Carlens S, Gilljam M, Remberger M, Aschan J, Christensson B, Dilber M S
Centre for Allogeneic Stem Cell Transplantation, Huddinge University Hospital, and Departments of, Huddinge, Sweden.
Exp Hematol. 2000 Oct;28(10):1137-46. doi: 10.1016/s0301-472x(00)00526-9.
In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance. Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation. Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum. While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch. Cell expansion in serum-free media would therefore be preferable.
We compared T-cell expansion rates in three commercially available serum-free culture media (X-VIVO 15, AIM-V, and Cellgro SCGM), with or without the addition of human serum (HS, 5%). We also aimed to evaluate how the in vitro expansion affected the composition of the various T-cell subsets. Buffy-coats from four healthy donors were expanded for 21 days. The media were compared to standard RPMI 1640 medium, supplemented with HS (5%) or FBS (10%). For retroviral transductions, the LN vector carrying the neomycin- resistance gene was used in four additional donors.
In our hands, X-VIVO 15 gave the highest rate of serum-free expansion (a median of 79-fold expansion, range 20-117). For serum-free expansion, activation with OKT3 for 21 days gave slightly higher expansion rates than a 5-day course (however, without statistical significance). When serum was added, this discrepancy was not seen. Cytokine analysis (IFN-gamma, IL-10, and IL-4) showed a distinct type1 cytokine pattern with elevated IFN-gamma levels during the whole period of culture. Flow cytometric analyses showed substantial inter-media, but also some inter-donor, variability in T-cell subset compositions. Transduction of cells with the LN vector and G418 selection resulted in a 14-fold increase (range 3-18) for serum-free X-VIVO 15 based cultures. Cell phenotypes remained unchanged by the transduction procedure as compared to nontransduced cells.
Among the tested serum-free media, X-VIVO 15 has shown to best support the in vitro expansion of T cells, resulting in equal percentages of CD4(+) and CD8(+) cells. These cells can easily be transduced and selected. There seem to be no significant benefits, regarding absolute cell numbers or T-cell subset compositions, with OKT3-stimulation for more than five days. The addition of low levels of HS increases the consistencies in the cell expansion rates for all media.
在异基因干细胞移植的背景下，经自杀基因改造的供体T细胞若能在体内被选择性灭活，则有可能实现对移植物抗白血病（GVL）/移植物抗宿主病（GVHD）平衡的最佳控制。逆转录病毒介导的T细胞转导需要体外细胞扩增，这通常通过白细胞介素-2（IL-2）和抗CD3刺激来实现。传统上，用于细胞扩增的培养基会添加胎牛血清（FBS）或人血清。虽然这些血清能促进细胞生长和存活，但它们含有未明确的成分，可能导致批次间结果不一致。因此，在无血清培养基中进行细胞扩增更为可取。
我们比较了三种市售无血清培养基（X-VIVO 15、AIM-V和Cellgro SCGM）在添加或不添加5%人血清（HS）情况下的T细胞扩增率。我们还旨在评估体外扩增如何影响各种T细胞亚群的组成。对来自四名健康供体的血沉棕黄层进行了21天的扩增。将这些培养基与添加了5%HS或10%FBS的标准RPMI 1640培养基进行比较。对于逆转录病毒转导，在另外四名供体中使用了携带新霉素抗性基因的LN载体。
在我们的实验中，X-VIVO 15实现了最高的无血清扩增率（中位数为79倍扩增，范围为20 - 117）。对于无血清扩增，用OKT3激活21天的扩增率略高于激活5天的扩增率（然而，无统计学意义）。添加血清后，这种差异消失。细胞因子分析（干扰素-γ、白细胞介素-10和白细胞介素-4）显示出明显的1型细胞因子模式，在整个培养期间干扰素-γ水平升高。流式细胞术分析表明，不同培养基之间以及不同供体之间，T细胞亚群组成存在显著差异。基于无血清X-VIVO 15培养基的培养物，用LN载体转导细胞并进行G418筛选后，细胞数量增加了14倍（范围为3 - 18）。与未转导的细胞相比，转导过程未改变细胞表型。
在测试过的无血清培养基中，X-VIVO 15最能支持T细胞的体外扩增，产生的CD4(+)和CD8(+)细胞百分比相等。这些细胞易于转导和筛选。对于绝对细胞数量或T细胞亚群组成而言，用OKT3刺激超过五天似乎没有显著益处。添加低水平的HS可提高所有培养基中细胞扩增率的一致性。
