An apparatus and method for coating micron-sized or sub-micron-sized particles such as living cells. The coating apparatus includes an encapsulation chamber enclosing a two-layer water-oil system for coating each islet cell with an aqueous polymeric coat. Islets together with an aqueous polymer solution are fed by a feed device that utilizes the principle of hydrodynamic focusing in order to ensure encapsulation of individual islets. The polymer in the aqueous coat is subsequently crosslinked by being exposed to laser light to produce structurally stable microcapsules of controllable thickness of the order of tens of microns. Encapsulated islets are removed from the encapsulation chamber by a valveless pump and recovered by filtration or centrifugation.

Description of the Invention

FIELD OF THE INVENTION

This invention relates generally to an apparatus and method for coating or encapsulating small particles, namely micro-sized particles and sub-micron particles, and, more particularly, to a device for encapsulation of living cells, e.g., pancreatic islet cells, intended for implantation into patients.

BACKGROUND OF THE INVENTION

More than one million people in the U.S. suffer from Type I diabetes, a disease in which pancreatic islets are no longer able to control glucose levels in the blood. The expectancy and quality of life of these patients is greatly compromised by diabetic complications that include retinopathies, renal failure, and vascular disease. An alternate therapeutic modality to regular insulin injections is transplantation of pancreatic islets from transgenic (allotransplantation) or nontransgenic (xenotransplantation) organisms. To suppress rejection by the recipient's immune system, transplanted islets are immunoisolated by enclosing them individually into microcapsules comprised of structurally stable, semipermeable membranes. The structural stability and selective permeability of the membrane ensures long-term viability and functionality of the islets.

Current methods for encapsulation generally include droplet generation, emulsion formation, polyelectrolyte multilayering, and direct polymerization from a surface-adsorbed initiator. Encapsulation methods are often restricted in terms of chemical composition, uniformity and thickness of the membrane, polymerization schemes, and applied stress to pancreatic islets. There is a need for an improved method for encapsulating islet cells.

SUMMARY OF THE INVENTION

A general object of the invention is to provide a cell encapsulation device with high yields of encapsulated pancreatic islets with long-term viability and functionality.

The general object of the invention can be attained, at least in part, through an apparatus for coating micron-sized or sub-micron-sized particles. The coating apparatus includes an encapsulation chamber including therein a first fluid layer of water or an aqueous solution disposed on a second fluid layer of a fluid incompatible with the first layer. The coating apparatus includes a particle feed tube with a particle passage in combination with a feed tube opening at a discharge end of the feed tube. The feed tube opening is disposed in the first fluid layer. A particle withdrawal tube includes a first end in combination with the encapsulation chamber and is connected at a second end to a filtration device.

The invention further comprehends an apparatus for coating micron-sized or sub-micron-sized particles. The coating apparatus includes an encapsulation chamber enclosing an aqueous fluid layer disposed on an oil fluid layer, and a particle feed device in combination with the encapsulation chamber by a particle feed tube. The particle feed tube includes a particle passage and a feed tube opening disposed in the aqueous fluid layer. The particle feed device includes a particle discharge channel having a particle discharge channel opening in combination with a first end of the particle passage. The particle feed device further includes a polymer solution injection channel adjacent the particle discharge channel and having an injection channel opening in combination with the first end of the particle passage. A particle withdrawal tube is connected at a first end to the encapsulation chamber and connected at a second end to a filtration device. A pump is in combination with the particle withdrawal tube.

The invention still further comprehends a method of coating micron-sized or sub-micron-sized particles. The method includes: mixing the particles with a polymer precursor solution in a particle feed tube; discharging the particles and the polymer precursor solution from the particle feed tube into an aqueous first fluid layer in an encapsulation chamber, the aqueous first fluid layer disposed on a second fluid layer formed of a fluid incompatible with the aqueous first fluid layer; removing the particles and at least a portion of the polymer precursor solution from the encapsulation chamber through a particle withdrawal tube; and polymerizing a polymer precursor of the polymer precursor solution within the particle withdrawal tube to coat the particles with a polymer material. Desirably, the mixing of the particles with a polymer precursor solution in the particle passage comprises hydrodynamically aligning the particles into a particle stream within the particle passage with at least one stream of the polymer precursor solution.

In one embodiment, removing the particles and at least a portion of the polymer precursor solution from the encapsulation chamber through a particle withdrawal tube comprises: placing an opening at an end of the particle withdrawal tube at a predetermined distance from an interface between the first fluid layer and the second fluid layer; drawing a stream of both the first fluid layer and the second fluid layer through the opening and into the particle withdrawal tube; and drawing the particles and the at least a portion of the polymer precursor solution through the opening and into the particle withdrawal tube.

The polymerization of the polymer precursor of the polymer precursor solution within the particle withdrawal tube to coat the particles with the polymer material includes applying light from a laser to the particles and the at least a portion of the polymer precursor solution within the particle withdrawal tube.

The device and method of this invention are particularly useful for encapsulation of pancreatic islet cells within a polymer film for implantation into patients suffering from Type I diabetes. The invention utilizes the method of selective withdrawal from a two-layer water-oil system for coating each islet with an aqueous polymeric coat. Islets together with an aqueous polymer solution are fed by the feed device that utilizes the principle of hydrodynamic focusing in order to ensure encapsulation of individual islets. The polymer in the aqueous coat is subsequently crosslinked by being exposed to laser light to produce structurally stable microcapsules of controllable thickness of the order of tens of microns. Encapsulated islets are removed from the encapsulation chamber by a valveless pump and recovered by filtration or centrifugation. The method and device of this invention ensure timely encapsulation of a number of islets, adequate for clinical trials, in microcapsules enclosed by semipermeable, hydrogel membranes of uniform thickness, that ensure long-term viability and functionality of the islets.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 (see Original Patent) generally illustrates a coating apparatus 100 (not to scale) according to one embodiment of this invention for coating micron-sized or sub-micron-sized particles. The apparatus 100 includes an encapsulation chamber 102 with a first fluid layer 104 disposed on a second fluid layer 106. The second fluid layer 106 includes a liquid that is incompatible with the first fluid layer 104, thereby allowing the first fluid layer 104 to sit atop of the second fluid layer 106. In one embodiment of this invention the first fluid layer 104 includes water or an aqueous solution. The second fluid layer 106 can include any liquid that in incompatible with, and thus separates from, water, such as an oil. In one particularly preferred embodiment of this invention, the second fluid layer 106 includes a chlorinated hydrocarbon oil such as sold under the name PAROIL, available from Dover Chemical Corporation, Dover, Ohio.

A particle feed device 110 is in combination with the encapsulation chamber 102 so as to introduce thereto the particles 112 to be coated. The particles 112 can be any particles, but the apparatus of this invention is particularly suited for coating micron-sized particles and smaller. In one embodiment of this invention, the particles 112 are living cells or cell aggregates, and the invention will be described below with reference to pancreatic islet cells as the particles 112.

The particle feed device 110 shown in FIG. 1 includes a particle discharge channel 114 adjacent and between a first polymer solution injection channel 116 and a second polymer solution injection channel 118. The particle discharge channel includes a particle discharge channel opening 120 through which the islet cells 112 enter a particle passage 134 of a particle feed tube 130 at a feed tube first end 132. Each of the polymer solution injection channels 116 and 118 includes an injection channel opening 122 and 124 (respectively) in combination with the first end 132 and through which a polymer solution enters the particle passage 134 of the particle feed tube 130.

FIG. 2 (see Original Patent) is a schematic of the feed device 110, which is desirably constructed of a plastic such as poly(methyl methacrylate) (PMMA) material. The islets 112 are injected from the central particle discharge channel 114 and focused hydrodynamically into an aligned single-islet-file stream through the particle passage 134, constrained by polymer precursor solution flows from the two lateral injection channels 116 and 118. The feed device of FIG. 2 follows principles discussed in Lee et al., "Hydrodynamic Focusing for a Micromachined Flow Cytometer," Trans. ASME 123, 672 (2001), which are hereby incorporated by reference.

The polymer solution used in the particle coating apparatus of this invention can be any suitable polymeric solution for coating the cells 112. Desirably, the polymeric solution includes a precursor of the polymer intended to coat the islet cells 112. In one embodiment of this invention the aqueous polymeric solution for the coating material is contained in both the polymer solution injected through the injection channels 116 and 118 and in the aqueous first fluid layer 104. Examples of suitable polymers or polymer precursors include PEG-based polymers, such as PEG-diacrylate.

Referring again to FIG. 1, the particle feed tube 130 includes a feed tube opening 136 at a feed tube second end 138 that is opposite the first end 132. The feed tube opening 136 is disposed in the aqueous first fluid layer 104 at a predetermined distance above a fluid interface 140 (e.g., a water-oil interface).

A particle withdrawal tube 142 is in combination with the encapsulation chamber 102 and has a first end 144 disposed at a predetermined distance below the fluid interface 140. An opposing second end 146 of the particle withdrawal tube 142 is connected to a filtration device 150. A pump 152 is in combination with the particle withdrawal tube 142 upstream of the filtration device 150. The first end 144 includes a withdrawal tube opening 148 disposed within the second fluid layer 106.

The pump 152 withdraws oil through the particle withdrawal tube 142 at a rate that defines the flow as laminar. The flow rate is controlled to secure hydrodynamic stresses levels much below the critical stress that proves to be damaging to the living cells of the islets 112. If the withdrawal tube 142 is placed below the fluid interface 140 at a distance less than a critical distance, a thin spout 154 of the first fluid layer 104 is entrained with the oil. Islets 112 arriving at the fluid interface 140, by design, exactly above the opening 148 of the withdrawal tube 142, are drawn in the spout 154 and enter the withdrawal tube 142. Desirably, the islet 112 diameter is greater than a diameter of the spout 154 at the opening 148, so that the balance of interfacial and viscous forces causes the spout 154 to break both above and below the entrained islet 112. At this point, a thin layer of the aqueous polymeric solution surrounds the entrained islet 112 within the withdrawal tube 142. The polymeric solution with the islets 112 flows as a string of circular cross section, along the axis of the withdrawal tube 142, while the oil flows in the annulus between the tube wall and the lateral surface of the string.

After the islets 112 have been entrained into the withdrawal tube 142 and coated with the polymer solution, the islets 112 are exposed to light from a light source 160, such as, for example, 514 nm light of an argon-ion laser, that is in light discharge alignment with a portion of the withdrawal tube 142. The light source 160 is used to polymerize the polymer precursor within the polymer solution to coat the islets 112. In one embodiment of this invention the cell culture medium of islets 112 injected through particle discharge channel 114 and/or the polymer solution through injection channel 116 and/or 118 includes eosin-Y as a photoinitiator, 1-vinyl-2-pyrrolidinone as an accelerator and triethanolamine as a coinititiator for polymerization. The light excites eosin-Y and initiates free-radical polymerization to produce, for example, a poly(ethylene glycol) (PEG) hydrogel from a PEG-diacrylate precursor.

As shown in FIG. 1, the withdrawal tube 142 includes a coiled portion 156 which is used to provide sufficient time within the light beam for polymer crosslinking and hydrogel formation. As will be appreciated, the frictional pressure drop associated with the flow in the coiled part of the withdrawal tube is a function of the Dean number, which is nothing but the Reynolds number multiplied by the ratio of the tube-to-coil-curvature radii. Using the apparatus of this invention, the thickness of the resulting microcapsule shell enclosing an individual islet is controllable to within tens of a micron.

The coating apparatus 100 of FIG. 1 also includes an oil reservoir 160 connected to the encapsulation chamber 102 by a pump 162. A level sensor 164 measures the level of the second fluid layer 106 and the layer 106 is adjusted according to need to maintain the desired distance between the fluid interface 140 and the withdrawal tube 142.

FIGS. 3A-D (see Original Patent) illustrate features and operation of the pump 152. The pump 152 is a valveless diffuser-nozzle pump that has no interior mechanical parts for transporting mammalian cells with minimal and preferably no damage. The pump 152 is used to generate a selective withdrawal flow and remove the encapsulated islets 112 from the encapsulation chamber 102. The pump 152 is a diaphragm pump that uses two diffusers 170 and 172 as flow directing elements. The pump 152 is desirably made of a glassy plastic material (e.g., PMMA) with a flexible top portion 174 (e.g., poly(dimethyl acrylate) (PDMA)) over an internal fluid cavity. A piezoelectric (PZT) patch is bonded to the flexible top 174 of the pumping chamber, e.g., with epoxy resin, and is used in connection with applied AC voltage to set the three-layer (PZT-epoxy-PDMA) plate into vibration at a frequency, below the frequency of the natural frequency of the PDMA plate. The vibrating plate drives fluid flow, and at the same time, the fluid increases the resistance to the vibration (fluid flow and plate vibration are coupled). If the action of the fluid is negligible, the plate will vibrate at the same frequency as the piezoelectric (excitation) force for small amplitude vibrations.

As shown in FIGS. 3C and 3D (see Original Patent), the pump cycle can be divided into a supply and a pump mode. In the supply mode, the fluid cavity volume increases and a larger amount of the fluid flows into the cavity through the input element, i.e., diffuser 170, than through the output element, i.e., diffuser 172, which acts as a nozzle. In the pump mode, when the fluid cavity volume decreases, a larger amount of fluid flows out of the cavity through the output element 172, which acts as a diffuser, than the input element 170, which acts as a nozzle. The result for the complete pump cycle is that the net volume is transported from the input to the output side of the pump.

The ability of the valveless pump 152 to direct the flow in a preferential direction is measured by the rectification efficiency -- see Original Patent.

The average flow rate through the pump 152 is given by -- see Original Patent.

The encapsulated islets 112 are recovered in filtration device 150 shown in FIG. 1, for example, by mechanical filtration or centrifugation. High-retention filtration devices, e.g., spin-filters, can be utilized. Centrifugation can also be utilized, especially if it is desirable to recover the encapsulated islets 112 in a cell culture medium for the survival of islet cells. In this case, a buffer (aqueous) medium is desirably fed to the centrifuge simultaneously with the encapsulated-islets-containing oil, such as into a disk centrifuge 200 shown in FIG. 5 (see Original Patent). In FIG. 5, the feed solution F, which includes the oil, water and islets, is introduced into the disk centrifuge 200. The heavy phase H (oil) and light phase L (water) separate within the centrifuge and are pumped out, as indicated by the respective arrows. The encapsulated islets are then recovered in the cell culture medium, which comprises the light phase.
 

Claim 1 of 19 Claims

1. An apparatus for coating micron-sized or sub-micron-sized particles, comprising: an encapsulation chamber including a first fluid layer of either water or an aqueous solution, the first fluid layer disposed on a second fluid layer of a fluid incompatible with the first layer; a particle feed tube including a particle passage in combination with a feed tube opening at a discharge end of the particle feed tube, the feed tube opening disposed in the first fluid layer; a particle feed device in communication with an end of the particle passage opposite the feed tube opening, the particle feed device comprising a particle discharge channel having a particle discharge channel opening in combination with the end of the particle passage, and a polymer solution injection channel having an injection channel opening in combination with the end of the particle passage; a particle withdrawal tube including a first end in combination with the encapsulation chamber and connected at a second end to a filtration device; and a valveless pump connected to the particle withdrawal tube between the first end and the second end.
 

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