(726d) Effects of Cryopreservation On the Cell and Biomaterial Components of An Encapsulated Cell System

Although microencapsulation is most widely known within the Tissue Engineering community, its use has become much more widespread in the last several years. Those investigating cancer therapies, stem cell therapies or attempting to treat genetic disease through more conventional cell therapies have all found encapsulation to provide an effective method of cell delivery as well as a degree of immune protection. One of the most promising materials for encapsulation is alginate as it is biocompatible and can be easily handled and gelled. While many focus their research on the development of the different therapies and cells that can be used in a microencapsulated system, it is also important to consider the ultimate goal: clinical use. In order to achieve widespread clinical use, any cell therapy will need to be manufactured on a large scale. This will require lengthy sterility testing as well as distribution. It is critical that these cell therapies reach the consumer in the same state that they began. Cryopreservation is the most promising way to accomplish this. A model tissue-engineered pancreatic substitute consisting of murine insulinomas encapsulated in alginate was preserved using two methods, conventional freezing and vitrification, in order to determine the effects of the preservation method on the biomaterial and cellular components of the encapsulated system in vitro and in vivo. While the cellular component of an encapsulated system may be the primary concern in preservation, the biomaterial integrity must also be considered. Conventional freezing utilizes low concentrations of cryoprotectants (CPAs) along with slow cooling and rapid warming. The low levels of CPAs are not as toxic to cells and may maintain cellular viability and function. However, the low concentrations of CPAs also allow ice formation. Ice formation occurs preferentially in the extracellular space which may affect the alginate matrix by creating ice pockets. Ice could decrease the mechanical strength of the alginate or change the overall morphology and performance. Vitrification was developed in order to eliminate ice formation by utilizing high concentrations of CPAs and rapid cooling and warming to achieve a glassy, or vitreous, state. While eliminating ice may help maintain the cross-linked alginate matrix, the high concentrations of CPAs are cytotoxic and must be added in multiple steps to minimize osmotic excursions. Both methods of cryopreservation were used to preserve an encapsulated cell system overnight before being tested in vitro or in vivo. In this study, murine insulinomas were encapsulated in either barium cross-linked alginate or calcium cross-linked alginate that was subsequently coated with poly-L-lysine and a final layer of alginate (calcium alginate/poly-L-lysine/alginate or APA). A mathematical model was used to determine addition and removal protocols for vitrification so as to minimize osmotic excursions. Post-thaw, the viability and function of the vitrified and conventionally frozen capsules were compared to those in fresh. Viable cell number was determined using alamarBlue? and insulin secretory function by introducing a step-change in glucose and measuring the amount of insulin released by ELISA. Biomaterial integrity was assessed using histology, NMR microimaging and mechanical testing. In order to determine the in vivo efficacy of the encapsulated system, ten week old, male Balb/c mice were induced to become diabetic using streptozotocin. After a diabetic state was confirmed, mice were implanted with fresh, conventionally frozen or vitrified APA beads. Blood glucose levels and animal weights were determined every 1-2 days until construct failure (two consecutive days of blood glucose levels above 250 mg/dL) or until two weeks after implantation, whichever occurred first. At this time, mice were sacrificed and APA beads were retrieved. Fluorescence imaging was done using LIVE/DEAD® staining. Insulin secretory function was determined as above and histology was used to assess biomaterial as well as inflammatory host response. Our results demonstrate that encapsulated systems can be cryopreserved so as to maintain cellular viability and biomaterial function. Furthermore, results reveal the advantages and drawbacks of each cryopreservation method used.