Two-step sedimentation process for selection of microcapsules containing cell aggregates.

Microencapsulation technologies are being developed to protect transplanted islets from immune rejection, to reduce or even eliminate the need for immunosuppression. However, unencapsulated cells increase the chances of rejection and empty beads increase transplant volumes. Thus, separation processes were investigated to remove these byproducts based on density differences. The densities of islet-sized mouse insulinoma 6 (MIN6) cell aggregates and acellular 5% alginate beads generated via emulsification and internal gelation were ~ 1.065 and 1.042 g/ml, respectively. The separation of empty beads from those containing aggregates was performed by sedimentation under unit gravity in continuous gradients of polysucrose and sodium diatrizoate with density ranges of 1.032-1.045, 1.035-1.044, or 1.039-1.042 g/ml. The 1.039-1.042 g/ml gradient enabled recoveries of ~ 80% of the aggregate-containing beads while the other gradients recovered only ~ 60%. The bottom fraction of the 1.039-1.042 g/ml gradient contained beads with ~ 6% of their volume occupied by cell aggregates. Separation of unencapsulated aggregates from the aggregate-containing beads was then achieved by centrifugation of this purified fraction in a 1.055 g/ml density solution. Thus, these sedimentation-based approaches can effectively remove the byproducts of cell encapsulation.

Stem cells and beta cell replacement therapy: a prospective health technology assessment study.

BACKGROUND: Although current beta cell replacement therapy is effective in stabilizing glycemic control in highly selected patients with refractory type 1 diabetes, many hurdles are inherent to this and other donor-based transplantation methods. One solution could be moving to stem cell-derived transplant tissue. This study investigates a novel stem cell-derived graft and implant technology and explores the circumstances of its cost-effectiveness compared to intensive insulin therapy. METHODS: We used a manufacturing optimization model based on work by Simaria et al. to model cost of the stem cell-based transplant doses and integrated its results into a cost-effectiveness model of diabetes treatments. The disease model simulated marginal differences in clinical effects and costs between the new technology and our comparator intensive insulin therapy. The form of beta cell replacement therapy was as a series of retrievable subcutaneous implant devices which protect the enclosed pancreatic progenitors cells from the immune system. This approach was presumed to be as effective as state of the art islet transplantation, aside from immunosuppression drawbacks. We investigated two different cell culture methods and several production and delivery scenarios. RESULTS: We found the likely range of treatment costs for this form of graft tissue for beta cell replacement therapy. Additionally our results show this technology could be cost-effective compared to intensive insulin therapy, at a willingness-to-pay threshold of $100,000 per quality-adjusted life year. However, results also indicate that mass production has by far the best chance of providing affordable graft tissue, while overall there seems to be considerable room for cost reductions. CONCLUSIONS: Such a technology can improve treatment access and quality of life for patients through increased graft supply and protection. Stem cell-based implants can be a feasible way of treating a wide range of patients with type 1 diabetes.

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.