WO2012167051A2

WO2012167051A2 - Microencapsulation device having microfluidic channels and related methods

Info

Publication number: WO2012167051A2
Application number: PCT/US2012/040423
Authority: WO
Inventors: Melur K. Ramasubramanian; Sameer TENDULKAR
Original assignee: North Carolina State University
Priority date: 2011-06-03
Filing date: 2012-06-01
Publication date: 2012-12-06
Also published as: WO2012167051A3

Abstract

Devices for the microencapsulation of biological units include a chamber for containing a plurality of biological units suspended in a liquid, and a plurality of liquid conduits in fluid communication with the chamber. Each of the plurality of liquid conduits has an encapsulation outlet. The device includes a compressed gas inlet, and a plurality of compressed gas conduits in fluid communication with the compressed gas inlet. Each of the plurality of compressed gas conduits has a compressed gas outlet that at least partially surrounds a respective encapsulation outlet. The encapsulation outlets and the compressed gas outlets are sized and configured to encapsulate the biological units from the solution when the biological units pass from the chamber through the plurality of fluid conduits and exit the encapsulation outlets.

Description

MICROENCAPSULATION DEVICE HAVING MICROFLUIDIC CHANNELS AND

RELATED METHODS

RELATED APPLICATIONS

[0001] This applications claims priority to U.S. Provisional Application No.

61/493,011 , filed June 3, 2011, the disclosure of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to devices for the microencapsulation of cells, and more particularly to multi-outlet configurations for increasing microencapsulation outputs.

BACKGROUND

[0003] Microencapsulation is an immunoisolation technique available for

immunoprotection of cells to be transplanted, which reduces or eliminates the use of immunosuppressive drugs. Uludag H, De Vos P, Tresco PA: Technology of mammalian cell encapsulation. Adv Drug Delivery Rev 42: 29-64, 2000. Although microencapsulation as a viable procedure to immunoisolate cells for transplantation was introduced more than thirty years ago.(Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 210: 908-910, 1980), it has had slow progress towards clinical application, for example, due to slow production rates and the appearance of fibrotic overgrowths around the capsules, which can result in endotoxin contamination, e.g., oxygen and nutrient deprivation of the enclosed cells.

[0004] One of the many prospective applications of this technology is the

development of a reliable bioartificial liver in the form of encapsulated hepatocytes, for providing temporary but adequate metabolic support to allow spontaneous liver regeneration, or as a bridge to orthotopic liver transplantation for patients with fulminant hepatic failure.

Joly A, Desjardins J-F, Fredmond B, et al. Survival, proliferation, and functions of porcine hepatocytes encapsulated in coated alginate beads: a step toward a reliable bioartificial liver. Transplantation 63: 795-803, 1997.

[0005] Examples of devices for microencapsulation include the air-syringe pump droplet generator (Wolters GH, Fritschy WM, Gerrits D, Van Schilfgaarde R: A versatile alginate droplet generator applicable for microencapsulation of pancreatic islets. J Appl Biomat 3 : 281-286, 1992) and the electrostatic bead generator (Hsu BR-S, Chen H-C, Fu S- H, Huang Y-Y, Huang H-S: The use of field effects to generate calcium alginate

microspheres and its application in cell transplantation. J Formos Med Assoc 93: 240-245, 1994.). Each of these devices is fitted with a single needle through which droplets of cells suspended in alginate solution are produced and cross-linked into spherical beads. Various methods for the production of encapsulated cells in increased numbers have been attempted, including the simultaneous production of multiple droplets in a multiple needle approach (De Vos P, De Haan BJ, Schilfgaarde R. Upscaling the production of microencapsulated pancreatic islets. Biomaterials 18: 1085-1090, 1997) or increasing the number of cells/mL of alginate suspension in the syringe to increase the probability of the formation of encapsulated cells. However, air-syringe pump droplet generators or electrostatic bead generators may be incapable of producing sufficient numbers of microcapsules in a short-time period to permit mass production of encapsulated and viable cells for transplantation in large animals and humans. Moreover, a prolonged process of encapsulation of cells may adversely affect the viability of the cells.

[0006] For example, in a study with four nozzles, the nozzles are fitted to a header plate wherein the cell in alginate is supplied and pushed through four hypodermic needles. De Vos P, De Haan BJ, Schilfgaarde R. Upscaling the production of microencapsulated pancreatic islets. Biomaterials 18: 1085-1090, 1997. However, this may not be scaled up effectively because of the support mass surrounding the hypodermic needle such as couplings and seals, which provides a spacing of about 1 cm between needles. An increase in the number of joints in the flow path through which the cell - alginate suspension travels may result in a higher possibility of stagnation, clogging, and contamination. Furthermore, alginate solutions used for encapsulation are viscous, making the process potentially fraught with the risk of clogging when small gauge needles are used to produce microcapsules of desirable size range (e.g., <800 microns in diameter). When needle clogging occurs, the process of unclogging the needle for resumption of encapsulation further increases the duration of microencapsulation of large batches of cells for therapeutic use. High density of parallel needles may not provide the access needed to clean clogged needles in the center of the array in a fairly dense grid. Multiple needles with a common flow header may not be viable and/or efficient.

[0007] Even using such an approach, production rates at several orders of magnitude higher may be desirable to meaningfully produce sufficient quantities of encapsulated and viable cells for transplantation in human subjects. For example, it has been estimated that for the 1 million islets needed for transplantation in a diabetic human subject, about 100 hours may be required to complete the encapsulation of this number of islets, assuming one islet/microcapsule and a single needle operation. However, in practice, it has actually been estimated that the duration of the process may be closer to 200 hours because of the additional steps involved in the encapsulation procedure, following the generation of the initial cell-containing alginate microspheres.

[0008] Moreover, it has been reported that by using the syringe method, the proportion of spheres that contain cells is only about 50%. Attempts may be made to increase the concentration of cells in the alginate suspension to increase the chance process of encapsulating a cell and thereby increasing the productivity. However, this may provide only a two-fold increase in productivity. Further, an increase in the number of cells/mL alginate could cause an increase in the number of beads of cells with imperfections, such as cell protrusion into the bead membrane. Protrusion of encapsulated tissue through the

microcapsule membrane may activate the cell-mediated host immune response leading to microcapsule transplant rejection. Sun AM. O'Shea GM. Goosen MF: Injectable

microencapsulated islet cells as a bioartificial pancreas. Appl Biochem Biotechnol 10: 87-99, 1984.

SUMMARY OF EMBODIMENTS OF THE INVENTION

[0009] In some embodiments, devices for the microencapsulation of biological units include a chamber for containing a plurality of biological units suspended in a liquid, and a plurality of liquid conduits in fluid communication with the chamber. Each of the plurality of liquid conduits has an encapsulation outlet. The device includes a compressed gas inlet, and a plurality of compressed gas conduits in fluid communication with the compressed gas inlet. Each of the plurality of compressed gas conduits has a compressed gas outlet that at least partially surrounds a respective encapsulation outlet. The encapsulation outlets and the compressed gas outlets are sized and configured to encapsulate the biological units from the solution when the biological units pass from the chamber through the plurality of fluid conduits and exit the encapsulation outlets.

[0010] In some embodiments, the compressed gas outlets substantially concentrically surround respective encapsulation outlets. At least a portion of the compressed gas conduits may substantially concentrically surround and may be coaxial with at least a portion of respective fluid conduits.

[0011] In some embodiments, the chamber, the plurality fluid conduits, and the plurality of compressed gas conduits are formed as a single unitary member. The single unitary member may include an polymeric material.

[0012] In some embodiments, the chamber includes at least one inlet. The chamber further may include at least one gas outlet that has a first open configuration configured for allowing gas to be released from the chamber when the biological units and the liquid are entering the chamber and a second closed configuration configured to substantially prevent the liquid from exiting the chamber. The at least one inlet of the cell-solution suspension chamber may be configured to connect to a variable flow pump.

[0013] In some embodiments, the biological unit is a cell or a protein. In some embodiments, the biological unit is suspended in an alginate.

[0014] In some embodiments according to the present invention, methods for the microencapsulation of biological units include flowing a plurality of biological units suspended in a liquid into a chamber, and flowing the plurality of biological units suspended in the chamber through a plurality of liquid conduits in fluid communication with the chamber. Each of the plurality of liquid conduits has an encapsulation outlet. The method further includes flowing a gas through a plurality of compressed gas conduits. Each of the plurality of compressed gas conduits has a compressed gas outlet that at least partially surrounds a respective encapsulation outlet so that the encapsulation outlets and the compressed gas outlets encapsulate the biological units from the solution when the biological units pass from the chamber through the plurality of fluid conduits and exit the encapsulation outlets.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the

description, serve to explain principles of the invention.

[0016] Figure 1 is a side view of a device according to some embodiments of the present invention.

[0017] Figure 2 is a side perspective view of the device of Figure 1.

[0018] Figure 3 is a side cross-sectional perspective view of the device in Figure 2 along cross sectional line 3-3.

[0019] Figure 4 is another side perspective view of the device of Figure 1.

[0020] Figure 5 is a side cross-sectional perspective view of the device in Figure 4 along cross-sectional line 5-5.

[0021] Figure 6 is an exploded perspective view of encapsulation nozzles of the device in Figure 1.

[0022] Figure 7 is a transparent perspective view of the device of Figure 1

illustrating exemplary flow patterns according to some embodiments of the present invention.

[0023] Figure 8 is a graph of the diameter of the droplets produced by the device in

Figure 1 as a function of frequency for different flow rates according to some embodiments of the present invention.

[0024] Figure 9 is a graph of the diameter of the droplets produced by the device in

Figure 1 as a function of frequency for different air pressure rates applied by the compressed gas supply according to some embodiments of the present invention.

[0025] Figure 10 is a graph of the diameter of the droplets produced by the device in

Figure 1 as a function of frequency for different alginate concentrations according to some embodiments of the present invention.

[0026] Figures 11A-11B are digital images of droplets formed at collection distances of 7.5 inches (Figure 11A) and 13 inches (Figure 11B) from the encapsulation nozzle of the device of Figure 1 according to some embodiments of the present invention.

[0027] Figure 12 is a digital image of droplets formed at a collection distance of 10 inches from the encapsulation nozzle of the device of Figure 1 according to some embodiments of the present invention.

[0028] Figures 13A-13B are digital images of protein (BSA) encapsulated hydrogel beads that are encapsulated using the device of Figure 1 according to some embodiments of the present invention.

[0029] Figures 14A-14C are digital images of rat pancreatic islets within the alginate capsules produced by the device in Figure 1 (Figure 14A), a parallel phase contrast image of rat pancreatic islets encapsulated with the device of Figure 1 (Figure 14B), and fluorescently labeled pancreatic islets from live and dead cells encapsulated with the device of Figure 1 and stained with carboxyfluorescein diacetate (CFDA; green) and propidium iodide (PI; red), respectively, and nuclear counterstain 4', 6-diamidino-2-phenylindole (DAPI; blue) (Figure 14C) according to some embodiments of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0030] The present invention now will be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0031] Like numbers refer to like elements throughout. In the figures, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity.

[0032] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y." As used herein, phrases such as "from about X to Y" mean "from about X to about Y."

[0033] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein, Well-known functions or constructions may not be described in detail for brevity and/or clarity.

[0034] It will be understood that when an element is referred to as being "on,"

"attached" to, "connected" to, "coupled" with, "contacting," etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[0035] Spatially relative terms, such as "under," "below," "lower," "over," "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features.

Thus, the exemplary term "under" can encompass both an orientation of "over" and "under." The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly," "downwardly," "vertical," "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0036] It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element discussed below could also be termed a "second" element without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0037] As illustrated in Figure 1, a microencapsulation system 10 includes a microfluidic device 100 and a container 200 for receiving encapsulated cells 210 from the device 100. The device 100 includes a chamber 110, nozzles 115 and a compressed air delivery unit 130.

[0038] The chamber 110 is in fluid communication with an inlet 120 for receiving a liquid having a biological unit suspended therein, such as a cell-solution suspension or protein-solution suspension, for example, from a pump 122 that is used to pump the fluid into the chamber 110. The chamber 110 may also include one or more outlets 124 for allowing overflow air and/or the fluid to escape from the chamber 110. In some embodiments, the outlets 124 may be connected to valves 126 to provide an open configuration for allowing gas to be released from the chamber 110 when the liquid is entering the chamber 110 and a closed configuration to prevent or substantially prevent the liquid from exiting the chamber 110. The chamber 110 is also connected to liquid conduits 112 and outlets 114 on the encapsulation nozzle 115. The compressed air delivery unit 130 includes a chamber 134, conduits 136, and an outlet 138 on the encapsulation nozzle 115. The compressed air delivery unit 130 further includes a compressed air inlet 132 that is connected to a compressed gas supply 142. The compressed gas conduits 136 include a portion 136A that substantially and concentrically surrounds a portion of the cell-solution conduits 112.

[0039] As can be seen, for example, in Figures 3, 5 and 7 the liquid, such as a cell- solution suspension, may be pumped from chamber 110 through the conduits 112 and out of the outlets 114. The compressed gas delivery unit 130 provides a gas, such as air, in a region that surrounds the outlets 114. As illustrated, for example, in Figure 6, the gas outlets 138 generally surround and/or are coaxial with the encapsulation outlet 114. In this configuration, the encapsulation outlets 114 in the gas outlets 138 are sized and configured such that, e.g. , cells in the cell-solution suspension may be encapsulated due to the size and shape of the outlets 114 and the gas flowing from the gas outlet 138. As the cell-solution suspension flows from the outlets 114, the gas from the gas outlets 138 shears off the solution and forms droplets of encapsulated cells. The droplets may be collected in, for example, a calcium chloride bath in the container 200.

[0040] In some embodiments, the device 100 including the chamber 110, the conduits

112, the encapsulated cell outlets 114, the gas conduits 136, and/or the gas outlets 138 are formed of a single unitary member. For example, the design for the device 100 may be generated using SolidWorks™ three-dimensional CAD program (Dassault Systemes

SolidWorks Corp., Massachusetts (USA)) and may be exported to a stereolithography apparatus (SLA) for fabrication using materials such as polymeric materials. For example, SLA can be used to generate a polymeric or plastic (photopolymer) mold, in which a silicon device, such as the device 100, could then be cast. Alternatively, SLA techniques may be used to fabricate the device 100 itself out of the of photopolymer elastomeric or other suitable SLA material. Examples of suitable photopolymers include those available from 3D Systems Corporation, South Carolina (USA), DSM Somos, Elgin, Illinois (USA) (for example Watershed 11120, NanoTool, ProtoTherm 12120) or VeroBlue FULLCURE 840

photopolymer.

[0041] Embodiments according to the invention may provide multiple cell encapsulation nozzles 115, each having a encapsulation outlet 114 and circumferential gas outlet 138, configured to encapsulate particles or biological units, such as cells or proteins (including therapeutic proteins) at each of the nozzles 115. As illustrated, multiple nozzles 115 may be used to provide encapsulated droplets using a common pump 122 and a common liquid source from the chamber 110 that is in fluid communication with the encapsulation outlets 114, together with a single compressed air supply 142 that is channeled through the inlet 132, the chamber 134 and the conduits 136. Thus, biological units, particles, cells and/or protein may be encapsulated via multiple nozzles 115 without requiring separate needle/syringes, each having a liquid source, for potentially increased production.

[0042] Although embodiments of the invention are illustrated with respect to the device 10 with eight nozzles 115, each having liquid conduits 112, encapsulated cell outlet 114, gas conduits 136 and gas outlets 138, it should be understood that other suitable configurations may be used. For example, different numbers of nozzles 115 with associated liquid conduits 112, encapsulated cell outlets 114, gas conduits 136 and gas outlets 138 may be used. For example, in some embodiments, a devices with sixteen, thirty-two, sixty-four or more nozzles using a common fluid chamber and compressed gas chamber may be provided. In addition, the nozzles 115 may be configured in a straight line as illustrated or the nozzles may be formed in a two-dimensional array.

[0043] With reference to Figure 6, the nozzles 115 may have encapsulation outlets

114 having an inner diameter of about 300 to about 500 microns, and the gas outlets 138 may have an inner diameter of about 1.5 to about 3 millimeters, or about 2 millimeters. Various sizes of encapsulated cells, proteins or microbeads (with or without cells) may be formed. In some embodiments, the droplets exiting the outlets 114 have a diameter of between about 400μηι to about 2mm or between about 300μηι to about 1mm. Moreover, the relative gas flow and liquid flow rates or ratios, the distance between the nozzles 115 and the collection chamber 200, and/or concentration of the encapsulation liquid (e.g., alginate) may be used to change or control the size of the droplets formed.

[0044] Any suitable cell-solution suspension may be used in the chamber 110 for cell encapsulation. For example, an alginate/cell solution suspension may be used. Exemplary cells that may be encapsulated include pancreatic cells or liver cells.

[0045] Embodiments according to the present invention may be used to encapsulate cells or other particles or biological units including therapeutic proteins. For example, it has been shown that alginate microspheres may be used to encapsulate therapeutic proteins for controlled drug delivery (Gombotz WR, Wee SF 1998. Protein release from alginate matrices. Adv Drug Deliv Rev 3 1 : 267-285.; Moya M, Lucas S, Francis-Sedlak M, Liu X, Garfmkel M, Opara EC, Brey E 2009. Sustained delivery of FGF-1 increases vascular density in contrast to bolus administration. Microvasc Res 78: 142- 147; Moya M, Huang J- J, Francis- Sedlak M, Kao S, Opara EC, Cheng M-H, Brey E 2010. Effect of FGF-1 -loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering biomaterials. Biomaterials 31 : 2816-2826). Thus, embodiments of the present invention may be used to encapsulate proteins or other suitable biological units or structures for pharmaceutical scale manufacture of microbeads for various purposes, including controlled drug delivery, [0046] Embodiments according to the present invention will now be described with respect to the following nonlimiting examples.

Examples

[0047] A microfluidic chip device such as the device 100 shown in Figures 1-7 may be capable of producing highly monodisperse droplets consists of a 3D air supply and multi- nozzle outlet for bead generation. An eight nozzle device such as the device 10 of Figures 1- 7 was constructed such that the encapsulated cell outlets 114 have a 380 micron inner diameter configured to produce hydrogel beads. The nozzles 115 are concentrically surrounded by air nozzles 138 having an inner diameter of 2mm.

[0048] There are two outlets 124 connected at the top to allow the air to escape through them as the alginate solution fills up the chamber 110. In order to generate hydrogel beads, alginate solution was introduced into the chamber 110 and compressed air was introduced in the air inlet 132. Once substantially all of the air escaped through the two outlets 124, the valves 126 were closed to prevent the alginate from rising in the outlets 124. A pump 122 (a variable flow pump 1 15 V (Thermo Fisher Scientific Inc., USA)), was used to pump alginate solution into the microfluidic device 10. Tygon® tubing (Fisher Scientific, USA) was used to connect the compressed gas supply 142 to the gas inlet 132 and to connect the pump 122 (e.g., a peristaltic/syringe pump) to the chamber 110. A pressure regulator was used to control the flow rate of air from the compressed gas supply 142. As the alginate solution was pumped into the microfluidic device 100, it filled up the chamber 110 and then started flowing out through the internal alginate nozzles 114. The air flowing through the outer nozzle 138 then sheared off the alginate solution and formed droplets, which were collected in a calcium chloride bath in the container 200 where the hydrogel beads crosslink to form microcapsules. These droplets thus formed are then further analyzed for shape, size, etc. under a high resolution microscope. The relative air/alginate flow ratios may be used to control the size of the droplets formed.

[0049] In this example, the microfluidic device 100 was designed using a

commercially available CAD package (SolidWorks 2008, Dassault Systemes SolidWorks Corp., MA USA). The CAD file was saved as a STL file which is the standard format for stereo lithography applications. After the CAD file was converted to STL file, it was analyzed for defects and features that may not form. It is then prepared for high resolution build using 3D Lightyear software for the Viper si2 SLA System (3D Systems Corporation, SC, USA). The parts were then built in the machine by UV curing of layers 0.002 inches thick into a vat of liquid polymer. As the part was built in a vat of liquid resin, appropriate supports were provided to support the structure. DSM Somos ProtoTherm 12120 polymer (3D Systems Corporation, SC, USA) was used as the liquid resin to build this device. After the build was complete, the excess liquid resin that was clinging to the parts was cleaned off by using a two step process. The first step included cleaning using a solvent called

'Polyflush,' which is a type of propanol which removes bulk of the uncured resin. In the second step, isopropyl alcohol was used to remove any Polyflush residue, which evaporates quickly and leaves the part clean and substantially residue-free. After the part was cleaned, it was then post-cured in a UV oven for an hour. Following post cure, the supports, which break off easily from the microfluidic device, were removed. After sanding, the parts may be sandblasted to provide a better surface finish.

[0050] FLUORESCENCE LABELING AND PROTEIN ENCAPSULATION

[0051] Alexa 568-carboxy was coupled to bovine serum albumin (BSA) by taking

435 μΐ of 2.3mg/ml of BSA in PBS in a 2ml flip cap vial, and adding 28.8μ1 of lmg/ml of ED AC (Sigma), 32.6 μΐ of lmg/ml of sulpho-NHS (Pierce), 3 μΐ of AlexaFlor568-carboxy- succinimide (in DMF). This mixture was allowed to react overnight at room temperature on stir plate and covered with aluminum foil. The above solution was dialyzed exhaustively with 4 buffer changes (1L PBS buffer solution) using a 3.5kDa dialysis tubing, 0.5-3ml capacity. The Bio-Rad protein assay was used to determine the concentration of BSA after dialysis.

[0052] ISOLATION OF ISLETS FROM RAT PANCREAS

[0053] Islets were isolated from the pancreas of Lewis rats (300 - 400g) using the protocol of collagenase digestion of pancreatic tissue (Lacy et al. (1967)) with modifications (Field et al. (1996)). Following euthanasia according to IACUC guidelines, the common bile duct was cannulated and 5mL of 0.25 mg/mL Liberase TL (Roche, Indianapolis) in HEPES- buffered Hanks balanced salt solution (HBSS) was infused to distend the pancreas prior to incubation at 37°C for 15 minutes. The digestion was stopped with the addition of 15mL ice- cold wash solution (HEPES-buffered HBSS with 10% fetal bovine serum (FBS)), and then shaken for 10 seconds to dissociate the digested pancreas. The digest was filtered through a 500μηι mesh filter and then washed three times with wash solution and centrifuged at 250 g for 3 minutes. Islets were then handpicked under a stereomicroscope, or purified on a Histopaque gradient prior to handpicking, and cultured overnight at 37°C, 5% C0₂ in RPMI- 1640 with 3.3mM glucose and 10% FBS at a concentration of 15 islets per mL.

[0054] MICROENCAPSULATION OF ISLETS

[0055] Islets were microencapsulated as previously described (Darrabie et al. (2005)), but instead using the 8-channel microfluidic device 100. Following purification, islets were suspended in 3%o alginate solution (ultrapure low- viscosity high-mannuronic acid (LVM) sodium alginate, NovaMatrix, Oslo, Norway) , and microspheres (< 600 μν ) containing one islet/microsphere were collected in 100 mM CaCl₂ bath where they were gelled during 15 minutes incubation. Following two washings with normal saline, the microspheres were incubated in 0.1% (w/v) Poly-L-Ornithine (PLO, Sigma- Aldrich, St. Louis, MO) for 10 minutes to provide them with perm-selectivity. After two washings in normal saline, the PLO-coated microcapsules were incubated in 0,25% alginate solution for 4 minutes followed by two saline washes. The microcapsules were then incubated in 55 mM sodium citrate for 10 minutes to liquefy the inner alginate core prior to two final washes with normal saline.

[0056] HISTOLOGICAL TESTS OF ENCAPSULATED ISLET VIABILITY

[0057] Following encapsulation, islets were fluorescently labeled for viability with carboxyfiuorescein diacetate (CFDA) and propidium iodide (PI) to demonstrate live and necrotic cells respectively. Briefly, capsules were incubated with CFDA in serum-free RPMI 1640 for 15 minutes at 37°C, followed by washes in normal saline and a two-minute incubation with PI, prior to fixation with 4% paraformaldehyde and nuclear counterstaining with 4', 6-diamidino-2-phenylindole (DAPI).

[0058] RESULTS

[0059] In this study, the effects of varying the flow rate of the aqueous phase, the shearing phase (by varying air pressure), alginate viscosity (by varying concentration), droplet formation time (through varying the distance between the nozzle tip and the gelation phase) were evaluated. Different droplet sizes and shapes were obtained by varying the flow rates of the aqueous phase (alginate) and the shearing phase (air), by changing the distance between the outlet nozzles and the collection plate and by varying the concentration of alginate by weight. Hydrogel beads with diameters ranging from 400μηι to 1mm may be produced with this microfluidic device. The one potential problem of microfluidic approaches to microencapsulation is the generation of satellite microparticles measuring, e.g., approximately 10 - 20 μιη in diameter, which may occur without adequate adjustments in the alginate flow rate and air pressure. The formation of satellite particles was observed under various conditions. However, these factors were controlled, and the formation of these satellite microparticles was reduced or even eliminated during microencapsulations with the device 100. The satellite particles that were smaller than the hydrogel beads are lighter and can generally be easily separated from the desired microsphere samples. All the graphs are made by ignoring the satellite particles. The hydrogel beads collected for each condition were allowed to crosslink for 15 minutes in the calcium chloride bath. After cross-linking, the microspheres were washed with water to remove excess calcium chloride and then stored in calcium-supplemented saline (saline + 0.25% CaCl₂) solution Moya et al. (2009). Small samples were randomly collected from the batch samples using transfer pipettes. Data for further study of geometry of the capsules was collected from smaller samples. Fifty diameter readings were taken from each of these samples using Olympus BH-2 UMA (Olympus Corporation, USA). The factors affecting the formation of hydrogel beads using the microfluidic device were assessed as follows:

[0060] Effect of flow rate of alginate:

[0061] It was observed that the size of the hydrogel beads decreases with reduction in the flow rate of alginate. Figure 8 is an illustration of the distribution of microbead size relative to alginate flow rate. As the flow rate of alginate is increased from 49.08ml/hr to 79.79ml/hr, the average diameter of the microspheres increases from 654 μηι to 707μηι.

[0062] Effect of change in air pressure:

[0063] The size of the alginate hydrogel beads is reduced with increase in the air pressure. Figure 9 illustrates the distribution of size with change in the air pressure from 5psi to 2psi. As the air pressure increased from 2psi to 5psi the average diameter of the hydrogel beads decreased from 624 μηι to 584 μηι.

[0064] Effect of Concentration of Alginate:

[0065] Figure 10 shows the effect of varying the alginate concentration at a fixed air pressure of 5 psi and alginate flow rate of 41.69ml/hr on size distribution. As the

concentration of alginate is increased from 1.5% to 3%, the average diameter of the hydrogel beads increased from 587μηι to 672 μηι.

[0066] Effect of Distance from Collection Plate:

[0067] As the distance of the collection plate from the outlet nozzles is reduced below

9.5 inches the shape of the beads changes from circular to tear drop to random shapes. The optimum distance range observed was 9.5 inches - 1 1.5 inches. As the distance increases beyond 11.5 inches the shape of the beads is spherical; however, it leads to the coalescence of the hydrogel beads from adjacent nozzles as they fall into the collection plate. Figures 11A- 11B and 12 were taken under the microscope at 5x zoom.

[0068] Encapsulation of Bovine Serum Albumin (BSA):

[0069] BSA was encapsulated in capsules and to demonstrate the encapsulation of protein using the microfluidic device 10. Figures 13A-13B shows fluorescence images of BSA encapsulated in alginate microcapsules. The bright red spots show the encapsulated protein. Figures 13A-13B were imaged at a magnification of ten times.

[0070] Encapsulation of Islet Cells:

[0071] Pancreatic islets isolated from normal Lewis rats were encapsulated using the high throughput microfluidic device 10. Under low magnification, pancreatic islets can be observed within the capsules as white spheroids approximately 100 - 200μηι in size, with one islet per capsule (Figure 14 A), and the encapsulation procedure does not affect viability of the pancreatic islets as demonstrated by the high number of live cells (green) compared to necrotic cells (red) within the islet (Figures 14A-14B).

[0072] DISCUSSION

[0073] Microencapsulation of islets prior to transplantation is designed to overcome the two major barriers to the use of islet transplants to treat Type 1 diabetic patients, which are inadequate availability of human islets and the need to use immunosuppressive drugs to prevent transplant rejection (Uludag et al. 2000; Opara et al. 2002; Lim and Sun, 1980; Lanza and Chick, 1997; Weir and Bonner- Weir, 1997; Leblond et al. 1999). Since the introduction of this technique (Lim and Sun (1980)), numerous studies have been performed and have yielded variable results in large animal and human studies (Soon-Shiong et al.,1994; Sun et al., 1996; Dufrane et al., 2006; Calafiore et al., 2006; Wang et al., 2008; Thanos and Elliot, 2009; Tuch et al., 2009), as many factors determine the outcome of encapsulated islet transplantation, including the length of time required to encapsulate enough islets for such studies. The length of time required to encapsulate sufficient quantities of islets for transplantation is a factor that affects the outcome of their transplantation, because a prolonged process of encapsulation results in decreased viability of the islets (Opara et al. (2010)). Good product quality control may be very difficult to achieve with the very slow microencapsulation devices currently available for encapsulating islets for transplantation. There is therefore a need for throughput microencapsulation devices to provide better product quality control if encapsulated islet transplantation will become a clinically viable procedure. The microfluidic approach in microencapsulation described herein according to some embodiments may be capable of producing large numbers of alginate microspheres to encapsulate cells and proteins. The device 100 may be capable of increasing by eight times the rate of production of microspheres compared to currently available devices, and may be scaled up even higher, for example, by a magnitude of 64 times or more. Consequently, the new microencapsulation approach could potentially reduce the 100 hours currently needed for the production of 1 million microencapsulated islets for human transplantation to less than 30 minutes. Indeed, millions of cells for many patients may be encapsulated in that same short duration, and the entire encapsulation process may be completed in less than 30 minutes. In addition to producing large quantities of encapsulated cells in a very short period of time, another potential advantage is that a given preparation of encapsulated cells may be subjected to the subsequent manual steps of perm-selective coating and washings at the same time, and this may have positive impact on the viability of the encapsulated cells resulting in increased product quality control.

[0074] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although a few exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. Therefore, it is to be understood that the foregoing is illustrative of the present invention and is not to be construed as limited to the specific embodiments disclosed, and that modifications to the disclosed embodiments, as well as other embodiments, are intended to be included within the scope of the appended claims. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A micro fiuidic device for the microencapsulation of biological units, the device comprising:

a chamber for containing a plurality of biological units suspended in a liquid;

a plurality of liquid conduits in fluid communication with the chamber, each of the plurality of liquid conduits having an encapsulation outlet;

a compressed gas inlet; and

a plurality of compressed gas conduits in fluid communication with the compressed gas inlet, each of the plurality of compressed gas conduits having a compressed gas outlet that at least partially surrounds a respective encapsulation outlet, wherein the encapsulation outlets and the compressed gas outlets are sized and configured to encapsulate the biological units from the solution when the biological units pass from the chamber through the plurality of fluid conduits and exit the encapsulation outlets.

2. The device of Claim 1, wherein the compressed gas outlets substantially concentrically surround respective encapsulation outlets.

3. The device of Claim 1, wherein at least a portion of the compressed gas conduits substantially concentrically surround and are coaxial with at least a portion of respective fluid conduits.

4. The device of Claim 1, wherein the chamber, the plurality fluid conduits, and the plurality of compressed gas conduits are formed as a single unitary member.

5. The device of Claim 3, wherein the single unitary member comprises an polymeric material.

6. The device of Claim 1, wherein the chamber further comprises at least one inlet.

7. The device of Claim 6, wherein the chamber turther comprises at least one gas outlet that has a first open configuration configured for allowing gas to be released from the chamber when the biological units and the liquid is entering the chamber and a second closed configuration configured to substantially prevent the liquid from exiting the chamber.

8. The device of Claim 7, wherein the at least one inlet of the cell-solution suspension chamber is configured to connect to a variable flow pump.

9. The device of Claim 1, wherein the biological unit is a cell.

10. The device of Claim 1, wherein the biological unit is a protein.

11. The device of Claim 1 , wherein the biological unit is suspended in an alginate.

12. A method for the microencapsulation of biological units, the method comprising:

flowing a plurality of biological units suspended in a liquid into a chamber;

flowing the plurality of biological units suspended in the chamber through a plurality of liquid conduits in fluid communication with the chamber, each of the plurality of liquid conduits having an encapsulation outlet;

flowing a gas through a plurality of compressed gas conduits, each of the plurality of compressed gas conduits having a compressed gas outlet that at least partially surrounds a respective encapsulation outlet so that the encapsulation outlets and the compressed gas outlets encapsulate the biological units from the solution when the biological units pass from the chamber through the plurality of fluid conduits and exit the encapsulation outlets.

13. The method of Claim 12, wherein flowing the gas comprises flowing the gas through compressed gas outlets that are substantially concentrically surrounding respective encapsulation outlets.

14. The method of Claim 12, further comprising forming the chamber, the plurality fluid conduits, and the plurality of compressed gas conduits as a single unitary member.

15. The device of Claim 14, wherein the single unitary member comprises an polymeric material.

16. The method of Claim 12, wherein the chamber further comprises at least one inlet, and the chamber further comprises at least one gas outlet, the method further comprising moving the at least one gas outlet between a first open configuration configured for allowing gas to be released from the chamber when the biological units and the liquid is entering the chamber and a second closed configuration configured to prevent the liquid from exiting the chamber.

17. The method of Claim 16, further comprising:

connecting the at least one inlet of the chamber to a variable flow pump; and pumping the fluid and biological unit into the chamber via the pump.

18. The method of Claim 12, wherein the biological unit is a cell.

19. The method of Claim 12, wherein the biological unit is a protein.

20. The method of Claim 12, wherein the biological unit is suspended in an alginate.