This video describes a method to immobilize mammalian cells, such as pancreatic islets in alginate beads using a simple stirred vessel. The principle of the method is to generate alginate droplets by water and oil emulsion, followed by internal gelation of the alginate droplets. The first step of the procedure consists in generating an emulsion where an aqueous phase containing alginate cells and calcium carbonate is dispersed in a mineral oil organic phase.
The second step is to acidify the emulsion by adding an oil-soluble acid, such as acetic acid, that rapidly partitions into the aqueous phase. The pH drop leads to the dissolution of the calcium carbonate, and to the internal gelation of the alginate droplets into beads. The final step consists in recovering the beads from the emulsion by adding an aqueous solution, separating the phases by centrifugation, followed by washing and filtering the beads.
The immobilized cells can then be cultured in vitro or used for transplantation. The method we described is an alternative to using nozzle-based cell encapsulators to generate alginate beads. This was derived from a method for enzymes and microbial cell immobilization, first reported by Poncelet and others in 1992.
The application that interests us most is alginate encapsulation as a means to immunoisolate transplanted cells, so as to reduce or even eliminate the need for anti-rejection drugs. The main advantages of the emulsion-based process over nozzle-based devices are its scalability and its robustness. Since the droplets are generated almost simultaneously, very large quantities of beads can be produced in very short periods of time, from either very dilute or very concentrated alginate solutions.
Moreover the process is very robust, and is not prone to failure, and even in presence of particles that could obstruct nozzles, and finally the process equipment is fairly simple and so it's accessible, very relatively low-cost to most laboratories. The key processing steps are emulsification to form the alginate droplets, and acidification to release the internal calcium source. The droplet size is influenced primarily by impeller design, agitation rate and agitation duration.
The mechanical properties of the beads and the cell survival are influenced by the extent and duration of acidification. In this video we demonstrate a method used to encapsulate cells in 5%alginate beads for transplantation. These parameters would likely require optimization for other potential applications.
To generate 5%alginate beads containing a half and half mixture of LVM and MVG alginates, weigh out 583 milligram LVM and 583 milligram MVG alginic acid powder. Place 20 milliliter process buffer on a magnetic stir plate. For transplantation applications we use a 10 millimolar HEPES buffer containing 170 millimolar sodium chloride at pH 7.4.
Progressively add the alginic acid powder to the solution. Leave the solution stirring overnight at a low speed. If necessary, fasten the flask to the stir plate.
The next day, if the alginate is incompletely dissolved, fasten the jar onto a rotary mixer and continue mixing overnight at 37 degrees Celsius. Once the alginate is completely dissolved, sterilize the solution by autoclaving for 30 minutes. Allow the temperature to fall below 50 degrees Celsius before opening the autoclave.
Prepare the calcium carbonate suspension by adding one gram of calcium carbonate to 20 milliliters process buffer. Autoclave the calcium carbonate suspension and the stirred vessel used for the emulsion process. Prior to use, remove any traces of condensed water from the vessel.
Immediately before the emulsion process, dissolve 44 microliter glacial acetic acid in 11 milliliter mineral oil placed in a 50 milliliter conical tube. A common mistake is incomplete dissolution of the acetic acid, avoid pipetting amounts smaller than 10 microliter, and ensure that the acid is completely dissolved by repeated vortexing. Allow all the solutions to reach room temperature before proceeding to cell encapsulation.
Place 10 milliliter mineral oil in the spinner flask, and start stirring at 250 RPM. If adherent cells such as beta-tc3 cells are used, trypsinize the cells. End the reaction by adding complete medium and take a sample for cell enumeration.
Determine the cell concentration manually or with an automated cell counter. Centrifuge the cells for seven minute at 300 g and then resuspend the cell pallet in complete medium. Repeat the centrifugation step and then resuspend the cells in the appropriate volume of complete medium to obtain 10 and 1/2 fold the desired final concentration in the beads.
Transfer 9.9 milliliter alginate solution into a flat-bottom tube, then add 1.1 milliliter of the cell stock, and 550 microliter of the calcium carbonate suspension. Mix the alginate, calcium carbonate and cell suspension by mild vortexing. Immediately transfer 10.5 milliliter of this mixture into the agitating oil using a syringe.
Increase the agitation rate and then start the timer. To determine the agitation rate, a standard curve relating the bead size to the agitation rate should first be generated. After 12 minutes add 10 milliliter of the oil and acetic acid solution to acidify the emulsion, release the calcium from the carbonate, and obtain gelled beads.
Allow eight minutes for this internal gelation step. Notice the color change of the emulsified droplets which contain the phenol red pH indicator. Reduce the agitation rate to 400 RPM, neutralize the acid by adding 40 milliliters of process buffer mixed with 10%medium, which leads to phase inversion.
Stop the agitation one minute later and transfer the mixture to conical tubes. Rinse the spinner flask with an additional 20 milliliter of medium, and add this to the tubes. Aspirate as much of the aqueous solution as possible before aspirating the oil phase.
Centrifuge the tubes for three minute at 630 g to accelerate bead settling and phase separation. Remove the oil and excess process buffer by aspirating with a Pasteur pipette. Wash the beads at least once with medium using 630 g centrifugation between washes.
Filter the bead suspension on 40-micron nylon cell strainers and aspirate excess liquid in the strainer from below. Transfer the beads into a known volume of medium using a spatula. Measure the bead volume and top up the medium to obtain the desired bead concentration.
A typical concentration is one milliliter beads per five milliliter total volume. From this point on, always handle the beads with large-bore pipettes to avoid damaging the beads. The encapsulated cells can now be transferred into T flasks, and used for in vitro culture or transplantation.
At the end of the emulsion process, alginate beads containing immobilized cells should be obtained. After the process, the bead size distribution and the cell survival should be routinely assessed. To determine the bead size distribution, the beads can be stained with toluidine blue, followed by image analysis.
A broad bead size distribution is expected from this process. To assess cell survival the beads can be incubated with live dead stains, such as calcein-AM and ethidium homodimer. Using the process described in this video, 76%beta-tc3 cell survival was measured.
After watching this video you should have a good understanding of how to immobilize mammalian cells in alginate beads using a simple stirred system. The basic protocol shown in this video should be suitable to immobilize a variety of cell types using a broad range of alginate types and calcitrations. We recommend generating a standard curve relating the average bead size to the agitation rate with every new lot of either alginate or oil.
The emulsification process that we've described is a promising alternative to nozzle-based cell encapsulators. It's a robust and simple method to immobilize mammalian cells in alginate beads. As we and others around the world develop nozzle cell therapies, such scale of methods will be needed for the many thousands of diabetic patients.
We have published promising results from transplanting a beta cell line in 5%alginate beads, and continue to explore the in vivo performance of this improved barrier to transplant rejection.
