This protocol aims to guide the fabrication of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a micron-sized hydrogel micro-column for use as an in vitro neuro-biological model or as a neuro-regenerative therapy. Trauma and neurodegenerative disease are particularly detrimental to patients due to the restricted neurogenic capacity in inhibitory environment that limit regeneration in the central nervous system. Engineered astrocytic bundles address the limitations of current treatment strategies by attempting to replicate key neuroanatomical features and glia-mediated developmental mechanisms to promoted targeted cell migration and axon pathfinding.
The architecture of these astrocytic scaffolds is primarily inspired by the aligned astrocytic processes in the glial tubes of the rostral migratory stream. Begin by preparing a 3%weight per volume agarose solution and DPBS in a sterile beaker. Maintain the heating and stirring to prevent the solution from solidifying prematurely.
Insert an acupuncture needle into the bottom opening of the bulb dispenser. Place a capillary tube over the exposed needle and secure the two by entering part of it into the rubber section of the bulb dispenser cylinder. Cap the cylinder to complete the assembly.
Transfer one milliliter of liquid agarose to the surface of an empty petri dish. While pinching the rubber cap of the bulb dispenser, insert one end of the capillary tube into the agarose, and release the pressure on the cap to draw agarose into the tube. Place the bulb, tube, needle assemblies on a petri dish and let the agarose solidify inside the capillary tubes for 5 minutes.
Then, pull the needle out of the capillary tube, leaving the micro-column still surrounding the needle. Use the tips of foreceps to nudge the micro-column to the end of the needle, then hold the needle over an open DPBS-containing petri dish and push the micro-column into the DPBS. To fabricate micro-column boats, which facilitate later handling of these constructs, begin by making a 4%weight per volume agarose solution, and keeping the solution heated and stirred.
Use forceps to move a micro-column to an empty petri dish. Then, while working under a stereoscope, use a micro scalpel to trim the micro-columns to the desired length, with the ends cut at a 45 degree angle. After trimming three more micro-columns, line up the four columns in parallel.
Load a pipet with 50 microliters of 4%agarose solution, and dispense a line of liquid over the micro-column array to connect and bundle the constructs into a boat. Allow to cool for 1 to 2 minutes to permit gelation of the 4%agarose. Then use fine forceps to pick up the micro-column boat by the connecting agarose bridge.
And move it to a petri dish containing DPBS. Transfer the micro-column boat to a biosafety cabinet and sterilize with exposure to ultraviolet light for thirty minutes. Inside a biosafety cabinet, prepare a 1 milligram per milliliter solution of rat tail type 1 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 micro-column boat to a 35 or 60 mL petri dish with forceps.
Using the stereoscope for guidance, situate the 10 mL tip of a micro-pipette at one end of each micro-column and suction to empty the lumen of DPBS and air bubbles. Charge a P10 micropipette with the collagen solution. Place the 10 mL tip against one end of each micro-column, and deliver enough solution to fill the lumen.
Then pipette a reservoir of ECM on either end of the micro-column. Once all columns have been filled, pipette coculture medium in a ring around the petri dish to prevent the columns from drying out. Incubate the dish containing the micro-columns 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 micro-column and transfer approximately 5 mL of cell solution into the lumen to fill it. After seating all of the micro-columns in a boat, incubate it at 37 degrees Celsius and 5%carbon dioxide for 1 hour to promote the attachment of astrocytes to the ECM. After the incubation period, carefully fill the dishes containing the seated micro-columns with 3 or 6 mLs of coculture media, for 35 or 60 mL petri dishes respectively.
Maintain the plated micro-columns and culture at 37 degrees Celsius and 5%carbon dioxide to promote the self assembly of the aligned of the astrocytic bundles, which should form a bundled, cable-like structure after six to ten hours. One hour after plating, astrocytes are spherical in shape and disperse throughout the construct. By five hours after plating, astrocytes initiate process extension and contraction.
At eight hours after plating, astrocytes have aligned and contracted. By 24 hours after plating, a dense bundle of astrocytic processes has formed in this example. This fully-formed astrocytic bundle, 24 hours after plating, shows the zippering effect, where the ends of the cable attach to distinct ends of the wall of the lumen.
This video shows bundling in action, 3 to 10 hours after plating. Immuno-labeling of the astrocytic scaffolds demonstrates the longitudinal alignment of astrocytic processes. In this confocal image of an astrocytic bundle, the astrocytic marker GFAP is read.
And the nuclei are seen in blue. This higher magnification of the bundle permits visualization of the cyto-architecture of the astrocytes within the micro-columns. After watching this video, you should be able to fabricate living scaffolds consisting of longitudinally aligned astrocytic bundles with a biologically inspired cytoarchitecture within a biomaterial encasement.
Following this procedure, a transplantation strategy can be designed to deliver the astrocytic bundles with or without the biomaterial encasement to injured brain tissue to promote directed cell migration from neurogenic regions and acts on guidance to the limiting CNS microenvironment. In addition, this technique can be expanded as biofidelic modeling platform glia-mediated neurobiological phenomenon benefited by the mimicry of the native three dimensional environment and the inherent experimental control of in vitro systems.
