Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

The overall goal of the following experiment is to introduce 3D spatial complexity within tissue engineering scaffolds by combining the use of additive manufacturing techniques and electro spinning. This is achieved by creating a non-degradable template made of polyethylene glycol, D acrl, or PEG DA, using a layer by layer photo curing technique called micro stereolithography. As a second step.

The templates are attached to a metallic substrate and a electro spinning is used to produce a biodegradable membrane that mimics the underlying morphology. Next, packaging procedures and degradation of the electros spun membranes are studied. Premature degradation of the stored membranes is avoided by controlling temperature and moisture.

The final step consists of placing the biodegradable membranes on a 3D wounded cornea model for studying their potential use as corneal regeneration devices. Results show the versatility of using additive manufacturing techniques and electro spinning for creating complex membranes containing micro pockets that mimic to a certain extent the natural stem cell niche. The main advantage of this technique is its versatility.

Since the non biodegradable molds can be created new, using a wide range of manufacturing techniques, This method would be of interest in both manufacturing and tissue regeneration fields, and it would help answer key questions related to stem cell behavior and the stem cell niche. This method of making micro fabricated scaffolds can provide insight into corneal regeneration. It can also be applied to other epithelial tissues with stem cell niches such as skin and oral mucosa, or to provide other 3D features and scaffolds for engineering of a wide range of tissues.

Both Leader Ortega and Thomas Patterson will be demonstrating the procedure. Begin this procedure by creating L one, the first layer. That will become the base of the structure using any suitable drawing program for a 1.2 centimeter black circle.

Next, create L two. The second layer, draw another 1.2 centimeter black circle, but include six to eight white 0.5 by 0.35 millimeter horseshoe shaped structures. Distribute the small white horseshoe shapes within the black circle structure.

Save L one and L two in JPEG format to set up for micro stereolithography. First, adjust and carefully clean the optics of the system. Put 300 microliters of polyethylene glycol di acrl, or PEG D mixture into a well of a 12 Well tissue culture plate.

The well should be pre-coated with Teflon or another non-stick material or easy removal of the structure. After curing switch on the blue laser and upload L one into the ALP three basic software. This software is a USP interface that provides the link between the PC and the digital micro mirror device.

Irradiate the first layer for 60 seconds. Add to the well 250 microliters more of the peg da. Upload L two and irradiate L two for 60 seconds.

Remove the uncured polymer and wash the rings with isopropanol overnight On the day before the electro spinning, create a static electro spinning collector by distributing the PEG DA rings on an electroplated 12 centimeter by 20 centimeter aluminum sheet. Attach the rings using conductive carbon tape. Prepare the poly lactide co glyco or PLGA solution and stir it overnight prior to use on the following day.

Place four five milliliter syringes with blunt ended 0.8 centimeter inner diameter needles on a syringe pump. Four syringes are used instead of a single syringe. To ensure more rapid electro spinning load 2.5 milliliters of the PLGA solution into each syringe.

For the success of this procedure, it is important to try and keep the laro pinine variables as constant as possible. Leave a distance between the needles and the collector of 15 centimeters. Set a flow rate of 30 microliters per minute and voltages ranging from 12 to 15 kilovolts for the electro spinning electro spin for one hour and 30 minutes.

When the electro spinning is done, carefully peel the PLGA electro sponge sheet from the collector supporting the PEG da rings. The PEG DA rings can be reused as templates for electro spinning. Use a circular hole punch to cut the electro spon scaffolds into 22 millimeter diameter circles.

Leaving the ring structure positioned in the center to prepare the PLGA membranes for long-term storage. Mount each in a small container such as a plastic petri dish and place it inside a medical grade bag. Place three bags of desiccant inside the medical grade bag.

Add to the bag a commercially available six spot humidity indicator card. This will detect any moisture accumulation during the storage period. Use a vacuum heat seal machine to vacuum and seal the bag.

Subsequently, the PLGA membranes are sent to an external company, the gamma or radiation prior to storage. In this experiment, rabbit limbal eggplants are isolated from rabbit eyes obtained from a farm where rabbits are bred for consumption. First disinfect the rabbit eyes using a 3%antiseptic solution.

Next, clean the eyes by removing any excess tissue surrounding the cornea. The limbal region can be identified as a thin circular area between the transparent cornea and the white sclera. Separate the limbal region from the rest of the cornea.

Cut the limbal region into segments of about 1.5 centimeters in length, disinfect the limbal segments in 1.5%antiseptic solution for one minute. After disinfection, use a scalpel blade to cut the limbal segments into small pieces. Store the small pieces of tissue in DMEM plus glut max media at 37 degrees Celsius and 5%carbon dioxide or maximum of one hour before use.

To begin this procedure, use a cell scraper to coat the ring membranes with 15 microliters of fibrillin glue. Distribute the fibrillin evenly while working under a dissecting microscope. Use a 25 gauge needle to place the limbal explants directly on the PLGA micro pockets.

Place the rings with the tissue explan on previously prepared denuded corneas. With the explan facing up and at air liquid interface conditions, the PLGA ring will quickly wet and adopt the shape of the cornea model. Maintain the organ culture models for four weeks in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide change the media every two days.

Micro stereolithography enables the fabrication of PEG DA rings of different sizes and the simultaneous incorporation of micro features. Rings can be reproducibly made and reused and are easily attached to a metal sheet that can be used as an electro spinning collector. In this example, the rings contain horseshoe micro features.

Electro spinning then produces PLGA replicas vacuum packing of the PLGA membranes significantly improves their long-term storage even at 37 degrees Celsius in deliberately moist conditions. The type of bag used also impacts membrane stability, scanning electron microscopy and examination of the three descants show no changes in fiber integrity or humidity of the intact fibers. Cell outgrowth from limbal implants or supported by both freshly made biodegradable PLGA rings and rings after six months of storage at minus 20 degrees Celsius.

When placing the PLGA membranes on 3D wounded cornea models, cell transfer was achieved after four weeks. H and e images show the new multi-layered epithelium formed by the cells coming out from the explants placed on the niches. The negative control confirmed the lack of formation of a new epithelium in the absence of any added cells.

In comparison to a conventional corneal epithelium observed in a fresh rabbit cornea used as a positive control. Immuno cyto chemistry demonstrated that the cells growing out from the implants were corneal epithelial cells since they were positive for cytokeratin three, a corneal differentiation marker. Since the electro spinning collectors containing the non biodegradable templates are reusable, the production of the scaffolds can be done in two hours when the protocol is optimized.

This technique allows researchers in the fields of tissue engineering to explore a new way of manufacturing complex tissue engineering membranes containing micro features in which to study cell behavior. After watching this video, you should have a good understanding of how to create a non-degradable micro fabricated template by using additive manufacturing techniques or similar approaches such as modeling and how to use electro spinning for adding complexity to your scaffolds.