Optimization of aqueous two-phase systems for human hematopoietic stem cells separation

Roksana Maria

Key information

Authors:

Roksana Maria (Roksana Maria Pirzgalska)

Supervisors:

Ana Margarida Nunes da Mata Pires de Azevedo (Ana Margarida Nunes da Mata Pires de Azevedo); Maria Raquel Murias dos Santos Aires Barros (Maria Raquel Murias dos Santos Aires Barros)

Published in

09/30/2011

Abstract

This thesis aims at the optimization of a novel separation method based on affinity aqueous two-phase system (ATPS) to isolate and purify human hematopoietic stem cells (HSC) from the whole umbilical cord blood (UCB). The preliminary experiments let to the selection of the prospective affinity ATPS possessing appropriate features for the specific HSC separation, namely a two-phase system composed of 5.6% polyethylene glycol (PEG; 8 000 Da) and 7.5% dextran (500 000 Da) supplemented with 0.15 M sodium chloride (NaCl) and the affinity ligand. A monoclonal antibody (mAb) against the CD34 antigen (anti-CD34) (common cell marker for primitive hematopoietic cells) have been used as a ligand to direct the partitioning of target CD34+ cells towards PEG-rich phase. Antibody (Ab) have been used in a free from, or copolymerized with thermosensitive polymer poly(N-isopropylacrylamide) (poly-NIPAM). The selected affinity ATPS was successfully used for the specific partitioning and recovery of CD34+ stem/progenitor cells from UCB. It was possible to achieve purification factors (PFs) up to 241 with only one step of partitioning experiment, that makes the implemented system an appealing alternative to traditional techniques for UCB processing, like classical ficoll density gradient centrifugation and magnetic sorting. The obtained data suggest that this novel selection method can be useful for the development of stem cell engineering, providing easy to apply and cost-effective method for purification of target populations. However, in order to verify the results and assess their statistical significance, all the experiments should be repeated.