Paruthiyil, S. | Search bioRxiv

bioRxiv2023

Development of a robotic cluster for automated and scalable cell therapy manufacturing

The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products. Here, we present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic clusters expansion module was demonstrated by activating and expanding human CD8+ T cells. The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies.

bioengineering↗

bioRxiv2023

Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization

Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the unicellular green alga Chlamydomonas reinhardtii, Cu import is dependent on Copper Response Regulator 1 (CRR1), the master regulator of Cu homeostasis. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble cysteine-rich protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulate the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high affinity Cu(I) uptake. Notably, the over-accumulation of Cu(I) in Zinc (Zn)-deficiency (20 times the quota) depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and is consistent with the lower substrate affinity of CTR2.

biochemistry↗