The overall goal of this procedure is to create a composite matrix made from natural biomaterials that can be used to deliver and differentiate stem cells to assist in wound healing. This is accomplished by first isolating adipose derived stem cells or a SC from a test animal. Next, the A SC are loaded onto pre-made chitosan microspheres.
Then FIBRILLATED collagen gel is cast, and the A-S-C-C-S-M beads are layered over the collagen gel. Finally, PEG fibrin gel is cast over the A-S-C-C-S-M collagen gel, and medium is added. Ultimately, results can be obtained that show the bidirectional simultaneous migration of A SC from the CSM bead into both collagen and PEG fibrin matrices.
Through this procedure, one single stem cell source can take differential cues from the collagen and PEG fibrin microenvironments, and become differentially stimulated to produce pleiotropic factors and form a network of pre vascular structures. The main advantage of this technique over existing methods like single material composites or decellularized skin substitutes, is that these other products do not actively address the revascularization of the product by the host. We first had the idea for this method when we noticed that isolated RAD A SE has the propensity to differentiate towards a vascular phenotype when cultured in a pig fibrin matrix.
This observation sparked the notion that a single stem cell source could be utilized simultaneously in a system that consisted of different biomaterials else. After isolating and culturing adipose derived stem cells or ASCs, preparing chitosan microspheres or CSMs and loading the ASCs into the CSMs, prepare polyethylene glycol, fibrin hydrogel by dissolving succinyl glut derate modified peg in four milliliters of tris buffered saline. Just before the experiment, use a 0.22 micron filter to sterilize the solution.
In a culture well of a six well plate mix, 500 microliters of fibrinogen stock and 250 microliters of PEG stock incubate for 20 minutes in a 5%carbon dioxide humidified incubator at 37 degrees Celsius. Next, mixed 250 microliters of previously prepared A-S-C-C-S-M with the pegylated fibrinogen solution, immediately add one milliliter of thrombin stock solution and using a pipette quickly tri rate once or twice immediately place the cell gel mixture into a 12 well plate and incubate in a 5%carbon dioxide humidified chamber at 37 degrees Celsius for 10 minutes. Next, wash the resulting peg fibrin twice with HBSS and incubate with alpha minimal essential medium supplemented with 10%FBS in a 5%carbon dioxide humidified incubator at 37 degrees Celsius using standard light microscopy techniques over an 11 day period.
Observe the migration of cells from the CSM into the gel mix, A-S-C-C-S-M with type one collagen extracted from rat tail tendons using two normal sodium hydroxide. Adjust the pH to 6.8 and fibrillate. Add the fibrillated collagen, A-S-C-C-S-M mixture to a 12 Well plate and incubate in a 5%carbon dioxide humidified incubator at 37 degrees Celsius for 30 minutes.
After checking that the fibrillation is complete, incubate the collagen A-S-C-C-S-M gels for up to 11 days in a 5%carbon dioxide humidified incubator at 37 degrees Celsius. Observe the migration of cells from CSM into the gel over an 11 day period using standard microscopy techniques to develop the bilayer construct to study the migratory and co induction properties of a single source of stem cells. Using two bio scaffolds.
Prepare both the collagen and the PEG fibrin gels with the following slight modifications, prepare one milliliter of 7.5 milligrams per milliliter of collagen. Then transfer the mixture to a six well tissue culture insert and incubate for 30 minutes at 37 degrees Celsius. After complete fibrillation, lay over the collagen surface, 200 microliters of five milligrams of A-S-C-C-S-M in culture medium.
After the microspheres have settled over the gel, prepare the PEG fibrin gel using 250 microliters of cell culture medium. Then layer the pegylated fibrinogen thrombin solution over the A-S-C-C-S-M collagen layers. Once completed, incubate the constructs for 30 minutes in a 5%carbon dioxide humidified incubator to achieve complete ation.
Finally, place one milliliter of medium in the upper chamber over the construct and three milliliters of medium in the lower chamber. Characterization of a SC embedded within this scaffold revealed that a SC loaded CSMs can be sandwiched in between a layer of collagen and peg fibrin simultaneously and differentially take cues from both extracellular environments to thrive under their new conditions. As shown here, collagen supported the ability of ASCs to remain stem cells as was demonstrated by their expression of stro one in green, as well as their fibroblast like morphology.
In contrast, PEG fibrin induced the ASCs to differentiate toward avascular phenotype as is demonstrated by their tube-like morphology shown here, complete with lumen and their endothelial cell specific expression of von Willow brandand factor shown here in red. In addition, as demonstrated here, these observed phenotypes appeared to occur early in culture and were maintained over 11 days. After watching this video, you should have a good understanding of how to isolate rat adipose-derived stem cells, load them onto chito and microspheres, and incorporate them into a bilayer hydrogel using FDA approved biomaterials.
Don't forget that the biomaterials and cells that are utilized in this protocol are derived from animals and humans and can be extremely hazardous if they're contaminated with pathogens from their host. And precautions such as personal protection equipment and aseptic cell culture techniques should always be taken while performing these procedures.
