Name: mammalian cell encapsulation in alginate beads using a simple stirred vessel
Uploaded: 2017-06-29T13:00:00
Description: Watch this Scientific Journal Video about Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel at JoVE.com

We thank Jill Osborne for her ground-laying work on the emulsification process and Lauren Wilkinson for technical support. We thank Dr. Igor La&#231;ik, Dr. Timothy J. Kieffer and Dr. James D. Johnson for their input and collaboration. We thank Diab&#232;te Qu&#233;bec, JDRF, Th&#233;Cell, the Centre qu&#233;b&#233;cois sur les mat&#233;riaux fonctionnels (CQMF), the Natural Sciences and Engineering Research Council (NSERC), the Centre for Human Islet Transplantation and Beta-cell Regeneration, the Canadian Stem Cell Network, the Michael Smith Foundation for Health Research, le Fonds qu&#233;b&#233;cois de la recherche sur la nature et les technologies and COST 865 for financial support.

1. Prepare the Alginate Solution, the CaCO3 Suspension and the Acidified Oil
<ol>
	<li>Prepare the process buffer and medium.
	<ol>
		<li>Prepare the typical process buffer used to generate high-concentration alginate beads using 10 mM HEPES, 170 mM NaCl. Adjust the pH to 7.4.</li>
		<li>Prepare the typical culture medium using Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 6 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin.</li>
	</ol>
	</l...

Various steps (depicted in Figure 2) during the internal gelation reaction can limit the overall kinetics. For calcium carbonate grains larger than ~2.5 &#181;m, the rate of carbonate dissolution has been shown to be rate-limiting26,44. The acidification step that leads to internal calcium release has also been shown to be the critical process variable affecting cell survival32. The conditions that lead to internal gelatio...

Mammalian cell encapsulation has been broadly studied as a means to protect transplanted cells from immune rejection1 or to provide a three-dimensional support for immobilized cell culture2,3,4. Pancreatic islet encapsulation in alginate beads has been used to reverse diabetes in allogeneic5,6 or xenogeneic7,8,9,10,11,12 rodents. Preclinical and clinical trials of encapsulated pancreatic islet transplantation to treat type 1 diabetes are ongoing13,14,15. For transplantation applications or larger-scale in vitro immobilized cell production, nozzle-based bead generators are generally used. Typically, a mixture of alginate and cells is pumped through a nozzle to form droplets that fall into an agitated solution containing divalent cations, resulting in the external gelation of the droplets. Coaxial gas flow16,17, nozzle vibration18, electrostatic repulsion19 or rotating wires20 facilitate droplet formation at the nozzle tip.
The main drawbacks of conventional bead generators are their limited throughput and the limited range of solution viscosities that will result in adequate bead formation21. At high flow rates, the fluid exiting the nozzle breaks up into droplets smaller than the nozzle diameter, decreasing size control. Multi-nozzle bead generators can be used to increase the throughput, but the uniform distribution of flow among the nozzles and the use of solutions &#62;0.2 Pas is problematic22. Lastly, all of the nozzle-based devices are expected to impart some damage to islets, since the diameter of the nozzles used is between 100 &#181;m and 500 &#181;m, while ~15% of human islets can be larger than 200 &#181;m23.
In this video, we describe an alternative method to encapsulate mammalian cells by forming droplets in a single emulsification step instead of drop-by-drop. Since bead production is performed in a simple stirred vessel, the method is suitable for small (~1 mL) to large-scale (103 L range) bead production with low equipment costs24. This method allows the production of beads with high sphericity using a broad range of alginate viscosities with short (e.g. 20 min) bead generation times. This method was originally developed by Poncelet et al.25,26 and used to immobilize DNA27, proteins28 including insulin29, and bacteria30. We have recently adapted these methods to the encapsulation of mammalian cells using pancreatic beta cell lines31,32 and primary pancreatic tissue32.
The principle of the method is to generate a water-in-oil emulsion consisting of alginate droplets in mineral oil, followed by internal gelation of the alginate droplets (Figure 1). First the encapsulant (e.g., cells) is dispersed in an alginate solution containing a fine grain calcium salt with low solubility at the initial process pH. The alginate mixture is then added to an agitated organic phase to create an emulsion, usually in the presence of a surfactant. In the case of mammalian cell encapsulation, components present in serum can act as surfactants. Next, the pH is reduced in order to solubilize the calcium salt by adding an oil-soluble acid that partitions into the aqueous phase. Acetic acid, with a mineral oil/water partition coefficient &#60;0.00533, should be pre-dissolved in oil, then added to the emulsion where it is mixed in the oil phase and rapidly partitions into the aqueous phase34. Figure 2 illustrates the chemical reactions and diffusion that take place during the acidification and internal gelation step. Finally, the encapsulated cells are recovered by phase inversion, phase separation accelerated by centrifugation, repeated washing steps and filtration. These steps can then be followed by bead and cell sampling for quality control analyses, in vitro cell culture and/or transplantation of the encapsulated cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55280/55280fig1.jpg" /> 
Figure 1: Schematic of the emulsification-based process to encapsulate mammalian cells. Beads are first produced by emulsifying an alginate, cell and CaCO3 mixture in mineral oil (steps 1 and 2 in the schematic), triggering internal gelation by adding acetic acid (step 3). The gelled beads are then separated from the oil by adding an aqueous buffer to trigger phase inversion (step 4), followed by centrifugation and oil aspiration (step 5), and then filtration (step 6). Finally, the beads collected on the filter are transferred into cell culture medium for in vitro culture or for transplantation. <a href="http://cloudflare.jove.com/files/ftp_upload/55280/55280fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55280/55280fig2.jpg" /> 
Figure 2: Reactions and diffusion steps occurring during internal gelation. (1) Acetic acid is added to the organic phase and is transported to the alginate droplets by convection. (2) The acetic acid partitions into the aqueous phase. (3) In the presence of water, the acid dissociates and diffuses to reach the CaCO3 grains depicted in dark blue. (4) The H+ ions are exchanged with the Ca2+ ions in CaCO3, releasing Ca2+ ions. (5) The calcium ions diffuse until they encounter unreacted alginate, leading to the ionotropic cross-linking of the alginate chains. <a href="http://cloudflare.jove.com/files/ftp_upload/55280/55280fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Contrary to conventional nozzle-based cell encapsulators, a broad bead size distribution is expected from this process due to the mechanism of droplet formation in stirred emulsification. For a subset of applications, this bead size distribution may be problematic. For example, a larger fraction of cells may be exposed at the bead surface in smaller beads. If nutrient (e.g. oxygen) limitations are a concern, these limitations may be exacerbated in larger beads. An advantage of the stirred emulsification method is that the average bead size can readily be adjusted by changing the agitation rate during the emulsification step. The broad bead size distribution can also be exploited to study the effect of bead size on encapsulated cell performance.
Mammalian cell encapsulation by emulsification and internal gelation is an interesting alternative for laboratories that are not equipped with a bead generator. Furthermore, this method gives users the option of reducing the processing time, or generating beads at very low or very high alginate concentrations.
The protocol outlined below describes how to encapsulate cells in 10.5 mL of 5% alginate solution prepared in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. The alginate consists of a 50:50 mixture of transplantation-grade LVM (low viscosity high mannuronic acid content) and MVG (medium viscosity high guluronic acid content) alginate. Calcium carbonate at a final concentration of 24 mM is used as the physical cross-linking agent. Light mineral oil constitutes the organic phase, while acetic acid is used to acidify the emulsion and trigger internal gelation. However, the alginate type and composition, as well as the process buffer selected depend on the desired application32. A variety of alginate types (see Table of materials) have been used to produce beads with this protocol.

<table border="0" cellpadding="0" cellspacing="0" width="990">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Reagents and consumables</td>
			<td style="width:164px;"></td>
			<td style="width:223px;"></td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LVM alginate (transplantation-grade)</td>
			<td style="width:164px;">Novamatrix</td>
			<td style="width:223px;">Non-applicable</td>
			<td style="width:348px;">Referred to as &#34;alginate #1&#34; in the results.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">MVG alginate (transplantation-grade)</td>
			<td style="width:164px;">Novamatrix</td>
			<td style="width:223px;">Non-applicable</td>
			<td style="width:348px;">Referred to as &#34;alginate #2&#34; in the results.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Alginate (cell culture-grade)</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">A0682 (low viscosity) or A2033 (medium viscosity)</td>
			<td style="width:348px;">A2033 is referred to as &#34;alginate #3&#34; in the results.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">DMEM</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:223px;">11995-065</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Fetal bovine serum, characterized, Canadian origin</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">SH3039603</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Glutamine</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:223px;">25030</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Penicillin and streptomycin</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">P4333-100ML</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">HEPES, cell culture tested</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">H4034-100G</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">NaCl</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">S271-1</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:256px;">Fine-grain CaCO3</td>
			<td style="width:164px;">Avantor Materials</td>
			<td style="width:223px;">1301-01</td>
			<td style="width:348px;">After preparing the CaCO3 suspension, sonicate and use within one month.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Light mineral oil</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">O121-4</td>
			<td style="width:348px;">Sterile filter through a 0.22 &#956;m pore size membrane prior to use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Glacial acetic acid</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">A38-500</td>
			<td style="width:348px;">Handle with caution: refer to MSDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Sterile spatulas</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">CLS3004-100EA</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Sterile nylon cell strainers, 40 &#181;m</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:223px;">08-771-1</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Serological pipettes (2 mL, 5 mL, 10 mL, 25 mL)</td>
			<td style="width:164px;">Sarstedt</td>
			<td style="width:223px;">86.1252.001, 86.1253.001, 86.1254.001 and 86.1685.001</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Pasteur pipettes</td>
			<td style="width:164px;">VWR</td>
			<td style="width:223px;">14673-043</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Toluidine Blue-O</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:223px;">T3260</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Equipment</td>
			<td style="width:164px;"></td>
			<td style="width:223px;"></td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:256px;">100 mL microcarrier spinner flasks</td>
			<td style="width:164px;">Bellco</td>
			<td style="width:223px;">1965-00100</td>
			<td style="width:348px;">The impeller configuration with recent models may not be suitable for adequate emulsification. A blade able to sweep the oil down to 0.5 cm from the bottom of the flask can be custom-made from a Teflon sheet.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Magnetic stir plate with adjustable speed</td>
			<td style="width:164px;">Bellco</td>
			<td style="width:223px;">7760-06005</td>
			<td style="width:348px;">The rotation speed should be calibrated (e.g. using a tachometer) prior to use.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:256px;">Cell counter</td>
			<td style="width:164px;">Innovatis</td>
			<td style="width:223px;">Cedex AS20</td>
			<td style="width:348px;">This system is now sold by Roche. This automated cell counter can also be replaced by manual cell enumeration after Trypan blue staining using a hemocytometer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">LED light box</td>
			<td style="width:164px;">Artograph</td>
			<td style="width:223px;">LightPad&#174; PRO</td>
			<td style="width:348px;">This item can be replaced by other types of illuminators.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:256px;">Handheld camera</td>
			<td style="width:164px;">Canon</td>
			<td style="width:223px;">PowerShot A590 IS</td>
			<td style="width:348px;">A variety of handheld cameras can be used to capture toluidine blue-o stained bead images. A ruler should be placed next to the Petri dish containing the beads prior to acquiring images.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:256px;">Fluorescence microscope with phase contrast and adequate fluorescence filters</td>
			<td style="width:164px;">Olympus</td>
			<td style="width:223px;">IX81</td>
			<td style="width:348px;">Several microscopy systems were used to image the beads. The results shown here were obtained with an IX81 microscope equipped with GFP and TRITC fluorescence filters. To capture entire beads, 4X to 20X objectives were used depending on the agitation rate. Live/dead staining images were typically captured with 20X to 40X objectives.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Image aquisition software</td>
			<td style="width:164px;">Molecular Devices</td>
			<td style="width:223px;">Metamorph</td>
			<td style="width:348px;">A variety of image acquisition software can be used to acquire phase contrast and fluorescence images.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Image analysis freeware</td>
			<td style="width:164px;">CellProfiler</td>
			<td style="width:223px;">Non-applicable</td>
			<td style="width:348px;">A variety of image analysis software can be used to identify beads as objects and analyze bead size (e.g. ImageJ).</td>
		</tr>
	</tbody>
</table>

mammalian cell encapsulation in alginate beads using a simple stirred vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

At the end of the emulsification and internal gelation process, a bead volume similar to the initial alginate and cell mixture volume should be recovered. The beads should be highly spherical with few defects (Figure 3). The beads should be sufficiently strong to withstand pipetting through large-bore pipettes. At high alginate concentrations, encapsulated oil or air droplets may be observed in the beads. This likely occurs due to the entrapment of oil in alg...

Watch this Scientific Journal Video about Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel at JoVE.com

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

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.

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