A mechanics theory for the exploration of a high-throughput, sterile 3D in vitro traumatic brain injury model.

Brain injuries resulting from mechanical trauma represent an ongoing global public health issue. Several in vitro and in vivo models for traumatic brain injury (TBI) continue to be developed for delineating the various complex pathophysiological processes involved in its onset and progression. Developing an in vitro TBI model that is based on cortical spheroids is especially of great interest currently because they can replicate key aspects of in vivo brain tissue, including its electrophysiology, physicochemical microenvironment, and extracellular matrix composition. Being able to mechanically deform the spheroids are a key requirement in any effective in vitro TBI model. The spheroids' shape and size, however, make mechanically loading them, especially in a high-throughput, sterile, and reproducible manner, quite challenging. To address this challenge, we present an idea for a spheroid-based, in vitro TBI model in which the spheroids are mechanically loaded by being spun by a centrifuge. (An experimental demonstration of this new idea will be published shortly elsewhere.) An issue that can limit its utility and scope is that imaging techniques used in 2D and 3D in vitro TBI models cannot be readily applied in it to determine spheroid strains. In order to address this issue, we developed a continuum mechanics-based theory to estimate the spheroids' strains when they are being spun at a constant angular velocity. The mechanics theory, while applicable here to a special case of the centrifuge-based TBI model, is also of general value since it can help with the further exploration and development of TBI models.

Discovery of surface biomarkers for cell mechanophenotype via an intracellular protein-based enrichment strategy.

Cellular mechanophenotype is often a defining characteristic of conditions like cancer malignancy/metastasis, cardiovascular disease, lung and liver fibrosis, and stem cell differentiation. However, acquiring living cells based on mechanophenotype is challenging for conventional cell sorters due to a lack of biomarkers. In this study, we demonstrate a workflow for surface protein discovery associated with cellular mechanophenotype. We sorted heterogeneous adipose-derived stem/stromal cells (ASCs) into groups with low vs. high lamin A/C, an intracellular protein linked to whole-cell mechanophenotype. Proteomic data of enriched groups identified surface protein candidates as potential biochemical proxies for ASC mechanophenotype. Select surface biomarkers were used for live-cell enrichment, with subsequent single-cell mechanical testing and lineage-specific differentiation. Ultimately, we identified CD44 to have a strong inverse correlation with whole-cell elastic modulus, with CD44lo cells exhibiting moduli three times greater than that of CD44hi cells. Functionally, these stiff and soft ASCs showed enhanced osteogenic and adipogenic differentiation potential, respectively. The described workflow can be replicated for any phenotype with a known correlated intracellular protein, allowing for the acquisition of live cells for further characterization, diagnostics, or therapeutics.

The Emerging Role of Lamin C as an Important LMNA Isoform in Mechanophenotype.

Lamin A and lamin C isoforms of the gene LMNA are major structural and mechanotransductive components of the nuclear lamina. Previous reports have proposed lamin A as the isoform with the most dominant contributions to cellular mechanophenotype. Recently, expression of lamin C has also been shown to strongly correlate to cellular elastic and viscoelastic properties. Nevertheless, LMNA isoforms exist as part of a network that collectively provides structural integrity to the nucleus and their expression is ultimately regulated in a cell-specific manner. Thus, they have importance in mechanotransduction and structural integrity of the nucleus as well as potential candidates for biomarkers of whole-cell mechanophenotype. Therefore, a fuller discussion of lamin isoforms as mechanophenotypic biomarkers should compare both individual and ratiometric isoform contributions toward whole-cell mechanophenotype across different cell types. In this perspective, we discuss the distinctions between the mechanophenotypic correlations of individual and ratiometric lamins A:B1, C:B1, (A + C):B1, and C:A across cells from different lineages, demonstrating that the collective contribution of ratiometric lamin (A + C):B1 isoforms exhibited the strongest correlation to whole-cell stiffness. Additionally, we highlight the potential roles of lamin isoform ratios as indicators of mechanophenotypic change in differentiation and disease to demonstrate that the contributions of individual and collective lamin isoforms can occur as both static and dynamic biomarkers of mechanophenotype.

Nuclear Lamin Protein C Is Linked to Lineage-Specific, Whole-Cell Mechanical Properties.

INTRODUCTION: Lamin proteins confer nuclear integrity and relay external mechanical cues that drive changes in gene expression. However, the influence these lamins have on whole-cell mechanical properties is unknown. We hypothesized that protein expression of lamins A, B1, and C would depend on the integrity of the actin cytoskeleton and correlate with cellular elasticity and viscoelasticity. METHODS: To test these hypotheses, we examined the protein expression of lamins A, B1, and C across five different cell lines with varied mechanical properties. Additionally, we treated representative "soft/stiff" cell types with cytochalasin D and LMNA siRNA to determine the effect of a more compliant whole-cell phenotype on lamin A, B1 and C protein expression. RESULTS: A positive, linear correlation existed between lamin C protein expression and average cell moduli/apparent viscosity. Though moderate correlations existed between lamin A/B1 protein expression and whole-cell mechanical properties, they were statistically insignificant. Inhibition of actin polymerization, via cytochalasin D treatment, resulted in reduced cell elasticity, viscoelasticity, and lamin A and C protein expression in "stiff" MG-63 cells. In "soft" HEK-293T cells, this treatment reduced cell elasticity and viscoelasticity but did not affect lamin B1 or C protein expression. Additionally, LMNA siRNA treatment of MG-63 cells decreased whole-cell elasticity and viscoelasticity. CONCLUSION: These findings suggest that lamin C protein expression is strongly associated with whole-cell mechanical properties and could potentially serve as a biomarker for mechanophenotype.

Adipose-derived stem cell fate is predicted by cellular mechanical properties.

Adipose-derived stem cells (ASCs) show great promise for tissue engineering applications and cell-based therapies because of their multipotency, relative abundance and immunosuppressive properties. However, ASCs must be isolated from heterogeneous cell populations present in adipose tissue. In this brief report, we provide a concise summary of the history and use of cellular mechanical properties as novel, label-free biomarkers to predict the differentiation potential of ASCs toward adipogenic, osteogenic and chondrogenic lineages. Additionally, we have found that passage number influences the mechanical properties of ASCs along with a discussion of potential environmental factors that could affect these properties. Altogether, this report provides evidence for the reliability of cellular mechanical properties as biomarkers for ASC differentiation potential and outlines how they can be used to sort ASCs with lineage-specific preferences for particular applications.

Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells.

The mechanical properties of adipose-derived stem cell (ASC) clones correlate with their ability to produce tissue-specific metabolites, a finding that has dramatic implications for cell-based regenerative therapies. Autologous ASCs are an attractive cell source due to their immunogenicity and multipotent characteristics. However, for practical applications ASCs must first be purified from other cell types, a critical step which has proven difficult using surface-marker approaches. Alternative enrichment strategies identifying broad categories of tissue-specific cells are necessary for translational applications. One possibility developed in our lab uses single-cell mechanical properties as predictive biomarkers of ASC clonal differentiation capability. Elastic and viscoelastic properties of undifferentiated ASCs were tested via atomic force microscopy and correlated with lineage-specific metabolite production. Cell sorting simulations based on these "mechanical biomarkers" indicated they were predictive of differentiation capability and could be used to enrich for tissue-specific cells, which if implemented could dramatically improve the quality of regenerated tissues.