Cellular mechanical perforation cartridge and methods of use thereof

Abstract

The present disclosure provides a cartridge for cell mechanical perforation, the cartridge holding a cell mechanical perforation filter. The cartridges described herein can include a first housing portion and a second housing portion, the first housing portion and the second housing portion together configured to hold the cell mechanical perforation filter. The first housing portion can include a fluid inlet detachably connected to a source of a cell mixture. The second housing portion can include a fluid outlet detachably connected to an output reservoir. The cartridges described herein can enable replacement of the cell mechanical perforation filter by detaching the first housing portion from the second housing portion. Furthermore, using the cartridges described herein, dead volume in the fluid inlet and / or the fluid outlet can be limited to no more than 10% of the volume through the cell mechanical perforation filter.

Description

Cross-reference to related applications

[0001] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 608,751, filed December 11, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure generally relates to cell mechanical perforation devices, and more specifically to a housing for holding a cell mechanical perforation filter for intracellular delivery of a payload, the housing being replaceably integrated into a cell processing system. Background Technology

[0003] Intracellular delivery of macromolecules such as nucleic acids, peptides, and proteins is a challenge for research and therapeutic applications. Delivery of these substances into cells can be controlled by rapidly mechanically deforming the cells to create transient cell membrane disruption, thereby allowing macromolecules to enter the cytosol (also referred to herein as mechanical perforation). Known techniques for cell mechanical deformation include the use of contractile microfluidic channels or porous surfaces (e.g., filters) that induce cell membrane disturbance. These microfluidic channels or filters have been incorporated into the housings of large cell processing systems for intracellular delivery. However, these cell processing systems require specialized equipment and machinery (e.g., pump systems or other pressure mechanisms) to drive cells through the channels or pores of the filters under appropriate conditions for mechanical perforation, which occupy space in already space-constrained environments. Furthermore, these channels and filters clog rapidly, necessitating frequent replacements of expensive parts and equipment within the system. Additionally, these cell processing systems require specially trained personnel to operate, increasing associated costs and hindering widespread adoption.

[0004] Cell processing systems, including filtration and washing systems, are widely used in laboratories and medical facilities to prepare cell samples for various therapies. For example, cell processing systems are used for cell separation, suspension, dilution, incubation, and / or washing. One or more of these functionalities may require a single system (i.e., a setup) to perform. Systems attempting to combine multiple of the aforementioned functionalities can be cumbersome and expensive. Summary of the Invention

[0005] This document describes a cartridge for cell mechanoperforation, a system comprising the cartridge, and a method for agitating cell membranes using the cartridge. The cartridge can be detachably connected to a cell mixture source and output reservoir, and one or more of the cartridges can belong to a larger fluid delivery (e.g., cell processing) system. The cartridge can hold a cell mechanoperforation filter used to agitate the cell membranes of the cell mixture passing through the cartridge from the fluid delivery system. In this way, the delivery cartridge can be easily replaced at any time within the fluid delivery system without the need for cumbersome and careful replacement of fragile small filters. The simple replacement of the cartridge requires less time, equipment, and personnel training. Furthermore, the cartridge can be easily replaced within the connected fluid delivery system without replacing other components of the fluid delivery system. By simplifying the replacement of the cell mechanoperforation filter, effective intracellular delivery of the payload into the cells can be maintained by avoiding the use of clogged filters.

[0006] The cartridge described herein can be configured to hold a cellular mechanically perforated filter between opposing shell portions of the cartridge. Having more than one shell portion allows the user to easily replace the filter held within the cartridge and to sterilize and reuse components of the cartridge. The shell portions of the cartridge provided herein can be configured such that they minimize dead volume within the cartridge and promote efficient flow through the filter held within the cartridge. The cartridge described herein can be used in both research-scale and clinical-scale systems and can therefore be configured to operate under a variety of pressure and flow conditions induced by connected fluid delivery systems.

[0007] Integrating the cell-mechanically perforated cassettes described herein into existing clinical-scale (i.e., process-scale) cell processing systems can enhance existing systems by enabling the intracellular delivery of payloads to various cell types processed by the system using simple, replaceable cassettes. In some examples, systems integrating cell-mechanically perforated filters can even be configured to allow several cassettes to be installed in parallel to increase system throughput. Research-scale (i.e., benchtop) systems with integrated cassettes can further enhance the intracellular delivery of payloads to cell types that are not yet fully understood. In both scenarios, payloads comprising mixtures of nucleic acids, small molecules, nanoparticles, etc., can be introduced into cells in a single run. Furthermore, in either case, the cassettes described herein can play a crucial role in transforming biologics into universal, available, and cost-effective cell therapy solutions.

[0008] In some examples, a housing for mechanoperforation of cells is provided, the housing comprising: a first housing portion having a fluid inlet detachably connected to a source of a cell mixture; a second housing portion connected to the first housing portion having a fluid outlet detachably connected to an output reservoir; and a mechanoperforation filter held by the first housing portion and the second housing portion and configured to operate at a rate of at least 0.5 mL / min / mm. 2 The volumetric flow rate of the porous surface area agitates the cell membrane of the cell mixture through the cellular mechanical perforation filter.

[0009] In some examples, a method for perturbing cell membranes is provided, the method comprising: detachably connecting a fluid inlet of a first housing portion of a housing to a cell mixture source, the housing including a second housing portion connected to the first housing portion and the second housing portion including a fluid outlet, wherein the connected first housing portion and the second housing portion hold a cell mechanical perforation filter; detachably connecting the fluid outlet to an output reservoir; receiving a cell mixture from the cell mixture source in the fluid inlet; and causing the cell mixture to flow at a rate of at least 0.5 mL / min / mm. 2 A volumetric flow rate of porous surface area passes through the cell mechanical perforation filter to agitate the cell membrane of the cell mixture; and the agitated cell mixture is delivered to the output reservoir via the fluid outlet.

[0010] In some examples, a method of assembling a cell mechanical perforation housing is provided, the method comprising: connecting a first housing portion of the housing to a second housing portion of the housing, the first housing portion including a fluid inlet and the second housing portion including a fluid outlet, wherein a cell mechanical perforation filter is maintained in fluid communication with the fluid inlet and the fluid outlet through the connected first housing portion and the second housing portion.

[0011] In some examples, a cell mechanoperforation system includes: a housing comprising: a first housing portion including a fluid inlet detachably connected to a source of a cell mixture; a second housing portion connected to the first housing portion including a fluid outlet; a cell mechanoperforation filter held by the connected first and second housing portions and configured to agitate cell membranes of the cell mixture from the source of the cell mixture; an output reservoir detachably connected to the second housing portion of the housing; and a pump configured to operate at a rate of at least 0.5 mL / min / mm. 2The volumetric flow rate of the porous surface area causes the cell mixture to move through the cell mechanical perforation filter.

[0012] In some examples, a method for perturbing cell membranes includes: detachably connecting a fluid inlet of a first housing portion of a housing to a cell mixture source, the housing including a second housing portion connected to the first housing portion and the second housing portion including a fluid outlet, wherein the connected first and second housing portions retain a cell mechanical perforation filter; detachably connecting the fluid outlet to an output reservoir; and receiving a cell mixture from the cell mixture source in the fluid inlet, the cell mixture containing at least 1.0 x 10⁻⁶ cells / mL. 8 The process involves passing the cell mixture through the cell mechanical perforation filter to agitate the cell membranes of the cell mixture, and delivering the agitated cell mixture to the output reservoir via the fluid outlet. Attached Figure Description

[0013] This application can be best understood by referring to the following description and the accompanying drawings included in the specification.

[0014] Figure 1A A cross-sectional view of an exemplary housing for mechanical cell perforation is shown according to some implementation schemes.

[0015] Figure 1B Exemplary housings for mechanical cell perforation are shown according to some implementation schemes.

[0016] Figure 1C This image shows a close-up cross-sectional view of a cellular mechanically perforated filter held within a housing, according to some implementation schemes.

[0017] Figure 2A A cross-sectional view of another exemplary housing for mechanical cell perforation is shown according to some implementation schemes.

[0018] Figure 2B Exemplary housings for mechanical cell perforation are shown according to some implementation schemes.

[0019] Figure 2C This image shows a close-up cross-sectional view of a cellular mechanically perforated filter held within a housing, according to some implementation schemes.

[0020] Figure 3A A cross-sectional view of another exemplary housing for mechanical cell perforation is shown according to some implementation schemes.

[0021] Figure 3B Exemplary housings for mechanical cell perforation are shown according to some implementation schemes.

[0022] Figure 3CAn exploded view of an exemplary housing for mechanical cell perforation, according to some embodiments, is shown, the housing holding a polymer filter and a mesh support.

[0023] Figure 3D An exploded view of an exemplary housing for mechanical cell perforation, according to some embodiments, is shown, the housing holding a silicon filter and sealing components.

[0024] Figure 4A The diagram illustrates a process drawing of an exemplary system comprising a housing for mechanical cell perforation, according to some implementation schemes.

[0025] Figure 4B A process diagram is shown for another exemplary system comprising a housing for mechanical cell perforation, according to some implementation schemes.

[0026] Figure 4C A process diagram is shown for another exemplary system comprising a housing for mechanical cell perforation, according to some implementation schemes.

[0027] Figure 4D A process diagram is shown for another exemplary system comprising a housing for mechanical cell perforation, according to some implementation schemes.

[0028] Figure 4E A process diagram is shown for another exemplary system comprising a housing for mechanical cell perforation, according to some implementation schemes.

[0029] Figure 5A The following describes an exemplary silicon cell mechanically perforated filter based on some implementation schemes.

[0030] Figure 5B This image shows a close-up view of a portion of an exemplary silicon cell mechanically perforated filter, according to some implementation schemes.

[0031] Figure 6A The following describes an exemplary polymer cell mechanical perforation filter based on some implementation schemes.

[0032] Figure 6B This demonstrates another exemplary polymer cell mechanical perforation filter according to some implementation schemes.

[0033] Figure 6C Exemplary mesh supports for polymer cell mechanical perforation filters are shown according to some embodiments.

[0034] Figure 7A The diagram illustrates an exemplary cellular mechanical perforation filter according to some implementation schemes.

[0035] Figure 7B This diagram illustrates another exemplary cellular mechanical perforation filter according to some implementation schemes.

[0036] Figure 7C This diagram illustrates another exemplary cellular mechanical perforation filter according to some implementation schemes.

[0037] Figure 8A The results demonstrate cell viability of cells maintained by a mechanically perforated filter integrated into a cell processing system, according to some implementation schemes.

[0038] Figure 8B The demonstration shows, according to some implementation schemes, the delivery of payloads into cells via a cell mechanically perforated filter held within a housing integrated into a cell processing system.

[0039] Figure 8C The presentation demonstrates, according to some implementation schemes, the expression results of the payload in cells held through a cell mechanical perforation filter integrated into a cell processing system.

[0040] Figure 9A The results demonstrate cell viability of cells maintained by a mechanically perforated filter within a housing integrated into another cell processing system, according to some implementation schemes.

[0041] Figure 9B The demonstration illustrates, according to some implementation schemes, the delivery of payloads into cells via a cell mechanically perforated filter held within a housing integrated into another cell processing system.

[0042] Figure 9C The presentation demonstrates, according to some implementation schemes, the expression results of the payload in cells held within a cellular mechanical perforation filter integrated into another cell processing system.

[0043] Figure 10A The results demonstrate cell viability of cells maintained by a mechanically perforated filter within a housing integrated into another cell processing system, according to some implementation schemes.

[0044] Figure 10B The demonstration illustrates, according to some implementation schemes, the delivery of payloads into cells via a cell mechanically perforated filter held within a housing integrated into another cell processing system.

[0045] Figure 10C The presentation demonstrates, according to some implementation schemes, the expression results of the payload in cells held within a cellular mechanical perforation filter integrated into another cell processing system.

[0046] Figure 11A The results demonstrate cell viability of cells maintained by a mechanically perforated filter within a housing integrated into another cell processing system, according to some implementation schemes.

[0047] Figure 11B The demonstration illustrates, according to some implementation schemes, the delivery of payloads into cells via a cell mechanically perforated filter held within a housing integrated into another cell processing system.

[0048] Figure 11C The presentation demonstrates, according to some implementation schemes, the expression results of the payload in cells held within a cellular mechanical perforation filter integrated into another cell processing system.

[0049] Figure 12A The results demonstrate the cell viability of activated T cells by maintaining a cell mechanical perforation filter within a housing integrated into a cell processing system, according to some implementation schemes.

[0050] Figure 12B The demonstration shows, according to some implementation schemes, the delivery of payloads to activated T cells via a cell mechanical perforation filter held within a cartridge integrated into a cell processing system.

[0051] Figure 12C The presentation demonstrates, according to some implementation schemes, the expression results of the payload in activated T cells held within a cellular mechanical perforation filter integrated into a cell processing system.

[0052] Figure 13A This study demonstrates control cell viability results for PBMCs with in-cell mechanical perforation filters maintained within a box installed in a desktop research-scale system, based on several implementation schemes.

[0053] Figure 13B This demonstrates control delivery results, based on some implementation schemes, of payloads to PBMCs held within a cassette-mounted cell mechanical perforation filter in a desktop research-scale system.

[0054] Figure 14A The cell viability results of PBMCs, which are maintained by a mechanically perforated filter of cells integrated into a cell processing system, are shown according to some implementation schemes.

[0055] Figure 14B This demonstrates, according to some implementation schemes, the delivery of payloads to a PBMC (Physically Perforated Cell Filter) held within a housing integrated into a cell processing system.

[0056] Figure 15A The results demonstrate cell viability of PBMCs with mechanically perforated cell filters maintained within a box mounted in a desktop research-scale system, according to several implementation schemes.

[0057] Figure 15BThis demonstrates, according to some implementation schemes, the delivery of payloads to PBMCs (Physically Perforated Cell Filters) held within a cassette mounted in a desktop research-scale system.

[0058] Figure 16A The results demonstrate cell viability of iPSCs held within a box-like structure with a mechanically perforated filter, installed in a desktop research-scale system, according to several implementation schemes.

[0059] Figure 16B This demonstrates, according to some implementation schemes, the delivery of payloads to iPSCs held within a cassette housing of cells via mechanically perforated filters, which are mounted in a desktop research-scale system. Detailed Implementation

[0060] This document describes a replaceable housing for a retaining filter used for cell mechanoperforation and a method for agitating cell membranes using the housing described herein. The housing can be integrated into various fluid delivery systems, such as research-scale desktop devices and clinical-scale cell processing systems. In any scale system, the housing described herein can be easily replaced by detachably attaching the housing to existing cell mixture source and output reservoirs. Therefore, instead of having to carefully handle fragile porous filters, the entire housing can be easily replaced when the cell mechanoperforation filter becomes clogged. The housing described herein is operable under a variety of pressure and flow rate conditions induced by different fluid delivery systems to agitate cell membranes and facilitate intracellular delivery of payloads. The housing may comprise two housing portions: a first housing portion detachably connected to a cell mixture source and a second housing portion detachably connected to an output reservoir. A cell mechanoperforation filter can be retained between the first and second housing portions. The fluid inlet and outlet of the housing can be configured to minimize dead volume within the housing as fluid passes through the filter. The housing portions can be detachably connected to each other to facilitate easy replacement of the internally retained filter. Several replaceable cartridges as described herein can be installed in a single fluid delivery system (e.g., in parallel) to increase the flux of the perturbed cell mixture.

[0061] The following public references are shown at least in Figures 1A-1C , Figures 2A-2C and Figures 3A-3D Several implementations described herein are exemplary replaceable housings for mechanical cell perforation. This document at least relates to Figures 4A-4E This document describes an exemplary system comprising a housing for mechanical perforation of cells. This article is about... Figures 5A-5B and Figures 6A-6C Exemplary cellular mechanical perforation filters (including silicon filters and polymer filters) are described (separately). This article is about Figures 7A-7C An exemplary support structure for a silicon filter is described. Finally, regarding... Figures 8A-8C , Figures 9A-9C , Figures 10A-10C , Figures 11A-11C , Figures 12A-12C , Figures 13A-13B , Figures 14A-14B , Figures 15A-15B and Figures 16A-16B The description shows experimental results on the cell mechanical perforation efficacy in systems containing the housings provided herein.

[0062] Replaceable housing for mechano-perforation of cells Figures 1A-1C , Figures 2A-2C and Figures 3A-3C Exemplary housings 100, 200, and 300 for mechanical cell perforation are shown. For simplicity, housing 100 and its features will be used primarily throughout the following description. Unless otherwise expressly stated, features described herein with respect to housing 100 should be understood to apply to housing 200 and vice versa. Similarly, features described herein with respect to housing 300 should be understood to apply to each of housings 100 and 200 (and vice versa), unless otherwise expressly stated.

[0063] The housing 100 may include a first housing portion 102 containing a fluid inlet 104 and a second housing portion 106 connected to the first housing portion 102 and containing a fluid outlet 108. The fluid inlet 104 of the first housing portion 102 may be detachably connected to a cell mixture source. The fluid outlet 108 may be detachably connected to an output reservoir.

[0064] The housing 100 may include a cell mechanically perforated filter 120 held by a first housing portion 102 and a second housing portion 106. As described herein, the cell mechanically perforated filter 120 may be configured to agitate the cell membranes of the cell mixture as it passes through the filter 120. The housing 100 may be configured such that the cell mechanically perforated filter 120 is disposed below the fluid inlet 104 within the housing, such that gravity may cause the cell mixture to be uniformly distributed to the filter 120.

[0065] The housing 100 can be configured to limit the dead volume of fluid passing through it. Limiting the dead volume through the housing is important because the payload and / or cell type passing through the housing may be difficult to obtain, expensive, or typically small in size. Therefore, maintaining the maximum fluid volume for system operation is important. For example, at least one of the fluid inlet 104 and fluid outlet 108 can be configured to limit the dead volume to no more than 10% of the volume passing through the cell mechanical perforation filter 120. In some embodiments, the housing 100 (i.e., the fluid inlet 104 and / or fluid outlet 108) can be configured to limit the dead volume to less than or equal to about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.2%. In some embodiments, the housing can be configured to limit the dead volume to greater than or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%. In some embodiments, the housing 100 may be configured to limit the dead volume to greater than 10%, such as about 12%, 15%, 18%, or 20%. In some embodiments, the housing 100 may be configured to limit the dead volume to about 0.1%-10%, 0.1%-1%, 1%-10%, 1%-5%, or 5%-10%.

[0066] Mechanical cell perforation requires a certain flow rate for effective cell membrane agitation. A flow rate that is too low may not adequately agitate the cell membrane. A flow rate that is too high may over-agitate and thus damage the cells, rendering the sample unusable. In some embodiments, the mechanical cell perforation filter 120 may be configured to receive at a flow rate of approximately 0.5–500 mL / min / mm. 2 Volumetric flow rate between porous surface areas. For example, the cell mechanical perforation filter 120 can be configured to receive approximately 0.5-100, 0.5-10, 50-500, 5-100, or 5-20 mL / min / mm. 2 The volumetric flow rate of the porous surface area. In some embodiments, the cellular mechanical perforation filter 120 may be configured to receive a flow rate greater than or equal to about 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, or 450 mL / min / mm. 2Volumetric flow rate of the porous surface area. In some embodiments, the cellular mechanical perforation filter 120 may be configured to receive flow rates less than or equal to about 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 mL / min / mm. 2 Volumetric flow rate of the porous surface area. In some embodiments, the cellular mechanical perforation filter 120 can be configured to receive greater than 500 mL / min / mm. 2 Porous surface area (e.g., approximately 550, 600, 650, 700, or 750 mL / min / mm²) 2 The volumetric flow rate (based on the porous surface area). In some embodiments, the cell mechanical perforation filter 120 can be configured to receive less than 50 mL / min / mm. 2 Porous surface area (e.g., approximately 0.1, 0.2, 0.3, or 0.4 mL / min / mm) 2 Volumetric flow rate (porous surface area).

[0067] The cartridge 100 can be used within or downstream of various cell processing systems (e.g., clinical-scale systems) that handle large volumes of fluid. Alternatively or additionally, the cartridge 100 can be integrated into research-scale systems that handle smaller volumes of fluid. Therefore, the cartridge 100 can be adapted to receive a wide range of fluid volumes. The cartridge 100 can be configured to receive a wide range of volumes, such as between at least 10 µL and 1000 L (e.g., about 100 mL). In some embodiments, the cartridge 100 can be configured to receive volumes between about 1-1000 µL, 1-100 µL, 1-1000 mL, 1-100 mL, or 1-1000 L. For example, the housing 100 can be configured to receive volumes greater than or equal to approximately 10 µL, 25 µL, 50 µL, 75 µL, 100 µL, 150 µL, 200 µL, 250 µL, 300 µL, 350 µL, 400 µL, 450 µL, 500 µL, 600 µL, 700 µL, 800 µL, 900 µL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 250 mL, 500 mL, 750 mL, 1 L, 2 L, 5 L, 10 L, 50 L, 100 L, or 500 L. In some embodiments, the housing 100 may be configured to receive volumes less than or equal to about 25 µL, 50 µL, 75 µL, 100 µL, 150 µL, 200 µL, 250 µL, 300 µL, 350 µL, 400 µL, 450 µL, 500 µL, 600 µL, 700 µL, 800 µL, 900 µL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 250 mL, 500 mL, 750 mL, 1 L, 2 L, 5 L, 10 L, 50 L, 100 L, 500 L, or 1000 L.

[0068] In some embodiments, the volumetric capacity of fluid inlet 104 and / or fluid outlet 108 can be between about 500 and 2000 µL. Volumetric capacity can be the amount of fluid that the inlet or outlet can hold at one time. For example, each of fluid inlet 104 and fluid outlet 108 can have a volumetric capacity between about 500 and 1000 µL. In some embodiments, the volumetric capacity of at least one of fluid inlet 104 and fluid outlet 108 can be greater than or equal to 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 µL. In some embodiments, the volumetric capacity of at least one of fluid inlet 104 and fluid outlet 108 can be less than or equal to 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 µL. Generally, fluid inlet 104 and fluid outlet 108 can have a volumetric capacity between about 1 and 2 mL. For example, the fluid inlet 104 and the fluid outlet 108 may have a total volumetric capacity greater than or equal to 1, 1.2, 1.5, or 1.8 mL. In some embodiments, the fluid inlet 104 and the fluid outlet 108 may have a total volumetric capacity less than or equal to 1.2, 1.5, 1.8, or 2 mL.

[0069] In some embodiments, the cartridge 100 for cell mechanoperforation can be configured to receive a constant volumetric flow rate. A constant volumetric flow rate allows the cell velocity to remain consistent as the cell mixture passes through the cartridge 100, thereby improving the cell mechanoperforation performance of the filter 120 held within the cartridge 100. Alternatively or additionally, the cartridge 100 can be configured to receive a pulsed flow rate. The flow rate can be controlled by at least one of a peristaltic pump, a syringe pump, and a pressurized reservoir. In some embodiments, the flow rate through the cartridge 100 can be manually controlled, such as by using a manual syringe.

[0070] The cartridge 100 can be easily replaced within a fluid delivery system (such as a cell processing system). For example, the cartridge 100 can be received by a cartridge receiving portion of the fluid delivery system. In other words, the cartridge 100 can be easily inserted into and removed from a corresponding portion of a larger fluid delivery system. In doing so, at least one of the fluid inlet 104 and the fluid outlet 108 can be detachably connected to a cell mixture source and an output reservoir (respectively). Exemplary fluid delivery systems may include those described herein. Figures 4A-4E A more detailed description of the cell processing system. For example, a fluid delivery system may include a filtration system, a panning system, a cell washing system, a separation system, or a cell therapy production system.

[0071] In some embodiments, housing 100 may be detachably connected to a cell mixture source. For example, fluid inlet 104 of the first housing portion 102 may be detachably connected to the cell mixture source using a Luer connector, a threaded connector, or a quick-release connector. In some embodiments, fluid inlet 104 may be detachably connected to the cell mixture source using one or more of pipes and the aforementioned connectors, or a tube weld.

[0072] In some embodiments, housing 100 may be detachably connected to an output reservoir. For example, the fluid outlet 108 of the second housing portion 106 may be detachably connected to the output reservoir using a Luer connector, a threaded connector, or a quick-release connector. In some embodiments, fluid outlet 108 may be detachably connected to the output reservoir using one or more of the aforementioned connectors or tubular fittings. The detachable connection of each of the fluid inlet 104 and fluid outlet 108 allows any user to easily install and remove housing 100 as described herein from existing equipment without specialized training.

[0073] In some embodiments, instead of using one or more connectors and / or pipes to detachably connect the fluid outlet 108 to the output reservoir, the fluid outlet 108 of the housing 100 can be detachably connected to the output reservoir by arranging the fluid outlet at an opening near the output reservoir, as described herein at least regarding Figure 4C As stated above.

[0074] In some embodiments, the housing 100 may be sterilizable. For example, the housing 100 may be sterilized using at least one of gamma irradiation and chemical sterilization. In some embodiments, the housing 100 may be sterilized independently or as part of a larger system, which may be integrated and / or fluidly connected to said larger system. For example, the housing 100 may be sterilized when connected to at least one of a cell mixture source and an output reservoir.

[0075] In some embodiments, the first housing portion 102 and the second housing portion 106 may be detachably connected. For example, the first housing portion 102 and the second housing portion 106 may be detachably connected using a threaded connection, press-fit, or snap-fit. When the first housing portion 102 and the second housing portion 106 are detachably connected, the cellular mechanical perforation filter 120 held within the housing 100 may be replaceable. Alternatively, in some embodiments, the first housing portion 102 and the second housing portion 106 may be permanently connected. For example, the first housing portion 102 and the second housing portion 106 may be permanently connected using a welded connection or a swaged connection.

[0076] As shown, a first (e.g., distal) end of the first housing portion 102 may be connected to a first (e.g., proximal) end of the second housing portion 106. In this example, the ends of each of the first housing portion 102 and the second housing portion 106 may be coplanar with the cellular mechanically perforated filter 120 held within the housing 100. In some embodiments, a first side of the first housing portion 102 may be connected to a first side of the second housing portion 106, with said side of each of the first housing portion 102 and the second housing portion 106 perpendicular to the cellular mechanically perforated filter 120.

[0077] Connecting the first housing portion 102 and the second housing portion 106 may include inserting at least a portion (e.g., an end region) of the first housing portion 102 into a corresponding receiving portion of the second housing portion 106 (or vice versa). For example, as Figure 1A As shown, the end of the first housing portion 102 can be inserted into the corresponding receiving portion of the second housing portion 106. Figure 1C One example is shown where the end of the second housing portion 106 can be inserted into a corresponding receiving portion of the first housing portion 102. In these examples, the outer surface of each of the end of the first housing portion 102 and the receiving portion of the second housing portion 106 may include corresponding threads or latches for connecting the first housing portion 102 and the second housing portion 106.

[0078] A cell mechanically perforated filter 120 can be disposed in a filter holding portion 110 within the housing 100. The filter holding portion 110 can be part of a first housing portion 102 and / or a second housing portion 106. For example, as... Figure 1A As shown, the end of the first housing portion 102 may include a filter holding portion 110, in which the filter 120 is disposed. Alternatively, as Figure 1CAs shown, the end of the second housing portion 106 may include a filter holding portion 110, in which a filter 120 is disposed.

[0079] In some embodiments, housing 100 may include at least one sealing element 112. Sealing element 112 may seal or secure the connection between the first housing portion 102 and the second housing portion 106. Sealing element 112 may include an O-ring, rubber diaphragm, or other flexible seal. Sealing element 112 may have an annular shape that allows fluid to pass through it.

[0080] The sealing element 112 can be flexible, but it can also resist deformation. In some examples, the sealing element 112 has a Shore hardness (durometer) between about 25 and 75 A (e.g., between about 40 and 60 A or about 45 and 55 A). Shore hardness refers to the hardness of a material measured using a Shore A scale. In some examples, the Shore hardness is greater than 25 A, 30 A, 35 A, 40 A, 45 A, 50 A, 55 A, 60 A, 65 A, or 70 A. In some examples, the Shore hardness is less than 75 A, 70 A, 65 A, 60 A, 55 A, 50 A, 45 A, 40 A, 35 A, or 30 A.

[0081] The inner diameter of the sealing component 112 allows the cell mixture to pass through the housing and filter. For example, the inner diameter can be between about 5-10 mm, such as between about 6-9 mm or about 7-8 mm. In some examples, the inner diameter of the sealing component 112 can be greater than about 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, or 9.5 mm. In some examples, the inner diameter of the sealing component 112 can be less than about 10 mm, 9.5 mm, 9 mm, 8.5 mm, 8 mm, 7.5 mm, 7 mm, 6.5 mm, 6 mm, or 5.5 mm.

[0082] The sealing member 112 may have a cross-section or thickness suitable for creating a seal between the first housing portion 102 and the second housing portion 106. For example, the sealing member 112 may have a cross-section between about 2 and 8 mm (e.g., between about 3 and 7 mm or about 4 and 6 mm). In some examples, the cross-section of the sealing member 112 is greater than about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, or 7.5 mm. In some examples, the cross-section of the sealing member 112 is less than about 8 mm, 7.5 mm, 7 mm, 6.5 mm, 6 mm, 5.5 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, or 2.5 mm.

[0083] The sealing member 112 can be configured to create a seal between the first housing portion 102 and the second housing portion 106 based on the degree of compression of the sealing member 112 between the first housing portion 102 and the second housing portion 106. For example, when properly attached, the first housing portion 102 and the second housing portion 106 can be configured to compress the sealing member 112 to about 20%-50%, 25%-45%, or about 30%-40% of its original cross-section. This parameter is also referred to herein as the compression ratio of the sealing member 112. In some examples, the compression ratio is greater than about 20%, 25%, 30%, 35%, 40%, or 45%. In some examples, the compression ratio is less than about 50%, 45%, 40%, 35%, 30%, or 25%. Those skilled in the art will appreciate that each of the parameters mentioned above together contributes to the proper sealing of the housing using the sealing member 112.

[0084] In some embodiments, the sealing member 112 may be arranged in the end region of the first housing portion 102 near the connected second housing portion 106, such as... Figure 1C As shown in the illustration. At least one sealing element 112 may be arranged above and / or below the cellular mechanical perforation filter 120. In the example mentioned above, such as Figure 1C As shown, the second housing portion 106 may include an end region of the cell mechanical perforation filter 120 adjacent to the first housing portion 102 and held therein. In some embodiments, a sealing member may additionally or alternatively be arranged in the end region of the second housing portion 106 adjacent to the first housing portion 102 (shown in...). Figure 1A (In this example, the end region of the first housing portion 102 may retain the cellular mechanically perforated filter 120. A sealing element 112 may be disposed between the fluid inlet 104 and the fluid outlet 108. In some embodiments, the sealing element may surround at least a portion of the fluid inlet 104 and / or the fluid outlet 108. In some embodiments, the housing 100 may not include the sealing element 112.)

[0085] Figures 1A-1C A housing 100 is shown, comprising a first housing portion 102 and a second housing portion 106 connected together. As shown, the widths of the first housing portion 102 and the second housing portion 106 may be substantially the same. In some embodiments, such as Figures 2A-2CAs shown in the illustrated box 200, the widths of the first housing portion 202 and the second housing portion 206 can be different. For example, the width of the first housing portion 202 can be greater than or less than the width of the second housing portion 206. The shape of the box 100 (i.e., the shape of each of the first housing portion 102 and the second housing portion 106) can be cylindrical, triangular, rectangular, or another polygonal shape.

[0086] In some embodiments, the width of the box 100 can be between approximately 5 and 50 mm. When the box 100 is cylindrical, such as at least in... Figure 1B As shown, the width can be the diameter of the box 100. In some embodiments, the width of the box 100 can be between about 5-20 mm, 5-10 mm, 10-50 mm, 10-20 mm, or 20-50 mm. In some embodiments, the width of the box 100 can be greater than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, or 45 mm. In some embodiments, the width of the box 100 can be less than or equal to about 10, 15, 20, 25, 30, 35, 40, 45, or 50 mm.

[0087] In some embodiments, the length of the box body 100 may be between approximately 15 and 70 mm. For example, the length of the box body 100 may be between approximately 15 and 30 mm, 25 and 70 mm, 25 and 50 mm, or 40 and 70 mm. In some embodiments, the length of the box body 100 may be greater than or equal to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 mm. In some embodiments, the length of the box body 100 may be less than or equal to 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mm.

[0088] The first housing portion 102 may include a region configured to connect to an external cell mixture source and a region configured to connect to a second housing portion 106. The region configured to connect to the cell mixture source may include a narrow width configured to connect to a conduit and / or connector. As shown, the region configured as the second housing portion 106 may have a wider width than the region configured to connect to the cell mixture source. Similarly, the second housing portion 106 may include a region configured to connect to an external output reservoir and / or disposed near the output reservoir and a region configured to connect to the first housing portion 102. The region configured to connect to or be disposed near the output reservoir may include a narrow width configured to connect to a conduit, connector, and / or disposed near and / or within the output reservoir. In some embodiments, the narrow width of this region of the second housing portion 106 may be configured to direct fluid output directly into the output reservoir without splashing or wasting fluid. Similar to the first housing portion 102, the second housing portion 106 may include a region configured to connect to the first housing portion 102, said region having a wider width than the region configured to connect to the output reservoir. As described herein, the greater width of each of the first housing portion 102 and the second housing portion 106 may be substantially the same or may be different. This greater width of the overall housing 100 allows the user to easily interact with the housing 100 to install the housing into a fluid delivery system and / or disconnect the first housing portion 102 from the second housing portion 106 (e.g., to replace the filter 120).

[0089] In some embodiments, the outer surface of the housing (e.g., the outer surface of at least one of the first housing portion 102 and the second housing portion 106) may include textured portions 114 for a user to grip, thereby facilitating the connection of the housing 100 to external components of the fluid delivery system and / or facilitating the connection of the first housing portion 102 to the second housing portion 106. For example, the first housing portion 102 may include textured portions that a user can grip to help connect the first housing portion 102 to a cell mixture source. Additionally or alternatively, the second housing portion 106 may include textured portions that a user can grip to help connect the second housing portion 106 to an output reservoir. The textured portions may include ridges, grooves, ribs, bumps, etc., that generate adhesive friction with the user's hand, such as at least on Figure 1B As shown in the image.

[0090] In some embodiments, the width of fluid inlet 104 and / or fluid outlet 108 may be between about 1 and 15 mm. For example, the width of fluid inlet 104 and / or fluid outlet 108 may be between about 1-10 mm, 1-5 mm, 5-15 mm, 5-10 mm, or 10-15 mm. In some embodiments, the width of fluid inlet 104 and / or fluid outlet 108 may be greater than or equal to about 1, 2, 5, 8, 10, or 12 mm. In some embodiments, the width of fluid inlet 104 and / or fluid outlet 108 may be less than or equal to about 2, 5, 8, 10, 12, or 15 mm. In some embodiments, the width of fluid inlet 104 may be substantially the same as the width of fluid outlet 108, as in... Figure 1A As shown in [the document]. Alternatively, as in [the document]... Figure 2A As shown, the widths of fluid inlet 204 and fluid outlet 208 can be different. For example, the width of fluid inlet 204 can be greater than or less than the width of fluid outlet 208.

[0091] The fluid inlet 104 and fluid outlet 108 may have circular cross-sections to facilitate efficient fluid flow through the housing 100 and minimize dead volume. Therefore, the width may be the diameter of the fluid inlet 104 and / or the fluid outlet 108. However, it should be understood that the cross-section of each of the fluid inlet 104 and fluid outlet 108 may alternatively have an oval, triangular, rectangular, or other polygonal shape.

[0092] like Figure 1A As shown, the fluid inlet 104 may include a tapered portion, the width of which increases as it approaches the cellular mechanical perforation filter 120 within the housing 100. For example, in Figure 1A In the configuration depicted, the fluid inlet 104 may include a cone-shaped portion whose width increases from a proximal end of the fluid inlet 104 to a distal end of the fluid inlet 104 near the cellular mechanical perforation filter 120. Similarly, the fluid outlet 108 may include a tapered portion whose width decreases as it moves away from the cellular mechanical perforation filter 120 within the housing 100. For example, in Figure 1A In the configuration depicted, fluid outlet 108 may include a cone whose width decreases from the proximal end of fluid outlet 108 near the cellular mechanical perforation filter 120 to the distal end of fluid outlet 108. The cones of fluid inlet 104 and / or fluid outlet 108 can help minimize dead volume in housing 100, enhance efficiency through filter 120, etc.

[0093] In some implementation schemes, such as in Figure 2AAs shown, fluid inlet 104 and / or fluid outlet 108 may not include a tapered portion. In other words, fluid inlet 104 and / or fluid outlet 108 may have a constant width (e.g., diameter) throughout their entire length.

[0094] The lengths of fluid inlet 104 and / or fluid outlet 108 can be between approximately 5 and 50 mm. In some embodiments, the lengths of fluid inlet 104 and / or fluid outlet 108 can be between approximately 5 and 20 mm, 5 and 10 mm, 10 and 50 mm, 10 and 20 mm, 20 and 50 mm, or 30 and 50 mm. In some embodiments, the lengths of fluid inlet 104 and / or fluid outlet 108 can be greater than or equal to approximately 5, 10, 15, 20, 25, 30, 35, 40, or 45 mm. In some embodiments, the lengths of fluid inlet 104 and / or fluid outlet 108 can be less than or equal to approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 mm. The lengths of fluid inlet 104 and fluid outlet 108 can be substantially the same, or they can be different. Generally, the lengths of fluid inlet 104 and fluid outlet 108 can be substantially equal to the length of housing 100.

[0095] As mentioned above, housing 200 may include any one or more features of housing 100 described herein. For example, housing 200 may include a first housing portion 202 having a fluid inlet 204 detachably connected to a source of the cell mixture. Housing 200 may include a second housing portion 206 connected to the first housing portion 202 and having a fluid outlet 208 detachably connected to an output reservoir. Housing 200 may include a cell mechanically perforated filter 220 held by the first housing portion 202 and the second housing portion 206. Filter 220 may be disposed within a filter holding portion 210 of the first housing portion 202 and / or the second housing portion 206. Sealing member 212 may be disposed between the first housing portion 202 and the second housing portion 206 to seal the connection between the two housing portions. The outer surface of at least one of the first housing portion 202 and the second housing portion 206 may include a textured portion 214 that a user can interact with to easily replace housing 200.

[0096] As mentioned above, housing 300 may include any one or more features of housings 100 and 200 described herein. For example, the fluid inlet 304 of housing 300 may include first housing portions 302a, 302b (collectively referred to herein as first housing portion 302), which include a fluid inlet 304 detachably connected to a source of the cell mixture. Housing 300 may include second housing portions 306a, 306b (collectively referred to herein as second housing portion 306), which are connected to the first housing portion 302 and include a fluid outlet 308 detachably connected to an output reservoir. Housing 300 may include a cell mechanically perforated filter 320 held by the first housing portion 302 and the second housing portion 306. A sealing member 312 may be disposed between the first housing portion 302 and the second housing portion 306 to seal the connection between the two housing portions holding the filter 320.

[0097] Fluid inlet 304 can be detachably connected to the cell mixture source using a Luer connector, threaded connector, or quick-release connector. In some embodiments, fluid inlet 304 can be detachably connected to housing 300 using one or more of the connectors mentioned above and conduit.

[0098] Fluid outlet 308 can be detachably connected to the output reservoir by arranging the fluid outlet near an opening in the output reservoir. Fluid outlet 308 may include a constriction 309 at its distal end, which guides fluid through fluid outlet 308 and into the output reservoir while minimizing any sample waste. Constriction 309 may have a width (e.g., diameter) at least 20% smaller than the width (diameter) of fluid outlet 308. For example, the width of constriction 309 may be at least 20%, 30%, 40%, 50%, 60%, 70%, or 80% smaller than the width of fluid outlet 308.

[0099] like Figures 3A-3D As shown, the housing 300 may have a larger external structure (e.g., a shell) that allows a user to easily grip the housing 300. The housing 300 can be manipulated more easily by creating a larger external shell surrounding the fluid inlet 304, the fluid outlet 308, and the filter 320 held therebetween. For example, a user can grip the housing 300 to insert it into and remove it from a larger fluid delivery system (e.g., a research-scale or process-scale system). The external structure of the housing 300 may consist of an outer first shell portion 302b and an outer second shell portion 306b, each described below.

[0100] The first housing portion 302 may include an inner layer portion (302a) and an outer layer portion (302b). The inner layer portion 302a may surround at least the fluid inlet 304 and the filter holding portion 310, such as Figure 3A As shown in the diagram. The outer layer portion 302b may include the remainder of the first housing portion 302, including portions that the user interacts with to operate the housing 300. The housing 300 may include openings or gaps between the inner layer portion 302a and the outer layer portion 302b of the first housing portion 302 to at least minimize the weight of the housing 300 and / or improve its usability.

[0101] The second housing portion 306 may also include an inner layer portion (306a) and an outer layer portion (306b). The inner layer portion 306a may surround at least the fluid outlet 308 and the filter retaining portion 310, as shown below. Figure 3A As shown in the diagram. The outer layer portion 306b may include the remainder of the second housing portion 306, including portions that a user can interact with to operate the housing 300. Similar to the first housing portion 302, the second housing portion 306 may include a perforation between the inner layer portion 306a and the outer layer portion 306b of the second housing portion 306 to at least minimize the weight of the housing 300 and / or the usability of the housing 300.

[0102] In housing 300, the first housing portion 302 can be detachably connected to the second housing portion 306, allowing a user to operatively connect and disconnect the housing portions (e.g., to insert and / or remove the filter 320 within housing 300). As in Figure 3B As shown, the outer second housing portion 306b may include one or more latches 311 configured to detachably connect housing portions 302b and 306b. The latches may be arranged circumferentially around the fluid inlet 304 and the fluid outlet 308. (As shown in...) Figure 3A As shown, the inner second housing portion 306a can be inserted into the socket of the inner first housing portion 302a (or vice versa), for example, to stabilize the connection between the housing portions.

[0103] In some implementation schemes, such as in Figure 3C As shown in the exploded view of housing 300, housing 300 can hold a mesh support 321 within a first housing portion 302 and a second housing portion 306. The mesh support 321 can be used in conjunction with a polymer filter 323 to support the filter. In some examples, in the previously described embodiments, the mesh support 321 can function as a sealing member 312.

[0104] As will be described in more detail below, the cell mechanical perforation filter 120 (and filter 220, collectively referred to herein as filter 120) may comprise a silicon filter or a polymer filter. For example, Figure 3D An exemplary housing 300 comprising a silicon filter 322 is depicted. In some embodiments, the cellular mechanically perforated filter 120 may comprise one or more materials such as synthetic or natural polymers, polycarbonate, silicon, silicon nitride, glass, metal, alloy, nitrocellulose, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, ceramics, and / or porcelain. The cellular mechanically perforated filter 120 may comprise a coating such as at least one of the following: gold, silver, platinum, Teflon, polyvinylpyrrolidone, adhesives, and surfactants. In some embodiments, the cellular mechanically perforated filter 120 may not comprise a coating.

[0105] The cell mechanical perforation filter 120 may contain no more than 300,000 pores. For example, the filter 120 may contain between approximately 500 and 400,000 pores, between 500 and 100,000 pores, between 10,000 and 400,000 pores, between 10,000 and 100,000 pores, between 50,000 and 400,000 pores, between 50,000 and 100,000 pores, or between 100,000 and 400,000 pores. In some implementations, the filter 120 may contain at least about 500, 1,000, 5,000, 10,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 200,000, 225,000, 250,000, 275,000, 300,000, 325,000, 350,000, or 375,000 pores. In some implementations, filter 120 may contain no more than about 1,000, 5,000, 10,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 200,000, 225,000, 250,000, 275,000, 300,000, 325,000, 350,000, 375,000, or 400,000 pores.

[0106] The integration of upstream cell processing devices and, more generally, the cell mechanical perforation filter 120 described herein into the cell processing system can cause pressure on the filter 120. Because the filter 120 includes a vulnerable porous surface for cell mechanical perforation, it is important that the filter 120 is configured and / or supported to withstand a variety of pressure conditions. In some embodiments, the cell mechanical perforation filter 120 may be configured to withstand pressures between approximately 1 and 50 psi. For example, the cell mechanical perforation filter 120 may be configured to withstand pressures between approximately 1 and 25 psi, 1 and 10 psi, 5 and 50 psi, 5 and 25 psi, 10 and 50 psi, 10 and 25 psi, or 25 and 50 psi. In some embodiments, the cellular mechanically perforated filter 120 may be configured to withstand pressures greater than or equal to about 1, 2, 4, 6, 8, 10, 12, 14, 15, 16, 18, 20, 22, 25, 28, 30, 35, 40, or 45 psi. In some embodiments, the cellular mechanically perforated filter 120 may be configured to withstand pressures less than or equal to about 2, 4, 6, 8, 10, 12, 14, 15, 16, 18, 20, 22, 25, 28, 30, 35, 40, 45, or 50 psi. In some embodiments, the cellular mechanically perforated filter 120 may be configured to withstand pressures greater than 50 psi (e.g., about 55, 60, 65, 70, or 75 psi).

[0107] In some embodiments, a system may be provided comprising a housing 100 and cells (or multiple cells, such as multiple cells of a cell mixture). As mentioned above, the housing 100 may be configured to deform cells from a cell mixture passing through the housing 100, causing perturbations within the cells, thereby allowing a payload to enter the cytosol. Cell deformation may be achieved via a cell mechanical perforation filter 120 for a time period ranging from about 1 µs to at least about 10 ms (e.g., about 1 µs, 10 µs, 50 µs, 100 µs, 500 µs, 1 ms, 2 ms, 5 ms, or 10 ms). Perturbations within the cells may be defined as gaps within the cells that allow material to move from outside the cells into the cells (e.g., pores, cracks, cavities, gaps, holes, fissures, fissures, perforations, etc.). Perturbations may be caused by pressure resulting from mechanical strain and / or shear forces. Perturbations may occur within the cell membrane. Perturbations may be transient. In some implementations, the perturbation in the cell can last for a duration between 1 second and 5 minutes. For example, the perturbation can last for at least 1 second, 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, or longer. In some implementations, the perturbation can last for less than 1 s, 10 s, 30 s, 1 min, 2 min, 3 min, 4 min, or 5 min.

[0108] In some implementations, the cell mechanical perforation filter 120 can be configured to receive a large number of cells in a single run. For example, the cell mechanical perforation filter 120 can be configured to receive cells containing approximately 1.0 x 10⁻⁶ cells in a single run. 8 With 1.0x10 12 A mixture of cells between [number] cells. In some embodiments, the cell mechanical perforation filter 120 may be configured to receive a mixture of cells containing approximately 1.0 x 10 [units]. 8 With 1.0x10 11 Cells between 1.0x10 8 With 1.0x10 10 Between 1.0 x 10 cells or 1.0 x 10 6 With 1.0x10 9 A mixture of cells between [number] cells. In some embodiments, the cell mechanical perforation filter 120 may be configured to receive a mixture of cells greater than or equal to about 1.0 x 10 [units]. 8 5.0 x 10 cells 8 1.0 x 10 cells 9 5.0 x 10 cells 9 1.0 x 10 cells 10 5.0 x 10 cells 10 1.0 x 10 cells 11 1 cell or 5.0x1011 Cells. In some embodiments, the cell mechanical perforation filter 120 may be configured to receive cells less than or equal to about 5.0 x 10⁻⁶. 8 1.0 x 10 cells 9 5.0 x 10 cells 9 1.0 x 10 cells 10 5.0 x 10 cells 10 1.0 x 10 cells 11 5.0 x 10 cells 11 1 cell or 1.0 x 10 12 Each cell.

[0109] In some embodiments, the cells (or cells) are cells obtained from or derived from an individual. In some embodiments, the individual is a mouse. In some embodiments, the individual is a non-human primate. In some embodiments, the individual is a human. In some embodiments, the individual is a mouse, dog, cat, horse, rat, goat, or rabbit.

[0110] In some embodiments, the cells (or cells) may be somatic cells, immortalized cells (e.g., HeLa cells, HEK cells, etc.), stem cells, or derivatives thereof. In some embodiments, one or more cells may be peripheral blood mononuclear cells (PBMCs) or derivatives thereof. In some embodiments, one or more cells may be immune cells. In some embodiments, one or more cells may be T cells, natural killer (NK) cells, monocytes, B cells, or dendritic cells (or mixtures thereof). In some embodiments, the cells may be unstimulated T cells or derivatives thereof. In some embodiments, the cells may be stimulated (or activated) T cells. In some embodiments, one or more cells may be stem cells, such as human stem cells. In some embodiments, one or more cells may be induced pluripotent stem cells, hematopoietic cells, or mesenchymal cells.

[0111] Cellular mechanical perforation system In some implementations, a cell mechanical perforation system comprising one or more cell mechanical perforation filters may be provided. Figures 4A-4E An exemplary cellular mechanical perforation system 430 is shown, comprising a housing 400. The housing 400 may include any one or more features of the housings 100, 200 described herein. Therefore, although in Figures 4A-4E Not explicitly shown, it should be understood that housing 400 can hold a cell mechanical perforation filter. The cell mechanical perforation filter can be configured to agitate the cell membranes of the cell mixture from cell mixture source 432.

[0112] like Figures 4A-4EAs shown, the cell mechanical perforation system 430 may include a housing 400 fluidly connected to a cell mixture source 432 and an output reservoir 434. For example, the housing may include a first housing portion (e.g., first housing portion 102, 202) having a fluid inlet detachably connected to the cell mixture source 432. The housing may include a second housing portion (e.g., second housing portion 106, 206) having a fluid outlet detachably connected to the output reservoir 434.

[0113] In some embodiments, the housing 400 can be easily replaced within the cell mechanical perforation system 430. For example, the housing 400 can be detachably connected to the cell mixture source 432 and / or the output reservoir 434. In some embodiments, the fluid inlets of the housing 400 (e.g., fluid inlets 104, 204) can be detachably connected to the cell mixture source 432 using at least one of a Luer connector, a threaded connector, or a quick-release connector. In some embodiments, such as at least in Figure 4B As shown, the fluid inlet of housing 400 can be detachably connected to cell mixture source 432 using one or more of the connectors mentioned above in combination with conduit 438. Conduit 438 may comprise one or more of polyvinyl chloride (PVC) and C-flex conduits. In some embodiments, the fluid inlet of housing 400 can be connected to cell mixture source 432 using a tubular weld connection.

[0114] In some embodiments, the fluid outlet of housing 400 (e.g., fluid inlets 104, 208) can be detachably connected to output reservoir 434 using a connector (such as a Luer connector, threaded connector, or quick-release connector). In some embodiments, such as at least in Figure 4B As shown, the fluid outlet of cartridge 400 can be detachably connected to output reservoir 434 using one or more of the connectors mentioned above in combination with conduit 438. Conduit 438 may comprise one or more of polyvinyl chloride (PVC) and C-flex conduits. In some embodiments, the fluid outlet of cartridge 400 can be connected to output reservoir 434 using a tubular welded connection. Any of the aforementioned connections between the cartridge and the cell mixture source and output reservoir is intended to be easily understood and used by the user, allowing the user to install and remove the cartridge within the fluid delivery system without requiring specialized training.

[0115] In some embodiments, the fluid outlet of the housing 400 can be detachably connected to the output reservoir 434 by arranging the fluid outlet at an opening near the output reservoir 434, such as... Figure 4CAs depicted in the illustration. For example, output reservoir 434 may include centrifuge tubes or other open containers configured to receive agitated cell mixtures from the fluid outlet of housing 400. In some embodiments, the fluid outlet of housing 400 may be at least partially disposed within the opening of output reservoir 434.

[0116] As described herein, the housing 400 may be sterilizable. For example, the housing 400 may be sterilizable using at least one of gamma irradiation and chemical sterilization. In some embodiments, one or more components of the system 430 may be sterilizable, for example, as a unit. For example, the housing 400 may be sterilizable when connected to at least one of the cell mixture source 432 and the output reservoir 434.

[0117] The cellular mechanical perforated filter held within the housing 400 may contain elements described in this article, respectively. Figures 1A-1C , Figures 2A-2C , Figures 4A-4B , Figures 5A-5B and Figures 6A-6C The filter 120, 220, 420, 520, and 620 shown herein may have any one or more of the features described. For example, the cell mechanically perforated filter held within the housing 400 may be a silicon filter (i.e., a silicon chip) or a polymer substrate. As described herein, the cell mechanically perforated filter may be replaceable within the housing 400. In some embodiments, the housing 400 may be configured to pass a cell mixture from cell mixture source 432 through the cell mechanically perforated filter within the housing 400 in a single run without replacing the filter.

[0118] In some embodiments, the cell mechanical perforation system 430 can operate at a constant volumetric flow rate. In other words, the pump 436 of system 430 can be configured to provide a constant volumetric flow rate to housing 400. In some embodiments, pump 436 can be configured to provide a pulsed volumetric flow rate to housing 400. In some embodiments, in addition to or instead of pump 436, the volumetric flow rate (pulsated or constant) can be controlled by a pressurized reservoir.

[0119] As described herein, the cell mechanical perforation filter held within the housing 400 can be configured to operate at a rate of 0.5–500 mL / min / mm. 2 The volumetric flow rate between the porous surface areas agitates the cell membrane. In other words, pump 436 can be configured to operate at a flow rate of approximately 0.5–500 mL / min / mm. 2The volumetric flow rate between the porous surface areas causes the cell mixture to move through the cellular mechanical perforation filter. In some embodiments, the pump 436 may be configured to operate at a flow rate between about 0.5-100 mL / min / mm² of porous surface area, 0.5-10 mL / min / mm² of porous surface area, 50-500 mL / min / mm² of porous surface area, 5-100 mL / min / mm² of porous surface area, or 5-20 mL / min / mm² of porous surface area. 2 The volumetric flow rate between the porous surface areas pumps the cell mixture through a cellular mechanically perforated filter. In some embodiments, pump 436 may be configured to pump the cell mixture through the cellular mechanically perforated filter at a volumetric flow rate greater than or equal to about 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, or 450 mL / min / mm. 2 Porous surface area. In some embodiments, pump 436 may be configured to pump the cell mixture through a cell mechanically perforated filter at a volumetric flow rate of less than or equal to about 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 mL / min / mm. 2 Porous surface area.

[0120] Cell mixture source 432 may contain a cell mixture (e.g., a cell suspension). The cell mixture may contain cells and a buffer solution. For example, the cell mixture may be contained in a physiological saline solution or a cell mixture in a physiological medium other than blood. The cells in the cell mixture may include a mixed cell population (e.g., whole blood) or a purified cell population (e.g., a purified population of nucleated or anucleated cells). As described above, the cells in the cell mixture may include, but are not limited to, anucleated cells (e.g., erythrocytes (such as red blood cells or reticulocytes), platelets, etc.), nucleated cells (e.g., immune cells, PBMCs, T cells, NK cells, iPSCs, HSCs, etc.), liposomes, exosomes, etc. The cell mixture may contain an aqueous solution including cell culture medium (e.g., DMEM, RMPI, OptiMEM, etc.), phosphate-buffered saline (PBS), salts, sugars, growth factors, surfactants, lubricants, amino acids, proteins, cell cycle inhibitors, vitamins, etc.

[0121] As described herein, the cassette 400 provided herein can be integrated with clinical-scale cell processing systems. These systems can supply the cassette with millions, hundreds of millions, billions, or even trillions of cells for cell mechanical perforation using a cell mechanical perforation filter held therein. Therefore, the filter described herein can be configured to receive large numbers of cells (e.g., billions of cells) and can perturb these cells with high efficiency and deliver a payload to them. Given that flow rates through porous surfaces are less controllable than, for example, microfluidic channels, the ability to perturb billions of cells in a cell mixture with high efficiency and deliver a payload to said cells using a filter held within the cassette described herein can be important for successful integration of the cassette described herein into cell processing systems of various scales. Furthermore, a single-cell mechanical perforation filter within the cassette described herein can be configured to process billions of cells in a single run without replacing the filter (or cassette).

[0122] In some implementations, the cell mixture may contain approximately 1.0 x 10⁻⁶ cells. 8 With 1.0x10 12 Between 1.0 x 10 cells. In some embodiments, the cell mixture may contain approximately 1.0 x 10 cells. 8 With 1.0x10 11 Cells between 1.0x10 8 With 1.0x10 10 Between 1.0 x 10 cells or 1.0 x 10 6 With 1.0x10 9 The cell mixture may contain between 1.0 x 10^6 cells. In some embodiments, the cell mixture may contain greater than or equal to about 1.0 x 10^6 cells. 8 5.0 x 10 cells 8 1.0 x 10 cells 9 5.0 x 10 cells 9 1.0 x 10 cells 10 5.0 x 10 cells 10 1.0 x 10 cells 11 1 cell or 5.0x10 11 100 cells. In some embodiments, the cell mixture may contain less than or equal to about 5.0 x 10⁻⁶ cells. 8 1.0 x 10 cells 9 5.0 x 10 cells 9 1.0 x 10 cells 10 5.0 x 10 cells 10 1.0 x 10 cells 11 5.0 x 10 cells 11 1 cell or 1.0 x 10 12 Each cell.

[0123] In some embodiments, the cell mixture has a volume of about 10 µL to about 5 L (at least about 10 µL, 100 µL, 500 µL, 1 mL, 5 mL, 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L or 5 L). In some embodiments, the cell mixture may have a volume between approximately 10-1000 µL, 10-500 µL, 10-100 µL, 10-50 µL, 50-1000 µL, 50-500 µL, 50-100 µL, 100-1000 µL, 100-500 µL, 500-1000 µL, 1-1000 mL, 1-500 mL, 1-100 mL, 1-50 mL, 1-10 mL, 10-1000 mL, 10-500 mL, 10-100 mL, 10-50 mL, 50-1000 mL, 50-500 mL, 50-400 mL, 50-300 mL, 50-200 mL, 50-100 Volumes range from 100-1000 mL, 100-500 mL, 100-400 mL, 100-300 mL, 100-200 mL, 500-1000 mL, or 1-5 L. As mentioned above, the filters described herein (and therefore the cell mechanical perforation systems comprising one or more filters) can be configured to handle a wide range of fluid volumes, depending on whether the filters are integrated with research-scale systems, clinical-scale systems, high-throughput screening systems, etc.

[0124] Cell mixture source 432 may comprise a fluid reservoir, flexible plastic bag, vial, bottle, container, ampoule, wide-mouth bottle, or other container suitable for containing the cell mixture. In another example, cell mixture source 432 may not comprise a separate container containing the cell mixture, instead, system 430 may be configured to receive a cell mixture from an upstream fluidly connected cell therapy generating system, cell washing system, filtration system, etc. For example, one or more of the systems mentioned above may process the cells by surface marker-based separation (e.g., Miltenyi), centrifugation / flow-based separation (e.g., panning), buffer exchange, cell washing, activation, amplification, or other biological changes before providing the mixture to system 430.

[0125] Exemplary filtration and / or panning systems that can be fluidly connected to housing 400 and may contain cell mixture source 432 may include, but are not limited to, tangential flow filtration (TFF) systems (e.g., Repligen KrosFlo® KR2i TFF system, Sartorius Ambr® Crossflow TFF system, Sartorius Sartoflow® Smart TFF system, etc.), standard filtration systems (e.g., Fresenius Cue® cell processing system), rotating membrane filtration (e.g., Fresenius KabiLovo® automated cell processing system), and panning systems (e.g., Gibco™ Cell Therapy System (CTS™) Rotea countercurrent centrifugation system, Terumo Elutra® cell separation system, etc.). Exemplary cell separation systems include, but are not limited to, Miltenyi CliniMACS Prodigy® instruments and StemCell cell separation systems (e.g., immunomagnetic cell separation systems such as EasySep™, RoboSep™, and StemSep™; immunodensity cell separation systems such as RosetteSep™ and SepMate™, etc.). Exemplary end-to-end cell therapy generation systems include, but are not limited to, the Lonza Cocoon® platform.

[0126] In some embodiments, the cartridge 400 may be integrated (e.g., as a component) into one or more of the aforementioned pumps, filtration systems, separation systems, cell therapy generation systems, and / or panning systems. For example, the cartridge 400 may be received by a cartridge receiving portion of a fluid delivery system. When the cartridge 400 is placed in the cartridge receiving portion of the fluid delivery system, the fluid delivery system may detachably connect the fluid inlet of the cartridge 400 to the cell mixture source 432 and / or detachably connect the fluid outlet of the cartridge 400 to the output reservoir 434.

[0127] In some embodiments, the cell mixture may comprise a cell suspension and a payload (e.g., a premixed cell suspension and payload). The type and concentration of the payload may depend on the cell type in the cell suspension and the intended cell therapy. In some embodiments, the payload comprises at least one of peptides, nucleic acids, small molecules, nanoparticles, and complexes thereof. For example, the payload may include, but is not limited to, one or more of the following: DNA, RNA (e.g., mRNA, siRNA, saRNA, tRNA, miRNA, etc.), proteins, small molecules, peptides, nanoparticles, viruses, synthetic materials, cell lysates containing antigens, and / or complexes (e.g., RNPs, protein CAS plus guide RNA, etc.). The payload may comprise multiple payloads (e.g., a mixture of nucleic acids, proteins, small molecules, etc.). The aforementioned payloads may function as: transcription factors, chemokines, cytokines, surface receptors, other intracellular or extracellular proteins, inhibitors or enhancers of any of the above, survival factors, cryoprotectants, prime editors, antibodies, enzymes, and / or any combination thereof.

[0128] In some embodiments, the payload may comprise at least one molecule having a molecular weight between about 100 Da and 10 MDa. For example, as described herein, the payload may comprise small molecules (about 100 Da) and / or large plasmids (about 10 MDa). For example, the molecular weight of the molecules in the payload may be between about 100 Da and 1 MDa, between 100 Da and 100 kDa, or between 100 Da and 1 kDa. In some embodiments, the payload may comprise at least one molecule having a molecular weight greater than or equal to about 100 Da, 500 Da, 1 kDa, 50 kDa, 100 kDa, 500 kDa, 1 MDa, or 5 MDa. In some embodiments, the payload may comprise at least one molecule having a molecular weight less than or equal to about 500 Da, 1 kDa, 50 kDa, 100 kDa, 500 kDa, 1 MDa, 5 MDa, or 10 MDa. As described herein, the payload may comprise a variety of molecules, particles, etc., having different molecular weights selected from those listed above.

[0129] Because the mechanical perforation mechanism described herein depends on the payload in the pores of the cell membrane, the diameter of the payload (e.g., particles, molecules, etc.) can be less than 100 nm. For example, the particle size can be between about 1-100 nm, 1-50 nm, or 1-10 nm. In some embodiments, the particle size (diameter) can be greater than or equal to 1, 5, 10, 25, 50, or 75 nm. In some embodiments, the particle size (diameter) can be less than or equal to 5, 10, 25, 50, 75, or 100 nm.

[0130] The concentration of the payload in the cell mixture can vary based on the type of payload and can be between about 1 nM and 1 mM. For example, the payload concentration can be between about 10 nM and 1 mM, 100 nM and 1 mM, 1 µm and 1 mM, 10 µm and 1 mM, or 100 µm and 1 mM. In some embodiments, the concentration of the payload in the cell mixture can be greater than or equal to 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 µm, 5 µm, 10 µm, 50 µm, 100 µm, 500 µm, or 1 mM. In some embodiments, the concentration of the payload in the cell mixture can be less than or equal to 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 µm, 5 µm, 10 µm, 50 µm, 100 µm, 500 µm, or 1 mM.

[0131] In some implementations, the payload may be included in the payload source 440. For example... Figure 4E As shown, payload source 440 may be fluidly connected to cell mixture source 432 to receive the cell mixture before passing the cell mixture and payload together into housing 400. Figure 4D As shown, payload source 440 may additionally or alternatively be fluidly connected to output reservoir 434 to provide payload to output reservoir 434 (also configured to receive a disturbed cell mixture from housing 400). In this example, the payload can be introduced into the disturbed cells of the cell suspension after the cell suspension has passed through a filter held within housing 400. Payload source 440 may comprise a fluid reservoir, flexible plastic bag, vial, bottle, container, ampoule, wide-mouth bottle, or other container suitable for containing the payload.

[0132] In some implementations, the payload may be contained in an output reservoir 434 that is fluidly connected to the housing 400 (e.g., it should be understood that it consists of at least one component). Figures 4A-4C(System 430 is shown in the diagram). That is, the mechanical perforation system 430 may not include an external payload source 440. In each of these foregoing embodiments, it is considered that a payload (e.g., included in cell mixture source 432, output reservoir 434, or payload source 440) may be combined with the cell mixture before, during, and / or after passing the cell mixture through the cell mechanical perforation filter within the housing 400.

[0133] One or more of the payload and / or cell mixture may contain solutions intended for prefilling the fluid pathways and / or reservoirs of system 430. For example, the payload may contain cell buffer and / or payload buffer. In some embodiments, the cell mechanical perforation system 430 may be used without prefilling (or wetting). For example, the fluid reservoirs and / or pathways of system 430 may be pre-wetted.

[0134] like Figure 4A As shown, the cell mechanoperforation system 430 may include a pump 436 configured to move a cell mixture through a cell mechanoperforation filter within a housing 400. In some embodiments, the pump 436 may be configured to move at a rate of approximately 0.5-500 mL / min / mm. 2 The volumetric flow rate between the porous surface areas causes the cell mixture to move through the cellular mechanical perforation filter. In some embodiments, system 430 may include multiple pumps, such as those configured to pump the payload from... Figure 4D The payload source 440 drives a pump in the output reservoir 434. Pump 436 can be configured to provide positive or negative pressure (e.g., vacuum) to move the cell mixture (i.e., cell suspension and / or payload) through system 430. For example, negative pressure from pump 436 can “pull” one or more of the aforementioned solutions through system 430, while positive pressure can “push” or drive the solution through system 430.

[0135] In some embodiments, pump 436 may comprise a peristaltic pump or a syringe pump. Pump 436 may include, but is not limited to, peristaltic pumps, syringe pumps, diaphragm pumps, positive displacement pumps, gear lobes, etc. More generally, the term "pump" may be further used herein to refer to manual syringes, pipettes, pistons, gas cylinders, and / or other fluid motivators (e.g., gravity, centripetal force, etc.). In some embodiments, pump 436 may be a pump for existing filtration systems, separation systems, panning systems, cell washing systems, and / or end-to-end cell therapy generation systems (described above).

[0136] Although not explicitly shown, it should be understood that pump 436 may be operatively coupled to at least one of housing 400, cell mixture source 432, and / or output reservoir 434 (i.e., operatively coupled to one or more fluid pathways fluidly connecting housing 400 to each of cell mixture source 432 and output reservoir 434) to move cell mixture from cell mixture source 432 through housing 400 into output reservoir 434. Where system 430 includes payload source 440, pump 436 may additionally or alternatively be coupled to payload source 440 (i.e., operatively coupled to one or more fluid pathways fluidly connected to payload source 440).

[0137] Pump 436 may be configured to provide a pressure (e.g., pulsed pressure, constant pressure, etc.) between at least 2 psi and 35 psi to the cellular mechanoperforation system 430. For example, pump 436 may provide a pressure between at least 2-20 psi, 2-10 psi, 10-35 psi, or 10-20 psi. In some embodiments, the pressure provided by pump 436 may be greater than or equal to about 1 psi, 5 psi, 10 psi, 15 psi, 20 psi, 25 psi, 30 psi, or 35 psi. In some embodiments, the pressure provided by pump 436 may be less than or equal to about 1 psi, 5 psi, 10 psi, 15 psi, 20 psi, 25 psi, 30 psi, or 35 psi. In some embodiments, system 430 may include one or more components (e.g., one or more valves, pressure dampers, etc.) configured to control or adjust (e.g., gradually reduce) the pressure provided by pump 436. In doing so, the pressure gradient across the filter within the housing 400 can be maintained within a desired range (e.g., between 2 psi and 20 psi). In some examples, the pressure gradient across the filter surface can be less than 20 psi, such as 18 psi, 15 psi, 14 psi, 12 psi, 10 psi, 8 psi, 5 psi, or 10 psi. In some examples, the pressure gradient across the filter surface can be between 2 and 20 psi, between 5 and 20 psi, between 8 and 20 psi, or between 10 and 20 psi. In some examples, the pressure gradient across the filter surface can be less than 2 psi, such as 1.5 psi, 1 psi, 0.5 psi, or 0.25 psi. In some implementations, the pressure gradient across the filter surface can be greater than 15 psi, such as 17.5 psi, 20 psi, 22.5 psi, 25 psi, 30 psi, or higher. The silicon filter described herein can be configured such that, under the aforementioned low pressure gradient and using the flow rate described herein, the filter surface can effectively agitate the cell membrane for payload delivery.

[0138] Pump 436 may be configured to pump (e.g., move) a total volume of at least 1 mL to 500 mL through system 430 (e.g., through housing 400) in a single run. For example, pump 436 may be configured to pump approximately 1-100 mL, 1-50 mL, 10-500 mL, 10-100 mL, or 100-500 mL through system 430 in a single run. In some embodiments, pump 436 may be configured to drive fluid volumes greater than or equal to approximately 1 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL through system 430. In some embodiments, pump 436 may be configured to drive fluid volumes less than or equal to approximately 1 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL through system 430. In some embodiments, pump 436 may be configured to pump less than 1 mL (e.g., a minimum volume of 100 µL, 250 µL, 500 µL, or 750 µL) of fluid through the system in a single run. In some embodiments, pump 436 may be configured to pump more than 500 mL (e.g., about 1 L, 2 L, 3 L, 4 L, or 5 L) of fluid through the system in a single run. In some embodiments, system 430 as described herein may be adapted for allogeneic production and / or bioprocessing, and therefore pump 436 may be configured to pump volumes greater than 5 L, such as 10 L, 100 L, or 1000 L.

[0139] In some embodiments, the entire cell mixture can pass through a mechanically perforated cell filter in about 30 minutes or less. In some embodiments, the entire cell mixture can pass through a mechanically perforated cell filter in no more than about 30 minutes, 25 minutes, 20 minutes, 15 minutes, 12 minutes, 10 minutes, 8 minutes, 5 minutes, 2 minutes, or 1 minute. In some embodiments, the entire cell mixture can pass through the filter in about 1-30 minutes, 1-25 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-5 minutes, 5-30 minutes, 5-25 minutes, 5-20 minutes, 5-15 minutes, 5-10 minutes, 10-30 minutes, 10-25 minutes, 10-20 minutes, or 10-15 minutes. The time it takes for the cell mixture to pass through the mechanically perforated cell filter can depend at least in part on pump 436 and / or one or more upstream cell processing systems.

[0140] In some embodiments, single cells of the cell mixture can pass through a cell mechanical perforation filter for approximately 1 microsecond to 10 milliseconds. For example, cells can pass through the cell mechanical perforation filter within 1 µs–1 ms, 10 µs–10 ms, 10 µs–1 ms, 100 µs–10 ms, or 100 µs–1 ms. In some embodiments, cells can pass through the cell mechanical perforation filter within approximately 1 µs, 5 µs, 10 µs, 50 µs, 100 µs, 500 µs, 1 ms, or 5 ms. In some embodiments, cells can pass through the cell mechanical perforation filter within approximately 5 µs, 10 µs, 50 µs, 100 µs, 500 µs, 1 ms, 5 ms, or 10 ms.

[0141] In some embodiments, the speed at which cells pass through the cellular mechanical perforation filter to cause cell membrane disturbance can be about 1 m / s. In some embodiments, the cell speed can be between 10 mm / s and 100 m / s. For example, the cell speed can be less than or equal to 10 mm / s, 20 mm / s, 50 mm / s, 1 m / s, 2 m / s, 5 m / s, 10 m / s, 20 m / s, 50 m / s, or 100 m / s. In some embodiments, the cell speed can be greater than or equal to 10 mm / s, 20 mm / s, 50 mm / s, 1 m / s, 2 m / s, 5 m / s, 10 m / s, 20 m / s, 50 m / s, or 100 m / s.

[0142] In some cases, the sample volume can minimize the residence time of cells in the output reservoir. Residence time is the time from when cells leave the filter to when cells leave the output reservoir (e.g., at...). Figures 4A-4C In the system 430 depicted in the figure) or when exposed to a payload (e.g., in Figure 4D The residence time of cells in the output reservoir (as described in system 430) can be less than 30 seconds (s), less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds. In some examples, the residence time is between about 0.01 seconds and about 30 seconds. Other characteristics of the system and its operating parameters (such as flow rate and cell concentration) may affect the residence time. Based on the description of such variables elsewhere in this application, those skilled in the art will readily recognize their ability to affect the residence time.

[0143] The mechanical perforation system 430 may include one or more output reservoirs 434 fluidly connected to the housing 400 to collect agitated cell mixtures. The one or more output reservoirs 434 may include a single output reservoir 434. Alternatively, the one or more output reservoirs 434 may include multiple output reservoirs 434 (hereinafter referred to as output reservoirs 434). The pump 436 mentioned above may be configured to move the agitated cell mixture into the output reservoir 434. The output reservoir 434 may include one or more flexible plastic bags, vials, containers, or other containers configured to collect the agitated cell mixture.

[0144] In some embodiments, the cell mechanoperforation system 430 may include multiple housings 400. For example, the system 430 may include any number of housings 400 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more housings). The multiple housings 400 may be arranged in parallel such that each housing 400 is fluidly connected to a cell mixture source 432 to receive a portion of the cell mixture and is fluidly connected to an output reservoir 434 to supply the reservoir 434 with a perturbed cell mixture. In this way, the cell mechanoperforation system can handle larger volumes of fluid. In some embodiments, the multiple housings 400 may be fluidly connected in series.

[0145] In some embodiments, system 430 may include one or more leukocyte-removing filters and / or cell sieves (not shown). For example, housing 400 may hold one or more leukocyte-removing filters upstream of a cell mechanically perforated filter, the leukocyte-removing filters being configured to remove leukocytes from the cell mixture before it passes through the filter. Similarly, cell sieves may be integrated within system 430 (e.g., held upstream of a cell mechanically perforated filter by the housing) and configured to remove larger clots (e.g., clots in the 40-70 µm size range) that might otherwise clog the filter. One or more leukocyte-removing filters and / or cell sieves may be fluidly connected to cell mixture source 432 to receive the cell mixture (e.g., cell suspension and optionally a payload). In some embodiments, system 430 may not include one or more leukocyte-removing filters. In some embodiments, system 430 may not include one or more cell sieves. For example, one or more of the leukocyte-removing filters and / or cell sieves may instead be integrated upstream of housing 400 within a fluid delivery system.

[0146] Exemplary Cellular Mechanical Perforation Filter As described herein, the housing can be configured to hold a cell mechanically perforated filter (e.g., filter 120, 220, 320). The cell mechanically perforated filters 120, 220, 320 can comprise a silicon filter or a polymer filter. Figures 5A-5B Showcasing the 520 silicon filter. Figures 6A-6B Polymer filter 620 is shown. Unless otherwise expressly stated, it should be understood that the features described herein with respect to silicon filter 520 should be construed as applicable to polymer filter 620, and vice versa. Furthermore, both silicon filter 520 and polymer filter 620 may include features described herein with respect to... Figures 1A-1C , Figures 2A-2C and Figures 3A-3D Any one or more features of the filters 120, 220, and 320.

[0147] The silicon filter 520 may include a filter surface 522 and a support structure 524 disposed on one side of the filter surface 522. For example... Figure 5A As shown, the support structure 524 can cover at least a portion of the filter surface 522. Figure 5B As shown, filter 520 may include a plurality of pores 526 extending through filter surface 522, the pores 526 being configured to agitate cell membranes as a mixture of cells passes through the plurality of pores 526.

[0148] The silicon filter 520 may comprise silicon, silicon oxide (e.g., silicon dioxide), silicon nitride, and / or silicon carbide. For example, the filter surface 522 may comprise one or more of the silicon materials mentioned above. Similarly, the support structure 524 may comprise one or more of the silicon materials mentioned above. The filter surface 522 and the support structure 524 may comprise the same or different silicon materials. The silicon filter 520 may be manufactured from a doped silicon wafer. For example, the silicon wafer used to manufacture the silicon filter 520 may be doped with boron, gallium, or phosphorus.

[0149] The polymer filter 620 may include a plurality of holes 626 traced to a polymer substrate, the holes extending through the filter surface of the filter 620. (As in...) Figure 6CAs shown, the polymer filter 620 may be accompanied by a support structure (such as a mesh 621) disposed on one side of the filter surface within the housing. In some embodiments, the mesh 621 may be disposed close to the filter surface, or the mesh 621 may be attached to the polymer filter surface to support it. The mesh 621 may comprise a permeable material through which the cell mixture can pass easily without perforating the cell membrane. The mesh may be made of a biocompatible, durable material capable of withstanding pressure and flow rate conditions exceeding those induced by the fluid delivery system described herein. For example, the mesh 621 may comprise stainless steel, titanium, cobalt, aluminum, or alloys thereof. At least a portion of the mesh 621 (e.g., the edges of the mesh 621) may be overmolded with a polymer to enhance the adhesion friction between the housing and the mesh. Exemplary polymers may include, but are not limited to, silicone, polyurethane, polyethylene glycol (PEG), etc.

[0150] The polymer filter 620 may comprise polycarbonate, polyester (PET), polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, and / or polytetrafluoroethylene. For example, the filter surface of the polymer filter 620 may comprise one or more of the polymer materials mentioned above. In some embodiments, the support structure (e.g., a mesh) may comprise one or more of the materials mentioned above. In some embodiments, the support structure and filter surface of the polymer filter 620 may comprise the same or different polymer materials. Figure 6A An exemplary track-etched polymer filter is shown, which contains 5 µm pores and is manufactured by it4ip. Figure 6B Another exemplary track-etched polymer filter, comprising 10 µm pores and manufactured by Steiltech, is shown. However, the disclosure provided herein is not intended to limit us to these exemplary polymer filters. Alternative pore sizes, filter thicknesses, etc., are described herein.

[0151] Cellular mechanically perforated filters (e.g., filters 520, 620) may be coated. One or more sides of the filter may be coated with a coating. For example, the filter may include a coating comprising one or more materials such as gold, silver, platinum, Teflon, polyvinylpyrrolidone, an adhesive coating, a surfactant, one or more proteins, adhesion molecules, antibodies, anticoagulants, factors regulating cell function, nucleic acids, lipids, carbohydrates, and / or transmembrane proteins. In some embodiments, the cellular mechanically perforated filter may not include a coating.

[0152] Cellular mechanical perforation filters (e.g., filters 520, 620) may contain hundreds, thousands, or tens of thousands (or tens of thousands) of pores for agitating the cell membrane (e.g., pores 526, 626, hereinafter collectively referred to as pore 526 for simplicity). For example, filter 520 may contain between approximately 500 and 400,000 pores, between 500 and 100,000 pores, between 10,000 and 400,000 pores, between 10,000 and 100,000 pores, between 50,000 and 400,000 pores, between 50,000 and 100,000 pores, or between 100,000 and 400,000 pores. In some implementations, the filter 520 may contain at least about 500, 1,000, 5,000, 10,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 200,000, 225,000, 250,000, 275,000, 300,000, 325,000, 350,000, or 375,000 pores. In some implementations, the filter 520 may contain no more than 1,000, 5,000, 10,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 200,000, 225,000, 250,000, 275,000, 300,000, 325,000, 350,000, 375,000, or 400,000 pores.

[0153] In some embodiments, a plurality of pores 526 extending through the filter surface 522 may comprise pores of uniform size or varying pore diameters throughout the surface. Different pore sizes can create varying effects on the cell membranes of different types of cells that can be disturbed through the plurality of pores 526. The plurality of pores 526 may comprise one or more cross-sectional shapes, including but not limited to circular, rectangular (e.g., square), elliptical, triangular, or other polygonal shapes. Because the filter surface 522 exhibits a certain thickness (described in more detail below), it should be understood that the plurality of pores 526 may exhibit a three-dimensional shape (e.g., prism) extending through the filter surface 522. For example, when the pore comprises a circular cross-section with a uniform diameter along the thickness of the filter surface 522, the three-dimensional shape of the pore may be cylindrical. When the pore comprises a triangular shape, the 3D shape of the pore may be a triangular prism. When the width of the pore varies (e.g., increases or decreases) along the thickness of the filter surface 522, the 3D shape of the pore may be conical.

[0154] In some embodiments, the width or size of each of the plurality of pores 526 may be between 2 µm and 20 µm. The width of the pore may depend on the type of one or more cells in the cell mixture passing through the plurality of pores 526. For example, the width may be between about 2-15 µm, 2-10 µm, 2-7 µm, 8-15 µm, 8-10 µm, or about 11-15 µm. In some embodiments, the pore width may be greater than or equal to about 2, 4, 6, 8, 10, 12, 14, 15, 16, or 18 µm. In some embodiments, the pore width may be less than or equal to about 4, 6, 8, 10, 12, 14, 15, 16, 18, or 20 µm. In some embodiments, each of the plurality of pores 526 may have a circular cross-section, and the width of the pore may be the diameter of the pore.

[0155] In some examples, multiple orifices 526 are of the same size. The consistency of orifice 526 can be critical to ensuring consistent flow rates and overall filter performance. For example, the width of the orifices (e.g., size) can be within approximately 25% of each other. This dimension may also be referred to as the orifice tolerance. In some examples, the orifice width can have a tolerance of approximately 1%–20%, 1%–15%, 1%–10%, or 1%–5%. In some examples, the orifice width has a tolerance greater than or equal to approximately 0.1%, 0.2%, 0.5%, 1%, 2%, or 5%. In some examples, the orifice width has a tolerance less than or equal to approximately 1%, 2%, 4%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, or 25%.

[0156] As mentioned above, the width of a given pore in a cell mechanical perforation filter (e.g., filters 520, 620) can depend on the cell type passing through the multiple pores. For example, the width of each pore can be smaller than the diameter of the cells in the mixture passing through the filter, such that forcing the cells through the pores under pressure causes disturbance of the cell membrane as the cells are constricted by the pores. For example, the pore width can be between 10% and 99% of the diameter of the cells in the cell suspension, such as approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cell diameter. It should be understood that reference to the diameter of the cells is intended to refer to the diameter of the cells in the cell mixture before passing through the filter (e.g., as the cells approach the filter), unless otherwise specified.

[0157] In some embodiments, the pore spacing between each of the plurality of pores 526 relative to the pore width (i.e., size) can be between 0.5:1 and 100:1. The pore spacing can be selected to allow the cell mixture to flow through the cellular mechanically perforated filter 520 without backflow, while also maintaining the structure of the cellular mechanically perforated filter and its ability to withstand various flow rate and pressure conditions. In some embodiments, the pore spacing between the pores relative to the pore width can be between about 0.5:1 and 5:1, 0.5:1 and 10:1, 0.5:1 and 25:1, or 0.5:1 and 50:1. In some embodiments, the pore spacing between the pores can be greater than or equal to about 0.5:1, 0.75:1, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, or 100:1. In some implementations, the hole spacing can be less than or equal to about 0.5:1, 0.75:1, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 75:1, or 100:1. In a non-limiting example, hole widths between 2 and 7 µm can have a 4:1 hole spacing, meaning that a 2 µm hole can be spaced 8 µm apart from another 2 µm hole. For example, in... Figures 6A-6B As shown in the filter 620, the pores 626 can be randomly distributed on the polymer filter surface.

[0158] Multiple pores 526 can cover between approximately 0.1% and approximately 60% of the total surface area of ​​the cell-mechanically perforated filters 520, 620. In some embodiments, the multiple pores 526 can comprise approximately 1.0 x 10⁻⁶ pores. 1 From approximately 1.0x10 10 The total surface area of ​​the filter is measured in pores per square millimeter. The number of pores per square millimeter may also be referred to herein as the pore density. In some embodiments, the porous surface may contain approximately 1.0 x 10⁻⁶ pores. 1 From approximately 1.0 x 10 15 Hole between / mm 2 The total surface area of ​​the filter. For example, a porous surface can contain approximately 1.0 x 10⁻⁶. 1 From approximately 1.0 x 10 6 Hole between / mm 2 1.0x10 3 From approximately 1.0 x 10 6 Hole between / mm 2 or approximately 1.0 x 10 4 From approximately 1.0 x 10 6 Hole between / mm 2 In some examples, porous surfaces may contain a density greater than or equal to approximately 1.0 x 10⁻⁶. 1Hole / mm 2 1.0 x 10 2 Hole / mm 2 1.0 x 10 3 Hole / mm 2 1.0 x 10 4 Hole / mm 2 1.0x10 5 Hole / mm 2 1.0 x 10 6 Hole / mm 2 1.0 x 10 8 Hole / mm 2 1.0 x 10 10 Hole / mm 2 1.0 x 10 12 Hole / mm 2 Or 1.0 x 10 14 Hole / mm 2 In some examples, porous surfaces may contain less than or equal to about 1.0 x 10⁻⁶ particles. 2 Hole / mm 2 1.0 x 10 3 Hole / mm 2 1.0 x 10 4 Hole / mm 2 1.0 x 10 5 Hole / mm 2 1.0 x 10 6 Hole / mm 2 1.0 x 10 8 Hole / mm 2 1.0 x 10 10 Hole / mm 2 1.0 x 10 12 Hole / mm 2 1.0 x 10 14 Hole / mm 2 Or 1.0 x 10 15 Hole / mm 2 .

[0159] In some embodiments, each of the plurality of orifices 526 may extend linearly through a filter surface (e.g., filter surface 522). For example, the orifices may extend through the filter in a direction collinear with the thickness dimension of the filter. In some embodiments, the linear extension of the orifices 526 through the filter surface 522 may be collinear with the flow direction of the cell mixture through the orifices 526.

[0160] In some embodiments, the thickness of the filter surface 522 can be between 0.1 µm and 100 µm. As mentioned above, the thickness of the filter surface 522 can be defined by an axis along which the cell mixture travels as it passes through a cellular mechanically perforated filter (e.g., through a plurality of pores 526). The thickness of the filter surface 522 can be chosen to minimize clogging within the pores while perturbing the cell membrane to the required pore length. Additionally, as described herein with respect to other features of the filter, the thickness of the filter surface 522 can be selected to withstand breakage during use. In some embodiments, the thickness of the filter surface 522 can be between about 0.1–10 µm, between 1–100 µm, between 1–10 µm, or between 10–100 µm. In some embodiments, the thickness of the filter surface 522 can be greater than or equal to about 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 50, 75, or 100 µm. In some implementations, the thickness of the filter surface 522 may be less than or equal to about 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 50, 75 or 100 µm.

[0161] Cellular mechanical perforation filters (e.g., filters 120, 220, 520, 620) may comprise circular, triangular, rectangular, elliptical, or other polygonal shapes. For example, as at least in Figures 7A-7C As shown, the filter surface 722 of filter 720 (which should be understood to be equivalent to filters 120, 220, 520 and 620) may include a rectangular (e.g., square) shape. Figure 7A The width of the filter surface 722 is indicated by reference numeral 726. As shown, the width 726 extends from a first side of the filter surface 722 to a second side opposite the first side. In some embodiments, the width of the filter surface 722 can be between 1 mm and 10 cm. For example, the width of the filter surface 722 can be between about 1 mm and 5 cm, 1 mm and 1 cm, 5 mm and 10 cm, 5 mm and 1 cm, 1 cm and 10 cm, 1 cm and 5 cm and 10 cm. In some embodiments, the width of the filter surface 722 can be greater than or equal to 1 mm, 2 mm, 5 mm, 8 mm, 1 cm, 2 cm, 5 cm, 8 cm, or 10 cm. In some embodiments, the width of the filter surface 722 can be less than or equal to 1 mm, 2 mm, 5 mm, 8 mm, 1 cm, 2 cm, 5 cm, 8 cm, or 10 cm.

[0162] In some implementations, the length of the filter surface 722 (in Figure 7AThe width of the filter surface 722 (denoted by reference numeral 728) can be substantially the same as the width of the filter surface 722. In some embodiments, the length of the filter surface 722 can be greater than or less than the width of the filter surface 722. Any of the exemplary ranges or values ​​of the width of the filter surface 722 above should be understood to apply to the length of the filter surface 722. In some embodiments, the filter surface 722 may include a circular shape (see, for example, Figure 5A and Figures 7B-7C In this case, the width can be understood as the diameter of the filter surface 722.

[0163] The shape and size of the filter (e.g., filter surface 722) can be selected to correspond to the upstream fluid inlet and / or downstream fluid outlet (e.g., fluid inlets 104, 204, 304 and fluid outlets 108, 208, 308 of housings 100, 200, 300, respectively). For example, the width of the filter surface 722 can be greater than or equal to the width of the fluid inlet and / or fluid outlet to limit the dead volume between the fluid path and the filter. In some embodiments, the shape of the filter surface 722 can correspond to the shape of the fluid inlet and / or fluid outlet. For example, if the fluid inlet and / or fluid outlet comprises a circular cross-section, the filter surface 722 can comprise a circular shape.

[0164] The filter surface 722 (and 522) is described with reference to single filters 520 and 720. However, as described herein, in some embodiments, the system may comprise multiple cellular mechanically perforated filters. For example, the filters may be arranged in parallel to create a filter array that together surrounds a larger filter surface. Any of the dimensions provided above with respect to filter surface 722 should be understood to apply to this filter surface comprised of multiple filters 720. For example, multiple cellular mechanically perforated filters 720 (each filter comprising a width of 1 mm) may be arranged in an array to produce a filter surface comprising a width on the order of 1-10 cm.

[0165] As described herein, the silicon filter 520 may include a support structure 524 disposed on one side of the filter surface 522. In some embodiments, the thickness of the support structure 524 may be between 20 µm and 1 mm. Similar to the filter surface 522, the thickness of the support structure 524 may be defined by an axis along which the cell mixture travels as it passes through the filter 520 (e.g., through a plurality of orifices 526). The thickness of the support structure 524 may be selected such that the support structure allows the silicon filter 520 to withstand breakage during use, but does not impede the flow of the cell mixture through the orifices of the filter surface 522. In some embodiments, the thickness of the support structure 524 may be between about 20 µm and 100 µm, between 50 µm and 1 mm, between 50 µm and 100 µm, between 0.1 and 1 mm, between 0.1 and 0.5 mm, or between 0.5 and 1 mm. In some embodiments, the thickness of the support structure 524 may be greater than or equal to about 20 µm, 50 µm, 80 µm, 0.1 mm, 0.2 mm, 0.5 mm, 0.8 mm, or 1 mm. In some embodiments, the thickness of the support structure 524 may be less than or equal to about 20 µm, 50 µm, 80 µm, 0.1 mm, 0.2 mm, 0.5 mm, 0.8 mm, or 1 mm.

[0166] In some embodiments, the support structure 524 may cover at least a portion of the filter surface 522, such as about 1% of the filter surface 522. The support structure 524 may cover at least a portion of the filter surface 522 to support the filter surface 522 and prevent it from breaking under various flow rate and pressure conditions on the filter 520. However, the support structure 524 must allow the cell mixture to pass through the filter surface 522 without backflow and / or filter blockage. Therefore, the support structure 524 may cover a portion of the filter surface 522. In some embodiments, the support structure 524 may cover between about 0.1% and 30%, between 0.1% and 10%, between 0.1% and 1%, between 1% and 30%, between 1% and 10%, or between 1% and 5% of the silicon filter surface 522. In some embodiments, the support structure 524 may cover greater than or equal to about 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, or 30% of the filter surface 522. In some embodiments, the support structure 524 may cover less than or equal to about 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, or 30% of the filter surface 522. In some embodiments, the support structure 524 may cover greater than 30% of the filter surface 522, such as about 35%, 40%, 45%, or 50% of the filter surface 522.

[0167] like Figures 7A-7C As shown, support structure 724 (e.g., support structure 524) may include one or more support members arranged on filter surface 722 and extending in one or more directions. For example, in Figures 7A-7C Each of the support structures 724 of the filter 720 shown in the image contains multiple support components.

[0168] As in Figures 7A-7B As shown, one or more support members can create two or more filter windows in the filter surface 722. Figure 7AAs shown, the support structure 724 may include a plurality of support members arranged on the filter surface 722, and the plurality of support members together may form at least one cross shape, the at least one cross shape separating a plurality of filter windows in the filter surface 722. For example, the support structure 724 may be composed of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more support members. The support members may form 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cross shapes on the filter surface 722. The support members may produce 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more filter windows in the filter surface 722. Figure 7A An exemplary filter 720 is shown, which includes a support structure 724 consisting of four support members forming four cross shapes, thereby creating nine filter windows in a filter surface 722.

[0169] The support structure 724 is not limited to one or more of the cross shapes mentioned above. For example, such as... Figure 7B As shown, the support members of the support structure 724 can create a central filter window in the filter surface 722. The support members creating the central filter window can include circular, oval, triangular, rectangular (e.g., square), or other polygonal shapes. In some embodiments, the support structure 724 including curved support members (e.g., circular support members, like the central filter window) can improve the filter's ability to withstand pressure and flow conditions induced during cell mechanical perforation, at least compared to a support structure 724 including one or more sharp corners or edges. One or more support members can extend from the central support member to the periphery of the filter surface 722. For example, 1, 2, 3, 4, 5, 6, 7, 8, or more support members can extend from the central support member to create one or more filter windows adjacent to the central filter window. In some examples, the support members arranged around the central filter window can be spaced apart from each other between approximately 30º and 180º, such as between approximately 45º and 120º or between approximately 60º and 90º. Figure 7B An exemplary filter 720 is shown, which includes four filter windows, namely one central window and three peripheral windows, the filter windows being generated by three support members extending from a central support member.

[0170] In some implementations, the support structure 724 may include one or more support members arranged independently as strips on the filter surface 722. Figure 7CAn exemplary filter 720 for cell mechanical perforation is shown, comprising a plurality of support members arranged on a filter surface 722. Each support member of the support structure 724 extends from one side of the filter surface 722 toward the opposite side of the aforementioned side of the filter surface 722. A given support member of the support structure 724 may extend almost entirely from one side, or may extend only a portion (e.g., Figure 7C (As shown in the diagram). For example, the width of the support structure 724 can be approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the width of the filter surface 722. The support members can be arranged in an alternating manner, such as... Figure 7C As shown, or alternatively, they may all extend from one side or the other side of the filter surface 722.

[0171] Such as at least in Figures 7A-7C As shown, the cellular mechanically perforated filter 720 may include a border surrounding the filter surface 722. The border may be made of a non-porous material; in other words, the border may not contain holes 526, 626 passing through it. This border can be used to hold the filter 720 within a housing (e.g., housing 100, 200, 300, etc.) without clogging the filter surface 722. In some embodiments, the filter (including the border) may include circular, triangular, rectangular, elliptical, or other polygonal shapes. In some embodiments, the shape of the filter 720 may mimic the shape of the filter surface 722. In some embodiments, such as... Figures 7B-7C As shown, the shapes of filter 720 and filter surface 722 can be different (e.g., the filter, including the border, can have a rectangular shape, while the filter surface 722 can have a circular shape). The size of the filter is not intended to be limited to the disclosure provided herein. In several non-limiting examples, filter 720 can be 2x2 mm, 5x5 mm, 10x10 mm, 12x12 mm, 14x14 mm, or any size in between.

[0172] In some embodiments, filter 520 may comprise one or more oxide layers. For example, the oxide layer may be disposed on filter surface 522 between filter surface 522 and support structure 524. Cellular mechanically perforated filter 520 may comprise an oxide layer to facilitate fabrication of each of filter surface 522 and support structure 524 from a silicon wafer. For example, fabrication of silicon filter 520 may include etching a silicon filter layer and a silicon support layer of a silicon wafer, the silicon layers being separated by an oxide layer. With an oxide layer between the filter layer and the support layer, each of filter surface 522 and support structure 524 can be reliably etched without etching the other side.

[0173] Methods for assembling cell-mechanical perforated cassettes In some embodiments, a method for assembling a housing for cell mechanical perforation may be provided. The method for assembly may be primarily referenced in... Figures 1A-1C The housing 100 shown herein is described. However, it should be understood that the method may be additionally or alternatively applied to assembling housings 200, 300. The method includes connecting a first housing portion 102 of housing 100 to a second housing portion 106 of housing 100. The first housing portion 102 and the second housing portion 106 may be detachably connected or permanently connected. The first housing portion 102 and the second housing portion 106 may be connected using press fitting, snap-fit, threaded connection, forging connection, or welding connection.

[0174] As described herein, the outer surface of at least one of the first housing portion 102 and the second housing portion 106 may include textured portions (e.g., ribs, grooves, etc.) that a user can interact with to help connect the first housing portion 102 and the second housing portion 106.

[0175] The method includes placing a cell-mechanically perforated filter 120 within a housing 100, such that a first housing portion 102 and a second housing portion 106 hold the filter 120. The filter 120 may be disposed within a filter holding portion 110 of the first housing portion 102 and / or the second housing portion 106. The first housing portion 102 may include a fluid inlet 104. The second housing portion 106 may include a fluid outlet 108. The filter 120 may be disposed within the housing 100 such that the filter is in fluid communication with the fluid inlet 104 and the fluid outlet 108.

[0176] In some embodiments, the assembly method may include arranging the sealing member 112 within the housing near the filter 120. For example, the sealing member 112 may be arranged above or below the filter 120 in the end region of the first housing portion 102 or the end region of the second housing portion 106.

[0177] The detachable connection of the first housing portion 102 and the second housing portion 106 allows for subsequent replacement of the cell mechanical perforation filter 120, improvement of the sterilization of the inner components of the housing 100, etc.

[0178] Methods for disturbing cell membranes In some embodiments, a method for perturbing a cell membrane using the housing described herein may be provided. The method includes any one or more steps of the method for assembling the housing described above. The method may primarily relate to... Figures 1A-1C The method is described using the box 100 shown in the figure. However, it should be understood that the method can be further applied to boxes 200, 300 and 400.

[0179] The method includes detachably connecting a fluid inlet 104 of the first housing portion 102 to a cell mixture source (e.g., cell mixture source 432). For example, the fluid inlet 104 may be detachably connected to the cell mixture source using one or more connectors, solder joints, and / or tubing. The method also includes detachably connecting a fluid outlet 108 to an output reservoir (e.g., output reservoir 434). Similarly, detachably connecting the fluid outlet 108 to the output reservoir may include using one or more connectors, solder joints, and / or tubing. In some embodiments, connecting the fluid outlet 108 to the output reservoir may include arranging the fluid outlet 108 near a receiving end of the output reservoir, such that fluid from the fluid outlet 108 can be delivered directly to the output reservoir with minimal fluid waste.

[0180] As described herein, the outer surface of at least one of the first housing portion 102 and the second housing portion 106 may include textured portions (e.g., ribs, grooves, etc.) that a user can hold to help connect the housing 100 to at least one of the cell mixture source and output reservoirs.

[0181] In some embodiments, detachably connecting the fluid inlet 104 to a cell mixture source and / or detachably connecting the fluid outlet 108 to an output reservoir may include placing the housing 100 within a housing receiving portion of the fluid delivery system. The fluid delivery system may include a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system. Inserting the housing 100 into the housing receiving portion of the fluid delivery system allows fluid inlet 104 to be fluidly connected to a cell mixture source and / or fluid outlet 108 to an output reservoir. In some embodiments, detachably connecting the fluid inlet 104 and / or the fluid outlet 108 to the respective cell mixture source and / or output reservoir may include using one or more connectors (e.g., Luer connectors, threaded connectors, quick-release connectors, etc.), tubing, and / or solder joints.

[0182] The method includes receiving a cell mixture from a cell mixture source at a fluid inlet 104. As described herein, the cell mixture may contain a cell suspension and a payload. In some embodiments, the cell mixture may contain only a cell suspension.

[0183] As described herein, cell mixtures may comprise somatic cells, immortalized cells (e.g., HeLa cells, HEK cells, etc.), stem cells, or derivatives thereof. In some embodiments, the cell mixture comprises PBMCs or derivatives thereof. In some embodiments, the cell mixture comprises immune cells. In some embodiments, the cell mixture comprises T cells, NK cells, monocytes, B cells, or dendritic cells. In some embodiments, the cell mixture comprises unstimulated T cells or derivatives thereof. In some embodiments, the cell mixture comprises stimulated (or activated) T cells. In some embodiments, the cell mixture comprises stem cells, such as human stem cells. In some embodiments, the cell mixture comprises induced pluripotent stem cells, hematopoietic cells, or mesenchymal cells. In some embodiments, the cell mixture comprises cells obtained from or derived from an individual (e.g., a human).

[0184] In some embodiments, the cell mixture contains a payload such that perturbing the cells in the cell mixture promotes the entry of the payload into the cells (i.e., the cytosol). In some embodiments, the method includes mixing the cell mixture (i.e., the cell suspension) with the payload before passing the cell mixture through a cell mechanical perforation filter 120. In some embodiments, the cell mixture may contain a premixed payload and a cell suspension. In some embodiments, the method includes introducing the payload into the cell mixture after cell perturbation, thereby promoting payload entry into the cells. The payload may contain at least one of peptides, nucleic acids, small molecules, nanoparticles, and complexes thereof. In some embodiments, the payload may contain a mixture consisting of more than one of nucleic acids, small molecules, nanoparticles, etc.

[0185] The method includes passing a cell mixture through a cell mechanical perforation filter 120 to agitate the cell membranes in the cell mixture. In some embodiments, passing the cell mixture through the filter 120 may include passing the cell mixture at a rate of about 0.5-500 mL / min / mm. 2 The volumetric flow rate between them passes through. In some embodiments, the housing 100 can be configured to pass the cell mixture through the cell mechanically perforated filter 120 in a single run without replacing the cell mechanically perforated filter 120. In some embodiments, effectively agitating millions of cells in the cell mixture requires only a single cell mechanically perforated filter.

[0186] The method includes delivering a disturbed cell mixture to an output reservoir via fluid outlet 108. After at least one run (i.e., one complete passage of the sample through the system), the method includes disconnecting cartridge 100 from the fluid delivery system. The method includes disconnecting first housing portion 102 from second housing portion 106 to sterilize and / or replace filter 120. In some embodiments, the method includes sterilizing cartridge 100. In some embodiments, cartridge 100 may be sterilized simultaneously with at least one component connected to the fluid delivery system (e.g., tubing, connector, etc.). Sterilization may include, for example, gamma irradiation and / or chemical sterilization. The method includes replacing cartridge 100 within the system.

[0187] In some embodiments, the method for perturbing the cell membrane can be used to perturb the cell membrane of a cell mixture containing a large number of cells (e.g., at least 100 million cells). For example, the cell mixture may contain millions, hundreds of millions, billions, or trillions of cells. In some embodiments, the method includes perturbing a cell membrane containing at least 1.0 x 10⁻⁶ cells. 8 Cells (e.g., in approximately 1.0 x 10⁻⁶) 8 With 1.0x10 12 A mixture of cells (between 1 and 10 cells) is passed through multiple pores. In some embodiments, at approximately 1.0 x 10⁻⁶ cells... 8 With 1.0x10 11 Cells between 1.0x10 8 With 1.0x10 10 Between 1.0 x 10 cells or 1.0 x 10 6 With 1.0x10 9 Cells can pass through multiple pores.

[0188] In some embodiments, the cell mixture passing through the cartridge 100 according to the method described herein can have a volume between at least 10 µL and 1000 L. For example, the cell mixture can have a volume between about 1-1000 µL, 1-100 µL, 1-1000 mL, 1-100 mL, or 1-1000 L. In some embodiments, the cell mixture can have a volume greater than or equal to about 10 µL, 50 µL, 100 µL, 500 µL, 1 mL, 5 mL, 10 mL, 20 mL, 50 mL, 100 mL, 500 mL, 1 L, 2 L, 5 L, 10 L, 50 L, 100 L, or 500 L. In some embodiments, the cell mixture may have a volume of less than or equal to about 50 µL, 100 µL, 500 µL, 1 mL, 5 mL, 10 mL, 20 mL, 50 mL, 100 mL, 500 mL, 1 L, 2 L, 5 L, 10 L, 50 L, 100 L, 500 L, or 1000 L.

[0189] In some embodiments, the cell mixture can pass through the housing 100 at a constant volumetric flow rate. In some embodiments, the cell mixture can pass through the housing 100 at a pulsed volumetric flow rate. For example, the flow rate of the cell mixture through the housing 100 can be controlled by at least one of a peristaltic pump, a syringe pump, and a pressurized reservoir.

[0190] In some embodiments, the cell mixture can be delivered at a rate of approximately 0.5–500 mL / min / mm. 2 The volumetric flow rate between the porous surface areas passes through the housing 100 (i.e., the cell mechanical perforation filter 120). In some embodiments, the cell mixture may pass through the housing 100 at a volumetric flow rate of approximately 0.5-100, 0.5-10, 50-500, 5-100, or 5-20 mL / min / mm. 2 Porous surface area. In some embodiments, the cell mixture can pass through the chamber 100 at a volumetric flow rate greater than or equal to about 0.5, 1, 2, 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, or 450 mL / min / mm. 2 Porous surface area. In some embodiments, the cell mixture can pass through the chamber 100 at a volumetric flow rate of less than or equal to about 1, 2, 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 mL / min / mm. 2 Porous surface area.

[0191] In some embodiments, the entire cell mixture can pass through housing 100 (e.g., cell mechanical perforation filter 120) in about 10 minutes or less. In some embodiments, the entire cell mixture can pass through the filter in no more than about 30 minutes, 25 minutes, 20 minutes, 15 minutes, 12 minutes, 10 minutes, 8 minutes, 5 minutes, 2 minutes, or 1 minute. In some embodiments, the entire cell mixture can pass through the filter in about 1-30 minutes, 1-25 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-5 minutes, 5-30 minutes, 5-25 minutes, 5-20 minutes, 5-15 minutes, 5-10 minutes, 10-30 minutes, 10-25 minutes, 10-20 minutes, or 10-15 minutes.

[0192] In some embodiments, a single cell of the cell mixture can pass through the pores of a multi-well array for approximately 1 microsecond to 10 milliseconds. For example, cells can pass through the pores within 1 µs–1 ms, 10 µs–10 ms, 10 µs–1 ms, 100 µs–10 ms, or 100 µs–1 ms. In some embodiments, cells can pass through the pores within approximately 1 µs, 5 µs, 10 µs, 50 µs, 100 µs, 500 µs, 1 ms, or 5 ms. In some embodiments, cells can pass through the pores within approximately 5 µs, 10 µs, 50 µs, 100 µs, 500 µs, 1 ms, 5 ms, or 10 ms.

[0193] In some embodiments, the speed at which cells pass through the multiple pores of the filter to cause cell membrane agitation can be about 1 m / s. In some embodiments, the cell speed can be between 10 mm / s and 100 m / s. For example, the cell speed can be less than or equal to 10 mm / s, 20 mm / s, 50 mm / s, 1 m / s, 2 m / s, 5 m / s, 10 m / s, 20 m / s, 50 m / s, or 100 m / s. In some embodiments, the cell speed can be greater than or equal to 10 mm / s, 20 mm / s, 50 mm / s, 1 m / s, 2 m / s, 5 m / s, 10 m / s, 20 m / s, 50 m / s, or 100 m / s.

[0194] In some embodiments, the method for agitating the cell membrane may include pre-filling the filter 120 before passing the cell mixture through the cell mechanical perforation filter 120. For example, the filter 120 may be pre-filled with a cell buffer (e.g., cell culture medium). In some embodiments, the cell mixture may contain a cell mixture for pre-filling. In some embodiments, the filter 120 may be pre-wetted. In some embodiments, the filter 120 may not require pre-filling.

[0195] In some cases, the sample volume allows for a minimum residence time of cells in the output reservoir. For example, the residence time of cells in the output reservoir can be less than 30 seconds (s), less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds. In some cases, the residence time is between approximately 0.01 seconds and approximately 30 seconds.

[0196] In some implementations, the methods mentioned above can be performed at temperatures between about 0°C and about 40°C. For example, the methods can be performed at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), above physiological temperature (e.g., about 37°C or higher), or at lower temperatures (e.g., about 0°C to 4°C).

[0197] The perturbed cell mixture, generated by passing the mixture through the housing 100, can be incubated (e.g., the cells may be frozen) for a certain duration. For example, the perturbed cell mixture can be incubated for approximately 0.0001 s to 20 min, such as 1 s, 30 s, 90 s, 2 min, 5 min, 10 min, 15 min, or 20 min. The perturbed cell mixture can be incubated at a temperature between 0°C and 40°C (e.g., at least 0°C, 5°C, 10°C, 20°C, 25°C, 30°C, 35°C, or 40°C). The perturbed cell mixture can be incubated in an output reservoir. In some embodiments, the perturbed cell mixture can be washed alternatively or additionally prior to incubation. Finally, in combination with one or more of the post-processing steps mentioned above and / or immediately following collection, the perturbed cell mixture can be injected into a patient to perform the intended cell therapy with the cells.

[0198] Manufacturing of silicon cell mechanical perforated filters In some embodiments, the silicon cell mechanically perforated filters described herein (e.g., filters 520, 720) can be formed from a silicon wafer comprising two silicon layers and an oxide layer buried between the two silicon layers. Multiple pores can be formed in one of the silicon layers of the wafer (i.e., the filter layer), and a support structure can be formed in the other silicon layer of the wafer (i.e., the support layer). After fabricating the support structure and pores, the buried oxide layer can be removed to expose the multiple pores in the filter layer and produce one or more silicon cell mechanically perforated filters. The formation of the pores and support structure can utilize known photoresist coatings and photolithography techniques (such as reactive ion etching) with specific parameters. Several silicon filters can be formed from a single silicon wafer; therefore, after removing the oxide layer, the method can include dicing the silicon wafer into multiple individual silicon filters.

[0199] In some embodiments, the silicon cell mechanically perforated filter described herein can be additionally or alternatively formed by masking a silicon wafer according to a desired support structure layout. The silicon wafer can then be oxidized in the remaining areas of the silicon wafer not masked for the support structure. Finally, multiple pores can be formed in the oxidized areas of the silicon wafer to produce the silicon cell mechanically perforated filter.

[0200] definition Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the meanings commonly understood in the art.

[0201] As used herein, the singular forms “a” and “the” are intended to include the plural forms as well, unless the context explicitly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and covers any and all possible combinations of one or more of the associated listed items. It should also be understood that the terms “includes,” “including,” “comprises,” and / or “comprising”, when used herein, specify the presence of the stated feature, integer, step, operation, element, component, and / or unit, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, units, and / or groups thereof.

[0202] The disclosed numerical ranges inherently support any range or value within the disclosed numerical ranges, including endpoints, even if no precise range limitation is stated verbatim in the specification, because this disclosure can be practiced throughout the disclosed numerical ranges.

[0203] in conclusion For purposes of explanation, the above description has been presented with reference to specific embodiments. However, the foregoing illustrative discussion is not intended to be exhaustive or to limit the invention to the exact forms disclosed. Many modifications and variations are possible in light of the foregoing teachings. These embodiments were chosen and described in order to best explain the principles of these techniques and their practical applications. Others skilled in the art will thus be able to best utilize these techniques and various embodiments with various modifications suitable for the particular intended use.

[0204] Although this disclosure and examples have been fully described with reference to the accompanying drawings, it should be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications should be understood to be included within the scope of this disclosure and examples as defined by the claims.

[0205] Exemplary Implementation The following implementations are exemplary and are not intended to limit the scope of any invention described herein.

[0206] Implementation Scheme 1. A housing for mechanical cell perforation, the housing comprising: A first housing portion includes a fluid inlet detachably connected to a source of the cell mixture; A second housing portion connected to the first housing portion, the second housing portion including a fluid outlet detachably connected to the output reservoir; and A cell mechanically perforated filter, the cell mechanically perforated filter being held by a first housing portion and a second housing portion and configured to operate at a rate of at least 0.5 mL / min / mm. 2 The volumetric flow rate of the porous surface area agitates the cell membrane of the cell mixture through the cellular mechanical perforation filter.

[0207] Implementation Scheme 2. The housing as described in Implementation Scheme 1, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

[0208] Implementation Scheme 3. The housing as described in Implementation Scheme 1 or 2, wherein the cell mechanical perforation filter is configured to operate at a rate of 0.5-500 mL / min / mm. 2 The volumetric flow rate between porous surfaces disturbs the cell membrane.

[0209] Implementation Scheme 4. The housing as described in any one of Implementation Schemes 1-3, wherein the cellular mechanical perforation filter is configured to perturb the inclusion of 1.0 x 10 8 With 1.0x10 12 The cell membrane of a mixture of cells between cells.

[0210] Implementation Scheme 5. The housing as described in any one of Implementation Schemes 1-4, wherein the housing is configured to receive a volume between 1 and 500 mL.

[0211] Implementation Scheme 6. The housing as described in any one of Implementation Schemes 1-5, wherein the housing is configured to pass the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

[0212] Implementation Scheme 7. The housing as described in any one of Implementation Schemes 1-6, wherein the housing is configured to allow the entire cell mixture to pass through the cell mechanical perforation filter within no more than 30 minutes.

[0213] Implementation Scheme 8. The housing as described in any one of Implementation Schemes 1-7, wherein the housing is configured to receive a constant volumetric flow rate controlled by at least one of a peristaltic pump, an injection pump, and a pressurized reservoir.

[0214] Implementation Scheme 9. A housing as described in any one of Implementation Schemes 1-8, wherein the housing is removably connected by a housing receiving portion of a fluid delivery system to at least one pair of the fluid inlet and the cell mixture source, and the fluid outlet and the output reservoir.

[0215] Implementation Scheme 10. The housing as described in Implementation Scheme 9, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

[0216] Implementation Scheme 11. The housing as described in any one of Implementation Schemes 1-10, wherein the fluid inlet is detachably connected to the cell mixture source using a Luer connector, threaded connector, or quick-release connector.

[0217] Implementation Scheme 12. The housing as described in any one of Implementation Schemes 1-10, wherein the fluid inlet is detachably connected to the cell mixture source via one or more tubes using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

[0218] Implementation Scheme 13. The housing as described in any one of Implementation Schemes 1-12, wherein the fluid outlet is detachably connected to the output reservoir using a Luer connector, threaded connector, or quick-release connector.

[0219] Implementation Scheme 14. The housing as described in any one of Implementation Schemes 1-12, wherein the fluid outlet is detachably connected to the output reservoir via one or more pipes using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

[0220] Implementation Scheme 15. The housing as described in any one of Implementation Schemes 1-12, wherein the fluid outlet is detachably connected to the output reservoir by arranging the fluid outlet at an opening near the output reservoir.

[0221] Implementation Scheme 16. The housing as described in any one of Implementation Schemes 1-15, wherein the first housing portion and the second housing portion are connected by welding, forging, threading, snap-fit ​​or press-fit.

[0222] Implementation Scheme 17. The housing as described in any one of Implementation Schemes 1-16, wherein the first housing portion and the second housing portion are detachably connected.

[0223] Implementation Scheme 18. The housing as described in any one of Implementation Schemes 1-17, wherein the cell mechanical perforation filter is replaceable.

[0224] Implementation Scheme 19. The housing as described in any one of Implementation Schemes 1-18, wherein the housing is capable of being sterilized by at least one of gamma irradiation and chemical sterilization.

[0225] Implementation Scheme 20. The housing as described in Implementation Scheme 19, wherein the housing is sterilizable when connected to at least one of the cell mixture source and the output reservoir.

[0226] Implementation Scheme 21. The housing as described in any one of Implementation Schemes 1-20, wherein the fluid inlet includes a tapered portion, the width of which increases from a proximal end of the fluid inlet to a distal end of the fluid inlet near the cellular mechanical perforation filter.

[0227] Implementation Scheme 22. The housing as described in Implementation Scheme 21, wherein the fluid inlet has a circular cross-section and the width is the diameter of the fluid inlet.

[0228] Implementation Scheme 23. The housing as described in any one of Implementation Schemes 1-22, wherein the fluid outlet includes a tapered portion, the width of which decreases from a proximal end of the fluid outlet near the cellular mechanical perforation filter to a distal end of the fluid outlet.

[0229] Implementation Scheme 24. The housing as described in Implementation Scheme 23, wherein the fluid outlet has a circular cross-section and the width is the diameter of the fluid outlet.

[0230] Implementation Scheme 25. The housing as described in any one of Implementation Schemes 1-24, the housing comprising a sealing member disposed in an end region of the first housing portion near the connected second housing portion.

[0231] Implementation Scheme 26. The housing as described in any one of Implementation Schemes 1-25, wherein the second housing portion includes an end region adjacent to the connected first housing portion, the end region of the second housing portion holding the cellular mechanical perforation filter.

[0232] Implementation Scheme 27. The housing as described in any one of Implementation Schemes 1-24, the housing comprising a sealing member disposed in an end region of the second housing portion near the connected first housing portion.

[0233] Implementation Scheme 28. The housing as described in any one of Implementation Schemes 1-24 and 27, wherein the first housing portion includes an end region adjacent to the connected second housing portion, the end region of the first housing portion holding the cellular mechanical perforation filter.

[0234] Implementation Scheme 29. The housing as described in any one of Implementation Schemes 1-28, wherein the outer surface of at least one of the first housing portion and the second housing portion includes a textured portion, the textured portion being configured to facilitate at least one of the following: connection of the first housing portion to the cell mixture source, connection of the second housing portion to the output reservoir, and connection of the first housing portion to the second housing portion.

[0235] Implementation Scheme 30. The housing as described in any one of Implementation Schemes 1-29, wherein the cell mechanical perforation filter comprises a silicon filter or a polymer filter.

[0236] Implementation Scheme 31. The housing as described in any one of Implementation Schemes 1-30, wherein the cell mechanical perforation filter comprises a silicon filter containing silicon, silicon oxide, silicon nitride and / or silicon carbide.

[0237] Implementation Scheme 32. The housing as described in any one of Implementation Schemes 1-30, wherein the cell mechanical perforation filter comprises a polymer filter containing polycarbonate, polyester (PET), polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF and / or polytetrafluoroethylene.

[0238] Implementation Scheme 33. The housing as described in any one of Implementation Schemes 1-32, wherein the cell mechanical perforation filter is coated with at least one of gold, silver, platinum, Teflon, polyvinylpyrrolidone, adhesive and surfactant.

[0239] Implementation Scheme 34. The housing as described in any one of Implementation Schemes 1-31 and 33, wherein the cellular mechanical perforation filter is doped with boron, gallium, or phosphorus.

[0240] Implementation Scheme 35. The housing as described in any one of Implementation Schemes 1-34, wherein the cell mechanical perforation filter is configured to withstand pressures between at least 1 and 50 psi.

[0241] Implementation Scheme 36. The housing as described in any one of Implementation Schemes 1-35, wherein the cell mechanical perforation filter comprises a filter surface and a plurality of pores extending through the filter surface, the plurality of pores being configured to agitate the cell membrane as the cell mixture passes through the plurality of pores.

[0242] Implementation Scheme 37. The housing as described in Implementation Scheme 36, wherein the plurality of holes comprises no more than 300,000 holes.

[0243] Implementation Scheme 38. The housing as described in Implementation Scheme 36 or 37, wherein the cell mechanical perforation filter includes a support structure disposed on one side of the filter surface.

[0244] Implementation Scheme 39. The housing as described in Implementation Scheme 38, wherein the support structure covers at least 1% of the filter surface.

[0245] Implementation Scheme 40. The housing as described in Implementation Scheme 38 or 39, wherein the support structure comprises a plurality of support members arranged on the filter surface and forming at least one cross or a plurality of strips.

[0246] Implementation Scheme 41. The housing as described in any one of Implementation Schemes 38-40, wherein the thickness of the support structure is between 20 µm and 3 mm.

[0247] Implementation Scheme 42. The housing as described in any one of Implementation Schemes 38-41, wherein the cellular mechanical perforation filter comprises an oxide layer disposed on the filter surface between the filter surface and the support structure.

[0248] Implementation Scheme 43. The housing as described in any one of Implementation Schemes 36-42, wherein each of the plurality of holes contains a width between 2 µm and 20 µm.

[0249] Implementation Scheme 44. The housing as described in Implementation Scheme 43, wherein the hole spacing between each of the plurality of holes is between 0.5:1 and 100:1 relative to the diameter of the hole.

[0250] Implementation Scheme 45. The housing as described in any one of Implementation Schemes 36-44, wherein the thickness of the filter surface is between 0.1 µm and 100 µm.

[0251] Implementation Scheme 46. The housing as described in any one of Implementation Schemes 36-45, wherein the width of the filter surface is between 1 mm and 10 cm.

[0252] Implementation Scheme 47. The housing as described in Implementation Scheme 46, wherein the width of at least one of the fluid inlet and the fluid outlet is at least 50% of the width of the filter surface.

[0253] Implementation Scheme 48. The housing as described in any one of Implementation Schemes 36-47, wherein at least one of the fluid inlet and the fluid outlet comprises a circular cross-section corresponding to the circular shape of the filter surface.

[0254] Implementation Scheme 49. A system comprising: The box body according to any one of embodiments 1-48; and cell.

[0255] Implementation Scheme 50. The system as described in Implementation Scheme 49, wherein the cell is a somatic cell, an immortalized cell, a stem cell, or a derivative thereof.

[0256] Implementation Scheme 51. The system as described in Implementation Scheme 49 or 50, wherein the cells are peripheral blood mononuclear cells (PBMCs) or derivatives thereof.

[0257] Implementation Scheme 52. The system as described in any one of Implementation Schemes 49-51, wherein the cells are immune cells.

[0258] Implementation Scheme 53. The system of any one of Implementation Schemes 49-52, wherein the cell is a T cell, a natural killer (NK) cell, a monocyte, a B cell, or a dendritic cell.

[0259] Implementation Scheme 54. The system as described in Implementation Scheme 49 or 50, wherein the cell is a stem cell.

[0260] Implementation Scheme 55. The system of any one of Implementation Schemes 49, 50 and 54, wherein the cell is a human stem cell.

[0261] Implementation Scheme 56. The system of any one of Implementation Schemes 49, 50, 54 and 55, wherein the cell is an induced pluripotent stem cell, a hematopoietic cell or a mesenchymal cell.

[0262] Implementation Scheme 57. The system of any one of Implementation Schemes 49-56, wherein the cells are obtained or derived from an individual.

[0263] Implementation Scheme 58. The system as described in Implementation Scheme 51, wherein the individual is a person.

[0264] Implementation Scheme 59. A method for perturbing a cell membrane, the method comprising: The fluid inlet of a first housing portion of the housing is detachably connected to a cell mixture source, the housing including a second housing portion connected to the first housing portion, and the second housing portion including a fluid outlet, wherein the connected first housing portion and the second housing portion retain a cell mechanical perforation filter; The fluid outlet is detachably connected to the output reservoir; The cell mixture from the cell mixture source is received at the fluid inlet; The cell mixture was prepared at a rate of at least 0.5 mL / min / mm. 2 A volumetric flow rate with a porous surface area passes through the cellular mechanically perforated filter to agitate the cell membrane of the cell mixture; and The disturbed cellular mixture is delivered to the output reservoir via the fluid outlet.

[0265] Implementation Scheme 60. The method of Implementation Scheme 59, wherein removably connecting the fluid inlet to the cell mixture source and / or removably connecting the fluid outlet to the output reservoir comprises placing the cartridge in a cartridge receiving portion of the fluid delivery system.

[0266] Implementation Scheme 61. The method of Implementation Scheme 60, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

[0267] Implementation Scheme 62. The method of any one of Implementation Schemes 59-61, wherein the cell mixture is passed through the cell mechanical perforation filter at a rate of 0.5-500 mL / min / mm. 2 The volumetric flow rate occurs between porous surfaces.

[0268] Implementation Scheme 63. The method of any one of Implementation Schemes 59-62, wherein the cell mixture is passed through the cell mechanical perforation filter at a constant volumetric flow rate controlled by at least one of a peristaltic pump, an injection pump, and a pressurized reservoir.

[0269] Implementation Scheme 64. The method of any one of Implementation Schemes 59-63, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

[0270] Implementation Scheme 65. The method of any one of Implementation Schemes 59-64, wherein the cell mixture comprises 1.0 x 10 8 With 1.0x10 12 Between the cells.

[0271] Implementation Scheme 66. The method of any one of Implementation Schemes 59-65, wherein the cell mixture has a volume between 1 and 500 mL.

[0272] Implementation Scheme 67. The method of any one of Implementation Schemes 59-66, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

[0273] Implementation Scheme 68. The method of any one of Implementation Schemes 59-67, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the entire cell mixture through the cell mechanical perforation filter within a time not exceeding 30 minutes.

[0274] Implementation Scheme 69. The method of any one of Implementation Schemes 59-68, wherein removably connecting the fluid inlet to the source of the cell mixture includes using a Luer connector, a threaded connector, or a quick-release connector.

[0275] Implementation Scheme 70. The method of any one of Implementation Schemes 59-68, wherein removably connecting the fluid inlet to the source of the cell mixture comprises using one or more tubes and Luer connectors, threaded connectors, quick-release connectors, or tubular weldments.

[0276] Implementation Scheme 71. The method of any one of Implementation Schemes 59-70, wherein detachably connecting the fluid outlet to the output reservoir includes using a Luer connector, a threaded connector, or a quick-release connector.

[0277] Implementation Scheme 72. The method of any one of Implementation Schemes 59-70, wherein detachably connecting the fluid outlet to the output reservoir comprises using one or more tubes and Luer connectors, threaded connectors, quick-release connectors, or tubular weldments.

[0278] Implementation Scheme 73. The method of any one of Implementation Schemes 59-70, wherein detachably connecting the fluid outlet to the output reservoir includes positioning the fluid outlet near an opening of the output reservoir.

[0279] Implementation Scheme 74. The method of any one of Implementation Schemes 59-73, wherein the first housing portion and the second housing portion are connected by welding, forging, threading, snap-fit ​​or press-fit.

[0280] Implementation Scheme 75. The method of any one of Implementation Schemes 59-74, wherein the cell mechanical perforation filter is replaceable.

[0281] Implementation Scheme 76. The method of any one of Implementation Schemes 59-75, the method comprising detachably connecting the first housing portion to the second housing portion.

[0282] Implementation Scheme 77. The method of any one of Implementation Schemes 59-76, wherein the housing is capable of being sterilized using at least one of gamma irradiation and chemical sterilization.

[0283] Implementation Scheme 78. The method of Implementation Scheme 77, wherein the housing is sterilizable when connected to at least one of the cell mixture source and the output reservoir.

[0284] Implementation Scheme 79. The method of any one of Implementation Schemes 59-78, wherein the outer surface of at least one of the first housing portion and the second housing portion includes a textured portion to connect the first housing portion to the cell mixture source, the second housing portion to the output reservoir, and at least one pair of the first housing portion and the second housing portion.

[0285] Implementation Scheme 80. The method of any one of Implementation Schemes 59-79, wherein the method comprises prefilling the cell mechanical perforation filter with a cell buffer before passing the cell mixture through the cell mechanical perforation filter.

[0286] Implementation Scheme 81. The method of any one of Implementation Schemes 59-80, wherein the cell mechanical perforation filter is configured to withstand pressures between at least 1 and 50 psi.

[0287] Implementation Scheme 82. The method of any one of Implementation Schemes 59-81, wherein the cell mixture comprises somatic cells, immortalized cells, stem cells or derivatives thereof.

[0288] Implementation Scheme 83. The method of any one of Implementation Schemes 59-82, wherein the cell mixture comprises peripheral blood mononuclear cells (PBMCs) or derivatives thereof.

[0289] Implementation Scheme 84. The method of any one of Implementation Schemes 59-83, wherein the cell mixture comprises immune cells.

[0290] Implementation Scheme 85. The method of any one of Implementation Schemes 59-84, wherein the cell mixture comprises T cells, natural killer (NK) cells, monocytes, B cells, or dendritic cells.

[0291] Implementation Scheme 86. The method of any one of Implementation Schemes 59-82, wherein the cell mixture comprises stem cells.

[0292] Implementation Scheme 87. The method of any one of Implementation Schemes 59-82 and 86, wherein the cell mixture comprises human stem cells.

[0293] Implementation Scheme 88. The method of any one of Implementation Schemes 59-82, 86 and 87, wherein the cell mixture comprises induced pluripotent stem cells, hematopoietic cells or mesenchymal cells.

[0294] Implementation Scheme 89. The method of any one of Implementation Schemes 59-88, wherein the cell mixture comprises cells obtained from or derived from an individual.

[0295] Implementation Scheme 90. The method as described in Implementation Scheme 89, wherein the individual is a person.

[0296] Implementation Scheme 91. The method of any one of Implementation Schemes 59-90, wherein the cell mixture comprises a payload.

[0297] Implementation Scheme 92. The method of Implementation Scheme 91, wherein the method comprises mixing the payload with the cell mixture before passing the cell mixture through the cell mechanical perforation filter.

[0298] Implementation Scheme 93. The method of implementation scheme 91 or 92, wherein the payload comprises at least one of peptides, nucleic acids, small molecules, nanoparticles and complexes thereof.

[0299] Implementation Scheme 94. The method of any one of Implementation Schemes 91-93, wherein the payload comprises more than one of peptides, nucleic acids, small molecules, nanoparticles and / or complexes thereof.

[0300] Implementation Scheme 95. The method of any one of Implementation Schemes 59-94, wherein the fluid inlet includes a tapered portion, the width of which increases from a proximal end of the fluid inlet to a distal end of the fluid inlet near the cellular mechanical perforation filter.

[0301] Implementation Scheme 96. The method of Implementation Scheme 95, wherein the fluid inlet comprises a circular cross-section and the width is the diameter of the fluid inlet.

[0302] Implementation Scheme 97. The method of any one of Implementation Schemes 59-96, wherein the fluid outlet comprises a tapered portion, the width of which decreases from a proximal end of the fluid outlet near the cellular mechanical perforation filter to a distal end of the fluid outlet.

[0303] Implementation Scheme 98. The method of Implementation Scheme 97, wherein the fluid outlet comprises a circular cross-section, and the width is the diameter of the fluid outlet.

[0304] Implementation Scheme 99. The method of any one of Implementation Schemes 59-98, wherein the housing includes a sealing member disposed in an end region of the first housing portion near the connected second housing portion.

[0305] Implementation Scheme 100. The method of any one of Implementation Schemes 59-99, wherein the second housing portion includes an end region adjacent to the connected first housing portion, the end region of the second housing portion holding the cell mechanical perforation filter.

[0306] Implementation Scheme 101. The method of any one of Implementation Schemes 59-98, wherein the housing includes a sealing member disposed in an end region of the second housing portion near the connected first housing portion.

[0307] Implementation Scheme 102. The method of any one of Implementation Schemes 59-98 and 101, wherein the first housing portion includes an end region adjacent to the connected second housing portion, the end region of the first housing portion holding the cell mechanical perforation filter.

[0308] Implementation Scheme 103. The method of any one of Implementation Schemes 59-102, wherein the cell mechanical perforation filter comprises a silicon filter or a polymer filter.

[0309] Implementation Scheme 104. The method of any one of Implementation Schemes 59-103, wherein the cell mechanical perforation filter comprises a silicon filter containing silicon, silicon oxide, silicon nitride and / or silicon carbide.

[0310] Implementation Scheme 105. The method of any one of Implementation Schemes 59-103, wherein the cell mechanical perforation filter comprises a polymer filter containing polycarbonate, polyester (PET), polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF and / or polytetrafluoroethylene.

[0311] Implementation Scheme 106. The method of any one of Implementation Schemes 59-105, wherein the cell mechanical perforation filter is coated with at least one of gold, silver, platinum, Teflon, polyvinylpyrrolidone, an adhesive, and a surfactant.

[0312] Implementation Scheme 107. The method of any one of Implementation Schemes 59-104 and 106, wherein the cell mechanical perforation filter is doped with boron, gallium or phosphorus.

[0313] Implementation Scheme 108. The method of any one of Implementation Schemes 59-107, wherein the cell mechanical perforation filter is configured to withstand pressures between at least 1 and 50 psi.

[0314] Implementation Scheme 109. The method of any one of Implementation Schemes 59-108, wherein the cell mechanical perforation filter comprises a filter surface and a plurality of pores extending through the filter surface, the plurality of pores being configured to agitate the cell membrane as the cell mixture passes through the plurality of pores.

[0315] Implementation Scheme 110. The method of implementation scheme 109, wherein the plurality of holes comprises no more than 300,000 holes.

[0316] Implementation Scheme 111. The method of implementation scheme 109 or 110, wherein the cell mechanical perforation filter includes a support structure disposed on one side of the filter surface.

[0317] Implementation Scheme 112. The method of Implementation Scheme 111, wherein the support structure covers at least 1% of the filter surface.

[0318] Implementation Scheme 113. The method of implementation scheme 111 or 112, wherein the support structure comprises a plurality of support members arranged on the filter surface and forming at least one cross or a plurality of strips.

[0319] Implementation Scheme 114. The method of any one of Implementation Schemes 111-113, wherein the thickness of the support structure is between 20 µm and 3 mm.

[0320] Implementation Scheme 115. The method of any one of Implementation Schemes 111-114, wherein the cell mechanical perforation filter comprises an oxide layer disposed on the filter surface between the filter surface and the support structure.

[0321] Implementation Scheme 116. The method of any one of Implementation Schemes 109-115, wherein each of the plurality of holes comprises a width between 2 µm and 20 µm.

[0322] Implementation Scheme 117. The method of Implementation Scheme 116, wherein the hole spacing between each of the plurality of holes is between 0.5:1 and 100:1 relative to the diameter of the hole.

[0323] Implementation Scheme 118. The method of any one of Implementation Schemes 109-117, wherein the thickness of the filter surface is between 0.1 µm and 100 µm.

[0324] Implementation Scheme 119. The method of any one of Implementation Schemes 109-118, wherein the width of the filter surface is between 1 mm and 10 cm.

[0325] Implementation Scheme 120. The method of Implementation Scheme 119, wherein the width of at least one of the fluid inlet and the fluid outlet is at least 50% of the width of the filter surface.

[0326] Implementation Scheme 121. The method of any one of Implementation Schemes 109-120, wherein at least one of the fluid inlet and the fluid outlet comprises a circular cross section corresponding to the circular shape of the filter surface.

[0327] Implementation Scheme 122. A method for assembling a cell mechanical perforation box, the method comprising: A first housing portion of the housing is connected to a second housing portion of the housing, the first housing portion including a fluid inlet and the second housing portion including a fluid outlet. The cell-mechanically perforated filter is maintained in fluid communication with the fluid inlet and the fluid outlet through the connected first housing portion and second housing portion.

[0328] Implementation Scheme 123. The method of implementation scheme 122, wherein connecting the first housing portion and the second housing portion includes detachably connecting the first housing portion and the second housing portion.

[0329] Implementation Scheme 124. The method as described in Implementation Scheme 122 or 123, wherein connecting the first housing portion and the second housing portion includes using a welded connection, a forged connection, a threaded connection, a snap-fit, or a press fit to connect the first housing portion and the second housing portion.

[0330] Implementation Scheme 125. The method of any one of Implementation Schemes 122-124, wherein the cell mechanical perforation filter is replaceable.

[0331] Implementation Scheme 126. The method of any one of Implementation Schemes 122-125, wherein the outer surface of at least one of the first housing portion and the second housing portion includes a textured portion to connect the first housing portion and the second housing portion.

[0332] Implementation Scheme 127. The method of any one of Implementation Schemes 122-126, the method comprising arranging a sealing member in an end region of the first housing portion near the connected second housing portion.

[0333] Implementation Scheme 128. The method of any one of Implementation Schemes 122-127, wherein the second housing portion includes an end region adjacent to the connected first housing portion, the end region of the second housing portion holding the cellular mechanical perforation filter.

[0334] Implementation Scheme 129. The method of any one of Implementation Schemes 122-126, the method comprising arranging a sealing member in an end region of the second housing portion near the connected first housing portion.

[0335] Implementation Scheme 130. The method of any one of Implementation Schemes 122-126 or 129, wherein the first housing portion includes an end region adjacent to the connected second housing portion, the end region of the first housing portion holding the cellular mechanical perforation filter.

[0336] Implementation Scheme 131. The method of any one of Implementation Schemes 122-130, wherein the housing is removably connected to at least one pair of the fluid inlet and the cell mixture source and the fluid outlet and the output reservoir by a housing receiving portion of the fluid delivery system.

[0337] Implementation Scheme 132. The method of implementation scheme 131, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

[0338] Implementation Scheme 133. The method as described in Implementation Scheme 131 or 132, wherein the fluid inlet is detachably connected to the cell mixture source using a Luer connector, threaded connector, or quick-release connector.

[0339] Implementation Scheme 134. The method as described in Implementation Scheme 131 or 132, wherein the fluid inlet is detachably connected to the cell mixture source via one or more tubes using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

[0340] Implementation Scheme 135. The method of any one of Implementation Schemes 131-134, wherein the fluid outlet is detachably connected to the output reservoir using a Luer connector, threaded connector or quick-release connector.

[0341] Implementation Scheme 136. The method of any one of Implementation Schemes 131-134, wherein the fluid outlet is detachably connected to the output reservoir via one or more pipes using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

[0342] Implementation Scheme 137. The method of any one of Implementation Schemes 131-134, wherein the fluid outlet is detachably connected to the output reservoir by arranging the fluid outlet at an opening near the output reservoir.

[0343] Implementation Scheme 138. The method of any one of Implementation Schemes 122-137, wherein the cell mechanical perforation box is sterilizable.

[0344] Implementation Scheme 139. The method of Implementation Scheme 138, wherein the cell mechanical perforation cassette is sterilizable when connected to at least one of the cell mixture source and the output reservoir.

[0345] Implementation Scheme 140. The method of any one of Implementation Schemes 122-139, wherein the cell mechanical perforation filter comprises a silicon filter or a polymer filter.

[0346] Implementation Scheme 141. The method of any one of Implementation Schemes 122-140, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

[0347] Implementation Scheme 142. The method of any one of Implementation Schemes 122-141, wherein the cell mechanical perforation filter is configured to operate at a rate of 0.5-500 mL / min / mm 2 The volumetric flow rate between porous surfaces disturbs the cell membrane.

[0348] Implementation Scheme 143. The method of any one of Implementation Schemes 122-142, wherein the cell mechanical perforation filter is configured such that the perturbation is contained in 1.0 x 10 8 With 1.0x10 12 The cell membrane of a mixture of cells between cells.

[0349] Implementation Scheme 144. The method of any one of Implementation Schemes 122-143, wherein the housing is configured to receive a volume between 1 and 500 mL.

[0350] Implementation Scheme 145. The method of any one of Implementation Schemes 122-144, wherein the housing is configured to pass the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

[0351] Implementation Scheme 146. The method of any one of Implementation Schemes 122-145, wherein the housing is configured to pass the entire cell mixture through the cell mechanical perforation filter within a time not exceeding 30 minutes.

[0352] Implementation Scheme 147. The method of any one of Implementation Schemes 122-146, wherein the housing is configured to receive a constant volumetric flow rate controlled by at least one of a peristaltic pump, an injection pump, and a pressurized reservoir.

[0353] Implementation Scheme 148. The method of any one of Implementation Schemes 122-147, wherein the cell mechanical perforation filter is configured to withstand pressures between at least 1 and 50 psi.

[0354] Implementation Scheme 149. The method of any one of Implementation Schemes 122-148, wherein the cell mechanical perforation filter comprises a filter surface and a plurality of pores extending through the filter surface, the plurality of pores being configured to agitate the cell membrane as the cell mixture passes through the plurality of pores.

[0355] Implementation Scheme 150. The method of implementation scheme 149, wherein the plurality of holes comprises no more than 300,000 holes.

[0356] Implementation Scheme 151. The method of implementation scheme 149 or 150, wherein the cell mechanical perforation filter includes a support structure disposed on one side of the filter surface.

[0357] Implementation Scheme 152. The method of Implementation Scheme 151, wherein the support structure covers at least 1% of the filter surface.

[0358] Implementation Scheme 153. The method as described in Implementation Scheme 151 or 152, wherein the thickness of the support structure is between 20 µm and 3 mm.

[0359] Implementation Scheme 154. The method of any one of Implementation Schemes 149-153, wherein each of the plurality of holes comprises a width between 2 µm and 20 µm.

[0360] Implementation Scheme 155. The method of implementation scheme 154, wherein the hole spacing between each of the plurality of holes is between 0.5:1 and 100:1 relative to the diameter of the hole.

[0361] Implementation Scheme 156. The method as described in Implementation Schemes 149-155, wherein the thickness of the filter surface is between 0.1 µm and 100 µm.

[0362] Implementation Scheme 157. The method of any one of Implementation Schemes 149-156, wherein the width of the filter surface is between 1 mm and 10 cm.

[0363] Implementation Scheme 158. The method of Implementation Scheme 157, wherein the width of at least one of the fluid inlet and the fluid outlet is at least 50% of the width of the filter surface.

[0364] Implementation Scheme 159. The method of any one of Implementation Schemes 149-158, wherein at least one of the fluid inlet and the fluid outlet comprises a circular cross-section corresponding to the circular shape of the filter surface.

[0365] Implementation Scheme 160. A cell mechanical perforation system, the cell mechanical perforation system comprising: The box body includes: A first housing portion includes a fluid inlet detachably connected to a source of the cell mixture; A second housing portion connected to the first housing portion, the second housing portion including a fluid outlet; and A cell mechanically perforated filter, the cell mechanically perforated filter being held by a connected first housing portion and a second housing portion and configured to agitate the cell membranes of a cell mixture from which the cell mixture originates; An output memory, the output memory being detachably connected to a second housing portion of the housing; and A pump configured to operate at a rate of at least 0.5 mL / min / mm 2 The volumetric flow rate of the porous surface area causes the cell mixture to move through the cell mechanical perforation filter.

[0366] Implementation Scheme 161. The system as described in Implementation Scheme 160, wherein the cell mechanical perforation system is capable of operating at a constant volumetric flow rate controlled by at least one of the pump and the pressurized reservoir.

[0367] Implementation Scheme 162. The system of implementation scheme 160 or 161, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

[0368] Implementation Scheme 163. The system of any one of Implementation Schemes 160-162, wherein the cell mechanical perforation filter is configured to operate at a rate of 0.5-500 mL / min / mm 2 The volumetric flow rate between porous surfaces disturbs the cell membrane.

[0369] Implementation Scheme 164. The system of any one of Implementation Schemes 160-163, wherein the cellular mechanical perforation filter is configured such that the perturbation is contained in 1.0 x 10 8 With 1.0x10 12 The cell membrane of a mixture of cells between cells.

[0370] Implementation Scheme 165. The system of any one of Implementation Schemes 160-164, wherein the housing is configured to receive a volume between 1 and 500 mL.

[0371] Implementation Scheme 166. The system of any one of Implementation Schemes 160-165, wherein the housing is configured to pass the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

[0372] Implementation Scheme 167. The system of any one of Implementation Schemes 160-166, wherein the housing is configured to pass the entire cell mixture through the cell mechanical perforation filter within no more than 30 minutes.

[0373] Implementation Scheme 168. The system of any one of Implementation Schemes 160-162, wherein the pump comprises a peristaltic pump or a syringe pump.

[0374] Implementation Scheme 169. The system of any one of Implementation Schemes 160-168, wherein the housing is removably connected by a housing receiving portion of a fluid delivery system to at least one pair of the fluid inlet and the cell mixture source and the fluid outlet and the output reservoir.

[0375] Implementation Scheme 170. The system of any one of Implementation Schemes 160-167 and 169, wherein the pump is a fluid delivery system.

[0376] Implementation Scheme 171. The system as described in Implementation Scheme 169 or 170, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

[0377] Implementation Scheme 172. The system of any one of Implementation Schemes 160-171, wherein the system comprises the source of the cell mixture.

[0378] Implementation Scheme 173. The system of any one of Implementation Schemes 160-172, wherein the system includes a payload source capable of fluid connection with the housing and containing a payload.

[0379] Implementation Scheme 174. The system of Implementation Scheme 173, wherein the payload source is fluidly connected to the cell mixture source to receive the cell mixture from the cell mixture source.

[0380] Implementation Scheme 175. The system of any one of Implementation Schemes 160-172, wherein the cell mixture source comprises a payload.

[0381] Implementation Scheme 176. The system of any one of Implementation Schemes 173-175, wherein the payload comprises at least one of peptides, nucleic acids, small molecules, nanoparticles and complexes thereof.

[0382] Implementation Scheme 177. The system of any one of Implementation Schemes 173-176, wherein the payload comprises more than one of peptides, nucleic acids, small molecules, nanoparticles and / or complexes thereof.

[0383] Implementation Scheme 178. The system of any one of Implementation Schemes 160-177, wherein the cell mixture comprises somatic cells, immortalized cells, stem cells or derivatives thereof.

[0384] Implementation Scheme 179. The system of any one of Implementation Schemes 160-178, wherein the cell mixture comprises peripheral blood mononuclear cells (PBMCs) or derivatives thereof.

[0385] Implementation Scheme 180. The system of any one of Implementation Schemes 160-179, wherein the cell mixture comprises immune cells.

[0386] Implementation Scheme 181. The system of any one of Implementation Schemes 160-180, wherein the cell mixture comprises T cells, NK cells, monocytes, B cells or dendritic cells.

[0387] Implementation Scheme 182. The system of any one of Implementation Schemes 160-178, wherein the cell mixture comprises stem cells.

[0388] Implementation Scheme 183. The system of any one of Implementation Schemes 160-178 and 182, wherein the cell mixture comprises human stem cells.

[0389] Implementation Scheme 184. The system of any one of Implementation Schemes 160-178, 182 and 183, wherein the cell mixture comprises induced pluripotent stem cells, hematopoietic cells or mesenchymal cells.

[0390] Implementation Scheme 185. The system of any one of Implementation Schemes 160-184, wherein the cell mixture comprises cells obtained from or derived from an individual.

[0391] Implementation Scheme 186. The system as described in Implementation Scheme 185, wherein the individual is a person.

[0392] Implementation Scheme 187. The system of any one of Implementation Schemes 160-186, wherein the fluid inlet is detachably connected to the cell mixture source using a Luer connector, threaded connector or quick-release connector.

[0393] Implementation Scheme 188. The system of any one of Implementation Schemes 160-186, wherein the fluid inlet is detachably connected to the cell mixture source via one or more tubes using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

[0394] Implementation Scheme 189. The system of any one of Implementation Schemes 160-188, wherein the fluid outlet is detachably connected to the output reservoir using a Luer connector, threaded connector or quick-release connector.

[0395] Implementation Scheme 190. The system of any one of Implementation Schemes 160-188, wherein the fluid outlet is detachably connected to the output reservoir via one or more pipes using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

[0396] Implementation Scheme 191. The system of any one of Implementation Schemes 160-188, wherein the fluid outlet is detachably connected to the output reservoir by arranging the fluid outlet at an opening near the output reservoir.

[0397] Implementation Scheme 192. The system of any one of Implementation Schemes 160-191, wherein the first housing portion and the second housing portion are connected by welding, forging, threading, snap-fit ​​or press-fit.

[0398] Implementation Scheme 193. The system of any one of Implementation Schemes 160-192, wherein the first housing portion and the second housing portion are detachably connected.

[0399] Implementation Scheme 194. The system of any one of Implementation Schemes 160-193, wherein the cell mechanical perforation filter is replaceable.

[0400] Implementation Scheme 195. The system of any one of Implementation Schemes 160-194, wherein the housing is capable of being sterilized by at least one of gamma irradiation and chemical sterilization.

[0401] Implementation Scheme 196. The system of Implementation Scheme 195, wherein the housing is sterilizable when connected to at least one of the cell mixture source and the output reservoir.

[0402] Implementation Scheme 197. The system of any one of Implementation Schemes 160-196, wherein the fluid inlet includes a tapered portion, the width of which increases from a proximal end of the fluid inlet to a distal end of the fluid inlet near the cellular mechanical perforation filter.

[0403] Implementation Scheme 198. The system of Implementation Scheme 197, wherein the fluid inlet comprises a circular cross-section and the width is the diameter of the fluid inlet.

[0404] Implementation Scheme 199. The system of any one of Implementation Schemes 160-198, wherein the fluid outlet includes a tapered portion, the width of which decreases from a proximal end of the fluid outlet near the cellular mechanical perforation filter to a distal end of the fluid outlet.

[0405] Implementation Scheme 200. The system of Implementation Scheme 199, wherein the fluid outlet has a circular cross-section and the width is the diameter of the fluid outlet.

[0406] Implementation Scheme 201. The system of any one of Implementation Schemes 160-200, wherein the housing includes a sealing member disposed in an end region of the first housing portion near the connected second housing portion.

[0407] Implementation Scheme 202. The system of any one of Implementation Schemes 160-201, wherein the second housing portion includes an end region adjacent to the connected first housing portion, the end region of the second housing portion holding the cellular mechanical perforation filter.

[0408] Implementation Scheme 203. The system of any one of Implementation Schemes 160-200, wherein the housing includes a sealing member disposed in an end region of the second housing portion near the connected first housing portion.

[0409] Implementation Scheme 204. The system of any one of Implementation Schemes 160-200 and 203, wherein the first housing portion includes an end region adjacent to the connected second housing portion, the end region of the first housing portion holding the cellular mechanical perforation filter.

[0410] Implementation Scheme 205. The system of any one of Implementation Schemes 160-204, wherein the outer surface of at least one of the first housing portion and the second housing portion includes a textured portion, the textured portion being configured to facilitate at least one of the following: connection of the first housing portion to the cell mixture source, connection of the second housing portion to the output reservoir, and connection of the first housing portion to the second housing portion.

[0411] Implementation Scheme 206. The system of any one of Implementation Schemes 160-205, wherein the cell mechanical perforation filter is configured to withstand pressures between at least 1 and 50 psi.

[0412] Implementation Scheme 207. The system of any one of Implementation Schemes 160-206, wherein the cell mechanical perforation filter comprises a silicon filter or a polymer filter.

[0413] Implementation Scheme 208. The system of any one of Implementation Schemes 160-207, wherein the cell mechanical perforation filter comprises a silicon filter containing silicon, silicon oxide, silicon nitride and / or silicon carbide.

[0414] Implementation Scheme 209. The system of any one of Implementation Schemes 160-207, wherein the cell mechanical perforation filter comprises a polymer filter containing polycarbonate, polyester (PET), polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF and / or polytetrafluoroethylene.

[0415] Implementation Scheme 210. The system of any one of Implementation Schemes 160-209, wherein the cell mechanical perforation filter is coated with at least one of gold, silver, platinum, Teflon, polyvinylpyrrolidone, adhesive and surfactant.

[0416] Implementation Scheme 211. The system of any one of Implementation Schemes 160-206 and 208, wherein the cellular mechanical perforation filter is doped with boron, gallium or phosphorus.

[0417] Implementation Scheme 212. The system of any one of Implementation Schemes 160-211, wherein the cell mechanical perforation filter comprises a filter surface and a plurality of pores extending through the filter surface, the plurality of pores being configured to agitate the cell membrane as the cell mixture passes through the plurality of pores.

[0418] Implementation Scheme 213. The system as described in Implementation Scheme 212, wherein the plurality of holes comprises no more than 300,000 holes.

[0419] Implementation Scheme 214. The system as described in Implementation Scheme 212 or 213, wherein the cell mechanical perforation filter includes a support structure disposed on one side of the filter surface.

[0420] Implementation Scheme 215. The system as described in Implementation Scheme 214, wherein the support structure covers at least 1% of the filter surface.

[0421] Implementation Scheme 216. The system as described in Implementation Scheme 214 or 215, wherein the support structure comprises a plurality of support members arranged on the filter surface and forming at least one cross or a plurality of strips.

[0422] Implementation Scheme 217. The system of any one of Implementation Schemes 214-216, wherein the thickness of the support structure is between 20 µm and 3 mm.

[0423] Implementation Scheme 218. The system of any one of Implementation Schemes 214-217, wherein the cell mechanical perforation filter comprises an oxide layer disposed on the filter surface between the filter surface and the support structure.

[0424] Implementation Scheme 219. The system of any one of Implementation Schemes 212-218, wherein each of the plurality of holes contains a width between 2 µm and 20 µm.

[0425] Implementation Scheme 220. The system of Implementation Scheme 219, wherein the hole spacing between each of the plurality of holes is between 0.5:1 and 100:1 relative to the diameter of the hole.

[0426] Implementation Scheme 221. The system of any one of Implementation Schemes 212-220, wherein the thickness of the filter surface is between 0.1 µm and 100 µm.

[0427] Implementation Scheme 222. The system of any one of Implementation Schemes 212-221, wherein the width of the filter surface is between 1 mm and 10 cm.

[0428] Implementation Scheme 223. The system of Implementation Scheme 222, wherein the width of at least one of the fluid inlet and the fluid outlet is at least 50% of the width of the filter surface.

[0429] Implementation Scheme 224. The system of any one of Implementation Schemes 212-223, wherein at least one of the fluid inlet and the fluid outlet comprises a circular cross-section corresponding to the circular shape of the filter surface.

[0430] Implementation Scheme 225. A method for perturbing a cell membrane, the method comprising: The fluid inlet of a first housing portion of the housing is detachably connected to a cell mixture source, the housing including a second housing portion connected to the first housing portion, and the second housing portion including a fluid outlet, wherein the connected first housing portion and the second housing portion retain a cell mechanical perforation filter; The fluid outlet is detachably connected to the output reservoir; A cell mixture from the cell mixture source is received at the fluid inlet, the cell mixture containing at least 1.0 x 10⁻⁶ cells / mL. 8 One cell; Passing the cell mixture through the cell mechanical perforation filter to agitate the cell membranes of the cell mixture; and The disturbed cellular mixture is delivered to the output reservoir via the fluid outlet.

[0431] Implementation Scheme 226. The method of Implementation Scheme 225, wherein removably connecting the fluid inlet to the cell mixture source and / or removably connecting the fluid outlet to the output reservoir comprises placing the housing in the housing receiving portion of the fluid delivery system.

[0432] Implementation Scheme 227. The method of Implementation Scheme 226, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

[0433] Implementation Scheme 228. The method of any one of Implementation Schemes 225-227, wherein the cell mixture is passed through the cell mechanical perforation filter at a rate of 0.5-500 mL / min / mm. 2 The volumetric flow rate occurs between porous surfaces.

[0434] Implementation Scheme 229. The method of any one of Implementation Schemes 225-228, wherein the cell mixture is passed through the cell mechanical perforation filter at a constant volumetric flow rate controlled by at least one of a peristaltic pump, an injection pump, and a pressurized reservoir.

[0435] Implementation Scheme 230. The method of any one of Implementation Schemes 225-228, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

[0436] Implementation Scheme 231. The method of any one of Implementation Schemes 225-230, wherein the cell mixture comprises 1.0 x 10 8 With 1.0x10 12 Between the cells.

[0437] Implementation Scheme 232. The method of any one of Implementation Schemes 225-231, wherein the cell mixture has a volume between 1 and 500 mL.

[0438] Implementation Scheme 233. The method of any one of Implementation Schemes 225-232, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

[0439] Implementation Scheme 234. The method of any one of Implementation Schemes 225-233, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the entire cell mixture through the cell mechanical perforation filter within a time not exceeding 30 minutes.

[0440] Implementation Scheme 235. The method of any one of Implementation Schemes 225-234, wherein detachably connecting the fluid inlet to the cell mixture source includes using a Luer connector, a threaded connector, or a quick-release connector.

[0441] Implementation Scheme 236. The method of any one of Implementation Schemes 225-234, wherein removably connecting the fluid inlet to the source of the cell mixture comprises using one or more tubes and Luer connectors, threaded connectors, quick-release connectors, or tubular weldments.

[0442] Implementation Scheme 237. The method of any one of Implementation Schemes 225-236, wherein detachably connecting the fluid outlet to the output reservoir includes using a Luer connector, a threaded connector, or a quick-release connector.

[0443] Implementation Scheme 238. The method of any one of Implementation Schemes 225-236, wherein detachably connecting the fluid outlet to the output reservoir comprises using one or more tubes and Luer connectors, threaded connectors, quick-release connectors, or tubular weldments.

[0444] Implementation Scheme 239. The method of any one of Implementation Schemes 225-236, wherein detachably connecting the fluid outlet to the output reservoir includes positioning the fluid outlet near an opening of the output reservoir.

[0445] Implementation Scheme 240. The method of any one of Implementation Schemes 225-239, wherein the first housing portion and the second housing portion are connected by welding, forging, threading, snap-fit ​​or press-fit.

[0446] Implementation Scheme 241. The method of any one of Implementation Schemes 225-240, wherein the cell mechanical perforation filter is replaceable.

[0447] Implementation Scheme 242. The method of any one of Implementation Schemes 225-241, the method comprising detachably connecting the first housing portion to the second housing portion.

[0448] Implementation Scheme 243. The method of any one of Implementation Schemes 225-242, wherein the housing is capable of being sterilized by at least one of gamma irradiation and chemical sterilization.

[0449] Implementation Scheme 244. The method of Implementation Scheme 243, wherein the housing is sterilizable when connected to at least one of the cell mixture source and the output reservoir.

[0450] Implementation Scheme 245. The method of any one of Implementation Schemes 225-244, wherein the outer surface of at least one of the first housing portion and the second housing portion includes a textured portion to connect the first housing portion to the cell mixture source, the second housing portion to the output reservoir, and at least one pair of the first housing portion and the second housing portion.

[0451] Implementation Scheme 246. The method of any one of Implementation Schemes 225-245, wherein the method comprises prefilling the cell mechanical perforation filter with a cell buffer before passing the cell mixture through the cell mechanical perforation filter.

[0452] Implementation Scheme 247. The method of any one of Implementation Schemes 225-246, wherein the cell mechanical perforation filter is configured to withstand pressures between at least 1 and 50 psi.

[0453] Implementation Scheme 248. The method of any one of Implementation Schemes 225-247, wherein the cell mixture comprises somatic cells, immortalized cells, stem cells or derivatives thereof.

[0454] Implementation Scheme 249. The method of any one of Implementation Schemes 225-248, wherein the cell mixture comprises peripheral blood mononuclear cells (PBMCs) or derivatives thereof.

[0455] Implementation Scheme 250. The method of any one of Implementation Schemes 225-249, wherein the cell mixture comprises immune cells.

[0456] Implementation Scheme 251. The method of any one of Implementation Schemes 225-250, wherein the cell mixture comprises T cells, natural killer (NK) cells, monocytes, B cells or dendritic cells.

[0457] Implementation Scheme 252. The method of any one of Implementation Schemes 225-248, wherein the cell mixture comprises stem cells.

[0458] Implementation Scheme 253. The method of any one of Implementation Schemes 225-248 and 252, wherein the cell mixture comprises human stem cells.

[0459] Implementation Scheme 254. The method of any one of Implementation Schemes 225-248, 252 and 253, wherein the cell mixture comprises induced pluripotent stem cells, hematopoietic cells or mesenchymal cells.

[0460] Implementation Scheme 255. The method of any one of Implementation Schemes 225-254, wherein the cell mixture comprises cells obtained from or derived from an individual.

[0461] Implementation Scheme 256. The method as described in Implementation Scheme 255, wherein the individual is a person.

[0462] Implementation Scheme 257. The method of any one of Implementation Schemes 225-256, wherein the cell mixture comprises a payload.

[0463] Implementation Scheme 258. The method of Implementation Scheme 257, wherein the method includes mixing the payload with the cell mixture before passing the cell mixture through the cell mechanical perforation filter.

[0464] Implementation Scheme 259. The method of implementation scheme 257 or 258, wherein the payload comprises at least one of peptides, nucleic acids, small molecules, nanoparticles and complexes thereof.

[0465] Implementation Scheme 260. The method of any one of Implementation Schemes 257-259, wherein the payload comprises more than one of peptides, nucleic acids, small molecules, nanoparticles and / or complexes thereof.

[0466] Implementation Scheme 261. The method of any one of Implementation Schemes 225-260, wherein the fluid inlet comprises a tapered portion, the width of which increases from a proximal end of the fluid inlet to a distal end of the fluid inlet near the cellular mechanical perforation filter.

[0467] Implementation Scheme 262. The method of Implementation Scheme 261, wherein the fluid inlet comprises a circular cross section and the width is the diameter of the fluid inlet.

[0468] Implementation Scheme 263. The method of any one of Implementation Schemes 225-262, wherein the fluid outlet comprises a tapered portion, the width of which decreases from a proximal end of the fluid outlet near the cellular mechanical perforation filter to a distal end of the fluid outlet.

[0469] Implementation Scheme 264. The method of Implementation Scheme 263, wherein the fluid outlet comprises a circular cross-section and the width is the diameter of the fluid outlet.

[0470] Implementation Scheme 265. The method of any one of Implementation Schemes 225-264, wherein the housing includes a sealing member disposed in an end region of the first housing portion near the connected second housing portion.

[0471] Implementation Scheme 266. The method of any one of Implementation Schemes 225-265, wherein the second housing portion includes an end region adjacent to the connected first housing portion, the end region of the second housing portion holding the cell mechanical perforation filter.

[0472] Implementation Scheme 267. The method of any one of Implementation Schemes 225-264, wherein the housing includes a sealing member disposed in an end region of the second housing portion near the connected first housing portion.

[0473] Implementation Scheme 268. The method of any one of Implementation Schemes 225-264 and 267, wherein the first housing portion includes an end region adjacent to the connected second housing portion, the end region of the first housing portion holding the cell mechanical perforation filter.

[0474] Implementation Scheme 269. The method of any one of Implementation Schemes 225-268, wherein the cell mechanical perforation filter comprises a silicon filter or a polymer filter.

[0475] Implementation Scheme 270. The method of any one of Implementation Schemes 225-269, wherein the cell mechanical perforation filter comprises a silicon filter containing silicon, silicon oxide, silicon nitride and / or silicon carbide.

[0476] Implementation Scheme 271. The method of any one of Implementation Schemes 225-269, wherein the cell mechanical perforation filter comprises a polymer filter containing polycarbonate, polyester (PET), polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF and / or polytetrafluoroethylene.

[0477] Implementation Scheme 272. The method of any one of Implementation Schemes 225-271, wherein the cell mechanical perforation filter is coated with at least one of gold, silver, platinum, Teflon, polyvinylpyrrolidone, adhesive and surfactant.

[0478] Implementation Scheme 273. The method of any one of Implementation Schemes 225-268 and 270, wherein the cell mechanical perforation filter is doped with boron, gallium or phosphorus.

[0479] Implementation Scheme 274. The method of any one of Implementation Schemes 225-273, wherein the cell mechanical perforation filter is configured to withstand pressures between at least 1 and 50 psi.

[0480] Implementation Scheme 275. The method of any one of Implementation Schemes 225-274, wherein the cell mechanical perforation filter comprises a filter surface and a plurality of pores extending through the filter surface, the plurality of pores being configured to agitate the cell membrane as the cell mixture passes through the plurality of pores.

[0481] Implementation Scheme 276. The method of implementation scheme 275, wherein the plurality of holes comprises no more than 300,000 holes.

[0482] Implementation Scheme 277. The method of implementation scheme 275 or 276, wherein the cell mechanical perforation filter includes a support structure disposed on one side of the filter surface.

[0483] Implementation Scheme 278. The method of Implementation Scheme 277, wherein the support structure covers at least 1% of the filter surface.

[0484] Implementation Scheme 279. The method of implementation scheme 277 or 278, wherein the support structure comprises a plurality of support members arranged on the filter surface and forming at least one cross or a plurality of strips.

[0485] Implementation Scheme 280. The method of any one of Implementation Schemes 277-279, wherein the thickness of the support structure is between 20 µm and 3 mm.

[0486] Implementation Scheme 281. The method of any one of Implementation Schemes 277-280, wherein the cell mechanical perforation filter comprises an oxide layer disposed on the filter surface between the filter surface and the support structure.

[0487] Implementation Scheme 282. The method of any one of Implementation Schemes 275-281, wherein each of the plurality of holes comprises a width between 2 µm and 20 µm.

[0488] Implementation Scheme 283. The method of implementation scheme 282, wherein the hole spacing between each of the plurality of holes is between 0.5:1 and 100:1 relative to the diameter of the hole.

[0489] Implementation Scheme 284. The method of any one of Implementation Schemes 275-283, wherein the thickness of the filter surface is between 0.1 µm and 100 µm.

[0490] Implementation Scheme 285. The method of any one of Implementation Schemes 275-284, wherein the width of the filter surface is between 1 mm and 10 cm.

[0491] Implementation Scheme 286. The method of implementation scheme 285, wherein the width of at least one of the fluid inlet and the fluid outlet is at least 50% of the width of the filter surface.

[0492] Implementation Scheme 287. The method of any one of Implementation Schemes 275-286, wherein at least one of the fluid inlet and the fluid outlet comprises a circular cross section corresponding to the circular shape of the filter surface.

[0493] Implementation Scheme 288. The housing as described in any one of Implementation Schemes 25-28, wherein the sealing member comprises at least one of the following: a Shore hardness between 25 and 75 A, an inner diameter between 5 and 10 mm, and a cross-section between 2 and 8 mm.

[0494] Implementation Scheme 289. The housing as described in any one of Implementation Schemes 25-28 and 288, wherein the first housing portion and the second housing portion are configured to compress the sealing member at a compression ratio between 20% and 50%.

[0495] Implementation Scheme 290. The method of any one of Implementation Schemes 99-102, wherein the sealing member comprises at least one of the following: a Shore hardness between 25 and 75 A, an inner diameter between 5 and 10 mm, and a cross-section between 2 and 8 mm.

[0496] Implementation Scheme 291. The method of any one of Implementation Schemes 99-102 and 290, wherein the first housing portion and the second housing portion are configured to compress the sealing member at a compression ratio between 20% and 50%.

[0497] Implementation Scheme 292. The method of any one of Implementation Schemes 127-130, wherein the sealing member comprises at least one of the following: a Shore hardness between 25 and 75 A, an inner diameter between 5 and 10 mm, and a cross-section between 2 and 8 mm.

[0498] Implementation Scheme 293. The method of any one of Implementation Schemes 127-130 and 292, wherein the first housing portion and the second housing portion are configured to compress the sealing member at a compression ratio between 20% and 50%.

[0499] Implementation Scheme 294. The system of any one of Implementation Schemes 201-204, wherein the sealing member comprises at least one of the following: a Shore hardness between 25 and 75 A, an inner diameter between 5 and 10 mm, and a cross-section between 2 and 8 mm.

[0500] Implementation Scheme 295. The system of any one of Implementation Schemes 201-204 and 294, wherein the first housing portion and the second housing portion are configured to compress the sealing member at a compression ratio between 20% and 50%.

[0501] Implementation Scheme 296. The method of any one of Implementation Schemes 265-268, wherein the sealing member comprises at least one of the following: a Shore hardness between 25 and 75 A, an inner diameter between 5 and 10 mm, and a cross-section between 2 and 8 mm.

[0502] Implementation Scheme 297. The method of any one of Implementation Schemes 265-268 and 296, wherein the first housing portion and the second housing portion are configured to compress the sealing member at a compression ratio between 20% and 50%.

[0503] Implementation Scheme 298. The housing as described in any one of Implementation Schemes 5-48, 288 and 289, wherein the volume is such that the residence time of the cell mixture after passing through the cell mechanical perforation filter is less than 30 seconds.

[0504] Implementation Scheme 299. The method of any one of Implementation Schemes 66-121, 290 and 291, wherein the volume is such that the residence time of the cell mixture after passing through the cell mechanical perforation filter is less than 30 seconds.

[0505] Implementation scheme 300. The method of any one of embodiments 144-159, 292 and 293, wherein the volume is such that the residence time of the cell mixture after passing through the cell mechanical perforation filter is less than 30 seconds.

[0506] Implementation scheme 301. The system of any one of embodiments 165-224, 294 and 295, wherein the volume is such that the residence time of the cell mixture after passing through the cell mechanical perforation filter is less than 30 seconds.

[0507] Implementation scheme 302. The method of any one of embodiments 232-287, 296 and 297, wherein the volume is such that the residence time of the cell mixture after passing through the cell mechanical perforation filter is less than 30 seconds.

[0508] Implementation scheme 303. The housing as described in any one of embodiments 1-48, 288, 289 and 298, wherein the pressure gradient across the cellular mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

[0509] Implementation scheme 304. The method of any one of embodiments 59-121, 290, 291 and 299, wherein the pressure gradient across the cell mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

[0510] Implementation scheme 305. The method of any one of implementation schemes 142-159, 292, 293 and 300, wherein the pressure gradient across the cell mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

[0511] Implementation scheme 306. The system of any one of implementation schemes 160-224, 294, 295 and 301, wherein the pressure gradient across the cell mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

[0512] Implementation scheme 307. The method of any one of implementation schemes 228-287, 296, 297 and 302, wherein the pressure gradient across the cell mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

[0513] Example Experiments demonstrating the effectiveness of a cell-mechanically perforated filter are conducted and described below. The cell-mechanically perforated filter is housed within the casing described herein and integrated into various cell processing systems or desktop research-scale systems.

[0514] Examples 1-4: The following setup is used to test the housing of the retaining filter in a closed system. The cell mixture source fluid is connected to the cell processing system described herein, such as Thermo Fisher Rotea, Fresenius Kabi Cue®, and Fresenius Kabi Lovo®. The cell processing system is fluidly connected to the housing (e.g., housing 300) that houses the cell mechanically perforated filter. The cell mechanically perforated filter in these embodiments has a 13 mm diameter. 2 Polymer track etching filter with large surface area. Connect the housing fluid to the output reservoir.

[0515] For the experiment, ¼ leukopak sample was recovered and PBMCs were isolated from the sample using Ficoll separation. The isolated PBMCs were fed to a cell processing system, where they were mixed with a payload to produce a cell mixture. The payload used in this experiment was dextran (0.1 mg / mL 3 kDa or 10 kDa Cascade Blue dextran). Dextran is a fluorescently labeled sugar polymer that can serve as a proxy for payloads such as mRNA, CRISPR RNP, etc., due to its similar delivery behavior. Measuring dextran delivery can represent the delivery efficiency of different payload types. The concentration of the cell mixture from the cell processing system was 20 x 10⁻⁶. 6 Cells / mL. The cell processing system delivers the cell mixture to the cartridge and through a cell mechanical perforation filter to agitate the cell membrane and facilitate intracellular delivery of the payload into the membrane. The agitated cell mixture (10 mL) is collected in an output reservoir. Flow cytometry is used to collect results, including cell viability and dextran delivery. These results are described below with reference to several examples. In the examples, different flow rates and cell processing systems were tested, and are summarized in more detail for each example.

[0516] Results of Example 1: Figures 8A-8C This demonstrates the results of testing the housing and the filters held within it when integrated with Thermo Fisher's Rotea®. Rotea® should be understood as representing a cell processing system that can be operated using a peristaltic pump and / or centrifuge mechanism. Figures 8A-8CThe samples tested included “non-contact” (NC) samples, “endocytosis” (endo) samples, and samples with four different flow rates. NC samples served as controls in the experiment, in which no payload was introduced. Endo samples were those in which cells were exposed to the payload but the cell mixture was not allowed to pass through the chamber. The four flow rates tested were: (1) 25 mL / min, (2) 50 mL / min, (3) 75 mL / min, and (4) 100 mL / min. For each flow rate tested, cell viability, enhancement efficiency, and dextran delivery were measured at the start and end of collection.

[0517] Figure 8A The cell viability of PBMCs passed through the chamber (and filter) at a flow rate between 25 and 50 mL / min is shown. Figure 8A As shown, all samples tested at various flow rates ranging from 25 mL / min to 100 mL / min exhibited high cell viability, exceeding 75%, and showed minimal variability between initial and final viability readings for each sample (the difference between initial and final viability readings for each sample was between approximately 0% and less than 5%). For example, cell viability was approximately 80%, 82%, or approximately 85% at different flow rates, depending on the flow rate. A flow rate of 25 mL / min showed the highest cell viability at approximately 85%. Results at all flow rates showed minimal variability (the difference was between approximately 0-5% at the different flow rates tested).

[0518] Figure 8B The enhanced efficiency of PBMCs at a flow rate between 25 and 50 mL / min through the housing (and filter) is shown. Figure 8C This indicates the presence of 3 kDa glucan in PBMCs. Figure 8B and Figure 8C As shown, the fortification efficiency and the presence of dextran demonstrate high delivery efficiency from payload to cargo. Fortification efficiencies ranged from approximately 30% to approximately 80% across all samples, with the 75 mL / min flow rate condition exhibiting the highest fortification efficiency (approximately 80%). In each sample, the final measured fortification efficiency was at least approximately 5% higher than the initially measured fortification efficiency. Regarding the data measured at the end for each sample, the fortification efficiency for the 25 mL / min and 50 mL / min samples was approximately 70% each. As mentioned above, the fortification efficiency for the 75 mL / min sample was approximately 80%. The fortification efficiency for the 100 mL / min sample was approximately 75%.

[0519] Results of Example 2: Figures 9A-9CThis section presents additional results regarding the test chamber and the filters held within it when integrated with Thermo Fisher's Rotea®. Results show controls and readings at the start and end of sample collection. For the samples tested in this example, the cell mixture was passed through the system at a flow rate of 75 mL / min.

[0520] like Figure 9A As shown, cell viability was high, exceeding approximately 80%, at both the beginning and end of collection. The difference between initial and final viability measurements was minimal. Figures 9B-9C As shown, the delivery of dextran to cells (e.g., 3kDa dextran) is high, at approximately 80%. The final delivery reading is approximately 5%–10% higher than the initial delivery reading.

[0521] Results of Example 3: Figures 10A-10C This demonstrates the results of testing the cartridge and the filters held within it when integrated with Fresenius Kabi's Cue®. Cue® should be understood as representing a cell processing system operable via an infusion pump. Results are shown for controls and readings at the start and end of sample collection. For the samples tested in this example, the cell mixture was passed through the system at a flow rate of 75 mL / min.

[0522] like Figure 10A As shown, although cell viability is only about 50%-60%, the readings at the beginning and end of collection are similar. Furthermore, as... Figures 10B-10C As shown, despite lower viability (at least compared to Examples 1 and 2 above), the delivery of dextran to living cells (e.g., 10 kDa dextran) was high, greater than about 80%. Similarly, the final delivery reading was about 5%-10% higher than the initial delivery reading.

[0523] Results of Example 4: Figures 11A-11C The results are shown when integrated with Fresenius Kabi's Lovo®, examining the test chamber and the filters held within it. Lovo® should be understood as representing a cell processing system operable via a peristaltic pump. Results are shown as a control and a single "enhanced" reading obtained after collecting the agitated cell mixture. For the sample tested in this example, the cell mixture was passed through the system at a flow rate of 75 mL / min.

[0524] like Figure 11A As shown, the cell viability at the time of collection was greater than approximately 80%. Figures 11B-11CAs shown, the dextran delivery (e.g., 3 kDa dextran) is at least lower than that of Examples 1-3 above, but still greater than 50%, at about 60%, thus still showing acceptable delivery efficiency.

[0525] Example 5: In another example, a housing for a mechanically perforated cell filter is tested in a closed system according to the following setup. A cell mixture source fluid is connected to the Fresenius Kabi Lovo® cell processing system. The cell processing system is fluidly connected to a housing (e.g., housing 300) containing the mechanically perforated cell filter. In these embodiments, the mechanically perforated cell filter is a silicon filter containing pores with a diameter of 6 µm. The housing is fluidly connected to an output reservoir.

[0526] T cells were activated for 3 days (72 hours) using CD3 / 28 bead activation. The activated T cells (D6-activated T cells) were fed to a cell processing system where they were mixed with a payload to provide a cell mixture. The payload used in this experiment was dextran (0.1 mg / mL 3 kDa and / or 10 kDa Cascade Blue dextran). As described herein, dextran is a fluorescently labeled glycopolymer that can act as a proxy for payloads such as mRNA, CRISPR RNP, etc., due to its similar delivery behavior. Measuring dextran delivery can be representative of measuring the delivery efficiency of different payload types. The concentration of the cell mixture from the cell processing system was 20 x 10⁻⁶. 6 Cells / mL. The cell processing system delivers the cell mixture to the cartridge and passes it through a mechanically perforated cell filter at a flow rate of 25 mL / min to agitate the cell membrane and facilitate intracellular delivery of the payload into the membrane. The agitated cell mixture (10 mL) is collected in the output reservoir. Results, including cell viability and dextran delivery, are collected using flow cytometry.

[0527] Figures 12A-12C The results are presented, including controls, initial readings taken as the cell mixture passes through the chamber, and final readings taken after collecting the disturbed cell mixture. Figure 12A As shown, the cell viability at the time of collection was between approximately 60% and 80% (e.g., approximately 65%, 70%, or 75%). Figures 12B-12C As shown, the dextran delivery (e.g., 3 kDa dextran) is high, at approximately 80% (both initial and final measurements).

[0528] Example 6: The objective of Example 6 was to determine the optimal flow rate for delivery and viability using process-scale technology (i.e., cell handling systems). In this experiment, a Fresenius Kabi Lovo™ peristaltic pump was used to propel cells through a cartridge containing a mechanically perforated filter. The cartridge was made of metal and contained a 10x10 mm 5-micron silicon filter. Cells passed through a 105-micron cell sieve, then through the cartridge and the filter. Pressure was measured before passing through the cartridge. Flow rate was measured after the cells passed through the cartridge. The input flow rate was set on the Lovo™ device.

[0529] PBMCs were isolated from Leukopak using Ficoll-based separation. Cells were then suspended in Opti-MEM solution at 20 million / mL. 3 kDa Cascade blue dextran was premixed in the cell suspension at a concentration of 100 μg / mL. Three flow rates (20, 40, 60 mL / min) were tested. 400 million cells were treated at 20 mL / min and 40 mL / min. Less than 100 million cells were left for processing for the final test at 60 M / mL. Approximately 1 mL of treated cells was collected in the “Start” sample. Approximately 1 mL of the final treated cells was collected in the “End” sample. The “Running” sample consisted of the remaining treated cells from the middle of the run. Filters were replaced after each test condition. Experimental controls were performed on a research-scale desktop system, including contactless (“NC”) samples, endocytosis (“endo”) samples, empty-enhanced (“EB”) samples, and 10 psi samples. NC samples were those in which no payload (i.e., dextran) was introduced into the cells, and the cells were not allowed to pass through the desktop system. An endo sample is one in which cells are exposed to the payload, but the cell mixture is not passed through the desktop system. An EB sample is one in which the payload is not introduced into the cells, but the cells are passed through the system.

[0530] Delivery of 3 kDa dextran was assessed using flow cytometry, and the results were plotted on... Figures 14A-14B middle. Figures 13A-13B The results are shown as a control using a desktop system. Propidium iodide staining was used in flow cytometry to measure cell viability. Figure 14A For example, the activity was slightly reduced when running at 40 mL / min compared to 20 mL / min. Figure 14B As shown, in-run delivery was improved by increasing the flow rate from 20 mL / min to 40 mL / min. Delivery at 60 mL / min was not comparable to other conditions due to the lower cell throughput.

[0531] Flow rate and pressure were also collected throughout the assay, as described above. For the 20 mL / min and 40 mL / min conditions, pressure accumulated to 20 PSI throughout the assay. For the 60 mL / min condition, pressure accumulated to 6 PSI due to the smaller number of cells being processed. The average measured flow rate for the 20 mL / min condition was 25 mL / min. The average measured flow rate for the 40 mL / min condition was 50 mL / min.

[0532] Example 7: Example 7 further demonstrates the use of the housing and filter described herein with a desktop research-scale system. PBMCs were isolated from Leukopak via Ficoll separation and frozen for storage. On the day of the experiment, the PBMCs were thawed and prepared for testing. Cells were suspended in Opti-Mem at 2 M / mL. 3 kDa Cascade blue dextran was premixed into the cell suspension at a concentration of 100 µg / mL. The housing was tested at pressures of 3, 6, 9, 12, and 15 PSI. Testing was conducted from low to high pressure. Three experimental controls were also performed, including endocytosis (“endo”) samples, unreinforced (“NB”) samples, and contactless (“NC”) samples, each described above. A new 5-micron 10x10 mm silicon filter was used for each pressure condition. The housing housing the filter further houses a 50A Shore A hardness sealing element (O-ring) with an 8.25 mm ID and a 5 mm cross-section (thickness / height). The housing is designed to seal the O-ring at a 32% compression ratio.

[0533] Delivery of 3 kDa dextran was assessed using flow cytometry, and the results were plotted on... Figures 15A-15B In flow cytometry, propidium iodide staining is used to measure cell viability. Figure 15A The highest cell viability, approximately 55%, was measured at the lowest pressure condition of 3 psi. Cell viability was measured between approximately 30% and 40% at the remaining pressure conditions. Cell viability did not change significantly with increasing pressure from 3 psi. Delivery ranged from approximately 45% to approximately 55% across the five pressure conditions, with the lowest delivery measured at 3 psi and the highest delivery measured at 6, 9, and 15 psi. Figure 15B Although the delivery was slightly lower, this was likely due to the low cell concentration used (2 M / mL).

[0534] Example 8: The purpose of Example 8 was to test the mechanical perforation of iPSCs using a research-scale desktop setup. For this experiment, iPSCs were cultured in mTESR plus medium. The iPSCs were harvested and resuspended in mTESR plus medium at a concentration of 2 million / mL. 3 kDa cascade blue dextran was premixed with the cells at a final concentration of 100 µg / mL. A 5x5 mm silica filter with a 10-micron diameter was used. The filter was loaded into a housing housing a 50A Shore A hardness sealing element (O-ring) with an 8.25 mm ID and a 5 mm cross-section. The housing was designed to seal the O-ring at a 32% compression ratio.

[0535] Delivery of 3 kDa dextran was assessed by flow cytometry, and the results are shown in... Figures 16A-16B In flow cytometry, propidium iodide staining is used to measure cell viability. Figure 16A The highest cell viability, slightly below 50%, was measured at 5 psi pressure. Cell viability was measured just above 40% at the other pressure conditions. Overall, cell viability did not vary significantly across pressure conditions. Delivery ranged from approximately 65% ​​to approximately 82% across the four pressure conditions, with the lowest delivery measured at 5 psi and the highest at 7 psi. Figure 15B ).

Claims

1. A housing for mechanical cell perforation, the housing comprising: A first housing portion includes a fluid inlet detachably connected to a source of the cell mixture; A second housing portion connected to the first housing portion, the second housing portion including a fluid outlet detachably connected to the output reservoir; and A cell mechanically perforated filter, the cell mechanically perforated filter being held by a first housing portion and a second housing portion and configured to operate at a rate of at least 0.5 mL / min / mm. 2 The volumetric flow rate of the porous surface area agitates the cell membrane of the cell mixture through the cellular mechanical perforation filter.

2. The housing of claim 1, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

3. The housing as claimed in claim 1 or 2, wherein the cell mechanical perforation filter is configured to operate at a flow rate of 0.5-500 mL / min / mm. 2 The volumetric flow rate between porous surfaces disturbs the cell membrane.

4. The housing as claimed in any one of claims 1-3, wherein the cellular mechanical perforation filter is configured to perturb the inclusion of 1.0 x 10 8 With 1.0x10 12 The cell membrane of a mixture of cells between cells.

5. The housing as claimed in any one of claims 1-4, wherein the housing is configured to receive a volume between 1 and 500 mL.

6. The housing as claimed in any one of claims 1-5, wherein the housing is configured to pass the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

7. The housing as claimed in any one of claims 1-6, wherein the housing is configured to allow the entire cell mixture to pass through the cell mechanical perforation filter within a time not exceeding 30 minutes.

8. The housing as claimed in any one of claims 1-7, wherein the housing is configured to receive a constant volumetric flow rate controlled by at least one of a peristaltic pump, an injection pump, and a pressurized reservoir.

9. The housing as claimed in any one of claims 1-8, wherein the housing is receivable by a housing receiving portion of a fluid delivery system to detachably connect the fluid inlet to at least one pair of the cell mixture source and the fluid outlet to the output reservoir.

10. The housing of claim 9, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

11. The housing as claimed in any one of claims 1-10, wherein the fluid inlet is detachably connected to the cell mixture source using a Luer connector, a threaded connector, or a quick-release connector.

12. The housing as claimed in any one of claims 1-10, wherein the fluid inlet is detachably connected to the cell mixture source via one or more tubes and using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

13. The housing as claimed in any one of claims 1-12, wherein the fluid outlet is detachably connected to the output reservoir using a Luer connector, a threaded connector, or a quick-release connector.

14. The housing as claimed in any one of claims 1-12, wherein the fluid outlet is detachably connected to the output reservoir via one or more pipes and using a Luer connector, threaded connector, quick-release connector, or tubular weldment.

15. The housing as claimed in any one of claims 1-12, wherein the fluid outlet is detachably connected to the output reservoir by arranging the fluid outlet at an opening near the output reservoir.

16. The housing as claimed in any one of claims 1-15, wherein the first housing portion and the second housing portion are connected by welding, forging, threading, snap-fit, or press-fit.

17. The housing as claimed in any one of claims 1-16, wherein the first housing portion and the second housing portion are detachably connected.

18. The housing as claimed in any one of claims 1-17, wherein the cell mechanical perforation filter is replaceable.

19. The housing as claimed in any one of claims 1-18, wherein the housing is capable of being sterilized using at least one of gamma irradiation and chemical sterilization.

20. The housing of claim 19, wherein the housing is sterilizable when connected to at least one of the cell mixture source and the output reservoir.

21. The housing according to any one of claims 1-20, wherein the fluid inlet comprises a tapered portion, the width of which increases from a proximal end of the fluid inlet to a distal end of the fluid inlet near the cellular mechanical perforation filter.

22. The housing of claim 21, wherein the fluid inlet has a circular cross-section and the width is the diameter of the fluid inlet.

23. The housing as claimed in any one of claims 1-22, wherein the fluid outlet comprises a tapered portion, the width of which decreases from a proximal end of the fluid outlet near the cellular mechanical perforation filter to a distal end of the fluid outlet.

24. The housing of claim 23, wherein the fluid outlet has a circular cross-section and the width is the diameter of the fluid outlet.

25. The housing as claimed in any one of claims 1-24, the housing comprising a sealing member disposed in an end region of the first housing portion near the connected second housing portion.

26. The housing as claimed in any one of claims 1-25, wherein the second housing portion includes an end region adjacent to the connected first housing portion, the end region of the second housing portion holding the cellular mechanical perforation filter.

27. The housing as claimed in any one of claims 1-24, the housing comprising a sealing member disposed in an end region of the second housing portion near the connected first housing portion.

28. The housing as claimed in any one of claims 1-24 and 27, wherein the first housing portion includes an end region adjacent to the connected second housing portion, the end region of the first housing portion holding the cellular mechanical perforation filter.

29. The housing as claimed in any one of claims 1-28, wherein the outer surface of at least one of the first housing portion and the second housing portion includes a textured portion, the textured portion being configured to facilitate at least one of the following: connection of the first housing portion to the cell mixture source, connection of the second housing portion to the output reservoir, and connection of the first housing portion to the second housing portion.

30. The housing according to any one of claims 1-29, wherein the cell mechanical perforation filter comprises a silicon filter or a polymer filter.

31. The housing according to any one of claims 1-30, wherein the cell mechanical perforation filter comprises a silicon filter containing silicon, silicon oxide, silicon nitride and / or silicon carbide.

32. The housing according to any one of claims 1-30, wherein the cell mechanical perforation filter comprises a polymer filter containing polycarbonate, polyester (PET), polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF and / or polytetrafluoroethylene.

33. The housing according to any one of claims 1-32, wherein the cell mechanical perforation filter is coated with at least one of gold, silver, platinum, Teflon, polyvinylpyrrolidone, adhesive and surfactant.

34. The housing according to any one of claims 1-31 and 33, wherein the cellular mechanical perforation filter is doped with boron, gallium or phosphorus.

35. The housing of any one of claims 1-34, wherein the cellular mechanical perforation filter is configured to withstand pressures between 1 and 50 psi.

36. The housing of any one of claims 1-35, wherein the cell mechanical perforation filter comprises a filter surface and a plurality of pores extending through the filter surface, the plurality of pores being configured to agitate the cell membrane as the cell mixture passes through the plurality of pores.

37. The housing of claim 36, wherein the plurality of holes comprises no more than 300,000 holes.

38. The housing of claim 36 or 37, wherein the cellular mechanical perforation filter includes a support structure disposed on one side of the filter surface.

39. The housing of claim 38, wherein the support structure covers at least 1% of the filter surface.

40. The housing as claimed in claim 38 or 39, wherein the support structure comprises a plurality of support members arranged on the filter surface and forming at least one cross or a plurality of strips.

41. The housing as claimed in any one of claims 38-40, wherein the thickness of the support structure is between 20 µm and 3 mm.

42. The housing according to any one of claims 38-41, wherein the cellular mechanical perforation filter comprises an oxide layer disposed on the filter surface between the filter surface and the support structure.

43. The housing as claimed in any one of claims 36-42, wherein each of the plurality of holes comprises a width between 2 µm and 20 µm.

44. The housing of claim 43, wherein the hole spacing between each of the plurality of holes is between 0.5:1 and 100:1 relative to the diameter of the hole.

45. The housing according to any one of claims 36-44, wherein the thickness of the filter surface is between 0.1 µm and 100 µm.

46. ​​The housing as claimed in any one of claims 36-45, wherein the width of the filter surface is between 1 mm and 10 cm.

47. The housing of claim 46, wherein the width of at least one of the fluid inlet and the fluid outlet is at least 50% of the width of the filter surface.

48. The housing as claimed in any one of claims 36-47, wherein at least one of the fluid inlet and the fluid outlet comprises a circular cross-section corresponding to the circular shape of the filter surface.

49. A system comprising: The box body according to any one of claims 1-48; and cell.

50. The system of claim 49, wherein the cell is a somatic cell, an immortalized cell, a stem cell, or a derivative thereof.

51. The system of claim 49 or 50, wherein the cell is a peripheral blood mononuclear cell (PBMC) or a derivative thereof.

52. The system of any one of claims 49-51, wherein the cell is an immune cell.

53. The system according to any one of claims 49-52, wherein the cell is a T cell, a natural killer (NK) cell, a monocyte, a B cell, or a dendritic cell.

54. The system of claim 49 or 50, wherein the cell is a stem cell.

55. The system of any one of claims 49, 50 and 54, wherein the cell is a human stem cell.

56. The system of any one of claims 49, 50, 54 and 55, wherein the cell is an induced pluripotent stem cell, a hematopoietic cell or a mesenchymal cell.

57. The system of any one of claims 49-56, wherein the cell is obtained from or derived from an individual.

58. The system of claim 51, wherein the individual is a person.

59. A method for perturbing a cell membrane, the method comprising: The fluid inlet of a first housing portion of the housing is detachably connected to a cell mixture source, the housing including a second housing portion connected to the first housing portion, and the second housing portion including a fluid outlet, wherein the connected first housing portion and the second housing portion retain a cell mechanical perforation filter; The fluid outlet is detachably connected to the output reservoir; The cell mixture from the cell mixture source is received at the fluid inlet; The cell mixture was prepared at a rate of at least 0.5 mL / min / mm. 2 A volumetric flow rate with a porous surface area passes through the cellular mechanically perforated filter to agitate the cell membrane of the cell mixture; and The disturbed cellular mixture is delivered to the output reservoir via the fluid outlet.

60. The method of claim 59, wherein removably connecting the fluid inlet to the cell mixture source and / or removably connecting the fluid outlet to the output reservoir comprises placing the housing in a housing receiving portion of the fluid delivery system.

61. The method of claim 59 or 60, wherein the cell mixture is passed through the cell mechanical perforation filter at a flow rate of 0.5-500 mL / min / mm. 2 The volumetric flow rate occurs between porous surfaces.

62. The method of any one of claims 59-61, wherein the cell mixture is passed through the cell mechanical perforation filter at a constant volumetric flow rate controlled by at least one of a peristaltic pump, an injection pump, and a pressurized reservoir.

63. The method of any one of claims 59-62, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

64. The method of any one of claims 59-63, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

65. The method of any one of claims 59-64, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the entire cell mixture through the cell mechanical perforation filter within a time not exceeding 30 minutes.

66. The method of any one of claims 59-65, wherein the method comprises prefilling the cell mechanical perforation filter with a cell buffer before passing the cell mixture through the cell mechanical perforation filter.

67. A method for assembling a cell mechanical perforation box, the method comprising: A first housing portion of the housing is connected to a second housing portion of the housing, the first housing portion including a fluid inlet and the second housing portion including a fluid outlet. The cell-mechanically perforated filter is maintained in fluid communication with the fluid inlet and the fluid outlet through the connected first housing portion and second housing portion.

68. The method of claim 67, wherein the cell mechanical perforation filter is replaceable.

69. The method of claim 67 or 68, the method comprising arranging a sealing member in an end region of the first housing portion near the connected second housing portion.

70. The method of any one of claims 67-69, the method comprising arranging a sealing member in an end region of the second housing portion near the connected first housing portion.

71. A cell mechanical perforation system, the cell mechanical perforation system comprising: The box body includes: A first housing portion includes a fluid inlet detachably connected to a source of the cell mixture; A second housing portion is connected to the first housing portion, and the second housing portion includes a fluid outlet; as well as A cell mechanically perforated filter, the cell mechanically perforated filter being held by a connected first housing portion and a second housing portion and configured to agitate the cell membranes of a cell mixture from which the cell mixture originates; An output storage device, wherein the output storage device is detachably connected to a second housing portion of the housing; as well as A pump configured to operate at a rate of at least 0.5 mL / min / mm 2 The volumetric flow rate of the porous surface area causes the cell mixture to move through the cell mechanical perforation filter.

72. The system of claim 71, wherein the cell mechanical perforation system is capable of operating at a constant volumetric flow rate controlled by at least one of the pump and the pressurized reservoir.

73. The system of claim 71 or 72, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

74. The system of any one of claims 71-73, wherein the cell mechanical perforation filter is configured to operate at a flow rate of 0.5-500 mL / min / mm. 2 The volumetric flow rate between porous surfaces disturbs the cell membrane.

75. The system of any one of claims 71-74, wherein the cellular mechanical perforation filter is configured such that the perturbation is contained within 1.0 x 10 8 With 1.0x10 12 The cell membrane of a mixture of cells between cells.

76. The system of any one of claims 71-75, wherein the housing is configured to receive a volume between 1 and 500 mL.

77. The system of any one of claims 71-76, wherein the housing is configured to pass the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

78. The system of any one of claims 71-77, wherein the housing is configured to pass the entire cell mixture through the cell mechanical perforation filter within 30 minutes.

79. The system of any one of claims 71-77, wherein the pump is a fluid delivery system.

80. The system of claim 79, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

81. The system of any one of claims 71-80, wherein the system comprises the source of the cell mixture.

82. The system of any one of claims 71-81, wherein the system comprises a payload source capable of fluid connection with the housing and containing a payload.

83. The system of claim 82, wherein the payload source is fluidly connected to the cell mixture source to receive the cell mixture from the cell mixture source.

84. The system of any one of claims 71-81, wherein the cell mixture source comprises a payload.

85. The system of any one of claims 82-84, wherein the payload comprises at least one of peptides, nucleic acids, small molecules, nanoparticles, and complexes thereof.

86. The system of any one of claims 82-85, wherein the payload comprises more than one of peptides, nucleic acids, small molecules, nanoparticles and / or complexes thereof.

87. A method for perturbing a cell membrane, the method comprising: The fluid inlet of a first housing portion of the housing is detachably connected to a cell mixture source, the housing including a second housing portion connected to the first housing portion, and the second housing portion including a fluid outlet, wherein the connected first housing portion and the second housing portion retain a cell mechanical perforation filter; The fluid outlet is detachably connected to the output reservoir; A cell mixture from the cell mixture source is received at the fluid inlet, the cell mixture containing at least 1.0 x 10⁻⁶ cells / mL. 8 One cell; The cell mixture is passed through the cell mechanical perforation filter to agitate the cell membranes of the cell mixture; as well as The disturbed cellular mixture is delivered to the output reservoir via the fluid outlet.

88. The method of claim 87, wherein removably connecting the fluid inlet to the cell mixture source and / or removably connecting the fluid outlet to the output reservoir comprises placing the housing in a housing receiving portion of the fluid delivery system.

89. The method of claim 88, wherein the fluid delivery system comprises a filtration system, a sifting system, a cell washing system, a separation system, or a cell therapy generation system.

90. The method of any one of claims 87-89, wherein the cell mixture is passed through the cell mechanical perforation filter at a flow rate of 0.5-500 mL / min / mm. 2 The volumetric flow rate occurs between porous surfaces.

91. The method of any one of claims 87-90, wherein the cell mixture is passed through the cell mechanical perforation filter at a constant volumetric flow rate controlled by at least one of a peristaltic pump, an injection pump, and a pressurized reservoir.

92. The method of any one of claims 87-91, wherein at least one of the fluid inlet and the fluid outlet is configured to limit the dead volume to no more than 10% of the volume passing through the cellular mechanical perforation filter.

93. The method of any one of claims 87-92, wherein the cell mixture comprises 1.0 x 10 8 With 1.0x10 12 Between the cells.

94. The method of any one of claims 87-93, wherein the cell mixture has a volume between 1 and 500 mL.

95. The method of any one of claims 87-94, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the cell mixture through the cell mechanical perforation filter in a single run without replacing the cell mechanical perforation filter.

96. The method of any one of claims 87-95, wherein passing the cell mixture through the cell mechanical perforation filter comprises passing the entire cell mixture through the cell mechanical perforation filter within a time not exceeding 30 minutes.

97. The housing as claimed in any one of claims 25-28, wherein the sealing member comprises at least one of the following: a Shore hardness between 25 and 75 A, an inner diameter between 5 and 10 mm, and a cross-section between 2 and 8 mm.

98. The housing as claimed in any one of claims 25-28 and 97, wherein the first housing portion and the second housing portion are configured to compress the sealing member at a compression ratio between 20% and 50%.

99. The housing as claimed in any one of claims 5-48, 97 and 98, wherein the volume is such that the residence time of the cell mixture after passing through the cell mechanical perforation filter is less than 30 seconds.

100. The method of any one of claims 59-66, wherein the volume of the cell mixture is such that the residence time of the cell mixture after passing through the cell mechanical perforation filter is less than 30 seconds.

101. The system of any one of claims 76-86, wherein the volume results in the cell mixture having a residence time of less than 30 seconds after passing through the cell mechanical perforation filter.

102. The method of any one of claims 94-96, wherein the volume results in the cell mixture having a residence time of less than 30 seconds after passing through the cell mechanical perforation filter.

103. The housing as claimed in any one of claims 1-48 and 97-99, wherein the pressure gradient across the cellular mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

104. The method of any one of claims 59-66 and 100, wherein the pressure gradient across the cell mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

105. The system of any one of claims 71-86 and 101, wherein the pressure gradient across the cellular mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

106. The method of any one of claims 87-96 and 102, wherein the pressure gradient across the cell mechanical perforation filter at the said volumetric flow rate is less than 20 psi.

Images