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>
	</li>
	<li>Prepare the alginate stock solution at 1.17 fold the final desired concentration in the beads.
	<ol>
		<li>First, weigh the appropriate amount of alginate powder. For example, to obtain a final concentration of 5% alginate with 50:50 LVM and MVG alginate, weigh out a total of 1.17 g sodium alginate by combining 0.583 g LVM alginate powder and 0.583 g MVG alginate powder.</li>
		<li>Place 20 mL process buffer into an autoclavable glass jar on a magnetic stir plate and agitate at ~200 rpm. Progressively add the sodium alginate powder to the solution.</li>
		<li>Leave the solution stirring overnight at a low speed. If necessary, fasten the flask to the stir plate.</li>
		<li>If the solution is incompletely dissolved, fasten the bottle onto a rotary mixer and continue mixing at 37 &#176;C for an additional 24 h.</li>
		<li>Sterilize the alginate solution by autoclaving the solution for 30 min in a vessel that is less than half full. Allow the temperature to decrease below 60 &#176;C before retrieving the solution.</li>
		<li>If necessary, remove particles by filtering the alginate solution shortly after autoclaving before it reaches room temperature, while the viscosity remains sufficiently low.</li>
	</ol>
	</li>
	<li>Prepare the calcium carbonate suspension at 21-fold the final desired concentration for internal gelation.
	<ol>
		<li>Weigh out the CaCO3 powder. For example, add 1 g CaCO3 to 20 mL process buffer to obtain 24 mM CaCO3 as the final gelling agent concentration.</li>
		<li>Autoclave the CaCO3 suspension for 30 min. 
		NOTE: The CaCO3 concentration will change over time due to the bicarbonate - CO2 equilibrium. Avoid using the same stock CaCO3 suspension for more than 10 encapsulation procedures.</li>
	</ol>
	</li>
	<li>Autoclave the stirred vessel used for the emulsification process. Prior to use, remove any traces of condensed water remaining in the spinner flask.</li>
	<li>Immediately before the emulsification process, prepare the acidified oil. Dissolve 44 &#181;L acetic acid per 11 mL mineral oil placed in a 50 mL conical tube. 
	NOTE: A common mistake is incomplete dissolution of the acetic acid. Avoid pipetting small amounts of acetic acid and ensure that the acetic acid is completely dissolved in oil by repeated vortexing. 
	CAUTION: acetic acid is a toxic and volatile acid. Handle this reagent under a fume hood and keep the acetic acid/oil solution in a closed container until the emulsification step. Refer to the MSDS information on this reagent for further information.</li>
	<li>Allow all the solutions to reach room temperature before proceeding to cell encapsulation.</li>
</ol>
2. Alginate Bead Generation by Emulsification and Internal Gelation
<ol>
	<li>Place 10 mL light mineral oil in the spinner flask and start agitation at a low rotation speed (e.g. 250 rpm for the spinner flask used in this video).</li>
	<li>If the cells used for the process are from adherent cultures, apply trypsin to suspend the cells. End the reaction by adding FBS-containing medium or trypsin inhibitor and collect a sample for cell enumeration.
	<ol>
		<li>Determine the cell concentration and viability after Trypan blue staining using a hemocytometer or an automated cell counter, as previously described32.</li>
	</ol>
	</li>
	<li>Centrifuge the cells for 7 min at 300 x g and wash them once in the desired medium for cell immobilization. Ensure that this medium contains emulsifiers such as either FBS or bovine serum albumin (BSA). For example, use DMEM containing 10% FBS.</li>
	<li>Re-suspend the cell pellet in the same medium to obtain 10.5-fold the desired final concentration in the beads.</li>
	<li>Add 1.1 mL of the 10.5-fold concentrated cell stock to 9.9 mL of the alginate solution. Then, add 550 &#181;L of CaCO3 suspension and mix by stirring with a sterile spatula to ensure an even distribution of the CaCO3.</li>
	<li>Immediately transfer 10.5 mL of the alginate, cell and CaCO3 mixture into the agitating oil using a syringe. 
	NOTE: For highly viscous solutions, aspirate and eject the mixture very slowly to avoid air bubbles. In some cases, it is preferable to stop the agitation while adding the mixture to the flask to avoid alginate entrainment onto the impeller shaft.</li>
	<li>Immediately after adding the alginate, cell and CaCO3 mixture to the oil, increase the agitation rate. 
	NOTE: For the 5% LVM:MVG alginate used here, a rotation speed of 1025 rpm was applied to generate beads of approximately 900 &#181;m diameter suitable for in vitro culture35. Higher rotation speeds and lower alginate concentrations will lead to lower average bead diameters, as described in previous publications25,32. Slight changes in impeller and vessel geometry, as well as alginate properties can greatly affect the average bead diameter. For each change in vessel configuration or alginate lot, a standard curve relating the surface area moment average bead diameter to the rotation speed should be generated32.</li>
	<li>Start the timer and emulsify the alginate for 12 min.</li>
	<li>Add 10 mL of the oil and acetic acid solution to acidify the emulsion, dissolve the CaCO3 and hence physically cross-link the alginate into gelled beads. Allow 8 min for this acidification step.</li>
</ol>
3. Bead Recovery
<ol>
	<li>Reduce the agitation rate to 400 rpm. Add 40 mL of process buffer mixed with 10% medium to increase the pH and to cause phase inversion.</li>
	<li>Stop the agitation 1 min later and transfer the mixture to 50 mL centrifuge tubes. Rinse the spinner flask with an additional 20 mL medium and add this to the tube. Aspirate as much aqueous phase as possible from the spinner flask before aspirating the oil phase to minimize bead contact with the oil phase. 
	NOTE: Use a large-bore pipette (e.g. 25 mL pipette) at this step and from this point on to avoid damaging the beads. For smaller volumes, the bead suspension can also be manipulated with cut pipette tips. To obtain such tips, cut the end of the pipette tip with scissors while wearing eye protection.</li>
	<li>Centrifuge the tubes for 3 min at 630 x g to accelerate bead settling and phase separation.</li>
	<li>Remove the oil and excess aqueous solution by aspirating with a Pasteur pipette.</li>
	<li>Wash the beads at least once with medium. Filter the bead suspension on 40 &#181;m nylon cell strainers, and aspirate excess liquid associated with the beads from below the strainer. Transfer the beads into a known volume of medium using a sterile spatula. 
	NOTE: Smaller or larger pore size filters can be used at this step, depending on the target minimum bead diameter. However, gravity filtration is recommended rather than pressure-driven filtration through sub-micron pore size filters to avoid damaging the beads.</li>
	<li>Measure the bead volume by volume displacement (volume after adding the beads, minus the volume prior to adding the beads) and top up the medium to obtain the desired bead:total volume ratio, which is typically 1:5, or 1 mL beads in 4 mL medium. From this point on, always use large bore pipettes to avoid damaging the beads.</li>
	<li>Transfer the encapsulated cells into T-flasks for in vitro culture or transplantation experiments.</li>
</ol>
4. Quality Control and Applications
NOTE: In order to ensure encapsulated cell and bead quality, the bead size distribution and cell survival after the process should be quantified. Reversing the gel to recover the cells from within the beads for further analysis is commonly performed.
<ol>
	<li>To assess the bead size distribution, stain the beads with toluidine blue-O.
	<ol>
		<li>Place 0.5 mL beads into 4 mL process buffer containing 10% complete medium.</li>
		<li>Add 500 &#181;L of a 1 g/L toluidine blue solution prepared in process buffer.</li>
		<li>Incubate for 60 min in a conical tube on a rotary shaker at 50 rpm.</li>
		<li>Add 5 mL process buffer containing 10% complete medium and immediately transfer the beads and solution into a Petri dish and acquire images on a low-magnification microscope or using a hand-held digital camera. If needed, place the Petri dish on a light box before acquiring images with a hand-held camera to enhance the contrast and avoid shadows.</li>
		<li>Perform image analysis to quantify the bead size distribution as previously described32, for example using image analysis freeware36.</li>
	</ol>
	</li>
	<li>To assess cell survival qualitatively, stain the cells using ethidium homodimer to identify dead cells and calcein AM to identify live cells.
	<ol>
		<li>Add 1 volume of beads to 4 volumes of process buffer containing 10% complete medium.</li>
		<li>Add the appropriate amount of calcein AM and ethidium homodimer stock solutions to obtain 4 &#181;M and 2 &#181;M concentrations, respectively. Generate single stain controls by adding only one of the reagents to some bead samples.</li>
		<li>Incubate the beads on ice for 20 min.</li>
		<li>Place the solution between a slide and coverslip using a spacer such as an o-ring to avoid compressing the beads.</li>
		<li>Proceed to fluorescence microscopy. Use the single stain controls to assess fluorescence bleed-through. Select appropriate fluorescence filters based on the excitation/emission wavelengths associated with calcein AM (494/517 nm) and ethidium homodimer bound to DNA (528/617 nm).</li>
	</ol>
	</li>
	<li>To recover the cells from the beads for further analysis, degel the alginate using citrate or another chelating solution.
	<ol>
		<li>Prepare a degelling solution containing 55 mM citrate, 10 mM HEPES and 95 mM NaCl at pH 7.4.</li>
		<li>Mix this solution with 10% medium, and then add 1 mL alginate beads to 9 mL of the mixture.</li>
		<li>Degel the beads on ice with 75 rpm agitation for 20 min. 
		NOTE: The cells can now be used for analysis or removed from the degelled alginate solution by centrifugation. For example, cell viability after degelling can be quantified by Trypan Blue staining. Alternatively, the cells can be centrifuged and washed, followed by mRNA, DNA and/or protein sampling and analysis.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;Reagents and consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LVM alginate (transplantation-grade)</td><td>Novamatrix</td><td>Non-applicable</td><td>Referred to as &quot;alginate #1&quot; in the results.</td></tr><tr><td>MVG alginate (transplantation-grade)</td><td>Novamatrix</td><td>Non-applicable</td><td>Referred to as &quot;alginate #2&quot; in the results.</td></tr><tr><td>Alginate (cell culture-grade)</td><td>Sigma</td><td>A0682 (low viscosity) or A2033 (medium viscosity)</td><td>A2033 is referred to as &quot;alginate #3&quot; in the results.</td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Fetal bovine serum, characterized, Canadian origin</td><td>Thermo Fisher Scientific</td><td>SH3039603</td><td></td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>Sigma</td><td>P4333-100ML</td><td></td></tr><tr><td>HEPES, cell culture tested</td><td>Sigma</td><td>H4034-100G</td><td></td></tr><tr><td>NaCl</td><td>Thermo Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>Fine-grain CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Avantor Materials</td><td>1301-01</td><td>After preparing the CaCO&lt;sub&gt;3&lt;/sub&gt; suspension, sonicate and use within one month.</td></tr><tr><td>Light mineral oil</td><td>Thermo Fisher Scientific</td><td>O121-4</td><td>Sterile filter through a 0.22 &amp;mu;m pore size membrane prior to use.</td></tr><tr><td>Glacial acetic acid</td><td>Thermo Fisher Scientific</td><td>A38-500</td><td>Handle with caution: refer to MSDS.</td></tr><tr><td>Sterile spatulas</td><td>Sigma</td><td>CLS3004-100EA</td><td></td></tr><tr><td>Sterile nylon cell strainers, 40 &amp;micro;m</td><td>Thermo Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>Serological pipettes (2 mL, 5 mL, 10 mL, 25 mL)</td><td>Sarstedt</td><td>86.1252.001, 86.1253.001, 86.1254.001 and 86.1685.001</td><td></td></tr><tr><td>Pasteur pipettes</td><td>VWR</td><td>14673-043</td><td></td></tr><tr><td>Toluidine Blue-O</td><td>Sigma</td><td>T3260</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 mL microcarrier spinner flasks</td><td>Bellco</td><td>1965-00100</td><td>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><td>Magnetic stir plate with adjustable speed</td><td>Bellco</td><td>7760-06005</td><td>The rotation speed should be calibrated (e.g. using a tachometer) prior to use.</td></tr><tr><td>Cell counter</td><td>Innovatis</td><td>Cedex AS20</td><td>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><td>LED light box</td><td>Artograph</td><td>LightPad&amp;reg; PRO</td><td>This item can be replaced by other types of illuminators.</td></tr><tr><td>Handheld camera</td><td>Canon</td><td>PowerShot A590 IS</td><td>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><td>Fluorescence microscope with phase contrast and adequate fluorescence filters</td><td>Olympus</td><td>IX81</td><td>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><td>Image aquisition software</td><td>Molecular Devices</td><td>Metamorph</td><td>A variety of image acquisition software can be used to acquire phase contrast and fluorescence images.</td></tr><tr><td>Image analysis freeware</td><td>CellProfiler</td><td>Non-applicable</td><td>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 ...

mammalian-cell-encapsulation-alginate-beads-using-simple-stirred

Research

JoVE Journal

Bioengineering

19.4K Views.  McGill University. 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.

Video: Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Developmental Biology

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Medicine

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Chemistry

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Biology

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

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Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

Fabrication of Amyloid-β-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation