Bartmann Christina, Rohde Eva, Schallmoser Katharina, Pürstner Peter, Lanzer Gerhard, Linkesch Werner, Strunk Dirk
Department of Internal Medicine, Division of Hematology and Stem Cell Transplantation, Medical University, Auenbrugger Platz 38, A-8036 Graz, Austria.
Transfusion. 2007 Aug;47(8):1426-35. doi: 10.1111/j.1537-2995.2007.01219.x.
Ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) is a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. Even large volumes of starting material and extended culture periods, however, do not necessarily produce 2 x 10(6) MSCs per kg per adult patient. A new two-step procedure has been devised to propagate more than 1 x 10(8) MSCs from small marrow volumes within fewer than 4 weeks.
The influence of log fold decreased MSC seeding (2500, 250, 25, 2.5/cm(2)) on clinical-scale expansion, MSC phenotype, and immunomodulatory function combined with multiplex cytokine display was analyzed. Maintenance of MSC characteristics was tested in fibroblast colony-forming unit and differentiation assays.
Reduced seeding density boosted MSC propagation. Low-density expanded MSCs were CD29+, CD73+, CD90+, CD105+, CD14-, CD34-, CD45-, HLA-DR-; retained their differentiation potential; and inhibited lymphocyte proliferation. This was accompanied by deregulated cytokine production. Seeding 0.7 x 10(6) to 1 x 10(6) MSCs derived from a 10- to 13-day primary culture at a low density of 28 to 40 per cm(2) permitted propagation of 1.5 x 10(8) to 3.7 x 10(8) functional MSCs within a 13- to 15-day secondary expansion step.
Primary seeding of only 10-mL marrow aspirates on approximately 0.2-m(2) culture area (Step 1) followed by expansion on 2.5 m(2) (Step 2) is sufficient to consistently generate at least 1.5 x 10(8) MSCs in fetal bovine serum-supplemented medium within less than 4 weeks. The efficiency of this two-step procedure for clinical-scale MSC propagation may facilitate rational clinical testing of MSC-based therapies.
多能间充质基质细胞（MSC）的体外扩增是在正在进行的临床试验中评估其治疗潜力的前提条件。然而，即使起始材料量很大且培养周期延长，也不一定能为每位成年患者每千克产生2×10⁶个MSC。已经设计出一种新的两步法，可在不到4周的时间内从小体积骨髓中扩增出超过1×10⁸个MSC。
分析了对数倍减少的MSC接种量（2500、250、25、2.5个/cm²）对临床规模扩增、MSC表型和免疫调节功能以及多重细胞因子展示的影响。在成纤维细胞集落形成单位和分化试验中测试了MSC特性的维持情况。
降低接种密度可促进MSC增殖。低密度扩增的MSC为CD29⁺、CD73⁺、CD90⁺、CD105⁺、CD14⁻、CD34⁻、CD45⁻、HLA-DR⁻；保留了它们的分化潜能；并抑制淋巴细胞增殖。这伴随着细胞因子产生失调。以28至40个/cm²的低密度接种来自10至13天原代培养的0.7×10⁶至1×10⁶个MSC，可在第13至15天的二次扩增步骤中扩增出1.5×10⁸至3.7×10⁸个功能性MSC。
在约0.2 m²培养面积上仅接种10 mL骨髓抽吸物（步骤1），然后在2.5 m²上进行扩增（步骤2），足以在补充胎牛血清的培养基中在不到4周的时间内持续产生至少1.5×10⁸个MSC。这种两步法用于临床规模MSC扩增的效率可能有助于基于MSC的疗法进行合理的临床试验。
