Patent Application
20180044640
The present invention relates to a method of producing a contractile cellular construct. In particular, the present invention relates to a method of producing a 3D contractile cellular construct for cell culture.
Pluripotent stem cells provide an unlimited ex vivo source of differentiated cells which provide unique opportunities to model disease, to establish personal predictive drug toxicology and target validation, ultimately to enable autologous cell-based therapies. An example of a differentiated cell type obtained from pluripotent stem cells are cardiomyocytes.
Mature adult cardiomyocytes in native heart are surrounded by an organization of supporting matrix and neighbouring cells with gradients of chemical, electrical and mechanical signals. Consequently, various approaches have been reported as in vitro cardiac models that better mimic the native heart tissue architecture and functions. These include 2-dimensional (2D) and 3-dimensional (3D) cell constructs formed by cells seeded and organized in either polymeric or biological scaffolds, or extracellular matrix (ECM) hydrogels such as collagen, matrigel, laminin, fibronectin, and fibrin, or decellularized natural matrix as well as the cellular tissues organized from stackable cell sheets. However, while these studies highlighted the utility of cardiac tissue constructs, the constructs containing exogenous scaffolds or ECM-based materials may interfere with direct cell-cell interaction, limiting the recapitulation of native microcellular structure. Furthermore, the use of animal cells or animal-derived extracellular matrix scaffolds poses a potential issue when considering the translational use of these constructs in human.
In addition, cardiac toxicity is a leading cause for drug attrition during the clinical development of pharmaceutical products and has resulted in numerous preventable patient deaths. Currently, several in vitro cardiac toxicity models based on human ESC or iPSC-derived cardiac cells have been used for drug tests, such as FLIPR® Tetra system and xCELLigence RTCA Cardio system. However, these in vitro toxicity screens either rely on costly, specially manufactured tissue culture plates and/or the characterization of single cardiac ion channels in cardiac cells, which do not accurately model pertinent biochemical characteristics of the human heart, thus limiting their pharmaceutical application.
There is therefore a need to provide an efficient in vitro culture system both for the production of functional differentiated cells, such as cardiomyocytes, and monitoring cell function profile that overcomes, or at least ameliorates, one or more of the disadvantages described above.
In one aspect, there is provided a method for producing a contractile cellular construct, comprising the steps of: a) seeding pre-selected cells onto a mold, wherein the pre-selected cells comprise signal emitting agents; and b) culturing the pre-selected cells to produce the contractile cellular construct.
In one aspect, there is provided a contractile cellular construct produced by the method as described herein.
In one aspect, there is provided a method of measuring the contractility of the contractile cellular construct as described herein, comprising:
a) measuring the location of the signal emitting agents in the contractile cellular construct, at two or more pre-selected times; and
b) determining the temporal change in the location of the signal emitting agents to produce a contraction profile and relaxation profile based upon the measurements of a).
In one aspect, there is provided a method for screening one or more agents for modulating the contractility of a contractile cellular construct, comprising:
a) contacting the contractile cellular construct as described herein with said one or more agents;
b) measuring the location of the signal emitting agents, comprised in the contractile cellular construct, at two or more pre-selected times;
c) determining the temporal change in the location of the signal emitting agents in said two or more pre-selected times, to produce a test contraction profile and test relaxation profile based upon the measurements of b);
d) comparing the test contraction profile and test relaxation profile of c) with a control contraction profile and control relaxation profile of a contractile cellular construct as described herein that has not been contacted with the one or more agents or has been contacted with the one or more agents at a different concentration to that of step a); wherein a differential profile between the test and control contraction or relaxation profiles demonstrates a modulating activity of said one or more agents on the contractile cellular construct.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
In one aspect the present invention refers to a method for producing a contractile cellular construct. The method comprises the steps of a) seeding pre-selected cells onto a mold, wherein the pre-selected cells comprise signal emitting agents; and b) culturing the pre-selected cells to produce the contractile cellular construct.
In one embodiment, prior to step a) the method further comprises: inducing a pluripotent stem cell into a pre-determined lineage of the pre-selected cells; isolating the induced pre-selected cells; and contacting the isolated pre-selected cells with signal emitting agents to produce pre-selected cells comprising signal emitting agents.
The pluripotent stem cell may be a human induced pluripotent stem cell (hiPSC). The hiPSC may be derived from a biological sample. The biological sample may be a sample of tissue or cells. The biological sample may include but is not limited to blood, blood plasma, serum, buccal smear, amniotic fluid, prenatal tissue, sweat, nasal swab or urine, organs, tissues, fractions, and cells isolated from mammals including humans. The sample may also comprise clinical isolates that may include sections of the biological sample including tissues (for example, sectional portions of an organ or tissue).
In some embodiments, prior to step a) the method further comprises: isolating the pre-selected cells from a biological sample; and contacting the isolated pre-selected cells with signal emitting agents to produce pre-selected cells comprising signal emitting agents.
The contractile cellular construct may comprise any cells having a contractile function and may be in vitro or ex vivo. In some embodiments, the contractile cellular construct may comprise muscle cells. The muscle cells may be selected from the group consisting of skeletal muscle cells, cardiac muscle cells and smooth muscle cells.
In another embodiment the contractile cellular construct may comprise cardiac cells with contractile function. For example, a construct comprising cardiac muscle cells, a cardiomyocyte-extracellular matrix (ECM) hydrogel construct, or a cardiomyocyte-polymer/biomaterial construct. The pre-selected cells may be cardiac cells. The cardiac cells may comprise one or more mammalian cells selected from the group consisting of cardiomyocytes, endocardial cells, cardiac adrenergic cells, endothelial cells, neuromuscular cells and cardiac fibroblasts. The cardiomyocytes may comprise one or more of ventricular cardiomyocytes, atrial cardiomyocytes and nodal cardiomyocytes.
In one embodiment, the cardiac cells may comprise cardiomyocytes expressing one or more markers selected from the group consisting of MYH6, α-sarcomeric actin, cTnT, Connexin 43, GATA4, Tbx5, MEF2c, sarcomeric MHC, sarcomeric actinin, Cardiac troponin I, atrial natriuretic peptide, Smooth muscle α-actin, desmin and NKX2.5.
In some embodiments, at least 70%, 80% or 90% of the cardiac cells are cardiomyocytes.
In one embodiment, the mold may be a substrate or scaffold, for example a biocompatible polymer substrate or scaffold. In one embodiment, the mold may be a non-rigid, flexible or resiliently deformable, polymer scaffold or substrate. The mold may be constructed from a material selected from the group consisting of a gel, agarose, polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polyisoprene, polybutadiene and silicone. The mold may also be a biocompatible polymer selected from the group consisting of matrigel, fibronectin, laminin and collagen. The mold may be 2D or 3D. In particular, the mold may be a 2D or 3D PDMS (polydimethylsiloxane) mold.
In one embodiment, the signal emitting agents may be selected from the group consisting of fluorochromes, fluorescent microspheres, fluorescent-labelled cells, luminescent particles, phosphorescent particles and magnetic particles.
In one embodiment, the culturing step b) of the method as described herein comprises culturing the pre-selected cells in a serum-free medium. The culturing step may be performed for 1 to 10 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 3 to 8 days or 3 to 7 days.
The cardiac construct may comprise extracellular matrix proteins characteristic of a cardiac construct. The extracellular matrix proteins may comprise various isoforms of laminin, collagen type I, collagen type IV, entactin, proteoglycans including but not limited to heparan sulfate, perlecan and fibronectin.
The contractile cellular construct may be a contractile cellular monolayer construct, a two-dimensional contractile cellular construct or a three-dimensional contractile cellular construct.
In another aspect, the present invention also provides a contractile cellular construct produced by the method as described herein. The cellular construct may be a three-dimensional cardiac construct.
In another aspect, the present invention also provides a method of measuring the contractility of the contractile cellular construct as described herein, comprising: a) measuring the location of the signal emitting agents in the contractile cellular construct, at two or more pre-selected times; and b) determining the temporal change in the location of the signal emitting agents to produce a contraction profile and relaxation profile based upon the measurements of a).
In one embodiment, the measuring step may comprise real-time video recording.
In one embodiment, the determining step may comprise image tracking analysis.
In some embodiments, the contraction profile may comprise at least one contraction parameter selected from the group consisting of contraction pattern, contraction amplitude, contraction time, contraction velocity and acceleration vector.
The relaxation profile may comprise at least one relaxation parameter selected from the group consisting of relaxation pattern, relaxation amplitude, relaxation time, relaxation velocity and acceleration vector.
In another aspect the present invention provides a method for screening one or more agents for modulating the contractility of a contractile cellular construct, comprising: a) contacting the contractile cellular construct as described herein with said one or more agents; b) measuring the location of the signal emitting agents, comprised in the contractile cellular construct, at two or more pre-selected times; c) determining the temporal change in the location of the signal emitting agents in said two or more pre-selected times, to produce a test contraction profile and test relaxation profile based upon the measurements of b); d) comparing the test contraction profile and test relaxation profile of c) with a control contraction profile and control relaxation profile of a contractile cellular construct as described herein that has not been contacted with the one or more agents or has been contacted with the one or more agents at a different concentration to that of step a); wherein a differential profile between the test and control contraction or relaxation profiles demonstrates a modulating activity of said one or more agents on the contractile cellular construct.
In one embodiment, the measuring step may be performed by real-time video recording. In another embodiment, the measuring step may be performed by image tracking analysis.
In another embodiment, the contraction profile comprises at least one contraction parameter selected from the group consisting of contraction pattern, contraction amplitude, contraction time, contraction velocity and acceleration vector. The relaxation profile may comprise at least one relaxation parameter selected from the group consisting of relaxation pattern, relaxation amplitude, relaxation time, relaxation velocity and acceleration vector.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
Materials and Methods
Manufacturing PDMS molds and tissue holders
Sylgard 184 silicone elastomer (Dow Corning) was used to prepare silicone molds and tissue holders. The custom-made casting molds were fabricated using 3D printing system (Stratasys, Objet). The PDMS pre-polymer solution containing a mixture of PDMS oligomers and a reticular agent from Sylgard 184 (10:1 mass ratio) was degassed under vacuum conditions before casting. The mixture was poured into the casting molds and cured at 80° C. overnight. Once demoulded, the PDMS substrate was carefully prepared with respect to the dimensions of cell culture wells dimensions. The PDMS substrates were then cleaned with ethanol and air-dried in laminar hood. The silicone molds and tissue holders were designed with dimensions to fit into wells of multi-well culture plates. For 96-well tissue culture plate (NUNC), PDMS mold diameter: 6 mm; Tissue channel: length/width/depth=5 mm×1.5 mm×1.5 mm; tissue holders: length/width=3 mm×2 mm (
Directed differentiation of hiPSCs into cardiac lineage cells under defined culture conditions
Cardiac differentiation of hiPSCs was carried out under serum-free condition. Typically, the patient derived iPSCs and healthy control iPSCs were plated on Matrigel-coated tissue culture plates in E8 medium to reach near confluence. The cells were washed with PBS, and exposed to medium consisting of RPMI 1640 supplemented with B27 minus insulin (Life Technologies) and CHIR99021 (12 μM, Tocris). After 24-hour treatment, the medium was replaced with RPMI 1640 supplemented with B27 minus insulin. Forty-eight hours later, the medium was changed to RPMI 1640 supplemented with IWP4 (5 μM, Stemgent). After 48-hour treatment, the medium was changed to RPMI 1640 supplemented with B27 minus insulin for two additional days followed by medium replacement to RPMI 1640 supplemented with B27 (Life Technologies) with medium change every two days.
For cardiac differentiation under xeno-free condition, hiPSCs were plated in E8 medium on Laminin521 (BioLamina)-coated culture plates to reach near confluence. The cells were treated with medium consisting of E5 supplemented with CHIR99021 (10 μM, Tocris), TGF β (1 ug/ml, R & D), Y-27632 (5 uM) and 1× concentrated lipid (Life Technologies) for 24 hours. The medium was changed to E5 supplemented with TGFβ (1 ug/ml) and 1× concentrated lipid for two days, then replaced with E5 supplemented with IWP4 (4 uM) and concentrated lipid. After 48-hour treatment, the medium was changed to RPMI 1640 supplemented with B27 Xeno (B27 Supplement CTS, Life Technologies) with medium change every two days.
Generation of 3D cardiac tissues under defined culture condition
To generate 3D cardiac tissues, 12-day differentiated cardiac cells were detached from culture wells using accutase (Stem Cell Technologies), suspended in RPMI-B27medium containing Y-27632 (5 uM) to reach a final concentration of 1.5×108 cells/mL. Casting molds were prepared by placing the tissue holder and PDMS molds in 96-well culture plates. For each tissue, 12 μL cell suspension was loaded into the cell loading channel. For florescence-labeled cardiac tissue, 12 μL cell suspension was mixed briefly with ˜20 FluoSpheres polystyrene microspheres (diameter 15 μm, Life Technologies) and pipetted into cell loading channels. The 96-well plates were placed in a 37° C., 5% CO2 culture incubator for 2 hours. 0.20 mL of cell culture medium was then added per well for continued culture. After one day of culture, media was changed to RPMI-B27 medium without Y-27632 and changed every day. After 3 days of culture, the cell constructs had formed tissues with sufficient mechanical properties to allow them to retain their integrity upon removal from PDMS molds and during manipulation with forceps.
Seeding cardiomyocytes in 384-well/96-well plates for drug screening
The 12-day differentiated cardiomyocytes were detached using accutase and suspended in RPMI-B27 medium containing Y-27632 (5 uM). The cell suspension was mixed with FluoSpheres polystyrene microspheres, and loaded into matrigel or fibronectin coated 384-well microplate (Greiner) or 96-well tissue culture plates at 0.25˜0.30 *106cells/50˜100 microspheres/cm2. After one day of culture, media was changed to RPMI-B27 medium without Y-27632 and changed every day. Cardiomyocyte monolayer was formed after one day, and started to contract synchronously 2 day post-seeding.
Assessment of cardiac tissue contractility
Cardiac tissue contractility was assessed based on the recorded video using IMARIS software. The cardiac tissues were labeled with fluorescence beads, as described above. The video was exported as image stacks for IMARIS tracking analysis. The software can identify the contraction and relaxation events based on the relative positions of the florescence beads during the entire tissue beating sequence. These positions were recorded, and the relevant contractility parameters were calculated from the temporal change of these positions.
Pharmaceutical tests
Toxicity studies were performed using a Zeiss fluorescence microscope, within a closed environment chamber maintaining constant 37° C. temperature and 5% CO2 humidified air for long time lapse imaging of live cells. An experiment design program in Zen software was used to create an automatic measurement program. For chronic drug response, videos were recorded for each well at every 1 h interval.
After 7 days of culture, the 3D cardiac tissues exhibiting good beating activity were subjected to measurement of drug toxicity. The cardiac tissues were equilibrated in RPMI 1640/B27 for one hour and incubated consecutively with increasing concentrations of drugs, including erythromycin, trovafloxacin, ampicillin, rosiglitazone, troglitazone, metformin, chromanol 293B, quinidine sulfate, and E-4031. The videos were recorded at cumulative concentrations 1 hr before measurement, and processed using IMARIS software.
After culture in 384-well microplate for 7 days, the cardiomyocytes were exposed to pentamidine under the following conditions: 1) cumulative concentrations (0.1, 1, 10, 100 μM) for 1 hr; 2) 1, 5, 10 μM for 2 days. The videos were recorded for each well at every 1 hr interval and processed using IMARIS software.
Toxicity assays based on ATP activity
Cardiac spheroids were incubated with various concentrations of compounds dissolved in culture medium for 1 hr, and cell viability was subsequently measured by CellTiter-Glo® 3D cell viability assay (Promega), which determines the number of viable cells in culture based on quantitation of the ATP present. Data is normalized to drug-free controls. Data from the same treatment on 3 occasions were averaged to represent the mean ATP measurement.
Flow cytometry
The differentiated cardiomyocytes were dissociated with Accutase for 6-10 minutes at 37° C., followed by gentle trituration to a single-cell suspension. The cells were processed for staining with anti-cTnT and analyzed with a BD LSR II.
RNA extraction, reverse transcription and polymerase chain reaction
Total RNA was isolated using Trizol reagent (Life Technologies) according to manufacturer's instructions. Before reverse transcription, RNA samples were treated with DNase I (Life Technologies) to remove contaminating genomic DNA. cDNA was synthesized using SuperScript III Reverse Transcriptase and Oligo [dT]18 primers according to the manufacturer's instructions (Life Technologies). PCR was carried out using Taq DNA Polymerase (Life Technologies) with gene specific primers.
Immunocytochemistry
The differentiated cardiomyocytes and tissues were fixed with 4% paraformaldehyde and immunostained with the antibodies as listed below: mouse anti-a-actinin, mouse anti-cTnT, mouse anti-MYH6, mouse anti-fibronectin, rabbit anti-collagen I, mouse anti-collagen IV, rabbit anti-laminin antibody (Abcam) and rabbit Polyclonal Nkx-2.5 (Life Technologies). Appropriate fluorescence (Alexa-Fluor-488/568)-tagged secondary antibodies were used for visualization (molecular probes, Eugene, USA). 4,6-diamidino-2-phenylindole (DAPI) counterstain was used for nuclear staining. The samples were observed under a Zeiss LSM510 laser scanning microscope and photographed and processed with LSM Image Browser software.
Results
Fabrication of PDMS master mold and tissue holder
Differentiation of hiPSCs into cardiomyocytes under serum free culture condition The quality of cardiomyocytes is critical for achieving functional beating tissue. In our study, the population for fabricating 3D tissue contained >90% cardiac cells. To achieve this, we differentiated hiPSCs into cardiomyocytes using a small molecule treatment approach. hiPSCs were seeded as single cells and cultured in E8 medium on matrigel-coated plates to reach confluence. Differentiation was induced in RPMI/B27 medium lacking insulin and containing CHIR99021, followed by inhibition of Wnt signaling with IWP4 (
Fabrication of 3D cardiac tissues under defined culture conditions
The 12-day differentiated cardiomyocytes were detached and loaded into spatially defined PDMS molds under defined serum-free conditions. Molds were fully immersed in culture medium and the culture was maintained in a 37° C., 5% CO2 humidified incubator. Over the course of 48 hours, the loaded cells started to aggregate into rod-shaped tissue constructs and exhibited progressive lateral and longitudinal condensation (
Fabrication of fluorescence labelled cardiac tissues for high throughput screening assays
One of the applications for the engineered cardiac tissues is for in vitro toxicity assays. In order to adapt this 3D model to a high throughput screening assay, fluorescence labels were incorporated into the cardiac tissues to enable real time monitoring of cardiac contractile motion. The fluorescence labels which can be used include fluorescent microspheres or cells. After optimization, it was found that microspheres of diameter comparable to that of the cell have negligible effects on tissue structure and contracting function, and also allows monitoring of the beating pattern by automated video-optical recording. The procedure is described below using FluoSpheres polystyrene microspheres (diameter: 15 μm) as an example. For 3D cardiac tissues: 12-day differentiated cardiomyocytes were detached, mixed with fluorescence microspheres (˜20 beads/tissue), and loaded into spatially defined PDMS molds under defined serum-free conditions. After three days, synchronized contractile 3D cardiac tissues were formed, and were either left inside or removed from the PDMS molds for continued culture (
Automatic analysis of contractile motion of 3D fluorescence labelled cardiac tissues
A methodology has also been developed to analyse the contractile motion of the 3D fluorescence labelled cardiac tissues to enable evaluation of cell tissue responses in screening assays. Real-time videos were taken of the cardiac tissues and IMARIS software was used to track and analyse the real-time positions of the fluorescent label. This software both enables processing of the data and provides selectable outputs for analysis. In this way, contraction and relaxation events can be identified based on the detection of florescence signals from the beads during the entire tissue beating sequence. Subsequently, the relevant contractility parameters can be calculated from the temporal change of these positions. Therefore, with this methodology, we are able to obtain quantitative information of 3D cardiac tissue contraction profile, including tissue contraction pattern, amplitude, time, velocity and acceleration vector, which is important in evaluating cardiac functional response in screening assays (as shown in
Pharmacological study using 3D fluorescence labelled cardiac tissues
The suitability of the formed 3D cardiac tissue models for pharmacological screening was validated. One-week fluorescence labelled contractile cardiac tissues were subjected to measurement of drug toxicity. The cardiac tissues were exposed to several cardioactive drugs known to block Ikr, prolong the QT interval, or induce Torsades de Pointes including E-4031, chromanol 293B, erythromycin and quinidine. Cell tissue responses were evaluated with real-time video recording and IMARIS software. Based on the fluorescence video recording, IMARIS software gives quantitative information including tissue contraction pattern, time, velocity and acceleration vector. Drugs exerted a decrease in contraction frequency of the cardiomyocytes and abnormal contraction patterns. Complete arrest of contraction was observed at 10 μM for erythromycin, 200 μM for chromanol 293B, 10 μM for E4031 and 10 μM for quinidine respectively (
Next, the toxicity effect of a panel of clinical drugs, including antibiotics and antidiabetics, was tested by exposing the 3D cardiac tissues to increasing concentrations of these drugs. For comparison purposes, the toxicity effect of these drugs were studied using ATP-based cell viability assays in parallel (
Pharmacological study using 2D fluorescence labelled cardiac tissues
The fluorescent labelling technique was extended to 2D high throughput format. Although advances in high throughput systems for drug screening have been made, current 2D cardiac models adaptable to high-throughput array formats, for example, 384-well plate are limited. Furthermore, most 2D models (using Ca channel sensitive dye or genetic modified cardiomyocytes) are not suitable for chronic toxicity evaluation, which play an important role in long-term patient treatment outcomes. Combined with the fluorescent labelling technique disclosed herein, the 2D fluorescence labelled cardiac model has several advantages: 1) Easily adaptable to 384-well plate or even smaller microplates. 2) Any type of cardiomyocyte can be used, including non- genetic/genetic modified cells, 3) Both short-term (mins, hrs) and long-term (days, weeks) drug effects can be evaluated.
Pentamidine toxicity effect was evaluated using the 2D fluorescence-labelled cardiomyocyte monolayer. Pentamidine is an antiprotozoal agent, which is used in the treatment of Pneumocystis carinii pneumonia. However, therapy with pentamidine is often accompanied by prolongation of the QT interval. In this study, treatment of the 2D cardiac monolayer with increasing concentrations of pentamidine for 1 hr showed pronounced changes in contraction speed (
Potential applications
This technique describes a novel protocol to fabricate 3D functional cardiac tissues under defined conditions. This approach allows uniform cell inclusion within constructs, creating 3D geometrically controlled microenvironments favourable for direct cell-cell self-organization of appropriate 3D ECM assembly with complex cell-matrix and cell-cell interactions that mimic functional properties of the corresponding tissue. Thus, this approach provides a simple model for recapitulating and better understanding physiologically relevant issues at native heart tissue level.
Some potential biomedical applications are listed as follows:
Cell-Based drug testing and high-throughput screening
Compared to other scaffold- or ECM-based 3D cell models, the present model recapitulates the in vivo cellular environment, better mimicking the native heart tissue architecture. As demonstrated in the experiments, the system allows simultaneous monitoring of cardiac tissue force generation, while reporting rapid changes in tissue contraction in response to drug stimuli. With its highly customizable design and fluorescence labelling method, this platform represents a unique approach to quantify the impact of drug on function of 3D cardiac tissues, thus showing great promise to be used for high-throughput, low-cost screening assays for pharmaceutical drug development.
Cardiovascular disease model
This technique allows for the generation of cardiac tissues from patients in the context of particular genetic identity, including individuals with sporadic forms of disease. These 3D cardiac tissue models will be ideally suited to test disease progression.
Basic study
By tailoring the mechanical modulus of the tissue holders, our approach can provide a simplified model with which to investigate directly the effect of different mechanical stresses on cardiomyocyte alignment and growth, and measuring the functional consequences of these interventions. This could also provide a platform to study the mechanical effect of infarct cardiac muscle on the surrounding healthy contractile tissues.
Cell-Based therapy
The 3D cardiac tissues generated from patient iPSCs under defined xeno-free conditions will have great potential for clinical cell-based therapy.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201503869P | May 2015 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2016/050228 | 5/16/2016 | WO | 00 |
