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首页> 外文期刊>Cytotherapy >An innovative method to generate a Good Manufacturing Practice-ready regulatory T-cell product from non-mobilized leukapheresis donors
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An innovative method to generate a Good Manufacturing Practice-ready regulatory T-cell product from non-mobilized leukapheresis donors

机译：一种从非动员的白血球捐献者生产符合Good Manufacturing Practice要求的调节性T细胞产品的创新方法

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Background aims. There is real and sustained interest in preparing T-regulatory cells from leukapheresis collections for cellular therapy through the use of simple, effective and reliable methods conforming to Good Manufacturing Practice (GMP). We describe a GMP-ready isolation procedure for CD25(+) products with the use of a sterile docking device, pigtail sampling, a laminar flow hood and the CliniMACS system that uses leukapheresis collections made by two apheresis machines. Methods. We used CD8/CD19 depletion followed by CD25-positive selection. The median number of CD4(+) cells recovered was 72.5 +/- 32.6 x 10(6), of which 60.5% +/- 17.8% were CD25(+)FOXP3(+) cells. Suppression of autologous CD25(-) cell proliferation by the cryopreserved CD25(+) products was 51.3% +/- 4.4%, 49.0% +/- 3.7% and 39.0% +/- 3.6% at CD25(+) :CD25(-) ratios of 1:1, 1:2 and 1:4 (n = 6), respectively, comparable to suppression by fresh CD25(+) products (53% +/- 6.2%, 51% +/- 3.3% and 39% +/- 7.1%). Results. We found Leukapheresis collections by Cobe Spectra contained more lymphocytes and platelets than collections by Spectra Optia apheresis machine (median, 9.2 x 10(9) versus 6.7 x 10(9); P = 0.04) and platelets (median, 610 x 10(9) versus 170 x 10(9); P = 0.04). The frequency of CD4(+)CD25(+)FOXP3(+) was significantly higher in the leukapheresis (4.85%; 95% confidence interval, 1.95% to 5.38%) than in peripheral blood (3.9%; 95% confidence interval, 2.63% to 6.45%) (P = 0.02). The CD8(-) and CD19-negative depletion step was associated with significant loss of total CD4(+) T cells (P = 0.001). Conclusions. Results suggest that functional CD25(+) products can be isolated with a GMP-ready method, and good recovery can be obtained with the use of an optimized cryopreservation protocol. These data and methods show the potential, possibilities and future work needed to isolate target cell populations in a reproducible, time-efficient and cost-efficient manner for clinical applications.
机译：背景目标。通过使用符合良好生产规范（GMP）的简单，有效和可靠的方法，从白细胞分离术收集物中制备T调节细胞用于细胞疗法，这是一个真正而持续的兴趣。我们描述了使用无菌对接设备，尾纤采样，层流罩和使用两台采血设备制造的白细胞去除术的CliniMACS系统对CD25（+）产品进行的GMP隔离程序。方法。我们使用了CD8 / CD19耗竭后再进行CD25阳性选择。回收的CD4（+）细胞的中位数为72.5 +/- 32.6 x 10（6），其中6025％+/- 17.8％是CD25（+）FOXP3（+）细胞。冷冻保存的CD25（+）产品对自体CD25（-）细胞增殖的抑制作用在CD25（+）处为51.3％+/- 4.4％，49.0％+/- 3.7％和39.0％+/- 3.6％：CD25（- ）分别为1：1、1：2和1：4（n = 6）的比率，与新鲜CD25（+）产品的抑制作用相当（53％+/- 6.2％，51％+/- 3.3％和39 ％+/- 7.1％）。结果。我们发现Cobe Spectra的白细胞分离术收集的淋巴细胞和血小板比Spectra Optia的单采血液分离机收集的淋巴细胞和血小板更多（中位数为9.2 x 10（9）对6.7 x 10（9）; P = 0.04）和血小板（中位数为610 x 10（9 ）与170 x 10（9）; P = 0.04）。白细胞分离术中CD4（+）CD25（+）FOXP3（+）的频率（4.85％; 95％置信区间为1.95％至5.38％）显着高于外周血（3.9％; 95％置信区间为2.63） ％至6.45％）（P = 0.02）。 CD8（-）和CD19阴性的耗竭步骤与总CD4（+）T细胞的大量损失有关（P = 0.001）。结论结果表明，可以使用GMP就绪方法分离功能性CD25（+）产品，并且可以使用优化的低温保存方案获得良好的回收率。这些数据和方法显示了以可重现，省时和有成本效益的方式分离用于临床应用的靶细胞群的潜力，可能性和未来的工作。

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