CAR-T cell therapy is leading the way in the field of cancer cell immunotherapies due to its high success rates. However, the manufacturing of CAR-T cells remains complex and expensive. T-cell enrichment from patient apheresis starting material is a key step in the manufacture but cellular impurities interfere with the ex vivo transduction of T-cells and their proliferation. Current enrichment methods including magnetic bead selection suffer from various limitations. We report here a bead-less T-cell enrichment process through a two-stage procedure based on inertial microfluidics. Using apheresis like starting material samples from healthy donors, the dual-stage process showed an efficient 87

The efforts to increase the active surface area of catalysts led to reduction of metal particle size, down to single metal atoms. This results in increasing importance of support-metal interactions. We demonstrate the mechanisms through which the support influences catalytic activity of nanoclusters: the support electronics, described by the O 2p energy level, and the support surface chemistry, determined by the density of Lewis base sites. Using Ru nanoclusters, our study shows that these parameters can be effectively captured within a single catalyst support descriptor (CSD). The apparent activation energy and turnover frequency (TOF) for the ammonia synthesis correlates strongly with CSD measured for the series Ru/MgO, Ru/Sc2O3, Ru/CeO2, Ru/La2O3, and Ru/Y2O3. Furthermore, the study demonstrates that CSD correlates linearly with the binding strength of N-Ru in nanocluster, thereby providing a direct link between the catalyst's surface chemistry and the nature of the support. The catalyst support descriptor developed in this study serves as a simple yet powerful tool for selecting the optimal support material to maximise the activity of metal nanoclusters without altering the metal itself.

Chimeric antigen receptor (CAR-T) cell therapy is a successful immunotherapy for treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (CLL) patient's samples. In healthy donors used to optimise the process, the lymphocyte purity was enriched from 65% (SD±0.2) to 91% (SD±0.06) and T-cell purity was enriched from 45% (SD±0.1) to 73% (SD±0.02). Leukemic samples had higher starting B-cell compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells was achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD±0.09), 89% (SD±0.1) and 74% (SD±0.09) respectively, and a 70% (SD±0.1) T-cell recovery. CLL samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the CAR-T manufacturing workflow.

Supplementary Figure Legends - PDF files 57K, Figure Legends

Fundamental knowledge about cell–surface interactions can be applied in the development of wound dressings and scaffolds to encourage wounds to heal. As surfaces produced with acid-functionalised monomers encourage keratinocyte adhesion, proliferation and migration, whilst amine functionalisation enhances fibroblast proliferation and migration in vitro, standard care wound dressings were plasma-coated with either acrylic acid or allylamine and applied to 6 mm excisional wounds on the backs of mice to test their effectiveness in vivo. At day 3, the rate of wound healing was increased in mice treated with dressings that were plasma-coated with allylamine compared to uncoated dressings, with a significantly reduced wound area. However, healing may be impaired following prolonged treatment with allylamine-functionalised dressings, with delayed re-epithelialisation and increased cellularisation of the wound site at later timepoints. Acrylic acid functionalisation, however, offered no early improvement in wound healing, but wounds treated with these dressings displayed increased collagen deposition at day 7 post wounding. These results suggest that plasma polymerisation may allow for the development of new dressings which can enhance wound closure by directing cell behaviour, but that the application of these dressings may require a timed approach to enhance specific phases of the wound healing response.

Supplementary Table 1 - PDF files 69K, Patient Data

Introduction: Stem cell therapies have been investigated as potential treatment modalities for chronic wounds however there has been limited success to date. Multipotent Adult Progenitor Cells (MAPCs©) have been identified as having potential as an allogenic stem cell product due to their high population doubling number and their characteristic dampening of T-cell proliferation. This helps to prevent autoimmunity and graft/cell rejection. Methods: We have developed a dressing, consisting of medical grade silicone coated with a heptylamine plasma polymer, which supports the growth and transfer of MAPCs to skin. To determine if the dressing can deliver functional stem cells into diabetic wounds, they were loaded with MAPCs and then placed over excisional wounds in both normal and diabetic mice. Results and discussion: Accelerated healing was observed in both the normal and diabetic wounds with wound gape being significantly smaller at day 3 when compared to controls. Wound analysis showed that treatment with the MAPC dressings dampened the inflammatory response with reduced numbers of neutrophils and macrophages observed. Additionally, an increase in pro-angiogenic VEGF and CD31 positive endothelial cells was observed indicating improved new blood vessel formation. The MAPC dressings had no effect on fibrosis with collagen I and III being equally affected in both control and treated wounds. Overall, the functionalized MAPC dressings improve healing responses particularly in diabetic mice with impaired healing responses and therefore, show potential for development as an advanced therapeutic approach for the treatment of chronic diabetic wounds.

Supplementary Figure 2 - PDF files 805K, Correlation of CD62L antibody responses and clinical parameters

Supplementary Table 2 - PDF files 77K, CD markers used in phenotype screen

Supplementary Figure 1 - PDF files 2472K, Expression of cell surface markers throughout CLL PBMC

Advances in cellular reprogramming have radically increased the use of patient-derived cells for neurological research in vitro. However, adherence of human neurons on tissue cultureware is unreliable over the extended periods required for electrophysiological maturation. Adherence issues are particularly prominent for transferable glass coverslips, hindering imaging and electrophysiological assays. Here, we assessed thin-film plasma polymer treatments, polymeric factors, and extracellular matrix coatings for extending the adherence of human neuronal cultures on glass. We find that positive-charged, amine-based plasma polymers improve the adherence of a range of human brain cells. Diaminopropane (DAP) treatment with laminin-based coating optimally supports long-term maturation of fundamental ion channel properties and synaptic activity of human neurons. As proof of concept, we demonstrated that DAP-treated glass is ideal for live imaging, patch-clamping, and optogenetics. A DAP-treated glass surface reduces the technical variability of human neuronal models and enhances electrophysiological maturation, allowing more reliable discoveries of treatments for neurological and psychiatric disorders.

Chimeric antigen receptor T (CAR-T) cell therapy is rapidly becoming a frontline cancer therapy. However, the manufacturing process is time-, labor- and cost-intensive, and it suffers from significant bottlenecks. Many CAR-T products fail to reach the viability release criteria set by regulators for commercial cell therapy products. This results in non-recoupable costs for the manufacturer and is detrimental to patients who may not receive their scheduled treatment or receive out-of-specification suboptimal formulation. It is demonstrated here that inertial microfluidics can, within minutes, efficiently deplete nonviable cells from low-viability CAR-T cell products. The percentage of viable cells increases from 40% (SD ± 0.12) to 71% (SD ± 0.09) for untransduced T cells and from 51% (SD ± 0.12) to 71% (SD ± 0.09) for CAR-T cells, which meets the clinical trials' release parameters. In addition, the processing of CAR-T cells formulated in CryStor yields a 91% reduction in the amount of the cryoprotectant dimethyl sulfoxide. Inertial microfluidic processing has no detrimental effects on the proliferation and cytotoxicity of CAR-T cells. Interestingly, ≈50% of T-regulatory and T-suppressor cells are depleted, suggesting the potential for inertial microfluidic processing to tune the phenotypical composition of T-cell products.

Stem cells have been shown to have potential as a new therapy for burns and promote wound healing through decreasing inflammation and increasing angiogenesis. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived stem cells with outstanding self-renewal and differentiation capacity. MAPC cells also secrete a wide range of cytokines which can affect cellular activities. This article aimed to examine the effects of MAPC cells treatment on burn injury repair using a mouse model of partial thickness burn injury. The immunomodulatory effect of MAPC cells was investigated in vitro using a simultaneous T-cell proliferation assay. Partial thickness burns were created on the dorsal surface of mice and MAPC cells were administered via intradermal injection to the wound margins 24 h post-burn injury. The burn tissues were analysed macroscopically to determine wound area and histologically assessed to determine wound width and rate of re-epithelialisation. Immunohistochemistry and ELISA were employed to assess cell proliferation, inflammation and angiogenesis and collagen deposition in the burn area. MAPC cells inhibit the proliferation of stimulated T cells in culture. Burns intradermally injected with MAPC cells showed a significant reduction in the macroscopic wound area, histologic wound width and had an increased rate of re-epithelialisation. Immunohistochemistry and ELISA analysis of burn tissues showed dampened inflammation evidenced by a reduction in neutrophilic infiltration and modulation of inflammatory cytokines. Angiogenesis within the burn area was also improved in MAPC cell treated mice. However, no significant effect of MAPC cell treatment was observed on extracellular matrix production. Treatment of burns with MAPC cells improved burn injury repair with reduced time to healing, decreased inflammation and increased angiogenesis. These findings demonstrate the promising effects of MAPC cells on burn injury repair and suggest MAPC cells as a candidate source for clinical cell therapies.

The production of methanol from glycerol over a basic oxide, such as MgO, using high reaction temperatures (320 °C) is a promising new approach to improving atom efficiency in the production of biofuels. The mechanism of this reaction involves the homolytic cleavage of the C3 feedstock, or its dehydration product hydroxyacetone, to produce a hydroxymethyl radical species which can then abstract an H atom from other species. Obtaining a detailed reaction mechanism for this type of chemistry is difficult due to the large number of products present when the system is operated at high conversions. In this contribution we show how DFT based modelling studies can provide new insights into likely reaction pathways, in particular the source of H atoms for the final step of converting hydroxymethyl radicals to methanol. We show that water is unlikely to be important in this stage of the process, C-H bonds of C2 and C3 species can give an energetically favourable pathway and that the disproportionation of hydroxymethyl radicals to methanol and formaldehyde produces a very favourable route. Experimental analysis of reaction products confirms the presence of formaldehyde. The calculations presented in this work also provide new insight into the role of the catalyst surface in the reaction showing that the base sites of the MgO(100) are able to deprotonate hydroxymethyl radicals but not methanol itself. In carrying out the calculations we also show how periodic DFT and QM/MM approaches can be used together to obtain a rounded picture of molecular adsorption to surfaces and homolytic bond cleavage which are both central to the reactions studied.

The CAR T cell manufacturing process is complex and expensive. In addition, the quality of the final product suffers from great variability, which is in part due to the personalized nature of the therapy. An important source of variability of final CAR T cells products is the starting cellular material for manufacture. The percentage of T-cells separated from a patient’s blood used for CAR T cell manufacture is highly variable and influenced by many factors. The percentage of ‘other’ cells typically range anywhere from 10 to 90%, and these cells interfere with the downstream T-cell modification and expansion processes, potentially preventing the production of high-quality end products. Microfluidic technologies which separate cells based on their physical attributes have previously shown great success in separating circulating cancer cells from blood but have not yet been applied in the CAR T cell manufacturing process. Early research has shown the possibility of depleting red blood cells and platelets, however little has been done in the specific purification of T-cells and depletion of other lymphocytic cells. This research therefore aimed to investigate the use of microfluidics in the separation and purification of T-cells towards addressing known bottlenecks/issues in the CAR T cells manufacturing process. We have designed and optimized microfluidic devices of various dimensions with enrichment and depletion outlets. We will report on the successful utilization of these devices in various critical steps of the manufacturing process. This abstract is also being presented as PO080. Citation Format: Mona T. Elsemary, Michelle F. Maritz, Louise Smith, Majid Warkiani, Benjamin Thierry. Microfluidic purification of T lymphocytes separated from blood for chimeric antigen receptor T-cell manufacturing [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR013.

Cellular migration plays a vital role in many physiological processes. To elucidate the role of surface nanotopography on the downstream signaling pathways underlying cell migration, model surfaces having well-defined hill-like surface nanotopography and uniform surface chemistry were designed and implemented using plasma polymerization and covalent attachment of nanoparticles of predetermined size. A scratch wound assay, immunostaining, and gene expression of focal adhesion (FA) proteins were performed to determine the influence of surface nanotopography on cell migration. The results of this study demonstrate that the gap closure between cell monolayers is faster on surfaces having greater nanoscale topography. The phenomenon is predominantly driven by cell migration and was independent from cell proliferation. Qualitative and quantitative assessment of proteins involved in the signaling pathways underlying cell migration showed significant modulation by surface nanotopography. Specifically, focal adhesion sites decreased with the increase in surface nanotopography scale while the expression of FA proteins increased. This implies that nanotopography mediated modulation of cell migration is directly governed by the recruitment of receptor and adapter proteins responsible for cell-surface interaction. The results of this study indicate that biomaterial devices and constructs having rationally designed surface nanotopography and chemistry could be utilized to regulate wound healing and tissue regeneration.

Background Stem cell therapies have been widely investigated for their healing effects. However, the translation of these therapies has been hampered by the requirement to deliver live allogeneic or autologous cells directly to the wound in a clinical setting. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived adherent stem cells that secrete a wide range of factors known to accelerate the wound healing process. The aim of this study was to determine the impact of MAPC cells secretome on healing outcomes without the presence of MAPC cells. Methods The effect of MAPC-conditioned medium (MAPC-CM) on the capacity of keratinocytes, fibroblasts and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay, respectively. The effect of MAPC-CM on collagen deposition and angiogenesis was also assessed using in vitro methods. Additionally, two excisional wounds were created on the dorsal surface of mice ( n = 8/group) and 100 μL of 20× MAPC-CM were intradermally injected to the wound margins. Wound tissues were collected at 3, 7 and 14 days post-wounding and stained with H&E for microscopic analysis. Immunohistochemistry was performed to investigate inflammation, angiogenesis and collagen deposition in the wounds. Results Skin fibroblasts, keratinocytes and endothelial cells treated with MAPC-CM all showed improved rates of scratch closure and increased cellular proliferation. Moreover, fibroblasts treated with MAPC-CM deposited more collagens I and III and endothelial cells treated with MAPC-CM showed increased capillary tube formation. Murine excisional wounds intradermally injected with MAPC-CM showed a significant reduction in the wound area and an increase in the rate of reepithelialisation. The results also showed that inflammatory cell infiltration was decreased while an increase in angiogenesis, as well as collagens I and III expressions, was observed. Conclusion These findings suggest that factors produced by MAPC cells can have an important effect on cutaneous wound healing by affecting skin cell proliferation and migration, balancing inflammation and improving the formation of extracellular matrix and angiogenesis. Development of stem cell-free therapy for the treatment of wounds may be a more clinically translatable approach for improving healing outcomes.

A series of ceria-based solid solution metal oxides were prepared by co-precipitation and evaluated as catalysts for glycerol cleavage, principally to methanol. The catalyst activity and selectivity to methanol were investigated with respect to the reducibility of the catalysts. Oxides comprising Ce-Pr and Ce-Zr were prepared, calcined and compared to CeO 2 , Pr 6 O 11 and ZrO 2 . The oxygen storage capacity of the catalysts was examined with analysis of Raman spectroscopic measurements and a temperature programmed reduction, oxidation and reduction cycle. The incorporation of Pr resulted in significant defects, as evidenced by Raman spectroscopy. The materials were evaluated as catalysts for the glycerol to methanol reaction, and it was found that an increased defect density or reducibility was beneficial. The space–time yield of methanol normalized to surface area over CeO 2 was found to be 0.052 mmol MeOH m −2 h −1 , and over CeZrO 2 and CePrO 2 , this was to 0.029 and 0.076 mmol MeOH m −2 h −1 , respectively. The inclusion of Pr reduced the surface area; however, the carbon mole selectivity to methanol and ethylene glycol remained relatively high, suggesting a shift in the reaction pathway compared to that over ceria. This article is part of a discussion meeting issue ‘Science to enable the circular economy'.

Healing of the skin and oral mucosa utilises similar mechanisms of tissue repair, however, scarring and the rate of wound closure is vastly superior in the oral cavity suggesting differences between these two environments. One key difference is the phenotype of dermal fibroblasts compared to fibroblasts of gingival tissues. Human gingival fibroblasts (hGFs) are undifferentiated cells with multi-differentiation and self-renewal capacities. This study aimed to examine if delivering hGFs or their secretome, contained in hGF-conditioned media (hGF-CM), would improve healing of the skin and recapitulate features of oral healing. Human fibroblasts, keratinocytes and endothelial cells were first treated with hGF-CM and showed improved migration, proliferation and angiogenic functions. A significant reduction in macroscopic wound area and histologic dermal wound width, as well as an increased rate of re-epithelialisation, were observed in both hGFs and hGF-CM treated murine excisional wounds. This improvement was associated with reduced inflammation, increased angiogenesis and elevated collagen deposition. These findings demonstrate that treatment of dermal wounds with either hGFs or hGF-CM may provide beneficial gingival-like properties to dermal wounds and may be a potential opportunity for improving healing of the skin.

Cryopreservation is an essential part of tissue banking and effective cryopreservation methods are critical for the development of cost-effective cell therapy products. Cell sheets are an attractive subset of cell therapy types, and cryopreservation has the potential to further drive down costs of allogeneic cell sheet therapy. This is currently a challenge as adhered cell monolayers are more susceptible to membrane damage during the freezing process. In this article, we investigate the performance of a surface-modified dressing for the cryopreservation of cells and strategies to improve cell recovery. Cryopreservation of multipotent adult progenitor cells (MAPC®) was performed on cells following their attachment to a surface for different periods of time. MAPC cells, given just 1 h to attach, washed off and were not recovered on the surface following thawing. Cells attached for longer periods, elongated further, and were more susceptible to damage from cryopreservation. A temporal window was identified that could allow cryopreservation on adherent surfaces where cells had attached to a surface without full elongation. By functionalizing the surface with coupled hyaluronic acid, cell spreading was initially retarded, thereby widening this temporal window. This approach demonstrates a novel method for enhancing the recovery of cryopreserved cell sheets on surfaces.