US20050220891A1 - Multi-layer cell encapsulation for tissue engineering - Google Patents

Multi-layer cell encapsulation for tissue engineering Download PDF

Info

Publication number
US20050220891A1
US20050220891A1 US11137483 US13748305A US2005220891A1 US 20050220891 A1 US20050220891 A1 US 20050220891A1 US 11137483 US11137483 US 11137483 US 13748305 A US13748305 A US 13748305A US 2005220891 A1 US2005220891 A1 US 2005220891A1
Authority
US
Grant status
Application
Patent type
Prior art keywords
microcapsule
cells
layer
microcapsules
inner
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11137483
Inventor
Hanry Yu
Kam Leong
Ser-Mien Chia
Andrew Wan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agency for Science Technology and Research, Singapore
National University of Singapore
Original Assignee
Agency for Science Technology and Research, Singapore
National University of Singapore
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062General methods for three-dimensional culture
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012Cell encapsulation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/067Hepatocytes
    • C12N5/0671Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • A61K2035/128Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00Supports or coatings for cell culture, characterised by material
    • C12N2533/30Synthetic polymers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00Supports or coatings for cell culture, characterised by material
    • C12N2533/50Proteins
    • C12N2533/54Collagen; Gelatin
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00Supports or coatings for cell culture, characterised by material
    • C12N2533/70Polysaccharides
    • C12N2533/72Chitin, chitosan
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S514/00Drug, bio-affecting and body treating compositions
    • Y10S514/962Capsule, e.g. gelatin
    • Y10S514/963Microcapsule-sustained or differential release
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S514/00Drug, bio-affecting and body treating compositions
    • Y10S514/97Containing designated ingredient to stabilize an active ingredient

Abstract

A multi-layered microcapsule has an inner extracellular matrix and an outer shell. The inner extracellular matrix includes a first inner layer of biopolymer and a second intermediate layer of polymer that provides partial immune-protection and holds the first layer in place. The outer shell can form an exoskeleton to provide mechanical stability. Each of the individual layers can be varied to optimize mechanical stability, cell function, and immuno-protection.

Description

    CROSS-REFERENCE TO RELATED APPLICATION
  • This application is a divisional application of U.S. application Ser. No. 09/975,273, filed Oct. 12, 2001, which claims benefit under 35 U.S.C. § 119(e) to U.S. application Ser. No. 60/239,259, filed Oct. 12, 2000, the disclosure of each of which is hereby incorporated by reference in its entirety.
  • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • This invention relates to cell encapsulation and, more particularly, to encapsulating living cells in a multi-layer polymeric membrane.
  • 2. Description of Related Art
  • Microcapsules for biological substances are composed of thin, semi-permeable membranes of cellular dimensions. Microcapsules can be prepared of various polymers and their contents can consist of enzymes, cells and other biological materials. Microcapsules are prepared in such a way as to prevent their contents from leaking out and causing an immunological reaction, but the microcapsules still allow the nutrients and metabolites to exchange freely. This method has found applications primarily in transplantation of foreign materials in vivo without immunosuppression. One example is microencapsulation of hepatocytes for use in bio-assisted liver devices (BLAD). The surface-to-volume ratio of a spherical microcapsule facilitates maximal transport of nutrients, gases, or metabolites exchange across the membrane. In addition, encapsulation of living cells allows better control of the microenvironment for optimal cellular functions via selection of suitable substrate and incorporation of controlled-release features into the local microenvironment. Other physical characteristics such as mass transport, mechanical and chemical stability can also be configured as desired without drastically affecting the functions of the living cells inside the microcapsules.
  • The commonly used techniques for cell encapsulation are complex coacervation and interfacial precipitation. Complex coacervation involves the electrostatic interaction of two oppositely charged polyelectrolytes. At the right matching charge density, the two poly-ions combine and migrate to form a colloid-rich or water-insoluble phase. The molecular weight and chain conformation parameters of the poly-ions may also play an important role in the complexation process. Interfacial precipitation simply relies on the solidification of a dissolved polymer upon contact with an aqueous phase.
  • One of the most extensively studied cell encapsulation schemes is one that involves an alginate-gelation complex coacervation method. In this system, alginate, a glycuranan extracted from the brown seaweed algae, can be chelated by calcium or other multivalent counter-ions to form a gel. These early in vivo results with the alginate-polylysine system have not been consistent because of the uncontrolled purity of alginate, and the incorporation of cells into the external membrane. As a result, a 2-step encapsulation was developed to further shield sensitive cells from the extra-capsular environment. The living cells were mixed with sodium alginate and extruded into calcium chloride to form calcium alginate gel droplets. These gel droplets were incorporated into larger alginate gel spheres and then reacted with a poly-amino acid such as poly-L-lysine to form a semi-permeable membrane. Incubating with sodium citrate liquefied the interior to form microcapsules. Unfortunately, the addition of sodium citrate appears to have affected the functions of the cells. Furthermore, the water-soluble alginate and poly-lysine were shown to be not particularly biocompatible as individual polymers, other matrices such as collagen may be better substrates for cellular functions than alginate.
  • To encapsulate living cells in natural matrices such as collagen, interfacial precipitation has been used. In this method, hydroxylethyl methacrylate-methylmethacrylate (HEMA-MMA) solution in dimethyl formamide and cell-suspension in collagen or Matrigel were extruded separately through two concentrically configured needles into a precipitating bath containing largely water with a floating layer of dodecane. Polyacrylates are water insoluble that enhances the in vivo stability of the microcapsules. The living cells encapsulated this way (especially with Matrigel) survive well. The interfacial precipitation requires a more elaborate setup than the complex coacervation to control the microcapsule sizes and minimize the contact of cells with organic solvents.
  • In U.S. application Ser. No. 09/414,964, filed Oct. 12, 1999, a negatively charged ter-polymer of hydroxyethyl methacrylate-methyl methacrylate-methacrylic acid (HEMA-MMA-MAA) is used to encapsulate cells within a positively charged collagen. The MAA added into the ter-polymer enhances the water solubility of the polymer, allowing the entire encapsulation to be carried out in an aqueous environment. Hence, the complex coacervation method is used while a simple setup provides for easy control of the microcapsule size. The resulting hepatocyte microcapsules exhibit enhanced cellular functions as well as desirable physical characteristics for use in bio-artificial liver. The microcapsules, however, were mechanically unstable as measured by nano-indentation method. After 4 days of static in vitro culture, the microcapsules became weak and breakable upon harsh handling. Attempts at improving the mechanical stability of the microcapsules resulted in tradeoffs with immune-barrier/mass transfer efficiencies and cellular function.
  • There remains a need for improved microcapsules that exhibit satisfactory mechanical stability in combination with improved immune-barrier/mass transfer efficiencies and cellular function.
  • SUMMARY OF THE INVENTION
  • The present invention, according to one aspect, is directed to a microcapsule for culturing cells, particularly anchorage-dependent cells. An inner, extra-cellular matrix surrounds the cells. The inner extracellular matrix can be formed from a biopolymer inner layer and a biocompatible synthetic polyelectrolyte outer layer, wherein the inner layer and the outer layer have charges sufficient to form a complex of the biopolymer and the polyelectrolyte. An outer shell of synthetic polymer surrounds and supports the extracellular matrix. The microcapsules are permeable to nutrients necessary to sustain normal metabolic functions of the cells and to toxins released by the cells.
  • According to another embodiment, a microcapsule for culturing anchorage-dependent cells comprises an inner extracellular matrix surrounding the cells and an outer shell surrounding and supporting the extracellular matrix. The outer shell comprises a macro-porous exoskeleton formed by complex coacervation with the extracellular matrix. The macro-porous exoskeleton preferably includes such biocompatible materials as alumina, alumina sol, or chitosan.
  • According to yet another embodiment, a microcapsule comprises an inner extracellular matrix surrounding living cells, a macro-porous exoskeleton surrounding and supporting the extracellular matrix, and an outer shell of synthetic polymer surrounding the macro-porous exoskeleton.
  • The microcapsule membrane preferably is permeable to molecules smaller than or equal to the size of albumin, to nutrients necessary to sustain normal metabolic functions of the bioactive cells, and to toxins released by the bioactive cells. The microcapsule membrane preferably is impermeable to immunoglobulins and macrophages.
  • The multi-layered microcapsule of the present invention systematically addresses all thee major aspects of the micro-encapsulation development: optimal ECM environment for high cell functions, good mechanical stability, and reliable immune-protection. Most previous efforts have been focused on immune-barrier development while keeping cell viability only. In most cases, cell functions were quite poor. For hepatocyte encapsulation, cell functions never exceeded that exhibited by the monolayer culture control. While some other microcapsules do exhibit good mechanical stability, the cell functions and mass transfer properties have been unsatisfactory. Therefore, the enhancement of cell functions due to encapsulation was not fully exploited; the mechanical stability was weak; and effective immune-barrier could not be ensured. The multi-layered cell encapsulation of the invention advantageously allows all three major properties of the microcapsules to be systematically tuned for required applications in tissue engineering.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • The present invention will now be described in more detail with reference to preferred embodiments of the invention, given only by way of example, and illustrated in the accompanying drawings in which:
  • FIG. 1 is a confocal micrograph of a Type-II microcapsule, which illustrates that the second ter-polymer shell covers the cells protruding from the inner ter-polymer shell to ensure a more thorough encapsulation;
  • FIGS. 2A and 2B are images of a Type-III microcapsule surface:
  • FIG. 2A is a scanning micrograph of a Type-III microcapsule using 0.02% chitosan as the exoskeleton biomaterial, and
  • FIG. 2B is a scanning micrograph of a Type-III microcapsule using 6 mM alumina sol-gel as the exoskeleton material, illustrating a macro-porous network formed by the condensation of positively charged alumina particles on the exterior of the microcapsule (scale bars represent 10 μm);
  • FIG. 3 is a scanning micrograph of a Type-IV microcapsule illustrating that the additional thin layer of ter-polymer resulted in a relatively smooth fourth layer that was sufficient to cover the exoskeleton beneath (scale bars represent 10 μm);
  • FIG. 4 is a permeability profile of the microcapsules: BSA (1%) was loaded into the four types of microcapsules and the rate of BSA released from the microcapsules was measured (Type-I: (∘), Type-II: (□), Type-III: (x), Type-IV: (⋄)); and
  • FIG. 5 illustrates a urea production profile of microencapsulated hepatocytes in Type-I (□), Type-II (Δ), Type-III (x), and Type-IV (∘) microcapsules, as well as a monolayer control (▪).
  • DETAILED DESCRIPTION OF THE INVENTION
  • A multi-layer microcapsule comprises an extracellular matrix having bioactive cells attached to a microcapsule membrane. The microcapsule membrane has a first inner layer of biopolymer, such as cationic collagen, anionic collagen, anionic esterified hyaluronic acid, or anionic amine-modified hyaluronic acid, and a second intermediate layer of polyelectrolyte synthetic polymer. As described herein, the microcapsule also has an outer shell to improve mechanical stability. The microcapsule may include additional layers, such as a fourth outermost layer of polyelectrolyte synthetic polymer. The layers can be individually tailored to meet the needs of a particular application.
  • As used herein, “Type-I microcapsule” refers to cells within a biopolymer, such as a positively charged collagen, encapsulated with a polyelectrolyte synthetic polymer, such as a negatively charged ter-polymer of hydroxyethyl methacrylate-methyl methacrylate-methacrylic acid (HEMA-MMA-MAA). Such microcapsules are described in U.S. application Ser. No. 09/414,964, filed Oct. 12, 1999, the disclosure of which hereby is incorporated by reference.
  • “Type-II microcapsule,” as used herein, refers to a microcapsule prepared by re-encapsulating a Type-I microcapsule in biopolymer- and polyelectrolyte synthetic polymer solutions.
  • “Type-III microcapsule,” as used herein, refers to a microcapsule prepared by re-suspending a Type-I microcapsule in an exoskeleton material to form a macro-porous network.
  • “Type-IV microcapsule,” as used herein, refers to a microcapsule prepared by re-encapsulating a Type-III microcapsule in a polyelectrolyte synthetic polymer solution.
  • Both naturally-occurring and modified biopolymers are suitable for use as biopolymers in the practice of the invention, as are both cationic and anionic biopolymers. In general, any commonly used substrates in cell studies can be used, non-limiting examples of which include collagen, cationic collagen, anionic collagen, anionic esterified hyaluronic acid, anionic amine-modified hyaluronic acid, fibronectin, and laminin. The biopolymers preferably are water-soluble and most often have a molecular weight of at least 20,000, preferably at least 75,000, more preferably at least 125,000, even more preferably at least 200,000, and yet even more preferably at least 250,000.
  • Whereas collagen has been used to encapsulate drugs, it has not found widespread use for encapsulating cells because, at neutral pH, there is insufficient charge density to form an encapsulating membrane. However, collagen modified to raise its pKi to at least about 9 is sufficiently positively charged at physiological pH to be complexed with oppositely-charged synthetic polyelectrolytes to form a coherent membrane. Collagen can be modified to form a more strongly basic polymer by converting the primary amino groups to tertiary amine groups or by esterification.
  • Anionic biopolymeric materials, such as hyaluronic acid (HA) and modified HA (esterified HA or amine-modified HA) are useful in the invention. In general, anionic biopolymers suitable for the practice of this invention will have a charge density of at least about 20%, preferably at least about 30%, and even more preferably at least about 50%. HA that is totally or partially esterified or reacted with a primary amine to render it less water-soluble will form a stronger complex with the polycationic outer layer than HA itself.
  • Preferred biopolymers for forming the inner layer of the encapsulating membrane are modified HA and modified collagen. Esterified collagen is particularly preferred as the inner layer. In general, the inner layer, though water-soluble, will be slightly hydrophobic.
  • Esterification or reaction to form tertiary amine groups on the biopolymer may be accomplished by reaction of the biopolymer with a wide variety of aliphatic reactants containing as many as about 18 carbon atoms in their chain. Such reactants include, inter alia, alcohols, primary amines and alcohol amines. Preferred reactants contain about 8 carbon atoms or less. For some purposes, use of reactants having only 2 or 3 carbon atoms may be preferred. Typical alcohols include methanol, ethanol, butanol and higher alcohols, whereas typical primary amines include methylamine, ethylamine and higher amines. Reactants with both alcohol and amine groups also can be used, such as ethanolamine. Reactants should be chosen so as to not impair the viability of the cells.
  • The outer layer of the membrane comprises a biocompatible synthetic polyelectrolyte having a charge opposite that of the biopolymer. Thus, when the biopolymer is polycationic (e.g., modified collagen), the synthetic polyelectrolyte used in the outer layer is polyanionic. Conversely, when the biopolymer is polyanionic (e.g., HA, modified HA, etc.), the synthetic polyelectrolyte used in the outer layer is polycationic. Suitable outer layer synthetic polyelectrolytes form a complex with the oppositely-charged biopolymer to form a membrane by the complex coacervation process and impart stability to the encapsulate. The charge density of the synthetic polymer typically will be from about 0.1% to about 20%, preferably is at least about 1%, and even more preferably is at least about 3%. Like the biopolymers, the synthetic polyelectrolytes preferably have a molecular weight of at least 20,000, preferably at least 75,000, more preferably at least 125,000, even more preferably at least 200,000, and yet even more preferably at least 250,000.
  • The biocompatible synthetic polyelectrolyte layer that is capable of forming, with the biopolymer of the inner layer, a membrane which allows environmentally-sensitive living cells, such as hepatocyte cells, to remain viable and, at the same time, protects the cells against immunological rejection by the host. A preferred class of biocompatible synthetic polyelectrolytes is acrylate polymers. Such polymers include acrylate polymers, copolymers and ter-polymers such as poly(acrylic acid), poly(methacrylic acid), poly(methacrylate), poly(methyl methacrylate), and acrylate copolymers and ter-polymers of acrylic acid, methacrylic acid, methacrylates, methyl methacrylates, hydroxyethyl methacrylic such as 2-hydroxyethyl methacrylate, hydroxypropyl-acrylate and the like, and blends thereof. Poly(dimethylaminoethyl methacrylate) (DMAEMA) and copolymers and ter-polymers of dimethylaminoethyl methacrylate with 2-hydroxyethyl methacrylate and/or hydroxypropylacrylate and methacrylate and/or methyl methacrylate are preferred cationic synthetic polymers. Copolymers or ter-polymers of acrylic acid and/or methacrylic acid with 2-hydroxyethyl methacrylic and/or hydroxypropylacrylate and methacrylate and/or methyl methacrylate are preferred anionic synthetic polymers. Each has exhibited biocompatibility when used in other biomaterials.
  • A preferred biocompatible synthetic polyelectrolyte outer layer is an acrylate ter-polymer of methacrylic acid (MAA), hydroxyethyl methacrylate (HEMA), and methyl methacrylate (MMA). The ter-polymer preferably comprises from about 10 mol % to about 30 mol %, more preferably from about 15 mol % to about 25 mol % MAA, from about 10 mol % to about 40 mol %, more preferably from about 20 mol % to about 30 mol % HEMA, and from about 20 mol % to about 60 mol %, more preferably from about 45 mol % to about 55 mol % MMA. In a preferred embodiment of the present invention, the ter-polymer is formed by polymerizing MAA, HEMA, and MMA monomers in about a 1:1:2 molar ratio.
  • The membrane of the encapsulated cell is selectively permeable. The cells encapsulated in accordance with the invention remain viable because the membrane is permeable to nutrients and other materials necessary to support the normal metabolic functions of the cells. Thus, ionic materials and oxygen, for example, pass through the membrane. The membrane also is permeable to products of the cells, such as hormones, and to metabolic byproducts. Thus, material produced by the cell can pass through the membrane from the interior of the microcapsule. In this way, material produced by the encapsulated cell can be introduced into the blood of a host, or can be introduced into a culture medium in which encapsulated cells are placed.
  • The membrane permeability essentially precludes entry of immunoglobulins, macrophages, and other immune system agents that cause rejection of cells by the host's immune system. According to a preferred embodiment of the invention, the membrane is impermeable to molecules greater than about 100 kDa, and preferably is impermeable to molecules greater than about 71 kDa. According to another preferred embodiment of the invention, the membrane is permeable to molecules greater than about 60 kDa and impermeable to molecules greater than about 150 kDa.
  • The composition of the outer layer can be modified to adjust the permeability and transport properties of the membrane. As an example, the permeability of the membrane to typically polar compounds found in biological systems can be increased by incorporating a hydrophilic copolymer, such as poly(2-hydroxyethyl methacrylate) (HEMA) or other hydroxy-containing acrylates, into the polyelectrolyte which forms the outer layer of the membrane. Increasing hydrophobicity of polyelectrolytes tends to cause decreased permeability.
  • In the preferred MAA/HEMA/MMA ter-polymer, HEMA provides hydrophilicity to render the ter-polymer water-soluble so that the entire encapsulation can be performed in the physiological aqueous buffer without the need for an organic solvent. MMA imparts mechanical strength, toughness, and elasticity to the microcapsules. MAA provides a negative charge to interact with a positively-charged inner layer. The inner layer preferably is an esterified collagen with net positive charge. The balance between the two charged polymers determines the physical characteristics of the microcapsules. Using a 10% ter-polymer and 1.5 mg/ml of modified collagen, for example, microcapsules can be formed having a thin outer layer (˜21 μm) and a semi-gel-like inner layer that minimizes impedance to mass transport across the membrane but remain stable as microcapsules for days. The semi-gel-like inner collagen layer is able to provide a “loose” extracellular matrix configuration that mimics the in vivo situation, therefore allowing the microcapsule to maintain higher levels of cell function. These characteristics of the microcapsules that satisfy most requirements for a bioartificial liver-assisted device (BLAD) were achieved through optimization of several parameters.
  • The permeability of the membrane also can be adjusted by selection of molecular weight or structure of the outer layer so as to preclude molecules having a preselected molecular weight or structure from passing through the membrane. As the molecular weight of the polyelectrolyte is increased, the membrane tends to be more permeable. Larger differences in charge densities between the inner biopolymer and the outer polyelectrolyte also tend to make the membrane more permeable. The mechanical stability of the membrane can be improved by increasing the molecular weight of the polyelectrolyte in the outer layer or by employing monomers in the polyelectrolyte that provide mechanical strength, such as MMA.
  • The membrane can be formed by complex coacervation by combining drops of a solution of biopolymer containing a cell suspension with a solution of synthetic polymer at physiological or neutral pHs of from about 6 to about 8 so as to avoid adversely affecting the viability of the cells. In such process, the biopolymer is dissolved in a suitable aqueous solvent that will not adversely affect the viable cells. Such solvents are well known and include buffered saline, culture medium and the like. Similarly, the synthetic polyelectrolyte is soluble in and dissolved in a suitable solvent that will not threaten the viability of the cells. Such solvents include aqueous solvents such as buffered saline, culture medium and the like. The solvent used for the biopolymer does not need to be the same solvent used for the synthetic polymer. Mild agitation of the polyelectrolytic solution can be utilized if desired.
  • In one suitable technique, a substrate polymer solution containing a cell suspension in a suitable diluent such as phosphate buffered saline (PBS) is added dropwise to a receiving solution containing synthetic polyelectrolyte of the opposite charge in PBS at ambient temperature. A cohesive membrane is formed at the interface of the two solutions to provide encapsulated cells. Advantageously, no organic solvent is required and no cross-linking reaction is necessary. Thus, the conditions of encapsulation are especially mild, yielding little cell mortality.
  • The proper matching of biopolymer and synthetic polyelectrolyte can be readily confirmed. A drop of a solution of biopolymer can be added to a solution of electrolyte. A proper match results in the rapid formation of a microcapsule or membrane by complex coacervation, which can be observed visually. The suitability of a given encapsulate regarding permeability can be readily determined by in vitro tests using standard cell culture media to determine if desired products are secreted, if unwanted immune components are excluded, and if viability of encapsulated cells is suitably maintained.
  • The concentrations of the polymer solutions, the size of the droplets added to the synthetic polyelectrolyte solution, and the rate at which the substrate polymer solution containing cell suspension is added to the synthetic polyelectrolyte solution can be adjusted to achieve an encapsulating membrane having the desired thickness of layers and desired size. Suitable concentrations for the biopolymer solution and for the synthetic polyelectrolyte solution will vary depending upon the specific polymers and solvents employed, but determination of such concentrations is easily within the skill of the art. While it is not possible to delineate concentrations for all possibilities, the concentration of the biopolymer often will be from about 0.1 to 2% whereas the concentration of the synthetic polyelectrolyte often will be from about 2 to 6%.
  • The thickness of the inner, substrate polymer layer, will depend on, inter alia, the viscosity of the biopolymer solution and the degree of penetration into the synthetic polyelectrolyte solution achieved by the substrate polymer solution droplets. The degree of penetration is related to the molecular weight of the polyions and the viscosity of the solutions. The thickness of the outer shell may vary over a wide range, depending on the material used (e.g., whether the microcapsule is Type-II, Type-III, or Type-IV as characterized herein) as well as the properties desired for a particular application. The outer shell most often has a thickness of from about 1 to about 20 μm.
  • The practice of this invention provides microspheres that may range in size from as small as about 30 μm to as large as several millimeters. The larger sizes are most suitable for cells that tend to aggregate such as islet of Langerhans cells and the like.
  • The number of cells within each microcapsule can be readily controlled and is a function of the density of the cell suspension within the biopolymer. For example, cells in PBS (which may be at densities of 103 to 106 cells per ml) can be mixed with the biopolymer to provide a variety of cell concentrations. Individual microcapsules can contain any desired number of cells, typically ranging from 1 to 200 cells or more. Collagen gel has been observed to exhibit a “skin effect” that is detrimental to mass transport, as a high concentration of collagen leads to gelation. Such “skin effect” is concentration- and temperature-dependent. Extra-cellular matrices like collagen or Matrigel have gelling temperatures of ˜22-35° C. depending on the concentration of these proteins. At 37° C., where hepatocytes are normally cultured in a bioreactor or transplantation is performed in vivo, the “skin effect” can be most pronounced. Since mass transport is among the most important considerations for the design of bioreactors in BLAD, it is desirable to employ the optimal concentration of collagen such that the “skin effect” is minimized while there still is enough collagen to complex with the synthetic polyanion forming stable microcapsules.
  • Albumin was used as a model molecule for the permeability optimization of the microcapsules. Albumin (MW ˜67,000 Da) is one of the secreted proteins of hepatocytes. It acts as a carrier to bind most metabolic wastes in the liver for removal from the blood. Another major scavenger protein is bilirubin (˜10,000 Da), which is smaller than albumin. Albumin was found to be freely permeable to the microcapsules. A known concentration (1% w/v) of albumin was added to collagen and microcapsules were formed. The microcapsules were equilibrated in a culture medium with the same concentration of albumin (1% w/v) at 37° C. for 2 hours to allow a possible “skin effect” to occur. Such equilibration before the permeability measurements is essential for detecting any “skin effect” from the gelling collagen. Pre-equilibration for up to 5 days indicated that the “skin effect” was marginally more pronounced than with the 2 hour pre-equilibration. The albumin released from the microcapsules into the fresh culture medium with no albumin added was thereafter monitored. With 1.5 mg/ml of the modified collagen, most of the encapsulated albumin was released from the microcapsules within 15 minutes. As the concentration of collagen in the microcapsule was increased to 4 mg/ml (˜0.4% w/v), the release of albumin was greatly inhibited. For collagen concentration below 1.5 mg/ml, hepatocytes could not be encapsulated, possibly due to insufficient positive charge from the diluted collagen. Therefore, 1.5 mg/ml of modified collagen was used for all other experiments.
  • One preferred ter-polymer composition is made up of 25 mol % HEMA, 25 mol % MAA and 50 mol % MMA at a concentration of 10% in PBS. When the ter-polymer composition was modified for higher negative charge at the expense of mechanical stability (e.g., 50 mol % MAA, 25 mol % HEMA, 25 mol % MMA), the urea-synthesis of the encapsulated hepatocytes decreases to levels below the monolayer control. The polymer composition and concentrations can be varied to achieve enhanced mechanical stability and other physical characteristics.
  • Because the membrane of the encapsulated cells of the invention precludes contact between the cells and the host's immune mediators, all types of living cells, including both naturally-occurring and genetically-engineered cells, may be encapsulated. The encapsulates are suitable for anchorage-independent cells and are particularly suitable for encapsulation of environmentally sensitive, anchorage-dependent living cells such as hepatocytes.
  • Encapsulated cells of the invention also are useful as, for example, a hormone-producing system. Use of cells microencapsulated in a selectively permeable biopolymeric membrane affords the opportunity to provide artificial organs and other methods for improving and restoring functions in people with physical disabilities.
  • An example of one type of hormone-producing cell is a cell of the anterior pituitary gland. Such cell excretes growth hormone, which inter alia stimulates skeletal growth. In accordance with the invention, encapsulated naturally-occurring anterior pituitary cells are useful in stimulating skeletal growth in a host. The encapsulated cells provide growth hormone produced by the cells and introduced to the blood of a host through the encapsulating membrane. Growth hormone also can be produced by genetically-engineered microorganisms. Such microorganisms, when encapsulated, may be used to provide growth hormone to a host.
  • Encapsulated cells that secrete hormones also may be suspended in a culture medium and will excrete hormone over an extended period. Encapsulated insulin-producing cells, for example, mammalian pancreatic alpha cells, beta cells, or intact islets of Langerhans, may also be used as an artificial pancreas. Such encapsulated cells can be implanted into a diabetic mammal and will function in vivo to excrete insulin and other hormones in response to host blood glucose concentration.
  • Other types of cells also may be beneficially encapsulated. For example, encapsulated neurotransmitter-secreting cells may be used to treat neurological disorders such as Parkinson's and Alzheimer's diseases. Similarly, chromaffin cell transplants may be used for alleviation of pain, especially chronic pain, and encapsulated chondrocytes may be used for repair of musculoskeletal defects. Skilled practitioners recognize the utility of encapsulating living cells, and will be able to identify still further cells suitable for encapsulation in accordance with the invention.
  • Even though the membrane may be permeable to proteases that can digest collagen and other biopolymers used to form the inner layer of the membrane, it has been found that the inner layer remains intact. Without being bound by any theory, it is believed that the proteases cannot digest the modified collagen, HA, modified HA, or other biopolymer when the biopolymer is complexed with the outer layer. This resistance can be analogized to the resistance to solubilization of type I collagen and to cross-linked collagen, such as is found in heart valve tissue. Again, without wishing to be bound by theory, it is postulated that the complexation shields or changes the conformation of the cleavage site (between glycine and leucine), thus making the resulting complexed biopolymer resistant to degradation.
  • The length of the period during which encapsulated cells remain intact will depend upon the properties of the medium in which the encapsulated cells are used and upon the composition of the biopolymer and of the synthetic polyelectrolyte. For example, encapsulated cells used in a culture medium might be expected to remain intact for a longer period than encapsulated cells introduced into a human or animal body. Also, the mechanical stability of the membrane can be improved by increasing the molecular weight of the synthetic polyelectrolyte. Skilled practitioners will be able to determine the length of the period during which encapsulated cells remain intact in various media.
  • Much effort on cell micro-encapsulation has focused on the materials and processes that make microcapsules for cell transplantation, cell-based drug delivery, and culture in bioreactors. There are a number of considerations for making microcapsules, but some are more important for certain applications than the others. For example, islet transplantation has more stringent requirements on immune isolation than microencapsulated hepatocyte cultured in an extra-corporeal bioreactor, which requires microcapsules with good mass transfer properties. This is because transplanted islets must function in vivo for extended periods of time, while the latter is often used for a few hours ex vivo. However, there are characteristics such as the good microenvironment for cell viability and functions, and the maximal permeability for oxygen and nutrient supply, which are important for all applications. The present invention provides a multi-layer microcapsule system based on such common characteristics, while allowing the other characteristics to be imparted by the individual layers of the microcapsules.
  • The four types of microcapsules (Types I-IV) according to preferred embodiments of the invention all share the same inner layer of modified collagen at 1.5 mg/ml. At such low concentration, the modified collagen does not completely gel but surrounds the cells loosely in a semi-gel state. Such a configuration provides a very good microenvironment for cellular functions. For the Type-II, -III, and -IV microcapsules, the additional layers most preferably should minimize the permeability impedance to the innermost layer to maintain good microenvironment for optimal cellular functions.
  • The four types of microcapsules (Types I-IV) described herein have some degrees of freedom for tuning the critical aspects of the microcapsule performance for different tissue engineering applications: good microenvironment for cellular functions, mechanical stability, complete encapsulation and selective permeability for potentially more reliable immune isolation. Type-I microcapsules provide a good microenvironment for cellular functions and exhibit good mass transfer properties, but are mechanically unstable and cannot ensure complete cell encapsulation. Type-I microcapsules are useful for applications such as the single-use bioreactor of an extra-corporeal device.
  • Type-II microcapsules have a more thorough cell encapsulation, which might provide a potentially more reliable immune isolation as well as better mechanical stability. Type-II microcapsules are more suitable for short-term applications such as hepatocyte transplantation where the transplanted hepatocytes are intended to stimulate the liver regeneration within a few weeks.
  • Type-III and -IV microcapsules provide significantly improved mechanical stability. Mechanical stability can be especially improved by using biocompatible materials such as alumina to form a macro-porous exoskeleton outside Type-I microcapsules. Polymers that carry positive charges, such as chitosan, can complex with the negatively charged ter-polymer shell. The micro-porosity of the exoskeleton layer can be controlled by the concentration of chitosan. As alumina possesses a relatively high point of zero charge (PZC) of 9.0, the particles of alumina sol at the physiological pH (7.2-7.4) possess positive charges. The positive charges on the alumina sol are neutralized by the negatively charged ter-polymer at pH 7.4. Due to this charge neutralization reaction, the alumina particles that originally repel each other due to their mutual positive charges begin to condense, resulting in the formation of a stable, macro-porous alumina network (FIG. 2B). Such an alumina sol-gel exoskeleton can be formed and optimized over a relatively wide range of concentrations. Type-III microcapsules are mechanically very stable over a period of 7 days as measured by nano-indentation assay. These mechanically stable microcapsules are expected to be suitable for culturing cells in a dynamic environment such as a fluidized bed bioreactor for large-scale cell culture or bio-artificial organs.
  • Type-IV microcapsules have an additional ter-polymer shell outside the exoskeleton of Type-III microcapsules. The negatively charged surfaces may minimize the adsorption of plasma proteins. With the relatively stable exoskeleton covered by a selectively permeable, negatively charged ter-polymer shell, the Type-IV microcapsules with two layers of built-in immune isolation features can allow plasma or even the whole blood to directly contact the microcapsules without the use of a hollow fiber membrane for immune isolation. The Type-IV microcapsules are expected to be useful in bio-artificial liver and in cell transplantation applications.
  • EXAMPLE
  • The following example is illustrative of preferred aspects of the invention and should not be construed to limit the scope of the present invention. All reagents were purchased from Sigma-Aldrich unless otherwise indicated.
  • Ter-Polymer Preparation
  • Ter-polymer of methacrylic acid (MAA), 2-hydroxyethyl methacrylate (HEMA), and methyl methacrylate (MMA) was synthesized by solution polymerization in 2-propanol=using 2,2′-azobisisobutyronitrile (AIBN) as initiator. The monomers were distilled under nitrogen at reduced pressure. The polymerization was performed with an initiator concentration of 0.1 mol % of monomers under nitrogen with a magnetic stirrer at 78° C. in an oil bath. The molar feed ratio of MAA, HEMA, and MMA was fixed at 25:25:50 or other ratios as desired and the ratio of total monomer to solvent at 1:6 (W/V). The reaction was allowed to proceed for overnight and quenched by cooling to room temperature. The polymer was precipitated by addition to a large excess of petroleum ether. The precipitate was re-dissolved in a minimum volume of ethanol, and re-precipitated in distilled water. Recovered polymer then was dissolved in a 1 M sodium hydroxide solution, and further purified by repeated dialysis against distilled water with MWCO of 3500, and lyophilized. The yield of the polymer was found to be ˜63%. The polymer composition was determined by proton NMR and the molar ratio of MAA, HEMA, and MMA was found to be 20.4:27.4:52.2 for the molar feed ratio of 25:25:50. The molecular weight of the ter-polymer before dialysis was determined by GPC (with THF as eluent) to be 30,000.
  • Modification of Collagen
  • Collagen can be modified to be cationic and anionic by the removal of either the negative or the positive charge from the collagen chains. In this case, cationic collagen was obtained through the modification of the carboxyl group by esterification with low molecular weight alcohol. 20 ml of stock solution (3 mg/ml) of collagen (Vitrogen 100, Collagen Corp., Palo Alto, Calif.) was first precipitated with 400 ml of acetone. The precipitated collagen was dissolved in 200 ml of 0.1 M HCl containing methanol (Merck), stirred at 4° C. for 6 days under sterile conditions. The lyophilized modified collagen can then be stored up to 6 months in −20° C. in the presence of desiccant. The modification was monitored by titration. Titration of the natural collagen gave a typical titration curve of a mixed acid or a dibasic acid, while that of modified collagen gave a typical titration curve of a week monobasic acid, indicating that most carboxyl groups have been esterified in modified collagen. In addition, neutralization of the modified collagen needs less sodium hydroxide than that of the natural collagen, indicating that the polymer chain of the modified collagen has less ionic groups because of the esterification of the carboxyl groups.
  • Isolation of Hepatocytes
  • Hepatocytes were harvested from male, Wistar rat, weighing from 250-300 g by a 2-step in situ collagenase perfusion. The rat was given 100 U/kg of heparin 30 minutes before anesthesia. Pentobarbital was administered at a dose of 30 mg/kg, intra-peritoneally at the start of the operation. After laparotomy, a portal cannula was placed and fixed in a position along the portal vein. A cut was rapidly made in the lower vena cava. In the first 2-3 minutes, pre-perfusion (with Ca2+-free perfusion buffer) was performed while the liver remained in situ. The perfusate flow was started at a rate of 50 ml per minute. While pre-perfusion was carried out, the liver was transferred to a petri-dish and placed in a position similar to its in situ site. After 10 minutes of pre-perfusion with Ca2+-free medium, the liver was then perfused with recirculating 0.05% collagenase buffer for another 10 minutes. This was terminated when the vena cava ruptured. The entire perfusion procedure was performed under oxygenation that greatly improved the cell viability. The cells were liberated from the connective vascular tissue and re-suspended in fresh growth medium. This was followed by incubation of the cell suspension in a 37° C. CO2 incubator for 30 minutes. The cell suspension was then filtered through a nylon mesh with a 60-μm pore size to further remove the connective tissue debris. The filtrate was then centrifuged at 50 g for 1 minute to obtain the cell pellet. The cells were collected and washed twice with growth medium. The viability of the hepatocytes was determined to be 90-95% in all cases using the conventional Trypan Blue exclusion test.
  • Preparation of Exoskeleton Materials
  • A. Preparation of Alumina Sol
  • Aluminum sec-butoxide (7.5 ml) was slowly added to 250 ml deionized water at 85° C. The suspension of white precipitate was magnetically stirred for half an hour, after which 0.15 ml of fuming hydrochloric acid (37%) was added to the suspension and kept in a stoppered vessel at 95-100° C. for 3 days. The resulting alumina sol was cooled to room temperature before use. The concentration of sol in term of aluminum (Al) concentration is 0.1168 M. The stock solution is further diluted with PBS to the desired working concentration (0.003 M to 0.006 M) before use.
  • B. Preparation of Chitosan
  • A stock solution of 2% chitosan (MW 400,000) was prepared by dissolving in 0.5% acetic acid at 95° C. The final working concentration of 0.01-0.02% was achieved through dilution of the stock with PBS.
  • Preparation of Microencapsulates
  • Micro-encapsulation was performed at room temperature with the aid of a syringe pump (IVAC P6000, Alaris Medical Systems, San Diego, Calif.). The microcapsules were incubated at 37° C. for one hour to allow the modified collagen to partially gel harvested by sedimentation and subsequently washed twice with 1× Phosphate Buffered Saline (PBS) for further studies (Type-I microcapsules).
  • Type-II microcapsules were prepared by re-encapsulating the Type-I microcapsules in 1.5 mg/ml of modified collagen solution and 10% ter-polymer solution using the same method as in the Type-I microcapsule preparation.
  • Type-III microcapsules were prepared by re-suspending the Type-I microcapsules in the respective exoskeleton materials for about 3 minutes for the formation of a macro-porous network, followed by extensive washing in PBS before in vitro culture.
  • Type-IV microcapsules were prepared by re-encapsulating the Type-III microcapsules in a 10% ter-polymer solution.
  • In Vitro Culture
  • The microcapsules were cultured for the required amount of time in Hepatozym Serum free medium (SFM, GIBCO Laboratories, Chagrin Falls, Ohio) in a 35 mm polystyrene dish in a humidified atmosphere with 5% CO2. The culture medium was supplemented with 10−7 M dexamethasone, 10 nM insulin (Boehringer Mainnhem), 20 ng/ml epidermal growth factor and 1% Penicillin and Streptomycin. After 1 day of culture, the microcapsules were incubated in the medium with 1 mM of NH4Cl for 90 minutes before the medium was collected for urea assay. The microcapsules were then cultured in fresh medium again.
  • Functional Analysis of the Microencapsulated Hepatocytes
  • The samples collected at each time point were assayed for the urea production calorimetrically with the Urea Nitrogen Diagnostic kit (Sigma Diagnostic). Data from three independent encapsulation experiments were analyzed and normalized against 106 cells.
  • Permeability Assay for the Microcapsules
  • Bovine serum albumin (BSA) was suspended in 1.5 mg/ml of modified collagen solution to reach a final concentration of 1% (w/v) and microcapsules of the four types (I-IV) were formed as previously described. The microcapsules were incubated at 37° C. for one hour for the gelation of the collagen. After the incubation, the microcapsules were transferred to a 2 ml PBS solution containing equal concentration of BSA, and allowed to equilibrate for 2 hours. The microcapsules were then washed with PBS and the BSA release profile over a 2-hour interval was obtained. BSA level was determined using the Detergent Compatibility (DC) protein assay (Bio-Rad Laboratories). The percentage of released BSA in PBS was plotted over time with respect to the BSA standards.
  • Light Microscope Imaging of the Microcapsules
  • The microcapsules were visualized in an inverted microscope (Olympus CK40, Tokyo, Japan) with phase-contrast optics. The numbers of hepatocytes within microcapsules were counted with the aid of a hemocytometer (Fuchs-Rosenthal).
  • An Olympus FLUOVIEW confocal microscope was used to image the ter-polymer and the encapsulated hepatocytes in transmitted mode. The thickness of the ter-polymer shell was measured with the software associated with the Olympus FLUOVIEW confocal microscope.
  • Scanning Electron Microscopy (SEM)
  • The microcapsules were fixed with 3% glutaraldehye on a coverglass coated with poly-L-lysine for an hour after which they were washed gently with 1×PBS for 5 minutes. The microcapsules were then post-fixed with osmium tetra-oxide for 1 hour and dehydration was accomplished using a graded series of ethanol (25%, 50%, 75%, 95%, and 100%). The microcapsules were then critical point dried for about 2 hours in absolute alcohol and mounted onto an aluminum stub and sputter coated with gold before viewing under a scanning electron microscope (Joel 5600 LV).
  • Assessment of Mechanical Stability by Nano-Indentation
  • Indentation measurement was done using a UMIS-2000 Nano-indenter (Australian Scientific Instruments). The three-faced pyramid indenter tip with an inclusion angle of 90° has well-defined geometry that enables the quantitative measurements of the mechanical properties located on the outermost shell or membrane of interest. The load and depth of penetration were measured by two LVDT (Linear Variable Differential Transformer) sensors independently. From the experimentally determined load-penetration data, hardness and modulus were determined through the following analysis:
    H=P/A
    E/(1−ν2)=(π1/2)/2·A −1/2 ·dP/dh
    where H is hardness of the specimen, P is indentation load, A is the true contact area at the maximum load, E is the elastic modulus of the microcapsules, and ν is Poisson's ratio. dP/dh (called unloading stiffness) essentially is the slope of unloading portion of the indentation load penetration data at the maximum indentation load. Average pressure that the microcapsules can withstand under a sharp point can be defined by applied load divided by contact area. The area of the indentation is therefore related to the depth of penetration, for an ideal sharp Berkovich indenter, is:
    A=24.56h 2
  • Microcapsules of various types were added onto a 13 mm coverglass coated with poly-L-lysine. The elastic modulus and hardness of the microcapsules were determined in the nano-indentation experiments with the maximum load of about 0.15 mN. This gave the penetration depth into the polymer of slightly less than 2 pm before rupture at day 7 for the Type-I microcapsule. The load was applied through a piezoelectric actuator in 15 steps to the maximum load. For each type of microcapsule, an average of 15 microcapsules were indented at the same load for each experiment. Data from three experiments were collected, with one indent on each microcapsule.
  • Results
  • Type-I microcapsules were developed with good microenvironment for enhanced cellular functions. Further development of other types of microcapsules should also maintain such a good microenvironment. To accomplish this, a semi-gel-like inner collagen layer (1.5 mg/ml) should surround the cells to provide a “loose” extra-cellular matrix configuration that mimics the in vivo situation. In Type-I microcapsules, the positively charged collagen layer was encapsulated by a thin layer (2-5 μm) of the negatively charged 10% ter-polymer shell. Other layers were added for specific applications with minimal additional thickness to avoid adverse effects to the functions of the microencapsulated cells.
  • Type II Microcapsules
  • Type-I microcapsules typically have a 2-5 μm thin shell, which does not ensure complete cell encapsulation. Like other single-shell microcapsules, the Type-I microcapsules have occasionally been observed with cells protruding out of the microcapsules (FIG. 1). To improve the reliability of immune isolation of the live cells within the microcapsules that are required for many applications, a two-step encapsulation method was employed. To ensure minimal impedance of the mass transfer properties and the functions of the encapsulated cells, the flow rate in the syringe pump was optimized such that the desired minimal thickness of the additional layers was achieved as measured by the confocal imaging. These particular Type-II microcapsules have four separate layers (two ter-polymer shells spaced by the two layers of the modified collagen) with the additional layers covering the protruding cells (FIG. 1) for a potentially more reliable immune isolation than the Type-I microcapsules.
  • Type III Microcapsules
  • Type-I microcapsules can maintain structural integrity in static cell culture vessels for a few days. The mechanical stability of the microcapsules requires further improvement for some applications, such as the more dynamic environment in a bioreactor. Therefore, it would be desirable to employ suitable materials that can form highly porous, but mechanically stable, layer(s) outside Type-I microcapsules. It is believed that such outer layer(s) could behave like an exoskeleton to confer mechanical stability while imposing no or little impedance to mass transfer properties of the microcapsules. Ideally, such an exoskeleton should be formed by complex coacervation to avoid exposure of live cells to organic solvents. Therefore, the exoskeleton materials should have net positive charge to interact with the negatively charged ter-polymer shell of the Type-I microcapsules.
  • Alumina and chitosan were tested for the ability to form macro-porous exoskeleton as examined by SEM. Such microcapsules with macro-porous exoskeleton are referred to herein as Type-III microcapsules. Chitosan can form a macro-porous exoskeleton outside Type-I microcapsules when used in a range of about 0.01-0.02% (w/v) (FIG. 2A). Lower concentrations of chitosan below this range were found not to afford sufficient material to cover the entire microcapsule surface. Higher concentrations of chitosan above this range were found to possess so much positive charges that the Type-I microcapsules disintegrated upon contact. Alumina sol also can form a macro-porous exoskeleton outside Type-I microcapsules when used in a range of about 3-6 mM (FIG. 2B).
  • Type IV Microcapsules
  • As many plasma proteins, such as serum albumin, are negatively charged (with pI<7) under physiological pH, some applications involving contact with blood would require a negatively charged outer-shell to minimize non-specific protein adsorption onto the microcapsules. Since Type-III microcapsules have a positively charged exoskeleton on the surface, another thin (2-5 μm) layer of the negatively charged ter-polymer shell can be formed outside the exoskeleton by complex coacervation. These Type-IV microcapsules have a selectively permeable, smooth and micro-porous fourth layer (FIG. 3) to ensure complete cell encapsulation for a potentially more reliable immune isolation than the Type-III microcapsules.
  • Permeability Profile of the Microcapsules
  • For all applications, the microcapsules need to be permeable to nutrients, oxygen and metabolic wastes. Some applications require selective permeability to allow nutrient exchange and at the same time to prevent the passage of large molecules, such as immunoglobulins, into the microcapsules. The permeability profiles of the Type-I, -II, -III, and -IV microcapsules were characterized to assess and optimize the suitability of different types of microcapsules for use in relevant applications. BSA with a MW of 66 kDa was encapsulated inside the microcapsules and the rate of release from the microcapsules was measured by spectrophotometry. Type-I microcapsules (∘) were used as a control since they release almost all the encapsulated BSA within 15 minutes (FIG. 4). Type-II (□), Type-III (x), and Type-IV (⋄) microcapsules all exhibited some degrees of reduction in permeability. Within about 15 minutes, Type-II microcapsules (□) had about 80% of the BSA released, while Type-III (x) and Type-IV (⋄) microcapsules had about 65% of the BSA released. By about 30-50 minutes, almost all the encapsulated BSA was released from all the microcapsules. Since Type-I microcapsules are impermeable to molecules larger than about 150 kDa, the other types of microcapsules should also be impermeable to larger molecules, such as immunoglobulins, which mediate the immune response.
  • Functional Profiles of the Microcapsules
  • As Type-II (□), Type-III (x), and Type-IV (⋄) microcapsules exhibited some degrees of permeability reduction (FIG. 4) when compared to Type-I microcapsules (∘), it is important to investigate the effects of permeability reduction on the functional status of the microencapsulated cells. The urea production profiles of the microencapsulated hepatocytes, which are very sensitive to the extra-cellular microenvironment, were characterized. All four types (I-IV) of microcapsules were found to exhibit similar levels of hepatocyte functions on day 1 (which is defined as 24 hours after the initial cell encapsulation) (FIG. 5). From day 1 onward, the urea production by the Type-II (Δ), Type-III (x), and Type-IV (∘) microcapsules decreased one after another from the level exhibited by the Type-I (□) microcapsules. The urea production by the Type-II microcapsules (Δ) started decreasing from day 1 to about 40% of Type-I microcapsules (□) on day 3 and then stabilized from day 3 onwards. The urea production by Type-III microcapsules (x) started decreasing from day 4 to about 50% of Type-I microcapsules (□) on day 5. The urea production by Type IV microcapsules (∘) decreased from day 3 to about 45% of Type-I microcapsules (□) on days 4-5. On days 6 and 7, all four types of the microcapsules exhibit similar levels of hepatocyte function, which is approximately the initial level of the monolayer control (▪).
  • Mechanical Stability of the Microcapsules
  • Type-III and -IV microcapsules were developed to improve mechanical stability. Nano-indentation with a pyramidal indenter tip with an inclusion angle of 90° was used to characterize the mechanical stability of the microcapsules over a period of 7 days (Table 1). Under the 0.15 mN load, which is the maximum load at rupture for Type-I microcapsules for the pyramidal indenter, the depth of penetration into Type-I microcapsule shell increases rapidly from 0.4±0.1 μm on day 1 to 0.6±0.1 μm on day 2 and 1.4˜0.8 μm on day 7. The depth of penetration of the pyramidal indenter tip into Type-II and Type-III microcapsule shells remained relatively stable, in the range of 0.4±0.1 μm to 0.6±0.1 μm, throughout the 7-day period. There is a large variation in the depth of penetration of the pyramidal indenter on day 7, which was not observed on days 1 and 2, or in any other types of microcapsules. The protruding cells not encapsulated by the ter-polymer shell might have contributed to such a large variation. The additional layers of either the ter-polymer shell or the exoskeleton could then stabilize Type-II and -III microcapsules over the 7-day period as compared to Type-I microcapsules.
    TABLE 1
    Depth of Penetration (μm ± standard error of means)
    Type-I Type-II Type-III
    Days Microcapsules Microcapsules Microcapsules
    1 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1
    2 0.6 ± 0.1 0.4 ± 0.1 0.6 ± 0.1
    7 1.4 ± 0.8 0.5 ± 0.1 0.4 ± 0.1
  • It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions and methods of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims (30)

  1. 1. A microcapsule for culturing anchorage-dependent cells comprising an inner extracellular matrix surrounding the cells and an outer shell of synthetic polymer surrounding and supporting the extracellular matrix; wherein said microcapsule is permeable to nutrients necessary to sustain normal metabolic functions of the cells and to toxins released by the cells; and wherein said outer shell has a thickness of from about 1 to about 20 μm.
  2. 2. The microcapsule of claim 1 wherein said inner extracellular matrix comprises a biopolymer inner layer and a biocompatible synthetic polyelectrolyte outer layer, wherein said inner layer and said outer layer have charges sufficient to form a complex of said biopolymer and said polyelectrolyte.
  3. 3. The microcapsule of claim 2 wherein said outer shell comprises (i) a biopolymer selected from the group consisting of cationic collagen modified to have a pKi of at least about 9, anionic esterified hyaluronic acid, anionic amine-modified hyaluronic acid, fibronectin, and laminin, and (ii) a biocompatible synthetic polyelectrolyte having an electrolytic charge opposite to that of the biopolymer.
  4. 4. The microcapsule of claim 3 wherein said biocompatible synthetic polyelectrolyte of said outer shell comprises an acrylate ter-polymer of methacrylic acid, hydroxyethyl methacrylate, and methyl methacrylate.
  5. 5. A microcapsule for culturing anchorage-dependent cells comprising an inner extracellular matrix surrounding the cells, a macro-porous exoskeleton surrounding and supporting the extracellular matrix; and an outer shell of synthetic polymer surrounding the macro-porous exoskeleton; wherein said microcapsule is permeable to nutrients necessary to sustain normal metabolic functions of the cells and to toxins released by the cells; and wherein said outer shell has a thickness of from about 1 to about 20 μm.
  6. 6. The microcapsule of claim 5 wherein said inner extracellular matrix comprises a biopolymer inner layer and a biocompatible synthetic polyelectrolyte outer layer, wherein said inner layer and said outer layer have charges sufficient to form a complex of said biopolymer and said polyelectrolyte.
  7. 7. The microcapsule of claim 5 wherein said macro-porous exoskeleton comprises at least one of alumina, alumina sol, and chitosan.
  8. 8. The microcapsule of claim 5 wherein said synthetic polymer of said outer shell comprises an acrylate ter-polymer of methacrylic acid, hydroxyethyl methacrylate, and methyl methacrylate.
  9. 9. A method of preparing a microcapsule having anchorage-dependent cells surrounded by an inner extracellular matrix and an outer shell of synthetic polymer surrounding and supporting the extracellular matrix, the process comprising: preparing an inner extracellular matrix having an inner layer and an outer layer, comprising extruding an inner layer biopolymer solution containing bioactive cells into a biocompatible synthetic polyelectrolyte outer layer; wherein said inner layer and said outer layer have charges sufficient to form a complex of said biopolymer and said polyelectrolyte; and forming an outer shell by encapsulating said inner extracellular matrix containing said cells with a synthetic polymer solution, wherein said outer shell thus-formed has a thickness of from about 1 to about 20 μm.
  10. 10. The method of claim 9 wherein said synthetic polymer solution of said outer shell comprises (i) a biopolymer selected from the group consisting of cationic collagen modified to have a pKi of at least about 9, anionic esterified hyaluronic acid, anionic amine-modified hyaluronic acid, fibronectin, and laminin, and (ii) a biocompatible synthetic polyelectrolyte having an electrolytic charge opposite to that of the biopolymer.
  11. 11. The method of claim 10 wherein said biocompatible synthetic polyelectrolyte of said outer shell comprises an acrylate ter-polymer of methacrylic acid, hydroxyethyl methacrylate, and methyl methacrylate.
  12. 12. A method of preparing a microcapsule having anchorage-dependent cells surrounded by an inner extracellular matrix and a macro-porous exoskeleton surrounding and supporting the extracellular matrix, the process comprising: preparing an inner extracellular matrix having an inner layer and an outer layer, comprising extruding an inner layer biopolymer solution containing bioactive cells into a biocompatible synthetic polyelectrolyte outer layer; wherein said inner layer and said outer layer have charges sufficient to form a complex of said biopolymer and said polyelectrolyte; and suspending said inner extracellular matrix containing said cells in an exoskeleton material having a charge opposite to that of the outer layer of said extracellular matrix to form a macro-porous exoskeleton over said extracellular matrix.
  13. 13. The method of claim 12 wherein said macro-porous exoskeleton comprises at least one of alumina, alumina sol, and chitosan.
  14. 14. The method of claim 12 further comprising forming an outer shell by encapsulating the microcapsule in a synthetic polymer solution.
  15. 15. The method of claim 14 wherein said synthetic polymer solution comprises an acrylate ter-polymer of methacrylic acid, hydroxyethyl methacrylate, and methyl methacrylate.
  16. 16. A method of culturing anchorage-dependent cells comprising applying agitation to the microcapsule of claim 1 after a predetermined time to rupture the outer shell, and removing the extracellular matrix to recover the cells.
  17. 17. A multi-layer microcapsule comprising bioactive cells attached to a microcapsule membrane; wherein said microcapsule membrane comprises (i) a first inner layer of biopolymer selected from the group consisting of cationic collagen, anionic collagen, anionic esterified hyaluronic acid, anionic amine-modified hyaluronic acid, fibronectin, and laminin; (ii) a second intermediate layer of polyelectrolyte synthetic polymer; and (iii) a third outer layer forming an exoskeleton to provide mechanical stability; wherein said first inner layer and said second intermediate layer are complexed via ionic charges; wherein said second intermediate layer and said third outer layer are complexed via ionic charges; wherein said microcapsule membrane is permeable to molecules smaller than or equal to the size of albumin, to nutrients necessary to sustain normal metabolic functions of the bioactive cells, and to toxins released by the bioactive cells; and wherein said microcapsule membrane is impermeable to immunoglobulins and macrophages.
  18. 18. The multi-layer microcapsule of claim 17 further comprising (iv) a fourth outer layer comprising a polyelectrolyte synthetic polymer surrounding said third layer, wherein said fourth outer layer is complexed with said third layer via ionic charges.
  19. 19. The multi-layer microcapsule of claim 17 wherein said second intermediate layer of polyelectrolyte synthetic polymer is an acrylate ter-polymer of methacrylic acid, hydroxyethyl methacrylate, and methyl methacrylate.
  20. 20. The multi-layer microcapsule of claim 17 wherein said third outer layer comprises a material selected from the group consisting of alumina, alumina sol, and chitosan.
  21. 21. The multi-layer microcapsule of claim 17 wherein said bioactive cells comprise a mixture of dividing cells and non-dividing cells.
  22. 22. The multi-layer microcapsule of claim 21 wherein said bioactive cells comprise a mixture of hepatocyte cells and non-parenchymal cells.
  23. 23. The multi-layer microcapsule of claim 4 wherein said third layer comprises a ceramic sol modified to be negatively charged, wherein said third layer is unstable at a physiological pH of 7.4 to provide a short-term controlled release of cells, cell aggregates, or tissue structures.
  24. 24. A process of preparing a bioartificial liver assist device comprising packing one or more of the biocompatible microcapsule of claim 1 in a bioreactor.
  25. 25. A process of preparing a bioartificial liver assist device comprising packing one or more of the biocompatible microcapsule of claim 5 in a bioreactor.
  26. 26. A bioartificial liver assist device comprising one or more of the biocompatible microcapsule of claim 1 contained within a bioreactor.
  27. 27. A bioartificial liver assist device comprising one or more of the biocompatible microcapsule of claim 5 contained within a bioreactor.
  28. 28. A method of preparing living cells for multi-dimensional imaging to study cells, tissue, or tissue constructs, the method comprising culturing at least one cell in the microcapsule of claim 1 and imaging the cell using microscopy.
  29. 29. A method of preparing living cells for transplantation comprising culturing at least one cell in the microcapsule of claim 1, harvesting the cell, and coupling the cell to a scaffold.
  30. 30. A method of analyzing cells comprising removing at least one cell from a biopsy sample, culturing the cell in the microcapsule of claim 1, and performing cytometry analysis.
US11137483 2000-10-12 2005-05-26 Multi-layer cell encapsulation for tissue engineering Abandoned US20050220891A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US23925900 true 2000-10-12 2000-10-12
US09975273 US6916640B2 (en) 2000-10-12 2001-10-12 Multi-layer cell encapsulation for tissue engineering
US11137483 US20050220891A1 (en) 2000-10-12 2005-05-26 Multi-layer cell encapsulation for tissue engineering

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11137483 US20050220891A1 (en) 2000-10-12 2005-05-26 Multi-layer cell encapsulation for tissue engineering
US12507900 US7943353B2 (en) 2000-10-12 2009-07-23 Multi-layer cell encapsulation for tissue engineering

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US09975273 Division US6916640B2 (en) 2000-10-12 2001-10-12 Multi-layer cell encapsulation for tissue engineering

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12507900 Division US7943353B2 (en) 2000-10-12 2009-07-23 Multi-layer cell encapsulation for tissue engineering

Publications (1)

Publication Number Publication Date
US20050220891A1 true true US20050220891A1 (en) 2005-10-06

Family

ID=22901354

Family Applications (4)

Application Number Title Priority Date Filing Date
US10398957 Expired - Fee Related US6905875B2 (en) 2000-10-12 2001-10-12 Non-disruptive three-dimensional culture and harvest system for anchorage-dependent cells
US09975273 Expired - Fee Related US6916640B2 (en) 2000-10-12 2001-10-12 Multi-layer cell encapsulation for tissue engineering
US11137483 Abandoned US20050220891A1 (en) 2000-10-12 2005-05-26 Multi-layer cell encapsulation for tissue engineering
US12507900 Expired - Fee Related US7943353B2 (en) 2000-10-12 2009-07-23 Multi-layer cell encapsulation for tissue engineering

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US10398957 Expired - Fee Related US6905875B2 (en) 2000-10-12 2001-10-12 Non-disruptive three-dimensional culture and harvest system for anchorage-dependent cells
US09975273 Expired - Fee Related US6916640B2 (en) 2000-10-12 2001-10-12 Multi-layer cell encapsulation for tissue engineering

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12507900 Expired - Fee Related US7943353B2 (en) 2000-10-12 2009-07-23 Multi-layer cell encapsulation for tissue engineering

Country Status (4)

Country Link
US (4) US6905875B2 (en)
EP (1) EP1326968B1 (en)
DE (2) DE60127983D1 (en)
WO (1) WO2002031135A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070149743A1 (en) * 2005-12-23 2007-06-28 Boston Scientific Scimed, Inc. Polymeric hybrid precursors, polymeric hybrid precursor composite matrices, medical devices, and methods
WO2007127231A2 (en) * 2006-04-24 2007-11-08 The Johns Hopkins University Magnetic resonance-detectable, ultrasound-detectable and/or radiopaque microcapsules and uses thereof
US8455088B2 (en) 2005-12-23 2013-06-04 Boston Scientific Scimed, Inc. Spun nanofiber, medical devices, and methods
US8956871B2 (en) 2012-11-12 2015-02-17 Industrial Technology Research Institute Cell culture system and serum-free method for cultivating cells
US9968446B2 (en) 2011-03-23 2018-05-15 The Regents Of The University Of California Tubular scaffold for fabrication of heart valves
US10016461B2 (en) 2012-12-03 2018-07-10 The Regents Of The University Of California Apparatus and process for growing a heart valve in three-dimensions

Families Citing this family (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1374893A1 (en) * 2002-06-17 2004-01-02 NovImmune S.A. Vaccination with immuno-isolated cells producing an immunomodulator
EP1610752B1 (en) 2003-01-31 2013-01-02 Boston Scientific Limited Localized drug delivery using drug-loaded nanocapsules and implantable device coated with the same
US7364585B2 (en) * 2003-08-11 2008-04-29 Boston Scientific Scimed, Inc. Medical devices comprising drug-loaded capsules for localized drug delivery
US7897384B2 (en) * 2003-09-08 2011-03-01 Ethicon, Inc. Chondrocyte therapeutic delivery system
US7927599B2 (en) 2003-09-08 2011-04-19 Ethicon, Inc. Chondrocyte therapeutic delivery system
US20050272153A1 (en) 2004-01-27 2005-12-08 Zou Xuenong Bone tissue engineering by ex vivo stem cells ongrowth into three-dimensional trabecular metal
US20060134779A1 (en) * 2004-03-10 2006-06-22 Banes Albert J Modulation of cell intrinsic strain to control cell modulus, matrix synthesis, secretion, organization, material properties and remodeling of tissue engineered constructs
US7700333B2 (en) * 2004-07-26 2010-04-20 Agency For Science Technology & Research Immobilization of cells in a matrix formed by biocompatible charged polymers under laminar flow conditions
US7704714B2 (en) * 2004-07-26 2010-04-27 Agency For Science, Technology & Research Encapsulation of cells in biologic compatible scaffolds by coacervation of charged polymers
CA2586400A1 (en) * 2004-11-11 2006-05-18 Agency For Science, Technology And Research Cell culture device
US7851189B2 (en) * 2005-03-07 2010-12-14 Boston Scientific Scimed, Inc. Microencapsulated compositions for endoluminal tissue engineering
CA3019254A1 (en) * 2006-02-07 2007-08-16 Spinalcyte, Llc Methods and compositions for repair of cartilage using an in vivo bioreactor
CN105296415A (en) * 2006-03-23 2016-02-03 普拉里斯坦有限公司 Methods for cell expansion and uses of cells and conditioned media produced thereby for therapy
US20070237749A1 (en) * 2006-04-07 2007-10-11 Wang Taylor G Multi-membrane immunoisolation system for cellular transplant
US20070238169A1 (en) * 2006-04-11 2007-10-11 The Board Of Trustees Of The Leland Stanford Junior University Cell sorter and culture system
US20090263849A1 (en) * 2006-04-21 2009-10-22 Drexel University Bioprinting Three-Dimensional Structure Onto Microscale Tissue Analog Devices for Pharmacokinetic Study and Other Uses
CN101448527B (en) * 2006-05-19 2013-12-18 香港大学 Cell-matrix microspheres, methods for preparation and applications
EP2029727B1 (en) * 2006-06-16 2012-04-11 FMC Biopolymer AS Alginate coated, collagen matrix cellular device, preparative methods, and uses thereof.
WO2008027989A3 (en) * 2006-08-29 2008-05-08 Univ Florida State Res Found Polymer mechanical damping composites and methods of production
JP5096486B2 (en) * 2006-12-13 2012-12-12 ビーエーエスエフ ソシエタス・ヨーロピアBasf Se Microcapsules
WO2008130529A1 (en) * 2007-04-16 2008-10-30 University Of Toledo Hybrid biomimetic particles, methods of making same and uses therefor
US20080281419A1 (en) * 2007-05-10 2008-11-13 Matheny Robert G Breast implants and compositions of extracellular matrix
US8257963B2 (en) * 2007-06-01 2012-09-04 Depuy Mitek, Inc. Chondrocyte container and method of use
EP2173858A4 (en) * 2007-07-02 2010-09-08 Univ Columbia Biologically derived composite tissue engineering
EP2185730A4 (en) 2007-08-23 2010-10-27 Intrexon Corp Methods and compositions for diagnosing disease
WO2009043052A1 (en) * 2007-09-27 2009-04-02 Columbia University Methods and systems for forming biocompatible materials
KR101666228B1 (en) 2007-09-28 2016-10-13 인트렉손 코포레이션 Therapeutic gene-switch constructs and bioreactors for the expression of biotherapeutic molecules, and uses thereof
CN101883842B (en) * 2007-10-11 2013-09-18 新加坡科技研究局 Forming cell structure with transient linker in cage
US20090137925A1 (en) * 2007-11-23 2009-05-28 Divya Cantor Impedance Spectroscopy Cervix Scanning Apparatus and Method
US8613776B2 (en) * 2007-12-27 2013-12-24 The Trustees Of Columbia University In The City Of New York Systems and methods for forming patterned extracellular matrix materials
US8114918B2 (en) 2008-08-15 2012-02-14 The Florida State University Research Foundation, Inc. Compacted polyelectrolyte complexes and articles
US20100166822A1 (en) * 2008-12-31 2010-07-01 Howmedica Osteonics Corp. Adhesive cartilage implant
US20110136162A1 (en) * 2009-08-31 2011-06-09 Drexel University Compositions and Methods for Functionalized Patterning of Tissue Engineering Substrates Including Bioprinting Cell-Laden Constructs for Multicompartment Tissue Chambers
WO2014037942A1 (en) * 2012-09-04 2014-03-13 Technion Research & Development Foundation Limited Use of decellularized extracellular matrix for encapsulating cells
US20160053231A1 (en) * 2013-04-10 2016-02-25 Tufts University Two and three dimensional decellularized ecm constructs and uses therefor
WO2015002724A4 (en) 2013-06-11 2015-03-26 President And Fellows Of Harvard College SC-β CELLS AND COMPOSITIONS AND METHODS FOR GENERATING THE SAME
US9919280B2 (en) 2014-11-24 2018-03-20 The Florida State University Research Foundation, Inc. Method of forming polyelectrolyte complex capsules
WO2016100921A1 (en) * 2014-12-18 2016-06-23 President And Fellows Of Harvard College METHODS FOR GENERATING STEM CELL-DERIVED β CELLS AND USES THEREOF
JP2018529706A (en) 2015-09-25 2018-10-11 マキシバックス エスアー Vaccination with immunoisolation cells producing an immunomodulator
WO2017152035A1 (en) * 2016-03-03 2017-09-08 Henry Ford Health System 3-d collagen scaffold-generated exosomes and uses thereof

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3959078A (en) * 1973-05-18 1976-05-25 Midwest Research Institute Enzyme immobilization with a thermochemical-photochemical bifunctional agent
US4293654A (en) * 1977-10-17 1981-10-06 Massachusetts Institute Of Technology Cell culture microcarriers
US4743545A (en) * 1984-08-09 1988-05-10 Torobin Leonard B Hollow porous microspheres containing biocatalyst
US4798786A (en) * 1982-05-06 1989-01-17 Stolle Research And Development Corporation Living cells encapsulated in crosslinked protein
US4994388A (en) * 1988-04-15 1991-02-19 Solohill Engineering, Inc. Collagen-coated polystyrene microcarrier beads
US5620883A (en) * 1994-04-01 1997-04-15 The Johns Hopkins University Living cells microencapsulated in a polymeric membrane having two layers
US5837234A (en) * 1995-06-07 1998-11-17 Cytotherapeutics, Inc. Bioartificial organ containing cells encapsulated in a permselective polyether suflfone membrane
US5840576A (en) * 1994-07-20 1998-11-24 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5846952A (en) * 1993-12-01 1998-12-08 Marine Polymer Technologies, Inc. Methods and compositions for poly-β-1-4-N-acetylglucosamine drug delivery
US5853747A (en) * 1994-06-27 1998-12-29 Institut De Recherche Biologique Therapeutic and dietetic uses of a brain phospholipid-based complex
US5908623A (en) * 1993-08-12 1999-06-01 Cytotherapeutics, Inc. Compositions and methods for the delivery of biologically active molecules using genetically altered cells contained in biocompatible immunoisolatory capsules
US6294381B1 (en) * 1996-10-04 2001-09-25 Johanna Olweus CD123+ dendritic cells in blood and lymphoid tissues
US20010049139A1 (en) * 2000-03-23 2001-12-06 Eric Lagasse Hepatic regeneration from hematopoietic stem cells
US20020028510A1 (en) * 2000-03-09 2002-03-07 Paul Sanberg Human cord blood as a source of neural tissue for repair of the brain and spinal cord
US20040136973A1 (en) * 2002-11-07 2004-07-15 Eliezer Huberman Human stem cell materials and methods

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007485A1 (en) 1989-11-09 1991-05-30 Bio-Metric Systems, Inc. Improved bioreactor surfaces and methods of making same
EP0529751A1 (en) 1991-08-09 1993-03-03 W.R. Grace &amp; Co.-Conn. Cell culture substrate, test material for cell culture and preparations thereof
US5858350A (en) * 1993-12-01 1999-01-12 Marine Polymer Technologies Methods and compositions for poly-β-1→4-N-acetylglucosamine cell therapy system

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3959078A (en) * 1973-05-18 1976-05-25 Midwest Research Institute Enzyme immobilization with a thermochemical-photochemical bifunctional agent
US4293654A (en) * 1977-10-17 1981-10-06 Massachusetts Institute Of Technology Cell culture microcarriers
US4798786A (en) * 1982-05-06 1989-01-17 Stolle Research And Development Corporation Living cells encapsulated in crosslinked protein
US4743545A (en) * 1984-08-09 1988-05-10 Torobin Leonard B Hollow porous microspheres containing biocatalyst
US4994388A (en) * 1988-04-15 1991-02-19 Solohill Engineering, Inc. Collagen-coated polystyrene microcarrier beads
US5908623A (en) * 1993-08-12 1999-06-01 Cytotherapeutics, Inc. Compositions and methods for the delivery of biologically active molecules using genetically altered cells contained in biocompatible immunoisolatory capsules
US5846952A (en) * 1993-12-01 1998-12-08 Marine Polymer Technologies, Inc. Methods and compositions for poly-β-1-4-N-acetylglucosamine drug delivery
US5620883A (en) * 1994-04-01 1997-04-15 The Johns Hopkins University Living cells microencapsulated in a polymeric membrane having two layers
US5853747A (en) * 1994-06-27 1998-12-29 Institut De Recherche Biologique Therapeutic and dietetic uses of a brain phospholipid-based complex
US5840576A (en) * 1994-07-20 1998-11-24 Cytotherapeutics, Inc. Methods and compositions of growth control for cells encapsulated within bioartificial organs
US5837234A (en) * 1995-06-07 1998-11-17 Cytotherapeutics, Inc. Bioartificial organ containing cells encapsulated in a permselective polyether suflfone membrane
US6294381B1 (en) * 1996-10-04 2001-09-25 Johanna Olweus CD123+ dendritic cells in blood and lymphoid tissues
US20020028510A1 (en) * 2000-03-09 2002-03-07 Paul Sanberg Human cord blood as a source of neural tissue for repair of the brain and spinal cord
US20010049139A1 (en) * 2000-03-23 2001-12-06 Eric Lagasse Hepatic regeneration from hematopoietic stem cells
US20040136973A1 (en) * 2002-11-07 2004-07-15 Eliezer Huberman Human stem cell materials and methods

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070149743A1 (en) * 2005-12-23 2007-06-28 Boston Scientific Scimed, Inc. Polymeric hybrid precursors, polymeric hybrid precursor composite matrices, medical devices, and methods
US7674864B2 (en) 2005-12-23 2010-03-09 Boston Scientific Scimed, Inc. Polymeric hybrid precursors, polymeric hybrid precursor composite matrices, medical devices, and methods
US8455088B2 (en) 2005-12-23 2013-06-04 Boston Scientific Scimed, Inc. Spun nanofiber, medical devices, and methods
WO2007127231A2 (en) * 2006-04-24 2007-11-08 The Johns Hopkins University Magnetic resonance-detectable, ultrasound-detectable and/or radiopaque microcapsules and uses thereof
WO2007127231A3 (en) * 2006-04-24 2008-12-04 Univ Johns Hopkins Magnetic resonance-detectable, ultrasound-detectable and/or radiopaque microcapsules and uses thereof
US9968446B2 (en) 2011-03-23 2018-05-15 The Regents Of The University Of California Tubular scaffold for fabrication of heart valves
US8956871B2 (en) 2012-11-12 2015-02-17 Industrial Technology Research Institute Cell culture system and serum-free method for cultivating cells
US10016461B2 (en) 2012-12-03 2018-07-10 The Regents Of The University Of California Apparatus and process for growing a heart valve in three-dimensions

Also Published As

Publication number Publication date Type
US7943353B2 (en) 2011-05-17 grant
EP1326968A1 (en) 2003-07-16 application
US6916640B2 (en) 2005-07-12 grant
US20020094569A1 (en) 2002-07-18 application
US20040023370A1 (en) 2004-02-05 application
EP1326968A4 (en) 2004-10-06 application
EP1326968B1 (en) 2007-04-18 grant
DE60127983D1 (en) 2007-05-31 grant
WO2002031135A1 (en) 2002-04-18 application
US20090286278A1 (en) 2009-11-19 application
US6905875B2 (en) 2005-06-14 grant
DE60127983T2 (en) 2008-01-17 grant

Similar Documents

Publication Publication Date Title
King et al. Alginate‐polylysine microcapsules of controlled membrane molecular weight cutoff for mammalian cell culture engineering
Kashyap et al. Hydrogels for pharmaceutical and biomedical applications
Lee et al. Biomedical applications of collagen
US6730298B2 (en) Tissue formation by injecting a cell-polymeric solution that gels in vivo
US5635609A (en) Particles prepared by transacylation reaction between an esterified polysaccharide and a polyamine, methods of preparation therefor and compositions containing same
US5874165A (en) Materials and method for the immobilization of bioactive species onto polymeric subtrates
La Flamme et al. Biocompatibility of nanoporous alumina membranes for immunoisolation
US5741685A (en) Parenchymal cells packaged in immunoprotective tissue for implantation
EP0301777A1 (en) Multiple membrane microencapsulation
US6231881B1 (en) Medium and matrix for long-term proliferation of cells
de Vos et al. Long-term biocompatibility, chemistry, and function of microencapsulated pancreatic islets
US5876742A (en) Biological tissue transplant coated with stabilized multilayer alginate coating suitable for transplantation and method of preparation thereof
US6126936A (en) Microcapsules and composite microreactors for immunoisolation of cells
Colton et al. Bioengineering in development of the hybrid artificial pancreas
US6565842B1 (en) Crosslinkable polypeptide compositions
US4352883A (en) Encapsulation of biological material
Khattak et al. Enhancing oxygen tension and cellular function in alginate cell encapsulation devices through the use of perfluorocarbons
Sun et al. Injectable microencapsulated islet cells as a bioartificial pancreas
Chua et al. Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold
US4391909A (en) Microcapsules containing viable tissue cells
US5578442A (en) Graft copolymers of polycationic species and water-soluble polymers, and use therefor
Kizilel et al. The bioartificial pancreas: progress and challenges
Panza et al. Treatment of rat pancreatic islets with reactive PEG
Sawhney et al. Interfacial photopolymerization of poly (ethylene glycol)-based hydrogels upon alginate-poly (l-lysine) microcapsules for enhanced biocompatibility
Ma et al. Generation of alginate-poly-l-lysine-alginate (APA) biomicrocapsules: the relationship between the membrane strength and the reaction conditions