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

Experimental autoimmune encephalomyelitis (EAE) is a model of the neuroimmune system responding to priming with central nervous system (CNS)-restricted antigens. It is an excellent model of post-vaccinal encephalitis and a useful model of many aspects of multiple sclerosis. EAE has been established in numerous species and is induced by priming with a large number of CNS-derived antigens. As a consequence, the pathogenesis, pathology and clinical signs vary significantly between experimental protocols. As I describe in this Timeline article, the reductionist approach taken in some lines of investigation of EAE resulted in a reliance on results obtained under a narrow range of conditions. Although such studies made important contributions to our molecular understanding of inflammation, T-cell activation, and MHC restriction, they did not advance as effectively our knowledge of the polyantigenic responses that usually occur in CNS immunopathology and autoimmunity.

The origin and application of experimental autoimmune encephalomyelitis

Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo types—small molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications a...

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Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts.

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.

/pdf/advances-in-mammalian-cell-line-development-technologies-for-3jh62zckao.pdf

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.

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

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

Efficient delivery of compounds and macromolecules into living cells is essential in many fields including basic research, applied drug discovery, and clinical gene therapy. Unfortunately, current delivery methods, such as cationic lipids and electroporation, are limited by the types of macromolecules and cells that can be employed, poor efficiency, and/or cell toxicity. To address these issues, novel methods were developed based on laser-mediated delivery of macromolecules into cells through optoinjection. An automated high-throughput instrument, the laser-enabled analysis and processing (LEAPTM) system, was utilized to elucidate and optimize several parameters that influence optoinjection efficiency and toxicity. Techniques employing direct cell irradiation (i.e., targeted to specific cell coordinates) and grid-based irradiation (i.e., without locating individual cells) were both successfully developed. With both techniques, it was determined that multiple, sequential low radiant exposures produced more favorable results than a single high radiant exposure. Various substances were efficiently optoinjected—including ions, small molecules, dextrans, siRNAs (small interfering RNAs), plasmids, proteins, and semiconductor nanocrystals—into numerous cell types. Notably, cells refractory to traditional delivery methods were efficiently optoinjected with lower toxicity. We establish the broad utility of optoinjection, and furthermore, are the first to demonstrate its implementation in an automated, high-throughput manner.

https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-11/issue-01/014034/Optoinjection-for-efficient-targeted-delivery-of-a-broad-range-of/10.1117/1.2168148.pdf

Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types

Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.

Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

Background:

Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAP™ (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection).



Methods:

LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy.



Results:

Live cell samples (adherent and suspension) could be purified to 90–100% purity with 50–90% yield, causing minimal cell damage depending on the cell type and plating density. Nearly one hundred percent of the targeted cells of all cell types examined could be successfully optoinjected with dextrans of 3–70 kDa, causing no visual damage to the cells. Indirect optoinjection effects were observed on untargeted cells within 5–60 μm to targeted areas under conditions used here.



Conclusions:

LEAP provides solutions in cell purification and targeted macromolecule delivery for traditional and challenging applications where other methods fall short. © 2006 International Society for Analytical Cytology

Scanning cytometry with a LEAP: Laser‐enabled analysis and processing of live cells in situ

The invention relates to methods and apparatuses for introducing and releasing substances into and out of cells, and more specifically to methods and apparatuses for transiently permeabilizing a living cell so that any one or more of a variety of substances, such as ions, proteins, and nucleic acids, can be loaded into or released out of the cell.

Elie G. Hanania

Papers

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

Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types

Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

Scanning cytometry with a LEAP: Laser‐enabled analysis and processing of live cells in situ

Method and apparatus for cell permeabilization