Advances in gene editing and cell therapies have recently led to outstanding clinical successes. However, the lack of a cost-effective manufacturing process prevents the democratization of these innovative medical tools. Due to the common use of viral vectors, the step of transfection in which cells are engineered to gain new functions, is a major bottleneck in making safe and affordable cell products. A promising opportunity lies in Single-Cell Transfection Technologies (SCTTs). SCTTs have demonstrated higher efficiency, safety and scalability than conventional transfection methods. They can also feature unique abilities such as substantial dosage control over the cargo delivery, single-cell addressability and integration in microdevices comprising multiple monitoring modalities. Unfortunately, the potential of SCTTs is not fully appreciated: they are most often restricted to research settings with little adoption in clinical settings. To encourage their adoption, we review and compare recent developments in SCTTs, and how they can enable selected clinical applications. To help bridge the gap between fundamental research and its translation to the clinic, we also describe how Good Manufacturing Practices (GMP) can be integrated in the design of SCTTs.
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http://dx.doi.org/10.1016/j.jconrel.2020.10.068 | DOI Listing |
Int J Mol Sci
January 2025
Clinical Division of General Anaesthesia and Intensive Care Medicine, Department of Anesthesia, Genera Intensive Care and Pain Therapy, Medical University Vienna, 1090 Vienna, Austria.
Drug development for human disease relies on preclinical model systems such as human cell cultures and animal experiments before therapeutic treatments can ultimately be tested on humans in clinical studies. We here describe the generation of a novel human cell line (HLMVEC/SVTERT289) that we generated by transfection of microvascular endothelial cells from healthy donor lung tissue with the catalytic domain of telomerase and the SV40 large T/small t-antigen. These cells exhibited satisfactory growth characteristics and largely maintained their native characteristics, including morphology, cell surface marker expression, angiogenic potential and the protein composition of secreted extracellular vesicles.
View Article and Find Full Text PDFBiotechnol Prog
January 2025
AdBIOPRO, VINNOVA Competence Centre for Advanced Bioproduction by Continuous Processing, Royal Institute of Technology (KTH), Stockholm, Sweden.
The possibility to produce recombinant adeno-associated virus (rAAV) by adherent HEK293T cells was studied in a stirred tank bioreactor (STR) culture of cell aggregates. A proof-of-concept of rAAV production was successfully demonstrated in a process where single cells were first expanded, then cell aggregates were formed by dilution into a different medium 1 day before triple plasmid transfection was conducted. An alternative approach for the STR inoculation using a seed taken from a high cell density perfusion (HCDP) culture was also investigated.
View Article and Find Full Text PDFACS Sens
January 2025
Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, P. R. China.
Accurate and efficient sorting of single target cells is crucial for downstream single-cell analysis, such as RNA sequencing, to uncover cellular heterogeneity and functional characteristics. However, conventional single-cell sorting techniques, such as manual micromanipulation or fluorescence-activated cell sorting, do not match current demands and are limited by low throughput, low sorting efficiency and precision, or limited cell viability. Here, we report an automated, highly efficient single-cell sorter, integrating laser-induced forward transfer (LIFT) with a high-throughput picoliter micropore array.
View Article and Find Full Text PDFJ Transl Med
January 2025
Department of Endocrine Medicine, Shanghai Sixth People's Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 201306, Chin, China.
Background And Objective: Mitochondria are crucial to the function of renal tubular cells, and their dynamic perturbation in many aspects is an important mechanism of diabetic kidney disease (DKD). Single-nucleus RNA sequencing (snRNA-seq) technology is a high-throughput sequencing analysis technique for RNA at the level of a single cell nucleus. Here, our DKD mouse kidney single-cell RNA sequencing conveys a more comprehensive mitochondrial profile, which helps us further understand the therapeutic response of this unique organelle family to drugs.
View Article and Find Full Text PDFCurrently, the most common approach for manufacturing GMP-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. The requirement for all three plasmids to be transfected into a single cell and the necessity for high quantities of input plasmid DNA, limits AAV production efficiency, introduces variability between production batches, and increases time and labor costs. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone.
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