Methods of electromechanical transfection

Abstract

Methods, devices, systems and kits for electromechanical cell transfection are provided, the devices including a first electrode, a second electrode and an active zone therebetween, where a potential difference applied to the first and second electrodes generates an electric field in the active zone sufficient to transfect at least a subset of cells flowing into the active zone.

Description

[Technical field]

[0001] Federally sponsored research This invention was made with support from the National Science Foundation under Phase II SBIR Grant No. 1747096 and Phase II SBIR Grant No. 1853194. The Government has certain rights in this invention. The present invention relates to a method of electromechanical transfection. [Background technology]

[0002] Immunotherapy is currently at the forefront of both basic science research and pharmaceutical clinical applications. This trend is driven in part by recent breakthroughs in targeted gene modification and the expanding use of CRISPR / Cas complex editing for therapeutic development. To identify therapeutic gene modifications, laboratories often must screen thousands of genetic variants, including modifications of endogenous genes and insertions of engineered genes. This drug discovery process is laborious and typically requires significant manual labor in laboratories, creating an industry-wide bottleneck due to the lack of suitable high-throughput technologies.

[0003] Biotechnology and pharmaceutical R&D activities are shifting towards automating almost every step of the process. Workflows include liquid handling robots with advanced laboratory management software, enabling high-throughput discovery. However, the transfection step is limited to low-throughput, inefficient techniques, and user-intensive systems that cannot be automated. An automated platform for transfection could not only significantly reduce process costs, but also increase cell viability and the amount of successfully engineered cells.

[0004] The unique strength of electroporation transfection is RNA delivery. Although existing viral technologies for DNA delivery appear to be comparable to electroporation transfection, there is a shortage of GMP-quality non-retroviral RNA viruses. Therefore, companies with electroporation platforms are targets for partnerships and acquisitions for the purpose of delivering mRNA into cells.

[0005] Current high-throughput gene transfer methods generally require viral transduction (e.g., lentiviral vectors), where viral particles infect cells and introduce the desired genetic modification. Although viral methods are amenable to high-throughput automated systems, they have production limitations and extend the timeline of research efforts. Viral vectors must be cloned and introduced into a viral production line, and then viral particles must be purified. This process can take laboratories several months, significantly impacting platform development timelines while also increasing drug discovery costs. In addition, viral transduction for gene transfer is not adaptable to genetic modification of all cell types, as some cells (e.g., certain immune cell subsets) are resistant to viral infection. Thus, there is an unmet need in the biotechnology industry for high-throughput automated systems for gene transfer that do not rely on viral transduction mechanisms. Summary of the Invention [Means for solving the problem]

[0006] We have demonstrated through the work described herein that a non-viral approach using electromechanical transfection, including the use of an electric field combined with high flow rates, is a scalable strategy for the development and manufacture of ex vivo cell therapies. Unlike purely electric field-based or purely mechanically-based poration methods, the combined effects of electromechanical transfection allow for the delivery of genetic material with high efficiency and low toxicity. This invention is the first to show that electromechanical transfection can be successfully used in human primary T cells. Utilizing a commercially available liquid handling system, rapid optimization of transduction into expanded T cells was observed with >90% efficiency and >80% viability. Validation of optimized electromechanical transfection parameters was evaluated in multiple use cases, including delivery to naive T cells, NK cells, B cells, monocytes, and macrophages, delivery of multiple payloads, and demonstration of 50-fold scale-up. Furthermore, transcriptome and ontology analyses indicate that highly efficient, high viability delivery by electromechanical transfection results in minimal gene dysregulation (only 2% change from baseline).The present invention demonstrates that non-viral electromechanical transfection is an efficient and scalable method for cell and gene therapy engineering and development.

[0007] Thus, in one aspect, the invention provides a method of introducing a composition into a plurality of mammalian cells suspended in a flowing liquid using any of the devices or systems of the invention (e.g., by electromechanical transfection). Specifically, the method of the invention includes providing a device including an entry zone with a first inlet and a first outlet, first and second electrodes, and an active zone with a second inlet and a second outlet. The method further includes selecting a combination of electric field (E), mean flow velocity (u), hydraulic diameter of the active zone (d), liquid conductivity (σ), liquid dynamic viscosity (μ), and liquid density (ρ) to achieve a concentration of at least 1×10 8 and 1×10 10 A dimensionless parameter Π with values ​​between 5where the dimensionless parameter Π 5 teeth,

number

[0008] In some embodiments, the composition is at least 1×10 per minute per active zone. 5 Cells, e.g. 10 5 cells / min~10 12 With a flux of cells / min, multiple cells, e.g., 10 per active zone 5 cells / min~10 6 cells / min, 5×10 5 cells / min~5×10 6 cells / min, 10 6 cells / min~10 7 cells / min, 5×10 6 cells / min~5×10 7 cells / min, 10 7 cells / min~10 8 cells / min, 5×10 7 cells / min~5×10 8 cells / min, 10 8 cells / min~10 9 cells / min, 5×10 8 cells / min~5×10 9 cells / min, 10 9 cells / min~10 9 cells / min, 5×10 9 cells / min~5×10 10 cells / min, or 10 10 cells / min~10 11 cells / min, e.g., about 10 3 cells / min, 5×10 3 cells / min, 10 4 cells / min, 5×10 4 cells / min, 10 5 cells / min, 5×10 5 cells / min, 10 6cells / min, 5×10 6 cells / min, 10 7 cells / min, 5×10 7 cells / min, 10 8 cells / min, 5×10 8 cells / min, 10 9 cells / min, 5×10 9 cells / min, 10 10 cells / min, 5×10 10 cells / min, or 10 11 cells / min, introduced.

[0009] In some embodiments, the entry zone and active zone are configured to provide an increased average flow velocity, for example relative to the average flow velocity in the entry zone.

[0010] In another aspect, the invention provides a method of introducing a composition into a plurality of cells suspended in a flowing liquid. The method includes providing a device including an entry zone having a first inlet and a first outlet, first and second electrodes, and an active zone including a second inlet and a second outlet. The method further includes passing the plurality of cells and the composition through the active zone while simultaneously delivering electrical energy from the first and second electrodes and simultaneously delivering mechanical energy at least in part from an average flow rate, the mechanical energy and electrical energy together introducing the composition into the plurality of cells with an efficiency, yield, and / or viability at least equal to that of electroporation or mechanical poration alone, and with less electrical or mechanical energy than electroporation requires to achieve the efficiency, yield, and / or viability.

[0011] In some embodiments, the composition is at least 1×10 per active zone. 5 Flux of cells / min, e.g., 10 per active zone 5 cells / min~10 6 cells / min, 5×10 5 cells / min~5×10 6 cells / min, 10 6 cells / min~10 7 cells / min, 5×106 cells / min~5×10 7 cells / min, 10 7 cells / min~10 8 cells / min, 5×10 7 cells / min~5×10 8 cells / min, 10 8 cells / min~10 9 cells / min, 5×10 8 cells / min~5×10 9 cells / min, 10 9 cells / min~10 9 cells / min, 5×10 9 cells / min~5×10 10 cells / min, or 10 10 cells / min~10 11 cells / min, e.g., about 10 3 cells / min, 5×10 3 cells / min, 10 4 cells / min, 5×10 4 cells / min, 10 5 cells / min, 5×10 5 cells / min, 10 6 cells / min, 5×10 6 cells / min, 10 7 cells / min, 5×10 7 cells / min, 10 8 cells / min, 5×10 8 cells / min, 10 9 cells / min, 5×10 9 cells / min, 10 10 cells / min, 5×10 10 cells / min, 10 11 cells / min, 5×10 11 cells / min, or 10 12 cells / min.

[0012] In some embodiments, the ratio of electrical energy provided by the electric field to mechanical energy provided to the flowing liquid by the product of the pressure drop in the active zone and the flow rate is greater than or equal to 10 3 :1 and 10 6 Between :1 and :10 3 :1 and 10 5 Between :1 and 10 4 :1 and 10 6 Between :1 and 103 :1 and 10 4 Between :1 and 10 5 :1 and 10 6 Between :1 (e.g., about 10 3 :1, 10 4 :1, 10 5 :1, or 10 6 :1). In some embodiments, the entry zone and the active zone are configured to provide an increase in average flow velocity.

[0013] Another aspect of the invention provides a method for introducing a composition to a plurality of cells suspended in a flowing liquid. The method includes providing a device including an entry zone having a first inlet and a first outlet, a first and second electrode, and an active zone having a hydraulic diameter (d), the active zone including a second inlet and a second outlet. The method further includes providing a test portion of the plurality of cells, both having a liquid conductivity (σ), a liquid dynamic viscosity (μ), and a liquid density (ρ), and a ratio of cells to composition, and a test composition, and passing the test portion and the test composition through the active zone at a mean flow rate (u) while applying an electric field (E), where at least one of (u), (E), (σ), (μ), and (ρ) is varied. The method further includes determining a dimensionless parameter Π, including a maximum yield, efficiency, and / or cell viability, for the introduction of the test composition to the test portion of the plurality of cells. 5 wherein:

number

[0014] In some embodiments, the range determination is repeated where the test portion of the plurality of cells and the test composition has a second ratio of cells to composition and / or where the active zone has a second hydraulic diameter (d).

[0015] In some embodiments, the method comprises varying the mean flow velocity (u) while holding the electric field (E) constant, or Π 5 This involves varying the electric field (E) while holding the mean flow velocity (u) constant during the range determination step.

[0016] In some embodiments, the composition is at least 1×10 per minute per active zone. 5 Cells, e.g., 10 per active zone 5 cells / min~10 12 Cells / min, e.g., 10 per active zone 5 cells / min~10 6 cells / min, 5×10 5 cells / min~5×10 6 cells / min, 10 6 cells / min~10 7 cells / min, 5×10 6 cells / min~5×10 7 cells / min, 10 7 cells / min~10 8 cells / min, 5×10 7 cells / min~5×10 8 cells / min, 10 8 cells / min~10 9 cells / min, 5×10 8 cells / min~5×10 9 cells / min, 10 9 cells / min~10 9 cells / min, 5×10 9 cells / min~5×10 10 cells / min, or 10 10 cells / min~10 11 Cells / min, e.g., about 10 per active zone 5 cells / min, 5×10 5 cells / min, 10 6 cells / min, 5×10 6 cells / min, 10 7 cells / min, 5×10 7 cells / min, 10 8 cells / min, 5×10 8 cells / min, 10 9 cells / min, 5×10 9 cells / min, 1010 cells / min, 5×10 10 cells / min, 10 11 cells / min, 5×10 11 cells / min, or 10 12 cells / min.

[0017] In some embodiments, the entry zone and active zone are configured to provide an increased mean flow velocity (u).

[0018] In another aspect, the invention provides a method of introducing a composition into a plurality of human immune cells isolated from a suspension in a flowing liquid taken from blood of a normal patient or donor. The method includes providing a device including an entry zone with a first inlet and a first outlet, first and second electrodes, and an active zone with a second inlet and a second outlet. The method further includes selecting a combination of electric field (E), mean flow velocity (u), hydraulic diameter (d) of the active zone, liquid conductivity (σ), liquid dynamic viscosity (μ) (e.g., as measured by a rotational viscometer), and liquid density (ρ) to achieve a composition of greater than 1×10 8 and 1×10 10 A dimensionless parameter Π with values ​​between 5 where the dimensionless parameter Π 5 teeth,

number

[0019] In some embodiments, the suspension is prepared using leukoreduction. In some embodiments, the composition contains at least 1×10 5 Cells / min, e.g. 10 per active zone 5 cells / min~10 12Cells / min, e.g. 10 per active zone 5 cells / min~10 6 cells / min, 5×10 5 cells / min~5×10 6 cells / min, 10 6 cells / min~10 7 cells / min, 5×10 6 cells / min~5×10 7 cells / min, 10 7 cells / min~10 8 cells / min, 5×10 7 cells / min~5×10 8 cells / min, 10 8 cells / min~10 9 cells / min, 5×10 8 cells / min~5×10 9 cells / min, 10 9 cells / min~10 9 cells / min, 5×10 9 cells / min~5×10 10 cells / min or 10 10 cells / min~10 11 Cells / min, e.g., about 10 per active zone 5 cells / min, 5×10 5 cells / min, 10 6 cells / min, 5×10 6 cells / min, 10 7 cells / min, 5×10 7 cells / min, 10 8 cells / min, 5×10 8 cells / min, 10 9 cells / min, 5×10 9 cells / min, 10 10 cells / min, 5×10 10 cells / min, 10 11 cells / min, 5×10 11 cells / min, or 10 12The composition is introduced into the plurality of cells at a flux of cells / minute. In some embodiments, the entry zone and active zone are configured to provide an increase in average flow rate, for example, relative to the flow rate through the entry zone. In some embodiments, the composition is introduced into the plurality of mammalian cells without altering desired cell surface markers. Cell surface markers include, but are not limited to, CD3, CD4, CD8, CD19, CD45RA, CD45RO, CD28, CD44, CD69, CD80, CD86, CD206, IL-2 receptor, CTLA4, OX40, PD-1, TIM3, CD56, TNFa, IFNg, LAG3, TCR alpha / beta, CD64, SIRP alpha / beta (CD172a / b), nestin, CD325 (N-cadherin), CD183 (CXCR 3), CD184 (CXCR4), CD197 (CCR7), CD27, CD11b, CCR7 (CD197), CD16, CD56, TIGIT, TRA-1-60, Nanog, TCR gamma / delta, OCT4, T-bet, GATA-3, FoxP3, IL-17, B220, CD25, IgM, PD-L1, IL-23, IL-12, CD11c, and F4 / 80.

[0020] In some embodiments of any of the preceding aspects, the active zone comprises a minimum hydraulic diameter of greater than 100 μm (e.g., between 100 μm and 10 mm, between 150 μm and 15 mm, between 200 μm and 10 mm, between 250 μm and 5 mm, between 500 μm and 10 mm, between 1 mm and 10 mm, between 1 mm and 50 mm, between 5 mm and 25 mm, or between 20 mm and 50 mm, e.g., about 0.5 mm, 1.0 mm, 1.5 mm, 2 mm, 5 mm, 10 mm, 15 mm, 25 mm, or 50 mm). In some embodiments, in any of the preceding aspects, the active zone has a minimum hydraulic diameter greater than the average cell diameter in the plurality of cells, e.g., at least 1.1 times the average cell diameter, e.g., 1-10 times the average cell diameter (e.g., 1-2 times, 2-3 times, 3-4 times, 4-5 times, 5-6 times, 6-7 times, 7-8 times, 8-9 times, or 1-10 times), or, e.g., 10-100 times the average cell diameter (e.g., 10-20 times, 20-30 times, 30-40 times, 40-50 times, 50-60 times, 60-70 times, 70-80 times, 80-90 times, or 10-100 times), or, e.g., 100-1,000 times the average cell diameter (e.g., 100-200 times, 200-300 times, 30 0-400x, 400-500x, 500-600x, 600-700x, 700-800x, 800-900x, or 100-1,000x), or, for example, 1,000-10,000x the average cell diameter (e.g., 1,000-2,000x, 2,000-3,000x, 3,000-4,000x, 4,000-5,000x, 5,000-6,000 times, 6,000-7,000 times, 7,000-8,000 times, 8,000-9,000 times, or 1,000-10,000 times), or greater than, for example, 10,000 times the average cell diameter, for example, 12,000 times, 15,000 times, 18,000 times, or 20,000 times the average cell diameter, including the minimum hydraulic diameter.

[0021] In some embodiments of any of the preceding aspects, the active zone has a substantially uniform cross-sectional area.

[0022] In some embodiments of any of the preceding aspects, the flow rate through the active zone is between 0.001 mL / min and 1,000 mL / min (e.g., between 0.001 mL / min and 0.05 mL / min, between 0.001 mL / min and 0.1 mL / min, between 0.001 mL / min and 1 mL / min, between 0.05 mL / min and 0.5 mL / min, between 0.05 mL / min and 5 mL / min, between 0.1 mL / min and 1 mL / min, between 0.5 mL / min and 2 mL / min, between 1 mL / min and 5 mL / min, Between 1mL / min and 10mL / min, Between 1mL / min and 100mL / min, Between 5mL / min and 25mL / min, Between 5mL / min and 150mL / min, Between 10mL / min and 100mL / min, Between 15mL / min and 150mL / min, Between 25mL / min and 100mL / min, Between 25mL / min and 200mL / min, Between 50mL / min and 150mL / min, Between 50mL / min and 250mL / min, Between 75mL / min and 200mL / min, Between 75mL / min and 350mL / min, Between 100mL / min and 250mL / min, Between 25mL / min and 200mL / min, Between 2 ...350mL / min and 350mL / min, Between 350mL / min and 400mL / min, Between 40mL / min and 50mL / min, Between 40mL / min and 50mL / min, Between 50mL / min and 150mL / min, Between 50mL / min and 250mL / min, Between 75mL / min and 350mL / min, Between 100mL / min and 250mL / min, Between 350mL / min and 400mL / min, Between 40mL / min and 50mL / min, Between 40mL / min and 50mL / min, Between L / min and 250 mL / min, between 100 mL / min and 400 mL / min, between 150 mL / min and 450 mL / min, between 200 mL / min and 500 mL / min, between 250 mL / min and 700 mL / min, between 300 mL / min and 1,000 mL / min, between 400 mL / min and 750 mL / min, between 500 mL / min and 1,000 mL / min, or between 750 mL / min and 1,000 mL / min, for example, about 0.001 mL / min, 0.01 mL / min, 0.05 mL / min, 0.1 mL / min, / min, 0.5 mL / min, 1 mL / min, 5 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, 90 mL / min, 100 mL / min, 150 mL / min, 200 mL / min, 250 mL / min, 300 mL / min, 350 mL / min, 400 mL / min, 450 mL / min, 500 mL / min, 600 mL / min, 700 mL / min, 800 mL / min, 900 mL / min, or 1,000 mL / min).

[0023] In some embodiments of any of the preceding aspects, the Reynolds number (ρ / μ) of the flowing liquid in the active zone (based on the hydraulic diameter (d) of the active zone) is between 10 and 3000 (e.g., between 10 and 2000, between 100 and 1600, between 100 and 1800, or between 183 and 1530).

[0024] In some embodiments, the average flow rate of the flowing liquid in suspension therein through the active zone is greater than 1×10 -2 between m / s and 10 m / s, for example between 0.01 and 1 m / s (for example between 0.01 and 0.05 m / s, between 0.05 and 0.1 m / s, between 0.1 and 0.5 m / s, between 0.5 and 1 m / s, between 1.5 and 2 m / s, between 1 and 2 m / s, between 2 and 3 m / s, between 3 and 4 m / s, between 4 and 5 m / s, between 5 and 6 m / s, between 6 and 7 m / s, between 7 and 8 m / s, between 8 and 9 m / s, or between 9 and 10 m / s), for example between 0.1 and 5 m / s. , between 0.4 and 1.4 m / s, between 0.65 and 1.3 m / s, or between 0.26 and 2.08 m / s, for example, about 0.1 m / s, 0.2 m / s, 0.3 m / s, 0.4 m / s, 0.5 m / s, 0.6 m / s, 0.7 m / s, 0.8 m / s, 0.9 m / s, 1.0 m / s, 1.1 m / s, 1.2 m / s, 1.3 m / s, 1.4 m / s, 1.5 m / s, 2 m / s, 3 m / s, 4 m / s, 5 m / s, 6 m / s, 7 m / s, 8 m / s, 9 m / s, or 10 m / s.

[0025] In some embodiments, the peak pressure of the flowing liquid in the suspension during passage through the active zone is greater than or equal to 1×10 -3 Pa and 9.5 x 10 4 Pa (for example, between 0.1 Pa and 10,000 Pa, between 1 Pa and 5,000 Pa, between 100 Pa and 3000 Pa, or between 136 Pa and 1600 Pa).

[0026] In some embodiments of any of the preceding aspects, the residence time of the plurality of cells suspended in the liquid in any active zone is between 0.1 ms and 50 ms (e.g., between 0.1 ms and 0.5 ms, between 0.5 ms and 5 ms, between 1 ms and 10 ms, between 1 ms and 15 ms, between 5 ms and 15 ms, between 10 ms and 20 ms, between 15 ms and 25 ms, between 20 ms and 30 ms, between 25 ms and 35 ms, between 30 ms and 40 ms, between 35 ms and 45 ms, or between 40 ms and 50 ms). For example, about 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, 1 ms, 1.5 ms, 2 ms, 2.5 ms, 3 ms, 3.5 ms, 4 ms, 4.5 ms, 5 ms, 5.5 ms, 6 ms, 6.5 ms, 7 ms, 7.5 ms, 8 ms, 8.5 ms, 9 ms, 9.5 ms, 10 ms, 10.5 ms, 11 ms, 11.5 ms, 12 ms, 12.5 ms, 13 ms, 13.5 ms, 14 ms, 14.5 ms, 15 ms, 20 ms, 25 ms, 30 ms, 35 ms, 40 ms, 45 ms, or 50 ms). In some embodiments, the dwell time is 5 to 20 ms (e.g., 6 to 18 ms, 8 to 15 ms, or 10 to 14 ms).

[0027] In some embodiments of any of the preceding aspects, the electric field is generated by a voltage pulse, where the voltage pulse energizes the first electrode with a particular applied voltage while energizing the second electrode with a particular applied voltage, thus applying a potential difference between the first and second electrodes, where the voltage pulse may be between -3 kV and 3 kV (e.g., between -3 kV and 1 kV), between -3 kV and 1 kV, between -3 kV and -1.5 kV, between -2 kV and 2 kV, between -1.5 kV and 1.5 kV, between -1.5 kV and 2.5 kV, between -1 kV and 1 kV, between -1 kV and between 2kV, between -0.5kV and 0.5kV, between -0.5kV and 1.5kV, between -0.5kV and 3kV, between -0.01kV and 2kV, between 0kV and 1kV, between 0kV and 2kV, between 0kV and 3kV, between 0.01kV and 0.1kV, between 0.01kV and 1kV, between 0.02kV and 0.2kV, between 0.03kV and 0.3kV, between 0.04kV and 0.4kV, between 0.05kV and 0.5kV, between 0.05kV and 1.5kV, between 0.06kV and 0.6kV, between 0.07kV and 0.7kV, between 0.08kV and between 0.8kV, between 0.09kV and 0.9kV, between 0.1kV and 0.7kV, between 0.1kV and 1kV, between 0.1kV and 2kV, between 0.1kV and 3kV, between 0.15kVtp5kV, between 0.2kV and 0.6kV, between 0.2kV and 2kV, between 0.25kV and 2.5kV, between 0.3kV and 3kV, between 0.5kV and 1kV, between 0.5kV and 3kV, between 0.6kV and 1.5kV, between 0.7kV and 1.8kV, between 0.8kV and 2kV, between 0.9kV and 3kV, between 1kV and 2kV, between 1.5kV and Between 2.5 kV or between 2 kV and 3 kV, for example, about 3 kV, -2.5 kV, -2 kV, -1.5 kV, -1 kV, -0.5 kV, -0.01 kV, 0 kV, 0.01 kV, 0.02 kV, 0.03 kV, 0.04 kV, 0.05 kV, 0.06 kV, 0.07 kV, 0.08 kV, 0.09 kV, 0.1 kV, 0.2 kV, 0.3 kV, 0.4 kV, 0.5 kV, 0.6 kV, 0.7 kV, 0.8 kV, 0.9 kV, 1 kV, 1.1 kV, 1.2 kV, 1.3 kV, 1.4 kV, 1.5 kV, 1.6 kV, 1.7 kV, 1.8 kV, 1.9 kV,The applied voltage may have an amplitude of 2 kV, 2.1 kV, 2.2 kV, 2.3 kV, 2.4 kV, 2.5 kV, 2.6 kV, 2.7 kV, 2.8 kV, 2.9 kV, or 3 kV. In some embodiments, the first electrode is energized with a particular applied voltage while the second electrode is held at ground (e.g., 0 kV), thereby applying a potential difference between the first and second electrodes. In some embodiments, the voltage pulse is between 0.01 ms and 1,000 ms (e.g., between 0.01 ms and 0.1 ms, between 0.01 ms and 1 ms, between 0.01 ms and 10 ms, between 0.05 ms and 0.5 ms, between 0.05 ms and 1 ms, between 0.1 ms and 1 ms, between 0.1 ms and 5 ms, between 0.1 ms and 500 ms, between 0.5 ms and 2 ms, between 1 ms and 5 ms, , between 1ms and 10ms, between 1ms and 25ms, between 1ms and 100ms, between 1ms and 1,000ms, between 5ms and 25ms, between 5ms and 150ms, between 10ms and 100ms, between 15ms and 150ms, between 25ms and 100ms, between 25ms and 200ms, between 50ms and 150ms, between 50ms and 250ms, between 75ms and 200ms, between 75 between 100 ms and 350 ms, between 100 ms and 250 ms, between 100 ms and 400 ms, between 150 ms and 450 ms, between 200 ms and 500 ms, between 250 ms and 700 ms, between 300 ms and 1,000 ms, between 400 ms and 750 ms, between 500 ms and 1,000 ms, or between 750 ms and 1,000 ms, e.g., about .01 ms, 0.05 ms, 0.1 ms, 0.5 ms, 1 ms, 5 ms, 10 ms, 15 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 150 ms, 200 ms, 250 ms, 300 ms, 350 ms, 400 ms, 450 ms, 500 ms, 600 ms, 700 ms, 800 ms, 900 ms, or 1,000 ms. In some embodiments, the voltage pulse has a duration of at least 1000 ms. In some embodiments, the voltage pulse has a frequency between 1 Hz and 50,000 Hz (e.g., between 1 Hz and 10 Hz, between 1 Hz and 100 Hz,Between 1Hz and 1,000Hz, Between 5Hz and 20Hz, Between 5Hz and 2,000Hz, Between 10Hz and 50Hz, Between 10Hz and 100Hz, Between 10Hz and 1,000Hz, Between 10Hz and 10,000Hz, Between 20Hz and 50Hz, Between 20Hz and 100Hz, Between 20Hz and 2,000Hz, Between 20Hz and 20,000Hz, Between 50Hz and 500Hz, Between 50Hz and 1,000Hz, Between 50Hz and 50,000Hz between 100Hz and 200Hz, between 100Hz and 500Hz, between 100Hz and 1,000Hz, between 100Hz and 10,000Hz, between 100Hz and 50, between 200Hz and 400Hz, between 200Hz and 750Hz, between 20Hz and 2,000Hz, between 500Hz and 1,000Hz, between 750Hz and 1,500Hz, between 750Hz and 10,000Hz, between 1,000Hz and 2,000Hz, between 1,000Hz and 5,0 between 1,000Hz and 10,000Hz, between 1,000Hz and 50,000Hz, between 5,000Hz and 10,000Hz, between 5,000Hz and 20,000Hz, between 5,000Hz and 50,000Hz, between 10,000Hz and 15,000Hz, between 10,000Hz and 25,000Hz, between 10,000Hz and 50,000Hz, between 20,000Hz and 30,000Hz, or between 20,000Hz and 50 The electrical current is applied to the first and second electrodes at a frequency between about 1 Hz, 5 Hz, 10 Hz, 20 Hz, 50 Hz, 75 Hz, 100 Hz, 150 Hz, 200 Hz, 300 Hz, 400 Hz, 500 Hz, 600 Hz, 700 Hz, 800 Hz, 900 Hz, 1,000 Hz, 2,000 Hz, 5,000 Hz, 10,000 Hz, 15,000 Hz, 20,000 Hz, 30,000 Hz, 40,000 Hz, or 50,000 Hz.

[0028] In some embodiments, the waveform of the voltage pulse is selected from the group consisting of DC, rectangular, pulse, bipolar, sinusoidal, ramp, asymmetric bipolar, any, and any superposition or combination thereof. In some embodiments, the electric field generated from the voltage pulse is between 100V / cm and 50,000V / cm (e.g., between 100V / cm and 500V / cm, between 100V / cm and 1,000V / cm, between 100V / cm and 2,000V / cm, between 100V / cm and 5,000V / cm, between 250V / cm and 2000V / cm, between 500V / cm and 2500V / cm, between 500V / cm and 5000V / cm, between 500V / cm and 1,500V / cm). / cm, between 300V / cm and 500V / cm, between 1000V / cm and 2,000V / cm, for example, about 100V / cm, 150V / cm, 200V / cm, 250V / cm, 300V / cm, 350V / cm, 400V / cm, 450V / cm, 500V / cm, 550V / cm, 600V / cm, 650V / cm, 700V / cm, 750V / cm, 800V / cm, 900V / cm, 1,000V / cm, or 2,000V / cm).

[0029] In some embodiments, the duty cycle of the voltage pulses is between 1% and 100% (e.g., between 1% and 10%, between 1% and 97%, between 2.5% and 20%, between 5% and 25%, between 5% and 40%, between 10% and 25%, between 10% and 50%, between 10% and 95%, between 15% and 60%, between 15% and 85%, between 20% and 40%, between 30% and 50%, between 40% and 60%). between 40% and 75%, between 50% and 85%, between 50% and 100%, between 75% and 100%, or between 90% and 100%, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%).

[0030] In some embodiments, the liquid has a viscosity of between 0.001 mS / cm and 500 mS / cm (e.g., between 0.001 mS / cm and 0.05 mS / cm, between 0.001 mS / cm and 0.1 mS / cm, between 0.001 mS / cm and 1 mS / cm, between 0.05 mS / cm and 0.5 mS / cm, between 0.05 mS / cm and 5 mS / cm, between 0.1 mS / cm and 1 mS / cm, between 0.1 mS / cm and 100 mS / cm, between 0.5 mS / cm and 2 mS / cm, between 1 mS / cm and 5 between mS / cm, between 1mS / cm and 10mS / cm, between 1mS / cm and 100mS / cm, between 1mS / cm and 500mS / cm, between 5mS / cm and 25mS / cm, between 5mS / cm and 150mS / cm, between 10mS / cm and 100mS / cm, between 10mS / cm and 250mS / cm, between 15mS / cm and 150mS / cm, between 25mS / cm and 100mS / cm, between 25mS / cm and 200mS / cm, between 50mS / cm and 150mS / cm, Between 50 mS / cm and 250 mS / cm, between 50 mS / cm and 500 mS / cm, between 75 mS / cm and 200 mS / cm, between 75 mS / cm and 350 mS / cm, between 100 mS / cm and 250 mS / cm, between mS / cm and 400 mS / cm, between 100 mS / cm and 500 mS / cm, between 150 mS / cm and 450 mS / cm, between 200 mS / cm and 500 mS / cm, between 300 mS / cm and 500 mS / cm, for example, about 0.001 mS / cm, 0.0 In some embodiments, the conductivity is 1 mS / cm, 0.05 mS / cm, 0.1 mS / cm, 0.5 mS / cm, 1 mS / cm, 5 mS / cm, 10 mS / cm, 15 mS / cm, 20 mS / cm, 30 mS / cm, 40 mS / cm, 50 mS / cm, 60 mS / cm, 70 mS / cm, 80 mS / cm, 90 mS / cm, 100 mS / cm, 150 mS / cm, 200 mS / cm, 250 mS / cm, 300 mS / cm, 350 mS / cm, 400 mS / cm, 450 mS / cm, or 500 mS / cm).

[0031] In some embodiments, the temperature of the plurality of cells suspended in the liquid is between 0° C. and 50° C. (between 0° C. and 5° C., between 2° C. and 15° C., between 3° C. and 30° C., between 4° C. and 10° C., between 4° C. and 25° C., between 5° C. and 30° C., between 7° C. and 35° C., between 10° C. and 25° C., between 10° C. and 40° C., between 15° C. and 50° C., between 20° C. and 40° C., between 25° C. and 50° C., or between 35° C. and and 45°C, for example, about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C).

[0032] In some embodiments, the method further comprises storing the plurality of cells suspended in the liquid in a harvest buffer after transfection. In some embodiments, the cells are in a concentration of between 0.1% and 99.9% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 99.9%, between 60% and 80%, between 75% and 99.9%, or between 85% and 99. 9%, for example, about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9%).

[0033] In some embodiments, the composition comprises a solubility of between 0.1% and 99.9% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 99.9%, between 60% and 80%, between 75% and 99.9%, or between 85% and 99.9%, For example, the antibody may be introduced into a plurality of cells with an efficiency of about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9%).

[0034] In some embodiments, the method provides a method for determining a concentration of 100% or more of a 100% or more ... , for example, about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%).In some embodiments, the method includes providing a concentration of between 0.1% and 500% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 85%, between 50% and 9 ... Between 0% and 75%, between 50% and 100%, between 60% and 80%, between 60% and 150%, between 75% and 100%, between 75% and 200%, between 85% and 150%, between 90% and 250%, between 100% and 200%, between 100% and 400%, between 150% and 300%, between 200% and 500%, or between 300% and 500%, for example, about 0.1%, 0.15%, 0.2%, 0.25% ,0.3%,0.35%,0.4%,0.45%,0.5%,0.55%,0.6%,0.65%,0.7%,0.75%,0.8%,0.85%,0.9%,0.95%,1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,15%,20%,25%,30%,35%,40%,45,50%,55%,60%,65%,70%,75%,80%,85%,90%,95%,99%,100%,1 50%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%), resulting in a yield of live engineered cells (e.g., recovery yield).

[0035] In some embodiments, the method includes providing a cellular lysate of between 0.1% and 100% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 100%, between 60% and 80%, between 60% and 90%, between 75% and 100%, or between 85% and 100%) immediately after transfection. 00%, e.g., between about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

[0036] In some embodiments, the composition delivered to the plurality of cells (e.g., electromechanically delivered to the plurality of cells) comprises at least one compound selected from the group consisting of a therapeutic agent, a vitamin, a nanoparticle, a charged molecule, an uncharged molecule, an engineered nuclease, DNA, RNA, a CRISPR-Cas complex, a CRISPR-Cas complex, a CRISPR-Cas complex, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a homing nuclease, a meganuclease (mns), a megaTAL, an enzyme, a transposon, a peptide, a protein, a virus, a polymer, a ribonucleoprotein (RNP), and a polysaccharide. In some embodiments, the composition comprises a concentration of between 0.0001 μM and 20 μM (e.g., 0.0001 μM to 0.001 μM, 0.001 μM to 0.01 μM, 0.001 μM to 5 μM, 0.005 μM to 0.1 μM, 0.01 μM to 0.1 μM, 0.01 μM to 1 μM, 0.1 μM to 1 μM, 0.1 μM to 5 μM, 1 μM to 10 μM, 1 μM to 15 μM, or 1 μM to 20 μM, e.g., about 0.0001 μM, 0.0005 μM, 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM , 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM, respectively). In some embodiments, the composition has a concentration between 0.0001 μg / mL and 1,000 μg / mL (e.g., 0.0001 μg / mL to 0.001 μg / mL, 0.001 μg / mL to 0.01 μg / mL, 0.001 μg / mL to 5 μg / mL, 0.005 μg / mL to 0.1 μg / mL, 0.01 μg / mL to 0.1 μg / mL, 0.01 μg / mL to 1 μg / mL, 0.1 μg / mL to 1 μg / mL, 0.1μg / mL~5μg / mL、1μg / mL~10μg / mL、1μg / mL~50μg / mL、1μg / mL~100μg / mL、2.5μg / mL~15μg / mL、5μg / mL~25μg / mL、5μg / mL~50μg / mL μg / mL、5μg / mL~500μg / mL、7.5μg / mL~75μg / mL、10μg / mL~100μg / mL、10μg / mL~1,000μg / mL、25μg / mL~50μg / mL、25μg / mL~25μg / mL 0μg / mL、25μg / mL~500μg / mL、50μg / mL~100μg / mL、50μg / mL~250μg / mL、50μg / mL~750μg / mL、100μg / mL~300μg / mL、100μg / mL ~1,000μg / mL、200μg / mL~400μg / mL、250μg / mL~500μg / mL、350μg / mL~500μg / mL、400μg / mL~1,000μg / mL、500μg / mL~750μg / mL mL, 650μg / mL to 1,000μg / mL, or 800μg / mL to 1,000μg / mL, for example about 0.0001μg / mL, 0.0005μg / mL, 0.001μg / mL, 0.005μg / mL, 0.01μg / mL, 0.02μg / mL, 0.03μg / mL, 0.04μg / mL, 0.05μg / mL, 0.06μg / mL, 0.07μg / mL, 0.08μg / mL, 0.09μg / mL, 0.1μg / mL, 0.2μg / mL L、0.3μg / mL、0.4μg / mL、0.5μg / mL、0.6μg / mL、0.7μg / mL、0.8μg / mL、0.9μg / mL、1μg / mL、1.5μg / mL、2μg / mL、2.5μg / mL、3μg / mL mL、3.5μg / mL、4μg / mL、4.5μg / mL、5μg / mL、5.5μg / mL、6μg / mL、6.5μg / mL、7μg / mL、7.5μg / mL、8μg / mL、8.5μg / mL、9μg / mL、9.It has a concentration in the liquid of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, 900 μg / mL, 950 μg / mL, or 1,000 μg / mL).

[0037] In some embodiments of any of the above aspects, the plurality of cells suspended in the liquid comprises eukaryotic cells (e.g., animal cells, e.g., human cells), prokaryotic cells (e.g., bacterial cells), plant cells, and / or synthetic cells. The cells can be primary cells (e.g., primary human cells), cells from a cell line (e.g., a human cell line), cells in suspension, adherent cells, stem cells, blood cells (e.g., peripheral blood mononuclear cells (PBMCs)), and / or immune cells (e.g., white blood cells (e.g., innate or adaptive immune cells)). In some embodiments, the cells (e.g., immune cells, e.g., T cells, B cells, natural killer cells, macrophages, monocytes, or antigen presenting cells) are unstimulated cells, stimulated cells, or activated cells. In some embodiments, the cells are adaptive immune cells and / or innate immune cells. In some embodiments, the plurality of cells is an antigen presenting cell (APC), a monocyte, a T cell, a B cell, a dendritic cell, a macrophage, a neutrophil, a NK cell, a Jurkat cell, a THP-1 cell, a human embryonic kidney (HEK-293) cell, a Chinese hamster ovary (e.g., CHO-K1) cell, an embryonic stem cell (ESC), a mesenchymal stem cell (MSC), or a hematopoietic stem cell (HSC). In some embodiments, the cell can be a primary human T cell, a primary human macrophage, a primary human monocyte, a primary human NK cell, or a primary human induced pluripotent stem cell (iPSC). In some embodiments of any of the methods described herein, the method further comprises storing the plurality of cells suspended in the liquid in a collection buffer after poration.

[0038] In some embodiments of any of the preceding aspects, the invention provides a kit including any of the devices or systems described herein, and a plurality of outer structures configured to encase a plurality of devices, e.g., a housing configured to encase a first electrode, a second electrode, and an active zone of at least one device, a first electrical input operably coupled to the first electrode, and a second electrical input operably coupled to the second electrode. In some embodiments, the plurality of outer structures are integral with the plurality of devices. In some embodiments, the plurality of outer structures are releasably coupled to the plurality of devices. In some embodiments, the housing further includes a thermal controller configured to increase the temperature of at least one device, the thermal controller being a heating element selected from the group consisting of a heat block, a liquid flow, a battery-powered heater, and a thin film heater. In some embodiments, the housing further includes a thermal controller configured to decrease the temperature of at least one device, the thermal controller being a cooling element selected from the group consisting of a liquid flow, an evaporative cooler, and a Peltier element.

[0039] In some embodiments of any of the preceding aspects, the invention provides a kit for introducing a composition into a plurality of cells suspended in a liquid, the kit comprising a plurality of devices as described herein and a plurality of outer structures configured to enclose the plurality of devices, each of the plurality of outer structures comprising a housing configured to enclose a first electrode, a second electrode, and an active zone of at least one of the devices, a first electrical input operably coupled to the first electrode, and a second electrical input operably coupled to the second electrode. In some embodiments, the plurality of outer structures are integral with the plurality of devices. In some embodiments, the plurality of outer structures are releasably coupled to the plurality of devices. In some embodiments, the housing further comprises a thermal controller configured to increase a temperature of at least one of the devices, the thermal controller being a heating element selected from the group consisting of a heat block, a liquid flow, a battery-powered heater, and a thin film heater. In some embodiments, the housing further comprises a thermal controller configured to decrease a temperature of at least one of the devices, the thermal controller being a cooling element selected from the group consisting of a liquid flow, an evaporative cooler, and a Peltier element.

[0040] In some embodiments of any of the preceding aspects, the invention provides a kit for electromechanically delivering a composition to a plurality of cells suspended in a liquid, the kit comprising a plurality of devices, each of the plurality of devices comprising a device of the above-mentioned embodiment, and a plurality of outer structures configured to surround the plurality of devices, each of the plurality of outer structures comprising a housing configured to surround the first electrode, the second electrode, and the active zone of at least one of the devices, a first electrical input operably coupled to the first electrode, and a second electrical input operably coupled to the second electrode. In some embodiments, the plurality of outer structures are integral with the plurality of devices. In some embodiments, the plurality of outer structures are releasably coupled to the plurality of devices. In some embodiments, the housing further comprises a thermal controller configured to increase the temperature of at least one of the devices, the thermal controller being a heating element selected from the group consisting of a heating block, a liquid flow, a battery-powered heater, and a thin film heater. In some embodiments, the housing further includes a thermal controller configured to reduce a temperature of the at least one device, the thermal controller being a cooling element selected from the group consisting of a liquid flow, an evaporative cooler, and a Peltier element.

[0041] In some embodiments of any of the preceding methods, the kit further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidly connected to a zone of the device, e.g., an entry zone, an activity zone, or a recovery zone. For example, the first reservoir is fluidly connected to the entry zone and the second reservoir is fluidly connected to the recovery zone (e.g., as a source of recovery buffer).

[0042] In some embodiments of any of the preceding aspects, a cross-section of the active zone is selected from the group consisting of a cylinder, an ellipse, a polygon, a star, a parallelogram, a trapezoid, and irregular.

[0043] In some cases, the hydraulic diameter of the entry zone or the hydraulic diameter of the recovery zone is between 0.01% and 100,000% of the hydraulic diameter of the active zone. For example, the hydraulic diameter of the entry zone or the hydraulic diameter of the recovery zone may be 0.01% to 1000% of the hydraulic diameter of the active zone, such as 0.01% to 1%, 0.1% to 10%, 5% to 25%, 10% to 50%, 10% to 1000%, 25% to 75%, 25% to 750%, or 50% to 1000% of the hydraulic diameter of the active zone. Alternatively, the hydraulic diameter of the entry zone or the hydraulic diameter of the recovery zone may be 100% to 100,000% of the hydraulic diameter of the active zone, for example, 100% to 1000%, 500% to 5,000%, 1,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000% of the hydraulic diameter of the active zone.

[0044] In some embodiments of any of the preceding aspects, the hydraulic diameter of the active zone is between 0.01 mm and 50 mm. In some embodiments, the length of the active zone is between 0.01 mm and 50 mm. In certain embodiments, the length of the active zone is between 0.01 mm and 25 mm. In some embodiments, the hydraulic diameter of either the first electrode or the second electrode is between 0.1 mm and 500 mm. In certain embodiments, none of the entry zone, collection zone, or active zone reduces any cross-sectional dimension of the plurality of cells suspended in the fluid, e.g., cells can pass through the device without deformation.

[0045] In further embodiments, the device includes an outer structure having a housing configured to encase the first electrode, the second electrode, and the active zone of the device. In some embodiments, the outer structure is integral with the device. In certain embodiments, the outer structure is releasably connected to the device.

[0046] In some embodiments of any of the preceding aspects, a cross-section of the active zone is selected from the group consisting of a cylinder, an ellipse, a polygon, a star, a parallelogram, a trapezoid, and irregular.

[0047] In some embodiments of any of the preceding aspects, the hydraulic diameter of the entry zone or the hydraulic diameter of the recovery zone is between 0.01% and 100,000% of the hydraulic diameter of the active zone. For example, the hydraulic diameter of the entry zone or the hydraulic diameter of the recovery zone may be between 0.01% and 1,000%, such as 0.01% and 1%, 0.1% and 10%, 5% and 25%, 10% and 50%, 10% and 1,000%, 25% and 75%, 25% and 750%, or 50% and 100% of the hydraulic diameter of the active zone. Alternatively, the hydraulic diameter of the entry zone or the hydraulic diameter of the recovery zone may be 100% to 100,000% of the hydraulic diameter of the active zone, for example, 100% to 1000%, 500% to 5,000%, 1,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000%.

[0048] In some embodiments of any of the preceding aspects, the hydraulic diameter of the active zone is between 0.01 mm and 50 mm. In some embodiments, the length of the active zone is between 0.005 mm and 50 mm. In certain embodiments, the length of the active zone is between 0.005 mm and 25 mm. In some embodiments, the hydraulic diameter of either the first electrode or the second electrode is between 0.1 mm and 500 mm. In certain embodiments, none of the entry zone, collection zone, or active zone reduces any cross-sectional dimension of the plurality of cells suspended in the fluid, e.g., cells can pass through the device without deformation.

[0049] In some embodiments of any of the preceding aspects, the first and / or second electrode is a porous or conductive fluid (eg, a liquid).

[0050] In further embodiments, the device includes an outer structure having a housing configured to encase the first electrode, the second electrode, and the active zone of the device. In some embodiments, the outer structure is integral with the device. In certain embodiments, the outer structure is releasably connected to the device.

[0051] In some embodiments of any of the preceding aspects, the invention provides a system for introducing a composition into a plurality of cells suspended in a flowing fluid by electromechanical transfection, the system comprising any of the devices described herein and an electrical potential source, wherein the first and second electrodes of the device are releasably connected to the electrical potential source, in which the plurality of cells suspended in the fluid are perforated upon entering the active zone.

[0052] In further embodiments, the device includes an outer structure having a housing configured to encase the first electrode, the second electrode, and the active zone of the device. In some embodiments, the outer structure includes a first electrical input operably coupled to the first electrode and a second electrical input operably coupled to the second electrode. In some embodiments, the releasable connection between the first or second electrical input and the source of electrical potential is selected from the group consisting of a clamp, a clip, a spring, a sheath, a wire brush, a mechanical connection, an inductive connection, or a combination thereof.

[0053] In some embodiments, the outer structure is integral to the device, hi certain embodiments, the outer structure is releasably connected to the device.

[0054] In some embodiments, the device, system, or method of any of the preceding aspects induces reversible pore formation. In certain embodiments, the electromechanical transfection is substantially non-thermally reversible electromechanical transfection.

[0055] In some embodiments, the releasable connection between the device and the electrical potential source is selected from the group consisting of a clamp, a clip, a spring, a sheath, a wire brush, a mechanical connection, an inductive connection, or a combination thereof, hi certain embodiments, the releasable connection between the device and the electrical potential source is a spring.

[0056] In some embodiments, the system further includes one or more reservoirs, e.g., a first reservoir and a second reservoir, fluidly connected to a zone of the device, e.g., an entry zone or a recovery zone. For example, the first reservoir can be fluidly connected to the entry zone and the second reservoir can be fluidly connected to the recovery zone.

[0057] In further embodiments, the system includes a fluid delivery source fluidly connected to the entry zone, the fluid delivery source configured to deliver a plurality of cells suspended in the fluid through the entry zone to the collection zone. In certain embodiments, the delivery rate from the fluid delivery source is between 0.001 mL / min and 1,000 mL / min, e.g., 25 mL / min. In certain embodiments, the residence time of any of the plurality of cells suspended in the fluid is between 0.5 ms and 50 ms. In certain embodiments, the conductivity of the fluid is between 0.001 mS / cm and 500 mS / cm, e.g., 1 to 20 mS / cm.

[0058] In further embodiments, the system includes a controller operably coupled to the potential source to deliver a voltage pulse to the first electrode and the second electrode to generate a potential difference between the first electrode and the second electrode. In some embodiments, the voltage pulse has an amplitude of up to 3 kV, e.g., 0.01 kV to 3 kV, e.g., 0.2 to 0.6 kV. In some cases, the duty cycle of the electromechanical transfection is 0.001% to 100%, e.g., 10 to 95%. In certain embodiments, the voltage pulse has a duration of 0.01 ms to 1,000 ms, e.g., 1 to 10 ms. In certain embodiments, the voltage pulse is applied to the first and second electrodes at a frequency between 1 Hz to 50,000 Hz, e.g., 100 to 500 Hz. The waveform of the voltage pulse may be DC, rectangular, pulsed, bipolar, sinusoidal, ramp, asymmetric bipolar, any, or a superposition or combination thereof. In certain embodiments, the electric field generated from the voltage pulse has a magnitude between 1 V / cm and 50,000 V / cm, e.g., 100 to 1,000 V / cm, e.g., 400 to 1,000 V / cm. In further embodiments, the system includes a housing (e.g., a housing structure) configured to accommodate the electromechanical transfection device described herein. In further embodiments, the housing (e.g., a housing structure) includes a thermal controller configured to increase or decrease the temperature of the housing or any component of the system. In some embodiments, the thermal controller is a heating element, e.g., a heating block, a liquid flow, a battery-powered heater, or a thin film heater. In other embodiments, the thermal controller is a cooling element, e.g., a liquid flow, an evaporative cooler, or a thermoelectric element, e.g., a Peltier element.

[0059] In further embodiments, the system includes multiple electromechanical transfection devices, for example in series or parallel. In certain embodiments, the system includes multiple outer structures for multiple electromechanical transfection devices.

[0060] In further embodiments, the method includes assessing the health of a portion of the plurality of cells suspended in the fluid. In certain embodiments, the assessing includes measuring the viability of the portion of the plurality of cells suspended in the fluid. In certain embodiments, the assessing includes measuring the transfection efficiency of the portion of the plurality of cells suspended in the fluid. In some embodiments, the assessing includes measuring the cell recovery of the portion of the plurality of cells suspended in the fluid. In certain embodiments, the assessing includes flow cytometry analysis of cell surface marker expression.

[0061] In some embodiments, the methods induce reversible electromechanical transfection, hi certain embodiments, the electromechanical transfection is substantially non-thermally reversible electromechanical transfection.

[0062] In some embodiments, cells suspended in a fluid having the composition are forced through an electric field in an active zone of the device by application of positive pressure, e.g., a pump, e.g., a syringe pump, a peristaltic pump, or a pressure source.

[0063] In certain embodiments, the cells in the plurality of cells in the sample are mammalian cells, eukaryotic cells, human cells, animal cells, plant cells, synthetic cells, primary cells, cell lines, suspension cells, adherent cells, unstimulated cells, stimulated cells, activated cells, immune cells, stem cells, blood cells, red blood cells, T cells, B cells, neutrophils, dendritic cells, antigen presenting cells (APCs), natural killer (NK) cells, monocytes, macrophages, or peripheral blood mononuclear cells (PBMCs), human embryonic kidney cells, e.g., K-293 cells, or Chinese hamster ovary (CHO) cells. In certain embodiments, the plurality of cells comprises Jurkat cells. In certain embodiments, the plurality of cells comprises primary human T cells. In certain embodiments, the plurality of cells comprises THP-1 cells. In certain embodiments, the plurality of cells comprises primary human macrophages. In certain embodiments, the plurality of cells comprises primary human monocytes. In certain embodiments, the plurality of cells comprises natural killer (NK) cells. In certain embodiments, the plurality of cells comprises Chinese hamster ovary cells. In certain embodiments, the plurality of cells comprises human embryonic kidney cells. In certain embodiments, the plurality of cells comprises B cells. In certain embodiments, the plurality of cells comprises primary human T cells. In certain embodiments, the plurality of cells comprises primary human monocytes. In certain embodiments, the plurality of cells comprises primary human macrophages. In certain embodiments, the plurality of cells comprises embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs). In certain embodiments, the plurality of cells comprises primary human induced pluripotent stem cells (iPSCs).

[0064] In some embodiments, the composition is a therapeutic agent, a vitamin, a nanoparticle, a charged therapeutic agent, a nanoparticle, a charged molecule, e.g., ions in solution, an uncharged molecule, a nucleic acid, e.g., DNA or RNA, a CRISPR-Cas complex, a protein, a polymer, a ribonucleoprotein (RNP), an artificial nuclease, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a homing nuclease, a meganuclease (MN), a megaTAL, an enzyme, a peptide, a transposon, or a polysaccharide, e.g., a dextran, e.g., dextran sulfate. Compositions that can be delivered to cells in suspension include nucleic acids (e.g., oligonucleotides, mRNA, or DNA), antibodies (or antibody fragments, e.g., bispecific fragments, trispecific fragments, Fab, F(ab')2, or single chain variable fragments (scFv)), amino acids, polypeptides (e.g., peptides or proteins), cells, bacteria, gene therapy drugs, genome-engineered therapeutic drugs, epigenome-engineered therapeutic drugs, carbohydrates, chemical agents, contrast agents, magnetic particles, polymeric beads, metal nanoparticles, metal microparticles, quantum dots, antioxidants, antibiotics, hormones, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, anti-inflammatory agents, antimicrobial agents, chemotherapeutic agents, exosomes, outer membrane vesicles, vaccines, viruses, bacteriophages, adjuvants, vitamins, minerals, organelles, and combinations thereof. In certain embodiments, the composition is a nucleic acid (e.g., oligonucleotides, mRNA, or DNA). In certain embodiments, the composition is a polypeptide (e.g., a peptide or protein).

[0065] In some embodiments, the composition has a concentration between 0.0001 μM and 20 μM (e.g., 0.0001 μM to 0.001 μM, 0.001 μM to 0.01 μM, 0.001 μM to 5 μM, 0.005 μM to 0.1 μM, 0.01 μM to 0.1 μM, 0.01 μM to 1 μM, 0.1 μM to 1 μM, 0.1 μM to 5 μM, 1 μM to 10 μM, 1 μM to 15 μM, or 1 μM to 20 μM, e.g., about 0.0001 μM, 0.0005 μM, 0.001 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, M, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0 .6μM,0.7μM,0.8μM,0.9μM,1μM,1.5μM,2μM,2.5μM,3μM,3.5μM,4μM,4.5μ M, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM) in the liquid.

[0066] In certain embodiments, the composition is between 0.0001μg / mL and 1,000μg / mL (for example, 0.0001μg / mL~1,000μg / mL) mL~5μg / mL、0.005μg / mL~0.1μg / mL、0.01μg / mL~0.1μg / mL、0.01μg / mL~1μg / mL、0.1μg / mL~1μg / mL、0.1μg / mL~5μg / mL、1μg / mL~10μg / mL、1μg / mL~50μl g / mL、1μg / mL~100μg / mL、2.5μg / mL~15μg / mL、5μg / mL~25μg / mL、5μg / mL~50μg / mL、5μg / mL~500μg / mL、7.5μg / mL~75μg / mL、10μg / mL~100μg / mL mL~1,000μg / mL、25μg / mL~50μg / mL、25μg / mL~250μg / mL、25μg / mL~500μg / mL、50μg / mL~100μg / mL、50μg / mL~250μg / mL、50μg / mL~750μg / mL、100μg / mL~ 300μg / mL, 100μg / mL~1,000μg / mL, 200μg / mL~400μg / mL, 250μg / mL~500μg / mL, 350μg / mL~500μg / mL, 400μg / mL~1,000μg / mL, 500μg / mL~750μg / mL, 650μg / mL~1,000μg / mL, or 800μg / mL~1,000μg / mL, e.g., about 0.0001μg / mL, 0.0005μg / mL, 0.001μg / mL, 0.005μg / mL, 0.01μg / mL, 0.02μg / mL, 0.03μg / mL 、0.04μg / mL、0.05μg / mL、0.06μg / mL、0.07μg / mL、0.08μg / mL、0.09μg / mL、0.1μg / mL、0.2μg / mL、0.3μg / mL、0.4μg / mL、0.5μg / mL、0.6μg / mL、0.7μg / mL 、0.8μg / mL、0.9μg / mL、1μg / mL、1.5μg / mL、2μg / mL、2.5μg / mL、3μg / mL、3.5μg / mL、4μg / mL、4.5μg / mL、5μg / mL、5.5μg / mL、6μg / mL、6.5μg / mL、7μg / mL.5μg / mL, 8μg / mL, 8.5μg / mL, 9μg / mL, 9.5μg / mL, 10μg / mL, 15μg / mL, 20μg / mL, 25μg / mL, 30μg / mL, 35μg / mL, 40μ g / mL, 45μg / mL, 50μg / mL, 55μg / mL, 60μg / mL, 65μg / mL, 70μg / mL, 75μg / mL, 80μg / mL, 85μg / mL, 90μg / mL, 95μg / m L, 100 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, 900 μg / mL, 950 μg / mL, or 1,000 μg / mL).

[0067] In further embodiments, the method includes a housing structure configured to house the electromechanical device described herein. In further embodiments, the housing structure includes a thermal controller configured to increase or decrease the temperature of the housing or any component of the system. In some embodiments, the thermal controller is a heating element, such as a heating block, a liquid flow, a battery-powered heater, or a thin film heater. In other embodiments, the thermal controller is a cooling element, such as a liquid flow, an evaporative cooler, or a thermoelectric element, such as a Peltier element. In certain embodiments, the temperature of the plurality of cells suspended in the fluid is between 0° C. and 50° C.

[0068] In further embodiments, the device comprises multiple electromechanical transfection devices, for example in series or parallel. In certain embodiments, the device comprises multiple outer structures for multiple devices.

[0069] In some embodiments, the method further comprises storing the plurality of cells suspended in the fluid in a recovery buffer after transfection. In certain embodiments, the transfected cells have a viability of between 0.1% and 99.9%, for example between 75% and 95%, after introduction of the composition. In other embodiments, the efficiency of introduction of the composition into the cells is between 0.1 and 99.9%, for example between 25% and 95%. In certain embodiments, the cell recovery rate is between 0.1% and 100%. In certain embodiments, the cell recovery yield is between 0.1% and 500%.

[0070] In another aspect, the invention provides a kit for introducing a composition into a plurality of cells suspended in a fluid by electromechanical transfection, the kit comprising a plurality of devices as described herein, a plurality of outer structures as described herein, and a transfection buffer.

[0071] In another aspect, the invention provides a kit for electromechanical transfection of a composition into a plurality of cells suspended in a fluid, the kit comprising a plurality of devices as described herein, a plurality of outer structures as described herein, and a transfection buffer.

[0072] In some embodiments of any of the preceding aspects, the outer structure is integral with the plurality of cellular devices. In certain embodiments, the outer structure is releasably coupled to the plurality of devices.

[0073] definition When values ​​are described as ranges, such disclosure will be understood to include disclosure of all possible subranges within such ranges, as well as the specific numerical values ​​falling within such ranges, whether or not a specific numerical value or specific subrange is explicitly stated.

[0074] As used herein, the term "about" refers to ±10% of the stated value.

[0075] As used herein, the term "average flow rate" refers to the volumetric flow rate (Q, in units of m 3 / s) is expressed as, for example, the cross-sectional area (A, unit m 2 ) and thus has the units m / s.

[0076] As used herein, the term "plurality" refers to more than one.

[0077] As used herein, the term "conductivity" refers to electrical conductance, i.e., the ability of electrically charged particles (e.g., ions) to move through a medium, e.g., ions of a salt, e.g., buffer ions, in a flowing liquid.

[0078] The term "substantially uniform" as used herein refers to a variation of ±5%.

[0079] The term "minimum hydraulic diameter," as used herein, refers to the smallest length equal to four times the cross-sectional area divided by the wetted perimeter (e.g., inner perimeter) of the cross section of, for example, a lumen (e.g., the lumen of an active zone or entry zone).

[0080] The term "cross-sectional area" means a cross-sectional area (eg, along a plane perpendicular to the longitudinal axis or flow direction), unless otherwise specified.

[0081] As used herein, the term "fluidically connected" refers to a direct connection between at least two device elements, e.g., electromechanical devices, reservoirs, etc., that allows fluid to move between such device elements without passing through an intervening element.

[0082] The term "fluid communication," as used herein, refers to an indirect connection between at least two device elements, e.g., active zones, reservoirs, etc., that allows fluid to move between such device elements, e.g., via intervening elements (e.g., intervening tubes, intervening channels, etc.).

[0083] The term "lumen," as used herein, refers to an internal cavity of a portion of a device of the present invention (eg, an active zone or entry zone) that allows fluid to pass therethrough.

[0084] The term "entry zone" as used herein refers to a portion of the device of the present invention through which a fluid and a plurality of cells suspended therein may pass prior to electromechanical transfection in the active zone. The entry zone may further comprise an additional reservoir in fluid communication with the active zone of the device of the present invention. When a potential difference is applied to the first and second electrodes of the device of the present invention, the electric field that may be generated within the entry zone of the device of the present invention is not high enough to cause poration of the cells.

[0085] The term "recovery zone," as used herein, constitutes a portion of the device of the invention through which fluid and a plurality of cells suspended therein may pass or reside following electromechanical transfection in the active zone. The recovery zone may include a portion of the device (e.g., a lumen, tube, channel, reservoir, etc.) that is downstream (e.g., immediately downstream, e.g., proximal to a second outlet) of the active zone. The recovery zone may further include an additional reservoir in fluid communication with the active zone.

[0086] The term "active zone," as used herein, refers to a portion of the device that is disposed between a first electrode and a second electrode, in fluid communication with the entry zone, and downstream of the entry zone (e.g., downstream of the first outlet). An electric field is delivered to fluid in the active zone.

[0087] As used herein, the term "transfection" refers to a process by which a payload can be introduced into a cell using means other than viral delivery methods, such as biological, chemical, electrical, mechanical, or physical methods.

[0088] The term "electroporation," as used herein, refers to a process that utilizes an applied electric field to create small pores in cell membranes through which a payload can be introduced into the cell (e.g., as a method of transfection).

[0089] As used herein, the term "electromechanical transfection" refers to a transfection process that can utilize a combination of an applied electric field and a mechanical pore-forming mechanism to introduce a payload into a cell. This method of introduction may reduce and / or stabilize the overall electric field exposure of the cells in the active zone, thereby improving cell viability and / or transfection efficiency, or both. The device of the present invention is configured to transfect cells via electromechanical transfection rather than electroporation alone. The method of the present invention allows the optimal combination of electrical energy (e.g., field strength) and mechanical energy (e.g., flow rate) to be determined for a given cell type.

[0090] The term "therapeutic dose" as used herein refers to an amount of transfected cells sufficient to achieve such treatment when administered to a patient for treating a condition, disorder, or pathology. The administration of a therapeutic dose can be alone, in combination with other agents, as part of a series of administrations, or a combination thereof. Treatment may result in a therapeutic benefit, such as a beneficial immune response, reduction or elimination of a disease state, such as a reduction in cancer cells or cancer biomarkers, a slowing or halting of the rate of replication of cancer cells, and the like. A therapeutic dose can be determined, for example, by monitoring the patient's condition (e.g., by clinical evaluation), clinical disease progression, the amount of disease or organ function biomarkers, such as the amount of white blood cells or red blood cells, and the like. A therapeutic dose can be any amount or concentration of cells described herein. [Brief description of the drawings]

[0091] [Figure 1] A schematic diagram of the device of the present invention is shown. Cells and payload are suspended in a dedicated buffer in a reservoir. As the cells and payload flow through the electromechanical transfection zone, they are exposed to both electrical energy and continuous fluid flow, inducing transient cell membrane disruption and simultaneous introduction of the genetic payload into the cells. The transfected cells are directly dispensed into growth medium for cell harvesting. [Diagram 2]A-D show correlation of electromechanical transfection related parameters with outcome data. Proliferating human T cells transfected with GFP reporter mRNA using the device, system, and method of the present invention. Cultures were assessed for cell viability (7AAD negative), transfection efficiency, and yield (live GFP+ cells observed from 1e6 input cells) after 24 hours. The mechanism of action of transfection in the introduction of GFP reporter mRNA into proliferating T cells by the method of the present invention is now visualized by plotting percent viability, efficiency, and yield against electromechanical transfection specific parameters π4 (FIG. 2A and FIG. 2B) and π5 (FIG. 2C and FIG. 2D). All analyses were completed using a Thermo Fisher Attune™ NxT flow cytometer; n=89 are depicted as individual data points. [Figure 3A] Figure 1 shows data comparing transfection using an electromechanical device according to the methods of the invention with two commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Expanded human T cells were treated with the electromechanical transfection system or the commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™) without payload. Representative data is shown 6 or 24 hours after treatment. Volcano plot showing significantly dysregulated genes (p<0.05) with a >1-fold change in expression after 6 hours. [Figure 3B]Figure 1 shows data comparing transfection using an electromechanical device according to the methods of the invention with two commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Expanded human T cells were treated with the electromechanical transfection system or the commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™) without payload. Representative data is shown 6 or 24 hours after treatment. Volcano plot showing significantly dysregulated genes (p<0.05) with a >1-fold change in expression after 6 hours. [Figure 3C] Figure 1 shows data comparing transfection using an electromechanical device according to the methods of the invention with two commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Expanded human T cells were treated with the electromechanical transfection system or the commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™) without payload. Representative data is shown 6 or 24 hours after treatment. Volcano plot showing significantly dysregulated genes (p<0.05) with a >1-fold change in expression after 6 hours. [Figure 3D] FIG. 1 shows data comparing transfection using an electromechanical device according to the methods of the present invention with two commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Expanded human T cells were treated with the electromechanical transfection system or the commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™) without payload. Representative data is shown 6 or 24 hours after treatment. FIG. 1 shows a graphical representation of genes showing baseline vs. expression aberrations at 6 hours. [Figure 3E]Figure 1 shows data comparing transfection using an electromechanical device according to the methods of the invention with two commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Expanded human T cells were treated with the electromechanical transfection system or the commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™) without payload. Representative data is shown 6 or 24 hours after treatment. Heatmap of selected up- and down-regulated genes at 6 and 24 hours. [Figure 3F] FIG. 1 shows data comparing transfection using an electromechanical device according to the methods of the invention with two commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Expanded human T cells were treated without payload using the electromechanical transfection system or the commercially available electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Representative data is shown 6 or 24 hours after treatment. FIG. 1 shows a heat map of gene ontology focused on T cell function. [Figure 4] A and B show the results of using the electromechanical transfection system according to the methods of the invention across different donors versus controls. Expanded human T cells from three unique donors were transfected with GFP reporter mRNA. Cultures were assessed for cell viability (7AAD negative) after 24 hours (A) and transfection efficiency (B). Bar graphs are mean ± SD with the following transfection sample sizes: donor #1 n=3, donor #2 n=22, donor #3 n=3. [Figure 5A]

[0023] Figure 1 shows the results of using the electromechanical transfection system and method of the present invention to transfect naive T cells. Naive T cells were transfected with GFP reporter mRNA. Cultures were assessed for proliferation potential measured up to 6 days after transfection. [Figure 5B]

[0023] Figure 1 shows the results of using the electromechanical transfection system and the method of the present invention to transfect naive T cells. Naive T cells were transfected with GFP reporter mRNA. Cultures were assessed for cell viability (trypan blue exclusion) measured up to 6 days after transfection. [Figure 5C] 1 shows the results of using the electromechanical transfection system and method of the present invention to transfect naive T cells. Naive T cells were transfected with GFP reporter mRNA. Naive T cells stained for expression of lineage markers CD45RA and CD45RO compared to controls. [Figure 5D] FIG. 1 shows the results of using the electromechanical transfection system and the method of the present invention to transfect naive T cells. Naive T cells were transfected with GFP reporter mRNA. Cell viability (7AAD negative) from 0.5M cell input measured 24 hours post-transfection is shown. Bar graphs are representative of n=2 transfections, mean±SD. [Figure 5E] FIG. 1 shows the results of using the electromechanical transfection system and the method of the present invention to transfect naive T cells. Naive T cells were transfected with GFP reporter mRNA. Transfection efficiency from 0.5M cell input measured 24 hours post-transfection is shown. Bar graphs are representative of n=2 transfections, mean±SD. [Figure 5F]FIG. 1 shows the results of using the electromechanical transfection system and method of the present invention to transfect naive T cells. Naive T cells were transfected with GFP reporter mRNA. The number of viable GFP+ cells (yield) from 0.5M cell input measured 24 hours post-transfection is shown. Bar graphs are representative of n=2 transfections, mean±SD. [Figure 6A] FIG. 1 shows how the methods and electromechanical transfection of the present invention described herein can be directly transferred from small-scale research transfection to large-scale cell production transfection. Schematic diagram showing how an electromechanical flow cell can be used in small-scale devices (e.g., integrated with liquid handling machines in a 96-well format), electromechanical transfection (e.g., arrays of devices arranged for batch-wise transfection) and large-scale electromechanical devices or systems (e.g., closed electromechanical transfection systems), allowing for direct translation from one scale to the other. [Figure 6B] We demonstrate how the methods and electromechanical transfection of the present invention described herein can be directly translated from small-scale research transfection to large-scale cell production transfection. Proliferating human T cells were transfected with a GFP mRNA reporter payload using a small-volume electromechanical device array platform (small-scale 10-100 μL transfection) and a large-volume flow electromechanical transfection system (large-scale >5 mL transfection). Cell viability (7AAD negative) and transfection efficiency were assessed after 24 hours. Bar graphs are mean ± SD small volume n=6 large volume n=2. [Figure 7]A and B show the gating strategy for determining total cell number and viability. In A, total cells are pre-gated on forward scatter (FSC) and side scatter (SSC) dot pots. This gate captures cells with a wide range of morphology for accurate analysis of the total cell population. In B, viability is determined by gating on 7-AAD-cells from within the total cell gate. An efficiency gate is determined based on untreated cells to eliminate background fluorescence (not shown). [Figure 8] A and B are heat maps of field strength versus flow rate, with viability (A) and efficiency (B) on the z-axis. Proliferating human T cells were transfected with GFP reporter mRNA using a large volume transfection system. Cultures were assessed for cell viability (A) (7AAD negative) and transfection efficiency (B) after 24 hours. All analyses were performed using a Thermo Fisher Attune™ NxT flow cytometer; n=96 are depicted as individual data points. [Figure 9] AC are volcano plots for expanded human T cells from the second of two unique donors treated without payload using electromechanical (A) or commercially available electroporation-based transfection systems: Neon™ (B) and 4D Nucleofector™ (C). The volcano plots show significantly dysregulated genes (p<0.05) with a >1-fold change in expression 6 hours after treatment. [Figure 10] A and B are bar graphs showing cell viability (7AAD negative) (A) and transfection efficiency (B) after 24 hours of expanded human T cells transfected with GFP reporter mRNA using either the electromechanical transfection system or commercial electroporation-based transfection systems (Neon™ and 4D Nucleofector™). Bars represent the mean ± SD, n=7. [Figure 11]A-C show the use of the system of the invention to deliver multiple payloads. Proliferating human T cells were transfected with GFP and mCherry reporter mRNA in a system comprising an array of devices of the invention. Cultures were assessed for cell viability (7AAD negative) (A) and transfection efficiency (B and C) 24 hours after parallel (B) or serial (48 hour separation) (C) delivery. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] The present invention provides a method for transfection of cells, such as mammalian cells, such as primary T cells, by electromechanical transfection with comparable or better volumes, comparable or better transfection efficiencies, comparable or better throughput, comparable or better recovery rates, comparable or better yields, and comparable or better cell viabilities compared to conventional cuvette-based electroporation approaches or commercially available electroporation instruments. In particular, systems and methods are provided that can perform electromechanical transfection in a flow-through mode, a continuous mode, or using multiple electromechanical transfection devices of the present invention to increase throughput and cell numbers. More particularly, the methods of the present invention allow for the application of less electrical energy than electroporation-based techniques, thus minimizing damage to transfected cells.

[0093] Non-viral cell transfection represents a promising evolution for both autologous and allogeneic cell therapy. Despite its advantages of decoupling cell therapy from the challenges of viral vector production, non-viral transfection has lagged behind viral methodologies in clinical applications. One of the best-known forms of non-viral transfection is electroporation, in which a high-energy electric field is applied to a quiescent cell suspension. Successful transfection by classical electroporation depends on the strength of the electric field experienced by each cell. However, too strong an electric field for a long period of time can cause irreversible cell membrane disruption, leading to cell death.

[0094] In order to improve the yield of electric field-assisted transfection, it is necessary to minimize the electrical energy applied to cells.The method of the present invention reduces the electrical energy required to allow cell membrane permeabilization by adding a mechanical component to the total energy applied to cells.This mechanical energy can be delivered, for example, via fluid flow, and reduces the high-energy electric field required for efficient delivery of genetic payloads, for example by inducing membrane disruption to deliver DNA, RNA, or CRISPR-RNP into the nucleus.

[0095] Electromechanical cell transfection uses an electric field coupled with mechanical stress, for example associated with moderate fluid flow rates, to permeabilize cells and introduce exogenous material. This technique differs from electroporation, which utilizes only an electric field to permeabilize cells and is typically performed with no or low fluid flow rates and minimal stress. In electromechanical cell transfection, pore formation is mediated by the combined effect of the electric field and mechanical energy input in the form of shear and normal stresses on the cells. Electromechanical cell transfection relies on the following parameters; the root mean square of the applied voltage (V RMS); the conductivity of the medium (i.e., electrical conductivity) s; the average fluid velocity u; the distance between the electrodes l; the dynamic viscosity of the fluid (measured, for example, by a rotational viscometer) μ; the channel diameter d; the cell diameter D; and the fluid density r. Applying the dimensional analysis by Buckingham Pi theorem, we obtain four dimensionless parameters. The first two parameters

number

number

number

number

[0096] The important physics of this process is expected to be governed by these four dimensionless groups and their combinations. 4The presence and importance of distinguishes this transfection mechanism from electroporation and purely mechanical transfection methods. In electroporation, the electric field and pulse conditions govern the transfection efficiency, and the process usually occurs in a static chamber with stationary fluid. Recently, flow-based electroporation has been attempted, in which the cell suspension moves at a constant velocity during the transfection process. However, in these systems, the flow is used to transport the cells to the transfection zone and does not affect the transfection itself as in electromechanical transfection. Although mechanical transfection methods, especially those using high flow rates, do depend on Re, the Π described here is not affected since no electric field is applied. 4 Thus, although superficially similar to electroporation, electromechanical transfection is a unique and novel technique for delivering foreign materials into cells.

[0097] The present invention presents a new transfection technique, electromechanical transfection. Electromechanical transfection utilizes continuous flow and electrical energy, which presents several advantages compared to electroporation, other viral and non-viral transfection methodologies. Dimensional analysis reveals that electromechanical transfection is optimized by balancing the effects of fluid flow and electric fields, setting this technique apart from previous methods that use electric fields. The presented physical model reveals the key parameters that drive the effect of this technique, primarily by replacing previous dimensionless parameters with a fifth dimensionless group.

number

[0098] The results of the transcriptome analysis described herein show that high delivery efficiency can be dissociated from significant gene dysregulation. Electromechanical transfection according to the present invention showed less than a 5% shift from baseline after 6 hours of treatment, whereas both commercially available electroporation-based transfection devices showed a greater than 5% shift from baseline in total gene dysregulation. Only 13% of the dysregulation induced by non-viral treatments was due to altered molecular function with the electromechanical device, whereas electroporation-based devices induced 19-24% dysregulation due to molecular function. This functional dysregulation was highlighted when markers of T cell exhaustion (CTLA4 and TIGIT) were found to be upregulated 6 hours after treatment with the electroporation-based device, but at baseline levels after treatment with the electromechanical device according to the method of the present invention. Analysis of transfection efficiency after 24 hours showed that the delivery efficiency index was not significantly different from that of commercially available electroporation-based devices from Thermo Fisher (Neon™) (e.g., 89.2% and 89.4%, respectively), with a reduction in gene dysregulation observed after electromechanical treatment. With the present invention, post-transfection survival rates of over 75% and delivery efficiencies of over 80% were observed in multiple use cases. These findings were also confirmed in multiple PBMC donors, with no significant differences in delivery efficiency and no significant differences in efficiency (GFP + The observed transfection efficiency was consistent with the expression of lineage-specific naïve cell markers (CD45RA+ / CD45RO - These findings are consistent with previous studies in which 100% of CD45RA cells were transfected with naïve CD4+ T cells 24 hours after electromechanical transfection. + / CD45RO - Our results demonstrate that high viability and delivery efficiency of >95% can be maintained while maintaining naive marker expression of 100-fold. Furthermore, the 50-fold scaled-up transfection results showed less than 2.5% change in both viability and delivery efficiency compared to the small-scale results. Taken together, these data suggest that cell engineering using non-viral electromechanical transfection methods is distinct from classical electroporation and represents a meaningful alternative to existing transfection methods. Electromechanical transfection can be leveraged with high-throughput automation for discovery or process development, and is also easily scaled up for manufacturing. The ability to scale out and up while maintaining cell health and high cell yields makes electromechanical transfection an attractive new solution for cell therapy development and manufacturing.

[0099] device In general, the devices of the invention are configured to be flow-through devices that can interface with existing liquid handling devices, pumps, or fluid transport devices, such as conventional pipette tip robots or large liquid handling systems, to provide continuous transfection of cells suspended in a liquid. The devices of the invention are configured such that transfection of cells occurs within the active zone via an electromechanical transfection mechanism that is distinct from the delivery mechanism in electroporation-based transfection systems. The devices of the invention typically feature two distinct regions, namely, an entry zone with a first inlet and a first outlet, and an active zone with a second inlet and a second outlet. The first and second electrodes are positioned to generate an electric field in the active zone. An example of an embodiment of the device of the invention is shown in FIG. 1. When a potential difference is applied to the first and second electrodes, a localized electric field is generated in the space between the two electrodes, e.g., the active zone, and cells exposed to the electric field are perforated. Individual devices of the invention may include two electrodes, as shown in FIG. 1; alternatively, individual devices of the invention may include three or more electrodes that define multiple active zones, thus allowing multiple transfections of cells suspended in a fluid. The devices of the invention may include multiple active zones between a first electrode and a second electrode, such that cells can experience different electric fields while flowing through a single device or multiple devices, e.g., as expressed by the different geometries of each of the multiple active zones.

[0100] In some cases, the first and second electrodes may be conductive wires, hollow cylinders, conductive thin films, metal foams, mesh electrodes, liquid diffusive membranes, conductive liquids, or any combination thereof may be included in the device. The electrodes may be arranged parallel to the fluid flow axis of the device, or may be arranged perpendicular to the fluid flow axis of the device. For example, the first and second electrodes may be hollow cylindrical electrodes arranged parallel to the fluid flow axis in the device such that the fluid flows through the electrodes, as in the device of FIG. 1. In alternatives, the first and / or second electrodes may be made of a porous conductor, e.g., a metal mesh, with pores aligned with the fluid flow axis of the device. In alternatives, the first and / or second electrodes may be a conductive fluid, e.g., a liquid. In some cases, the first and second electrodes may be configured as a helix, e.g., a double helix, of a solid conductor, e.g., a wire, around the active zone. In this configuration, the hydraulic diameter of the active zone remains substantially uniform, but the first and second electrodes vary in position along the length of the active zone. The first and second electrodes are in fluid communication with an active zone, and when a potential difference is applied to the electrodes, an electric field is generated that rotates as cells suspended in the fluid move through the device of the invention. In certain embodiments, the first and second electrodes are embedded in the device of the invention, with the active zone disposed in or near fluid communication with the active zone such that a fluid carrying the cells in suspension contacts a portion of the electrode, and the electric field is generated in the active zone.

[0101] When configured as a hollow cylindrical electrode, the diameter of the electrode is about 0.1 mm to 5 mm, for example, about 0.1 mm to 1 mm, 0.5 mm to 1.5 mm, 1 mm to 2 mm, 1.5 mm to 2.5 mm, 2 mm to 3 mm, 2.5 mm to 3.5 mm, 3 mm to 4 mm, 3.5 mm to 4.5 mm, or 4 mm to 5 mm, for example, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, The electrode may be 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, or 5 mm. An exemplary electrode outer diameter is 1.3 mm, which corresponds to a 16 gauge electrode.

[0102] The active zone can fluidly and / or electrically connect the first and second electrodes of the device of the invention, and when the electrodes are energized, a local electric field is generated between them. The active zone can be fluidly connected to a collection zone downstream of the active zone. The cross-sectional shape of the active zone can be any suitable shape that allows cells to pass through the active zone and the electric field within the active zone. The cross-sectional shape can be, for example, circular, elliptical, polygonal, such as a square, rectangular, triangular, n-sided (e.g., a regular or irregular polygon having 4, 5, 6, 7, 8, 9, 10, or more sides), star, parallelogram, trapezoid, or irregular, such as an ellipse, or curved. In some cases, the active zone is a flow path having a substantially uniform cross-sectional dimension along its length, for example, the active zone can have a circular cross-section, with a constant diameter from the fluid connection with the entry zone to the outlet of the active zone (e.g., the second outlet) or the fluid connection with the collection zone. In this configuration, the resulting electric field is more uniform, thus allowing for more predictable electric field exposure of cells suspended in fluid. Alternatively, the hydraulic diameter of the active zone may be varied along its length. For example, the hydraulic diameter of the active zone may increase or decrease along its length, or there may be a change in more than one dimension along its length, for example, the hydraulic diameter, e.g., the diameter, may increase or decrease by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or at most about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In this configuration, the active zone may have a cross section of a truncated cone, with the diameter increasing from the top opening to the bottom opening, or decreasing from the top opening to the bottom opening. In some cases, the devices of the invention can include multiple active zones fluidly connected in series, each having either a uniform or non-uniform cross-section, and each having a different cross-sectional shape. As a non-limiting example, the devices of the invention can include multiple active zones connected in series, each of the multiple active zones having a cylindrical cross-section with a different hydraulic diameter, e.g., each having a different diameter.

[0103] In some embodiments, the hydraulic diameter of the active zone is between 0.005 mm and 50 mm, e.g., between 0.005 mm and 0.05 mm, between 0.01 mm and 0.1 mm, between 0.05 mm and 0.5 mm, between 0.1 mm and 1 mm, between 0.5 mm and 1 mm, between 0.5 mm and 2 mm, between 0.7 mm and 1.5 mm, between 1 mm and 5 mm, between 3 mm and 7 mm, between 5 mm and 10 mm, between 7 mm and 12 mm, between 10 mm, ~15mm, 13mm~18mm, 15mm~20mm, 22mm~30mm, 25mm~35mm, 30mm~40mm, 35mm~45mm, or 40mm~50mm, for example, about 0.005mm, 0.006mm, 0.007mm, 0.008mm, 0.009mm, 0.01mm, 0.02mm, 0.03mm, 0.04mm, 0.05mm, 0.06mm , 0.07mm, 0.08mm, 0.09mm, 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm,1mm,2m m,3mm,4mm,5mm,6mm,7mm,8mm,9mm,10mm,11mm,12mm,13mm,14mm,15mm,16mm,17mm,18mm,19mm,20 In general, the diameter of the active zone may be sized such that it does not have a constriction that would contact the cell and deform the cell membrane with the channel wall, e.g., such that mechanical deformation due to squeezing the cell does not induce cell poration, e.g., cells can pass freely through the active zone.

[0104] In some cases, the length of the active zone is between 0.005 mm and 50 mm, e.g., 0.005 mm to 50 mm, 0.005 mm to 0.05 mm, 0.01 mm to 0.1 mm, 0.05 mm to 0.5 mm, 0.1 mm to 1 mm, 0.5 mm to 2 mm, 1 mm to 5 mm, 3 mm to 7 mm, 4 mm to 8 mm, 5 mm to 10 mm, 7 mm to 12 mm, 10 mm to 15 mm, 1 3mm to 18mm, 15mm to 20mm, 22mm to 30mm, 25mm to 35mm, 30mm to 40mm, 35mm to 45mm, or 40mm to 50mm, for example, about 0.005mm, 0.006mm, 0.007mm, 0.008mm, 0.009mm, 0.01mm, 0.02mm, 0.03mm, 0.04mm, 0.05mm, 0.06mm, 0.07mm mm, 0.08mm, 0.09mm, 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm,1mm,2mm,3 mm,4mm,5mm,6mm,7mm,8mm,9mm,10mm,11mm,12mm,13mm,14mm,15mm,16mm,17mm,18mm,19mm,20mm , 21mm, 22mm, 23mm, 24mm, 25mm, 26mm, 27mm, 28mm, 29mm, 30mm, 31mm, 32mm, 33mm, 34mm, 35mm, 36mm, 37mm, 38mm, 39mm, 40mm, 41mm, 42mm, 43mm, 44mm, 45mm, 46mm, 47mm, 48mm, 49mm, or 50mm.

[0105] The hydraulic diameter of the entry zone and / or recovery zone may independently be substantially the same as the hydraulic diameter of the active zone. Alternatively, the hydraulic diameter of the entry zone and / or recovery zone may independently be smaller or larger than the hydraulic diameter of the active zone. For example, when the hydraulic diameter of the entry zone and / or recovery zone is independently configured to be smaller than the hydraulic diameter of the active zone, the hydraulic diameter of the entry zone and / or recovery zone may be between 0.01% and 100%, between 0.01% and 1%, between 0.1% and 10%, between 1% and 5%, between 1% and 10%, between 5% and 25%, between 5% and 10%, between 10% and 25%, between 10% and 50%, between 25% and 75%, or between 50% and 100%, for example, about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, The concentration may be 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0106] Alternatively, if the hydraulic diameter of the entry zone and / or recovery zone is independently configured to be larger than the hydraulic diameter of the active zone, the hydraulic diameter of the entry zone and / or recovery zone may be between 100% and 100,000%, e.g., between 100% and 1000%, between 100% and 250%, between 100% and 500%, between 250% and 750%, between 500% and 1,000%, between 500% and 5,000%, between 1,000% and 10,000%, between 5,000% and 25,000%, between 10,000% and 50,000%, between 25,000% and 75,000%, or between 50,000% and 100,000%, e.g., about 100%, 150%, 175%, 200%, 225%, 250%, 300%, 250%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%, 2,000%, 3,000%, 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 15,000%, 20,0 The option may be 00%, 25,000%, 30,000%, 35,000%, 40,000%, 45,000%, 50,000%, 55,000%, 60,000%, 65,000%, 70,000%, 75,000%, 80,000%, 85,000%, 90,000%, 95,000%, or 100,000%.

[0107] The device of the invention may also include one or more reservoirs for fluidic reagents, e.g., buffers, or samples, e.g., suspensions of cells and compositions to be introduced to the cells. For example, the device of the invention may include reservoirs for cells suspended in fluid to flow into the entry zone and active zone, and / or reservoirs for holding transfected cells. Similarly, there may be reservoirs for flowing liquid into additional components of the device, such as additional inlets intersecting the first or second electrodes. A single reservoir may also be connected to multiple devices of the invention, e.g., when introducing the same liquid into two or more individual devices of the invention configured to transfect cells in parallel or in series. Alternatively, the device of the invention may be configured to mate with a source of liquid that is an external reservoir, such as a vial, tube, pouch, etc. Similarly, the device may be configured to mate with a separate part that houses the reservoir. The reservoir may be of any suitable size, for example to contain 10 mL to 5000 mL, e.g., 10 mL to 3000 mL, 25 mL to 100 mL, 100 mL to 1000 mL, 40 mL to 300 mL, 1 mL to 100 mL, 10 mL to 500 mL, 250 mL to 750 mL, 250 mL to 1000 mL, or 1000 mL to 5000 mL. If multiple reservoirs are present, each reservoir may be the same or different size.

[0108] In addition to the components described above, the devices of the invention may include additional components. For example, the first and second electrodes of the devices of the invention may include one or more additional fluid inlets to allow for the introduction of non-sample fluids, such as buffers, to the appropriate regions of the device. For example, the collection region of the devices of the invention may include additional inlets and outlets for circulating a collection buffer that helps provide a growth environment for the cells after the transfection process.

[0109] Systems and Kits One or more electromechanical transfection devices of the present invention can be combined with various external components, such as power sources, pumps, reservoirs (e.g., bags), controllers, reagents, liquids, and / or samples, in the form of a system. In some embodiments, the system of the present invention includes a plurality of devices of the present invention and a potential source releasably connected to the first and second electrodes of the device(s) of the present invention. In this configuration, the device of the present invention is connected to the potential source, the first electrode is energized, and the second electrode is held at earth. This creates a localized electric field in the active zone, transfecting cells passing through the device. Electromechanical systems incorporating the devices of the present invention can induce reversible poration of cells passing through the devices and systems of the present invention. For example, the devices and systems of the present invention can induce substantially non-thermal reversible poration.

[0110] In some cases, the releasable connection to the first and second electrodes can include any practical electromechanical connection that can maintain consistent electrical contact between the potential source and the first and second electrodes. Examples of electrical connections include, but are not limited to, clamps, clips, such as alligator clips, springs, such as leaf springs, external sheaths or sleeves, wire brushes, flexible conductors, pogo pins, mechanical connections, inductive connections, or combinations thereof. Other types of electrical connections are also known in the art. The device of the present invention can be attached to the opening of the conductive grid such that the first and second electrodes of the device can contact the conductive grid. In particular, the conductive grid includes a spring-loaded electrode, such as an electrode connected to a spring, such that when the device of the present invention is placed in the opening of the conductive grid, the spring-loaded electrode displaces and compresses the spring (which further provides a restoring force for the first and second electrodes of the device of the present invention), thus ensuring electrical contact between the device of the present invention and the potential source.

[0111] The electric potential source is configured to deliver an applied voltage to one or more electrodes to provide a potential difference between the electrodes and establish a uniform electric field in the active zone. In a two-electrode circuit or the like, the applied voltage is delivered to a first electrode and the second electrode is held at earth. Without wishing to be bound by any particular theory, the applied voltage delivered to the electrodes is delivered at a particular amplitude, a particular frequency, a particular pulse shape, a particular duration, a particular number of pulse applications, and a particular duty cycle. These parameters, in combination with the shape of the active zone, result in a particular electric field in the active zone that will be experienced by the cells suspended in the fluid. The electrical parameters described herein can be optimized for a particular cell line and / or composition delivered to a particular cell line. The application of the electric potential to the electrodes of the device of the present invention can be initiated and / or controlled by a controller, e.g., a programmable computer, operatively linked to the electric potential source.

[0112] The geometry of the device of the invention, e.g., the cross-sectional shape and dimensions of the active zone, together with the potential parameters described herein, control the shape and strength of the resulting electric field in the active zone. Typically, a device having an active zone with a uniform cross-section will exhibit a uniform electric field along its length. To adjust the electric field produced in the active zone, the active zone can include multiple different hydraulic diameters and / or different cross-sectional shapes along its length. As a non-limiting example, the device of the invention may include multiple serially connected active zones, each of the multiple active zones having a circular cross-section with a different hydraulic diameter, e.g., each having a different diameter. In this configuration, each of the different diameter circular cross-sections of the active zone acts as an independent active zone, inducing a different electric field for each change in dimension at the same applied voltage, e.g., a constant DC voltage.

[0113] In some cases, a device of the invention can include multiple active zones fluidly connected in series, each having either uniform or non-uniform cross-section, each having a different cross-sectional shape. Alternatively, a system of the invention can include multiple devices of the invention in a parallel configuration, each device operating independently of the other to increase the overall throughput of electromechanical transfection.

[0114] In some cases, the amplitude of the applied voltage is between -3 kV and 3 kV, e.g., between -3 kV and -0.1 kV, between -2 kV and -0.1 kV, between -1 kV and -0.1 kV, between -0.1 kV and -0.01 kV, between 0.01 kV and 3 kV, e.g., between 0.01 kV and 0.1 kV, between 0.02 kV and 0.2 kV, between 0.03 kV and 0.3 kV, between 0.04 kV and 0.4 kV, 0.05kV~0.5kV, 0.06kV~0.6kV, 0.07kV~0.7kV, 0.08kV~0.8kV, 0.09kV~0.9kV, 0.1kV ~1kV, 0.1kV~2.0kV, 0.1kV~3kV, 0.15kV~1.5kV, 0.2kV~2kV, 0.25kV~2.5kV, or 0.3kV~3 kV, for example, 0.01 to 1 kV, 0.1 kV to 0.7 kV, or 0.2 to 0.6 kV, for example, about 0.01 kV, 0.02 kV, 0.03 kV, 0.04 kV, 0.05 kV, 0.06 kV, 0.07 kV, 0.08 kV, 0.09 kV, 0.1 kV, 0.2 kV, 0.3 kV, 0.4 kV, 0.5 kV, 0.6 kV , 0.7kV, 0.8kV, 0.9kV, 1kV, 1.1kV, 1.2kV, 1.3kV, 1.4kV, 1.5kV, 1.6kV, 1.7kV, 1.8kV, 1.9kV, 2kV, 2.1kV, 2.2kV, 2.3kV, 2.4kV, 2.5kV, 2.6kV, 2.7kV, 2.8kV, 2.9kV, or 3kV.

[0115] In some cases, the frequency of the applied voltage may be between 1 Hz and 50,000 Hz, e.g., between 1 Hz and 1,000 Hz, between 1 Hz and 500 Hz, between 100 Hz and 500 Hz, between 100 Hz and 5,000 Hz, between 500 Hz and 10,000 Hz, between 1000 Hz and 25,000 Hz, or between 5,000 Hz and 50,000 Hz, e.g., between 10 Hz and 1000 Hz, between 10 Hz and 500 Hz, between 500 Hz and 750 Hz, or between 100 Hz and 5 00Hz, for example, about 1Hz, 2Hz, 3Hz, 4Hz, 5Hz, 6Hz, 7Hz, 8Hz, 9Hz, 10Hz, 20Hz, 30Hz, 40Hz, 50Hz, 60Hz, 70Hz, 80Hz, 90Hz, 100Hz, 110Hz, 120Hz, 130Hz, 140Hz, 150Hz, 160Hz, 170Hz, 180Hz, 190Hz, 200Hz, 210Hz, 220Hz, 230Hz, 240Hz, 25 0Hz,260Hz,270Hz,280Hz,290Hz300Hz,310Hz,320Hz,330Hz,340Hz,350Hz,360Hz,370Hz,380Hz,390Hz,400H z, 410Hz, 420Hz, 430Hz, 440Hz, 450Hz, 460Hz, 470Hz, 480Hz, 490Hz, 500Hz, 510Hz, 520Hz, 530Hz, 540Hz, 550Hz, 600Hz, 700Hz, 800Hz, 900Hz, 1,000Hz, 2,000Hz, 3,000Hz, 4,000Hz, 5,000Hz, 6,000Hz, 7,000Hz, 8,000Hz, 9,000Hz, 10,000Hz, 15,000Hz, 20,000Hz, 25,000Hz, 30,000Hz, 35,000Hz, 40,000Hz, 45,000Hz, or 50,000Hz.

[0116] In some embodiments, the shape of the applied pulse, e.g., waveform, can be a square wave, a pulse, a bipolar wave, a sine wave, a ramp, an asymmetric bipolar wave, or any. Other voltage waveforms are known in the art. The selected waveform can be applied in any practical voltage pattern, including, but not limited to, high voltage-low voltage, low voltage-high voltage, direct current (DC), alternating current (AC), unipolar, positive (+) polarity only, negative (-) polarity only, (+) / (-) polarity, (-) / (+) polarity, or superpositions or combinations thereof. Those skilled in the art will appreciate that these pulse parameters depend on the electrical properties of the composition delivered to the cells, depending on the cell line.

[0117] The applied voltage pulse may be 0.01 ms to 1,000 ms, for example, 0.01 ms to 1 ms, 0.1 ms to 10 ms, 0.1 ms to 15 ms, 1 ms to 10 ms, 1 ms to 50 ms, 10 ms to 100 ms, 25 ms to 200 ms, 50 ms to 400 ms, 100 ms to 600 ms, 300 ms to 800 ms, or 500 ms to 1,000 ms, for example, about 0.01 ms to 100 ms, 0.1 ms to 50 ms, or 1 ms to 10 ms, for example, 0.01 ms, 0.02 ms, 0.03 ms, 0.04 ms, 0.05 ms, 0.06 ms, 0.07 ms, 0.08 ms, 0.09 ms, 0.1 ms, 0.2 ms, 0.3 ms, 0 The pulse width can be delivered to the active zone for a duration of 0.4ms, 0.5ms, 0.6ms, 0.7ms, 0.8ms, 0.9ms, 1ms, 2ms, 3ms, 4ms, 5ms, 6ms, 7ms, 8ms, 9ms, 10ms, 11ms, 12ms, 13ms, 14ms, 15ms, 20ms, 30ms, 40ms, 50ms, 60ms, 70ms, 80ms, 90ms, 100ms, 150ms, 200ms, 250ms, 300ms, 350ms, 400ms, 450ms, 500ms, 550ms, 600ms, 650ms, 700ms, 750ms, 800ms, 850ms, 900ms, 950ms, or 1,000ms.

[0118] In some cases, the number of applied voltage pulses delivered may be 1 or more, e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 100 or more, e.g., 1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11, 7-12, or 9-13, e.g., 0.01-1,000, e.g., 1-10, 1-50, 5-10, 5-15, 10-100, 25-200, 50-400, 10 It can be 0 to 600, 300 to 800, or 500 to 1,000, for example 1 to 100, 1 to 50, or 1 to 10, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 or more.

[0119] In some cases, the number of applied voltage pulses delivered can be 1 or more. For example, in some cases, the number of applied voltage pulses can be 1,000 to 1,000,000, e.g., 1,000 to 10,000 (e.g., 1,000 to 2,000, 2,000 to 3,000, 3,000 to 4,000, 4,000 to 5,000, 5,000 to 6,000, 6,000 to 7,000, 7,000 to 8,000, 8,000 to 9,000, or 9,000 to 10,000, e.g., 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or ...5,000, 6,000, 7,000, 8,000, 9,000, or 10,000, e.g., 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000, e.g. ,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), 10,000-100,000 (e.g., 10,000-20,000, 20,000-30,000, 30,000-40,000, 40,000-50,000, 50,000-60,000, 60,000-70,000, 70,000-80,000, 80,000-90,000, or 90, 000 to 100,000, for example 10,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, 80,000, 90,000, or 100,000), or 100,000 to 1,000,000 (for example 100,000 to 200,000, 200,000 to 300,000, 300,000 to 400,000, 400,000 to 500,000, 500, 000 to 600,000, 600,000 to 700,000, 700,000 to 800,000, 800,000 to 900,000, to 900,000 to 1,000,000, for example, about 100,000, 200,000, 250,000, 300,000, 400,000, 500,000, 600,000, 700,000, 750,000, 800,000, 900,000, or 1,000,000).

[0120] Pulses of applied voltage may be delivered with a duty cycle of 1%-100%, e.g., 1%-10%, 2.5%-20%, 5%-40%, 10%-60%, 30%-80%, or 50%-100%, e.g., 0.01%-100%, 0.1%-99%, 1%-97%, or 10%-95%, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, in some cases.

[0121] The device(s) of the invention, when the electrodes are connected to a potential source and energized, generate a local electric field in the active zone, which in combination with mechanical energy (e.g., from flow) transfects cells passing through. In some cases, the electric field generated in the active zone can be between 2V / cm and 50,000V / cm, e.g., between 2V / cm and 1,000V / cm, between 100V / cm and 1,000V / cm, between 100V / cm and 5,000V / cm, between 400V / cm and 2,000V / cm, between 400V / cm and 1,000V / cm, between 500V / cm and 10,000V / cm, between 1,000V / cm and 25,000V / cm. 000V / cm, or 5,000V / cm to 50,000V / cm, for example, 2V / cm to 20,000V / cm, 5V / cm to 10,000V / cm, or 100V / cm to 1,000V / cm, for example, about 2V / cm, 3V / cm, 4V / cm, 5V / cm, 6V / cm, 7V / cm, 8V / cm, 9V / cm, 10V / cm, 20V / cm, 30 V / cm, 40V / cm, 50V / cm, 60V / cm, 70V / cm, 80V / cm, 90V / cm, 100V / cm, 200V / cm, 300V / cm, 400V / cm, 5 00V / cm, 600V / cm, 700V / cm, 800V / cm, 900V / cm, 1,000V / cm, 2,000V / cm, 3,000V / cm, 4,000V / cm, 5 ,000 V / cm, 6,000 V / cm, 7,000 V / cm, 8,000 V / cm, 9,000 V / cm, 10,000 V / cm, 15,000 V / cm, 20,000 V / cm, 25,000 V / cm, 30,000 V / cm, 35,000 V / cm, 40,000 V / cm, 45,000 V / cm, or 50,000 V / cm.

[0122] The system of the invention typically includes a fluid delivery source configured to pump a plurality of cells suspended in a fluid through an entry zone to an active zone (e.g., through a first electrode) and from the active zone (e.g., through a second electrode), e.g., to a recovery zone. The fluid delivery source typically includes a pump, including but not limited to a high pressure source, a syringe pump, a micropump, or a peristaltic pump. Alternatively, the fluid may be pumped by displacement of a working fluid against a reservoir of the fluid to be pumped, or by air displacement. Other fluid delivery sources are known in the art. In some cases, the fluid delivery source is configured to flow cells suspended in the fluid by application of positive pressure. Without wishing to be bound by a particular theory, the flow rate at which the cells in suspension are flowed through the device of the invention and the particular geometry of the active zone of the device of the invention determine the residence time of the cells in the electric field of the active zone.

[0123] In some cases, the volumetric flow rate of the fluid delivered from the fluid delivery source is between 0.001 mL / min and 1,000 mL / min per active zone, e.g., between 0.001 mL / min and 1,000 mL / min per active zone, between 0.001 mL / min and 0.1 mL / min, between 0.01 mL / min and 1 mL / min, between 0.1 mL / min and 10 mL / min, between 1 mL / min and 50 mL / min, between 10 mL / min and 100 mL / min, between 25 mL / min and 200 mL / min, between 50 mL / min and 400 mL / min, between 100 mL / min and 600 mL / min. , 300 mL / min to 800 mL / min, or 500 mL / min to 1,000 mL / min, for example, about 0.001 mL / min, 0.002 mL / min, 0.003 mL / min, 0.004 mL / min, 0.005 mL / min, 0.006 mL / min, 0.007 mL / min, 0.008 mL / min, 0.009 mL / min, 0.01 mL / min, 0.02 mL / min, 0.03 mL / min, 0.04 mL / min, 0.05 mL / min, 0.06 mL / min, 0.07 mL / min, 0.08 mL / min, 0.09 mL / min, 0.1mL / min, 0.2mL / min, 0.3mL / min, 0.4mL / min, 0.5mL / min, 0.6mL / min, 0.7mL / min, 0.8mL / min, 0.9mL / min, 1mL / min, 2mL / min, 3mL / min, 4mL / min, 5mL / min, 6mL / min, 7mL / min, 8mL / min, 9mL / min, 10mL / min, 15mL / min, 20mL / min, 25mL / min, 30mL / min, 35mL / min, 40mL / min, 45mL / min, 50mL / min, 55mL / min, 60mL / min, 65mL , 70 mL / min, 75 mL / min, 80 mL / min, 85 mL / min, 90 mL / min, 95 mL / min, 100 mL / min, 150 mL / min, 200 mL / min, 250 mL / min, 300 mL / min, 350 mL / min, 400 mL / min, 450 mL / min, 500 mL / min, 550 mL / min, 600 mL / min, 650 mL / min, 700 mL / min, 750 mL / min, 800 mL / min, 850 mL / min, 900 mL / min, 950 mL / min, or 1000 mL / min. In certain embodiments, the flow rate is between 10 mL / min and 100 mL / min per active zone, for example, about 10 mL / min, 20 mL / min, 30 mL / min, 40 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, 90 mL / min, or 100 mL / min per active zone.

[0124] In some cases, the Reynolds number of the liquid during passage through the active zone is between 10 and 3,000 (e.g., 10-100, 25-200, 50-400, 100-600 mL / min, 300 mL / min to 800 mL / min, 500-1,000, 800-1,500, 1,200-2,000, 1,800-2,500, or 2,400-3,000, e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000, 1,500, 2,000, 2,050, or 3,000).

[0125] In some cases, the peak pressure of the liquid during passage through the active zone is as high as 1×10 -3 Pa and 9.5 x 10 4Pa, for example, between 0.001 and 9,500 (for example, 0.001 Pa to 0.1 Pa, 0.01 Pa to 1 Pa, 0.1 Pa to 10 Pa, 1 Pa to 50 Pa, 10 Pa to 100 Pa, 25 Pa to 200 Pa, 50 Pa to 400 Pa, 100 Pa to 600 Pa, 300 Pa to 800 Pa, or 500 Pa to 1,000 Pa, 1,000 Pa to 6,000 Pa, 3,000 Pa to 8,000 Pa, 5,000 Pa to 9,000 Pa, or 7,500 Pa to 9,500 Pa, for example, about 0.001 Pa, 0.002 Pa, 0.003 Pa, 0.00 4Pa, 0.005Pa, 0.006Pa, 0.007Pa, 0.008Pa, 0.009Pa, 0.01Pa, 0.02Pa, 0.03Pa, 0.04Pa, 0.05Pa, 0.06Pa, 0.07Pa, 0.08Pa, 0.09Pa, 0.1Pa, 0.2Pa, 0.3Pa , 0.4Pa, 0.5Pa, 0.6Pa, 0.7Pa, 0.8Pa, 0.9Pa, 1Pa, 2Pa, 3Pa, 4Pa, 5Pa, 6Pa, 7Pa, 8Pa, 9Pa, 10Pa, 15Pa, 20Pa, 25Pa, 30Pa, 35Pa, 40Pa, 45Pa, 50Pa, 55Pa, 6 0Pa, 65Pa, 70Pa, 75Pa, 80Pa, 85Pa, 90Pa, 95Pa, 100Pa, 150Pa, 200Pa, 250Pa, 300Pa, 350Pa, 400Pa, 450Pa, 500Pa, 550Pa, 600Pa, 650Pa, 700Pa, 750Pa, 8 00Pa, 850Pa, 900Pa, 950Pa, 1,000Pa, 1,100Pa, 1,500Pa, 2,000Pa, 2,500Pa, 3,000Pa, 3,500Pa, 4,000Pa, 4,500Pa, 5,000Pa, 5,500Pa, 6,000Pa, 6,500 Pa, 7,000 Pa, 7,500 Pa, 8,000 Pa, 8,500 Pa, 9,000 Pa, or 9,500 Pa, or for example, about 3,300 Pa (e.g., 2,500 to 4,000 Pa, e.g., 2,500 Pa to 3,000 Pa, 2,800 to 3,300 Pa, 3,100 Pa to 3,400 Pa), for example, about 2,800 Pa, 2,900 Pa, 3,000 Pa, 3,100 Pa, 3,200 Pa, 3,300 Pa, 3,400 Pa, or 3,500 Pa. In some cases, the average flow velocity of the liquid while passing through the active zone is about 1×10-2 m / s and 10 m / s, for example between 0.01 and 1 m / s (for example between 0.01 and 0.05 m / s, between 0.05 and 0.1 m / s, between 0.1 and 0.5 m / s, between 0.5 and 1 m / s, between 1.5 and 2 m / s, between 1 and 2 m / s, between 2 and 3 m / s, between 3 and 4 m / s, between 4 and 5 m / s, between 5 and 6 m / s, between 6 and 7 m / s, between 7 and 8 m / s, between 8 and 9 m / s, or between 9 and 10 m / s), for example between 0.1 and 5 m / s. between 0.4 and 1.4 m / s, between 0.65 and 1.3 m / s, or between 0.26 and 2.08 m / s, for example, about 0.1 m / s, 0.2 m / s, 0.3 m / s, 0.4 m / s, 0.5 m / s, 0.6 m / s, 0.7 m / s, 0.8 m / s, 0.9 m / s, 1.0 m / s, 1.1 m / s, 1.2 m / s, 1.3 m / s, 1.4 m / s, 1.5 m / s, 2 m / s, 3 m / s, 4 m / s, 5 m / s, 6 m / s, 7 m / s, 8 m / s, 9 m / s, or 10 m / s.

[0126] The residence time of a cell in the active zone of the device of the present invention may be from 0.5 ms to 50 ms, for example, from 0.5 ms to 5 ms, from 1 ms to 10 ms, from 5 ms to 15 ms, from 10 ms to 20 ms, from 15 ms to 25 ms, from 20 ms to 30 ms, from 25 ms to 35 ms, from 30 ms to 40 ms, from 35 ms to 45 ms, or from 40 ms to 50 ms, for example, about 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, 1 ms, 1.5 ms, 2 The dwell time may be 5-20 ms (e.g., 6-18 ms, 8-15 ms, or 5-14 ms).

[0127] The systems of the invention typically feature a housing that contains and supports the device(s) of the invention and the necessary electrical connections (e.g., electrode connections). The housing may be configured to hold and energize a single device of the invention, or may be configured to hold and energize multiple devices of the invention simultaneously. The housing may include a thermal controller that can regulate the temperature of the device of the invention during transfection, or thermally regulate a component of the system, e.g., a fluid, e.g., a buffer or suspension containing cells. The thermal controller may be configured to heat the device of the invention or a component of its system, or may be configured to cool the device of the invention or a component of its system, or may be configured to perform both operations. When configured to heat the device of the invention, or a component of its system, suitable thermal control devices include, but are not limited to, a heating block or mantle, liquid heating, e.g., an immersion bath or a circulating fluid bath, a battery-powered heater, or a resistive heater, e.g., a thin film heater, e.g., a heat tape. When configured to cool the components of the device of the invention, or a system, suitable thermal control devices include, but are not limited to, liquid cooling, e.g., an immersion or circulating fluid bath, an evaporative cooler, or thermoelectric, e.g., a Peltier cooler. For example, when implemented with liquid cooling, the device of the invention or a housing configured to hold the device of the invention is either in direct contact with tubing that circulates chilled fluid or is surrounded by a cooling jacket that includes tubing that circulates chilled fluid. Other heating and cooling elements are known in the art.

[0128] In some embodiments, the housing (e.g., cartridge) is configured for use with and / or insertion into an automatically closing system used to deliver cell therapy to a patient in a clinical or hospital setting.

[0129] In some embodiments, the housing (e.g., cartridge) further comprises a cooling / heating area / enclosure for storage of cell suspensions and / or buffers before, during, or after electromechanical transfection of the specimen. In some embodiments, the system (e.g., device and housing) is externally powered.

[0130] In some embodiments, the device of the invention includes a touch screen user interface or other alternative user interface(s) that allows the user to select parameters such as flow rate, waveform, applied potential, transfected volume, time delay, cooling function, heating function, transfection state, progress, and other parameters used to optimize the electromechanical transfection or electromechanical protocol. In some embodiments, the user interface also includes pre-set parameter selections that allow the user to operate the system with specific parameters and conditions previously validated by the user or recommended by the manufacturer. In some embodiments, the user interface may be connected to programming that allows automatic running of the system and / or execution of algorithms to optimize transfection for a given sample of a known cell type and payload combination. In some embodiments, the optimization algorithm has the ability to independently or autonomously adjust the electromechanical parameters if the user selects this feature. In some embodiments, the optimization algorithm allows for continuous adjustment of parameters used in the electromechanical transfection process that may depend on the type of cell, the conductivity of the cell suspension, the volume of the cell suspension, the dynamic viscosity, the life of the transfection cartridge(s), the physical state of the suspension, or the state of the transfection device(s).

[0131] In some embodiments, the optimization algorithm has the ability to perform predictive analysis based on known input cell type parameters and adjust the electromechanical parameters accordingly. Measured input parameters include, but are not limited to, suspension conductivity, suspension temperature, suspension dynamic viscosity, cell morphology, cell size, and cell impedance. In some embodiments, the optimization algorithm adjusts the electromechanical parameters based on electrical signals in any of the devices of the present invention. In some embodiments, the optimization algorithm adjusts the electromechanical parameters based on detected flow parameters in any of the devices of the present invention. In some embodiments, the optimization algorithm adjusts the transfection parameters based on unique dimensionless input parameters. In some embodiments, the optimization algorithm has the ability to adjust the electromechanical transfection parameters based on unique multivariate combinations of parameters that predict high viability outcomes, high efficiency outcomes, or consistent viability and efficiency outcomes.

[0132] The system of the present invention may include one or more outer structures configured to cover the electrodes of one or more devices of the present invention, for example, to reduce end user exposure to live electrical connections. Typically, an electromechanical system includes one outer structure that covers its electrodes and active zones. The outer structure may be a non-conductive material, for example, a non-conductive polymer, and includes structural features for electromechanically engaging with components of the device, for example, electrodes or active zones. The outer structure may include one or more recesses, cutouts, or similar openings in the structure to accommodate the device. The outer structure may be a component that can be removed from the device. It may be configured to be. For example, the outer structure may include two separate components connected by a hinge, for example, a living hinge, so that it can be folded onto the device of the present invention. Alternatively, the outer structure may be one or more separate parts that can be joined using appropriate interlocking features to form a single structure. In these embodiments, the outer structure may be affixed to the device of the present invention using any suitable fastener, for example, a snap, latch, button, or clip, which may be integrated into the outer structure or may be externally connected to the outer structure. Other suitable fastener types are known in the art. In some embodiments, the outer structure includes one or more alignment features, such as pins, grooves, or tabs, that ensure proper alignment of one or more portions of the outer structure. In some cases, the outer structure is configured to be permanently connected to the device of the present invention.

[0133] In some embodiments, a housing (e.g., a cartridge, e.g., an outer structure) encloses one or more devices used in the previously described inventions or continuous flow electromechanical transfection. In some embodiments, the housing (e.g., a cartridge) is configured to allow for use with and / or insertion into an automatically closing system for delivery of cell therapy to a patient. In some embodiments, the housing further includes a cooling / heating area / enclosure for storage of cell suspensions and / or buffers before, during, and after electromechanical transfection of a specimen. In some embodiments, the system (e.g., one or more devices and the housing) is externally powered.

[0134] In some embodiments, the system also includes optimization algorithms that have the ability to independently or autonomously adjust the electromechanical parameters if the user selects this feature. These optimization algorithms allow for continuous adjustment of parameters used in the transfection process that may depend on cell type, electrical conductivity, suspension volume, dynamic viscosity, electromechanical cartridge life, suspension physical state, or electromechanical device state.

[0135] In any of the embodiments of the outer structure described herein, the outer structure provides an electrical connection between an external potential source and the electrodes of the device of the present invention. For example, the outer structure can include one or more electrical inputs for electrical connection, such as spades, banana plugs, or bayonets, such as BNC, connectors, that facilitate electrical connection between the potential source and the electrodes of the device of the present invention inside the outer structure.

[0136] The device and external structure of the present invention can be combined in a kit with additional external components such as reagents, e.g., buffers, e.g., transfection buffers or harvesting buffers, and / or samples. In certain embodiments, the transfection buffer comprises a composition suitable for electromechanical transfection of cells. In some embodiments, the transfection buffer comprises a suitable concentration of one or more salts (e.g., potassium chloride, sodium chloride, potassium phosphate, potassium dihydrogen phosphate) or sugars (e.g., dextrose or myo-inositol) at a concentration of 0.1 to 200 mM (e.g., 0.1 to 1.0 mM, 1.0 mM to 10 mM, or 10 mM to 100 mM), or any combination thereof.

[0137] Buffers and media The devices and systems of the present invention can be used with transfection buffers or cell culture growth media that contain additives that support transfection. To control the conductivity of the transfection buffer and / or cell culture growth media, KCl, MgCl 2 , NaCl, glucose, Na 2 HPO 4 , NaH 2 PO 4 , Ca(NO 3 ) 2 , mannitol, succinate, dextrose, hydroxyethylpiperazineethanesulfonic acid (HEPES), trehalose, CaCl 2 , dimethyl sulfoxide (DMSO), K 2 HPO 4 , K.H. 2 PO 4 , ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), KOH, NaOH, K 2 SO 4 , Na 2 SO 4 , histidine buffer, citrate buffer, phosphate-buffered saline (PBS), ATP-disodium salt, and NaHCO 3To control the dynamic viscosity of the transfection buffer and / or cell culture growth medium used, certain additives such as Ficoll, dextran, polyethylene glycol (PEG), methylcellulose (Methocel), collagen I, and Matrigel can be added.

[0138] method The present invention features a method for introducing a composition, e.g., a genetic payload, into at least a portion of a plurality of cells suspended in a fluid using the electromechanical transfection device described herein. The method described herein can be used to significantly increase the throughput of delivery of compositions to cell types that are often considered to be a bottleneck in the fields of genetic engineering research and genetically modified cell therapy treatment. In particular, the method described herein can be applied to many more cell types than typical transfection methods, e.g., lentiviral transfection, or commercially available cell transfection instruments, e.g., electroporation-based instruments, to significantly increase cell recovery numbers, transfection efficiency, and cell viability after transfection.

[0139] The composition is introduced to at least a portion of the plurality of cells suspended in the fluid by passing the fluid, along with the suspended cells, through a device of the invention, e.g., an electromechanical transfection device, as described herein, with the fluid. The composition and cells suspended in the fluid can be delivered through the device of the invention, e.g., by application of positive pressure from a fluid source, e.g., a peristaltic pump, a digital pipette, or a pump connected to an automated liquid handling source. The composition and cells suspended in the fluid pass from an entry zone to an active zone, e.g., an active zone arranged to pass through an electric field generated by two electrodes. As the composition and cells suspended in the fluid pass through the active zone, a potential difference is applied to the first and second electrodes, generating an electric field that electrically energizes the cells in the active zone, thereby exposing the cells to the electric field. The cells in the fluid are simultaneously exposed to mechanical energy from the flow. Exposure of the cells to the generated electric field, in combination with the mechanical energy of the flow, increases the temporary permeability of the plurality of cells, thereby introducing the composition into at least a portion of the plurality of cells. In certain embodiments, the electric field and the flow are dimensionless parameters,

number

[0140] In some embodiments, the ratio of electrical energy delivered to the flowing liquid by the electric field to the mechanical energy delivered by the pressure drop in the active zone is greater than or equal to 10 3 :1 to 10 6 Between :1, e.g. 10 3 :1 and 10 5 Between :1 and 10 4 :1 and 10 6 Between :1 and 10 3 :1 and 10 4 Between :1 or 10 5 :1 and 10 6 :1 (for example, about 10 3 :1, 104 :1, 10 5 :1, or 10 6 :1).

[0141] In some embodiments, the methods of the invention first involve passing a test portion of a plurality of cells (e.g., a test portion from a larger plurality of cells) and a test composition through an active zone according to any method described herein. 5 An optimal range of Π can be determined, e.g., that corresponds to maximum cell viability, transfection efficiency, and / or engineered cell yield. 5 A range of Π corresponding to maximum cell viability, transfection efficiency, and / or engineered cell yield can be found. 5 To find the range of Π, a test portion (e.g., having a certain ratio of cells to composition) may be passed through the active zone at an average flow velocity (u) while applying an electric field (E) while varying one or more of (u), (E), liquid conductivity (σ), liquid dynamic viscosity (μ), and liquid density (ρ). This can be repeated several times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) with the same and / or different ratios of cells to composition. Alternatively, or in addition, the test can be repeated with active zones having different hydraulic diameters. Π 5 After determining the appropriate range(s) of (u), (E), (σ), (μ), and (ρ), a plurality of cells may be passed through the active zone (of appropriate hydraulic diameter) at any combination of (u), (E), (σ), (μ), and (ρ) to introduce the composition to the plurality of cells. Any one or any combination of the variables (u), (E), (σ), (μ), and (ρ) may be varied.

[0142] In certain embodiments, the test moiety can be passed through the active zone while applying a constant electric field (E) at the electrodes and varying the average flow rate (u), or the average flow rate can be kept constant while the electric field is varied (e.g., by varying the voltage between the electrodes). Either or both of these steps can be repeated one or more times, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.

[0143] In some embodiments of the method, the phenotypic markers of a cell related to the health of the cell, or the expression of a particular surface marker, or a particular cellular characteristic, e.g., a characteristic required for therapeutic function, may not change relative to a baseline measurement of the phenotypic markers of a cell, or other measurement of the health, function, etc. of the cell upon exiting the active zone of the device of the invention. In certain embodiments, the plurality of cells have no measurable change in a particular phenotypic marker related to the health or desired function (e.g., expression) of the cell upon exiting the active zone. For example, a baseline or control measurement for establishing the phenotype of a cell may be a measurement of the expression of a cell surface marker in a cell that has not been transfected with the device of the invention. To assess the change in the cellular phenotype, a corresponding identical measurement of the expression of the same cellular marker on a cell that has been transfected with the device of the invention may be used. The cellular phenotype is assessed by flow cytometric analysis of cell surface marker expression to confirm that there is minimal or no change in the cellular phenotype following electromechanical transfection. Examples of cell surface markers to be evaluated include CD3, CD4, CD8, CD19, CD45RA, CD45RO, CD28, CD44, CD69, CD80, CD86, CD206, IL-2 receptor, CTLA4, OX40, PD-1, and TIM3, CD56, TNFa, IFNg, LAG3, TCRα / β, CD64, SIRPα / β (CD172a / b), nestin, CD325 (N-cadherin), CD183 ( Examples of antibodies that may be used include, but are not limited to, CXCR3), CD184 (CXCR4), CD197 (CCR7), CD27, CD11b, CCR7 (CD197), CD16, CD56, TIGIT, TRA-1-60, Nanog, TCR gamma / delta, OCT4, T-bet, GATA-3, FoxP3, IL-17, B220, CD25, IgM, PD-L1, IL-23, IL-12, CD11c, and F4 / 80. Cell morphology is assessed (e.g., using bright field or fluorescent microscopy) to ensure there is no phenotypic change following electromechanical transfection.

[0144] In some embodiments, after the composition is introduced into at least a portion of the plurality of cells, the plurality of cells are stored in a recovery buffer. The recovery buffer is configured to promote eventual closure of holes formed in the plurality of cells. The recovery buffer typically comprises a cell culture medium that may contain other components for nourishing and growing the cells, such as serum, minerals, etc. One skilled in the art can understand that the choice of recovery buffer depends on the cell type to be subjected to electromechanical transfection.

[0145] The cell flux, i.e., the number of cells processed per minute per active zone, e.g., the number of cells transfected per minute, is typically greater than 10 3 cells / min~10 11 Cells / min, e.g., 10 3 cells / min~10 4 cells / min, 5×10 3 cells / min~5×10 4 cells / min, 10 5 cells / min~10 5 cells / min, 5×10 5 cells / min~5×10 6 cells / min, 10 6 cells / min~10 7 cells / min, 5×10 6 cells / min~5×10 7 cells / min, 10 7 cells / min~10 8 cells / min, 5×10 7 cells / min~5×10 8 cells / min, 10 8 cells / min~10 9 cells / min, 5×10 8 cells / min~5×10 9 cells / min, 10 9 cells / min~10 9 cells / min, 5×10 9 cells / min~5×10 10 cells / min, or 10 10 cells / min~10 11 cells / min range, e.g., about 10 3 cells / min, 5×10 3 cells / min, 10 4 cells / min, 5×10 4cells / min, 10 5 cells / min, 5×10 5 cells / min, 10 6 cells / min, 5×10 6 cells / min, 10 7 cells / min, 5×10 7 cells / min, 10 8 cells / min, 5×10 8 cells / min, 10 9 cells / min, 5×10 9 cells / min, 10 10 cells / min, 5×10 10 cells / min, or 10 11 In some of the methods, the composition is at least 1×10 cells per minute per active zone. 5 Cells, e.g. 10 5 cells / min~10 5 cells / min, 5×10 5 cells / min~5×10 6 cells / min, 10 6 cells / min~10 7 cells / min, 5×10 6 cells / min~5×10 7 cells / min, 10 7 cells / min~10 8 cells / min, 5×10 7 cells / min~5×10 8 cells / min, 10 8 cells / min~10 9 cells / min, 5×10 8 cells / min~5×10 9 cells / min, 10 9 cells / min~10 9 cells / min, 5×10 9 cells / min~5×10 10 cells / min, 10 10 cells / min~10 11 cells / min, or 10 11 cells / min~10 12 cells / min, e.g., about 10 3 cells / min, 5×10 3 cells / min, 10 4 cells / min, 5×10 4 cells / min, 10 5 cells / min, 5×10 5 cells / min, 10 6 cells / min, 5×106 cells / min, 10 7 cells / min, 5×10 7 cells / min, 10 8 cells / min, 5×10 8 cells / min, 10 9 cells / min, 5×10 9 cells / min, 10 10 cells / min, 5×10 10 cells / min, 10 11 cells / min, or 10 12 The antibody is delivered to multiple cells at a flux of 100,000 cells / min.

[0146] In some embodiments of the methods described herein, the volume of fluid with suspended cells (e.g., replacement volume) flowed through an active zone of a device of the invention and the composition introduced to the cells may be from 0.001 mL to 2000 mL, 0.001 mL to 1000 mL, e.g., 0.001 mL to 0.1 mL, 0.01 mL to 1 mL, 0.01 mL to 750 mL, 0.01 mL to 1500 mL, 0.1 mL to 5 mL, 0.1 mL to 500 mL, 0.1 mL to 2000 mL, 1 mL to 10 mL, 1 mL to 1000 mL, 2 mL to 2000 mL, 2.5mL to 20mL, 5mL to 40mL, 10mL to 60mL, 10mL to 1000mL, 20mL to 2000mL, 30mL to 80mL, 50mL to 200mL, 100mL to 500mL, or 250mL to 750mL, 500mL to 1000mL, 500mL to 2000mL, 750mL to 1500mL, or 1000mL to 2000mL, for example, 0.01mL to 100mL, 0.1mL to 99mL, 1mL to 97mL, or 10mL to 95mL, for example, 0.0025mL to 10mL, 0.01mL to 1mL, or 0.025mL ~0.1mL, for example, about 0.001mL, 0.0025mL, 0.005mL, 0.0075mL, 0.01mL, 0.025mL, 0.05mL, 0.075mL, 0.1mL, 0.25mL, 0.5mL, 0.75mL,1mL,2mL,3mL,4mL,5mL,6 mL,7mL,8mL,9mL,10mL,15mL,20mL,25mL,30mL,35mL,40mL,45mL,50mL,55mL,60mL,65mL,70mL,75mL,80mL,85mL,90mL,95mL,100mL,150mL,200mL,2 It may be 50mL, 300mL, 350mL, 400mL, 450mL, 500mL, 550mL, 600mL, 650mL, 700mL, 750mL, 800mL, 850mL, 900mL, 950mL, 1000mL, 1050mL, 1100mL, 1150mL, 1200mL, 1250mL, 1300mL, 1350mL, 1400mL, 1450mL, 1500mL, 1550mL, 1600mL, 1650mL, 1700mL, 1750mL, 1800mL, 1850mL, 1900mL, 1950mL, or 2000mL.

[0147] In some embodiments, the volume of fluid flowed through the active zone of the device of the invention (e.g., displacement volume), displacement rate, or other controlled parameters may or may not affect the transfection efficiency of multiple cells. In some embodiments, the device of the invention is configured for use with an automated fluid handling platform capable of processing multiple cells in volumes of about 10-200 μL per reaction. In some embodiments, the device of the invention is part of a system capable of processing volumes up to several liters per reaction. In some embodiments, the automated fluid handling platform is configured for use with one or more fluid delivery sources (e.g., pumps, e.g., syringe pumps, micropumps, or peristaltic pumps) that deliver the volume of fluid flowed through the active zone of the device of the invention. In some embodiments, the volume of fluid flowed through the active zone of the device of the invention may be delivered by displacement of the actuating fluid relative to a reservoir of the fluid to be delivered or by air displacement. In some embodiments, the fluid delivery source is configured to flow cells suspended in the fluid by application of positive pressure.

[0148] In certain embodiments, the electrical conductivity of the fluid in which the cells are suspended can affect the electromechanical transfection of the cells in suspension. The electrical conductivity of the fluid in which the cells are suspended can be 0, 0.001 mS to 500 mS, e.g., 0, 0.001 mS to 0.1 mS, 0.01 mS to 1 mS, 0.1 mS to 10 mS, 1 mS to 50 mS, 10 mS to 100 mS, 25 mS to 200 mS, 50 mS to 400 mS, or 100 mS to 500 mS, e.g., 0. 0.01mS to 100mS, 0.1mS to 50mS, or 1 to 20mS, for example, about 0.001mS, 0.002mS, 0.003mS, 0.004mS, 0.005mS, 0.006mS, 0.007mS, 0.008mS, 0.009mS, 0.01mS, 0.02mS, 0.03mS, 0.04mS, 0. 05mS,0.06mS,0.07mS,0.08mS,0.09mS,0.1mS,0.2mS,0.3mS,0.4mS,0.5mS,0.6mS, 0.7mS,0.8mS,0.9mS,1mS,2mS,3mS,4mS,5mS,6mS,7mS,8mS,9mS,10mS,15mS,20mS,2 It may be 5mS, 30mS, 35mS, 40mS, 45mS, 50mS, 55mS, 60mS, 65mS, 70mS, 75mS, 80mS, 85mS, 90mS, 95mS, 100mS, 150mS, 200mS, 250mS, 300mS, 350mS, 400mS, 450mS, or 500mS.

[0149] The methods of the invention can be used to culture mammalian cells, eukaryotic, prokaryotic, synthetic cells, human cells, animal cells, plant cells, primary cells, cell lines, suspension cells, adherent cells, unstimulated cells, stimulated cells, or activated cells, immune cells, solid tumor cells, stem cells (e.g., stem cells (e.g., primary human induced pluripotent stem cells, e.g., iPSCs, embryonic stem cells, e.g., ESCs, mesenchymal stem cells, e.g., MSCs, or hematopoietic stem cells, e.g., hematopoietic stem cells), blood cells (e.g., red blood cells), T cells (e.g., primary human T cells), B cells, antigen presenting cells (APCs), The compositions can be delivered to a variety of cell types, including, but not limited to, natural killer (NK) cells (e.g., primary human NK cells), monocytes (e.g., primary human monocytes), macrophages (e.g., primary human macrophages), and peripheral blood mononuclear cells (PBMCs), neutrophils, dendritic cells, human embryonic kidney (e.g., HEK-293) cells, or Chinese hamster ovary (e.g., CHO-K1) cells. Typical cell numbers that can be transfected are 10 to 10 per active zone. 4 10 from cells 12 cells (e.g., 10 4 10 from cells 5 cells, 10 4 10 from cells 5 cells, 10 4 10 from cells 5 cells, 10 4 10 from cells 5 cells, 10 4 10 from cells 5 cells, 10 4 10 from cells 5 cells), 10 4 10 from cells 5 cells, 10 4 10 from cells 6 cells, 10 4 10 from cells 7 cells, 5 x 10 4 5 × 10 cells 5 cells, 10 5 10 from cells 6 cells, 10 5 10 from cells 7 cells, 2.5 x 10 5 cells ~10 6 cells, 5 x 10 5cells ~5×10 6 cells, 10 6 cells ~10 7 cells, 10 6 cells ~10 8 cells, 10 6 cells ~10 12 cells, 5 x 10 6 cells ~5×10 7 cells, 10 7 cells ~10 8 cells, 10 7 cells ~10 9 cells, 10 7 cells ~10 12 cells, 5 x 10 7 cells ~5×10 8 cells, 10 8 cells ~10 9 cells, 10 8 cells ~10 10 cells, 10 8 cells ~10 12 cells, 5 x 10 8 cells ~5×10 9 cells, 10 9 cells ~10 10 cells, 10 9 cells ~10 11 cells, 10 10 cells ~10 11 cells, 10 10 cells ~10 12 cells, or 10 11 cells ~10 12 Cells, e.g., about 10 4 cells, 2.5 x 10 4 cells, 5 x 10 4 cells, 10 5 cells, 2.5 x 10 5 cells, 5 x 10 5 cells, 10 6 cells, 2.5 x 10 6 cells, 5 x 10 6 cells, 10 7 cells, 2.5 x 10 7 cells, 5 x 10 7 cells, 10 8 cells, 2.5 x 10 8 cells, 5 x 10 8 cells, 10 9 cells, 2.5 x 10 9 cells, 5 x 109 cells, 10 10 cells, 5 x 10 10 cells, 10 11 cells, or 10 12 The cell may be a human cell.

[0150] The method of the present invention described herein can deliver various compositions to cells suspended in fluid.Compositions that can be delivered to cells include therapeutic agents, vitamins, nanoparticles, charged molecules, such as ions in solution, uncharged molecules, nucleic acids, such as DNA or RNA, CRISP, such as DNA or RNA, CRISPR-Cas complexes, proteins, polymers, ribonucleoproteins (RNPs), artificial nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), homing nucleases, meganucleases (MNs), megaTALs, enzymes, peptides, transposons, or polysaccharides, such as dextran, such as dextran sulfate. Exemplary compositions that can be delivered to cells in suspension include, but are not limited to, nucleic acids, oligonucleotides, antibodies (or antibody fragments, e.g., bispecific fragments, trispecific fragments, Fab, F(ab')2, or single chain variable fragments (scFv)), amino acids, peptides, proteins, gene therapy drugs, genome engineering therapeutic drugs, epigenome engineering therapeutic drugs, carbohydrates, chemical drugs, contrast agents, magnetic particles, polymer beads, metal nanoparticles, metal microparticles, quantum dots, antioxidants, antibiotics, hormones, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, anti-inflammatory agents, antimicrobial agents, chemotherapeutic agents, exosomes, outer membrane vesicles, vaccines, viruses, bacteriophages, adjuvants, minerals, and combinations thereof. The composition to be delivered can include a single compound, such as a compound described herein. Alternatively, the composition to be delivered may include multiple compounds or components that target different genes.

[0151] The typical concentration of the liquid composition is 0.0001μg / mL~1000μg / mL, (for example, 0.0001μg / mL~0.001μg / mL, 0.001μg / mL~0.01μg / mL, 0.001μg / mL~5μg / mL, 0.005μg / mL~0.1μg / mL L、0.01μg / mL~0.1μg / mL、0.01μg / mL~1μg / mL、0.1μg / mL~1μg / mL、0.1μg / mL~5μg / mL、1μg / mL~10μg / mL、1μg / mL~50μg / mL、1μg / mL~100μg / mL、2.5μg / mL ~15μg / mL、5μg / mL~25μg / mL、5μg / mL~50μg / mL、5μg / mL~500μg / mL、7.5μg / mL~75μg / mL、10μg / mL~100μg / mL、10μg / mL~1,000μg / mL、25μg / mL~50μg / mL 、25μg / mL~250μg / mL、25μg / mL~500μg / mL、50μg / mL~100μg / mL、50μg / mL~250μg / mL、50μg / mL~750μg / mL、100μg / mL~300μg / mL、100μg / mL~1,000μg / mL、 200μg / mL to 400μg / mL, 250μg / mL to 500μg / mL, 350μg / mL to 500μg / mL, 400μg / mL to 1,000μg / mL, 500μg / mL to 750μg / mL, 650μg / mL to 1,000μg / mL, or 800μg / mL to 1,000μg / mL, for example, about 0.0001μg / mL, 0.0005μg / mL, 0.001μg / mL, 0.005μg / mL, 0.01μg / mL, 0.02μg / mL, 0.03μg / mL, 0.04μg / mL, 0.05μg / mL, 0.06μg / mL, 0 .07μg / mL、0.08μg / mL、0.09μg / mL、0.1μg / mL、0.2μg / mL、0.3μg / mL、0.4μg / mL、0.5μg / mL、0.6μg / mL、0.7μg / mL、0.8μg / mL、0.9μg / mL、1μg / mL、1.5μg / mL mL、2μg / mL、2.5μg / mL、3μg / mL、3.5μg / mL、4μg / mL、4.5μg / mL、5μg / mL、5.5μg / mL、6μg / mL、6.5μg / mL、7μg / mL、7.5μg / mL、8μg / mL、8.5μg / mL、9μg / mL.5µg / mL、10µg / mL、15µg / mL、20µg / mL、25µg / mL、30µg / mL、35µg / mL、40µg / mL、45µg / mL、50µg / mL、55µ g / mL、60µg / mL、65µg / mL、70µg / mL、75µg / mL、80µg / mL、85µg / mL、90µg / mL、95µg / mL、100µg / mL、200µ g / mL、250µg / mL、300µg / mL、350µg / mL、400µg / mL、450µg / mL、500µg / mL、550µg / mL、600µg / mL、650µg / mL、700µg / mL、750µg / mL、800µg / mL、850µg / mL、9 00µg / mL, 950µg / mL, 1,000µg / mL).

[0152] The concentrations of the respective active ingredients are 0.0001µM and 20µM and the range(s are 0.0001µM~0.001µM). M~0.01μM、0.001μM~5μM、0.005μM~0.1μM、0.01μM~0.1μM、0.01μM~1μM、0 .1µM~1µM, 0.1µM~5µM, 1µM~10µM, 1µM~15µM µM, 0.0005µM, 0.001µM, 0.005µM, 0.01µM, 0.02µM, 0.03µM, 0.04µM, 0.05µ M、0.06µM、0.07µM,0.08µM,0.09µM,0.1µM,0.2µM,0.3µM,0.4µM,0.5µM, 0.6μM,0.7μM,0.8μM,0.9μM,1μM,1.5μM,2μM,2.5μM,3μM,3.5μM,4μM4. 5µM, 5µM, 5.5µM, 6µM, 6.5µM, 7µM, 7.5µM, 8µM, 8.5µM, 9µM, 9.5µM, 10µM 1µM, 12µM, 13µM, 14µM, 15µM, 16µM, 17µM, 18µM, 19µM, and 20µM.

[0153] In some cases, the temperature of the fluid containing the suspended cells and the composition is controlled using a thermal control device integrated into the housing supporting the device of the invention. The temperature of the fluid is controlled to reduce the effect of Joule heating from the electric field generated in the active zone, since too high a temperature can compromise the viability of the cells after electromechanical transfection. The temperature of the fluid can be between 0°C and 40°C, e.g., between 0°C and 10°C, between 1°C and 5°C, between 2°C and 15°C, between 3°C and 20°C, between 4°C and 25°C, between 5°C and 30°C, between 7°C and 35°C, between 9°C and 40°C, between 10°C and 38°C, between 15°C and 40°C, between 20°C and 40°C, between 25°C and 40°C, or between 35°C and 40°C, e.g., about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 38°C, 15°C, 40°C, 20°C, 40°C, 25°C, 40°C, or 35°C, ... , 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0154] Cells transfected using the methods of the present invention are more efficiently transfected and have higher viability than using typical transfection methods, such as lentiviral transfection, or commercially available cell transfection equipment. For example, the transfection efficiency, i.e., the efficiency of successfully delivering the composition to the cells, for the methods described herein may be between 0.1% and 99.9%, e.g., between 0.1% and 5%, 1% and 10%, 2.5% and 20%, 5% and 40%, 10% and 60%, 30% and 80%, or 50% and 99.9%, e.g., between 0.1% and 5%, 1% and 10%, 2.5% and 20%, 5% and 40%, 10% and 60%, 30% and 80%, or 50% and 99, 10% and 90%, 25% and 85%, e.g., about 0.1% and 99.9 ... .1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.9%.

[0155] The cell viability of cells suspended in a fluid after introduction of a composition using the methods of the invention described herein, i.e., the number or percentage of healthy cells after the electromechanical transfection process, can be between 0.1% and 99.9%, e.g., between 0.1% and 5%, 1% and 10%, 2.5% and 20%, 5% and 40%, 10% and 60%, 30% and 80%, or 50% and 99.9%, e.g., between 10% and 90%, 25% and 85%, e.g., about 0.1%, 0.15%, 0.2 %, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.9%.

[0156] The recovery yield, i.e., the percentage of live engineered cells recovered after electromechanical transfection, can range from 0.1% to 500%, e.g., 0.1% to 5%, 1% to 10%, 2.5% to 20%, 5% to 40%, 10% to 60%, 30% to 80%, 50% to 99.9%, 75% to 150%, 100% to 200%, 150% to 250%, 20% to 30% and 40% at 24 h after transfection. 00% to 300%, 250% to 350%, 300% to 400%, 350% to 450%, or 400% to 500%, for example, about 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%,3%,4%,5%,6%,7%,8%,9%,10%,15%,20%,25%,30%,35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%,95%,99.9%,100%,110%,120%,130%,140%,150%,160%,170%,180%,190%,200%,21 It may be 0%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%.

[0157] In some embodiments, the method provides for a concentration of between 0.1% and 100% (e.g., between 0.1% and 5%, between 1% and 10%, between 2.5% and 20%, between 5% and 40%, between 10% and 30%, between 10% and 60%, between 10% and 90%, between 25% and 40%, between 25% and 85%, between 30% and 50%, between 30% and 80%, between 40% and 65%, between 50% and 75%, between 50% and 100%, between 60% and 80%, between 75% and 100%, between 85% and 100%, e.g., about 0.1%, 0.15%, 0.2% , 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%), resulting in a cell recovery yield, i.e., the percentage of live engineered cells recovered following electromechanical transfection.

[0158] One of skill in the art will appreciate that optimal conditions will vary depending on cell type and other factors: for each new cell type, parameters such as waveform, electric field, pulse time, buffer exposure time, buffer temperature, post-treatment conditions, etc. can be adjusted as necessary. EXAMPLES

[0159] Example 1 - Application of electromechanical transfection Electromechanical transfection performed using an automated liquid handler is demonstrated herein. In this example, a flow cell was designed to be integrated with the liquid handling system, allowing the delivery of electrical and mechanical energy to the cell suspension (Figure 1). The flow cell includes a pipette tip designed with a reservoir, allowing the pick-up and dispensing of cells and payload suspended in a fluid buffer. As the cells and payload suspended in a fluid buffer pass through the flow cell at a defined flow rate, a precise electric field is delivered across the flow cell by contact with electrodes positioned across the flow cell area. These cells are dispensed into a 96-well plate containing growth medium and cultured for 24 hours. Biological analysis is then performed and output metrics determined by the flow cytometer (an example of gating can be seen in Figure 7).

[0160] To use electromechanical transfection effectively, optimal conditions and parameters must be determined for the targeted cell-payload combination. These conditions and parameters include flow rate and electric field characteristics. To determine optimal conditions for applying electromechanical transfection for mRNA-based payload delivery to primary T cells, plate-based matrix experiments were performed on a fully automated platform that includes an array of electromechanical devices integrated with a commercially available liquid handling system (PerkinElmer JANUS® G3 System, Waltham, MA). Utilizing this technology, up to 96 independently programmed combinations of electromechanical transfection parameters can be delivered in a batch-wise manner. In this set of experiments, reporter mRNA payloads were delivered to day 9 expanded human T cells, 24 hours after thawing. Primary human T cells were expanded with soluble anti-CD3 and anti-CD28 antibodies and resuspended in transfection buffer. Samples were harvested for culture and downstream analysis immediately after processing. Cell viability, viable cell count, and transfection efficiency were measured using flow cytometry (Thermo Fisher Attune™ NxT), and GFP (green fluorescent protein) mRNA was used to measure transfection efficiency into proliferating T cells (Figure 8). This series of experiments demonstrated that the transfected cells had high viability (>70% viable cells) and high transfection efficiency (>90% GFP mRNA). + ) were obtained, resulting in multiple conditions (11 in total) that exhibited both.

[0161] The most relevant cell transfection parameters, such as viability, transfection efficiency and cell yield, were determined by Π 1 , Π 2 ,Re,Π 4 , and combinations of these (Figure 2). A fifth dimensionless group, a combination of all four, appears to govern the main physics associated with cell transfection. This dimensionless group is defined as:

number

[0162] Example 2 - Transcriptional profiling To further evaluate the effect of introducing genetic payloads into T cells using electromechanical transfection, we performed a transcriptomic analysis to evaluate the transcriptional changes that occur after treatment (Figure 3A-F). In addition, commercially available non-viral electroporation-based systems were included in the comparison: Thermo Fisher's Neon™ Transfection System (referred to as "Neon™") and Lonza's 4D Nucleofector™ (referred to as "4D Nucleofector™"). Each system was evaluated with a 100 μL reaction containing 5 M cells and a proprietary program and buffer specific to the device. Program information for each device is listed in the Materials section. For each device, cells were treated in the absence of payload and compared to a donor control that was not treated with anything. For this analysis, cells from two donors were treated in duplicate with each device. Significant (p<0.05) gene dysregulation greater than 1-fold at 6 hours after cell treatment is shown as red (upregulated genes) or green (downregulated genes) in the volcano plots in Figures 3A-C (replicate data from a second donor is shown in Figure 9). The electromechanical system showed a near-baseline gene expression profile after 6 hours, with only 2% of genes dysregulated by the electromechanical transfection process (Figure 3D). The Neon™ static transfection system showed a low dysregulation profile after 6 hours, with only 6% of genes dysregulated by this electroporation-based process (Figure 3D). The 4D Nucleofector™ showed significant dysregulation after 6 hours, with 47% of all genes dysregulated by this electroporation-based transfection process (Figure 3D).

[0163] The functional capacity of the cell product may directly affect the efficacy of the cells to induce a desired immunological response. For example, differentiation and exhaustion of edited cells have been shown to lead to limited efficiency of T cell therapy. To better understand the impact of cell processing on T cell function, upregulation of genes commonly associated with T cell function was assessed at 6 and 24 h time points (Figure 3E). These included inflammatory cytokines (IFNγ and IL-2) and activating receptors (CD69 and CD27), which were selected to assess process-driven cell activation. Additionally, exhaustion receptors (CTLA4 and TIGIT) were selected as indicators of process impact on downstream cell function in treated cells. No upregulation of inflammatory cytokines, activating receptors, or exhaustion markers was observed in electromechanically treated cells (Figure 3E). Conversely, electroporation induced upregulation of these genes; the Neon™ transfection system upregulated CTLA4, and the 4D Nucleofector™ upregulated inflammatory cytokines, activation receptors, and exhaustion markers in treated cells (Figure 3E).

[0164] To further explore the impact on whole cell health and function, gene ontology focused on molecular function, biological process, and protein class was assessed using the Protein Analysis Through Evolutionary Relationships (PANTHER) classification system. At 6 hours, electromechanical transfection showed that 6% of the total dysregulations were associated with protein class, 13% were associated with molecular function, and 18% of the total dysregulations were associated with biological process (Figure 3F). With the Neon™ electroporation system, 10% of the total dysregulations were associated with protein class, 19% were associated with molecular function, and 36% of the total dysregulations were associated with biological process (Figure 3F). With transfection using the 4D Nucleofector™, 13% of the total dysregulations were associated with protein class, 24% were associated with molecular function, and 52% of the total dysregulations were associated with biological process (Figure 3F).

[0165] To correlate the transcriptome data with post-treatment viability and delivery efficiency, cells from the same donor were transfected with reporter mRNA payloads using the same programs and conditions for the electromechanical, Neon™, and 4D Nucleofector™ platforms (Figures 11A-11C). The electromechanical system showed similarly high transfection cell viability, ~80%, as the Neon™ system, while the 4D Nucleofector™ system showed low transfection cell viability, ~45% (Figure 11A). In terms of delivery, both the electromechanical and Neon™ systems achieved high delivery efficiency, ~90%, whereas the 4D Nucleofector™ system had a moderate delivery efficiency, ~50% (Figure 11B). Taken together with the transcriptome analysis, it is clear that non-viral delivery efficiency is independent of poor health of the post-treatment cell product. Furthermore, electromechanical transfection compares favorably with existing electroporation-based transfection devices in all metrics, including gene dysregulation, viability, efficiency, and cell health output.

[0166] Example 3 - Introduction of multiple mRNAs into primary human T cells Experiments were performed to evaluate the ability of the electromechanical transfection method to deliver multiple payloads to a single cell in parallel (i.e., co-delivery in a single treatment) and serial (i.e., staggered treatments at 48 h intervals). Parallel treatments were performed on the same day with a cell mix containing two mRNAs, including GFP reporter mRNA and mCherry reporter mRNA, whereas serial treatments were performed every other day with a cell mix containing a single mRNA at each time point, administering GFP reporter mRNA first and mCherry mRNA 48 h later. The mRNAs expressed fluorescent reporter genes to track delivery efficiency at the single cell level (Figures 11A-11C). The viability of primary T cells 24 h after treatment with electromechanical transfection was approximately 80% for both methods (Figure 11A), indicating that parallel and serial transfections are not detrimental to cell health, and that transfections can be repeated with staggered times without significant loss of cell viability using the electromechanical technique. However, different expression profiles were observed for the two methods (Figure 11B). The parallel method achieved a dual transfection efficiency of 94.2% in single cells, whereas the serial transfection achieved a transfection efficiency of 82.3% (Figure 11C). When mRNAs were co-transfected in parallel, a clean 1:1 expression was observed, with very few cells (1%) expressing only a single fluorescent reporter (Figure 11B). In contrast, serial transfection resulted in 3.3% of the population being single positive for GFP and 11.3% of the population being single positive for mCherry (Figure 11C).

[0167] Example 4 - Multiple T cell donors Donor heterogeneity is a constant in all cell therapy manufacturing and development pipelines, and therefore it is imperative to evaluate output metrics across multiple donors. For clinical manufacturing, raw materials containing T cells from various donors may require recharacterization and comparability testing. Therefore, it is crucial for cell therapy development to show that the results achieved in the optimization efforts above are applicable to T cells obtained from various starting materials (Figure 4A-4B). Cells were isolated from three different healthy PBMC donors (demographics are in Table 1 below), expanded, and then transfected with GFP mRNA via electromechanical transfection. All donors in this study met the starting phenotypic and viability criteria outlined in the Materials and Methods section. This experiment showed consistent results with cell material from multiple donors with less than 10% change in viability from no-transfection controls (Figure 4A). Furthermore, the transduction efficiency of GFP mRNA was less variable (greater than 84%) for all three donors (Figure 4B).

[0168] Table 1 - Expanded human T cells from three unique donors were transfected with GFP reporter mRNA using electromechanical techniques. This demographic table compares three unique donors. [Table 1]

[0169] Example 5 - mRNA delivery into naive human T cells Although non-activated naive T cells have attracted attention in the field of cell engineering due to their relative simplicity in preparation and handling prior to transfection, this cell type has been underutilized in the fields of cell and gene therapy due to historical challenges associated with the introduction of genetic information and the inability to perform retroviral transduction without first activating the T cells. To evaluate the performance of electromechanical transfection for this traditionally difficult-to-transfect cellular state, isolated naive T cells (CD3 + / CD4+ / CD45RA + / CD45RO - ) were transfected with mRNA encoding GFP (Figure 5A-F). The naive T cells were expanded with soluble anti-CD3 / anti-CD28 activation reagents and monitored for 6 days after transfection. The proliferation rate of these cells after transfection was comparable to that of untreated control cells up to 6 days after activation (Figure 5A), and there was no significant loss of viability (Figure 5B). Furthermore, staining of the cells with the naive T cell markers CD45RA and CD45RO (Figure 5B) revealed no change in the phenotype of the transfected cells, indicating that the cells were naive CD45RA + / CD45RO - The viability of transfected naive T cells was comparable to that of untreated cells, 95.4% and 98.3%, respectively (Figure 5D). Delivery efficiency was 96.7% (Figure 5E), corresponding to a high total yield (Figure 5F).

[0170] Example 6 - Scale-up of production volume It is generally accepted that standard electroporation requires further optimization in the process of scaling up from research to manufacturing volumes due to changes in both electrode and cuvette geometries. To address this issue, numerous workarounds have emerged in the field of electroporation-based transfection, including the application of microfluidics, batch-based automation, and nanostructures. So far, these solutions have not been able to meet the requirements of both high-throughput development and large-scale manufacturing in the evolving cell and gene therapy industry. Electromechanical transfection technology utilizes the same flow cells previously described for low-volume production systems (Figures 6A-6B) configured in a large-volume production platform (e.g., electromechanical systems and devices configured to continuously redeliver new cell suspensions). Due to its continuous flow nature, electromechanical transfection in large-volume platforms scales up over time. Thus, to process large volumes, one simply needs to operate for a proportionately longer time. Large-volume electromechanical transfection solutions can process up to 100 mL of liquid in approximately 2-3 minutes, transferring cell samples from an input bag to an output bag. Fluid flow is controlled via a peristaltic pump (Masterflex® L / S). Because electromechanical systems utilize the exact same transfection mechanisms and components regardless of scale, the same parameters identified during optimization on our small-scale system directly apply to larger volume systems (Figure 6A). To demonstrate implementation at 5 mL (50-fold scale-up from the data above), 50M primary human T cells were transfected at 10x with 1 mg of mRNA. 6Cells were transfected at a density of 1000 µg / mL (Figure 6B). As a result, no significant decrease in cell viability was observed 24 h after electromechanical transfection, with viabilities of 73.5% via the small platform and 71.0% via the large platform (Figure 6B). The observed delivery efficiencies were also similar 24 h after electromechanical transfection, with 94.3% for the small platform and 92.2% for the large platform (Figure 6B). Thus, electromechanical transfection can be easily scaled up to clinically relevant throughput.

[0171] Example 7 - Methods and Materials The following methods and materials were used to collect the data discussed in Examples 1-6.

[0172] T cell culture and expansion Human peripheral blood cells (PBMCs) were purchased from STEMCELL Technologies™ (#70025). 100M PBMCs were thawed in 100mL X-VIVO™ 10 medium (#04-380Q) from Lonza supplemented with recombinant human IL-2 protein (#202-IL) from R&D Systems™. After 24 hours of culture after thawing, PBMCs were activated with ImmunoCult™ human CD3 / CD28 T cell activation reagent from STEMCELL Technologies (#10971) for 3 days according to the manufacturer's protocol. On day 4, cells were pelleted (500×g, 5 min) and transferred to a G-Rex100® (#80500) from Wilson Wolf with 500mL fresh X-VIVO™ 10 medium and recombinant human IL-2. Fresh recombinant human IL-2 was added every 3 days and cultured in G-Rex® culture for a total of 12 days. Cells were then frozen in aliquots for future use using Bulldog Bio's Bambanker™ Cell Freezing Medium (#BB01). Aliquots of T cells thawed after expansion were cultured at a density of 1e6 / mL in RPMI 1640 medium from Thermo Fisher (#11875119) supplemented with 10% fetal bovine serum (FBS) from Sigma-Aldrich® (#F-4135), penicillin-streptomycin solution from Corning® (#30-002-CI), and recombinant IL-2. Naïve primary human T cells (CD3 + / CD4 + / CD45RA + ) were also obtained from STEMCELL Technologies™ (#70029) and cultured as above in RPMI supplemented with 10% FBS and IL-2. Cells were cultured in a standard cell culture device at 37°C and 5% CO 2 Cell viability and size were monitored during cell culture using a Countess™ II (Thermo Fisher).

[0173] Electromechanical transfection Transfection was performed with mRNA encoding GFP (#L7601) or mCherry (#L7203) commercially available from TriLink® Biotechnologies. T cells were counted, pelleted (500 × g, 5 min), and cultured at 10–50 × 10 6 The cells were resuspended in a transfection buffer compatible with the present invention at a density of 100 / mL. Payload was added up to 10% volume and the cell:payload solution was mixed by pipetting.

[0174] Cell processing with the small-volume electromechanical device array was performed on a PerkinElmer JANUS® G3 BioTx Pro Plus Workstation equipped with an 8-chip Varispan™ head. The cell solution was transferred to a mixing plate cooled to 4°C in a 96-well plate and aspirated through the tips of the electromechanical device above the microfluidic channels. The solution was then dispensed at a constant flow rate through the same tips while a specific electric field was applied to these tips through an electric delivery manifold. The cells were delivered directly into the cell medium for collection in the 96 deep-well plate.

[0175] Cell processing using the large volume electromechanical transfection system was performed using a prototype in which fluid flow was controlled using a Cole-Palmer Masterflex® L / S peristaltic pump and PharMed® BPT tubing (L / S 13:#06508-13) through an electromechanical transfection device placed between an input bag and an output bag connected by tubing. The cell:payload solution was transferred to the input reservoir and pumped through the channel at a constant flow rate while applying a specific electric field. The cells were then immediately transferred to cell culture medium and collected in the output reservoir.

[0176] For both platforms, the final density of the harvested fluid was 1×10 6The transfection buffer was incubated at 37°C in 5% CO in a standard cell culture vessel. 2 and cultured.

[0177] Neon™ Transfection Thermo Fisher Neon™ Transfection System was used according to the manufacturer's instructions. Briefly, 5M day 9 expanded T cells were resuspended in T buffer and loaded into a 100 μL Neon™ pipette tip. The protocol performed was the Neon™ T cell microporation protocol (2100V 1 pulse 20 ms). Treated cells were transferred to tissue culture vessels.

[0178] 4D Nucleofector™ Transfection The Lonza 4D Nucleofector™ system was used according to the manufacturer's instructions. Briefly, 5M day 9 expanded T cells were resuspended in freshly prepared Human T Cell Nucleofection Solution and loaded into a 100 μL Lonza certified cuvette. The protocol run was the Amaxa™ 4D Nucleofector™ protocol for unstimulated human T cells (EO115). Treated cells were transferred to tissue culture vessels.

[0179] Flow cytometry analysis Viability and efficiency metrics were assessed using a Thermo Fisher Attune™ Nxt flow cytometer. 200 μL of cultured cells were pelleted (500 × g, 5 min) and suspended in Dulbecco's Phosphate Buffered Saline (DPBS) from Fisher Scientific (#14190250) supplemented with 7-AAD viability solution from eBiosciences (#00-6993-50). Cells were then analyzed by volumetric measurement using an Attune™ Nxt autosampler. Total cells were gated on a dot plot of forward scatter (FSC) vs. side scatter (SSC). Viable cells (7-AAD) were then analyzed to measure delivery efficiency by expression of a fluorescent reporter. - ) were gated. Total cell number and yield were calculated from the applied dilution based on the total volume of cell culture (7.5x per mL). Naive T cell marker staining was performed with BioLegend® APC / Cy7 mouse anti-human CD45RA antibody (#304128) and BV510 mouse anti-human CD45RO antibody (#304232).

[0180] Transcriptome analysis Whole cell pellets were harvested after 6 and 24 hours of cell culture and stored at -80°C. RNA extraction, cDNA synthesis, next generation sequencing, and preliminary raw data normalized to controls were all completed by GENEWIZ®. Normalized data were analyzed (Excel-Microsoft) and graphed (GraphPad-Prism 8) in-house. Gene ontology analysis used the PANTHER (Protein Analysis Through Evolutionary Relationships) classification system.

[0181] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between definitions in this specification and the documents incorporated herein, the definitions set forth in this specification shall apply.

[0182] While the present disclosure has been described in relation to particular embodiments thereof, it will be understood that the present disclosure is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the present disclosure in accordance with the principles of the present disclosure, within known or customary practice in the art to which the present disclosure pertains, including departures from the present disclosure that may be applicable to the essential features described hereinabove, and in accordance with the scope of the claims.

[0183] Other embodiments are within the scope of the claims.

Claims

1. 1. A method for introducing a composition into a plurality of mammalian cells suspended in a flowing liquid, the method comprising: (a) providing a device comprising: i) an entry zone comprising a first inlet and a first outlet; ii) first and second electrodes; and iii) an active zone with a second inlet and a second outlet; (b) Select a combination of electric field (E), mean flow velocity (u), hydraulic diameter of the active zone (d), liquid conductivity (σ), liquid dynamic viscosity (μ), and liquid density (ρ) to obtain a liquid conductivity of 1×10 ８ and 1 x 10 １０ A dimensionless parameter Π with values ​​between ５ where the dimensionless parameter Π ５ teeth, [0010] and giving, represented by (c) passing said plurality of mammalian cells and said composition through said active zone while providing said selected combination of (E), (u), (d), (σ), (μ), and (ρ), thereby introducing said composition into said plurality of mammalian cells; The method includes:

2. The composition is at least 1×10 per minute per active zone ５ The method of claim 1 , wherein the vector is introduced into the plurality of mammalian cells at a cellular flux.

3. The method of claim 1 , wherein the entry zone and the active zone are configured to provide an increased average flow velocity.

4. The method of claim 1 , wherein the composition is introduced into the plurality of mammalian cells without altering a desired cell surface marker.

5. 1. A method for introducing a composition into a plurality of cells suspended in a flowing liquid, the method comprising: (a) providing a device comprising: i) an entry zone comprising a first inlet and a first outlet; ii) first and second electrodes; and iii) an active zone with a second inlet and a second outlet; (b) passing the plurality of cells and the composition through the active zone while delivering electrical energy from the first and second electrodes and at least partially mechanical energy from an average flow rate, the mechanical energy and the electrical energy together resulting in introducing the composition into the plurality of cells with an efficiency, viability, and / or yield at least equal to that of electroporation or mechanical poration alone, and with less electrical or mechanical energy than electroporation or mechanical poration would require to achieve that efficiency, viability, and / or yield; The method includes:

6. The composition is at least 1×10 per minute per active zone ５ The method of claim 5 , wherein the antibody is introduced into the plurality of cells at a cellular flux.

7. The ratio of the electrical energy delivered to the flowing liquid by the electric field to the mechanical energy delivered by the pressure drop in the active zone is greater than or equal to 10 ３ : 1 to 10 ６ 7. The method of claim 5 or 6, wherein the ratio of the β-amino acid to the β-amino acid is between 0.5:

1.

8. The method of claim 5 , wherein the entry zone and the active zone are configured to provide an increased average flow velocity.

9. 1. A method for introducing a composition into a plurality of cells suspended in a flowing liquid, the method comprising: (a) providing a device comprising: i) an entry zone having a first inlet and a first outlet; and ii) first and second electrodes; iii) an active zone having a hydraulic diameter (d) with a second inlet and a second outlet; (b) providing a test portion of the plurality of cells, both having a liquid conductivity (σ), liquid dynamic viscosity (μ), and liquid density (ρ), and a cell to composition ratio, and a test composition, and passing the test portion and the test composition through the active zone at a mean flow velocity (u) while applying an electric field (E), where at least one of (u), (E), (σ), (μ), and (ρ) is changed; (c) a dimensionless parameter Π, comprising maximum cell viability, transfection efficiency, and / or engineered cell yield, for introduction of the test composition into the test portion of the plurality of cells. ５ Identifying a range of [0025] Identifying, (d) a Π that includes at least one of the maximum cell viability, the transfection efficiency, or the engineered cell yield. ５ passing the plurality of cells and the composition through the active zone with a combination of (u), (E), (σ), (μ), and (ρ) corresponding to values ​​of The method includes:

10. 10. The method of claim 9, further comprising repeating step (b) with the test portion of cells and the test composition having a second ratio of cells to composition and / or the active zone having a second hydraulic diameter (d).

11. 11. The method of claim 9 or 10, wherein the electric field (E) is held constant while the mean flow velocity (u) is varied, or the mean flow velocity (u) is held constant while the electric field (E) is varied.

12. The composition is at least 1×10 per second per active zone ５ The method of claim 9 , wherein the antibody is introduced into the plurality of cells at a cellular flux.

13. 10. The method of claim 9, wherein the entry zone and the active zone are configured to provide an increased mean flow velocity (u).

14. 1. A method for introducing a composition into a plurality of human immune cells from a suspension of blood taken from a normal patient or donor and suspended in a flowing liquid, the method comprising: (a) providing a device comprising: i) an entry zone comprising a first inlet and a first outlet; ii) first and second electrodes; and iii) an active zone with a second inlet and a second outlet; (b) Select a combination of electric field (E), mean flow velocity (u), hydraulic diameter of the active zone (d), liquid conductivity (σ), liquid dynamic viscosity (μ), and liquid density (ρ) to obtain a liquid conductivity of 1×10 ８ and 1 x 10 １０ A dimensionless parameter Π with values ​​between ５ where the dimensionless parameter Π ５ teeth, [0030] and giving, represented by (c) passing the plurality of human immune cells and the composition through the active zone while providing the selected combination of (E), (u), (d), (σ), (μ), and (ρ), thereby introducing the composition into the plurality of human immune cells to produce a therapeutic dose of transfected cells; The method includes:

15. The method of claim 14, wherein the suspension is prepared using leukoreduction.

16. The composition is at least 1×10 per minute per active zone ５ The method of claim 14, wherein the human immune cells are introduced into the plurality of human immune cells with a flux of cells.

17. The method of claim 14 , wherein the entry zone and the active zone are configured to provide an increased mean flow velocity.

18. 15. The method of claim 14, wherein the composition is introduced into the plurality of human immune cells without altering a desired cellular characteristic.

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