title: Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method.
abstract: 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 MgCl2during 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.
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. Electronic address: ikaur@mdanderson.org.
authors.forename: Indreshpal
authors.initials: I
authors.lastname: Kaur
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Zulovich
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Marissa
authors.initials: M
authors.lastname: Gonzalez
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Kara M
authors.initials: KM
authors.lastname: McGee
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Nirmali
authors.initials: N
authors.lastname: Ponweera
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Daljit
authors.initials: D
authors.lastname: Thandi
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Enrique F
authors.initials: EF
authors.lastname: Alvarez
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Kathy
authors.initials: K
authors.lastname: Annandale
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Flagge
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Katayoun
authors.initials: K
authors.lastname: Rezvani
authors.affiliation: Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Shpall
mesh_terms: D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D002470:Cell Survival; D015925:Cryopreservation; D004912:Erythrocytes / Q000166:cytology* / Q000502:physiology; D005312:Fetal Blood / Q000166:cytology*; D005362:Ficoll / Q000737:chemistry*; D005434:Flow Cytometry; D005615:Freezing / Q000009:adverse effects; D006098:Granulocytes / Q000166:cytology / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D009000:Monocytes / Q000166:cytology / Q000502:physiology
chemical_list: D005362:Ficoll
keywords: Ficoll; MNC enrichment; cord blood