hematopoietic compartment:I. Krogh's model. Biophys, 2001,81: 675-84.
[69] Donald P, Lennon et al .Cultivation of Rat Marrow-Derived Mesenchymal Stem Cells in Reduced Oxygen Tension: Effects on In Vitro and In Vivo Osteochondrogenesis. Journal of Cellular Physiology, 2001, 187: 345-355.
[70] Warren L, Grayson, Feng Zhao et al. Effects of Hypoxia on Human Mesenchymal Stem Cell Exoansion and Plasticity in 3D Constructs. Cellular Physiology, 2006, 207:331-339.
[71] Moussavi-Harami F, Duwayri Y, Martin JA, et al. Oxygen effects on senescence in chondrocytes and mesenchymal stem cells:consequences for tissue engineering. Iowa Orthop J, 2004, 24:15-20.
[72] Toshihiko Ezashi, Padmalaya Das, R. Michael Roberts. Low O_2 tensions and the prevention of differentiation of hES cells.PNAS,2005, 102(13): 4783-4788.
[73] M. Lash , H.G. Bohlen. Perivascular and tissue PO_2 in contracting rat spinotrapezius muscle. AJP - Heart and Circulatory Physiology, 252(6): 1192-1202.
[74] Packer, Fuehr. Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature, 1977, 267:423-425.
[75] Minchenko A, Bauer T, Salceda S et al. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest, 1994, 71 (3):374-9.
[76] Yoko Horino, Shigeru Takahashi, Takashi Miura. Prolonged hypoxia accelerates the posttranscriptional process of collagen synthesis in cultured fibroblasts. Life Sciences, 2002, 71 (15) :3031-3034.
[77] Deren JA, Kaplan FS, Brighton CT. Alkaline phosphatase production by periosteal cells at various oxygen tension in vitro. Clin Orthop, 1990, (252):307-312.
[78] Zhu LL , Wu LY , Yew DT et al. Effects of hypoxia on the proliferation and differentiation of NSCs. Mol Neurobiol,2005,31(13):231-242.
[79] Lorenz S, Marie C, Lee S H et al. Enhanced proliferation , survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. Neurosc , 2000, 20(19) : 7377-7383.
[80] Zhao HQ, Zhu LL, Zhao T et al. Effects of hypoxia on the proliferation of embryonic stem cell. Chin J Appl Physiol, 2004,20(3):209-213.
[81]赵彤,张翠萍,朱玲玲.低氧促进神经干细胞向多巴胺能神经元分化.Acta Physiologica Sinica,2007.59(3):273-277.
[82]Morrison SJ,Csete M,Groves AK et al.Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.J Neurosci,2000,20:7370-7376.
[83]Eun Ji Gang,Seung Hyun Hong,Ju Ah Jeong et al.In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells.Biochemical and Biophysical Research Communications,2004,321:102-108.
[84]Dolores Baksha,John E.Daviesa,Peter W.Zandstra.Adult human bone marrow - derived mesenchymal progenitorcells are capable of adhesion-independent survival and expansion.Experimental Hematology,2003,31:723-732.
[85]Collins P C.,Nielsen L K.,Patel S D et al.Characterization of Hematopoietic Cell Expansion,Oxygen Uptake,and Glycolysis in a Controlled,Stirred-Tank Bioreactor System.Biotechnol.Prog,1998,14:466-472.
[86]D.Schop,F.W.Janssen,E.Borgartl et al.Expansion of mesenchymal stem cells using a microcarrier-based cultivation system:growth and metabolism.J Tissue Eng Regen Med,2008,2(2-3):126-35.
[87]Chert Zhaolie,Pirklutkemeyer,Kai Iding.High-density culture of recombinant chinese hamster ovary cells producing prothrom in protein-free medium.Biotechnology Letters,2001,23(1):767-770.
[88]Schwarz R P,Goodwin T J,Wolf D A.Cell culture for three-dimensional modeling in rotating-wall vessels:an application of simulated microgravity.J.Tissue Cult.Method,1992,14(2):51-58.
[89]Liu Y,Liu T,Fan X,Ma X,Cui Z:Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel.J Biotechnol 2006,124:592-601.
[90]王佃亮,韩梅胜.动物细胞培养用生物反应器及相关技术.中国生物工程杂志,2003,11:24-27.
[91]候旭,黄华,万昌秀等.组织工程生物反应器及其仿生力学环境探讨.航天医学与医学工程,2006,19(2):153-156.
[92]朱琳.气升式环流中空纤维膜生物反应器内骨髓间充质干细胞的培养与扩增:硕士学位论文.大连:大连理工大学,2007.
[93]Feng Zhao,Pragyansri Pathi.Effects of Oxygen Transport on 3-D Human Mesenchymal Stem Cell Metabolic Activity in Perfusion and Static Cultures:Experiments and Mathematical Model.Biotechnol.Prog,2005,21:1269-1280.
[94]Pankaj Godara,Clive D McFarland,Robert E Nordon.Mini-review Design of bioreactors for mesenchymal stem cell tissue engineering.Chem Technol Biotechnol,2008,83:408-420.
[95]张前程,张凤宝,姚康德.动物细胞培养生物反应器研究进展.化工发展,2002,21(8):560-563.
[96]安东莫泽尔.生物工艺技术学.曲音波等译.长沙:湖南省科学技术出版社,1993.
[97]Nyberg SL,Platt JL,Shirabe K et al.Immunoprotection of xenocytes in a hollow fiber bioartificial liver.ASAIO J,1992,38(3):463-7.
[98]Richard A.Knazekl,Pietro M et al.Cell Culture on Artificial Capillaries:An Approach to Tissue Growth in vitro.Science,1972,178(4056):65-67.
[99]Catapano G.Mass transfer limitations to the performance of membrane bioartificial liver support devices.The International Journal of Artificial Organs,1996,19(1):18-31.
[100]Randall E.McClelland,Robin N.Coger.Use of Micropathways to Improve Oxygen Transport in a Hepatic System.Biomechanical Engineering,2000,122(3):268-273.
[101]Stephen P.Wolfe,Edward Hsu,Lola M.Reid et al.A Novel Multi-Coaxial Hollow Fiber Bioreactor for Adherent Cell Types.Part 1:Hydrodynamic Studies.Biotechnology and Bioengineering,2002,77(1):83-90.
[102]Hironori Yamazoe,Hiroo Iwata.Efficient generation of dopaminergic neurons from mouse embryonic stem cells enclosed in hollow fibers.Biomaterials,2006,27:4871-4880.
[103]王常勇,王永红,郭希民等.应用旋转生物反应器和微载体技术体外大规模扩增人骨髓间充质干细胞.中华实验外科杂志,2002,19(4):378.
[104]吴清法,吴祖泽,董波.微载体悬浮培养成人骨髓间充质干细胞.中国实验血液学杂志,2003,1(1):15-21.
[105]王飚,王智明,伍锦华等.旋转生物反应器内微载体扩增骨髓间充质干细胞组织工程修复兔下颌骨缺损.中国美容整形外科杂志,2007,18(2):143-146.
[106]蒋丹丹,胡静波,周燕等.兔骨髓间充质干细胞在Cytodex 3微载体悬浮培养系统中的贴附条件优化.生物医学工程学杂志,,2007,24(4):884-888.
[107]邱垂源.人MSC灌注生物反应器培养与生物材料生物相容性研究:硕士学位论文.广州:暨南大学,2007.
[108]MeinelL,Karageorgiou,VFajardoR,etal.Bone tissue engineering using humnamesenehymal stem cells:effets of scaffold material and medium flow.Ann BiomedEng.2004,32(1):112-122.
[109]RdaisieM,EulothM,YangL,etal.High-density seeding of myoeyte cells for cardiac tissue engineering.Bioteehnol Bioeng,2003,82(4):403-414.
[110]Kuhn D M,Balkis M,Chandra J,et al.Uses and limitations of the XTT assay in studies of Candida growth and metaboliam[J].J Clin Microbiol,2003,41(1):506-508.
[111]Natanel Korin,Avishay Bransky,Uri Dinnar and Shulamit Levenberg.A parametric study of human fibroblasts culture in amicrochannel.Bioreactor.Lab Chip,2007,7:611-617.
[112]Lorenz Meinel,Vassilis Karageorgiou,Robert Fajardo,et al.Bone Tissue Engineering Using Human Mesenchymal Stem Cells:Effects of Scaffold Material and Medium Flow.Annals of Biomedical Engineering,Vol.32(1):112-122.
[113]Feng Zhao,Teng Ma.Perfusion Bioreactor System for Human Mesenchymal Stem Cell Tissue Engineering:Dynamic Cell Seeding and Construct Development.Biiotechnology and Bioengineering,2005,4(91):482-493.
[114]Michael Scott Kallos.The development of bioreactor protocols for the large scale expansion of mammalian neural stem cells(PhD).Canada,University of Calgary,1999
[115]赵虎.成骨细胞在新型生物反应器内的大规模扩增:硕士学位论文.大连:大连理工大学,2006.
[116]王艳芳.生物反应器内培养兔骨髓间充质干细胞:硕士学位论文.大连:大连理工大学,2006.
[117]李洪鹏,许建中,周强,等.流体剪切应力对人骨髓间充质干细胞增值及细胞周期的影响.第三军医大学学报,2005,27(16):1637-1639.
[118]Riddle RC,Taylor AF,Genetos DC,et al.MAP kinase and calciums ignaling mediate fluid flow-induced human mesenchymal s tem cell proliferation.Am J Phys iol Cell Physiol,2006,290(3):776-84.
[119]余勤、连俊兰、王艳.大鼠骨髓间充质干细胞体外培养扩增及生物学特性研究.浙江中医药大学学报,2006,30(2):189-191.
[120]艾国平,粟永萍,闫国和.骨髓间充质干细胞的分离与培养.第三军医大学学报.2001,23(5):553-555.
[121]Donaldp,Lennon.Cultivation of Rat Marrow-Derived Mesenchymal Stem Cells in Reduced Oxygen Tension:Effects on In Vitro and In Vivo Osteochondrogenesis.Journal of cellular physiology,2001,187:345-355.
[122]D'Ippolito G,Diabira S,Howard GA,et al.Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells.Bone,2006;39:513-522.
[123]Patel S D,Papoutsakis E T,Winter J N,et al.The lactate issue revisited:novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures.Biotechnol Prog.2000,16(5):885-892.
[124]王佃亮,韩梅胜.动物细胞培养用生物反应器及相关技术.中国生物工程杂志.2003,11:24-27.
[125]李胜富,黄定强,卢晓风等.兔骨髓间充质干细胞定向诱导分化为脂肪细胞及成骨细胞的研究.生物医学工程学杂志.J Biomed Eng,2003,20(2):209-213.
[126]李冬梅,金连弘,张宇等.骨髓间充质干细胞定向诱导成类软骨细胞的实验研究.解剖科学进展,2004,10(4):311-314.