P-259

Fabrication of monolayered islet cell sheets from cryopreserved islet cells

K. Ohashi¹, S. Mukobata², R. Uoth¹, H. Sakai², T. Okano^1
1 Tokyo Women's Medical University, Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan; ² CellSeed Inc., Tokyo, Japan

Background: Toward the establishment of novel islet-based therapies, our group has recently developed technologies for creating a monolayered contiguous sheet made from freshly dispersed islet cells. Previous experiments had shown that islet cell sheet is transplantable and engraftable in subcutaneous site. The use of a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dishes with laminin-5 coating is an important feature for the creationof islet cell sheets. The present study was conducted to assess the value of dispersedand cryopreserved islet cells for creating islet cell sheets.

Methods: Specially coated plastic dishes were prepared by covalently immobilizing PIPAAm onto the culture plastic, followed by coatingwith laminin-5 as described previously (Shimizu et al, Biomaterials, 2009). Dispersed rat islet cells were obtained from Lewis rats and were resuspended with UW solution with 10% DMSO. After cryopreservation for 1 month, cells were thawed and plated on the PIPAAm dish.

Results: After being thawed, 85.1 ± 7.1% of the cells were recovered. Viability of the thawed islet cells assessed by trypan blue exclusion test was found to be 53.1 ± 10.9%. The viable dispersed islet cells favorably attached to the PIPAAm dish. At day 3 after the start of the culture, the culture temperature was reduced to be 20 °C for 30 min, and cells were able to be harvested as a contiguous cell sheet because of the temperature-dependent change of the culture surfaces.

Conclusions: The present data indicates that dispersed islet cells, which were appropriately frozen and thawed, were able to preserve their adhesive character and form a monolayer on the PIPAAm dish. These findings suggest that dispersed and cryopreserved islet cells would be an appropriate source of cells for creating functional islet cell sheets.

P-259

Fabrication of monolayered islet cell sheets from cryopreserved islet cells

K. Ohashi¹, S. Mukobata², R. Uoth¹, H. Sakai², T. Okano^1
1 Tokyo Women's Medical University, Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan; ² CellSeed Inc., Tokyo, Japan

Background: Toward the establishment of novel islet-based therapies, our group has recently developed technologies for creating a monolayered contiguous sheet made from freshly dispersed islet cells. Previous experiments had shown that islet cell sheet is transplantable and engraftable in subcutaneous site. The use of a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dishes with laminin-5 coating is an important feature for the creationof islet cell sheets. The present study was conducted to assess the value of dispersedand cryopreserved islet cells for creating islet cell sheets.

Methods: Specially coated plastic dishes were prepared by covalently immobilizing PIPAAm onto the culture plastic, followed by coatingwith laminin-5 as described previously (Shimizu et al, Biomaterials, 2009). Dispersed rat islet cells were obtained from Lewis rats and were resuspended with UW solution with 10% DMSO. After cryopreservation for 1 month, cells were thawed and plated on the PIPAAm dish.

Results: After being thawed, 85.1 ± 7.1% of the cells were recovered. Viability of the thawed islet cells assessed by trypan blue exclusion test was found to be 53.1 ± 10.9%. The viable dispersed islet cells favorably attached to the PIPAAm dish. At day 3 after the start of the culture, the culture temperature was reduced to be 20 °C for 30 min, and cells were able to be harvested as a contiguous cell sheet because of the temperature-dependent change of the culture surfaces.

Conclusions: The present data indicates that dispersed islet cells, which were appropriately frozen and thawed, were able to preserve their adhesive character and form a monolayer on the PIPAAm dish. These findings suggest that dispersed and cryopreserved islet cells would be an appropriate source of cells for creating functional islet cell sheets.