Acellular RAFT TEs were prepared in 24-well plates using rat-tail type I collagen and neutralizing solution (TAP Biosystems). Briefly, collagen solution (80% of total volume) was
mixed with 10xMEM (10%), neutralizing solution (5.9%) and DMEM (4.1%). The mixture was held on ice for 30 minutes prior to seeding 2.4 ml mixture into each well. This was allowed to set for 30 minutes using an electronic heater (TAP Biosystems, Royston, Cambridge, UK) prior to gentle wicking of water from collagen hydrogels using hydrophilic porous absorbers (TAP Biosystems).
The cell suspension containing a mixed population of CSSC and
HLE was divided equally onto the surface of 4 RAFT TEs (24-well) and cultured at 37 °C and 5% CO2 up to 13 days with CSSC media replaced daily or at least every second day.
Human limbal epithelial cells (HLE) and corneal stromal stem cells (CSSC) reside in close proximity in vivo in the corneal limbal stem cell niche. However, HLE are typically cultured in vitro without supporting niche cells. Here, we re-create the cell-cell juxtaposition of the native environment in vitro, to provide a tool for investigation of epithelial-stromal cell interactions and to optimize HLE culture conditions for potential therapeutic application. RAFT (Real Architecture For 3D Tissue) tissue equivalents (TEs) were used as a 3-dimensional substrate for co-culturing HLE and CSSC. Our results demonstrate that a monolayer of HLE that maintained expression of p63a, ABCB5, CK8 and CK15 (HLE markers), formed on the surface of RAFT TEs within 13 days of culture. CSSC remained in close proximity to HLE and maintained expression of mesenchymal stem cell markers. This simple technique has a short preparation time of only 15 days with the onset of HLE layering and differentiation observed. Furthermore, co-cultivation of HLE with another niche cell type (CSSC) directly on RAFT TEs, eliminates the requirement for animal-derived feeder cells. RAFT TEs may be useful for future therapeutic delivery of multiple cell types to restore the limbal niche following ocular surface injury or disease.