Topological defects in self-assembled patterns of mesenchymal stromal cells in vitro are predictive attributes of condensation and chondrogenesis.

Mesenchymal stromal cells (MSCs) are promising therapeutic agents for cartilage regeneration, including the potential of cells to promote chondrogenesis in vivo. However, process development and regulatory approval of MSCs as cell therapy products benefit from facile in vitro approaches that can predict potency for a given production run. Current standard in vitro approaches include a 21 day 3D differentiation assay followed by quantification of cartilage matrix proteins. We propose a novel biophysical marker that is cell population-based and can be measured from in vitro monolayer culture of MSCs. We hypothesized that the self-assembly pattern that emerges from collective-cell behavior would predict chondrogenesis motivated by our observation that certain features in this pattern, namely, topological defects, corresponded to mesenchymal condensations. Indeed, we observed a strong predictive correlation between the degree-of-order of the pattern at day 9 of the monolayer culture and chondrogenic potential later estimated from in vitro 3D chondrogenic differentiation at day 21. These findings provide the rationale and the proof-of-concept for using self-assembly patterns to monitor chondrogenic commitment of cell populations. Such correlations across multiple MSC donors and production batches suggest that self-assembly patterns can be used as a candidate biophysical attribute to predict quality and efficacy for MSCs employed therapeutically for cartilage regeneration.

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells.

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.

Single-cell assays using integrated continuous-flow microfluidics.

The recent maturation of continuous-flow microfluidic technologies has coincided with transformative new methods to profile single cells, including their genetic types, protein expression and enzyme activities. Continuous-flow high-throughput single-cell screening and sorting can reveal relationships across cellular phenotypes (e.g., enzyme activity and secretion) and genetic fingerprints. This technology provides unique opportunities, as well as experimental and computational challenges, for integrative approaches that can process large amounts of single-cell data. In this chapter, we discuss recent advances in integrated continuous-flow microfluidic approaches with a focus on measurements and statistical analysis of single-cell enzyme activity and their applications in quantitative biology, synthetic biology, and diagnosis.

Ultrafast Single-Cell Level Enzymatic Tumor Profiling.

In the context of tumor analysis, the implementation of precision medicine requires on-time clinical measurements, which requires rapid large-scale single-cell screening that obtains cell population distributions and functions in tumors to determine disease progression for therapeutics. In this study, a high-throughput screening (HTS) platform integrating optical fluorescence detectors and a computational method was developed as a droplet-based microfluidic flow cytometer (Droplet-µFC) to comprehensively analyze multiple proteolytic activities of a patient-derived tumor (with ∼0.5-2 M cells) at single-cell resolution within 2 h. The data-driven analytical method identified distinct cell types and status through protease profiling with high precision. Multiple protease activities of single cells harvested from a tumor were thus determined with a throughput of ∼100 cells per second. This platform was used to screen protease activities of a wide range of cell types, forming a library. With the development of advanced computational clustering and cell mapping, rapid quantitative tumor profiling with a comprehensive description of cell population distributions and functions could be obtained for clinical treatments.

Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology.

Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 µL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.