Advanced manufacturing process design for Mesenchymal Stromal Cell therapies

For decades, the potential immunomodulatory effects of Mesenchymal Stromal Cells (MSCs) have prompted numerous cell-therapy clinical investigations targeting various diseases such as graft-versus-host disease and autoimmune diseases. Despite their ubiquitous usage in clinical trials, significant challenges related to their manufacturing and biological variabilities have led to poorly reproducible outcomes of therapeutic efficacy. Therefore, identification of validated critical quality attributes (CQAs) correlative to therapeutic function is of great interest to the MSC community. Such CQAs would also permit identification of critical process parameters (CPPs) to achieve and maintain MSC quality while producing a high yield. In this study, we designed and tested a “smart” feedback-controlled hollow fiber-based bioreactor for maintaining nutrient and waste levels for human umbilical cord tissue-derived MSC expansions. The bioreactor platform is a semi-autonomous system complete with in-line sensors, modeling, data-driven controllers, and an automated sampling platform. The small-scale system reduced costs, labor, time, and perturbations and improved yields of MSC products using a hollow fiber cartridge that closely models the basic design of the large-scale Quantum Cell Expansion System. Our feedback-controlled bioreactor responded to in-line glucose and lactate levels while recorded pH and dissolved oxygen measurements. This information was fed into a controller, which auto-calculates cell growth rates based on our developed mathematical model, and subsequently regulated media feed rates to support cell growth and nutrient requirements. Compared to the manual expansion process, the automated expansion processes showed higher yields and comparative therapeutic potency of MSCs, indicated by indolamine 2,3-dioxygenase assay and T cell proliferation assay. Future directions of our study propose to correlate metabolites and secreted proteins in culture media as putative CQAs that can be used as in-line predictors of MSC yield and therapeutic potency. Moreover, we aim to maintain a metabolic and secretory profile throughout MSC expansions enabled by real-time modulation of CPPs and scale up of the “smart” bioreactor. The proposed bioprocess for MSC products can be adapted and applied to industrial cell therapy manufacturing and can enable high-yield and high-quality products while minimizing variabilities. Please click Additional Files below to see the full abstract

Processing and Property Investigation of Single-Walled Carbon Nanotube (SWNT) Buckypaper/Epoxy Resin Matrix Nanocomposites

Abstract Due to their extraordinary high modulus and strength, single-walled carbon nanotubes (SWNTs) are considered by many researchers as the most promising reinforcement materials for use in high performance composites. However, fabricating SWNT-reinforced composites with good tube dispersion and high SWNT loading is a great challenge since SWNTs have a strong tendency to form bundles or ropes and rapidly increase the viscosity during processing. In this research, a new method was developed to fabricate nanocomposites with preformed SWNT networks and high tube loading. SWNTs were first dispersed in water-based suspension with the aid of surfactant and sonication. Through a filtration process, SWNTs were fabricated into thin membranes called buckypapers to form networks of SWNT ropes. The tube/resin impregnation of the produced buckypaper was realized by infiltrating acetone diluted epoxy resin (Epon 862/EPI Cure W system) along the thickness direction. A hot press molding process was used for curing to produce the final nanocomposites of multiple layer buckypapers with high SWNT loading (up to 39 wt%). Dynamic mechanical analysis (DMA) results show that the storage moduli of the resulting nanocomposites were as high as 15 Gpa. The DMA results also indicate that the nanotubes had a strong influence on the composites damping properties. The through thickness permeability of the resulting buckypapers with nanoscale pore structure was also measured. AFM and SEM observations show that the SWNTs have a good dispersion in the buckypaper and nanocomposites. The research results show that the proposed buckypaper/resin infiltration approach is capable of fabricating nanocomposites with controllable nanostructure and high SWNT loading, which are important for developing high performance nanotube-based composites.

Investigation of Salt Tolerance Mechanisms across a Root Developmental Gradient in Almond Rootstocks

The intensive use of groundwater in agriculture under the current climate conditions leads to acceleration of soil salinization. Given that almond is a salt-sensitive crop, selection of salt-tolerant rootstocks can help maintain productivity under salinity stress. Selection for tolerant rootstocks at an early growth stage can reduce the investment of time and resources. However, salinity-sensitive markers and salinity tolerance mechanisms of almond species to assist this selection process are largely unknown. We established a microscopy-based approach to investigate mechanisms of stress tolerance in and identified cellular, root anatomical, and molecular traits associated with rootstocks exhibiting salt tolerance. We characterized three almond rootstocks: Empyrean-1 (E1), Controller-5 (C5), and Krymsk-86 (K86). Based on cellular and molecular evidence, our results show that E1 has a higher capacity for salt exclusion by a combination of upregulating ion transporter expression and enhanced deposition of suberin and lignin in the root apoplastic barriers, exodermis, and endodermis, in response to salt stress. Expression analyses revealed differential regulation of cation transporters, stress signaling, and biopolymer synthesis genes in the different rootstocks. This foundational study reveals the mechanisms of salinity tolerance in almond rootstocks from cellular and structural perspectives across a root developmental gradient and provides insights for future screens targeting stress response