Transplantation of non-autologous β-cells is currently regarded as a promising therapy for the treatment of type 1 diabetes, caused by massive β-cell destruction, consequently resulting in insulin shortage. To evade the host’s immune responses, new materials are being developed to encapsulate and shield implanted β-cells. Several immunoprotective material formulations have been developed, but their clinical translation is challenged by their impermanent nature, development fibrous capsules upon implantation1, which is insufficient to produce therapeutical signifficance2. Additionally, such materials should be capable of inhibiting the diffusion of large immune molecules (i.e., IgG) whilst enabling diffusion of small molecules (i.e. Insulin and glucose) in a semi-permeable fashion3. In this work, we have developed non-immunogenic, immunoprotective, and enzymatically crosslinked, hollow, polyethylene glycol-tyramine (PEG-TA) microgels for β-cell delivery therapies.
Polyethylene glycol 8 arm – tyramine (PEG-TA) conjugates were synthesized in a two-step reaction and optimized for enzymatic crosslinking using horseradish peroxide and non-cytotoxic levels of hydrogen peroxide4. Droplet generation was optimized for production of micrometer-sized hollow PEG-TA microgels laden with beta cells using microfluidics5. Hollow microgels containing MIN6 pancreatic cells were extensively characterized in vitro based on variables such as cytocompatibility, cyto-immunity, permselectivity, and glucose responsiveness. Furthermore, in vivo performance was assessed by implanting diabetic STZ-mice to investigate immunoprotectiveness of the implant and its ability to re-establish normoglycemia.
RESULTS & DISCUSSION:
Consistent production of ~120-150 µm hollow microcapsules with ~20 µm shell thickness was achieved and characterized. Immunoprotection was confirmed by exposing cell-laden gels by absence of diffusion of IgG-FITC molecules into the microgels and by co-culture with natural killer NK-92 cells without inducing cell death.
Encapsulated cells were shown to sustain viability and capable of secreting insulin in vitro over a period of one month without showing significant shell burst. Immunological assessment by multiplexed ELISA analysis on live blood immune reactivity was performed, demonstrating little pro-inflammatory cytokine release in response to microgel presence, confirmed by gene expression analysis and immunostainings of inflammatory markers.
Intraperitoneal implantation of β-cell laden microgels in diabetic mice showed restoration of normoglycemia within the first 5 days and good tissue integration, with histology revealing alive aggregates at the time of sacrifice (14 days)
Consistent high throughput microfluidic production of immunoprotective PEG-TA microgels was achieved. The produced microgels revealed good suitability for shielding and delivering non-autologous beta-cells within a living host.
ACKNOWEDGEMENTS: Financial support was received from the European Research Council (ERC, Starting Grant, #759425) and JDRF.
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2Golpanian S, Schulman IH, Ebert RF. Stem Cells Translational Medicine 5: 186–191, 2016; 3B, Lewińska D, Biomaterials Science, 8, 1536-1574, 2020;
4Kamperman T, Henke S, Zoetebier B, Ruiterkamp N, Wang R, Pouran B, Weinans H, Karperien M, Leijten J. Journal of Materials Chemistry B 5(25), 2017;
5van Loo SR, Salehi S, Henke S, Shamloo A, Kamperman T, Karperien M, Leijten L.Materials Today Bio, 6:100047 2020