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.

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

Research

JoVE Journal

Bioengineering

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

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 ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...