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1.
Clinical‐scale expansion of adipose‐derived stromal cells starting from stromal vascular fraction in a single‐use bioreactor: proof of concept for autologous applications
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Mélanie Gadelorge Marion Bourdens Nicolas Espagnolle Clémence Bardiaux Julie Murrell Lenaig Savary Sylvain Ribaud Benoît Chaput Luc Sensebé 《Journal of tissue engineering and regenerative medicine》2018,12(1):129-141
Adipose‐derived stromal cells (ASCs) are adult multipotent cells increasingly used for cell therapy due to their differentiation potential, their paracrine effect and their convenience. ASCs are currently selected from stromal vascular fractions (SVFs) of adipose tissue and expanded in 2D flasks following good manufacturing practices. This process is limited in surface area, labour‐intensive and expensive, especially for autologous applications requiring selection and expansion steps for every patient. Closed and automated bioreactors offer an alternative for scalable and cost‐effective production of ASCs. This study investigated a single‐use stirred‐tank bioreactor that can expand ASCs from SVFs on microcarriers. A preliminary microcarrier screening in static and spinner flask conditions was performed to evaluate the best candidate for adhesion, amplification and harvest. The selected microcarrier was used for process development in the bioreactor. The first experiments showed poor selectivity and growth of the ASCs from the SVF (n = 2). The process was then adjusted by two means: (1) decreasing the platelet lysate in the medium for enhancing cell adherence; and (2) adding a shear protectant (Pluronic F68). Following these modifications, we demonstrated that the number of population doublings of ASCs from SVFs was not significantly different between the bioreactor and the 2D controls (n = 3). In addition, the ASC characterization after culture showed that cells maintained their clonogenic potential, phenotype, differentiation potential and immunosuppressive capacities. This study provides the proof of concept that isolation and amplification of functional ASCs from SVFs can be performed in a stirred‐tank bioreactor combined with microcarriers. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
2.
Sicilia T. Grady Lorraine Britton Katrin Hinrichs Alan J. Nixon Ashlee E. Watts 《Journal of tissue engineering and regenerative medicine》2019,13(2):191-202
Mesenchymal stem cells (MSCs) improve the osteoarthritis condition, but the fate of MSCs after intra‐articular injection is unclear. We used fluorescent nanoparticles (quantum dots [QDs]) to track equine MSCs (QD‐labelled MSCs [QD‐MSCs]) in vivo after intra‐articular injection into normal and osteoarthritic joints. One week after injection of QD‐MSCs, unlabelled MSCs, or vehicle, we determined the presence of QD‐MSCs in synovium and articular cartilage histologically. In vitro, we evaluated the persistence of QDs in MSCs and whether QDs affected proliferation, immunophenotype, or differentiation. In joints injected with QD‐MSCs, labelled cells were identified on the synovial membrane and significantly less often on articular cartilage, without differences between normal and osteoarthritic joints. Joints injected with QD‐MSCs and MSCs had increased synovial total nucleated cell count and protein compared with vehicle‐injected joints. In vitro, QDs persisted in nonproliferating cells for up to 8 weeks (length of the study), but QD fluorescence was essentially absent from proliferating cells within two passages (approximately 3 to 5 days). QD labelling did not affect MSC differentiation into chondrocytes, adipocytes, and osteocytes. QD‐MSCs had slightly different immunophenotype from control cells, but whether this was due to an effect of the QDs or to drift during culture is unknown. QD‐MSCs can be visualized in histological sections 1 week after intra‐articular injection and are more frequently found in the synovial membrane versus cartilage in both normal and osteoarthritic joints. QDs do not alter MSC viability and differentiation potential in vitro. However, QDs are not optimal markers for long‐term tracking of MSCs, especially under proliferative conditions. 相似文献
3.
微载体悬浮培养成人骨髓间充质干细胞 总被引:15,自引:1,他引:15
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 相似文献
4.
Sébastien Sart Abdelmounaim Errachid Yves‐Jacques Schneider Spiros N. Agathos 《Journal of tissue engineering and regenerative medicine》2013,7(7):537-551
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. 相似文献
5.
The effect of serum on the proliferation of bone marrow‐derived mesenchymal stem cells from aged donors and donors with or without chronic heart failure
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Hideki Kubo Kanae Takamura Noritoshi Nagaya Hajime Ohgushi 《Journal of tissue engineering and regenerative medicine》2018,12(1):e395-e397
Many clinical studies of regenerative medicine using bone marrow‐derived mesenchymal stem cells (MSCs) have been conducted globally. We initiated clinical studies using MSCs in 2001 and have now treated over 100 cases with patients aged 0–92 years. In a few cases involving patients with chronic heart failure (CHF), we observed that MSCs proliferated poorly. This contrasts with cell therapy studies wherein MSCs of patients with CHF were used for treatment. The effects of serum on the proliferation of MSCs from donors with normal heart function and with CHF have not been reported. Moreover, whether cell therapy is effective for elderly patients remains uncertain. Therefore, characterization of MSCs from aged donors and/or donors with CHF is urgently required. We retrospectively analysed the population doubling times (PDTs) of MSCs between the first and second passages. Although we had data for many samples of well‐expanded MSCs from aged donors, a positive correlation was observed between donor age and PDT. A trend towards reduced variance in PDTs was observed in MSCs supplemented with fetal bovine serum (FBS) compared with those supplemented with autologous serum. When autologous serum was used, the average PDT of MSCs from donors with CHF was significantly longer than that of MSCs from donors without CHF. In contrast, when FBS was used, similar PDTs were observed in MSCs from donors with and without CHF. Thus, FBS promotes MSC expansion even from donors with CHF and MSC‐based regenerative medicine might be feasible even for elderly patients. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
6.
Electrical stimulation of adipose‐derived mesenchymal stem cells and endothelial cells co‐cultured in a conductive scaffold for potential orthopaedic applications
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Jieyu Zhang Koon Gee Neoh En‐Tang Kang 《Journal of tissue engineering and regenerative medicine》2018,12(4):878-889
Electrical stimulation (ES) has emerged as a useful tool to regulate cell behaviour, but the effect of ES on mesenchymal stem cell (MSC)/vasculogenic cell co‐culture has not been investigated. Herein, human adipose‐derived MSCs (AD‐MSCs) and umbilical vein endothelial cells (HUVECs) were co‐cultured in an electrically conductive polypyrrole/chitosan scaffold. Compared with AD‐MSC monoculture, calcium deposition in the co‐culture without and with ES (200 μA for 4 h/day) was 139% and 346% higher, respectively, after 7 days. As the application of ES to AD‐MSC monoculture only increased calcium deposition by 56% compared with that without ES after 7 days, these results indicate that ES and co‐culture with HUVECs have synergistic effects on AD‐MSCs' osteogenic differentiation. ES application also significantly enhanced CD31 expression of HUVECs. In HUVEC monoculture, application of ES increased CD31 expression by 224%, whereas the corresponding increase in AD‐MSC/HUVEC co‐culture with ES application was 62%. The gene expression results indicate that ES enhanced the cellular functions in AD‐MSC and HUVEC monoculture via autocrine bone morphogenetic protein‐2 (BMP‐2) and vascular endothelial growth factor (VEGF), respectively. In co‐culture, crosstalk between AD‐MSCs and HUVECs due to paracrine BMP‐2 and VEGF enhanced the cellular functions compared with the respective monoculture. With application of ES to the AD‐MSC/HUVEC co‐culture, autocrine signalling was enhanced, resulting in further promotion of cellular functions. These findings illustrate that co‐culturing AD‐MSC/HUVEC in a conductive scaffold with ES offers potential benefits for bone defect therapy. 相似文献
7.
Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability
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Ashley B. Allen Emily B. Butts Hazel Y. Stevens Robert E. Guldberg 《Journal of tissue engineering and regenerative medicine》2017,11(10):2876-2884
Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC‐seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell‐seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL‐supplemented media resulted in rMSC death, by a process attributable to heat‐labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
8.
Barbara Dozza Giuliana Gobbi Enrico Lucarelli Michela Pierini Claudia Di Bella Tommaso Frisoni Pier Luigi Tazzari Francesca Ricci Prisco Mirandola Cecilia Carubbi Sandro Giannini Davide Donati Marco Vitale 《Journal of tissue engineering and regenerative medicine》2014,8(6):483-492
Mesenchymal stem cells (MSCs) and platelet‐rich plasma (PRP) are currently used alone or in combination for therapeutic applications especially for bone repair. We tested whether MSCs can be isolated from bone marrow (BM) aspirate using a commercially available kit commonly used to obtain PRP from peripheral blood (PB). Results revealed that mononuclear cells and platelets from both PB and BM could be efficiently isolated by obtaining a mononuclear and platelet rich fraction (PB‐MPRF and BM‐MPRF, respectively). Starting with comparable volumes, the number of platelets increased 1.5‐fold in BM‐MPRF compared to PB‐MPRF. The number of clonogenic cells in BM‐MPRF samples was significantly higher than whole BM samples as revealed by CFU‐F assay (54.92 ± 8.55 CFU‐F/1.5 x 105 nucleated cells and 32.50 ± 12.43 CFU‐F/1.5 x 105 nucleated cells, respectively). Cells isolated from BM‐MPRF after in vitro expansion fulfilled the definition of MSCs by phenotypic criteria, and differentiated along osteogenic, adipogenic and chondrogenic lineages following induction. Results showed that the kit isolated MSCs and platelets from BM aspirate. Isolated MSCs were further expanded in a laboratory and BM‐MPRF was used clinically following BM withdrawal for rapid intra‐operative cell therapy for the treatment of bone defects. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
9.
Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors
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Yuichiro Ueda Asifiqbal Kadari Katharina Günther Süleyman Ergün Antoine Heron Aletta C. Schnitzler Martha Rook Frank Edenhofer 《Journal of tissue engineering and regenerative medicine》2018,12(2):e1076-e1087
The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell‐based therapies and cell‐loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two‐step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA‐1‐60, TRA‐1‐81, SSEA‐4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16‐fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10‐fold increase was achieved. Taken together, 16 × 106 hiPSCs were expanded into 2 × 109 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell‐based therapies and brings their clinical potential closer to fruition. 相似文献
10.
BACKGROUND: Single‐donor or pooled platelet lysates (PL) can substitute for fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) expansion. However, for clinical applications of MSCs, the use of virally inactivated PL would be desirable. Recently, we have developed a solvent/detergent (S/D)‐treated human PL preparation (S/D‐PL) rich in growth factors. The capacity to use this virally inactivated preparation for MSC expansion needs to be evaluated. STUDY DESIGN AND METHODS: Platelet concentrates were treated by S/D (1% tri‐n‐butyl phosphate and 1% Triton X‐45), extracted by oil, purified by C18 hydrophobic interaction chromatography, and sterile filtered. S/D‐PL was compared to FBS as a medium supplement (10% vol/vol) for isolating, maintaining, and expanding adipose tissue–derived MSCs (AT‐MSCs). Cell morphology; proliferation kinetics; immunophenotype; differentiation capacity toward the chondrogenic, osteogenic, and osteogenic lineages; and cytokine antibody array were assessed. RESULTS: AT‐MSCs had a typical spindle morphology and proliferated in S/D‐PL at least as well as in FBS. Immunophenotype at Passage 7 was characteristic of MSCs and similar for both culture conditions. Differentiation capacity into the three lineages was maintained and chondrogenesis was enhanced by S/D‐PL. In a 120 human cytokine antibody array analysis, 73 cytokines were detected in S/D‐PL, including 22 with a concentration higher than in FBS. CONCLUSION: S/D‐PL is an alternative to FBS for AT‐MSC maintenance and expansion, does not compromise the differentiation capacity nor the immunophenotype, and may accelerate chondrogenesis. S/D‐PL protocols for MSC clinical scale‐up may represent a major step toward challenging new use in stem cell therapies. 相似文献
11.
成人骨髓间充质干细胞中各亚群的比较研究 总被引:7,自引:4,他引:7
为了观察成人骨髓间充质干细胞在体外培养中的形态,比较不同亚群细胞的免疫表型和细胞周期等生物学性状的差异,取正常人骨髓单个核细胞,进行间充质干细胞(mesenchymal stem cell,MSC)培养,倒置相差显微镜观察MSC不同亚群细胞的形态;流式细胞术检测MSC不同亚群细胞的表型并进行细胞周期分析;用10μm滤膜将不同群体细胞分离,分别接种半固体培养体系。结果表明:①MSC在体外培养中明显分为两群,成纤维样梭形细胞为成熟的MSC,即mMSC;体积很小的圆形细胞为RS细胞(rapidly MSC self-renewing cells);②99%的RS细胞处于G0/G1期,mMSC中处于G0/G1期的细胞占90%。③两群细胞的系定向抗原均为阴性(CD34、CD45、CD3、CD19、CD33、HLA-DR、CD38等),而CD90、CD105、C166、CD29、CD44、CD49e、CD54、CD13呈阳性表达,但RS细胞表面这些抗原表达的阳性率和平均荧光强度都明显低于mMSC。而CD117和KDR的表达则显著高于mMSC。④半固体培养4—5周后两种细胞群体均未见造血细胞集落形成。结论:MSC是一个异质性的细胞群体,其中包含的RS细胞可能是一种更原始的中胚层前体细胞,具有更强的自我更新和多向分化潜能。 相似文献
12.
Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co‐culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs,human induced pluripotent stem cell‐derived MSCs and human embryonic stem cell‐derived MSCs
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Wenchuan Chen Xian Liu Qianmin Chen Chongyun Bao Liang Zhao Zhimin Zhu Hockin H.K. Xu 《Journal of tissue engineering and regenerative medicine》2018,12(1):191-203
Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co‐culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co‐cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC‐MSCs) and embryonic stem cells (hESC‐MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary‐like structures were successfully formed on CPC in vitro in all four co‐cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co‐cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co‐cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co‐cultured with hUCMSCs, hiPSC‐MSCs and hESC‐MSCs achieved new bone and vessel density similar to hUVECs co‐cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC‐MSCs and hESC‐MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co‐cultures of hUVECs with hUCMSCs, hiPSC‐MSCs, hESC‐MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co‐culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd. 相似文献
13.
M. Haack‐Sorensen T. Friis L. Bindslev S. Mortensen H. E. Johnsen J. Kastrup 《Scandinavian journal of clinical and laboratory investigation》2013,73(3):192-203
Objective. Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. Material and methods. Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP‐accepted medium (EMEA medium) or in one of four other media. Results. FACS analysis showed that the plastic‐adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary‐like structures. Conclusions. We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease. 相似文献
14.
Eun‐Ju Kang Young‐Hyurk Lee Min‐Jeong Kim Yeon‐Mi Lee B. Mohana Kumar Byeong‐Gyun Jeon Sun‐A Ock Hyun‐Joon Kim Gyu‐Jin Rho 《Journal of tissue engineering and regenerative medicine》2013,7(3):169-182
The present study compared mesenchymal stem cells derived from umbilical cord matrix (UCM‐MSCs) with bone marrow (BM‐MSCs) of miniature pigs on their phenotypic profiles and ability to differentiate in vitro into osteocytes, adipocytes and neuron‐like cells. This study further evaluated the therapeutic potential of UCM‐MSCs in a mouse Parkinson's disease (PD) model. Differences in expression of some cell surface and cytoplasm specific markers were evident between UCM‐MSCs and BM‐MSCs. However, the expression profile indicated the primitive nature of UCM‐MSCs, along with their less or non‐immunogenic features, compared with BM‐MSCs. In vitro differentiation results showed that BM‐MSCs had a higher tendency to form osteocytes and adipocytes, whereas UCM‐MSCs possessed an increased potential to transform into immature or mature neuron‐like cells. Based on these findings, UCM‐MSCs were transplanted into the right substantia nigra (SN) of a mouse PD model. Transplantation of UCM‐MSCs partially recovered the mouse PD model by showing an improvement in basic motor behaviour, as assessed by rotarod and bridge tests. These observations were further supported by the expression of markers, including nestin, tyrosine hydroxylase (TH), neuronal growth factor (NGF), vascular endothelial growth factor (VEGF) and interleukin‐6 (IL‐6), at the site of cell transplantation. Our findings of xenotransplantation have collectively suggested the potential utility of UCM‐MSCs in developing viable therapeutic strategies for PD. Copyright © 2011 John Wiley & Sons, Ltd. 相似文献
15.
成人和胎儿骨髓间充质干细胞的比较研究 总被引:39,自引:0,他引:39
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。 相似文献
16.
Yifan Yuan Michael S. Kallos Christopher Hunter Arindom Sen 《Journal of tissue engineering and regenerative medicine》2014,8(3):210-225
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. 相似文献
17.
Human platelet lysate successfully promotes proliferation and subsequent chondrogenic differentiation of adipose‐derived stem cells: a comparison with articular chondrocytes
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F. Hildner M. J. Eder K. Hofer J. Aberl H. Redl M. van Griensven C. Gabriel A. Peterbauer‐Scherb 《Journal of tissue engineering and regenerative medicine》2015,9(7):808-818
Fetal calf serum (FCS) bears a potential risk for carrying diseases and eliciting immune reactions. Nevertheless, it still represents the gold standard as medium supplement in cell culture. In the present study, human platelet lysate (PL) was tested as an alternative to FCS for the expansion and subsequent chondrogenic differentiation of human adipose‐derived stem cells (ASCs). ASCs were expanded with 10% FCS (group F) or 5% PL (group P). Subsequently, three‐dimensional (3D) micromass pellets were created and cultured for 5 weeks in chondrogenic differentiation medium. Additionally, the de‐ and redifferentiation potential of human articular chondrocytes (HACs) was evaluated and compared to ASCs. Both HACs and ASCs cultured with PL showed strongly enhanced proliferation rates. Redifferentiation of HACs was possible for cells expanded up to 3.3 population doublings (PD). At this stage, PL‐expanded HACs demonstrated better redifferentiation potential than FCS‐expanded cells. ASCs could also be differentiated following extended passaging. Glycosaminoglycan (GAG) quantification and qRT–PCR of 10 cartilage related markers demonstrated a tendency for increased chondrogenic differentiation of PL‐expanded ASCs compared to cells expanded with FCS. Histologically, collagen type II but also collagen type X was mainly present in group P. The present study demonstrates that PL strongly induces proliferation of ASCs, while the chondrogenic differentiation potential is retained. HACs also showed enhanced proliferation and even better redifferentiation when previously expanded with PL. This suggests that PL is superior to FCS as a supplement for the expansion of ASCs and HACs, particularly with regard to chondrogenic (re)differentiation. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
18.
D. Schop R. van Dijkhuizen‐Radersma E. Borgart F. W. Janssen H. Rozemuller H‐J. Prins J. D. de Bruijn 《Journal of tissue engineering and regenerative medicine》2010,4(2):131-140
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. 相似文献
19.
Maitane Pérez-Ilzarbe María Díez-Campelo Pablo Aranda Soraya Tabera Tania Lopez Consuelo del Cañizo Juana Merino Cristina Moreno Enrique J. Andreu Felipe Prósper José Antonio Pérez-Simón 《Transfusion》2009,49(9):1901-1910
BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells. Based on their properties, several clinical trials have been designed to explore their potential therapeutic effect. Fetal calf serum (FCS, commonly used for in vitro expansion) is an undesirable source of xenogeneic antigens and bears the risk of transmitting contaminations. As an alternative for FCS, platelet lysate (PL) and both autologous and allogeneic human serum have been proposed. The aim of this study is to compare the culture of bone marrow (BM)-derived MSCs in the presence of different serum supplements to determine the effect on cell growth, differentiation potential, and immunologic function.
STUDY DESIGN AND METHODS: MSCs from BM of healthy volunteer donors were grown in the presence of 10% FCS supplemented with 1 ng/mL basic fibroblast growth factor (bFGF), 10% human serum supplemented with 1 ng/mL bFGF, 5% PL, and PL 5% supplemented with 1 ng/mL bFGF (PL plus bFGF).
RESULTS: MSCs that expanded in either medium showed a comparable morphology, phenotype, and proliferative and differentiation capacity. While the presence of MSCs in vitro significantly decreased CD3/CD28-mediated T-cell activation, this effect was significantly higher in MSCs cultured with human serum. Production of interferon-γ was inhibited by cocultured media with MSCs while MSCs also induced a significant inhibition of cell cycle in T cells.
DISCUSSION: In conclusion, PL or autologous serum could offer an alternative to the use of FCS in MSC expansion for clinical use maintaining the same growing potential, phenotype, immunomodulatory properties, and differentiation potential. 相似文献
STUDY DESIGN AND METHODS: MSCs from BM of healthy volunteer donors were grown in the presence of 10% FCS supplemented with 1 ng/mL basic fibroblast growth factor (bFGF), 10% human serum supplemented with 1 ng/mL bFGF, 5% PL, and PL 5% supplemented with 1 ng/mL bFGF (PL plus bFGF).
RESULTS: MSCs that expanded in either medium showed a comparable morphology, phenotype, and proliferative and differentiation capacity. While the presence of MSCs in vitro significantly decreased CD3/CD28-mediated T-cell activation, this effect was significantly higher in MSCs cultured with human serum. Production of interferon-γ was inhibited by cocultured media with MSCs while MSCs also induced a significant inhibition of cell cycle in T cells.
DISCUSSION: In conclusion, PL or autologous serum could offer an alternative to the use of FCS in MSC expansion for clinical use maintaining the same growing potential, phenotype, immunomodulatory properties, and differentiation potential. 相似文献
20.
Chung Eun Yeum Eun Young Park Seong‐Beom Lee Heung‐Jae Chun Gue‐Tae Chae 《Journal of tissue engineering and regenerative medicine》2013,7(4):279-291
Mesenchymal stem cells (MSCs) are known to be effective in wound healing, but not much has been reported on quantitative correlations between MSCs injected into the wound site and MSCs that actually participate in wound healing. This study traced MSCs participating in wound healing by using small intestinal submucosa (SIS) as a cell carrier, identified their moving path and calculated the number of MSCs involved in wound healing. First, MSCs were isolated from the nude mouse and 1 × 106 cells were seeded onto the centre of the SIS. MSC‐seeded SIS complexes were injected onto full‐thickness skin wounds made on the dorsum of nude mice. Tracing of MSC‐seeded SIS complex transplanted to the wound site revealed that 27.6% of the MSCs were migrated to the wound site at the first attempt. Second, repeated injection of additional MSCs did not increase the number of MSCs participating in wound healing beyond a certain constant maximum amount. The number of MSCs present in the wound site remains constant in the range 2–3 × 105 from day 1 to day 10. The expression of skin regeneration‐related growth factors was confirmed by real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA). MSCs participating in wound healing were found not only to suppress inflammation of the wound but also to increase the skin regeneration‐related growth factors that enable the recovery of the skin. An optimal number of about 3 × 105 MSCs injected into the site was found to adapt themselves to the skin wound‐healing process effectively. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献