Corneal regenerative medicine in recent times has taken a focus on the recapitulation of the limbal epithelial stem cell (LESC) niche. Located peripheral to the central cornea, this pool of stem cells is vital for the preservation of sight throughout adult life. The limbus as an anatomical feature has striking topographical characteristics which are readily observed using clinical ophthalmological equipment. Despite this accessibility, it has remained a challenge to recapitulate its true structure in vitro in a manner which is truly biomimetic both in terms of shape and tissue physical properties. Current methods include structure mimicry techniques, such as lithography  and tissue mechanics replication which can involve biological (or biological-like) substrate engineering . In an approach to unify surface shape and mechanical properties, soft polymeric wrinkling  was utilized to form a topographical surface for LESC culture and fate control.
To produce the substrates, chips of polydimethylsiloxane were exposed to a dual oxidation treatment using strong acid and low temperature oxygen plasma. Subsequent to this, the surfaces were coated with 10% GelMa containing 0.25% LAP through UV crosslinking. Afterwards, substrates were re-hydrated to produce the wrinkled-like surfaces. Human LESC’s were isolated from human corneas (NHSBT) using a 2mg/ml Collagenase IV digest of dissected limbal region sections at 37⁰C overnight. The grown monolayer cells were detached and seeded on the sterilised substrates for up to 7 days culture before fixation. Cell response was characterized using immunofluorescence of key limbal markers: ABCG2, p63, Vimentin, and of key corneal markers: CK3, CK12, Nestin. Imaging was taken using a confocal microscopy. The depth-resolved morphology of the elastomer was examined using Optical Coherence Tomography (OCT).
The substrates have demonstrated regular aligned wrinkles with the dimension (70.86±25.39µm) close to limbal crypt width. OCT images showed consistent crypt depth around 17.14±4.64 µm across the whole substrates. Limbal cells responded to the topographic features well by the clear ordering and alignment with elongation of cells in plane along wrinkle propagation physically. The phenotype change toward the substrate was demonstrated through the changes in marker expression, in particular the marked increase of CK3 and Nestin expression in comparison to monolayer controls in conjunction to a decrease in ABCG2 expression. It is worthy to note that the time in culture on the substrate affected marker expression, stem cell markers were preserved at day 3 but showed transition to corneal commitment at day 7.
In this study, we present a novel fabrication technique to produce biocompatible smart material with physically instructive nature of the topography which is able to replicate the shape of key limbal anatomical features whilst provide mechanobiological signals to exert cell fate control, driving commitment of LESC’s towards epithelial cells.
Kang, K B. et al., Nat. Sci. Rep. 9,1, 1-8, 2019.
Tan, Y. et at., Macro-Mol. Bio. 100441, 2021.
Dimmock, R L et al., Rec. Prog. In Mat. 2,1,2020."