The overall goal of this protocol is to investigate the cellular interactions between human corneal stromal cells and neuronal cells in their natural extracellular matrix. The protocol can be expanded to study corneal diseases and dystrophies, such as keratoconus, diabetic keratopathy, and Fuchs'dystrophy. This method can help answer key questions in the human cornea biology field, by identifying the underlying mechanisms and defects in the stroma nerve interactions during a specific corneal disease or dystrophy.
The main advantage of this technique is that cells are allowed to secrete their own extracellular matrix in vitro that provides a better and more accurate model of the actual human corneal tissue in vivo. The implications of this technique extend toward therapy of corneal diseases, because assessment of cell-cell and cell extracellular matrix interactions will ensure accurate representation of the corneal tissue, and allow more targeted drug development. Now, this method can provide insight into corneal nerve damage and regeneration.
It can also be applied to other systems, such as drug screening and development. Upon receipt of human cornea tissue samples, transfer the tissues into a 21.5 square centimeter dish containing two milliliters of sterile Dulbecco's phosphate buffered saline. Position the cornea dome face up and use a razor blade at a 45 degree angle to scrape off the epithelial layer.
Next, flip the cornea dome face down and thoroughly scrape off the endothelium from the stroma. Wash the stromal tissue with DPBS. Keep the stromal pieces wet with DPBS, and use a number 10 surgical blade to cut the stromal tissues into small pieces.
Use sterile forceps to transfer the stromal pieces into a T25 culture flask without media. Allow the explants to adhere to the bottom of the flask at 37 degrees Celsius for about 30 to 40 minutes. After precisely cutting the stromal tissue, carefully transfer the flask to the incubator.
It is important to allow 30 to 40 minutes, and then add media slowly. This step is critical to allow complete adherence of the explants, and prevent their detachment. Once adhered, slowly add Eagle's Minimum Essential Medium, containing 10%fetal bovine serum and 1%antibiotic-antimycotic to the flask, without disturbing the explants.
Gently place the flask in the incubator at 37 degrees Celsius and 5%carbon dioxide. Leave the explants undisturbed until the cells begin isolating and migrating in the flask. After approximately two to four weeks, cells should reach 100%confluence.
Upon 100%confluence, passage the cells by removing the tissue culture medium, and washing the cells with DPBS. Add trypsin to the cells, and then incubate at 37 degrees Celsius. After approximately five minutes, view the cells under a light microscopy to confirm that they have detached.
Halt proteolysis by adding fresh medium and transfer the cell suspension into a 15 milliliter conical tube. Centrifuge the tubes at 1000 x g to pellet the cells. Remove the supernatant carefully without disturbing the pellet and suspend the cells in fresh medium.
After performing a cell count, seed approximately one times 10 to the six cells in T75 culture flask with fresh medium for further expansion. To assemble 3D construct, first passage and count human corneal fibroblasts, then seed approximately one times 10 to the six HCF 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 and 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 eight times 10 to the third 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 reach the differentiation phase, add 1.5 milliliters of serum-free medium containing two 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.
This is a high magnification transmission electron microscopy image of the 3D self-assembled construct showing the polycarbonate membrane and the corneal stromal cells seated on top. This image shows the differentiated neuronal cells seated on top, which are exhibiting a stromal-neuronal cell arrangement. The arrows in the image show the neuronal cells.
While attempting this procedure, it's important to remember to keep corneal tissue wet at all times, when cutting explants, and maintain surgical instruments and blades sterile. After watching this video, you should have a good understanding of how to set up and use this in vitro model to investigate the cell interactions between human corneal stromal cells and neuronal cells in their natural extracellular matrix. Don't forget that working with BDNF, retinoic acid, and surgical blades can be extremely hazardous, and precautions, such as wearing suitable protective clothing, should always be taken while performing this procedure.
