Alginate Encapsulation as a Novel Strategy for the Cryopreservation of Neurospheres

Abstract

Primary cultures of brain cell neurospheres are valuable in vitro models for neurotoxicology and brain cell research. Such applications would greatly benefit from the development of efficient cryopreservation protocols that assure the availability of viable and genetically stable stocks of functional neurospheres. In this work we aimed at developing an integrated strategy allowing for long-term culture and cryopreservation of brain cell neurospheres with high viability and reduced recovery time postthawing. Microencapsulation in clinical-grade, ultrahigh viscous, highly purified alginate uniformly cross-linked with Ba(2+) was evaluated as the main strategy to avoid the commonly observed loss of cell-cell and cell-matrix interactions with consequent aggregate's fragmentation and decrease in cell viability that occurs postthawing. Brain cells isolated from 16-day-old fetal rats were cultured in spinner vessels as neurospheres, encapsulated at the 5th day of culture, and cryopreserved at day 19. Culture characterization and assessment of postthawing recovery, concerning cell metabolism, aggregate's cell type composition, and neuron-astrocyte interactions were performed through analysis of membrane integrity, metabolic activity assays, and immunohistochemistry. Our results show that the encapsulation process does not affect cell viability's central metabolism; neither cell differentiation nor cell extensions into cell networks are usually observed between neurons and astrocytes within the neurosphere structure. Neurosphere encapsulation resulted in reduced recovery time postthawing and significantly less fragmentation. Further, the use of serum-free CryoStor™ solution provided further protection for both nonencapsulated and encapsulated aggregates compared with serum-supplemented culture medium as the cryopreservation medium.

Storage Temperatures for Cold-Chain Delivery in Cell Therapy: A Study of Alginate-Encapsulated Liver Cell Spheroids Stored at −80°C or −170°C for Up to 1 Year

“…18,19,20 However, our current work provided no evidence for this. Indeed, ELS stored at -80°C showed progressive injury over the 12 months, which argues against any evidence for a major physical protection by encapsulation during storage at temperatures such as -80°C.…”

Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation

“…In the commercialization model of the only human FDA-approved clinical trial with ES cells, the cells used for the treatment of all the patients were produced in one large batch that was thoroughly validated and cryopreserved (Alper, 2009). A similar model could be used with an encapsulated cell product, particularly if the cells and/ or beads could be cryopreserved (Hardikar et al, 2000;Mahler et al, 2003;Malpique et al, 2009;Murua et al, 2009;Rialland et al, 2000). Immobilized culture of ES cells in alginate beads has been proposed as a means to efficiently scale-up embryoid body formation, the first step in several differentiation protocols (Magyar et al, 2001).…”

Bioactive surfaces from seaweed-derived alginates for the cultivation of human stem cells

“…The composition of the polymers, however, depends on the choice of algae species (Smidsrød and Skjåk-Braek 1990), environmental conditions (Storz et al 2009), or individual parts of the algae (stipe, leaf) (Dalheim et al 2016). The gelation of alginate solutions occurs by ionotropic gelation under gentle conditions (Malpique et al 2010) (Smidsrød and Skjåk-Braek 1990). Since the alginate gelation can be conducted at physiological pH and temperatures, it is therefore particularly suited for applications in regenerative medicine and tissue engineering (Mettler et al 2013).…”