Kaur Indreshpal, Zulovich Jane M, Gonzalez Marissa, McGee Kara M, Ponweera Nirmali, Thandi Daljit, Alvarez Enrique F, Annandale Kathy, Flagge Frank, Rezvani Katayoun, Shpall Elizabeth
Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Cytotherapy. 2017 Mar;19(3):433-439. doi: 10.1016/j.jcyt.2016.11.010. Epub 2016 Dec 26.
Umbilical cord blood (CB) is being used as a source of hematopoietic stem cells (HSCs) and immune cells to treat many disorders. Because these cells are present in low numbers in CB, investigators have developed strategies to expand HSCs and other immune cells such as natural killer (NK) cells. The initial step in this process is to enrich mononuclear cells (MNCs) while depleting unwanted cells. The manual method of MNC enrichment is routinely used by many centers; however, it is an open system, time-consuming and operator dependent. For clinical manufacturing, it is important to have a closed system to avoid microbial contamination.
In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units.
Using Sepax, we observed higher recovery of total nucleated cells (TNC), CD34 cells, NK cells and monocytes when compared to manual enrichment, despite similar TNC and CD34 viability with the two methods. Even though the depletion of red blood cells, granulocytes and platelets was superior using the manual method, significantly higher CFU-GM were obtained in MNCs enriched using Sepax compared to the manual method. This is likely related to the fact that the automated Sepax significantly shortened the processing time (Sepax: 74 - 175 minutes versus manual method: 180 - 290 minutes). The use of DNAse and MgCl during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery.
We optimized enrichment of MNCs from cryopreserved CB products in a closed system using the Sepax which is a walk away and automated processing system.
脐带血（CB）正被用作造血干细胞（HSC）和免疫细胞的来源以治疗多种疾病。由于这些细胞在脐带血中的数量较少，研究人员已开发出扩增造血干细胞和其他免疫细胞（如自然杀伤细胞（NK））的策略。此过程的第一步是富集单核细胞（MNC），同时去除不需要的细胞。许多中心常规使用手动富集单核细胞的方法；然而，这是一个开放系统，耗时且依赖操作人员。对于临床生产而言，拥有一个封闭系统以避免微生物污染很重要。
在本研究中，我们优化了一种用于从冷冻保存的脐带血单位中富集单核细胞的自动化封闭系统（Sepax）。
使用Sepax时，与手动富集相比，我们观察到总核细胞（TNC）、CD34细胞、NK细胞和单核细胞的回收率更高，尽管两种方法的TNC和CD34活力相似。尽管使用手动方法去除红细胞、粒细胞和血小板的效果更好，但与手动方法相比，使用Sepax富集的单核细胞中获得的集落形成单位 - 粒细胞 - 巨噬细胞（CFU - GM）显著更高。这可能与自动化的Sepax显著缩短处理时间有关（Sepax：74 - 175分钟，而手动方法：180 - 290分钟）。在Sepax解冻和洗涤过程中使用DNA酶和氯化镁可防止细胞结块和活力丧失，从而提高解冻后细胞回收率。
我们使用Sepax在封闭系统中优化了从冷冻保存的脐带血产品中富集单核细胞的方法，Sepax是一种无需人工干预的自动化处理系统。
