This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.
This method can help resolve major challenges in pancreatic islet transplantation, such as immuno-rejection, post-transplantation islet revascularization, and survival. The main advantages of this technique is that this easy to adopt approach enables surface engineering of living cells by reshaping the cellular landscape of pancreatic islet it will improve graft function, survival and ultimately the therapeutic efficacy of islet transplantation. The implications of this technique extend towards cell-based therapies because the therapeutic outcome of cell-based therapies is also limited by low cell retention and poor survival.
Demonstrating this procedure is Jingyi Yang a graduate student from my laboratory. First, weight out 6.9 grams of Heparin and dissolve it in 2 milliliters of ice cold deionized water in an Eppendorf tube. Dissolve 0.23 grams of NHS in 0.5 milliliters of ice cold deionized water in an Eppendorf tube.
Then, dissolve 0.77 grams of EDC in 0.5 milliliters of ice cold deionized water in a 50 milliliter conical tube. Mix the NHS and EDC solutions in a 50 milliliter conical tube. After leaving the mixed solution at room temperature for 30 minutes, centrifuge it at 12, 000 RPM for 10 minutes to remove the excess EDC and NHS.
Following this, add 30 milliliters of cold Ethanol to the solution and centrifuge at 12, 000 RPM for 10 minutes. Now, add 1 milliliter of 10%starPEG MI aid to a 50 milliliter conical tube. Then add 0.67 milliliters of 10%Heparin NHS solution to the same 50 milliliter conical tube.
Place the mixed solution in an incubator at 25 degrees Celsius for 20 minutes to obtain a viscous clear solution. Handpick 200 previously extracted mouse islets under a dissection microscope and transfer to a 1.5 milliliter tube. Add 500 microliters of PBS to the islets and centrifuge at 1, 200 RPM for one minute at four degrees Centigrade.
Remove the supernatant, add 250 microliters of the Heparin PEG solution, and place the mixture on ice for 10 minutes. Pipette up and down to allow the islets to mix well with the Heparin PEG solution. Following this, centrifuge the mixture at 1, 200 RPM for one minute.
Then, remove the supernatant and add 500 microliters of PBS. Pipette up and down to mix well. After centrifuging at 1, 200 RPM for one minute, remove the supernatant and keep the islets for further use.
Next, add one to two milliliters of RPM I 1640, supplemented with 10%FBS to the coated islets. And transfer these islets into a non-charged sterile bacterial culture dish. Finally, observe the morphology and fluorescent signals of the FAM-Heparin-PEG-coated islets under an inverted fluorescence microscope.
Characteristic Heparin peaks, corresponding to the HYDROXAL groups, were observed in the Heparin-PEG nanocoating FTIR spectrum. The decrease in amplitude of these peaks represents conjugation between the star-PEG MI aid amide groups and the Heparin carbonal group. The peak corresponding to the amide carbonal stretching vibration was also reduced, indicating sufficient reaction between the Heparin carboxylate groups with the succynimidal suxsonate and the star-PEG MI aid amine.
Scanning an electron microscopy data shows the highly interconnected porous structure of the Heparin-PEG nanocoating, suggesting that it could be suitable for cell survival during in vivo delivery. A thin layer of nanocoating, shown by green fluorescence, was evenly deposited across the surface of the coated islets, without causing evident changes on islet volume or size. Heparin-PEG-coated mouse islet exhibited robust islet viability in culture.
Significantly more advanced vascular formation was evident from islet endothelial cells that were co-cultured with Heparin-PEG-coated islets, indicated by elongated micro-vessel-like structures and network-like vascular structures. A low level of insulin secretion was observed in all treatment groups when the islets were perfused with a physiological salt solution, supplemented with a sub-stimulatory level of glucose. When the islets were stimulated with a super-physiological level of glucose, an increase in insulin secretion was observed.
Once mastered, the coating can be done in a minute if it's performed properly to preserve islet viability.
