Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS: Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS: The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS: These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.

A scalable system for generation of mesenchymal stem cells derived from induced pluripotent cells employing bioreactors and degradable microcarriers.

Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3-fold expansion of ihMSCs was achieved over 8 days. Complete recovery by 5-minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost-effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential.

Automated mesenchymal stem cell segmentation and machine learning-based phenotype classification using morphometric and textural analysis.

Purpose: Mesenchymal stem cells (MSCs) have demonstrated clinically relevant therapeutic effects for treatment of trauma and chronic diseases. The proliferative potential, immunomodulatory characteristics, and multipotentiality of MSCs in monolayer culture is reflected by their morphological phenotype. Standard techniques to evaluate culture viability are subjective, destructive, or time-consuming. We present an image analysis approach to objectively determine morphological phenotype of MSCs for prediction of culture efficacy. Approach: The algorithm was trained using phase-contrast micrographs acquired during the early and mid-logarithmic stages of MSC expansion. Cell regions are localized using edge detection, thresholding, and morphological operations, followed by cell marker identification using H-minima transform within each region to differentiate individual cells from cell clusters. Clusters are segmented using marker-controlled watershed to obtain single cells. Morphometric and textural features are extracted to classify cells based on phenotype using machine learning. Results: Algorithm performance was validated using an independent test dataset of 186 MSCs in 36 culture images. Results show 88% sensitivity and 86% precision for overall cell detection and a mean Sorensen-Dice coefficient of 0.849 ± 0.106 for segmentation per image. The algorithm exhibited an area under the curve of 0.816 ( CI 95 = 0.769 to 0.886) and 0.787 ( CI 95 = 0.716 to 0.851) for classifying MSCs according to their phenotype at early and mid-logarithmic expansion, respectively. Conclusions: The proposed method shows potential to segment and classify low and moderately dense MSCs based on phenotype with high accuracy and robustness. It enables quantifiable and consistent morphology-based quality assessment for various culture protocols to facilitate cytotherapy development.

Separating full-length protein from aggregating proteolytic products using filter flow-through purification.

Separation of full-length protein from proteolytic products is challenging, since the properties used to isolate the protein can also be present in proteolytic products. Many separation techniques risk non-specific protein adhesion and/or require a lot of time, enabling continued proteolysis and aggregation after lysis. We demonstrate that proteolytic products aggregate for two different proteins. As a result, full-length protein can be rapidly separated from these fragments by filter flow-through purification, resulting in a substantial protein purity enhancement. This rapid approach is likely to be useful for intrinsically disordered proteins, whose repetitive sequence composition and flexible nature can facilitate aggregation.

A quantitative comparison of human HT-1080 fibrosarcoma cells and primary human dermal fibroblasts identifies a 3D migration mechanism with properties unique to the transformed phenotype.

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels ("synthetic extracellular matrix" or "synthetic ECM"). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced ß1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with ß1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

A synthetic strategy for mimicking the extracellular matrix provides new insight about tumor cell migration.

Understanding the role of the tumor microenvironment during cancer progression and metastasis is complicated by interactions between cells, the extracellular matrix (ECM), and a variety of biomolecules. Using a synthetic strategy, we investigated proteolytic modes of migration for HT-1080 fibrosarcoma cells in an environment that limited confounding extracellular influences. A large percentage of HT-1080s migrated through a Rho kinase (ROCK)-dependent rounded morphology with a leading edge protrusion that defined the direction of migration, and migration was only weakly dependent on the adhesive peptide RGDS. HT-1080s migrating in thiol-ene hydrogels are more rounded and exhibit much more invasive behavior than dermal fibroblasts. Our results indicate that HT-1080s have the capacity to migrate through a mechanism that is distinct from mesenchymal cells, with significant amoeboid character even when utilizing a proteolytic migration strategy. The migration mode observed here provides insight into the invasiveness of metastatic cells in vivo and demonstrates the potential of a synthetic strategy for investigating complex biological problems.

Wilms tumor in a child with L-2-hydroxyglutaric aciduria.

We report a male infant with L-2-hydroxyglutaric aciduria and Wilms tumor. L-2-hydroxyglutaric aciduria is a rare, autosomal-recessive, inborn error of metabolism characterized by a variable degree of progressive encephalopathy. Of the fewer than 100 cases reported in the literature, at least 9 patients have developed tumors of the central nervous system. To our knowledge, the present case is the 1st example of an extracranial tumor associated with L-2-hydroxyglutaric aciduria. This observation potentially widens the tumor spectrum in this metabolic disorder and may lead to further insight into the relationship between L-2-hydroxyglutaric acid and cellular transformation.

Arsenic levels in wipe samples collected from play structures constructed with CCA-treated wood: impact on exposure estimates.

Lumber treated with chromated copper arsenate (CCA) has been used in residential outdoor wood structures and playgrounds. The U.S. EPA has conducted a probabilistic assessment of children's exposure to arsenic from CCA-treated structures using the Stochastic Human Exposure and Dose Simulation model for the wood preservative scenario (SHEDS-Wood). The EPA assessment relied on data from an experimental study using adult volunteers and designed to measure arsenic in maximum hand and wipe loadings. Analyses using arsenic handloading data from a study of children playing on CCA-treated play structures in Edmonton, Canada, indicate that the maximum handloading values significantly overestimate the exposure that occurs during actual play. The objective of our paper is to assess whether the dislodgeable arsenic residues from structures in the Edmonton study are comparable to those observed in other studies and whether they support the conclusion that the values derived by EPA using modeled maximum loading values overestimate hand exposures. We compared dislodgeable arsenic residue data from structures in the playgrounds in the Edmonton study to levels observed in studies used in EPA's assessment. Our analysis showed that the dislodgeable arsenic levels in the Edmonton playground structures are similar to those in the studies used by EPA. Hence, the exposure estimates derived using the handloading data from children playing on CCA-treated structures are more representative of children's actual exposures than the overestimates derived by EPA using modeled maximum values. Handloading data from children playing on CCA-treated structures should be used to reduce the uncertainty of modeled estimates derived using the SHEDS-Wood model.

Simulated inhalation levels of fragrance materials in a surrogate air freshener formulation.

This study measured postapplication exposure levels of fragrance materials in a surrogate air freshener formulation in an environmentally-controlled exposure room (ECER). A five-s spray was released to simulate normal consumer use conditions. Time-course airborne fragrance material levels were sampled with Tenax tubes, and aerosol size distributions were monitored with a TSI 3320 aerodynamic particle sizer. Triplicate experiments were performed for each of the control/test substances. The control substance (unfragranced formulation) experiments indicated that the airborne fragrance materials were not detected, suggesting that the base propellant formulation did not interfere with the sampling procedure or analytical results. The test substance experiments found that the higher the volatility of the fragrance material, the higherthe airborne fragrance concentration within the ECER. In the adult breathing zone height, the maximum concentrations of the nine fragrance materials ranged from 108 to 347 microg/m3 during the first minute postapplication. In the child breathing zone height, the maximum fragrance material concentrations ranged from 125 to 362 microg/m3 during 2-6 min postapplication. Particle size distributions indicated that approximately 60-70% of the generated aerosols were less than 1 microm aerodynamic diameter. Initial peak particle mass concentrations (<5 microm) were 800-1000 microg/m3 during the first minute postapplication. Following initial peak concentrations, there was approximately 10-15 min of fluctuation, and then particle levels decayed gradually and exponentiallyto near background levels. Exposure to the test formulation would originate from two components: particle-bound and vapor-phase fragrance materials. Particle-bound fragrance exposure accounted for approximately 47% and 72% of the total exposures during the first minute postapplication period in the adult and child breathing zone heights, respectively.