Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

The overall goal of this procedure is to demonstrate the engineering process of 3D brain-like tissues. This is achieved by first preparing the porous scaffolds from silk solution using a salt leaching technique. The second step is to precut the scaffolds into donut shapes and prepare them for cell culture by sterilization and coating with poly de lycine or PDL.

Next, the prepared scaffolds are seeded with freshly isolated primary rat cortical neurons. The final step is collagen embedding of the scaffolds and transfer to the culture plates. Ultimately, immunofluorescence microscopy is used to show the neural growth and development of neural networks.

The implications of this technique extend toward therapy or diagnosis of brain affecting diseases because this technique can be used as a basis for developing new research models of Parkinson or Alzheimer. Before beginning, prepare the 6%silk solution as described in the text protocol and store it at four degrees Celsius. To prepare porous scaffolds, first sieve, the granular sodium chloride and collect granules between 500 and 600 micrometers in diameter.

Pour 30 milliliters of the silk solution into the polytetrafluoroethylene or PTFE mold, and then scatter 60 grams of sift sodium chloride evenly over. The solution. Incubate for 48 hours at room temperature to polymerize the silk.

Place the scaffold containing mold in an oven set at 60 degrees Celsius for one hour to finalize the cross-linking of silk and to evaporate any remaining liquid. Next, immerse the mold and scaffold combination in a two liter beaker of distilled water for 48 hours. To leach out the salt, change the water two to three times per day.

Remove the scaffold from the mold and cut it into small pieces. First with a five millimeter diameter biopsy punch. Then cut the scaffold to be two millimeters in height and remove the center from each piece with a two millimeter biopsy punch.

Autoclave the scaffolds in a wet cycle for 20 minutes. Inside a cell culture hood, place a pair of sterilized forceps, cell culture media, and the autoclave scaffolds. Remove a 96 well plate from its wrapping and place inside to seed the scaffolds.

First place one sterile scaffold per well. In the 96 well plate add cell culture medium to immerse the scaffolds and incubate at 37 degrees Celsius in a tissue culture incubator to equilibrate them for at least 30 minutes. Following incubation, aspirate the excess medium from the scaffolds.

Then add two times 10 to the six rat cortical neuronal cells in 100 microliters of neuro basal medium per scaffold. Return the plate to the incubator and leave overnight to allow the cells to attach to the scaffold. On the following morning.

Carefully aspirate the non-attached cells and replace with 200 microliters of fresh cell culture.Medium. Incubate a 37 degree Celsius for a brief period. Next, aspirate the medium from the wells using sterile forceps.

Transfer the scaffolds containing cells to empty wells of the 96. Well plate then to each scaffold at 100 microliters of freshly diluted ice cold, three milligrams per milliliter rat tail collagen solution. Return the cells to the 37 degree Celsius incubator to allow polymerization of collagen for 30 minutes.

Remove the plate from the incubator and add 100 microliters of prewarm cell culture. Medium per well return. Turn the plate to the incubator for the desired period of cell growth, replacing half of the medium from each well with fresh medium every day for up to one week.

In these highly porous scaffolds, neuronal cells can be cultured to a high density shown. Here are the cell viability results. One day after seeding fluorescent di acetate labeled live cells appear in green and propidium iodide and silk scaffold.

In red, note the high percentage of live cells seen under low magnification. This figure shows com compartmentalization of neuronal cell bodies and axonal processes within the donut shaped scaffold. Neuronal cell bodies remain attached to the silk scaffold as seen here one day after seeding cells are immuno stained for beta three tubulin and appear green.

As expected, the cells have densely populated the scaffold by day seven of culture. In contrast, the collagen matrix in the central hollow of the scaffold provide support for extensive axonal growth. Shown here is the axonal meshwork.

Seven days after seeding. This movie shows a confocal Z scan of the central collagen window of the scaffold containing the neural network cells were labeled by staining for beta three tubulin. After watching this video, you should have good understanding of how to set up the 3D brain-like tissue constructs.