US 11,835,433 B2
Non-invasive, in vitro functional tissue assay systems
Heribert Bohlen, Cologne (DE); Eugen Kolossov, Cologne (DE); Ralf Kettenhofen, Bonn (DE); Melanie Scholz, Cologne (DE); and Leo Fink, Cologne (DE)
Assigned to Evotec International GmbH, Hamburg (DE)
Filed by Evotec International GmbH, Hamburg (DE)
Filed on Oct. 15, 2020, as Appl. No. 17/071,574.
Application 17/071,574 is a continuation of application No. 15/954,413, filed on Apr. 16, 2018, abandoned.
Application 15/954,413 is a continuation of application No. 11/547,871, granted, now 9,945,840, issued on Apr. 17, 2018, previously published as PCT/EP2005/003662, filed on Apr. 7, 2005.
Claims priority of application No. 04 008 497 (EP), filed on Apr. 7, 2004.
Prior Publication US 2021/0025869 A1, Jan. 28, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/50 (2006.01); G01N 33/543 (2006.01); G01N 33/483 (2006.01)
CPC G01N 33/5008 (2013.01) [G01N 33/4836 (2013.01); G01N 33/5014 (2013.01); G01N 33/5023 (2013.01); G01N 33/5058 (2013.01); G01N 33/5073 (2013.01); G01N 33/5088 (2013.01); G01N 33/5438 (2013.01); G01N 2800/52 (2013.01)] 15 Claims
 
1. An in vitro method for screening at least one test substance for an effect on an isolated population of cardiomyocyte cells, comprising:
providing an electrode array comprising one or more isolated populations of cardiomyocyte 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 non-cardiomyocyte cells by using a selection system that is lethal to the undifferentiated cells and/or non-cardiomyocyte cells by expressing a selectable marker gene that renders cells of the first cell type resistant to the lethal effect,
contacting the populations of cardiomyocyte cells in an electrode array with the at least one test substance; and
measuring the electrical activity of the contacted cardiomyocyte 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 and/or beating frequency,
(x) contractility,
(xi) analysis of AV-dissociation contractility,
(xii) conductivity and/or impedance,
(xiii) nitrous oxide-effects, or
(xiv) morphological changes; and
selecting a test substance that has an effect on at least one parameter in the measuring step as compared to cardiomyocyte 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 cardiomyocyte cells.