BACKGROUND AIMS: 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. METHODS: In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. RESULTS: 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 MgCl2 during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. DISCUSSION: 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.
BACKGROUND AIMS: 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. METHODS: In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. RESULTS: 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 MgCl2 during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. DISCUSSION: 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.
Authors: Vandana Keskar; Anup Sood; Evelina Loghin; Ernest Kovacs; R Scott Duthie; Shutong Liu; Jee Hyun Park; Chrystal Chadwick; Reginald Smith; Martin Brown; David F Stroncek; Steven L Highfill Journal: J Immunother Date: 2020-10 Impact factor: 4.912