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Method of preparing basement membrane, method of constructing basement membrane specimen, reconstituted artificial tissue using the basement membrane specimen and process for producing the same

外国特許コード F110005700
整理番号 Y0222US2
掲載日 2011年9月12日
出願国 アメリカ合衆国
出願番号 59976006
公報番号 20070065807
公報番号 7906332
出願日 平成18年11月15日(2006.11.15)
公報発行日 平成19年3月22日(2007.3.22)
公報発行日 平成23年3月15日(2011.3.15)
国際出願番号 JP2002009841
国際公開番号 WO2003026712
国際出願日 平成14年9月25日(2002.9.25)
国際公開日 平成15年4月3日(2003.4.3)
優先権データ
  • 特願2001-292510 (2001.9.25) JP
  • 特願2001-292675 (2001.9.25) JP
  • 特願2001-292676 (2001.9.25) JP
  • 特願2001-292677 (2001.9.25) JP
  • 特願2002-278243 (2002.9.24) JP
  • 特願2002-278244 (2002.9.24) JP
  • 10/809,218 (2004.3.25) US
  • 2002JP009841 (2002.9.25) WO
発明の名称 (英語) Method of preparing basement membrane, method of constructing basement membrane specimen, reconstituted artificial tissue using the basement membrane specimen and process for producing the same
発明の概要(英語) The object of the present invention is to provide the following: a method for preparing a basement membrane which is extracellular matrices having a function to control morphology, differentiation, proliferation, motility, function expression and the like of cells; a method for constructing a specimen of a basement membrane; a process for producing a reconstituted artificial tissue; a basement membrane specimen or an artificial tissue which can be transplanted while maintaining the structure of a basement membrane.
A basement membrane having a barrier function is formed by culturing alveolar epithelial cells or vascular endothelial cells on a fibrous collagen matrix coated with a polymer having a sugar chain which can localize a receptor having an activity to accumulate a basement membrane component on the basal surface of the cells having an ability to form a basement membrane.
A reconstructed artificial tissue is obtained by seeding and culturing desired homogeneous or heterogeneous cells having an ability to form a basement membrane on the basement membrane specimen constructed by the following process: the cells having an ability to form a basement membrane adhered onto a support structure through a basement membrane are treated with a surface active agent; the lipid component of cells is lysed; the mixture of an alkaline solution and a protease inhibitor is used to lyse the protein remained on the surface of the basement membrane of the cells.
A protein support structure is temporarily adhered to plastic surface by hydrophobic bonding.
従来技術、競合技術の概要(英語) BACKGROUND ART
An epithelial tissue, which is a cell layer covering the inside and outside surface of an animal body, such as an epidermis, a corneal epithelium, an alveolar epithelium, a mucosal epithelium of digestive system, renal glomerular epithelium, hepatic parenchymal cells and the like, prevents the invasion of an exogenous material (microorganism, allergen, chemical substance, etc.) from the external world.
The outer interface of epithelial cells that institutes said epithelial tissue is called apical, and the inside undersurface is called basal.
Just beneath said basal surface, there is a thin film structure of 50-100 nm thickness called a basement membrane comprised of extracellular matrices (ECM) such as proteins, proteoglycans and the like (not including cells).
A basement membrane is considered to be an essential structure for immature epithelial cells to proliferate, to differentiate into mature cells, and to express its original morphology or function.
In other words, without a basement membrane, an epithelial tissue cannot maintain itself or achieve its original performance.
Although an epithelial cell layer of multilayer or monolayer prevents the invasion of an exogenous material from the external world as a barrier, a basement membrane itself also acts as a physical barrier.
Thus, epithelial cells comprising an epithelial tissue collaborate with a basement membrane to form a solid barrier and to protect the internal vital activity.
A basement membrane, which is a specific membranous structure of extracellular matrices formed on the interface of parenchymal cells, such as epithelial cells, endothelial cells, muscle cells, adipocytes, Schwann cells and the like, and connective tissue, is universally found in respective tissue/organ of a living body, however, some basement membranes (?) are highly specialized such as a renal glomerular capillary loop, a nervous synapse membrane and the like.
Therefore, not only its function to adhere cells to an interstitium, but also its function to selectively permeate a substance/cells, and to induce the differentiation of cells are also disclosed.
In renal glomerulus, negative electric charge of a basement membrane is considered to be responsible for the filtration function of kidney, and said negative electric charge is traditionally known to be charged by heparan sulfate proteoglycan (HSPG) which is currently called perlecan.
HSPG is widely distributed not only to a renal glomerular basement membrane but also to various basement membranes as its basic component in the same manner as type IV collagen, laminin, entactin and the like.
An extracellular matrix, especially abasement membrane, is now gradually known to be deeply involved not only in physiological phenomena such as generation or differentiation of an individual as mentioned above, but also in formation of pathology such as proliferative metastasis of cancer, inflammation and the like.
Therefore, clarification of the function of its constituent protein has been an important task.
For example, laminin, which is a main glycoprotein of a basement membrane, is a complex comprised of three subunits alpha , beta , and gamma , fifteen types of its isoforms are known, and they are expressed tissue-specifically and at each step of development.
Laminin is a complicated macromolecule of 900,000 molecular weight having various bioactivities, and over 20 types of laminin receptors are reported.
The interaction between a component of a basement membrane, which is a thin extracellular matrix layer wherein cells can be adhered, and epithelial cells influences the cell function such as migration, proliferation, differentiation and the like (Crouch et al., Basement membrane.
In The Lung (ed. R. G. Crystal and J. B. West), pp 53.1-53.23. Philadelphia: Lippincott-Raven. 1996).
As for the main components of a basement membrane, laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), and entactin are known as mentioned above (Curr. Opin. Cell Biol. 6, 674-681, 1994), and mesenchymal cells are considered to play an important role for the synthesis of a basement membrane component including isoform of laminin and type IV collagen (Matrix Biol. 14, 209-211, 1994; J. Biol. Chem. 268, 26033-26036, 1993), however, the role of epithelial cells is also important.
HSPG is believed to have been derived from epithelial cells, however, laminin, type IV collagen, and entactin are synthesized in vivo by both of epithelial cells and mesenchymal cells (Development 120, 2003-2014, 1994; Gastroenterology 102, 1835-1845, 1992).
Many attempts have been made to construct an epithelial tissue model in vitro showing a continuous lamina densa.
Tissue models of intestine (J. Cell Biol. 133, 417-430, 1996) and skin (J. Invest. Dermatol. 105, 597-601, 1995; J. Invest. Dermatol. 109, 527-533, 1997; Dev.
Dynam. 197. 255-267, 1993) and the like have been studied, and some of basement membrane components derived from mesenchymal cells have been found to play an important role in the formation of a basement membrane.
Several methods to constitute a basement membrane by culturing epithelial cells, and to constitute epithelial cells wherein a basement membrane structure is present just beneath the basal surface have been reported.
For example, the present inventors have reported that a basement membrane can be formed in vitro by coculturing alveolar epithelial cells and pulmonary fibroblasts (Cell Struc. Func., 22: 603-614, 1997).
It has been reported that: if pulmonary fibroblasts, being embedded in type I collagen gel, were cultured, the collagen gel was contracted and became more solid by pulmonary fibroblasts, and extracellular matrices being secreted and adsorbed to collagen fiber around the cells and deposited; such contracted collagen gel by fibroblasts is called a pseudointerstitium since it is similar to an interstitium in vivo; and if type II alveolar epithelial cell lines (SV40-T2) were cultured on such a pseudointerstitial tissue for approximately 14 days (T2-Fgel), basement membrane components such as type IV collagen, laminin and the like in extracellular matrices secreted by pulmonary fibroblasts were diffused in a culture medium and reached to the basal surface of the above-mentioned type II alveolar epithelial cell lines, and used as a material for the constitution of a basement membrane, and as a result, a basement membrane structure was formed.
It is also reported that dilute neutral collagen solution was incubated at 37 deg. C. in 5% CO2, and collagen fiber was formed, then air-dried collagen fibrous matrix (fib) which was air-dried in aseptic condition was used as a alternative for the above-mentioned pseudointerstitium in a same manner as the above-mentioned coculture of alveolar epithelial cells and pulmonary fibroblasts to form a basement membrane (Eur. J. Cell Biol., 78:867-875, 1999; J. Cell Sci., 113:859-868, 2000).
In this process, if the concentration of collagen solution is high, there will be less or no gap in fibrous collagen matrix formed, and if epithelial cells are cultured for a long term (10 days-2 weeks) for the purpose of forming a basement membrane, cells are detached and floated (e.g. Becton Dickinson, Fibrous collagen coat culture insert), therefore, the concentration of collagen solution is considered to be optimum at 0.3-0.5 mg/ml (Eur. J. Cell. Biol., 78:867-875, 1999; J. Cell Sci., 113:859-868, 2000).
Type II alveolar epithelial cell lines (SV40-T2) were cultured on fibrous collagen matrix wherein Matrigel the registered trademark of Becton Dickinson) was added, instead of using collagen matrix wherein fibrous cells were embedded.
In this case, Matrigel functioned as an exogenous resource of basement membrane components.
Matrigel is a mixture of basement membrane components extracted from Engelbreth-Holm-Swarm tumor matrix (J. Ekp. Med. 145, 204-220, 1977), and contains laminin-1, entactin, type IV collagen, and perlecan, as well as various cytokines that possibly influence the ECM synthesis (Exp. Cell Res. 202, 1-8, 1992).
In order to trace the Matrigel components incorporated in a basement membrane, Matrigel was labeled with biotin, and the process wherein the formation of a basement membrane was accelerated depending on the amount of Matrigel, and a basement membrane matrix being secreted in punctiform manner deposited in a sheet form, then a basement membrane development was observed by immunofluorescent staining of basement membrane components such as laminin, entactin, type IV collagen, perlecan, and the electron micrographic monitoring.
As a result, it has been found that stable exogenous laminin-1 and entactin are largely involved in the complete development of a basement membrane by the above-mentioned epithelial cells in vitro at the lower surface of alveolar epithelial cells (J. Cell Sci., 113:859-868, 2000).
Further, an artificial skin formation promoting agent and skin basement membrane stabilizing agent comprising matrix metalloproteinase inhibitor or matrix metalloproteinase inhibitor and matrix protein production promoting agent; as well as a production method of artificial skin comprising adding matrix metalloproteinase inhibitor or matrix metalloproteinase inhibitor and matrix protein production promoting agent to an artificial skin formation medium are known (Japanese Laid-Open Patent Application No. 2001-269398).
The present inventors made a study on a process to form a basement membrane structure just beneath the basal surface of epithelial cells on a fibrous collagen matrix by the coculture with fibroblasts-embedded collagen gel, and by the culture in the presence of TGF-beta or Matrigel.
In the case of type II alveolar epithelial cells, it was confirmed that a basement membrane was formed in the following cases as shown in FIG. 1: the case wherein type II alveolar epithelial cells were cultured on pulmonary fibroblast-embedded collagen matrix in upper wells of culture inserts (collagen gel wherein fibrous cells are embedded) (T2-Fgel); the case wherein they were cultured on fibrous collagen substratum on upper wells in coculture with alveolar fibroblasts-embedded collagen matrix in lower wells (T2-fib-Fcm); the case wherein they were cultured on fibrous collagen substratum in upper wells in the presence of Matrigel coat on lower wells (T2-fib-MG); the case wherein they are cultured on fibrous collagen substratum in upper wells in the presence of growth factor TGF-beta in upper and lower wells (T2-fib-TGFbeta ).
However, it was also confirmed that type II alveolar epithelial cells were unable to constitute a basement membrane without the supply of, for example, a fibroblast, exogenous basement membrane components from Matrigel or a growth factor TGF-beta , since the endogenous basement membrane components from type II alveolar epithelial cells are not enough for the cells to assemble a basement membrane structure effectively.
However, following problems still remained: a problem of frequent troubles that, when fibroblasts secreting basement membrane components and a growth factor of TGF-beta are used, gel contraction occurs during the culture, and the fibroblasts are peeled off from plastic membrane together with alveolar epithelial cells; a problem that reagents used to peel alveolar epithelial cells for the preparation of basement membrane specimen tend to remain inside the gel if fibroblasts are embedded, and the wash procedure of such reagents is complicated; a problem that if some parts of cells remain, it may become an antigen; a problem that culture protocol for the formation of a basement membrane itself is complicated.
Further, there has been also a problem that if fibroblasts alternative such as Matrigel and the like or a growth factor TGF-beta is used as a resource of basement membrane component, said Matrigel and said growth factor TGFbeta are expensive, and it is not advantageous in terms of the cost.
On the other hand, constitution of a basement membrane by endothelial cells (EC) was also considered.
Although a basement membrane which is present just beneath the basal surface of endothelial cells also contributes to the expression and the maintenance of functions in endothelial cells, and a basement membrane of endothelial cells plays a role of a barrier when inflammatory cells invade into tissue from blood vessel, or when cancer cells metastasize, a basement membrane of vascular endothelial cells cannot be easily formed as for the case of epithelial cells.
In the formation of a basement membrane by vascular endothelial cells, unlike the case of type II alveolar epithelial cells, as shown in FIG. 2, a basement membrane was not formed in the following cases with the exception of the case of (EC-Fgel) wherein the culture was carried out on fibroblast-embedded collagen matrix in upper wells; the case wherein the culture was carried out on fibrous collagen substratum in (on) upper wells in the presence of pulmonary fibroblasts-embedded collagen matrix in lower wells (EC-fib-Fcm); the case wherein the culture was carried out on fibrous collagen substratum in upper wells in the presence of Matrigel coat in lower wells (EC-fib-MG); the case wherein the culture was carried out on fibrous collagen matrix in upper wells (EC-fib).
In the meantime, the present inventors have reported that epithelial cells can be automatically detached from abasement membrane if alveolar epithelial cells which formed the above-mentioned basement membrane are treated with 0.18 M of hydrogen peroxide solution for 10 minutes, continued to culture for an additional day (Cell Struc. Func., 22, 603-614, 1997).
In such process, however, it has been found to be a problem that there are cases that artificial human tissue having sufficient physiological activity such as function expression and maintenance of cells even if homogeneous or heterogeneous cells having a certain ability to form a basement membrane are seeded and cultured on said basement membrane since there are some cases wherein the detachment of cells from a basement membrane is insufficient, and a part of a basement membrane is damaged.
The object of the present invention 1 is to provide: a method for preparing a basement membrane which is extracellular matrices having a function to control morphology, differentiation, proliferation, motility, function expression and the like of cells, particularly a method for preparing a basement membrane with which cells having an ability to form a basement membrane such as epithelial cells, endothelial cells and the like can effectively activate an endogenous basement membrane component; a tissue model which can be obtained by the above-mentioned method for preparing a basement membrane; a test tissue kit including said tissue model.
The object of the present invention 2 is to easily and in the short term provide a basement membrane specimen having a function to control morphology, differentiation, proliferation, motility, function expression and the like of cells when a certain homogeneous or heterogeneous cells having an ability to form a basement membrane are seeded and cultured.
The object of the present invention 3 is to provide a process for producing a reconstituted artificial tissue having versatility wherein desired artificial tissue can be produced easily and efficiently in the short term at any time and any place when needed by seeding and culturing certain cells which are homogeneous or heterogeneous to cells which formed a basement membrane using a basement membrane specimen having a function to control morphology, differentiation, proliferation, motility, function expression and the like of cells as a common base material for tissue construction.
Another object of the present invention 3 is to provide a reconstituted artificial tissue such as a tissue model, an organ model and the like, which can be obtained by said process for producing an artificial tissue, which has cell layers and a basement membrane structure with barrier function original to a living body, and which can be advantageously applied to pharmacological test, toxicity test or the like of chemical substances.
The object of the present invention 4 is to provide a basement membrane specimen which is extracellular matrices having a function to control morphology, differentiation, proliferation, motility, function expression and the like of cells, with much higher versatility since it is possible to transplant while maintaining the structure of a basement membrane, and which is formed on a protein support structure which is temporarily adhered to plastic surface in order to be physically detached from plastic surface when needed while it is adsorbed and fixed on plastic surface when a basement membrane and an artificial tissue are prepared.
The examples include: a basement membrane specimen formed on a collagen fiber; an artificial tissue and an artificial organ such as an artificial blood vessel, an artificial lung, an artificial liver, an artificial kidney, an artificial skin, an artificial cornea and the like; for example, an artificial tissue on a collagen fiber which formed a basement membrane structure on a matrix just beneath the cells.
Basement membrane components secreted from the cells having an ability to be assembled into a basement membrane by such as epithelial cells, endothelial cells and the like, or from fibroblasts, cannot automatically form a basement membrane structure by themselves, and needs a receptor which is considered to be localized on the surface of the cells having an ability to form a basement membrane such as epithelial cells, endothelial cells and the like, or particularly on the basal surface of said cells.
The identity of said receptor, however, is not clearly known at the moment including the concern whether it is a single protein.
The present inventors, in the course of a keen study on the mechanism of a basement membrane formation, obtained the knowledge that type II alveolar epithelial cells or vascular endothelial cells express receptors for sugar chains on their basal surface since type II alveolar epithelial cells or vascular endothelial cells can be adhered to a polymer having a certain sugar chain in vitro, namely a sugar chain which can localize a receptor having an activity to accumulate a basement membrane component on the basal surface of the cells having an ability to form a basement membrane, e.g. a sugar-chain coat having beta -D-glucopyranosyl nonreducing end or 2-acetoamide-2-deoxy-beta -D-glucopyranosyl nonreducing end.
Secondly, as a result of culturing type II alveolar epithelial cells or vascular endothelial cells on fibrous collagen substratum coated with the above-mentioned polymer, the present inventors have found that a basement membrane having a barrier function similar to the one seen in vivo is formed just beneath the type II alveolar epithelial cells or vascular endothelial cells.
It was also found that although the supply of a basement membrane component such as a Matrigel and the like, and the addition of TGF-beta was not necessary for the formation of said basement membrane, if Matrigel was added, construction of a basement membrane was significantly accelerated, the culture term was long enough with a week, and the basement membrane became several fold thick.
The knowledge that the above-mentioned fact is a result of that the receptor against the above-mentioned sugar chain was involved to progress the basement membrane formation was obtained.
The present invention 1 has been completed based on such knowledge.

特許請求の範囲(英語) [claim1]
1. A method for constructing a basement membrane specimen wherein the method comprises the following steps: (a) culturing cells having an ability to form a basement membrane on a support structure coated with a sugar-chain coat which can localize a receptor having an activity to accumulate a basement membrane component on the basal surface of the cells having an ability to form a basement membrane, wherein the sugar chain coat has a beta -D-glucopyranosyl non-reducing end or a 2-acetoamide-2-deoxy-beta -D-glucopyranosyl non-reducing end and,
wherein the cells are adhered onto the support structure through the basement membrane;
(b) removing cells from the basement membrane on the support structure by subjecting the cells adhered onto the support structure through the basement membrane to a delipidating treatment using a solvent having the ability to lyse lipid of the cells;
(c) conducting a treatment to remove proteinous and nucleic acid residues contained by the cells using an alkaline solution after or at the same time as the delipidating treatment, whereby the cells are removed and the basement membrane is exposed on the support structure.
[claim2]
2. The method for constructing a basement membrane specimen according to claim 1, wherein the solvent having the ability to lyse lipid in step (b) is a surface active agent.
[claim3]
3. The method for constructing a basement membrane specimen according to claim 2, wherein the surface active agent is Triton X-100.
[claim4]
4. The method for constructing a basement membrane specimen according to claim 1, wherein the alkaline solution in step (c) is an alkaline solution with pH 8-14.
[claim5]
5. The method for constructing a basement membrane specimen according to claim 4, wherein the alkaline solution is an alkaline solution with pH 9-10.
[claim6]
6. The method for constructing a basement membrane specimen according to claim 1, wherein the support structure is a collagen gel containing embedded fibroblasts.
[claim7]
7. The method for constructing a basement membrane specimen according to claim 1, wherein the cells have an ability to form the basement membrane in the presence of a matrix metalloproteinase inhibitor.
[claim8]
8. The method for constructing a basement membrane specimen according to claim 1, wherein the cells are hyperexpressing cells.
  • 発明者/出願人(英語)
  • MOCHITATE KATSUMI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • NATIONAL INSTITUTE FOR ENVIRONMENTAL STUDIES
国際特許分類(IPC)
米国特許分類/主・副
  • 435/395
  • 435/325
  • 435/357
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