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EP1675943A2 - Synthese, a l'echelle du nanolitre, de biomateriaux en reseaux et criblage de ceux-ci - Google Patents

Synthese, a l'echelle du nanolitre, de biomateriaux en reseaux et criblage de ceux-ci

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Publication number
EP1675943A2
EP1675943A2 EP04788758A EP04788758A EP1675943A2 EP 1675943 A2 EP1675943 A2 EP 1675943A2 EP 04788758 A EP04788758 A EP 04788758A EP 04788758 A EP04788758 A EP 04788758A EP 1675943 A2 EP1675943 A2 EP 1675943A2
Authority
EP
European Patent Office
Prior art keywords
cells
diacrylate
polymer
monomer
cell
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP04788758A
Other languages
German (de)
English (en)
Other versions
EP1675943A4 (fr
Inventor
Daniel G. Anderson
Shulamit Levenberg
Robert S. Langer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Massachusetts Institute of Technology
Original Assignee
Massachusetts Institute of Technology
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Filing date
Publication date
Priority claimed from US10/843,707 external-priority patent/US20050019747A1/en
Application filed by Massachusetts Institute of Technology filed Critical Massachusetts Institute of Technology
Publication of EP1675943A2 publication Critical patent/EP1675943A2/fr
Publication of EP1675943A4 publication Critical patent/EP1675943A4/fr
Withdrawn legal-status Critical Current

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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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • 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
    • C12N2533/30—Synthetic polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention pertains to the use of embryonic stem cells, and, more specifically, to the differentiation, isolation, and characterization of human embryonic epithelial cells.
  • Tissue engineered constructs, ex- vivo cell isolation, bio-reactors and cell encapsulation require some type of interaction between cells and supporting material for growth, function, and/or delivery (R.P. Lanzo, et al., "Principles of tissue engineering", Academic Press, ed. 2 nd (2000)).
  • Much research is currently focused on the development of biomaterials that provide optimal cellular substrates, including the development of bioactive materials through the incorporation of ligands, and encapsulation of DNA and growth factors (R.R. Chen, et al., Pharmaceutical Research 20, 1103-1112 (2003); S.E. Sakiyama-Elbert, et al., Annual Review of Materials Research 31, 183-201 (2001)).
  • embryonic epithelial cell refers to a partially differentiated cell that may differentiate to an epithelial cell under appropriate in vivo or in vitro conditions.
  • Embryonic epithelial cells may be identified by expression of genes or production of proteins characteristic of epithelial cells, for example, cytokeratin.
  • Cytokeratms are a family of proteins that are found in epithelial tissue in various parts of the body. Different tissues may include one or more of over two dozen cytokeratms. For example, cytokeratin 7 is found in lung and breast epithelium but not colon and prostate epithelium. Cytokeratin 20 is found in gastric and intestinal epithelium.
  • alkyl as used herein refers to saturated, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom.
  • alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert- butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.
  • alkoxy refers to an alkyl groups, as previously defined, attached to the parent molecular moiety through an oxygen atom.
  • alkenyl denotes a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom.
  • Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1- methyl-2-buten-l-yl, and the like.
  • alkynyl as used herein refers to a monovalent group derived form a hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom.
  • alkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.
  • alkylamino, di ⁇ lkylamino, and trialkylamino as used herein refers to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom.
  • alkylamino refers to a group having the structure -NHR' wherein R r is an alkyl group, as previously defined; and the term dialkylamino refers to a group having the structure -NR'R", wherein R' and R" are each independently selected from the group consisting of alkyl groups.
  • trialkylamino refers to a group having the structure -NR'R' ⁇ .'", wherein R', R", and R'" are each independently selected from the group consisting of alkyl groups. Additionally, R', R", and/or R'" taken together may optionally be - (CH 2 )r where k is an integer from 2 to 6.
  • Example include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino, trimethylamino, and propylamino.
  • alkylthioether and thioalkoxyl refer to an alkyl group, as previously defined, attached to the parent molecular moiety through a sulfur atom.
  • aryl refers to carbocyclic ring system having at least one aromatic ring including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.
  • Aryl groups can be unsubstituted or substituted with substituents selected from the group consisting of branched and unbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide.
  • substituted aryl groups include tetrafluorophenyl and pentafluorophenyl.
  • carboxylic acid as used herein refers to a group of formula -C0 2 H.
  • halo and halogen refer to an atom selected from fluorine, chlorine, bromine, and iodine.
  • heterocychc refers to a non-aromatic partially unsaturated or fully saturated 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms in size and bi- and tri-cyclic ring systems which may include aromatic six-membered aryl or aromatic heterocyclic groups fused to a non-aromatic ring.
  • heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • aromatic heterocyclic refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from sulfur, oxygen, and nitrogen; zero, one, or two ring atoms are additional heteroatoms independently selected from sulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
  • heterocyclic and aromatic heterocyclic groups that may be included in the compounds of the invention include: 3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine, 4-(bis-(4-fluorophenyl)methyl)piperazine, 4- (diphenylmethyl)piperazine, 4-(ethoxycarbonyl)piperazine, 4- (ethoxycarbonylmethyl)piperazine, 4-(phenylmethyl)piperazine, 4-( 1 - phenylethy ⁇ )piperazine, 4-( 1 , 1 -dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2- propenyl) amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine, 4-(2- chlorophenyl)piperazine, 4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine, 4-(
  • carbamoyl refers to an amide group of the formula -CONH 2 .
  • hydrocarbon refers to any chemical group comprising hydrogen and carbon. The hydrocarbon may be substituted or unsubstitued. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.
  • Illustrative hydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, and the like.
  • all valencies must be satisfied in making any substitutions.
  • the terms substituted, whether preceded by the term "optionally" or not, and substituent, as used herein, refer to the ability, as appreciated by one skilled in this art, to change one functional group for another functional group provided that the valency of all atoms is maintained.
  • the substituent may be either the same or different at every position.
  • the substituents may also be further substituted (e.g., an aryl group substituent may have another substituent off it, such as another aryl group, which is further substituted with fluorine at one or more positions).
  • ureido refers to a urea groups of the formula - H- CO-NH 2 . Summary of the Invention In one aspect, the invention is a population of embryonic epithelial cells produced in vitro from embryonic stem cells.
  • the invention is a population of cytokeratin 7-positive cell produced in vitro from embryonic stem cells.
  • the invention is a population of cytokeratin or cytokeratin 7- positive cells produced by the step of exposing a population of embryonic stem cells to retinoic acid. The population may be exposed to retinoic acid in the presence of serum, and the population of embryonic stem cells may be seeded on a cell support substrate.
  • the invention is a composition comprising a cell support substrate and human embryonic epithelial cells supported by the cell support substrate.
  • the composition may further include retinoic acid or serum.
  • the invention is a method of enriching a population of embryonic stem cells with epithelial-like cells.
  • the method includes providing a population of human embryonic stem cells and culturing the stem cells on an acrylate polymer in a culture-medium including retinoic acid.
  • the culture medium may include serum.
  • Providing a population of human embryonic stem cells may include culturing embryonic stem cells under conditions where embryoid bodies are formed and dissociating the embryoid bodies.
  • the invention is a method of screening cell-polymer interactions.
  • the method includes depositing monomers as a plurality of discrete elements on a substrate, causing the deposited monomers to polymerize to create an array of discrete polymer elements on the substrate, incubating the substrate in a cell- containing cell culture medium, and characterizing a predetermined cell behavior on each element.
  • a portion of the polymer elements may include a homopolymer, and the substrate may be coated with a cytophobic material before depositing.
  • Exemplary cytophobic materials include poly(hydroxyethyl methacrylate), poly(alkylene glycol), co-polymers including an alkylene glycol monomer, polymers derivatized with a poly(alkylene glycol), and a hydrogel.
  • the cell culture medium may include a growth factor or serum.
  • a portion of the polymer elements may be co-polymers of at least two monomer species.
  • the cell behavior may be one or more of adhesion, proliferation, metabolic behavior, differentiation, production of a predetermined protein, expression of a predetermined gene, or an amount of any of these (e.g., an amount of proliferation, the amount of predetermined protein that is produced, etc.).
  • the invention is a method of controlling cell behavior. The method includes selecting a first polymer in combination with which a predetermined cell exhibits a particular cell behavior, selecting a second polymer differing from the first polymer in cross-link density or electron density, and seeding the predetermined cell on the second polymer.
  • the second polymer may differ from the first in a density of acrylate groups, a density of methacrylate groups, a density of ester groups, a density of ether groups, the presence of an electron donating group, identity of a heteroatom, the substitution on a heteroatom, the presence of a predetermined substituent, the presence of predetermined heteroatom, or any combination of these.
  • the invention is a method of controlling a behavior of human embryonic stem cells. The method includes exposing human embryonic stem cells to a synthetic polymer. The polymer is selected to promote a predetermined behavior of the cells.
  • the invention is a method of controlling a behavior of human embryonic stem cells.
  • the method includes exposing human embryonic stem cells to a synthetic polymer that is not a polycation, polystyrene, a poly(lactide), or a copolymer including lactide monomers.
  • the invention is a method of controlling cell behavior. The method includes selecting a first monomer in combination with the polymer of which cells exhibit a particular cell behavior, selecting a second monomer, that, when co- polymerized with the first monomer, modifies the cell behavior, co-polymerizing the first and the second monomer to produce a co-polymer, and seeding cells on the co- polymer.
  • Seeding the cells on the co-polymer may include incubating the co-polymer in a cell-containing cell culture medium containing a growth factor.
  • the growth factor modifies the cell behavior of the cells in comparison to the behavior of cell seeded on the co-polymer in the absence of the growth factor.
  • the first and second monomers may be co-polymerized on a cytophobic surface.
  • Seeding cells may include culturing embryonic stem cells under conditions where embryoid bodies are formed, dissociating the embryoid bodies, adding the dissociated cells to a culture medium, and incubating the co-polymer in the cell-containing culture medium.
  • the cell- containing culture medium may include serum.
  • Seeding cells on the co-polymer may include incubating the co-polymer in a cell-containing cell culture medium including retinoic acid.
  • the invention is a method of controlling cell behavior. The method includes selecting a first monomer, in combination with the polymer of which cells exhibit a particular cell behavior, selecting a growth factor that modifies that cell behavior when the cells are seeded on the polymer of the first monomer, polymerizing the first monomer to produce a polymer, and incubating the polymer in a cell- containing culture medium containing a growth factor.
  • the cell-containing culture medium may include serum.
  • the growth factor may be retinoic acid.
  • the invention is a method of controlling cell behavior.
  • the method includes selecting cells characterized by a predetermined level of expression of a first gene, selecting a monomer, in combination with a polymer of which the cells exhibit a level of expression of the first gene different from the predetermined level, polymerizing the monomer to produce a polymer, and seeding the cells on the polymer.
  • the method includes selecting cells characterized by a pre-determined level of a first protein, selecting a monomer, in combination with the polymer of which the cells exhibit a level of expression of the first protein different from the predetermined level, polymerizing the monomer to produce a polymer and seeding the cells on the polymer.
  • the cells may be human embryonic stem cells.
  • the invention is a method of supporting growth of C2C12 cells in vitro.
  • the method includes culturing the C2C12 cells on a polymer produced from one or more of 1,4 butanediol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, phenylene diacrylate 1,3, propoxylated neopentyl glycol diacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, triethylene glycol diacrylate, triethylene glycol dimethacrylate, tripropylene glycol diacrylate, caprolactone 2-(methacryloyloxy)ethyl ester, 5-ethyl-5-(hydroxymethyl)- ⁇ , ⁇ - dimethyl-l,3-dioxane-2-ethanol diacrylate, 1,6-hexanediol prop
  • Figure 1 A is a schematic of an exemplary polymer microarray produced using the techniques of the invention
  • Figure IB is a schematic of an alternative polymer microarray produced using the techniques of the invention
  • Figure 2A depicts monomers employed to make microarrays according to an embodiment of the invention
  • Figure 2B is a diagram indicating the distribution of monomers in the array to form copolymers
  • Figure 2C is an image of a polymer array in triplicate provided by an Arrayworx reader (red box: 70% 1; yellow box: 70% 6);
  • Figure 2D is a DIC light micrograph of a typical polymer element overlayed with a few fluorescent cells (red);
  • Figure 3 is a schematic view of an exemplary apparatus for use with the invention
  • Figure 4A is an image of a polymer array in triplicate incubated with hES EB day 6 cells in the presence of retinoic acid for 6 days and then stained for cytokeratin 7 (green) and viment
  • Figures 6A, C-E are images of hES cells grown on a polymer array in the absence of retinoic acid for 6 days and then stained for cytokeratin 7 (green) and vimentin (red) (polymer spots and unstained cells are blue);
  • Figures 6B, F-H are images of hES cells grown on a polymer array in the presence of retinoic acid for 6 days and then stained for cytokeratin 7 (green) and vimentin (red);
  • Figures 6I-K are an image of hES cells grown on a polymer array in the absence of retinoic acid for 24 hours and then stained for cytokeratin 7 (green) and vimentin (red);
  • Figures 6L-N are images of hES cells grown on a polymer array in the presence of retinoic acid for 24 hours and then stained for cytokeratin 7 (green) and vimentin (red);
  • Figure 7 provides images and data for hES cells grown on "hit" polymer arrays (
  • the invention provides a method of enriching a population of embryonic stem cells with epithelial-like cells.
  • the method includes providing a population of human embryonic stem cells and culturing the stem cells on an acrylate polymer in a culture medium including retinoic acid.
  • Microarrays of polymers may be employed to select polymers which facilitate proliferation and differentiation of the cells.
  • Polymer Microarrays exploits polymer microarrays such as those disclosed in U.S. Patent Applications Nos. 10/214,723 and 09/803,319, published as 2004- 0028804 and 2002-0142304, respectively.
  • the techniques of the invention may be exploited to produce a cell-compatible, miniaturized polymer array characterized by the ability to synthesize a large number of materials in nanoliter volumes, polymer elements that are attached to the microarray in a manner that would be compatible with those materials and resistant to the aqueous conditions necessary for cell-based testing, inhibition of cell growth in the spaces between different polymers to allow material effects on cells to be independent of neighboring materials, and a format that allows simple, simultaneous assay of multiple cellular markers.
  • a substrate surface is treated to render it cytophobic, for example, by coating it first with epoxide and then with poly(hydroxyethyl methacrylate) (pHEMA).
  • pHEMA inhibits cell growth (J. Folkman, et al., Nature 273, 345-349 (1978)), and a monomer deposited on a pHEMA surface may interpenetrate and potentially become fixed in place upon polymerization.
  • Other polymers that may be used to form cytophobic surfaces include poly alkylene glycols such as poly(ethylene glycol) and its co-polymers. Alternatively, polymers derivatized with poly(ethylene glycol) or other poly(alkylene glycols) may be employed.
  • Polymer elements are produced on the surface by depositing an array of monomers and then polymerizing them in situ.
  • the polymer elements may be associated with the substrate surface via non-covalent interactions such as chemical adsorption, hydrogen bonding, surface interpenetration, ionic bonding, van der Waals forces, hydrophobic interactions, dipole-dipole interactions, mechanical interlocking, and combinations of these; however, the polymer elements may also be associated with the substrate surface via covalent interactions.
  • the base can be a glass, plastic, metal, or ceramic, but can also be made of any other suitable material.
  • Figure 1 A shows an embodiment of an array of polymer elements 2 disposed on a surface 4 of substrate 6.
  • Figure IB illustrates an embodiment in which a coating 8 is disposed on substrate 6, and polymer elements 2 are disposed on surface 4, which is the surface of the coating.
  • the substrate surface material should be chosen to maximize adherence of the polymer elements while controlling spreading of the deposited monomer.
  • a cytophobic coating will prevent migration of cells from one polymer element to another.
  • An epoxy coating interposed between the cytophobic coating and the base may increase the adherence of the coating to the base.
  • monomers are deposited on the surface and polymerized to form a microarray of polymer elements.
  • liquid monomers diluted in 25% dimethylformamide (DMF) are deposited on the substrate.
  • the solvent decreases the viscosity of the monomers and facilitates deposition of a precise amount of monomer.
  • the amount of solvent or the solvent itself may be changed to alter the viscosity as needed.
  • Alternative solvents include but are not limited to dimethylsulfoxide, chloroform, dichlorobenzene, and other chlorinated solvents.
  • the monomer is part of a biocompatible polymer.
  • biodegradable and non-biodegradable biocompatible polymers are known in the field of polymeric biomaterials, controlled drug release and tissue engineering (see, for example, U.S. Patents Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404 to Vacanti; 6,095,148; 5,837,752 to Shastri; 5,902,599 to Anseth;
  • biocompatible polymer classes that may be incorporated into polymer elements 2 using the techniques of the invention include polyamides, polyphosphazenes, polypropylfumarates, synthetic poly(amino acids), polyethers, polyacetals, polycyanoacrylates, polyurethanes, polycarbonates, polyanhydrides, poly(ortho esters), polyhydroxyacids, polyesters, polyacrylates, ethylene-vinyl acetate polymers, cellulose acetates, polystyrenes, poly(vinyl chloride), poly(vinyl fluoride), poly(vinyl imidazole), poly(vinyl alcohol), and chlorosulphonated polyolefins.
  • biodegradable refers to materials that are enzymatically or chemically (e.g., hydrolytically) degraded in vivo into simpler chemical species.
  • Monomers that are used to produce these polymers are easily purchased from companies such as Polysciences, Sigma, Scientific Polymer Products, and Monomer- Polymer & Dajac Laboratories. These monomers may be combined in an array to form a wide variety of co-polymers. The monomers may polymerize by chain polymerization.
  • Exemplary monomers subject to radical chain polymerization include ethylene, vinyl derivatives of ethylene, including but not limited to vinyl acetate, vinyl chloride, vinyl alcohol, and vinyl benzene (styrene), vinylidine derivatives of ethylene, including but not limited to vinylidine chloride, acrylates, methacrylates, acrylonitriles, acrylamides, acrylic acid, and methacrylic acid, fluoropolymers, dienes, including but not limited to butadiene, isoprene, and their derivatives, and aromatic monomers such as phenylene and its derivatives, such as phenylene vinylene.
  • vinyl derivatives of ethylene including but not limited to vinyl acetate, vinyl chloride, vinyl alcohol, and vinyl benzene (styrene)
  • vinylidine derivatives of ethylene including but not limited to vinylidine chloride
  • acrylates, methacrylates, acrylonitriles, acrylamides, acrylic acid, and methacrylic acid fluoropolymers
  • Monomers such as ⁇ - olefins, 1,1-dialkyl olefins, vinyl ethers, aldehydes, and ketones may be polymerized by anionic chain polymerization, cationic chain polymerization, or both. Additional monomers can be found in George Odian's Principles of Polymerization, (3rd Edition, 1991, New York, John Wiley and Sons), the entire contents of which are incorporated herein by reference. One skilled in the art will recognize that the techniques of the invention may also be exploited to produce microarrays by step polymerization. The reaction conditions for a variety of polyesters, polyamides, polyurethanes, and other condensation polymers are well known in the art (see Odian, 1991).
  • Such reactions may be easily adapted to produce microarrays on substrates.
  • neat monomers are deposited as a liquid or in a solution with a solvent such as DMSO or chloroform to prevent premature precipitation of the polymer.
  • a solvent such as DMSO or chloroform
  • Non-volatile solvents are preferred to reduce evaporation.
  • a catalyst for example, sulfuric acid or p-toluenesulfonic acid, may be used to increase the rate of reaction.
  • the substrate may be heated or placed in a low pressure atmosphere to drive off the condensation product and drive the reaction.
  • the low volume and high surface area of the droplets should facilitate the removal of the condensation product without the use of purging gases or high vacuum conditions.
  • Monomers that require chemical initiators may also be used.
  • the monomer solutions should be cooled during deposition and then warmed to initiate polymerization. It may be desirable to use a less viscous solvent than would be employed to deposit the microarray at room temperature.
  • monomers may be deposited in a microarray and then exposed to an ozone atmosphere to initiate polymerization.
  • the molecular weight of the resultant polymer may be controlled by adjusting the properties of the solvent. Modifying the viscosity of the solvent changes the polymerization rate and the resulting molecular weight distribution. Some solvents provide a more favorable environment for radicals and intermediate products formed during polymerization and allow polymerization to continue for a longer time before termination.
  • the molecular weight of the polymer may be controlled by varying the concentration of monomer in the stock solution or the ratios of difunctional monomers to unifunctional monomers. Increased concentrations of difunctional monomers will increase the degree of cross-linking in the chains.
  • Monofunctional monomers may be modified to form difunctional monomers by reacting them with a linker chain. Appropriate linkers and chemical reactions will be evident to one skilled in the art. For example, dicarboxylic acids are reactive with a wide variety of functional groups commonly incorporated into vinyl monomers, including alcohols, amines, and amides.
  • acrylate monomers are used to produce the polymer arrays of the invention.
  • a variety of acrylate-based polymers have been used for tissue engineering, surgical glues, and drug delivery (J.P. Fisher, et al., Annu. Rev. Mater. Res. 31, 171-181 (2001)).
  • acrylate monomers having the structure
  • Ri may be methyl or hydrogen.
  • R 2 , R 2 ', and R 2 " may include alkyl, aryl, heterocycles, cycloalkyl, aromatic heterocycles, multicycloalkyl, hydroxyl, ester, ether, halide, carboxylic acid, amino, alkylamino, dialkylamino, trialkylamino, amido, carbamoyl thioether, thiol, alkoxy, or ureido groups.
  • R 2 , R 2 ', and R 2 " may also include branches or substituents including alkyl, aryl, heterocycles, cycloalkyl, aromatic heterocycles, multicycloalkyl, hydroxyl, ester, ether, halide, carboxylic acid, amino, alkylamino, dialkylamino, trialkylamino, amido, carbamoyl, thioether, thiol, alkoxy, or ureido groups.
  • monomers are sufficiently stable that they can be deposited on the slide and sit for a moment, e.g., 30 seconds to 1 or 2 minutes, before being polymerized after exposure to UV light.
  • Exemplary acrylate monomers including bifunctional and multifunctional acrylates for use with the invention are listed in Table 1 and shown in Figure 2A. These may be purchased from Sigma-Aldrich (Milwaukee, WI), Scientific Polymer Products (Onterio, NY), and Polysciences (Warrington, PA). In one embodiment, these monomers are diluted by 25% with DMF " before spotting to reduce their viscosity and ensure reproducible deposition onto the substrate (see Examples). One skilled in the art will recognize that mixtures of multifunctional and monofunctional monomers may be used to control the degree of cross-linking in the polymer.
  • Branched monomers also change electron density by allowing more ether groups to fit in an R 2 group of a certain length, by changing the packing density of the resulting polymer, or both.
  • the use of cyclic moieties and aromatic moieties also changes the electron density of R 2 .
  • An Ri methyl group contributes more electron density to the ester group that a hydrogen atom.
  • the cross-link density of the polymer may be adjusted by varying the proportion of monofunctional, bifunctional, and other multifunctional monomers.
  • the use of a co-monomer enables fine tuning of the electron density of the polymer. Both the composition and the amount of the co-monomer may be varied to adjust the hydrophobicity or hydrophilicity of the resulting polymer.
  • the monomers can be formed into a polymer microarray on the substrate surface using a range of techniques known in the art.
  • the elements of the microarray are formed by depositing small drops of each monomer solution at discrete locations on the substrate surface, preferably by using an automated liquid handling device.
  • the monomers of the invention are initially provided as diluted liquids or solutions of dissolved solids. Once the stock solutions of the polymeric biomaterials have been prepared, a predetermined volume of each biomaterial stock solution is placed in the separate reservoirs of the robotic liquid handling device.
  • the drops may be deposited on the substrate surface using a microarray of pins (e.g., ChipMaker2TM pins, available from TeleChem International, Inc. of Sunnyvale, CA).
  • a range of pins exist that take a sample volume up by capillary action and deposit a spot volume of 1 to 10 nl or more.
  • These pins may be controlled by a robotic liquid handling device that controls the speed and travel pattern of the pins as well as automatic washing cycles and pauses between deposition steps.
  • the device carrying the pins may be programmed to change the amount and length of washing cycles between deposition steps and adjust the speed with which the pins are transported from the monomer supply to the substrate at which the monomer is deposited.
  • the path over which the pins are transported may be optimized.
  • the drops may be deposited on the substrate surface using syringe pumps controlled by micro-solenoid ink-jet valves that deliver volumes greater than about 10 nl (e.g., using printheads based on the SYNQUADTM technology, available from Cartesian Technologies, Inc. of Irvine, CA).
  • the drops may be deposited on the substrate surface using piezoelectric ink-jet fluid technology that deposits smaller drops with volumes between about 0.1 and 1 nl (e.g., using the MICROJETTM printhead available from MicroFab Technologies, Inc. of Piano, TX).
  • Alternative techniques may be employed to deposit smaller or larger drops.
  • pins may be pre-tapped to release a large drop and then tapped on the substrate to release a smaller drop, just as a paintbrush is tapped on the side of the can to remove excess paint and prevent messy drips on the painted surface.
  • small drops they should be polymerized shortly after deposition, before the solvent evaporates.
  • a portion of an array may be deposited and polymerized before deposition of a second portion of the array.
  • the drops are arranged as a rectangular microarray on a glass slide. The size of the array may be determined by the user and will depend on the size of the elements of the array, the spacing between the elements and the size of the substrate surface.
  • the rectangular microarray may, for example, be an 18 x 40, an 18 x 54 or a 22 x 64 microarray; however, smaller, larger and alternatively shaped microarrays (e.g., square, triangular, circular, elliptical, etc.) may be used.
  • the shape of the microarray and the arrangement and spacing of polymer elements within it may depend on the analytical methods used to examine the arrayed polymers. For example, a particular sensor may require a specific shape or distribution of polymer elements.
  • robotic controls to move the pins enables any distribution and arrangement of spots regardless of symmetry.
  • two or more identical arrays are deposited alongside one another so that experiments on the polymers may be repeated.
  • each element of the microarray is formed by depositing a single drop taken from one of the monomer stock solutions. In another embodiment, some or all of the elements are formed by depositing at least two drops taken from one of the monomer stock solutions. In yet another embodiment, some or all of the elements are fo ⁇ ned by depositing at least two drops taken from at least two different monomer stock solutions. In an alternative embodiment, stock solutions of mixed monomers are prepared. In one embodiment, the dimensions of the elements of the microarray are substantially the same; however, in certain embodiments of the present invention, the dimensions of the elements of the microarray may differ from one element to the next.
  • the vertical dimension means the vertical dimension of the element when viewed from a direction that is parallel to the substrate surface (i.e., from the side).
  • the “horizontal dimension”, as that term is used herein, means the horizontal dimension of the element when viewed from a direction that is perpendicular to the substrate surface (i.e., from above).
  • the vertical dimensions of elements of the microarray of the present invention are such that each element may comprise hundreds or even thousands of layers of polymer molecules. When viewed from above or from the side, the elements may be circular, oblong, elliptical, square or rectangular. For example, the overall shape of the elements may be sphere-like or disk-like.
  • the drops are deposited at intervals that range from about 300 to about 1200 ⁇ m. In one embodiment, the drops are deposited at about 720 ⁇ m intervals; however, the drops may be deposited at smaller or larger intervals.
  • the size and density of the elements depends on the application. Smaller elements, e.g., spaced at intervals of 1 ⁇ m or less, may be preferred for chemical analysis to further increase the number of compounds that can be analyzed in one batch. For example, 100 million elements, spaced at 0.1 ⁇ m intervals, can fit in an area of a square millimeter. In other embodiments, the array may have a density of one or fewer polymer elements per square centimeter.
  • the density, vertical dimension, and horizontal dimension of the elements will be optimized for the particular manufacturing technique and the variable being tested.
  • polymer arrays of 576 spots 24 x 24
  • the elements of the microarray are deposited on the substrate surface as drops that range in volume from 0.1 to 100 nl. However, smaller and larger volumes may be deposited on the substrate surface.
  • the ultimate dimensions of the drops depend on the application. For example, for cell attachment, the vertical dimension of the elements should be between about 50 and 500 ⁇ m, and the horizontal dimension of the deposited drops should be between 300 and 600 ⁇ m.
  • the element should be large enough to minimize edge effects, but, for a single cell, the element may not need to be any larger than 10 ⁇ m across.
  • the drop volume and monomer viscosity may be adjusted so that the polymer element is thinner than 50 ⁇ m or even essentially flat.
  • the primary limits on drop size are the ability to detect and deposit tiny drops. For some applications, it may be desirable to deposit drops as thin as a few 10s of nanometers.
  • Microinjectors and robots can produce arrays of miniscule droplets, but the viscosity of the precursor must be carefully controlled to prevent clogging. Ink-jet printers may be used to reproducibly deposit drops of a specified size.
  • the precursor should not polymerize before deposition and perhaps clog the dispenser.
  • Thicker polymer elements may be produced by depositing a larger volume of precursor solution or by depositing several layers at each location. Bigger drops are easily deposited by e.g., using bigger pins (e.g., from TeleChem International, Inc., Sunnyvale, CA). Drop size may need to be optimized for a variety of factors, including the space required by seeded cells, the ability of the pins to handle a particular volume of monomer solution depending on factors such as the viscosity of the solution and the reproducibility of drop deposition, and the volatility of the monomer or any solvent. After the monomer has been deposited on the surface, it is polymerized.
  • the microarray is exposed to UN light, which initiates polymerization.
  • a chemical initiator is used, the microarray is exposed to conditions under which the initiator will start reacting with the monomer.
  • exemplary radical initiators include, but are not limited to, azobisisobutylnitrile (AIB ⁇ ), 2,2-dimethoxy-2-phenyl-acetophenone (DPMA), benzoyl peroxide, acetyl peroxide, and lauryl peroxide. Redox and thermal initiators may also be exploited.
  • peroxides may be combined with a reducing agent such as Fe 2+ , Cr 2"1" , V 2+ , Ti 3+ , Co 2+ , Cu + , and amines such as N,N- dialkylaniline.
  • a reducing agent such as Fe 2+ , Cr 2"1" , V 2+ , Ti 3+ , Co 2+ , Cu + , and amines such as N,N- dialkylaniline.
  • a reducing agent such as Fe 2+ , Cr 2"1" , V 2+ , Ti 3+ , Co 2+ , Cu +
  • amines such as N,N- dialkylaniline.
  • These initiators may be mixed with the monomer solutions and co- deposited. Because such initiators are often sensitive to temperature, they should be deposited at depressed temperatures. The temperature is then raised to start polymerization. A monomer that polymerizes in air should be deposited under nitrogen or argon and then exposed to air to start polymerization.
  • the polymer microarray is placed in an evacuated desiccator at about 25 °C for 12 to 48 hrs to remove any residual solvent. Alternatively, or additionally, the microarray may be washed to remove the solvent.
  • the substrate surface or the array is modified after the polymer array has been deposited. Self assembled monolayer (SAM) systems may be chosen that react with the base layer but not with the various polymers. Alternatively, the polymer array may be deposited directly on the substrate and the uncovered surface modified afterwards using standard organosilane chemistry.
  • SAM Self assembled monolayer
  • One aspect of the present invention involves the recognition that an endless variety of polymers can be obtained according to the present invention by varying the compositions of the stock solutions that are initially added to the robotic liquid handling device and/or by layering drops taken from these stock solutions in a series of sequential deposition steps. To produce bulk quantities of polymers would require large amounts of monomer and solvents which would then have to be disposed of properly. Small amounts of stock solutions of the desired monomers can be used for multiple tests, enabling a large number of monomers to be mixed in several different proportions in a single experiment. In addition, fewer stock solutions are required than to deposit polymerized polymers in the array.
  • the composition of the polymers themselves may be analyzed spectrophotometrically, for example, by fluorescence, infrared, or Raman spectroscopy.
  • a microarray of biocompatible polymers provided according to the invention may be seeded with cells.
  • the invention is appropriate for use with a wide range of cell types and is not limited to any specific cell type.
  • cell types include but are not limited to bone or cartilage forming cells such as chondrocytes and fibroblasts, other connective tissue cells such as epithelial and endothelial cells, cancer cells, hepatocytes, islet cells, smooth muscle cells, skeletal muscle cells, heart muscle cells, kidney cells, intestinal cells, other organ cells, lymphocytes, blood vessel cells, and stem cells such as or mesenchymal stem cells.
  • mammalian cells For therapeutic applications, it is preferable to practice the invention with mammalian cells, and more preferably human cells.
  • non-mammalian cells such as bacterial cells (e.g., E. coli), yeast cells (e.g., S. cerevisiae) and plant cells may also be used with the present invention.
  • Embryonic stem cells (ES) are also suited for use with the invention.
  • Embryonic stem (ES) cells including human ES (hES) cells, are a promising source for cell transplantation due to their unique ability to give rise to all somatic cell lineages when they undergo differentiation (Dushnik-Levinson, M., et al., "Embryogenesis in vitro: study of differentiation of embryonic stem cells," Biol Neonate 61, 77-83 (1995); Thomson, J.A., et al., "Embryonic stem cell lines derived from human blastocysts," Science 282, 1145-1147 (1998); Wobus, A.M., “Potential of embryonic stem cells," Mol Aspects Med 22, 149-164 (2001); Stocum, D.L., “Stem cells in regenerative biology and medicine,” Wound Repair Regen 9, 429-442 (2001)).
  • ES embryoid bodies
  • EBs embryoid bodies
  • the invention provides a method of screening polymers for suitability as substrates for stem cells proliferation and differentiation.
  • the cells are first cultured in a suitable growth medium, as would be obvious to one of ordinary skill in the art. See, for example, Current Protocols in Cell Biology, Ed. by Bonifacino et al., John Wiley & Sons Inc., New York, NY, 2000 (incorporated herein by reference).
  • a microarray of biocompatible polymers prepared as above is then placed in a suitable container (e.g., a 25 mm by 150 mm round suspension culture dish or a TEFLONTM trough) and incubated with a solution of the cultured cells.
  • the cells are present at a concentration that ranges from about 10,000 to 500,000 cells/cm 3 .
  • the incubation time and conditions (e.g., temperature, C0 2 and 0 2 levels, growth medium, etc.) will depend on the nature of the cells that are under evaluation. For most cell types, the choice of conditions will be obvious to one skilled in the art.
  • the incubation time should be sufficiently long to allow the cells to adhere to the elements of the polymeric biomaterial microarray.
  • the environmental conditions will need to be optimized in a series of screening experiments.
  • a growth factor may be added to the medium in which the cells are incubated with the polymer array.
  • parallel experiments are conducted with and without the growth factor to determine if the growth factor modifies the response of the cells to a particular polymer.
  • a cell type may proliferate on a particular polymer in the presence of a growth factor but not otherwise, or vice versa, or the growth factor may have no affect on cell proliferation.
  • Exemplary growth factors include but are not limited to activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic protein, platelet derived growth factor, hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor ( ⁇ - or ⁇ -TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).
  • ACT activin A
  • RA retinoic acid
  • epidermal growth factor epidermal growth factor
  • bone morphogenetic protein platelet derived growth factor
  • IGF insulin-like growth factors
  • IGF insulin-like growth factors
  • the cellular behavior of the seeded cells is assayed for each element of the microarray.
  • the invention employs a wide range of cell-based assays that enable the investigation of a variety of aspects of cellular behavior. Exemplary cell-based assays are discussed in our commonly owned application U.S.S.N. 09/803,319, entitled “Uses and Methods of Making Microarrays of Polymeric Biomaterials," the entire contents of which are incorporated herein by reference.
  • the cellular behaviors that can potentially be investigated according to the invention include but are not limited to cellular adhesion, proliferation, differentiation, metabolic behavior (e.g., activity level, metabolic state, DNA synthesis, apoptosis, contraction, mitosis, exocytosis, synthesis, endocytosis, migration), gene expression, protein expression, and the degree or amount of any of these.
  • metabolic behavior e.g., activity level, metabolic state, DNA synthesis, apoptosis, contraction, mitosis, exocytosis, synthesis, endocytosis, migration
  • gene expression protein expression
  • biocompatible polymers that enhance the proliferation of a given cell type.
  • biocompatible polymers that enhance the adhesion and proliferation of chondrocytes could be used as scaffolds in the preparation of engineered cartilage.
  • polymeric biomaterials that support differentiation of neural stem cells into glial cells or neurons may be useful as scaffolds in the regeneration of neural tissue. Different growth factors or growth media may be tested to enhance this effect.
  • the cell's interactions with a selection or library of chemicals may be evaluated by producing an array with one polymer on which a variety of small molecules, DNA, biomolecules, etc. are immobilized.
  • any of the cell-based assays known in the art may be used according to the present invention to screen for desirable interactions between the biocompatible polymers of the microarray and a given cell type.
  • the cells When they are assayed, the cells may be fixed or living.
  • Preferred assays employ living cells and involve fluorescent or chemiluminescent indicators, most preferably fluorescent indicators.
  • a variety of fixed and living cell-based assays that involve fluorescent and or chemiluminescent indicators are known in the art. For a review of cell-based assays, see Current Protocols in Cell Biology, Ed.
  • Cell-based assays screen for interactions at the cellular level using cellular targets and are to be contrasted with molecular-based assays that screen for interactions at a molecular level using molecular targets.
  • a cellular environment e.g., expression of a gene of interest
  • the experimenter does not require prior knowledge of the specifics of the interactions involved (e.g., the nature of the surface receptor or cytoplasmic cascade that triggers expression of the gene of interest).
  • cytokeratin is a marker for epidermal cells while desmin is a marker for muscle cells, and nestin and GFAP production may be used to identify cells that are differentiating as nerve cells.
  • the presence of alpha feto protein may be used to confirm the differentiation of cells towards liver cells, and vimentin assays may be used to confirm that cells are differentiating as mesodermal cells. Actin indicates contractile activity in cells.
  • markers may be used to identify expression of a predetermined gene, whether cells have fully differentiated, or whether there are still precursor cells seeded on the polymeric biomaterials.
  • genetic markers associated with particular cell types or cell behaviors may be used to characterize the seeded cells. For example, expression of the neurofilament heavy chain gene is associated with brain tissue, while expression of the alpha-1 anti-trypsin gene is associated with liver tissue.
  • Other genetic markers are listed in Schuldiner, et al., PNAS, 97: 11307-11312, 2000, the entire contents of which are incorporated herein by reference.
  • any of the cell-based assays known in the art may be used according to the present invention to screen for desirable interactions between the polymeric biomaterials of the microarray and a given cell type.
  • the cells may be fixed or living.
  • Preferred assays employ living cells and involve fluorescent or chemiluminescent indicators, most preferably fluorescent indicators.
  • fluorescent or chemiluminescent indicators most preferably fluorescent indicators.
  • a variety of fixed and living cell-based assays that involve fluorescent and/or chemiluminescent indicators are known in the art.
  • cell-based assays that can be used according to the present invention include but are not limited to assays that involve the use of phase contrast microscopy alone or in combination with cell staining; immunocytochemistry with fluorescent- labeled antibodies; fluorescence in situ hybridization (FISH) of nucleic acids; gene expression assays that involve fused promoter/reporter sequences that encode fluorescent or chemiluminescent reporter proteins; in situ PCR with fluorescently labeled oligonucleotide primers; fluorescence resonance energy transfer (FRET) based assays that probe the proximity of two or more molecular labels; and fused gene assays that enable the cellular localization of a protein of interest.
  • FISH fluorescence in situ hybridization
  • FRET fluorescence resonance energy transfer
  • One method of fluorescence immunocytochemistry involves the first step of hybridizing primary antibodies to the desired cellular target. Then, secondary antibodies conjugated with fluorescent dyes and targeted to the primary antibodies are used to tag the complex. The complex is visualized by exciting the dyes with a wavelength of light matched to the dye's excitation spectrum.
  • fluorescent dyes such as fluorescein and rhodamine are known in the art. Appropriate antibodies are well described in the art, and a variety of labeled and unlabeled primary and secondary antibodies are available commercially (e.g., from Sigma). Colocalization of biological moieties in a cell may be performed using different sets of antibodies for each cellular target.
  • one cellular component can be targeted with a mouse monoclonal antibody and another component with a rabbit polyclonal antibody. These are designated as primary antibodies. Subsequently, secondary antibodies to the mouse antibody or the rabbit antibody, conjugated to different fluorescent dyes having different emission wavelengths, are used to visualize the cellular target.
  • An ideal combination of dyes for labeling multiple components within a cell would have well-resolved emission spectra. In addition, it would be desirable for this combination of dyes to have strong absorption at a coincident excitation wavelength.
  • fluorescent immunocytochemistry can be used to assay for cellular adhesion, gene expression, and cell proliferation.
  • fluorescent molecules such as the Hoechst dyes (e.g., benzoxanthene yellow or DAPI (4,6-diamidino-2-phenylindole)) that target and stain DNA directly and non-specifically can be used to estimate the total cell population on each element of a seeded microarray of the invention.
  • Hoechst dyes e.g., benzoxanthene yellow or DAPI (4,6-diamidino-2-phenylindole)
  • FISH Fluorescence in situ hybridization
  • FISH typically involves the fluorescent tagging of an oligonucleotide probe to detect a specific complementary DNA or RNA sequence.
  • FISH Fluorescence in situ hybridization
  • Fluorescence resonance energy transfer provides a method for detecting the proximity of two or more biological compounds by detecting the long-range resonance energy transfer that can occur between two organic fluorescent dyes if the spacing between them is less than approximately 100 A. Conversely, this effect can be used to determine that two or more biological compounds are not in proximity to each other.
  • FRET Fluorescence resonance energy transfer
  • Cell-based assays that use promoter/reporter genes are designed to assay for expression of a gene of interest. Typically, this is achieved by transforming a given cell type with a plasmid comprising the promoter region of the gene of interest fused to the reporter sequence of a fluorescent or chemiluminescent protein. If the cytoplasmic cascade that normally leads to expression of the gene of interest and involves binding of a promoter moiety to the promoter sequence of the gene of interest is triggered, the transformed cells will begin to produce the reporter protein.
  • Reporter genes that are known in the art include the genes that code for the family of blue, cyan, green, yellow, and red fluorescent proteins; the gene that codes for luciferase, a protein that emits light in the presence of the substrate luciferin; and the genes that code for ⁇ -galactosidase and ⁇ -glucuronidase (proteins that hydrolyze colorless galactosides and glucuronides respectively to yield colored products).
  • a variety of vectors that contain fused promoter/reporter genes are available commercially (e.g., from Clontech Laboratories, Inc. of Palo Alto, CA).
  • an automated device may be used to analyze the cell- based assays for each element of the polymeric biomaterial microarray.
  • the devices may be manually or automatically operated.
  • an automated device that detects multicolored luminescent indicators can be used to acquire an image of the microarray and resolve it spectrally.
  • the device can detect samples by imaging or scanning. Imaging is preferred since it is faster than scanning. Imaging involves capturing the complete fluorescent or chemiluminescent data in its entirety. Collecting fluorescent or chemiluminescent data by scanning involves moving the sample relative to the imaging device.
  • An exemplary device may include three parts: 1) a light source, 2) a monochromator to spectrally resolve the image, or a set of narrow band filters, and 3) a detector array. The light source is only required for the detection of fluorescent indicators.
  • the light source may be derived from the output of a white light source such as a xenon lamp or a deuterium lamp that is passed through a monochromator to extract out the desired wavelengths.
  • a white light source such as a xenon lamp or a deuterium lamp that is passed through a monochromator to extract out the desired wavelengths.
  • filters could be used to extract the desired wavelengths.
  • any number of continuous wave gas lasers can be used. These include, but are not limited to, any of the argon ion laser lines (e.g., 457, 488, 514 nm, etc.), a HeCd laser, or a HeNe laser.
  • solid state diode lasers could be used. To spectrally resolve two different fluorescent or chemiluminescent indicators, light from the microarray may be passed through an image-subtracting double monochromator.
  • the fluorescent or chemiluminescent light from the microarray may be passed through two single monochromators with the second one reversed from the first.
  • the double monochromator consists of two gratings or two prisms and a slit between the two gratings.
  • the first grating spreads the colors spatially.
  • the slit selects a small band of colors, and the second grating recreates the image.
  • the fluorescent or chemiluminescent images may be recorded using a camera fitted with a charge-coupled device (CCD).
  • a CCD is a light sensitive silicon solid state device composed of many small pixels. The light falling on a pixel is converted into a charge pulse which is then measured by the CCD electronics and represented by a number.
  • a digital image is the collection of such light intensity numbers for all of the pixels from the CCD.
  • a computer can reconstruct the image by varying the light intensity for each spot on the computer monitor in the proper order.
  • digital images can be stored on disk, transmitted over a computer network and analyzed using powerful image processing techniques.
  • Any two-dimensional detector or CCD can be used.
  • CCDs and two- dimensional detectors are available commercially (e.g., from Hamamatsu Corp. of Bridgewater, NJ).
  • a variety of automated imaging systems that combine CCDs with computers and image processing software are also available commercially (e.g., the ARRAYWORXSTM microarray scanner available from Applied Precision, Inc. of Issaquah, WA).
  • the fluorescent or chemiluminescent light is detected by scanning the microarray of the present invention.
  • An apparatus using the scanning method of detection collects light data from the sample relative to a detection device by moving either the microarray or the detection device.
  • the microarray may be scanned by moving the detection device.
  • the light from the microarray may be passed thought a single monochromator, a grating or a prism.
  • filters could be used to resolve the colors spectrally.
  • the detector is preferably a diode array which records the light that is emitted at a particular spatial position.
  • polymers and growth factors and polymer growth factor combinations may be identified that promote a specific level of cell activity.
  • a particular monomer may facilitate one level of activity when co-polymerized with monomer A and a different level of activity when co- polymerized with monomer B.
  • the invention may be used to identify polymer-growth factor combinations that promote particular differentiation pathways.
  • a particular polymer in combination with retinoic acid may promote differentiation of stem cells into epithelial-like cells. Substitution of a different growth factor, or a different polymer, may induce the stem cells to follow a different path.
  • the polymer arrays of the invention may be more finely tuned by the addition of cell membrane components, adhesion peptides, or other materials. These materials may be used to promote differentiation along a particular path or to prevent de- differentiation of cells such as chondrocytes that are particularly prone to de- differentiation. Examples
  • Example 1 Production of a Polymer Array
  • the use of robotic fluid handling for the production of DNA, protein, and small molecule microarrays is well defined (G. MacBeath, et al., Journal of the American Chemical Society 121, 7967-7968 (1999); G. MacBeath, et al., Science 289, 1760-1763 (2000); M. Schena, et al., Science 270, 467-470 (1995)).
  • the deposition of structurally diverse acrylate monomers to produce a uniform, cell- compatible polymer microarray required significant modification of existing robotic technology.
  • Epoxy coated glass slides (Xenopore, Hawthorne, NJ) were dip coated into 4%> (w/v) poly (hydroxyethyl methacrylate) (pHEMA, Aldrich, Milwaukee, WI) solution in ethanol and dried for 3 days prior to use.
  • Monomers ( Figure 2 A) were purchased from Aldrich, Scientific Polymers (Onterio, NY), and Polysciences (Warrington, PA). Stock solutions were prepared at a ratio of (v/v) 75% monomer, 25%) DMF, and 1% (w/v) DPMA. These were then mixed pair-wise in 384 well black polypropylene plates at a ratio of 70:30 (v/v).
  • Monomers were mixed in all possible combinations with the exception of monomer 17, which was substituted with monomer 25 to increase polymer hydrophilicity.
  • Monomers were printed using CMP9B or CMP6B pins (Telechem International, Sunnyvale, CA) with a Pixsys 5500 robot (Cartesian, Ann Arbor, MI) in humid argon.
  • Printing of acrylate monomers required several modifications to existing printing methods: 1) incorporation of 25% dimethyl formamide to reduce viscosity, 2) substantially increasing washing and preprinting steps, and 3) modification of pin speed and size.
  • Figure 3 shows an exemplary apparatus for producing arrays for use with the invention.
  • Pins 10 were initially washed in DMF in reservoir 12 with agitation for about 10 seconds, and placed in a vacuum apparatus 14 to remove the DMF. Four pins 10 were used, but the block 15 that retains the pins can hold 32. The receptacles for the unused 28 pins in the vacuum were easily stopped with tape to decrease the pressure in the vacuum. The pins 10 were dipped in the appropriate monomer solutions in tray 16 for about 3 seconds and tapped on a slide in row 18 to remove excess monomer solution. Pins 10 were tapped multiple times (20-30 times) using multiple tapping sites to remove excess from the pins until there was sufficient solution on the pin to deposit reproducibly. The pins were then translated to the slides in array 20 on which the arrays were produced and allowed to deposit monomer on each slide.
  • the slides in array 20 were transferred under a UN lamp 22 and the pins were rinsed for about 10s.
  • the process was then repeated, starting with the initial washing step.
  • the table 30 translates along the x axis, and the robot arm 32 translates the pins along the x and y axes.
  • All 24 polymers composed of 70% of a particular monomer were produced as a 6 x 4 group on the array, as highlighted by the red and yellow boxes (Figure 2C).
  • Three blocks of 576 polymers were produced on each slide, with a center-to-center spacing of 740 microns ( Figure 2B).
  • Example 2 Cell Culture H9 cells (Thomson, J.A., et al., "Embryonic stem cells lines derived from human blastocysts", Science 282, 1145-1147 (1998)) were grown as described in Spradling, A., et al., “Stem cells find their niche", Nature 414, 98-104 (2001), the entire contents of which are incorporated herein by reference.
  • C2C12 cells were grown as described in Yaffee, D. & Saxel, O., "Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle", Nature 270, 725-7 (1977).
  • hES cells H9 clone were grown on mouse embryo fibroblasts (Cell Essential) in KnockOut Medium (Gibco-BRL, Gaithersburg, MD), a modified version of Dulbeco's modified Eagle's medium optimized for ES cells (Itskovitz-Eldor, et. al., (2000) Mol. Med. 6, 88-95, the contents of which are incorporated herein by reference). Tissue cover plates were covered with 0.1% gelatin (Sigma). Culture were grown in 5%> C0 2 and were routinely passaged every 5-6 days after disaggregating with 1 mg/ml collagenase type IV (Gibco-BRL).
  • hES colonies were digested using either 1 mg/ml collagenase type IV or trypsin/EDTA (0.1%/lmM) and transferred to petri dishes to allow their aggregation and prevent adherence to the plate.
  • Embryoid bodies were trypsinized after 6 days according to Levenberg, S., et al., "Differentiation of Human Embryonic Stem Cells on Three Dimensional Polymer Scaffold", Proc. Nat. Acad. Sci., 100:12741-12746 (2003).
  • EB's were dissociated with 0.025% / 0.01% trypsin /EDTA and washed with PBS containing 5% FBS.
  • Chips were added to the growth media (KO DMEM, 20 % heat inactivated fetal bovine serum, L-Glutamine, B-Mercaptoethanol, minimal essential amino acids (Invitrogen, Carlsbad, CA), and 1 ⁇ M retinoic acid (Aldrich) when indicated), and then seeded onto chips in 26 x 100mm Teflon dishes. Chips were incubated at 37°C with 5% C0 2 and media was changed after 1 day, and then every 2 days thereafter.
  • KO DMEM 20 % heat inactivated fetal bovine serum, L-Glutamine, B-Mercaptoethanol, minimal essential amino acids (Invitrogen, Carlsbad, CA), and 1 ⁇ M retinoic acid (Aldrich) when indicated
  • Example 3 Immunoliistochemistry Chips were washed, fixed in 4% paraformaldehyde for 8 minutes, blocked with 10% goat serum (Zymed, San Francisco, CA) and permeablized with 0.2% triton X-100 for 30 minutes. Primary antibodies, Ms anti-Cytokeratin 7, Ms anti-Myogenin (Dako, Carpinteria, CA), Rb anti- Vimentin (Biomeda, Foster City, CA) in PBS with 3% goat serum were incubated on the chips for 1 hr. Chips were washed 3 times in 1% goat serum PBS.
  • Example 4 Evaluation of Cell-Polymer Interactions of hES Cells
  • acrylate-based polymers have been used for tissue engineering, surgical glues, and drug delivery (Stocum, D.L., "Stem cells in regenerative biology and medicine", Wound Repair Regen 9, 429-442 (2001)).
  • monomers commercially available, and these can be polymerized quickly using a light-activated radical initiator.
  • a cell-compatible, miniaturized, polymer array To maximize throughput and minimize use of expensive reagents and cells, we developed a cell-compatible, miniaturized, polymer array.
  • pHEMA poly(hydroxyethyl methacrylate)
  • Example 5 Focus on hES Cells and Favorable Polymers To more thoroughly study polymers of interest and their effects on hES differentiation we created polymer arrays with 24 polymers of interest identified in the first screen ( Figure 5). Each "hit” array contained 1,728 polymer spots; 24 polymers materials with 72 replicates per array. These were seeded with fewer cells, only 4 million, to more clearly identify polymer effects.
  • Example 6 C2C12-Polymer Interactions To examine the whether polymer effects on cell growth are observed in other
  • Example 7 Differentiation of hES Cells in the Presence of Retinoic Acid hES cells were cultured on gelatin-coated glass slides using the techniques described in Example 2.
  • the growth media was KO DMEM, 20 %> heat inactivated fetal bovine serum, L-Glutamine, B-Mercaptoethanol, minimal essential amino acids (Invitrogen, Carlsbad, CA).
  • 1 ⁇ M retinoic acid (Aldrich) or 300nM AM580 (a retinoic acid analog, available from Sigma) were added as indicated in Figure 9. Cells were stained for cytokeratin 7 (red) and DNA (blue) using the techniques described in Examples 3 and 4.
  • Actin green was identified by staining with fiuorophore-labeled phalloidin (Molecular Probes, Alexa Fluor 488). Cytokeratin 7-positive cells are found even when no growth factor is added, while the addition of a growth factor increases the development of cytokeratin 7-positive cells.

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Abstract

Une population de cellules épithéliales embryonnaires est produite in vitro à partir de cellules souches embryonnaires. Dans une forme d'exécution, au moins 45 % des cellules expriment de la cytokératine, par exemple, de la cytokératine-7. L'invention concerne un procédé de criblage d'interactions cellule-polymère, procédé comprenant : le dépôt de monomères, sous forme d'une pluralité d'éléments discrets, sur un substrat, provoquant ainsi la polymérisation des monomères déposés, et créant ainsi un réseau d'éléments polymères discrets sur le substrat ; l'incubation du substrat dans un milieu de culture contenant des cellules ; et la caractérisation d'un comportement cellulaire prédéterminé sur chaque élément polymère.
EP04788758A 2003-09-15 2004-09-15 Synthese, a l'echelle du nanolitre, de biomateriaux en reseaux et criblage de ceux-ci Withdrawn EP1675943A4 (fr)

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US50316503P 2003-09-15 2003-09-15
US57018704P 2004-05-12 2004-05-12
US10/843,707 US20050019747A1 (en) 2002-08-07 2004-05-12 Nanoliter-scale synthesis of arrayed biomaterials and screening thereof
PCT/US2004/030095 WO2005028619A2 (fr) 2003-09-15 2004-09-15 Synthese, a l'echelle du nanolitre, de biomateriaux en reseaux et criblage de ceux-ci

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EP2240536B1 (fr) 2008-01-30 2019-09-04 Asterias Biotherapeutics, Inc. Surfaces synthétiques pour la culture de cellules progénitrices d'oligodendrocytes issues de cellules souches
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MX353900B (es) 2008-11-07 2018-02-01 Massachusetts Inst Technology Lipidoides de aminoalcohol y usos de los mismos.
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EP3403647A1 (fr) 2009-12-01 2018-11-21 Translate Bio, Inc. Administration d'arnm pour l'augmentation des protéines et des enzymes dans des maladies génétiques humaines
WO2012027675A2 (fr) 2010-08-26 2012-03-01 Massachusetts Institute Of Technology Poly(bêta-amino-alcools), leur préparation et utilisations de ceux-ci
US9238716B2 (en) 2011-03-28 2016-01-19 Massachusetts Institute Of Technology Conjugated lipomers and uses thereof
US20140206753A1 (en) 2011-06-08 2014-07-24 Shire Human Genetic Therapies, Inc. Lipid nanoparticle compositions and methods for mrna delivery
EP2859102A4 (fr) 2012-06-08 2016-05-11 Shire Human Genetic Therapies Polynucléotides résistant à la nucléase et leurs utilisations
WO2014028487A1 (fr) 2012-08-13 2014-02-20 Massachusetts Institute Of Technology Lipidoïdes contenant des amines et leurs utilisations
ES2981185T3 (es) 2013-03-14 2024-10-07 Translate Bio Inc Métodos para la purificación de ARN mensajero
CA2904151C (fr) 2013-03-14 2023-09-12 Shire Human Genetic Therapies, Inc. Composition a base d'arnm du gene cftr et procedes et utilisations associes
WO2014179562A1 (fr) 2013-05-01 2014-11-06 Massachusetts Institute Of Technology Dérivés de 1,3,5-triazinane-2,4,6-trione et leurs utilisations
MX2016005238A (es) 2013-10-22 2016-08-12 Shire Human Genetic Therapies Formulaciones de lipidos para la administracion de acido ribonucleico mensajero.
BR112016009014B1 (pt) 2013-10-22 2024-02-06 Translate Bio, Inc USO DE COMPOSIÇÃO COMPREENDENDO mRNA PARA DEFICIÊNCIA DE ARGININOSSUCINATO SINTETASE
WO2015061491A1 (fr) 2013-10-22 2015-04-30 Shire Human Genetic Therapies, Inc. Thérapie à l'arnm pour la phénylcétonurie
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UA121863C2 (uk) 2014-06-24 2020-08-10 Транслейт Байо, Інк. Стереохімічно збагачені композиції для доставки нуклеїнових кислот
US9840479B2 (en) 2014-07-02 2017-12-12 Massachusetts Institute Of Technology Polyamine-fatty acid derived lipidoids and uses thereof
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MX2019010155A (es) 2017-02-27 2020-12-10 Translate Bio Inc Arnm de cftr optimizado por codón novedoso.
MA49138A (fr) 2017-05-16 2020-03-25 Translate Bio Inc Traitement de la fibrose kystique par administration d'arnm à codons optimisés codant pour la cftr
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FR2825370A1 (fr) * 2001-05-31 2002-12-06 Inst Nat Sante Rech Med Keratinocytes obtenus a partir de cellules souches embryonnaires de mammiferes

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