Ex vivo expansion of natural killer cells for clinical applications
Ex vivo expansion of natural killer cells for clinical applications
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DOI：
10.1080/14653240310004548
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发表时间：
2004-01-01
期刊：
影响因子：
4.5
通讯作者：
Martinson, J
中科院分区：
文献类型：
--
作者：
Klingemann, HG;Martinson, J
背景NK细胞的免疫治疗受到无法获得足够数量的适合操作和扩增的纯NK细胞的限制。本研究的目的是分离CD 56（+）细胞（CD 3（-）/CD 56（+），CD 3（+）/CD 56（+）），并在符合当前良好生产规范的培养条件下扩增它们。用于分离含有CD 3（-）/56（+）NK（60.6 +/-10.8%）和CD 3（+）/56（+）NK T细胞（30.4 +/-8.6%）的CD 56（+）群体，以启动扩增研究。将分离的CD 56（+）细胞在补充有10%人AB血清和500 U/mL重组人IL-2或500 U/mL IL-2加10 ng/mL重组人IL-15的X-Vivo 10无血清培养基中培养14天。培养物喂新鲜的培养基和细胞因子，每3 - 4天，并进行了评价细胞扩增，表型，和细胞毒性在培养period.ResultsSignificant扩增的CD 56细胞只发生在培养的第二周。尽管观察到平均两个对数扩增，但取决于供体，甚至当在不同场合测试相同供体时，存在实质性的细胞扩增变异性。在低E：T比率下选择和扩增的CD 56（+）细胞的细胞毒性显著高于起始群体，但与在相同条件下扩增2周的未分离的PBMC相当。IL-15（与IL-2组合）在1：1的E：T比率下比单独的IL-2诱导更高的杀伤。由于CD 3细胞没有预先耗尽，因此CD 3（+）CD 56（+）细胞的扩增是CD 3（-）CD 56（+）细胞的2 - 3倍。表达FcgRIII（CD 16）的NK细胞可以介导Ab依赖性细胞毒性，并且可以有助于增强MAb治疗的功效。在本实验条件下，CD 56（+）/CD 3（-）/CD 16（+）细胞的扩增量仅为中等，以CD 56（+）/CD 3（+）/CD 16（+）细胞为主。
BackgroundImmunotherapy with NK cells has been limited by the inability to obtain sufficient numbers of pure NK cells suitable for manipulation and expansion. The goal of this study was to isolate CD56(+) cells (CD3(-)/CD56(+), CD3(+)/CD56(+)) and expand them under culture conditions compliant with current good manufacturing practices.MethodsMagnetic cell-selection technology, using paramagnetic CD56 microbeads and cell selection columns, was used to isolate a CD56(+) population containing both CD3(-)/56(+) NK (60.6 +/- 10.8%) and CD3(+)/56(+) NK T cells (30.4 +/- 8.6%) to initiate the expansion studies. The isolated CD56(+) cells were cultured in X-Vivo10 serum-free media supplemented with 10% human AB serum and 500 U/mL recombinant human IL-2 or 500 U/mL IL-2 plus 10 ng/mL recombinant human IL-15 for 14 days. Cultures were fed fresh media and cytokines every 3 - 4 days, and were evaluated for cell expansion, phenotype, and cytotoxicity at the end of the culture period.ResultsSignificant expansion of CD56 cells occurred only during the second week of culture. Although an average of two log expansions was observed, there was substantial cell-expansion variability, depending on the donor, and even when the same donor was tested on different occasions. The cytotoxicity of selected and expanded CD56(+) cells at a low E: T ratio was significantly higher than the starting population, but was comparable to non-separated PBMC expanded for 2 weeks under the same conditions. IL-15 ( in combination with IL-2) induced higher killing at the 1: 1 E: T ratio than IL-2 alone. Since CD3 cells were not depleted upfront, the expansion of CD3(+) CD56(+) cells was 2 - 3 times that of CD3(-) CD56(+) cells. NK cells that express the FcgRIII (CD16) can mediate Ab-dependent cellular cytotoxicity, and can contribute to enhanced efficacy of MAb treatment. Under the given culture conditions, only moderate expansion of CD56(+)/CD3(-)/ CD16(+) cells occurred, with the majority of cells being CD56(+)/CD3(+)/ CD16(+) cells.DiscussionOur studies suggest that the positive magnetic cell-separation method provides a good basis for obtaining enriched CD56(+) cells but expansion conditions need to be optimized.