Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding

Twite, Amy A.; Lau, Katherine H.W.J.; Kashani, Moein Navvab; Priest, Craig; Nieva, Jorgé; Gottlieb, David; Pawell, Ryan S.

doi:10.1101/343426

Cited by 4 publications

(6 citation statements)

References 38 publications

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“…Microfluidics refers to the manipulation of fluids at micro‐scale and such technology has been used extensively in cell processing and diagnostics. [ 63 ] A large number of microfluidic platforms have been created for transfection with kinds of mechanisms involving microinjection, [ 64 ] cell squeezing, [ 65 ] shear stresses, [ 66 ] vortex shedding, [ 67 ] deterministic mechanoporation, [ 68 ] compressive forces, [ 69 ] laser, [ 70 ] and electrical fields. [ 71 ] Microfluidic technology has also been combined with biochemical polymers to enhance transfection efficiency.…”

Section: Emerging Transfection Methodsmentioning

confidence: 99%

Materials for Improving Immune Cell Transfection

et al. 2021

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recognize and bind to tumor-associated antigens such as CD19 and epidermal growth factor receptor (EGFR) before releasing cytotoxic granules and effector cytokines to destroy cancer cells. [3] CAR-T therapy has shown remarkable success against CD19 expressing B cell hematological malignancies and, thus far, five products (Kymriah from Novartis; Yescarta and Tecartus from Kite Pharma/Gilead Sciences; and Breyanzi and Abecma from Bristol-Myers Squibb) have been approved by the US Food and Drug Administration (FDA) for clinical use. [4,5] Currently, there are at least 500 clinical trials registered on ClinicalTrials.gov database using engineered immune cells to treat various cancers including that of the brain, lungs, and skin.Despite the clinical success of CAR-T cells against B cell leukemia, CAR-T therapy still face tremendous challenges in manufacturing, safety, and affordability before it can become a viable clinical option. [6] One of the biggest technical difficulties is to transfect sensitive, primary T cells whereby biomolecules like oligonucleotides and proteins have to be delivered intracellularly and into the nuclei of cells. FDA-approved gold standard viruses and bulk electroporation suffer from low transfection efficiency while also perturbing the critical biological attributes of cells such as proliferation, metabolism, and gene expression. [7] This increases the time and costs for cell expansion, and without an efficient and cost-friendly transfection technology, the price (between USD 0.4-0.5 million per patient) for FDA-approved CAR treatments (Kymriah and Yescarta) will remain unaffordable and untimely. [8] The limitations in conventional transfection techniques have motivated the development of micro-and nanoplatforms such as microfluidics, nanoparticles, and high-aspect-ratio nanostructures to improve immune cell viability and throughput during transfection.Herein, we first provide an overview of CAR-T cell manufacturing, with emphasis on the science of transfection and limitations of traditional technology using viruses and bulk electroporation. There will also be discussion of other cell-based cancer immunotherapy using other types of promising immune cell types. Next, we describe emerging transfection platforms and companies that have been established to overcome gaps in CAR-T transfection. We then provide a list of assays constituting the polyfunctionalities of immune cells that we believe will help the field better assess the robustness and suitability of their transfection methods. Finally, we end off with existing challenges in CAR-T transfection and how overcoming these challenges can significantly enhance the clinical impact of CAR-T therapy.Chimeric antigen receptor T cell (CAR-T) therapy holds great promise for preventing and treating deadly diseases such as cancer. However, it remains challenging to transfect and engineer primary immune cells for clinical cell manufacturing. Conventional tools using viral vectors and bulk electroporation suffer from low efficiency while posing risks ...

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Section: Emerging Transfection Methodsmentioning

confidence: 99%

Materials for Improving Immune Cell Transfection

et al. 2021

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“…reported a microfluidic vortex shedding device for transfecting human primary T lymphocytes ( Figure a). [ 48 ] When the fluid passed the cylinders in the microfluidic channels, fluctuating vortices were generated behind the cylindrical structures. The induced vortices disrupted the lipid membrane of the cells, allowing the entry of external molecules.…”

Section: Mechanical Plasma Membrane Disruption‐mediated Intracellular...mentioning

confidence: 99%

Microfluidic and Nanofluidic Intracellular Delivery

Hur

Chung

2021

Advanced Science

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Innate cell function can be artificially engineered and reprogrammed by introducing biomolecules, such as DNAs, RNAs, plasmid DNAs, proteins, or nanomaterials, into the cytosol or nucleus. This process of delivering exogenous cargos into living cells is referred to as intracellular delivery. For instance, clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 gene editing begins with internalizing Cas9 protein and guide RNA into cells, and chimeric antigen receptor‐T (CAR‐T) cells are prepared by delivering CAR genes into T lymphocytes for cancer immunotherapies. To deliver external biomolecules into cells, tools, including viral vectors, and electroporation have been traditionally used; however, they are suboptimal for achieving high levels of intracellular delivery while preserving cell viability, phenotype, and function. Notably, as emerging solutions, microfluidic and nanofluidic approaches have shown remarkable potential for addressing this open challenge. This review provides an overview of recent advances in microfluidic and nanofluidic intracellular delivery strategies and discusses new opportunities and challenges for clinical applications. Furthermore, key considerations for future efforts to develop microfluidics‐ and nanofluidics‐enabled next‐generation intracellular delivery platforms are outlined.

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“…Various physical strategies have been reported to generate transient nanopores on the cell membranes. [ 151–158 ] For example, Chung and co‐workers developed a hydroporation strategy to induce rapid cell deformation using fluid inertia in microfluidic platforms with cross‐ or T‐shaped channels. [ 151–153 ] Figure 5a shows the most recent design of microfluidic hydroporation.…”

Section: Programming Living Cellsmentioning

confidence: 99%

Recent Advances in Microfluidic Platforms for Programming Cell‐Based Living Materials

2021

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Cell‐based living materials, including single cells, cell‐laden fibers, cell sheets, organoids, and organs, have attracted intensive interests owing to their widespread applications in cancer therapy, regenerative medicine, drug development, and so on. Significant progress in materials, microfabrication, and cell biology have promoted the development of numerous promising microfluidic platforms for programming these cell‐based living materials with a high‐throughput, scalable, and efficient manner. In this review, the recent progress of novel microfluidic platforms for programming cell‐based living materials is presented. First, the unique features, categories, and materials and related fabrication methods of microfluidic platforms are briefly introduced. From the viewpoint of the design principles of the microfluidic platforms, the recent significant advances of programming single cells, cell‐laden fibers, cell sheets, organoids, and organs in turns are then highlighted. Last, by providing personal perspectives on challenges and future trends, this review aims to motivate researchers from the fields of materials and engineering to work together with biologists and physicians to promote the development of cell‐based living materials for human healthcare‐related applications.

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Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding

Cited by 4 publications

References 38 publications

Materials for Improving Immune Cell Transfection

Materials for Improving Immune Cell Transfection

Microfluidic and Nanofluidic Intracellular Delivery

Recent Advances in Microfluidic Platforms for Programming Cell‐Based Living Materials

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