Adaptive T cell processing through integrated process analytical technologies

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Viability enrichment of final drug product using counter-flow centrifugation

The CAR-T manufacturing process is sensitive to donor variability, which impacts the final product quality, including cell viability. A counterflow centrifugation cell washer offers a solution to this problem by utilizing physical differences between viable and non-viable cells to elutriate the smaller and less dense non-viable cell population while preserving the viable cells population in the washing chamber. The Thermofisher CTS Rotea Counterflow Centrifugation System was evaluated against a dead-end centrifuge to wash low viability donors at the end of cell culture. The counterflow centrifuge cell washer improved the viability of the final drug product by 15% against the dead-end centrifuge washer. The ability to enrich low viability cell cultures during final harvest mitigates the risk of low viability final drug products and facilitates the stability of product quality

The Role of the Aging Microenvironment on Cancer Cell Functions

Advanced age is the main risk factor for a range of human pathologies, including cardiovascular, neurodegenerative and neoplastic syndromes. Ageing involves the accumulation of damage to macromolecules (i.e. nuclear DNA damage, misfolded proteins, and mitochondrial DNA deletions). Ageing-induced damage leads to the malfunction of organelles and promotes cellular defects and tissue dysfunction. Age-related pathologies and deficits are, in part, a result of the accumulation of senescent cells in the aging organism. Cellular senescence is a state of irreversible growth arrest that occurs as a result of oncogenic stimulus or genomic stress. This includes telomere erosion, DNA double-strand breaks, genetic defects and overexpression of oncogenes, such as KRAS. Growth arrest prevents the perpetuation of cellular damage from one generation to the next and thus provides a potent tumor-suppressive mechanism to cells exposed to oncogenic stimuli. Despite their anti-tumorigenicity, senescent cells can contribute to neoplastic progression by promoting a pro-inflammatory environment. Transcriptional changes that accompany senescence promote a robust increase in mRNA, translation and the secretion of cytokines, chemokines, growth factors and proteases. This complex senescence-associated secretory phenotype (SASP) promotes tissue remodeling and stimulates a malignant phenotype and tumor progression in neighboring epithelial cells. In particular, this pro-inflammatory stimulus elicits aggressive cancer behavior, including enhanced invasion, proliferation, loss of cell-to-cell contacts and an apparent epithelial-mesenchymal transition (EMT). The molecular mechanism underlying this aggressive tumor cell behavior, in particular its transition to an invasive and motile phenotype, remains largely unknown. In this work, I create an in vitro model of cellular aging by enabling DNA double strand breaks and inducing senescence. I further characterize the proliferative potential, secretory profile and organization of chromatin with presenescent and senescent fibroblasts. Next, I demonstrate that SASP from senescent stromal fibroblasts promote spontaneous morphological changes accompanied by an aggressive migratory behavior in originally non-motile human breast cancer cells. This phenotypic switch is coordinated, in space and time, by a dramatic reorganization of the actin and microtubule filament networks, a discrete polarization of EB1 comets, and an unconventional front-to-back inversion of nucleus-MTOC polarity. SASP-induced morphological/migratory changes are critically dependent on microtubule integrity and dynamics, and are coordinated by the inhibition of RhoA and cell contractility. RhoA/ROCK inhibition reduces focal adhesions and traction forces, while promoting a novel gliding mode of migration. Finally, I show that factors secreted by senescent stromal cells substantially alter the nuclear shape and architecture of stimulated cancer cells, whereby nuclei form deep invaginations and show large-scale volume shrinkage. These nuclear-shape changes are highly dependent on intact lamin A/C-mediated connections and the reduction in acto-myosin tension. Loss of cellular tension causes the decrease in lateral pulling force exerted on the nucleus, leading to the formation of nuclear lobulations. I establish that acto-myosin tension is both necessary and sufficient to induce nuclear-shape changes. I also demonstrate that SASP stimulation is sufficient to elicit substantial transcriptional changes in carcinoma cells