Preparation of cell aggregates incorporating gelatin hydrogel microspheres of sugar-responsive water solubilization.

The objective of this study is to design hydrogel microspheres of a cell scaffold, which not only function as a scaffold to form cell aggregates of three-dimensional culture but also can disappear to release growth factors in the well-controlled manner by noncytotoxic stimulation in any timing. The hydrogel microspheres were prepared by a water-in-oil emulsion method from m-aminophenylboronic acid (APBA)-introduced gelatin (APBA-gelatin) with or without poly(vinyl alcohol) (PVA) mixing. Irrespective of the PVA concentration, the microspheres with the same diameter were prepared. The microspheres were water solubilized only by adding sorbitol of a sugar although the solubilization extent depended on the PVA concentration. When cocultured with the microspheres, mesenchymal stem cells formed cell aggregates homogeneously incorporating the microspheres. Upon adding sorbitol in the culture medium, mixed APBA-gelatin-PVA hydrogel microspheres disappeared with time in the cell aggregates. The microspheres containing basic fibroblast growth factor or bone morphogenetic protein-2 released the respective growth factor accompanied with the microspheres disappearance. It is concluded that the present microspheres of sugar-responsive water solubilization are promising scaffold of cell aggregates and have an ability to allow growth factors to be released in the cell aggregates when it is required.

Enhanced survival and insulin secretion of insulinoma cell aggregates by incorporating gelatin hydrogel microspheres.

INTRODUCTION: The objective of this study is to evaluate the survival and glucose-induced insulin secretion of rat-derived insulinoma cells (INS-1) from their aggregates incorporating different size of gelatin hydrogel microspheres comparing with microspheres-free cell aggregates. METHODS: The gelatin hydrogel microspheres were prepared by the conventional w/o emulsion method. The INS-1 cells were cultured in a V-bottomed well, combining with or without the gelatin hydrogel microspheres to form their aggregates with or without microspheres. RESULTS: When the cell viability, the live cell number, the reductase activity, and the insulin secretion of cell aggregates were evaluated 7 or 14 days after incubation, the cell aggregates incorporating gelatin hydrogel microspheres showed higher cell viability, reductase activity and a larger number of live cells. The cell aggregates incorporating larger size and number of gelatin hydrogel microspheres secreted a larger amount of insulin, compared with those incorporating smaller size and number of microspheres or without microspheres. CONCLUSION: It is conceivable that the incorporation of gelatin hydrogel microspheres in cell aggregates is promising to improve their survival and insulin secretion function.

Insulin secretion of mixed insulinoma aggregates-gelatin hydrogel microspheres after subcutaneous transplantation.

INTRODUCTION: The objective of this study is to evaluate the insulin secretion of mixed aggregates of insulinoma cells (INS-1) and gelatin hydrogel microspheres after their subcutaneous transplantation. METHODS: Gelatin hydrogel microspheres were prepared by the conventional w/o emulsion method. Cell aggregates mixed with or without the hydrogel microspheres were encapsulated into a pouched-device of polytetrafluoroethylene membrane. An agarose hydrogel or MedGel™ incorporating basic fibroblast growth factor (bFGF) was subcutaneously implanted to induce vascularization. After the vascularization induction, cell aggregates encapsulated in the pouched-device was transplanted. RESULTS: The vascularization had the potential to enable transplanted cell aggregates to enhance the level of insulin secretion compared with those of no vascularization induction. In addition, the insulin secretion of cell aggregates was significantly promoted by the mixing of gelatin hydrogel microspheres even in the pouched-device encapsulated state. CONCLUSION: It is possible that the microspheres mixing gives cells in aggregates better survival condition, resulting in promoted insulin secretion.

Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research.

We previously reported that tumor vessel-redirected T cells, which were genetically engineered with chimeric antigen receptor (CAR) specific for vascular endothelial growth factor receptor 2 (VEGFR2), demonstrated significant antitumor effects in various murine solid tumor models. In the present study, we prepared anti-VEGFR2 CAR-T cells by CAR-coding mRNA electroporation (mRNA-EP) and analyzed their immunological characteristics and functions for use in clinical research. The expression of anti-VEGFR2 CAR on murine and human T cells was detected with approximately 100% efficiency for a few days, after peaking 6-12 hours after mRNA-EP. Triple transfer of murine anti-VEGFR2 CAR-T cells into B16BL6 tumor-bearing mice demonstrated an antitumor effect comparable to that for the single transfer of CAR-T cells engineered with retroviral vector. The mRNA-EP did not cause any damage or defects to human T-cell characteristics, as determined by viability, growth, and phenotypic parameters. Additionally, two kinds of human anti-VEGFR2 CAR-T cells, which expressed different CAR construction, differentiated to effector phase with cytokine secretion and cytotoxic activity in antigen-specific manner. These results indicate that our anti-VEGFR2 CAR-T cells prepared by mRNA-EP have the potential in terms of quality and performance to offer the prospect of safety and efficacy in clinical research as cellular medicine.