Microencapsulation

March 20, 2000

How to makeRandy's best Microencapsules

First of all, I should forewarn you that if you want to be successful with the method, you will need to do a lot of work. Depending on your goals, it's probably not sufficient just to run the islets through the electrostatic droplet generator. This will result in only a fraction of the islets being completely covered and you'll induce rejection. Following is the entire method for double coating islets to ensure complete coverage. I give details only for the electrostatic drop generation and "halo" formation here. The method you use for the second atomization is less critical. If you want to coat cells other than islets, I would recommend using a simpler method for imparting the first alginate coating. The electrostatic drop generator is designed to make small droplets without excessive shear which would damage islets. Most cells are more tolerant of shear.

Alginate

First, you will need two different purified alginate formulations. Details for preparing high mannuronate- and high guluronate-alginate are posted on our website. For practice, you can use the appropriate store-bought alginates without purification.

Electrostatic Droplet Generator

You can view the patent (with useful illustrations) at http://www.delphion.com/details?pn=US05639467

To set up the generator, attach a wire (preferably an automobile ignition wire or the like) to the van de Graaff dome. You can use electrician's tape to affix it. Connect the other end of the wire to a 20g 1-1/2" blunt needle. The needle is fitted on a 1cc tuberculin syringe which contains the islets suspended in 0.8% high guluronate rich alginate in saline with 0.5 mM sodium citrate and 10 mM HEPES, pH 7. Concentration of islets should be about 1-5%. The islets may need to be pre-screened to get rid of any clumps or large particles which might clog the 20g needle.

The syringe is mounted vertically in a sturdy plastic jig with the needle pointing down. A second 1 cc syringe is mounted above the islet-containing syringe with its plunger aimed down at the plunger of the islet-containing syringe. The upper syringe is initially fully depressed and the 2 syringes are aligned so that the plungers are in contact. Thus, when the upper syringe plunger is extended, it will press down on the lower syringe, dispensing the islet suspension. To drive the upper syringe, use some non-elastic spaghetti tubing (such as Teflon, polyethylene or polypropylene) to connect the upper syringe to a third syringe mounted in a syringe drive. The syringe in the syringe drive should initially be fully extended and filled with distilled water. The tubing should also be filled with water and there should be absolutely no air bubbles in the syringes or in the tubing, as these will compress and cause the plungers to chatter.

When you turn on the syringe drive, it will slowly depress the syringe which is mounted in it. In turn, the hydraulically connected syringe mounted above the islet-containing syringe will extend and force the plunger of the islet-containing syringe down, dispensing the islets. The slower the flow rate, the smaller the droplets will be. A flow rate of about 200 microliters per minute should produce very small droplets. The process is very sensitive to viscosity and gelling properties of the alginate. If the resultant beads are teardrop shaped, your alginate concentration is probably too low. If they're big or if the stream sputters, the concentration is probably too high.

Beneath the blunt needle through which the islet suspension is being dispensed is a dish containing isotonic (1.70%) calcium chloride dihydrate buffered with 10 mM HEPES. A stainless wire is immersed in the calcium solution and connected to a good ground. The dish is preferably mounted on a platform with adjustable height so that the distance between the needle and the calcium solution can be adjusted. The distance should be something on the order of an inch (this results in a voltage differential of about 8 kV). Adjust the current by changing the van de Graaff belt speed. These settings can be established beforehand using an alginate solution without islets to obtain the finest stream (and thus the smallest droplets) possible.

Ion Exchange

Wash the encapsulted islets with a couple changes of isotonic (9.25%) sucrose, 10 mM HEPES, pH 7 to get rid of free calcium ions. Suspend in a 1:5 dilution of 120 mM barium chloride in buffered isotonic sucrose. Incubate for 10 or 15 minutes with gentle agitation to allow exchange of mannuronic-bound calcium by barium. Wash with several changes of isotonic sucrose to get rid of all the free ions.

Halo Formation

After the last wash, reduce the volume of the suspension to 4 times the pellet volume. Add a volume of 4% high mannuronate alginate in buffered isotonic sucrose with 0.5 mM sodium citrate equal to 5 times the total volume of the suspension. The alginate should be added quickly with constant vigorous stirring to prevent clump formation. A "halo" of soft gel will form around the encapsulated islets as exchanged barium diffuses outward.

Overcoating

Atomize the resulting suspension by, for example, an air knife or a spinning disk droplet generator and capture in buffered isotonic calcium chloride. The "blanks" from this final step should be very small and can be separated from the overcoated islets by velocity sedimentation. A couple methods for atomization can be found on the IBM patent server at http://www.delphion.com/details?pn=US05643594 (spinning disk) and http://www.delphion.com/details?pn=US05521079(air knife). Read the patent http://www.delphion.com/details?pn=US05578314 for more details on ovrcoating.

It's a lot of effort, but it works and it's therefore worth it.

What you will need

  • Electrostatic generator: Any Van de Graff generator with variable belt speed will work. The 30" high generator in Edmund (catalog #Q52-587) has a rheostat to adjust belt speed. You only need about 7,000 volts, which will be determined by the spacing between the dish in which you collect your capsules and the needle through which the islet suspension is extruded (an inch or so is good). Adjusting the belt speed will afford control over the current flow, which will effect the stream acceleration rate and extent of local ionization of air. I would recommend getting an extra belt (Edmund catalog #Q52-806 if you decide on the 30" generator).
  • syringe drive
  • syringes
  • 20g blunt needles
  • Petri dishes
  • electrical wire (an automobile ignition wire is nice but not necessary)
  • some small bore PVC, Teflon or polyethylene tubing
  • some miscellaneous common syringe fittings
  • PVC electric tape
  • There's only one custom component required, which you can easily scrap together. That's a holder that will mount a couple syringes "back-to-back", so that when water is pumped into one, the plunger moves out and presses against the plunger of the second syringe, dispensing its contents. A couple long bolts (6" or so), a couple plates of plastic or metal and a hand drill are all that are needed to build it.
  • The only other thing needed is reagents: calcium chloride, HEPES buffer, NaCl, BaCl2, sucrose and some good alginate.

- Randy