P-245 Poster of distinction

Laminin coating for creating transplantable rat islet cell sheets

S. Mukobata¹, K. Ohashi², R. Utoh², H. Sakai¹, T. Okano^2
1 CellSeed Inc., Tokyo, Japan; ² Tokyo Women's Medical University, Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan

Background: Our experimental approach toward the development of novel treatments for diabetes mellitus (DM) has allowed us to create a monolayered contiguous sheet (islet cell sheet), which was transplanted subcutaneously (Shimizu et al, Biomaterials, 2009) . For fabricating an islet cell sheet, dispersed islet cells were seeded onto a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dish coated with rat laminin-5. Since increasing the number of cells attached on the PIPAAm dish may increase the function of fabricated islet cell sheets, the present study explored various isotypes of human laminins for coating materials on PIPAAm surfaces, and for determining the status of rat islet cells cultured on the dish surfaces.

Methods: Dispersed islet cells were obtained from Lewis rats. Temperature responsive culture surfaces were prepared by covalently immobilizing PIPAAm onto plastic dishes. The polymer surfaces were then coated with rat laminin-5(RL-5), human laminin-411 (HL-411), human laminin-211 (HL-211), or human laminin-322 (HL-322). Dispersed rat islet cells were plated onthe PIPAAm dishes at a ratio of 5 x 10⁶ cells/cm².

Results: Plating efficiencies determined at day 1 were 76.6%, 34.5%, 27.8%, and 82.8% in the RL-5, HL-411, HL-211, and HL-322 groups, respectively. Confluencies of the cells determined at day 3 were 87.1%, 20.7%, 16.4%, and 92.5% in the RL-5, HL-411, HL-211, andHL-322 groups, respectively. By reducing culture temperature from 37 °C to 20 °C for 30 min in RL-5 and HL-322 groups, cells were able to harvested as an islet cell sheet.

Conclusions: The present data indicates that surfaces coated with HL-322 and RL-5 were found to significantly stimulate islet cells to adhere and expand on the PIPAAm culture surfaces, leading to the formation of a confluent monolayered structure.

P-245 Poster of distinction

Laminin coating for creating transplantable rat islet cell sheets

S. Mukobata¹, K. Ohashi², R. Utoh², H. Sakai¹, T. Okano^2
1 CellSeed Inc., Tokyo, Japan; ² Tokyo Women's Medical University, Institute of Advanced Biomedical Engineering and Science, Tokyo, Japan

Background: Our experimental approach toward the development of novel treatments for diabetes mellitus (DM) has allowed us to create a monolayered contiguous sheet (islet cell sheet), which was transplanted subcutaneously (Shimizu et al, Biomaterials, 2009) . For fabricating an islet cell sheet, dispersed islet cells were seeded onto a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), grafted culture dish coated with rat laminin-5. Since increasing the number of cells attached on the PIPAAm dish may increase the function of fabricated islet cell sheets, the present study explored various isotypes of human laminins for coating materials on PIPAAm surfaces, and for determining the status of rat islet cells cultured on the dish surfaces.

Methods: Dispersed islet cells were obtained from Lewis rats. Temperature responsive culture surfaces were prepared by covalently immobilizing PIPAAm onto plastic dishes. The polymer surfaces were then coated with rat laminin-5(RL-5), human laminin-411 (HL-411), human laminin-211 (HL-211), or human laminin-322 (HL-322). Dispersed rat islet cells were plated onthe PIPAAm dishes at a ratio of 5 x 10⁶ cells/cm².

Results: Plating efficiencies determined at day 1 were 76.6%, 34.5%, 27.8%, and 82.8% in the RL-5, HL-411, HL-211, and HL-322 groups, respectively. Confluencies of the cells determined at day 3 were 87.1%, 20.7%, 16.4%, and 92.5% in the RL-5, HL-411, HL-211, andHL-322 groups, respectively. By reducing culture temperature from 37 °C to 20 °C for 30 min in RL-5 and HL-322 groups, cells were able to harvested as an islet cell sheet.

Conclusions: The present data indicates that surfaces coated with HL-322 and RL-5 were found to significantly stimulate islet cells to adhere and expand on the PIPAAm culture surfaces, leading to the formation of a confluent monolayered structure.