JPH07505786A - Methods for culturing viable cells and regulating compound concentrations in body fluids - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for culturing viable cells and regulating compound concentration in body fluids Background of the invention field of invention The invention generally relates to copolymers having specific tensile strength, maximum elongation, and water absorption properties. It is made up of A non-porous and semi-permeable biocompatible material that is impermeable to cells and particulate matter, but Concerning the cultivation of viable cells in the presence of a synthetic film. The method of the invention In this case, external nutrients and culture medium are allowed to enter the cell environment through the film. Permeates the cell's products from the inside out from the cell's environment through the film. Ru.

Background explanation Standard immunosuppression, for example in diabetic patients, is through the use of cyclosporine. This poses acute risk issues, such as nephrotoxicity, especially in young In the case of urinary patients, it is extremely difficult to prove validity.

The four methods of immunoisolation that have been used to date are:

(1) Extravascular diffusion chamber.

(2) Intravascular diffusion chamber.

(3) Intravascular ultrafiltration chamber.

(4) Encapsulation with microcapsules.

All of these approaches have undesirable features summarized below.

■, they induce a host fibrotic response against the graft, e.g. arginine Like microcapsule encapsulation of acids, it creates instability of the material.

■, protein deposits, blood clotting, or fibrous ingrowth that feeds through the membrane pores. Diffusion of nutrients across prior art semipermeable membranes requires osmotic There is a limit due to the decrease in

■ Permeability of glucose and insulin across semipermeable membrane barriers of the prior art and Diffusion has a lag time that results in a cell response to the host's blood glucose concentration. This causes a delay in the reaction.

The membranes and methods used in prior art implants were With the possible exception of microcapsules, semipermeable membranes with micropores have been used. Kaka Membranes that can It has been manufactured from a certain impermeable polymer. Millipore [F] and Nucleo Bore ■, or microporous polycarbonate membranes used in prior art, essentially Made from a virtually impermeable polymer to support long-term cell viability There isn't. Other semipermeable membranes are for the transport of glucose and insulin across the membrane. It showed low hemocompatibility as well as low osmotic proficiency. What is noteworthy is that the minute Porous membranes accommodate high permeate fluxes in pressure-based processes such as ultrafiltration but at the same time at a concentration such as in the in vivo method of the present invention. It has very low permeability in the process. Blood membrane with micropores When left in direct contact with body fluids, such as fluids, they accumulate a fibrin layer and membrane is a major barrier to mass transportation through

Generally block or segmented copolymers of polyether urethanes exhibits good biocompatibility along with high strength and elasticity. unique of this nature The combination is due in part to the biphasic morphology of the polyurethane molecule. typical port In urethanes, aggregated aromatic or aliphatic urethane or urea segments constitute a hard glassy or semicrystalline phase, while the glass transition temperature ( Tg), the oligomeric segments form a liquid-like, rubbery soft phase or contains soft segments. The morphology of polyurethane is hard segment and soft segment. the chemistry of the segment, the difference in segment polarity, the hard segment content, and the hard segment chemistry. and a number of factors including the molecular weight of the soft segment.

Soft segment formation in both polyurethaneureas and polyurethanes chemistry affects the degree of phase separation in a polymer, which in turn affects overall and surface properties and subsequently biocompatibility. Substances disclosed in this patent and polyurethaneureas, which are similar only with respect to the composition of the hard segment, are species It has been shown to be resistant to degradation in various applications (PaynLe r et al., “Stability of polyurethaneurea, mitratan, against hydrolysis, X-ray photoelectron Research by spectroscopy (The Hydrolytic 5tability of 　Mitrathane.

a Po1urethanurea, An X-ray Photoel eckeon 5pectroscopy 5tudy) JJ, Bio+ned, Mater, Res, 22.687-698 (1988); 5zych er et al., “Blood Compatible Polyurethane Elast v-(Blood Compat ible Polyurethane Elastomers) J J, Bio Mater, Appl, 2.290-313 (1987)).

Suitability of natural and synthetic polymeric membranes for the separation of low molecular weight gas and liquid mixtures. Its use has been reported in numerous review articles. For simple low molecular weight (MW) penetrants Many studies have been reported on the permeability of membranes that The composition of the rubbery or crystalline-rubbery copolymers varies.

The polyurethane multipolymer film, unlike the materials disclosed herewith, is resistant to water and It has been shown to be permeable to water and salt. If one block or segment is one lath-like or crystalline (hard segment), the other rubber-like or liquid-like (soft segment) A thermoplastic segmented block copolymer with soft segments) In this case, molecular penetration occurs primarily through the soft segment. relatively impervious Hard segments that are permeable can cause swelling of soft segments. conditions, due to strong intermolecular interactions with similar segments on adjacent molecules. and provide physical integrity to the polymer.

Okkema et al. are suitable as blood contact surfaces and are useful in the present invention. polyethylene oxy, with a hard segment content of 55% by weight, which is too high for (PEO), polytetramethylene oxide (PTMO), and PEO/PTM Opened a series of polyether polyurethanes based on soft segments of O mixtures. (Okkema et al., “Total analysis of polyurethane modified with polyethylene oxide”). Body and surface characteristics and characteristics when in contact with blood (Bulk, 5 surface, a nd Blood-Contacting Properties of Pol yurethanes Modified with Polyethylen e 0xide) JJ, Biomater.

Sci, Polymer, 1st edition (1) 43-62 (1989)).

Takahaya et al. disclosed the preparation of mentated poly(ether urethane urea) (SPUU) (Ta Kahara et al., [having hydrophilic and hydrophobic soft segment components (S Mobility of surface molecules of PUUS) and platelet reactivity (Surface Molec ular Mobility and Platelet Reactivit y of (SPUUS) with+1ydrophilic and Hy drophobic 5oft Segn+ent Components)J J.

Biomater Sci, Polymer, 1st edition (1) 17-29 (19 89)). Platelet adhesion and dynamic contacts measured after adsorption of bovine serum albumin The angle is 5puu with a hydrophilic soft segment and a non-adhesive surface. It was shown that

Chen et al. investigated the relationship (Chen et al., "Synthesis of polyether-based polyurethanes" and characterization and permeability (Synthesis).

Characterization and Per+neation Pro parties of Polyuretha nes)”, J, Appl, Poly+n, Sci, 16.2105-211 4 (1972)).

Of particular interest is that it consists of a hard segment and a soft segment, the former being Composed of elastomers, which are block copolymers that act as physical cross-links. The purpose of this study is to investigate the transport of water and low molecular weight salts through polymeric membranes.

U.S. Pat. No. 3,804,786 to Sekmakas describes the use of hydrated cations resins, especially polyurethanes with hydroxyl groups that have tertiary amine functionality. by reaction of resinous polyepoxide with polyisocyanate ester to provide discloses a polyurethane resin prepared by These resins are used as electrodes at the cathode. Useful as a part.

U.S. Patent No. 3.826.768 to 5uzuki and 0sonol containing polyurethanes made from polyols in water under specified conditions. Possible by dispersing the isocyanate ester contained in the ester and the organic isocyanate ester. A process for preparing a urethane composition is disclosed.

No. 3,852,090 to Leonard et al. Discloses the use of urethane films for waterproofing substrates for objects.

U.S. Pat. No. 4,124,572 to Mao describes Thermoplastic polyurethane. Elastomers produced in this way is used for automobile-related products, cow ear tags, coatings, and coatings. Useful for coated fabrics.

U.S. Pat. No. 4,183,836 to Wolfe Jr. Dispersion of polyurethane and aliphatic diisocyanate ester By reacting with an active hydrogen compound, a carboxyl group and a free isocyanate group are formed. and then form a prepolymer containing a tertiary amine and The preparation of polyurethanes by dispersing them in an aqueous medium containing diamines and diamines It shows.

No. 4,190,566 to Nol et al. a substantially linear molecular structure and lateral polyalkylene oxides. The present invention relates to a nonionic hydrated polyurethane having an oxide polyether chain.

U.S. Pat. No. 4,202,880 to Fildes et al. namely, a drug and a linear and hydrophilic polyoxyalkylene-polyurethane block. A sustained release delivery method is disclosed that includes a copolymer and optionally a buffer.

A single hydrophilic soft segment is used. Only hard segments are hydrophobic It is gender.

U.S. Pat. No. 4,202,957 to Bunk et al. polyurethane resin, which is suitable for injection molding due to its greater heat resistance. Discloses elastomers based on riether.

U.S. Pat. No. 4,224,432 to Pechbold et al. Furan, alkylene oxide, organic polyisocyanate, and aliphatic polymer A polymer containing the reaction product of a polymer with a chain extender that is a polyol or a polyamine. Discloses urethane.

U.S. Pat. No. 4,367,327 to Holker et al. breathable polyurethane film for waterproofing. This poly Urethane film is a low molecular weight diiso containing a compound with two functional groups. Contains a hard segment consisting of cyanate ester and polyethylene glycol. It contains soft segments stoichiometrically. The mechanical properties of the film are , improved by crosslinking with triisocyanate ester.

U.S. Pat. No. 4,849,458 to Reed et al. Discloses a hydrophilic segmented polyurethane-urea that exhibits high tensile strength and elongation. ing. This polymer forms a transparent film that is permeable to water vapor .

Many of the materials mentioned above are segmented polyurethane elastomers. those Some have even found biomedical applications without virtually any modification. There is. However, despite their widespread use, many biological The substance has been independently developed for non-medical use. In fact, most polyurethane Tan materials were developed to meet a large number of industrial needs. most significant An example is Dupot, a polyurethane used in the fabrication of components of cardiovascular support devices. It is LYCRA 5PANDBX■ (LYCRA 5PANDBX■) from Co., Ltd. child The material was later given the trade name Biomer ■ (B[OMER@) segmented polyurethane. It was sold under

AVCOTHANE-51■ is available in two commercially available Polymers, both of which are widely used in coating textiles, include silicone and and polyurethane. Abcocein-510 is an intra-aortic balloon. It is used in biomedical devices such as

A unique improvement introduced for its biomedical applications is the highly purified starting material cleanliness for use of quality materials, filtration of produced solutions, and fabrication of surfaces in contact with blood. These are the conditions. Abcocein-610, another biomedical polyurethane ■CARDIOMAT-6100 and Angeoff Also called ANG (10FLEX), it is now known as a blood pump or and heart valves with three leaflets.

PELLETHANE, a thermoplastic substance, was initially introduced into blood vessels. It was applied to the manufacture of cannulae and later to the manufacture of catheters. this substance Initially, over their polyester-based counterparts, Developed as an extrusion molding resin that exhibits excellent stability.

Many polyurethanes and polyurethaneureas are commercially available, and some of them As we discussed in the preamble, they are all related to nutrients and cellular products. , which allows the passage of cellular waste products but is not beneficial for cell proliferation and production of cell products. cells by not allowing the passage of potentially harmful immunological or microbiological substances. Develop membranes with the permeability, strength, flexibility, and biocompatibility required for growth. Not formed.

The present invention will be better understood by the following detailed description taken in conjunction with the accompanying drawings. A more complete evaluation of the invention and its attendant benefits will be readily understood. .

Other aspects, benefits, and features of the invention will become apparent to those skilled in the art in the following discussion. It will be.

Summary of the invention The present invention provides a method for culturing living, viable cells. about 5 to 45% by weight of the cells under effective conditions for cell growth. at least one hard segment and about 95-55% by weight of an aliphatic polyol , an aliphatic polyamine, an aromatic polyamine, and a mixture thereof. A small number (one hydrophilic oligomer or one hydrophobic oligomer selected from the group) at least one soft segment comprising an aliphatic oligomer. Cultured in the presence of a nonporous, semipermeable, biocompatible film composed of a copolymer including that the film is greater than about 350 psi and about 10,000 psi tensile strength up to and an ultimate elongation of more than about 300% and up to about 1,500%. The sum of the volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment is greater than about 100% and up to about 2.000% of the volume of the dry polymer; The polymer has a water absorption capacity of greater than about 50% and up to about 95% by volume of the polymer. It is a film, and molecules with a molecular weight of about 6,000 to 600,000 can penetrate it. is virtually impermeable to cells and particulate matter, as well as nutrients and Penetrates the cells and media components through the film into the cell environment and filters the cell products. cell, characterized by being a film that penetrates out of the cell's environment through the membrane Regarding culture methods.

The present invention also describes defects in which endogenous defects result in abnormal concentrations of certain compounds in body fluids. The concentration of the compound in the body fluids of patients suffering from the disease can be determined to reduce the harmful immune response. The method essentially eliminates the presence of endogenous defect-free cells in the patient's cells. cells, at least a portion of which is a non-porous, semi-permeable material that substantially encapsulates the cells. encapsulation in a biocompatible device comprising a biocompatible film, the film comprising: 5-45% by weight of at least one hard segment and 95-55% by weight of fat. Aliphatic polyols, aliphatic polyamines, aromatic polyamines, and mixtures thereof. and at least one soft segment selected from the group consisting of a copolymer having a pressure of greater than about 350 psi and about 10.0 psi. Tensile strength up to 000 psi and greater than about 300% up to about 1.500% The volume fraction of absorbed water and the hydrophilic volume fraction of the soft segment have an ultimate elongation rate of The sum of the dry polymer volume is greater than about 100% and up to about 2.000% of the volume of the dry polymer. and is greater than about 50% and up to about 95% of the volume of the wetted polymer. It has water absorption properties and molecules with a molecular weight of about 6.000 to 600.000 can penetrate. , a film that is substantially impermeable to cells and particulate matter. characterized in that the device containing the cells is placed in a patient's body where the cells come into contact with body fluids of the patient. It involves transplanting the transplanted cells to a site within the body, and the transplanted cells are directly exposed to the compound. contact for interaction and act to regulate the concentration of the compound in body fluids The present invention relates to a method of regulation that involves keeping cells alive at the site of transplantation.

This article also refers to kits for correcting metabolic defects in patients. wherein at least a portion of the device comprises at least one hard segment of about 5-45% ment, about 95 to 55% by weight of aliphatic polyol, and aliphatic polyamine. and aromatic polyamines, and mixtures thereof. Also contains a hydrophilic oligomer, a hydrophobic oligomer, or an aliphatic oligomer. a non-porous copolymer comprising at least one soft segment of and a biocompatible implantable device comprising a semipermeable biocompatible film. and the film has a pressure greater than about 350 psi and up to about 10,000 psi. It has a tensile strength and an ultimate elongation of more than about 300% and up to about 1.500%, and is absorbent. The sum of the volume fraction of water collected and the volume fraction of hydrophilicity in the soft segment is greater than about 100% and up to about 2.000% of the volume of the combined wetted polymer has a water absorption capacity of greater than about 50% up to about 95% by volume, and has a molecular weight of about Molecules up to 6,000 to 600,000 can penetrate, but cells and particulate matter The film is characterized by being substantially impermeable to objects, including syringes. the needle and the pre-selected metabolically defect-free cells into the device. The device is then implanted into a patient suffering from a metabolic defect, allowing the cells to interact with the patient's body fluids and be able to alleviate symptoms associated with the defect A kit is provided containing instructions on how to use the kit.

The present invention has a tensile strength and an ultimate elongation rate of greater than about 300% and up to about 1.500%. at least a portion of the device is made of a biocompatible, hydrophilic, segmented polymer. Cells in the presence of a device containing a film containing a urethane block copolymer A method for culturing is provided.

In one aspect of the invention, the in vivo method comprises Applicable to cure metabolic defects that produce harmful or undesirable compounds in body fluids and when the compound comes into contact with cells for direct interaction, it is harmless or It is converted into the preferred compound and then returned to body fluids. This means that phenylketo This is true in the case of urine disease, in which genetically engineered cells are created in the child's bloodstream. Phenylalanine is a harmless product, and tyrosine or phenylpyruvin Can be converted to acid.

In another aspect, the method uses cells, including cells that produce the product. ing. An example of this type of method is high blood glucose concentrations in diabetic patients. This is a way to combat this. Insulin-producing cells are transferred once according to the present invention. Once implanted, the cells will produce insulin when activated by increased blood levels of glucose. It produces phosphorus, which penetrates into the bloodstream and is distributed to target cells, where it is absorbed by the cells. Promotes glucose uptake.

A further part of the invention provides a kit for correcting a metabolic defect in a patient. , at least a portion of the device comprises about 5-45% by weight of at least one hard segment. and about 95 to 55% by weight of an aliphatic polyol, an aliphatic polyamine, and At least one selected from the group consisting of aromatic polyamines and mixtures thereof. Contains one hydrophilic oligomer, one hydrophobic oligomer, or one aliphatic oligomer a non-porous and copolymer comprising at least one soft segment; a biocompatible device including a semipermeable biocompatible film, the film being , about 350 ps, and up to about 000 psi. Other techniques that have attempted to restore the function of defective biological systems include Production that is not where it should be, such as the administration of insulin or other hormones by law. administration of products or incomplete products and ex vi of human body fluids such as blood. VO treatment and external ex vivo to accomplish therapeutic actions on body fluids implantation of a catheter-like device that remains connected to the system through the skin; I have relied on The present invention is intended for continuous treatment of human diseases with genetic causes. , provides a novel and unobtrusive method.

In the more general method of the invention, the method comprises A method of culturing cells to grow living, viable cells. about 5 to 45% by weight of at least one hard segment under effective conditions of , about 95-55% by weight of aliphatic polyols and aliphatic polyamines as well as aromatic at least one selected from the group consisting of polyamines of the group consisting of polyamines, and mixtures thereof. Contains hydrophilic oligomers, hydrophobic oligomers, or aliphatic oligomers. A non-porous and semi-permeable copolymer comprising at least one soft segment. culturing in the presence of a biocompatible film of about 30% 50ps beam thicker (tensile strength up to about 10.0OOpsi, about 300% greater) It has an ultimate elongation rate of up to about 1.500%, and the volume fraction of absorbed water and soft segment The sum of the hydrophilic volume fraction of the ment and the hydrophilic volume fraction is greater than about 100% of the dry polymer volume. up to about 2.000% and greater than about 50% of the volume of the wetted polymer. It is a film that has a water absorption of up to 95%, and has a molecular weight of about 6.000~ Up to 600,000 molecules can penetrate, but it is not effective against cells and particulate matter. The film is qualitatively impermeable and even retains external nutrients and components of the medium. Penetrates into the environment of the cell through the film, and transports the products of the cell from inside through the film. The method includes a method characterized in that the film penetrates outside the environment of the cell.

This method of culturing viable living cells requires nutrients and substrates to be The product of the cell is passed from the medium outside the membrane or membrane into the area of the cell, and vice versa. The cells are transported from their environment into the medium on the other side of the film or membrane.

The properties of this non-porous and semi-permeable biocompatible film or membrane are This film allows molecules to pass through, but at the same time, this film is unique in that it allows cells and other microorganisms to pass through. It remains virtually impermeable to particulate matter. Therefore, the composition of the film The films are prepared by varying the They can have a predetermined cutoff molecular weight that prevents them from being transported into the environment. Ru. This film is therefore recommended by patients for complement and exogenous cell transplantation. preparations to preempt the passage of immune molecules, such as normally produced complement analogs Can be done.

Any drug that can "cure" endogenous functional defects that have biochemical causes Cells of the same type can also be used in the practice of the present invention.

Substrates, their proportions, polymers, preparation methods and films for different applications The method for preparing the properties of “Copolymers and Their Nonporous Polymers” by Kathleen White and Kathleen White. Semipermeable membranes and their use for the permeation of molecules in a defined molecular weight range SOM 20011) and that such information is Portions of the text disclosing information are incorporated into this text by reference.

The preferred polymers used in the method of the invention are based on empirical and systematic applications. The implementation of the roach determines the specific permeability and (or or) can be synthesized to have specific molecular weight blocking. this method The empirical property of is the property of the event of permeability through a dense membrane, i.e. the solubility characteristics of specific penetrating or non-penetrating substances, including their molecular size and structure; It depends on the quality. In this text, we describe the polymerization according to the invention. provides a systematic approach to producing body membranes, which can be tailored for specific applications. It can be used to adjust the properties of the membrane. This is briefly explained in the next paragraph. simply stated.

The permeation of solutes through dense polymer membranes is dependent on the diffusivity and solubility of the permeating substances during model coalescence. It is mostly determined by If the model coalescence absorbs a significant amount of solvent, Transmission is determined by the diffusivity and solubility of the penetrating substance in the model coalescence swollen with the solvent. Will.

Absorption of a solvent, such as water, by model coalescence is caused by the fact that the polymer is resistant to the solvent. Requires having some affinity. Furthermore, by definition, the solvent is the solute/ It should be able to dissolve the penetrant. Therefore, the absorption of solvent by the membrane is It is possible to make the environment inside the mold coalesce closer to a state of pure solvent than when it is in a dry state. This can increase the contribution of the solubility factor to the permeability.

In general, in addition to increasing the solubility of permeating substances in model coalescence, low molecular weight Solvents often serve as plasticizers for model coalescence. Plasticization is a plasticizer This affects the degree of dissolution of the polymer.

Furthermore, as the value of plasticizer/solvent increases, the glass transition temperature of the mixture generally will decline. The glass transition temperature decreases because the plasticizer itself enters between adjacent chains. promotes the relative movement of polymer chains by increasing the space between molecules. This suggests that progress can be made. In addition to the preamble, the plasticizer/solvent may be Due to the specific interaction between the plasticizer/solvent and the polymer-polymer phase May reduce the degree of interaction. Water that occurs in certain model combinations of the present invention The decrease in the crystallinity of soft segments due to summation is an illustration of the latter mechanism. It is.

In the case of isotropic polymeric membranes, significant solvent absorption/swelling occurs due to For example, the X-axis, Y-axis, and Z along each of the axes results in a measurable increase in the physical dimensions of the membrane. It will be. This means that the same number of polymer molecules are still contained within the larger total volume. In the swollen state, the polymer chains have increased intermolecular Gives direct evidence of distance. This increased space in the polymer chains and Facilitated motion refers to the rate of penetration by increasing the contribution of the diffusivity to the rate of penetration. can increase sex.

Therefore, absorption of solvent by model coalescence depends on both the diffusivity and solubility of a particular permeating substance. The permeability of the membrane can be increased by increasing the . Specific penetration rate One depreparation method of the present invention to obtain molecular weight blocking and/or molecular weight blocking is to modify the composition of the membrane. It is to change objects and forms. This also increases the amount of solvent absorbed. Additionally, the increased magnitude of solubility and diffusivity resulting from greater solvent absorption It will bring about additional benefits.

In some instances, it is not possible to make accurate quantitative predictions of the permeability of the resulting membrane. Although it is not always possible to We found that there are both physical and quantitative relationships. Furthermore, it is expected that The permeability of the membrane was determined by the method described by the inventors in this text. can be achieved. The property-to-structure relationships provided in this text are Synthetic steps and membranous forms interact to adjust the permeability of the Use through permeability measurements until the desired value for use is obtained. Can be done.

In the examples provided below, the osmotic material is a water-soluble polymer and the solvent is water. or a water-soluble liquid. Those skilled in the art will understand, for example, that water-insoluble Others by modifying the soft segments to promote solvent absorption Understand that similar approaches suitable for solvent/permeant systems can be applied Will.

Table 1. Relationship between properties and structure of membrane polymers ・Increase in penetration rate (10++) ・Increase in blocking molecular weight (++) ・Increase in elongation modulus (＋＋＋) ・Increase in wet strength (++) (+++) indicates a strong positive effect; (-) indicates a weak negative effect.

The hard segments of the copolymers of the present invention have a molecular weight of about 160-10.000. , more preferably a molecular weight of about 200 to 2.000. Its ingredients are also It has a preferred molecular weight as shown in Table 2 below.

Table 2. Molecular weight aliphatic isocyanate ester preferred as hard segment component 150-270 100-500 chain extender 6 (120018-500 Both hard and soft segments can be used in a wide range of molecular weights. However, Table 3 below lists some typical components of the soft segment. Typical practical and preferred molecular weight ranges are shown.

Table 3. Preferred molecular weight polytetramethyleneoxy as a component of the soft segment 1,000-9,000 500-50,000 polypropylene oxide Poly 1,000-5.000 500-50.000 polyethylene oxide Polytetramethylene soxide 1,000-2,000 500-50,000 -polyethylene oxide Amine-capped polypro 600-6.000 200-1.000,00 0 pyrene-polyethylene Polycarbonate 300-3.000 200-50,000 Amine Cap polytetra 500-2.000 20 (1-50,000 ramethyleneoxy de Silicone polyethylene oxide 500-5.000 200-1,000,00 0 sites Polybutadiene 500-3.000 200-50,000 Polyisobutylene: / 1,000-5.000 500-10,000 copolymer hard segment The content of It is a combination of a hydrophilic oligomer, a hydrophobic oligomer, and an aliphatic oligomer. It may be a combination.

In one preferred embodiment, the copolymer has about 9-30% by weight hard segments. more preferably 10 to 28% by weight of hard segments. Ru. Similarly, the typical content of soft segments is about 91-70% by weight, and It is preferably about 90 to 72% by weight.

However, other proportions of hard and soft segments are also possible in this invention. suitable for implementation.

Polymers made from this composition will have the properties listed in Table 4 below.

Table 41 Characteristics of the film of the present invention Features range Tensile strength: approx. 350 ps, greater than i, approx. 10,000 psi, elongation at break Hydrophilic soft segment greater than about 300% and up to about 1.500% wet weight More than 50% and up to about 95% water absorption only More than about 50% of wet weight to about 95% % The present invention also provides non-porous and semi-permeable materials comprising the block copolymers of the present invention. provides a material-compatible film. In a preferred embodiment, the film is a Formed from copolymers. In another preferred embodiment, the film retains coated on the body. In yet another preferred embodiment, the film is part of an integrated matrix, and the matrix is composed of the same or similar polymers. Consists of the body.

In particularly preferred embodiments, the non-porous films of the invention are flexible sheets or and in the form of hollow membranes or hollow fibers. Typically, flexible sheets are , a long retractable notebook approximately 10 to 15 inches wide and approximately 1 to 6 feet long. It is prepared as However, other dimensions can also be selected. Special What is important is the thickness of the sheet, which is approximately 5 to 100 microns. More preferably about 19 to 25 mm when used without a retainer or reinforcement. It's Kron.

This flexible sort can be obtained from the block copolymers of the present invention, as is well known in the art. method, typically by casting, more preferably by web or spouting. Prepared by casting on a pour liner. As previously shown, this composition It can be coated onto a substrate as a film. Reinforcing webs, e.g. textiles If permanently retained on the film or membrane, the film or membrane may be thinner, e.g. It can be made as thick as microns, but when used without holding The thickness can only be reduced to about 5-10 microns.

From the polymers of the present invention, knife-overs on jet paper, webs, or liners are prepared. - Knife-over-roll casting of dried film When producing membranes in the form of a film, they are coated on a continuous coating line in approximately 1 It has a slight thickness of ~100 microns.

With two forced air furnaces, e.g. 2 with a web width of up to 15 inches A 0 foot long continuous web coater can be used. one special In some embodiments, the air intake ducts are equipped with high efficiency particulate air (IIEPA) ducts. The coater can be modified for clean operation by fitting the filter. Ru. Add filtered polymer solution or filtered reactive prepolymer solution. A nitrogen-purged coater box can be used for dispensing.

However, other devices are also suitable.

All but trace amounts of casting solvents, such as formamide, are HE Can be removed by the coater's hot air oven fitted with a PA filter . After casting the membrane, the membrane and substrate are further dried to reduce the residual solvent content It is measured by chromatography and should be lower than about JOOppm. Perfect The thickness of the cast film dried is, for example, 0.0001 inch (2.5 μM). It can be measured visually using a micrometer with a sensitivity of Wear.

The membranes of the invention can be used, for example, in solutions, dispersions, 100% solids prepolymer liquids, polymer – Any form resulting from a process that utilizes a liquid that is subsequently converted to a solid, such as a melt. You can also take the form. Converted shapes can also be mold cut or heat sealed. , solvent or adhesive bonding, or some other commonly used manufacturing method. and can be further transformed. For example, in a hollow tube configuration, the membrane Generally prepared to have a diameter of about 0.5 to 10 mm, more preferably about 1 to 3 mm. , with a thickness of about 1 to 100 microns, more preferably about 19 to 25 microns. Prepare properly. Hollow membranes can be easily prepared into long rollable shapes. , cut to lengths of about 0.75 to 31 inches, more preferably about 0.5 to 6 inches. Cut into.

Any drug that can "cure" endogenous functional defects that have biochemical causes Ip cells are also suitable for use in this text. This method is suitable for many different treatments. a wide range of genetically engineered or mutated cell types It can be implemented using

One example of an application of this method is the treatment of diabetes. Another example is Phenylalani It does not produce enzymes that convert chlorine into tyrosine or phenylpyruvate, which are harmless to the human body. Application of this method for the treatment of phenylketonuria using cells capable of It is.

The methods of the invention address the microvasculature complications associated with type I diabetes and other metabolic diseases. This will reduce the incidence and severity of the disease.

The method uses a device containing appropriate cells to correct the defect in the patient and further To, (1) Provide protection for cultured cells from macrophages and immune cells, (2) maintain cells for a long period of time, (3) allow free passage of nutrients, secretagogues, and cell products; (4) allow body fluids and (5) provides a biocompatible surface for patients and tissues; (5) provides easy surgical implantation and (6) in vivo provided in this text. The equipment used in the method is easily fixed in place and easily removed when needed. Provide the performance to achieve the goal.

This method addresses allograft tissue, a major obstacle to long-term survival of cell grafts. or overcome immune rejection of xenograft tissue.

Viable foreign cells can be introduced into the human body by a variety of devices. Kaka An example of such a device is given by Robert S. Ward and Robert Kuhn. “Biocompatible, Therapeutic, Implantable device (Biocompatible Therapeutic 1m Plantable Device) J (serial number 07) /874.342), in which living, viable exogenous cells are transferred to the human body. The text paragraphs describing various devices for implantation into incorporated into the text.

One method of the invention is to perform a function that is deficient in the patient to whom the cells are transplanted. in vivo of a device containing live, viable cells capable of It depends on the port that can be used. For example, the method of the present invention may provide diabetic patients with The surin-producing cells are made into a biocompatible implantable device, at least in part contains a film or membrane as mentioned in this text, surrounds the cell, and It is carried out by transplanting cells into a device that isolates them within the human body. Ru. Therefore, this method involves isolating and growing cells in a certain location in the human body. can be carried out in which cells are exposed to human body fluids such as blood in direct contact with They are placed in close contact for continued interaction. For diabetics, insulin The producing cells are transplanted into or around blood vessels, the intestinal lumen, or other parts of the body. It interacts with the patient's blood. like insulin produced by cells proteins or hormones are transported through nonporous and semipermeable films or membranes. It can penetrate the outside of the device and enter the bloodstream, from where it can reach target cells.

Any glucose present in the blood is utilized and metabolized there, so the human body will be able to enter that cell. Therefore, the number of patients affected by diabetes is The undesirably high blood concentration of lucose decreases.

In a preferred embodiment, the living viable cells produce insulin. In addition, hormone production can be increased in response to blood glucose levels. can be adjusted. However, other cells can also be used .

The use of nonporous, self-retaining semipermeable membranes, such as those used in this text, production of nutrients, secretagogues, and cells while maintaining immune isolation for the cells. Let things pass. The method uses durable, dense, water-swollen membranes or Although the system uses permeable to products produced by

In a particular embodiment of the in vitro method of the invention, the cells produce Contains cells that secrete substances. Its products are thyroid hormone and pancreatic hormone. , hormones like other hormones, or groups produced following modification of genes. It is possible that the protein is a modified protein.

In another preferred embodiment, the cells include insulin producing cells. moreover In a preferred embodiment, the production of insulin by the cells is based on glucose in the medium. It can be adjusted by changing the concentration.

Examples of cells that can be used to produce insulin in vitro are: , unmodified mammalian islet of Langerhans cells and insulin-producing prokaryotes. recombinant cells or eukaryotic cells, resulting from homologous recombination or mutation; A eukaryotic insulin-producing cell that is regulated by glucose.

Generally, to carry out the methods of the invention, cells are selected from prokaryotic and eukaryotic cells. You can choose. Among these, immortalized cells and living cells Cells can be selected from the group consisting of cells and primary cultured cells. these are, Methods well known in the art that do not require further discussion in this text, or or from commercially available sources. For example, Gazdar, A, D.

and Chtck, W, 1. , and Die, H., et al. Continuously cloned insulin and insulin Matostatin-secreting cell line (Continuous, C1ona1. In5u lin and Somatostatin-3ecreting Ce1l L ines Established From a Transplantable e Rat l5let Ce1l Tumor) J, Proc, Natl, Acad, Sci.

USA, 77, 3519-3523 (1980), and 5anter re, R, F, and Cook.

“Simian Virus-40” by R, A, Cr1ses, RoM, D et al. in a cloned cell line of transformed hamster pancreatic beta cells. Insulin synthesis in C1onal Ce1l Lineof 51m1an Virus 40-Transfo rmed tamster Pancreatic Beta Ce1ls) J.A. Proc, Natl, Acad, Sci, USA, 78.4339-4343 (1981).

In particularly preferred embodiments of the method of the invention, the apparatus used herein is substantially thin. Enclose the cells.

In the most preferred embodiment of the method of the invention, the cells encapsulated within the device are immobilized in a hydrogel containing more than % water.

The hydrogel maintains a uniform distribution of cells within the device. This means that intracellular products , ensuring optimal diffusion out of the implanted device.

Suspending cells within a hydrogel was also found to increase cell viability. has been done.

When the method of the invention is carried out in vivo, the method further comprises: It involves the implantation of a device containing cells into a patient's body, where the culture step In vivo, there is virtually no harmful immune response. It is done.

As already indicated, the film or membrane used in this method can be the passage of immune molecules into the cellular environment that can be triggered by the presence of sexually active cells. impede

One method of the invention is that the cells perform a function that is deficient in the patient to whom they are transplanted. in vivo of a device containing living, viable cells capable of is dependent on porting. For example, the methods of the present invention may provide diabetic patients with: The device must be biocompatible, implantable, and at least a portion of the device must meet the requirements of this text. The device surrounds the cells and protects them. Insulin-producing cells in a device that is installed in isolation within the human body. It can be performed by transplantation. Therefore, this method does not require isolation within the human body. This can be done by growing cells in blood, where the cells are It is placed in direct and continuous interaction with body fluids such as Diabetes In the case of the patient, the insulin-producing cells are inter alia vascular, intestinal, or They are implanted in areas such as around organs and are designed to interact with the patient's blood. Ru. Proteins or proteins, such as insulin, produced by cells Lumon permeates out of the device through a non-porous but semi-permeable film or membrane and It enters the fluid and from there can reach the target cells. char that exists in the blood Glucose can also enter the cells of the human body for utilization and metabolism there. It will be possible. Therefore, the unfavorable glucose levels suffered by diabetes The high blood concentration gradually decreases.

In a preferred embodiment, the living viable cells produce insulin. Not only can that hormone production also affect the glucose concentration in the blood. It can be adjusted accordingly. This suggests that recombinant insulin-producing mammals cells and their analogs and their ability to express proteins such as insulin. This is true in particular uses of retroviral vectors. However, others cells can also be used.

According to recent estimates, approximately 400,000 Americans are responsible for producing insulin. and/or insulin-dependent type I sugars characterized by defective release. I am suffering from urinary disease.

The method of the invention passes molecules up to a predetermined molecular weight, but allows cells and other particulates to pass through. A device having at least one permeable region that does not allow the passage of child substances, the device comprising: , of the glucose concentration in the region of the device in contact with the semipermeable film or membrane. By releasing insulin in response to changes, it can provide almost normal blood sugar levels. An “artificial device” containing a biocompatible device containing insulin-producing cells. It is useful in the treatment of diabetes by transplantation of the "pancreas."

In particularly preferred embodiments, the cells to be transplanted are the patient's own defective cells. They are genetically engineered to overcome defects prior to transplantation. Ru.

The method of the invention involves implanting a cell-filled device at multiple sites within a patient's body. It can be implemented by Examples of this site include, among other things, the interior and inside and around the large apparatus, inside the vagina, inside the skin, under the skin, inside the sinuses, and around the abdomen. Within the membrane tissue and within the blood vessels.

When carrying out this method by subcutaneously implanting this device, the cell The size and nature of the products produced or their presence in body fluids and exposure to cells. what needs to be done to affect the release of a product (or products) by the cell Depending on the size and nature of the tumor, it can be implanted anywhere in the patient's body.

One example is the area adjacent to the site where lymph returns to the circulation. It's growth hormone. Hormones or other protein products may be prepared according to the methods of the present invention. For example, this can be done by implanting the device subcutaneously under the arm. For other transplants Areas include within the subepithelial fat pad and within the intestinal tract and its analogs.

In a preferred embodiment, the method of the invention comprises at least one film comprising a film of the invention. A device such as a catheter, which has a section, is moved into a patient's blood vessel, such as an artery. It is carried out by planting.

An example of insulin production that is available but not regulated is pancreatic cancer. tumor cells, such as hamster insulinoma and rat insulinoma It's Ma. Cell lines such as these can be iteratively selected and/or mutated. and transform them into glucose-regulatable insulinomas. .

The methods of the invention can prevent metabolic defects in a subject in a number of ways. . One example is when the subject's cells have sufficient amounts of certain compounds, such as hormones, such as insulin, or thyroxy This is an example of a protein that does not produce coagulation factors or coagulation factors. A second example is when a patient changes metabolites. Undesirable levels of metabolites can accumulate in the circulation due to inability to That is. Another example is when the medium is in direct interactive contact with the cells. Harmful or desirable components that are transformed into harmless or desirable components that are returned to the earth. It is made up of ingredients that are not present. Thus, for example, the enzymes present in this cell metabolizes the media components and renders them harmless.

In another aspect, the invention provides methods for producing certain compounds in the substantial absence of adverse immunological reactions. said in the body fluids of a subject suffering from an endogenous defect that causes abnormal levels of the substance in the body fluids. Provides a method for regulating the levels of a compound, which method includes the steps: Cells lacking the endogenous defect of the patient's cells are surrounded in a device, where the At least one portion of the device is a non-porous, semi-permeable material substantially surrounding said cells. 5 to 45% by weight of the film. and about 95 to 55% by weight of at least one soft segment. The soft segment is made of a copolymer consisting of a fatty segment. consisting of family polyols, aliphatic and aromatic polyamines and mixtures thereof at least one hydrophilic, hydrophobic or amphipathic oligomer selected from the group the film is greater than about 350 psi and about 10,000 psi; Tensile strength up to, greater than about 300% and ultimate elongation up to about 1.500% The sum of the volume fraction of absorbed water and the volume fraction of hydrophilicity of the soft segment is Exceeding about 100% and up to about 2.000% of the volume of dry polymer and wet poly water absorption of greater than about 50% and up to about 95% of the volume of the polymer; The film is transparent to molecules with a molecular weight of about 6.000 to 600.000. permeable and substantially impermeable to cells and certain substances; The device consisting of cells is placed in a region of the subject's body where the cells are in contact with body fluids of the subject. and wherein said cells are in direct interactive contact with said compound. to keep the cells alive at the site of implantation and to regulate their levels in body fluids. Let it work: Consisting of

In the most preferred embodiment of this method, the body fluid consists of blood and the endogenous defect is A level that is substantially higher than the normal level of the compound in the blood. in other aspects , the endogenous defect is a level that is substantially lower than the normal level of said compound. . These are hormonal imbalances, whether there is an excess of hormones or a defect This is the case. An endogenous defect causes substantially higher than normal levels of certain compounds in the blood. An example is when the level of glucose in the blood is low due to lack of insulin. This is the case when it increases. Substantially higher than normal levels of compounds with endogenous defects If the levels are low, as in the case of thyroxine, growth hormone, etc. This is the case when there is a deficiency of hormones that need to be renewed.

Preferred cell types for the practice of this method are The compensation of enzyme action is inversely adjustable by the level of compounds present in it. It is Ipu.

As in the case of in vitro methods, examples of cells suitable for the practice of the invention include permanent cells. cleft-competent cells, living tissue cells, and transiently cultured cells. insulin cells deficient in cells, e.g. native islets of Langerhans, or transgenic techniques. For a method of treating glucose-regulated insulin-producing eukaryotic cells resulting from In this case, homologous recombination or mutation can be used. These details vesicles are known in the art and may be the same or wild species from different species. Produced by cloning viable genes or using transgenic technology can do. Furthermore, cells can be mutated by means other than cloning. can be produced.

Examples of these are radiation, mutagenesis and selection.

In one aspect of the invention, the method is applied to diabetic patients and the cells are Consists of glucose-regulated, insulin-producing cells.

The cells can be the subject's own insulin-deficient cells, and they isolated from the body and then genetically engineered or otherwise mutated to produce glucose-regulatable, insulin-producing cells and then convert them into This same subject can be transplanted.

Typically, the cells used in cases of diabetes are glucose-regulatable, i. The mammalian islets of Langerhans contain insulin-producing cells.

Although the invention has been generally described, it can be understood by reference to specific embodiments. It will be better understood. These examples are included herein for illustrative purposes only. and are not intended to limit the invention or any aspect thereof unless otherwise specified. .

Example In order to act as an implantable immunological barrier, a suitable membrane material must meet a number of criteria. Must be satisfied: (1) the material has sufficient strength and at least must be sufficiently resistant to biodegradation to function well for one year .

(2) The material is sensitive to and surrounding the living cells located within it. It must be non-toxic to the surrounding tissue.

(3) The material transports sufficient nutrients and oxygen through its membrane to support the cells within it. must support the maintenance and/or proliferation of

(4) The material supports physiological signals in both directions, e.g., glucose that feeds islet cells. and the cell products in question, -1 e.g. insulin produced by islets. It must be possible to transport it quickly.

(5) The membrane protects the cells inside it from the cell's immune system and thus preventing the passage of immunoglobulins, e.g. IgG, and thus complement-mediated cells. It must be ensured that lysis of the cells cannot occur.

Studies on the transport of molecules across PTG membranes The experiments described below demonstrate that the present invention It was designed to test the transport of methylcellulose across a urethane membrane. cross the membrane The diffusion of glucose, insulin, and serum components in various chemicals was examined.

Example 1: Transport of glucose across the membrane To examine the diffusion of substances across the membrane Two methods were tested.

In the first method, co-pending, co-filed U.S. Patent Application No. 07/874, 33 No. 6 (inventors: Robert S., Ward and Kathleen Whi te) (incorporated herein by reference). , sheets of membrane were cast from the copolymer. The membranes were then each placed in a volume of 5 ml. It was placed in a two-compartment diffusion chamber to separate the two compartments. phosphate slow A buffer solution (PBS) is placed on one side and a test solution containing the test compound is placed on the other side. I put it on the side of

For this study of glucose diffusion experiments, 10 mg/m of glucose in PBS l solution was used.

Diffusion of glucose across the membrane occurred within a short time. Glucose rapidly crosses the membrane and equilibrium was reached within about 2 hours. Sufficient glucose for up to 0.5 hours The membrane being tested is considered suitable if it traverses the membrane within a period of . Membrane tested allowed passage of more than 1500 μg glucose/ml in the first 30 minutes.

This is a fast transport of glucose across the membrane.

Example 2: Transport of proteins through membrane tubes Membrane tubes instead of membrane tubes, simultaneously Pending, co-filed U.S. Patent Application No. 07/874.336 (Inventor: Robe rt S, Ward and Kathleen A.

White) (add here by citation) did. In this tube, add the test sample containing a trace amount of protein labeled with 125-1. It was filled with a 1 mg/m+ solution of protein. Both ends of this tube are sealed using a heat sealing device. After sealing, it was centrifuged in a 1.5 ml Eppendorf. Placed in 1 ml of PBS in a tube. Aliquots of PBS at various times and the amount of radioactivity in each sample was determined. 20 equal volumes for each sample % TCA, mix the sample, and collect the precipitate as a pellet by centrifugation. Ta. The radioactivity present in the pellet was determined. TCA precipitable, therefore TCA precipitable Only protein bound counts were used for diffusion rate calculations. at a given time The amount of protein that crosses a known cross-sectional area of the membrane is the same as that of the protein placed in the tube. can be calculated based on the specific activity. The permeability coefficient p is shown in Table 5. with understanding As can be seen, transmittance is not simply a function of molecular size.

Table 5 = Permeability of membranes used in in vivo studies: tubular and islet permeable substances Molecular weight (dalton) Pg-Cm/Cm2 days/g/mi glucose 180 965 0 Insulin 6000 2300 Glucagon 3500 400 Angiotensin I 1400 90 Aprotinin 6500 25 Albumin 60000 10 ℃gG 150000 0 Example 3: Insulin transport across the membrane tube For this experiment, Example 2 was used. A small amount of +25-I-labeled insulin was added to the tube of the membrane used. Loaded with 0.5 mg/ml of insulin in FBS containing phosphorus. cut the tube 1. In a 5 ml centrifuge tube containing 1. On+l PBS. I put it in. Samples were collected and analyzed as in Example 2. I cross the membrane. The amount of insulin was increased within the first 30 minutes at a rate of 2-1 lxlO-10 mol/Cm2. The amount is calculated based on its original specific activity as a function of time. Equilibration in approximately 60-120 minutes, as evidenced by flattening of the insulin curve. reached. Thus, the membrane of the invention is shown to be suitable for the transport of insulin. It was. This membrane also exhibited excellent strength and handling properties.

In summary, the membranes of the present invention support molecules the size of both glucose and insulin. certified as being suitable for applications where the The diffusion characteristics were shown to be clear. In this case, both glucose and insulin glucose and insulin are transported across the membrane at a reasonable rate. Both reach equilibrium across the membrane in 1-2 hours.

Cell source: In tubes or insulets constructed of dense membrane material Research aimed at testing the ability of cells to survive and function in was carried out using isolated cell lines or isolated porcine islets. ATC two cell lines and was used for these studies. These are: Established lymphoblastoid from RAJI (Burkitt's lymphoma) human cells. These cells are grown in suspension culture and their maintenance and Absolutely dependent on the presence of serum for growth. These cells were transferred to 10% of fetal calves. (routinely cultured in RPMI 1640 medium supplemented with sheep serum (FBS)), and MOPC-3IC cells (these are mouse plasma cells grown in suspension culture). It is an IgG that secretes cytomas. RPMI supplemented with 10% FBS 1640 medium). Crowther et al. (Cr other, N. J., Gotfredson, C. FoMoody. A, J., and Grenel, C. “Porcine islet isolation, cell composition and and secretion response (PorcineIslet l5olation, Cellu lar Composition and 5ecretary Re5pon se)”.

Horsn, Mthabol, Res. 21: 590-595. (198 9) and Ricordi et al., (Ricordi, C., Finke, B. 1L, and Lacy, P,E, “Bloc Isolation of Islets from the Adult Porcine Pancreas. Method (A Method for The Mass l5olationof l5lets fro II the Adult Pig Pancreas) ”.Diabels 35: 649-653, (1986)) It was prepared by the method. After isolation, the islets were soaked in 10% horse serum and 11.5+oM glue. They were routinely cultured in vitro in RPMI containing lucose. Cell lines were kept at 37°C. The islets were cultured at room temperature. All cultures were under 5% Cot. carried out.

In vivo culture research: ■、Toxicity research: RAJ or MOPC cells (3X10'/well) were placed in a 6-well tissue culture plate. into each well of the plate. Each well contains 5 ml of 10% PBS RPMI 1640 medium was added. Either nothing is placed in each well (control) or or a hollow tube constructed from 10-inch HMU22866 (test), or Insert a rod of SP negative control plastic reference standard containing the same surface area. It was. This latter substance is a well-recognized non-toxic substance. After vaccination 1. 5.　 On days 7 and 16, the number of cells in each well was determined. Number of cells in culture A 7 day plot was generated and used to determine the growth rate of the cells. The difference will eventually be ) 1M022866 membrane material has no effect on the cells being cultured. The substance is proven to be non-toxic. Islets do not grow in culture. Kojima for 2 weeks A decrease in the number of islets or insulin secretion when cultured for a period of was not observed in wells containing membranes compared to those without. this As clearly demonstrated by the studies of It is not toxic to either.

■、Cell survival: Cells that survive and proliferate within the membrane when the only source of nutrients is the medium outside the membrane apparatus The study was designed to examine the capacity of the system and islets. for these studies , both the heat-sealed tube and the insulet J device. used. Cells were placed into the device as follows. Fill the tube with medium containing cells The ends were then sealed after removing all the air. When experiments require short tubes If necessary, you can simply heat-seal long tubes at appropriate intervals to make them smaller. It was possible to compartmentalize it into segments. When using in-thread, two 2 Insert 6 gauge needles through the thick membrane "O-ring" at approximately 30° angles to each other. Ta. Using a syringe, place the cell suspension into the in-thread. The second needle is a device to allow removal of all air from the The tube segment or insula is then Place the plate into a chamber in a 6-well plate and add 5 ml of medium. did. Medium was replaced weekly. Cell survival and proliferation were visually inspected (these The tube is optically clear). Although this method does not give quantitative results, cell proliferation it is obvious. The number of cells increased significantly over time. By 6 weeks, the tubes are almost full of cells. It was almost confluent.

The cells continued to proliferate for about 4 months (the longest period we have tested). ). At the end of this period, these tubes contain an almost solid mass of cells and become thin A "bulging" phenomenon was observed in the cluster of cells.

In these studies, the only nutrients required for growth and maintenance are It was supplied by diffusion. MOPC cells synthesize and secrete immunoglobulins . At both times we can detect immunoglobulins in the outer medium. I couldn't do it. This means that immunoglobulins do not cross the membrane barrier. showed, corroborating earlier research. It is possible to detect an increase in IgG concentration within the device. came.

Islets do not grow in culture. Therefore, the islet preparation placed inside the membrane device does not increase the number of cells in the cell. However, continuous monitoring of islet survival can do. These experiments were carried out using a living organism that supplied fresh medium only to the outside of the tube. We established that islets can survive for at least 6 months in external culture. external The culture medium is assayed for the presence of insulin and in such an assay It has been discovered that insulin secretion and diffusion occur throughout these periods. Ta.

Example 4: Cell line research A 10 inch long membrane tube was prepared as described in Example 1 and autoclaved. and filled with 7×10” Raji or MOPC cells. The membrane was then A 1 inch intravenous hot water treatment was applied. Swiss - five 1 inch segments implanted intraperitoneally into Webster or nude mice. At 2, 4, and 12 weeks 3 mice were sacrificed, their grafts were removed, and intramembranous viability was determined. It was determined.

Upon explantation, the membrane appeared to be intact. Structural changes can be detected by regular examination or microscopic examination. It could not be observed by any speculum examination. By 4 weeks, the membranes are large It was completely surrounded by a well-vascularized fat pad. surrounding organization No abnormality was observed in the. Similarly, in mice, physiological changes occur following transplantation. No results were observed. Open the explanted membrane with a scalpel and remove the endogenous factors contained inside. Sex-defective cells were tested for viability. Cells recovered from within the membrane are treated with Tribunbu Due to its ability to eliminate roe and to grow under in vitro culture conditions after harvest, was determined to be viable. Cells in the tube before implantation and after tube removal When examined, the cells are not only alive but also proliferating extensively. It was revealed that. This includes immunocompromised (nude) mice and normal mice. The membrane successfully protects the cell line from the host mouse's immune system. proved that. Furthermore, if Raji cells escape from the membrane apparatus, lymphoma observed in nude mouse studies. This was not observed, so the membrane It not only protects the internal cells from the host immune system, but also protects the host from its internal It can be concluded that it protects from cells.

Example 5: Kojima system RPMI medium or RP+III medium containing Matrigel Underground Pig Islands (15,000, 40,000, or 50 for each device) ．． 000) into a membrane tube or in-thread as described in Example 1. Ta.

The device was then implanted intraperitoneally or subcutaneously into normal BALB/C mice. . After 3 months, the devices were removed and tested for islet cell viability. fruit It was not possible to determine the number of islet cells present at the end of the experiment. However, I We were able to demonstrate the presence of living islet cells within the device. moreover, When such a device is placed into an in vitro culture, insulin can be measured in the culture medium. I was able to This is because the islet cells survive and function in the animal after a period of 3 months. This could only happen if it continued to function. These studies were conducted in normal mice. Because these studies were carried out in proved that.

Example 6: Treatment of diabetic mice To demonstrate the ability of porcine islets to function within membranes in vivo, diabetic mice were implanted with a device prepared as described in Example 1. Diabetes is BALB/ C mice were induced by injection of streptocytosine (STZ). Animal's Fasting blood glucose level greater than 300 mg/dl for 3 consecutive measurements Animals were considered diabetic when they were older. Each so and ooo pig Devices containing islets were implanted intraperitoneally or subcutaneously into diabetic mice.

When fasting blood glucose levels in these animals returned to below 200 mg/d1, and the animal was considered cured. Control diabetic animals received a diet that did not contain islets. The location was transplanted. Blood glucose levels were monitored weekly.

Within two weeks after transplantation, animals began to decrease blood glucose levels.

The figure shows typical results for animals implanted at 4 or 7 weeks. At time 0 Represents fasting blood glucose levels. As you can understand, the "control J" for diabetes is 3 Those with fasting blood glucose levels greater than 00 mg/di is approximately 100 mg/dI for normal or transplanted mice. Pig inside the device The presence of islets clearly regulates blood glucose levels in transplanted diabetic mice. Ta. Glucose tolerance test tests against the physiological stress of increasing glucose levels more closely demonstrate the animal's ability to respond to These are 3mg of gram at time O. Lucose levels are performed by intraperitoneal injection into mice. 30.60 ．． Blood glucose levels were determined at 90, 120, and 180 minutes.

As can be seen in Figure 1, normal mice have slightly lower glucose levels. rise and return to normal by 120 minutes post-injection. In contrast, diabetic mice increases and shows no tendency to return to pre-opposition during the course of the experiment. Contains pig islets Diabetic animals implanted with a device that Showing a similar initial rise. However, in contrast, these animals During this period, glucose levels return to more normal levels.

This experiment clearly demonstrates that porcine islets are present in the membrane apparatus in normal mice. can survive, continue to function, and improve their diabetic condition. true In this sense, this device functions as an artificial pancreas.

The membrane materials used in the practice of this invention are non-toxic to cells and serum dependent. In persistent cell lines, long-term It has been demonstrated that they can survive and proliferate within the membrane for a period of time. two serums Dependent cell lines, Raji and MOPC-31C, grow inside the intermembrane until June I let it happen. Not only did they survive, but they also grew normally. These details Devices containing cells were also placed into normal mice. These cells are 4 months old survived and multiplied longer. They apparently protect cells from the host immune system Ta. In addition, they demonstrated that the membrane protected the host from internal cells. The islets are cultured inside a device made of this membrane material. and the islets survived in vitro for a period longer than 6 months. throughout this period , they normally release insulin primarily and in response to secretagogue opposition. continued to function.

Optimal diffusion of intracellular products from within the membrane graft to the exterior depends, in part, on the uniformity of cells within the membrane. It was discovered that this can be achieved by a uniform distribution. Using hydrophilic natural polymers It is known in the art to encapsulate mammalian cells. in general , mammalian cells, for example, by cross-linking using alginate and polylysine. and are becoming microencapsulated. using certain cross-linked hydrophilic polymers. One drawback to microencapsulation using microencapsulation is that the cross-linked polymer There is a tendency to

Alginates and some vegetable gums are also suitable as encapsulating agents. It is known that it is possible to form a strong water gel. Gel with moisture content higher than 99.5% has been manufactured. The high moisture content of these gels makes them ideal for hybrid prostheses. The present invention provides a gel that has high permeability to various permeable substances that are important for the purpose of use. very Even at high moisture levels, these gels are viscous and thus dispersed within the gel. Cells can be fixed. However, some acrylic hydrogen One drawback to the use of gels, especially when microencapsulating gels directly into tissue, is that Their biodegradability when transplanted as

By placing these high water hydrogels within the membrane of the present invention, It was discovered that the gel does not biodegrade rapidly once implanted. thus , a preferred embodiment of the invention provides ≧about 35% water inside the dense semi-permeable polymeric membrane. , preferably using a hydrophilic gel with a water content of 290%. The dense of the present invention The membrane provides immunoisolation and the necessary selective permeability. while also providing an absolute barrier to cells. water swelling game This method fixes the cell position within a dense membrane (e.g., in the form of a hollow fiber) and punches it. (bunching). Furthermore, the high water content of water-swollen gels provide low resistance to permeation of species that leave or reach the cells Ru.

Furthermore, cells fixed within hydrogels exhibit improved survival within implantable devices. observed to exhibit ability.

The dense membrane also provides a biostable protective layer against water-swollen gels, thus prevent or reduce the biodegradability of

Suitable membranes include, but are not limited to: ≧about 20% water, preferably preferably ≧about 50% water, but less equilibrium water than a water-swollen gel. Hydrophilic or amphiphilic polyurethane. Moisture was measured at 37°C and Expressed as a percentage of the wet weight of the sample after a large weight gain has occurred. This value is Weigh the wet polymer and again store it under standard laboratory conditions, e.g., about 23°C and by weighing after drying the polymer at equilibrium at 50% relative humidity. to be determined.

Suitable water-swellable gels include alginates (e.g., sodium alginate, alginate ammonia phosphate, potassium alginate, propylene glycol alginate, lugin), guar gum, dragacant gum, locust bean gum, mentocel, kisa tantan rubber, polyethylene oxide, polyethylene oxide, dextran, aluminum Acrylate, methacrylate, polyvinyl alcohol, polyvinylpyrrolidone and and a combination of the above.

Those rubbers and resins that can be “crosslinked” can be used crosslinked or in linear form. can do. For example, sodium alginate [use as is] or, alternatively, can be converted to its insoluble calcium form.

The most preferred hydrogel is calcium alginate with a water content greater than about 90%. It is.

For all of the embodiments of the present invention, the hydrogel is comprised of greater than about 35% water. It is preferred to suspend the cells using Hydrogel immobilizes cells within the membrane, This ensures uniform cell distribution within the membrane.

The most preferred embodiment of the present invention is that the hollow fibers are filled with calcium alginate. It consists of a dense membrane implant in the form of hollow fibers with a water content of greater than about 90%.

However, dense membrane geometries encompass sheets, larger diameter tubes, etc. It will be understood that it can be of any form.

Although the present invention has been fully described, it will be apparent to those skilled in the art that the spirit and spirit of the invention have been fully described. Variations and modifications are possible without departing from the scope and scope.

and-mouth 4rβ medieval times International Survey 11 Everyone PCT/US 93103843 Continuation of front page (51) Int, C1,6 identification symbol Internal reference number C12N 5/10 (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN.

TD, TG), AT, AU, BB, BG, BR, CA.

CH, DE, DK, ES, FI, GB, HU, JP, KP, KR, KZ, L K, LU, MG, MN, MW, NL, No, PL, RO, RU , SD, SE, SK, UA.

N FI (72) Inventor Ward, Robert Niss.

United States of America, California 94549゜Lough Eyet, Lowell Lane East 323 (72) Inventor Monaghan, John United States, California 94563° Orinda, Taraburo Dry 19 (72) Inventor Kuhn, Robert United States, California 94941° Mill Valley, Millside Ray N 55

Claims (50)

[Claims] 1. live, viable cells under conditions effective for continued cell survival Consists of culturing in the presence of a non-porous, semi-permeable, biocompatible film. , the film comprises about 5-45% by weight of at least one hard segment, and From a copolymer consisting of about 95-55% by weight of at least one soft segment The soft segment is composed of aliphatic polyols, aliphatic and aromatic at least one selected from the group consisting of polyamines and mixtures thereof. consisting of hydrophilic, hydrophobic or amphiphilic oligomers; the film has about 350 tensile strength greater than psi and up to about 10,000 psi, greater than about 300% The ultimate elongation rate up to about 1,500% and the volume fraction of absorbed water and The sum of the hydrophilic volume fractions of the soft segment and the soft segment is approximately 100% of the volume of the dry polymer. and up to about 2,000% and more than about 50% of the wet polymer volume and about 9 has a water absorption rate of up to greater than 5%, and the film has a water absorption rate of about 6,000 to Permeable to molecules with molecular weights up to 600,000 and highly sensitive to cells and certain substantially impermeable to substances; said film is in the presence of a nutrient medium; , said medium consists of nutritional substances and components of the medium, said nutritional substances and components of the medium The cells enter the environment of the cell through the film, and the cell products are released from within. permeating through the film to the outside of the cell ring; A method of culturing living, viable cells. 2. 2. The method of claim 1, wherein the cells consist of cells that secrete the product. 3. 3. The method of claim 2, wherein the cells consist of cells that secrete insulin. 4. Insulin production by cells is regulated by changes in glucose levels in the culture medium A method according to claim 3, wherein the method is possible. 5. Native mammalian islets of Langerhans cells, insulin-producing recombinant prokaryotes Glucose-regulated proteins resulting from biological cells and homologous recombination or mutations 3. selected from the group consisting of insulin-producing eukaryotic cells. Method. 6. the claimed cell is selected from the group consisting of prokaryotic cells and eukaryotic cells; The method described in Scope 1. 7. Cells endowed with permanent dividing capacity, cells of living tissues and cells of primary culture 7. The method of claim 6, wherein the method is selected from the group consisting of: 8. 6. The method of claim 1, wherein the device substantially surrounds the cell. 9. When the medium is in direct interactive contact with cells, there is no need to return it to the medium. from a harmful or undesirable component that is transformed into a harmful or undesirable component The method according to claim 1. 10. further comprising implanting the device comprising the cells into the subject's body; Said culturing step is carried out in the substantial absence of deleterious immunological responses in vivo. , The method according to claim 8. 11. If the cells are the subject's own defective cells and the cells are genetically modified prior to transplantation. 11. The method of claim 10, wherein the method is operated to overcome deficiencies. 12. Devices consisting of cells can be inserted into the dermis, subcutaneously, intracavitally, intraperitoneally, intravascularly, in organs, or is implanted nearby, in or near the omental sac, or intravaginally. How to put it on. 13. The body is exposed to abnormal levels of certain compounds in the virtual absence of harmful immunological responses. Regulating the levels of said compounds in the body fluids of subjects suffering from endogenous defects occurring in the fluids A method for treating cells lacking an endogenous defect in a patient's cells, comprising: enclosed within an implantable device, wherein at least one portion of said device is surrounded by said device; Consists of a non-porous, semi-permeable, biocompatible film that substantially surrounds the cells. , the film comprises about 5-45% by weight of at least one hard segment, and From a copolymer consisting of about 95-55% by weight of at least one soft segment The soft segment is composed of aliphatic polyols, aliphatic and aromatic at least one selected from the group consisting of polyamines and mixtures thereof. consisting of hydrophilic, hydrophobic or amphiphilic oligomers; the film has about 350 tensile strength greater than psi and up to about 10,000 psi, greater than about 300% The ultimate elongation and absorbed water volume fraction up to approximately 1,500% and The total hydrophilic volume fraction of the soft segments is approximately 100% of the volume of the dry polymer. and up to about 2,000% and about 50% and about 95% of the wet polymer volume %, and said film has a water absorption rate of about 6.000-6. Permeable to molecules with a molecular weight of up to virtually impermeable to quality; The device comprising the cells is placed in the body of a subject in which the cells are in contact with body fluids of the subject. and said cells are in direct interactive contact with said compound. keep the cells alive at the transplant site and regulate their levels in body fluids. make it act like; The method comprising: 14. The body fluid consists of blood, and the intrinsic defect is due to the normality of the compounds in the blood. 14. The method of claim 13, wherein the level is substantially higher than the level of. 15. The effect of cells on the level of said compound in body fluids is reversed by the level of the compound. 13. The method of claim 12, which is adjustable. 16. Cells endowed with permanent dividing capacity, cells of living tissues, and primary cultures 14. The method of claim 13, wherein the cell is selected from the group consisting of. 17. If the patient is diabetic and the cells are glucose regulated and insulin producing 14. The method of claim 13, comprising living cells. 18. Cells were isolated from native mammalian islets of Langerhans, and from homologous recombination or is a group of glucose-regulated, insulin-producing eukaryotic cells that result from mutations. 18. The method of claim 17, wherein the method is selected from: 19. The cells are the subject's own insulin-deficient cells, and the cells are the subject's own insulin-deficient cells. and genetically engineered to produce glucose-regulated insulin before transplantation. 18. The method according to claim 17, wherein 20. Harmless return to body fluids when they are in direct interactive contact with cells Are harmful or undesirable compounds transformed into desirable or desired compounds? 14. The method according to claim 13, comprising: 21. 14. The method of claim 13, wherein the cells consist of product producing cells. 22. a biodegradable, implantable device, wherein at least one portion of said device is consisting of a non-porous, semi-permeable, biocompatible film, said film having a 45% by weight of at least one hard segment and about 95-55% by weight of at least one hard segment. formed from a copolymer consisting of at least one soft segment; The quality segment consists of aliphatic polyols, aliphatic and aromatic polyamines and At least one hydrophilic, hydrophobic, or both hydrophilic and hydrophobic compound selected from the group consisting of a mixture of comprising a hydrophilic oligomer; the film is greater than about 350 psi and about Tensile strength up to 10,000 psi, greater than about 300% and about 1,500% Ultimate elongation and absorbed water volume fraction and hydrophilicity of the soft segment up to The sum of the volume fractions of up to about 100% and about 2,000% of the dry polymer volume and exceeds about 50% and up to about 95% of the volume of the wet polymer. and the film has an absorption rate of about 6,000 to 600,000 molecules. permeable to large amounts of molecules and virtually impermeable to cells and certain substances hyperactive; syringe; needle; and Preselected cells lacking the metabolic defect are inserted into the device and the metabolic defect is detected. The device is implanted into the body of a subject suffering from the disease, and the cells are thus absorbed into the subject's body fluids. to be able to interact with and alleviate the symptoms associated with said defect. Instructions for use of the kit for; A kit for correcting a metabolic defect in a subject, comprising: 23. The insulin production kit according to claim 22, further comprising a cell culture medium. to. 24. said living viable cells consist of more than about 35% water; 2. The method of claim 1, wherein the cells are suspended in a gel. 25. 25. The method of claim 24, wherein the hydrogel is an alginate. 26. Claim 24, wherein the hydrogel is polyethylene glycol. the method of. 27. The method according to claim 24, wherein the cells consist of cells that secrete products. Law. 28. 28. The method of claim 27, wherein said cells consist of insulin producing cells. . 29. Insulin production by cells is regulated by changes in glucose levels in the culture medium. 29. The method of claim 28, wherein the method is configurable. 30. Cells were extracted from native mammalian islets of Langerhans, an insulin-producing recombinant Glucose regulation resulting from karyotic cells and coffin isogenetic recombination or mutations Claim 28 selected from the group consisting of insulin-producing eukaryotic cells. How to put it on. 31. Claiming that the cell is selected from the group consisting of prokaryotic cells and eukaryotic cells. The method according to item 24. 32. Cells endowed with permanent dividing capacity, cells of living tissues and primary cultures 32. The method of claim 31, wherein the cell is selected from the group consisting of cells. 33. 25. The method of claim 24, wherein the device substantially surrounds the cell. 34. returning the medium when the medium is in direct interactive contact with the cells; Are harmful or undesirable components transformed into harmless or desirable components? 25. The method according to claim 24, comprising: 35. further comprising implanting the device comprising the cells into the subject's body; Said culturing step is carried out in the substantial absence of deleterious immunological responses in vivo. , A method according to claim 33. 36. If the cells are the subject's own defective cells and the cells are genetically modified prior to transplantation. 36. The method of claim 35, wherein the method is operated to overcome deficiencies. 37. Devices consisting of cells can be inserted into the dermis, subcutaneously, intracavitally, intraperitoneally, intravascularly, in organs, or is implanted nearby, in or near the omental sac, or intravaginally. How to put it on. 38. The body is exposed to abnormal levels of certain compounds in the virtual absence of harmful immunological responses. Regulating the levels of said compounds in the body fluids of subjects suffering from endogenous defects occurring in the fluids A method for placing cells lacking an endogenous defect in a patient's cells into a hydrogel. suspended, wherein the hydrogel consists of greater than about 35% water; The cells are surrounded in a biodegradable, implantable device, where at least one of said devices is The other portion is a non-porous, semi-permeable, biocompatible material that substantially surrounds the cells. comprising about 5 to 45% by weight of at least one hard material. segment, and about 95-55% by weight of at least one soft segment. The soft segment is formed from a copolymer consisting of an aliphatic polyol, selected from the group consisting of aliphatic and aromatic polyamines and mixtures thereof; at least one hydrophilic, hydrophobic or amphipathic oligomer; The film has a tensile strength greater than about 350 psi and up to about 10,000 psi , an ultimate elongation of greater than about 300% and up to about 1,500% and an absorbed The sum of the water volume fraction and the hydrophilic volume fraction of the soft segment is more than about 100% of the volume and up to about 2,000% of the volume of the wet polymer having a water absorption rate of greater than 50% and up to about 95%; is permeable to molecules of molecular weight from about 6,000 to 600,000 and substantially impermeable to cells and certain substances; implanting the device into a region of the subject's body where the cells are in contact with body fluids of the subject; and at the site of implantation where said cells are in direct interactive contact with said compound. causing said cells to survive and to regulate their levels in body fluids; The method comprising: 39. 39. The method of claim 38, wherein the hydrogel is an alginate. 40. Claim 38, wherein the hydrogel is polyethylene glycol. the method of. 41. The body fluid consists of blood, and the intrinsic defect is due to the normality of the compounds in the blood. 39. The method of claim 38, wherein the level is substantially higher than the level of. 42. The effect of cells on the level of said compound in body fluids is reversed by the level of the compound. 39. The method of claim 38, wherein the method is adjustable. 43. Cells endowed with permanent dividing capacity, cells of living tissues, and primary cultures 39. The method of claim 38, wherein the cell is selected from the group consisting of. 44. If the patient is diabetic and the cells are glucose regulated and insulin producing 39. The method of claim 38, comprising living cells. 45. Cells were isolated from native mammalian islets of Langerhans, and from homologous recombination or is a group of glucose-regulated, insulin-producing eukaryotic cells that result from mutations. 45. The method of claim 44, wherein the method is selected from: 46. The cells are the subject's own insulin-deficient cells, and the cells are the subject's own insulin-deficient cells. and genetically engineered to produce glucose-regulated insulin before transplantation. 45. The method of claim 44, wherein 47. Harmless return to body fluids when they are in direct interactive contact with cells Are harmful or undesirable compounds transformed into desirable or desired compounds? 39. The method of claim 38, comprising: 48. 39. The method of claim 38, wherein the cell comprises a product producing cell. 49. Hydrogels comprising greater than about 35% water; biodegradable, implantable devices; wherein at least one portion of the device is a non-porous, semi-permeable, biocompatible material. a film, said film comprising about 5 to 45% by weight of at least one hard cell. and about 95-55% by weight of at least one soft segment. The soft segment is made of a copolymer of aliphatic polyol, selected from the group consisting of aliphatic and aromatic polyamines and mixtures thereof consisting of at least one hydrophilic, hydrophobic or amphipathic oligomer; The film has a tensile strength of greater than about 350 psi and up to about 10,000 psi; Ultimate elongation and absorbed water greater than about 300% and up to about 1,500% The sum of the volume fraction of the soft segment and the hydrophilic volume fraction of the dry polymer body and up to about 2,000% and about 5% of the wet polymer volume. and the film has a water absorption rate of greater than 0% and up to about 95%; Permeable to molecules with a molecular weight of about 6,000 to 600,000 and finely fused. substantially impermeable to cells and certain substances; syringes; needle; and Preselected cells lacking metabolic defects are suspended in the hydrogel and placed in the device. and implanting said device into the body of a subject suffering from a metabolic defect; so that the cells interact with the subject's body fluids and alleviate symptoms associated with the defect. Instructions for use of the kit to enable A kit for correcting metabolic defects in a subject. 50. The insulin production kit according to claim 49, further comprising a cell culture medium. to.