My Name is Goldie Ana, and I'm an undergraduate student working in Dr.Eric Bras lab at the Illinois Institute of Technology. In this video presentation, I'll be describing techniques for the use of algin microbeads in biomedical engineering, specifically in the field of cell and tissue engineering. All the work described here has been done in collaboration with Dr.Emmanuel OPA lab at the Wake Forest Institute for regenerative medicine.
I will present techniques for fabricating gin microbeads for use in both drug delivery and cell encapsulation. Finally, I'm gonna demonstrate an novel procedure that we've developed in our lab for generating multi-layered algebra microcaps that can be used for the dual purpose of cell and protein. Encapsulation Alginate is composed of beta D onic acid and alpha luronic acid residues in varying sequences.
Alginates solution cross links to form a gel in the presence of a multivalent cation, such as calcium two plus, which is most commonly used. Researchers have investigated the use of algen microbeads for cell and protein encapsulation. The properties of algen hydrogels can be varied based on their synthesis conditions with inherently different properties, which, for example, can be used to tune release kinetics.
Alginate microbeads have been used for many wide ranging applications, including protein encapsulation, drug delivery, cell encapsulation, and as an ingredient in food science such as molecular gastronomy. We begin by making up an inner layer alginate solution. Add 25 millimolar of heis 118 millimolar of sodium chloride, 5.6 millimolar of potassium chloride, and 2.5 millimolar of magnesium chloride to deionized water shake thoroughly to ensure All reagents have dissolved.
Adjust the pH to 7.4, which is consistent with physiological conditions. Measure out all powder to yield the desired concentration and transfer into the prepared inter allergen Solution. Place the Solution on a vortex machine until all the powder has dissolved, resulting in a clear viscous solution.
Prepare the cross-linking solution by adding 10 millimolar of heis buffer and 100 millimolar of calcium chloride into deionized water. Thoroughly shake the solution and adjust its pH to 7.4. Add 10 milliliters of cross-linking solution to a beaker.
Transfer the alginate into a syringe fitted with a needle of the desired gauge. Position the needle perpendicular to the crosslinking solution and slowly eject droplets into the crosslinking solution, allowing the formation of spherical beads In order to fabricate a large Volume of microbeads consistent in size. A two channel error micro encapsulation can be utilized.
Load the algenate solution into the syringe and turn on the air jacket valve, followed by the alginate jacket valve, allowing droplets to be ejected into a flask containing cross-linking Solution. Allow the beads to incubate In the solution for 15 minutes. The microbeads are to be washed three times with a solution of 22 millimolar.
Calcium Chloride and normal saline algen Microcaps have been researched extensively for the encapsulation of cells. For example, one proposed method of treating type one diabetes involves transplanting donor eyelids into a patient in order to prevent rejection of the autologous cells. However, patients must take immunosuppressive drugs that compromise their immune system.
Encapsulating alis within a bin. Material such as alginic may obviate the need for immunosuppressive drugs. A polyol ornithine or PLO coating is added around the alginic microbe would serve as a semipermeable membrane.
A thin monolayer of alginate is added around the PLO coating that helps limit the inflammatory response. The positively charged PLOI ironically interacts with the alginate resulting in a coating around the bead that serves as a perm selective membrane. A monolayer of alginate is then created around the PLO coating in order to mask its positive charge and limit the inflammatory response.
Post transplantation, create a 0.1%solution of poly l ornithine by dissolving PLO in normal saline. Add the PLO solution to the alginate microbeads place on a vortex for 30 minutes to allow the algen microbeads sufficient time to interact with the PLO solution. Remove the surrounding liquid and perform three washes for two minutes each with 22 millimolar calcium chloride in normal saline Solution.
Add a solution of alginate to the PLO coated microbeads and place on a vortex for five minutes. Perform Three washes for two minutes each, using a 0.9 normal saline solution to remove any unbound alginic. After completing the washes, you can store the beads by placing them in a calcium chloride solution.
One of the limitations of cell Is that many cells die due to a lack of a patent blood supply that delivers oxygen, glucose, and other vital nutrients to the cells upon transplantation angiogenesis, the growth of blood vessels from preexisting blood vessels can be stimulated by delivering therapeutic angiogenic proteins such as fibroblasts growth factor one or FGF one, or vascular endothelial growth factor or vegf. Our lab has previously shown that a consistent low level of release of FGF one promotes stable vascularization compared to a high bolus dose. We have developed a novel procedure to create multi-layered algen microbeads, the sizable outer alginic layer instead of simply a thin mono layer that is traditionally used.
Our drug delivery system serves the dual purpose of cell and protein encapsulation, where, for example, s can be encapsulated in the inner core and angiogenic proteins such as FGF one in the outer alginic region. The release of therapeutic proteins would cost the formation of new blood vessels towards the transplant sites, thereby increasing the viability of the island cells. Multi-layered allergen at microbeads serve the dual purpose of cell and protein encapsulation.
A sizable outer allergen region instead of a mono monolayer that is conventionally used is created around the PLO coating and can be used as a region for protein encapsulation. The release of therapeutic proteins from the outer alginic region would serve to cause the sprouting of vessels towards the transplant site. This would, in theory, provide a patent blood supply that distributes vital nutrients to the encapsulated cells, thereby increasing the cell's viability.
The procedure to make multi-layered algenate microbeads is similar to the one previously discussed. Algenate microbeads containing cells are synthesized, followed by the construction of A PLO coating. However, in the final step, the outer layer is created by cross-linking the alginate in a calcium chloride solution instead of with Saline.
To create Multi-layered ginnet microbeads begin by transferring the PLO coated microbeads contained in solution to a cell strainer watt away excess liquid, transfer the beads onto a flat surface such as perfil Or a Petri dish, transfer the outer allergen solution on top of the microbeads and allow it to incubate for 45 minutes. Then remove excess outer allergen solution using a pipette, transfer the microbeads into a solution of calcium chloride, thereby cross-linking the alginate to form the outer layer. Perform three washes for two minutes each.
Using a two millimolar calcium chloride and saline solution panel. A shows The surface of an ginnet microbead, which is contrasted with panel B, where a sizable outer allergen layer can be seen. Panel C shows an image of a multilayered algenate microbead with fluorescently labeled protein encapsulated in the outer layer.
The size of the outer algenate layer can be varied based on the composition and concentration of algenate used it used the release profile of FGF one from multilayered alginate. Microbeads can be varied based on the conditions used to synthesize the outer layer. All conditions provide a burst release within the first four hours, but continuing to release FGF one for up to 30 days.
The long-term release of FGF one exhibited has a potential to stimulate persistent vascularization that would serve to increase the viability of the encapsulated cells. That brings us to The end of our presentation. We hope that you find it to be informative.You.
