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 (Video) | JoVE

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.

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 ...

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