从最小血容量中快速分离高产的单个核细胞:一种用于免疫治疗的简化和稳健的方法

Sudhir Bhatia, Gudrun Baersch
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摘要

目的:目前,单个核细胞(MNC)的分离是使用密度梯度方法进行的,这种方法既费力又耗时,需要至少10ml的灰白色被毛或血液。这种方法不适用于从小体积(如100µl)中分离MNCs。因此,我们的目标是开发一种简单、快速、经济有效的方法,从微量血液和灰褐色被毛中分离MNCs。材料和方法:我们利用MNC分离器从100µl和500µl的血液和灰白色被毛中分离出MNCs,通过简单的混合和离心。将分离的细胞进行培养,利用配体特异性磁珠从培养细胞中分离CD4和CD45特异性细胞。结果:在15次实验中,MNC分离器成功地从小体积血液和白皮毛中分离出了MNC。细胞培养物建立并保持3年无污染,细胞保持健康。利用磁珠成功分离CD4和CD45细胞,并成功进行冷冻和解冻。结论:在这项研究中,我们介绍了一种简单、经济、可靠的方法,用于从小体积的灰白色皮毛和血液中分离MNCs,适用于免疫学应用和磁珠细胞分离。这种方法是用户友好的,可以在世界各地的细胞培养实验室中采用,可能为开发新型抗体开辟新的途径,使用磁珠分离各种细胞群,并获得大量用于免疫疗法开发的t细胞,如CAR - t细胞疗法。关键词:单核细胞分离,黄皮,血液,CD4阳性细胞,磁珠
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Rapid Isolation of Mononuclear Cells with High Yield from Minimal Blood Volumes: A Simplified and Robust Approach for Immunotherapeutic Applications
Objective: Currently, mononuclear cell (MNC) isolation is performed using density gradient methods, which are laborious and time-consuming, requiring a minimum of 10 ml of buffy coat or blood. This method is inadequate for isolating MNCs from small volumes, such as 100 µl. Therefore, we aimed to develop a straightforward, rapid, and cost-effective method for isolating MNCs from minute volumes of blood and buffy coat. Material and Methods: We utilized the MNC isolator to isolate MNCs from microvolumes, such as 100 µl and 500 µl of blood and buffy coat, by simple mixing and centrifugation. The isolated cells were cultured, and ligand-specific magnetic beads were employed to isolate CD4 and CD45 specific cells from the cultured cells. Results: In 15 experiments, the MNC isolator successfully isolated MNCs from small volumes of blood and buffy coat. The cell cultures were established and maintained for up to 3 years without contamination, with the cells remaining healthy. Successful isolations of CD4 and CD45 cells using magnetic beads were achieved, and the cells were successfully frozen and thawed. Conclusion: In this study, we have introduced a simple, cost-effective, and robust method for isolating MNCs from small volumes of buffy coat and blood, suitable for immunological applications and magnetic bead cell isolation. This method is user-friendly and can be adopted in cell culture laboratories worldwide, potentially opening new avenues for the development of novel antibodies, isolation of various cell populations using magnetic beads, and obtaining a large number of T-cells for immunotherapy development, such as CAR T-cell therapies. Keywords: mononuclear cell isolation, buffy coat, blood, CD4 positive cells, magnetic beads
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