This study outlines a method for creating a 3D co-culture model of human corneal fibroblasts and neurons. The model utilizes vitamin C and retinoic acid to stimulate cell differentiation and extracellular matrix assembly.
Take human corneal fibroblasts.
Transfer them to a membrane insert within a multi-well plate containing media.
Add media to the membrane insert. Incubate for fibroblast adherence to the insert.
Next, replace the media with media containing vitamin C in the membrane insert and the reservoir.
Incubate. Vitamin C stimulates the fibroblasts to secrete and assemble a 3D extracellular matrix.
Add a human neuroblastoma cell suspension over the fibroblasts.
Incubate, allowing neuroblastoma cell adhesion.
Now, add media containing retinoic acid over the cells and supplement the reservoir with fresh media.
Incubate. Retinoic acid triggers neuroblastoma cell differentiation into neural progenitor cells.
Next, remove the media. Introduce fresh media to the reservoir. Add media containing a neurotrophic factor to the membrane insert and incubate.
Neurotrophic factors trigger progenitor cell differentiation into neurons, generating a 3D co-culture model of human corneal fibroblasts and neurons.
To assemble 3D constructs, first, passage and count human corneal fibroblasts. Then, seed approximately 1 times 10 to the 6 HCFs onto a polycarbonate membrane insert with 0.4-micron pores. Add 1.5 milliliters of fresh medium to the top of the construct. Then, pipette another 1.5 milliliters of the same media to the bottom of each construct.
After 24 hours, add 1.5 milliliters of medium supplemented with 0.5 millimolar vitamin C to the top and bottom of each construct. Incubate at 37 degrees Celsius in 5% carbon dioxide for three weeks, adding fresh media containing vitamin C every other day. This will stimulate the cells to secrete and assemble their own extracellular matrix. After three weeks, add 8 times 10 to the 3 SH-SY5Y human neuroblastoma cells on the top of the previously assembled 3D constructs.
It is critical to calculate exact cell number in this step and to allow 24 hours before initiating cell differentiation.
After 24 hours of incubation, initiate SH-SY5Y cell differentiation by stimulating the cells with 1.5 milliliters of EMEM with 10% FBS and 10 micromolar retinoic acid. Then, add 1.5 milliliters of the medium containing vitamin C to the bottom of the construct. Retinoic acid is light-sensitive so this step should be performed in the dark.
After five days, when the cells reached the differentiation phase, add 1.5 milliliters of serum-free medium containing 2 nanomolar BDNF and 1% antibiotic-antimycotic to the top of the construct. Add fresh vitamin C-supplemented medium to the bottom of the construct. Culture the cells for two additional days before processing for further analysis.
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