In vitro Expansion of Natural Regulatory T Lymphocytes Useful for Cell Therapy in Allotransplantation

Immunotherapy with regulatory T lymphocytes is considered to be an attractive new therapeutic modality to prevent allograft rejection. The success of this new therapy is critically dependent on the preparation of highly effective and enough number of regulatory T cells. Here, we tried to establish a proper strategy for the ex vivo expansion of regulatory T cells and evaluated their characteristics. CD4+CD25h+CD62L+ T cells were isolated from the recipient mice and weekly stimulated with various stimuli in the presence of IL-2. The most efficient protocol for the expansion of regulatory T cells maintaining Foxp3 expression and regulatory activity was the three cycles stimulation with donor bone marrow-derived dendritic cells (BM-DCs) which yielded around 400 fold expansion of regulatory T cells. The in vitro-expanded regulatory T cells expressing lymph node homing receptors on their cell surface, were composed of polyclonal population, and did not acquire the ability to produce effector cytokines. Importantly, these expanded regulatory T cells induced a modest prolongation of skin allograft survival when combined with transient T cell depletion in recipient mice. These data indicate that our protocol could be used to obtain an effective population of natural regulatory T cells available for the regulatory T cell therapy to prevent allograft rejection.

Experimental Micro Encapsulation of Pancreatic Islets with Air-driven Droplet Generator and Alginate / 대한이식학회지

PURPOSE: Transplantation of microencapsulated islets is proposed as an ideal therapy for the treatment of type 1 diabetes mellitus without immunosuppression. This is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. The aim of this study is to establish an ideal condition of microencapsulation by using an air-driven droplet generator and alginate in vitro. METHODS: Islets were prepared from Sprague Dawley rat and semi SPF-micro pig. Alginate concentrations were changed from 1.5% to 3.0%, and inflow rate of alginate was varied from 10 mL/hr to 40 mL/hr. CO2 flow rate was regulated from 2.0 L/min to 4.0 L/min. Viability was checked by dithizone and FDA/PI staining. Secretory function was tested with glucose challenge and insulin stimulation index was investigated. RESULTS: The optimal conditions for islet encapsulation were revealed with alginate inflow rate of 10 mL/hr, CO2 flow rate of 2.0 L/min in concentration of 2% alginate. In concentration of 2.5% alginate, alginate inflow rate of 20 mL/hr, CO2 flow rate 3.0 L/min was ideal, and alginate inflow rate of 40 mL/hr, CO2 flow rate of 4.0 L/min showed good conditions of microcapsules in concentration of 3% alginate. Viability of encapsulated islets was higher than 90% in both rat and porcine. In terms of insulin secretion, encapsulated islets secreted insulin in response to glucose in static culture medium. However there was no normal response to low and high glucose challenge with stimulation index of less than 2.0. CONCLUSION: Microencapsulation of islets in rat and pig was successful with air-driven droplet generator and alginate in vitro. Further studies about biocompatibility and glucose control in vivo should be followed to be a useful tool for treatment of diabetes mellitus patients in clinical setting.