Patent Publication Number: US-2004048358-A1

Title: System and method for encapsulating biological material by applying electrostatic charge to capsules

Description:
PRIORITY APPLICATION  
     [0001] This application is a Divisional application of U.S. patent application Ser. No. 09/900,466, filed Jul. 5, 2001, which claimed the benefit of U.S. Provisional Patent Application Serial No. 60/215,947, filed Jul. 5, 2000, which is incorporated herein by reference. 
    
    
     
       FIELD OF THE INVETION  
       [0002] This invention describes a method and system for the consistent and effective encapsulation of viable biological material. (e.g., individual living cells, cell clusters, or organ tissue) with a polymeric coating material.  
       BACKGROUND OF THE INVENTION  
       [0003] Transplantation of organ tissue (e.g., pancreatic islets) into genetically dissimilar hosts has gained a significant interest in the treatment for functional deficiencies of secretory and other biological organs. Transplantation, however, generally requires the continuous use of immunosuppressive agents by the transplant recipient in order to forestall rejection of the transplanted tissue by the recipient&#39;s immune system. Unfortunately, these immunosuppressive agents can deprive the recipient of adequate protective immune function against diseases.  
       [0004] A potential solution that avoids the need for such immunosuppressive agents is the encapsulation of the tissue material so as to protect the transplanted tissue from the recipient&#39;s immune system. Encapsulation generally eliminates the need for immunosuppressive agents to prevent adverse immune system response and rejection of the implant. Encapsulation with a sufficiently semi-permeable protective barrier coating not only generally prevents an immune response, but also provides for diffusion of oxygen into the encapsulated material along with the transfer of nutrients, ions, glucose, and hormones, as well as the excretion of metabolic waste. This maintains the health of the encapsulated tissue material.  
       [0005] One promising approach for the encapsulation of tissue material such as pancreatic islets involves the use of coatings formed of a non-fibrogenic alginate, a gelatinous substance that can be derived from certain kinds of kelp. The islets are suspended in a viscous, liquid alginate, which is then atomized by any of a number of different arrangements into droplets of suitable size to encapsulate the islets. Once the droplets come into contact with a gelling solution, such as calcium chloride or barium chloride, a single layer alginate coating is created around the islets. Examples of this approach for creating single layer alginate coatings using an electrostatic coating process are shown in U.S. Pat. No. 4,789,550 (Hommel et al.), U.S. Pat. No. 4,956,128 (Hommel et al.), U.S. Pat. No. 5,429,821. (Dorian et al.), U.S. Pat. No. 5,639,467 (Dorian et al.), U.S. Pat. No. 5,656,468 (Dorian et al.) and U.S. Pat. No. 5,693,514 (Dorian et al.). An example for creating a single layer alginate coating using an air knife process is shown in U.S. Pat. No. 5,521,079 (Dorian et al.). A pressurized process for coating droplets is described in U.S. Pat. No. 5,260,002 (Wang) and U.S. Pat. No. 5,462,866 (Wang). Other examples for creating a single layer alginate coating using a spinning disk arrangement are shown in U.S. Pat. No. 5,643,594 (Dorian et al.) and U.S. Pat. No. 6,001,387 (Cochrum). Examples for creating a single layer alginate coating using a piezoelectric nozzle are shown in U.S. Pat. No. 5,286,496 (Batich et al.), U.S. Pat. No. 5,648,099 (Batich et al.) and U.S. Pat. No. 6,033,888 (Batich et al.)  
       [0006] A problem common to all of these techniques for creating single layer alginate coatings is the formation of non-encapsulated or partially encapsulated islets. Any non-encapsulated biological material or capsules that are only partially coated or that have too thin of a coating will lead to an adverse immunological response when transplanted into the recipient. One way to decrease this problem is to increase the diameter of the single-coating so that the capsules have diameters in the range of 700-800 microns. Unfortunately, these large-sized capsules tend to be less effective when transplanted into a recipient because the larger diameter diminishes the ability of oxygen to penetrate completely into the interior of the capsule. It has also been found that large-sized capsules tend to increase the potential for macrophage attack by the recipient&#39;s immune system and can limit the potential transplantation sites as compared to smaller-sized capsules.  
       [0007] It has been discovered that encapsulation of tissue material such as pancreatic islets with a second coating of a cross-linkable polymer can provide substantially complete coverage of the islets in order to minimize or eliminate the possibility of adverse immune reactions, while at the same time providing a capsule having a dimension on the order of 400-500 microns. The multiple coatings of the individual capsules containing the core of living tissue serve as an additional means for assisting in the resistance to chemical, mechanical, or immune destruction by the host. The smaller-sized capsule is believed to permit oxygen to better permeate into the interior of the capsule as oxygen can normally permeate up to about 200-250 microns into encapsulated tissue material.  
       [0008] U.S. Pat. Nos. 5,470,731 (Cochrum) and 5,531,997 (Cochrum) describe a double layer coating for tissue that comprises a first layer of a gellable organic polymer and a cationic polymer and a second water-soluble, semi-permeable layer chemically bonded to the first layer. U.S. Pat. No. 6,020,200 (Enevold) describes a dual layer coating having a stabilized outer layer formed of a cross-linked polymer matrix. U.S. Pat. No. 5,227,298 (Weber at al.) describes a double walled alginate coating. U.S. Pat. No. 5,578,314 (Cochrum et al.) teaches such a method for applying multiple layers of alginate onto biological material (e.g., pancreatic islets). In this method, a first layer of a multiple layer alginate coating is applied using a solution of an alginate containing a high ratio of guluronate to mannuronate, and the second layer is applied using a solution of an alginate containing a high ratio of mannuronate to guluronate. U.S. Pat. No. 5,876,742 (Cochrum et al.) teaches a multiple layer alginate coating where an intermediary halo layer of a soft gel is formed between the inner and outer alginate coating layers.  
       [0009] While the use of multiple layer alginate coatings solves many of the problems associated with single layer coatings, the existing techniques for generating such multiple layer alginate coatings are not well suited to large scale manufacturing systems that can consistently and reliably produce large amounts of encapsulated material. In order to obtain amounts of encapsulated islets, for example, necessary for a single human transplantation procedure, as many as 500,000 to 1,000,000 encapsulated islet equivalents (one islet equivalent is equal to a cell cluster of islets having a diameter of 150 microns), or at least 8000 islet equivalents per kilogram of body weight may be required.  
       [0010] Several problems with the existing techniques have generally prevented the large-scale manufacture of encapsulated islets to meet these needs. First, the existing techniques tend to generate a very large number of empty capsules or “blanks.” While such blanks can be created in either the first coating process or the second process, the problem is most noticeable where a droplet is produced during the second coating process that does not contain an islet. Second, the existing techniques also tend to create encapsulated islets in which multiple single coated islets either stick together during the coating process or end up with more than about ten islets being contained within the same second coating encapsulation, conditions which are referred to as “clumping.” When clumping occurs during the coating process, the entire batch of capsules being processed can be destroyed. Single-coated capsules can bind together into clumps that are subsequently coated a second time during the encapsulation process. Depending upon the number of single-coated capsules in a particular clump, the coated clumps do not function as effectively as a double-coated capsule containing only one or up to four single-coated capsules, most likely because the size of the resultant capsule of clumped single-coated capsules is too large.  
       [0011] Most importantly, the entire encapsulation process for biological materials is a time and stress sensitive process. The longer that living tissue or cells are exposed to the process or the stresses created by the encapsulation process, the less viable and effective the resulting encapsulated tissue material will be. Moreover, it has been discovered that encapsulated islet cells, for example, have a limited viability of only a few days in cell culture, both prior to encapsulation and after encapsulation and prior to transplantation. The existing techniques for double alginate coating have been limited to processing relatively small batches of cell clusters, on the order of only tens of thousands of islets per batch. Even with these small batches, the process can take several hours, exposing the cells to stress during the entire period of the coating process. The ability to process only small batches of islets requires the encapsulation process to be repeated numerous times in order to obtain the requisite number of islets for a single transplantation, thereby potentially extending the time that the encapsulated islets must be maintained in cell culture prior to transplantation. Small batches are also inefficient in that a certain number of the islets will be lost during each process. When small batches are processed, the number of islets lost represents a larger percentage of the total islets processed than if larger batches could be processed.  
       [0012] Although multiple layer alginate coatings for encapsulating tissue material have offered promise as a potential alternative for protecting tissue implants without the use of immunosuppressive agents, the existing techniques are not well suited for the large-scale manufacturing required to generate the necessary volume of encapsulated tissue material in a short period of time for a successful transplant operation. It would be desirable to provide for a manufacturing method and system for the consistent and effective encapsulation of large batches of biological material that can overcome these limitations.  
       SUMMARY OF INVENTION  
       [0013] A method and system for the consistent and effective encapsulation of large batches of biological material applies an electrostatic charge to capsules having a first layer coating prior to creating a second layer coating so as to singulate and separate the single-coated capsules during a mixing process prior to the second layer coating process. The single layer coated capsules are suspended in a liquid carrier medium for purposes of applying the electrostatic charge. Preferably, the liquid carrier medium is low viscosity and physiologically balanced to the biological material. The electrostatically charged carrier medium with the single layer coated capsules is then introduced into an alginate solution as part of a mixing process performed prior to the second layer coating process. Preferably, both the first layer coating process and the second layer coating process utilizes a spinning disk encapsulation apparatus having a central cup with at least one groove in an interior surface that causes droplets to exit from the center cup into an outer collection chamber containing a gelling solution in one or more relatively well-defined singulated lines.  
       [0014] In a preferred method of the present invention, cell clusters of biological material are suspended in a first alginate solution to form a first alginate suspension. Droplets are formed from the first alginate suspension, with at least some of the droplets containing cell clusters. The droplets are then gelled to form capsules having a first layer coating surrounding at least a portion of the cells. These single-coated capsules are suspended in a liquid carrier medium that is preferably low viscosity and physiologically balanced to the biological material to form a carrier suspension. An electrostatic charge is applied to the carrier suspension prior to introducing the carrier suspension into a second alginate solution to create a singulated flowstream of the carrier suspension containing the single-coated capsules. As part of the mixing process, the second alginate solution is agitated by a vortex mixer as the singulated flowstream of the carrier medium containing the single-coated capsules is introduced into the second alginate solution to create a second alginate suspension. As a result of the process of applying the electrostatic charge and agitating the second alginate solution, the capsules do not clump as they are introduced into the second alginate solution to create the second alginate suspension. After this mixing process is complete, the second alginate suspension containing the separated single-coated capsules are formed into droplets and then gelled to form a second layer coating around the capsules.  
       [0015] In a preferred system of the present invention, a series of apparatus and processing techniques are arranged to effectively and consistently encapsulate biological material. The system includes a system for atomizing a first alginate suspension containing cell clusters of the biological material in a first alginate solution to form droplets, the majority of the droplets containing at least one cell cluster. The droplets are gelled in order to form single-coated capsules having a first layer coating surrounding at least a portion of the cell clusters. The single-coated capsules are placed in a liquid carrier medium for the purpose of applying the electrostatic charge prior to the second layer coating process. Preferably, a conductive collar is used for applying the electrostatic charge to the liquid carrier medium containing the single-coated capsules. As the carrier medium containing the single-coated cells is introduced into a second alginate solution to create a second alginate suspension, the electrostatic charge separates the single-coated capsules in a fluid stream of the carrier medium. The second alginate solution is agitated by a vortex mixer as the carrier medium is introduced. Together, the electrostatic charge and the agitation of the second alginate solution prevent the single-coated capsules from clumping together in the second alginate solution as the second alginate suspension is formed. After this mixing process is complete, a system for atomizing and gelling the separated capsules in the second alginate suspension is used to form a second layer coating around the capsules.  
       [0016] Preferably, the system for atomizing and gelling droplets to create both the first and second layer coatings is a spinning disk encapsulation apparatus having a center cup into which droplets of the appropriate alginate suspension are introduced and an outer collection chamber containing a gelling solution for gelling the droplets of the alginate suspension so as to form a layer coating around the capsules. In order to achieve more effective operation for the coating process, the center cup preferably includes at least one groove defined on an inner wall of the center cup. When the droplets of the alginate suspension are introduced into the center cup, the grooves cause the droplets to travel from the center cup into the outer collection chamber in one or more relatively well-defined singulated lines exiting from the center cup. A singulated line is a line of moving liquid and/or droplets that does not mingle with other lines. The grooves in the central cup provide the ability to control the location of the singulated lines of droplets, as well as the uniformity of the size and shape of the droplets created by the spinning disk encapsulation apparatus.  
       [0017] The present invention overcomes the problem of clumping that has traditionally been associated with scaling up the coating of biological material and reduces the problems of blanks created in the second coating process. The challenge of successfully mixing and double coating living tissue in large quantities without damaging the tissue is complex. Single-coated capsules suspended in a second alginate solution behave somewhat like tapioca beads in pudding. Mixing or stirring with mechanical mixing techniques to disperse the capsules in the second alginate solution is either ineffective because the mixing is not vigorous, or is damaging to the tissue material because the mixing is too vigorous. If the viscosity of the second alginate solution is reduced or if the volume is increased so as to more easily mechanically mix the capsules, the effectiveness of the subsequent coating process is effected and the number of blanks produced in the droplet generation stage of the subsequent coating process increases significantly. The present invention recognizes that the way to avoid the clumping problem is to utilize a mixing process that avoids clumping in the first place by singulating and separating the single-coated capsules such that the capsules do not touch one another as they are introduced into the second alginate solution.  
       [0018] Specifically, the present invention provides a method and system for the effective and consistent encapsulation of viable (i.e., living or physiologically active) clusters of biological material (preferably, pancreatic islets also known as islets of Langerhans) with a polymeric material (preferably, a biocompatible semi-permeable alginate) to form a gelled capsule, which can be transplanted into genetically dissimilar hosts. At least two layers of polymer (e.g., alginate) are applied. With the current invention, the failures and defects in the previously described methods are eliminated and it is possible to produce high quality double layer capsules in high volumes, while minimizing the processing time and stress for the biological material being encapsulated. Unlike existing double layer alginate encapsulation techniques, the present invention allows for double layer encapsulation of all of the cell clusters from a donor organ as part of one large-scale batch process. In contrast to existing techniques which typically produce blanks during the second coating process at a ratio of at least 10:1 to encapsulated cells, and more commonly at a blank ratio of 50:1, the present invention produces blanks during the second coating process at a ratio of less than 10:1 and more typically at a blank ratio of 6:1 or better.  
       [0019] One embodiment of a method of the present invention includes providing a composition that includes a liquid carrier medium and polymeric capsules that include biological material in a first polymeric coating; applying an electrostatic charge to the composition of carrier medium and polymeric capsules; producing a flowing stream of droplets of the composition, wherein a relatively high proportion of the droplets include the polymeric capsules; and introducing the flowing stream of droplets directly into a second polymeric coating composition to form a suspension. These steps form the electrostatic mixing process described in greater detail below. Preferably, the method subsequently includes atomizing and contacting the suspension with a gelling solution to gel the second polymeric coating composition and encapsulate the polymeric capsules having a first polymeric coating in a second polymeric coating. This process can be used to provide one or more polymeric coatings to biological material by repeating the steps of applying an electrostatic charge, introducing the flowing stream of droplets into a polymeric coating composition to create a suspension, and atomizing and contacting the suspension with a gelling solution.  
       [0020] This step of introducing the single-coated material (i.e., single-coated capsules, which are the polymeric capsules that include biological material in a first polymeric coating) into the second polymeric coating composition using an electrostatic charge is not carried out in conventional methods. Typically, in conventional methods the single-coated capsules are simply mixed with the second polymeric coating composition by mechanical means. In conventional methods, this suspension of single-coated capsules is then atomized by electrostatic charge or other means and collected in a gelling solution (as opposed to a polymeric coating composition). The step of the present invention of dispersing the single-coated capsules into the second polymeric coating composition using an electrostatic mixing process provides significant advantage in that it reduces the aggregation and clumping of the single-coated capsules and enhances the yield of the double-coated capsules (i.e., the capsules with a first and second polymer coating).  
       [0021] A preferred embodiment of the present invention is used for encapsulating pancreatic islets, although the present invention is equally applicable to encapsulation of clusters of other cells ranging in size from single cells to cell clusters as large as several hundred microns. When used with pancreatic islets, the carrier medium is a physiological saline solution and the polymeric capsules including pancreatic islets in a first polymeric coating preferably include an alginate. An electrostatic charge at a voltage of about 1 kV to about 100 kV (preferably, 5 kV to 30 kV) is applied to the composition of the carrier medium and the polymeric capsules containing the pancreatic islets to create an electrostatic charge on a flowing stream of droplets of the composition, wherein a relatively high proportion of the droplets include pancreatic islet clusters. The flowing stream of droplets is introduced directly into a second polymeric coating composition contained in a vortex mixer to form a suspension. This suspension is then atomized and gelled by introducing the suspension into an encapsulation apparatus including a spinning disk atomizer and an outer collection chamber that includes a gelling solution to gel the second polymeric coating composition and encapsulate the polymeric capsules in a second polymeric coating, which includes an alginate.  
       [0022] The preferred embodiment of the spinning disk atomizer includes a central cup having an opening; a reservoir portion; at least one inner wall portion connecting the reservoir portion to the opening; and at least one groove formed in the inner wall portion. Preferably, the at least one groove extends substantially between the reservoir portion and the opening. In a preferred embodiment, the at least one inner wall comprises a first inner wall frustoconical surface tapered outward and having a plurality of grooves defined on the frustoconical surface and a second inner wall defined between the frustoconical surface and the reservoir having a second set of grooves defined in the second inner wall, at least a portion of the first set of grooves aligned with the second set of grooves. The preferred embodiment of the spinning disk atomizer directs the path of travel of the capsules from the center cup into an outer collection chamber in one or more relatively well-defined singulated lines exiting from the center cup, thereby improving the consistency of the coating process, while minimizing damage to the biological material potentially caused by collisions between droplets exiting the center cup. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0023]FIG. 1 is an overall schematic diagram of the method and system of the present invention.  
     [0024]FIG. 2 is a schematic of an electrostatic singularizing apparatus and mixer for use in the present invention.  
     [0025]FIG. 3 is a detailed schematic of the electrostatically singularized droplets formed in the electrostatic mixing process shown in FIG. 2.  
     [0026]FIG. 4 is a representation of an encapsulation apparatus including a spinning disk atomizer.  
     [0027] FIGS.  5 A- 5 D are schematics of a spinning disk atomizer.  
     [0028]FIG. 6 is a detailed cross-section of a preferred embodiment of the conductive charging collar of the electrostatic mixing apparatus.  
     [0029]FIG. 7 is a front view of a preferred embodiment of a stand for the electrostatic mixing apparatus.  
     [0030]FIGS. 8A and 8B are front views of a preferred embodiment of a syringe adapter for use in the preferred embodiment of the spinning disk atomized. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
     [0031] The present invention provides a system and method for the encapsulation of biological material (e.g., pancreatic islets) that allows for the efficient production of amounts necessary for transplantation. Significantly, with the present invention, biological material encapsulated by a single coating of a polymeric material can be further encapsulated with a second coating with little or no aggregation or clumping together of the capsules. The use of multiple layers ensures substantially complete coverage of the implanted biological material so as to minimize or eliminate the possibility of fibrogenic and/or immune reactions that are destructive to the implant&#39;s long-term functionality in the host. Applying additional coatings increases the probability that all parts of the living material are encapsulated. For example, if 5% of the capsules have imperfect coverage with only one coating, a second coating with similar failure rate will reduce the percentage of incomplete encapsulation to 0.25% (0.05×0.05=0.0025).  
     [0032] Referring now to FIG. 1, the overall process of double coating a capsule containing biological material will be described. Unlike conventional double layer coating techniques which include only two steps in which droplets of material are formed (one for forming each coating layer), the present invention incorporates three steps: a first layer coating process as shown generally at  10 , an electrostatic mixing process as shown generally at  20 , and a second layer coating process as shown generally at  30 . While the present invention takes advantage of the combination of all three of these processes  10 ,  20  and  30 , it will be understood that alternative arrangements for accomplishing any of the processes in combination with the other processes as described. For example, any of the known techniques for atomizing and gelling droplets or otherwise creating polymeric material encapsulating viable biological material could be used for either the first coating process  10  and/or the second coating process  30  in connection with the electrostatic mixing process  20 . Alternatively, the preferred spinning disk encapsulation apparatus could be used without the electrostatic mixing process to apply one or more polymeric coating layers for coating capsules containing biological material.  
     [0033] A preferred method of the first coating process  10  is an improvement on a first coating process as disclosed in more detail in U.S. Pat. No. 5,578,314 (Cochrum et al.). Typically, the first coating layer is formed by gently combining the biological material  104  into a first polymeric coating composition  102 , preferably a suspension of a relatively higher ratio of guluronate relative to mannuronate (“high G/M” alginate). Then, the first polymeric coating composition  102  and biological material  104  are dispersed into droplets using, for example, a spinning cup or disk atomizer  300  to form droplets  120 . As described in greater detail below with respect to FIGS. 4 and 5, the spinning disk apparatus of the preferred embodiment is an improvement on the spinning disk apparatus as disclosed in U.S. Pat. No. 5,578,314 (Cochrum et al.). The droplets  120  are collected in a CaCl 2  or BaCl 2  gelling solution  112 , for example, to cross-link or gel the polymer and form single-coated capsules  122 . Other potential gelling solutions could include MgCl 2  and ZnCl 2 , or any equivalent divalent cation solution. With pancreatic islets, each capsule  122  may include one or more islets. Preferably, each capsule contains no greater than about 10 pancreatic islets, and more preferably, the majority of capsules contain no greater than about 4 pancreatic islets per capsule.  
     [0034] In the preferred embodiment of the process flow as shown at  130  between the first coating process  10  and the electrostatic mixing process  20 , the single-coated capsules  122  are then mixed with a CaCl 2  solution (preferably, about 2 mM to about 6 mM), removed, mixed with a CaCl 2  solution (preferably, about 6 mM to about 12 mM), removed, and finally mixed with a physiologically balanced saline solution in about the same volumetric proportion to the volume of the capsules. The entire process  130  is completed relatively quickly (preferably, between about 10 to 30 minutes). Unlike the techniques disclosed in U.S. Pat. No. 5,578,314 (Cochrum et al.), the process flow  130  uses significantly lower concentrations of the CaCl 2  solution and maintains the capsules in the process for a shorter period of time which reduces or eliminates damage to the tissue.  
     [0035] Prior to the present invention, an attempt was made to overcome the prior art problems of aggregation or clumping together of the capsules that occurs in the second coating process by using a dilute suspension of the single-coated capsules in sucrose and dispersing the suspension into a large quantity of a second alginate solution. This resulted in a suspension that could be used in the spinning disk, electrostatic, or air-assist atomizer, or other means for forming droplets without aggregating the capsules and clogging of the apparatus for small quantities of capsules. Unfortunately, it was discovered that using a sucrose solution created a significant biological stress for the encapsulated islets. In addition, the method produced only small quantities of double-coated capsules along with a large number of capsules not containing the single-coated material (i.e., blanks). While it may be possible to select and separate out the blanks after the second coating process is complete, this process can be time consuming and detrimental to the viability of the living tissue. The present invention overcomes many of these problems.  
     [0036] According to the present invention and referring again to FIG. 1, prior to applying the second coating layer, an electrostatic mixing process  20  is used to singulate and separate the single-coated capsule  122 . The single-coated capsules  122  in a saline solution used as a liquid carrier medium  200  are mixed with a second polymeric coating composition  214 . The liquid carrier medium  200  preferably is a physiologically balanced, low viscosity solution that has an osmolarity and pH balanced for the particular characteristics of the biological material being encapsulated. In the case of islets, for example, physiologically buffered saline solution. The viscosity of the liquid carrier medium  200  is preferably similar to water. By low viscosity as used in the present invention, it will be understood that the carrier medium  200  has a viscosity of preferably zero or close to zero; however, a viscosity low enough to permit the carrier medium  200  to form the flow stream  204  could also be utilized.  
     [0037] The electrostatic mixing process  20  is preferably accomplished by dispensing the single-coated capsules in carrier medium  200  from syringe  201  mounted in a conductive charging collar  203 . Alternatively, the single-coated capsules can be dispensed from a metallic conductive orifice  202  such as a needle, preferably having a diameter of about 0.1 mm to about 2 mm. An electrostatic charge is applied to the conductive collar  203  or conductive orifice  202 , preferably at a voltage of about 1 kV to about 100 kV (more preferably, about 5 kV to about 30 kV, and most preferably, about 20 kV). This is described in greater detail below with respect to FIGS. 2 and 3.  
     [0038] The high voltage applied to the conductive charging collar  203  forms an attenuated varicose liquid stream  204  that forms a “varicose bulge” due to the presence of a capsule and breaks up into droplets  210 . These droplets  210 , which preferably include only one capsule  122  per droplet  210 , fall into a second polymeric coating material  214 , preferably an alginate with a relatively higher ratio of mannuronate relative to guluronate (“high M/G” alginate) as disclosed in U.S. Pat. No. 5,578,314 (Cochrum et al.). The second polymeric coating composition  214  is preferably at ground potential as shown at  216 . The high voltage, which is applied by a charging collar  203 , serves to force the liquid from the syringe  201  into a conical shape, which then forms a ligament structure punctuated by bulges or a varicose structure formed by the capsules  122 . The thinning of the liquid stream  204  serves to separate the capsules  122  into an approximate linear sequence. The bulges produce a radius of curvature in the streamwise direction, which causes the liquid to collapse in the streamwise or longitudinal direction under the force of surface tension into either a thin layer of saline solution around the individual capsules  122  before they are driven into the second polymeric coating composition  214  by the electrostatic forces or into a sphere of saline solution. Although, ideally this results in singularized capsules (i.e., only one single-coated capsule per droplet), there may be droplets  210  that include more than one capsule  122 . Preferably, at least ninety percent (90%) of the droplets  210  contains no greater than about 4 capsules per droplet. More preferably, a majority of the droplets contains no greater than about 2 capsules per droplet.  
     [0039] Preferably, the droplets  210 , which contain a saline carrier  200  and single-coated capsules  122 , are introduced in the second polymeric coating material  214  with mixing, typically caused by a vortex mixer  213  to assist the single-coated capsules  122  from aggregating in the solution  214 . While a vortex mixer  213  is preferred, it will be recognized that other forms of mixing such as vibratory, shaking or ultrasonic could also be utilized to agitate the second polymeric coating material.  214 . Less preferably, a mechanical mixing arrangement such as a stirring or folding arrangement could be used, although the mechanical operation of a mixing apparatus within the second polymeric coating material  214  may tend to increase the potential for damage to the biological material in the capsules. Together with the electrostatic separation charge supplied to the capsules  122  by the charging collar, a uniformly dispersed suspension  222  of single-coated biological material in the second coating material  214  is provided.  
     [0040] The second coating material  214  containing the separated capsules  122  is transferred at process step  230  into the second coating process  30 . The electrostatic mixing process of the invention allows for separated capsules  122  to be present in coating material  214  at concentrations much higher than was previously possible. Since the concentration of separated capsules  122  is so high, less coating material  214  is required and, the number of blanks generated during second coating process  30  is greatly reduced. Using conventional techniques, in the most favorable cases, the second coating process would produce blanks at a ratio of at least 10:1 to encapsulated cells, and more commonly at a blank ratio of 50:1. In contrast, the preferred embodiment of the present invention produces blanks during the second coating process at a ratio of less than 10:1 and more typically at a blank ratio of 6:1 or better.  
     [0041] In the second coating process  30 , the second polymeric coating composition  214  containing the single-coated capsules  122  is formed into droplets  320  using a spinning disk atomizer  300  or other mechanical or electrostatic dispensing means that are subsequently gelled to form double coated capsules  322  containing the previously single-layer encapsulated living biological material. Preferably, the encapsulation apparatus  300  includes a spinning disk atomizer  302  that is used in the second encapsulation process  30 . The atomizer  302  receives single-coated capsules  122  suspended in the second polymeric coating composition  214  from a syringe  304  and catheter  306 . This syringe  304  and catheter  306  are positioned above the spinning disk atomizer  302 . The tip of the catheter  306  is typically placed very close to the bottom of a reservoir  504  in a spinning center cup  502  of the spinning disk atomizer  302 .  
     [0042] Preferably, an automated motion control apparatus is used to adjust the tip of the catheter  306  relative to the bottom of the reservoir  504 . In this embodiment, a conductive replica of the catheter tip  306  is inserted on the syringe  304  to measure when the catheter tip  306  would be at the bottom of the reservoir  504  by sensing electrical conductivity between the conductive replica of the catheter tip  306  and the center cup  502 . An automated motion control system with a corresponding position sensor/detector then selectively steps the syringe adapter  340  holding the syringe  304  such that the catheter tip  306  is stepped away from the bottom of the reservoir  504  a predetermined distance. Preferably, this distance is greater than about 500-600 microns or the largest diameter of the capsules to be created), but less than about 1500 microns. The optimum standoff distance of the catheter tip  306  from the bottom of the reservoir  504  in this range can be determined without undue experimentation by a person skilled in the art dependent upon the particular characteristics of the capsules, biological material, rotational speed(s) of the spinning disk atomizer  302  and qualities of the polymeric coatings.  
     [0043] A drive mechanism  308  is preferably used to feed the mixture at a fixed flow rate. The spinning disk atomizer  302  is then rotated at an appropriate speed. Through centrifugal motion, spherical droplets  320  of the suspension material  222  of about 50 microns to about 500 microns in diameter are produced. The suspension material  222  rises up the walls of the center spinning cup  502  of the spinning disk atomizer  302  by centrifugal force and forms an outward radial flow of the suspension material  214 , preferably under directional impetus from grooves in the wall of the spinning cup  502 , as will be described. The droplets  320  are then collected in an outer collection chamber  310 , preferably, an annular collection dish. The outer collection chamber  310  is filled with a gelling solution  312  as described in U.S. Pat. No. 5,578,314 (Cochrum et al.), for example. Typically, the gelling solution  312  is a calcium chloride or barium chloride solution. Other potential gelling solutions could include MgCl 2  and ZnCl 2 , or any equivalent divalent cation solution.  
     [0044] After the second coating step  30 , the suspension with the encapsulated islets is then collected and placed into sterile containers (e.g., 50-ml conical tubes) to settle. After the double-coated capsules  322  have settled, approximately two-thirds of the gelling solution  312  is removed. The double-coated capsules  322  are then combined into a single tube and allowed to settle again. This process typically does not require more than about 10 minutes. The remainder of the gelling solution is carefully removed, replaced with a lesser concentration of buffered calcium chloride solution (preferably, between 1-5 mM) and allowed to settle for about 10-30 minutes. After this, the calcium chloride solution is removed, cell culture media is added to the double-coated capsules, and this mixture is plated out and put into an incubator.  
     [0045] An advantage of the present invention is that the multilayer coating is applied in a short time so that the living biological material is not stressed and a high degree of viability is maintained. In a preferred embodiment, the total processing time for batches of at least 100,000 cell clusters is less than two to three hours. Also, the present invention allows for the coating of relatively large-scale batches of at least 100,000 cell clusters per batch and optimally more than 500,000 cell clusters per batch. Furthermore, effective singularizing of the single-coated capsules allows for the application of a second polymeric layer of a substantially uniform thickness to a large number of the individual capsules in a convenient, controllable, and reproducible manner. Multilayered capsules containing islets produced with this approach have been shown to function normally for more than one year in mice.  
     [0046] Thus, significantly, the present invention reduces the amount of aggregation of the single-coated capsules during the second coating process and allows the subsequent coatings to be applied to a high percentage of the capsules and allows for good bonding between coating layers. The thickness and permeability of the coatings may be controlled by the selection of the various process parameters. The concentration of the capsules in the alginate suspensions can be maintained at a higher density without aggregating or gelling. Most importantly, all of this is accomplished in such a way as to provide for the consistent and effective manufacture of large-scale batches of cell clusters. The fact that the present invention can process large-scale batches of cell clusters allows for double layer encapsulation of all of the cell clusters from a donor organ as part of one large-scale batch process. The ability to process an entire donor organ in a single process is desirable from a number of perspectives, including quality control, efficiency and regulatory approval.  
     [0047] The following sections provide more details about each of the portions of the process and apparatus of the present invention. In the following description, the process is described in terms of encapsulating pancreatic islets; however, it can be applied to a variety of biological materials as will be understood by one of skill in the art. Such biological materials include living tissues, cells, cell lines, and other biologically active substances intended to be implanted into the body of a host. Examples include, but are not limited to, pancreatic islets, hepatic cells, neural cells, renal cortex cells, vascular endothelial cells, thyroid cells, adrenal cells, thymic cells, and ovarian cells. Preferably, the biological material includes pancreatic islets. For purposes of the present invention, the biological material to be encapsulated will be generally referred to as cell clusters, recognizing that such a cell cluster may contain as little as one cell or a very large number of cells, but preferably such cell clusters are arranged to yield a double-coated capsule having an average final resulting diameter on the order of no more than about 350-600 microns.  
     [0048] Preferred Initial Encapsulation Process  
     [0049] Islets from a donor are typically maintained in sterile cell culture dishes. These islets are collected, allowed to settle, washed, and placed in a saline solution. This can be done by placing them in conical tubes (e.g., of about 50-ml. volume), for example. Cell culture media is carefully removed, for example using a pipette. The collected cells are combined into one tube and allowed to settle and are then washed with a suitable buffer. For example, a solution containing 0.9 wt-% sodium chloride buffered with 10 mM HEPES (N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulfonic acid]) (pH 7.2) can be used. The islets are again allowed to settle in the conical tube and the washing process is repeated, typically three times (with about 30 milliliters (ml) for each washing). The islets are then transferred to a shorter conical tube after the third wash and excess saline solution is removed until the volume of saline is approximately equal to twice that of the islets. The saline drawn off in this manner is discarded.  
     [0050] The islets are coated with a first polymeric coating. This can be done using a variety of suitable polymeric materials. Such coating materials are preferably sufficiently permeable to permit effective diffusion of nutrients and other essential biological materials into the transplanted material and passage of transplant tissue products therefrom into the host system. Typically and preferably, the polymeric coating materials for the first coating are water-soluble, natural or synthetic polysaccharides, typically in the form of gums. Examples include, but are not limited to, alginates, guar gum, gum arabic, charageenan, chitosan, pectin, tragacanth gum, xanthan gum, or acids thereof. Preferably, the first coating material is an alginate containing a relatively high level of guluronate relative to mannuronate (“high G/M” alginate or simply “G alginate” coating composition), preferably wherein the mannuronate alginate to guluronate alginate is in a ratio of greater than about 50:50 as disclosed in U.S. Pat. No. 5,578,314 (Cochrum et al.). More preferably, the polymer composition is prepared according to Example 1 of U.S. Pat. No. 5,578,314 (Cochrum et al.).  
     [0051] The islets can be coated with such polymeric materials in a variety of ways. In one embodiment, the saline and islets are drawn up with a syringe of appropriate volume with an appropriate size catheter (e.g., 14-gauge catheter), for example, to collect all of the saline plus islets. The suspension is drawn completely into the syringe taking care not to introduce air bubbles. The G alginate (preferably, diluted to about 1.5%) is placed into a second syringe of appropriate size, which is then attached to one side of a Y-syringe connector which has been primed with alginate. The syringes containing the islets and saline are attached to the other side of the Y-connector. The islets in saline and the alginate are mixed by emptying their respective syringes simultaneously through the Y-connector into a container (e.g., a settling tube). The mixing of the alginate and islets in the container is continued by any suitable mechanical agitator (e.g., a vortex mixer), taking care not to introduce air bubbles, until a uniform mixture has been achieved.  
     [0052] In a preferred embodiment, the islets are transferred to a syringe  100  with an equal volume of saline. An amount of G-alginate with a weight approximately equal to the weight of the islets and saline is placed in a container (not shown) and the syringe  100  is used to draw the G-alginate into the islets. Gently mixing of the G-alginate with the islets can be accomplished by any of a variety of techniques. Preferably, the G-alginate and islets are gently mixed by manually rolling or otherwise manipulating the syringe  100  to obtain a uniform mix and color of the first alginate suspension. The relative volumes of the islets plus saline and the first polymeric coating composition are selected to produce a mixture having a sufficient viscosity for adequate mixing and coating and to provide the desired thickness and morphology of the coating. Preferably, for a 1% alginate solution, the viscosity is about 15 centipoise (cps) to about 5000 cps. A preferred range of solution for the first alginate solution is between 1% and 2%. These relative amounts can be determined without undue experimentation by one of skill in the art. It will be recognized that other forms of dispersing the cell clusters with the first polymeric coating solution can also be used, such as mechanical dispersion or liquid dispersion.  
     [0053] The process of coating includes a variety of techniques to atomize and gel this first coating composition. For example, the methods described in U.S. Pat. No. 5,578,314 (Cochrum et al.) and U.S. Pat. No. 5,643,594 (Dorian et al.) can be used if desired to prepare capsules containing islets with a first polymeric coating thereon. Alternatively, any of the other known techniques previously discussed for atomization and droplet formation, as well as for gelling and coating the capsules. Preferably, a spinning cup or disk atomizer  300  as described below in connection with FIGS. 4 and 5 is used to form droplets. The droplets are typically collected in a CaCl 2  or BaCl 2  gelling solution, for example, to cross-link or gel the polymer and form single-coated capsules.  
     [0054] Electrostatic Mixing Process  
     [0055] The single-coated living biological material is encapsulated with a second polymeric coating. This can be done using a variety of suitable polymeric materials. As with the first coating material, these coating materials are preferably sufficiently permeable to permit effective diffusion of nutrients and other essential biological materials into the transplanted material and passage of transplant tissue products therefrom into the host system. Typically and preferably, the polymeric coating materials for the second coating are water-soluble, natural or synthetic polysaccharides, typically in the form of gums. Examples include, but are not limited to, alginates, guar gum, gum arabic, charageenan, chitosan, pectin, tragacanth gum, xanthan gum, or acids thereof. Preferably, the second coating material is an alginate containing a relatively high level of mannuronate alginate relative to guluronate alginate (“high MIG alginate” or simply “M alginate” coating composition), preferably wherein the guluronate alginate to mannuronate alginate is in a ratio of greater than about 50:50 as disclosed in U.S. Pat. No. 5,578,314 (Cochrum et A.).  
     [0056] Prior to application of the second coating, the single-coated capsules are collected and washed with a suitable agent, typically three times (with about 30 ml for each washing). For example, a solution containing 0.9 wt-% sodium chloride and 3 mM calcium chloride (pH 7.2) buffered with 10 mM HEPES can be used. The single-coated capsules are then mixed with a suitable carrier for introduction into the second polymeric coating composition. For example, the carrier can include 0.9 wt-% sodium chloride buffered with 10 mM HEPES.  
     [0057] In one embodiment of the electrostatic mixing process, as shown in FIG. 2, the suspension of single-coated capsules in a carrier (e.g., physiological saline) is placed in a syringe  201  or other suitable means. The mixture is gently agitated to ensure that the mixture is uniform. Any entrained air bubbles are preferably allowed to float to the top surface. The syringe containing the single-coated capsules preferably has a conductive orifice as a result of the tip of syringe  201  being mounted within a stainless steel charging collar  203  or fitted with a conductive orifice  202  or other conductive material. The orifice  202  preferably has a diameter of about 0.2 mm to about 5 mm, and more preferably about 1.5 mm to about 2 mm. As shown best in FIG. 6, the charging collar  203  is located about a syringe  201  for purposes of applying the electrostatic separation charge. Preferably, the conductive charging collar  203  includes a slightly tapered center channel  250  through which the carrier medium  200  containing the single-coated capsules is forced. Preferably, the orifice  202  or charging collar  203  is connected to a high voltage source to provide a DC voltage of about 1 kilovolt (kV) to about 100 kV (more preferably, about 5 kV to about 30 kV). In a particularly preferred embodiment, the electrostatic voltage is set at approximately 20 kV.  
     [0058] In a preferred embodiment as shown in FIG. 7, the charging collar  203  includes a flange portion  252  about the center of the charging collar  203  that is supports the charging collar  203  with the syringe  201  mounted therein in a slot  262  of a base  264  of a stand apparatus  260 . Preferably, a stabilized mounting column  266  of the stand apparatus  260  is electrically isolated from at least a pair of mounting arms  268  by a corresponding pair of insulators  270 . A syringe driver  272  is slidably mounted on a channel  274  defined in a face bracket  276  that is connected between base  264  and a top one of the mounting arms  268 . The syringe driver  272  may be selectively moved and locked in a desired position by a wing nut or equivalent releasably locking mechanical arrangement. The syringe driver  272  is preferably fluidly coupled to an automated pumping apparatus (not shown) that precisely controls an amount of fluid driven into the syringe driver  272 . Alternatively, a stepper motor or other automated motion control apparatus could be used to drive the syringe  201  to expel the first polymeric suspension  200 .  
     [0059] In this electrostatic mixing process, the syringe driver  272  is used to force the suspension at a preselected constant flow rate through the orifice  202  or charging collar  203 . The charge induced in the liquid forces the liquid out the orifice and into a sinuous threadlike fiber or ligament. The process of drawing out the liquid into the conical shape is shown schematically in FIG. 3. The high voltage on the collar is adjusted to produce a single sinuous stream of liquid, given a preset flow rate. For a particularly preferred embodiment, the voltage and flow rate are set at 20 kV and 2.75 ml/min in a preferred embodiment, and an approximate current of 0.1 mA (milliampere). It will be seen that both the flow voltage and flow rate associated with the electrostatic mixing process  20  of the preferred embodiment of the present invention are significantly different than flow rates (typically less than 0.5 ml/min) used in conventional electrostatic droplet generators for polymeric coating materials having similar properties to the properties of the coating materials described herein.  
     [0060] Referring to FIG. 2, and as shown in greater detail in FIG. 3, the high voltage applied to the conductive orifice  202  or charging collar  203  forms an attenuated varicose liquid stream  204  that forms a “varicose bulge”  206  due to the presence of a capsule  122  and breaks up into droplets  210 . These droplets  210 , which preferably include only one capsule  122  fall into a second polymeric coating material  214 .  
     [0061] The second polymeric coating composition, preferably at ground potential, in a container  212  of a vortex mixer  213  is located beneath the electrostatic discharge orifice to collect the droplets  210 . The vertical distance from the top of the container  212 , or the surface of the second coating composition  214  contained therein, to the exit of the syringe  201  is adjusted to produce an adequate electrostatic field, and to allow complete separation of the droplets  210  as they fall from the syringe. This distance can be experimentally determined without undue experimentation by one of skill in the art. It can range from millimeters to meters.  
     [0062] As shown in FIG. 2, the droplets  210  impact the moving surface of the second polymeric coating composition  214  and are swept into the composition through the agitation action of the mixer  213 . The high relative potential on the capsules  122  in the droplets  210  provides the additional force needed to drive the capsules into the viscous polymeric composition. Since liquid alginate is semi-conductive, a residual charge will likely remain in the single-coated capsules (“G” alginate) even after they are engulfed by the high mannuronate alginate (“M” alginate). This residual charge is believed to serve to help prevent aggregation of the capsules. Thus, the mixing step  20  of this invention facilitates singularizing the capsules and then separating the capsules to disperse them effectively into the second polymeric coating composition, thereby forming a “suspension” or “suspension material.” 
     [0063] The relative volumes of the single-coated islets plus carrier and the second polymeric coating composition are selected to produce a mixture having a sufficient viscosity for adequate mixing and coating and to provide the desired thickness and morphology of the coating. Unlike the prior art techniques that required larger amounts of the second polymeric coating, the electrostatic mixing process  20  of the present invention requires much smaller quantities of the second polymeric coating solution  214 . For a given volume of single-coated capsules  122  in saline solution  200  (e.g., a volume of capsules  122  in approximately one to two times, and preferably about 1.5 times, the same volume of saline solution  200 , the weight of second alginate solution  214  required for effective encapsulation is between about two to three times the combined weight of the capsule  122  and saline  200 , and, more preferably, about 2.2 times the combined weight of the capsules  122  and saline  200 . The amounts and volumes of the second alginate solution  214  are preferably chosen to yield a range of about 2.5% to 4% of the M-alginate, and more preferably about 2.8%.  
     [0064] The mixer  213  is allowed to run for a sufficient amount of time (e.g., at least approximately 30 seconds) to ensure that complete mixing of the capsules, saline, and the second polymeric coating composition takes place. Preferably, the mixer  213  is bolted or otherwise secured to a workbench to insure more uniform agitation action. With other encapsulation polymers and biological material, the time may be longer or shorter. This could be determined experimentally. One skilled in the art would easily determine what level of mixing is appropriate for the process at hand. Failure to completely mix the suspension may not result in a homogeneous suspension, which can be detrimental to the second coating process.  
     [0065] Preferred Encapsulation Process  
     [0066] Once the single-coated islets are mixed with the second polymeric coating composition, the single-coated islets are encapsulated with a second coating. FIG. 4 illustrates an encapsulation apparatus  300  including a spinning disk atomizer  302  that is preferably used in the second encapsulation process. The atomizer  302  receives single-coated islets suspended in the second polymeric coating composition (referred to collectively as the “suspension material” or “suspension” from the previous electrostatic mixing process), from a syringe  304  and catheter  306  (e.g., 20-gauge catheter). This syringe  304  and catheter  306  are positioned above the spinning disk atomizer  302 . The tip of the catheter  306  is typically placed very close to the bottom of the reservoir of the spinning disk atomizer  302  (typically, about 0.7 mm from the bottom of the spinning disk reservoir), as shown in FIG. 4. A drive mechanism  308  (e.g., a syringe pump) is preferably used to feed the mixture at a fixed flow rate (e.g., between 0.5 ml/minute and 5 ml/minute and preferably about 1.2 ml/minute for the particular configuration of the size and shape of the atomizer  302  of the preferred embodiment).  
     [0067] Once the single-coated capsules in the second suspension  222  have just filled the bottom of a reservoir portion (as described below with reference to FIG. 5), the spinning disk atomizer  302  is then rotated at an appropriate speed. Through centrifugal motion, spherical droplets of the suspension material of about 50 microns to about 500 microns in diameter are produced. This is referred to herein as atomization. The suspension material rises up the walls of the spinning disk atomizer by centrifugal force and forms an outward radial flow of the suspension material from grooves in the wall (described in greater detail below). The droplets  320  are then collected in an outer collection chamber  310 , preferably, an annular collection dish. The collection dish is preferably formed of a polypropelene material, although other plastic or metal materials could be used. The outer collection chamber  310  is filled with a gelling solution  312  as described in U.S. Pat. No. 5,578,314 (Cochrum et al.), for example. Typically, the gelling solution  312  is a calcium chloride solution. Preferably, this includes about 150 ml of 10 mM HEPES buffer with 120 mM calcium chloride solution (pH 7.2).  
     [0068] The spinning disk atomizer  302  is rotated until the syringe  304  is empty. The outer annular collection chamber  310  is also rotated at a speed sufficient to attain a sufficient depth of the gelling solution in the inside of the outer wall. The direction of rotation of the annular collection chamber  310  is preferably the same direction as the direction of rotation of the spinning disk atomizer  302 . Preferably, a wall of the outer collection chamber  310  is slightly angled at an angle equivalent to an angle of the gelling solution  312  tends to maintain when the outer collection chamber  310  is rotated at the desired operating speed.  
     [0069] The spinning disk atomizer  302  may be operated at different rotational speeds depending on the size of drops required and the diameter of the center reservoir, the viscosity and surface tension of the polymeric composition, and other process parameters. One skilled in the art would know that the fundamental correlations for rotating atomizers and/or experimental trials could be used to establish the desired operating conditions. Also, one skilled in the art would recognize that the tangential component of the drop velocity of the droplet should be similar to the tangential speed of the outer collection chamber  310 .  
     [0070] Preferably, the spinning disk atomizer  302  is rotated at a rate of about 2000 revolutions per minute (rpm) to about 8000 rpm, and more preferably, at a rate of about 4000 rpm. Preferably, the outer collection chamber  310  is rotated at a speed of between 300 to 500 rpm. In a preferred embodiment, the distance between the edge of the atomizer  302  and the outer collection chamber is between 5-20 cm, although the optimum distance will depend upon the speeds at which both the spinning disk atomizer  302  and the outer collection chamber  310  are rotated.  
     [0071] The suspension with the encapsulated islets  322  is then collected and placed into sterile containers (e.g., 50-ml conical tubes) to settle. After the double-coated capsules have settled, approximately two-thirds of the gelling solution is removed. The double-coated capsules are then combined into a single tube and allowed to settle again. This process typically does not require more than about 10 minutes. The remainder of the gelling solution is carefully removed, replaced with 30 ml of 10 mM HEPES buffer mixed with 0.9 wt-% sodium chloride and 3 mM calcium chloride solution (pH 7.2), and allowed to settle for about 30 minutes.  
     [0072] As shown in FIG. 4 and FIGS. 8 a  and  8   b , a preferred embodiment of the encapsulation apparatus  300  utilizes an adjustable syringe adapter  340  to accommodate different size syringes  100 ,  304  in a stand apparatus  330 . The stand apparatus  330  includes a support column  332  and a pair of support arms  334  that are preferably selectively shiftable on the support column to accommodate different sizes of syringes  100 ,  304 . The adapter  340  is preferably seated in an aperture (not shown) defined on a lower one of the support arms  334 , although other mechanical arrangements for mounting the adapter  340  to the support arm  334  are contemplated. The adapter  340  includes an upper portion  342  with a tab  344  and a bottom portion  346  with a tab  348  each having a corresponding aperture  350 ,  452  for accommodating a syringe of a particular size. The distance between the upper tab  344  and the bottom tab  348  is preferably selectable, such as by including an upper channel  354  and a bottom channel  356  with a mating extension bar  358  releasably secured to each portion  342 ,  346  of the adapter  340 . Any of a number of alternative mechanical arrangements for selectively positioning the height of the upper tab  344  relative to the bottom tab  348  could also be utilized. In one embodiment for islet cells as shown in FIGS. 8 a  and  8   b , an adapter  340  is provided for each of a number of different size syringes, such as a 3 ml (for up to about 100,000 cell clusters), 12 ml (for up to about 350,000 cell clusters), 35 ml (for up to about 750,000 cell clusters) and 60 ml (for up to about 1,200,000 cell clusters).  
     [0073] Spinning Disk Atomizer  
     [0074] Attention is now directed to the particular construction of an exemplary spinning disk atomizer  500  as illustrated in FIGS.  5 A- 5 D. A more expanded description of an exemplary spinning disk atomizer of this type without the uniquely modified central spinning cup of the present invention is shown and described in U.S. Pat. No. 5,643,594 (Dorian et al.) and U.S. Pat. No. 6,001,387 (Cochrum), the disclosures of which are hereby incorporated by reference. While described with particularity, the atomizer illustrated in the accompanying figures and discussed below is intended to be exemplary only. Modifications to the spinning disk atomizer  500  as well as to the remainder of the encapsulation apparatus are certainly possible without departing from the scope of the invention. It is preferred that the spinning disk atomizer  500  is used for both the first coating process  10  and the second coating process  30 , although it will be understood that other atomization and gelling techniques could be used for one or the other of these coating processes with lesser effectiveness.  
     [0075]FIG. 5A illustrates a side elevation view of the atomizer  500 . The atomizer  500  is preferably made from stainless steel or similar material. As shown more clearly in FIG. 5B, the atomizer  500  forms a cup-shaped receptacle  502  for receiving the suspension material. Access to the receptacle  502  is provided via an opening  506  (see FIGS. 5A and 5C) formed in a first end  523  of the atomizer  500 . As is evident in FIG. 5C, a lip  512  surrounds the opening  506 .  
     [0076] Formed within the bottom of center cup receptacle  502  is a reservoir portion  504  (also shown in FIG. 5C) adapted to receive and hold the suspension material. The reservoir portion  504  may have most any shape but is typically semi-spherical or, alternatively, slightly tapered, e.g., conically shaped having a cone angle of about 2 degrees. The receptacle  502  preferably tapers between the receptacle opening  506  and the reservoir portion  504 . That is, a first wall portion, e.g., frustoconical surface  508 , may extend from the opening  506  to the reservoir portion  504 .  
     [0077] The receptacle  502  may further be defined by one or more grooves  510  extending along the frustoconical surface  508  as best shown in FIGS. 5B and 5C. Preferably, four evenly spaced grooves  510 , i.e., every 90 degrees, are included. However, other configurations may include other numbers of grooves having regular, e.g., even, or irregular spacing. The grooves  510  are preferably cut through the lip  512  as illustrated in FIG. 5C, i.e., the grooves  510  “break through” the lip  512 . Alternatively, the grooves  510  may be feathered out prior to breaking through the lip  512 .  
     [0078] In a preferred embodiment, a second set of grooves  514  generally aligned with and corresponding to the grooves  510  are preferably located just above the reservoir portion  504  in a second wall portion  522  below frustoconical first wall portion  508  as shown in FIGS. 5B, 5C, and  5 D. The second wall portion  522  is generally cylindrical in shape. Alternatively, the second wall portion  522  may be slightly tapered, e.g., conically shaped having a cone angle of about 2 degrees. While shown as larger in size, the grooves  514  may actually be of identical or, alternatively, smaller or larger in size than the grooves  510 . The grooves  514  may extend along the second wall portion  522  from the frustoconical surface  508  to the bottom of the reservoir portion. Alternatively, but less preferably, the grooves  514  may run out or terminate prior to the bottom of the reservoir portion  504  as generally indicated in FIG. 5B.  
     [0079] Preferably, the grooves  514  and  510  form indentations, e.g., semi-cylindrical channels, along the sides of the reservoir portion  504  and the frustoconical surface  508 , respectively. The grooves may be formed by any of a number of various machining processes including EDM techniques and conventional drilling/milling. The transition between intersecting grooves  510  and  514  is preferably smooth, i.e., there are no sharp edges or obstructions that may interfere with fluid flow between the reservoir portion  504  and the opening  506 . While illustrated and described as semi-cylindrical channels, the grooves  510  and  514  may be of most any other machinable cross-sectional shape.  
     [0080] A second end  524  (see FIG. 5A) of the atomizer  500  includes a threaded portion  526  adapted to permit coupling of the atomizer to a receiving structure as further described bellow.  
     [0081] Dimensions of an atomizer  500  in accordance with one embodiment of the invention will now be described. Of course, these dimensions are exemplary only and are by no means limiting. In practice, dimensions of the atomizer are highly interrelated and are typically selected based on various factors including, for example, properties of the suspension material.  
     [0082] In one embodiment, the receptacle opening  506  may have an outer diameter  516  (see FIG. 5B) of about 1 inch (25 mm) and a lip thickness  518  (see FIG. 5C) of about 0.030 inch (0.77 mm). The lip thickness  505  at the point at which grooves  510  meet the lip  512  is about 0.008 inch (0.2 mm). The receptacle  502  may further taper to a reservoir diameter  520  (see FIG. 5D) of about 0.25 inch (6.3 mm) which may extend to a depth of second wall portion  522  (see FIG. 5B) of about 0.42 inch (10.6 mm).  
     [0083] Extending along at least part of the depth of second wall portion  522  are the grooves  514 . The grooves  514  may have a radius  515  (see FIG. 5D) of about 0.40 inch (1.0 mm). The grooves  510 , on the other hand, preferably have a radius of about 0.015 inch (0.41 mm). The frustoconical surface  508  may form an angle  509  (see FIG. 5B) of about 25 degrees, yielding a cone angle of about 50 degrees.  
     [0084] Referring back to FIG. 4, the spinning disk atomizer  302  is illustrated as being positioned within a clearance opening in the annular collection chamber  310 . The threaded portion ( 526 , FIG. 5A) of the atomizer  302  is adapted to couple to a drive motor  312  (FIG. 4). Once coupled, the atomizer  302  may spin independently of the collection chamber  310  under the driving power of the motor  314 . The annular collection chamber  310  may optionally be driven by an independent external motor  316  as also shown in FIG. 4.  
     [0085] Referring again to FIG. 4, during operation, the atomizer  302  is filled via the catheter  306  with the suspension material until the level reaches the top (i.e., the point at which the grooves meet the reservoir) of the reservoir ( 504 , FIG. 5B). The drive motor  314  is then activated and the atomizer  302  begins to rotate. While the rotational speed of the atomizer  302  may vary depending on factors such as atomizer size and configuration, suspension material viscosity, etc., speeds in the range of about 2000 rpm to about 8000 rpm have been found to produce satisfactory results. As the atomizer  302  spins, friction between the atomizer and the suspension material accelerates the latter, causing it to rise up out of the reservoir portion ( 504 , FIG. 5B) and onto the frustoconical wall portion ( 508 , FIG. 5B). The wall portions and the grooves ( 508  and  522 ,  510  and  514 , FIG. 5B) assist the flow of the suspension material upwardly and outwardly. When the suspension material reaches the lip ( 512 , FIG. 5C), ligaments or spherical drops form and are projected radially outward.  
     [0086] Due to centrifugal forces, the ligaments are dynamically unstable. Surface tension ultimately causes the ligaments to format spherical drops of nearly uniform size. These drops preferably have a diameter that ranges from about 100 μm to about 350 μm. The actual diameter is dependent on many factors though, including rotational speed, flow rate to the atomizer, surface tension and viscosity of the suspension material, and the size of the biological material encapsulated to name a few.  
     [0087] The complete disclosures of the patents, patent documents, and publications cited herein are incorporated by reference in their entirety as if each were individually incorporated. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.