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.

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.

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.

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.

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.

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.

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.].

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.

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.

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.

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.

Single Step Double-walled Nanoencapsulation (SSDN).

A quick fabrication method for making double-walled (DW) polymeric nanospheres is presented. The process uses sequential precipitation of two polymers. By choosing an appropriate solvent and non-solvent polymer pair, and engineering two sequential phase inversions which induces first precipitation of the core polymer followed by precipitation of the shell polymer, DW nanospheres can be created instantaneously. A series of DW formulations were prepared with various core and shell polymers, then characterized using laser diffraction particle sizing, scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry (DSC). Atomic force microscopy (AFM) imaging confirmed existence of a single core polymer coated with a second polymer. Insulin (3.3% loading) was used as a model drug to assess its release profile from core (PLGA) and shell (PBMAD) polymers and resulted with a tri-phase release profile in vitro for two months. Current approaches for producing DW nanoparticles (NPs) are limited by the complexity and time involved. Additional issues include aggregation and entrapment of multiple spheres and the undesired formation of heterogeneous coatings. Therefore, the technique presented here is advantageous because it can produce NPs with distinct, core-shell morphologies through a rapid, spontaneous, self-assembly process. This method not only produces DW NPs, but can also be used to encapsulate therapeutic drug. Furthermore, modification of this process to other core and shell polymers is feasible using the general guidelines provided in this paper.

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.

Cell Mimicking Microparticles Influence the Organization, Growth, and Mechanophenotype of Stem Cell Spheroids.

Substrate stiffness is known to alter cell behavior and drive stem cell differentiation, though most research in this area has been restricted to traditional, two-dimensional culture systems rather than more physiologically relevant, three-dimensional (3D) platforms. In this study, we utilized polymer-based, cell mimicking microparticles (CMMPs) to deliver distinct, stable mechanical cues to human adipose derived stem cells in 3D spheroid culture to examine changes in adipogenic differentiation response and mechanophenotype. After 21 days of adipogenic induction, spheroids containing CMMPs (composite spheroids) stiffened in accordance with CMMP elasticity such that spheroids containing the stiffest, ~ 10 kPa, CMMPs were over 27% stiffer than those incorporating the most compliant, ~ 0.25 kPa CMMPs. Adipogenically induced, cell-only spheroids were over 180% larger and 50% more compliant than matched controls. Interestingly, composite spheroids cultured without chemical induction factors dissociated when presented with CMMPs stiffer than ~ 1 kPa, while adipogenic induction factors mitigated this behavior. Gene expression for PPARG and FABP4 were upregulated more than 45-fold in adipogenically induced samples compared to controls but were unaffected by CMMP elasticity, attributed to insufficient cell-CMMP contacts throughout the composite spheroid. In summary, mechanically tuned CMMPs influenced whole-spheroid mechanophenotype and stability but minimally affected differentiation response.

Regenerative Potential and Inflammation-Induced Secretion Profile of Human Adipose-Derived Stromal Vascular Cells Are Influenced by Donor Variability and Prior Breast Cancer Diagnosis.

Adipose tissue contains a heterogeneous population of stromal vascular fraction (SVF) cells that work synergistically with resident cell types to enhance tissue healing. Ease of access and processing paired with therapeutic promise make SVF cells an attractive option for autologous applications in regenerative medicine. However, inherent variability in SVF cell therapeutic potential from one patient to another hinders prognosis determination for any one person. This study investigated the regenerative properties and inflammation responses of thirteen, medically diverse human donors. Using non-expanded primary lipoaspirate samples, SVF cells were assessed for robustness of several parameters integral to tissue regeneration, including yield, viability, self-renewal capacity, proliferation, differentiation potential, and immunomodulatory cytokine secretion. Each parameter was selected either for its role in regenerative potential, defined here as the ability to heal tissues through stem cell repopulation and subsequent multipotent differentiation, or for its potential role in wound healing through trophic immunomodulatory activity. These data were then analyzed for consistent and predictable patterns between and across measurements, while also investigating the influence of the donors' relevant medical histories, particularly if the donor was in remission following breast cancer treatment. Analyses identified positive correlations among the expression of three cytokines: interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1. The expression of these cytokines also positively related to self-renewal capacity. These results are potentially relevant for establishing expectations in both preclinical experiments and targeted clinical treatment strategies that use stem cells from patients with diverse medical histories.

Concise Review: Fabrication, Customization, and Application of Cell Mimicking Microparticles in Stem Cell Science.

Stem and non-stem cell behavior is heavily influenced by the surrounding microenvironment, which includes other cells, matrix, and potentially biomaterials. Researchers have been successful in developing scaffolds and encapsulation techniques to provide stem cells with mechanical, topographical, and chemical cues to selectively direct them toward a desired differentiation pathway. However, most of these systems fail to present truly physiological replications of the in vivo microenvironments that stem cells are typically exposed to in tissues. Thus, cell mimicking microparticles (CMMPs) have been developed to more accurately recapitulate the properties of surrounding cells while still offering ways to tailor what stimuli are presented. This nascent field holds the promise of reducing, or even eliminating, the need for live cells in select, regenerative medicine therapies, and diagnostic applications. Recent, CMMP-based studies show great promise for the technology, yet only reproduce a small subset of cellular characteristics from among those possible: size, morphology, topography, mechanical properties, surface molecules, and tailored chemical release to name the most prominent. This Review summarizes the strengths, weaknesses, and ideal applications of micro/nanoparticle fabrication and customization methods relevant to cell mimicking and provides an outlook on the future of this technology. Moving forward, researchers should seek to combine multiple techniques to yield CMMPs that replicate as many cellular characteristics as possible, with an emphasis on those that most strongly influence the desired therapeutic effects. The level of flexibility in customizing CMMP properties allows them to substitute for cells in a variety of regenerative medicine, drug delivery, and diagnostic systems. Stem Cells Translational Medicine 2018;7:232-240.