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CELL SORTING METHOD

Foreign code F180009566
File No. (S2017-0495-N0)
Posted date Nov 5, 2018
Country WIPO
International application number 2018JP008615
International publication number WO 2018164133
Date of international filing Mar 6, 2018
Date of international publication Sep 13, 2018
Priority data
  • P2017-043744 (Mar 8, 2017) JP
Title CELL SORTING METHOD
Abstract Provided is a novel simplified and easy cell separation method. Also provided are: a cell sorting method, comprising (a) performing a treatment for increasing the difference in cell membrane fluidity between a target cell and a non-target cell and (b) sorting the target cell utilizing the difference in cell membrane fluidity; a kit for cell sorting; a culture medium for use in cell sorting; a reagent for performing a treatment for increasing the difference in cell membrane fluidity between a target cell and a non-target cell; and a method for quantifying the adhesion of a cell to a substrate.
Outline of related art and contending technology BACKGROUND ART
Takebe et al. develop liver initial occurrence of a phenomenon relating to the processes 3 one of the cells (formed in) are identified, they may be mixed in a liver in vitro (organ type) to create the original group of successful (non-patent document 1:Nature 2013, Patent Document 1:WO2013/047639 A1: a method of producing the tissues and organs) of the original group is liver. for the expression of a liver function after implantation, for patients with organ dysfunction have been spotlighted as a therapy epoch. in addition, the method of the same intestine, kidney, pancreas, brain, lung, cancer, such as the heart, can be extended to a variety of tissues (JP-2:WO2015/012158 A1, JP-3:WO2015/129822 A1) from, the original group of the base to construct the principle of the organ, is expected to be applied to various fields and where., is applied to the actual organ regeneration for medical, the safety and the homogeneity of the organ to establish a method to induce the original group is necessary. however, as a point of major problem with the present system, cells in the raw material 'safety' and the 'uniformity' of the original group is a problem such as a human organ. iPS cells from stem cells by adjusting the material and a plurality of cells, is capable of inducing, iPS undifferentiated cells, such as midway in the undifferentiated progenitor cells of the non-target cells may be mixed therein and, safety and uniformity problems have been left for example., this remaining undifferentiated cells and to cancer, the risk to grow although it has been shown that, in the experiment by a simple and easy user level is an undifferentiated cell separation method is not established (non-patent document 2: the Cunningham et al., Nat.Biothechnol. 2012)., and endodermal cells in the midway of the non-target cells to differentiate into the gut and intestinal tissue may be formed in the remaining, stromal cells in the same manner as the remaining excess fibrotic tissue can be formed although the risk of such as pointed out, such a tissue progenitor cells are selectively removed from the cell source method does not exist in the actual laboratory level., a number of donor cells of the organ using the original group of researchers that function depends on the stoichiometry multilaterally analyze the experience-and the method, on the industrial applications has been a crucial barrier and reproduced as a cell sorting. medical research, drug discovery is widely used in the field of research or Fluorescence activated cell sorting (FACS), is Magnetic-activated cell sorting (MACS) (Non-Patent Document 3: corresponding to surface antigens Maecker et al., Nature Immunology 2010). the present method is applied to a magnetic material and a fluorescent antibody reaction, fluorescence and magnetic force can be isolated selectively only emits cells. however, the selection of fluorescent antibodies to the identification of surface antigens, and the enormous amount of time to purchase further FACS the cost also., the actual field of regenerative medicine in the case where the assumed to be used, solid organ such as a large amount greater than required for the treatment of (1010-) the accuracy of the cell sorting is required, the sorting takes a long time because of the damage to the cells with difficult to use in a further., and the remaining markers is also an issue of great risk assessment for the remaining unlabeled and where., low cost, the development of rapid cell selection methods have been expected.
Scope of claims (In Japanese)請求の範囲
[請求項1]
a)標的細胞と非標的細胞との細胞膜流動性の差を拡大させる処理を行うこと,及び
b) 細胞膜流動性の差を利用して,標的細胞を選別すること
を含む,細胞選別法.
[請求項2]
未分化細胞を除去するために用いられる請求項1記載の方法.
[請求項3]
分化細胞を濃縮するために用いられる請求項1記載の方法.
[請求項4]
細胞集団を構成する細胞を均質化するために用いられる請求項1記載の方法.
[請求項5]
標的細胞と非標的細胞との細胞膜流動性の差を拡大させる処理が,細胞膜流動性を細胞種特異的に変化させることができる物質を培地に添加することである請求項1~4のいずれかに記載の方法.
[請求項6]
細胞膜流動性の差を拡大することにより、基質に対する細胞の接着性の差を拡大させ、この差を利用して、標的細胞を選別する請求項1~5のいずれかに記載の方法。
[請求項7]
細胞膜流動性を細胞種特異的に変化させることができる物質が、ポリフェノール、分化誘導因子、インヒビター、増殖因子、薬剤又はアミノ酸・界面活性剤のいずれかである請求項5又は6に記載の方法。
[請求項8]
細胞膜流動性を細胞種特異的に変化させることができる物質が、下記の群から選択される少なくとも1つの化合物である請求項7記載の方法。
(1) ポリフェノール群:resveratrol、epigallocatechin gallate (EGCG)、curcumin及びgenistein
(2) 分化誘導因子群:activin-A、wint-3a、sodium butylate、basic fibroblast growth factor (bFGF)、oncostatin M (OSM)、dexamethasone (DEX)、hepatocyte growth factor (HGF)、CHIR-99021及びforskolin
(3) インヒビター群:Y-27632 (rock inhibitor)、(s)-(-)-blebbistatin、IWP2、A83-01、LY294002、SB-431542、NVP-BHG、Cyclopamine-KAAD、及びPD-0325901
(4) 増殖因子群:FGF4、LDN-193189、insulin like growth factor (IGF)、bone morphogenetic protein (BMP)2、transforming growth factor (TGF)β2、BMP4、FGF-7、platelet-derived growth factor (PDGF)β3、epidermal growth factor (EGF)、exendin-4、human neuregulin (hHRG)β3、retionic acid (RA)、L-Ascorbic acid 2-phosphate (AA2P)、ascorbic acid、insulin-transferrin-selenium ethanolamine solution (ITS-X)、及びinsulin
(4) 薬剤群:rifampicin、prostaglandin E2 (PGE2)及びpeniciline/streptomycine solution
(5) アミノ酸・界面活性剤群:2-mercaptoethanol、3-mercaptopropane-1,2-diol (thioglycerol)、L-proline、L-glutamine、non-essential amino acid mixture(NEAA)、sodium pyruvate、trypsin-EDTA及びphosphatidylinositol (PI)
[請求項9]
標的細胞と非標的細胞との細胞膜流動性の差を拡大させる処理を行うための試薬を含む,細胞選別用キット.
[請求項10]
標的細胞と非標的細胞との細胞膜流動性の差を拡大させる処理を行うための試薬を含む,細胞選別用培地.
[請求項11]
細胞膜流動性を細胞種特異的に変化させることができる物質を含む,標的細胞と非標的細胞との細胞膜流動性の差を拡大させる処理を行うための試薬.
[請求項12]
基質に接着した細胞の割合(接着率)を培養時間毎に測定することを含む,基質に対する細胞の接着性を定量する方法.
[請求項13]
細胞を提供するドナー間の差を調べるために測定を行う請求項12記載の方法.
[請求項14]
細胞種間の差を調べるために測定を行う請求項12記載の方法.
[請求項15]
a1) 未分化細胞を分化誘導するにあたり,未分化細胞の分化前と分化後との細胞膜流動性の差を拡大させる処理を行なうこと,及び
b1)細胞膜流動性の差を利用して,分化細胞を選別すること
を含む,細胞の分化誘導方法.
[請求項16]
a1) 未分化細胞を分化誘導するにあたり,未分化細胞の分化前と分化後との細胞膜流動性の差を拡大させる処理を行なうこと,及び
b1)細胞膜流動性の差を利用して,分化細胞を選別すること
を含む,分化細胞の調製方法.
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • PUBLIC UNIVERSITY CORPORATION YOKOHAMA CITY UNIVERSITY
  • Inventor
  • TAKEBE, Takanori
  • TANIGUCHI, Hideki
  • MATSUZAKI, Takahisa
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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