Top > Search of International Patents > CELL CULTURE SUBSTRATE, CANCER CELL AGGREGATE AND METHOD FOR MANUFACTURING SAME USING SAID SUBSTRATE, AND DRUG SCREENING METHOD USING SAID CANCER CELL AGGREGATE

CELL CULTURE SUBSTRATE, CANCER CELL AGGREGATE AND METHOD FOR MANUFACTURING SAME USING SAID SUBSTRATE, AND DRUG SCREENING METHOD USING SAID CANCER CELL AGGREGATE

Foreign code F180009554
File No. (S2017-0598-N0)
Posted date Nov 2, 2018
Country WIPO
International application number 2018JP014119
International publication number WO 2018182044
Date of international filing Apr 2, 2018
Date of international publication Oct 4, 2018
Priority data
  • P2017-072512 (Mar 31, 2017) JP
Title CELL CULTURE SUBSTRATE, CANCER CELL AGGREGATE AND METHOD FOR MANUFACTURING SAME USING SAID SUBSTRATE, AND DRUG SCREENING METHOD USING SAID CANCER CELL AGGREGATE
Abstract [Problem] An objective of the present invention is the in-vitro production of a cancer cell population retaining native biological properties, such as morphological polarity and tissue motion polarity, of the original cancer tissue.
[Solution] The present invention pertains to a cell culture substrate comprising a base material and a biocompatible polymer layer, the cell culture substrate having, at prescribed intervals on the substrate surface, a plurality of rough sections having a prescribed surface structure having a prescribed shape and not covered by the biocompatibile polymer layer. The present invention makes it possible to use a very simple process, namely culturing cancer cells on a cell culture substrate having a prescribed structure, to obtain, in a living state, a cancer cell aggregate having the same morphological polarity and tissue motion polarity as is observed in-vivo, whereby the heretofore impossible live imaging of micro tumors in-vitro is made possible. Since it is considered that said cancer cell aggregate would reproduce the in-vivo sequence of cancer development, proliferation, invasion, metastasis and recurrence, the cancer cell aggregate could be used as a research tool for cancer research or for screening cancer drugs.
Outline of related art and contending technology BACKGROUND ART
Cancer, still cannot completely overcome one of the disease. That the increase of the cost of drug development, and super senior involved in the increase of the number of cancer patients resulting in an increase in medical costs, a national financial has been a factor to strangle the. In such a situation, effective and inexpensive development of cancer therapeutic agents of the next generation, it is assumed that urgent problem.
In the study of cancer diseases, biological properties at the cellular level has been solved can be said that the progress of the other hand, a number of cancer cells is formed on the biological level of cancerous tissue kinetics characteristics, observed by the absence of the art processes, in spite of the importance are not of interest, as a result most of the not yet been elucidated. Therefore, the target cancerous tissue in vitro and the molecular basis of the physiological examination, in particular cancerous tissue as to elucidate the characteristics of the biological dynamics is, in the development of cancer therapeutic agents of the next generation, have a significant expected.
Cancer research has been carried out in conventional molecular level cancer tissue physiological considerations, primarily, ex vivo biological tissue sample was extracted and was observed with the pathological diagnosis, in the above embodiment can be estimated. On the other hand, a population of cancer cells in vitro in vivo live imaging technology, the technical difficulty and therefore has not been developed, the pathophysiological dynamics of the cancer tissue is rarely considered that it is not, a new anti-cancer agent or basic research of cancer development is performed, in the current.
Many epithelial system in cancer cells, invasion, metastasis (epithelial mesenchymal transformation) the EMT must be known. Epithelial carcinoma cancer cell system loses traits by the EMT system, such as invasive potential and capable of being in motion and acquire mesenchymal transformation invasion, metastasis and is considered to be the cause, recent studies have pancreatic ductal adenocarcinoma cells in the EMT does not require invasion, metastasis was found to occur (non-patent document 1). Not through the EMT a new cancer invasion, cell population as a transformation and movement (collective cell migration) of interest, the observed in vitro in vivo cancer cell population is a need for a technique has been increasingly in demand.
In vitro, a two-dimensional conventional single layer 2 is not in cell culture, to mimic the in vivo environment as more, has a special structure using a two-dimensional cell culture substrate 3 to form a mass, a so-called three-dimensional culture of cells 3 have been reported (for example Patent Document 1, such as 2). However, the three-dimensional cell culture 3 are formed by most of the cell mass, vigorous motion and the polarity of the polarity of the inflatable does not show morphological spheroids growth (spheroid mass of cancer cells) and, as a feature of the malignant tumor of the cancer cells in vivo in the invasive growth reflecting the population of the present invention is far.
In addition, normal epithelial cell growth and survival of the scaffold such as adhesion to the extracellular matrix is essential, cannot be adequately adhered to the scaffold (anoikis) epithelial cells are apoptotic anoikis referred to as the die. On the other hand, causes a cancer cell is EMT epitheliaf system, resistant to anoikis subject to cell death, the floating vessel from other tissue and metastasize HCCs are known.
Cancer epithelial cancer cell infiltration of anoikis-resistance, and easiness of transition from the involved, epithelial cancer cells to determine the resistance of the system the meaning of anoikis is large. However, a culture of epithelial cancer cells in the conventional system can be directed to the cell culture, the presence or absence of cancer cells are epithelial system regardless of anoikis resistance since adhesive, anoikis epithelial cancer cells in vitro to evaluate the resistance of the system is difficult.
Scope of claims (In Japanese)請求の範囲
[請求項1]
 基板と生体適合性ポリマー層とを有する細胞培養基材であって、
該基材は、生体適合性ポリマー層で覆われていない複数の粗面部分を基材表面上に有し、
ここで
粗面部分の形状は、径が20μm~100μmのスポットであるか、又は幅が3μm~30μmのグルーブであり、粗面部分の形状がグルーブの場合、粗面部分はその端部において他の粗面部分と連結されていてもよく、
2つの隣接する粗面部分間の距離は少なくとも10μm以上であり、
粗面部分はその表面に高さ20nm~200nmの凹凸構造を持つ、前記細胞培養基材。
[請求項2]
 粗面部分の界面展開面積比(Sdr)が0.002以上である、請求項1に記載の細胞培養基材。
[請求項3]
 2つの隣接する粗面部分間の距離が10~1200μmである、請求項1又は2に記載の細胞培養基材。
[請求項4]
 粗面部分表面の算術平均粗さ(Ra)が4nm以上、最大高さ粗さ(Rz)が30nm以上、及び/又は山頂点算術平均曲(Spc)が300以上である、請求項1~3のいずれか一項に記載の細胞培養基材。
[請求項5]
 生体適合性ポリマーが生物学的材料との非特異的吸着を抑制する両親媒性ポリマーである、請求項1~4のいずれか一項に記載の細胞培養基材。
[請求項6]
 生体適合性ポリマーが2-メタクリロイルオキシエチルホスホリルコリンである、請求項1~5のいずれか一項に記載の細胞培養基材。
[請求項7]
 以下の(a)~(e)の特徴を持つ、接着性がん細胞から構成される、分離された生きたがん細胞集合体。
(a)細胞内細胞構造を有する
(b)非スフェロイド状形態を示す
(c)表面にα-チューブリンの膜状発現がある
(d)形態学的極性を有する
(e)組織運動極性を有する
[請求項8]
 さらに以下の(f)~(k)のうちの少なくとも1つの特徴を持つ、請求項7に記載のがん細胞集合体。
(f)生きたがん細胞を可逆的に放出し、取り込む能力がある
(g)表面に繊毛を有する
(h)糸状仮足又は葉状仮足様形態を示す
(i)死細胞を取り込む能力がある
(j)細胞デブリ吸引力を有する
(k)表面がホスファチジルセリン陽性である
[請求項9]
 請求項7又は8に記載のがん細胞集合体が3次元構造を有する細胞培養基材に接着してなる、複合体。
[請求項10]
 請求項1~6のいずれか一項に記載の細胞培養基材を用いて接着性がん細胞を培養する工程を含む、請求項7又は8に記載のがん細胞集合体の製造方法。
[請求項11]
 請求項1~6のいずれか一項に記載の細胞培養基材を用いて接着性がん細胞を培養する工程を含む、請求項9に記載の複合体の製造方法。
[請求項12]
 請求項7又は8に記載のがん細胞集合体と被験物質を共存させる工程、
 以下のがん細胞集合体の特徴
(a)細胞内細胞構造を有する
(b)非スフェロイド状形態を示す
(c)表面にα-チューブリンの膜状発現がある
(d)形態学的極性を有する
(e)組織運動極性を有する
(f)生きたがん細胞を可逆的に放出し、取り込む能力がある
(g)表面に繊毛を有する
(h)糸状仮足又は葉状仮足様形態を示す
(i)死細胞を取り込む能力がある
(j)細胞デブリ吸引力を有する
(k)表面がホスファチジルセリン陽性である
のうちの少なくとも1つを観察し、被験物質と共存させないがん細胞集合体のそれと比較する工程、及び
被験物質共存下で上記特徴の減弱又は喪失がより強く観察された場合、被験物質は抗がん作用を有すると判定する工程
を含む、がんの予防及び/又は治療のための薬剤のスクリーニング方法。
[請求項13]
 請求項7又は8に記載のがん細胞集合体と被験物質を共存させる工程、
がん細胞集合体又はその仮足の長さ又は大きさを測定し、被験物質と共存させないがん細胞集合体のそれと比較する工程、及び
被験物質共存下でがん細胞集合体又はその仮足がより短くなった又はより小さくなった場合、被験物質は抗がん作用を有すると判定する工程
を含む、がんの予防及び/又は治療のための薬剤のスクリーニング方法。
[請求項14]
 薬剤が、がんの浸潤及び/又は転移を抑制する薬剤である、請求項12又は13に記載のスクリーニング方法。
[請求項15]
 薬剤が、がんの免疫回避機構を抑制及び/又は解除する薬剤である、請求項12又は13に記載のスクリーニング方法。
[請求項16]
 請求項1~6のいずれか一項に記載の細胞培養基材を用いて被験上皮系癌細胞を培養する工程、及び
上皮系癌細胞が細胞培養基材に接着せずに増殖した場合に上皮系癌細胞はアノイキス耐性を有すると判定する工程
を含む、上皮系癌細胞のアノイキス耐性を判定する方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • HOKKAIDO UNIVERSITY
  • Inventor
  • MIYATAKE Yukiko
  • SHIGETOMI Kaori
  • OKAJIMA Takaharu
  • KASAHARA Masanori
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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