Abstract
Purpose :
Human corneal stromal stem cells (CSSC) delivered in fibrinogen to the corneal stroma suppress corneal scarring in a mouse wound healing model. Additionally, CSSC have been reported to promote regression of human scars. Treatment with CSSC requires cultures at a specific density and passage. A broad application of stem cell-based corneal therapy would be facilitated by availability of a standardized stable reagent suitable for storage and shipment to venues not equipped with GMP cell culture facilities. Our study examined the hypothesis that delivering CSSC embedded in compressed collagen gels could satisfy these needs.
Methods :
CSSC were isolated from limbal stroma of human corneal rims and applied to wounded mouse corneas in fibrinogen as previously described (PMID: 25504883). CSSC in passage 3 were embedded in acid-soluble rat tendon collagen, gelled at 37 C and compressed using RAFTtm absorbers (Lonza). Corneal scarring was assessed visually in masked fashion two weeks after wounding and expression of fibrotic mRNA in wounded corneas was assessed by qPCR. Gels loaded with CSSC were cryopreserved in 20% FBS-10%DMSO in liquid nitrogen. Significance was determined with t-tests with Dunn’s post-hoc analysis using p<0.05 as a criterion.
Results :
Collagen gels containing CSSC could be compressed to 100 µm thickness from which 2 mm disks were punched using a trephine. The disks adhered securely to de-epithelialized mouse cornea with fibrin-based tissue adhesive, and embedded cells remained viable and visible. After 48 hr in vivo, neither gels nor CSSC were present on the ocular surface. Visible scarring was effectively prevented by as few as 500 CSSC in the gels (p<0.001, n=8) and expression of collagen 3A mRNA was reduced (p<0.05, n=3). CSSC in collagen blocked scarring more effectively on a per-cell basis than CSSC in fibrinogen (p<0.01, n=8). Gels were dimensionally stable and CSSC in the compressed gels remained >80% viable after 1 week in culture media. The collagen-embedded cells retained the ability to suppress corneal scarring after 2 weeks of conventional cryopreservation (p<0.01, n=6).
Conclusions :
Stem cell-based therapy has high potential for prevention as well as regression of corneal scarring. The current study demonstrates a method of applying CSSC to cornea that facilitates storage and handling of cells in a manner that may allow off-the-shelf delivery of stem cells for corneal scarring.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.