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

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 millil