Microencapsulation technology: a powerful tool for human embryonic stem cells expansion and cryopreservation

Dissertation presented to obtain a Master degree in Biotechnology at the Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaHuman embryonic stem cells (hESCs) are known by their ability to either self-renewal and differentiate into any adult cell type. These properties confer to hESCs a huge applicability for cell therapy, tissue engineering and drug screening. However, successful implementation of hESCs-based technologies requires the production of large numbers of well characterized cells and their efficient long-term storage. In this study, alginate microencapsulation technology was used in order to develop an efficient, scalable and integrated 3D culture system for expansion and cryopreservation of pluripotent hESCs. Three strategies were outlined: microencapsulation of hESCs as single cells, cell aggregates and cells immobilized on microcarriers. Encapsulation of hESCs immobilized on microcarriers was the best strategy to expand and cryopreserve pluripotent hESCs. The culture of encapsulated hESCs-microcarriers in spinner vessels assured an approximately 20-fold increase in cell concentration. Moreover, this strategy improved twice cell survival after cryopreservation by a slow-freezing rate procedure, comparatively with non-encapsulated culture. Microencapsulation also protected hESC aggregates from damage caused by stirring, allowed the control of aggregates size and the maintenance of cells pluripotency for two weeks. This work demonstrates that microencapsulation technology is a powerful tool to enhance growth and post-thawing recovery of pluripotent hESCs. The 3D culture systems developed herein represent a promising vehicle to assist the transition of hESCs to the clinical and industrial fields.This work was performed in the scope of the project - Integrated strategy for expansion, neuronal differentiation and cryopreservation of human embryonic stem cells (PTDC/BIO/72755/2006) funded by FCT (Fundação para a Ciência e Tecnologia