Table 5.
Relative timeframe for completion of each step in the assembly, stimulation, and characterization of the corneal tissue model. The amount of time may vary significantly depending on the user and the total number of constructs generated. These timeframes are based on 12 constructs per experiment.
| Stage | Application | Active Time | Total Time |
|---|---|---|---|
| Scaffold Preparation | PDMS molds | 30 minutes | 1 day with overnight incubation |
| Silk isolation | 3-4 hours | 5-6 days | |
| Patterned, RGD-conjugated silk films | 3 hours | 2.5 days with 48 hour incubation | |
| Nonpatterned silk film with epithelial growth factor stamp | 2-3 hours | 2.5 days with 48 incubation | |
| Silk sponge | 2 hours | 5 days | |
| Cell Culture | Culture hCECs | 2 hours | 1 week from frozen stock + 2 days on silk film = ~9 days |
| Culture hCSSCs | 2 hours | 2-3 days from frozen stock + 2 days on silk film = ~5 days | |
| Culture hiNSCs | 5 hours | 1 week to culture MEF feeder plate + 0.5 day to inactivate + 1-2 weeks of hiNSC on MEFs + 10 day sensory differentiation = ~3-4 weeks | |
| Cultivation | Assemble construct | 2-3 hours | 0.5 day |
| Maintenance of construct | 12 hours | 4 weeks with media changes every other day | |
| Chemical Stimulation | Topical application of chemical to construct | 1 hour | 1 day with overnight incubation |
| Characterization | MTT toxicity assay | 1.5 hours | 4 hours |
| Conditioned media isolation and ELISA | 3 hours | 1.5 days with overnight incubation | |
| IHC | 2 hours + imaging | 2.5 days | |
| Cell lysis and protein isolation | 30 minutes | 2 hours | |
| Measuring protein concentration by BCA assay | 30 minutes | 1 hour | |
| Western blot | 1 hour | 1.5 days with overnight incubation |