Design of Optimal Pancreatic Islet Spheroids Using Hanging-Drop Method on the Functioned Surface

Abstract

For the treatment of type I insulin-dependent diabetes mellitus (IDDM), islet transplantation is considered as the promising strategy. However, because of detrimental conditions after transplantation, the function of the transplanted islets is not efficient enough to control the blood glucose level. Researchers developed islet spheroids to overcome this, and improve transplantation outcome as a result. Here, we investigated the islet spheroids formed using the functioned surface and increased the transfection efficiency of polymeric gene carrier. Rat pancreatic islets were dissociated into single cells by treating 0.25% trypsin-EDTA. And 5 × 105 islet single cells were seeded in a 12-well culture plate. Transfection complexes which consist of 5 μg of DNA and corresponding amount of PEI were treated to each well and filled with extra culture medium to make 2 ml of total volume. Transfected single cells were reaggregated into spheroids using hanging drop method on the polystyrene petri dish. To improve the glucose sensitivity, polydopamine patterned superhydrophobic surface was adopted on the process. Single cells were resuspended in the culture medium (500 cells/30 μl) to form 30 μl drops onto the hydrophilic polydopamine coated part of the surface. The surface was then inverted and drops were incubated for 4-5 days. The lower dish was filled with sufficient PBS to prevent the drops from drying. Intact islets and islet spheroids show no morphologic differences. Also, viability of islet spheroids was not damaged. Transfection efficiency of IL-10 using polyethyleneimine (PEI) increased to approximately 4.5-fold, compared to that of intact islets. This is due to the increase in total surface area that contacts to DNA-carrier complexes. If the incubation environments became more three dimension-like culture, glucose sensitivity tended to be increased. However, between islet spheroids formed with two different surfaces, there was no significant difference. Formation of islet spheroids using hanging drop method would be advantageous in terms of reduced, controlled size and high transfection efficiency. Also, by using hanging drop method, glucose sensitivity of islet spheroids increased compared to those formed with two dimensional culture method which is the conventional cell aggregation methods using intermittent pipetting. With the surface which has greater contact angle, I could reduce the formation duration of islet spheroids.