Encapsulation of Equine Endothelial Colony Forming Cells in Highly Uniform, Injectable Hydrogel Microspheres for Local Cell Delivery

Abstract

A common challenge in cell therapy is the inability to routinely maintain survival and localization of injected therapeutic cells. Delivering cells by direct injection increases the flexibility of clinical applications, but may cause low cell viability and retention rates due to the high shear forces in the needle and mechanical wash out. In this study, we encapsulated endothelial colony forming cells (ECFCs) in poly(ethylene glycol)-fibrinogen (PF) hydrogel microspheres using a custom-built microfluidic device; this system supports rapid encapsulation of high cell concentrations (10 million cells per mL) and resulting cell-laden microspheres are highly uniform in shape and size. The encapsulated ECFCs were shown to have >95% viability and continued to rapidly proliferate. Expression of cell markers (von Willebrand factor, CD105, and CD14), the ability to form tubules on basement membrane matrix, and the ability to take up low-density lipoprotein were similar between pre- and post-encapsulated cells. Viability of encapsulated ECFCs was maintained after shear through 18-23-gauge needles. Ex vivo and in vivo cell delivery studies were performed by encapsulating and injecting autologous equine ECFCs subcutaneously into distal limb full-thickness wounds of adult horses. Injected ECFCs were visualized by labeling with fluorescent nanodots before encapsulation. One week after injection, confocal microscopy analysis of biopsies of the leading edges of the wounds showed that the encapsulated ECFCs migrated into the surrounding host tissue indicating successful retention and survival of the delivered ECFCs. Rapid, scalable cell encapsulation into PF microspheres was demonstrated to be practical for use in large animal cell therapy and is a clinically relevant method to maintain cell retention and survival after local injection.

Rapid Production of Cell‐Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform

“…It takes ≈1 s for photocrosslinking, whereas other platforms take up to 20 s or longer per microsphere . As previously reported, using this platform 4 million cells were encapsulated in 400 µL of PF precursor solution in 24 min, resulting in ≈1500 cell‐laden microspheres with diameters of 800 µm . A range of power output (0.5–3 W, 10%–100%) of the light source has been tested.…”

“…Tight control over the size and shape of microspheres is critical for various applications, such as high‐throughput drug screening, cell production, bioprinting using microspheres as building blocks, and cell delivery . For regenerative medicine, high uniformity of microspheres prepared from multiple batches is needed to ensure smooth and consistent cell delivery by injection.…”

“…[18,19] As previously reported, using this platform 4 million cells were encapsulated in 400 µL of PF precursor solution in 24 min, resulting in ≈1500 cellladen microspheres with diameters of 800 µm. [17] A range of power output (0.5-3 W, 10%-100%) of the light source has been tested. In order to achieve rapid photocrosslinking, a minimum of 2.8 W cm −2 light was necessary to form microspheres with stable boundaries and structural integrity, and a mirror was placed behind the device to reflect the light for higher crosslinking efficiency.…”

“…Tight control over the size and shape of microspheres is critical for various applications, such as high-throughput drug screening, [36] cell production, [37] bioprinting using microspheres as building blocks, [38] and cell delivery. [17] For regenerative medicine, high uniformity of microspheres prepared from multiple batches is needed to ensure smooth and consistent cell delivery by injection. For high-throughput drug screening, tight control over size and shape of microspheres enables a better comparison of drug effects and reduces the number of required replicates.…”

“…Furthermore, pressure differences and changes in flow stability within these smaller channels make it more challenging to encapsulate cells at high densities or in cell clusters, which tend to clog the microfluidic channels and junctions . High cell density is critical for therapeutic cell delivery as we have shown in previous work, where millions of cells were used for large animal cell therapy . To minimize the delivered volume and fabrication time while achieving the desired therapeutic dose, high cell densities were required.…”