Systems and methods for modeling disease and assessing adverse side effects of therapeutics therefor
AssigneeNovoHeart Limited
InventorRonald A. Li
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Abstract
The disclosure provides a substrate for the ordered growth and development of cardiomyocytes such as ventricular cardiomyocytes derived from pluripotent stem cells along with methods of culturing the cells on the substrates for use in assays such as cardiotoxicity assays. Further provided are methods for assessing cardiotoxicity using one or more criteria disclosed herein
Claims59
A method of assessing the cardiotoxicity of a compound comprising:
- (a) contacting an anisotropic layer of cardiac cells on a microfabricated substrate with a compound;
- (b) stimulating the anisotropic layer of cells at one or more points;
- (c) detecting an electrical signal propagation in the anisotropic layer of cells;
- and
- (d) determining whether the compound exhibits cardiotoxicity.
A method of identifying cardiomyocytes suitable for use in an in vitro cardiotoxicity assay comprising:
- (a) contacting a cardiomyocyte derived from a pluripotent stem cell with a microfabricated substrate;
- and
- (b) identifying cardiomyocytes suitable for use in an in vitro cardiotoxicity assay as cardiomyocytes exhibiting at least two of the following properties:
- (i) a radiating conduction pattern with an anisotropic ratio of at least 1.8 in response to electrical point stimulation;
- (ii) at least 70% cardiac troponin T+;
- (iii) a maximum capturing frequency of at least 1.5 Hz;
- (iv) an absence of reentrant events in response to steady-state pacing from 0.5 Hz through to 3 Hz (10V, 10 ms) with 0.5 Hz increments until the loss of 1:1 capture;
- or
- (v) an absence of induced spiral waves in response to programmed electrical stimulation comprising a first train of eight electrical pulses (S1) followed by a second train of electrical pulses (S2) with progressively shortened S1-S2 intervals.
A method of producing an anisotropic layer of cardiac cells comprising:
- (a) dissociating pluripotent stem cell cultures of at least 80% confluence to yield individual cells;
- (b) culturing individual cells in suspension under hypoxic conditions of about 5% O2 and about 5% CO2 for about 8 days to yield cultured cells, wherein the culturing comprises:
- (i) incubating the cells in mTeSR™-1 medium supplemented with 10 μM Rho kinase inhibitor, 1 ng/mL bone morphogenetic protein-4 and 40 μg/mL matrigel for about 24 hours;
- (ii) further incubating the cells in StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid, 2 mM GlutaMAX™, 10 ng/mL BMP4 and 10 ng/mL Activin-A for about 3 days;
- (iii) continuing to incubate the cells in StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid, 2 mM GlutaMAX™, and 5 μM of a Wnt inhibitor for about 3 days;
- (iv) transferring the cells to StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid under ambient O2, 5% CO2 conditions on day 8 to yield cardiospheres;
- (c) maintaining the cardiospheres in StemPro®-34 SFM complete medium comprising 50 μg/mL ascorbic acid until day 15-22, with medium replenishment every 3-4 days;
- (d) dissociating the cardiospheres by exposure to 1 mg/mL collagenase for 30 minutes, followed by exposure to 0.05% trypsin for 3 minutes;
- (e) seeding the cardiospheres onto a matrigel-coated microfabricated substrate to form an anisotropic layer of cardiac cells;
- (f) incubating the anisotropic layer of cardiac cells in high-glucose (4.5 g/L) DMEM medium comprising 10% heat-inactivated fetal bovine serum, 1× GlutaMAX™, and 1×MEM non-essential amino acids for 2 days;
- and
- (g) replenishing the medium every 2 days with RPMI 1640 medium for at least 7 days, thereby producing an anisotropic layer of cardiac cells.
A method of manufacturing a microfabricated substrate comprising:
- (a) generating an elastomeric mold using photolithography to yield a mold having a series of channels oriented along the same axis in which each channel is 3-10 μm deep and 2-50 μm wide with a spacing between channels of 3-10 μm;
- (b) embossing the features of step (a) onto plastic using heat to generate a substrate;
- (c) exposing the substrate to UVO treatment for about 8 minutes;
- and
- (d) producing the microfabricated substrate by sterilizing the substrate in a chemical sterilizing agent.
A system that models a cardiac disease comprising a bio-hybrid material comprising:
- (a) human cardiomyocytes, and (b) a microfabricated substrate for cell attachment.
A method of screening for a therapeutic for cardiac disease comprising (a) incubating an anisotropic layer of cardiac cells under conditions maintaining cell viability;
- (b) contacting the cardiac cells with a candidate therapeutic for cardiac disease;
- and (c) measuring the effect of the candidate therapeutic for cardiac disease on the cardiac cells, wherein the candidate therapeutic for cardiac disease is identified as a therapeutic for cardiac disease if the candidate therapeutic for cardiac disease reduces a symptom of a cardiac disease in the cardiac cells.
A method for assessing the cardiotoxic effect of a compound comprising (a) contacting an anisotropic layer of cardiac cells with a compound;
- and (b) measuring the cardiotoxic effect of the compound on the cardiac cells.
Dependencies are inferred from the claim text.