Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

1. Primary HCFs (human corneal fibroblasts) Culture and 3D Constructs Assembly

1. To assemble 3D constructs from explants previously grown in EMEM 10% FBS 1% Antibiotic/Antimycotic, passage and count HCFs.
2. Seed approximately 1 x 10⁶ cells/well of primary HCFs on polycarbonate membrane inserts with 0.4-µm pores from the 3D constructs, along with 1.5 mL of fresh EMEM 10% FBS 1% Antibiotic/Antimycotic to the top of the construct. Add another 1.5 mL of the same media to the bottom of each construct.
3. After 24 h of cell seeding, culture the cells with EMEM 10% FBS 1% Antibiotic/Antimycotic, stimulated with 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C) by adding 1.5 mL on the top and 1.5 mL on the bottom of each construct.
4. Maintain the cultures in an incubator at 37 °C, 5 % CO₂ for 3 weeks and supply fresh media every other day during the entire study period. No further passage is needed.

2. Cell Differentiation and Co-cultures

1. After 3 weeks, add 8 x 10³ cells/well of SH-SY5Y human neuroblastoma cells on the top of the previously assembled 3D constructs containing 1.5 mL of the EMEM containing 10% FBS, 1% Antibiotic/Antimycotic, and 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C).
   1. Allow the SH-SY5Y neuroblastoma cells to grow on top of the cornea stromal cells for at least 24 h before initiating cell differentiation.
2. Initiate SH-SY5Y cell differentiation by stimulating the cells with EMEM containing 1% FBS and 1.5 mL of 10 µM Retinoic Acid on top of the construct. Add 1.5 mL of the EMEM 10% FBS 1% Antibiotic/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C) to the bottom of the construct. Note that the Retinoic Acid step should be performed in the dark.
   1. Continue to culture the cells in this differentiation media for 5 days.
3. When the cells reach the differentiation phase, switch the cells to 1.5 mL of serum-free media containing 2 nM of Brain-derived neurotrophic factor (BDNF) and 1% Antibiotic/Antimycotic added to the EMEM, by adding to the top of the construct. Add 1.5 mL of the EMEM 10% FBS 1% Antibiotic/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C) to the bottom of the construct. Note that the BDNF step should be performed in the dark.
4. Culture the cells for 2 additional days before processing for any further analysis.