Table 4.

Troubleshooting guide for construct assembly and characterization.

Problem	Possible Cause	Solution
hCECs do not attach to silk films and are floating or adhered to cell culture plate	• Lack of collagen coating on silk film • Older passage of hCECs • Addition of media to well prior to cell adhesion	• Always prepare fresh stamping solution and growth factors prior to coating of film. • Use passages of <5 of hCECs. These cells are particularly sensitive to aging and thawing. • Visualize by microscope proper cell adherence to film prior to addition of media to well.
Lack of confluent hCEC layer	• Older passage of hCECs • Absence of epithelial growth factors in media • Poor cell proliferation	• Utilize only passages of <5 of hCECs. These cells are particularly sensitive to aging and thawing. • Prepare fresh media containing standard epithelial growth factors, e.g. EGF, BPE, etc. • May substitute a corneal epithelial cell line, e.g. HCE-TJ, or limbal epithelial stem cell source to improve epithelial barrier integrity.
Poor hiNSC proliferation on the MEF feeder plate	• Older passage of MEFs • Lack of confluent feeder layer • Absence of fresh FGF in media	• Utilize only passages of <5 of MEFs. • Seed MEFs at a high density and allow for 100% confluence prior to inactivation. • Prepare fresh media containing FGF immediately prior to use.
hCSSCs do not attach to RGD-coated films and are floating or adhered to cell culture plate	• Low pH of the film may cause cell death • Lack of RGD-functionalization to film • Addition of media to petri dish prior to cell adhesion	• Ensure that the MES solution pH is neutral (pH 6.5-7). Wash films in PBS prior to use in cell culture. • Utilize freshly dissolved RGD peptide to crosslink to films. • Visualize by microscope proper cell adherence to film prior to addition of media to petri dish.
Frequent bacterial or fungal contamination	• Contamination of scaffolds prior to seeding • Lack of aseptic technique during cell culture	• Autoclave silk sponges after cutting and immediately prior to use in cell culture. Sterilize silk films under UV-light (30 min each side) and immerse in 70% v/v EtOH in distilled water prior to RGD-coupling. Filter-sterilize (0.2 μm) crosslinking solutions and RGD-peptide prior to incubation with sterile films. Autoclave waffle-shaped PDMS mold prior to inclusion in system. • Adopt standard aseptic approaches during construct assembly and maintenance.
Low total protein isolated	• Inadequate cell lysis • Protein degradation • Cell loss during cultivation	• Ensure that the isolated cell layer is submerged in the lysis buffer. • Include protease inhibitors in lysis buffer and maintain samples on ice during homogenization and incubation steps. Aliquot samples to reduce freeze-thaw cycles. • Monitor media color between media changes. A slight change should be noticeable indicating active metabolism. Ensure that cells properly attach to scaffolds prior to assembly of the construct.
Unusually high total protein isolated	• Inadequate removal of media prior to construct lysis • Bacterial or fungal contamination	• Be sure to gently wash or immerse construct in PBS prior to isolation. • Monitor media color between media changes. A severe change in color to yellow or cloudiness of the media are common indicators of contamination. Visually inspect constructs for contamination.
Poor IHC images	• High silk autofluorescence in the green channel • Lack of antibody binding	• Choose secondary antibodies with emissions wavelengths outside of the green channel range, e.g. λem=480-638 nm) • Incubate primary and secondary antibodies with construct overnight at 4°C with rocking to allow perfusion into the tissue.