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METHOD FOR PRODUCING SPHEROIDS AND CULTURE MEDIUM USED IN METHOD

Foreign code F160008886
File No. (S2015-0789-N0)
Posted date Oct 25, 2016
Country WIPO
International application number 2016JP057307
International publication number WO 2016143808
Date of international filing Mar 9, 2016
Date of international publication Sep 15, 2016
Priority data
  • P2015-046420 (Mar 9, 2015) JP
Title METHOD FOR PRODUCING SPHEROIDS AND CULTURE MEDIUM USED IN METHOD
Abstract The purpose of the present invention is to provide a method with which spheroids of good quality can be produced in large quantities by a simple method. Another purpose of the present invention is to provide a method with which spheroids can be easily produced without using a growth factor or an adhesion factor, an intercellular matrix, or a special culture system. The present invention provides a method for producing spheroids, characterized in that cells are cultured using a glucose-containing culture medium in which the glucose substantially comprises l-glucose. Also provided is a method for producing spheroids in a culture medium free of a growth factor, an adhesion factor and an intercellular matrix, using a culture system that does not employ a three-dimensional support.
Outline of related art and contending technology BACKGROUND ART
A commercially available culture dish or multiwell plate or the like in the culture vessel was used, cells are incubated in a single layer two-dimensionally, some of the functions of cells in vivo may not be sufficiently expressed. On the other hand, 3 cells were grown in a two-dimensional, spherical or ovoid in the three-dimensional culture method is to take the form of spheroids, nutrients and oxygen, waste products such as carbon dioxide as metabolites or physiological gradient is thought to occur within the spheroid, present in the environment in vivo than cells in close proximity to appear in cell state can be, such as evaluating the efficacy of the drug discovery screening and tumor assessments, the functional expression of the production of cellular products such as rapidly as an effective method has been increasingly used.
As a method for forming a spheroidal, hanging drop culture method up to now (non-patent document 1) or gel (non-patent document 2) of culture in, was used a low-adhesion culture (non-patent document 3), (scaffold) nanoimprinting precision processing technology to prepare a scaffold by using special dish culture (non-patent document 4), the addition of growth factors such as Matrigel (non-patent document 5), various methods have been proposed.
These methods however, have low reproducibility, and the expensive special device requires a container, or, the shape of the container as it is or an obstacle for cell observation, growth factors and adhesion factors, it has been found that a medium that does not component such as a problem that silicon is necessary.
As a specific culture method is as described below have been proposed. Is hanging drop culture method, culture using the surface tension of the droplets of the cell suspension in the culture conditions and in the spot (non-patent document 1). By this method, the formation of spheroids having a uniform size but, in order to exchange the medium, making it difficult and long-term culture, the method is not suitable for generating a large amount of spheroids, which is problematic.
Using a low-adhesion culture dish spheroids, cells adhering to the surface of the dish to form spheroids by preventing culture methods but, once formed and facilitates fusion of spheroids are gradually, there is a problem in stability (non-patent document 6).
Such as U-Shape is due to the use of culture dish, to avoid the merged spheroids, can be to allow formation of a single spheroid. Then, the bottom or bottom V U is devised a method using a special container, which is commercially available. The method, but the formation of spheroids of uniform size, tends to become non-dense cell, do not have a spherical or elliptical shape of the bowl-shaped tends to be scattered. In order to make the dense spheroids, but the addition of a growth factor to the medium, the nature of the as spheroids tend to become insufficient.
Is rotated by culturing spheroid culture method, a roller culture and also requires a special device, non-uniform size and shape of spheroids, the spheroids affect the reproducibility of experiments (non-patent document 1).
Then performing a special processing by fine spheroid culture method as special container, the bottom surface of a dish, a wide area of the smaller size of the cell adhesion surface are arranged in non-cell-adhesive surface, the bond to the bottom surface of a dish of cells by unstable to cause the formation of spheroids and a method, a lattice-like structure of the bottom surface of a dish nano-sized fine working of the spheroidal cell adhesion by inhibiting the like have been proposed a method for forming a, which is commercially available. These methods may be employed, is the formation of spheroids having a uniform shape. However, then performing a special processing on the dish is extremely costly, not yet published or recommended Matrigel and other components may be added as a special additive or the like, it should be noted that in vivo of a substrate which is a fine formation of spheroids tend to be insufficient. In further microscopic observation, a special processing of the bottom surface of a dish become an obstacle such as the existence of (non-patent document 4) and a disadvantage.
Abnormal cells in vivo, cancer cells and the course, undifferentiated cells such as differentiation in over the course of, or cells from proximity to each other in the form of a non-uniform state and functions has been known. The method for manufacturing a conventional spheroids, the spheroids approximately 100μm in diameter or more, proliferating cells in the peripheral portion, the center of the necrotic-like cell clusters exhibit typical spheroids formed. However, the above-described in vivo between cells and showing a state the precise spheroidal, a large amount, with good reproducibility, it is difficult to stably in vitro was formed.
Is a conventional spheroid culture method, with the elapse of days in culture rapidly lost and spheroids, the spheroids can be used good quality thus results in a very short timing, a disadvantage in that the easy to use. In addition, in the case of culture methods will not be spheroidal, dense and between cells, it was difficult for the formation of spheroids of good quality.
Made of spheroids, in culture, cell agglomerates 3 by forming a three-dimensional structure, and a state close to appear more in vivo, cells can exhibit specific functions considered. In addition, the evaluation of the efficacy in drug discovery or tumor assessments, such as the production of cellular products in the functional expression of evaluation or the like, a comparable in vivo cell is a cell exhibits a function to stably produce cell clump is desirable. At this time, as much as possible in a simple manner, producing a large number of spheroids can be of good quality has been desired.
The inventors so far, not a naturally occurring 2-NBDLG(2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-2-deoxy-L-glucose L- fluorescent-labeled glucose derivatives; Patent Document 1) to, and collected from a live human cancer cells is applied to the cancerous tissue, cancer cells selectively capture fluoresce 2-NBDLG, non-cancer cells can be identified as found (Japanese Patent Application 2014-015735, and Japanese Patent Application 2014-193424).
The inventors of the present invention also relates to, in the case of using cultured cancer cells, the manner in which the ends of the abnormal cells that exhibit the size of the nuclei in the spheroids including a group of cells to grant the capture 2-NBDLG reported (patent document 2). That is, in the same manner as derived from living cells, carcinoma cells in culture is also formed on the first spheroidal capture 2-NBDLG is reported. However, such a large amount of spheroids can be formed with high reproducibility in the conventional method even by additives such as Matrigel was difficult.
Scope of claims (In Japanese)[請求項1]
グルコース含有培地であってグルコースが実質的にL-グルコースから構成される培地を用いて細胞を培養することを特徴とするスフェロイドの作製方法。
[請求項2]
L-グルコースの割合が、D-グルコースおよびL-グルコースからなる全グルコースの90%以上である請求項1に記載の方法。
[請求項3]
前記細胞が、がん細胞由来の細胞である請求項1または2に記載の方法。
[請求項4]
前記培地での培養が3日以上である請求項1~3のいずれか一つに記載の方法。
[請求項5]
前記培地が、成長因子および/または接着因子を添加しない培地である、請求項1~4のいずれか一つに記載の方法。
[請求項6]
前記培地が、細胞間マトリックスを添加しない培地である、請求項1~5のいずれか一つに記載の方法。
[請求項7]
前記培養が立体的な支持体を用いない培養である請求項1~6のいずれか一つに記載の方法。
[請求項8]
以下の工程:
(a)D-グルコースを含有する培地にて細胞を培養する工程、
(b)培地中のD-グルコースをL-グルコースに置き換える工程、
(c)グルコース含有培地であってグルコースが実質的にL-グルコースから構成される培地を用いて細胞を培養する工程、
を含む、スフェロイドの作製方法。
[請求項9]
前記工程(b)が、培地中のD-グルコース濃度を段階的に下げるとともに、L-グルコース濃度を段階的に上げて細胞を培養する工程である、請求項8に記載の方法。
[請求項10]
工程(c)におけるグルコース含有培地中のL-グルコースの割合が、D-グルコースおよびL-グルコースからなる全グルコースの90%以上である請求項8または9に記載の方法。
[請求項11]
前記工程(c)における培養が3日以上である、請求項8~10のいずれか一つに記載の方法。
[請求項12]
前記細胞が、がん細胞由来の細胞である請求項8~11のいずれか一つに記載の方法。
[請求項13]
前記工程(c)の培地が、成長因子および/または接着因子を添加しない培地である、請求項8~12のいずれか一つに記載の方法。
[請求項14]
前記工程(c)の培地が、細胞間マトリックスを添加しない培地である、請求項8~13のいずれか一つに記載の方法。
[請求項15]
前記工程(a)~(c)の培地が、成長因子、接着因子、細胞間マトリックスのいずれも添加しない培地である、請求項8~14のいずれか一つに記載の方法。
[請求項16]
前記培養が立体的な支持体を用いない培養である請求項8~15のいずれか一つに記載の方法。
[請求項17]
請求項1~16のいずれか一つに記載の方法により作製されたスフェロイド。
[請求項18]
前記スフェロイドが細胞間が緻密な状態なスフェロイドである請求項17に記載のスフェロイド。
[請求項19]
グルコース含有培地であってグルコースが実質的にL-グルコースから構成されるスフェロイド培養培地。
[請求項20]
L-グルコースの割合が、D-グルコースおよびL-グルコースからなる全グルコースの90%以上である請求項19に記載の培地。
[請求項21]
前記培地が成長因子および/または接着因子を添加しない培地である、請求項19または20に記載の培地。
[請求項22]
前記培地が細胞間マトリックスを添加しない培地である、請求項19~21のいずれか一つに記載の培地。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • HIROSAKI UNIVERSITY
  • Inventor
  • YAMADA, Katsuya
  • ONO, Kouki
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 DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR 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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