This technique is a dependable way to deliver any content such as drugs, proteins, or cells to specific locations in tissues or animals. You can track the delivery and tune the release of the contents to fit the experiment you desire. It can be used to treat numerous diseases from wound healing to ocular and cardiac diseases.
The main advantage of this method is the ability to deliver therapeutic cells using alginate microcapsules to the target sites without causing any immune reactions and maintain a sustained delivery of fresh secreted therapeutics for a longer period of time. Begin by culturing retinal epithelial cells according to manuscript directions. Passage the cells when they reach 70 to 80%confluency using standard tissue culture procedures.
To encapsulate the cells, mix sodium alginate with deionized water for a final concentration of 2%weight to volume and purify the mixture via filtration with a 0.2 micrometer sterile syringe filter. Trypsinize, centrifuge, and wash the cells with 10 millimolar HEPES buffered saline solution. Use a hemocytometer to count the cells and adjust the final cell concentration to one million per one milliliter of alginate solution.
Load 300 microliter aliquots of the alginate and cells mixture into a three milliliter syringe and attach it to a syringe pump. Prepare a gelling bath according to manuscript directions and place it in a sterile 50 milliliter beaker below the syringe tip at a seven millimeter needle to bath spraying distance. Adjust the voltage to eight kilovolts and the flow rate to 30 millimeters per hour to produce microcapsules that are about 150 micrometers in size.
Connect the black ground clip to the copper wire that is halfway submerged in the gelling bath and connect the clip of the red anode wire to the needle. It takes approximately 30 minutes to prepare encapsulated cells out of each one milliliter batch of the alginate and cell mixture. When formed, wash the microcapsules with washing solution twice and incubate them with 10%FBS supplemented DMEM media in a humidified incubator at 37 degrees Celsius and 5%CO2.
After a 24-hour incubation, prepare a 500 microliter sample of microcapsules for staining by washing them twice in washing solution. Use a LIVE/DEAD assay kit to stain them for viability. To ensure that the microcapsules are appropriately sized, dilute them in serum-free media.
Carefully aspirate them with a 2.5 microliter Hamilton syringe. Slowly eject one microliter drops onto a microscope slide and determine the integrity of the microcapsules using an upright Brightfield microscope. In an appropriately anesthetized mouse, apply ocular lubricant to maintain eye moisture and use a 26 gauge 3/8 inch beveled syringe needle to establish an initial eye puncture just below the limbus.
Make sure to approach the eye from a nasal direction at a 45 degree angle keeping the beveled tip upward to avoid lens perforation. Next, ensure that the entire needle tip is inserted into the eye for guide hole generation, but be careful not to travel too far and puncture the temporal retina. Once this is complete, slowly retract the needle.
Within the micromanipulator, place a 27 gauge blunt tip 2.5 microliter Hamilton syringe that's been filled with capsule solution and line the needle up with the initial guide hole. Hold the animal's head in a stable position and deliberately maneuver the needle towards the eye. To ensure a correct needle position within the vitreous, insert the syringe until the needle tip can be visualized behind the lens.
Then inject the capsule solution slowly over the course of 25 to 30 seconds. And once completed, gradually retract the blunt tip syringe in order to minimize injury to the eye. If necessary, adjust the capsule size by adjusting the voltage and flow rate according to manuscript directions.
The encapsulation of the cells in alginate was confirmed with Brightfield imaging and a LIVE/DEAD assay demonstrated that 90%of cells were viable post-encapsulation. Long-term viability of the cells was ensured by dissolving the capsules in sodium citrate and replating. Intravitreal injection was performed under visual inspection which allowed for the visualization of capsules in the vitreous.
When attempting this procedure, do not use PBS for any washing steps because PBS dissolves formed alginate microcapsules. After its development, our lab is used as a delivery system to treat forms of heart failure and hypertension. This technique is considered a promising tool for the future treatment of different cardiovascular and neurological diseases.
