Methods and devices for microencapsulation of cells

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DESCRIPTION

Methods and devices for microencapsulation of cells

BACKGROUND OF THE INVENTION

The present invention relates to the field of microencapsulation of cells, and more specifically to the production of microencapsulated cells.

Microencapsulation is an immunoisolation technique available for the 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 twenty years ago (Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 210: 908910, 1980), has had a slow evolution towards clinical application, for example due to slow production rates and the appearance of fibrotic hypertrophy around the capsules, which can result in endotoxin contamination, for example lack oxygen and nutrients from the enclosed cells.

One of the many potential applications of this technology is the development of a reliable bioartificial liver in the form of encapsulated hepatocytes, to provide temporary but adequate metabolic support to allow spontaneous liver regeneration, or as a bridge to orthotopic liver transplantation for patients with impaired liver failure. fulminant liver. Joly A, Desjardins JF, 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.

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 HC, Fu SH, Huang YY, Huang HS: 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 equipped with a single needle through which droplets of cells suspended in alginate solution are produced and crosslinked into spherical beads. Various methods have been tried for the production of encapsulated cells in increasing numbers, including the simultaneous production of multiple droplets in a multi-needle approach (De Vos P, De Haan BJ, Schilfgaarde R. Upscaling the production of microencapsulated pancreatic islets. Biomaterials 18: 1085-1090, 1997), or by increasing the number of cells / ml of alginate suspension in the syringe to increase the likelihood of encapsulated cell formation. However, air syringe pump droplet generators or electrostatic bead generators may be unable to produce sufficient numbers of microcapsules in a short period of time to allow mass production of viable, encapsulated cells for transplantation in large animals. and human beings. Furthermore, a prolonged cell encapsulation procedure can adversely affect the viability of the cells.

For example, in a study with four nozzles, the nozzles are fitted to a collector plate in which 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 cannot be scaled up efficiently due to the supporting mass surrounding the hypodermic needle, such as couplings and seals, which provide a gap of about 1 cm between the needles. An increase in the number of junctions in the flow path through which the alginate-cell suspension travels can result in a greater possibility of stagnation, plugging, and contamination. In addition, the alginate solutions used for encapsulation are viscous, making the process potentially burdened with the risk of plugging when small-gauge needles are used to produce microcapsules of desirable size range (eg, <800 microns in diameter). . When needle clogging occurs, the process of unclogging the needle to resume encapsulation further increases the duration of microencapsulation of large batches of cells for therapeutic use. A high density of parallel needles may not provide the necessary access to clean plugged needles in the center of the array on a fairly dense grid. Multiple needles with a common flow manifold may not be viable and / or efficient.

Even using such an approach, production rates of several orders of magnitude higher may be desirable to significantly produce sufficient amounts of encapsulated and viable cells for transplantation into human subjects. For example, it has been estimated that for the one million islets required for transplantation in a diabetic human subject, it takes about 100 hours to complete encapsulation of this number of islets, assuming one islet / microcapsule and a single needle operation. . However, in practice, it has currently been estimated that the duration of the procedure may be close to 200 hours due to the additional steps involved in the encapsulation procedure, after the generation of the initial alginate microspheres containing the cells.

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Furthermore, it has been disclosed that, using the syringe method, the proportion of spheres containing cells is only about 50%. Attempts can be made to increase the concentration of cells in the alginate suspension to increase the timeliness of the encapsulation process of a cell, and thereby increase productivity. However, this can provide only a two-fold increase in productivity. Furthermore, an increase in the number of cells / ml of alginate could cause an increase in the number of cell beads with blemishes, such as cell protrusion in the bead membrane. Protrusion of encapsulated tissue through the microcapsule membrane can activate the host's cell-mediated immune response, leading to microcapsule transplantation 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 THE INVENTION

Embodiments of the present invention provide methods, systems, and devices for the microencapsulation of cells. In some embodiments, a first chamber is provided to contain a cell suspension-solution. A plate covers one end of the first chamber. The plate has a plurality of openings. A second chamber is provided to receive the encapsulated cells. The second chamber is separated from the first chamber by the plate. Cells from the first chamber are encapsulated by passing through the openings in the plate and into the second chamber when pressure is applied to the suspension of cell solution.

In other embodiments, the method for microencapsulating cells includes providing a suspension of cells-solution in a first chamber having a plate covering one end, the plate comprising a plurality of openings, and passing the suspension of cell solution through the openings in the plate into a second chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic drawing of a cell encapsulation device according to embodiments of the invention.

Figure 2 is a cross-sectional side view illustrating a perforation for encapsulating a cell according to embodiments of the invention.

Figures 3 and 4 are perspective views of a perforated plate for encapsulating cells according to embodiments of the invention.

Figures 5, 6 and 7 are perspective views of a cell encapsulation device according to further embodiments of the invention.

Figure 8 is a perspective view of a multi-tank device according to further embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be more fully described hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. However, this invention should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this description will be thorough and complete, and will fully disclose the scope of the invention to those skilled in the art.

Embodiments of the invention provide methods and systems for the microencapsulation of cells. Cell microencapsulation includes techniques designed to trap viable cells within the confines of semi-permeable membranes that are permeable to small molecules, such as nutrients and oxygen, which are essential for cell survival, but impervious to large molecules, such as antibodies and immune cells. . Such techniques may be of interest in cell therapy for a variety of pathophysiological states, including anemia, dwarfism, renal failure, liver failure, CNS and pituitary failure, and diabetes mellitus. However, other applications of encapsulated cell technology are possible in addition to cell transplantation. Uludag H, De Vos P, Tresco PA: Technology of mammalian cell encapsulation. Adv Drug Delivery Rev 42: 29-64, 2000. Other applications of encapsulated cell technology may include large-scale production of cell-derived molecules in biotechnology, clonal selection of desired cell phenotypes, in vitro cultures of cells dependent on very close cell-to-cell contact, cell cultures in vivo, reproductive technology, and the cytotoxicity assay.

Examples of encapsulated cells include cells encapsulated in alginate spheres that are ionically cross-linked (gelled) with Ca ++ to immobilize living cells. Capsules in the diameter range of 0.05-1.5mm are generally referred to as microcapsules. As explained here, the success of the cell microencapsulation technique can be determined by evaluating the properties of the capsules, and the

ES 2 389 441 T3 function of encapsulated cells. Smidsrod O, Skjak-Braek G: Alginate as immobilization matrix for cells. Trends Biotechnol 8 (3): 71-78, 1990. Opara EC, Kendall WF. Immunoisolation techniques for islet cell transplantation. Expert Opin Biol Therapy 2: 503-511, 2002.

As shown in Figure 1, a cell microencapsulation device 100 includes a pre-potting tank 115 and a post-potting tank 139, separated by a perforated plate 141. The pre-potting tank 115 includes a suspension. 127 cell solution containing the cells to be encapsulated, such as Hep G2 cells, suspended in an encapsulating solution, such as alginate. Post-encapsulation tank 139 includes a post-encapsulation solution 137, such as a sodium chloride solution, which can facilitate crosslinking of the encapsulation solution around cells.

Briefly, pressure is exerted on the cell / solution suspension 127 to force the suspension 127 through the openings in the perforated plate 141 to produce encapsulated cells 125. The generated encapsulated cells 125 are received in tank 139 of post-encapsulation and continue until dissolution 137, which facilitates crosslinking of the encapsulation material.

Although embodiments of the present invention are described herein with respect to the microencapsulation of Hep G2 cells in an alginate solution, other cells and solutions can be used. For example, normal hepatocytes can be encapsulated for use as a bioartificial liver. Parenchymal hepatocytes can also be isolated from live animals and subsequently encapsulated. Other encapsulated cells include islets, pituitary cells, and adrenal cells. Hep G2 is a single cell line derived from human hepatocellular carcinoma. Ultra-purified, low-viscosity, high-mannuronic acid (UPA) alginate, with low endotoxin content, is also available from Pronova / FMC (Philadelphia, PA). Hep G2 produces the normal biological macromolecules such as albumin, alpha-fetoprotein, apotransferrin, alpha2-macroglobulin, haptoglobin, ceruloplasmin, fibrinogen, plasminogen, complement (C3, C4), and a host of other proteins normally produced by parenchymal hepatocytes (US Pat. No. 4,393,133), and may be a good hepatocyte model. Therefore, the mass production of encapsulated Hep G2 cells can provide a valid basis for the development of a bioartificial liver system involving the use of isolated normal hepatocytes.

As described herein, encapsulated cell viability can be assessed using molecular probes to determine cell viability and proliferation, and metabolic techniques can be used to assess cell function. The function of encapsulated cells can be compared to that of non-encapsulated cells. Furthermore, ELISA assays can be used to determine the identity of specific proteins (albumin, transferrin, and afetoprotein) synthesized and exported abroad by encapsulated cells. Biomaterials for encapsulation can be compared to determine the biocompatibility, mechanical strength, and durability of the microcapsules. Based on the evaluation of these microcapsule property parameters, appropriate adjustments can be made to the microencapsulation process to suit the properties of the encapsulation material, including viscosity, density, and the like. The desired perme-selectivity can be obtained. For example, encapsulation materials can be selected that allow the secretion of high molecular weight proteins (MW> 150 kDa) made by encapsulated cells, while preventing the entry of immune cells into the capsules.

Referring to Figure 1, the perforated plate 141 includes a plurality of apertures that can be perforated, for example, using electron beam perforation techniques, on the surface of the plate 141 adjacent to the post-potting tank 139. The openings can be shaped and positioned in a manner to facilitate the smooth flow of the cell suspension / solution when subjected to pressure. As illustrated in Figure 1, a piston 113 can be used to exert pressure on the cell suspension 127 / solution, to cause the encapsulated cells to pass through the perforated plate 141. A piston actuator 109 can be in contact. complete with solution / cell suspension 127, and can be electromechanically actuated with a servolinear actuator and controller that can be programmed to generate the desired amplitude and frequency of motion. The piston actuator 109 may be a piezoelectric piston actuator, although other types of actuators and / or configurations may be used to exert pressure on the cell / solution suspension 127. For example, the piston actuator 109 and the piston 113 can be omitted, and the pre-potting tank 115 can be provided with a seal with a lid with inlet or inlets for the inlet of compressed air to exert pressure on cell suspension 127 / solution. A source 103 of compressed air, regulated by a pressure regulating valve 107 and a pressure pulse generator 105, can apply force to the cell / solution suspension 127. To exert pressure on the cell / solution suspension 127, a space 111 of compressed air may be provided.

In some embodiments, the level of the cell / solution suspension 127 is maintained using a non-contact, ultrasonic level sensor positioned within the lid of the pre-potting tank 115 or piston 113. The flow of the solution and cells through input 117 can be controlled with feedback from the level signal. The compressed air in the compressed air space 111 can be used to provide additional space to pressurize the cell / solution suspension 127 and / or to pass the cell / solution suspension 127 through the holes in the bottom of the cell. vessel. The air cushion in the compressed air space 111

ES 2 389 441 T3 above the cell / solution 127 suspension can be pulsed, for example, using the pressure pulse generator 105, to control droplet formation by preventing a continuous stream of fluid through the orifices. Pulsed pressure can be applied to produce encapsulated cells 125 of uniform size. Such pulsed pressure from a compressed air source 103 can be applied without the need for piston 113. Also, aggressive pressure relief may not be necessary for successful microencapsulation production.

The air gap 111 can be reduced or eliminated, and the pressurization of the cell / solution suspension 127 can be maintained by creating pressure pulses using the piston actuator 109, such as a piezoelectric transducer, to drive the piston 113 from the side. superior using high forces and small displacement.

With continued reference to Figure 1, an inlet 117 is provided that can be used for the inlet of the solution and cells into the pre-encapsulation tank 115. For example, inlet 117 may include high pressure valve inlets for a supply of solution, such as alginate, and a supply of cells suspended in a suitable medium, such as saline. To pump the solution into the pre-encapsulation tank through inlet 117, peristaltic pumps and / or Moyno cavity pumps can be used. Moyno cavity and / or peristaltic pumps can be used to impart low shear stress to the conveyed fluid, and can also provide a controlled sterile environment. These pumps can also handle neat liquids, including viscous and non-viscous fluids to viscous fluids with suspended solids.

Plate 141 may be provided with planar vibratory motion to facilitate droplet release and produce a steady stream of microencapsulated cells 125. Planar vibratory motion may be provided by a plate actuator 119, such as a piezoelectric actuator, which is attached to plate 141 as shown in Figure 1. Any suitable agitation mechanism can be used to avoid sedimentation of the cells at the bottom of the tank and to maintain uniform distribution of cells in the cell suspension / solution 127, including a large agitator that rotates slowly for mass movement, and / or smaller high speed mixers to impart high shear stresses in a localized area to break up cell clusters.

In some embodiments, a continuous mode of production can be provided such that a suitable "bead lifter", such as a transporter 129, is used to transport crosslinked microencapsulated cells 125 and transfer the cells 125 to a holding tank 131 containing a wash solution 133, continuously. The configuration of carrier 129 can be selected such that the beads reside in solution 137 in post-encapsulation tank 139 for the required crosslinking time. For example, crosslinking may require between about 5 and about 15 minutes. In some embodiments, post-encapsulation solution 139 flows under perforated plate 141 such that the beads continue to move for the required gelation time before being lifted out of the crosslinking bath.

As illustrated, incline conveyor 129 has a flexible surface submerged in post-encapsulation tank 139 that is continuously moved to transport encapsulated cells 125 from the bottom of conveyor 129 and ending up on top of conveyor 129 after the crosslinking time. wanted. However, other configurations can also be used, such as horizontal conveyors. The surface of the carrier 129 may contain fully cross-linked encapsulated cells 125 that can be removed by light suction through an opening 145 in the dwell tank 131. The encapsulated cells 125 are stored in a collection area 135 of the dwell tank 131 in the wash solution 133. The wash solution 133 can be a saline bath.

In other embodiments, the dwell tank 131 and conveyor 129 can be omitted, and the encapsulated cells 125 can be produced batchwise.

After formation of the microencapsulated cells, various solutions can be applied to provide additional layers to the microencapsulated cell. For example, microencapsulated cells can be coated with poly-L-lysine or polyornithine, followed by an outer coating with alginate to form a semi-permeable membrane around the initial encapsulated core, which can be an alginate core. In addition, the central alginate can be re-liquefied by chelation with sodium citrate solution.

In some embodiments, the various tanks 115, 139, and 131 may be made of smooth 316 stainless steel cylindrical vessel. The flat perforated plate 141 can be integrated into one or more of the tanks 115, 139, or 131, or it can be provided as a separate installation. The internal diameter of the various tanks 115, 139 and / or 131 can be about 3.5 inches (88.9 mm), and they can have a wall thickness of about 1/4 inch (6.35 mm). The holes can be drilled using electron beam drilling techniques in the bottom of the pre-potting tank 115, or in a separately provided plate 141. Various microperforation procedures can be used, including non-traditional procedures. For example, electron discharge micromachining (EDM), laser micro-drilling, and electron beam drilling can be used. By way of example, in certain filter applications, 6 million holes, each having a diameter of 0.006 inches, were drilled in a 24-inch diameter 0.125-inch thick stainless steel disc.

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To control aspects of the generation of the microencapsulated cells, various parameters can be adjusted. For example, the geometry of the openings in plate 141 can be selected to generate microencapsulated cells of a certain shape or size.

Shown in Figure 2 is a cross-sectional side view of an exemplary opening 20 that may be formed in plate 141. Opening 20 has a top surface 21 that is adjacent to pre-encapsulation tank 115 in Figure 1, and a surface bottom 22 that is adjacent to post-encapsulation tank 139. Referring to Figure 2, the top surface 21 has an inlet 23 with a contoured edge 27 to receive the cell suspension 127 / solution (Figure 1). The lower surface 22 includes an outlet 25 with a straight edge 29 which is connected to the inlet 23 via a straight portion 24. An enlarged view of the upper surface 21 and the lower surface 22 is provided in Figures 3 and 4. As shown in Figure 2, opening 20 is sized so that a cell and a desired amount of solution (eg, alginate solution) can be forced through inlet 23 around the contoured edge. 27 and through straight portion 24 so that a microencapsulated cell is formed and exits through outlet 25.

Consequently, various parameters of the aperture 20 shown in Figure 2 can be adjusted sensitively to the size of the cells and / or the size of the encapsulated material that is desired, such as the shape of the contoured edge 27, the size of the inlet 23, the length of the straight portion 24, the size of the outlet 25, and the size of the trailing edge 29. For example, inlet 23 and / or outlet 25 may provide a greater cross-sectional area than straight portion 24 of the channel.

In some embodiments, opening 20 has a cross section of inlet 23 of about 0.5mm in diameter, a tapered tapered section along contoured edge 27 along the first about 0.5mm in depth, followed by a straight portion 24 having a length between about 1mm and 3mm and having the required diameter to produce a desired droplet size, and a final flared outlet 25 of 0.5mm. The diameter of the straight portion 24 can vary based on the size of the cell to be encapsulated. For example, Hep G2 cells have a diameter of about 20 pm, whereas an islet cell can be as large as 200 pm, or typically between about 70 pm and 200 pm. Consequently, the diameter of the straight portion 24 should be about 20 pm to about 40 pm for a Hep G2 cell, and about 150 pm to about 250 pm for an islet cell. Other configurations are possible. For example, in some embodiments, the straight edge portion 29 of the outlet 25 is absent and is replaced by a contoured or chamfered edge.

In some embodiments, 113 µm diameter holes are drilled into a 0.55 mm thick stainless steel plate using electron beam drilling techniques. In some embodiments, a one millimeter diameter shell per hole is allowed for a single 63.5 mm diameter plate, resulting in approximately 4900 holes. In this way, a large number of encapsulated cells can be produced in parallel.

In addition to the examples given above, between about 3000 and about 5000 apertures can be provided on a plate. Each opening may have a chamfered inlet that has a cross-sectional diameter of about 0.5mm, an outlet that has a cross-sectional diameter of about 0.5mm, and a portion of the channel connecting the inlet and the outlet having a cross-sectional diameter between about 50 pm and about 500 pm.

Other variable parameters within the scope of the invention arise from the operations of the device. Operational parameters include the viscosity of the cell suspension / solution (e.g., alginate), the concentration of cells (e.g., Hep G2 cells) in the suspension, the fluid levels of the pre-potting tank 115, the tank 139 of post-potting, and the dwelling tank 131 (shown in Figure 1), the flow rate of the solution in the pre-potting tank 115, the internal stirring speeds of the pre-potting tank stirrers, the air pressure (such as in compressed air space 111), and the pulsation frequency of the force exerted on the cell / solution suspension 127. For example, the viscosity of the cell suspension / solution (eg, alginate) can be between about 20 and about 200 mPa.s. The concentration of cells (eg Hep G2 cells) in the suspension can be between about 500 and about 10,000 cells / ml. The fluid levels in the pre-encapsulation tank 115 can be between about 10% and about 100% of the tank height; the fluid levels in the post-potting tank 139 can be between about 30% and about 80% of the tank height; and the fluid levels in the dwell tank 131 may be between about 30% and about 80% of the tank height. The flow rate of the solution in the pre-encapsulation tank 115 is between about 0.5 and about 10 liters / min. The internal stirring speed can be between about 1 Hz and about 100 Hz. The air pressure in the compressed air space 111 can be between about 5 kPa and about 600 kPa. The pulsation frequency of the force exerted on the cell / solution suspension 127 can be between about 1 Hz and about 1000 Hz.

Additional parameters refer to the ability of the system to prevent microencapsulated cells or spheres of the encapsulating solution (e.g. alginate) exiting a perforated plate from coalescing, and

ES 2 389 441 T3 to prevent encapsulated cells from being carried into the air. For example, as illustrated in Figure 1, the spacing from the bottom of the perforated plate 141 to the surface of the solution bath 137 can be selected to prevent the exiting microencapsulated cells 125 from coalescing or moving away in the solution. air. In some embodiments, the space between plate 141 and solution 137 can be closed to minimize the distance from plate 141 to the top of solution 137, or a mist of calcium chloride can be maintained immediately below the perforated plate. to facilitate crosslinking as soon as possible. Furthermore, the cell-containing microspheres can begin crosslinking shortly after leaving the perforations and before they have a chance to drift and cross-interact and possibly coalesce. Thus, it may be desirable to facilitate direct flow of microencapsulated cells 125 into bath solution 137 using electrostatic forces. For example, electrostatic forces can be used to pull the droplets away from the perforated plate 141. For example, a spray direction can be applied using electrostatic forces by constructing pre-encapsulation tank 115 and perforated plate 141 with non-conductive materials and forming a high voltage electrical circuit using cell / solution suspension 127 and bath solution 137. (for example, alginate and sodium chloride, respectively).

Optimal conditions can be determined or estimated for the production of spherical beads, such as alginate beads, with minimal variation in shape and size. It may be possible to successfully characterize the device in generating empty alginate microspheres prior to producing encapsulated cells. Subsequently, various additional optimal conditions can be determined, including the pre-encapsulation solution / cells ratio, air pressure, and flow rate of the pre-encapsulation solution for the production of standard size microspheres (diameter range 50-500 mm).

The parameters explained here can be studied and the systems can be designed from the formation of flow patterns through openings and / or nozzles of various sizes. For example, to examine various two-phase and viscous flow capabilities to design the shape of the opening, modeling software such as FLUENT, a Computational Fluid Dynamics (CFD) Code (Fluent Incorporated Network Services,) can be used. Lebanon, NH, USA). Dimensions for the desired size of microspheres can be selected, and the droplet generation process can be simulated. Various parameters such as viscosity, concentration and distance between the plate and the solution of the post-encapsulation tank, for example a calcium chloride bath, can also be studied parametrically using a CFD model of the procedure. Such analysis can be used to design devices according to embodiments of the present invention. CFD results can be validated experimentally. The manufacturing process can be characterized in terms of production speed and the quality of the microcapsules.

In some embodiments, the surfaces that may be in contact with the cells and the suspension solution may be treated or shaped to provide a smooth surface and to reduce the attraction of the cells to the surfaces. For example, various surfaces that can be in contact with cells and the solution in which the cells are suspended can be coated with a thin layer of diamond-like carbon using chemical vapor deposition or a plasma coating technique to provide a smooth flow of alginate and cells, especially through narrow perforated plate pathways. These surfaces can be applied using techniques known to those of skill in the art.

According to embodiments of the present invention, larger numbers of cells encapsulated in microspheres, such as alginate microspheres, can be produced compared to conventional techniques. In some embodiments, the production speed can be increased by about 5000 times or more compared to the conventional devices described herein. For example, certain conventional devices require 100 or more hours for the production of one million microencapsulated islets for transplantation into humans. Embodiments of the present invention can reduce the production time of one million microencapsulated islets to less than about two minutes. The entire encapsulation procedure of one million microencapsulated islets can be completed in less than forty-five minutes, including all post-processing steps, such as the crosslinking residence time of about five to about fifteen minutes, washing for about three to about five minutes, Polyornithine coating procedures with a residence time of about five to about ten minutes followed by washing the encapsulated cells, re-coating the encapsulated cells with alginate, and a chelation process to liquefy the nucleus in about ten to about fifteen minutes. Furthermore, a reduced production time can result in a positive impact on the viability of the encapsulated cells.

Although embodiments of the present invention have been described with respect to the device shown in Figure 1, other configurations can be used. The various tanks can be provided as cylinders or other shapes, such as various polyhedra, cubes, or irregular shapes. For example, higher production rates can be obtained by further piercing a laterally curved surface of a spherical cylinder and pressurizing an enclosed potting solution / cell suspension. The potting solution / cell suspension can be subjected to pulsating pressure at the same time as the cylinder is rotated. The pressure pulsation can be generated by an eccentrically rotating solid cylindrical surface, which can produce a narrow groove and a wide region at a predetermined frequency. The production speed is generally proportional to the length of the cylinder.

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Referring to Figures 5 and 6, a device 200 includes an upper portion 200a for containing the pre-potting tank (not shown) and a post-potting tank 208. The upper portion 200a of the device 200 without the post-potting tank 208 is shown in Figure 7. Device 200 includes an upper frame 210a having a piston 214 to provide pressure to a liquid in the pre-encapsulation tank, a large coaxial shaft 204 to rotate a large stirrer 212, and two smaller shafts 202 to rotate two stirrers 206 More smalls. A lower frame 210b has a perforated plate 218 between the upper portion 200a and the post-potting tank 208. Frame 210b may be connected to vibratory actuators to vibrate perforated plate 218 as described above. The pre-encapsulation tank is not shown for clarity. However, it should be understood that a pre-potting tank may be provided in the region between the upper frame 210a and the lower frame 210b.

As shown in Figure 7, the coaxial shaft 204 includes an inner portion 204a and an outer portion 204b. Inner shaft 204a is connected to piston 214 to provide a pressure pulsating movement. The outer shaft 204b provides rotational movement to rotate the large agitator 212. The agitator 212 can rotate at a relatively slower speed, and the smaller agitators 206 can rotate at a relatively faster speed. The agitators 206 and 212 agitate the cell suspension / solution so that the cells are more evenly spread throughout the suspension. Clustering of cells can be reduced. In particular, agitators 206 can reduce clumping of cells in piston 214.

As illustrated in Figures 6 and 7, the piston 214 of Figure 5 can be removed. Apertures 216 are provided for the introduction and expulsion of air, including compressed air to provide pressure to the cell suspension / solution as explained above.

Another device 300 for use in batch mode processing is shown in Figure 8. Device 300 includes an indexer plate 302 to contain post-potting tanks 304. One or more of the post-potting tanks 304 can be connected to a separate pre-potting tank by means of a perforated plate via flange 306. For example, the pre-potting tank can be the pre-potting tank 208 described. in Figures 5, 6 and 7.

In some embodiments, a single pre-potting tank is connected to a selected one of the post-potting tanks 304. The cells can then be encapsulated using the encapsulation methods and devices discussed here. When the selected post-potting tank 304 has received a given number of encapsulated cells (for example, the selected tank 304 is full), the plate 302 can be rotated so that the pre-potting tank connects to another of post-encapsulation tanks 304. In some embodiments, the post-encapsulation tanks 304 can be replaced with additional tanks for processing, such as tanks filled with calcium chloride solution. For example, an automatic arm can remove and replace tanks 304 that have been filled to a certain capacity with encapsulated cells. In this configuration, a number of tanks 304 can be filled with encapsulated cells to allow post-processing time, such as the time that may be required for cross-linking, and the production of microencapsulated cells can occur substantially continuously. Other multi-tank configurations can be used. For example, post-potting tanks 304 can be provided on a linear conveyor belt.

According to embodiments of the present invention, the microencapsulated microspheres and / or cells can be produced with minimal variation in shape and size. Optimal conditions can be determined as explained here, including alginate / cell ratio, air jacket pressure (e.g. pressure of compressed air space 111 shown in Figure 1), alginate flow rate for production of standard size spherical alginate microcapsules (diameters 300-500 pm, preferably smaller) containing Hep G2 cells. The size of the microcapsules can be controlled. For example, larger capsules can be associated with increased cell death, given the impeded diffusion of nutrients and oxygen to cells in the center of the interior of the capsule. Also, the size of the microcapsules can have an impact on the kinetics of product release from encapsulated cells. Opara EC, Kendall WF. Immunoisolation techniques for islet cell transplantation. Expert Opin Biol Therapy 2: 503-511, 2002. However, the incidence of improper encapsulation can be inversely proportional to the diameter of the capsule. De Vos P, Hamel AF, Tatarkiewicz K: Considerations for successful transplantation of encapsulated pancreatic islets. Diabetologia 45: 159173, 2002. Van Schilfgaarde R, De Vos P: Factors influencing the properties and performance of microcapsules for immunoprotection of pancreatic islets. J Mol Med 77: 199-205, 1999. An optimal microcapsule size range of 500-600 microns in diameter can be achieved without significantly compromising the suitability of the capsules.

The parameters can be adjusted to decrease the degree of imperfection in the microcapsule shape, such as appendage formation, which is associated with fibrotic reactions around alginate spheres. One factor that affects the size, shape, and degree of unsuitability in alginate microspheres is the shear stress involved in the encapsulation process. For example, when the shear stresses during the production of the alginate spheres are very high, unwanted tiny satellite micro-artifacts are generated inside and outside the alginate beads. Certain effects of shear stresses on extruded non-Newtonian viscosity fluids are known. You can carry out the analysis of the Code of Dynamics of

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Computational Fluids (CFD) to quantify slow shear stresses in the procedure and design procedure parameters to keep maximum shear stresses under tolerable maximums.

In certain cases, fibrotic hypertrophy can result in oxygen and nutrient starvation of the enclosed cells. This problem can be reduced by using purified alginate. Sun Y, Ma X, Zhou D, Vacek I, Sun AM. Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immunosuppression. J Clin Invest 98: 1417-1422, 1996. Soon-Shiong P, Heintz RE, Merideth N, Yao QX, Yao Z, Zheng T, Murphy M, Molloney MK, Mendez R, Sandford PA. Insulin independence in type 1 diabetic patient after encapsulated islet transplantation. Lancet 343: 950-951, 1994. Hasse C, Zielke A, Klock G, et al. First successful xenotransplantation of microencapsulated human parathyroid tissue in experimental hypoparathyroidism: long-term function without immunosuppression. J Microencapsulation 14: 617-626, 1997. Lanza RP, Chick WL. Transplantation of encapsulated cells and tissues. Surgery 121: 1-9, 1997. De Vos P, Van Straaten JFM, Nieuwenhuizen AG, de Grrot M, Ploeg RJ, De Haan BJ, Van Schilfgaarde R. Why do microencapsulated islet grafts fail in the absence of fibrotic overgrowth? Diabetes 48: 1381-1388, 1999. Zimmermann U, Mimietz Ser M, et al. Hydrogel-based non-autologous cell and tissue therapy. BioTechniques 29: 564-581, 2000. In addition to the purity, the size, durability and any imperfections in the shape of the alginate microcapsules may be important factors to consider in the use of islet microcapsules for transplantation. De Vos P, De Haan B, Wolters GHJ, Schilfgaarde RV. Factors influencing the adequacy of microencapsulation of rat pancreatic islets. Transplantation 62: 888-893, 1996. De Vos P, De Haan B, Pater J, Schilfgaarde RV. Association between capsule diameter, adequacy of encapsulation, and survival of microencapsulated rat islet allografts. Transplantation 62: 893899, 1996. Lanza RP, Jackson R, Sullivan A, Ringeling J, McGrath C, Kuhntreber W, Chick WL. Xenotransplantation of cells using biodegradable microcapsules. Transplantation 67: 1105-1111, 1999. Leblond FA, Simard G, Henley N, et al. Studies on smaller (~ 315 μ) microcapsules: Feasibility and safety of intrahepatic implantations of small alginate poly-L-lysine microcapsules. Cell Transplant 8: 327-337, 1999. Darrabie M, Freeman BK, Kendall WF, Hobbs HA, Opara EC. Durability of polylysine alginate microcapsules. J Biomed Mater Res 54: 396-399, 2001. In certain embodiments, the microencapsulation process involves the formation of gelled alginate spheres containing cells, followed by a first coating with poly-L-lysine, and an outer coating with alginate to form a semi-permeable membrane around the initial alginate core. Opara EC. The therapeutic potential of islet cell transplants in the treatment of diabetes. Expert Opin Investig Drugs 7: 1-11, 1998. Without being bound by any particular theory, it may be important that the outer alginate coating process be completed because poly-lysine tends to promote fibroblast adhesion, causing the capsules to become completely covered by cell growth along with other inflammatory reactions, within a week of transplantation. Fan MY, Lum ZP, Fu XW, et al. Reversal of diabetes in BB rats by transplantation of encapsulated pancreatic islets. Diabetes 39: 519-522, 1990. In preliminary studies, it appears that poly-L-ornithine may be less immunogenic than poly-L-lysine. Hobbs HA, Kendall WF, Darrabie M, Collins B, Bridges S, Opara EC. Substitution of polyornithine for polylysine in alginate microcapsules. Diabetes 49 (Suppl 1): A111, 2000. In addition, chelation (liquefaction) of the cross-linked alginate may be necessary for enhanced diffusion of permissible molecules, such as insulin, across the semipermeable membrane. Garfinkel MR, Harland RC, Opara, EC. Optimization of the microencapsulated islet for transplantation. J Surg Res 76: 7-10, 1998. The use of barium as a crosslinking cation can result in oxygen deprivation of encapsulated cells (Schrezenmeir J, Kirchgessner J, Gero L, et al. Effect of microencapsulation on oxygen distribution in islets organs. Transplantation 57: 1308-1314, 1994) because barium cross-linked alginate microcapsules cannot be liquefied. Zimmermann U, Mimietz Ser M, et al. Hydrogel-based non-autologous cell and tissue therapy. BioTechniques 29: 564-581, 2000. Other researchers have shown that by increasing the incubation time during the poly-lysine coating, the thickness of the poly-lysine membrane can be increased, and the exclusion of molecular sizes of the microcapsules. Brissova M, Petro M, Lacik I, Powers AC, Wang T. Evaluation of microcapsule Permeability via inverse size exclusion chromatography. Anal Biochem 242: 104-111, 1996.

The mechanical strength and durability of the microcapsules can also be determined or estimated as part of the device parameter optimization procedure. The permeability properties of the microcapsules can be selected using standard pore size exclusion techniques. Powers AC, Brissova M, Lacik I, et al. Permeability assessment of capsules for islet transplantation. Ann NY Acad Sci 831: 208216, 1997. To determine the behavior of microcapsules produced according to embodiments of the present invention, various data can be used in these parameters, including data generated with a single needle device.

The following non-limiting examples are provided to illustrate embodiments of the present invention in detail. The following examples, to provide the use of molecular probing techniques for rapid assessment of cell viability after encapsulation, a single cell line may be used in place of a tissue composed of cells, such as the islet.

Example 1

Production of encapsulated Hep G2 cells

Encapsulated Hep G2 cells can be produced, for example, using the encapsulation device 100 shown in Figure 1. The encapsulating solution (in cell suspension 127 / solution) is alginate

ES 2 389 441 T3 of 1.5% ultra-purified sodium (Kelco / Monsanto, San Diego). Post-encapsulation solution 139 is calcium chloride. Wash solution 133 is salt bath.

Example 2

Post-production and assay stages of encapsulated Hep G2 cells

The beads can be coated first with about 0.05% to 0.1% polyornithine for permeselectivity, followed by a second coating with about 0.1% to 0.3% alginate. Encapsulated Hep G2 cell viability assays can be performed using the LIVE / DEAD® Viability / Cytotoxicity Kit Assay (L-3224, Molecular Probes, Inc., Eugene, OR). This assay provides a two-color fluorescence assessment of cell viability that is based on the simultaneous determination of live and dead cells with two probes that measure two recognized parameters of cell viability, namely intracellular esterase activity and integrity of the plasma membrane. The fluorescence images obtained after this assay identify dead cells in red, and live cells in green.

Example 3

Assays of a-fetoprotein

The cells of Example 2 can be tested using the enzyme-linked immunosorbent assay (ELISA) technique to demonstrate that α-fetoprotein (MW = 76,000 kDa), a key protein made by hepatocytes, can be synthesized and secreted out after culture. of encapsulated Hep G2 cells. Unencapsulated and microencapsulated Hep G2 cells can be cultured in minimal essential medium (MEM) supplemented with 20% normal rat serum. Samples (0.5 ml) of the culture medium can be taken after 1, 2, 3, 4 and 9 days, for the α-fetoprotein (AFP) assay using the Dot-Blot apparatus (Bio-Rad) with standard procedures. AFP can be detected using rabbit anti-human AFP antibody. The primary antibody can be visualized using HRP-conjugated anti-rabbit antibody and a chemiluminescence kit. The amounts of AFP in the culture media of both unencapsulated and encapsulated Hep G2 cells can be measured after a short culture (24 hours).

Example 4

Assays of metabolic activity

Isolated encapsulated rat islets, or other encapsulated cells such as those produced in Example 2, can be used to assess the function of microencapsulated cells. The test is based on the evaluation of the metabolic activity of cells in the absence or in the presence of a metabolic fuel such as glucose. The assay can be easily applied to any cell type for immediate assessment of cell function. After encapsulating isolated rat islets, capsules containing an islet / capsule can be selected for experiments. The intensity of the fluorescence increases above the basal level (without glucose) as oxygen utilization is increased during glucose metabolism by the encapsulated islets, thus showing that the encapsulated islets are viable.

Example 5

Microencapsulation procedure

Experiments can be performed with ultra-purified, low viscosity, high mannuronic acid alginate (Kelco / Monsanto, San Diego, CA). Small droplets (300-500 pm diameter) of 1.5% alginate with or without Hep G2 cells can be generated in large quantities using the procedures described in Example 1. More specifically, the microcapsules can be gelled as they fall into a 1.1% calcium chloride solution, in which they are allowed to incubate for 15 minutes resulting in solid spherical alginate beads, which are then washed in saline. normal. These beads can be coated with poly-Lornithine by incubation in 0.1% solution of this amino acid polymer for 6-20 minutes (which was varied to manipulate the permeability of the capsule), followed by washing in normal saline. Further coating of the microcapsules can be accomplished by incubating them in 0.25% alginate solution for 4 minutes, followed by another wash with saline. The initial alginate bead can then be liquefied by incubation in 55 mM sodium citrate solution for 7 minutes before two final washes with saline. Quality control for microcapsules can be carried out by visual examination under the microscope and by measuring their mechanical strength and durability.

Example 6

Determination of the physical characteristics of the microcapsules

The sizes and numbers of capsules and cells produced using the techniques explained in Example 5 can be measured using an inverted microscope that is equipped with a Pixera camera and linked to a computer program (UTHSCSA Image Tool, University of Texas) for the determination. of the number and size of

ES 2 389 441 T3 particles. Also, the shape of the capsules, and imperfections in the microcapsules, such as the formation of appendages, and the protrusion of the cells through the membrane of the capsules, can be evaluated and photographed using the microscope and its formation system. of pictures.

Example 7

Determination of the mechanical resistance and durability of the microcapsules

The mechanical strength of the microencapsulated cells produced as described in Example 5 can be evaluated by measuring the resistance of the microcapsules to mechanical stress, using various procedures. For example, fluorescein isothiocyanate (FITC) labeled dextran (MW 50-150 kDa) can be microencapsulated as depicted above. The microcapsules can then be mixed with 3mm glass beads and continuously shaken for a fixed time up to 48 hours. The percentage of capsules ruptured can be calculated by measuring the fluorescence in the supernatant, and in the residual intact capsules after thorough washing in saline before dissolution. Another procedure includes a manual method (for example hand-picking under a stereomicroscope), in which the percentage of broken capsules can be obtained using a ratio of 250 glass beads per 1000 microcapsules. Leblond FA, Tessier J, Halle JP. Quantitative method for the evaluation of biomicrocapsule resistance to mechanical stress. Biomaterials 17: 2097-2102, 1996. The durability of the microcapsules can be determined using in vitro incubations in RPMI 1640 culture medium supplemented with serum and maintained for periods of up to 6 months at 37 ° C. Hobbs HA, Kendall WF, Darrabie M, Opara EC. Prevention of morphological changes in alginate microcapsules for islet xenotransplantation. J Investig Med 49: 572-575, 2001.

Example 8

Functional Viability of Encapsulated Hep G2 Cells

Molecular probes can be used to assess the quality of encapsulated cells to determine cell viability and proliferation, and metabolic techniques can be used for function assessment. The function of encapsulated Hep G2 cells can be compared to that of unencapsulated cells. In addition, ELISA assays can be used to determine the identity and quantity of specific proteins (albumin, α-fetoprotein, and transferrin) synthesized and exported by encapsulated Hep G2 cells.

Example 9

Feasibility tests

The LIVE / DEAD® Viability / Cytotoxicity Kit Assay (L-3224, Molecular Probes, Inc., Eugene, OR) is a fluorescence-based cell viability assessment method, and can be used in place of exclusion with trypan blue, 51 Cr release, and similar methods to determine cell viability and cytotoxicity. After encapsulation, the viability of the encapsulated cells can be determined by performing the LIVE / DEAD® Cell Assay immediately after encapsulation using a sample of the capsules, as previously explained. The ability of cells to proliferate can be measured by repeating the LIVE / DEAD® assay on aliquots of the encapsulated cells every other day for 7 days of culture, and performing the cell proliferation assay, as described above. An assay can be carried out which is based on the metabolic activity of the cells and which is linked to the consumption of oxygen by the encapsulated cells.

Example 10

Cell proliferation assay

The CellTitre 96RAQueous Solution Cell Proliferation Kit Assay (Promega Corporation, Madison, WI) can be used for the following evaluation. This is a colorimetric method to determine the number of viable cells in proliferation or cytotoxicity assays. The kit reagent contains a new tetrazolium compound [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium inner salt; MTS (a)] and an electron coupling reagent (phenazine ethosulfate; PES). PES has improved chemical stability, allowing it to combine with MTS to form a stable solution. This assay can be a reliable surrogate for the [3H] thymidine incorporation procedure. Cory AH, Owen TC, Barltrop JA, Cory JG. Use of an aqueous soluble tetrazolium / formazan assay for cell growth assays in culture. Cancer Commun 3: 207-212, 1991. Riss TL, Moravec RA. Comparison of MTT, XTT, and a novel tetrazolium compound for MTS for in-vitro proliferation and chemosensitivity assays. Mol Biol Cell (Suppl) 3: 184a, 1992.

Example 11

Oxygen biosensor assay

The following procedure tests the function of the encapsulated cells. The principle of this fluorescence assay is based on the ability of living cells to use oxygen, and provides an instant assessment of the metabolic function of cells. Unencapsulated (control) and encapsulated cells can be

ES 2 389 441 T3 place in microplates containing RPMI 1640 culture medium, with sensors (eg BD Blosensor system) at the bottom of the wells. Oxygen diffuses from the atmosphere into the environment, and is consumed by living cells. As oxygen is depleted, the biosensor fluoresces, providing a linear signal that can be directly correlated with cell viability, to which viability index scores can be assigned. Scores can be based on the rate of stimulation of oxygen utilization in 11.1 mM glucose, compared to the rate in the absence of glucose; high = 4; good = 3; acceptable = 2; bad = 1; no oxygen consumption = 0. Blank microplates containing the test materials but excluding cells can be mounted and used to correct for any background oxygen consumption in the test system. As a positive control for this assay, sodium sulfite can be used.

An acceptable level of viable cells obtained as determined by the LIVE / DEAD cell assay immediately after encapsulation can be about 80% or greater. The number of viable cells may increase as the duration of the culture increases, which may be indicative of the cells' ability to proliferate. This phenomenon can be confirmed by the cell proliferation assay. Encapsulated Hep G2 cells can also be shown to be metabolically viable using the Biosensor Oxygen Consumption Assay.

Example 12

Determination of encapsulated Hep G2 cell products

ELISA assays can be carried out with culture media obtained after incubation of encapsulated Hep G2 cells, in order to identify and quantify specific proteins synthesized and exported outside the capsules. Specifically, assays can be carried out to determine the ability of encapsulated Hep G2 cells to synthesize and export important proteins such as albumin (MW = 66 kDa), α-fetoprotein (MW = 70 kDa), and transferrin (MW = 76 KDa), which can be secreted outside the capsules. Using standard microencapsulation procedure, outlined here, the microcapsule pore size exclusion limit can be approximately 75 kDa. Antibodies / kits to measure these proteins formed and secreted by Hep G2 cells are available from Sigma Co. (St. Louis, MO). Large molecular weight proteins, such as ceruloplasmin (135 kDa), IgG (150 kDa), fibrinogen (340 kDa), and alpha2-macroglobulin (720 kDa), which are also formed by Hep G2 cells, may exceed the limit of pore size of the microcapsules obtained under certain conditions. Adjustments can be made in the concentration of the poly-ornithine solution, as well as the incubation time of the alginate beads in the poly-ornithine solution, in order to manipulate the pore size of the semipermeable membrane. Brissova M, Petro M, Lacik I, Powers AC, Wang T. Evaluation of microcapsule permeability via inverse size exclusion chromatography. Anal Biochem 242: 104-111, 1996. Powers AC, Brissova M, Lacik I, et al. Permeability assessment of capsules for islet transplantation. Ann NY Acad Sci 831: 208-216, 1997. These settings may exclude immune cells, but possibly allow the exit of additional products from the encapsulated Hep G2 cells, including small molecular weight immunoglobulins, such as IgG (MW = 150 kDa). .

Encapsulated Hep G2 cells may be able to synthesize protein in a manner similar to their non-encapsulated counterparts. With adjustments in the perme-selectivity of the microcapsule membrane, molecular products (proteins) with molecular weights less than 150 kDa can be efficiently excreted by encapsulated Hep G2 cells.

Quantitative data can be expressed as mean + standard error, and for comparative data between sets of experiments, the data can be normalized by given volume or number of capsules or cells. Statistical evaluation of data requiring multiple comparisons can be carried out using an analysis of variance (ANOVA) computer program (GraphPad, San Diego, CA), and, depending on the ANOVA result, the Bonferroni correction will be used. to evaluate the significance of the difference between the samples. In comparisons of the significance of the difference between the means of two groups of data, the Student's t test can be used. A value of p <0.05 can be accepted as significant.

In the above examples, poly-L-lysine can be substituted for poly-L-ornithine as the semipermeable coating of the initial alginate bead. When imperfections occur by exposing the polyamino acid membrane of the microencapsulated cell, poly-ornithine may be less immunogenic than poly-lysine. Hobbs HA, Kendall WF, Darrabie M, Collins B, Bridges S, Opara EC. Substitution of polyornithine for polylysine in alginate microcapsules. Diabetes 49 (Suppl 1): A111, 2000. Additionally, poly-ornithine coating can provide lower permeselectivity for alginate microcapsules, and can result in microcapsules that are less prone to swelling than poly-lysine coated capsules, thereby obviating the need for sulfate treatment. sodium from poly-ornithine coated alginate capsules. The inner alginate core of the microcapsules can be liquefied to enhance the diffusion of oxygen, nutrients and hormones, for a more desirable function of the encapsulated islet cells. Garfinkel MR, Harland RC, Opara, EC. Optimization of the microencapsulated islet for transplantation. J Surg Res 76: 7-10, 1998. The swelling of the microcapsules can cause the destruction of the hollow alginate microcapsules coated with poly-lysine, by altering their sizes and shapes over time [19, 27]. Darrabie M, Freeman BK, Kendall WF, Hobbs HA, Opara EC. Durability of polylysine alginate microcapsules. J Biomed Mater Res 54: 396-399, 2001. Hobbs HA, Kendall WF,

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Darrabie M, Opara EC. Prevention of morphological changes in alginate microcapsules for islet xenotransplantation. J Investig Med 49: 572-575, 2001. The swelling of microcapsules induced by colloidal osmotic pressure can be reduced.