WO2015035996A1 - Produits de culture cellulaire pour cultures cellulaires adhérentes et leur fabrication - Google Patents

Produits de culture cellulaire pour cultures cellulaires adhérentes et leur fabrication Download PDF

Info

Publication number
WO2015035996A1
WO2015035996A1 PCT/DK2014/000046 DK2014000046W WO2015035996A1 WO 2015035996 A1 WO2015035996 A1 WO 2015035996A1 DK 2014000046 W DK2014000046 W DK 2014000046W WO 2015035996 A1 WO2015035996 A1 WO 2015035996A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell culture
coating
self
cyclodextrin
modified
Prior art date
Application number
PCT/DK2014/000046
Other languages
English (en)
Inventor
Kim Lambertsen Larsen
Ludmilla LUMHOLDT
Jørgen ROSENKILDE
Christian Clausen
Original Assignee
Amphidex A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amphidex A/S filed Critical Amphidex A/S
Publication of WO2015035996A1 publication Critical patent/WO2015035996A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D105/00—Coating compositions based on polysaccharides or on their derivatives, not provided for in groups C09D101/00 or C09D103/00
    • C09D105/16—Cyclodextrin; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to products for adherent cell cultures and manufacture thereof.
  • cells in liquid media can be introduced into a cell culture vessel such as a cell culture flask or a multi-well cell culture plate, the cell culture vessel placed into a suitable environment such as an incubator, and the cells allowed to settle onto a surface of the cell culture vessel where they attach, grow, and divide.
  • a suitable environment such as an incubator
  • some cells perform better than others when growing on a flat surface.
  • Some cells require specialized surfaces in order to maintain a more natural phenotype, and to provide optimal in vitro data.
  • the conditions under which the cells are cultured affects the characteristics of cells in culture, and therefore affect the value of data obtained from cells in culture. There is a need in the industry to provide cell culture surfaces and conditions to provide data that is more highly correlated with in vivo cell behaviour. Summary of the invention
  • one object of the present invention is to provide a product for adherent cell culture suitable for different types of cell cultures.
  • a specific object of the present invention is to provide a product for adherent cell culture suitable for pluripotent stem cells, such as human pluripotent stem cells.
  • Another specific object of the present invention is to provide a product for adherent cell culture suitable for mesenchymal stem cells, such as human mesenchymal stem cells.
  • One aspect of the invention relates to the use of an adsorption solution comprising at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent for coating a cell culture surface, wherein at least one of the hydroxyl groups (-OH) of said cyclodextrin is modified to -0-(CO) n Ri, -NR 1 R 2 , -SR ⁇ -N-COR1, or -O-(CO) n OR 1 ; wherein Ri, and R 2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; Ri and R 2 , are optionally linked with a bridge member Y n , thereby forming one or more rings, Y n being a bond or a Ci_i 2 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4
  • Another aspect relates to a product for adherent cell culture comprising:
  • a first coating comprising one or more modified self-assembling or self- aggregating cyclodextrins, wherein at least one of the hydroxyl groups (- OH) of said cyclodextrin is modified to -0-(CO) friendshipRi , -NRi R 2 , -SRi , -N-COR1 , or -O-(CO) n ORi;
  • Ri, and R 2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; Ri and R 2 , are optionally linked with a bridge member Y n , thereby forming one or more rings, Y n being a bond or a C 1-12 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 hetero
  • Yet another aspect relates to a method of making product for adherent cell culture comprising:
  • adsorption solution comprises at least one modified self-assembling or self- aggregating cyclodextrin and at least one solvent, wherein at least one of the hydroxyl groups (-OH) of said cyclodextrin is modified to -0-(CO) n Ri, - NR 1 R2, -SRi, -N-COR1, or -0-(CO) n ORi; wherein R ⁇ and R 2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; Ri and R 2 , are optionally linked with a bridge member Y n , thereby forming one or more rings, Y n being a bond or a C-i- 12 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having
  • Figure 1 shows microscopic images of human MSC cell adhesion on cell culture surfaces treated with different CD concentrations in the adsorption solution compared to a control
  • Figure 2 shows microscopic images of human MSC cell adhesion on a surface treated with 2 mg/ml CD concentration in the adsorption solution compared to a control
  • Figure 3 shows microscopic images of human iPS cell adhesion on cell culture surfaces treated with a coating comprising an extracellular matrix material (Pluripro®) (control), compared to adhesion on cell culture surfaces treated with a double coating, the inner coating being the CD coating and the outer coating comprising an extracellular matrix material (Pluripro®),
  • Pluripro® extracellular matrix material
  • Figure 4 shows flow cytometry analysis for standard iPS cell surface markers of the images shown in figure 3
  • Figure 5 shows microscopic images of human iPS (two cell lines: Biol and Bio2) cell adhesion on cell culture surfaces in a setup according to table 1 , and
  • Figure 6 shows a microscopic image of human iPS cell adhesion on cell culture surfaces treated with a double coating, the inner coating being an extracellular matrix material (Pluripro®), and the outer coating being the CD coating with Y-27632 (Rock inhibitor) encapsulated therein as an inclusion complex (30 ⁇ Y-27632 in the adsorption solution). Furthermore, two control images are shown, as well as a graph showing results of the cell counting analysis.
  • Pluripro® extracellular matrix material
  • Y-27632 Rock inhibitor
  • one object of the present invention is to provide a product for adherent cell culture suitable for different types of cell cultures.
  • a specific object of the present invention is to provide a product for adherent cell culture suitable for pluripotent stem cells, such as human pluripotent stem cells.
  • Another specific object of the present invention is to provide a product for adherent cell culture suitable for mesenchymal stem cells, such as human mesenchymal stem cells.
  • the above objects are solved by coating a cell culture surface with an adsorption solution comprising at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent.
  • One aspect of the invention relates to the use of an adsorption solution comprising at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent for coating a cell culture surface, wherein at least one of the hydroxyl groups (-OH) of said cyclodextrin is modified to -O-(CO) n Ri , -NR 1 R 2 , -SRi, -N : CORi , or -O-(CO) n ORi; wherein R- 1 , and R 2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; Ri and R 2 , are optionally linked with a bridge member Y n , thereby forming one or more rings, Y n being a bond or a d- 12 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring
  • self-assembling cyclodextrins relates to cyclodextrins which undergo spontaneous assembling into ordered structures.
  • self-aggregating cyclodextrins relates to cyclodextrins which undergo spontaneous assembling into non- ordered/disorganized structures.
  • the terminology “self-assembling” and “self-aggregating” are generally accepted within the field of chemistry and are as examples described in the books 1) "Supramolecular Chemistry", WILEY 1999, Chapter 7.2 - Biochemical Self-Assembly; Jonathan W. Steed, Jerry L. Atwood and 2) "Supramolecular Chemistry", Oxford Chemistry Primers 2000, Chapter 6 - Self-Assembly; Paul D. Beer, Philip A. Gale and David K. Smith.
  • cyclodextrins examples include self-assembling cyclodextrins and/or self-aggregating cyclodextrins.
  • cyclodextrin and CD are used interchangeably throughout this application.
  • the CD is made up of a-1 ,4 linked glucopyranose units. The number of glucopyranose determines the type of cyclodextrin, with the most common having 6, 7 or 8 units called a-CD (alpha-CD), ⁇ -CD (beta- CD) and ⁇ -CD (gamma-CD), respectively.
  • the modified CD is a modified ⁇ -CD.
  • multi-layers of self- assembling cyclodextrins can be formed on the cell culture surface.
  • the term "cell culture surface” include plastic or polymeric substrates which may form cell culture vessels or containers (products having a cell culture surface), and include those comprising or composed of polyester, polyurethane, polyacrylate, polyacrylamide, polystyrene, polypropylene, polymethyl methacrylate, polyolefin, cyclic polyolefin, polyvinyl chloride, polymethyl pentene, polyethylene, polycarbonate, polysulfone, polystyrene copolymers (e.g., SAN and ABS), polypropylene copolymers, ethylene/vinyl acetate copolymer, polyamides, fluoropolymers, polyvinylidene fluoride, polytetrafluoroethylene, silicones, and elastomers, including silicone, hydrocarbon, fluorocarbon elastomers or any other suitable surface. Additional embodiments include plastic or polymeric substrates which have been pre-coated or pre-treated.
  • the term "cell culture surface” also includes inorganic substrates which may form cell culture vessels or containers (products having a cell culture surface) and include those comprising or composed of inorganic materials such as metals, semiconductor materials, glass and ceramic materials.
  • metals that can be used as surface or substrate materials include oxides of gold, platinum, nickel, palladium, aluminum, chromium, steel, and gallium arsenide.
  • Semiconductor materials used for substrate or surface material can include silicon and germanium.
  • Glass and ceramic materials that can be used for surface or substrate material can include quartz, glass, porcelain, alkaline earth aluminoborosilicate glass and other mixed oxides.
  • Further examples of inorganic substrate materials include graphite, zinc selenide, mica, silica, lithium niobate, and inorganic single crystal materials.
  • cell culture vessels or containers include labware or cell culture vessels made from a substrate or combination of substrates, where any part of the labware or cell culture vessel has been coated with a cell culture coating of the present invention.
  • Labware includes slides, cell culture plates, multi-well plates, 6-well plates, 2-well plates, 24-well plates, 48- well plates, 96-well plates, 384-well plates, 136 well plates, flasks, multi-layer flasks, bioreactors, cell culture inserts such as the Transwell® made by Corning Incorporated, and other surfaces or containers or vessels useful for cell culture.
  • the formed micelles or aggregates are thermodynamically unstable, and that this is the driving force for adsorption of the self-assembling cyclodextrin onto a solid material surface.
  • the more unstable the suspension the larger the driving force for adsorption onto a solid material surface.
  • an unstable adsorption solution is optimal for coating.
  • the combination of the one or more solvents with specific cyclodextrins with radicals promotes self-assembling and formation of micelles or aggregates.
  • the purpose of Ri and R 2 is to provide self- assembling or self-aggregating layers of said cyclodextrin onto a surface.
  • the adsorption solution comprises at least one modified self-assembling cyclodextrin and at least two solvents, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to - 0-(CO) n R, wherein R is a straight or branched aliphatic chain of 1-50 carbons, and n is an integer from 0 to 1 , wherein at least one solvent is polar.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least two solvents, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -0-(CO) n Ri , wherein is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -0-(CO) n Ri, wherein R- ⁇ is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -0-(CO) n Ri, wherein Ri is a straight or branched aliphatic chain of 1-50 carbons, and n is an integer from 0 to 1.
  • the adsorption solution comprises at least one modified self-assembling cyclodextrin and at least two solvents, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -0-(NH) n R, wherein R is a straight or branched aliphatic chain of 1-50 carbons, such as 2-20 carbons, preferably 4-12 carbons, such as 4-8 carbons, and n is an integer from 0 to 1 , wherein at least one solvent is polar.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -NR ⁇ , wherein Ri and R 2 , independently of one another, is a straight or branched aliphatic chain of 1-50 carbons, such as 2-20 carbons, preferably 4-12 carbons, such as 4-8 carbons.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -NR-
  • Y n being a bond or a C 1-12 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatoms selected from the group consisting of N, O, S, S(0)i -2 or carbonyl, and wherein n is an integer between 1 and 12.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -SR ⁇ wherein Ri is a straight or branched aliphatic chain of 1-50 carbons, such as 2-20 carbons, preferably 4-12 carbons, such as 4-8 carbons.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -SRi, wherein Ri is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -N-COR 1 , wherein RT is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -N-COR 1 , wherein Ri is a straight or branched aliphatic chain of 1-50 carbons, such as 2-20 carbons, preferably 4-12 carbons, such as 4-8 carbons.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -0-(CO) n ORi , wherein Ri is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl.
  • the adsorption solution comprises at least one modified self-assembling or self-aggregating cyclodextrin and at least one solvent, characterized in that at least one of the hydroxyl groups (-OH) of the cyclodextrin is modified to -O-iCO ⁇ OR L wherein is a straight or branched aliphatic chain of 1-50 carbons, such as 2-20 carbons, preferably 4-12 carbons, such as 4-8 carbons.
  • the adsorption solution comprises at least one solvent, wherein at least one solvent is polar.
  • the adsorption solution comprises at least one solvent, wherein at least one solvent is apolar.
  • the adsorption solution comprises at least two solvents.
  • the adsorption solution comprises at least two solvents, wherein at least one solvent is polar.
  • At least one solvent is polar.
  • Form a ring means that the atoms mentioned are connected through a bond such that the ring structure is formed.
  • ring is used synonymously with the term “cyclic”.
  • Alkyl group means a saturated linear, branched or cyclic hydrocarbon group including, for example, methyl, ethyl, isopropyl, t-butyl, heptyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, and the like.
  • Preferred alkyls are lower alkyls, i.e. alkyls having 1 to 10 carbon atoms, such as 1 , 2, 3, 4, 5, 6, 7 or 8 carbon atoms.
  • a cyclic alkyl/cycloalkyl means a saturated carbocyclic compound consisting of one or two rings, of three to eight carbons per ring, which can optionally be substituted with one or two substituents selected from the group consisting of hydroxy, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, aryl-aminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylamino-carbonyl, alkylcarbonylamino and arylcarbonylamino.
  • the alkyl group may also be understood as a heteroalkyl.
  • a heteroalkyl is a saturated linear, branched or cyclic hydrocarbon group (including, for example, methyl, ethyl, isopropyl, t- butyl, heptyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, and the like) wherein one or more carbon atoms are substituted for a heteroatom selected from N, O, S, S(0)i -2, Si or P and which can optionally be substituted with one or more substituents selected from the group consisting of hydroxyl, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfony
  • Heteroalkyls of the present invention may be branched or unbranched or forming a ring and may range from one (1) to fifty (50) carbon atoms in length wherein 50% or less, of said carbon atoms may be substituted for N, NH(Q-4), O, S, S(0).sub.1-2, Si, P, CI, Br:
  • a cyclic heteroalkyl/heterocyclyl means a saturated cyclic compound or part of a compound, consisting of one to more rings, of three to eight atoms per ring, incorporating one, two, three or four ring heteroatoms, selected from N, O or S(0).sub.1-2, and which can optionally be substituted with one or two substituents selected from the group consisting of hydroxyl, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl,
  • heterocyclyls of the present invention include, but are not limited to piperazine and piperidine which may thus be heterocyciyi substituents as defined herein. Such substituents may also be denoted piperazino and piperidino respectively.
  • a further heterocyciyi of the present invention is thiophene.
  • alkoxy groups are hexyloxy, pentoxy, n-butoxy, propoxy, i-propoxy, methoxy and ethoxy, in particular ethoxy and n-butoxy.
  • the radical R may also be an optically active organic radical containing one or more asymmetrical carbon atoms.
  • Halogen preferably represents chlorine, but may also be bromine, fluorine or iodine.
  • Alkenyl group means a non-saturated linear, branched or cyclic hydrocarbon group including, for example, methylene or ethylene.
  • Preferred alkenyls are lower alkenyls, i.e. alkenyls having 1 to 10 carbon atoms, such as 1 , 2, 3, 4, 5 or 8 carbon atoms.
  • a cyclic alkenyl/cycloalkenyl means a non-saturated carbocyclic compound consisting of one or two rings, of three to eight carbons per ring, which can optionally be substituted with one or two substituents selected from the group consisting of hydroxy, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, aryl-aminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylamino-carbonyl, alkylcarbonylamino and arylcarbonylamino.
  • the alkenyl group may also be understood as a heteroalkenyl.
  • a heteroalkenyl is a non-saturated linear, branched or cyclic hydrocarbon group (including, for example, methyl, ethyl, isopropyl, t-butyl, heptyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, and the like) wherein one or more carbon atoms are substituted for a heteroatom selected from N, O, S, S(0)i -2 , Si or P and which can optionally be substituted with one or more substituents selected from the group consisting of hydroxyl, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl,
  • Heteroalkenyls of the present invention may be branched or unbranched or forming a ring and may range from one (1) to fifty (50) carbon atoms in length wherein 50% or less, of said carbon atoms may be substituted for N, NH(Q-4), O, S, S(0).sub.1-2, Si, P, CI, Br.
  • a cyclic heteroalkenyl means a non-saturated cyclic compound or part of a compound, consisting of one or more rings, of three to eight atoms per ring, incorporating one, two, three or four ring heteroatoms, selected from N, 0 or S(0)i -2, and which can optionally be substituted with one or two substituents selected from the group consisting of hydroxyl, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminofarbonyl, arylaminocarbonyl,
  • Alkynyl group means a non-saturated linear or branched hydrocarbon group including, for example, ethynyl or propynyl.
  • Preferred alkynyls are lower alkynyls, i.e. alkynyls having 1 to 10 carbon atoms, such as , 2, 3, 4, 5 or 9 carbon atoms.
  • the acyl radical has the formula RCO, where R represents an alkyl group that is attached to the CO group with a single bond.
  • R represents an alkyl group that is attached to the CO group with a single bond.
  • Examples of acyl radicals are alkanoyl, aroyl, lower alkoxycarbonyl, or N,N-di-lower alkylcarbamoyl, preferably lower alkanoyl.
  • Acyl in acyloxy represents lower alkanoyl, aroyl, lower alkoxycarbonyl, or N,N-di-lower alkylcarbamoyl, preferably lower alkanoyl.
  • Lower alkanoyl is preferably acetyl, propionyl, butyryl, or pivaloyl, especially acetyl.
  • Aroyl is preferably benzoyl; and also e.g. benzoyl substituted by one or two of lower alkyl, lower alkoxy, halogen or trifluoromethyl; aroyl is also e.g. thienoyl, pyrroloyl, 2-, 3- or 4-pyridylcarbonyl, advantageously nicotinoyl.
  • Lower alkanoyloxy is preferably acetoxy; and also e.g. pivaloyloxy or propionyloxy.
  • Aroyloxy is preferably benzoyloxy; and also e.g.
  • Heteroaroyloxy is preferably 2-, 3- or 4-pyridylcarbonyloxy, advantageously nicotinoyloxy.
  • Aryl represents a hydrocarbon comprising at least one aromatic ring, and may contain from 5 to 18, preferably from 6 to 14, more preferably from 6 to 10, and most preferably 6 carbon atoms.
  • Typical aryl groups include phenyl, naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenylenyl, and fluorenyl groups.
  • Particularly preferred aryl groups include phenyl, naphthyl and fluorenyl, with phenyl being most preferable.
  • aryl represents a carbocyclic or heterocyclic aromatic radical comprising e.g.
  • phenyl optionally substituted phenyl, naphthyl, pyridyl, thienyl, indolyl or furyl, preferably phenyl, naphthyl, pyridyl, thienyl, indolyl or furyl, and especially phenyl.
  • substituents are halogen, alkyl, alkenyl, alkoxy, cyano and aryl.
  • a carbocyclic aromatic radical represents preferably phenyl or phenyl substituted by one or two substituents selected from lower alkyl, lower alkoxy, hydroxy, acyloxy, nitro, amino, halogen, trifluoromethyl, cyano, carboxy, carboxy functionalized in form of a pharmaceutically acceptable ester or amide, lower alkanoyl, aroyl, lower alkylsulfonyl, sulfamoyl, N-lower alkylsulfamoyl and N,N-di-lower alkylsulfamoyl; also 1- or 2-naphthyl, optionally substituted by lower alkyl, lower alkoxy, cyano or halogen.
  • a heterocyclic aromatic radical represents particularly thienyl, indolyl, pyridyl, furyl; and also e.g. a said heterocyclic radical monosubstituted by lower alkyl, lower alkoxy, cyano or halogen.
  • Thienyl represents 2- or 3-thienyl, preferably 2-thienyl.
  • Pyridyl represents 2- , 3- or 4-pyridyl, preferably 3- or 4-pyridyl advantageously 3-pyridyl.
  • Furyl represents 2- or 3-furyl, preferably 3-furyl.
  • Indolyl represents preferably 3-indolyl.
  • Heteroaryl means an aromatic cyclic compound or part of a compound having one or more rings, of four to eight atoms per ring, incorporating one, two, three or four heteroatoms (selected from nitrogen, oxygen, or sulfur) within the ring which can optionally be substituted with one or two substituents selected from the group consisting of hydroxy, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonlamino and arylcarbonylamino.
  • Substituted lower alkyl means a lower alkyl having one to three substituents selected from the group consisting of hydroxyl, alkoxy, amino, amido, carboxyl, acyl, halogen, cyano, nitro and thiol.
  • lower referred to above and hereinafter in connection with organic radicals or compounds, respectively, preferably defines such with up to and including 10, preferably up to and including 7 and advantageously one or two carbon atoms.
  • a lower alkyl group preferably contains 1-4 carbon atoms and represents for example ethyl, propyl, butyl or advantageously methyl.
  • a lower alkenyl group preferably contains 2-4 carbon atoms and represents for example allyl or crotyl.
  • a lower alkoxy group preferably contains 1-4 carbon atoms and represents for example methoxy, propoxy, isopropoxy or advantageously ethoxy.
  • the boiling point of the solvents is below 50 degrees Celsius, such as in the range of 20-140 degrees Celsius, e.g. 130 degrees Celsius, such as in the range of 30-120 degrees Celsius, e.g. 110 degrees Celsius, such as in the range of 40-100 degrees Celsius, e.g. 95 degrees Celsius, such as in the range of 45-90 degrees Celsius, e.g. 80 degrees Celsius, such as in the range of 50-75 degrees Celsius, e.g. 65 degrees Celsius.
  • the solvents for the adsorption solution are selected from the group consisting of water, ethanol, methanol, butanol, isopropanol, tetrahydrofuran and acetone.
  • the adsorption solution comprises water and ethanol.
  • the solvents for the adsorption solution are selected from the group consisting of water, ethanol, methanol, butanol, isopropanol, tetrahydrofuran, chloroform, propanol, methyl acetate and acetone.
  • the Snyder polarity index ( ⁇ ') of the solvent system in the adsorption solution is decreased by 2 to 35% upon mixing the solvent having the highest individual Snyder polarity index with the one or more additional solvents, such as decreased by 2 to 20% or decreased by 21 to 35%, e.g. decreased by 5 to 15% or decreased by 25 to 30%.
  • the Snyder polarity index ( ⁇ ') of the solvent system in the adsorption solution is decreased by 2 to 85% upon mixing the solvent having the highest individual Snyder polarity index with the one or more additional solvents, such as decreased by 5 to 70% or decreased by 10 to 65%, e.g. decreased by 15 to 60% or decreased by 35 to 50%.
  • the term "Snyder polarity index” relates to the index defined by Snyder in Journal of Chromatography, 92 (1974) 223-230.
  • the Snyder polarity index ( ⁇ ') of the solvent system is calculated by the following equation: P -aA(P'A) + bB(P'B) + Cc(P'c); wherein the letters a-c refer to the solvent volume fractions of the solvents A-C, and ( ⁇ -c) refer to the Snyder polarity index of the individual solvents A-C.
  • the equation is not limited to three solvents.
  • the Snyder polarity index ( ⁇ ') of the solvent system in the adsorption solution is less than 9 in order to promote the formation of micelles or aggregates of the self- assembling cyclodextrin in said adsorption solution, such as in the range of 0-8.9, e.g. 0-8.5, such as in the range of 0-7.9, e.g. 0.5-7.5, such as in the range of 1 -6.9, e.g. 1 .5-6.5, such as in the range of 2-5.9, e.g. 2.5-5.5, such as in the range of 3.5-4.9, e.g. 4.
  • the concentration of the at least one modified self-assembling or self-aggregating cyclodextrin in the adsorption solution is at least 0.2 pg/ml, such as in the range of 0.2 g/ml to 50 mg/ml, e.g. at least 0.5 pg/ml, such as in the range of 1 .0 Mg/ml to 45 mg/ml, e.g. at least 5 Mg/ml, such as in the range of 2 Mg/ml to 40 mg/ml, e.g. at least 10 Mg/ml, such as in the range of 15 M /ml to 35 mg/ml, e.g.
  • Mg/ml such as in the range of 20 pg/ml to 30 mg/ml, e.g. at least 25 Q/ml. such as in the range of 50 g/ml to 30 mg/ml, e.g. at least 75 Mg/ml, such as in the range of 100 Mg/ml to 25 mg/ml, e.g. at least 150 Mg/ml, such as in the range of 200 Mg/ml to 25 mg/ml, e.g. at least 200 Mg/ml, such as in the range of 250 g/ml to 20 mg/ml, e.g.
  • Mg/ml such as in the range of 300 ⁇ sg/m ⁇ to 15 mg/ml, e.g. at least 350 Mg/ml, such as in the range of 400 Mg/ l to 10 mg/ml, e.g. at least 500 Mg/ml, such as in the range of 650 Mg/ l to 9 mg/ml, e.g. at least 700 g/ l, such as in the range of 750 /ml to 8 mg/ml, e.g. at least 800 M /ml, such as in the range of 900 Mg/ml to 7 mg/ml, e.g. at least 1 mg/ml, such as in the range of 2 mg/ml to 6 mg/ml, e.g. at least 5 mg/ml.
  • the inventors have found that a coating concentration of 2 mg modified self-assembling or self-aggregating cyclodextrin per ml adsorption solution leads to a more complex cellular multilayer than lower concentrations.
  • the concentration of the at least one modified self-assembling or self-aggregating cyclodextrin in the adsorption solution is at least 2 mg/ml, such as in the range of 2-50 mg/ml, e.g. at least 3 mg/ml, such as in the range of 4-45 mg/ml, e.g. at least 4 mg/ml, such as in the range of 5-40 mg/ml, e.g. at least 5 mg/ml, such as in the range of 6-35 mg/ml, e.g. at least 6 mg/ml, such as in the range of 7-30 mg/ml, e.g.
  • At least 7 mg/ml such as in the range of 8-25 mg/ml, e.g. at least 8 mg/ml, such as in the range of 9-20 mg/ml, e.g. at least 9 mg/ml, e.g. at least 10 mg/ml.
  • cell adhesion may occur by focal adhesions, connecting the ECM to actin filaments of the cell, and by hemidesmosomes, connecting the ECM to intermediate filaments such as keratin.
  • This cell-to-ECM adhesion may be regulated by specific cell- surface cellular adhesion molecules (CAM) known as integrins.
  • CAM cell- surface cellular adhesion molecules
  • Integrins are cell-surface proteins that bind cells to ECM structures, such as fibronectin and laminin, and also to integrin proteins on the surface of other cells. Fibronectins bind to ECM macromolecules and facilitate their binding to transmembrane integrins. The attachment of fibronectin to the extracellular domain initiates intracellular signalling pathways as well as association with the cellular cytoskeleton via a set of adaptor molecules such as actin.
  • the cell culture surface is pre-coated with a coating comprising an extracellular matrix material, such as extracellular matrix protein and/or extracellular polysaccharide.
  • extracellular matrix material such as extracellular matrix protein and/or extracellular polysaccharide.
  • the coating for pre-coating comprises p- catenin/P30Q antagonists, Activin/TG Fp ligands or mixtures thereof.
  • the cell culture surface is pre-coated with a culture media as disclosed in WO2013054112, hereby incorporated by reference.
  • extracellular matrix protein refers to a protein which constitutes an extracellular matrix. Examples thereof include, but are not limited to, osteopontin, vitronectin, fibronectin, von Willebrand Factor, collagen, laminin, tenascin, fibrinogen, thrombospondin, angiostatin, plasmin, and VCAM-1.
  • extracellular matrix refers to a complex aggregate of biological polymers, which fills the extracellular space in tissue, in accordance with the meaning commonly used in the art (for example, see “Dictionary of Molecular Cell Biology", page 323, Tokyo Kagaku Dozin Co., Ltd., 1997).
  • Synthetic extracellular matrix materials such as ProNectin (Sigma Z378666), may also be used. Mixtures of different extracellular matrix materials may be used, if desired.
  • the extracellular matrix material will normally be coated onto a cell culture vessel, but may (in addition or alternatively) be supplied in solution.
  • Suitable extracellular matrix material may comprise commercially available substrates such as Pluripro Matrix(TM) (Cell Guidance Systems, Cambridge UK) or CeiiStart(TM) (Invitrogen, Paisley, UK).
  • the extracellular matrix compositions as described herein may comprise serum, or may be serum-free and/or serum-replacement free, as described elsewhere herein.
  • the culture media and methods disclosed herein are useful for expanding a population of pluripotent stem cells, whilst maintaining the pluripotency of the cells and without problematic differentiation of the cells.
  • the cell culture surface is post-coated with a coating comprising an extracellular matrix material.
  • the coating for post-coating comprises p- catenin/P30Q antagonists, Activin/TG Fp ligands or mixtures thereof.
  • the cell culture surface is post-coated with a culture media as disclosed in WO2013054112, hereby incorporated by reference.
  • a coating comprising an extracellular matrix material can be used both as a pre- and a post-coating in relation to the self-assembling or self-aggregating cyclodextrin coating.
  • the cell culture surface is post-coated with a coating comprising Pluripro®.
  • the cell culture surface is pre-coated with a coating comprising Pluripro®.
  • the adsorption solution, and hence the resulting coating of the present invention allows for a further way of regulating the cell culture. This is due to the ability of cyclodextrins to form complexes with guest molecules.
  • the inclusion complexes vary from inclusion of small molecules e.g. benzene to large macromolecular structures, e.g. proteins. With larger macromolecular structures, the CD can only form an inclusion complex with a small part of the structure, such as a hydrophobic group which is able to sterically fit into the cavity.
  • the adsorption solution further comprises one or more types of guest molecules (for complexing with the cyclodextrin).
  • Suitable types of guest molecules are molecules with a regulatory effect on the cell culture, such as, but not limited to, survival factors, growth factors, attachment and matrix factors, and antibiotics.
  • At least one of the guest molecules is a survival factor for the cells to be cultured.
  • the survival factor is a Rho-associated kinase inhibitor. In yet another embodiment, the survival factor is Y-27632.
  • the cell culture surface is for culturing pluripotent stem cells, such as human pluripotent stem cells.
  • 'Pluripotent' stem cells are those that have the potential to differentiate into cells of all three germ layers (endoderm, mesoderm and ectoderm) under appropriate conditions, but not extra-embryonic tissue.
  • Pluripotent stem cells are not totipotent, i.e. they cannot form an entire organism, such as a fetus.
  • Induced pluripotent stem cells commonly abbreviated as iPS cells or iPSCs are a type of pluripotent stem cell artificially derived from a non- pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of certain genes and transcription factors.
  • iPS cells induced pluripotent stem cells
  • Human iPS cells derived in this way have all of the properties of human embryonic stem (ES) cells, such as the ability to grow indefinitely and form many or all somatic cell types of the body, but without the use of embryos or the need for somatic cell nuclear transfer to derive pluripotent cells from an individual of choice.
  • Human iPS cells are increasingly becoming a tool for the establishment of patient-specific drug screenings systems in the pharmaceutical industry.
  • Pluripotent stem cells may be obtained using well-known methods.
  • Various types of pluripotent stem cells may be used as described herein, whether obtained from embryonic, fetal or adult tissue (see for example, Thomson et al. 1998 Science 282 1061 -62; Mali et al. 2008.
  • Pluripotent stem cells may be cloned directly from an organism for use pluripotent stem cells, but established pluripotent stem cell lines will typically be used.
  • an initial population of pluripotent stem cell lines may be the progeny of previously-isolated pluripotent stem cell lines or may be the progeny of an established pluripotent stem cell line, such that the use of tissue samples is avoided.
  • MSCs Mesenchymal stem cells, or MSCs, are multipotent stromal cells that can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells).
  • MSCs mesenchymal stem cells
  • HSCs hematopoietic stem cells
  • MSCs from other sources are also able to be expanded in vitro rapidly with sustained stable phenotype and differentiation potential toward several mesenchymal lineages, such as fat, cartilage, and bone.
  • the cell culture surface is for culturing mesenchymal stem cells, such as human mesenchymal stem cells.
  • a product for adherent cell culture comprising:
  • a first coating comprising one or more modified self-assembling or self- aggregating cyclodextrins, wherein at least one of the hydroxyl groups (- OH) of said cyclodextrin is modified to -O-(CO) n Ri, -NR ⁇ , -SRT, -N-COR1 , or -O-(CO) n ORi; wherein R-i, and R 2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; and R 2 , are optionally linked with a bridge member Y N , thereby forming one or more rings, Y N being a bond or a CM 2 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatoms selected from the
  • An advantage of the first coating comprising one or more modified self- assembling or self-aggregating cyclodextrins is that it provides a smooth hydrophilic surface.
  • Such a surface is suitable for direct coating of active ingredients with a regulatory effect on the cell culture, such as, but not limited to, survival factors, growth factors, attachment and matrix factors, and antibiotics.
  • the first coating contains at least 0.01 g/m 2 of one or more modified self-assembling cyclodextrins, wherein at least one of the hydroxyl groups (-OH) of said cyclodextrin is modified to -O-(CO) n R, wherein R is a straight or branched aliphatic chain of 1-50 carbons, such as 2-20 carbons, preferably 4-12 carbons, such as 4-8 carbons, and n is an integer from 0 to 1.
  • the chains may comprise halogens, alcohols, ethers, amines, carboxylic acids or other functional groups.
  • the first coating contains at least 0.003 g/m 2 of one or more modified self-assembling or self-aggregating cyclodextrins, wherein at least one of the hydroxyl groups (-OH) of said cyclodextrin is modified to -0-(CO) n Ri , -NR 1 R 2 , -SRL -N-COR 1 f or -0-(CO) n ORi ; wherein R 1 t R 2 are independently of one another being selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; and R 2 , optionally linked with a bridge member Y n , thereby forming one or more rings.
  • Y n being a bond or a C 1- 2 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure having 1-3 rings, 3-8 ring members in each and 0 to 4 heteroatoms, or a heteroalkyl comprising 1 to 12 heteroatoms selected from the group consisting of N, O, S, S(0)i -2 or carbonyl, and wherein n is an integer between 1 and 12.
  • the first coating contains at least 0.003 g/m 2 of one or more modified cyclodextrins, wherein at least one of the hydroxyl groups (-OH) of said cyclodextrin is modified to -0-(CO) n R, wherein R is a straight or branched aliphatic chain of 1-50 carbons, such as 2-20 carbons, preferably 4-12 carbons, such as 4-8 carbons, and n is an integer from 0 to 1 .
  • the at least one modified hydroxyl group is a primary hydroxyl group.
  • the at least one modified hydroxyl group is a secondary hydroxyl group.
  • the modified self- assembling cyclodextrin is amphiphilic.
  • the term "amphiphilic" relates to a cyclodextrin possessing both hydrophilic and lipophilic properties.
  • the first coating contains at least 0.02 g/m 2 of one or more modified self-assembling cyclodextrins, such as at least 0.03 g/m 2 , e.g. at least 0.04 g/m 2 , 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 g/m 2 .
  • the first coating contains at least 0.01 to 0.5 g/m 2 of one or more modified self-assembling cyclodextrins, such as 0.02 to 0.4 g/m 2 , e.g. 0.03 to 0.3 g/m 2 , such as 0.04 to 0.2 g/m 2 , e.g. 0.05 to 0.1 g/m 2 .
  • the first coating contains at least 0.003 to 0.1 g/m 2 of one or more modified self-assembling or self-aggregating cyclodextrins, such as 0.004 to 0.09 g/m 2 , e.g. 0.005 to 0.08 g/m 2 , such as 0.006 to 0.07 g/m 2 , e.g. 0.007 to 0.06 g/m 2 , such as 0.008 to 0.06 g/m 2 , e.g. 0.009 to 0.05 g/m 2 , such as 0.01 to 0.04 g/m 2 , e.g. 0.02 to 0.03 g/m 2 .
  • modified self-assembling or self-aggregating cyclodextrins such as 0.004 to 0.09 g/m 2 , e.g. 0.005 to 0.08 g/m 2 , such as 0.006 to 0.07 g/m 2 , e.g. 0.007 to 0.06 g/m 2 ,
  • At least 1 % of the self-assembling cyclodextrin derivatives are complexed with at least one guest molecule, such as in the range of 5-100%, e.g. 99%, such as in the range of 15-95%, e.g. 90%, such as in the range of 25-85%, e.g. 80%, such as in the range of 35-75%, e.g. 70%, such as in the range of 45-65%, e.g. 60%, such as in the range of 55-60%, e.g. 50% of the self-assembling cyclodextrin derivatives are complexed with at least one guest molecule.
  • Yet another aspect relates to a method of making products for adherent cell culture comprising:
  • adsorption solution comprises at least one modified self-assembling or self- aggregating cyclodextrin and at least one solvent, wherein at least one of the hydroxyl groups (-OH) of said cyclodextrin is modified to -0-(CO) n Ri , - NR ⁇ , -SRi, -N-COR 1 , or -0-(CO) n ORi ;
  • Ri , and R 2 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and heteroaryl; and R 2 , are optionally linked with a bridge member Y n , thereby forming one or more rings, Y n being a bond or a CM2 alkyl or an aryl, a carbocyclic, a heterocyclic or a heteroaromatic structure
  • the main object of this study was to test a modified self-assembling or self- aggregating cyclodextrin for its supportive capability in relation to culturing iPSCs and human mesenchymal stem cells.
  • C4 adsorption solution (Modified CD synthesized from butyryl chloride) is prepared according to the method disclosed in WO2011 134469, which is hereby incorporated by reference.
  • Beta-CD was purchased from Wacker Chemie AG (Munich, Germany). Beta-CD was dried at 1 10 degrees Celsius for 24 hours prior to use. Butyryl chloride and hydrochloric acid was purchased from Sigma Aldrich (Saint Louis, USA) and used as received. Pyridine was purchased from Sigma Aldrich and dried overnight using molecular sieves prior to use (4 Angstrom, Sigma Aldrich). Ethanol 96% v/v was purchased from Kemethyl (Koge, DK) and used as received.
  • Beta-CD was derivatized using a modified method of Gallois-Montbrun et al. ["Direct synthesis of novel amphiphilic cyclodextrin". Journal of Inclusion Phenomena and Macrocyclic Chemistry, 57:131-135, 2007]. 10 g of dried Beta-CD was added slowly to 200 ml pyridine under stirring. After dissolving the Beta-CD, 6.5 ml of alkyl chloride was added. After 30 minutes of reaction, 1 ml of water was added. The pyridine with product was then added drop wise to 4 litres of 2M HCI in order to precipitate the product. After precipitation the product was collected by filtration and re- dissolved in 100 ml 96 % EtOH. The EtOH solution was added drop wise to 2 L demineralized water. The precipitate was collected and freeze dried overnight.
  • Pluripro® coating solution was purchased from Cell Guidance System, and is a mixture of extracellular proteins supporting adherence of induced pluripotent stem cells.
  • Mesenchymal stem cells were provided by Bioneer A/S.
  • Adsorption solution is transferred to cell culture surface, and removed after 6 hours incubation at 37 degrees Celsius;
  • the potential of encapsulating compounds in the coating layer then provides an opportunity to directed culture of stem cells, potential leading to new differentiation technology.
  • the first experimental study was set-up as with the mesenchymal stem cells, testing the same concentrations of the adsorption solution.
  • a coating of modified self-assembling or self-aggregating cyclodextrin alone on a standard polystyrene cell culture surface did not support the culture of human iPS cells. No cells adhered to the coated surfaces as observed under microscopic investigations. This may be because human iPS cells are highly dependent on biological signals from the culture surface, and conventional cultures of these cells require a coating of an extracellular protein mixture.
  • modified self-assembling or self-aggregating cyclodextrin as a way to functionalize cell culture processes by incorporating compounds in the CD cavities, another approach was selected for improved culture of human iPSCs.
  • Adsorption solution is transferred to a cell culture surface, and removed after 6 hours incubation at 37 degrees Celsius;
  • Pluripro® coating solution is added to the coated cell culture surface, and removed after 18 hours incubation at 37 degrees Celsius;
  • iPSCs are seeded on the double coated cell culture surface
  • human iPS cells are able to adhere and proliferate on the double coated polystyrene plastic surfaces (microscopic investigation after 3 days).
  • the cells displayed normal morphology compared to the control cultures.
  • Pluripro® coating solution is added to the cell culture surface, and removed after 18 hours incubation at 37 degrees Celsius;
  • Adsorption solution is transferred to the coated cell culture surface, and removed after 6 hours incubation at 37 degrees Celsius;
  • iPSCs are seeded on the double coated cell culture surface
  • Group 3 4, 8 and 9, the CD layer is used as the top-layer. Two procedures for obtaining this double coating were tested; one in which the CD layer was allowed to dry and one where the CD layer was still wet and washed afterwards.
  • Group 1 , 2, 6 and 7 are control groups (either CD or Pluripro as the only coating - where Pluripro is the standard). The adherence and proliferation of the iPS cells was analyzed after 3 days in culture. CD alone did not support cell adherence of iPS cells as observed in orevious exDeriments (see above . The ⁇ iPS cell line adhered and proliferated with equal efficiency on both Pluripto/CD coating and CD/Pluripro coating (group 5).
  • Flow cytometry analysis was used for analyzing the surface marker expression profile as described for the previous experiment. This analysis confirms the analysis from the previous experiment and furthermore demonstrates that using the CD as a top layer does not affect the surface marker profile of the iPS cells compared to the standard culture coating conditions on Pluripro alone.
  • the conclusions from the adherence and proliferation studies demonstrated that Pluripro/CD coating structure support the adherence and proliferation of human iPS cell lines.
  • the flow cytometry analysis furthermore demonstrates that the stem cell phenotype is not affected by having CD incorporated as a coating layer in the standard Pluripro coating.
  • the CD layer can be used as a top-layer, which opens up the possibility for release of compounds to the cells in culture.
  • the final experimental set-up was aiming at making a preliminary investigation on the encapsulation of the small molecule Y-27632, a Rho- associated kinase inhibitor.
  • This small molecule is in standard iPS cell culture processes used as a survival factor for the cells, when the cells are seeded onto new plates. In these processes, the molecule is added to the culture medium in a concentration of 10 ⁇ . Although the addition of this molecule improves the initial adherence and proliferation after seeding iPS cells, a large number of cells are not adhering and this critical culture process step needs improvement. This molecule was used as a test compound in the preliminary encapsulation study.
  • Pluripro® coating solution is added to the cell culture surface, and removed after 18 hours incubation at 37 degrees Celsius;
  • Adsorption solution comprising Y-27632 is transferred to the coated cell culture surface, and removed after 6 hours incubation at 37 degrees Celsius;
  • iPSCs are seeded on the double coated cell culture surface
  • the microscopic images illustrate the adherence efficiency for the iPS cell seeded in configurations with 30 ⁇ Y-27632 - either as encapsulated or as a standard additive to the medium. No difference was observed by changing the Y-27632 concentration to 10 ⁇ or 60 ⁇ . However apoptotic events were observed under microscopic investigations in cultures where 60 ⁇ was used as an additive, while cells displayed a healthy morphology with 60 ⁇ encapsulated in CD. The cell counting analysis confirmed the microscopic observations (figure 6).
  • C4 cyclodextrins from Amphidex is biocompatible with human mesenchymal stem cells and human induced pluripotent stem cells.
  • CD coating alone can be used for culturing MSCs. Encapsulating compounds for guided MSC differentiation is thus a potential that could be investigated in future studies.
  • the protocol using the biological matrix, Pluripro, and CD as a top-layer presents an opportunity for improved culture technologies/protocols for the culture of pluripotent stem cells (both iPSCs and embryonic stem cells).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des produits pour cultures cellulaires adhérentes et leur fabrication.
PCT/DK2014/000046 2013-09-11 2014-09-04 Produits de culture cellulaire pour cultures cellulaires adhérentes et leur fabrication WO2015035996A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA201370504 2013-09-11
DKPA201370504 2013-09-11

Publications (1)

Publication Number Publication Date
WO2015035996A1 true WO2015035996A1 (fr) 2015-03-19

Family

ID=51570226

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2014/000046 WO2015035996A1 (fr) 2013-09-11 2014-09-04 Produits de culture cellulaire pour cultures cellulaires adhérentes et leur fabrication

Country Status (1)

Country Link
WO (1) WO2015035996A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009149A1 (fr) * 1997-08-01 1999-02-25 Massachusetts Institute Of Technology Matrices de polymeres tridimensionnelles
US20040106199A1 (en) * 2002-12-02 2004-06-03 Eliseev Alexey V. Charged cyclodextrin derivatives and their use in plant cell and tissue culture growth media
US20100316688A1 (en) * 2009-06-11 2010-12-16 Bamdad Cynthia C Methods for culturing stem and progenitor cells
WO2011134469A1 (fr) 2010-04-26 2011-11-03 Aalborg Universitet Procédé de fonctionnalisation d'une surface de matériau solide avec des cyclodextrines à auto-assemblage ou à auto-ségrégation et produits associés
WO2013054112A1 (fr) 2011-10-10 2013-04-18 Cell Guidance Systems Limited Milieux de culture pour cellules souches pluripotentes
WO2014122345A1 (fr) * 2013-02-05 2014-08-14 Universidade De Santiago De Compostela Hydrogels de poloxamine et leur utilisation pour la régénération ou la réparation osseuse

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009149A1 (fr) * 1997-08-01 1999-02-25 Massachusetts Institute Of Technology Matrices de polymeres tridimensionnelles
US20040106199A1 (en) * 2002-12-02 2004-06-03 Eliseev Alexey V. Charged cyclodextrin derivatives and their use in plant cell and tissue culture growth media
US20100316688A1 (en) * 2009-06-11 2010-12-16 Bamdad Cynthia C Methods for culturing stem and progenitor cells
WO2011134469A1 (fr) 2010-04-26 2011-11-03 Aalborg Universitet Procédé de fonctionnalisation d'une surface de matériau solide avec des cyclodextrines à auto-assemblage ou à auto-ségrégation et produits associés
WO2013054112A1 (fr) 2011-10-10 2013-04-18 Cell Guidance Systems Limited Milieux de culture pour cellules souches pluripotentes
WO2014122345A1 (fr) * 2013-02-05 2014-08-14 Universidade De Santiago De Compostela Hydrogels de poloxamine et leur utilisation pour la régénération ou la réparation osseuse

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"Dictionary of Molecular Cell Biology", 1997, TOKYO KAGAKU DOZIN CO., LTD., pages: 323
GALLOIS-MONTBRUN ET AL.: "Direct synthesis of novel amphiphilic cyclodextrin", JOURNAL OF INCLUSION PHENOMENA AND MACROCYCLIC CHEMISTRY, vol. 57, 2007, pages 131 - 135
JIANG ET AL., NATURE, vol. 418, no. 6893, 2002, pages 41 - 49
JONATHAN W. STEED; JERRY L; ATWOOD: "Supramolecular Chemistry", 1999, WILEY, article "Biochemical Self-Assembly"
MALI ET AL., STEM CELLS, vol. 26, no. 8, 2008, pages 1998 - 2005
PAUL D. BEET; PHILIP A; GALE; DAVID K. SMITH: "Supramolecular Chemistry", 2000, OXFORD CHEMISTRY PRIMERS
SIMÕES SUSANA M N ET AL: "Poloxamine-cyclodextrin-simvastatin supramolecular systems promote osteoblast differentiation of mesenchymal stem cells.", MACROMOLECULAR BIOSCIENCE JUN 2013, vol. 13, no. 6, June 2013 (2013-06-01), pages 723 - 734, XP055153186, ISSN: 1616-5195 *
SNYDER, JOURNAL OF CHROMATOGRAPHY, vol. 92, 1974, pages 223 - 230
THOMSON ET AL., SCIENCE, vol. 282, 1998, pages 1061 - 62
WANG T ET AL: "Bone marrow stem cells implantation with alpha-cyclodextrin/MPEG-PCL-MPEG hydrogel improves cardiac function after myocardial infarction", ACTA BIOMATERIALIA, ELSEVIER, AMSTERDAM, NL, vol. 5, no. 8, 1 October 2009 (2009-10-01), pages 2939 - 2944, XP026625147, ISSN: 1742-7061, [retrieved on 20090506], DOI: 10.1016/J.ACTBIO.2009.04.040 *

Similar Documents

Publication Publication Date Title
Bhattacharya et al. Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture
Chen et al. Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction
Dubbin et al. Dual-stage crosslinking of a gel-phase bioink improves cell viability and homogeneity for 3D bioprinting
Yeh et al. The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes
Ladet et al. Multi-membrane chitosan hydrogels as chondrocytic cell bioreactors
Pek et al. The effect of matrix stiffness on mesenchymal stem cell differentiation in a 3D thixotropic gel
JP6513023B2 (ja) 三次元細胞培養
Tsai et al. Enhancement of human adipose-derived stem cell spheroid differentiation in an in situ enzyme-crosslinked gelatin hydrogel
Rowlands et al. Directing osteogenic and myogenic differentiation of MSCs: interplay of stiffness and adhesive ligand presentation
Turner et al. Human hepatoblast phenotype maintained by hyaluronan hydrogels
Wang et al. Graphene sheet-induced global maturation of cardiomyocytes derived from human induced pluripotent stem cells
Wang et al. Self-assembling peptide hydrogel scaffolds support stem cell-based hair follicle regeneration
ES2742444T3 (es) Diferenciación de células madre pluripotentes y células progenitoras cardíacas en fibras de cardiomiocito estriadas usando laminianas LN-15, LN-521 y LN-221
Ren et al. Hyaluronic acid hydrogel with adjustable stiffness for mesenchymal stem cell 3D culture via related molecular mechanisms to maintain stemness and induce cartilage differentiation
De Luca et al. Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling
TW200902716A (en) Complexes of hyaluronans, other matrix components, hormones and growth factors for maintenance, expansion and differentiation of hepatic cells
ITGE20120102A1 (it) Procedimento di precondizionamento chimico di materiale cellulare per ottenere la riprogrammazione epigenetica chimica e l'espressione di pluripotenzialità
Abbasgholizadeh et al. A highly conductive 3D cardiac patch fabricated using cardiac myocytes reprogrammed from human adipogenic mesenchymal stem cells
CA3154587A1 (fr) Production de cellules de muscle squelettique et de tissu de muscle squelettique a partir de cellules souches pluripotentes
WO2021117900A1 (fr) Composition et utilisation correspondante
JPWO2019131940A1 (ja) 多能性幹細胞凝集抑制剤
EP3970795A1 (fr) Procédé de purification de cellules de crête neurale ou de cellules épithéliales cornéennes
Hickman et al. The importance and clinical relevance of surfaces in tissue culture
Narakornsak et al. Sesamin encouraging effects on chondrogenic differentiation of human amniotic fluid-derived mesenchymal stem cells
Sun et al. Human urinal cell reprogramming: synthetic 3D peptide hydrogels enhance induced pluripotent stem cell population homogeneity

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14766897

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 14766897

Country of ref document: EP

Kind code of ref document: A1