JP6644395B2 - Methods for improving the ability of normal human hepatocytes to remove ammonia - Google Patents

Description

  The present invention relates to human hepatocytes for transplantation cultured in a medium.

  Liver transplantation is an extremely effective treatment for liver failure, but the shortage of donors is severe and no clue has been found at present. Therefore, hepatocyte transplantation is considered instead of liver transplantation. However, the preparation of a clinical research protocol for human hepatocyte transplantation has not yet been sufficiently elucidated, and the protocol for bone marrow cell transplantation cannot be directly applied.

  It is known that the liver has a high regenerative ability in vivo, and it can be said that partial liver transplantation is a treatment that applies this high regenerative capacity, but on the other hand, hepatocytes separated and cultured are separated from normal liver Even proliferated hepatocytes have inferior proliferative ability as compared to in vivo. Furthermore, after the separation, the ability of the cells to remove ammonia may decrease.

  Patent Document 1 discloses that a human hepatocyte for transplantation comprises a step of injecting an isolated human hepatocyte into an immunodeficient mouse, a step of expanding the hepatocyte, and a step of collecting the human hepatocyte. Methods for growing are described.

  Non-patent Document 1 describes a method for inducing and culturing differentiation of human iPS cells into hepatocytes by culturing hepatic stem progenitor cells in a laminin-containing medium.

Japanese Unexamined Patent Publication No. 2010-528661

Takayama K., Nagamoto Y., Mimura N., Tashiro K., Sakurai F., Tachibana M., Hayakawa T., Kawabata K., Mizuguchi H. Long-term self-renewal of human ES / iPS-derived hepatoblast- like cells on human Laminin 111-coated dishes. Stem Cell Rep., 1, 322-335 (2013)

  However, the above-mentioned technique does not sufficiently increase the number of human hepatocytes for transplantation, and cannot be said to be sufficient in terms of maintaining the function of hepatocytes after culture.

  The present invention has been made in view of such a problem, and an object of the present invention is to provide a human hepatocyte that can be efficiently proliferated and has a sufficient hepatocyte function even after culture.

  The human hepatocytes according to the present invention are human hepatocytes cultured in a medium containing myricetin.

  ADVANTAGE OF THE INVENTION According to this invention, while being able to proliferate efficiently, the human hepatocyte which the hepatocyte function was fully ensured also after culture | cultivation is obtained.

FIG. 1A shows the morphology of human hepatocytes before culture in the presence of myricetin, and FIG. 1B shows the morphology of human hepatocytes when cultured in the presence of myricetin for 7 days. It is a figure which shows the ammonia removal ability of a cell. It is a figure which shows the expression of a liver related gene. FIG. 4A is a view showing a change in appearance of a mouse when human hepatocytes are transplanted into a mouse liver that is immunodeficient and mimics a human urea circuit abnormality. FIG. FIG. FIG. 5A shows transplanted human hepatocytes in immunodeficient mouse liver tissue, and FIG. 5B shows the amount of human albumin in the serum of the mouse.

  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating the understanding of the principle of the present invention, and the scope of the present invention is described below. The present invention is not limited to the embodiments, and other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.

  The human hepatocytes according to the present embodiment are human hepatocytes cultured in a medium containing myricetin.

  Myricetin (myricetin) is 3,5,7-Trihydroxy-2- (3,4,5-trihydroxyphenyl) -4-chromenone, has a structure represented by the following formula 1, and is a natural flavonol that is a flavonoid. It is a kind.

  The medium is not particularly limited, but is, for example, a medium containing fetal bovine serum, insulin, dexamethasone, penicillin, and streptomyces, and for example, William's E medium can be used.

  The concentration of myricetin in the medium is not particularly limited, but is, for example, 1 to 30 μmol / L.

  The type of human hepatocytes cultured in a medium containing myricetin is not particularly limited, and for example, hepatocytes, whole hepatocytes, mesenchymal stem cells, hepatocytes differentiated from iPS cells, and the like may be used. However, it is preferably a hepatocyte.

  Either an autologous cell or another autologous cell can be suitably used as a cell collection source, and either fresh or frozen, even in a stored state, can be suitably used. When using frozen hepatocytes, it is preferable to inject either the preserved cord blood transplant or the preserved autologous stem cell transplant and then inject the whole preservation solution after thawing, or to suspend and inject the transfusion solution after centrifugal washing. It is possible. Frozen hepatocytes have the advantage that they can be used at any time and that they can be checked for quality such as bacterial contamination while frozen.

  The human hepatocytes according to the present embodiment are for transplantation, and the transplantation destination is not particularly limited.For example, inborn error of metabolism, chronic liver failure, or transplantation to a patient with acute liver failure Is possible. Inborn errors of metabolism include familial hypercholesterolemia, α1-antitrypsin deficiency, Crigler-Najal syndrome, factor deficiency, glycogen disease, Refsum disease (peroxisome disease), progressive familial intrahepatic disease Cholestatic disease and the like. Chronic liver failure cases include viral hepatitis. Acute hepatic failure is hepatic failure such as drug-induced or viral. The place of transplantation is via the portal vein for inborn errors of metabolism, but it is also possible to transplant into the spleen for chronic liver failure and acute liver failure.

Transplanted cell number, particularly limited as not disease type varies by the patient's age and the like, for example may be a 4x10 ⁷ cells ～3.9X10 ¹⁰ pieces in a single dose.

  In the method for producing human hepatocytes according to the present embodiment, for example, hepatic parenchymal cells can be cultured in a medium containing myricetin from the beginning. After culturing for a period of time, it is also possible to add myricetin to the medium and culture in a medium containing myricetin. The number of culture days in a medium containing myricetin is not particularly limited, and is, for example, 5 to 10 days.

  The human hepatocytes according to the present invention are human hepatocytes cultured in a medium containing myricetin, but are not limited to such embodiments, and human hepatocytes cultured in a medium containing a myricetin derivative It is also possible to use

  The myricetin derivative is a compound derived from the myricetin as a basic skeleton and is not particularly limited, but is specifically represented by the following formula 2.

Here, Xa, Xb, Xc, Xd, Xe, Xf, X1 and 2 are each independently O or S,
Ra, Rb, Rc, Rd, Re and Rf are each independently H, an alkyl group having 1 to 10 carbon atoms substituted with 1 to 5 Ry, or a dialkyl ether group having 2 to 20 carbon atoms. Wherein each alkyl having 1 to 10 carbon atoms of the dialkyl ether group is substituted with 1 to 5 Ry,
Ry is Rq, or an alkyl group having 2 to 10 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms, or a tricycloalkyl group alkyl group having 8 to 14 carbon atoms. A phenyl group, a naphthyl group or an aromatic group having 14 carbon atoms,
R1, R2, R3 and R4 each independently represent H or an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, an alkynyl group having 2 to 10 carbon atoms, or a cycloalkyl having 3 to 10 carbon atoms. Group, a phenyl group, a naphthyl group, an aromatic group having 14 carbon atoms,
Rq is CN, OH or a halogen atom.

1. Human hepatocyte culture Normal human frozen hepatocytes (Lot # FLO, EJW) were purchased from Celsis, In Vitro Technologies (Baltimore, MD). After thawing according to a conventional method, 1 × 10 ⁶ hepatocytes were subjected to 1 micro-mol / L dexamethasone, 1 micro-mol / L insulin, 2 mmol / L L-glutamine, 100 U / ml penicillin, 100 U / ml streptomycin, Suspended in 2 mL of William's E medium (Sigma, W1878) containing 10% fetal bovine serum, and seeded on a 6-well culture plate (BD Biocoat, type I collagen-coated 6-well plate, Becton Dickinson, Cat code 2352227), 5 Cultured at 37 ° C.,% CO ₂ . After culturing for 20 hours, add myricetin (Sigma-Aldrich, M6760) dissolved in dimethylsulfoxide at a concentration of 20 mmoL / L to the culture medium to a final concentration of 1, 3, 10, 30 micro-mol / L, and add it for 7 days. The cells were cultured (FIGS. 1A, B). FIG. 1A shows the morphology of human hepatic parenchymal cells before culturing in the presence of myricetin; Ctrl; control 1 (no addition of myricetin), DMSO; 1 micro-mol / L myricetin, 3 μM; 3 micro-mol / L myricetin, 10 μM; 10 micro-mol / L myricetin, 30 μM; 30 micro-mol / L myricetin. FIG. 1B shows the morphology of human hepatic parenchymal cells when cultured for 7 days in the presence of myricetin; Ctrl; control 1 (no addition of myricetin); DMSO; control 2 (only addition of DMSO, the solvent when adding myricetin) 1 μM; myricetin 1 micro-mol / L, 3 μM; myricetin 3 micro-mol / L, 10 μM; myricetin 10 micro-mol / L, 30 μM; myricetin 30 micro-mol / L.

2. Cell characteristic analysis 2-1. The NH ₄ Cl so as to 2 mmol / L at a final concentration of ammonia removal capacity above medium was added, the ammonia concentration in the medium up to 4 hours every hour was measured (bromocresol green method), the slope of the decreasing linear density Was used to determine the ability of the cells to remove ammonia (fmol / hr / cell) (FIG. 2).

2-2. Gene expression Cells were collected from the culture plate, RNA was extracted, and cDNA was prepared by reverse transcription. Using this cDNA, liver related genes (albumin, CYP3A4, α1 antitrypsin, tyrosine aminotransferase, tryptophan 2,3 dioxygenase, HNF4a, cytokeratin 18, creatine phosphate synthase 1, ornithine transcarbamylase) and internal standard The expression of the GAPDH gene was examined by quantitative PCR (FIG. 3).

3. Transplantation experiment into mouse liver Immunodeficient SCID mouse (CB-17 / IcrHsd-Prkdcscid, Japan SLC) male and SCID mouse ornithine transcarbamylase deficient mouse (spf-ash, Kumamoto University, National Center for Child Health and Development) female Were crossed to obtain offspring. After confirming ornithine transcarbamylase deficiency by the polymerase chain reaction (PCR) method, 2 × 10 ⁵ human hepatocytes cultured for 4 days in the presence of 5 micro-mol / L myricetin in 4-day-old neonatal liver (20 micro-L as a physiological saline suspension) was directly penetrated and implanted (using a 27 gauge injection needle). As controls, human hepatocytes cultured in physiological saline alone or in a medium without the addition of myricetin for 10 days were administered. Thereafter, the survival days of the mice were determined. Table 1 shows the results.

  FIG. 4A is a diagram showing changes in mouse appearance when human hepatocytes were transplanted into the liver of an ornithine transcarbamylase-deficient immunodeficient mouse (OTCD-CB-17 / IcrHsd-Prkdcscid), three weeks after human hepatocyte transplantation. This shows the condition in the eyes. FIG. 4B is a diagram showing human hepatocyte engraftment images in liver tissue when human hepatocytes are transplanted into the liver of an ornithine transcarbamylase-deficient immunodeficient mouse. Each square in the liver tissue photograph indicates the right field of view. . Here, Ctrl; untreated mice (negative control), Non treated hHPCs; transplantation of cultured human hepatocytes without myricetin, Treated hHPCs; transplantation of cultured human hepatocytes with myricetin.

In addition, human hepatocytes cultured for 10 days in the presence of 5 micro-mol / L myricetin or 2 × 10 ⁵ human hepatocytes cultured for 10 days in the absence of myricetin were transplanted into immunodeficient SCID mouse neonatal liver, Sera were sacrificed daily and serum and liver were collected, and human hepatocytes in liver tissue (FIG. 5A) and the amount of human albumin in serum (FIG. 5B) were examined. FIG. 5A is a diagram showing the evaluation of engraftment of human hepatocytes transplanted into the liver of an immunodeficient mouse (CB-17 / IcrHsd-Prkdcscid). Each square in the photograph indicates a visual field on the right side. Here, Ctrl; untreated mouse liver tissue (negative control), hLiver; human liver tissue (positive control), Non treated hHPCs; transplantation of cultured human hepatocytes without myricetin, Treated hHPCs; transplantation of cultured hepatocytes with myricetin It is. Immunohistochemical staining involves fixing liver tissue with 10% neutral formalin, making sections, reacting with rabbit anti-human albumin antibody (Inter-Cell Technologies, Jupiter, FL), and horseradish peroxidase (HRP) -conjugated. It was detected with a secondary antibody. FIG. 5B shows the amount of human albumin in mouse blood when human hepatocytes were transplanted into the liver of an immunodeficient mouse (CB-17 / IcrHsd-Prkdcscid). The measurement of the amount of human albumin was performed using the Albumin ELISA Quantitation Kit, Human (Bethyl, E80-129). As shown in FIG. 5A, the cultured human hepatocytes supplemented with myricetin exhibited excellent engraftment after transplantation. Further, as shown in FIG. 5B, the cultured human hepatocytes added with myricetin were superior in increasing the amount of albumin after transplantation.

Claims (1)

A normal human hepatocyte is cultured in a medium containing 1 to 10 μmol / L myricetin so that the normal human hepatocyte has an ammonia removing ability of 50 to 100 fmol / hr / cell. A method for improving the ability of hepatocytes to remove ammonia.