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.

Temporal responsiveness of adipose-derived stem/stromal cell immune plasticity.

We determined the role of time in adipose-derived stem/stromal cell (ASC) response to a model inflammatory environment. ASCs and other mesenchymal stem/stromal cells exhibit immune plasticity. We evaluated the persistence of pro- and anti-inflammatory phenotypes for ASCs exposed to a sustained or pulse inflammatory stimulus. Using qPCR, flow cytometry, and immunocytochemistry, we monitored the temporal expression and up-regulation patterns of a pro-inflammatory gene (caspase 1), a pleiotropic gene/protein (interleukin 6, IL-6), and an anti-inflammatory gene/protein (indoleamine 2, 3-dioxygenase, IDO1) after exposing ASCs to the cytokines tumor necrosis factor-α and interferon-γ. In response to sustained cytokine stimulation, we discovered that time played a role in the balance of pro- and anti-inflammatory ASC phenotypes. IL-6 was present at all time points for both cytokine-stimulated and non-stimulated conditions, whereas IDO1 was heterogeneously up-regulated in stimulated conditions at later time points. After a pulse stimulus, ASC immunoresponse remained consistent for 96-168 h. As a final measure of immune plasticity, we cultured cytokine-stimulated ASCs with blood-derived macrophages to observe macrophage polarization. While the presence of ASCs altered macrophage phenotype, there was no dependency on the length of ASC cytokine exposure time.

Single-Cell Microgels for Diagnostics and Therapeutics.

Cell encapsulation within hydrogel droplets is transforming what is feasible in multiple fields of biomedical science such as tissue engineering and regenerative medicine, in vitro modeling, and cell-based therapies. Recent advances have allowed researchers to miniaturize material encapsulation complexes down to single-cell scales, where each complex, termed a single-cell microgel, contains only one cell surrounded by a hydrogel matrix while remaining <100 µm in size. With this achievement, studies requiring single-cell resolution are now possible, similar to those done using liquid droplet encapsulation. Of particular note, applications involving long-term in vitro cultures, modular bioinks, high-throughput screenings, and formation of 3D cellular microenvironments can be tuned independently to suit the needs of individual cells and experimental goals. In this progress report, an overview of established materials and techniques used to fabricate single-cell microgels, as well as insight into potential alternatives is provided. This focused review is concluded by discussing applications that have already benefited from single-cell microgel technologies, as well as prospective applications on the cusp of achieving important new capabilities.

Mass-Added Density Modulation for Sorting Cells Based on Differential Surface Protein Levels.

Cell sorting is a powerful tool in basic research and therapeutic enrichment. However, common cell sorting methods, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) have significant limitations, such as generally low cell yields or restriction to binary separation, respectively. To address these limitations, we developed a two-step cell sorting method called mass-added density centrifugation (MADC) to enable nonbinary separation of large cell numbers based on surface protein levels. In the first MADC step (mass-adding), antibody-directed massive microparticles bind target surface proteins to modulate single-cell density proportionally to target protein level. Second, microparticle-laden cells are subjected to discontinuous density gradient centrifugation, whereby they separate into discrete density bands which can be isolated for downstream use. MADC will prove especially advantageous for obtaining sufficient cell numbers for protein analyses from large source populations, and it is a fast process that can facilitate live cell enrichment for therapies that require tens of millions of cells. Here, we demonstrate MADC's utility for both live and fixed cell sorts of multiple cell types based on abundance of an example target protein, CD44. CD44 quantity in separated cell groups was assayed with western blots and correlated with modulated cell density. This novel sorting method enables rapid, nonbinary isolation of large quantities of cells based on surface protein levels and should prove useful in both basic science and therapeutic applications. © 2020 International Society for Advancement of Cytometry.

Synthesis and Characterization of a Magnetically Active 19F Molecular Beacon.

Gene expression is used extensively to describe cellular characteristics and behaviors; however, most methods of assessing gene expression are unsuitable for living samples, requiring destructive processes such as fixation or lysis. Recently, molecular beacons have become a viable tool for live-cell imaging of mRNA molecules in situ. Historically, beacon-mediated imaging has been limited to fluorescence-based approaches. We propose the design and synthesis of a novel molecular beacon for magnetic resonance detection of any desired target nucleotide sequence. The biologically compatible synthesis incorporates commonly used bioconjugation reactions in aqueous conditions and is accessible for laboratories without extensive synthesis capabilities. The resulting beacon uses fluorine (19F) as a reporter, which is broadened, or turned "off", via paramagnetic relaxation enhancement from a stabilized nitroxide radical spin label when the beacon is not bound to its nucleic acid target. Therefore, the 19F NMR signal of the beacon is quenched in its hairpin conformation when the spin label and the 19F substituent are held in proximity, but the signal is recovered upon beacon hybridization to its specific complementary nucleotide sequence by physical separation of the radical from the 19F reporter. This study establishes a path for magnetic resonance-based assessment of specific mRNA expression, providing new possibilities for applying molecular beacon technology in living systems.

High-Throughput Assessment of Cellular Mechanical Properties.

Traditionally, cell analysis has focused on using molecular biomarkers for basic research, cell preparation, and clinical diagnostics; however, new microtechnologies are enabling evaluation of the mechanical properties of cells at throughputs that make them amenable to widespread use. We review the current understanding of how the mechanical characteristics of cells relate to underlying molecular and architectural changes, describe how these changes evolve with cell-state and disease processes, and propose promising biomedical applications that will be facilitated by the increased throughput of mechanical testing: from diagnosing cancer and monitoring immune states to preparing cells for regenerative medicine. We provide background about techniques that laid the groundwork for the quantitative understanding of cell mechanics and discuss current efforts to develop robust techniques for rapid analysis that aim to implement mechanophenotyping as a routine tool in biomedicine. Looking forward, we describe additional milestones that will facilitate broad adoption, as well as new directions not only in mechanically assessing cells but also in perturbing them to passively engineer cell state.

Adipose-derived stem cells retain their regenerative potential after methotrexate treatment.

In musculoskeletal tissues like bone, chemotherapy can impair progenitor cell differentiation and proliferation, resulting in decreased bone growth and mineralization throughout a patient׳s lifetime. In the current study, we investigated the effects of chemotherapeutics on adipose-derived stem cell (ASC) function to determine whether this cell source could be a candidate for repairing, or even preventing, chemotherapy-induced tissue damage. Dose-dependent proliferation rates of ASCs and normal human fibroblasts (NHFs) were quantified after treatment with cytarabine (CY), etoposide (ETO), methotrexate (MTX), and vincristine (VIN) using a fluorescence-based assay. The influence of MTX on the multipotency of ASCs and freshly isolated stromal vascular fraction (SVF) cells was also evaluated using lineage-specific stains and spectrophotometry. ASC and NHF proliferation were equally inhibited by exposure to CY and ETO; however, when treated with MTX and VIN, ASCs exhibited greater resistance. This was especially apparent for MTX-treated samples, with ASC proliferation showing no inhibition for clinically relevant MTX doses ranging from 0.1 to 50 µM. Additional experiments revealed that the differentiation potential of ASCs was not affected by MTX treatment and that upregulation of dihydrofolate reductase possibly contributed to this response. Moreover, SVF cells, which include ASCs, exhibited similar resistance to MTX impairment, with respect to cellular proliferation, clonogenicity, and differentiation capability. Therefore, we have shown that the regenerative properties of ASCs resist the cytotoxicity of MTX, identifying these cells as a potential key for repairing musculoskeletal damage in patients undergoing chemotherapy.

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.

3D Viscoelastic traction force microscopy.

Native cell-material interactions occur on materials differing in their structural composition, chemistry, and physical compliance. While the last two decades have shown the importance of traction forces during cell-material interactions, they have been almost exclusively presented on purely elastic in vitro materials. Yet, most bodily tissue materials exhibit some level of viscoelasticity, which could play an important role in how cells sense and transduce tractions. To expand the realm of cell traction measurements and to encompass all materials from elastic to viscoelastic, this paper presents a general, and comprehensive approach for quantifying 3D cell tractions in viscoelastic materials. This methodology includes the experimental characterization of the time-dependent material properties for any viscoelastic material with the subsequent mathematical implementation of the determined material model into a 3D traction force microscopy (3D TFM) framework. Utilizing this new 3D viscoelastic TFM (3D VTFM) approach, we quantify the influence of viscosity on the overall material traction calculations and quantify the error associated with omitting time-dependent material effects, as is the case for all other TFM formulations. We anticipate that the 3D VTFM technique will open up new avenues of cell-material investigations on even more physiologically relevant time-dependent materials including collagen and fibrin gels.

Microparticles with tunable, cell-like properties for quantitative acoustic mechanophenotyping.

Mechanical properties of biological cells have been shown to correlate with their biomolecular state and function, and therefore methods to measure these properties at scale are of interest. Emerging microfluidic technologies can measure the mechanical properties of cells at rates over 20,000 cells/s, which is more than four orders of magnitude faster than conventional instrumentation. However, precise and repeatable means to calibrate and test these new tools remain lacking, since cells themselves are by nature variable. Commonly, microfluidic tools use rigid polymer microspheres for calibration because they are widely available in cell-similar sizes, but conventional microspheres do not fully capture the physiological range of other mechanical properties that are equally important to device function (e.g., elastic modulus and density). Here, we present for the first time development of monodisperse polyacrylamide microparticles with both tunable elasticity and tunable density. Using these size, elasticity, and density tunable particles, we characterized a custom acoustic microfluidic device that makes single-cell measurements of mechanical properties. We then applied the approach to measure the distribution of the acoustic properties within samples of human leukocytes and showed that the system successfully discriminates lymphocytes from other leukocytes. This initial demonstration shows how the tunable microparticles with properties within the physiologically relevant range can be used in conjunction with microfluidic devices for efficient high-throughput measurements of mechanical properties at single-cell resolution.

Chondrogenesis of Adipose-Derived Stem Cells Using an Arrayed Spheroid Format.

Objective: The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling these systems up for high-throughput testing is technically challenging and wasteful given the necessary cell numbers and reagent volumes. However, adopting microscale spheroid cultures for this purpose shows promise. Spheroid systems work with only thousands of cells and microliters of medium. Methods: Molded agarose microwells were fabricated using 2% w/v molten agarose and then equilibrated in medium prior to introducing cells. ASCs were seeded at 50, 500, 5k cells/microwell; 5k, 50k, cells/well plate; and 50k and 250k cells/15 mL centrifuge tube to compare chondrogenic responses across spheroid and micromass sizes. Cells were cultured in control or chondrogenic induction media. ASCs coalesced into spheroids/pellets and were cultured at 37 °C and 5% CO2 for 21 days with media changes every other day. Results: All culture conditions supported growth of ASCs and formation of viable cell spheroids/micromasses. More robust growth was observed in chondrogenic conditions. Sulfated glycosaminoglycans and collagen II, molecules characteristics of chondrogenesis, were prevalent in both 5000-cell spheroids and 250,000-cell micromasses. Deposition of collagen I, characteristic of fibrocartilage, was more prevalent in the large micromasses than small spheroids. Conclusions: Chondrogenic differentiation was consistently induced using high-throughput spheroid formats, particularly when seeding at cell densities of 5000 cells/spheroid. This opens possibilities for highly arrayed experiments investigating tissue repair and remodeling during or after exposure to drugs, toxins, or other chemicals. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00746-8.

Force sensors for measuring microenvironmental forces during mesenchymal condensation.

Mechanical forces are an essential element to early tissue formation. However, few techniques exist that can quantify the mechanical microenvironment present within cell-dense neotissues and organoid structures. Here is a versatile approach to measure microscale, cellular forces during mesenchymal condensation using specially tailored, hyper-compliant microparticles (HCMPs). Through monitoring of HCMP deformation over both space and time, measurements of the mechanical forces that cells exert, and have exerted on them, during tissue formation are acquired. The current study uses this technology to track changes in the mechanical microenvironment as mesenchymal stem cells self-assemble into spheroids and condense into cohesive units. An array analysis approach, using a high-content imaging system, shows that cells exert a wide range of tensile and compressive forces during the first few hours of self-assembly, followed by a period of relative equilibrium. Cellular interactions with HCMPs are further examined by applying collagen coating, which allows for increased tensile forces to be exerted compared to non-coated HCMPs. Importantly, the hyper-compliant nature of our force sensors allows for increased precision over less compliant versions of the same particle. This sensitivity resolves small changes in the microenvironment even at the earliest stages of development and morphogenesis. The overall experimental platform provides a versatile means for measuring direct and indirect spatiotemporal forces in cell-dense biological systems.

Quantification of Antibody Persistence for Cell Surface Protein Labeling.

INTRODUCTION: Antibodies are an essential research tool for labeling surface proteins but can potentially influence the behavior of proteins and cells to which they bind. Because of this, researchers and clinicians are interested in the persistence of these antibodies, particularly for live-cell applications. We developed an easily adoptable method for researchers to characterize antibody removal timelines for any cell-antibody combination, with the benefit of studying broad, hypothesized mechanisms of antibody removal. METHODS: We developed a method using four experimental conditions to elucidate the contributions of possible factors influencing antibody removal: cell proliferation, internalization, permanent dissociation, and environmental perturbation. This method was tested on adipose-derived stem cells and a human lung fibroblast cell line with anti-CD44, CD90, and CD105 antibodies. The persistence of the primary antibody was probed using a fluorescent secondary antibody daily over 10 days. Relative contributions by the antibody removal mechanisms were quantified based on differences between the four culture conditions. RESULTS: Greater than 90% of each antibody tested was no longer present on the surface of the two cell types after 5 days, with removal observed in as little as 1 day post-labeling. Anti-CD90 antibody was primarily removed by environmental perturbation, anti-CD105 antibody by internalization, and anti-CD44 antibody by a combination of all four factors. CONCLUSIONS: Antibody removal mechanism depended on the specific antibody tested, while removal timelines for the same antibody depended more on cell type. This method should be broadly relevant to researchers interested in quantifying an initial timeframe for uninhibited use of antibody-labeled cells. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00670-3.

Spatial mapping of the biomechanical properties of the pericellular matrix of articular cartilage measured in situ via atomic force microscopy.

In articular cartilage, chondrocytes are surrounded by a narrow region called the pericellular matrix (PCM), which is biochemically, structurally, and mechanically distinct from the bulk extracellular matrix (ECM). Although multiple techniques have been used to measure the mechanical properties of the PCM using isolated chondrons (the PCM with enclosed cells), few studies have measured the biomechanical properties of the PCM in situ. The objective of this study was to quantify the in situ mechanical properties of the PCM and ECM of human, porcine, and murine articular cartilage using atomic force microscopy (AFM). Microscale elastic moduli were quantitatively measured for a region of interest using stiffness mapping, or force-volume mapping, via AFM. This technique was first validated by means of elastomeric models (polyacrylamide or polydimethylsiloxane) of a soft inclusion surrounded by a stiff medium. The elastic properties of the PCM were evaluated for regions surrounding cell voids in the middle/deep zone of sectioned articular cartilage samples. ECM elastic properties were evaluated in regions visually devoid of PCM. Stiffness mapping successfully depicted the spatial arrangement of moduli in both model and cartilage surfaces. The modulus of the PCM was significantly lower than that of the ECM in human, porcine, and murine articular cartilage, with a ratio of PCM to ECM properties of approximately 0.35 for all species. These findings are consistent with previous studies of mechanically isolated chondrons, and suggest that stiffness mapping via AFM can provide a means of determining microscale inhomogeneities in the mechanical properties of articular cartilage in situ.

Generating Cell Type-Specific Protein Signatures from Non-symptomatic and Diseased Tissues.

Here we demonstrate a technique to generate proteomic signatures of specific cell types within heterogeneous populations. While our method is broadly applicable across biological systems, we have limited the current work to study neural cell types isolated from human, post-mortem Alzheimer's disease (AD) and aged-matched non-symptomatic (NS) brains. Motivating the need for this tool, we conducted an initial meta-analysis of current, human AD proteomics studies. While the results broadly corroborated major neurodegenerative disease hypotheses, cell type-specific predictions were limited. By adapting our Formaldehyde-fixed Intracellular Target-Sorted Antigen Retrieval (FITSAR) method for proteomics and applying this technique to characterize AD and NS brains, we generated enriched neuron and astrocyte proteomic profiles for a sample set of donors (available at www.fitsarpro.appspot.com ). Results showed the feasibility for using FITSAR to evaluate cell-type specific hypotheses. Our overall methodological approach provides an accessible platform to determine protein presence in specific cell types and emphasizes the need for protein-compatible techniques to resolve systems complicated by cellular heterogeneity.

Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting.

Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications. Despite this, throughputs, yields, viabilities, and processing times of common sorting methods like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are underreported. In the current study, we set out to quantify the ability of these sorting methods to separate defined mixtures of alkaline phosphatase liver/bone/kidney (ALPL)-expressing and non-expressing cell types. Results showed that initial MACS runs performed using manufacturer's recommended antibody and microbead concentrations produced inaccurate ALPL+ vs. ALPL- cell splits compared to FACS when ALPL+ cells were present in larger proportions (>~25%). Accuracy at all proportions could be achieved by using substantially higher concentrations of labeling reagents. Importantly, MACS sorts resulted in only 7-9% cell loss compared to ~70% cell loss for FACS. Additionally, MACS processing was 4-6 times faster than FACS for single, low proportion samples but took similar time for single, high-proportion samples. When processing multiple samples, MACS was always faster overall due to its ability to run samples in parallel. Average cell viability for all groups remained high (>83%), regardless of sorting method. Despite requiring substantial optimization, the ability of MACS to isolate increased cell numbers in less time than FACS may prove valuable in both basic science and translational, cell-based applications.

Integration of hyper-compliant microparticles into a 3D melanoma tumor model.

Multicellular spheroids provide a physiologically relevant platform to study the microenvironment of tumors and therapeutic applications, such as microparticle-based drug delivery. The goal of this study was to investigate the incorporation/penetration of compliant polyacrylamide microparticles (MPs), into either cancer or normal human cell spheroids. Incorporation of collagen-1-coated MPs (stiffness: 0.1 and 9 kPa; diameter: 15-30 µm) into spheroids (diameter ∼100 µm) was tracked for up to 22 h. Results indicated that cells within melanoma spheroids were more influenced by MP mechanical properties than cells within normal cell spheroids. Melanoma spheroids had a greater propensity to incorporate and displace the more compliant MPs over time. Mature spheroids composed of either cell type were able to recognize and integrate MPs. While many tumor models exist to study drug delivery and efficacy, the study of uptake and incorporation of cell-sized MPs into established spheroids/tissues or tumors has been limited. The ability of hyper-compliant MPs to successfully penetrate 3D tumor models with natural extracellular matrix deposition provides a novel platform for potential delivery of drugs and other therapeutics into the core of tumors and micrometastases.

Correction to: Translating Mechanobiology to the Clinic: A Panel Discussion from the 2018 CMBE Conference.

[This corrects the article DOI: 10.1007/s12195-018-0556-5.].

Shape-Preserved Transformation of Biological Cells into Synthetic Hydrogel Microparticles.

The synthesis of materials that can mimic the mechanical, and ultimately functional, properties of biological cells can broadly impact the development of biomimetic materials, as well as engineered tissues and therapeutics. Yet, it is challenging to synthesize, for example, microparticles that share both the anisotropic shapes and the elastic properties of living cells. Here, a cell-directed route to replicate cellular structures into synthetic hydrogels such as polyethylene glycol (PEG) is described. First, the internal and external surfaces of chemically fixed cells are replicated in a conformal layer of silica using a sol-gel process. The template is subsequently removed to render shape-preserved, mesoporous silica replicas. Infiltration and cross-linking of PEG precursors and dissolution of the silica result in a soft hydrogel replica of the cellular template as demonstrated using erythrocytes, HeLa, and neuronal cultured cells. The elastic modulus can be tuned over an order of magnitude (≈10-100 kPa) though with a high degree of variability. Furthermore, synthesis without removing the biotemplate results in stimuli-responsive particles that swell/deswell in response to environmental cues. Overall, this work provides a foundation to develop soft particles with nearly limitless architectural complexity derived from dynamic biological templates.

Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes.

The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or "biomarkers," of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations.