An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level
November 2nd, 2020
This paper presents methods for growing cardiac myocytes with different shapes, which represent different pathologies, and sorting these adherent cardiac myocytes based on their morphology at a single cell level. The proposed platform provides a novel approach to high throughput and drug screening for different types of heart failure.
We propose a novel platform for investigating the effects of cell shape on gene expression, using methods for growing and sorting adherences with different morphologies at the single cell level. The main advantage of this technique is that it facilitates the high throughput study of cell shape in vitro as comparing cells with different shapes in vivo is technically demanding. To set up a patterned cardiomyocyte culture, transfer a cardiomyocyte suspension at the aspect ratio of interest to a 15 milliliter tube for counting and dilute the cells to a one times 10 to the five cells per milliliter of appropriate plating medium concentration.
Add two milliliters of cells onto a fibronectin coated micropatterned chip submerged in two milliliters of warm plating medium inside a 35 milliliter Grenier Petri dish and place the dish in a 37 degrees Celsius and 5%carbon dioxide incubator for 18 hours. The next day, check the chip by light microscopy to confirm that most of the cells have attached. Remove the medium from the dish to remove any debris and or dead cells, and starting from the center and moving towards the sides in a drop-wise fashion, gently add PBS to the cells.
After the third wash, replace the PBS with four milliliter maintenance medium and return the cells to the cell culture incubator. After 72 hours, gently flush the chip surface two times with two milliliters of warm Dulbecco's PBS per wash as demonstrated and use forceps to immediately transfer the washed chip to a new sterile 35 milliliter Grenier Petri dish. Quickly add 1.5 microliters of vibrant dicycle green diluted 1000 fold in DPBS to the chip.
Fix a chamber over the chip and place the dish onto the dish holder of the cell picker stage. Insert the magnetic cap and locate the crosshair in the live view window. Focus on the crosshair and, in the scanning and sorting window, select the calibration for automated injection and calibrate.
Replace the dish supernatant with 1.5 milliliters of a one-to-one triple E in DPBS solution to loosen the cells from the fibronectin and open the scanning tab. To scan the entire chip, focus on the top left corner of the chip in the field of view and click get current microscope position. Next, focus on the bottom right corner of the chip and click get current microscope position.
In the popup window, click set sharpest plane and click go to the top right and go to the bottom left corner buttons. Then, click finish to start scanning. When the scanning is complete, open the analyzing tab and click show map to select the single cells that pass the study criteria.
Center the glass microcapillary in the middle of the microscope live view and open the pump tab. To create a vacuum, retract the plunger from a 50 milliliter number one syringe four milliliters and open the sorting tab. To allow a single cell to be picked, set the valve two to be opened for 120 milliseconds and valve one to be opened for 20 milliseconds after a time-lapse of 200 milliseconds.
To allow the picked cell to be delivered successfully to its PCR tube containing lysis buffer, set the valve one to be opened for 20 milliseconds and valve two to be opened for 10 milliseconds after a time-lapse of 10 milliseconds. When the valve settings have been adjusted, click compute the path. The software will compute the fastest path from cell to cell for picking up and injecting the selected cells throughout the chip.
Then, focus the microscope on a pattern on the chip surface. Use the joystick to move the microcapillary down carefully so that the sharpest image of the tip of the microcapillary can be obtained without touching the chip surface and click set. A new window will open, showing the microcapillary cross-section.
To have the software record the tip offset of the capillary in the X, Y, and Z coordinates, click on the exact center of the capillary. Then, click start sorting to launch the sorting. In this representative analysis of pre-amplified cDNA from a picked single cell, a clear band in the gel like densitometry plot was observed corresponding to the peak at 1, 852 base pairs in the electropherogram.
The average size of fragments was 1, 588 base pairs with a small number of fragments that were shorter than 300 base pairs, indicating the generation of a good cDNA library. Immunofluorescent staining and analysis can also be performed to evaluate the sarcomere structure within the patterned cardiomyocytes. This platform paves the way for high throughput study and drug screening of different types of heart failure.
Overview
This study introduces a novel platform for growing and sorting cardiac myocytes based on their morphology at the single cell level. This method enables high throughput drug screening for various types of heart failure.
Key Study Components
Area of Science
- Cardiac myocyte culture
- Cell morphology analysis
- Drug screening methodologies
Background
- Cardiac myocytes exhibit diverse shapes that correlate with different pathologies.
- Investigating cell shape effects on gene expression is challenging in vivo.
- High throughput techniques are essential for effective drug screening.
- Current methods lack efficiency in sorting and analyzing single cells based on morphology.
Purpose of Study
- To develop a platform for studying the impact of cell shape on gene expression.
- To facilitate the sorting of cardiac myocytes based on their morphology.
- To enhance drug screening processes for heart failure treatments.
Methods Used
- Cardiomyocyte suspension preparation and dilution.
- Use of fibronectin-coated micropatterned chips for cell culture.
- Light microscopy for cell attachment confirmation.
- Automated scanning and sorting of single cells using a microcapillary system.
Main Results
- Successful attachment and maintenance of patterned cardiomyocytes.
- Generation of a good cDNA library from sorted single cells.
- Immunofluorescent staining revealed sarcomere structures.
- The platform supports high throughput studies and drug screening.
Conclusions
- The developed platform offers a novel approach to studying cardiac myocytes.
- It enables efficient sorting and analysis of cells based on morphology.
- This method can significantly advance research in heart failure therapies.
Frequently Asked Questions
Executive Industry Relevance
This platform enables systematic investigation of how cardiac myocyte morphology influences gene expression, addressing a critical gap in understanding shape-dependent transcriptomic regulation in heart failure. By linking defined geometrical morphotypes to pathological phenotypes such as hypertrophic and dilated cardiomyopathy, it provides a mechanistic bridge between cellular structure and disease-relevant signaling. The approach supports early-stage target validation and phenotypic screening by generating reproducible, morphology-stratified single-cell transcriptomic data for de-risking therapeutic hypotheses.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by correlating specific cardiomyocyte aspect ratios with gene expression profiles linked to pathological remodeling.
- Operational Value: Facilitates biological de-risking through standardized, morphology-controlled single-cell isolation and transcriptomic profiling.
- Predictive Value: Supports portfolio triage by identifying shape-dependent molecular signatures that may predict compound efficacy across heart failure subtypes.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems with precise length:width aspect ratios for downstream compound screening in heart failure models.
- Operational Value: Ensures assay standardization and reproducibility via micropatterned fibronectin chips that enforce consistent cell morphologies across replicates.
- Scalability: Supports high throughput screening through automated single-cell picking and injection into lysis buffer for parallel processing.
Translational & Preclinical Research
- Translational Continuity: Connects discovery-phase morphology-stratified transcriptomics to preclinical validation by modeling HCM and DCM-associated geometries.
- Biomarker Alignment: Enables identification of shape-dependent transcriptomic signatures that may serve as predictive indicators of treatment response.
- Risk-Adjusted Advancement: Informs go/no-go decisions by revealing how cell shape modulates pathway activation in response to pharmacological perturbation.
Pipeline & Workflow Integration
The method integrates into the discovery continuum by enabling hypothesis-driven analysis of cell shape effects prior to lead identification, with outputs informing preclinical model selection and mechanistic de-risking strategies.
- Discovery Biology: Supports mechanistic de-risking by isolating the variable of cell shape to clarify its role in transcriptional regulation under normal and pathological conditions.
- Screening: Delivers assay-ready, morphology-defined cardiomyocyte populations for reliable compound evaluation in heart failure drug discovery.
- Analytics: Generates quantitative single-cell transcriptomic measurements that allow comparison of gene expression across distinct geometrical morphotypes.
- Translational Research: Provides disease-relevant systems modeling concentric and eccentric hypertrophy through controlled aspect ratios, enhancing preclinical continuity.
- Enterprise Reuse: Establishes a reusable platform for iterative screening campaigns across multiple heart failure phenotypes and compound libraries.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing ambiguity in how cellular morphology influences signaling pathways.
- Operational Value: Enhances reproducibility and standardization through micropatterned culture and automated single-cell handling.
- Strategic Value: Improves capital efficiency by enabling early detection of shape-dependent biological responses that may indicate clinical translatability.
- Portfolio Impact: Supports risk-adjusted prioritization of compounds based on their effects across morphology-stratified cardiomyocyte models.
Implementation Considerations
- Requires expertise in cardiac cell culture, micropatterning techniques, and single-cell manipulation systems.
- Dependent on access to fibronectin-coated micropatterned chips, fluorescence microscopy, and automated microcapillary-based cell pickers.
- Necessitates cross-team standardization of aspect ratio definitions and transcriptomic analysis pipelines for consistent interpretation.
- Involves adaptation considerations when extending the platform to other cell types or disease models beyond cardiac myocytes.
- Practical limitations include the need for optimization of cell density and incubation times to ensure uniform attachment across micropatterns.
