This protocol describes the fabrication of micro gel rods that interlink into micropore scaffolds. These scaffolds can be utilized in combination with cells providing the required space for efficient cell cell interaction. The two different types of micro gel rods interlink upon contact.
At the high aspect ratio leads to larger pores while maintaining the scaffold stability using less synthetic material compared to spherical micro gels. Micropores scaffolds would significantly enhance infiltration and interaction of endogenous cells to repair damaged tissue. The large pores will facilitate blood vessel formation to exchange nutrients to the growing tissue.
The reduction of scaffolded material is beneficial as more open spaces provided for tissue formation, which is important for both in vitro and in vivo applications. A critical aspect is the microfitic on chipulation technique. To ensure continuous production of micro gel rods, the product must be effectively transported out of the tube without clogging.
To begin, insert the needles into the polyethylene tubes and remove the gas from the syringe and the tube. Then insert an additional polyethylene tube into the outlet for product collection. Next, place all the glass syringes and the syringe pumps and insert each tubing end into the corresponding inlet.
Then, focus the microscope on the oil water cross section. Start the first oil syringe pump to fill the channel with oil first, to prevent channel wetting by the dispersed phase. Then, decrease the first oil flow rate to 30 microliters per hour and start the pre polymer syringe pump until the dispersed aqueous phase can be observed at the cross section.
Next, set the pre polymer flow rate to 30 microliters per hour and focus the microscope on the outlet. Then, start the second oil syringe pump and wait until the flow regime is stable. Place the outlet tube in a collection container and set the UV irradiation system such that the irradiance is in the range of 900 to 1000 milli watts per centimeter squared.
And the irradiation spot is the straight channel part before the outlet. Before UV irradiation, adjust the flow rates of the pre polymer and the first oil to achieve the desired aspect ratio in the range of 3.0 to 4.5, and set the irradiation time of the dispersed phase to approximately 2.3 seconds, depending on the size of the irradiation spot. Then start UV irradiation, and if necessary, adjust the flow rates again according to the previous subsection.
Next, change the collection container and note the product collection start time and flow rates. To end collection, remove the collection container noting the time. Afterward, stop irradiation and all the syringe pumps.
Subsequently wash the product five times each with n-Hexane, isopropanol, and deionized water. Then remove the supernatant after rod sedimentation. Transfer the first component dispersion into a conical 1.5 or two milliliter transparent vial.
Then add the second component in a controlled manner in a continuous operation with a 100 microliters pipette and mix the contents directly using the pipette to take up liquid and add it again. Then add G R G D S P C solution to the interlinked structure to modify all remaining epoxy groups with the cell adhesive peptide bearing a free amine and thyal and leave at room temperature overnight. Next, remove the unreacted molecules by washing with deionized water and remove the supernatant.
After reducing the water level, open the vial and de eradiate with UV light of wavelength, 250 to 300 nanometer. Then close the vial and transfer the vial onto a clean bench. Afterward, wash with sterile water.
Replace the water in the vial with cell culture media and allow for equilibration for five minutes. Next, transfer the macro porous scaffold into a cell culture well plate for the experiment by pouring or using a spatula. Confocal microscopy revealed that the 3D macro porous construct was composed of interlinked amine and epoxy functionalized micro gel rods.
The construct exhibits a compact geometry of approximately 10, 000 micro gel rods formed within two or three seconds. Effective Young's modulus of amine and epoxy micro gel rods along with amine and epoxy micro gel spheres is measured by nano indentation. To detect active functional groups, fluorescein iso thiocyanate can be used to visualize primary amino groups and fluorescein amine isomer one can be employed to label epoxy groups.
The amine micro gel rods have dimensions with an average length of 553 micrometers and an average width of 193 micrometers in deionized water, resulting in an aspect ratio of approximately 3.0. The mean values of the macro pores and the scaffolds composed of micro gel rods are 100 micrometers, with 90%of the pore sizes ranging from 30 micrometers to over 150 micrometers. The sphere like micro gels, result in clusters with pore sizes between approximately 10 to 55 micrometers, with a mean value of around 22 micrometers.
The described micro gel rods are designed to produce interlinked micropore scaffolds by random mixing. A controlled mixing procedure could allow for the fabrication of more tailored scaffold geometries in the future. The stiffness of the micro gels and the biochemical cues can be varied based on the sales requirements.
By combining different building blocks a wide variety of geometries and properties can be achieved within one scaffold. This way we can target specific cells efficiently to form multi-cellular tissues.
