JP2022121166A - Cell sheet to be transplanted to liver surface - Google Patents

Abstract

To provide a cell sheet to be transplanted to a liver surface, the cell sheet being stratified including hepatocytes, the cell sheet capable of expressing its functions for as long as 8 weeks after transplanted.SOLUTION: The present invention discloses a cell sheet to be transplanted to a liver surface, the cell sheet being stratified comprising a hepatocytic layer and a feeder cell layer. The feeder cell may be a fibroblast. In the inventive cell sheet, the hepatocyte may be a human hepatocyte that is collected from an immunodeficient nonhuman animal having a hepatic dysfunction with a human hepatocyte engrafted thereinto. The present invention also discloses a pharmaceutical composition for transplantation to a liver surface, comprising the inventive cell sheet. The inventive pharmaceutical composition can be used for the prevention and/or treatment of at least one disease selected from liver disease, blood disease and metabolic disease.SELECTED DRAWING: None

Description

There is an application for application of Article 30, Paragraph 2 of the Patent Law (1) February 20, 2020 https://www. micenavi. jp/jsrm2020/search/detail_program/id:982 ▲2 ▼ May 18, 2020 Announced through the 19th Annual General Meeting of the Japanese Society for Regenerative Medicine ▲3 ▼ October 8, 2020 The 16th Hiroshima Presented in Abstract of Liver Project Research Center Symposium (4) October 10, 2020 16th Hiroshima Liver Project Research Center Symposium WEB (https://us02web.zoom.us/j/87002351236) and TKP Garden City PREMIUM Presented at the North Exit of Hiroshima Station

TECHNICAL FIELD The present invention relates to a cell sheet containing hepatocytes and a pharmaceutical composition containing the cell sheet for prevention and treatment of liver diseases and the like, and more specifically, a cell sheet containing hepatocytes for transplantation onto the surface of the liver. and a pharmaceutical composition for prevention and treatment of liver diseases, etc., containing the cell sheet.

Liver transplantation is effective not only for liver diseases caused by dysfunction of the liver itself, such as liver failure, but also for blood diseases such as hemophilia, which are caused by abnormalities in proteins produced by the liver, and metabolic diseases such as urea cycle disorders. established as a treatment. However, living-donor liver transplantation has donor limitations due to histocompatibility and other conditions. Therefore, secondary liver formation by heterotopic transplantation of hepatocyte sheets is expected as one of the effective treatments for liver diseases.

Patent Literature 1 discloses a liver tissue cell sheet for living body transplantation that can maintain the function of liver tissue cells for a long period of time. In the detailed description of the invention of Patent Document 1, subcutaneous tissue, intraperitoneal tissue, liver and muscle in vivo are listed as transplantation sites. However, in the examples of Patent Document 1, the site where the human liver tissue cell sheet is transplanted is only the subcutaneous area where the vascular network is previously induced with bFGF.

However, in order to support the function of the liver weakened by disease, a large amount of human liver tissue cell sheets must be engrafted for a long period of time to express the function. It is true that the subcutaneous site is attractive as a site for transplantation of hepatic tissue cell sheets because the degree of invasiveness of treatment is low. There are many problems from the viewpoint of QOL. On the other hand, on the liver surface, highly developed sinusoids for blood circulation to hepatocytes exist just below the liver capsule, and nutrient-rich gastrointestinal venous blood is available through the sinusoids. Furthermore, since the liver is the largest visceral organ, there is a large surface area available for transplantation. Therefore, there is a need in the field of liver regenerative medicine for the development of a hepatocyte sheet for transplantation onto the liver surface.

Patent Literature 2 discloses transplantation of a hepatocyte-like cell sheet derived from human mesenchymal stem cells onto the liver surface. Examples in Patent Document 2 disclose that acute liver dysfunction was suppressed for 8 or 10 days after transplantation. It has been suggested that serum protein markers (such as α1-antitrypsin (α1-AT)) and cytokines produced by human mesenchymal stem cell-derived hepatocyte-like cells act effectively on liver regeneration ( Patent Document 2, paragraph 0182). However, Patent Document 2 cannot be said to disclose or suggest a cell sheet capable of expressing liver function for a long period of 8 weeks or longer after transplantation.

International publication WO2007/080990 pamphlet Japanese Patent No. 6008297

An object of the present invention is to develop a layered cell sheet containing hepatocytes for transplantation onto the surface of the liver, which is capable of expressing its function for a long period of 8 weeks or more after transplantation. . Another subject of the present invention is to develop a pharmaceutical composition for transplantation on the liver surface containing the cell sheet of the present invention.

The inventors of the present invention conducted intensive studies to solve the above problems, and found that a cell sheet containing a hepatocyte layer and a feeder cell layer can exhibit functions for a long period of 12 weeks or more by transplanting it to the surface of the liver. It was discovered that it is, and came to complete this invention.

That is, the present invention is as follows.
(1) A layered cell sheet comprising a hepatocyte layer and a feeder cell layer for transplantation onto the liver surface.
(2) The cell sheet according to (1), wherein the feeder cells are fibroblasts.
(3) The cell sheet according to (1) or (2), wherein the hepatocytes are human hepatocytes collected from an immunodeficient non-human animal with liver damage engrafted with human hepatocytes.
(4) The cell sheet according to (3), wherein the non-human animal is genetically liver-damaged.
(5) The cell sheet according to (4), wherein the non-human animal is a uPA ^+/+ /SCID ^+/+ mouse.
(6) A pharmaceutical composition for transplantation onto the liver surface, comprising the cell sheet according to any one of (1) to (5).
(7) The pharmaceutical composition according to (6), which is for prevention and/or treatment of at least one disease selected from liver disease, blood disease and metabolic disease.
(8) Use of the cell sheet according to any one of (1) to (5) for prevention and treatment of at least one disease selected from liver disease, blood disease and metabolic disease.
(9) Using the cell sheet according to any one of (1) to (5) for the production of a pharmaceutical composition for the prevention and treatment of at least one disease selected from liver diseases, blood diseases and metabolic diseases Use of.
(10) transplanting an effective amount of the cell sheet of any one of (1) to (5) or the pharmaceutical composition of (6) or (7) into the liver of a subject with reduced function; A method of improving liver function in said subject, comprising:
(11) The method according to (11), wherein the subject has at least one disease selected from liver disease, hematologic disease and metabolic disease.

The stratified cell sheet containing the hepatocyte layer and the feeder cell layer provided by the present invention can exhibit its function for a long period of 12 weeks or longer by being transplanted to the surface of the liver.

The upper row shows the first day (Day 1) and the third day of culture of PXB mouse-derived human hepatocytes (hereinafter referred to as "PXB cells") in a 6-well temperature-responsive culture plate. A phase-contrast micrograph of an eye (Day 3), a micrograph of a cell sheet of only PXB cells detached from the culture plate at room temperature on the third day of culture, and a tissue section of the cell sheet stained with hematoxylin and eosin (H&E). Photo. The lower row shows the first day of co-culture (PXB / TIG-118) in which human skin-derived fibroblast TIG118 cells were seeded in a 6-well temperature-responsive culture plate and 4 days later, PXB cells were further seeded ( Phase-contrast micrographs of Day 1) and the third day of co-culture (Day 3), and micrographs of the co-cultured cell sheet detached from the culture plate due to the temperature shift to room temperature on the third day of co-culture, and hematoxylin and A photograph of a tissue section of the co-cultured cell sheet stained with eosin (H&E). A culture of PXB cells only (PXB-only) and a co-culture (PXB/TIG-118) in which human skin-derived fibroblast TIG118 cells were seeded and PXB cells were further seeded 4 days later were subcutaneously or scratched in mice, respectively. A line graph of changes in body weight over time after transplantation onto the treated liver surface (liver surface). The vertical axis is the relative value of the average body weight of the mice, with the average body weight of the mice on the day of transplantation set to 1. The horizontal axis is the transplantation period (unit: weeks). Error bars represent the standard deviation of the measurements for each of the four mice. A culture of PXB cells only (PXB-only) and a co-culture (PXB/TIG-118) further seeded with PXB cells 4 days after seeding human skin-derived fibroblast TIG118 cells were subcutaneously or liver surface (liver Table) is a line graph of changes in human ALB (albumin) concentration over time after transplantation. The vertical axis is blood human albumin concentration (logarithmic scale, unit: ng/mL), and the horizontal axis is transplantation period (unit: weeks). Error bars represent the standard deviation of measurements from 4 mice.

One embodiment of the present invention provides a layered cell sheet comprising a hepatocyte layer and a feeder cell layer for implantation onto the liver surface.

A "cell sheet" of the present invention refers to a substantially planar structure comprising hepatocytes of the present invention or hepatocytes and feeder cells of the present invention. In the culture of hepatocytes and feeder cells, the feeder cells are spread on the culture substrate, and the hepatocytes are placed thereon, so that there are at least two layers of cells. Moreover, when cells detach, feeder cells lose their anchorage for spreading and can be attracted and contract. When the feeder cells contract, the hepatocytes that were on the spread flattened feeder cells are not completely covered by the contracted and rounded feeder cells and fold over, resulting in 3 or more cell layers. . The "stratified cell sheet" of the present invention refers to a cell sheet consisting of at least two cell layers, a layer of feeder cells and a layer of hepatocytes. The "layered cell sheet" of the present invention may be obtained by stacking two or more of the "cell sheet comprising at least two cell layers, a layer of feeder cells and a layer of hepatocytes".

Sakai, Y.; et al., (PLoS ONE 8(7): e70970. doi: 10.1371/journal.pone.0070970), the "layered cell sheet" of the present invention comprises "a layer of feeder cells and a layer of hepatocytes. When one cell sheet consists of at least two cell layers, the dimensions of the cell sheet are, for example, one well of a 6-well culture plate as a culture container, for example, a diameter of 7 to 20 mm and a thickness. may be about 10 to 25 μm.

The layered cell sheet of the present invention is used for long-term maintenance by transplanting it onto the liver surface of a subject in need of transplantation. The subject may be a human or non-human animal. When the subject is an animal other than human, it is preferable to use an animal that does not elicit an immune response to human cells in order to avoid an immune response to human cells transplanted as a cell sheet. More preferred are animals engrafted with human cells, preferably human liver cells. Examples of preferred non-human animals include liver-damaged (e.g., transgenic mice overexpressing urokinase-type plasminogen activator (hereinafter "uPA") under the control of an albumin enhancer/promoter), and hyperimmune Including, but not limited to, chimeric mice in which pups of uPA ^+/+ /SCID ^+/+ mice with both traits of deficiency (eg, SCID mice) are transplanted with human thawed hepatocytes. The cell sheet of the present invention "capable of expressing its function over a long period of time" means at least 8 weeks, 10 weeks, 12 weeks, 15 weeks, 18 weeks, 21 weeks, 24 weeks, 27 weeks, 30 weeks, and 33 weeks after transplantation. , 36 weeks, 39 weeks, 42 weeks, 45 weeks, 48 weeks, 52 weeks or longer, or 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, It means that the cell sheet continues to survive at the transplanted site for a longer period of time while expressing its function. Here, the expression that the cell sheet of the present invention "expresses a function" means that the hepatocytes contained in the cell sheet produce and secrete proteins, synthesize and secrete bile, and/or Performing at least one of the liver's functions including, but not limited to, detoxifying and breaking down substances. When the hepatocytes of the present invention express a gene that was not expressed in the donor hepatocytes by exogenous gene transfer or genome editing, the cell sheet of the present invention can also produce the gene product. included in expressing Whether or not the cell sheet of the present invention exerts its function can be determined quantitatively or qualitatively by ELISA, Western blotting, or other immunological or biochemical techniques for the proteins, bile, harmful substances, etc. in blood. can be determined by detecting Such immunological or biochemical techniques are well known to those skilled in the art of the present invention.

"Hepatocytes" of the present invention include all cell types that can be isolated from all or part of a normal liver, including liver parenchymal cells, liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, pit cells, Hepatic non-parenchymal cells such as bile duct epithelial cells and mesothelial cells are included. The hepatocytes contained in the cell sheet of the present invention are fresh hepatocytes collected from the liver of a healthy human donor (hereinafter referred to as "fresh hepatocytes"), as well as hepatocytes obtained by culturing the fresh hepatocytes. (hereinafter referred to as "cultured hepatocytes"), fresh hepatocytes or cultured hepatocytes that have been cryopreserved and then thawed (hereinafter referred to as "thawed hepatocytes") or thawed hepatocytes Cultured hepatocytes (hereinafter referred to as "thawed cultured hepatocytes") may also be used. The hepatocytes of the present invention, particularly the cultured hepatocytes or thawed cultured hepatocytes, may express genes that have not been expressed in the donor hepatocytes due to foreign gene transfer or genome editing. The "hepatocytes" of the present invention are cells selectively enriched and/or depleted of cells of one or more specific cell types contained in the fresh hepatocytes, or selectively enriched or depleted Cells cultured later, cells that have been selectively concentrated or removed and then frozen and then thawed, or cells that have been further cultured from the thawed cells may be used. The "hepatocytes" of the present invention may be a mixture of selectively enriched cells of two or more specific cell types contained in the fresh hepatocytes at different mixing ratios, or different arrangements or configurations. may be arranged on a cell sheet. In addition to liver-derived hepatocytes, the hepatocytes of the present invention may be derived from iPS cells, pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, and other undifferentiated cells capable of differentiating into hepatocytes. can.

The "feeder cells" of the present invention produce physiologically active substances, growth factors, etc. necessary for the survival and function of the hepatocytes of the present invention in culture and/or after transplantation, and/or Refers to cells that are co-cultured with the hepatocytes of the present invention in order to support the survival and function of the hepatocytes of the present invention, such as functioning as scaffolds for cells. The "feeder cells" of the present invention may be seeded in the same culture vessel prior to culturing the hepatocytes of the present invention. Prior to co-culturing with the hepatocytes of the present invention, the feeder cells of the present invention are added with a cell growth inhibitor (e.g., mitomycin C, etc.), irradiated with radiation (e.g., γ-rays, etc.), or otherwise treated to lose the ability to proliferate. I don't mind being treated. The "feeder cells" of the present invention are desirably cells that can adhere to a culture vessel for culturing hepatocytes. Including, but not limited to, non-human cultured cells or established cell lines.

Except for the part in contact with the diaphragm, the liver is covered with a liver capsule consisting of a serous membrane composed of a single layer of squamous epithelium and an underlying fibrous connective tissue, and hepatocytes exist inside the liver capsule. The "liver surface" in the present invention means the liver surface in a state where the liver capsule completely seals the underlying hepatocytes and is physically inaccessible from the outside of the liver capsule (hereinafter, "intact liver surface"). ), and the liver surface where hepatocytes can physically contact from the outside of the liver capsule by partially removing the liver capsule and forming an opening in the liver capsule (hereinafter referred to as "opening in the liver capsule"). formed liver surface"). The "liver surface having openings formed in the liver capsule" in the present invention refers to the liver surface having a large number of substantially groove-shaped openings formed in the liver capsule, which are regular or irregular in length, width, depth and direction. may be The surface of the liver onto which the cell sheet of the present invention is to be transplanted may be any area of the liver provided that the transplantation is not hindered. For example, in the case of mice, the ventral surface of the left lateral lobe and the dorsal surface of the left medial lobe (when the abdomen of the mouse is opened upward, the upper surface of the left lateral lobe and the back surface of the left medial lobe, respectively); It can be the liver surface of any area where the cell sheet can be surgically implanted, for example the ventral surface of the left lateral lobe area and the dorsal surface of the left medial lobe area. The generally grooved opening in the liver capsule can be formed, for example, by scratching the outside of the liver capsule with a cotton swab or other instrument. Alternatively, the "liver surface in which openings are formed in the liver capsule" in the present invention means that one or more circular, polygonal and/or irregular shaped openings are formed in the liver capsule, instead of the large number of substantially grooved openings. It may be a formed liver surface. One or more circular, polygonal and/or irregular shaped openings in the liver capsule can be formed, for example, by excising the liver capsule with scissors or other cutting tools. Alternatively, the liver capsule openings may be a combination of the multiple generally grooved openings and one or more of the circular, polygonal and/or irregularly shaped openings. The sum of the area of the liver capsule in which the large number of substantially groove-shaped openings are formed and the area of the one or more openings having circular, polygonal and/or irregular shapes is The area may be the same as or smaller than the area to be applied. When the opening is formed in the liver capsule, a hemostatic agent may be used to prevent bleeding from capillaries directly under the liver capsule and/or capillaries near hepatocytes. When the transplant operation of the cell sheet of the present invention is completed, the abdomen is closed after confirming that the bleeding has stopped. The hemostatic agent is a fibrin glue-based hemostatic agent that is also used for the tissue adhesive (e.g., Tacoseal (registered trademark) tissue adhesive sheet (CSL Behring Co., Ltd.) and Volheal (registered trademark, Teijin Pharma Limited), etc. In addition, collagen-based hemostatic agents (eg, Helistat (Hakuho Co., Ltd.)) and plant starch-based hemostatic agents (eg, Bird Arista AH (Medicon Co., Ltd.)) are included, but not limited to these.

One embodiment of the present invention provides the cell sheet of the present invention, wherein said feeder cells are fibroblasts.

Fibroblasts are derived from skin and other connective tissues, and are cells that produce extracellular matrix constituent proteins such as collagen and fibronectin. In culture, they elongate in a spindle shape and adhere to the substrate of the culture vessel. . The fibroblasts of the present invention may be any fibroblasts that can function as feeder cells in culture, form a feeder cell layer, and constitute a stratified cell sheet together with a hepatocyte layer. do not have. The fibroblasts of the present invention may be primary cultured cells, cultured cells that can be subcultured but are not immortalized cells, or established cell lines. Fibroblast primary culture cells include human skin-derived normal diploid fibroblast TIG-118 cells, Hs68 cells, ASF-4 cells, and mouse embryonic skin-derived fibroblast MEF cells. Cultured cells that can be replaced but are not immortalized cells include mouse embryo skin-derived STO cells and the like, and established cell lines include mouse embryo skin-derived NIH3T3 cells, the STO cell-derived SNL76/7 cells, and the like. be. When the subject is a human, it is preferable to use subject-derived fibroblasts as feeder cells.

Among fibroblasts, TIG-118 cells are known to be preferred feeder cells for supporting the function of hepatocytes. Sakai, Y.; (PLoS ONE 8(7): e70970. doi: 10.1371/journal.pone.0070970), temperature-responsive cell culture equipment for cell sheet recovery (UpCell (registered trademark), Cellseed Co., Ltd.) was used. A cell sheet obtained by co-culturing HepaRG (registered trademark) cells, which are terminally differentiated hepatocytes derived from a human hepatocyte progenitor cell line, and TIG118 cells in a culture system for 2 days, followed by low-temperature treatment and peeling from the culture vessel. is smaller than the cell sheet obtained by culturing HepaRG cells alone for 2 days, but becomes thicker and forms a three-dimensional tissue in which HepaRG cells overlap. In the co-cultured cell sheet obtained by co-culturing in this way, the production amounts of albumin and α1-antitrypsin per number of seeded HepaRG cells increased about 1.2-fold compared to HepaRG cells alone.

The cell sheet of the present invention may contain an extracellular matrix in addition to hepatocytes and feeder cells. The extracellular matrix may be produced by hepatocytes and/or feeder cells, or may be artificially added.

One embodiment of the present invention provides the cell sheet of the invention, wherein the hepatocytes are human hepatocytes harvested from an immunodeficient non-human animal with liver damage engrafted with human hepatocytes.

In the present invention, "immunodeficient non-human animal having liver damage engrafted with human hepatocytes" means that the hepatocytes of the non-human animal are dead or alive, but the liver function of the non-human animal is impaired. A chimeric animal in which human hepatocytes are engrafted in a non-human animal with liver damage, which is present in such a small number that it cannot be fully performed, and the hepatocytes of the non-human animal are replaced by human hepatocytes. Point. The liver disorder is a liver disorder in which only hepatocytes of the non-human animal are abnormal. The chimeric animal also develops immunodeficiency and thus can be engrafted with human hepatocytes.

One embodiment of the present invention provides the cell sheet of the present invention, wherein said non-human animal has a genetic liver disorder.

Liver damage in the present invention is such that the cell lineage of hepatocytes is not differentiated, or even if once differentiated as hepatocytes, they die, or even if they survive, the liver function of the non-human animal is not fully performed. No, or any hereditary liver disorder. The genetic liver disorders of the present invention may be naturally occurring or mutagen-induced mutant animals, or mutant animals produced by genetic engineering experiments. Mutant animals produced by genetic manipulation experiments are, for example, knockout animals in which genes essential for hepatocyte differentiation and/or survival are disrupted by genome editing technology, or transcriptional control of genes that are specifically expressed in hepatocytes. There are transgenic animals that express the suicide gene under the control of the region. The hepatocyte-specifically expressed gene includes, but is not limited to, transcription regulatory sequences such as albumin promoters and enhancers. Said suicide genes include, for example, toxins (e.g., diphtheria toxin A chain) and enzymes that convert nontoxic prodrugs into toxic products (e.g., thymidine kinase, carboxylesterase, carboxypeptidase, cytochrome P450 isoenzymes, deoxyribonucleotide kinase, nitro reductase, etc.).

One embodiment of the invention provides the cell sheet of the invention, wherein said non-human animal is a uPA ^+/+ /SCID ^+/+ mouse.

The uPA ^+/+ mice are autosomal integrated with a transgene that overexpresses a urokinase-type plasminogen activator under the control of an albumin enhancer/promoter (hereinafter referred to as "uPA"). SCID ^+/+ mice are homozygous individuals of a spontaneous severe immunodeficiency mutation (hereinafter referred to as “SCID”) lacking both T-cell and B-cell function. Therefore, uPA ^+/+ /SCID ^+/+ mice are homozygotes for both uPA and SCID.Hepatocytes used in the cell sheet of the present invention are uPA ^+/+ /SCID ^+/+ mice. It may be a chimeric mouse in which human hepatocytes are transplanted into a juvenile animal before liver damage becomes severe.Such a chimeric mouse can be obtained commercially as a PXB mouse from Phoenix Bio Co., Ltd., for example. PXB mice are chimeric mice created by transsplenic transplantation of 2- to 3-week-old uPA ^+/+ /SCID ^+/+ mice with normal human hepatocytes from informed donors. Hepatocytes collected from PXB mice whose replacement rate with human hepatocytes reached 70% or more after 12 weeks of age were removed by immunosurgical treatment using anti-mouse antibody and complement. After concentrating the human-derived hepatocytes, they can be used to produce the cell sheet of the present invention (hereinafter referred to as "PXB cells").

The cell sheet of the present invention is produced by the steps of seeding hepatocytes in a culture vessel in which a drug for cell sheet detachment is immobilized, and applying a stimulus to the culture vessel to detach the drug for cell sheet detachment from the culture vessel. It may be produced by a production method including a step of exfoliating a cell sheet containing hepatocytes. Furthermore, the cell sheet of the present invention is produced by a step of seeding feeder cells in a culture vessel in which a cell sheet detachment agent is immobilized, a step of seeding hepatocytes in the culture vessel after the step of seeding the feeder cells, and It may be produced by a production method including a step of exfoliating the cell sheet containing the hepatocytes and feeder cells by applying a stimulus to the culture vessel to cause the cell sheet exfoliation agent to detach from the culture vessel.

In the step of seeding hepatocytes, hepatocytes are seeded in a culture vessel at a predetermined seeding density. The predetermined seeding density refers to a cell density that reaches a confluent state 2 to 5 days after the start of culture, preferably 3 to 4 days after the start of culture. Specifically, it means seeding 5×10 ⁵ to 2×10 ⁶ hepatocytes, or about 1×10 ⁶ hepatocytes per well of a 6-well plate. The medium in which the hepatocytes are cultured is, for example, Dulbecco's Modified Eagle's Medium or other basal medium supplemented with additives selected from the group consisting of L-glutamine, EGF, hydrocortisone, insulin, transferrin and selenium, and antibiotics such as penicillin and streptomycin. A medium containing the substance and 10% fetal bovine serum, or a commercially available medium for PXB-cells (Phoenix Bio Co., Ltd., Higashi-Hiroshima City, Hiroshima Prefecture), Medium 670 (Biopredic International, KAA Co., Ltd.) Shi, Kyoto) or equivalent serum-free media, but are not limited to these. When transplanted into humans, it is desirable not to bring the cell sheet of the present invention into contact with fetal bovine serum or other animal-derived components. These media are used to culture hepatocytes under conditions of 37° C., 5% CO ₂ and saturated steam. When a temperature-responsive polymer is used as the cell sheet detachment agent, a medium and a culture vessel heated to a temperature sufficiently higher than the lower critical solution temperature (T) of the temperature-responsive polymer are used.

In the step of seeding the feeder cells in the production of the cell sheet of the present invention, the feeder cells of the present invention are seeded in a culture vessel at a predetermined seeding density. Cells selected as feeder cells often have the property of spreading on the surface of the culture vessel, so a seeding density of 1/10 to 1/100 of the hepatocytes per surface area of the culture vessel is desirable. The period until the feeder cells spread over the entire surface of the culture vessel and become confluent may be 3 to 5 days, or about 4 days. For example, when TIG118 cells, which are human skin-derived fibroblasts, are selected as feeder cells, specifically, 1×10 ⁴ to 2×10 ⁵ cells per well of a 6-well plate, for example, 2. 6×10 ⁴ to 1.5×10 ⁵ feeder cells can be seeded. Media for culturing feeder cells include, but are not limited to, Eagle's minimum essential medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum, for example, in the case of TIG118 cells. These media are used to culture at 37° C., 5% CO ₂ and saturated steam conditions. When a temperature-responsive polymer is used as the cell sheet detachment agent, a medium and a culture vessel heated to a temperature sufficiently higher than the lower critical solution temperature (T) of the temperature-responsive polymer are used. Furthermore, attention should be paid to the temperature in the step of seeding hepatocytes as well.

In the production of the cell sheet of the present invention, the step of seeding the hepatocytes in the culture vessel after the step of seeding the feeder cells is such that after seeding the feeder cells, the feeder cells spread over the entire surface of the culture vessel to reach confluency. Hepatocytes are seeded after a period of time until the Specifically, hepatocytes are seeded 3 to 5 days after seeding the feeder cells, or about 4 days later. If PXB cells are selected as hepatocytes for seeding, 1.0×10 ⁶ PXB cells can be seeded per well of a 6-well plate. When seeding hepatocytes, the feeder cell medium can be removed and replaced with hepatocyte medium.

When producing the cell sheet of the present invention, an agent for exfoliating the hepatocytes and feeder cells from the culture vessel without impairing the cell-cell adhesion and cell-substrate adhesion of these cells (hereinafter referred to as "cell sheet In some cases, a culture vessel in which a detachment agent is immobilized is used. When the drug is exposed to a predetermined stimulus, the hepatocytes and feeder cells are bound to the drug and are released from the culture vessel. Cells can be detached from the culture vessel. Even after the "cell sheet" of the present invention is detached from the culture vessel, the cell sheet detachment agent is applied to the hepatocytes, the hepatocytes and feeder cells, and the extracellular matrix secreted by these cells. are sometimes combined. Therefore, the "cell sheet" of the present invention may contain the drug for cell sheet detachment.

The agent for cell sheet detachment contains a stimulus-responsive polymer that changes the degree of adhesion to cells by a given stimulus such as light or temperature. It does not interfere with adhesion to the culture vessel surface and survival. However, when exposed to the stimulus, the hepatocytes and/or feeder cells of the present invention and the extracellular matrix produced by these cells are detached from the culture container, so that the cell sheet of these cells is detached from the culture container. . Examples of stimuli-responsive polymers include, but are not limited to, temperature-responsive polymers, pH-responsive polymers, ion-responsive polymers, photo-responsive polymers, and the like. Among these, temperature-responsive polymers are preferable as agents for cell sheet detachment because they are exposed to stimuli without changing the composition of cell sheet culture medium.

When the stimulus-responsive polymer is a temperature-responsive polymer, a pH-responsive polymer, an ion-responsive polymer, or a photo-responsive polymer, the stimulus for detaching the cell sheet detachment agent from the culture vessel is temperature, pH, These are the ionic composition of the culture medium and the light stimulus that permeates the culture vessel.

As the temperature-responsive polymer, for example, a polymer that exhibits cell adhesiveness at the temperature for culturing cells and exhibits cell non-adhesiveness at the temperature for detaching the formed cell sheet can be used. For example, at temperatures below the lower critical solution temperature, a temperature-responsive polymer has an improved affinity for the surrounding water, and the polymer absorbs water and swells, making it difficult for cells to adhere to the surface (cell non-adhesiveness). At a temperature equal to or higher than the lower critical solution temperature, water is desorbed from the polymer, causing the polymer to shrink, thereby making it easier for cells to adhere to the surface (cell adhesiveness). It is preferable to use a temperature-responsive polymer having a lower critical solution temperature (T) of 0° C. or higher and 42° C. or lower, preferably 4° C. or higher and 37° C. or lower.

Suitable temperature-responsive polymers include, for example, acrylic or methacrylic polymers, more specifically poly-N-isopropylacrylamide (PNIPAAm) (T=32° C.), poly-Nn-propylacrylamide ( T = 21 ° C.), poly-Nn-propyl methacrylamide (T = 32 ° C.), poly-N-ethoxyethylacrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T=about 35° C.), poly-N,N-diethylacrylamide (T=32° C.), and the like. Further, it may be a copolymer obtained by combining two or more kinds of monomers for forming these polymers and polymerizing them.

A pH-responsive polymer or an ion-responsive polymer suitable for the cell sheet to be formed can be appropriately selected.

As the cell sheet detachment agent, only one type of stimuli-responsive polymer may be used, or a plurality of types of stimuli-responsive polymers may be mixed and used.

It is preferable that the drug for cell sheet detachment be immobilized on the surface of the culture vessel with which the cells to be cultured come into contact. Further, the cell sheet detachment agent may be formed on substantially the entire bottom of the cell culture vessel, or may be formed on a part of the bottom of the cell culture vessel.

As a method for immobilizing the drug for cell sheet detachment on the surface of the culture vessel, for example, a polymer solution is placed on the surface of the culture vessel by an immersion method, a spray method, a spin coating method, or the like, and then the solvent is removed (dried). including but not limited to methods. From the viewpoint of uniformity, the polymer liquid is preferably applied using a bar coater. Alternatively, for handling the detached cell sheet, for example, a cell sheet detachment agent can be immobilized on the culture vessel in a predetermined pattern so that the cell sheet is gradually detached from the edge. The surface of the culture vessel can be subjected to UV ozone treatment, plasma treatment, electron beam treatment, corona treatment and other treatments. The application amount of the cell sheet detachment agent and other conditions are not particularly limited as long as the hepatocytes and/or feeder cells used in the cell sheet of the present invention are uniformly detached from the culture vessel by a predetermined stimulus.

The material of the culture vessel can be, for example, glass or plastic, but is not limited to these. Examples of plastic include, but are not limited to, polystyrene, polypropylene, polyvinyl chloride, polyethylene, cyclic polyolefin, polyethylene terephthalate, polycarbonate, silicone resin, or acrylic resin. As the acrylic resin, polymethyl methacrylate (PMMA) is preferable from the viewpoint of versatility.

A specific example of the culture vessel in which the drug for cell sheet detachment is immobilized is UpCell (registered trademark, Cellseed Co., Ltd.), a temperature-responsive cell cultureware for cell sheet recovery, which uses a temperature-responsive polymer as the drug for cell sheet detachment. Including but not limited to.

In addition, the culture vessel for producing the cell sheet of the present invention may contain an agent for manipulating the arrangement or arrangement of the hepatocytes and feeder cells of the present invention on the cell sheet (hereinafter referred to as "agent for controlling cell arrangement"). ) may be included on the surface of the chemical for cell sheet detachment. Therefore, even after the "cell sheet" of the present invention is detached from the culture vessel, the cell arrangement control bound to the hepatocytes, the hepatocytes and the feeder cells, and the extracellular matrix secreted by these cells. May contain drugs for

The agent for controlling cell arrangement includes, but is not limited to, a phospholipid bilayer membrane that coats the cell sheet detachment agent and a cell-specific labeling molecule embedded in the bilayer membrane. . For example, Togo, S.; (APL Bioeng. 4, 016103 (2020); doi: 10.1063/1.5123749) reported that 1-stearoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (SOPC ), and as cell-specific labeling molecules embedded in phospholipid bilayer membranes, polyethylene glycol (PEG) or its derivatives linked to single-stranded DNA, and extracellular cell adhesion molecules (e.g., E-cadherin) A fusion protein of a domain and a metal complex-binding peptide tag such as a histidine tag and a phospholipid derivative of the metal complex (for example, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1 -carboxypentyl)-iminodiacetic acid)succinyl]nickel salt). Another example of the cell-specific labeling molecule is a combination of an antibody that specifically binds to the cells contained in the cell sheet of the present invention and a secondary antibody that binds to the antibody, or a combination that specifically binds to the cells. and a conjugate in which biotin or a sugar chain is bound to an antibody that binds to a protein that specifically binds to the biotin (e.g., avidin or streptavidin) or a lectin that specifically binds to the sugar chain. is not limited to The application amount and other conditions of the agent for controlling cell arrangement are not particularly limited as long as the arrangement or arrangement on the cell sheet of the hepatocytes and/or feeder cells of the present invention can be manipulated.

The cell sheet of the present invention can be applied by applying fibrin glue or other tissue adhesives (for example, Tacoseal (registered trademark) tissue adhesive sheet (for example, Tacoseal (registered trademark) tissue adhesive sheet ( CSL Behring Co., Ltd.) and Volheal (registered trademark, Teijin Pharma Co., Ltd.) The dosage and other conditions of the tissue adhesive are determined by attaching the cell sheet of the present invention to the liver surface and fixing it to the liver surface. It is not particularly limited as long as it can be used.

The cell sheet of the present invention scoops up the cell sheet detached from the culture vessel, moves it, and protects the cell sheet during the operation of attaching it to the liver surface, and / or after attaching it to the liver surface. Protective agents for protecting cell sheets in the host (e.g., extracellular matrix components, tissue adhesives, polyglycolic acid, and/or polyethylene, including but not limited to hyaluronic acid, collagen, laminin, fibronectin) glycol). The dosage and other conditions of the protective agent are to protect the cell sheet during the operation of scooping up the cell sheet detached from the culture vessel, moving it, and attaching it to the liver surface, and/or attaching it to the liver surface. It is not particularly limited as long as it can protect the cell sheet in the host body after the treatment.

One embodiment of the present invention provides a pharmaceutical composition for implantation on the liver surface, comprising the cell sheet of the present invention.

A "pharmaceutical composition" of the present invention comprises an effective amount of any cell sheet of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be used for transplantation medicine as a sheet for transplantation on the surface of the liver with openings formed in the liver capsule. The pharmaceutical composition of the present invention can be cryopreserved in the state of a cell sheet, provided that the pharmaceutical composition can be exfoliated from the culture vessel immediately before transplantation, or can exert its efficacy at the time of transplantation.

The active ingredient of the pharmaceutical composition of the present invention is a layered cell sheet containing a hepatocyte layer and a feeder cell layer. The effective dose of the pharmaceutical composition of the present invention varies depending on the disease to be treated or prevented, but is about 1×10 ⁶ to about 1×10 ⁹ cells contained in the exfoliated cell sheet. Accordingly, a single dose of the pharmaceutical composition of the present invention, ie, the number of transplanted hepatocytes, is about 1×10 ⁶ to about 1×10 ⁹ . The cell sheet, which is the active ingredient of the pharmaceutical composition of the present invention, contains feeder cells in addition to hepatocytes. Furthermore, it may contain a culture medium, a preservation medium, or a protective agent surrounding the cell sheet. The cell sheet may contain feeder cells that are about 1/100 to about 1/5 the number of hepatocytes. The cell sheet may contain an extracellular matrix, and a cell sheet detachment drug or other drug attached to the cell sheet when the cell sheet is detached from the culture vessel. However, at the time of implantation on the liver surface, the weight of the active ingredient, hepatocytes, may be from about 0.1% to about 50% of the weight of the cell sheet unit dosage form. Pharmaceutical compositions of the invention are preferably formulated in unit dosage form, each dose typically containing from about 0.5 mg to about 100 mg of a compound of the invention. The term "unit dose form" generally refers to physical discrete units containing predetermined quantities of active ingredients in association with suitable pharmaceutical excipients and used one or more throughout the dosage regimen. to produce the desired therapeutic effect. However, in the pharmaceutical composition of the present invention, the cell sheet transplanted in a single transplant operation is maintained for a long period of time by being applied to the surface of the liver having an opening formed in the liver capsule and administered.

One embodiment of the present invention provides the pharmaceutical composition of the present invention for prevention and/or treatment of at least one disease selected from liver disease, hematologic disease and metabolic disease.

One embodiment of the present invention provides the use of the cell sheet of the present invention for prevention and treatment of at least one disease selected from liver disease, hematologic disease and metabolic disease.

One embodiment of the present invention provides use of the cell sheet of the present invention for the production of a pharmaceutical composition for the prevention and treatment of at least one disease selected from liver diseases, blood diseases and metabolic diseases.

In the present invention, "liver disease" includes acute hepatitis, chronic hepatitis, fatty liver, liver fibrosis, cirrhosis, portal hypertension, regenerative failure, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), liver Insufficiency, including, but not limited to, hepatic insufficiency associated with impaired hepatic blood flow.

In the present invention, the term "blood disease" refers to a blood disease caused by abnormal liver function such as abnormal protein production in the liver, including but not limited to von Willebrand's disease and hemophilia.

In the present invention, "metabolic disease" means Wilson's disease, alpha 1 antitrypsin deficiency, tyrosinemia, urea cycle deficiency (OTCD, CPS1D, argininosuccinate synthase deficiency), organic acidemia (methylmalonic acidemia A metabolic disease caused by abnormal liver function, including, but not limited to, abnormal production of enzymes and other proteins in the liver.

One embodiment of the present invention comprises transplanting an effective amount of the cell sheet of the present invention or the pharmaceutical composition of the present invention into the liver of a subject with reduced liver function, improving liver function in the subject. provide a way.

One embodiment of the present invention provides the method of improving liver function of the present invention, wherein the subject has at least one disease selected from liver disease, blood disease and metabolic disease.

In this specification, unless otherwise specified, the adnominal "about" for a numerical value refers to a numerical range equal to or greater than a numerical value that is 10% less than the numerical value and less than or equal to a numerical value that is 10% greater than the numerical value.

All documents mentioned herein are hereby incorporated by reference in their entirety.

The embodiments of the invention described below are for illustrative purposes only and are not intended to limit the scope of the invention. The technical scope of the present invention is limited only by the description of the claims.

(1) Materials and Methods (1.1) Human Hepatocyte Chimeric Mouse Human hepatocytes used for culture are derived from PXB mice (Phoenix Bio Inc.), which are human hepatocyte chimeric mice. PXB mice are human hepatocyte chimeric mice in which 2-3 week old uPA ^+/+ /SCID ^+/+ mice were transplanted via the spleen with normal human hepatocytes from informed donors. PXB mice were obtained that were over 12 weeks old and had a replacement rate of 70% or more with human hepatocytes.

(1.2) Recovery of hepatocytes from human hepatocyte chimeric mice Isolation of hepatocytes from human hepatocyte chimeric mice was performed using a two-step collagenase perfusion method. Livers of the chimeric mice were treated with 200 mg/mL ethylene glycol tetraacetic acid (EGTA), 1 mg/mL glucose, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 10 μg/mL. of Ca ²⁺ and Mg ²⁺ -free Hanks Balanced Salt Solution (CMF-HBSS) containing gentamycin at 38° C. at a flow rate of 1.5 mL per minute. The perfusate is switched to CMF-HBSS containing 0.05% collagenase (Wako Pure Chemical), 0.6 mg/mL CaCl ₂ , 10 mM HEPES and 10 μg/mL gentamycin for an additional 17-23 minutes. Perfusion was performed at a flow rate of 1.5 mL per minute. Livers were excised, transferred to dishes, and hepatocytes were gently dissociated in CMF-HBSS containing 10% bovine albumin, 10 mM HEPES and 10 μg/mL gentamycin. The dissociated cells were centrifuged (50×g, 2 min) three times and then mixed with Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 20 mM HEPES, 44 mM NaHCO ₃ and antibiotics. The pellet was suspended in medium consisting of substances (100 IU/ml penicillin G and 100 μg/ml streptomycin). Cell number and viability were assessed using the trypan blue exclusion test.

(1.3) Enrichment of PXB cells Human hepatocytes isolated from PXB mice are mixed with mouse-derived hepatocytes. Washed and incubated with Dynabeads (Dynal Biotech) conjugated with sheep anti-rat IgG antibody in tubes on ice for 30 minutes. The tube was placed on a magnetic stand DynalMPC-1 for 1-2 minutes to remove 66Z-binding mouse hepatocytes. Enriched human hepatocytes were collected as 66Z-negative cells.

(1.4) Cultivation of PXB cells The enriched human hepatocytes were suspended in a PXB-cell medium (Phoenix Bio Inc.) and placed in a 6-well temperature-responsive culture plate (Up Cell (registered trademark) CS3004, Cellseed Inc.). ) were seeded with 1×10 ⁶ cells per well. In the following, the cell culture conditions were 95% saturated water vapor, 37°C, and 5% _CO2 . After 3 days of culture, they were transplanted into mice.

(1.5) Co-culture of human fibroblasts and PXB cells Human skin-derived fibroblasts TIG118 cells (National Research and Development Agency National Institutes of Biomedical Innovation, Health and Nutrition JCRB cell bank, cell number: JCRB053) were added to 10% FBS. and seeded at 1.5×10 ⁵ per well in a 6-well temperature-responsive culture plate (Up Cell (registered trademark) CS3004, Cellseed Co., Ltd.). After 4 days, the concentrated PXB cells were suspended in a PXB-cell medium (Phoenix Bio Inc.), and the TIG118 cells were spread on a 6-well temperature-responsive culture plate (Up Cell (registered trademark) CS3004). 1×10 ⁶ cells were sown per. Cell sheets were harvested after 3 days of co-cultivation.

(1.6) Collection of cell sheet The temperature-responsive culture plate was allowed to stand at room temperature for 2 to 3 hours, and the cell sheet was peeled off from the culture plate. The exfoliated cell sheet was scooped up with a sterilized circular glass plate and used for transplantation into mice.

(1.7) Transplantation of Cell Sheets into Mice Prior to transplantation of cell sheets, uPA ^+/+ /SCID mice were subcutaneously administered an analgesic (butorphanol tartrate). In the case of liver surface transplantation, the abdomen of the mouse is opened upward under isoflurane anesthesia, and the surface of the liver (upper surface of the left lateral lobe and the back surface of the left inner lobe) is scratched with a cotton swab, and the cell sheet is sterilized after hemostasis. It was scooped up with a pre-formed round glass plate and attached to the surface of the liver. In the case of subcutaneous transplantation, the abdomen of the mouse was opened upward under isoflurane anesthesia, and the cell sheet was attached to the back side of the skin (the side in contact with the peritoneum). After the operation, it was confirmed that hemostasis had been achieved, and the abdomen was closed.

(1.8) Method for Measuring Blood Albumin Blood was collected by orbital venous plexus collection under anesthesia. ELISA method (Human Album ELISA Quantitation Kit, Bethyl Laboratories, Montgomery, TX) or latex agglutination immunoturbidimetry (LZ test 'Eiken' U-ALB, Eiken Chemical Co., Ltd.) using an automatic analyzer BioMajesty™ (JCA-BM6050, JEOL Ltd.).

(2) Results (2.1) Preparation of cell sheet containing PXB cells The upper part of Fig. 1 shows the first day of culture (PXB-only) of PXB cells in a 6-well temperature-responsive culture plate (Day 1). and a phase-contrast micrograph on the third day of culture (Day 3), and a micrograph of a cell sheet of a culture of only PXB cells detached from the culture plate at room temperature on the third day of culture and stained with hematoxylin and eosin (H&E). 2 shows a photograph of a tissue section of the cell sheet. As shown in the upper part of FIG. 1, when only PXB cells were seeded, the cells did not spread on the substrate on the first day of culture, but on the third day of culture, some cells spread on the substrate. has become accepted. When the temperature-responsive culture plate seeded with cells was allowed to stand at room temperature for 2 to 3 hours, the extracellular matrix produced by the cells and the cells were detached as a single cell sheet. A microscopic examination of the hematoxylin and eosin (H&E)-stained tissue section of the cell sheet revealed that the PXB cells formed an almost monolayered cell sheet.

(2.2) Preparation of co-culture cell sheet of human skin-derived fibroblast TIG118 cells and PXB cells The lower part of FIG. PXB / TIG-118) on the first day of culture (Day 1) and the third day of culture (Day 3), and the co-cultured cell sheet detached from the culture plate at room temperature on the third day of culture. and a tissue section photograph of the co-cultured cell sheet stained with hematoxylin and eosin (H&E). As shown in the lower part of FIG. 1, when PXB cells were seeded on the 4th day after TIG118 cells were seeded, most of the PXB cells had spread over the TIG118 cells even on the first day after seeding the PXB cells. PXB cells reached a confluent state on the third day of culture. When the co-cultured temperature-responsive culture plate was allowed to stand at room temperature for 2 to 3 hours, TIG118 cells and PXB cells were detached as a single cell sheet. The detached co-cultured cell sheet was thicker than the cultured cell sheet of PXB cells alone. Microscopic examination of tissue sections of co-cultured cell sheets stained with hematoxylin and eosin (H&E) revealed not only that PXB cells were overlaid on TIG118 cells, but also that the cytoplasm of PXB cells increased and they became more mature as hepatocytes. , suggesting that TIG118 cells, as feeder cells, support the liver function of PXB cells.

(2.3) Effect of transplantation of cultured cell sheets and co-cultured cell sheets containing only PXB cells Cultured cell sheets containing only PXB cells were placed subcutaneously or on the liver surface (hepatic surface) of 5-week-old uPA ^+/+ /SCID ^+/+ male mice. (PXB-only) or co-cultured cell sheet (PXB/TIG-118) was transplanted weekly until 12 weeks after transplantation, the body weight and blood human albumin concentration of the mice were measured over time. 3. Figure 2 shows a subcutaneous or liver surface (liver surface) culture of PXB cells only (PXB-only) and a co-culture (PXB/TIG-118) in which human skin-derived fibroblast TIG118 cells were seeded and then the PXB cells were seeded. Table) is a line graph of changes in body weight over time after transplantation. The vertical axis represents the relative value of the average mouse body weight on the day of transplantation, and the horizontal axis represents the transplantation period (unit: weeks). represents the standard deviation of the measurement results. Figure 3 shows a culture of PXB cells only (PXB-only) and a co-culture (PXB/TIG-118) in which human skin-derived fibroblast TIG118 cells were seeded and then the PXB cells were seeded subcutaneously or on the liver surface (liver surface). ) is a semi-logarithmic line graph of changes in human ALB (albumin) concentration over time after transplantation into . The vertical axis represents the blood human albumin concentration (logarithmic scale, unit: ng/mL), the horizontal axis represents the transplantation period (unit: weeks), and the error bars represent the standard deviation of the measurement results for each of the four mice.

As shown in FIG. 2, a cultured cell sheet containing only PXB cells (PXB-only) or a co-cultured cell sheet (PXB/TIG-118) was transplanted subcutaneously or on the surface of the liver (liver surface) of uPA ^+/+ /SCID mice. However, there was little difference in changes in body weight of the mice. However, as shown in FIG. 3, in mice subcutaneously transplanted with a cultured cell sheet containing only PXB cells, the blood concentration of human albumin was lower than that in mice under other transplantation conditions two weeks after transplantation, and continued to decrease thereafter. In the mice subcutaneously transplanted with the co-cultured cell sheet of TIG118 cells and PXB cells, there was almost no difference in the blood concentration of human albumin between the mice transplanted to the surface of the liver and the mice 2 to 4 weeks after transplantation. However, from 5 weeks after the transplantation, the human albumin concentration in the blood of the mice subcutaneously transplanted with the co-cultured cell sheet became lower than that of the mice transplanted onto the surface of the liver, and continued to decrease thereafter. On the other hand, in the mice transplanted to the surface of the liver, the human albumin blood concentration in the mice transplanted with the co-cultured cell sheet was lower than that in the mice transplanted with the cultured cell sheet containing only hepatocytes at 5 weeks after transplantation. Both continued to produce human albumin in the blood without significant changes.

From the results shown in FIG. 3 above, the liver surface transplantation of the cultured cell sheet of only PXB cells has the best results in the functional expression of the transplanted hepatocytes, followed by the liver surface transplantation of the co-cultured cell sheet of TIG118 cells and PXB cells. It was an achievement. In subcutaneous transplantation, both the cultured cell sheet containing only hepatocytes and the co-cultured cell sheet showed a marked decrease in the blood concentration of human albumin over time. That is, subcutaneous transplantation of hepatocytes provided only temporary effects at best.

The cell sheet of the present invention can exhibit its function for 12 weeks or more after transplantation, which is far longer than that of known cell sheets such as hepatocytes transplanted onto the surface of the liver. Therefore, the cell sheet of the present invention and pharmaceutical compositions containing the cell sheet are useful in the field of liver regenerative medicine.

Claims (7)

A layered cell sheet comprising a hepatocyte layer and a feeder cell layer for implantation on the liver surface. The cell sheet according to claim 1, wherein said feeder cells are fibroblasts. 3. The cell sheet according to claim 1, wherein the hepatocytes are human hepatocytes collected from an immunodeficient non-human animal having liver damage engrafted with human hepatocytes. 4. The cell sheet according to claim 3, wherein said non-human animal has a genetic liver disorder. 5. The cell sheet according to claim 4, wherein said non-human animal is a uPA ^+/+ /SCID ^+/+ mouse. A pharmaceutical composition for transplantation onto the liver surface, comprising the cell sheet according to any one of claims 1 to 5. 7. The pharmaceutical composition according to claim 6, which is for prevention and/or treatment of at least one disease selected from liver disease, hematological disease and metabolic disease.

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