An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns
Transcript
Inside a biosafety cabinet, prepare a 1 milligram per milliliter solution of rat tail type I collagen in co-culture medium and then place it on ice. Adjust the pH of the ECM solution with acid or base as necessary, until the pH is stable in the 7.2 to 7.4 range. Transfer the microcolumn boat to a 35 or 60-milliliter Petri dish with forceps. Using the stereoscope for guidance, situate the 10-microliter tip of a micropipette at one end of each microcolumn and suction to empty the lumen of DPBS and air bubbles.
Charge a P10 micropipette with the collagen solution. Place the 10-microliter tip against one end of each microcolumn, and deliver enough solution to fill the lumen, then pipette a reservoir of ECM on either end of the microcolumn. Once all columns have been filled, pipette co-culture medium in a ring around the Petri dish to prevent the columns from drying out.
Incubate the dish containing the microcolumns at 37 degrees Celsius and 5% carbon dioxide for at least one hour to promote polymerization of collagen before adding cells. Afterwards, place the tip of a P10 micropipette at one end of a microcolumn, and transfer approximately 5 microliters of cell solution into the lumen to fill it. After seeding all the microcolumns in a boat, incubate it at 37 degrees Celsius and 5% carbon dioxide for one hour to promote the attachment of astrocytes to the ECM.
After the incubation period, carefully fill the dishes containing the seeded microcolumns with 3 or 6 milliliters of co-culture media for 35 or 60-millimeter Petri dishes, respectively. Maintain the plated microcolumns in culture at 37 degrees Celsius and 5% carbon dioxide to promote the self-assembly of the aligned astrocytic bundles, which should form a bundled cable-like structure after 6 to 10 hours.
