Poster N8 - Sacrificial Electrospun Dipeptide for Encapsulation of Cell Therapies

Introduction: Cell based therapies are promising approaches with both academic and industry interest due to the broad range of disease applications, but the advances in stem cell research and genetic engineering that make the field so attractive are held back by biomaterial and engineering challenges. Hydrogel microencapsulation approaches excel at immunoisolation and suppressing fibrotic response, but are difficult to scale into safe, retrievable treatments. Current polymer macroencapsulation devices that address retrievability struggle to balance transport with protection and prevent thick fibrotic capsule formation. The objective of this study is to develop a 3D printable hydrogel material with controlled submicron porosity to increase the rate of transport across an immunoprotective barrier while also enabling increased flexibility in device design. Here, we present a UV crosslinked hydrogel embedded with easily removable nanofibronic porogens. The improved transport properties, in conjunction with light-induced polymerization, is compatible with 3D printing to obtain a wide range of sizes and shapes for the versatility of artificial organ designs.

Materials and

Methods: Several types of porogens were tested to produce nano-microporous hydrogels including calcium carbonate nanoparticles, two types of ionic surfactants, and electrospun alginate. The final molecule selected for further testing was Fmoc-diphenylalanine (Fmoc-FF), a pH sensitive dipeptide capable of self assembly. Solutions of Fmoc-FF nanofibers were electrospun directly into a solution of dilute HCl to form nano to micro scale fibers. The fibers were collected then suspended into a solution of acrylamide monomer, bisacrylamide (crosslinker), and LAP (photoinitiator). The fibers were dissolved and removed from polymerized gels by incubating the gels in a basic buffer. The gels were also placed into a custom electrophoresis device to drive out remaining peptide molecules using electric field. Gel total and macro porosity were measured gravimetrically. Preliminary evaluation of gel porosity performed was through comparison of electrophoresis of BSA. Diffusion of low molecular weight dextran was measured using a diffusion cell. Oxygen permeability was measured using a dissolved oxygen probe by mounting gels directly on the probe end. The probe was placed in low oxygen environment to deplete the gel of oxygen, then placed in an oxygenated environment for data collection. The steady state current from the sensor was recorded and used to calculate the oxygen permeability. Molecular weight cut off was also investigated by imaging penetration of fluorescent dextrans (MW 3-70kDa) driven with hydrostatic pressure into gels mounted in a custom fluidic chip.

Results, Conclusions, and Discussions: Electrospun fibers incorporated with rhodamine dye were imaged in polymerized gel with confocal microscopy and measured to be about 450 nm in diameter on average. From the gravimetric data, removal of the fibers from polyacrylamide gel results in 50% added macroporosity. Preliminary BSA electrophoresis testing showed that the protein band in gel prepared with Fmoc-FF porogens travels up to 2.7 times further than the control under the same conditions. The effective diffusion coefficient of 3kDa dextran in gels prepared with Fmoc-FF porogens was 80% greater than that in control gels. The oxygen permeability of porous gels was 67% greater than control gels. Dextran penetration testing showed that only 3kDa dextran enters control gels, while dextrans up to 40kDa readily enters gels that were prepared with porogens while 70kDa dextran was still excluded.

The approach presented here allows for the preparation of a dispersion of peptide nanofibers that are stable in polyacrylamide matrices and can be easily dissolved and removed. The resulting highly porous hydrogels have significantly improved transport of oxygen and small molecules while still excluding molecules larger than 70kDa. Further, the improved transport properties, in conjunction with light-induced polymerization, is compatible with 3D printing to obtain a wide range of sizes and shapes for the versatility of artificial organ designs. 3D printing allows for designs that optimizing overall device size, surface topology, and shape for mitigating the foreign body response, one of the main challenges of encapsulated cell therapies. Printed micropores can also increase surface area, influence the foreign body response, and induce vascularization. Additionally, although this study uses polyacrylamide, the methods are compatible with other biomaterials capable of free radical polymerization allowing for further tuning of mechanical properties, biofouling resistance, or inclusion of immunomodulatory components. While the method described here excludes immune cells, it does not prevent transport of cytokines, complement system proteins, or damage associated molecular patterns. Additional immunomodulatory cells types or molecules are likely required for long term implant viability. Future work will apply 3D printing to produce cell culture chambers to explore the effect of our highly porous gel on encapsulated spheroid viability.

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