In vitro comparison of the photothermal anticancer activity of graphene nanoparticles and carbon nanotubes.

From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

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Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(07)00182-5.pdf

Selection methods for high-producing mammalian cell lines.

Collective cell migration plays essential roles in a wide spectrum of biological processes, such as embryogenesis, tissue regeneration, and cancer metastasis. Numerous wound-healing assays based on mechanical, chemical, optical, and electrical approaches have been developed to create model “wounds” in cell monolayers to study the collective cell migration processes. These approaches can result in different microenvironments for cells to migrate and possess diverse assay characteristics in terms of simplicity, throughput, reproducibility, and multiplexability. In this review, we provide an overview of advances in wound-healing assays and discuss their advantages and limitations in studying collective cell migration.

/pdf/advances-in-wound-healing-assays-for-probing-collective-cell-183gonzv72.pdf

Advances in Wound-Healing Assays for Probing Collective Cell Migration:

Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system.

Background

Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield.



Methods

An automated closed-loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser-based semiconductor manufacturing equipment. Laser-enabled analysis and processing has combined high-throughput in situ cell imaging with laser-mediated cell manipulation via large field-of-view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility.



Results

Photothermal purification required approximately 108 W/cm2 at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 109 W/cm2 at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification required more than 1010 W/cm2 independent of wavelength, resulting in immediate cell lysis. Each approach resulted in high efficiency purification (>99%) after a single operation, as demonstrated with eight cell types. An automated closed-loop process to re-image and irradiate remaining targets in situ was implemented, resulting in improved purification (99.5–100%) without decreasing cell yield or affecting sterility in this closed system. Efficient purification was demonstrated with B- and T-cell mixtures over a wide range of contaminating cell percentages (0.1–99%) and cell densities (104-106/cm2). Efficient cloning of 293T cells based on fluorescence with green fluorescent protein after plasmid transfection was also demonstrated.



Conclusions

In situ laser-mediated purification was achieved with nonadherent and adherent cells on the automated laser-enabled analysis and processing platform. Closed-loop processing routinely enabled greater than 99.5% purity with a greater than 90% cell yield in sample sizes ranging from 101 to 108 cells. Throughput ranged from approximately 103 to 105 total cells/s for contaminating percentages ranging from 99% to 0.1%, respectively. © 2004 Wiley-Liss, Inc.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.a.20079

Glenn C. Sasaki

Papers

High‐throughput laser‐mediated in situ cell purification with high purity and yield