Acoustophoretic rapid media exchange and continuous-flow electrotransfection of primary human T cells for applications in automated cellular therapy manufacturing

Hsi, Peter; Christianson, Rebecca J.; Dubay, Ryan; Lissandrello, Charles; Fiering, Jason; Balestrini, Jenna L.; Tandon, Vishal R

doi:10.1039/c9lc00458k

Cited by 21 publications

(29 citation statements)

References 60 publications

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“…Isolation and culture of primary human T cells. T-cell isolation was performed as described in our previous work 32 . De-identified, unpurified buffy coats from healthy human donors were obtained from Research Blood Components (Watertown, MA).…”

Section: Methodsmentioning

confidence: 99%

“…Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll centrifugation in Ficoll-Paque PLUS (1.077 g/ml) density gradient media (GE Healthcare) according to the Flow cytometry. Flow cytometry was performed as described in our previous work 32 . T cell samples were pelleted, washed, and resuspended in autoMACS Rinsing Solution (Miltenyi Biotec) containing 1:20 dilution of MACS BSA Stock Solution (Miltenyi Biotec) and 1:1000 Sytox Live/Dead stain (ThermoFisher) as per manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…This aids in maintaining high cell recovery and viability. This type of flow configuration has been used successfully in the past to transfect HEK-293A, HeLa, neuro-2A, and HEK-293 mammalian cell lines 30 and yeast cells 31 , and by our group to deliver mRNA to primary human T cells 32 . Compared with these previous efforts, our device is designed for orders-of-magnitude greater throughput for clinical-scale processing (enabled by increased channel cross-sectional dimensions and a sturdier material set), is fabricated from hard plastics compatible with the transition to mass-manufacturing, and has been optimized for primary human T cells rather than model mammalian cell lines.…”

Section: Openmentioning

confidence: 99%

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High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing

Lissandrello

Santos

Hsi

et al. 2020

Sci Rep

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Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95% transfection efficiency with minimum impact on cell viability and expansion potential. We additionally demonstrated processing of samples comprising up to 500 million T cells at a rate of 20 million cells/min. We anticipate that our device will help to streamline the production of autologous therapies requiring on the order of 10$$^8$$ 8 –10$$^9$$ 9 cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Openmentioning

confidence: 99%

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High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing

Lissandrello

Santos

Hsi

et al. 2020

Sci Rep

Self Cite

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“…For example, Gupta et al (1995) separated PS and LDPE particles in a glycerol-water mixture [97], and Petterson et al ( 2007) separated PS and PMMA particles of the same size suspended in water to which cesium chloride was added, and also demonstrated separation of 2 µm, 5 µm, 8 µm and 10 µm PS particles by size [73]. Acoustophoretic media exchange devices have been developed to transfer particles en masse from one fluid to another fluid, and may be useful for diverting MPs into fluids that facilitate separation by polymer type [98].…”

Section: Acoustophoresismentioning

confidence: 99%

Field-Portable Microplastic Sensing in Aqueous Environments: A Perspective on Emerging Techniques

Blevins

Allen

Colson

et al. 2021

Sensors

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Microplastics (MPs) have been found in aqueous environments ranging from rural ponds and lakes to the deep ocean. Despite the ubiquity of MPs, our ability to characterize MPs in the environment is limited by the lack of technologies for rapidly and accurately identifying and quantifying MPs. Although standards exist for MP sample collection and preparation, methods of MP analysis vary considerably and produce data with a broad range of data content and quality. The need for extensive analysis-specific sample preparation in current technology approaches has hindered the emergence of a single technique which can operate on aqueous samples in the field, rather than on dried laboratory preparations. In this perspective, we consider MP measurement technologies with a focus on both their eventual field-deployability and their respective data products (e.g., MP particle count, size, and/or polymer type). We present preliminary demonstrations of several prospective MP measurement techniques, with an eye towards developing a solution or solutions that can transition from the laboratory to the field. Specifically, experimental results are presented from multiple prototype systems that measure various physical properties of MPs: pyrolysis-differential mobility spectroscopy, short-wave infrared imaging, aqueous Nile Red labeling and counting, acoustophoresis, ultrasound, impedance spectroscopy, and dielectrophoresis.

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“…5 Recently, several research groups have developed methods for continuous medium exchange in microfluidic systems. These methods are based on particle trapping, [6][7][8][9][10] dragging particles through a liquid interface, [11][12][13][14][15][16][17][18][19] continuous buffer exchange, 20 droplet merging, [21][22][23] and fluid relocation. 24 The forces utilized to achieve medium exchange are manifold.…”

Section: Introductionmentioning

confidence: 99%

Acoustofluidic Medium Exchange for Preparation of Electrocompetent Bacteria Using Channel Wall Trapping

Gerlt

Ruppen

Leuthner

et al. 2021

Preprint

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Transformation, i.e. reprogramming of bacteria by delivering exogenous genetic material (such as DNA) into the cytoplasm, is a key process in molecular engineering and modern biotechnology in general. Transformation is often performed by electroporation, i.e. creating pores in the membrane using electric shocks in a low conductivity environment. However, cell preparation for electroporation can be cumbersome as it requires the exchange of growth medium (high-conductivity) for low-conductivity medium, typically performed via multiple time-intensive centrifugation steps. To simplify and miniaturize this step, we developed an acoustofluidic device capable of trapping the bacterium Escherichia coli non-invasively for subsequent exchange of medium, which is challenging in acoustofludic devices due to detrimental acoustic streaming effects. With an improved etching process, we were able to produce a thin wall between two microfluidic channels, which, upon excitation, can generate streaming fields that complement the acoustic radiation force and therefore can be utilized for trapping of bacteria. Our novel design robustly traps Escherichia coli at a flow rate of 10 µL minute-1 and has a cell recovery performance of 47 ± 3 % after washing the trapped cells. To verify that the performance of the medium exchange device is sufficient, we tested the electrocompetence of the recovered cells in a standard transformation procedure and found a transformation efficiency of 8∙105 CFU per µg of plasmid DNA. Our device is a viable low-volume alternative to centrifugation-based methods and opens the door for miniaturization of a plethora of microbiological and molecular engineering protocols.

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Acoustophoretic rapid media exchange and continuous-flow electrotransfection of primary human T cells for applications in automated cellular therapy manufacturing

Abstract: Our novel device acoustophoretically transfers cells from culture media to electroporation media and then electroporates them using integrated electrodes.

Cited by 21 publications

References 60 publications

High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing

High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing

Field-Portable Microplastic Sensing in Aqueous Environments: A Perspective on Emerging Techniques

Acoustofluidic Medium Exchange for Preparation of Electrocompetent Bacteria Using Channel Wall Trapping

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