Poly(ethylene glycol)-Based Hydrogel Microcarriers Alter Secretory Activity of Genetically Modified Mesenchymal Stromal Cells.

In order to scale up culture therapeutic cells, such as mesenchymal stromal cells (MSCs), culture in suspension bioreactors using microcarriers (µCs) is preferred. However, the impact of microcarrier type on the resulting MSC secretory activity has not been investigated. In this study, two poly(ethylene glycol) hydrogel formulations with different swelling ratios (named "stiffer" and "softer") were fabricated as µC substrates to culture MSCs and MSCs genetically modified to express the interleukin-1 receptor antagonist (IL-1Ra-MSCs). Changes in cell number, secretory and angiogenic activity, and changes in MAPK signaling were evaluated when cultured on hydrogel µCs, as well as on tissue culture plastic-based Synthemax µCs. We demonstrated that culture on stiffer µCs increased secretion of IL-1Ra compared to culture on Synthemax µCs by IL-1Ra-MSCs by 1.2- to 1.6-fold, as well as their in vitro angiogenic activity, compared to culture on Synthemax µCs, while culture on both stiffer and softer µCs altered the secretion of several other factors compared to culture on Synthemax µCs. Changes in angiogenic activity corresponded with increased gene expression and secretion of hepatocyte growth factor by MSCs cultured on softer µCs by 2.5- to 6-fold compared to MSCs cultured on Synthemax µCs. Quantification of phosphoprotein signaling with the MAPK pathway revealed broad reduction of pathway activation by IL-1Ra-MSCs cultured on both stiffer and softer µCs compared to Synthemax, where phosphorylated c-Jun, ATF2, and MEK1 were reduced specifically on softer µCs. Overall, this study showed that µC surfaces can influence the secretory activity of genetically modified MSCs and identified associated changes in MAPK pathway signaling, which is a known central regulator of cytokine secretion.

Development and characterization of Factor Xa-responsive materials for applications in cell culture and biologics delivery.

Stimuli-responsive biomaterials may be used to better control the release of bioactive molecules or cells for applications involving drug delivery and controlled cell release. In this study, we developed a Factor Xa (FXa)-responsive biomaterial capable of controlled release of pharmaceutical agents and cells from in vitro culture. FXa-cleavable substrates were formed as hydrogels that degraded in response to FXa enzyme over several hours. Hydrogels were shown to release both heparin and a model protein in response to FXa. Additionally, RGD-functionalized FXa-degradable hydrogels were used to culture mesenchymal stromal cells (MSCs), enabling FXa-mediated cell dissociation from hydrogels in a manner that preserved multicellular structures. Harvesting MSCs using FXa-mediated dissociation did not influence their differentiation capacity or indoleamine 2,3-dioxygenase (IDO) activity (a measure of immunomodulatory capacity). In all, this FXa-degradable hydrogel is a novel responsive biomaterial system that may be used for on-demand drug delivery, as well as for improving processes for in vitro culture of therapeutic cells.

Culture Substrates for Improved Manufacture of Mesenchymal Stromal Cell Therapies.

Recent developments in mesenchymal stromal cell (MSC) therapies have increased the demand for tools to improve their manufacture, including the selection of optimal culture substrate materials. While many clinical manufacturers use planar tissue culture plastic (TCP) surfaces for MSC production, others have begun exploring the use of alternative culture substrates that present a variety of spatial, mechanical, and biochemical cues that influence cell expansion and resulting cell quality. In this review, the effects of culture and material properties distinct from traditional planar TCP surfaces on MSC proliferation, surface marker expression, and commonly used indications for therapeutic potency are examined. The different properties summarized include the use of alternative culture formats such as cellular aggregates or 3D scaffolds, as well as the effects of culture substrate stiffness and presentation of specific adhesive ligands and topographical cues. Specific substrate properties can be related to greater cell expansion and improvement in specific therapeutic functionalities, demonstrating the utility of culture materials in further improving the clinical-scale manufacture of highly secretory MSC products.

Localized Sampling Enables Monitoring of Cell State via Inline Electrospray Ionization Mass Spectrometry.

Nascent advanced therapies, including regenerative medicine and cell and gene therapies, rely on the production of cells in bioreactors that are highly heterogeneous in both space and time. Unfortunately, advanced therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. This can be attributed largely to a void in existing process analytical technologies (PATs) capable of characterizing the secreted critical quality attribute (CQA) biomolecules that correlate with the final product quality. The Dynamic Sampling Platform (DSP) is a PAT for cell bioreactor monitoring that can be coupled to a suite of sensor techniques to provide real-time feedback on spatial and temporal CQA content in situ. In this study, DSP is coupled with electrospray ionization mass spectrometry and direct-from-culture sampling to obtain measures of CQA content in bulk media and the cell microenvironment throughout the entire cell culture process (≈3 weeks). Post hoc analysis of this real-time data reveals that sampling from the microenvironment enables cell state monitoring (e.g., confluence, differentiation). These results demonstrate that an effective PAT should incorporate both spatial and temporal resolution to serve as an effective input for feedback control in biomanufacturing.

Sub-nanoliter metabolomics via mass spectrometry to characterize volume-limited samples.

The human metabolome provides a window into the mechanisms and biomarkers of various diseases. However, because of limited availability, many sample types are still difficult to study by metabolomic analyses. Here, we present a mass spectrometry (MS)-based metabolomics strategy that only consumes sub-nanoliter sample volumes. The approach consists of combining a customized metabolomics workflow with a pulsed MS ion generation method, known as triboelectric nanogenerator inductive nanoelectrospray ionization (TENGi nanoESI) MS. Samples tested with this approach include exhaled breath condensate collected from cystic fibrosis patients as well as in vitro-cultured human mesenchymal stromal cells. Both test samples are only available in minimum amounts. Experiments show that picoliter-volume spray pulses suffice to generate high-quality spectral fingerprints, which increase the information density produced per unit sample volume. This TENGi nanoESI strategy has the potential to fill in the gap in metabolomics where liquid chromatography-MS-based analyses cannot be applied. Our method opens up avenues for future investigations into understanding metabolic changes caused by diseases or external stimuli.

Hydrogel Culture Surface Stiffness Modulates Mesenchymal Stromal Cell Secretome and Alters Senescence.

Current cell culture surfaces used for the expansion and production of mesenchymal stromal cells (MSCs) are not optimized for the production of highly secretory and nonsenescent cells. In this study, we used poly (ethylene glycol) hydrogel substrates with tunable mechanical and biochemical properties to screen the effect of culture surfaces on pro-regenerative secretome by multiplex enzyme-linked immunosorbent assay, proliferation by PicoGreen DNA analysis, and senescence by senescence-associated ß-galactosidase activity. We demonstrate that MSCs cultured on 30 kPa hydrogels, regardless of biochemical functionalization, broadly enhanced the secretion of immunomodulatory and regenerative factors versus stiffer 100 kPa or tissue culture plastic surfaces, but did not support robust proliferation. In contrast, culture on 100 kPa hydrogel surfaces promoted proliferation at a similar level and did not substantially alter the amount of secreted factors as compared with tissue culture plastic. Culture on integrin-engaging, cadherin-engaging, and hyaluronic acid-containing 30 kPa substrates enhanced MSC-conditioned media (CM) angiogenic activity in a human umbilical vein endothelial cell tube formation assay and human THP-1 monocyte chemoattraction in a transwell assay. However, 30 kPa substrate culture did not impact the myogenic activity of MSC CM in a C2C12 myoblast tube formation assay. Culture on selected 100 kPa surfaces enhanced CM angiogenic activity and monocyte chemotaxis, but not myogenic activity. Serial culture on 100 kPa RGD hydrogel surfaces significantly reduced senescence in MSCs versus tissue culture plastic, while maintaining the capacity of the cells to enhance their secretome in response to 30 kPa surfaces. Thus, hydrogel substrates that exhibit stiffness orders of magnitude lower than standard tissue culture plastic can serve as novel surfaces for the production of MSCs with an improved therapeutic secretory capacity and reduced senescence. Impact statement The success of mesenchymal stromal cell (MSC)-based therapies is dependent on the manufacture of a large number of cells with high therapeutic potency. Among the culture surfaces tested in this study, we demonstrate that substrate stiffness rather than biochemical functionalization predominantly guides changes in MSC proliferation and secretory capacity. We have identified substrate parameters to support MSC proliferation, enhance secretion of paracrine factors, and to reduce replicative senescence. By maximizing secretory capacity and reducing senescence through the choice of hydrogel culture materials, these findings have great potential to improve the large-scale production of therapeutic MSCs.

Multiomics characterization of mesenchymal stromal cells cultured in monolayer and as aggregates.

Mesenchymal stromal cells (MSCs) have failed to consistently demonstrate their therapeutic efficacy in clinical trials, due in part to variability in culture conditions used for their production. Of various culture conditions used for MSC production, aggregate culture has been shown to improve secretory capacity (a putative mechanism of action in vivo) compared with standard monolayer culture. The purpose of this study was to perform multiomics characterization of MSCs cultured in monolayer and as aggregates to identify aspects of cell physiology that differ between these culture conditions to begin to understand cellular-level changes that might be related to secretory capacity. Targeted secretome characterization was performed on multiple batches of MSC-conditioned media, while nontargeted proteome and metabolome characterization was performed and integrated to identify cellular processes differentially regulated between culture conditions. Secretome characterization revealed a reduction in MSC batch variability when cultured as aggregates. Proteome and metabolome characterization showed upregulation of multiple protein and lipid metabolic pathways, downregulation of several cytoskeletal processes, and differential regulation of extracellular matrix synthesis. Integration of proteome and metabolome characterization revealed individual lipid metabolites and vesicle-trafficking proteins as key features for discriminating between culture conditions. Overall, this study identifies several aspects of MSC physiology that are altered by aggregate culture. Further exploration of these processes and pathways is needed to determine their potential role in regulating cell secretory capacity.

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis.

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvß3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.

Vascular Targeting of a Gold Nanoparticle to Breast Cancer Metastasis.

The vast majority of breast cancer deaths are due to metastatic disease. Although deep tissue targeting of nanoparticles is suitable for some primary tumors, vascular targeting may be a more attractive strategy for micrometastasis. This study combined a vascular targeting strategy with the enhanced targeting capabilities of a nanoparticle to evaluate the ability of a gold nanoparticle (AuNP) to specifically target the early spread of metastatic disease. As a ligand for the vascular targeting strategy, we utilized a peptide targeting alpha(v) beta(3) integrin, which is functionally linked to the development of micrometastases at a distal site. By employing a straightforward radiolabeling method to incorporate Technetium-99m into the AuNPs, we used the high sensitivity of radionuclide imaging to monitor the longitudinal accumulation of the nanoparticles in metastatic sites. Animal and histological studies showed that vascular targeting of the nanoparticle facilitated highly accurate targeting of micrometastasis in the 4T1 mouse model of breast cancer metastasis using radionuclide imaging and a low dose of the nanoparticle. Because of the efficient targeting scheme, 14% of the injected AuNP deposited at metastatic sites in the lungs within 60 min after injection, indicating that the vascular bed of metastasis is a viable target site for nanoparticles.