WO2021075451A1 - Blood-tissue barrier in vitro model and method for evaluating blood-tissue barrier transportability of drug - Google Patents

Blood-tissue barrier in vitro model and method for evaluating blood-tissue barrier transportability of drug Download PDF

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WO2021075451A1
WO2021075451A1 PCT/JP2020/038721 JP2020038721W WO2021075451A1 WO 2021075451 A1 WO2021075451 A1 WO 2021075451A1 JP 2020038721 W JP2020038721 W JP 2020038721W WO 2021075451 A1 WO2021075451 A1 WO 2021075451A1
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drug
blood
model
vitro model
tissue barrier
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俊明 竹澤
美秋 宇津
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国立研究開発法人農業・食品産業技術総合研究機構
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Priority to JP2021552405A priority Critical patent/JP7336154B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to an in vitro model of blood tissue barrier and a method for evaluating blood tissue barrier transferability of a drug.
  • the present application claims priority based on Japanese Patent Application No. 2019-190832 filed in Japan on October 18, 2019, the contents of which are incorporated herein by reference.
  • vascular endothelium that can extrapolate the permeability of chemical substances in the capillaries of normal human tissues such as the blood-brain barrier, skin, and liver sinusoids, and the capillaries of focal tissues such as cancer and inflammation.
  • the development of a culture model has been renowned.
  • various culture models have been developed by culturing vascular endothelial cells on a porous membrane (see, for example, Patent Documents 1 to 4 and Non-Patent Document 1).
  • Patent Document 1 proposes an in vitro model of the blood-brain barrier constructed by proliferating endothelial cells of cerebral microvessels on a porous individual support until it becomes a confluent monolayer.
  • Patent Document 2 proposes a blood-brain barrier reconstruction model by co-culture of an immortalized brain capillary endothelial cell line derived from a transgenic rat and an immortalized astrocyte cell line derived from the same rat.
  • Patent Document 3 a blood-brain barrier in vitro model constructed by a co-culture system of brain capillary endothelial cells, astrocytes, and pericytes, and a pathological blood-brain barrier constructed by using a culture solution corresponding to a predetermined pathological condition are used.
  • An in vitro model has been proposed.
  • Patent Document 4 three types of cell layers, vascular endothelial cells, pericytes, and astrocytes, form a layer structure in a state where they can directly interact with each other, thereby reproducing the anatomical structure more accurately.
  • An in vitro model of the blood-brain barrier has been proposed.
  • Non-Patent Document 1 the world's first in vitro reconstruction model of the blood-brain barrier has been developed.
  • Pericytes from brain microvessels strength the barrier integrity in primary cultures of rat brain Hayashi K, Nakaoke R, Kataoka Y, Niwa M.
  • the present invention has been made in view of the above circumstances, and is an in vitro model of the blood tissue barrier of various human-type tissues including brain tissue, which can be easily constructed, and the blood tissue having simple and excellent reproducibility.
  • the present invention includes the following aspects.
  • [1] The collagen vitrigel membrane, human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane, organ-derived cells arranged on the lower surface of the collagen vitrigel membrane, and a culture solution are included.
  • a blood tissue barrier in vitro model in which human vascular endothelial cells are cells that can differentiate into vascular endothelial cells of the organ depending on the organ-derived cells.
  • the human vascular endothelial cells include a collagen vitrigel membrane, human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane, and an acclimatized culture solution of organ-derived cells, and the human vascular endothelial cells become the organ-derived cells.
  • An in vitro model of the blood tissue barrier which is a cell that can differentiate into vascular endothelial cells of the organ in a dependent manner.
  • the drug is added to the upper part, and after a certain period of time, the amount of the drug leaked to the lower part of the collagen in vitro gel membrane is measured and compared to evaluate the pathological tissue selective migration of the drug.
  • an in vitro model of a blood tissue barrier of various human tissues including brain tissue which can be easily constructed, and a simple and excellent reproducible blood tissue barrier in vitro model of a drug
  • a method for evaluating blood tissue barrier transferability can be provided.
  • FIG. 100 It is a schematic diagram of the blood tissue barrier in vitro model 100 of the embodiment. It is a schematic diagram of the blood tissue barrier in vitro model 101 of the embodiment. It is a figure which shows the manufacturing process of the vascular endothelial culture model in Example 1.
  • FIG. It is a photograph of the cell in the vascular endothelial culture model in Example 1. It is a measurement result of the transendothelium electric resistance value of the skin vascular endothelial culture model in Example 2. It is the result of examination of the effect of the conditioned culture solution derived from the "organ-like plate type" model of skin blood vessels in Example 3. It is a measurement result of the transendothelium electric resistance value of the inflammatory cutaneous vascular endothelial model in Example 4.
  • Example 5 It is the permeability evaluation result of the fluorescent substance of the inflammatory skin vascular endothelial culture model in Example 5. It is a measurement result of the transendothelium electric resistance value of the tissue-specific vascular endothelial culture model in Example 6.
  • A It is a photograph of the parent strain (Parent) and the clone cell line (clone A) of hCMEC / D3 in Example 7.
  • B It is a measurement result of the transendothelium electrical resistance value of hCMEC / D3 (Partent).
  • C It is a measurement result of the transendothelial electrical resistance value of hCMEC / D3 (clone A).
  • the present invention comprises a collagen vitrigel membrane, human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane, organ-derived cells arranged on the lower surface of the collagen vitrigel membrane, and a culture solution.
  • human vascular endothelial cells are cells that can differentiate into vascular endothelial cells of the organ depending on the organ-derived cells.
  • FIG. 1 is a schematic view of the blood tissue barrier in vitro model 100 of the first embodiment.
  • the incubator 1 contains the culture solution 2, and the collagen vitrigel membrane 3 is immersed in the culture solution 2 by a hanger 4 for suspending the collagen vitrigel membrane 3.
  • Human vascular endothelial cells 5 are arranged on the upper surface of the collagen vitrigel membrane 3
  • organ-derived cells 6 are arranged on the lower surface of the collagen vitrigel membrane 3, due to the porosity of the collagen vitrigel membrane 3. Cross talk between both cells is possible.
  • the human vascular endothelial cell 5 is a cell that is in a proliferative state and has not yet undergone terminal differentiation, and is a cell that can differentiate into the vascular endothelial cell of the organ depending on the organ-derived cell 6.
  • human vascular endothelial cells 5 are cells that differentiate into cerebral vascular endothelial cells constituting the blood-brain barrier.
  • human vascular endothelial cells include human neonatal capsule-derived microvascular endothelial cells, various human stem cells (iPS cells, ES cells, etc.), and hCMEC / D3 cells (SV-40 rage T antigen-introduced human brain microvascular endothelium). Cell line) and the like.
  • the conventional blood-brain barrier model having a barrier function is not an easily operable culture model because it is constructed by a co-culture system of vascular endothelial cells and heterologous cells such as pericytes and astrocytes.
  • a method for producing a human blood-brain barrier model a vector containing a temperature-sensitive SV-40 large T antigen is isolated and cultured from the human blood-brain barrier, and brain microvascular endothelial cells, astrocytes, and astrosites are used.
  • a method of introducing it into pericytes and producing it has been reported, it has not been easy to control the temperature of the culture.
  • the blood tissue barrier in vitro model can be easily constructed by using the above-mentioned human vascular endothelial cells.
  • the blood tissue barrier in vitro model 100 of this embodiment can be applied to the blood tissue barrier in vitro model of various human tissues including brain tissue.
  • Applicable tissues include the blood-brain barrier, skin, liver sinusoids and the like.
  • organ-derived cells 6 placed on the collagen vitrigel membrane 3 it is possible to support these blood tissue barrier in vitro models.
  • Corresponding organ-derived cells 6 include kidney-derived cells, digestive organ-derived cells such as small intestine, urinary organ-derived cells such as bladder, skeletal muscle-derived cells, myocardial-derived cells, smooth muscle-derived cells, adipose-derived cells, lung-derived cells, and pancreas-derived cells.
  • Examples thereof include cells, adrenal-derived cells, thyroid-derived cells, skin-derived cells, brain-derived cells, liver-derived cells and the like. More specifically, human skin-derived fibroblasts, C6 cells (cell line derived from rat nerve glial cell tumor), HepG2-NIAS cells (human hepatoma cell line) and the like can be mentioned.
  • the "bitrigel” is a conventional hydrogel after the free water in the hydrogel is completely removed and then the bound water is partially removed to promote the trademarkization. It refers to a gel in a stable state obtained by rehydration, and has been named "vitrigel (registered trademark)" by the present inventor. In the present specification, when the term “Vitrigel” is used, the term “(registered trademark)” may be omitted. Further, as a more preferable raw material among collagen, native collagen or atelocollagen can be exemplified, and native collagen is further preferable.
  • the culture solution 2 contained in the incubator 1 includes DMEM, Minimum Essential Medium (MEM), RPMI-1640, Glasgow Medium Eagle (BME), Dulvecco's Modified Eagle's Medium / NutritionM. -12), Glasgow Minimium Essential Medium (Glasgow MEM) and the like.
  • MEM Minimum Essential Medium
  • BME Glasgow Medium Eagle
  • BME Dulvecco's Modified Eagle's Medium / NutritionM. -12
  • Glasgow Minimium Essential Medium Glasgow MEM
  • serum-free culture is performed from the viewpoint of avoiding non-specific adsorption of the compound to be evaluated to the protein. Liquid is preferred.
  • FIG. 2 is a schematic view of the blood tissue barrier in vitro model 101 of the second embodiment. Unlike the blood tissue barrier in vitro model 100 of the first embodiment, the organ-derived cells 6 are not arranged on the lower surface of the collagen vitrigel membrane 3, and the conditioned culture solution 20 is used instead of the culture solution 2.
  • Examples of the conditioned culture solution 20 include the culture solution used for culturing the organ-derived cells 6 for a certain period of time.
  • the culture solution used in the blood tissue barrier in vitro model 100 of the first embodiment may be used in this embodiment.
  • the culture solution used in the hanger in the blood tissue barrier in vitro model 100 is directly transferred into the hanger in the blood tissue barrier in vitro model 101, and the blood tissue barrier is transferred.
  • the culture solution outside the hanger in the in vitro model 100 may be directly transferred to the outside of the hanger in the blood tissue barrier in vitro model 101, or the culture solution in the hanger in the blood tissue barrier in vitro model 100 may be transferred to the outside of the hanger in the blood tissue barrier in vitro model 101.
  • the culture solution outside the hanger in the blood tissue barrier in vitro model 100 may be transferred into the hanger in the blood tissue barrier in vitro model 101, or the culture solution inside and outside the hanger in the blood tissue barrier in vitro model 100 may be mixed and used to mix the culture solution inside and outside the hanger in the blood tissue barrier in vitro model 101. You may move to.
  • the conditioned culture solution 20 By using the conditioned culture solution 20, it is possible to save the trouble of arranging the organ-derived cells 6 on the lower surface of the collagen vitrigel membrane 3. Further, as will be described later, when the blood tissue barrier in vitro model 101 of the present embodiment is used in the method for evaluating the transferability of a drug to the blood tissue barrier, non-specific adsorption of the compound to be evaluated to the organ-derived cell 6 is performed. It can be avoided. Further, from the viewpoint of avoiding non-specific adsorption of the compound to be evaluated to the protein, it is preferable to use a serum-free culture solution as the acclimation culture solution 20.
  • the present invention uses the blood tissue barrier in vitro model to add a drug to the upper part of the collagen vitrigel membrane, and after a certain period of time, the drug leaked to the lower part of the collagen vitrigel membrane.
  • a method for evaluating the transferability of a drug to the blood tissue barrier to measure the amount of the drug is provided.
  • the present invention uses the blood tissue barrier in vitro model to prepare an in vitro model of a normal state and an in vitro model of a pathological condition, and in both in vitro models, a drug is applied to the upper part of the collagen vitrigel membrane. After a certain period of time after addition, the amount of the drug leaked to the lower part of the collagen in vitro gel membrane was measured and compared to evaluate the pathological tissue selective migration of the drug. Provide a sex evaluation method.
  • an in vitro model of a pathological condition can be prepared by using a culture solution corresponding to the pathological condition, or by using cells derived from a patient suffering from a disease. Then, by examining the tissue transferability of the drug in each of the in vitro model of the normal state and the in vitro model of the pathological condition, it is possible to screen the drug that selectively migrates to the pathological tissue.
  • Example 1 Preparation of vascular endothelial culture model using human neonatal foramen-derived microvascular endothelial cells (HMVEC) and fibroblasts derived from the same (human dermal fibroblast; HDF)
  • HMVEC human neonatal foramen-derived microvascular endothelial cells
  • HDF human dermal fibroblast
  • Pre-cultured HDF purchased from Kurabo Industries, KF-4009 was collected and mixed with the culture medium such that 1.3 ⁇ 10 5 cells / mL, to prepare a suspension of HDF.
  • As the culture medium 10% fetal bovine serum (FBS), 20 mM HEPES, 100 units / mL penicillin, 100 ⁇ g / mL streptomycin-containing DMEM (purchased from Thermo Fisher Scientific, 11885-084) was used.
  • FBS fetal bovine serum
  • HEPES 100 units / mL penicillin
  • streptomycin-containing DMEM purchased from Therm
  • the culture medium was 5% FBS, 5 ng / mL recombinant human FGF-b, 50 ⁇ g / mL ascorbic acid, 1 ⁇ g / mL hydrocortisone hemicouccinate, 10 mM L-glutamine, 15 ng / mL recombinant human IGF-1, 5 ng / mL recombinant human EGF.
  • a model in which HMVEC and HDF are cultured on both sides of the insert is an "organ-like plate type” model of skin blood vessels, and a model in which only one type of cells of HMVEC or HDF is cultured is a "tissue sheet type”. Defined as a model.
  • the “organ-like plate type” model see (A) to (C) in FIG. 4
  • the “tissue sheet type” model of HMVEC are used for day0, day2, and day6 (FIG. 4). See (D) to (F) of 4.).
  • a "tissue sheet type” model of HDF was observed.
  • Example 2 Measurement of changes over time in transendothelial electrical resistance (TEER) values of a cutaneous vascular endothelial culture model (1)
  • the TER values of the three models prepared in Example 1 are set to day0, day1, and so on. Measurements were made on day2, day3, and day6. Before the measurement, the culture broth inside and outside the chamber of each model was removed, and 0.5 mL of the HMVEC culture broth that had been returned to room temperature in advance was added into the chamber and 1.5 mL outside the chamber.
  • TEER transendothelial electrical resistance
  • the "organ-like plate type” model showed higher TER values than day1, and the TER values of day1, day2, day3, and day6 were 22.62 ⁇ 3.06 ⁇ ⁇ cm 2 , 31.50 ⁇ 2, respectively. It was .46 ⁇ ⁇ cm 2 , 42.63 ⁇ 3.94 ⁇ ⁇ cm 2 , and 57.24 ⁇ 7.29 ⁇ ⁇ cm 2 . From the above, it was confirmed that the "organ-like plate type” model has a high endothelial barrier function.
  • Example 3 Examination of the effect of the conditioned medium (CM) derived from the "organ-like plate type” model of skin blood vessels (1)
  • the HMVEC "tissue sheet type” model of day 6 used in Example 2 The culture medium inside and outside the chamber was removed, and CM inside and outside the chamber of the "organ-like plate type” model of day 6 also used in Example 2 was added and cultured for 24 hours (see FIG. 6 (A)). ..
  • the TER value of the HMVEC "tissue sheet type” model (HMVEC + culture solution) to which the culture solution was added was 14.5 ⁇ 1.51 ⁇ ⁇ cm 2.
  • Example 4 Measurement of TER value of inflamed cutaneous vascular endothelial model (1) Cultivation inside and outside the chamber of the HMVEC "tissue sheet type” model and "organ-like plate type” model of day 6 used in Example 2. The liquid was removed, and 0.5 mL of VascuLife® Basal Medium containing no additives, which had been returned to room temperature in advance, was added into the chamber and 1.5 mL outside the chamber. (2) Next, an electrode was set in the chamber and the TER value was measured. The measured value at this time was taken as the value at the start of measurement (0 seconds).
  • VascuLife (registered trademark) Basal Medium in the chamber was removed, and VascuLife (registered trademark) Basal Medium containing histamine dihydrochloride (purchased from Tokyo Chemical Industry Co., Ltd., H0146) was added.
  • the TEER measurement was started, and the TEER value was continuously measured every 10 seconds for 180 seconds.
  • the value at the start of measurement was about 50 ⁇ ⁇ cm 2 , but it decreased to about 32 ⁇ ⁇ cm 2 with the addition of 1 nM histamine, and this change was controlled without adding histamine. It was a statistically significant change in comparison ( *** P ⁇ 0.001, tested by Tukey-Kramer method after two-way ANOVA).
  • Example 5 Evaluation of fluorescence substance permeability of inflamed skin vascular endothelial culture model (1) The culture medium inside and outside the chamber of the HMVEC “tissue sheet type” model of day 6 used in Example 2 was removed. Hank's Balanced Salt Solution (HBSS, purchased from Thermo Fisher Scientific, 14025-092), which had been returned to room temperature in advance, was washed twice by adding 0.5 mL into the chamber and 1.5 mL outside the chamber. (2) Next, 1.8 mL of HBSS was added to the outside of the chamber and 0.5 mL of HBSS containing 100 ⁇ M histamine was added to the inside of the chamber, and the mixture was incubated at room temperature for 10 minutes.
  • HBSS Hank's Balanced Salt Solution
  • P- app dQ / dt x 1 / A x 1 / C 0 dQ / dt; Transition amount per unit time ( ⁇ g / sec), A; Surface area (cm 2 ), C 0 ; Sodium fluorescein (molecular weight 376), FD-4 (molecular weight 376), by addition of 100 ⁇ M histamine initial concentration of each fluorescent substance. molecular weight of 4,000), and FD-40 (also P app of any molecules of molecular weight 40,000) was increased (FIG. 8 (a) - (C) see).
  • an in vitro model of a normal state and an in vitro model of a pathological condition are prepared, and a drug having permeability selectively in the pathological tissue is screened by comparing and evaluating the transferability of the drug in both in vitro models. can do.
  • Example 6 Preparation of tissue-specific vascular endothelial culture model and measurement of TEER value (1) HMVEC and rat in the same manner as the "organ-like plate type" model using HMVEC and HDF prepared in Example 1.
  • Grioma cell line C6 (provided by RIKEN BioResource Center, RCB2854) or HMVEC and liver cancer cell line HepG2-NIAS (cell line in which HepG2 provided by RIKEN Bioresource Center was conditioned and re-deposited, RCB4679)
  • the "organ-like plate type" model used was prepared.
  • DMEM containing 10% FBS, 20 mM HEPES, 100 units / mL penicillin, and 100 ⁇ g / mL streptomycin was used.
  • the change in the TEER value over time was measured by the same method as in Example 2. The results are shown in FIG. Not only skin-derived HDF (see FIG. 9 (A)) but also brain-derived C6 (see FIG. 9 (B)) and liver-derived HepG2-NIAS (see FIG. 9 (C)) can be used. It was confirmed that a vascular endothelial culture model could be prepared.
  • hCMEC / D3 clone cell line and measurement of TEER value hCMEC / D3 is a human vascular endothelial cell line that has high proliferative ability and is widely used, but has a low endothelial barrier function, so that it is a blood tissue barrier model. Not suitable for building. Therefore, a cloned cell line having an endothelial barrier function was selected from the cells, and its inflammatory responsiveness was confirmed.
  • Clone A was cultured in a collagen vitrigel membrane chamber for 10 days, 100 ⁇ M of histamine (His) was added into the chamber, and the TER value was measured every 1 minute. As a result, 15 minutes after the addition of histamine, the value at the start of the measurement was measured. Decreased by about 70% (see FIG. 11). This change was statistically significant compared to Control ( * *** P ⁇ 0.001 two-way ANOVA followed by Tukey-Kramer test). On the other hand, pretreatment with Y-27632, an inhibitor of ROCK, which is a downstream factor of histamine receptor, significantly suppressed the decrease in TER value due to histamine ( ### P ⁇ 0.001, binary). Tested by Tukey-Kramer method after ANOVA), it was suggested that clone A is a cell line with excellent histamine responsiveness (see FIG. 11).
  • Clone A is cultured in a collagen vitrigel membrane chamber for 16 days, and 100 ⁇ M of histamine (His) and four kinds of fluorescent substances (Sodium fluorescein, FD-4, FD-10, and FD-40) are added to the chamber. After incubating for 1 hour, the amount of fluorescent substance transferred to the outside of the chamber was measured. As a result, histamine enhanced only the permeability of FD-4 (molecular weight 4,000) and FD-10 (molecular weight 10,000) (see FIG. 12). In particular, the increase in permeability of FD-10 was a statistically significant change ( * P ⁇ 0.05, tested by t-test).
  • histamine-induced increase in vascular permeability in clone A has molecular size selectivity.
  • this cloned cell line is suitable for constructing a model for evaluating the tissue transfer of a drug. ..
  • Example 8 Examination of the effect of CM derived from the "tissue sheet type" model of HDF (1)
  • the HDF cultured in advance was collected and mixed with the culture solution so as to have 8.0 ⁇ 10 4 cells / mL.
  • a suspension of HDF was prepared.
  • the culture medium is 5% FBS, 5 ng / mL recombinant human FGF-b, 50 ⁇ g / mL ascorbic acid, 1 ⁇ g / mL hydrocortisone heparinate, 10 mM L-glutamine, 15 ng / mL recombinant human IGF-1, 5 ng / mL recombinant.
  • Human EGF 5 ng / mL recombinant human VEGF, 0.75 units / mL heparin sulfate, 30 mg / mL gentamicin, 15 ⁇ g / mL Amhotericin B-containing VascuLife® Basal Medium was used.
  • the recovered CM (HDF-CM in and HDF-CM out) or culture solution was added from day 0 to day 7 inside and outside the chamber of the HMVEC “tissue sheet type” model prepared in Example 1.
  • Day 4 and day 7 measured the TER value.
  • the TEER value of the HMVEC "tissue sheet type" model (HMVEC + culture solution) to which the culture solution was added was 18.0 ⁇ 1 in day 4 (see FIG. 13 (C)) and day 7 (see FIG. 13 (D)), respectively. It was .41 ⁇ ⁇ cm 2 and 17.6 ⁇ 3.17 ⁇ ⁇ cm 2 .
  • the TER values of the same model (HMVEC + (HDF-CM in)) to which HDF-CM in was added were 33.5 ⁇ 1.01 ⁇ ⁇ cm 2 and 33.0 ⁇ 2. There was 67 ⁇ ⁇ cm 2 .
  • the TEER value of the same model (HMVEC + (HDF-CM out)) with HDF-CM out added is 33.9 ⁇ 3.68 ⁇ ⁇ cm 2 and 32.7 ⁇ 2.19 ⁇ ⁇ cm 2 on day 4 and day 7, respectively. Met.
  • the TER value when HDF-CM in and HDF-CM out were added was statistically significantly higher in both day 4 and day 7 than when the culture medium was added ( ** P).
  • Example 3 Comparing the above results with Example 3, it was shown that the CM of the HDF "tissue sheet type" model has a stronger effect of strengthening the endothelial barrier function than the CM of the "organ-like plate type” model.
  • a tissue specificity using an easily constructable blood tissue barrier in vitro model of various human tissues including brain tissue and the blood tissue barrier in vitro model having simple and excellent reproducibility can be provided.

Abstract

This blood-tissue barrier in vitro model contains: a collagen vitrigel membrane; human vascular endothelial cells disposed on the top surface of this collagen vitrigel membrane; organ-derived cells disposed on the bottom surface of this collagen vitrigel membrane; and a culture solution. The human vascular endothelial cells are dependent on the organ-derived cells and are capable of differentiating into the vascular endothelial cells of that organ.

Description

血液組織関門インビトロモデル、及び薬物の血液組織関門移行性評価方法Blood tissue barrier in vitro model and method for evaluating blood tissue barrier migration of drugs
　本発明は、血液組織関門インビトロモデル、及び薬物の血液組織関門移行性評価方法に関する。
　本願は、２０１９年１０月１８日に、日本に出願された特願２０１９－１９０８３２号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to an in vitro model of blood tissue barrier and a method for evaluating blood tissue barrier transferability of a drug.
The present application claims priority based on Japanese Patent Application No. 2019-190832 filed in Japan on October 18, 2019, the contents of which are incorporated herein by reference.
　創薬及び医療の分野では、血液脳関門、皮膚、肝類洞等のヒト正常組織の毛細血管や、癌や炎症等の病巣組織の毛細血管における化学物質の透過性を外挿できる血管内皮の培養モデルの開発が切望されてきた。
　特に、血液脳関門については、多孔質膜上で血管内皮細胞を培養することで、様々な培養モデルが開発されてきた（例えば、特許文献１～４及び非特許文献１参照。）。 In the fields of drug discovery and medicine, vascular endothelium that can extrapolate the permeability of chemical substances in the capillaries of normal human tissues such as the blood-brain barrier, skin, and liver sinusoids, and the capillaries of focal tissues such as cancer and inflammation. The development of a culture model has been coveted.
In particular, for the blood-brain barrier, various culture models have been developed by culturing vascular endothelial cells on a porous membrane (see, for example, Patent Documents 1 to 4 and Non-Patent Document 1).
　バリア機能を有する血管内皮の培養モデルとして、特許文献１で脳微小血管の内皮細胞を多孔質の個体支持体上で、コンフルエント単層となるまで増殖させて構築した血液脳関門のインビトロモデルが提案されている。
　特許文献２では、トランスジェニックラット由来の不死化脳毛細血管内皮細胞株と同ラット由来の不死化アストロサイト細胞株との共培養による血液脳関門再構築モデルが提案されている。
　特許文献３では、脳毛細血管内皮細胞、アストロサイト、及びペリサイトの共培養系で構築した血液脳関門インビトロモデル、及び所定の病態条件に対応する培養液を利用して構築した病態血液脳関門インビトロモデルが提案されている。
　特許文献４では、血管内皮細胞、ペリサイト、及びアストロサイトの３種類の細胞層が、直接的な相互作用が可能な状態で層構造を形成することで、解剖学的構造をより正確に再現した血液脳関門インビトロモデルが提案されている。
　非特許文献１では、世界で初めて血液脳関門のｉｎ　ｖｉｔｒｏ再構築系モデルが開発されている。 As a culture model of vascular endothelium having a barrier function, Patent Document 1 proposes an in vitro model of the blood-brain barrier constructed by proliferating endothelial cells of cerebral microvessels on a porous individual support until it becomes a confluent monolayer. Has been done.
Patent Document 2 proposes a blood-brain barrier reconstruction model by co-culture of an immortalized brain capillary endothelial cell line derived from a transgenic rat and an immortalized astrocyte cell line derived from the same rat.
In Patent Document 3, a blood-brain barrier in vitro model constructed by a co-culture system of brain capillary endothelial cells, astrocytes, and pericytes, and a pathological blood-brain barrier constructed by using a culture solution corresponding to a predetermined pathological condition are used. An in vitro model has been proposed.
In Patent Document 4, three types of cell layers, vascular endothelial cells, pericytes, and astrocytes, form a layer structure in a state where they can directly interact with each other, thereby reproducing the anatomical structure more accurately. An in vitro model of the blood-brain barrier has been proposed.
In Non-Patent Document 1, the world's first in vitro reconstruction model of the blood-brain barrier has been developed.
国際公開第１９９１／０５０３８号International Publication No. 1991/05038 特開２００１－２３８６８１号公報Japanese Unexamined Patent Publication No. 2001-238681 特許第５１１３３３２号公報Japanese Patent No. 5113332 国際公開第２０１７／１７９３７５号International Publication No. 2017/179375
　しかし、上述した先行技術文献においては、（１）操作性が容易な培養モデルであること、（２）ヒト型の培養モデルであること、（３）化学物質の透過性試験までを行うにあたり、簡便な操作性と優れた再現性を有すること等が満足されていない技術的な課題を有するのみならず、ヒト型の様々な組織の血管内皮の培養モデルが開発されていないことから、これまでの培養モデルはいずれも製薬会社等で動物実験を代替して普及する段階には達していない。 However, in the above-mentioned prior art literature, (1) a culture model with easy operability, (2) a human-type culture model, and (3) a chemical substance permeability test are performed. Not only does it have technical problems that are not satisfied with its easy operability and excellent reproducibility, but also because culture models of vascular endothelium of various human-type tissues have not been developed so far. None of these culture models have reached the stage where they are used by pharmaceutical companies as a substitute for animal experiments.
　本発明は、上記事情を鑑みてなされたものであり、容易に構築可能な、脳組織を含めヒト型の様々な組織の血液組織関門インビトロモデル、及び簡便かつ優れた再現性を有する当該血液組織関門インビトロモデルを利用した、薬物の血液組織関門移行性評価方法を提供する。 The present invention has been made in view of the above circumstances, and is an in vitro model of the blood tissue barrier of various human-type tissues including brain tissue, which can be easily constructed, and the blood tissue having simple and excellent reproducibility. We provide a method for evaluating the transferability of a drug to blood tissue using an in vitro model.
　本発明は以下の態様を含む。
［１］コラーゲンビトリゲル膜と、前記コラーゲンビトリゲル膜の上面に配置されたヒト血管内皮細胞と、前記コラーゲンビトリゲル膜の下面に配置された臓器由来細胞と、培養液と、を含み、前記ヒト血管内皮細胞は、前記臓器由来細胞に依存して、前記臓器の血管内皮細胞に分化し得る細胞である、血液組織関門インビトロモデル。
［２］前記培養液は、無血清培養液である、［１］に記載の血液組織関門インビトロモデル。
［３］コラーゲンビトリゲル膜と、前記コラーゲンビトリゲル膜の上面に配置されたヒト血管内皮細胞と、臓器由来細胞の馴化培養液と、を含み、前記ヒト血管内皮細胞は、前記臓器由来細胞に依存して、前記臓器の血管内皮細胞に分化し得る細胞である、血液組織関門インビトロモデル。
［４］前記馴化培養液は、無血清培養液である、［３］に記載の血液組織関門インビトロモデル。
［５］［１］～［４］のいずれか一つに記載の血液組織関門インビトロモデルを用いて、前記コラーゲンビトリゲル膜の上方部に薬剤を添加し、一定時間後に、前記コラーゲンビトリゲル膜の下方部に漏れ出た、薬物の量を測定する、薬物の血液組織関門移行性評価方法。
［６］［１］～［４］のいずれか一つに記載の血液組織関門インビトロモデルにおいて、正常状態のインビトロモデルと病態のインビトロモデルを用意し、両インビトロモデルにおいて、前記コラーゲンビトリゲル膜の上方部に薬剤を添加し、一定時間後に、前記コラーゲンビトリゲル膜の下方部に漏れ出た、薬物の量をそれぞれ測定し、比較することにより、薬物の病態組織選択的移行性を評価する、薬物の血液組織関門移行性評価方法。 The present invention includes the following aspects.
[1] The collagen vitrigel membrane, human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane, organ-derived cells arranged on the lower surface of the collagen vitrigel membrane, and a culture solution are included. A blood tissue barrier in vitro model in which human vascular endothelial cells are cells that can differentiate into vascular endothelial cells of the organ depending on the organ-derived cells.
[2] The blood tissue barrier in vitro model according to [1], wherein the culture solution is a serum-free culture solution.
[3] The human vascular endothelial cells include a collagen vitrigel membrane, human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane, and an acclimatized culture solution of organ-derived cells, and the human vascular endothelial cells become the organ-derived cells. An in vitro model of the blood tissue barrier, which is a cell that can differentiate into vascular endothelial cells of the organ in a dependent manner.
[4] The blood tissue barrier in vitro model according to [3], wherein the conditioned culture medium is a serum-free culture medium.
[5] Using the blood tissue barrier in vitro model according to any one of [1] to [4], a drug is added to the upper part of the collagen vitrigel membrane, and after a certain period of time, the collagen vitrigel membrane is used. A method for evaluating the transferability of a drug to the blood tissue barrier, which measures the amount of the drug leaked to the lower part of the drug.
[6] In the blood tissue barrier in vitro model according to any one of [1] to [4], an in vitro model of a normal state and an in vitro model of a pathological condition are prepared. The drug is added to the upper part, and after a certain period of time, the amount of the drug leaked to the lower part of the collagen in vitro gel membrane is measured and compared to evaluate the pathological tissue selective migration of the drug. A method for evaluating the transferability of a drug to the blood tissue barrier.
　本発明によれば、容易に構築可能な、脳組織を含めヒト型の様々な組織の血液組織関門インビトロモデル、及び簡便かつ優れた再現性を有する当該血液組織関門インビトロモデルを利用した、薬物の血液組織関門移行性評価方法を提供することができる。 According to the present invention, an in vitro model of a blood tissue barrier of various human tissues including brain tissue, which can be easily constructed, and a simple and excellent reproducible blood tissue barrier in vitro model of a drug A method for evaluating blood tissue barrier transferability can be provided.
実施形態の血液組織関門インビトロモデル１００の概略図である。It is a schematic diagram of the blood tissue barrier in vitro model 100 of the embodiment. 実施形態の血液組織関門インビトロモデル１０１の概略図である。It is a schematic diagram of the blood tissue barrier in vitro model 101 of the embodiment. 実施例１における血管内皮培養モデルの作製工程を示す図である。It is a figure which shows the manufacturing process of the vascular endothelial culture model in Example 1. FIG. 実施例１における血管内皮培養モデルにおける細胞の写真である。It is a photograph of the cell in the vascular endothelial culture model in Example 1. 実施例２における皮膚血管内皮培養モデルの経内皮電気抵抗値の測定結果である。It is a measurement result of the transendothelium electric resistance value of the skin vascular endothelial culture model in Example 2. 実施例３における皮膚血管の「器官様プレート型」モデル由来の馴化培養液の効果の検討結果である。It is the result of examination of the effect of the conditioned culture solution derived from the "organ-like plate type" model of skin blood vessels in Example 3. 実施例４における炎症皮膚血管内皮モデルの経内皮電気抵抗値の測定結果である。It is a measurement result of the transendothelium electric resistance value of the inflammatory cutaneous vascular endothelial model in Example 4. 実施例５における炎症皮膚血管内皮培養モデルの蛍光物質の透過性評価結果である。It is the permeability evaluation result of the fluorescent substance of the inflammatory skin vascular endothelial culture model in Example 5. 実施例６における組織特異的血管内皮培養モデルの経内皮電気抵抗値の測定結果である。It is a measurement result of the transendothelium electric resistance value of the tissue-specific vascular endothelial culture model in Example 6. （Ａ）実施例７におけるｈＣＭＥＣ／Ｄ３の親株（Ｐａｒｅｎｔ）及びクローン細胞株（クローンＡ）の写真である。（Ｂ）ｈＣＭＥＣ／Ｄ３（Ｐａｒｅｎｔ）の経内皮電気抵抗値の測定結果である。（Ｃ）ｈＣＭＥＣ／Ｄ３（クローンＡ）の経内皮電気抵抗値の測定結果である。(A) It is a photograph of the parent strain (Parent) and the clone cell line (clone A) of hCMEC / D3 in Example 7. (B) It is a measurement result of the transendothelium electrical resistance value of hCMEC / D3 (Partent). (C) It is a measurement result of the transendothelial electrical resistance value of hCMEC / D3 (clone A). 実施例７におけるヒスタミン添加後のｈＣＭＥＣ／Ｄ３（クローンＡ）の経内皮電気抵抗値の測定結果である。It is a measurement result of the transendothelial electrical resistance value of hCMEC / D3 (clone A) after the addition of histamine in Example 7. 実施例７におけるヒスタミン添加後のｈＣＭＥＣ／Ｄ３（クローンＡ）の蛍光物質の透過性評価結果である。It is the transparency evaluation result of the fluorescent substance of hCMEC / D3 (clone A) after the addition of histamine in Example 7. （Ａ）実施例８における馴化培養液回収の説明図である。（Ｂ）実施例８における馴化培養液回収の説明図である。（Ｃ）馴化培養液を加えたＨＭＶＥＣの「組織シート型」モデルにおけるｄａｙ４の経内皮電気抵抗値の測定結果である。（Ｄ）馴化培養液を加えたＨＭＶＥＣの「組織シート型」モデルにおけるｄａｙ７の経内皮電気抵抗値の測定結果である。(A) It is explanatory drawing of the acclimatization culture solution recovery in Example 8. (B) It is explanatory drawing of the acclimatization culture solution recovery in Example 8. (C) It is a measurement result of the transendothelial electrical resistance value of day4 in the "tissue sheet type" model of HMVEC to which the conditioned culture solution was added. (D) It is a measurement result of the transendothelial electrical resistance value of day7 in the "tissue sheet type" model of HMVEC to which the conditioned culture solution was added.
　以下、場合により図面を参照しつつ、本発明の実施形態について詳細に説明する。なお、各図における寸法比は、説明のため誇張している部分があり、必ずしも実際の寸法比とは一致しない。 Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings in some cases. The dimensional ratio in each figure is exaggerated for explanation and does not necessarily match the actual dimensional ratio.
≪血液組織関門インビトロモデル≫
　１実施形態において、本発明は、コラーゲンビトリゲル膜と、前記コラーゲンビトリゲル膜の上面に配置されたヒト血管内皮細胞と、前記コラーゲンビトリゲル膜の下面に配置された臓器由来細胞と、培養液と、を含み、前記ヒト血管内皮細胞は、前記臓器由来細胞に依存して、前記臓器の血管内皮細胞に分化し得る細胞である、血液組織関門インビトロモデルを提供する。 ≪Blood tissue barrier in vitro model≫
In one embodiment, the present invention comprises a collagen vitrigel membrane, human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane, organ-derived cells arranged on the lower surface of the collagen vitrigel membrane, and a culture solution. To provide an in vitro model of blood tissue barrier, wherein the human vascular endothelial cells are cells that can differentiate into vascular endothelial cells of the organ depending on the organ-derived cells.
　図１は、第１実施形態の血液組織関門インビトロモデル１００の概略図である。培養器１は、培養液２を収容しており、コラーゲンビトリゲル膜３を吊るすハンガー４によって、コラーゲンビトリゲル膜３を培養液２中に浸漬している。
　コラーゲンビトリゲル膜３の上面には、ヒト血管内皮細胞５が配置されており、コラーゲンビトリゲル膜３の下面には、臓器由来細胞６が配置されており、コラーゲンビトリゲル膜３の多孔性により両細胞間のクロストークが可能となっている。 FIG. 1 is a schematic view of the blood tissue barrier in vitro model 100 of the first embodiment. The incubator 1 contains the culture solution 2, and the collagen vitrigel membrane 3 is immersed in the culture solution 2 by a hanger 4 for suspending the collagen vitrigel membrane 3.
Human vascular endothelial cells 5 are arranged on the upper surface of the collagen vitrigel membrane 3, and organ-derived cells 6 are arranged on the lower surface of the collagen vitrigel membrane 3, due to the porosity of the collagen vitrigel membrane 3. Cross talk between both cells is possible.
　ヒト血管内皮細胞５は、増殖状態にあって未だ終末分化していない細胞であり、臓器由来細胞６に依存して、前記臓器の血管内皮細胞に分化し得る細胞である。例えば、臓器由来細胞６として、脳のアストロサイトを用いる場合、ヒト血管内皮細胞５は、血液脳関門を構成する脳血管内皮細胞に分化する細胞である。
　ヒト血管内皮細胞５としては、ヒト新生児***由来微小血管内皮細胞、各種ヒト幹細胞（ｉＰＳ細胞、ＥＳ細胞、等）、ｈＣＭＥＣ／Ｄ３細胞（ＳＶ－４０ ｌａｒｇｅ　Ｔ抗原が導入されたヒト脳微小血管内皮細胞株）等が挙げられる。 The human vascular endothelial cell 5 is a cell that is in a proliferative state and has not yet undergone terminal differentiation, and is a cell that can differentiate into the vascular endothelial cell of the organ depending on the organ-derived cell 6. For example, when brain astrocytes are used as the organ-derived cells 6, human vascular endothelial cells 5 are cells that differentiate into cerebral vascular endothelial cells constituting the blood-brain barrier.
Examples of human vascular endothelial cells include human neonatal capsule-derived microvascular endothelial cells, various human stem cells (iPS cells, ES cells, etc.), and hCMEC / D3 cells (SV-40 rage T antigen-introduced human brain microvascular endothelium). Cell line) and the like.
　従来のバリア機能を有する血液脳関門モデルは、血管内皮細胞とペリサイトとアストロサイト等の異種細胞との共培養系で構築されているため、容易に操作可能な培養モデルではなかった。
　また、ヒト型の血液脳関門モデルを生産する方法としては、温度感受性ＳＶ－４０ ｌａｒｇｅ　Ｔ抗原を含有するベクターを、ヒトの血液脳関門から単離培養した脳微小血管内皮細胞、アストロサイト、及びペリサイトに導入して作製する方法が報告されているが、培養の温度管理が容易ではなかった。
　本実施形態においては、上述したヒト血管内皮細胞を用いることで容易に血液組織関門インビトロモデルを構築することができる。 The conventional blood-brain barrier model having a barrier function is not an easily operable culture model because it is constructed by a co-culture system of vascular endothelial cells and heterologous cells such as pericytes and astrocytes.
In addition, as a method for producing a human blood-brain barrier model, a vector containing a temperature-sensitive SV-40 large T antigen is isolated and cultured from the human blood-brain barrier, and brain microvascular endothelial cells, astrocytes, and astrosites are used. Although a method of introducing it into pericytes and producing it has been reported, it has not been easy to control the temperature of the culture.
In the present embodiment, the blood tissue barrier in vitro model can be easily constructed by using the above-mentioned human vascular endothelial cells.
　本実施形態の血液組織関門インビトロモデル１００は、脳組織を含めヒト型の様々な組織の血液組織関門インビトロモデルに適用可能である。適用可能な組織としては、血液脳関門、皮膚、肝類洞等が挙げられる。コラーゲンビトリゲル膜３に配置する臓器由来細胞６を変えることで、これらの血液組織関門インビトロモデルに対応可能である。対応する臓器由来細胞６としては、腎臓由来細胞、小腸等消化器官由来細胞、膀胱等泌尿器由来細胞、骨格筋由来細胞、心筋由来細胞、平滑筋由来細胞、脂肪由来細胞、肺由来細胞、膵臓由来細胞、副腎由来細胞、甲状腺由来細胞、皮膚由来細胞、脳由来細胞、又は肝臓由来細胞等が挙げられる。より具体的には、ヒト皮膚由来線維芽細胞、Ｃ６細胞（ラット神経グリア細胞腫瘍に由来する細胞株）、ＨｅｐＧ２－ＮＩＡＳ細胞（ヒト肝がん細胞株）等が挙げられる。 The blood tissue barrier in vitro model 100 of this embodiment can be applied to the blood tissue barrier in vitro model of various human tissues including brain tissue. Applicable tissues include the blood-brain barrier, skin, liver sinusoids and the like. By changing the organ-derived cells 6 placed on the collagen vitrigel membrane 3, it is possible to support these blood tissue barrier in vitro models. Corresponding organ-derived cells 6 include kidney-derived cells, digestive organ-derived cells such as small intestine, urinary organ-derived cells such as bladder, skeletal muscle-derived cells, myocardial-derived cells, smooth muscle-derived cells, adipose-derived cells, lung-derived cells, and pancreas-derived cells. Examples thereof include cells, adrenal-derived cells, thyroid-derived cells, skin-derived cells, brain-derived cells, liver-derived cells and the like. More specifically, human skin-derived fibroblasts, C6 cells (cell line derived from rat nerve glial cell tumor), HepG2-NIAS cells (human hepatoma cell line) and the like can be mentioned.
　コラーゲンビトリゲル膜３において、「ビトリゲル」とは、従来のハイドロゲルを、ハイドロゲル内の自由水を完全に除去した後に結合水を部分除去することで、ガラス化（ｖｉｔｒｉｆｉｃａｔｉｏｎ）を進行させた後に再水和して得られる安定した状態にあるゲルのことを指し、本発明者によって、「ビトリゲル（ｖｉｔｒｉｇｅｌ）（登録商標）」と命名されている。
　本明細書において、用語「ビトリゲル」を用いる際には、用語「（登録商標）」を省略して用いる場合がある。
　また、コラーゲンの中でもより好ましい原料としては、ネイティブコラーゲン又はアテロコラーゲンを例示でき、ネイティブコラーゲンがさらに好ましい。 In the collagen vitrigel film 3, the "bitrigel" is a conventional hydrogel after the free water in the hydrogel is completely removed and then the bound water is partially removed to promote the trademarkization. It refers to a gel in a stable state obtained by rehydration, and has been named "vitrigel (registered trademark)" by the present inventor.
In the present specification, when the term "Vitrigel" is used, the term "(registered trademark)" may be omitted.
Further, as a more preferable raw material among collagen, native collagen or atelocollagen can be exemplified, and native collagen is further preferable.
　培養器１が収容する培養液２としては、ＤＭＥＭ、Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ（ＭＥＭ）、ＲＰＭＩ－１６４０、Ｂａｓａｌ　Ｍｅｄｉｕｍ　Ｅａｇｌｅ（ＢＭＥ）、Ｄｕｌｂｅｃｃｏ’ｓ　Ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ’ｓ　Ｍｅｄｉｕｍ：Ｎｕｔｒｉｅｎｔ　Ｍｉｘｔｕｒｅ　Ｆ－１２（ＤＭＥＭ／Ｆ－１２）、Ｇｌａｓｇｏｗ　Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ（Ｇｌａｓｇｏｗ ＭＥＭ）等が挙げられる。後述するように、本実施形態の血液組織関門インビトロモデル１００を薬物の血液組織関門移行性評価方法に用いる場合には、評価対象の化合物のタンパク質への非特異的吸着回避の観点から無血清培養液が好ましい。 The culture solution 2 contained in the incubator 1 includes DMEM, Minimum Essential Medium (MEM), RPMI-1640, Glasgow Medium Eagle (BME), Dulvecco's Modified Eagle's Medium / NutritionM. -12), Glasgow Minimium Essential Medium (Glasgow MEM) and the like. As will be described later, when the blood tissue barrier in vitro model 100 of the present embodiment is used as a method for evaluating the transferability of a drug to the blood tissue barrier, serum-free culture is performed from the viewpoint of avoiding non-specific adsorption of the compound to be evaluated to the protein. Liquid is preferred.
　図２は、第２実施形態の血液組織関門インビトロモデル１０１の概略図である。第１実施形態の血液組織関門インビトロモデル１００と異なり、コラーゲンビトリゲル膜３の下面には、臓器由来細胞６は配置されず、培養液２の代わりに、馴化培養液２０が用いられている。 FIG. 2 is a schematic view of the blood tissue barrier in vitro model 101 of the second embodiment. Unlike the blood tissue barrier in vitro model 100 of the first embodiment, the organ-derived cells 6 are not arranged on the lower surface of the collagen vitrigel membrane 3, and the conditioned culture solution 20 is used instead of the culture solution 2.
　馴化培養液２０としては、臓器由来細胞６を一定期間培養するのに用いられた培養液が挙げられる。また、第１実施形態の血液組織関門インビトロモデル１００で用いられた培養液を本実施形態で用いてもよい。第１実施形態の血液組織関門インビトロモデル１００で用いられた培養液を用いる場合、血液組織関門インビトロモデル１００におけるハンガー内の培養液を血液組織関門インビトロモデル１０１におけるハンガー内にそのまま移し、血液組織関門インビトロモデル１００におけるハンガー外の培養液を血液組織関門インビトロモデル１０１におけるハンガー外にそのまま移してもよく、血液組織関門インビトロモデル１００におけるハンガー内の培養液を血液組織関門インビトロモデル１０１におけるハンガー外に移し、血液組織関門インビトロモデル１００におけるハンガー外の培養液を血液組織関門インビトロモデル１０１におけるハンガー内に移してもよく、血液組織関門インビトロモデル１００におけるハンガー内外の培養液を混ぜて血液組織関門インビトロモデル１０１に移してもよい。
　馴化培養液２０を用いることで、コラーゲンビトリゲル膜３の下面に、臓器由来細胞６を配置する手間が省ける。また、後述するように、本実施形態の血液組織関門インビトロモデル１０１を、薬物の血液組織関門移行性評価方法に用いる場合には、評価対象の化合物の臓器由来細胞６への非特異的吸着を回避することができる。更に、評価対象の化合物のタンパク質への非特異的吸着回避の観点から、馴化培養液２０として、無血清培養液を用いることが好ましい。 Examples of the conditioned culture solution 20 include the culture solution used for culturing the organ-derived cells 6 for a certain period of time. In addition, the culture solution used in the blood tissue barrier in vitro model 100 of the first embodiment may be used in this embodiment. When the culture solution used in the blood tissue barrier in vitro model 100 of the first embodiment is used, the culture solution in the hanger in the blood tissue barrier in vitro model 100 is directly transferred into the hanger in the blood tissue barrier in vitro model 101, and the blood tissue barrier is transferred. The culture solution outside the hanger in the in vitro model 100 may be directly transferred to the outside of the hanger in the blood tissue barrier in vitro model 101, or the culture solution in the hanger in the blood tissue barrier in vitro model 100 may be transferred to the outside of the hanger in the blood tissue barrier in vitro model 101. , The culture solution outside the hanger in the blood tissue barrier in vitro model 100 may be transferred into the hanger in the blood tissue barrier in vitro model 101, or the culture solution inside and outside the hanger in the blood tissue barrier in vitro model 100 may be mixed and used to mix the culture solution inside and outside the hanger in the blood tissue barrier in vitro model 101. You may move to.
By using the conditioned culture solution 20, it is possible to save the trouble of arranging the organ-derived cells 6 on the lower surface of the collagen vitrigel membrane 3. Further, as will be described later, when the blood tissue barrier in vitro model 101 of the present embodiment is used in the method for evaluating the transferability of a drug to the blood tissue barrier, non-specific adsorption of the compound to be evaluated to the organ-derived cell 6 is performed. It can be avoided. Further, from the viewpoint of avoiding non-specific adsorption of the compound to be evaluated to the protein, it is preferable to use a serum-free culture solution as the acclimation culture solution 20.
≪薬物の血液組織関門移行性評価方法≫
　１実施形態において、本発明は、上記血液組織関門インビトロモデルを用いて、コラーゲンビトリゲル膜の上方部に薬剤を添加し、一定時間後に、前記コラーゲンビトリゲル膜の下方部に漏れ出た、薬物の量を測定する、薬物の血液組織関門移行性評価方法を提供する。 ≪Evaluation method for drug transfer to blood tissue barrier≫
In one embodiment, the present invention uses the blood tissue barrier in vitro model to add a drug to the upper part of the collagen vitrigel membrane, and after a certain period of time, the drug leaked to the lower part of the collagen vitrigel membrane. Provided is a method for evaluating the transferability of a drug to the blood tissue barrier to measure the amount of the drug.
　係る方法において、血管の内腔側を外挿するコラーゲンビトリゲル膜の上方部から、組織側を外挿するコラーゲンビトリゲル膜の下方部に漏れ出た、薬物の量を測定することにより、所望する組織での作用を期待する薬物が、血液組織関門移行性を有するかを容易に判断することができる。また、係る方法を用いることで、血液組織関門自体に作用する薬物をスクリーニングすることもできる。 In such a method, desired by measuring the amount of drug leaked from the upper part of the collagen vitrigel membrane extrapolating the lumen side of the blood vessel to the lower part of the collagen vitrigel membrane extrapolating the tissue side. It is possible to easily determine whether a drug that is expected to act on a tissue having a blood tissue barrier transfer property. In addition, by using such a method, it is possible to screen for a drug that acts on the blood tissue barrier itself.
　また、１実施形態において、本発明は、上記血液組織関門インビトロモデルを用いて、正常状態のインビトロモデルと病態のインビトロモデルを用意し、両インビトロモデルにおいて、コラーゲンビトリゲル膜の上方部に薬剤を添加し、一定時間後に、コラーゲンビトリゲル膜の下方部に漏れ出た、薬物の量をそれぞれ測定し、比較することにより、薬物の病態組織選択的移行性を評価する、薬物の血液組織関門移行性評価方法を提供する。
　上述した血液組織関門インビトロモデルにおいて、病態条件に対応する培養液を用いる、又は、疾患を患っている患者由来の細胞を用いる等により、病態のインビトロモデルを作製することができる。
　そして、正常状態のインビトロモデルと病態のインビトロモデル、それぞれにおける薬物の組織移行性を検討することにより、病態組織選択的に移行する薬物をスクリーニングすることができる。 Further, in one embodiment, the present invention uses the blood tissue barrier in vitro model to prepare an in vitro model of a normal state and an in vitro model of a pathological condition, and in both in vitro models, a drug is applied to the upper part of the collagen vitrigel membrane. After a certain period of time after addition, the amount of the drug leaked to the lower part of the collagen in vitro gel membrane was measured and compared to evaluate the pathological tissue selective migration of the drug. Provide a sex evaluation method.
In the blood tissue barrier in vitro model described above, an in vitro model of a pathological condition can be prepared by using a culture solution corresponding to the pathological condition, or by using cells derived from a patient suffering from a disease.
Then, by examining the tissue transferability of the drug in each of the in vitro model of the normal state and the in vitro model of the pathological condition, it is possible to screen the drug that selectively migrates to the pathological tissue.
　以下、実施例により本発明を説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.
［実施例１］ヒト新生児***由来微小血管内皮細胞（ｈｕｍａｎ　ｍｉｃｒｏｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｃｅｌｌｓ;ＨＭＶＥＣ）、及び同由来線維芽細胞（ｈｕｍａｎ　ｄｅｒｍａｌ　ｆｉｂｒｏｂｌａｓｔ；ＨＤＦ）を用いた血管内皮培養モデルの作製
（１）予め培養したＨＤＦ(クラボウより購入、ＫＦ－４００９)を回収し、１．３×１０５細胞／ｍＬとなるように培養液と混合し、ＨＤＦの懸濁液を調製した。培養液は、１０％ウシ胎児血清(Ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍ；ＦＢＳ)、２０ｍＭ　ＨＥＰＥＳ、１００ｕｎｉｔｓ／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシン含有ＤＭＥＭ（ＴｈｅｒｍｏＦｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃより購入、１１８８５－０８４)を用いた。
（２）次いで、オプションリング（関東化学株式会社より購入、０８３６９－９６）を装着したａｄ－ＭＥＤビトリゲル（登録商標）２（１２ウエル）、０８３６３－９６）のチャンバー裏面に、（１）で調製したＨＤＦの懸濁液を４．０×１０４細胞／ｃｍ２となるように０．３ｍＬ加え、培養した（図３のｄａｙ－１参照。）。
（３）次いで、（２）の翌日（図３のｄａｙ０参照。）に予め培養したＨＭＶＥＣ(クラボウより購入、ＫＥ－４２０９)を回収し、１．４×１０５細胞／ｍＬとなるように培養液と混合し、ＨＭＶＥＣの懸濁液を調製した。培養液は、５％ＦＢＳ、５ｎｇ／ｍＬ組み換えヒトＦＧＦ－ｂ、５０μｇ／ｍＬアスコルビン酸、１μｇ／ｍＬヘミコハク酸ヒドロコルチゾン、１０ｍＭ　Ｌ－グルタミン、１５ｎｇ／ｍＬ組み換えヒトＩＧＦ－１、５ｎｇ／ｍＬ組み換えヒトＥＧＦ、５ｎｇ／ｍＬ組み換えヒトＶＥＧＦ、０．７５ｕｎｉｔｓ／ｍＬヘパリン硫酸、３０ｍｇ／ｍＬゲンタマイシン、１５μｇ／ｍＬアムホテリシンＢ含有ＶａｓｃｕＬｉｆｅ（登録商標）Ｂａｓａｌ　Ｍｅｄｉｕｍ(Ｌｉｆｅｌｉｎｅ Ｃｅｌｌ Ｔｅｃｈｎｏｌｏｇｙより購入、ＬＭ－０００２)を用いた。
（４）次いで、（２）で用意したチャンバーを１２ウェルプレートにセットし、チャンバーの内側に（３）で調製したＨＭＶＥＣの懸濁液を７．０×１０４細胞／ｃｍ２となるように０．５ｍＬ加えた。次いで、チャンバーの外側にＨＭＶＥＣの培養液を１．５ｍＬ加えた。
　なお、図３に示すように、ＨＭＶＥＣとＨＤＦをインサート両面に培養したモデルを皮膚血管の「器官様プレート型」モデル、ＨＭＶＥＣ又はＨＤＦの１種類の細胞のみを培養したモデルを「組織シート型」モデルと定義する。
（５）次いで、位相差顕微鏡を用いて、ｄａｙ０、ｄａｙ２、ｄａｙ６に「器官様プレート型」モデル（図４の（Ａ）～（Ｃ）参照。）、ＨＭＶＥＣの「組織シート型」モデル（図４の（Ｄ）～（Ｆ）参照。）。ＨＤＦの「組織シート型」モデル（図４の（Ｇ）～（Ｉ）参照。）を観察した。 [Example 1] Preparation of vascular endothelial culture model using human neonatal foramen-derived microvascular endothelial cells (HMVEC) and fibroblasts derived from the same (human dermal fibroblast; HDF) (1) Pre-cultured HDF (purchased from Kurabo Industries, KF-4009) was collected and mixed with the culture medium such that 1.3 × 10 5 cells / mL, to prepare a suspension of HDF. As the culture medium, 10% fetal bovine serum (FBS), 20 mM HEPES, 100 units / mL penicillin, 100 μg / mL streptomycin-containing DMEM (purchased from Thermo Fisher Scientific, 11885-084) was used.
(2) Next, prepare in (1) on the back surface of the chamber of ad-MED Vitrigel (registered trademark) 2 (12 wells), 08363-96) equipped with an option ring (purchased from Kanto Chemical Co., Inc., 08369-96). The suspension of HDF was added in an amount of 0.3 mL so as to have a concentration of 4.0 × 10 4 cells / cm 2, and cultured (see day-1 in FIG. 3).
(3) Then, the culture so that the next day (purchased from Kurabo Industries, KE-4209) pre-cultured HMVEC in (day 0 reference. In FIG. 3) was collected, 1.4 × 10 5 cells / mL of (2) The mixture was mixed with the liquid to prepare a suspension of HMVEC. The culture medium was 5% FBS, 5 ng / mL recombinant human FGF-b, 50 μg / mL ascorbic acid, 1 μg / mL hydrocortisone hemicouccinate, 10 mM L-glutamine, 15 ng / mL recombinant human IGF-1, 5 ng / mL recombinant human EGF. , 5 ng / mL recombinant human VEGF, 0.75 units / mL heparin sulfate, 30 mg / mL gentamicin, 15 μg / mL amhotericin B-containing VascuLife® Basal Medium (purchased from Lifeline Cell Technology, LM-0002) was used.
(4) Next, the chamber prepared in (2) was set on a 12-well plate, and the suspension of HMVEC prepared in (3) was placed inside the chamber so as to be 7.0 × 10 4 cells / cm 2. 0.5 mL was added. Then, 1.5 mL of HMVEC culture solution was added to the outside of the chamber.
As shown in FIG. 3, a model in which HMVEC and HDF are cultured on both sides of the insert is an "organ-like plate type" model of skin blood vessels, and a model in which only one type of cells of HMVEC or HDF is cultured is a "tissue sheet type". Defined as a model.
(5) Next, using a phase-contrast microscope, the “organ-like plate type” model (see (A) to (C) in FIG. 4) and the “tissue sheet type” model of HMVEC are used for day0, day2, and day6 (FIG. 4). See (D) to (F) of 4.). A "tissue sheet type" model of HDF (see (G)-(I) in FIG. 4) was observed.
［実施例２］皮膚血管内皮培養モデルの経内皮電気抵抗(ｔｒａｎｓｅｎｄｏｔｈｅｌｉａｌ　ｅｌｅｃｔｒｉｃａｌ　ｒｅｓｉｓｔａｎｃｅ；ＴＥＥＲ)値の経時的変化の測定
（１）実施例１で作製した３種類のモデルのＴＥＥＲ値をｄａｙ０、ｄａｙ１、ｄａｙ２、ｄａｙ３、及びｄａｙ６に測定した。測定前に、各モデルのチャンバー内及びチャンバー外に入っている培養液を除去し、予め室温に戻しておいたＨＭＶＥＣの培養液をチャンバー内に０．５ｍＬ、チャンバー外に１．５ｍＬ加えた。
（２）次いで、ＴＥＥＲ測定装置（関東化学株式会社より購入、４０２２５－９７）につないだ専用電極（関東化学株式会社より購入、１４１３６－９７）を各モデルのチャンバーにセットし、ＴＥＥＲ値を記録した。ネガティブコントロールであるＨＤＦの「組織シート型」モデルのＴＥＥＲ値は、ｄａｙ３が最大値(１１．６９±０．２７Ω・ｃｍ２)であった（図５参照。）。これに対し、ＨＭＶＥＣの「組織シート型」モデルのｄａｙ３のＴＥＥＲ値は、２３．５３±０．４５Ω・ｃｍ２であった。一方、「器官様プレート型」モデルは、ｄａｙ１より高いＴＥＥＲ値を示し、ｄａｙ１、ｄａｙ２、ｄａｙ３、及びｄａｙ６のＴＥＥＲ値は、それぞれ、２２．６２±３．０６Ω・ｃｍ２、３１．５０±２．４６Ω・ｃｍ２、４２．６３±３．９４Ω・ｃｍ２、及び５７．２４±７．２９Ω・ｃｍ２であった。以上より、「器官様プレート型」モデルは、高い内皮バリア機能を有することが確認された。 [Example 2] Measurement of changes over time in transendothelial electrical resistance (TEER) values of a cutaneous vascular endothelial culture model (1) The TER values of the three models prepared in Example 1 are set to day0, day1, and so on. Measurements were made on day2, day3, and day6. Before the measurement, the culture broth inside and outside the chamber of each model was removed, and 0.5 mL of the HMVEC culture broth that had been returned to room temperature in advance was added into the chamber and 1.5 mL outside the chamber.
(2) Next, set the dedicated electrode (purchased from Kanto Chemical Co., Inc., 14136-97) connected to the TEER measuring device (purchased from Kanto Chemical Co., Inc., 40225-97) in the chamber of each model, and record the TEER value. did. The TEER value of the "tissue sheet type" model of HDF, which is a negative control, was the maximum value (11.69 ± 0.27 Ω · cm 2 ) for day 3 (see FIG. 5). In contrast, the TEER value of day 3 of the HMVEC "tissue sheet type" model was 23.53 ± 0.45 Ω · cm 2 . On the other hand, the "organ-like plate type" model showed higher TER values than day1, and the TER values of day1, day2, day3, and day6 were 22.62 ± 3.06Ω · cm 2 , 31.50 ± 2, respectively. It was .46 Ω · cm 2 , 42.63 ± 3.94 Ω · cm 2 , and 57.24 ± 7.29 Ω · cm 2 . From the above, it was confirmed that the "organ-like plate type" model has a high endothelial barrier function.
［実施例３］皮膚血管の「器官様プレート型」モデル由来の馴化培養液(ｃｏｎｄｉｔｉｏｎｅｄ ｍｅｄｉｕｍ；ＣＭ)の効果の検討
（１）実施例２で用いたｄａｙ６のＨＭＶＥＣの「組織シート型」モデルのチャンバー内、及び外の培養液を除去し、同じく実施例２で用いたｄａｙ６の「器官様プレート型」モデルのチャンバー内及び外のＣＭを加えて２４時間培養した（図６（Ａ）参照）。
（２）次いで、ＴＥＥＲ値を測定したところ、培養液を加えたＨＭＶＥＣの「組織シート型」モデル (ＨＭＶＥＣ＋培養液) のＴＥＥＲ値は１４．５±１．５１Ω・ｃｍ２であったのに対し、ＣＭを加えた同モデル (ＨＭＶＥＣ＋ＣＭ) のＴＥＥＲ値は２７．０４±７．７７ Ω・ｃｍ２であり、統計学的に有意な差を認めた（**Ｐ＜０．０１、ｔ検定により検定）（図６（Ｂ）参照） 。以上より、「器官様プレート型」モデルのＣＭには内皮バリア機能を強化する作用があることが確認された。 [Example 3] Examination of the effect of the conditioned medium (CM) derived from the "organ-like plate type" model of skin blood vessels (1) The HMVEC "tissue sheet type" model of day 6 used in Example 2 The culture medium inside and outside the chamber was removed, and CM inside and outside the chamber of the "organ-like plate type" model of day 6 also used in Example 2 was added and cultured for 24 hours (see FIG. 6 (A)). ..
(2) Next, when the TER value was measured, the TER value of the HMVEC "tissue sheet type" model (HMVEC + culture solution) to which the culture solution was added was 14.5 ± 1.51 Ω · cm 2. The TEER value of the same model (HMVEC + CM) with CM added was 27.04 ± 7.77 Ω · cm 2 , and a statistically significant difference was observed ( ** P <0.01, by t-test). Test) (see FIG. 6 (B)). From the above, it was confirmed that the CM of the "organ-like plate type" model has an action of strengthening the endothelial barrier function.
［実施例４］炎症皮膚血管内皮モデルのＴＥＥＲ値の測定
（１）実施例２で用いたｄａｙ６のＨＭＶＥＣの「組織シート型」モデル、及び「器官様プレート型」モデルのチャンバー内及び外の培養液を除去し、予め室温に戻しておいた添加剤を含まないＶａｓｃｕＬｉｆｅ（登録商標）Ｂａｓａｌ　Ｍｅｄｉｕｍをチャンバー内に０．５ｍＬ、チャンバー外に１．５ｍＬ加えた。
（２）次いで、チャンバーに電極をセットし、ＴＥＥＲ値を測定した。この時の測定値を、測定開始時（０秒） の値とした。
（３）次いで、チャンバー内のＶａｓｃｕＬｉｆｅ（登録商標）Ｂａｓａｌ　Ｍｅｄｉｕｍを除去し、ヒスタミン二塩酸塩（東京化成工業株式会社より購入、Ｈ０１４６）を含有するＶａｓｃｕＬｉｆｅ（登録商標）Ｂａｓａｌ　Ｍｅｄｉｕｍを加えた。次いで、ＴＥＥＲ測定を開始し、１０秒ごとに１８０秒間ＴＥＥＲ値を連続測定した。「器官様プレート型」モデルでは測定開始時の値が約５０Ω・ｃｍ２であったのに対し、１ｎＭヒスタミンの添加により約３２Ω・ｃｍ２まで減少し、この変化はヒスタミンを加えていないコントロールと比較して統計学的に有意な変化であった（***Ｐ＜０．００１、二元配置分散分析の後Ｔｕｋｅｙ－Ｋｒａｍｅｒ法により検定）。さらに、１μＭ ヒスタミンの添加では約２３Ω・ｃｍ２まで減少したが、この変化はコントロールおよび１ｎＭヒスタミン添加の両方と比較して統計学的に有意な変化であった（***Ｐ＜０．００１、###Ｐ＜０．００１、二元配置分散分析の後Ｔｕｋｅｙ－Ｋｒａｍｅｒ法により検定）（図７（Ａ）参照）。一方、ＨＭＶＥＣの「組織シート型」モデルでは測定開始時の値が約５．７Ω・ｃｍ２であったのに対し、１００ μＭヒスタミンの添加で約３．２Ω・ｃｍ２まで減少したが、この変化はコントロールと比較して統計学的に有意な差ではなかった（二元配置分散分析により検定）（図７（Ｂ）参照）。以上より、「器官様プレート型」モデルは「組織シート型」モデルよりも低濃度のヒスタミンに濃度依存的に応答し、ＴＥＥＲ値の変化を統計学的な有意な差をもって検出できることが示された。 [Example 4] Measurement of TER value of inflamed cutaneous vascular endothelial model (1) Cultivation inside and outside the chamber of the HMVEC "tissue sheet type" model and "organ-like plate type" model of day 6 used in Example 2. The liquid was removed, and 0.5 mL of VascuLife® Basal Medium containing no additives, which had been returned to room temperature in advance, was added into the chamber and 1.5 mL outside the chamber.
(2) Next, an electrode was set in the chamber and the TER value was measured. The measured value at this time was taken as the value at the start of measurement (0 seconds).
(3) Next, VascuLife (registered trademark) Basal Medium in the chamber was removed, and VascuLife (registered trademark) Basal Medium containing histamine dihydrochloride (purchased from Tokyo Chemical Industry Co., Ltd., H0146) was added. Then, the TEER measurement was started, and the TEER value was continuously measured every 10 seconds for 180 seconds. In the "organ-like plate type" model, the value at the start of measurement was about 50 Ω · cm 2 , but it decreased to about 32 Ω · cm 2 with the addition of 1 nM histamine, and this change was controlled without adding histamine. It was a statistically significant change in comparison ( *** P <0.001, tested by Tukey-Kramer method after two-way ANOVA). In addition, the addition of 1 μM histamine reduced it to about 23 Ω · cm 2 , which was a statistically significant change compared to both control and 1 nM histamine addition ( *** P <0.001). , ### P <0.001, tested by Tukey-Kramer method after two-way ANOVA) (see FIG. 7 (A)). On the other hand, in the HMVEC "tissue sheet type" model, the value at the start of measurement was about 5.7 Ω · cm 2 , but it decreased to about 3.2 Ω · cm 2 with the addition of 100 μM histamine. The changes were not statistically significant differences compared to the controls (tested by two-way ANOVA) (see Figure 7 (B)). From the above, it was shown that the "organ-like plate type" model responds to lower concentrations of histamine in a concentration-dependent manner than the "tissue sheet type" model, and changes in TEER value can be detected with a statistically significant difference. ..
［実施例５］炎症皮膚血管内皮培養モデルの蛍光物質の透過性評価
（１）実施例２で用いたｄａｙ ６のＨＭＶＥＣの「組織シート型」モデルのチャンバー内及び外の培養液を除去し、予め室温に戻しておいたＨａｎｋ’ｓ Ｂａｌａｎｃｅｄ Ｓａｌｔ Ｓｏｌｕｔｉｏｎ （ＨＢＳＳ、ＴｈｅｒｍｏＦｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃより購入、１４０２５－０９２) をチャンバー内に０．５ｍＬ、チャンバー外に１．５ ｍＬ加えて２回洗浄した。
（２）次いで、ＨＢＳＳをチャンバー外に１．８ｍＬ、１００μＭヒスタミン含有ＨＢＳＳをチャンバー内に０．５ｍＬ加え、室温で１０分間インキュベートした。
（３）次いで、 チャンバー内の溶液を除去し、１００μＭヒスタミン及び１０μｇ/ｍＬ Ｓｏｄｉｕｍ ｆｌｕｏｒｅｓｃｅｉｎ (Ｕｒａｎｉｎｅ)（東京化成工業株式会社より購入、Ｆ００９６）又は５００μｇ/ｍＬ Ｆｌｕｏｒｅｓｃｅｉｎ ｉｓｏｔｈｉｏｃｙａｎａｔｅ－ｄｅｘｔｒａｎ (ＦＤ)－４（Ｓｉｇｍａ－Ａｌｄｒｉｃｈより購入、ＦＤ４－１００ＭＧ）又は５００μｇ/ｍＬ ＦＤ－４０（Ｓｉｇｍａ－Ａｌｄｒｉｃｈより購入、ＦＤ４０Ｓ－１００ＭＧ）を含有するＨＢＳＳをチャンバー内に０．５ｍＬ加え、室温で６０分間インキュベートした。
（４）次いで、チャンバー外の溶液１００μＬを９６ウェルプレートに移し、マイクロプレートリーダーＳｐｅｃｔｒａＭａｘ Ｇｅｍｉｎｉ ＸＳ Ｍｉｃｒｏｐｌａｔｅ Ｒｅａｄｅｒ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓより購入）を用いて蛍光強度を測定した(λｅｘｃｉｔａｔｉｏｎ／λｅｍｉｓｓｉｏｎ＝４９０ｎｍ／５２０ｎｍ) 。あわせて、各蛍光物質の希釈系列を用いて検量線を作製し、チャンバー外へ移行した蛍光物質の量を求めた。
（５）次いで、次式に基づいて各蛍光物質の見かけの透過係数Ｐａｐｐ（ｃｍ/ｓｅｃ） を求めた。
Ｐａｐｐ＝ｄＱ／ｄｔ×１／Ａ×１／Ｃ０
ｄＱ／ｄｔ; 単位時間あたりの移行量(μｇ／ｓｅｃ)、Ａ; 表面積 (ｃｍ２)、Ｃ０; 各蛍光物質の初濃度
１００μＭヒスタミンの添加により、Ｓｏｄｉｕｍ ｆｌｕｏｒｅｓｃｅｉｎ（分子量３７６）、ＦＤ－４（分子量４，０００）、及びＦＤ－４０（分子量４０，０００）のいずれの分子のＰａｐｐも上昇した（図８（Ａ）－（Ｃ）参照）。特に、ＦＤ－４についてはＣｏｎｔｒｏｌのＰａｐｐが３．１５×１０－６±９．８３×１０－７ｃｍ／ｓｅｃであったのに対し、１００μＭヒスタミンの添加により約２倍の５．８８×１０－６±５．５２×１０－７ｃｍ／ｓｅｃまで上昇し、この変化は統計学的に有意な変化であった（*Ｐ＜０．０５、ｔ検定により検定）。以上より、皮膚血管の「器官様プレート型」モデルではヒスタミン誘発の炎症によって分子量４，０００程度の中分子の透過性が上昇することが確認された。
　係る結果から、評価に用いた分子量の異なる上記３種の化合物は、病態組織選択的透過性がそれぞれ異なる化合物ということができる。即ち、本発明によれば、正常状態のインビトロモデルと病態のインビトロモデルを用意し、両インビトロモデルにおいて、薬物の移行性を比較評価することにより、病態組織選択的に透過性を有する薬物をスクリーニングすることができる。 [Example 5] Evaluation of fluorescence substance permeability of inflamed skin vascular endothelial culture model (1) The culture medium inside and outside the chamber of the HMVEC “tissue sheet type” model of day 6 used in Example 2 was removed. Hank's Balanced Salt Solution (HBSS, purchased from Thermo Fisher Scientific, 14025-092), which had been returned to room temperature in advance, was washed twice by adding 0.5 mL into the chamber and 1.5 mL outside the chamber.
(2) Next, 1.8 mL of HBSS was added to the outside of the chamber and 0.5 mL of HBSS containing 100 μM histamine was added to the inside of the chamber, and the mixture was incubated at room temperature for 10 minutes.
(3) Next, the solution in the chamber is removed, and 100 μM histamine and 10 μg / mL Fluorescein (Uranine) (purchased from Tokyo Kasei Kogyo Co., Ltd., F0906) or 500 μg / mL Fluorescein isothiocyanate-dextran (FD) -4 (Sigma). -HBSS containing FD-40 (purchased from Aldrich, FD4-100MG) or 500 μg / mL FD-40 (purchased from Sigma-Aldrich, FD40S-100MG) was added into the chamber in an amount of 0.5 mL and incubated at room temperature for 60 minutes.
(4) Next, 100 μL of the solution outside the chamber was transferred to a 96-well plate, and the fluorescence intensity was measured using a microplate reader SpectraMax Gemini XS Microplatate Reader (purchased from Molecular Devices) (λ excitation / λ mechanism = 490 nm / 520 nm). .. At the same time, a calibration curve was prepared using the dilution series of each fluorescent substance, and the amount of the fluorescent substance transferred to the outside of the chamber was determined.
(5) Next, the apparent permeability coefficient P- app (cm / sec) of each fluorescent substance was determined based on the following equation.
P- app = dQ / dt x 1 / A x 1 / C 0
dQ / dt; Transition amount per unit time (μg / sec), A; Surface area (cm 2 ), C 0 ; Sodium fluorescein (molecular weight 376), FD-4 (molecular weight 376), by addition of 100 μM histamine initial concentration of each fluorescent substance. molecular weight of 4,000), and FD-40 (also P app of any molecules of molecular weight 40,000) was increased (FIG. 8 (a) - (C) see). In particular, for FD-4, the P- app of Control was 3.15 × 10-6 ± 9.83 × 10-7 cm / sec, whereas the addition of 100 μM histamine doubled it to 5.88 ×. It increased to 10-6 ± 5.52 × 10-7 cm / sec, and this change was a statistically significant change ( * P <0.05, tested by t-test). From the above, it was confirmed that in the "organ-like plate type" model of cutaneous blood vessels, the permeability of medium molecules having a molecular weight of about 4,000 increases due to histamine-induced inflammation.
From the above results, it can be said that the above three compounds having different molecular weights used in the evaluation are compounds having different pathological tissue selective permeability. That is, according to the present invention, an in vitro model of a normal state and an in vitro model of a pathological condition are prepared, and a drug having permeability selectively in the pathological tissue is screened by comparing and evaluating the transferability of the drug in both in vitro models. can do.
［実施例６］組織特異的血管内皮培養モデルの作製及びＴＥＥＲ値の測定
（１）実施例１で作製したＨＭＶＥＣとＨＤＦを用いた「器官様プレート型」モデルと同様の方法で、ＨＭＶＥＣとラットグリオーマ細胞株Ｃ６（理化学研究所バイオリソースセンターより提供、ＲＣＢ２８５４）又はＨＭＶＥＣと肝がん細胞株ＨｅｐＧ２－ＮＩＡＳ（理化学研究所バイオリソースセンターより提供されたＨｅｐＧ２を馴化培養し再寄託した細胞株、ＲＣＢ４６７９）を用いた「器官様プレート型」モデルを作製した。Ｃ６とＨｅｐＧ２－ＮＩＡＳの培養液は、１０％ ＦＢＳ、２０ｍＭ ＨＥＰＥＳ、１００ ｕｎｉｔｓ/ｍＬ ペニシリン、１００ μｇ/ｍＬ ストレプトマイシン含有ＤＭＥＭを用いた。
（２）次いで、実施例２と同様の方法で経時的なＴＥＥＲ値の変化を測定した。結果を図９に示す。皮膚由来のＨＤＦ（図９（Ａ）参照。）のみならず、脳由来のＣ６（図９（Ｂ）参照。）、肝臓由来のＨｅｐＧ２－ＮＩＡＳ（図９（Ｃ）参照。）を用いても血管内皮培養モデルが作製できることが確認された。 [Example 6] Preparation of tissue-specific vascular endothelial culture model and measurement of TEER value (1) HMVEC and rat in the same manner as the "organ-like plate type" model using HMVEC and HDF prepared in Example 1. Grioma cell line C6 (provided by RIKEN BioResource Center, RCB2854) or HMVEC and liver cancer cell line HepG2-NIAS (cell line in which HepG2 provided by RIKEN Bioresource Center was conditioned and re-deposited, RCB4679) The "organ-like plate type" model used was prepared. As the culture medium of C6 and HepG2-NIAS, DMEM containing 10% FBS, 20 mM HEPES, 100 units / mL penicillin, and 100 μg / mL streptomycin was used.
(2) Next, the change in the TEER value over time was measured by the same method as in Example 2. The results are shown in FIG. Not only skin-derived HDF (see FIG. 9 (A)) but also brain-derived C6 (see FIG. 9 (B)) and liver-derived HepG2-NIAS (see FIG. 9 (C)) can be used. It was confirmed that a vascular endothelial culture model could be prepared.
［実施例７］ｈＣＭＥＣ／Ｄ３クローン細胞株の作製及びＴＥＥＲ値の測定
　ｈＣＭＥＣ／Ｄ３は、増殖能が高く汎用されているヒト血管内皮細胞株であるが、内皮バリア機能が低いため血液組織関門モデルの構築には不適である。そこで、本細胞から内皮バリア機能を有するクローン細胞株を選別し、その炎症応答性を確認した。 [Example 7] Preparation of hCMEC / D3 clone cell line and measurement of TEER value hCMEC / D3 is a human vascular endothelial cell line that has high proliferative ability and is widely used, but has a low endothelial barrier function, so that it is a blood tissue barrier model. Not suitable for building. Therefore, a cloned cell line having an endothelial barrier function was selected from the cells, and its inflammatory responsiveness was confirmed.
　Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ社より購入し、培養を開始したｈＣＭＥＣ／Ｄ３（Ｐａｒｅｎｔ）をコラーゲンビトリゲル膜チャンバー内で培養し、経時的にＴＥＥＲ値を測定すると、７日間培養しても５Ω・ｃｍ２未満であったことから、本細胞は、内皮バリア機能がほとんどないことが示唆された（図１０（Ａ）及び（Ｂ）参照。）。そこで、本細胞を限界希釈法によりクローニングしたところ、内皮バリア機能を有するクローンＡを得ることができた（図１０（Ａ）及び（Ｃ）参照。）。 Purchased from Merck Millipore Corp., a hCMEC / D3 culture was started (Parent) were cultured in collagen arbitration Rigel film chamber, as measured over time TEER values, be cultured for 7 days was less than 5 [Omega · cm 2 This suggests that the cells have almost no endothelial barrier function (see FIGS. 10 (A) and 10 (B)). Therefore, when the cells were cloned by the limiting dilution method, clone A having an endothelial barrier function could be obtained (see FIGS. 10 (A) and 10 (C)).
　クローンＡをコラーゲンビトリゲル膜チャンバー内で１０日間培養し、チャンバー内にヒスタミン（Ｈｉｓ）１００μＭを加えて１分おきにＴＥＥＲ値を測定したところ、測定開始時の値に対し、ヒスタミン添加１５分後には約７０％減少した（図１１参照。）。この変化はＣｏｎｔｒｏｌと比較して統計学的に有意な変化であった（***Ｐ＜０．００１二元配置分散分析の後Ｔｕｋｅｙ－Ｋｒａｍｅｒ法により検定）。一方、ヒスタミン受容体の下流因子であるＲＯＣＫの阻害薬であるＹ－２７６３２で前処理すると、ヒスタミンによるＴＥＥＲ値の減少が有意に抑制されたことから（###Ｐ＜０．００１、二元配置分散分析の後Ｔｕｋｅｙ－Ｋｒａｍｅｒ法により検定）、クローンＡはヒスタミン応答性に優れた細胞株であることが示唆された（図１１参照。）。 Clone A was cultured in a collagen vitrigel membrane chamber for 10 days, 100 μM of histamine (His) was added into the chamber, and the TER value was measured every 1 minute. As a result, 15 minutes after the addition of histamine, the value at the start of the measurement was measured. Decreased by about 70% (see FIG. 11). This change was statistically significant compared to Control ( * *** P <0.001 two-way ANOVA followed by Tukey-Kramer test). On the other hand, pretreatment with Y-27632, an inhibitor of ROCK, which is a downstream factor of histamine receptor, significantly suppressed the decrease in TER value due to histamine ( ### P <0.001, binary). Tested by Tukey-Kramer method after ANOVA), it was suggested that clone A is a cell line with excellent histamine responsiveness (see FIG. 11).
　クローンＡをコラーゲンビトリゲル膜チャンバー内で１６日間培養し、チャンバー内にヒスタミン（Ｈｉｓ）１００μＭ、及び４種類の蛍光物質（Ｓｏｄｉｕｍ ｆｌｕｏｒｅｓｃｅｉｎ、ＦＤ－４、ＦＤ－１０、及びＦＤ－４０）を加えて１時間インキュベートし、チャンバー外へ移行した蛍光物質量を測定した。この結果、ヒスタミンによってＦＤ－４（分子量４，０００）及びＦＤ－１０（分子量１０，０００）の透過性のみが亢進した（図１２参照。）。特にＦＤ－１０の透過性の亢進は統計学的に有意な変化であった（*Ｐ＜０．０５、ｔ検定により検定）。よって、クローンＡにおいてヒスタミンが誘発する血管透過性の亢進には分子サイズ選択性があることが示唆された。
　これらの結果、生理的な濃度のヒスタミンによる内皮バリア機能変化を定量的に評価することができたことから、本クローン細胞株は、薬物の組織移行性評価モデルの構築に適していると考えられる。 Clone A is cultured in a collagen vitrigel membrane chamber for 16 days, and 100 μM of histamine (His) and four kinds of fluorescent substances (Sodium fluorescein, FD-4, FD-10, and FD-40) are added to the chamber. After incubating for 1 hour, the amount of fluorescent substance transferred to the outside of the chamber was measured. As a result, histamine enhanced only the permeability of FD-4 (molecular weight 4,000) and FD-10 (molecular weight 10,000) (see FIG. 12). In particular, the increase in permeability of FD-10 was a statistically significant change ( * P <0.05, tested by t-test). Therefore, it was suggested that histamine-induced increase in vascular permeability in clone A has molecular size selectivity.
As a result, it was possible to quantitatively evaluate the change in endothelial barrier function due to the physiological concentration of histamine, and it is considered that this cloned cell line is suitable for constructing a model for evaluating the tissue transfer of a drug. ..
［実施例８］ＨＤＦの「組織シート型」モデル由来のＣＭの効果の検討
（１）予め培養したＨＤＦを回収し、８．０×１０４細胞／ｍＬとなるように培養液と混合し、ＨＤＦの懸濁液を調製した。培養液は、５％ ＦＢＳ、５ｎｇ/ｍＬ 組み換えヒトＦＧＦ－ｂ、５０μｇ/ｍＬ アスコルビン酸、１μｇ/ｍＬ ヘミコハク酸ヒドロコルチゾン、１０ ｍＭ Ｌ－グルタミン、１５ ｎｇ/ｍＬ 組み換えヒトＩＧＦ－１、５ｎｇ/ｍＬ組み換えヒトＥＧＦ、５ｎｇ/ｍＬ 組み換えヒトＶＥＧＦ、０．７５ｕｎｉｔｓ/ｍＬ ヘパリン硫酸、３０ｍｇ/ｍＬ ゲンタマイシン、１５μｇ/ｍＬ アムホテリシンＢ含有ＶａｓｃｕＬｉｆｅ（登録商標）Ｂａｓａｌ　Ｍｅｄｉｕｍを用いた。 [Example 8] Examination of the effect of CM derived from the "tissue sheet type" model of HDF (1) The HDF cultured in advance was collected and mixed with the culture solution so as to have 8.0 × 10 4 cells / mL. A suspension of HDF was prepared. The culture medium is 5% FBS, 5 ng / mL recombinant human FGF-b, 50 μg / mL ascorbic acid, 1 μg / mL hydrocortisone heparinate, 10 mM L-glutamine, 15 ng / mL recombinant human IGF-1, 5 ng / mL recombinant. Human EGF, 5 ng / mL recombinant human VEGF, 0.75 units / mL heparin sulfate, 30 mg / mL gentamicin, 15 μg / mL Amhotericin B-containing VascuLife® Basal Medium was used.
（２）次いで、１２穴プレートに設置したコラーゲンビトリゲル膜チャンバーの外側に１．５ｍＬの培養液を注入し、コラーゲンビトリゲル膜チャンバー内に（１）で調製したＨＤＦの懸濁液を４．０×１０４細胞／ｃｍ２となるように０．５ｍＬ加えて６日間培養した。この間、毎日培養液を交換した。培養４日目、５日目、及び６日目のチャンバー内及び外のＣＭを回収して混合した(図１３（Ａ）参照。)。 (2) Next, 1.5 mL of the culture solution was injected outside the collagen vitrigel membrane chamber placed on the 12-well plate, and the HDF suspension prepared in (1) was placed in the collagen vitrigel membrane chamber. 0.5 mL was added so as to be 0 × 10 4 cells / cm 2, and the cells were cultured for 6 days. During this time, the culture medium was changed daily. CMs inside and outside the chamber on days 4, 5, and 6 of the culture were collected and mixed (see FIG. 13 (A)).
（３）次いで、予め培養したＨＤＦを回収し、１．３×１０５細胞／ｍＬとなるように培養液と混合し、ＨＤＦの懸濁液を調製した。  
（４）また、オプションリングを装着したコラーゲンビトリゲル膜チャンバーのチャンバー裏面に、（３）で調製したＨＤＦの懸濁液を４．０×１０４細胞／ｃｍ２となるように０．３ｍＬ加えて培養した。細胞が接着した２時間後にオプションリングを外して１２穴プレートに設置し、コラーゲンビトリゲル膜チャンバーの外側に１．５ｍＬ、内側に０．５ｍＬの培養液を注入して６日間培養した。この間、毎日培養液を交換した。培養４日目、５日目、及び６日目のチャンバー内及び外のＣＭを回収して混合した（図１３（Ｂ）参照。）。 (3) Then, to recover the pre-cultured HDF, mixed with the culture medium such that 1.3 × 10 5 cells / mL, to prepare a suspension of HDF.
(4) In addition, 0.3 mL of the HDF suspension prepared in (3) was added to the back surface of the collagen vitrigel membrane chamber equipped with the option ring so as to be 4.0 × 10 4 cells / cm 2. Was cultured. Two hours after the cells adhered, the option ring was removed and placed on a 12-well plate, and 1.5 mL of the culture solution was injected outside the collagen vitrigel membrane chamber and 0.5 mL was injected inside, and the cells were cultured for 6 days. During this time, the culture medium was changed daily. CMs inside and outside the chamber on days 4, 5, and 6 of the culture were collected and mixed (see FIG. 13 (B)).
（５）次いで、実施例１で作製したＨＭＶＥＣの「組織シート型」モデルのチャンバー内及び外に、回収したＣＭ(ＨＤＦ－ＣＭ ｉｎ及びＨＤＦ－ＣＭ ｏｕｔ)又は培養液をｄａｙ０からｄａｙ７まで添加し、ｄａｙ４及びｄａｙ７においてＴＥＥＲ値を測定した。培養液を加えたＨＭＶＥＣの「組織シート型」モデル（ＨＭＶＥＣ＋培養液) のＴＥＥＲ値はｄａｙ４（図１３（Ｃ）参照。）及びｄａｙ７（図１３（Ｄ）参照。）においてそれぞれ１８．０±１．４１Ω・ｃｍ２及び１７．６±３．１７Ω・ｃｍ２であった。これに対し、ＨＤＦ－ＣＭ ｉｎを加えた同モデル（ＨＭＶＥＣ＋（ＨＤＦ－ＣＭ ｉｎ））のＴＥＥＲ値は、ｄａｙ４及びｄａｙ７において、それぞれ３３．５±１．０１Ω・ｃｍ２及び３３．０±２．６７Ω・ｃｍ２あった。またＨＤＦ－ＣＭ ｏｕｔを加えた同モデル(ＨＭＶＥＣ＋（ＨＤＦ－ＣＭ ｏｕｔ）)のＴＥＥＲ値は、ｄａｙ４及びｄａｙ７においてそれぞれ３３．９±３．６８Ω・ｃｍ２及び３２．７±２．１９Ω・ｃｍ２であった。ＨＤＦ－ＣＭ ｉｎ及びＨＤＦ－ＣＭ ｏｕｔを加えた時のＴＥＥＲ値は、ｄａｙ４及びｄａｙ７のいずれにおいても培養液を加えた時と比較して統計学的に有意に高い値であった（**Ｐ＜０．０１、***Ｐ＜０．００１、Ｔｕｋｅｙ－Ｋｒａｍｅｒ法により検定）。以上の結果を実施例３と比較すると、「器官様プレート型」モデルのＣＭよりもＨＤＦの「組織シート型」モデルのＣＭの方が内皮バリア機能を強化する作用が強いことが示された。 (5) Next, the recovered CM (HDF-CM in and HDF-CM out) or culture solution was added from day 0 to day 7 inside and outside the chamber of the HMVEC “tissue sheet type” model prepared in Example 1. , Day 4 and day 7 measured the TER value. The TEER value of the HMVEC "tissue sheet type" model (HMVEC + culture solution) to which the culture solution was added was 18.0 ± 1 in day 4 (see FIG. 13 (C)) and day 7 (see FIG. 13 (D)), respectively. It was .41 Ω · cm 2 and 17.6 ± 3.17 Ω · cm 2 . On the other hand, the TER values of the same model (HMVEC + (HDF-CM in)) to which HDF-CM in was added were 33.5 ± 1.01 Ω · cm 2 and 33.0 ± 2. There was 67Ω ・ cm 2 . The TEER value of the same model (HMVEC + (HDF-CM out)) with HDF-CM out added is 33.9 ± 3.68 Ω · cm 2 and 32.7 ± 2.19 Ω · cm 2 on day 4 and day 7, respectively. Met. The TER value when HDF-CM in and HDF-CM out were added was statistically significantly higher in both day 4 and day 7 than when the culture medium was added ( ** P). <0.01, *** P <0.001, tested by Tukey-Kramer method). Comparing the above results with Example 3, it was shown that the CM of the HDF "tissue sheet type" model has a stronger effect of strengthening the endothelial barrier function than the CM of the "organ-like plate type" model.
　本発明によれば、容易に構築可能な、脳組織を含めヒト型の様々な組織の血液組織関門インビトロモデル、及び簡便かつ優れた再現性を有する当該血液組織関門インビトロモデルを利用した、組織特異的な血管透過性化学物質の評価方法を提供することができる。 According to the present invention, a tissue specificity using an easily constructable blood tissue barrier in vitro model of various human tissues including brain tissue and the blood tissue barrier in vitro model having simple and excellent reproducibility. A method for evaluating a vascular permeable chemical substance can be provided.
　１００，１０１…血液組織関門インビトロモデル、１…培養器、２…培養液、３…コラーゲンビトリゲル膜、４…ハンガー、５…ヒト血管内皮細胞、６…臓器由来細胞。 100, 101 ... Blood tissue barrier in vitro model, 1 ... Incubator, 2 ... Culture solution, 3 ... Collagen Vitrigel membrane, 4 ... Hanger, 5 ... Human vascular endothelial cells, 6 ... Organ-derived cells.

Claims (6)

  1. 　コラーゲンビトリゲル膜と、
    　前記コラーゲンビトリゲル膜の上面に配置されたヒト血管内皮細胞と、
    　前記コラーゲンビトリゲル膜の下面に配置された臓器由来細胞と、培養液と、を含み、
    　前記ヒト血管内皮細胞は、前記臓器由来細胞に依存して、前記臓器の血管内皮細胞に分化し得る細胞である、血液組織関門インビトロモデル。     Collagen Vitrigel membrane and
    Human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane and
    Includes organ-derived cells arranged on the lower surface of the collagen vitrigel membrane and a culture medium.
    A blood tissue barrier in vitro model in which the human vascular endothelial cells are cells that can differentiate into vascular endothelial cells of the organ depending on the organ-derived cells.
  2. 　前記培養液は、無血清培養液である、請求項１に記載の血液組織関門インビトロモデル。 The blood tissue barrier in vitro model according to claim 1, wherein the culture solution is a serum-free culture solution.
  3. 　コラーゲンビトリゲル膜と、
    　前記コラーゲンビトリゲル膜の上面に配置されたヒト血管内皮細胞と、
    　臓器由来細胞の馴化培養液と、を含み、
    　前記ヒト血管内皮細胞は、前記臓器由来細胞に依存して、前記臓器の血管内皮細胞に分化し得る細胞である、血液組織関門インビトロモデル。     Collagen Vitrigel membrane and
    Human vascular endothelial cells arranged on the upper surface of the collagen vitrigel membrane and
    Contains conditioned culture medium of organ-derived cells,
    A blood tissue barrier in vitro model in which the human vascular endothelial cells are cells that can differentiate into vascular endothelial cells of the organ depending on the organ-derived cells.
  4. 　前記馴化培養液は、無血清培養液である、請求項３に記載の血液組織関門インビトロモデル。 The blood tissue barrier in vitro model according to claim 3, wherein the conditioned culture solution is a serum-free culture solution.
  5. 　請求項１～４のいずれか一項に記載の血液組織関門インビトロモデルを用いて、前記コラーゲンビトリゲル膜の上方部に薬剤を添加し、一定時間後に、前記コラーゲンビトリゲル膜の下方部に漏れ出た、薬物の量を測定する、薬物の血液組織関門移行性評価方法。 Using the blood tissue barrier in vitro model according to any one of claims 1 to 4, a drug is added to the upper part of the collagen vitrigel membrane, and after a certain period of time, the drug leaks to the lower part of the collagen vitrigel membrane. A method for evaluating the transferability of a drug to the blood tissue barrier, which measures the amount of the drug.
  6. 　請求項１～４のいずれか一項に記載の血液組織関門インビトロモデルにおいて、正常状態のインビトロモデルと病態のインビトロモデルを用意し、
    　両インビトロモデルにおいて、前記コラーゲンビトリゲル膜の上方部に薬剤を添加し、一定時間後に、前記コラーゲンビトリゲル膜の下方部に漏れ出た、薬物の量をそれぞれ測定し、比較することにより、薬物の病態組織選択的移行性を評価する、薬物の血液組織関門移行性評価方法。     In the blood tissue barrier in vitro model according to any one of claims 1 to 4, an in vitro model of a normal state and an in vitro model of a pathological condition are prepared.
    In both in vitro models, the drug was added to the upper part of the collagen vitrigel membrane, and after a certain period of time, the amount of the drug leaked to the lower part of the collagen vitrigel membrane was measured and compared. A method for evaluating the transferability of a drug to the blood tissue barrier, which evaluates the pathological tissue-selective migration of the drug.
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