Tumor stiffness has been associated with malignancy and increased risk for metastasis. Extensive research has been done investigating breast cancer cell lines’ responsiveness to surfaces of varying rigidities as well as examining the biophysical properties of breast cancer tumor samples. However, there is a critical gap regarding the relationship between cells’ mechanosensitivity in conjunction to biophysical properties of their extracellular matrix environment. To explore this relationship, we will analyze single-cell mechanosensitivity in comparison to tumor rigidity via shearwave ultrasound elastogrophy (SWE). Given the putative affiliation, we hypothesize that cells expressing invasive mechanosensitivity profiles will correlate with stiffer tumor regions. Using collagen gels containing different cell types, we derived biopsy-sized samples allowing us to optimize single-cell mechanosensitivity analysis. Cells were stained using different dyes corresponding to invasiveness. Subsequently, we analyzed their morphology. Morphological identification within organoid environments would allow for single-cell analysis without the aggression of tissue digestion, though preliminary results suggest high heterogeneity may not allow for confident cell identification solely on morphology. Thus, inquisition into cell viability and integrity was explored by analyzing the effects of tissue digestion with HyQtase on single-cells. Cell count and live-dead stain via flow cytometry allowed for analysis of single-cell viability. Lastly, cell integrity was evaluated by a 2D adhesion assay of isolated cells. The live/dead stain revealed that digestion resulted in isolation of approximately 10% of the original 500,000 cell population with 90–97% of the isolated population being live-cells (invasive and non-invasive respectively). Furthermore, the adhesion assay showed that these isolated single cells retained the ability to adhere to new surfaces, with no difference between the invasive and non-invasive cell types. These results show that cells are able to retain mechanosensitive properties following enzymatic digestion. However, they also suggest our digestion procedure is not aggressive enough to isolate invasive subpopulations that are more strongly imbedded in the original tissues. Development of these novel techniques will allow for accurate and confident analysis of precious human biopsy samples. Insight into the relationship between single-cell mechanosensitivity and tumor biophysical properties could elucidate pathways for metastasis inhibition and prevention.
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A Model for Studying the Biomechanical Effects of Varying Ratios of Collagen Types I and III on Cardiomyocytes
Purpose
To develop a novel model composed solely of Col I and Col III with the lower and upper limits set to include the ratios of Col I and Col III at 3:1 and 9:1 in which the structural and mechanical behavior of the resident CM can be studied. Further, the progression of fibrosis due to change in ratios of Col I:Col III was tested.
Methods
Collagen gels with varying Col I:Col III ratios to represent a healthy (3:1) and diseased myocardial tissue were prepared by manually casting them in wells. Absorbance assay was performed to confirm the gelation of the gels. Rheometric analysis was performed on each of the collagen gels prepared to determine the varying stiffnesses and rheological parameters of the gels made with varying ratios of Col I:Col III. Second Harmonic Generation (SHG) was performed to observe the 3D characterization of the collagen samples. Scanning Electron microscopy was used for acquiring cross sectional images of the lyophilized collagen gels. AC16 CM (human) cell lines were cultured in the prepared gels to study cell morphology and behavior as a result of the varying collagen ratios. Cellular proliferation was studied by performing a Cell Trace Violet Assay and the applied force on each cell was measured by means of Finite Element Analysis (FEA) on CM from each sample.
Results
Second harmonic generation microscopy used to image Col I, displayed a decrease in acquired image intensity with an increase in the non-second harmonic Col III in 3:1 gels. SEM showed a fiber-rich structure in the 3:1 gels with well-distributed pores unlike the 9:1 gels or the 1:0 controls. Rheological analysis showed a decrease in substrate stiffness with an increase of Col III, in comparison with other cases. CM cultured within 3:1 gels exhibited an elongated rod-like morphology with an average end-to-end length of 86 ± 28.8 µm characteristic of healthy CM, accompanied by higher cell growth in comparison with other cases. Finite element analysis used to estimate the forces exerted on CM cultured in the 3:1 gels, showed that the forces were well dispersed, and not concentrated within the center of cells, in comparison with other cases.
Conclusion
This study model can be adopted to simulate various biomechanical environments in which cells crosstalk with the Collagen-matrix in diseased pathologies to generate insights on strategies for prevention of fibrosis.
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- NSF-PAR ID:
- 10276593
- Date Published:
- Journal Name:
- Cardiovascular engineering and technology
- Volume:
- 12
- ISSN:
- 1869-4098
- Page Range / eLocation ID:
- 311-24
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Results Our assay was able to support angiogenesis in a comparable manner to Matrigel in soft 2 kPa gels while enabling tunability to study the effects of stiffness on sprout formation. Matrigel and 2 kPa groups contained significantly more samples with sprouts when compared to the stiffer 10 and 20 kPa gels. Growth factor treatment did not have as obvious an effect, although the 20 kPa PDGF + FGF‐treated group had significantly longer vessels than the vascular endothelial growth factor‐treated group.
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null (Ed.)Hydrogels are a class of biomaterials used for a wide range of biomedical applications, including as a three-dimensional (3D) scaffold for cell culture that mimics the extracellular matrix (ECM) of native tissues. To understand the role of the ECM in the modulation of cardiac cell function, alginate was used to fabricate crosslinked gels with stiffness values that resembled embryonic (2.66 ± 0.84 kPa), physiologic (8.98 ± 1.29 kPa) and fibrotic (18.27 ± 3.17 kPa) cardiac tissues. The average pore diameter and hydrogel swelling were seen to decrease with increasing substrate stiffness. Cardiomyocytes cultured within soft embryonic gels demonstrated enhanced cell spreading, elongation, and network formation, while a progressive increase in gel stiffness diminished these behaviors. Cell viability decreased with increasing hydrogel stiffness. Furthermore, cells in fibrotic gels showed enhanced protein expression of the characteristic cardiac stress biomarker, Troponin-I, while reduced protein expression of the cardiac gap junction protein, Connexin-43, in comparison to cells within embryonic gels. The results from this study demonstrate the role that 3D substrate stiffness has on cardiac tissue formation and its implications in the development of complex matrix remodeling-based conditions, such as myocardial fibrosis.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/https://doi.org/10.1007/s13239-020-00514-7
