WO2012099482A2 - Device, method and system for preparing microcapsules - Google Patents
Device, method and system for preparing microcapsules Download PDFInfo
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- WO2012099482A2 WO2012099482A2 PCT/PT2012/000003 PT2012000003W WO2012099482A2 WO 2012099482 A2 WO2012099482 A2 WO 2012099482A2 PT 2012000003 W PT2012000003 W PT 2012000003W WO 2012099482 A2 WO2012099482 A2 WO 2012099482A2
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- tubing
- needle
- microcapsules
- syringe
- horizontal
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/0052—Preparation of gels
- B01J13/0065—Preparation of gels containing an organic phase
Definitions
- the present invention relates to the encapsulation of bioactive agents and cells into hydrogel microcapsules with homogenous size distribution. More particularly, this invention relates to the generation of homogenous size micro droplets of polymer/cells mixture when injected into cross-flowing oil stream inside elastic tubing .
- microcapsulation of living cells inside microcapsules is of great importance for cell therapy, cell culture and tissue engineering applications.
- the technology of cell microencapsulation is based on the immobilization of cells within a polymer matrix, often alginate, surrounded by a poly (lysine) coat.
- the hydrogel microcapsules protect encapsulated cells from immunological reactions against cells by forming a barrier, which can block immunoglobulin and white blood cells.
- the microcapsules produced in homogenous size distribution and spherical shapes can be used as cell carriers and can facilitate cell delivery through syringe injection.
- the microcapsules can provide a l suitable 3D environment for in vitro cell culture. Microcapsules with a mean diameter between 300 and 600 ⁇ are generally considered optimal for cell encapsulation.
- capsules can be prepared with some degree of success, the majority of these methods present several problems and limitations, such as difficulty to control the microcapsule diameter and poor overall balance of capsule properties in terms of stability, permeability, and biocompatibility, among others.
- the microfluidic microencapsulation method has emerged as a recent technology. Microfluidic devices have been applied for the formation of water-in-oil (w/o) multiphase flow regime having monodisperse droplets (S. Abraham, Y. H. Park, J. K. Lee, C. S. Ha, I. Kim, Microfluidic synthesis of reversibly swelling porous polymeric microcapsules with controlled morphology. Adv. Mater., 2008, 20:2177- 2182) .
- microfluidic devices are highly labour intensive and requires sophisticated microfabrication techniques.
- a crystalline silicon wafer or glass slide coated with a photo-sensitive polymer (photoresist) is used.
- a mask containing a pattern designed in the micro-scale is applied over the photoresist, followed by electron beam or UV irradiation.
- a carefully monitored etching process is made on the substrates to obtain reproducible etching depth.
- the etched substrate must be drilled for fluid intake and outflow and connectors are mounted to those drilled holes on the fragile substrate.
- the tubing device of the present invention does not require all those microfabrication and drilling steps.
- the elastic tubing used in the present invention commercially available in a wide range of standard internal diameters, provides convenient flow regimes.
- This tubing device only requires the assembling of one central part, a blunt-end needle supported by a holder in vertical position.
- the overall assembled device is considerably flexible and can be mounted to peristaltic and syringe pumps readily providing tubing connectors.
- the present invention is well suited for laboratory and pilot applications.
- microcapsules harbouring living cells have also found applications in cell culture in vitro and tissue engineering fields. There is a considerable interest in production of mono-size microcapsules or microbeads of uniform size for the encapsulation of living cells.
- the microcapsule For successful clinical or laboratory use, the microcapsule must be produced in a narrow size distribution and spherical morphology.
- the device producing microcapsules must be reusable, easily cleanable and sterilizable by convenient techniques such as autoclaving.
- microfluidics technology has provided a new route to prepare microcapsules with controlled morphology and uniform size.
- Most of microfluidics systems have been intended for drug encapsulation purposes .
- Over the past few years there has been several studies reporting this technique for cell encapsulation.
- the preparation complexity of microfabrication procedures may challenge its practical application in cell microencapsulation applications.
- the aim of the present invention is the development of a cell microencapsulating device based on microdroplet formation of a polymer/cell mixture when injected into cross-flowing oil in a narrow tubing channel.
- the device is autoclavable and produced by using biocompatible materials; its design allows easy connection with syringe and peristaltic pumps with proper syringe and syringe needle connectors.
- the present invention relates to a microencapsulation device based on micro droplet generation by injecting a hydrogel mixture into a oil stream inside a tubing.
- the device can produce microdroplets in a narrow size distribution under constant injection rate of polymer/cell or polymer/ bioactive agent mixtures and oil flow maintained by a syringe and peristaltic pump, respectively.
- the described microdroplet generator is capable of dispensing a polymer solution into oil flow of tubing by secure positioning of a blunt-ended hypodermic needle into the centre of channel space vertically.
- the stable junction of needle and seal at puncture site of tubing is provided by an elastic needle holder, which can fix the needle in a vertical position and wrap-seal around tubing tightly.
- the microdroplet generator for cell or bioactive agent encapsulation is composed of a platform, elastic tubing, blunt-ended hypodermic needle, needle holder and adapters for syringe and tubing ends.
- the platform preferably made from a poly (tetrafluoroethylene) (PTFE) block, possesses a horizontal open semicircular hollow space for holding and orienting the tubing in a straight position and two adjustable clips for fixing the tubing in place.
- the hypodermic needle is inserted halfway the tube diameter through a small hole and the junction needle/tubing is stabilized and assured by the needle holder.
- Said needle holder has a hollow cylindrical part which allows the needle to pass through the channel and supports the Luer hub of needle at the top.
- the cylindrical part of the needle holder is made concave at the bottom so that it can fit and grip the tubing for better sealing of the hole. Additionally, the tubing holder stabilizes the tubing and needle by its semicircular wings, which can be glued on the tubing surface .
- the polymer solution injection in the hypodermic needle is made by the syringe pump through an extender tubing, which is connected to the needle and syringe by Luer lock-tubing and syringe-tubing adapters, respectively.
- the oil perfusion to the inlet of tubing which is horizontally fixed on a platform, is performed by a peristaltic pump.
- the outlet of tubing is connected to a collector vessel, which contains a gelling solution with a emulsifying agent for microcapsule formation.
- the solution in the collector vessel can be conventionally stirred by using a magnetic stirrer during gelling process .
- Biocompatible surface active agents like Tween-80 ® can be used in the gelling solution to prevent aggregation of microdroplets of the polymer/cells mixture when exiting trough the outlet of tubing and entering into contact with the gelling solution.
- a gel forming polymer solution is used to form microcapsules that can encapsulate various materials .
- the material must be of a size small enough to be suitable for encapsulation by the droplet method of this invention but can vary widely in diameter from less than a 100 micron to several millimetres.
- One of the most critical aspects of the present invention is the ability of the device to generate micro-droplets with a uniform distribution and tuneable sizes. The present process allows viable cells to be encapsulated in microcapsules.
- the process of this invention is particularly well suited for use in encapsulating biological materials.
- the biological materials to be encapsulated can be tissue, organelle, plant or animal cells, bacteria, among others.
- the present invention uses a murine ATDC5 chondrocyte cell line in the cell encapsulation experiments.
- the cells to be encapsulated are not limited to chondrocytic cells but include other cell types, such as primary cultures (pancreatic islets, hepatocytes, fibroblasts, osteoblasts, articular chondrocytes, mesenchymal stem cells) as well as established cell lines.
- the gel-forming polymer can be any non-toxic water- soluble gel-forming polymer, which forms a gel upon contact with a gelling inducer (multivalent ions or charged polymers) .
- the gel-forming polymer can be a water-soluble polysaccharide. Suitable polysaccharides include sodium alginate, guar gum, gum arabic, carrageenan, pectin, xanthan gum, gellan gum, chitosan. Upon contact with gel inducers the polysaccharide molecules form a water-insoluble shape-retaining gel membrane microcapsule.
- polysaccharides include, for example, hyaluronic acid, chondroitin sulfate, dextran sulfate, heparin, heparin sulfate, heparan sulfate. These polymers can form capsules by polyelectrolyte complexation.
- the gel forming polymers that can be used with the present invention is not limited to completely aqueous solutions of hydrophilic polymers but also amphiphilic polymers, which have molecular self- organization properties in physiological solutions.
- hydrophobic polymers that dissolve in organic solvents can be processed with the device by providing an immiscible aqueous solution as continuous phase in tubing in order to obtain an emulsion of oil-in-water .
- Figure 1 illustrates the tubing microdroplet apparatus in an exploded and in an assembled isometric view.
- Figure 2 illustrates the cross-section of the central part of the device at anterior position where the needle is inserted to tubing and sealed by needle holder.
- Figure 3 illustrates the encapsulating apparatus including the tubing that generate the microcapsules together with perfusion devices through a peristaltic pump and the polymer/cells injection through a syringe pump.
- Figure 4 presents a photograph of cell microscopy fluorescence (murine ATDC5 chondrocyte cell line) encapsulated in carboxymethyl xanthan microcapsule showing that the encapsulated cells remain viable after 21 days of culture.
- the microencapsulator apparatus (1) is mainly composed of a horizontal tubing (5) and a vertical needle (7) supported by a tubing platform (2) and needle holder (6) , respectively.
- the vertical part of this device consists of a needle holder (6) , a blunt-ended needle (7) and an adapter (8) , which connects both Luer lock hub part of needle (coloured plastic part with an universal fitting system) and a tubing extender (9) .
- a syringe-tubing adapter (10) makes the connection with the syringe (11) , which is mounted to a syringe pump
- the horizontal tubing into which the needle is inserted is supported by a platform (2) .
- the platform contains a semicircular open-channel (3) and adjustable clips (4) for fixation of tubing in stretched and horizontal form.
- PTFE polypropylene
- Novosil ® biocompatible and heat resistant silicone elastomer tubing
- the diameter of tubing and the needle gauge size can be varied according to the specific needs of encapsulation process.
- the invention here described uses a tubing of 1 mm internal diameter and a needle with gauge size of 21G to produce microdroplets from viscous polysaccharide solution, which is providing a stable cell encapsulation, near 400 ⁇ .
- the micro droplet size can be reduced by using a needle with smaller gauge number and tubing with narrower internal diameters. Conversely, the size of microdroplets can be increased by vice versa.
- the blunt-ended needle is inserted into the horizontal tubing through a needle holder.
- the insertion of needle can be facilitated by puncturing a narrow hole (with a diameter inferior to that of the needle) before the insertion process.
- the needle can easily move into the centre of tubing channel by extending the hole needle holder and tubing wall, as elastic materials are used.
- This insertion method physically seals the needle at the insertion site.
- the needle holder can be wrapped around tubing for better fixation.
- the needle holder has dual function: fixing the needle in vertical position preventing its dislocation from puncture site and provides an effective sealing on the tubing preventing leakage of liquids. The sealing is best provided by applying a moderate layer of silicone glue on the wings of needle holder before inserting needle into the tubing.
- the extender tubing (9) provides a flexible connection between the syringe (11) , which is mounted between the syringe pump (12) and the needle (7) , using Luer hub lock-tubing (8) and syringe-tubing adaptor (10) .
- the sterile mineral oil which can be autoclavable and bioinert, is reserved in a Schott Bottle (13) which is mounted to a pipetting needle with a Luer hub and membrane filter (0.22 ⁇ ) in its cap for maintaining the atmospheric pressure in vessel in a sterile condition.
- the oil is pumped out from the reservoir and carried into the tubing device by using a peristaltic pump via sterile tubing with stoppers (15) .
- the shear force of the oil flow inside tubing device then acts on the tip of needle forming microdroplets of polymer/cells.
- the microdroplets in oil can be carried into the gelling inducer solution vessel (17) using a relatively wider and inert tubing (preferably PTFE) (16) which provides minimum microdroplet interaction on surface.
- the microdroplets gellify when they exit the tubing and enter into gelling inducer solution.
- the mild but enough stirring rate is essential in order to prevent aggregation of gelling microcapsules. In this stage, a harsh stirring can damage the premature microcapsule walls, especially when a magnetic bar is used for stirring.
- the use of a biocompatible emulsifiying agent like Tween 80 ® at low concentration can effectively prevent aggregation of microcapsules in gelling inducer solution.
- the gelling inducer solution composition can change depending of the gelation method pretended (eg. ionic crosslinking, ionic complexations or self-assembly gelation techniques) .
- the gelling solution can be prepared by using bivalent salts like CaCl 2 for anionic hydrogels at an effective concentration. It is essential that the ionic strength of crosslinking solution must be maintained in physiological ion concentration (0.9% NaCl) and pH at 7.4 for viability of encapsulated cells.
- the polyionic solution may be constituted by an oppositely charged polyelectrolyte relatively to the polyelectrolyte microdroplets .
- the complexation reaction takes place when the oppositely charged polymers react (e.g. interactions polysacharide-polysaccharide; polysacharide-protein/peptides) .
- the pH should be slightly lower than isoelectric point, while the pH of polysaccharide should be maintained at physiological pH.
- the gelling inducer solution for self- assembled biopolymers can be any kind of physiological solution, (e.g. phosphate buffered saline or cell culture medium) , which may induce the gel formation through self- assembly process.
- Example 1 - This example demonstrates the preparation of microcapsules by ionic crosslinking of the polyanionic biopolymer (carboxymethyl xanthan gum) in the presence of counterions.
- the mixture of bioactive material cell suspension of murine ATDC5 chondrocytes
- carboxymethyl xanthan 5%) in culture medium is extruded through the droplet forming device into a gelling solution containing 1.5% CaCl 2 and 0.9% NaCl .
- the Ca +2 cations cross- link the carboxymethyl xanthan matrix almost instantaneously to form gel beads.
- the beads are then treated with poly-L- lysine to strengthen the outer surface of the microcapsules .
- the microcapsules present spherical shape with an average diameter of 500 ⁇ ( Figure 4) . Live/dead assay of encapsulated cells shows that the cells remain viable in the microcapsules up to 21 days of culture.
- Example 2 - This example demonstrates a mild cell encapsulation method based on triggering of self-assembly of amphiphile constructed by pH and ion sensitive polysaccharide (xanthan gum) , which is conjugated to hydrophobic palmitic acid.
- the mixture of bioactive material cell suspension of murine ATDC5 chondrocytes
- palmitoyl xanthan (1%) is extruded through the droplet forming device into phosphate buffer solution (PBS) .
- PBS phosphate buffer solution
- the palmitoyl xanthan droplets self-assemble into capsular gel structure.
- the formed hollow microcapsules are then treated with poly-L- lysine to strengthen the outer surface of the microcapsules .
- microcapsules present spherical shape with an average diameter of 450 ⁇ .
- the ability of palmitoyl-xanthan microcapsules to sustain viability and proliferation of encapsulated cells was confirmed by the AlamarBlue ® and cells deoxyribonucleic acid (DNA) quantification assays.
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Abstract
The invention discloses a novel device for the formation of microcapsules based on micro-droplet generation inside silicone tubing, which is punctured with a blunt-ended syringe needle vertically (1). The device uses droplet generation by vertical injection of hydrophilic polymer/cell mixture into hydrophobic oil flow, which is horizontally maintained in the silicone tubing. The injection of polymer/cell mixture into a stream of mineral oil results in the generation of spherical droplet and in the formation of a water- in-oil emulsion due to the immiscibility of the two phases. Subsequently, the micro-droplets in oil phase are converted into stable microcapsules by gelation in a separate chamber which is loaded with ionic cross- linking solution at physiological ionic strength and pH. The utility of the microcapsules generated by the device of present invention is virtually unlimited in the fields of regenerative medicine, controlled delivery of growth factor or drug.
Description
DESCRIPTION
DEVICE, METHOD AND SYSTEM FOR PREPARING MICROCAPSULES
FIELD OF INVENTION
The present invention relates to the encapsulation of bioactive agents and cells into hydrogel microcapsules with homogenous size distribution. More particularly, this invention relates to the generation of homogenous size micro droplets of polymer/cells mixture when injected into cross-flowing oil stream inside elastic tubing .
STATE OF THE ARTE
The encapsulation of living cells inside microcapsules is of great importance for cell therapy, cell culture and tissue engineering applications. The technology of cell microencapsulation is based on the immobilization of cells within a polymer matrix, often alginate, surrounded by a poly (lysine) coat. The hydrogel microcapsules protect encapsulated cells from immunological reactions against cells by forming a barrier, which can block immunoglobulin and white blood cells. The microcapsules produced in homogenous size distribution and spherical shapes can be used as cell carriers and can facilitate cell delivery through syringe injection. Besides, the microcapsules can provide a l
suitable 3D environment for in vitro cell culture. Microcapsules with a mean diameter between 300 and 600 μπι are generally considered optimal for cell encapsulation.
Several methods for microcapsule manufacture have been described in the literature. These methods include the use of different droplet-forming systems, such as the extrusion with the droplet-forming technique (with or without syringe pump) {K. Ohkawa, T. Kitagawa, H. Yamamoto, Preparation and characterization of chitosan- gellan hybrid capsules formed by self-assembly at an aqueous solution interface, Macromol. Mater. Eng., 2004, 289:33-40) electrospray processes ( Y. Fukuia, T. Maruyama, Y. Iwa atsua, A. Fujiia, T. Tanakaa, Y. Ohmukaia, H. Matsuyam, Preparation of monodispersed polyelectrolyte microcapsules with high encapsulation efficiency by an electrospray technique, Coll. Surf. A, 2010, 370 (1-3) : 28-34) ; coextrusion techniques
(coextrusion nozzle) ( . V. Sefton, J.R. Hwang, J. E. Babensee, Selected aspects of microencapsulation of mammalian cells in HEMA-MMA. Bioartif. Organs, 1997, 831: 260-270) ; or more sophisticated devices such as microfluidics {S. -K. Hsiung, C. -T. Chen, G. -B. Lee, Micro-droplet formation utilizing microfluidic flow focusing and controllable moving-wall chopping techniques. J. Micromec. Microeng. , 2006, 16:2403-2410).
Although capsules can be prepared with some degree of success, the majority of these methods present several problems and limitations, such as difficulty to control the microcapsule diameter and poor overall balance of
capsule properties in terms of stability, permeability, and biocompatibility, among others. The microfluidic microencapsulation method has emerged as a recent technology. Microfluidic devices have been applied for the formation of water-in-oil (w/o) multiphase flow regime having monodisperse droplets (S. Abraham, Y. H. Park, J. K. Lee, C. S. Ha, I. Kim, Microfluidic synthesis of reversibly swelling porous polymeric microcapsules with controlled morphology. Adv. Mater., 2008, 20:2177- 2182) . Very recently, the micofludics technology has been applied for encapsulation of mammalian cells into polysaccharide microcapsules (L. Capretto, S. Mazzitelli, G. Luca, C. Nastruzzi, Preparation and characterization of polysaccharidic microbeads by a microfluidic technique: Application to the encapsulation of Sertoli cells. Acta Biomater. , 2010, 6:429-435) .
On the other hand, the production of microfluidic devices is highly labour intensive and requires sophisticated microfabrication techniques. Typically, in these techniques, a crystalline silicon wafer or glass slide coated with a photo- sensitive polymer (photoresist) is used. A mask containing a pattern designed in the micro-scale is applied over the photoresist, followed by electron beam or UV irradiation. Finally, a carefully monitored etching process is made on the substrates to obtain reproducible etching depth. Then, the etched substrate must be drilled for fluid intake and outflow and connectors are mounted to those drilled holes on the fragile substrate. There has been much interest in the development of encapsulation methods that are rapid,
reliable and easy to operate while offering control over the properties of the microcapsules.
The tubing device of the present invention does not require all those microfabrication and drilling steps. The elastic tubing used in the present invention, commercially available in a wide range of standard internal diameters, provides convenient flow regimes. This tubing device only requires the assembling of one central part, a blunt-end needle supported by a holder in vertical position. The overall assembled device is considerably flexible and can be mounted to peristaltic and syringe pumps readily providing tubing connectors. The present invention is well suited for laboratory and pilot applications.
Transplantation of encapsulated cells in microbeads or microcapsules is a promising approach for treatments of metabolic diseases and endocrine disorders (F. Lim, A. M. Sun, Microencapsulated Islets as bioartificial endocrine pancreas, Science, 1980, 210 : 908-910; M. V. Sefton, W. T. K. Stevenson, Microencapsulation of live animal cells using Polyacrylates, Adv. Polym. Sci., 1993, 107:143-197).
The microcapsules harbouring living cells have also found applications in cell culture in vitro and tissue engineering fields. There is a considerable interest in production of mono-size microcapsules or microbeads of uniform size for the encapsulation of living cells. For successful clinical or laboratory use, the microcapsule
must be produced in a narrow size distribution and spherical morphology. Preferably, the device producing microcapsules must be reusable, easily cleanable and sterilizable by convenient techniques such as autoclaving.
Recently, the microfluidics technology has provided a new route to prepare microcapsules with controlled morphology and uniform size. Most of microfluidics systems have been intended for drug encapsulation purposes . Over the past few years there has been several studies reporting this technique for cell encapsulation. In these systems, however, the preparation complexity of microfabrication procedures may challenge its practical application in cell microencapsulation applications.
The aim of the present invention is the development of a cell microencapsulating device based on microdroplet formation of a polymer/cell mixture when injected into cross-flowing oil in a narrow tubing channel. The device is autoclavable and produced by using biocompatible materials; its design allows easy connection with syringe and peristaltic pumps with proper syringe and syringe needle connectors.
SUMARY THE INVENTION
The present invention relates to a microencapsulation device based on micro droplet
generation by injecting a hydrogel mixture into a oil stream inside a tubing. The device can produce microdroplets in a narrow size distribution under constant injection rate of polymer/cell or polymer/ bioactive agent mixtures and oil flow maintained by a syringe and peristaltic pump, respectively.
The described microdroplet generator is capable of dispensing a polymer solution into oil flow of tubing by secure positioning of a blunt-ended hypodermic needle into the centre of channel space vertically. The stable junction of needle and seal at puncture site of tubing is provided by an elastic needle holder, which can fix the needle in a vertical position and wrap-seal around tubing tightly.
The microdroplet generator for cell or bioactive agent encapsulation is composed of a platform, elastic tubing, blunt-ended hypodermic needle, needle holder and adapters for syringe and tubing ends. The platform, preferably made from a poly (tetrafluoroethylene) (PTFE) block, possesses a horizontal open semicircular hollow space for holding and orienting the tubing in a straight position and two adjustable clips for fixing the tubing in place. The hypodermic needle is inserted halfway the tube diameter through a small hole and the junction needle/tubing is stabilized and assured by the needle holder. Said needle holder has a hollow cylindrical part which allows the needle to pass through the channel and supports the Luer hub of needle at the top. The cylindrical part of the needle holder is made concave at
the bottom so that it can fit and grip the tubing for better sealing of the hole. Additionally, the tubing holder stabilizes the tubing and needle by its semicircular wings, which can be glued on the tubing surface .
The polymer solution injection in the hypodermic needle is made by the syringe pump through an extender tubing, which is connected to the needle and syringe by Luer lock-tubing and syringe-tubing adapters, respectively.
The oil perfusion to the inlet of tubing, which is horizontally fixed on a platform, is performed by a peristaltic pump. The outlet of tubing is connected to a collector vessel, which contains a gelling solution with a emulsifying agent for microcapsule formation. The solution in the collector vessel can be conventionally stirred by using a magnetic stirrer during gelling process .
Biocompatible surface active agents like Tween-80® can be used in the gelling solution to prevent aggregation of microdroplets of the polymer/cells mixture when exiting trough the outlet of tubing and entering into contact with the gelling solution. In accordance with the present invention, a gel forming polymer solution is used to form microcapsules that can encapsulate various materials . The material must be of a size small enough to be suitable for encapsulation by the droplet method of this invention but can vary widely in
diameter from less than a 100 micron to several millimetres. One of the most critical aspects of the present invention is the ability of the device to generate micro-droplets with a uniform distribution and tuneable sizes. The present process allows viable cells to be encapsulated in microcapsules. Thus, it will be appreciated that the process of this invention is particularly well suited for use in encapsulating biological materials. Thus, one of the preferred embodiments of this invention falls in the context of encapsulating biological materials . The biological materials to be encapsulated can be tissue, organelle, plant or animal cells, bacteria, among others. The present invention uses a murine ATDC5 chondrocyte cell line in the cell encapsulation experiments. The cells to be encapsulated are not limited to chondrocytic cells but include other cell types, such as primary cultures (pancreatic islets, hepatocytes, fibroblasts, osteoblasts, articular chondrocytes, mesenchymal stem cells) as well as established cell lines.
The gel-forming polymer can be any non-toxic water- soluble gel-forming polymer, which forms a gel upon contact with a gelling inducer (multivalent ions or charged polymers) . The gel-forming polymer can be a water-soluble polysaccharide. Suitable polysaccharides include sodium alginate, guar gum, gum arabic, carrageenan, pectin, xanthan gum, gellan gum, chitosan. Upon contact with gel inducers the polysaccharide molecules form a water-insoluble shape-retaining gel membrane microcapsule. Other polysaccharides include, for
example, hyaluronic acid, chondroitin sulfate, dextran sulfate, heparin, heparin sulfate, heparan sulfate. These polymers can form capsules by polyelectrolyte complexation. The gel forming polymers that can be used with the present invention is not limited to completely aqueous solutions of hydrophilic polymers but also amphiphilic polymers, which have molecular self- organization properties in physiological solutions. In addition, hydrophobic polymers that dissolve in organic solvents can be processed with the device by providing an immiscible aqueous solution as continuous phase in tubing in order to obtain an emulsion of oil-in-water .
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the tubing microdroplet apparatus in an exploded and in an assembled isometric view.
Figure 2 illustrates the cross-section of the central part of the device at anterior position where the needle is inserted to tubing and sealed by needle holder.
Figure 3 illustrates the encapsulating apparatus including the tubing that generate the microcapsules together with perfusion devices through a peristaltic pump and the polymer/cells injection through a syringe pump.
Figure 4 presents a photograph of cell microscopy fluorescence (murine ATDC5 chondrocyte cell line) encapsulated in carboxymethyl xanthan microcapsule showing that the encapsulated cells remain viable after 21 days of culture.
DETAILED DESCRIPTION OF INVENTION
The microencapsulator apparatus (1) is mainly composed of a horizontal tubing (5) and a vertical needle (7) supported by a tubing platform (2) and needle holder (6) , respectively.
The vertical part of this device consists of a needle holder (6) , a blunt-ended needle (7) and an adapter (8) , which connects both Luer lock hub part of needle (coloured plastic part with an universal fitting system) and a tubing extender (9) . At the end of extender tubing a syringe-tubing adapter (10) makes the connection with the syringe (11) , which is mounted to a syringe pump
(12) . The horizontal tubing into which the needle is inserted is supported by a platform (2) . The platform contains a semicircular open-channel (3) and adjustable clips (4) for fixation of tubing in stretched and horizontal form. For sterilization of the device by convenient autoclaving, the poly (tetrafluoroethylene)
(PTFE) platform and biocompatible and heat resistant silicone elastomer tubing (Novosil®) are selected. The
diameter of tubing and the needle gauge size can be varied according to the specific needs of encapsulation process. The invention here described uses a tubing of 1 mm internal diameter and a needle with gauge size of 21G to produce microdroplets from viscous polysaccharide solution, which is providing a stable cell encapsulation, near 400 μιη. The micro droplet size can be reduced by using a needle with smaller gauge number and tubing with narrower internal diameters. Conversely, the size of microdroplets can be increased by vice versa.
The blunt-ended needle is inserted into the horizontal tubing through a needle holder. The insertion of needle can be facilitated by puncturing a narrow hole (with a diameter inferior to that of the needle) before the insertion process. The needle can easily move into the centre of tubing channel by extending the hole needle holder and tubing wall, as elastic materials are used. This insertion method physically seals the needle at the insertion site. After insertion, the needle holder can be wrapped around tubing for better fixation. The needle holder has dual function: fixing the needle in vertical position preventing its dislocation from puncture site and provides an effective sealing on the tubing preventing leakage of liquids. The sealing is best provided by applying a moderate layer of silicone glue on the wings of needle holder before inserting needle into the tubing. Consequently, the wrapping of the wings around the tubing and curing the silicone pre-polymer allows a permanent seal around the puncture site.
The extender tubing (9) provides a flexible connection between the syringe (11) , which is mounted between the syringe pump (12) and the needle (7) , using Luer hub lock-tubing (8) and syringe-tubing adaptor (10) . By enabling an easy operation for connecting syringe and Luer hub of needle any possible dislocation and damage of assembly will be prevented.
The sterile mineral oil, which can be autoclavable and bioinert, is reserved in a Schott Bottle (13) which is mounted to a pipetting needle with a Luer hub and membrane filter (0.22 μιη) in its cap for maintaining the atmospheric pressure in vessel in a sterile condition. The oil is pumped out from the reservoir and carried into the tubing device by using a peristaltic pump via sterile tubing with stoppers (15) . The shear force of the oil flow inside tubing device then acts on the tip of needle forming microdroplets of polymer/cells. The microdroplets in oil can be carried into the gelling inducer solution vessel (17) using a relatively wider and inert tubing (preferably PTFE) (16) which provides minimum microdroplet interaction on surface. The microdroplets gellify when they exit the tubing and enter into gelling inducer solution. The mild but enough stirring rate is essential in order to prevent aggregation of gelling microcapsules. In this stage, a harsh stirring can damage the premature microcapsule walls, especially when a magnetic bar is used for stirring. The use of a biocompatible emulsifiying agent like Tween 80® at low concentration can effectively prevent aggregation of microcapsules in gelling inducer solution. The gelling
inducer solution composition can change depending of the gelation method pretended (eg. ionic crosslinking, ionic complexations or self-assembly gelation techniques) . The gelling solution can be prepared by using bivalent salts like CaCl2 for anionic hydrogels at an effective concentration. It is essential that the ionic strength of crosslinking solution must be maintained in physiological ion concentration (0.9% NaCl) and pH at 7.4 for viability of encapsulated cells.
The polyionic solution may be constituted by an oppositely charged polyelectrolyte relatively to the polyelectrolyte microdroplets . The complexation reaction takes place when the oppositely charged polymers react (e.g. interactions polysacharide-polysaccharide; polysacharide-protein/peptides) . In the case of peptides/proteins complexation with polysaccharide, the pH should be slightly lower than isoelectric point, while the pH of polysaccharide should be maintained at physiological pH. The gelling inducer solution for self- assembled biopolymers can be any kind of physiological solution, (e.g. phosphate buffered saline or cell culture medium) , which may induce the gel formation through self- assembly process.
The description of this invention is complemented through the following examples that are intended to provide a better understanding of the same, although these examples should not be addressed with a restrictive nature .
Example 1 - This example demonstrates the preparation of microcapsules by ionic crosslinking of the polyanionic biopolymer (carboxymethyl xanthan gum) in the presence of counterions. In this method, the mixture of bioactive material (cell suspension of murine ATDC5 chondrocytes) and carboxymethyl xanthan (5%) in culture medium is extruded through the droplet forming device into a gelling solution containing 1.5% CaCl2 and 0.9% NaCl . The Ca+2 cations cross- link the carboxymethyl xanthan matrix almost instantaneously to form gel beads. The beads are then treated with poly-L- lysine to strengthen the outer surface of the microcapsules . The microcapsules present spherical shape with an average diameter of 500 μπι (Figure 4) . Live/dead assay of encapsulated cells shows that the cells remain viable in the microcapsules up to 21 days of culture.
Example 2 - This example demonstrates a mild cell encapsulation method based on triggering of self-assembly of amphiphile constructed by pH and ion sensitive polysaccharide (xanthan gum) , which is conjugated to hydrophobic palmitic acid. In this method, the mixture of bioactive material (cell suspension of murine ATDC5 chondrocytes) and palmitoyl xanthan (1%) is extruded through the droplet forming device into phosphate buffer solution (PBS) . In this solution the palmitoyl xanthan droplets self-assemble into capsular gel structure. The formed hollow microcapsules are then treated with poly-L- lysine to strengthen the outer surface of the microcapsules . The microcapsules present spherical shape with an average diameter of 450 μπι. The ability of
palmitoyl-xanthan microcapsules to sustain viability and proliferation of encapsulated cells was confirmed by the AlamarBlue® and cells deoxyribonucleic acid (DNA) quantification assays.
Claims
1. Microencapsulation device characterized in that it comprises a horizontal elastic tubing (5) supported by a tubing platform (2) and a vertical part comprising a needle holder (6) , a blunt-ended needle (7) and an adapter (8) , which should be connected between both Luer lock hub part of needle and a tubing extender (9) , being said needle (7) inserted in the horizontal tubing through said needle holder (6) that allows a tight sealing between the pierced tubing and needle by having its wings glued with silicon prepolymer containing a curing agent.
2. Microencapsulation device according to claim 1 characterized in that it comprises a platform (2) for aligning the tubing on horizontal position with a semicircular open half-channel (3) and fixing by two clips (4) at each side of said platform for fixation of tubing in stretched and horizontal form.
3. Microencapsulation device according to claim 1 and 2 characterized in that it comprises an extender tubing (9) which enables an easy operation and connection between the syringe (11) mounted to a syringe pump (12) and needle inserted into the horizontal tubing through needle holder (6) the connection of extender tubing to needle and syringe being achieved by the Luer hub lock-tubing (8) and syringe-tubing adaptor (10) , respectively, allowing a minimal distortion to the assembled device (1) , which needs the needle to be in vertical position without any break in seal between tubing and needle holder .
4. Microencapsulation device, according to the preceding claims, characterized in that it generates microdroplets in the tubing device, which are carried by larger PTFE tubing and released into a mildly stirring solution which has a gelling solution with a emulsifying agent at physiological conditions.
5. Microencapsulation device, according to the preceding claims, characterized in that its main components are fabricated in inert, biocompatible and autoclavable materials, allowing an easy and quick sterilization process .
6. Method for preparing microcapsules characterized in that it uses droplet generation by vertical injection of a hydrophilic mixture of hydrogel-polymer/cell into a hydrophobic oil flow which is maintained in the horizontal silicone tubing of the device described in claim 1, the injection of hydrogel-cell mixture into a stream of mineral oil resulting in spherical droplet generation and a water-in-oil emulsion formation, and said droplets in oil phase being converted into stable microcapsules by gelation in a separate chamber which is loaded with ionic cross -linking solution at physiological ionic strength and pH .
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Cited By (3)
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WO2016018678A1 (en) * | 2014-07-30 | 2016-02-04 | The Regents Of The University Of California | Methods for bacteriophage detection |
CN109499492A (en) * | 2018-11-30 | 2019-03-22 | 南昌大学 | A kind of preparation facilities and preparation method thereof for the snake oil gel containing allantoin |
WO2022179983A1 (en) * | 2021-02-26 | 2022-09-01 | Microcaps Ag | Capsules with a hydrogel matrix |
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CN108354181A (en) * | 2018-03-23 | 2018-08-03 | 刘洪明 | A kind of preparation method and system of three probiotic microcapsules |
CN114921343B (en) * | 2022-06-28 | 2023-09-05 | 中国科学院苏州生物医学工程技术研究所 | Cell gel microsphere generating device based on high-voltage pulse electric field |
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GB9814619D0 (en) * | 1998-07-06 | 1998-09-02 | Cole Polytechnique Fudurale De | Materials and methods relating to encapsulation |
EP1203084A1 (en) * | 1999-07-30 | 2002-05-08 | University College London | Microencapsulated nitric oxide synthase source |
CN101695646A (en) * | 2009-10-28 | 2010-04-21 | 中国海洋大学 | Device and method for preparing gel microspheres with uniform grain sizes |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016018678A1 (en) * | 2014-07-30 | 2016-02-04 | The Regents Of The University Of California | Methods for bacteriophage detection |
CN109499492A (en) * | 2018-11-30 | 2019-03-22 | 南昌大学 | A kind of preparation facilities and preparation method thereof for the snake oil gel containing allantoin |
CN109499492B (en) * | 2018-11-30 | 2023-10-31 | 南昌大学 | Preparation device and preparation method of allantoin-coated snake oil gel |
WO2022179983A1 (en) * | 2021-02-26 | 2022-09-01 | Microcaps Ag | Capsules with a hydrogel matrix |
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PT105489B (en) | 2014-05-13 |
WO2012099482A3 (en) | 2012-11-01 |
PT105489A (en) | 2012-07-18 |
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