Incapsulated hepatocyte composition. RU patent 2500390.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a microcapsules for preventing or treating hepatic disorders. The microcapsules for preventing or treating hepatic disorders containing a capsule coating, an encapsulating suspension of a therapeutically effective hepatocyte count in a physical contact with a hepatocyte-stimulating amount of erythropoietin. A method for preparing microcapsules involving preparing the suspension of the therapeutically effective hepatocyte count and the hepatocyte-stimulating amount of erythropoietin to bring them in physical contact with each other, and encapsulating the suspension of hepatocytes and erythropoietin in a biologically compatible capsule shell so that to form a microcapsule. A method for preventing or treating a hepatic disorder in an individual in need thereof involving administering the microcapsules in the individual in need thereof. The method for introducing the hepatocytes in the individual involving administering the microcapsules in the individual. A method for hepatocyte culture in a culture medium involving hepatocyte culture in the microcapsules in the appropriate culture medium.

EFFECT: microcapsules are effective for preventing or treating the hepatic disorders.

20 cl, 1 dwg, 1 ex

The present invention relates to containing cells of the liver and erythropoietin, processes for obtaining the above microcapsules and methods of treatment of the patient, including the application of the said microcapsules the patient.

Although the liver healthy individual is able to regenerate through the restoration or replacement of the damaged or diseased tissue, unfortunately, after the death of or serious damage to a certain number of cells of the liver due to disease or injury, it may refuse the whole body. Such a failure, whether acute or chronic heart failure may be a cause of disease and death. Therapy of liver diseases includes conventional methods, such as the introduction of drugs. However, the present level of technology also includes a liver transplant or its parts. In addition, widely known transplantation populations of cells of a liver patients, for example, as described in the WO 2004/009766 A2. However, there is still a big problem in the development of therapies for the treatment of acute or chronic liver disease with the delivery of liver cells to the needy in this patient, where methods are optimized in terms of viability, engraftment, proliferation and differentiation of the transplanted liver cells in the target tissue.

In the Haque et al. (Biotechnology Letters 27 (5) (2005), 317-322)) described in vitro study of microcapsules of alginate-chitosan as an alternative to transplantation of liver cells for the treatment of liver failure. In Chandrasekaran et al. (Tissue Engineering 12 (7), (2006)) describes the precursor cells of the liver cells, placed in an electrostatically generated granules.

Despite the efforts to develop therapeutic systems, effectively delivering liver cells in the target tissue of the patient, there remains a need to provide more reliable therapeutic methods of treatment of diseases of the liver, in particular, methods to ensure a high viability and physiological activity of liver cells in the body of the target.

This guide solves this problem by providing microcapsules containing shell capsules, preferably biologically compatible shell capsules, wraps suspension therapeutically efficient quantity of the liver cells in physical contact with a stimulating the liver cells and the amount of erythropoietin. In a preferred embodiment, the invention relates to containing shell of a capsule and the kernel, where the shell of a capsule in the kernel encapsulates the suspension of a therapeutically efficient quantity of the liver cells and stimulating the liver cells and the amount of erythropoietin, in particular, where erythropoietin (referred to below as the EPO) and liver cells are in physical contact with each other to provide a stimulating effect of EPO on the liver cells.

Thus, in a preferred embodiment, the invention provides for the granting of microcapsules containing shell capsules, preferably obtained from biocompatible substance capsule shell and core surrounded by a specified shell of a capsule. In one of the preferred options for the implementation of the present invention core of suspension contains a therapeutically efficient quantity of the liver cells in physical contact with a stimulating the liver cells and the amount of erythropoietin.

In another preferred embodiment, the kernel contains a matrix, preferably obtained from biocompatible substance matrix, where suspension therapeutically efficient quantity of the liver cells placed in the specified matrix in physical contact with a stimulating the liver cells and the amount of erythropoietin. Preferably substance matrix is the same as the substance of the shell of a capsule. In another embodiment biocompatible substance matrix may represent a substance different from the substance of the shell of a capsule.

Thus, the present invention relates to the leadership of encapsulation together erythropoietin and liver cells in biologically compatible shell of a capsule to erythropoietin was in physical contact with the cells of the liver to develop at least one of the species of the biological action on cells of the liver, in particular, stimulation of the cells of the liver.

One of the advantages of the present invention is that liver cells encapsulated in the shell of a capsule and, thus, does not cause any adverse reactions, in particular allergic or immune reaction from the patient who preferably are transplanted microcapsule. In addition, a close contact with erythropoietin cell stimulates the liver specified liver cells to perform their biological function is to provide the patient biological functions of the liver.

In particularly preferred embodiment liver cells are contained in in such concentration to be in physical contact with each other, providing even more pronounced effect upon stimulation with erythropoietin. Thus, especially in a preferred embodiment, the invention provides that liver cells contained in microcapsules, are in physical contact with each other and are in physical contact with with erythropoietin.

In the context of the present invention, the expression "the liver cells are in physical contact with with erythropoietin" means that erythropoietin is able to exercise at least one of their biological functions at the cell of the liver, in particular is capable of attaining and reversible or irreversible contact erythropoietin receptors, located on the cage of the liver.

In the context of the present invention, the expression "the liver cells, which are in physical contact with each other" means that the cells of the liver are according to the present invention in such close proximity that cells relate to each other and provide a stable environment very similar to the natural physiological state of the liver.

In the context of the present invention, the term "stimulation of the cells of the liver" means that erythropoietin increases the biological functionality of the liver cells, preferably from the patient who transplanted microcapsule increases the viability of the cells of the liver, increases their stability during storage and/or increases their potential for successful realization of their biological function after transplantation of the individual.

In the context of the present invention "biological compatibility" means that the substance, in particular substance capsule shell and/or substance matrix able to maintain viable integrated liver cells and allows the interaction with erythropoietin and liver cells. In particularly preferred embodiment, the term "biologically compatible" means that the substance allows, preferably a long-term, the implantation of the patient, while preserving, however, the functions placed liver cells without calling any unwanted local or systemic effect of the individual, in particular of allergic and immune reactions. In particularly preferred embodiment, the term "biologically compatible" means that the substance is able to provide as a substrate supporting the activity of liver cells, including stimulation of molecular and mechanically similar system between the liver cells and EPO, preferably with the purpose of optimization of the liver regeneration without identifying any adverse effects in cells and the individual.

In the context of the present invention, the term "erythropoietin" means hormone that controls erythropoiesis or blood cell formation, preferably substance which, in the appropriate dosage, controls the growth, differentiation and maturation of stem cells from to erythrocytes.

Erythropoietin is a glycoprotein that contains 166 amino acids, three plots of glycosylation, and with a molecular weight of approximately 34,000 Yes. During the EPO-induced differentiation of progenitor cells red blood cells, induced synthesis of globin, increased synthesis of a complex of heme, and increases the number of receptors ferritin. Thus, the cell can absorb more iron and synthesize functional hemoglobin. In the Mature erythrocytes and hemoglobin binds oxygen. Thus, blood contained in hemoglobin play a key role in supplying the organism with oxygen. These processes initiated by the interaction of the EPO with the appropriate receptor on the cell surface of progenitor cells red blood cells (Graber and Krantz, Ann. Rev. Med. 29(1978), 51-56).

In the context of the present invention, the term "erythropoietin" includes erythropoietin wild type, in particular human erythropoietin, and its derivatives. In the context of the present invention derivative of erythropoietin are recombinant proteins erythropoietin, characterized by at least a change of one amino acid, in particular the deletion, addition, or replacement of one amino acids compared with EPO wild-type and/or proteins erythropoietin with excellent glycosylation pattern compared with erythropoietin wild type. In particularly preferred embodiment derivative erythropoietin also represent a fusion proteins or truncated proteins erythropoietin wild-type or their derivatives. In particularly preferred embodiment derivative erythropoietin is also a erythropoietin wild type, containing a great glycosylation pattern compared with the pattern of glycosylation of the wild type.

In respect of the present invention under "derivatives" also understand such derivative of erythropoietin, which, while retaining the basic structure of erythropoietin, are collected by means of substitution of one or more atoms or molecular groups, or residues, in particular, by the substitution of sugar chains, such as ethylene glycol, and/or amino acid sequences, which differ from the natural protein erythropoietin, human or animal at least one situation, but in fact have a high degree of homology at the level of amino acids and comparable biological activity. Derivatives of erythropoietin, which can be used in the present invention, for example, are known from WO 94/25055, EP 0148605 B1 or WO 95/05465.

In particular, the "homology" means the identity of a sequence of at least 80%, preferably at least 85% and specifically preferably at least more than 90%, 95%, 97% and 99%. Thus a well-known specialist in this field, the term "homology" refers to the degree of relationship between two or more polypeptide molecules. It is determined by the coincidence between sequences. This match can mean or identical match, or conservative amino acid substitution.

In respect of the invention, a "derivative" includes merged proteins, in which the functional domains of another protein are on the N-end station or on the C-leaf plot. In one of the embodiments of the invention this another protein, for example, can be a GM-CSF, VEGF, PIGF, a statin or another factor having a stimulating effect on the endothelial precursor cells. Additional embodiment other protein can also be a factor having a stimulating effect on the liver cells.

The differences between the derivative of erythropoietin and natural, or with erythropoietin to wild-type can appear, for example, as a result of mutations, such as deletions, substitutions, insertions, add, replace the grounds and/or recombinant nucleic acid sequences coding for the amino acid sequences of erythropoietin. Obviously, such differences can also be variations of the natural sequence, such as the sequence of another organism, or sequence, mutated by nature, or mutations, selectively introduced in nucleic acid sequences coding for erythropoietin, known in the region by conventional methods, such as chemicals and/or physical means. Thus, in relation to an invention, a "derivative" includes mutant molecules erythropoietin, or in other words erythropoietin.

According to the invention, you can also use the analogs of peptides or proteins erythropoietin. In respect of the present invention, the term "equivalents" includes compounds that do not contain any amino acid sequence of the identical amino acid sequence of erythropoietin, but having a three-dimensional structure largely similar to the three-dimensional structure of erythropoietin so that they are comparable biological activity. For example, erythropoietin analogues may constitute compounds containing amino acid residues with a suitable conformation responsible for binding erythropoietin to its receptors, and, thus, able to stimulate the basic properties of the surface bonding field of erythropoietin. The compounds of this class are described, for example, Wrighton et al., Science, 273 (1996), 458.

Used in the invention of EPO can be obtained in various ways, for example through the allocation of human urine or urine or plasma (including serum) of patients suffering from aplastic anemia (Miyake et al., J.B.C. 252 (1977), 5558). For example, the EPO person can be also obtained from tissue culture cells of kidney cancer man (JA Unexamined Application 55790/1979), cells of lymphoblasts person with the ability to produce EPO rights (JA Unexamined Application 40411/1982), and of culture received merge cells cell line human. EPO also can receive ways gene technology, using suitable DNA or RNA encoding corresponding amino acid sequence EPO to obtain the desired protein through genetic engineering, for example, in bacteria, yeast or in the plant cell line of the animal or human. For example, such methods are described in the EP 0148605 B2 or EP 0205564 B2 and EP 0411678 B1.

In the context of the present invention in a preferred embodiment derivative erythropoietin represent the functional and confirmed by clinical tests derived EPO, preferably selected from the group consisting of epoetina, also called , , or , epoetina Delta, also called , , also called , PD-, CERA (continuous erythropoietin receptor antagonist) and -epoetina beta, also called .

In a preferred embodiment of the present invention micro capsule is a preferable spherical pellets, resulting from biocompatible substance shell capsules containing placed in her therapeutically effective amount of liver cells in physical contact with a stimulating the liver cells and the amount of erythropoietin. In the context of the present invention preferably micro capsule is a pellet, preferably with a diameter of 10 microns up to 10 mm, preferably from 140 microns up to 10 mm, preferably from 50 microns to 1 mm, preferably from 60 microns up to 800 microns, preferably from 100 to 700 microns. In a preferred embodiment of the present invention microcapsules according to the present invention contain liver cells, erythropoietin and biocompatible substance shells of a capsule. Preferably in addition to the above three elements provide coverage microcapsules and/or biologically compatible matrix in the nucleus of the microcapsule.

Thus, especially in a preferred embodiment, the invention relates to that contains a therapeutically efficient quantity of liver cells are encapsulated in a biologically compatible matter matrix in physical contact with a stimulating the liver cells and the amount of erythropoietin.

In particularly preferred embodiment liver cells are placed into the shell of a capsule in the form of suspension, preferably in the form of suspension of cell culture. Preferably a cell suspension liver is in the form of liver cells contained in the environment for the cultivation of cells or in the physiologically acceptable aqueous solution. Preferably a cell suspension liver is a suspension of liver cells in the environment for the cultivation of cells.

In particularly preferred embodiment liver cells in the form of suspension placed in contained in the shell matrix, preferably in the form of suspension cell culture, preferably in the form of liver cells contained in the environment for the cultivation of cells or in the physiologically acceptable aqueous solution.

In particularly preferred embodiment, the invention relates to , where the cells of the liver selected from the group consisting of progenitor cells of the liver cells, the stem cells of the liver, , hepatocytes and endothelial cells.

Additionally, in a preferred embodiment, the invention relates to , where liver cells derived from the liver of an adult liver embryo, fetal liver, the liver newborn or cultures of cells of the liver. Preferably liver cells represent the living cells of the liver. In a preferred embodiment liver cells can be obtained from living or dead, in particular the recently-deceased, donor.

Additionally, in a preferred embodiment, the invention relates to , where liver cells are cells of the human liver, a liver cell is not a man primacy, pig liver cells, liver cells dogs liver cells cat, rabbit liver cells, liver cells mouse or rat liver cells.

In particularly preferred embodiment, the invention relates to , where the average diameter of microcapsules is from 100 up to 700 microns, preferably from 200, 300, 400, 500, 600 or 650 700 microns.

In particularly preferred embodiment, the invention relates to , where a therapeutically effective amount of the liver cells ranges from 4 to 10 10 8 , preferably from 5 to 10 10 7 liver cells/ml of suspension.

In particularly preferred embodiment, the invention relates to , where the average diameter of the liver cells ranges from 8 to 14 microns, preferably from 9 to 12 microns.

Additionally, in a preferred embodiment, the invention relates to , where a stimulating the liver cells and the amount of erythropoietin is 10 -7 to 10 -2 , preferably from 10 -7 to 10 -3 , preferably from 10 -7 to 10 -5 and preferably from 10 -6 to 10 -5 U/ml of suspension.

Additionally, in a preferred embodiment, the invention relates to where EPO erythropoietin is a wild-type or recombinant erythropoietin.

Additionally, in a preferred embodiment, the invention relates to , where the substance of capsule shell is selected from the group consisting of alginate, alginate-chitosan (AC), alginate-poly-L-lysine (APA), polymer and PEG-hydrogel.

Additionally, in a preferred embodiment, the invention relates to , where the substance matrix is selected from the group consisting of alginate, alginate-chitosan (AC), alginate-poly-L-lysine (APA), polymer and PEG-hydrogel.

Additionally, in a preferred embodiment, the invention relates to , where in addition to liver cells in is at least one additional type of cells.

Additionally, in a preferred embodiment of the present invention provides that micro capsule in addition to liver cells and EPO contains at least one additional factor of growth, preferably selected from the group consisting of HGH (growth factor persons), VEGF (vascular endothelial growth factor), CSF (colony-stimulating factor), , complex SCF (Skp, containing F-box complex), SDF (factor-1 stromal cells), NGF (nerve growth factor), PIGF (protein biosynthesis anchor-class " f"), inhibitor HMGCoA reductase inhibitor of ACE (angiotensin converting enzyme), inhibitor AT-1 and donor NO.

In particularly preferred embodiment, the invention relates to where microcapsules are covered.

In particularly preferred embodiment of the present invention coating is a polymer coating, sugar coating of sugar alcohol and/or fatty or waxy coating.

Additionally, in a preferred embodiment, the invention relates to a method for obtaining the microcapsules, which includes:

a) the suspension of a therapeutically efficient quantity of the liver cells and stimulating the liver cells and the number of erythropoietin,

b) mixing suspension of the liver cells and erythropoietin to bring them into physical contact with each other and

c) encapsulation suspension of the liver cells and erythropoietin in the substance of capsule shell, so to form a microcapsule.

Additionally, in a preferred embodiment, the invention relates to a method where the microcapsules are received on stage c), cryopreserved.

Additionally, in a preferred embodiment, the invention relates to a method of prophylaxis or therapeutic treatment of liver disease in need of the individual, including the introduction of microcapsules of the present invention to the needy in this individual.

In particularly preferred embodiment, the invention relates to a method, where liver disease is a hepatitis, cirrhosis, congenital metabolic disorders, acute liver failure, acute liver infection, acute chemical toxicity, chronic liver failure, cholangitis, biliary cirrhosis, alagille syndrome, deficiency of alpha-1-antitrypsin deficiency, autoimmune hepatitis, bile ducts, liver cancer, liver disease, fatty dystrophy of the liver, , gallbladder stones, Gilbert's syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C and other viral hepatitis infection, Porphyrius, primary sclerosing holangit, Reye's syndrome, sarcoidosis, , disease accumulation of glycogen type 1 or Wilson's disease.

Additionally, in a preferred embodiment, the invention relates to a method, where the introduction of conduct through the introduction of microcapsules under capsule liver, spleen, liver, liver parenchyma or artery, or in the portal vein.

In particularly preferred embodiment of the present invention introduction of a through the introduction of need of the individual, where introduction is a local, or parenteral introduction. In a preferred embodiment local introduction is a preference dermal, inhalation, vaginal or intranasal introduction. In a preferred embodiment enteral introduction of a through the mouth, through the gastric nutrient probe or rectally. In a preferred embodiment parenteral administration of conduct by means of injections or infusions, preferably intravenous, intra-arterial, intramuscular, , subcutaneous, , , , or introduction. Additionally, in a preferred embodiment parenteral introduction, transdermal, or inhalation introduction.

Additionally, in a preferred embodiment, the invention relates to a method of culturing the cells of the liver in the environment for cultivation, preferably in vitro, including the cultivation of microcapsules of the present invention in a suitable for cultivation environment and in terms appropriate to maintain or improve the viability of the cells of the liver.

In particularly preferred embodiment, the invention relates to a method to maintain or improve the energy state of the liver cells in the environment for cultivation, preferably in vitro, including the cultivation of microcapsules of the present invention in a suitable environment for the cultivation and in terms appropriate to maintain or improve the energy state of the liver cells.

Additional preferred embodiments of the present invention are subject to subparagraphs claims.

In figure 1 as an indicator of the energy state of the cells illustrated by the attitude of the ATP/ADP after cultivation of liver cells are encapsulated with EPO or without EPO.

Example 1

Liver cells in the presence of EPO (5 x 10 -3 units/ml), encapsulated with in concentration from 4 to 10 10 8 cells/ml in granules with a mean diameter from 100 up to 700 microns, are more viable than the cells of the liver, identical encapsulated without EPO. The ratio of the ATP/ADP used as an indicator of the energy state of the cells. If during the experiment, the attitude of the ATP/ADP increases with higher speed or remains greater than untreated cells, but these findings support the hypothesis.

Encapsulation of liver cells from a single source material cryopreserved cells of the liver people conducted using the device electrostatic generation of granules. Liver cells suspended in an appropriate environment for the cultivation and mixed with 2% solution of sodium alginate in 1:1 ratio. The resulting 1% solution of alginate, containing liver cells in a concentration of 4 million cells/ml administered 1 syringe equipped with 24 caliber. at the base of the needle dug into 23 caliber for use as the positive electrode electrostatic alloy wheels. The syringe was injected into the syringe pump (Braintree Scientific BS-8000, Braintree, MA) and had to drop, leaving fell perpendicularly to the surface of 125 mm solution of calcium chloride (CaCl2 ) (accepted or buffer Cytonet 4 with 1% albumin human, with 5 x 10 -3 and 5 x 10 -6 U/ml of EPO or without EPO) in 250 ml of analytical glass. The distance from the tip of the to the surface CaCl 2 installed approximately 2.5 see the Performance of the pump installed in the range from 0.75 to 1.5 ml/min Earthed electrode immersed in collecting CaCl 2 bath. Created electrostatic potential between the tip and bath with CaCl 2 , using high-voltage DC source (Spellman model RHR30PF30, Hauppauge, NY) in the range from 3.8 to 6 kV. Size of granules (500 microns) controlled regulation of the applied potential. When the syringe pump included the presence of a high electrostatic potential, speaker solution of sodium alginate has pulled in the form of small droplets, which are in contact with a solution CaCl2 in solid calcium alginate.

After encapsulation liver cells alginate pellets contained in the environment, William E supplemented with 10% serum, 244 units/ml penicillin, 0,244 mg/ml and 5.5 mm glutamine, 0,195 units/ml insulin, 0,017 mcg/ml glucagon, 0.73 mkg/ml prednisolone, 0,54 mcg/ml of dexamethasone and cultivated for 2 hours.

Extract from the capsules conducted by placing granules 100 mm citrate. When alginate has dissolved, the cells twice washed with PBS and besieged by centrifugation at 200xG for one minute. Metabolites extracted by means of placement of the cells in 4% chloric acid and homogenization within 20 seconds in the manual . Then the sample was centrifuged at 14000xG for three minutes and removed to metabolites. Chloric acid was neutralized KOH and deleted insoluble perchlorate potassium by centrifugation. Then to determine the ATP, ADP sample were analyzed by HPLC method described in the "Measurements of ATP in Mammalian Cells" (Manfredi et al. Methods 2002 (4), 317-326). To determine the level of ATP/ADP used three parallel tablet.

Results

The ratio of the ATP/ADP is an indicator of energy and reflects the influence of all biochemical pathways on the metabolic state of the cell. To maintain a relationship ATP/ADP (see figure)observed in liver cells encapsulated with EPO, clearly demonstrates that cells are able to support the production of energy required for any function of the cells. In this experiment, the ratio of the ATP/ADP supported in the group treated with EPO, but has not been included in the untreated controls. Despite the fact that the group treated with EPO, was able to maintain a ratio of ATP/ADP ratio ATP/ADP in controls dropped below 0,5. This demonstrates that the EPO can transmit signals via cascade managed receptor EPO, providing cells trophic stimulation.

1. Microcapsules for the prevention or therapeutic treatment of diseases of the liver, containing shell of a capsule that encapsulate the suspension of a therapeutically efficient quantity of the liver cells in physical contact with a stimulating the liver cells and the amount of erythropoietin.

2. Microcapsules according to claim 1, where the cells of the liver selected from the group consisting of progenitor cells of the liver cells, the stem cells of a liver, , endothelial cells and hepatocytes.

3. Microcapsules according to claim 1 or 2, where the cells of the liver is derived from the liver of an adult or cultures of cells of the liver.

4. Microcapsules according to claim 1 or 2, where the liver cells are cells of the human liver, a liver cell is not a man primacy, pig liver cells, liver cells dogs liver cells cat, rabbit liver cells, liver cells mouse or rat liver cells.

5. Microcapsules according to claim 1 or 2, where the average diameter of microcapsules is from 100 up to 700 microns.

6. Microcapsules according to claim 1 or 2, where the average diameter of the liver cells ranges from 8 to 14 microns.

7. Microcapsules according to claim 1 or 2, where a therapeutically effective amount of the liver cells ranges from 4 to 10 10 8 liver cells/ml of suspension.

10. Microcapsules according to claim 1 or 2, where the shell of a capsule obtained from biocompatible substance, selected from the group consisting of alginate, alginate-chitosan (AU), alginate-poly-L-lysine (APA), polymer and PEG-hydrogel.

11. Microcapsules according to claim 1 or 2, where, in addition to liver cells in is at least one additional type of cell, selected from the group consisting of stellate endotheliocytes liver, biliary cells, hematopoietic cells, monocytes, macrophages, lymphocytes, and endothelial cells.

12. Microcapsules according to claim 1 or 2, where the microcapsules are covered.

13. Microcapsules according to claim 1 or 2, where advanced micro capsule contains biologically compatible matrix containing cells of the liver and erythropoietin.

14. Method of obtaining of microcapsules by any of the preceding paragraphs, including (a) obtaining suspension therapeutically efficient quantity of the liver cells and stimulating the liver cells and the number of erythropoietin, b) mixing suspension of the liver cells and erythropoietin to bring them into physical contact with each other and c) encapsulation suspension of the liver cells and erythropoietin in biologically compatible matter capsule shell, so to form a microcapsule.

15. The method according to paragraph 14, where microcapsules received in stage C), cryopreserved.

16. A method of prevention or therapeutic treatment of liver disease in need of the individual, including the introduction of microcapsules need of the individual for any one of claims 1 to 13.

17. The method according to article 16, where liver disease is a hepatitis, cirrhosis, congenital metabolic disorders, acute liver failure, acute liver infection, acute chemical toxicity, chronic liver failure, cholangitis, biliary cirrhosis, alagille syndrome, deficiency of alpha-1-antitrypsin deficiency, autoimmune hepatitis, bile ducts, liver cancer, liver disease, fatty dystrophy of the liver, , gallbladder stones, Gilbert's syndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C and other caused by a virus infection, hepatitis, Porphyrius, primary sclerosing holangit, Reye's syndrome, sarcoidosis, , disease accumulation of glycogen type 1 or Wilson's disease.

18. The method according to item 16 or 17, where the introduction of conduct through the introduction of microcapsules under capsule liver, spleen, liver, liver parenchyma or artery, or in the portal vein.

19. The way the liver cells individual, including the introduction of microcapsules individual on any one of claims 1 to 13.

20. The method of cultivation of liver cells in the environment for cultivation, including the cultivation of liver cells in microcapsules on any one of claims 1 to 13 in a suitable medium for cultivation.