Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism

Adult stem cells, or mesenchymal stromal cells (MSCs), are of great potential for cell therapy and tissue‐engineering applications. However, for therapeutic use, these cells need to be isolated from tissue or a biopsy and efficiently expanded, as they cannot be harvested in sufficient quantities from the body. In our opinion, efficient expansion of MSCs can be achieved in a microcarrier‐based cultivation system. This study selected a suitable microcarrier for human bone marrow‐derived stromal cells (HBMSCs), optimized cell‐seeding strategies by varying serum concentrations, and optimized dynamic expansion of the HBMSCs in a microcarrier‐based spinner flask cultivation system by applying various feeding regimes. Cytodex 1 microcarriers in combination with a low‐serum concentration (0–5%) in the medium resulted in the highest seeding efficiency for the HBMSCs. Subsequently, significant expansion of the HBMSCs on these carriers has been observed. The highest number of HBMSCs population doublings (4.8 doublings) was obtained by a combination of 50% medium refreshment combined with addition of 30% medium containing microcarriers every 3 days. Exponential cell growth was observed for at least 9 days after seeding, provided that sufficient nutrients (such as glucose) were present, metabolite concentrations (such as ammonia) were kept below growth‐inhibitory concentrations and adequate surface area was present for the cells. After dynamic expansion of the HBMSCs, the cells retained their differentiation potential and their cell surface markers, indicating that HBMSCs expansion on Cytodex 1 microcarriers did not alter the phenotypic properties of the cells. Copyright © 2009 John Wiley & Sons, Ltd.     

微载体悬浮培养成人骨髓间充质干细胞

本研究采用微载体旋转培养系统和常规静止培养系统对成人骨髓间充质干细胞 (mesenchymalstemcell,MSC)的培养进行比较。MSC是贴壁依赖性细胞 ,旋转培养系统采用CultiSpherG大孔微载体 ,浓度为 1g L ,常规静止培养在 12孔培养板中进行。两系统细胞接种密度均为 5× 10 4 cells ml。结果 :旋转培养 7天后达到最大活细胞密度5 .15 0× 10 5cells ml,常规静止培养第 5天就达到最大活细胞密度 1.6 75× 10 5cells ml。在微载体旋转培养中生成乳酸12 .0 6mmol L ,而常规静止培养中生成乳酸 13.10mmol L ,葡萄糖消耗分别为 7.38mmol L和 5 .37mmol L。在微载体旋转培养中平均乳酸产率为 1.6 3,远低于常规静止培养的平均乳酸产率 2 .4 4。这些表明 ,在微载体悬浮培养条件下 ,MSC生长更为旺盛 ,细胞产量更高 ,葡萄糖消耗和能量利用率优于常规静止培养。微载体悬浮培养 12天后 ,MSC依然保持其干细胞特性。结论 :微载体培养系统是扩增组织工程种子细胞的有效方法     

Modulation of mesenchymal stem cell actin organization on conventional microcarriers for proliferation and differentiation in stirred bioreactors

Tissue engineering applications require an appropriate combination of a cell population, biochemical factors and scaffold materials. In this field, mesenchymal stem cells (MSCs) emerge as an attractive cell population, due to their ready availability and their potential to be differentiated into various mesodermal cell types. Commercially available microcarriers have been recently recognized as an efficient tool for the propagation of such cells compared to traditional monolayer culture, enabling efficient scale‐up and serving as a cell delivery system. The organization of actin as well as the induction of its effectors was previously shown to affect dramatically both proliferation and differentiation of MSCs in monolayer culture. To achieve mass scale production of differentiated cells derived from MSCs in scalable stirred bioreactors, this work aims at rationally screening microcarriers based on the characterization of actin organization. First, among the various supports tested, gelatin‐based microcarriers were found to be most suitable for MSC expansion, due to their best‐adapted actin organization compared to monolayer cultures. Secondly, the proper actin organization on Cultispher‐S was closely linked to its ability to bind serum adhesion molecules enabling Rho GTPase activation. Finally, by modulating actin behaviour, it was feasible to efficiently guide MSC differentiation on microcarriers. Taken together, these results show that controlling actin behaviour is a good strategy toward mass scale sequential expansion followed by differentiation of MSCs in a microcarrier based bioreactor. Copyright © 2012 John Wiley & Sons, Ltd.     

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.     

成人和胎儿骨髓间充质干细胞的比较研究

目的 比较成人和胎儿骨髓间充质干细胞（MSC）的表型和生物学性状差异，为临床选择使用MSC提供实验依据。方法 取正常人和胎儿骨髓单个核细胞，在SF培养基中进行MSC培养，测定生长曲线。电镜观察MSC形态，利用流式细胞仪进行SMC表型测定和细胞周期分析；SA方法测定Ⅰ，Ⅲ型胶原和vWF因子表达。通过碱性磷酸酶染色，苏丹黑染色及骨钙蛋白和脂蛋白酯酶mRNA的表达等来检测细胞向成骨，成脂肪细胞分化情况。结果 从成人和胎儿骨髓中可培养出MSC，并保持多向分化潜能。两者在细胞形态，生长特性，表面抗原表达等方面是相似的。胎儿骨髓MSC的扩增潜力及多向分化能力明显强于成人MSC。成人骨髓MSC的粘附功能则强于胎儿。结论 从成人及胎儿骨髓中可分离培养出MSC，在体外有效扩增且保持其低分化状态和多向分化能力。胎儿MSC较成人MSC更原始，具有更大的多向分化和体外扩增潜能，可作为组织工程的种子细胞；而成人MSC支持造血，促进造血功能恢复和重建造血的功能则强于胎儿，具有更广泛的临床移植应用前景。     

异种关节软骨与MSCs体外共同培养的实验研究

目的提供一种新的种子细胞获得方法,解决软骨缺损修复过程中软骨数量不足的困难。方法人骨髓间充质干细胞体外扩增诱导分化后与兔的关节软骨细胞按不同比例共同培养利用real-time PCR技术分析细胞表型的表达情况。结果实验组2种细胞共培养4周后,混合培养显示正反馈调节软骨细胞的增殖,软骨细胞外基质、SOX9、Ⅱ型胶原基因表达增加,软骨细胞生成明显增加。结论间充质干细胞以旁分泌或自分泌的方式调节间充质干细胞向软骨细胞的转化。     

骨髓间充质干细胞对K562细胞增殖及凋亡的影响

本研究比较K562细胞与相同数量以及不同数量的人骨髓间充质干细胞（MSC）黏附培养前后K562细胞增殖、细胞周期和凋亡的变化,以探讨MSC对人白血病K562细胞生长的影响。通过建立正常人骨髓MSC的体外培养体系及其与K562细胞共培养的体系测定K562细胞的生长曲线;将不同数量的MSC与K562细胞共培养测定K562细胞的增殖率曲线;应用流式细胞术检测K562细胞周期以及凋亡的变化。结果显示：与单独K562细胞培养相比,K562细胞与相同数量MSC共培养后,生长受抑;K562细胞与不同数量MSC共培养后,MSC对K562细胞的抗增殖作用呈剂量依赖性;细胞周期分析发现,K562细胞与MSC共培养后,G0／G1期以及G2／M期的细胞增加,S期的细胞减少。共培养24、48、72小时后流式细胞仪检测发现,K562细胞的凋亡率下降。结论：正常人骨髓MSC能使导致K562细胞生长抑制,阻止K562细胞周期的运行,凋亡率下降,且在一定范围内,随着MSC数量的增加,对K562细胞的抗增殖作用增强。     

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.     

Improved expansion of human bone marrow‐derived mesenchymal stem cells in microcarrier‐based suspension culture

Human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM‐MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier‐based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM‐MSCs in stirred suspension bioreactors using CultiSpher‐S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post‐inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM‐MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200‐fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM‐MSCs in culture, which in turn should facilitate the development of new stem cell‐based therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

9.4TMR波谱检测间充质干细胞成骨分化代谢特征的研究

目的 研究间充质干细胞向成骨细胞分化的高分辨MR波谐（MRS）代谢特征。材料与方法 培养获取间胎儿脐带充质干细胞,建立体外诱导音充质干细胞向成骨细胞分化的模型,利用甲醇/氯仿获取细胞代谢提取物,经后处理后利用9.4T高分辨率MRS检测代谢提取物频谱特征。     

Osteogenic capacity of human BM‐MSCs,AT‐MSCs and their co‐cultures using HUVECs in FBS and PL supplemented media

Human bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and human adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) are the most frequently used stem cells in tissue engineering. Due to major clinical demands, it is necessary to find an optimally safe and efficient way for large‐scale expansion of these cells. Considering the nutritional source in the culture medium and method, this study aimed to analyze the effects of FBS‐ and PL‐supplemented media on osteogenesis in stem cell mono‐ and co‐cultures with human umbilical vein endothelial cells (HUVECs). Results showed that cell metabolic activity and proliferation increased in PL‐ compared to FBS‐supplemented media in mono‐ and co‐cultures for both BM‐MSCs and AT‐MSCs. In addition, calcium deposition was cell type dependent and decreased for BM‐MSCs but increased for AT‐MSCs in PL‐supplemented medium in both mono‐ and co‐cultures. Based on the effects of co‐cultures, BM‐MSCs/HUVECs enhanced osteogenesis compared to BM‐MSCs monocultures in both FBS‐ and PL‐supplemented media whereas AT‐MSCs/HUVECs showed similar results compared to AT‐MSCs monocultures. Copyright © 2013 John Wiley & Sons, Ltd.     

高糖高脂对人脐带间充质干细胞增殖及凋亡的影响

背景:研究证实,间充质干细胞移植进入糖尿病患者体内后,疗效受多种因素影响,其中血糖、血脂起着关键作用。目的:模拟糖尿病患者体内高糖高脂环境,进一步验证高糖高脂对体外培养脐带间充质干细胞的影响。方法:葡萄糖和软质酸不同浓度分别体外干预人脐带间充质干细胞,分为对照组、低糖组、低脂组、低糖低脂组、高糖组、高脂组及高糖高脂组。干预48h后通过CCK-8方法酶标仪测定细胞增殖,Annexin-V/PI方法流式细胞仪检测各组细胞的凋亡情况。结果与结论:在高糖高脂的条件下,短期培养后细胞增殖及凋亡明显改变,与对照组相比,人脐带间充质干细胞增殖率明显减低(P<0.01)、凋亡率明显增高(P<0.01),且随着血糖血脂浓度的增加,人脐带间充质干细胞增殖率明显受到抑制,凋亡率明显上升;低糖低脂组和高脂组均抑制细胞增殖(均P<0.05),促进细胞凋亡(均P<0.01),高糖则促进人脐带间充质干细胞生长(P<0.01)。结果提示,糖尿病患者体内处于高糖高脂状态,将抑制脐带间充质干细胞的生长。在间充质干细胞移植之前,控制患者血糖血脂水平,将更有利于干细胞在体内发挥作用。     

人脐带间充质干细胞半连续灌注培养：灌注启动时间及灌注频率和灌注体积

背景：人脐带间充质干细胞作为新的种子细胞来源,如何迅速提高其增殖以满足临床所需的数量和质量,是迫切需要解决的现实问题。目的：探讨不同的灌注启动时间、灌注频率和灌注体积对人脐带间充质干细胞扩增和代谢的影响,建立高密度培养的半连续灌注培养工艺。方法：培养传代人脐带间充质干细胞,以批培养方式为参照,半连续灌注培养分成3组。①灌注启动时间组：批培养24h和48h,分别开始换液,每2d更换75％。②灌注频率组：批培养48h开始换液,频率分别为每1,2,3d,换液体积比为75％。⑧灌注体积组：批培养48h开始换液,频率为每2d,换液体积比分别为25％,50％,75％,100％。结果与结论：相对于其他培养条件,灌注启动时间为48h、频率为每天、体积比为75％的静态培养条件下,稀释率较大,细胞密度较大,葡萄糖比消耗速率和乳酸比生成速率较高,谷氨酰胺比消耗速率较低,氨比生成速率较高,乳酸对葡萄糖的代谢系数较低,谷氨酰胺的代谢系数较高。这表明稀释率大,不仅葡萄糖和谷氨酰胺充足,而且细胞所需的微量元素和未知的因子也充足,满足了细胞生长的要求;同时排除乳酸和氨等对细胞的影响,去除条件培养基中某些因子对细胞的交互影响,提高了细胞的增殖。     

低氧对间充质干细胞的影响

氧对于几乎所有的生命都是必需的,它维持能量代谢及细胞的功能,但机体多种细胞是处于生理或病理性低氧状态中,而体外细胞实验一般是在20%氧浓度下进行,并不符合生理状态。直到最近,关于低氧对于间充质干细胞（mesenchymal stem cells,MSC）的影响有了一系列的研究。体外实验表明,适度低氧下MSC的生长和存活能力更好,氧浓度可以影响MSC向成骨、成软骨、成脂的分化倾向,低氧能提高特异性受体和配体相结合所介导的迁移。低氧可影响胚胎干细胞、造血干细胞、神经干细胞的生理学特性也是一证据。干细胞对缺氧反应的分子机制主要涉及低氧诱导因子-1（hypoxic inducible factor-1,HIF-1）信号通路。本文综述了低氧对MSC的存活、增殖、分化和迁移的影响,以及涉及HIF-1等的分子机制。     

间充质干细胞靶向转运胸苷激酶基因联合更昔洛韦杀伤鼻咽癌细胞

背景：研究证实,间充质干细胞能通过基因修饰成为肿瘤治疗的靶向载体。目的：观察转染胸苷激酶基因的胸苷激酶-间充质干细胞联合更昔洛韦对鼻咽癌细胞的靶向杀伤作用。方法：应用LipofectamineTM2000将表达胸苷激酶基因的重组质粒pGL3-EGFP-胸苷激酶转染至SD大鼠间充质干细胞,观察其增殖能力。应用Transwell小室观察胸苷激酶-间充质干细胞的归巢特性;将胸苷激酶-间充质干细胞植入裸鼠,观察其致瘤性;用胸苷激酶-间充质干细胞/更昔洛韦干预人鼻咽癌细胞5-8F,观察其对细胞的杀伤作用及旁观者效应。结果与结论：荧光显微镜观察及RT-PCR检测结果提示实验成功将胸苷激酶基因转染至间充质干细胞,CCK-8检测结果显示胸苷激酶-间充质干细胞与间充质干细胞增殖能力差异无显著性意义（P〉0.05）。Transwell小室迁移实验显示胸苷激酶-间充质干细胞具有归巢特性,裸鼠移植瘤实验显示胸苷激酶-间充质干细胞无致瘤性。CCK-8检测检测发现胸苷激酶-间充质干细胞/更昔洛韦具有旁观者效应,可明显降低5-8F的生存率（P〈0.01）。提示胸苷激酶-间充质干细胞/更昔洛韦对鼻咽癌细胞具有靶向迁移及杀伤作用,间充质干细胞可作为治疗鼻咽癌的理想靶向转运载体。     

碱性成纤维生长因子和人重组骨形态发生蛋白2诱导分化兔骨髓间充质干细胞

背景:骨髓间充质干细胞依赖特定的生长和分化因子能分化为骨、软骨、脂肪等不同组织。人重组骨形态发生蛋白2有明显的骨诱导作用,能诱导未分化的骨髓间充质干细胞不可逆地形成骨与软骨,并最终导致新骨生成;碱性成纤维生长因子亦参与调控骨髓间充质干细胞的软骨与骨的分化。目的:观察碱性成纤维生长因子和人重组骨形态发生蛋白2对兔骨髓间充质干细胞增殖和分化的影响,以便寻求合适的骨形成诱导方法以代替常规的成骨培养体系。设计:随机对照实验。单位:解放军第四军医大学唐都医院骨肿瘤研究所。材料:培养的兔骨髓间充质干细胞。方法:实验于2004-06/12在第四军医大学唐都医院骨肿瘤研究所暨全军骨科中心完成。①体外培养的兔骨髓间充质干细胞分别经3种不同的生长因子(100μg/L人重组骨形态发生蛋白2,100μg/L碱性成纤维生长因子,100μg/L人重组骨形态发生蛋白2加100μg/L碱性成纤维生长因子)和成骨培养体系处理。②其增殖和分化通过四甲基偶氮唑盐比色法,碱性磷酸酶、骨钙素检测和钙结节VonKossa染色观察。主要观察指标:通过检测增殖率和碱性磷酸酶活性比较不同生长因子对兔骨髓间充质干细胞增殖和分化的影响。结果:①人重组骨形态发生蛋白2能促进兔骨髓间充质干细胞的增殖和分化,特别是其分化。②碱性成纤维生长因子能刺激兔骨髓间充质干细胞的增殖,与对照组相比,细胞增值率提高近100%;虽然对兔骨髓间充质干细胞分化无明显影响,但其能促进人重组骨形态发生蛋白2对兔骨髓间充质干细胞分化。结论:碱性成纤维生长因子和人重组骨形态发生蛋白2是兔骨髓间充质干细胞有效的分化因子,两者在体外都能促进兔骨髓间充质干细胞的增殖和分化。碱性成纤维生长因子和人重组骨形态发生蛋白2能协同作用加速骨诱导及骨形成,可以用作分化骨组织工程中的种子细胞。     

血管内皮细胞生长因子在兔骨髓基质干细胞内的表达及影响

目的:构建血管内皮细胞生长因子pcDNA3/hVEGF165真核表达载体,并用脂质体法转染骨髓基质干细胞(mesenchymalstemcells,MSCs),观察血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)转染后对骨髓MSCs的生长、转化的影响。方法:采用贴壁法分离骨髓MSCs,脂质体法转染。100g/L胎牛血清DMEM培养MSCs,计数法绘制生长曲线,特殊染色观察MSCs的转化,免疫组织化学法观察转染后VEGF的表达。结果:转染了pcDNA3/hVEGF165的骨髓MSCs胞浆内出现VEGF阳性颗粒,而转染了pcDNA3组及未转染组中未出现阳性颗粒,生长曲线在3组中无明显差别。特殊染色出现的时间亦无明显差别。而且,注射有转染了pcDNA3/hVEGF165的MSCs的大白兔局部皮下血管数量增加,其他两组未发现。结论:本实验成功构建了pcDNA3/hVEGF165真核表达载体并能够在MSCs细胞内表达VEGF,其表达产物具有血管内皮增殖刺激活性。     

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix‐derived mesenchymal stem/stromal cells

Umbilical cord matrix (UCM)‐derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non‐invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large‐scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate‐based culture supplement (UltraGRO^TM, AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC‐like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO^TM‐supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO^TM‐supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13‐fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic‐free microcarrier‐based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.     

猪骨髓间质干细胞的分离培养及分化潜能的鉴定

目的：建立猪骨髓间质干细胞(MSCs)的体外分离培养和鉴定的方法，探讨体外培养的间充质干细胞的一些生物学特点，为利用猪的实验研究提供实验基础。方法：猪的髂嵴穿刺吸取骨髓，经密度梯度离心得到骨髓单个核细胞，接种后形成单层贴壁的成纤维样的细胞。检测细胞周期，多向诱导分化鉴定分离的细胞。结果：体外培养的原代MSCs12～14d达到融合，传代后仍具有分化成骨的能力，细胞周期显示有80％的细胞处于GO／G1期。结论：体外培养猪的MSCs具有分化成骨的潜能，生长稳定，传代后仍保持未分化状态．猪骨髓间充质干细胞分离培养体系的建立为基础研究和组织工程提供了一个有价值的动物模型。     

骨髓间充质干细胞的研究前景

除造血干细胞以外,骨髓中还含有另一类干细胞,被称为间充质干细胞（ｍｅｓｅｎｃｈｙｍａｌ ｓｔｅｍ ｃｅｌｌ,ＭＳＣ）在不同的诱导条件下,这类细胞可分化为多种造血以外组织特别是中胚层和神经外胚层来源组织细胞,如成骨细胞、成软骨细胞、脂肪细胞、成肌细胞和星形神经胶质细胞等。骨髓间充质干细胞易于分离和培养扩增,还易于外源基因的导入和表达,有望成为一种新的细胞治疗和基因治疗的靶细胞,在多种造血以外组织的缺