Bahnson A B, Dunigan J T, Baysal B E, Mohney T, Atchison R W, Nimgaonkar M T, Ball E D, Barranger J A
Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, PA 15261, USA.
J Virol Methods. 1995 Aug;54(2-3):131-43. doi: 10.1016/0166-0934(95)00035-s.
Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.
多年来，离心法一直被用于增强多种不同类型病毒对培养细胞的感染，但直到最近才证明其对逆转录病毒有效（Ho等人，(1993)《白细胞生物学杂志》53, 208 - 212；Kotani等人，(1994)《人类基因治疗》5, 19 - 28）。在基因治疗中，离心法被作为一种增加逆转录病毒载体转导的手段进行研究，该载体用于将基因转移到有可能移植并植入患有遗传疾病的人类患者体内的细胞中。研究发现，离心法显著提高了对贴壁小鼠成纤维细胞和非贴壁人类造血细胞（包括原代富集的CD34 +细胞）的转导速率。后者的样本包括能够在清髓患者中重建造血功能的细胞。作为优化该方法的一个步骤，研究表明有效的转导是：(1) 在室温下实现；(2) 与离心时间和高达10,000 g的相对离心力直接相关；(3) 使用试管中的非贴壁细胞靶点时，对于体积≥0.5 ml的上清液，转导与上清液体积无关，但对于平底培养板中贴壁细胞靶点的覆盖，转导依赖于体积；(4) 使用非贴壁细胞时，转导与每管中的细胞数量成反比。这些结果支持了离心法增加病毒与细胞可逆结合的观点，并且与Hodgkin等人报道的结果（Hodgkin等人，(1988)《病毒学方法杂志》22, 215 - 230）一起，这些数据支持了一个模型，即在结合的可逆阶段，离心场抵消了导致解离的扩散力。
