Non-invasive, in vitro functional tissue assay systems

• US 9,945,840 B2
• Filed: 04/07/2005
• Issued: 04/17/2018
• Est. Priority Date: 04/07/2004
• Status: Active Grant

• Current Assignee: Evotec International GmbH (Evotec SE)
• Original Assignee: Axiogenesis AG
• Inventors: Bohlen, Heribert, Kolossov, Eugen, Kettenhofen, Ralf, Scholz, Melanie, Fink, Leo
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US11/547,871
• Publication Number:
  US 20080132422A1
• Time in Patent Office: 4,758 Days
• Field of Search: 435 613
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/4836 using multielectrode arrays
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/5088 of vertebrates
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

Provided are functional cell and tissue assay systems based on substrate-integrated multifunctional microelectrode arrays implementing stem cell technology. The system covers normal and pathogenic characteristics.

Claims

1. An in vitro method for screening a test substance for an effect on a cardiomyocyte, comprising:

(a) providing cardiomyocytes having been obtained by differentiating mouse or human pluripotent stem cells;

wherein the stem cells have been transfected with at least one vector, wherein the at least one vector comprises a first nucleotide sequence encoding a selectable marker and the at least one vector comprises a second nucleotide sequence encoding a reporter gene;

wherein the transfected stem cells comprise the first nucleotide sequence operably linked to at least one cardiac cell-specific regulatory sequence and comprise the second nucleotide sequence operably linked to a cardiac cell-specific regulatory sequence, and

wherein the cardiomyocytes have been selected based on expression of the selectable marker;

(b) contacting cardiomyocytes of step (a) with a test substance, wherein the cardiomyocytes have been placed on an electrode array;

(c) measuring the electrical activity of the contacted cardiomyocytes 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;

(d) selecting a test substance that has an effect on at least one parameter in step (c) as compared to cardiomyocytes which were not contacted with a test substance and wherein a change in at least one parameter measured in step (c) indicates that the test substance has an effect on a cardiomyocyte.

2. The assay method of claim 1, wherein said pluripotent stem cells are embryonic stem (ES) cells.

3. The method of claim 1, wherein said electrode array is a multi- or microelectrode array (MEA).

4. The method of claim 3, wherein the electrode array is coated with fibronectin.

5. The method of claim 1, 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.

6. The method of claim 1, wherein test substance is one of a collection of test substances.

7. The method of claim 6, wherein said collection of test substances comprises about 10³ to about 10⁵ substances.

8. The method of claim 1, wherein three or more measurements are taken.

9. The method of claim 1, further comprising isolating the measured cardiomyocytes.

10. The method of claim 8, wherein said measurements are taken at different positions within the array.

11. The method of claim 1, wherein said selection step positively screens a test substance.

12. The method of claim 1, wherein said selection step screens out a test substance.