NON-INVASIVE, IN VITRO FUNCTIONAL TISSUE ASSAY SYSTEMS
Provided are functional cell contacts for amplifier and tissue assay systems based on substrate-integrated multifunctional microelectrode arrays implementing stem cell technology. The system covers normal and pathogenic characteristics.
1 - 33 . (canceled)
34 . An in vitro method for screening at least one test substance for an effect on an isolated population of cells, comprising:
providing an electrode array comprising one or more isolated populations of cells having been obtained by differentiating mouse or human pluripotent stem cells, wherein the stem cells have been genetically altered to comprise a selectable marker operably linked to a regulatory sequence specific for a first cell type, wherein the one or more isolated population of cells have been differentiated and depleted of undifferentiated cells and/or of undesired cell types by using a selection system that is lethal to the undesired cells and cell types by expressing a selectable marker gene that renders cells of the first cell type resistant to the lethal effect,
contacting the populations of cells in an electrode array with the at least one test substance; and
measuring the electrical activity of the contacted cells with the electrode array and analyzing at least one parameter selected from the group consisting of:
(i) Na+ channel activity,
(ii) Ca2+/K+ channel activity,
(iii) K+ channel activity;
(iv) amplitude and/or field potential duration,
(v) chronotropy,
(vi) arrhythmia,
(vii) pH-value,
(viii) oxygen partial pressure,
(ix) beating arrest,
(x) contractility,
(xi) analysis of AV-dissociation contractility,
(xii) conductivity and/or impedance,
(xiii) nitrous oxide-effects, or
(ix) morphological changes; and
selecting a test substance that has an effect on at least one parameter in the measuring step as compared to cells of the same type which were not contacted with a test substance and wherein a change in said at least one parameter indicates that the test substance has an effect on the populations of cells.
35 . The method of claim 34 , wherein said cells are selected from the group consisting of neuronal cells, glial cells, cardiomyocytes, glucose-responsive insulin secreting pancreatic beta cells, hepatocytes, astrocytes, oligodendrocytes, chondrocytes, osteoblasts, retinal pigment epithelial cells, fibroblasts, keratinocytes, dendritic cells, hair follicle cells, renal duct epithelial cells, vascular endothelial cells, testicular progenitors, smooth muscle cells, and skeletal muscle cell.
36 . The method of claim 34 , wherein said pluripotent stem cells are embryonic stem (ES) cells.
37 . The method of claim 34 , wherein the selectable marker confers resistance to puromycin, neomycin, or hygromycin.
38 . The method of claim 34 , wherein said electrode array comprises one or more electrodes.
39 . The method of claim 34 , wherein said electrode array is a multi- or microelectrode array (MEA).
40 . The method of claim 38 , wherein the electrode array is coated with fibronectin.
41 . The method of claim 34 , wherein the cells are cardiomyocytes.
42 . The method of claim 34 , wherein the cells are neuronal cells.
43 . The method of claim 34 , wherein the cells are neural cells.
44 . The method of claim 34 , wherein the cells are fibroblasts.
45 . The method of claim 34 , wherein the cells are keratinocytes.
46 . The method of claim 34 , wherein the cells are smooth muscle cells.
47 . The method of claim 34 , wherein the cells are skeletal muscle cells.
48 . The method of claim 41 , wherein at least one of the parameters analyzed is selected from the group consisting of: beating frequency, mean contractility, maximum contraction and mean area of contraction is analyzed.
49 . The method of claim 34 , wherein test substance is one of a collection of test substances.
50 . The method of claim 34 , wherein said collection of test substances comprises about 10 3 to about 10 5 substances.
51 . The method of claim 34 , wherein three or more measurements are taken.
52 . The method of claim 34 , further comprising removing the measured cells.
53 . The method of claim 51 , wherein said measurements are taken at different positions within the array.
54 . The method of claim 34 , wherein said selection step positively screens a test sub stance.
55 . The method of claim 34 , wherein said selection step screens out a test substance.
56 . The method of claim 34 , wherein said selection step determines the toxicity of the test substance on the cells.