Centrifuge devices and methods for intracellular delivery

Abstract

Provided herein are centrifuge devices and methods of use thereof for transfecting cells to facilitate delivery of a payload to the cells. The centrifuge devices described herein may be contained or embodied within a centrifuge tube compatible with existing centrifuge equipment. The centrifuge devices described herein may comprise a cell constricting component fluidly connected to an input reservoir and a collection reservoir. The centrifuge devices and methods of use thereof may facilitate the passage of a mixture comprising cells through the cell constricting component upon reaching a centripetal force or acceleration suitable for mechanoporation of cells.

Description

CENTRIFUGE DEVICES AND METHODS FOR INTRACELLULAR DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 514,337, filed July 18, 2023, the entire contents of which are incorporated herein by reference.FIELD

[0002] This disclosure relates generally to centrifuge devices, and more specifically to centrifuge devices that facilitate intracellular delivery of a pay load to cells.BACKGROUND

[0003] Intracellular delivery of macromolecules such as nucleic acids, peptides and proteins is a challenge in research and therapeutic applications. The delivery of the aforementioned materials into cells can be controlled by rapidly mechanically deforming the cell to produce transient cell membrane disruptions that allow the macromolecules to enter the cell cytosol. Known techniques of mechanical deformation of cells include the use of constricting microfluidic channels or porous surfaces (e.g., filters) that cause perturbations in the cell membranes. These microfluidic channels or filters are typically embodied in cartridges that require open systems compatible with specialized pump systems, syringe drives, or other pressure mechanisms (e.g., gas cylinders) to drive cells through the channels or pores of the filters.

[0004] Centrifuge devices are one of the most common pieces of lab equipment and are known to be simple to use and operate. Centrifuges are used extensively in research and clinical settings for separation, purification, and isolation of organelles, cells, and cell components, as well as more complex mixtures of nucleic acids, proteins, and viruses. One of the most common uses of centrifuges is to separate red blood cells from the remaining components of a whole blood cell sample.SUMMARY

[0005] Described herein are centrifuge devices and methods of intracellular delivery of a payload to cells using the centrifuge devices. The disclosed centrifuge devices and methodsof use thereof may provide a simple, compact centrifuge tube device compatible with existing centrifuge equipment. The centrifuge devices may include a cell constricting component such as a cell perturbation filter or microfluidic channel(s) fluidly connected to an input reservoir and a collection reservoir. The centrifuge devices described herein may additionally comprise a force-regulated mechanism configured to control the flow of a cell mixture from the input reservoir into the cell constricting component. For example, the force-regulated mechanism may comprise a valve that can actuate the flow of the mixture into the cell constricting component when a threshold centripetal force or acceleration in the centrifuge device is reached. The centrifuge devices can be adapted to fit within a conventional centrifuge tube, or in some instances, may be embodied in a specialized cell perturbation centrifuge device.

[0006] As mentioned above, known techniques for delivering payloads into cells include mechanically deforming the cell membrane using microfluidic channels and / or pores. Existing systems that implement said microfluidic channels / pores typically use specially designed pumping and / or pressure-driving equipment (e.g., gas cylinders) that necessitate an open system to drive cells through the cartridge and cause mechanoporation of the cells, and furthermore increase the amount of bulky equipment in lab or clinical areas already scarce for space. The safety and portability of such existing systems can be complicated based on high pressure requirements of the pressure-driving equipment. Moreover, the complexity and sterility of these existing systems is complicated due to the need for an open system that facilitates compatibility between the external pressure-driving equipment and the cartridge / system comprising the microfluidic channels or pores. Furthermore, existing cartridge systems may require separate, standalone modules and / or processes to perform individual tasks in cell processing (e.g., mixing, which may be performed manually by technicians). Adding to the number of devices, systems, and processes increases the number of technicians needed to set-up and operate the various devices. Additional technicians and equipment may cause an increase in the processing time of cells as well as an increase in cost. Accordingly, improved systems for mechanoporation of cells are needed.

[0007] The centrifuge devices described herein may solve one or more of the aforementioned problems with existing mechanoporation devices and systems by providing a simple, compact, closed-system device compatible with conventional centrifuge devices. The centrifuge devices may leverage the centripetal force or acceleration conditions induced by centrifuges to control flow and therefore perturb cell membranes. For example, a force- regulated mechanism of the centrifuge device may be configured to facilitate the passage of amixture comprising cells through a cell constricting component when the centripetal force or acceleration within the centrifuge device meets known force conditions suitable for mechanoporation of cells. Moreover, use of the centrifuge devices described herein may be as simple as adding a mixture comprising cells (and a payload / cargo) into the input reservoir of the centrifuge device, loading the device into the centrifuge, running the centrifuge at a predetermined speed, and removing the sample from the centrifuge tube. No external pressure-driving equipment, such as pressurized gas sources, are needed to facilitate cell mechanoporation. Thus, implementation and operation of the centrifuge devices herein can be comparatively simple at least in comparison to existing mechanoporation systems and devices.

[0008] The centrifuge devices and methods of use thereof as provided herein have determined a unique combination of small-scale mechanical design features and geometries of the centrifuge device in combination with suitable operating parameters of the centrifuge that achieve force / acceleration conditions suitable for intracellular delivery a payload into cells. As mentioned above, known microfluidic systems for intracellular delivery typically require gas pressures that are challenging to identify and replicate with equipment such as conventional lab centrifuges. Moreover, filter-based intracellular delivery systems heretofore have failed to conceptualize flow-controlling mechanisms that can facilitate cellular flow through a filter once proper mechanoporation conditions are achieved. The disclosed centrifuge devices and methods of use can solve one or more of the aforementioned problems while yielding sufficient delivery and minimizing damage to or pelleting of the cell mixture.

[0009] Using the centrifuge devices provided herein, several cell samples may be processed in parallel with a single centrifuge. Moreover, with use of the centrifuge devices provided herein, cell processing can be simplified, and scarce clinical and lab space can be preserved by minimizing excess equipment and instead utilizing existing lab centrifuges. Reducing the complexity of and amount of equipment required for cell processing may also reduce barriers of entry to cell therapies by reducing costs associated with large equipment and complex systems. Moreover, eliminating the need for external pressure-driving equipment can minimize any risk introduced with contacting the cell mixture with external gas sources.

[0010] In some embodiments, a centrifuge device for delivering a payload to cells is provided, comprising: a housing configured to be disposed in a vessel, the housingcomprising an input reservoir configured to receive a mixture comprising cells, and a cell constricting component receiving portion disposed distal to the input reservoir; and a cell constricting component disposed within the cell constricting component receiving portion of the housing and in fluid communication with the input reservoir and a collection reservoir, wherein the cell constricting component is configured to perturb membranes of cells and to deposit a perturbed cell mixture into the collection reservoir during centrifugation.

[0011] In some embodiments, a cell perturbation centrifuge tube for delivering a payload to cells is provided, comprising: a centrifuge tube comprising an input reservoir configured to receive a mixture comprising cells, a cell constricting component receiving portion disposed distal to the input reservoir, and a collection reservoir disposed distal to the cell constricting component receiving portion; and a cell constricting component disposed within the cell constricting component receiving portion of the centrifuge tube and fluidly connected to the input reservoir and the collection reservoir, wherein the cell constricting component is configured to perturb membranes of cells and to deposit a perturbed cell mixture into the collection reservoir during centrifugation.

[0012] In some embodiments, a method for delivering a payload into cells is provided, comprising: adding a mixture comprising cells to an input reservoir of a cell perturbation centrifuge tube; and centrifuging, using a centrifuge, the cell perturbation centrifuge tube to cause the mixture comprising cells to pass through a cell constricting component and deposit into a collection reservoir of the cell perturbation centrifuge tube as a perturbed cell mixture.BRIEF DESCRIPTION OF THE FIGURES

[0013] Various aspects of the disclosed systems and methods are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosed systems and methods will be obtained by reference to the detailed description of illustrative embodiments and the accompanying drawings.

[0014] FIG. 1A illustrates a front view of a first exemplary centrifuge device, in accordance with some embodiments.

[0015] FIG. IB illustrates a perspective view of the first exemplary centrifuge device, in accordance with some embodiments.

[0016] FIG. 2A illustrates a front view of a second exemplary centrifuge device, in accordance with some embodiments.

[0017] FIG. 2B illustrates a perspective view of the second exemplary centrifuge device, in accordance with some embodiments.

[0018] FIG. 2C illustrates a burstable membrane of the second exemplary centrifuge device, in accordance with some embodiments.

[0019] FIG. 3A illustrates a cross-sectional view of third exemplary centrifuge device, in accordance with some embodiments.

[0020] FIG. 3B illustrates an exploded view of the third exemplary centrifuge device, in accordance with some embodiments.

[0021] FIG. 3C illustrates a detailed cross-sectional view of the third exemplary centrifuge device, in accordance with some embodiments.

[0022] FIG. 4A illustrates a cross-sectional view of a fourth exemplary centrifuge device, in accordance with some embodiments.

[0023] FIG. 4B illustrates a detailed cross-sectional view of the fourth exemplary centrifuge device, in accordance with some embodiments.

[0024] FIG. 4C illustrates a perspective view of the fourth exemplary centrifuge device, in accordance with some embodiments.

[0025] FIG. 5A illustrates a front view of a fifth exemplary centrifuge device, in accordance with some embodiments.

[0026] FIG. 5B illustrates a cross-sectional view of the fifth exemplary centrifuge device, in accordance with some embodiments.

[0027] FIG. 5C illustrates a detailed cross-sectional view of the fifth exemplary centrifuge device, in accordance with some embodiments.

[0028] FIG. 6A illustrates a front view of a sixth exemplary centrifuge device, in accordance with some embodiments.

[0029] FIG. 6B illustrates an exploded view of the sixth exemplary centrifuge device, in accordance with some embodiments.

[0030] FIG. 6C illustrates a detailed cross-sectional view of the sixth exemplary centrifuge device, in accordance with some embodiments.

[0031] FIG. 6D illustrates a detailed cross-sectional view of the sixth exemplary centrifuge device, in accordance with some embodiments.

[0032] FIG. 7A illustrates a cross-sectional view of a seventh exemplary centrifuge device, in accordance with some embodiments.

[0033] FIG. 7B illustrates a cross-sectional view of the seventh exemplary centrifuge device, in accordance with some embodiments.

[0034] FIG. 7C illustrates a plunger body of the seventh exemplary centrifuge device, in accordance with some embodiments.

[0035] FIG. 7D illustrates a detailed cross-sectional view of the seventh exemplary centrifuge device, in accordance with some embodiments.

[0036] FIG. 7E illustrates a detailed cross-sectional view of the seventh exemplary centrifuge device, in accordance with some embodiments.

[0037] FIG. 8A illustrates a cross-sectional view of an eighth exemplary centrifuge device, in accordance with some embodiments.

[0038] FIG. 8B illustrates an exploded view of the eighth exemplary centrifuge device, in accordance with some embodiments.

[0039] FIG. 8C illustrates a perspective view of the eighth exemplary centrifuge device, in accordance with some embodiments.

[0040] FIG. 8D illustrates a detailed cross-sectional view of the eighth exemplary centrifuge device, in accordance with some embodiments.

[0041] FIG. 9A illustrates a cross-sectional view of a ninth exemplary centrifuge device, in accordance with some embodiments.

[0042] FIG. 9B illustrates a detailed cross-sectional view of the ninth exemplary centrifuge device, in accordance with some embodiments.

[0043] FIG. 9C illustrates a detailed cross-sectional view of the ninth exemplary centrifuge device, in accordance with some embodiments.

[0044] FIG. 10A illustrates a cross-sectional view of a tenth exemplary centrifuge device, in accordance with some embodiments.

[0045] FIG. 10B illustrates an exploded view of the tenth exemplary centrifuge device, in accordance with some embodiments.

[0046] FIG. 10C illustrates a detailed cross-sectional view of the tenth exemplary centrifuge device, in accordance with some embodiments.

[0047] FIG. 11A illustrates a cross-sectional view of an eleventh exemplary centrifuge device, in accordance with some embodiments.

[0048] FIG. 11B illustrates a cross-sectional view of the eleventh exemplary centrifuge device, in accordance with some embodiments.

[0049] FIG. 11C illustrates a first component of the eleventh exemplary centrifuge device, in accordance with some embodiments.

[0050] FIG. 1 ID illustrates a detailed cross-sectional view of a second component of the eleventh exemplary centrifuge device, in accordance with some embodiments.

[0051] FIG. 12A illustrates a front view of a twelfth exemplary centrifuge device, in accordance with some embodiments.

[0052] FIG. 12B illustrates a front view of the twelfth exemplary centrifuge device, in accordance with some embodiments.

[0053] FIG. 12C illustrates a perspective view of the twelfth exemplary centrifuge device with a spring component, in accordance with some embodiments.

[0054] FIG. 12D illustrates a detailed view of the twelfth exemplary centrifuge device, in accordance with some embodiments.

[0055] FIG. 12E illustrates a detailed view of the twelfth exemplary centrifuge device, in accordance with some embodiments.

[0056] FIG. 12F illustrates a detailed view of the twelfth exemplary centrifuge device, in accordance with some embodiments.

[0057] FIG. 13A illustrates a cross-sectional view of a thirteenth exemplary centrifuge device, in accordance with some embodiments.

[0058] FIG. 13B illustrates a magnetic plunger body of the thirteenth exemplary centrifuge device, in accordance with some embodiments.

[0059] FIG. 13C illustrates a detailed cross-sectional view of the thirteenth exemplary centrifuge device, in accordance with some embodiments.

[0060] FIG. 14A illustrates a perspective view of a fourteenth exemplary centrifuge device, in accordance with some embodiments.

[0061] FIG. 14B illustrates a perspective view of the fourteenth exemplary centrifuge device, in accordance with some embodiments.

[0062] FIG. 15 illustrates an exemplary method for delivering a pay load into cells using a centrifuge device, in accordance with some embodiments.

[0063] FIG. 16A illustrates viability of PBMCs processed at relative centrifugal forces (RCFs) between 80-150 RCF using the fifth exemplary centrifuge device shown in FIGS. 5A- 5C, in accordance with some embodiments.

[0064] FIG. 16B illustrates delivery of Dextran to PBMCs processed at RCFs between 80-150 RCF using the fifth exemplary centrifuge device shown in FIGS. 5A-5C, in accordance with some embodiments.

[0065] FIG. 17A illustrates viability of iPSCs processed using the fifth exemplary centrifuge device shown in FIGS. 5A-5C, in accordance with some embodiments.

[0066] FIG. 17B illustrates delivery of 3 kDa Dextran to iPSCs processed using the fifth exemplary centrifuge device shown in FIGS. 5A-5C, in accordance with some embodiments.

[0067] FIG. 17C illustrates delivery of 10 kDa Dextran to iPSCs processed using the fifth exemplary centrifuge device shown in FIGS. 5A-5C, in accordance with some embodiments.

[0068] FIG. 18 illustrates delivery of Dextran to iPSCs processed using a centrifuge device having a metal plunger and a centrifuge device having a plastic plunger, both centrifuge devices as shown in FIGS. 5A-5C, in accordance with some embodiments.

[0069] FIG. 19A illustrates viability of PBMCs processed at target RCFs between 200- 800 RCF using the fifth exemplary centrifuge device shown in FIGS. 5A-5C, in accordance with some embodiments.

[0070] FIG. 19B illustrates delivery of 3 kDa Dextran to PBMCs processed at target RCFs between 200-800 RCF using the fifth exemplary centrifuge device shown in FIGS. 5A- 5C, in accordance with some embodiments.DETAILED DESCRIPTION

[0071] Provided herein are centrifuge devices and methods of using the centrifuge devices for intracellular delivery of a payload. The centrifuge devices described herein may be compatible with existing centrifuge equipment. The centrifuge devices may have a simple, compact design paired with simple usability. For example, the centrifuge devices may comprise a housing adapted to be contained within a centrifuge tube or, in some instances, may be embodied within a specialized cell perturbation centrifuge tube itself. The centrifuge devices may comprise a cell constricting component such as a cell perturbation filter or microfluidic channel(s) fluidly connected to an input reservoir and a collection reservoir. Use of the centrifuge devices described herein may be as simple as adding a cell mixture (e.g., cells and a payload) into the input reservoir of the centrifuge device, loading the centrifuge device into the centrifuge, running the centrifuge at a predetermined speed, and removing the sample from the centrifuge tube.

[0072] The centrifuge devices may comprise a force-regulated mechanism that can control the flow of the mixture from the input reservoir, through the cell constricting component, and into the collection reservoir. For example, the centrifuge devices may leverage centripetal force or acceleration conditions induced by centrifuges to control flow of cells and therefore cause cell perturbation by the constricting component (e.g., filter, channels, etc.). In some instances, the centrifuge devices described herein may additionally or alternatively control the fluid flow using one or more mechanical design features, such as force-regulated mechanisms (e.g., burstable membranes, spring valves, etc.) and / or magnetic mechanisms, each of which are described in greater detail below.

[0073] As mentioned above, existing devices and methods for mechanically deforming cells to cause perturbation of the cell membranes use specialized equipment designed for pumping and mixing the materials prior to mechanoporation of the cells. The additional equipment adds to the number of devices and systems crowding already scarce clinical and lab space and may utilize additional technicians to operate said devices. Moreover, the external pressure-driving equipment necessitates an open system, therefore increasing risk ofcontamination of the sample. The centrifuge devices and methods of use thereof as described herein can simplify cell processing systems by providing a simple, compact, closed-system disposable centrifuge device for mechanoporation that is housed or embodied within a centrifuge tube. The centrifuge devices can leverage existing centrifuge equipment, thus reducing any barriers to entry in cell therapies. The centrifuge devices do not require external gas pressure sources to facilitate mechanoporation, instead relying on force and acceleration induced by conventional centrifuge devices. Most clinical and research lab spaces include centrifuge equipment, use of the equipment is known to be simple, and most (if not all) technicians understand how to properly use the centrifuge equipment. Thus, implementation and use of the centrifuge devices described herein can be simple, fast, and repeatable.

[0074] The centrifuge devices described herein have determined how to utilize the centripetal force and acceleration conditions induced during centrifugation to facilitate cell mechanoporation. Centrifugation to achieve cell mechanoporation may be challenging due to the likelihood of cell damage, over-perturbation, under-perturbation, and / or cell pelleting in the tube. Moreover, it may be challenging to ascertain and control the optimal conditions (e.g., timing, speed) at which the cells should pass through the cell constricting component. Finally, it may be challenging to achieve mechanical design features for controlling mechanoporation at a small scale. The centrifuge devices and methods of use thereof as disclosed herein have achieved a combination of simple design features and operating parameters that provide force and acceleration conditions lending to efficacious cell mechanoporation for intracellular delivery of a pay load into cells. Using the centrifuge devices described herein, passage of a mixture comprising cells through the cell constricting component may be actuated when the force or acceleration conditions are suitable for mechanoporation, and the necessary cell speed through the mechanoporation mechanism may be maintained. Moreover, reliance on external pressure-driving equipment for mechanoporation can be eliminated, thus allowing for a closed-system that maintains sterility of the mixture and minimizes complexity of the system.

[0075] As used herein, a cell constricting component may comprise one or more constrictions, or narrowing, that deforms the cells that pass through the component. The use of the term cell constricting component in this disclosure can be understood to encompass mechanoporation devices known to one of ordinary skill in the art, such as a cell perturbation filters (e.g., filter), a microfluidic device containing one or more channels with constrictions or confinements in parallel or series, post configurations to induce perturbations,constrictions created through depth changes in fluidic channels, a microneedle or microneedle array, etc.

[0076] In some embodiments, a centrifuge device for delivering a payload to cells is provided. The centrifuge devices described herein may comprise an input reservoir, collection reservoir, and cell constricting component in fluid communication with each of the input reservoir and the collection reservoir. Thus, during centrifugation, the cell constricting component may perturb membranes of cells from a mixture contained in the input reservoir and deposit a perturbed cell mixture into the collection reservoir. Various centrifuge devices that can achieve cell mechanoporation are illustrated in FIGS. 1A-1B, 2A-2C, 3A-3C, 4A- 4C, 5A-5C, 6A-6D, 7A-7E, 8A-8D, 9A-9C, 10A-10C, 11A-11D, 12A-12F, 13A-13C, and 14A-14B, each of which will be described in greater detail below.

[0077] As mentioned above, the cell constricting component may comprise a filter, otherwise referred to as a cell perturbation filter, a microfluidic device comprising one or more channels, a microneedle array, etc. The various examples illustrated in the Figures may be described with respect to a particular type of cell constricting component (e.g., a cell perturbation filter) and cell constricting component receiving portion (e.g., filter receiving portion). However, it is to be understood that additional or alternative cell constricting elements, such as microfluidic devices comprising channels, microneedle(s), etc. (described in greater detail above) may be reasonably incorporated in the centrifuge devices described herein. Moreover, the filter receiving portion may be configured to receive additional or alternative cell constricting components, such as microfluidic devices comprising channels, microneedle(s), etc.

[0078] FIGS. 1A-1B illustrate a centrifuge device in accordance with some embodiments. As shown, the centrifuge device may comprise a housing 100 disposed within (or configured to be disposed within) a vessel, such as centrifuge tube 150. The centrifuge device may comprise an input reservoir 102, a filter receiving portion 104 disposed distal to the input reservoir 102, and collection reservoir 106 disposed distal to the filter receiving portion 104. The centrifuge device may comprise a cell perturbation filter 108 disposed within the filter receiving portion 104 of the housing 102. Thus, as described above, the cell perturbation filter 108 may be in fluid communication with each of the input reservoir 102 and collection reservoir 106.

[0079] In some embodiments, at least the input reservoir 102 and cell perturbation filter 108 (e.g., disposed within filter receiving portion 104) may be contained within housing 100. In some embodiments, the collection reservoir 106 may additionally be contained within housing 100. In some embodiments, collection reservoir 106 may alternatively be disposed within centrifuge tube 150. That is, a distal portion (e.g., end) of centrifuge tube 150 may serve as the collection reservoir 106. In some embodiments, collection reservoir 106 may be partially embodied in housing 100 and in centrifuge tube 150. In any instance, housing 100 may comprise at least one opening that fluidly connects the cell perturbation filter 108 to the collection reservoir 106. For example, the at least one opening can deposit the perturbed cell mixture from passage through the cell perturbation filter to the end of centrifuge tube 150.

[0080] The input reservoir 102 may be configured to receive a mixture comprising cells. In some embodiments, the mixture may comprise a cell suspension. In some embodiments, the mixture may comprise a cell suspension and a payload. Alternatively, the payload may be disposed in the collection reservoir 106. For example, the centrifuge device may be provided with the payload pre-loaded in the collection reservoir 106. Alternatively, use of the device may require a user to load the pay load into the collection reservoir 106 prior to centrifugation.

[0081] In some embodiments, housing 100 may comprise a lip 110 at a proximal end of the housing 100 that is configured contact a proximal end of centrifuge tube 150 when the housing 100 is disposed within centrifuge tube 150, as shown in FIGS. 1A-1B. For example, the lip 110 may limit advancement of housing 100 into the centrifuge tube 150. Moreover, when sealing the centrifuge device, a cap may be secured to the lip 110 of housing 100. In some embodiments, housing 100 may not have a lip 110. For example, the housing 100 may be inserted into centrifuge tube 150 such that substantially all of housing 100 is within the centrifuge tube 150. The fit between the housing 100 and centrifuge tube 150 may be threaded, a press-fit, a snap-fit, etc. In some embodiments, an external surface of housing 100 and / or internal surface of centrifuge tube 150 may comprise one or more latches or threads to facilitate a secure connection between the components. Although described with respect to a centrifuge tube, it is to be understood that other types of vessels comprising shapes / structures that may be different from a standard centrifuge tube may be used and are understood to be encompassed in the scope of the disclosure.

[0082] FIGS. 1A-1B illustrate an embodiment of a centrifuge device that may not comprise a flow control mechanism. Rather, the centrifuge device may simply comprise an elongated neck portion and a tapered portion within housing 100. Said elongated neck portion and / or tapered portion may introduce a simple pressure differential useful in controlling the pressure head experienced by the fluid as it flows into the cell perturbation filter 108 in the centrifuge devices described herein. Although illustrated together in a single embodiment, it is to be understood that each of the elongated neck and tapered portion may be incorporated into a centrifuge device individually. Moreover, centrifuge devices without an elongated neck and / or tapered portion are understood to be encompassed in the scope of this disclosure. For example, the input reservoir 102 may comprise a simple fluid reservoir (e.g., a cylindrical fluid reservoir) that is fluidly connected to a filter receiving portion 104.

[0083] In some embodiments, input reservoir 102 may comprise a proximal portion 102a and a distal portion 102b. The proximal portion 102a may comprise a cross-sectional dimension that is greater than a cross-sectional dimension of the distal portion 102b. For example, each of proximal portion 102a and distal portion 102b may comprise a cylindrical shape, and a cross-sectional diameter of the proximal portion 102a may be greater than a cross-sectional diameter of the distal portion 102b.

[0084] In some embodiments, the proximal portion 102a and / or distal portion 102b of input reservoir 102 may be dimensioned such that a majority of the mixture volume prior to centrifugation is contained within the proximal portion 102a. For example, the ratio of volume in the proximal portion 102a in relation to the distal portion 102b may be 90:10, 85:15, 80:20, 75:25, 70:30, 60:40, etc. Said volume ratios may be achieved by varying dimensions such as height (e.g., length) and / or width (e.g., diameter) of each of proximal portion 102a and distal portion 102b, as will be understood by one of ordinary skill in the art.

[0085] In some embodiments, input reservoir 102a may comprise a tapered portion. The tapered portion of input reservoir 102 may be within the proximal portion 102a of the input reservoir between the proximal portion 102a and the distal portion 102b, as shown in FIG. 1A. The tapered portion may be tapered from the proximal portion 102a toward the distal portion 102b. In some embodiments, the input reservoir 102 may be tapered about 45° from a longitudinal axis coaxial with a central axis of the centrifuge tube 150. In some embodiments, the taper angle may be less than or greater than 45°, such as 15°, 30°, 60°, or 75°.Centrifuge Devices with Force-regulated Mechanisms

[0086] In some embodiments, the centrifuge devices described herein may comprise one or more force-regulated mechanisms configured to control the flow of the mixture within the centrifuge device. For example, the force-regulated mechanisms described herein may be configured to control the flow of the mixture comprising cells from the input reservoir 102 into the cell perturbation filter 108. The force-regulated mechanisms described herein may be configured to actuate upon reaching a threshold (e.g., minimum) force or acceleration. In some embodiments, the force-regulated mechanisms described herein may additionally or alternatively be configured to close upon reaching a second threshold (e.g., maximum) force or acceleration. The threshold force and / or acceleration(s) may correspond to known force / acceleration conditions effective for mechanoporation of cells. As will be described in greater detail below, force-regulated mechanisms may include but are not limited to blow-off valves, spring valves, plungers, and / or combinations thereof.

[0087] FIGS. 2A-2B illustrate an embodiment of a centrifuge device that comprises a blow-off valve, and more specifically, a burstable membrane. The burstable membrane 212 may be configured to release the mixture comprising cells from the input reservoir 202 when a threshold force or acceleration in the centrifuge device is reached during centrifugation. The centrifuge device illustrated in FIGS. 2A-2B may comprise any one or more features of centrifuge devices described herein at least with respect to FIGS. 1A-1C. For example, the centrifuge device may comprise a housing 200 comprising an input reservoir 202 and a filter receiving portion 204. The housing 200 may be configured to be disposed within a centrifuge tube 250. The centrifuge device may comprise a collection reservoir 206 contained within the housing 200 and / or centrifuge tube 250. The centrifuge device may comprise a cell perturbation filter 208 disposed within the filter receiving portion 204 of the housing 200.

[0088] In some embodiments, the centrifuge device described with respect to FIGS. 2A- 2C may comprise an input reservoir 202 comprising an elongated neck and / or a tapered portion, as shown and described above at least with respect to FIGS. 1A-1C. However, it is to be understood that such features of the input reservoir 202, although illustrated in the Figures, are not required for the functionality of the device. For example, as described above, the input reservoir 202 may comprise a simple shape, such as a cylindrical shape.

[0089] As described herein, the centrifuge device illustrated in FIGS. 2A-2B may differ from that which is illustrated in and described with respect to FIGS. 1A-1C at least because the centrifuge device may comprise a burstable membrane 212. FIG. 2C illustrates anexemplary burstable membrane 212. The burstable membrane 212 may release the mixture from the input reservoir 202 upon reaching a threshold force or acceleration in the centrifuge device during centrifugation. In some embodiments, the threshold force / acceleration may correspond with pressure conditions suitable for cell mechanoporation. The burstable membrane 212 may be disposed proximal to (e.g., above) the cell perturbation filter 208 within the centrifuge device, such that, upon reaching the threshold force or acceleration during centrifugation, the mixture may be released from the input reservoir 202 to pass through the cell perturbation filter 208.

[0090] Other types of blow-off valves that may be used in addition to or instead of the burstable membrane described above may include balls, plugs, cap-actuated valves, snaps, etc. For example, a snap component may initially block the flow of the mixture through a fluid pathway fluidly connected to the cell perturbation filter, and upon meeting or exceeding a threshold force / acceleration, the snap component may detach to allow flow of the mixture through the fluid pathway. In a similar manner, a burstable valve of a centrifuge device may comprise a plug initially disposed within a fluid pathway and configured to be removed from (e.g., unplug, or pop-off) the fluid pathway to allow the mixture to flow through the pathway and to the cell perturbation filter.

[0091] FIGS. 3A-3C illustrate an embodiment of a centrifuge device that comprises a spring valve. The spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 302 and into the cell perturbation filter 308. The centrifuge device illustrated in FIGS. 3A-3C may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A-1B and 2A-2C. For example, the centrifuge device may comprise a housing 300 comprising an input reservoir 302 and a filter receiving portion 304. The housing 300 may be configured to be disposed within a centrifuge tube 350. The centrifuge device may comprise a collection reservoir 306 contained within the housing 300 and / or centrifuge tube 350. The centrifuge device may comprise a cell perturbation filter 308 disposed within the filter receiving portion 304 of the housing 300.

[0092] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 302 into cell perturbation filter 308. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 302 when centrifuged to allow the mixture to flow into cell perturbation filter 308. The spring valve may be configured to release the mixture comprising cells from inputreservoir 302 when a threshold force or acceleration in the centrifuge device is reached.Example mechanics of the spring valve are described herein with respect to FIGS. 3A-3C.

[0093] In some embodiments, housing 300 may be made up of one or more individual components, or bodies. For example, housing 300 may comprise a first body 314, a second body 316, and a third body 318. The first body 314 may comprise input reservoir 302. In some embodiments, the first body 314 may comprise an outer portion 314a and an inner portion 314b. The inner portion 314b may be configured to be received in the second body 316, as shown. In some embodiments, the second body 316 may be configured to block flow of the mixture comprising cells from input reservoir 302 before centrifugation. In some embodiments, filter receiving portion 304 (and therefore cell perturbation filter 308) may be disposed within first body 314, within second body 316, between first body 314 and second body 316, and / or between first body 314 and second body 316.

[0094] The third body 318 may be configured to receive the spring 313, the first body 314, and / or the second body 316. For example, as shown in FIGS. 3A-3C, third body 318 may be disposed distal to at least a portion of first body 314 and / or second body 316. The third body 318 may be configured to be attached to first body 314, for example via a press-fit, snap-fit, threading, heat staking, snaps, adhesives, or other means of connection. In some embodiments, third body 318 may be attachable to first body 314 to tune a release force or acceleration of the spring valve. For example, third body 318 and first body 314 may be attachable via a threaded or other type of connection. The extent to which first body 314 and third body 318 are threaded together may dictate the release force of spring valve comprising spring 313. A more threaded connection may correspond with a more tightly wound (e.g., more pre-compressed) spring 313, and thus a lower release force. On the other hand, a less threaded connection may correspond with a looser (e.g., less pre-compressed) spring 313, and thus a greater release force. The release force is associated with the amount of force necessary to instigate flow. A more pre-compressed spring will require more force to compress further and instigate flow (or open a valve), whereas a less pre-compressed spring will require less force to compress further and instigate flow (or open a valve). A similar concept can be applied to other types of connections, as listed above.

[0095] The second body 316 may be configured to translate with the spring 313 when the device is centrifuged. For example, the second body 316 may translate in a vertical downward motion when, during centrifugation, the centrifuge device reaches a thresholdforce / acceleration, which may be regulated by spring 313. Spring 313 may controllably contract upon reaching a threshold force / acceleration as the device is centrifuged. As the second body 314 translates downward, the mixture may be released from input reservoir 302 (e.g., contained within first body 314), and may pass through cell perturbation filter 308 to be disposed within collection reservoir 306. Each of second body 316 and third body 318 may comprise one or more openings configured to allow the mixture to pass through the bodies and into the collection reservoir 306 after mechanoporation. For example, the one or more openings of second body 316 may flank either side of an inner blocking portion of the second body 316.

[0096] Although the fluid control mechanism is described above primarily with respect to the spring valve, one of ordinary skill in the art will understand that several components of the centrifuge device may contribute to the valve opening (and in some instances, closing). For example, the weight of the body disposed above the spring (e.g., second body 316 and spring 313) that compresses the spring during centrifugation may contribute to the fluid control mechanism. The centripetal force and acceleration during acceleration can amplify the weight of the body causing compression of the spring. Moreover, the volume of the mixture disposed in the centrifuge device may also contribute to the fluid control mechanism. The fluid control mechanism may be a fine-tuned balance between the fluid volume and body weight against the resistance of the spring. As centrifugation elapses, the force from the fluid volume and / or body weight can eventually overcome the resistance of the spring to allow the valve to open.

[0097] Although illustrated and described with respect to several components, it is to be understood that one or more of the aforementioned bodies (e.g., first body 314, second body 316, and / or third body 318) may be reasonably combined to achieve the aforementioned spring valve mechanics with spring 313. Moreover, the configuration, orientation, shape, and / or size of each of the components of the centrifuge device illustrated in FIGS. 3A-3C are not intended to be limited by that which is illustrated, and rather may be modified to achieve similar functionalities, as will be understood by one of ordinary skill in the art. For example, the centrifuge devices illustrated in at least FIGS. 4A-4B, 5A-5C, 6A-6D, 7A-7D, 8A-8D, 9A-9D, 10A-10C, and / or 11A-11D may utilize similar spring mechanics with varying configurations of one or more bodies similar to first body 314, second body 316, and / or third body 318 to achieve mechanoporation of cells, as will be described in greater detail below.

[0098] FIGS. 4A-4B illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 4A-4B may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A-1B, FIGS. 2A-2C, and / or FIGS. 3A-3C. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 402 and into the cell perturbation filter 408. Moreover, the centrifuge device may comprise a housing 400 comprising an input reservoir 402 and a filter receiving portion 404. The housing 400 may be configured to be disposed within a centrifuge tube 450. The centrifuge device may comprise a collection reservoir 406 contained within the housing 400 and / or centrifuge tube 450. The centrifuge device may comprise a cell perturbation filter 408 disposed within the filter receiving portion 404 of the housing 400. In some embodiments, housing 400 may be made up of one or more individual components, or bodies. For example, housing 400 may comprise a first body 414, a second body 416, and a third body 418. As shown, the first body 414 may comprise input reservoir 402, and the second body 416 may be configured to block flow of the mixture from input reservoir 402 before centrifugation.

[0099] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 402 into cell perturbation filter 408. For example, the spring 413 may be configured to release the mixture comprising cells from input reservoir 402 when centrifuged to allow the mixture to flow into cell perturbation filter 408. The spring valve may be configured to release the mixture comprising cells from input reservoir 402 when a threshold force or acceleration in the centrifuge device is reached. As described herein, third body 418 may be attachable (e.g., via threads) to first body 414 to tune a release acceleration or force of the spring valve comprising spring 413. The extent to which first body 414 and third body 418 are threaded together may dictate the release force of the spring valve.

[0100] The centrifuge device illustrated in FIGS. 4A-4B may differ from that illustrated at least in FIGS. 3A-3C in that filter receiving portion 404 may comprise a separate body disposed between first body 414 and a portion of second body 416. Thus, cell perturbation filter 408 may be disposed between first body 414 and second body 416 such that cell perturbation filter 408 can be in fluid communication with input reservoir 402 and collection reservoir 406.

[0101] In some embodiments, second body 416 may comprise an extended fluid pathway 416a. The fluid pathway 416a may direct the mixture from the cell perturbation filter 408 to the collection reservoir 406. In doing so, the fluid pathway 416a may also ensure that the cell mixture does not come into contact with the spring valve. For example, the fluid pathway 416a may comprise a tapered portion followed by an elongated portion that can guide the mixture to the collection reservoir 406.

[0102] Second body 416 may comprise one or more openings configured to allow the mixture to pass from the cell perturbation filter 408 and into the collection reservoir 406 after mechanoporation. For example, an inner blocking portion of the second body 416 may block fluid travel from input reservoir 406 and into cell perturbation filter 408. However, when a threshold force or acceleration is reached during centrifugation, the second body 416 (and therefore the inner blocking portion of second body 416) may translate downward to allow the mixture to flow through the one or more openings in second body 416.

[0103] As described herein, the collection reservoir 406 may be contained within the centrifuge tube 450. As shown in FIGS. 4A-4B, in some embodiments, centrifuge tube 450 may comprise an insert or other component configured to raise the bottom surface of the centrifuge tube 450 and therefore decrease the travel distance of the mixture from the cell perturbation filter 408 to the collection reservoir 406.

[0104] FIGS. 5A-5C illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 5A-5C may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A-1B, FIGS. 2A-2C, FIGS. 3A-3C, and / or FIGS. 4A-4B. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 502 and into the cell perturbation filter 508. Moreover, the centrifuge device may comprise a housing 500 comprising an input reservoir 502 and a filter receiving portion 504. The housing 500 may be configured to be disposed within a centrifuge tube 550. The centrifuge device may comprise a collection reservoir 506 contained within the housing 500 and / or centrifuge tube 550. The centrifuge device may comprise a cell perturbation filter 508 disposed within the filter receiving portion 504 of the housing 500.

[0105] In some embodiments, housing 500 may be made up of one or more individual components, or bodies. For example, housing 500 may comprise a first body 514, a second body 516, and a third body 518. As shown, the first body 514 may comprise input reservoir502, and the second body 516 may be configured to block flow of the mixture from input reservoir 502 before centrifugation. In some embodiments, filter receiving portion 504 may comprise a separate body disposed between first body 514 and second body 516. Thus, cell perturbation filter 508 may be disposed between first body 514 and second body 516 such that cell perturbation filter 508 can be in fluid communication with input reservoir 502 and collection reservoir 506.

[0106] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 502 into cell perturbation filter 508. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 502 when centrifuged to allow the mixture to flow into cell perturbation filter 508. The spring valve may be configured to release the mixture comprising cells from input reservoir 502 when a threshold force or acceleration in the centrifuge device is reached. As described herein, third body 518 may be attachable (e.g., via threads) to first body 514 to tune a release acceleration or force of the spring valve comprising spring 513. The extent to which first body 514 and third body 518 are threaded together may dictate the release force of the spring valve.

[0107] The centrifuge device illustrated in FIGS. 5A-5C may differ from that illustrated at least in FIGS. 3A-3C and / or FIGS. 4A-4B in that the centrifuge device may comprise one or more pins 520 configured to connect first body 514 and the separate body comprising filter receiving portion 504. It is to be understood that the connection between components within the centrifuge device, whether it be the first body 514 and the filter receiving portion 504, or any other body, may be other types of connections such as a snap-fit, press-fit, threads, etc.

[0108] As shown in FIG. 5C, in some embodiments, the distal portion 502b of input reservoir 502 may comprise a flared portion configured to facilitate the flow of the mixture from the input reservoir 502 into the cell perturbation filter 508. An outward flare at the end of distal portion 502b of input reservoir 502 may allow the mixture to engage with the full surface area of cell perturbation filter 508. However, such a feature may be optional in the centrifuge devices described herein. For example, the size of the cell perturbation filter 508 incorporated in the centrifuge devices provided herein may alternatively be varied to coincide with a cross-sectional dimension of the distal portion 502b of the input reservoir 502.

[0109] In some embodiments, the centrifuge devices described herein may comprise one or more seals, such as “o-rings” disposed between different components of the housing. Forexample, as shown at least in FIG. 5C, the housing 500 may comprise one or more o-rings disposed distal and proximal to cell perturbation filter 508. Said o-rings may be optional; however, including seals such as the o-rings may be beneficial in enhancing the interface(s) between various components of the housing 500.

[0110] FIGS. 6A-6D illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 6A-6D may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A-1B, FIGS. 2A-2C, FIGS. 3A-3C, FIGS. 4A-4B, and / or FIGS. 5A-5C. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 602 and into the cell perturbation filter 608. Moreover, the centrifuge device may comprise a housing 600 comprising an input reservoir 602 and a filter receiving portion 604. The housing 600 may be configured to be disposed within a centrifuge tube 650. The centrifuge device may comprise a collection reservoir 606 contained within the housing 600 and / or centrifuge tube 650. The centrifuge device may comprise a cell perturbation filter 608 disposed within the filter receiving portion 604 of the housing 600. In some embodiments, housing 600 may be made up of one or more individual components, or bodies. For example, housing 600 may comprise a first body 614, a second body 616, and a third body 618. As shown, the first body 614 may comprise input reservoir 602 and the second body 616 may be configured to block flow of the mixture from input reservoir 602 before centrifugation.

[0111] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 602 into cell perturbation filter 608. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 602 when centrifuged to allow the mixture to flow into cell perturbation filter 608. The spring valve may be configured to release the mixture comprising cells from input reservoir 602 when a threshold force or acceleration in the centrifuge device is reached. As described herein, third body 618 may be attachable (e.g., via threads) to first body 614 to tune a release acceleration or force of the spring valve comprising spring 613. The extent to which first body 614 and third body 618 are threaded together may dictate the release force of the spring valve. Accordingly, the spring valve may be adjusted (e.g., by adjusting the threading of first body 614 and third body 618) to modify the release force of the spring valve. The spring valve may additionally or alternatively be adjusted by adjusting the spring constant of the spring valve.

[0112] The centrifuge device illustrated in FIGS. 6A-6D may differ from that illustrated at least in FIGS. 3A-3C, FIGS. 4A-4B, and / or FIGS. 5A-5C in that the third body 618 may comprise the filter receiving portion 604. Thus, cell perturbation filter 608 may be disposed within the third body 618. In some embodiments, the second body may be disposed between first body 614 and the cell perturbation filter 608 disposed within third body 618. Thus, second body 616 may comprise one or more openings configured to fluidly connect the input reservoir 602 to cell perturbation filter 608. Specifically, the device may function such that the second body 616 initially blocks the flow of the mixture from input reservoir 602 and into cell perturbation filter 608. However, upon reaching a threshold force or acceleration during centrifugation, the second body 616 may be configured to translate vertically downward to allow the mixture to flow through the one or more openings in the second body 616 and into the cell perturbation filter 608.

[0113] FIGS. 7A-7E illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 7A-7E may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A-1B, FIGS. 2A-2C, FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, and / or FIGS. 6A-6D. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 702 and into the cell perturbation filter 708. Moreover, the centrifuge device may comprise a housing 700 comprising an input reservoir 702 and a filter receiving portion 704. The housing 700 may be configured to be disposed within a centrifuge tube 750. The centrifuge device may comprise a collection reservoir 706 contained within the housing 700 and / or centrifuge tube 750. The centrifuge device may comprise a cell perturbation filter 708 disposed within the filter receiving portion 704 of the housing 700. In some embodiments, housing 700 may be made up of one or more individual components, or bodies. For example, housing 700 may comprise a first body 714, a second body 716, and a third body 718. As shown, the first body 714 may comprise input reservoir 702, and the second body 716 may be configured to block flow of the mixture from input reservoir 702 before centrifugation. The third body 718 may comprise the filter receiving portion 704 and therefore the cell perturbation filter 708.

[0114] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 702 into cell perturbation filter 708. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 702 when centrifuged to allow the mixture to flow into cell perturbation filter708. The spring valve may be configured to release the mixture comprising cells from input reservoir 702 when a threshold force or acceleration in the centrifuge device is reached. The mechanics of the spring valve may be substantially the same as that which is described herein with respect to the centrifuge devices illustrated in FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A- 5C, and / or FIGS. 6A-6D. As described herein, third body 718 may be attachable (e.g., via threads) to first body 714 to tune a release acceleration or force of the spring valve comprising spring 713. The extent to which first body 714 and third body 718 are threaded together may dictate the release force of the spring valve.

[0115] The centrifuge device illustrated in FIGS. 7A-7E may differ from that illustrated at least in FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, and / or FIGS. 6A-6D in that a portion of the first body 714 may not be disposed within the second body 716. Rather, the second body 722 may act as a plunger disposed substantially proximal to spring 713 within third body 718. In conjunction with the spring valve comprising spring 713, the second body 716 may be configured to release the mixture comprising cells from input reservoir 702 to allow the mixture to flow into cell perturbation filter 708. Specifically, the second body 716 may initially block the flow of the mixture from input reservoir 702 and into cell perturbation filter 708, as shown in FIG. 7D. In some embodiments, at least a portion of the second body 716 may extend into the input reservoir 702. Upon reaching a threshold force or acceleration during centrifugation, the second body 716 may translate vertically downward to allow the mixture to flow through a fluid pathway in the third body 718 and into the cell perturbation filter 708, as shown in FIG. 7E.

[0116] FIGS. 8A-8D illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 8A-8D may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A-1B, FIGS. 2A-2C, FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, and / or FIGS. 7A-7E. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 802 and into the cell perturbation filter 808. Moreover, the centrifuge device may comprise a housing 800 comprising an input reservoir 802 and a filter receiving portion 804. The housing 800 may be configured to be disposed within a centrifuge tube 850. The centrifuge device may comprise a collection reservoir 806 contained within the housing 800 and / or centrifuge tube 850. The centrifuge device may comprise a cell perturbation filter 808 disposed within the filter receiving portion 804 of the housing 800. In some embodiments, housing 800 may be made up of one or more individual components, orbodies. For example, housing 800 may comprise a first body 814, a second body 816, and a third body 818. As shown, the first body 814 and / or a proximal portion 816b of the second body 816 may comprise input reservoir 802. The third body 818 may comprise the filter receiving portion 804 and therefore the cell perturbation filter 808. In some embodiments, the filter receiving portion 804 may be disposed between second body 816 and third body 818.

[0117] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 802 into cell perturbation filter 808. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 802 when centrifuged to allow the mixture to flow into cell perturbation filter 808. The spring valve may be configured to release the mixture comprising cells from input reservoir 802 when a threshold force or acceleration in the centrifuge device is reached. As described herein, third body 818 may be attachable (e.g., via threads) to first body 814 to tune a release acceleration or force of the spring valve comprising spring 813. The extent to which first body 814 and third body 818 are threaded together may dictate the release acceleration or force of the spring valve.

[0118] The centrifuge device illustrated in FIGS. 8A-8D may differ from that illustrated at least in FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, and / or FIGS. 7A-7E in that the first body 814 may comprise a plunger portion 814c. In some embodiments, the plunger portion 814c may extend from a proximal end of the first body 814 into the input reservoir 802 contained within the first body 814. The plunger portion 814c may be configured to block a fluid pathway 816a extending within second body 816, such that, prior to centrifugation, the mixture is contained within input reservoir 802 (e.g., within first body 814 and / or a proximal portion 816b of second body 816). Upon reaching a threshold force or acceleration during centrifugation, the second body 816 may be configured to translate vertically downward (e.g., regulated by the spring valve) to allow the mixture to flow through the fluid pathway 816b within second body 816 and into the cell perturbation filter 808, as shown in FIG. 8D. Thus, in combination with the plunger portion 814c of first body 814, the second boy 816 may be configured to block flow of the mixture from input reservoir 802 before centrifugation.

[0119] FIGS. 9A-9C illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 9A-9C may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A-1B, FIGS.2A-2C, FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, FIGS. 7A-7E, and / or FIGS. 8A-8D. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 902 and into the cell perturbation filter 908. Moreover, the centrifuge device may comprise a housing 900 comprising an input reservoir 902 and a filter receiving portion 904. The housing 900 may be configured to be disposed within a centrifuge tube 950. The centrifuge device may comprise a collection reservoir 906 contained within the housing 900 and / or centrifuge tube 950. The centrifuge device may comprise a cell perturbation filter 908 disposed within the filter receiving portion 904 of the housing 900. In some embodiments, housing 900 may be made up of one or more individual components, or bodies. For example, housing 900 may comprise a first body 914 and a second body 916. As shown, the input reservoir 902 may be contained within a portion of first body 914 and / or within second body 916. The first body 914 may comprise the filter receiving portion 904 and therefore the cell perturbation filter 908.

[0120] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 902 into cell perturbation filter 908. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 902 when centrifuged to allow the mixture to flow into cell perturbation filter 908. The spring valve may be configured to release the mixture comprising cells from input reservoir 902 when a threshold force or acceleration in the centrifuge device is reached.

[0121] The centrifuge device illustrated in FIGS. 9A-9C may differ from that illustrated at least in FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, FIGS. 7A-7E, and / or FIGS. 8A-8D in that the second body 916 may be configured to translate vertically downward (regulated by the spring valve comprising spring 913) to facilitate the flow of the mixture from the input reservoir 902 to the cell perturbation filter 908. Thus, the second body 916 may be configured to block the flow of the mixture from input reservoir 902 before centrifugation. In some embodiments, second body 916 may comprise a fluid pathway configured to fluidly connect with a fluid pathway in the first body 914 when the second body 916 translates to a predetermined position, as shown. The fluid pathway within the first body 914 may be fluidly connected to the cell perturbation filter 908.

[0122] In some embodiments, the fluid pathway in first body 914 may be configured such that the flow of the mixture into the cell perturbation filter 908 from the input reservoir 902 can be controlled. For example, the fluid pathway may comprise one or more flow-limitinggeometries configured to control the flow of fluid through the fluid pathway. Flow-restricting geometries may include but are not limited to constrictions, turns, obstacles, etc.

[0123] FIGS. 10A-10C illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 10A-10C may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A- 1B, FIGS. 2A-2C, FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, FIGS. 7A-7E, FIGS. 8A-8D, and / or FIGS. 9A-9C. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 1002 and into the cell perturbation filter 1008. Moreover, the centrifuge device may comprise a housing 1000 comprising an input reservoir 1002 and a filter receiving portion 1004. The housing 1000 may be configured to be disposed within a centrifuge tube 1050. The centrifuge device may comprise a collection reservoir 1006 contained within the housing 1000 and / or centrifuge tube 1050. The centrifuge device may comprise a cell perturbation filter 1008 disposed within the filter receiving portion 1004 of the housing 1000. In some embodiments, housing 1000 may be made up of one or more individual components, or bodies. For example, housing 1000 may comprise a first body 1014, a second body 1016, a third body 1018, and a fourth body 1022. As shown, the input reservoir 1002 may be contained within the first body 1014, and the second body 1016 may be configured to block flow of the mixture from input reservoir 1002 before centrifugation. The third body 1018 may comprise the filter receiving portion 1004 and therefore the cell perturbation filter 1008. The fourth body 1022 may receive a portion of the spring 1013. In some embodiments, the fourth body 1022 may comprise at least a portion of the collection reservoir 1006.

[0124] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 1002 into cell perturbation filter 1008. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 1002 when centrifuged to allow the mixture to flow into cell perturbation filter 1008. The spring valve may be configured to release the mixture comprising cells from input reservoir 1002 when a threshold force or acceleration in the centrifuge device is reached. As described herein, third body 1018 may be attachable (e.g., via threads) to first body 1014 to tune a release acceleration or force of the spring valve comprising spring 1013. The extent to which first body 1014 and third body 1018 are threaded together may dictate the release force of the spring valve.

[0125] The centrifuge device illustrated in FIGS. 10A-10C may differ from that illustrated at least in FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, FIGS. 7A-7E, FIGS. 8A-8D, and / or FIGS. 9A-9C in that the filter receiving portion 1004 and therefore the cell perturbation filter 1008 may be disposed between the first body 1014 comprising the input reservoir 1002 and the second body 1016. In other words, the second body 1016 may be disposed distal to filter receiving portion 1004 and therefore cell perturbation filter 1008.

[0126] FIGS. 11A-1 ID illustrate another embodiment of a centrifuge device that comprises a spring valve. The centrifuge device illustrated in FIGS. 11A-1 ID may comprise any one or more features of the centrifuge devices described above with respect to FIGS. 1A- 1B, FIGS. 2A-2C, FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, FIGS. 7A-7E, FIGS. 8A-8D, FIGS. 9A-9C, and / or FIGS. 10A-10C. For example, the spring valve may be configured to control the flow of the mixture comprising cells from the input reservoir 1102 and into the cell perturbation filter 1108. Moreover, the centrifuge device may comprise a housing 1100 comprising an input reservoir 1102 and a filter receiving portion 1104. The housing 1100 may be configured to be disposed within a centrifuge tube. The centrifuge device may comprise a collection reservoir 1106 contained within the housing 1100 and / or the centrifuge tube. The centrifuge device may comprise a cell perturbation filter 1108 disposed within the filter receiving portion 1104 of the housing 1100. In some embodiments, housing 1100 may be made up of one or more individual components, or bodies. For example, housing 1100 may comprise a first body 1114 and a second body 1116. As shown, the input reservoir 1102 may be contained within a portion of the first body 1114 and / or within a portion of the second body 1116. The second body 1116 may be configured to block flow of the mixture from input reservoir 1102 before centrifugation. The first body 1114 may comprise the filter receiving portion 1104 and therefore the cell perturbation filter 1108.

[0127] As described herein, the spring valve may control the flow of fluid (e.g., the mixture comprising cells) from input reservoir 1102 into cell perturbation filter 1108. For example, the spring valve may be configured to release the mixture comprising cells from input reservoir 1102 when centrifuged to allow the mixture to flow into cell perturbation filter 1108. The spring valve may be configured to release the mixture comprising cells from input reservoir 1102 when a threshold force or acceleration in the centrifuge device is reached.

[0128] The centrifuge device illustrated in FIGS. 11A-1 ID may differ from that illustrated at least in FIGS. 3A-3C, FIGS. 4A-4B, FIGS. 5A-5C, FIGS. 6A-6D, FIGS. 7A-7E, 1FIGS. 8A-8D, FIGS. 9A-9C, and / or FIGS. 10A-10C in that the mechanism of second body 1116 translating vertically downward may comprise rotation of the second body 1116. For example, an outer surface of second body 1116 and an inner surface of first body 1114 may comprise corresponding helical surfaces, such that second body 1116 can translate downward within first body 1114 during centrifugation. In some embodiments, the rate at which second body 1116 translates within first body 1114 during centrifugation may be regulated by the spring valve comprising spring 1113.

[0129] As shown, second body 1116 may comprise a fluid pathway. In some embodiments, first body 1114 may also comprise a fluid pathway that is fluidly connected to cell perturbation filter 1108. The fluid pathways of each of first body 1114 and second body 1116 may be configured to fluidly connect when the second body 1116 reaches a predetermined position within first body 1114. Upon reaching a predetermined position and therefore once fluidly connected, the mixture contained within input reservoir 1102 may flow into the cell perturbation filter 1108.

[0130] Although not explicitly illustrated herein, in some embodiments, the centrifuge devices described herein may additionally or alternatively comprise mechanisms configured to limit the maximum force or acceleration experienced within the cell perturbation centrifuge tube. For example, mechanisms may include fluid constricting pathways, one or more flow valves, etc. that prohibit the flow of the mixture through the centrifuge device upon reaching a maximum (e.g., second threshold) force / acceleration. Maximum force / acceleration mechanisms may additionally or alternatively be incorporated to provide consistent fluid flow through the device.

[0131] As described herein, centrifuge devices may comprise force-regulated mechanisms configured to actuate upon reaching a first threshold force / acceleration and close upon reaching a second threshold (e.g., maximum) force / acceleration. FIGS. 12A-12F illustrate a centrifuge device that comprises a plunger body 1224 configured to control the flow of the mixture comprising cells from the input reservoir 1202 and into the cell perturbation filter 1208. The centrifuge device may comprise a housing 1200 comprising an input reservoir 1202 and a filter receiving portion 1204. The housing 1200 may be configured to be disposed within a centrifuge tube 1250. The centrifuge device may comprise a collection reservoir 1206 contained within the housing 1200 and / or the centrifuge tube 1250.The centrifuge device may comprise a cell perturbation filter 1208 disposed within the filter receiving portion 1204 of the housing 1200.

[0132] The input reservoir 1202 within housing 1200 may comprise one or more constrictions. In some embodiments, the input reservoir 1202 may comprise a tapered portion disposed proximal to the one or more constrictions. In some embodiments, the plunger body 1224 may be disposed within input reservoir 1202. For example, plunger body 1224 may be sized such that it can be disposed within the tapered portion of input reservoir 1202, within the one or more constrictions, and / or within a portion of input reservoir 1202 distal to the one or more constrictions.

[0133] In some embodiments, the distal portion of input reservoir 1202 may comprise one or more latches 1215. The one or more latches 1215 may extend from a wall of input reservoir 1202 into a central portion of input reservoir 1202. The one or more latches 1215 may be configured to control translation of the plunger body 1224. For example, the one or more latches 1215 may be spring-biased, or flexible, such that the one or more latches 1215 can flex to counter movement of plunger body 1224 when the plunger body 1224 translates downward. In some embodiments, as shown at least in FIG. 2C, the centrifuge device may additionally or alternatively comprise a spring valve. The spring valve may be configured to function in a similar manner as described herein with respect to the one or more latches 1215. For example, the spring valve may comprise a spring 1213 and may be configured to control vertical translation of plunger body 1224.

[0134] In some embodiments, plunger body 1224 may be disposed at least partially within input reservoir 1202 and may be configured to release the mixture comprising cells from input reservoir 1202 to allow the mixture to flow into cell perturbation filter 1208. The plunger body 1224 may be configured to release the mixture comprising cells from input reservoir 1202 when a first threshold force or acceleration in the centrifuge is reached. The plunger body 1224 may be configured to prevent the flow of the mixture comprising cells from the input reservoir 1202 when a second threshold force / acceleration is reached.

[0135] FIGS. 12D-12F illustrate example interactions between plunger body 1224 and the one or more latches 1215. At a low force or acceleration in the centrifuge device (e.g., prior to centrifugation), the plunger body 1224 may be in a closed position, shown in FIG. 12D. In said closed position, at least a distal portion of plunger body 1224 may be configured to block fluid flow into the distal portion of input reservoir 1202 (e.g., and therefore into cellperturbation filter 1208). When the centrifuge device reaches a first threshold force / acceleration, the plunger body 1224 may translate vertically downward to a first predetermined position (e.g., at least partially controlled by the one or more latches 1215), as shown in FIG. 12E. In this position of the plunger body 1224, the mixture may be able to travel from the proximal portion of input reservoir 1202, through the one or more constrictions, and into the distal portion of input reservoir 1202 prior to traveling into the cell perturbation filter 1208. Finally, if the centrifuge device reaches a second threshold force / acceleration during centrifugation, the plunger body 1224 may be configured to translate vertically downward to a second predetermined position, as shown in FIG. 12F. In this second position of the plunger body 1224, at least a proximal portion of plunger body 1224 may be configured to block fluid flow into the distal portion of input reservoir 1202 (e.g., and therefore into cell perturbation filter 1208). The centrifuge device may fluctuate between one or more of the aforementioned positions during centrifugation, dependent on the force or acceleration experienced in the centrifuge device. Controlling the force and / or acceleration in a manner such as described above may be beneficial in ensuring the mixture comprising cells is at or around an optimal force or acceleration for cell mechanoporation.

[0136] Other types of spring valves that may be included in centrifuge devices described herein may comprise elastic elements, spring- actuated balls, plugs, etc. For example, a centrifuge device comprising a valve including an elastic element, such as a bending beam, may function by in that the beam may initially block a fluid pathway via the bending beam, and upon exceeding a threshold force / acceleration during centrifugation, the beam may bend to allow the mixture to flow through the fluid pathway to the cell perturbation filter. The bending beam may return to the initial position in the instance the strength of the beam exceeds the threshold force or acceleration. In another embodiment, a centrifuge device may comprise a ball disposed above and initially blocking the fluid pathway that can pivot between an open and closed position (e.g., in a linear fashion) to control the flow of the mixture through the fluid pathway and to the cell perturbation filter.Centrifuge Devices with Magnetic Mechanisms

[0137] In some embodiments, the centrifuge devices described herein may comprise magnetic mechanisms, such as a magnet valve. FIGS. 13A-13C illustrate a centrifuge device that comprises a magnet valve configured to control the flow of the mixture comprising cells from the input reservoir 1302 and into the cell perturbation filter 1308. For example, the magnet valve may comprise magnets 1326 and may be configured to release the mixturecomprising cells from input reservoir 1302 during centrifugation to allow the mixture to flow into cell perturbation filter 1308. The centrifuge device may comprise a housing 1300 comprising an input reservoir 1302 and a filter receiving portion 1304. The housing 1300 may be configured to be disposed within a centrifuge tube 1350. The centrifuge device may comprise a collection reservoir 1306 contained within the housing 1300 and / or the centrifuge tube 1350. The centrifuge device may comprise a cell perturbation filter 1308 disposed within the filter receiving portion 1304 of the housing 1300.

[0138] In some embodiments, the magnet valve may comprise one or more pairs of magnets 1326 configured to control movement of a plunger body 1324 disposed between the one or more pairs of magnets. For example, a first magnet 1326 may be disposed proximal to plunger body 1324 and a second magnet 1326 may be disposed distal to plunger body 1324. In some embodiments, each of the first magnet 1326 and the second magnet 1326 may be annular in shape such that they can surround at least a portion of the input reservoir 1302. Other magnet shapes sized to be incorporated within housing 1300 may also be suitable for use, as will be understood by one of ordinary skill in the art. For example, in some embodiments, the magnet valve may comprise 2, 3, 4, or more pairs of magnets 1326, configured to accommodate (e.g., surround) input reservoir 1302.

[0139] Initially during use, the magnetic force between each magnet in a pair of magnets 1326 may exceed a centripetal force experienced in the centrifuge device based on at least in part on the weight of the mixture disposed within the input reservoir 1302, the weight of plunger body 1324, and / or the weight of magnets 1326. During centrifugation, the weight of the mixture in the input reservoir 1302 and the weights of plunger body 1324 and magnets 1326 can be amplified. When during centrifugation this combination of forces exceeds the magnetic force of magnets 1326, the magnets may detach, therefore causing the plunger body 1324 (e.g., and the distal one or more magnets 1326) to translate vertically downward a predetermined distance. The force required to facilitate passage of the mixture into the cell perturbation filter 1308 may be tunable, for example, by using magnets 1326 of different strengths, placing magnets 1326 within a pair of magnets 1326 at different distances apart from one another, adding / removing other bodies (e.g., shims) between magnets within pairs of magnets 1326, etc., as will be understood by one of ordinary skill in the art.

[0140] As shown in FIGS. 13A-13C, in some embodiments, the plunger body 1324 may comprise one or more openings configured to allow fluid flow through the plunger body 1324and into cell perturbation filter 1308 upon exceeding the magnetic force. For example, the one or more openings may not initially be fluidly connected to the input reservoir 1302, however, upon the plunger body 1324 translating a predetermined distance downward, the one or more openings may fluidly connect to the input reservoir 1302 (as shown in FIG.13C). Thus, the mixture comprising cells may flow from the input reservoir 1302, through the one or more openings in plunger body 1324, and into the cell perturbation filter 1308 fluidly connected to the one or more openings. In some embodiments, the plunger body 1324 may additionally or alternatively be sized such that, upon translating downward, the mixture may be able to flow to the cell perturbation filter 1308 by flowing along one or more sides of the plunger body 1324 within the housing 1300.Centrifuge Devices with Moveable Filters

[0141] In some embodiments, the centrifuge devices described herein may comprise moveable cell perturbation filters. For example, the device may be configured such that, during centrifugation, the cell perturbation filter may translate vertically downward to allow the flow of the mixture comprising cells to pass through the cell perturbation filter. FIGS. 14A-14B illustrate an embodiment of a centrifuge device in which the cell perturbation filter 1408 is moveable. The centrifuge device may comprise a housing 1400 comprising an input reservoir 1402 and a filter receiving portion 1404. The housing 1400 may be configured to be disposed within a centrifuge tube 1450. In some embodiments, the housing 1400 may be the centrifuge tube 1450. The centrifuge device may comprise a collection reservoir 1406 contained within the housing 1400. The centrifuge device may comprise a cell perturbation filter 1408 disposed within the filter receiving portion 1404 of the housing 1400.

[0142] In some embodiments, the housing 1400 may comprise an outer body (e.g., the centrifuge tube 1450) and an inner moveable body disposed within the outer body. The outer body may comprise the input reservoir 1402. The inner moveable body may comprise at least the filter receiving portion 1404 (e.g., and therefore the cell perturbation filter 1408) and the collection reservoir 1406. For example, the cell perturbation filter 1408 disposed within filter receiving portion 1404 may be at a distal end of the inner body. In some embodiments, the filter receiving portion 1404 and cell perturbation filter 1408 may be disposed elsewhere within the body, leaving necessary space for collection reservoir 1406 proximal to the cell perturbation filter 1408.

[0143] During use of the centrifuge device comprising housing 1400, a user may initially add a mixture comprising cells to the input reservoir 1402. Following addition of the mixture to the input reservoir 1402, the inner body comprising the collection reservoir 1406 and the cell perturbation filter 1408 disposed within filter receiving portion 1404 may be placed within the input reservoir 1402 such that the inner body is positioned substantially above the mixture within the input reservoir 1402. The inner body may translate vertically downward, dictated by the force / acceleration conditions induced during centrifugation. As the body translates downward, the mixture comprising cells may pass through the cell perturbation filter 1408 and into the collection reservoir 1406 within the inner body. In some embodiments, in a final position after centrifugation, the inner body may be in substantially the same position within housing 1400 that initially comprised the input reservoir 1402, as shown in FIG. 14B.

[0144] Although not explicitly illustrated herein, in some embodiments, the centrifuge devices described herein with respect to the various Figures may comprise a plunger body configured to be disposed within the input reservoir above the mixture once the mixture has been added to the input reservoir. The plunger body may be used to maintain a consistent pressure throughout the volume of fluid during centrifugation. The plunger body may be configured to translate vertically (e.g., downward) during centrifugation. In doing so, the plunger body may aid in increasing acceleration / force through the cell constricting element during centrifugation.

[0145] Although not explicitly illustrated herein, in some embodiments, the centrifuge devices described herein with respect to the various Figures may comprise one or more timing mechanisms (e.g., timers) configured to actuate flow from the input reservoir and into the cell constricting element. For example, timers may include use of a chemical agent, sensor (e.g., accelerometer), etc. that can allow passage of the mixture into the cell constricting element when a predetermined amount of time has elapsed. For example, timing mechanisms may be configured to open a fluid pathway within the centrifuge device upon a threshold amount of time elapsing.

[0146] Although various features of centrifuge devices are described herein with reference to specific embodiments and / or figures, it is to be understood that any combination of features of the centrifuge devices described herein could be reasonably achieved and is understood to be covered in the scope of this disclosure.Characteristics of Centrifuge Devices

[0147] In some embodiments, the housing of the centrifuge devices described herein may have a volume capacity of at least 100 pL. For example, the volume capacity may be about 25 pL, 50 pL, 75 pL, 100 pL, 125 pL, 150 pL, 175 pL, 200 pL, 250 pL, 300 pL, 400 pL, 500 pL, 1000 pL or more. In the instance the housing comprises multiple components, or bodies, as described herein, the volume capacity may be reasonably achieved by each of the bodies alone or in combination, as will be understood by one of ordinary skill in the art.

[0148] In some embodiments, the housing of the centrifuge devices described herein may comprise one or more of polycarbonate, polypropylene, polyethylene, polytetrafluoroethylene (PTFE), polymethyl methacrylate (PMMA), or mixtures thereof. In some embodiments, the housing may comprise one or more metals, such as stainless steel, titanium, or mixtures thereof. In the instance the centrifuge device comprises a spring valve, as described herein, said spring valve (and spring) may comprise medical-grade metals, such as stainless steel or titanium. Each of the one or more materials selected for use in the centrifuge devices described herein may be biocompatible, at least because materials may contact the mixture comprising cells.

[0149] In some embodiments, the centrifuge tube used with the centrifuge devices described herein may comprise a conical end or a round end.

[0150] In some embodiments, the porous surface or channels of the cell constricting component comprises uniformly sized pores (and / or channels). In some embodiments, the porous surface of the cell constricting component may comprise varying pore sizes throughout the surface and / or may comprise channels of varying sizes. The pores / channels (hereinafter referred to collectively as pores for simplicity, unless stated otherwise) of the cell constricting component may comprise one or more cross-sectional shapes including but not limited to circular, rectangular (e.g., square), elliptical, triangular, or another polygonal shape. In the instance the cross-section of the pores comprises a circular shape, the diameter of each of the one or more pores may be between 0.1 pm and 20 pm. For example, the diameter of a given pore may be about 0.1 pm, 0.5 pm, 1 pm, 2 pm, 5 pm, 10 pm, 15 pm, or 20 pm. The diameter of the each of the pores of the cell constricting component may be based on the diameter of the cells in the cell suspension. For example, the diameter of each of the pores may be a smaller diameter than a cell of a cell suspension passing through the filter, such that forcing the cell through the pore due to centripetal force causes a perturbation in (ordeformation of) the membrane of the cell as the cell is constricted by the pore. The pore diameter may be between 10% and 99% of the diameter of the cells in a cell suspension, such as about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the diameter of the cells. Each of the aforementioned characteristics of pores of a porous surface are understood to be attributable to, e.g., channels of a microfluidic device. It is to be understood that reference to a diameter of a cell in the cell suspension means the diameter of the cell in the cell suspension prior to being passed through the filter, e.g., as the cell approaches the filter, unless otherwise specified.

[0151] In some embodiments, the cell constricting component of the centrifuge devices described herein may be a cell perturbation filter, such as a micro-sieved (MS) filter or a track-etched (TE) filter. The porous surface of the cell perturbation filter may comprise a range of total pores. For example, the pores may cover between about 0.1% and about 60% of the total surface area of the filter. In some embodiments, the porous surface may comprise between about l.OxlO1to about l.OxlO10pores per millimeter of surface area. The porous surface may in some embodiments comprise between about l.OxlO1to about 1.0 xlO15pores per millimeter of surface area. The cell perturbation filter may have a cross-sectional shape comprising a circular, triangular, rectangular, elliptical, or other polygonal shape.

[0152] The cell constricting component may comprise a thickness, or length, defined by the axis by which the mixture travels along as it passes through the cell constricting component. The thickness of the cell constricting component may be between about 0.1 pm and 1 mm. For example, the thickness of the cell constricting component may be less than or equal to about 0.1 pm, 0.5 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm, 500 pm, or 1 mm. In some embodiments, the thickness of the cell constricting component may be greater than or equal to about 0.1 pm, 0.5 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm, 500 pm, or 1 mm. Thus, given the thickness of the filter, the pores of the filter may comprise a 3-dimensional shape. In the instance a pore comprises a circular cross-section with a uniform diameter along the thickness of the filter, the 3-dimensional shape of the pore may comprise a cylinder. In the instance the diameter of the pore varies (e.g., increases or decreases) along the thickness of the filter, the 3-dimensional shape of the pore may be conical.

[0153] In some embodiments, the cell constricting component of the centrifuge devices described herein (e.g., a cell perturbation filter) may comprise one or more materials such as synthetic or natural polymers, polycarbonate, silicon, silicon nitride, glass, metal, alloy,cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluorethylene (PTFE), mixed cellulose ester, porcelain, and / or ceramic. The cell constricting component may comprise a coating, the coating comprising one or more materials such as Teflon, polyvinylpyrrolidone, an adhesive coating, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors that modulate cellular function, nucleic acids, lipids, carbohydrates, and / or transmembrane proteins. In some embodiments, the cell constricting component may not comprise a coating.

[0154] In some embodiments, the cell constricting component of the centrifuge devices described herein may comprise a leukocyte reduction filter or cell strainer. For example, a leukocyte reduction filter or cell strainer may be disposed within the centrifuge device upstream of a microfluidic device, filter, or other component configured to perturb the cell membrane.

[0155] In some embodiments, the centrifuge devices described herein may be provided in a system. For example, the system may comprise a centrifuge device (e.g., a housing and a cell constricting component) and a centrifuge tube. In some embodiments, the centrifuge device may be provided as already installed within the centrifuge tube. In some embodiments, the centrifuge device and the centrifuge tube may be provided separately, such that the user may be required to install (e.g., insert) the centrifuge device into the centrifuge tube. The centrifuge device and the centrifuge tube may connect via a snap-fit, press-fit, or other connection means known to one of ordinary skill in the art.

[0156] In some embodiments, the centrifuge devices described herein may be incorporated into a centrifuge tube. For example, a centrifuge tube (e.g., a cell perturbation centrifuge tube) may be provided that comprises any one or more features of the centrifuge devices described herein, as well as known standard features of a centrifuge tube. For example, the outer shape, size, volume capacity, etc. of a standard centrifuge tube (e.g., a 1.5 mL, 3 mL, 15 mL, 50 mL, or other size therebetween) may be incorporated in the cell perturbation centrifuge tubes described herein.

[0157] In some embodiments, the centrifuge tube may comprise an input reservoir, a cell constricting component disposed within a cell constricting component receiving portion, and a collection reservoir. Each of the input reservoir, cell constricting component receiving portion, cell constricting component, and collection reservoir, may comprise one or more features of the respective components of the centrifuge devices described herein. Forexample, the input reservoir may be configured to receive a mixture comprising cells. The cell constricting component may be disposed within a cell constricting component receiving portion of the centrifuge tube. The cell constricting component receiving portion may be disposed distal to the input reservoir, and the collection reservoir may be disposed distal to the cell constricting component receiving portion. Thus, the cell constricting component may be fluidly connected to the input reservoir and the collection reservoir. As described herein, the cell constricting component may be configured to perturb membranes of cells to produce a perturbed cell mixture that can be deposited into the collection reservoir during centrifugation.

[0158] As described herein, the cell perturbation centrifuge device may embody one or more of the input reservoir, cell constricting component receiving portion, and / or collection reservoir. For example, at least a portion of a wall of the centrifuge tube may comprise at least a portion of a wall of the input reservoir. In other words, the centrifuge device may comprise at least a portion of the input reservoir. In some embodiments, the cell perturbation centrifuge tube may comprise a separate housing disposed within the centrifuge tube. The separate housing can comprise at least a portion of the input reservoir (as described above with respect to the centrifuge devices illustrated in the Figures).

[0159] In a similar manner, at least a portion of a wall of the centrifuge tube may comprise at least a portion of a wall of the cell constricting component receiving portion. Stated otherwise, the centrifuge tube may comprise at least a portion of the cell constricting component receiving portion. In the instance the cell perturbation centrifuge tube comprises a housing disposed within the centrifuge tube, the housing may comprise at least a portion of the cell constricting component receiving portion. For example, the housing may comprise the cell constricting component receiving portion in addition to or instead of the input reservoir. In some embodiments, the housing may comprise one or more components, or bodies. For example, a first body may comprise at least a portion of the input reservoir, and a second body may comprise at least a portion of the cell constricting component receiving portion.

[0160] In some embodiments, at least a portion of a wall of the centrifuge tube may comprise at least a portion of a wall of the collection reservoir. In other words, the centrifuge tube may comprise at least a portion of the collection reservoir. In some embodiments, the cell perturbation centrifuge tube may comprise a separate housing, and the housing maycomprise at least a portion of the collection reservoir. An example housing of the cell perturbation centrifuge tube may comprise the collection reservoir in addition to or instead of the input reservoir and / or cell constricting component receiving portion. As described herein, the housing may comprise one or more bodies, such as a first body comprising at least a portion of the input reservoir, a second body comprising at least a portion of the cell constricting component receiving portion, and a third body comprising at least a portion of the collection reservoir. In some embodiments, one or more of the input reservoir, cell constricting component receiving portion, and collection reservoir may be incorporated in a single housing or body of a multi-body housing.

[0161] The aforementioned configurations of the input reservoir, cell constricting component receiving portion (and therefore cell constricting component), and collection reservoir within the cell perturbation centrifuge tube are not intended to be exhaustive. Rather, various configurations of the cell perturbation centrifuge tube components will be apparent to one of ordinary skill in the art based on the disclosure provided herein.

[0162] The cell perturbation centrifuge tubes described herein may comprise one or more features configured to control fluid flow to the cell constricting component, as described herein with respect to the centrifuge devices. For example, the input reservoir may comprise an elongated neck portion and / or a tapered portion configured to control the pressure head of the mixture in the input reservoir. In some embodiments, the cell perturbation centrifuge tube may comprise one or more of the pressure-regulated valves and / or magnet valves described herein with respect to the centrifuge devices, the valves configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

[0163] For example, the pressure-regulated valve may comprise a burstable membrane configured to release the mixture comprising cells from the input reservoir when a threshold pressure in the cell perturbation centrifuge tube is reached during centrifugation. In some embodiments, the pressure-regulated valve may comprise a spring valve configured to release the mixture comprising cells from the input reservoir when a threshold pressure in the cell perturbation centrifuge tube is reached.

[0164] In some embodiments, the cell perturbation centrifuge tubes may additionally or alternatively comprise a magnet valve configured to release the mixture comprising cells from the input reservoir during centrifugation to allow the mixture comprising cells to flow into the cell constricting component. The magnet valve may be configured to allow themixture comprising cells to flow into the cell constricting component when a magnetic strength of the magnet valve is exceeded.

[0165] The cell perturbation centrifuge tubes provided herein may comprise a volume capacity, one or more materials, etc. as described above with respect to the centrifuge devices. For example, the volume capacity of the cell perturbation centrifuge tube may be at least 100 pL. In some embodiments, the cell perturbation centrifuge tube may comprise one or more materials described herein with respect to the centrifuge devices, including but not limited to polycarbonate, polypropylene, polyethylene, polytetrafluoroethylene (PTFE), and / or polymethyl methacrylate (PMMA). The cell constricting component disposed within the cell perturbation centrifuge tube described herein may be a cell perturbation filter, such as a micro-sieved or a track-etched filter, as described above. Other characteristics understood to be applicable to the cell constricting component disposed in the cell perturbation centrifuge tubes provided herein are described in greater detail above.Methods for Intracellular Delivery

[0166] In some embodiments, methods for intracellular delivery of a payload to cells are described herein. For example, the centrifuge devices described herein may be used to perturb the membranes of cells to allow a payload to be delivered into the cell cytosol. FIG. 15 illustrates an exemplary method 1500 for delivering a pay load into cells using the centrifuge devices described herein.

[0167] It is to be understood that the methods for intracellular delivery provided herein may be executed using any exemplary centrifuge device described herein and / or centrifuge devices comprising combinations of features of exemplary centrifuge devices described herein. For example, method 1500 may include use of a centrifuge device that comprises a housing insertable to a centrifuge tube. Alternatively, method 1500 may include use of a centrifuge device that is embodied within a centrifuge tube. Each of these envisaged centrifuge devices and the various embodiments thereof are collectively referred to herein as cell perturbation centrifuge tubes.

[0168] The method may comprise preparing the mixture to be added into the centrifuge device. In some embodiments, the mixture may comprise cells in the form of a cell suspension (e.g., a cell sample and a buffer solution). In some embodiments, the mixture may comprise a cell suspension and a payload.

[0169] The payload, including its concentration, may be dependent on the type of cells in the cell suspension and the intended cell therapy. The pay load may include but is not limited to one or more of DNA, RNA (e.g., mRNA, siRNA, saRNA, tRNA, miRNA, etc.), proteins, small molecules, fluorophores, peptides, nanoparticles, viruses, synthetic materials, a cell lysate comprising an antigen, and / or complexes (e.g., RNP, protein CAS plus guide RNA, etc.). The aforementioned payloads may function biologically 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 combinations of the above.

[0170] The concentration of the payload in a cell suspension may vary based on the type of payload and may be less than or equal to 0.01 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL, or 2 mg / mL. In some embodiments, the concentration of the payload in a cell suspension may be greater than or equal to 0.01 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL, or 2 mg / mL.

[0171] As described herein, the cell suspension may comprise cells and a buffer solution. For example, the cells may comprise a mixture of cells in a physiological saline solution or physiological medium other than blood. The cells in the cell suspension may include but are not limited to a mixed cell population (e.g., whole blood) or a purified cell population (e.g., a purified nucleate or anucleate cell population). The cells in the cell suspension may include but are not limited to anucleate cells, (e.g., red blood cells such as erythrocytes or reticulocytes, platelets, etc.), nucleate cells (e.g., immune cells, PBMCs, T cells, NK cells, iPSCs, HSCs, etc.), liposomes, exosomes, etc. The cell suspension may comprise an aqueous solution including a 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. The cell suspension may comprise a cell concentration between about LOxlO1cells / mL and LOxlO12cells / mL.

[0172] In the instance the cell perturbation centrifuge tube used in the methods described herein comprises an assembly of a centrifuge tube and separate housing, the method may include inserting the housing into the centrifuge tube. The housing may comprise at least the input reservoir and the cell constricting component receiving portion (e.g., with a cell constricting component disposed in the cell constricting component receiving portion) of thecell perturbation centrifuge tube. Inserting the housing into the centrifuge tube may comprise inserting the housing into the centrifuge tube until a lip at a proximal end of the housing contacts a proximal end of the centrifuge tube. In some embodiments, the connection between the housing and centrifuge tube may be a press-fit, snap-fit, threaded connection, etc. The connection may be removable such that the user can remove the housing from the centrifuge tube as necessary (e.g., after centrifugation).

[0173] In some embodiments, the cell perturbation centrifuge tube may comprise an assembly of a housing and a separate centrifuge tube, as described above, however the device may be provided pre-assembled.

[0174] At step 1510, the method may comprise adding the mixture to the input reservoir of a cell perturbation centrifuge tube. Adding the mixture may comprise using a pipette or other similar tool to transfer the mixture to the input reservoir. In the instance the mixture comprises only the cell suspension, the method may additionally include adding the payload to a collection reservoir of the cell perturbation centrifuge tube. In some embodiments, the cell perturbation centrifuge tube may be provided such that the payload is already loaded (e.g., pre-loaded) in the centrifuge tube (e.g., in the collection reservoir of the centrifuge tube).

[0175] At step 1520, the method may comprise sealing the input reservoir of the centrifuge tube. Sealing the tube may comprise securing, or closing, a cap to a proximal end of the cell perturbation centrifuge tube. The cap may be a removable cap. The cap may hinge from a portion of the proximal end of the cell perturbation centrifuge tube or may be a separate unit from the cell perturbation centrifuge tube. The cap may be secured to the cell perturbation centrifuge tube, for example, via threading, a snap-fit, a press-fit, or other fitting known to one of ordinary skill in the art.

[0176] Also at step 1520, the method may comprise loading the cell perturbation centrifuge tube into a centrifuge. The centrifuge may comprise a fixed angle rotor, swing out rotor, vertical rotor, etc. that is configured to receive the cell perturbation centrifuge tube. It is to be understood that the method described herein is not limited to a single centrifuge tube. For example, the centrifuge used with the cell perturbation centrifuge tubes described herein may be configured to receive a plurality of centrifuge tubes at once, such that several cell samples in individual cell perturbation centrifuge tubes can be processed at once.

[0177] As shown in step 1525 in method 1500, the method may optionally comprise setting one or more parameters, such as a release force (e.g., relative centrifugal force, or RCF) of the cell perturbation centrifuge tube. For cell perturbation centrifuge tubes comprising a spring valve, as described above with respect to at least FIGS. 6A-6D, setting the release force of the cell perturbation tube may include adjusting the spring valve. For example, the spring valve may be composed of one or more opposing, threaded bodies, and the extent to which the bodies are threaded together may dictate the release force of the cell perturbation centrifuge tube. In another example, adjusting the spring valve can include adjusting a spring constant of the spring valve.

[0178] At step 1530, the method may comprise centrifuging, using a centrifuge, the cell perturbation centrifuge tube. Centrifuging the centrifuge tube may cause the mixture comprising cells to pass through a cell constricting component in the cell perturbation centrifuge tube, otherwise referred to as mechanoporation of the cells. Upon passing through the cell constricting component, the perturbed cell mixture may be deposited into a collection reservoir of the cell perturbation centrifuge tube.

[0179] As shown in step 1540 in method 1500, the method may optionally comprise providing operating parameters to the centrifuge prior to running the centrifuge. In some embodiments, the operating parameters may already be programmed to the centrifuge prior to execution of the methods described herein. Example operating parameters may include gravitational force, ramp-up time, and runtime of the centrifuge. The ramp-up time may define how quickly the centrifuge speeds up to reach a specified setpoint. The centrifuge may be configured to start and / or stop running automatically. In some embodiments, the centrifuge may be configured to run in accordance with the programmed parameters upon a user manually starting the centrifuge. In some embodiments, a user may be required to manually stop operation of the centrifuge. In some embodiments, the operating parameters may include operating temperature in the centrifuge. Temperature may be set during execution of the method using the centrifuge to optimize delivery of the payload into the cell.

[0180] Operating parameters for the centrifugation process described herein may be selected such that they facilitate passage of the mixture comprising cells through the cell constricting component in the cell perturbation centrifuge tube. Operating parameters that may be programmed by a user may include runtime, gravitational force (e.g., G-force or relative centrifugal force (RCF)), or acceleration, and / or ramp-up time of the centrifuge,examples of which are provided below. Additional operating parameters may include the target release RCF of the spring valve.

[0181] In some embodiments, a target G-force must be met by the centrifuge to actuate the mechanoporation process within the cell constricting component. In some embodiments, the gravitational force (e.g., G-force) may be between about 10-10,000 G. In some embodiments, the gravitational force may be between about 10-9,000 G, 10-7,500 G, 10- 5,000 G, 10-1,000 G, 10-500 G, 10-300 G, 100-10,000 G, 100-7,500 G, 100-5,000 G, 100- 1,000 G, 100-500 G, 100-300 G, 250-10,000 G, 250-7,500 G, 250-5,000 G, 250-1,000 G, 250-500 G, 500-10,000 G, 500-7,500 G, 500-5,000 G, or 500-1,000 G. The gravitational force may be less than or equal to about 10,000 G, 7,500 G, 5,000 G, 2,500 G, 1,000 G, 500 G, 300 G, or 100 G. The gravitational force may be greater than or equal to about 10 G, 50 G, 100 G, 250 G, 500 G, 1,000 G, or 2,500 G.

[0182] In some embodiments, mechanoporation of the cells may be actuated within a threshold range around a target G-force. For example, mechanoporation of cells may occur within 5% (+ / -) of a target G-force. In some embodiments, mechanoporation may be actuated within about 1%, 2%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, or 30% etc. of a target G-force.

[0183] In some embodiments, the target relative centrifugal force (RCF) of the centrifuge may be selected to enable mechanical disruption of cells via the cell constricting component and deliver the pay load to the cell while retaining cell viability. In some embodiments, the RCF may be between about 20-800 RCF. In some embodiments, the RCF may be between about 50-150 RCF, 60-130 RCF, or 80-100 RCF. In some embodiments, the RCF may be less than or equal to about 800 RCF, 750 RCF, 700 RCF, 650 RCF, 600 RCF, 550 RCF, 500 RCF, 450 RCF, 400 RCF, 350 RCF, 300 RCF, 250 RCF, 200 RCF, 180 RCF, 130 RCF, 110 RCF, 100 RCF, 80 RCF, 60 RCF, or 50 RCF. In some embodiments, the RCF may be greater than or equal to about 20 RCF, 50 RCF, 60 RCF, 80 RCF, 100 RCF, 110 RCF, 130 RCF, 180 RCF, 200 RCF, 250 RCF, 300 RCF, 350 RCF, 400 RCF, 450 RCF, 500 RCF, 550 RCF, 600 RCF, 650 RCF, 700 RCF, or 750 RCF.

[0184] In some embodiments, the target release relative centrifugal force (RCF) of the spring valve may be selected to enable release of cells to be exposed to mechanical disruption of the cells via the cell constricting component, and to deliver the payload to the cell while retaining cell viability. In some embodiments, the spring valve release RCF may besubstantially the same as the target RCF set on the centrifuge. In some embodiments, the spring valve release RCF may be between about 20-200 RCF. In some embodiments, the spring valve release RCF may be between about 50-150 RCF, 60-130 RCF, or 80-100 RCF. In some embodiments, the spring valve release RCF may be less than or equal to about 200 RCF, 180 RCF, 130 RCF, 110 RCF, 100 RCF, 80 RCF, 60 RCF, or 50 RCF. In some embodiments, the spring valve release RCF may be greater than or equal to about 20 RCF, 50 RCF, 60 RCF, 80 RCF, 100 RCF, 110 RCF, 130 RCF, or 180 RCF.

[0185] In some embodiments, the runtime may be less than or equal to 5 minutes. In some embodiments, the runtime may be less than or equal to 1 minute. For example, the runtime may be about 5 minutes, 4 minutes, 3 minutes, 2 minutes, 90 seconds, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or less. In some embodiments, the runtime may be between about 15-90s, 15-60s, 15-45s, 15-30s, 30-90s, 30-60s, 30-45s, 45- 90s, or 45-60s. In some embodiments, the runtime may be between about 1-3 min, 1-2 min, 1-1.5 min, 1.5-3 min, 1.5-2 min, or 2-3 min. The runtime may be greater than or equal to about 10s, 15s, 30s, 45s, 60s, 90s, 2 min, 3 min, or 4 min.

[0186] These parameters (e.g., centrifuge RCF / G-force, spring valve release RCF, runtime, ramp time, etc.) that govern the centrifugation process may be imperative in combination with the device design to properly transfect the cells in the mixture. For example, successful mechanoporation of the cells may require a predetermined centripetal force, spring valve release RCF, speed, and / or acceleration to be met before, during, and / or after the cells pass through the cell constricting component.

[0187] For example, the timing in which the mixture comprising cells is passed through the cell constricting component may be about 1 second. In some embodiments, the centrifuge devices described herein may be configured to pass the mixture through the cell constricting component in less than or equal to about .1 s, 0.2 s, 0.5 s, 1 s, or 2 s. At larger volume samples, the mixture may pass through the cell constricting component in more than a second. For example, the mixture may pass through the filter in 10 s, 20 s, 30 s, 45 s, 60 s, or more.

[0188] The speed of the cells through the cell constricting component to induce cell membrane perturbation may be about 1 m / s. In some embodiments, the speed of the cells may be between 10 mm / s and 100 m / s. For example, the speed of the cells may 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 100m / s. In some embodiments, the speed of the cells may 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.

[0189] The cells in the mixture may be deformed from the cell constricting component for a period of time ranging from about 1 ps to at least about 10 ms, such as about 1 ps, 10 ps, 50 ps, 100 ps, 500 ps, 1 ms, 2 ms, 5 ms, or 10 ms. The perturbation in the cell can be defined as a breach in the cell that allows material from outside the cell to move into the cell (e.g., a hole, tear, cavity, aperture, pore, break, gap, perforation, etc.). The perturbation may be caused by centripetal force, acceleration, mechanical strain, and / or shear forces. The perturbation may be in the cell membrane. The perturbation may be transient. In some embodiments, the perturbation in the cell may persist for a duration between 1 second and 5 minutes. For example, the perturbation may persist for at least 1 s, 10 s, 30 s, 1 min, 2 min, 3 min, 4 min, 5 min, or more. In some embodiments, the perturbation may persist for less than 1 s, 10 s, 30 s, 1 min, 2 min, 3 min, 4 min, or 5 min.

[0190] In some examples, the cell perturbation centrifuge tube can include one or more tapered, narrow, and / or bending fluid pathways that limit the flow of fluid through the cell perturbation centrifuge tube. Additionally or alternatively, the cell perturbation centrifuge tube can include one or more burstable membranes, springs, threaded connections, etc. that restrict the flow of fluid through the cell perturbation centrifuge tube. Examples of various elements that may influence fluidic resistance within the cell perturbation centrifuge tube are described in greater detail above. In these examples, the parameters of the centrifuge may be adjusted to account for the fluidic resistance within the centrifuge tube and in turn enable successful mechanoporation of the cells. For example, a higher RCF of the centrifuge may be used with a cell perturbation centrifuge tube having greater fluidic resistance therein to maintain the cell speed through the cell constricting component that is necessary for efficacious mechanoporation of the cell.

[0191] As mentioned above, the time elapsed to reach said target G-force, the time elapsed during mechanoporation, and / or the time elapsed after mechanoporation may also be important in determining operating parameters. For example, if too much time is spent in ramping up to the target G-force and / or ramping down from the target G-force after mechanoporation, the cell sample may pellet, which may not be desirable. In another example, the timing (and G-force) may affect the cell speed and / or forces achieved during mechanoporation and thus in turn affect the efficacy of the mechanoporation.

[0192] In some embodiments, if one or more of the gravitational force, RCF, and runtime are not met, the efficacy of the cell mechanoporation could decrease, or worse, the cell sample could be damaged. For example, as mentioned above, the cell sample may pellet in the collection reservoir, which could render the sample unusable. In some instances, incorrect operating parameters and / or device design could cause cell death, over-perturbation of the cell membrane, or inadequate perturbation of the cell membrane. Thus, it is clear that there are several variables contributing to the challenge of transfecting cell membranes using cell constricting components and centrifuge devices. However, using the cell perturbation centrifuge tubes in combination with the operating parameters provided herein, mechanoporation of cells with a high efficacy can be achieved.

[0193] Returning to method 1500, at step 1550, the method may include (after centrifugation) removing the perturbed cell mixture from the collection reservoir of the centrifuge tube. In the instance the cell perturbation centrifuge tube comprises an assembly of a housing and a centrifuge tube, the collection reservoir may be in the centrifuge tube, in the housing, or partially in the housing and the centrifuge tube. In some embodiments, removing the perturbed cell mixture from the collection reservoir may comprise using a pipette or other similar tool to remove the mixture. As would be understood by one of ordinary skill in the art, removing the perturbed cell mixture from the collection reservoir in the cell perturbation centrifuge tube may first necessitate opening the cell perturbation centrifuge tube to access the collection reservoir in the cell perturbation centrifuge tube. Opening the cell perturbation centrifuge tube may comprise removing a cap from the proximal end of the centrifuge tube.

[0194] In some embodiments, before or after removing the perturbed cell mixture from the collection reservoir, the method may include unloading the cell perturbation centrifuge tube from the centrifuge.

[0195] In some embodiments, the method may include incubating the perturbed cell mixture. The perturbed cell mixture may be incubated within the cell perturbation centrifuge tube and / or within a separate vesicle in which the perturbed cell mixture has been transported to after centrifugation. For example, the mixture may be incubated for about 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 mixture may be incubated at a temperature between 0 °C and 40 °C, such as at least 0 °C, 5 °C, 10 °C, 20 °C, 25 °C, 30 °C, 35 °C, or 40 °C. In some embodiments, the method may include washing the perturbed cell mixture prior to incubation. In some embodiments, either following incubationor immediately after collection, the method may include injecting the perturbed cell mixture to a patient to execute the intended cell therapy with the cells.

[0196] In some embodiments, the method may include disposing of the cell perturbation centrifuge tube. In some embodiments, the whole device may be disposed of. In some embodiments, one or more components of the device (e.g., in the instance the device comprises an assembly of components, such as a housing and a centrifuge tube) may be recycled and / or sterilized for reuse. In one example, the housing may be removed from the centrifuge tube, and the housing may be disposed of whereas the centrifuge tube may be disposed of in a different manner (e.g., recycled) or sterilized for reuse.

[0197] In some embodiments, the aforementioned method may be performed at a temperature between about 0°C and about 40 °C. For example, the method may be carried out at room temperature (e.g., about 20 °C), physiological temperature (e.g., about 37 °C), higher than physiological temperature (e.g., about 37 °C or more), or at a reduced temperature (e.g., about 0 °C to 4 °C).

[0198] The centrifuge devices and methods of use thereof as described herein may result in a pay load delivery after centrifugation that is between about 20-70%. For example, the payload delivery after centrifugation may be between about 30-60%, about 40-60%, or about 30-50%. In some examples, the payload delivery may be greater than or equal to 20%, 30%, 40%, 45%, 50%, 55%, 60%, or 65%. In some examples, the payload delivery may be less than or equal to about 70%, 65%, 60%, 55%, 50%, 45%, 40%, or 30%.

[0199] The centrifuge devices and methods of use thereof as described herein may result in a cell viability after centrifugation that is between about 40-90%. For example, the cell viability after centrifugation may be between about 50-80%, 50-70%, or 60-80%. In some examples, the cell viability may be greater than or equal to about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%. In some examples, the cell viability may be less than or equal to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%.EXAMPLES

[0200] The following examples are exemplary and are not intended to limit the scope of any invention or inventions described herein.

[0201] A series of experiments were performed to demonstrate the efficacy and functionality of the centrifuge devices described herein across various cell types andpayloads. In each of the following examples, the device used was the centrifuge device illustrated in FIGS. 5A-5C and described above. The centrifuge equipment used was an Eppendorf 591 OR centrifuge.Example 1: Payload Delivery to PBMCs at RCFs between 80-150 RCF

[0202] Objective: The objective of the first experiment was to demonstrate the efficacy of using a device to deliver dextran to peripheral blood mononuclear cells (PBMCs). Spring valve compression was varied within the device to release the sample at the same target RCF as that which was set on the centrifuge. The goal of the study was to determine how increasing relative centrifugal force (RCF) of the centrifuge and device fluid release in unison can affect delivery of the pay load and cellular viability.

[0203] Methods: PBMCs were isolated from a leukopak using a Ficoll-based separation method and were then frozen. PBMCs were prepared from frozen stock and suspended in Opti-mem at 20 M / mL. 3 kDa Cascade blue Dextran was mixed with cells at a total concentration of 200 pg / mL. Dextran is a fluorescently labeled sugar polymer that can be a proxy for payloads such as mRNA, CRISPR RNP, etc. because it has similar delivery behavior. Four devices were assembled, and the spring valve of each device was set to enable mechanical disruption at varied RCF values (80 RCF, 110 RCF, 130 RCF, and 150 RCF). The RCF of the spring valve was set by threading the opposing bodies of the device making up the spring valve together to varying degrees. The extent to which these opposing bodies of the device were threaded together dictated the spring valve RCF. The centrifuge was set to the same respective RCF. A silicon chip with 5-micron wide constrictions was loaded into each device. 300 pL of pre-mixed cells and payload were loaded into each device and the devices were set to deploy at the specified RCF values. The devices were spun on the centrifuge for 1 minute at the varied RCF values. After centrifugation, the cells were collected, and full growth media was added. Non-Contact (NC) and endocytosis (Endo) controls were also performed to assess starting viability and delivery from endocytosis. The NC control is a sample in which no payload was introduced to the cells, and the cells were not passed through the silicon chip. The Endo control is a sample in which the cells were exposed to the payload, but the cell mixture was not passed through the silicon chip. Flow cytometry analysis was performed using the Attune NxT Cytometer to assess Dextran delivery and cellular viability.

[0204] Results: FIG. 16A demonstrates cell viability for PBMCs spun at varying RCFs and with the spring valves set to release at RCFs between 80-150 RCF. As shown in FIG. 16A, a decrease in viability was observed as the RCF values were increased. PBMC viability ranged between about 60% and 80%, with the highest viability being observed for 80 RCF (about 80% viability), and the lowest viability being observed for 130 RCF (about 60% viability). Each of 110 RCF and 150 RCF resulted in a PBMC viability between about 65- 70%.

[0205] FIG. 16B demonstrates payload delivery for PBMCs spun at these various RCFs. As shown in FIG. 16B, an increase in 3 kDa Dextran delivery was observed as the RCF values were increased. Dextran delivery ranged between about 20% and 50%, with the highest delivery being observed for 130 RCF (about 50% delivery), and the lowest delivery being observed for 80 RCF (about 20% delivery). Each of 110 RCF and 150 RCF resulted in a Dextran delivery between about 30-40%.Example 2: Payload Delivery to iPSCs

[0206] Objective: The objective of the second experiment was to demonstrate the efficacy of using a device to deliver Dextran to induced pluripotent stem cells (iPSCs). Spring valve compression was varied within the device to release the sample at the same target RCF as that which was set on the centrifuge. The goal of the study was to determine how increasing relative centrifugal force (RCF) of the centrifuge and device fluid release in unison can affect delivery of the pay load and cellular viability.

[0207] Methods: IPSCs were cultured in 6-well plates with mTeSR Plus medium. Cells were collected and premixed with 200 pg / mL of either 3kDa or lOkDa Dextran. The final concentration of cells was 20 M / mL. 300 pL of cells and payload were loaded into four devices, and the spring valve of each device was set to deploy at one of the following RCF values: 50 RCF, 60 RCF, 80 RCF, and 100 RCF. As noted above in Example 1, the RCF of the spring valve was set by threading the opposing bodies of the device making up the spring valve together to varying degrees. A silicon chip with 10-micron wide circular constrictions was loaded into each device. The devices were spun on the centrifuge for 1 minute at the varied RCF values. After centrifugation, the cells were collected, and full growth media was added. Non-Contact (NC) and endocytosis (Endo) controls were also performed to assess starting viability and delivery from endocytosis. Flow cytometry was performed, as described with respect to Example 1, to assess Dextran delivery and cellular viability.

[0208] Results: FIG. 17A demonstrates cell viability for iPSCs spun at RCFs and with the spring valve set to release at RCFs between 50-100 RCF. As shown in FIG. 17A, a minor decrease in viability was observed as RCF values increased. iPSC viability ranged from about 50% to about 70%, with the highest viability being observed for 80 RCF (about 69% viability) and the lowest viability being observed for 100 RCF (about 51% viability). 50 RCF resulted in an iPSC viability of about 67%, and 80 RCF resulted in an iPSC viability of about 56%.

[0209] FIGS. 17B-17C demonstrate payload delivery for iPSCs spun at these various RCFs. Specifically, FIG. 17B demonstrated delivery of 3 kDa Dextran to iPSCs, and FIG. 17C demonstrates delivery of 10 kDa Dextran to iPSCs. As shown in FIGS. 17B-17C, increasing RCF caused an increase of both 3 kDa Dextran and 10 kDa Dextran delivery.

[0210] With reference to FIG. 17B, 3 kDa Dextran delivery ranged between about 30- 60%, with the highest delivery of 3 kDa Dextran being observed for 100 RCF (about 56% delivery), and the lowest delivery of 3 kDa Dextran being observed for 50 RCF (about 31% delivery). 60 RCF resulted in about 35% delivery of 3 kDa Dextran, and 80 RCF resulted in about 40% delivery of 3 kDa Dextran.

[0211] The results of 10 kDa Dextran delivery to iPSCs was similar to 3 kDa Dextran delivery. With reference to FIG. 17C, 10 kDa Dextran delivery ranged between about 30- 60%, with the highest delivery of 10 kDa Dextran being observed for 100 RCF (about 58% delivery), and the lowest delivery of 10 kDa Dextran being observed for 50 RCF (about 33% delivery). 60 RCF resulted in about 37% delivery of 10 kDa Dextran, and 80 RCF resulted in about 41% delivery of 10 kDa Dextran.Example 3: Centrifuge device loading volume & device design

[0212] Objective: The objective of the third experiment was to determine the effect of loading volume and device design on payload delivery to iPSCs. Loading volume was varied to study how increasing loading volume can affect delivery of the payload. The material of the plunger within the device was also varied to study how mass of the plunger can affect delivery of the payload.

[0213] Methods: iPSCs were cultured, prepared, and analyzed on the Attune, as previously described with reference to Examples 1 and 2. Ten devices were loaded with silicon chips with 10-micron wide constrictions and the spring valve was set to deploy at the same RCF value of 100 RCF. The centrifuge was also set to the target 100 RCF. The loadingvolumes were varied from 50-1000 pF (50 pF, 100 pF, 300 pF, 600 pF, and 1000 |aL). Half the devices were assembled with a plastic plunger having a mass of 1.0 g, and half the devices were assembled with a metal plunger having a mass of 9.4 g. All devices were spun on the centrifuge for 1 minute. After centrifugation, the cells were collected, and full growth media was added. Non-Contact (NC) and endocytosis (Endo) controls were also performed to assess starting delivery from endocytosis. Flow cytometry was performed, as described with respect to Example 1, to assess Dextran delivery.

[0214] Results: FIG. 18 demonstrates pay load delivery for iPSCs at various loading volumes between 50-1000 pL. An increase in delivery of 3 kDa Dextran was shown as loading volumes increased. Dextran delivery ranged between about 20% and 50%, with the highest delivery being observed for a loading volume of 600 pF with a metal plunger (about 50% delivery), and the lowest loading volume being observed for a loading volume of 50 pL with a metal plunger (about 20% delivery). As shown in FIG. 18, increasing the mass of the plunger caused increased delivery at higher volume conditions. A loading volume of 50 pF resulted in about the same Dextran delivery when using a metal plunger versus a plastic plunger. Similarly, a loading volume of 100 pF resulted in a Dextran delivery of about 20% for both metal and plastic plungers. A loading volume of 300 pF resulted in a Dextran delivery of about 40% using a metal plunger, and about 35% using a plastic plunger. As noted above, a loading volume of 600 pF resulted in a Dextran delivery of about 50% using a metal plunger, whereas the resulting Dextran delivery using a plastic plunger was only about 30%. A loading volume of 1000 pF resulted in a Dextran delivery of about 45% using a metal plunger, whereas the resulting Dextran delivery using a plastic plunger was only about 35%.Example 4: Payload Delivery to PBMCs at RCFs between 200-800 RCF

[0215] Objective: The objective of the fourth experiment was to see if increasing relative centrifugal force (RCF) of the centrifuge beyond the RCF deployment setting of the device caused different device performance. Spring valve compression was kept constant across samples and the centrifuge target of the RCF was set to higher values than that of the spring valve. The goal of the study was to determine how increasing relative centrifugal force (RCF) of the centrifuge while maintaining a constant RCF of the device fluid can affect delivery of the pay load and cellular viability. It was hypothesized that if the mechanism allowing flow to occur through the device functions as expected, the cells will pass through the membrane the moment the speed of the centrifuge reaches the RCF setpoint of the spring valve of thedevice. Therefore, increasing RCF of the centrifuge passed the target RCF of the device was predicted to have a negligible effect on cellular delivery and viability.

[0216] Methods: PBMCs were isolated from a leukopak using a Ficoll-based separation method and were then frozen. PBMCs were prepared from frozen stock and suspended in Opti-mem at 20 M / mL. 3kDa Dextran was mixed with cells at a total concentration of 100 pg / mL. Three devices were assembled, and the spring valve of each device was set to enable mechanical disruption at an RCF determined to be equivalent to 15 psi of air pressure, which was below or equal to 200 RCF As noted above in Examples 1 and 2, the RCF of the spring valve was set by threading the opposing bodies of the device making up the spring valve together to varying degrees. The centrifuge was set varied RCFs for the different devices (200 RCF, 400 RCF, and 800 RCF). A silicon chip with 5-micron wide constrictions was loaded into each device. 300 pL of pre-mixed cells and payload were loaded into each device and the devices were set to deploy at the specified RCF values. The devices were spun on the centrifuge for 1 minute at the varied RCF values. After centrifugation, the cells were collected, and full growth media was added. Non-Contact (NC) and endocytosis (Endo) controls were also performed to assess starting viability and delivery from endocytosis. The NC control is a sample in which no payload was introduced to the cells, and the cells were not passed through the silicon chip. The Endo control is a sample in which the cells were exposed to the payload, but the cell mixture was not passed through the silicon chip. Flow cytometry analysis was performed using the Attune NxT Cytometer to assess Dextran delivery and cellular viability.

[0217] Results: FIG. 19A demonstrates cell viability for PBMCs spun at varying RCFs and with the spring valves set to release at an RCF determined to be equivalent to 15 psi of air pressure, which was below or equal to 200 RCF. As shown in FIG. 19A, a minimal increase in viability was observed as the RCF of the centrifuge were increased. PBMC viability ranged between about 70% and 80%, with the highest viability being observed for 800 RCF (about 80% viability), and the lowest viability being observed for 200 RCF (about 70-75% viability). The 400 RCF centrifuge setting resulted in a PBMC viability between about 75-80%.

[0218] FIG. 19B demonstrates payload delivery for PBMCs spun at these various RCFs. As shown in FIG. 16B, the delivery of AF488 3kDa Dextran was substantially the same across the RCFs of 200 RCF, 400 RCF, and 800 RCF. Dextran delivery ranged betweenabout 10% and 12%. 200 RCF and 400 RCF demonstrated a delivery of about 12% delivery, whereas 800 RCF demonstrated a delivery of about 10-11%. This evidence supports the theory that varying spring valve compression within the orbital booster can cause different performance of the device. Varying target RCF of the centrifuge (without varying spring valve compression) minimally affects the performance of the device once it is beyond the RCF that the spring valve releases. This is because, once the spring valve has reached its target release force, within a few seconds the cells have moved through the mechanoporation filter and sit at the bottom of the device. All the while, the centrifuge typically takes a few minutes to ramp from 0 RCF to 800 RCF.EMBODIMENTS

[0219] The following embodiments are exemplary and are not intended to limit the scope of any invention described herein.

[0220] Embodiment 1. A centrifuge device for delivering a payload to cells, comprising: a housing configured to be disposed in a vessel, the housing comprising an input reservoir configured to receive a mixture comprising cells, and a cell constricting component receiving portion disposed distal to the input reservoir; and a cell constricting component disposed within the cell constricting component receiving portion of the housing and in fluid communication with the input reservoir and a collection reservoir, wherein the cell constricting component is configured to perturb membranes of cells and to deposit a perturbed cell mixture into the collection reservoir during centrifugation.

[0221] Embodiment 2. The centrifuge device of embodiment 1, wherein the cell constricting component comprises a cell perturbation filter or a microfluidic device comprising one or more channels.

[0222] Embodiment 3. The centrifuge device of embodiment 1 or 2, wherein the collection reservoir is connected to the cell constricting component through an opening in the housing.

[0223] Embodiment 4. The centrifuge device of embodiment 3, wherein in the opening is configured to deposit the perturbed cell mixture into an end of the centrifuge tube.

[0224] Embodiment 5. The centrifuge device of any one of embodiments 1-4, wherein the housing comprises the collection reservoir.

[0225] Embodiment 6. The centrifuge device of any one of embodiments 1-5, wherein the collection reservoir contains a payload.

[0226] Embodiment 7. The centrifuge device of any one of embodiments 1-6, wherein the input reservoir is configured to receive a mixture comprising the cells and the payload.

[0227] Embodiment 8. The centrifuge device of any one of embodiments 1-7, wherein the housing comprises a lip at a proximal end of the housing that is configured to contact a proximal end of the centrifuge tube when the housing is disposed in the centrifuge tube.

[0228] Embodiment 9. The centrifuge device of any one of embodiments 1-8, wherein the input reservoir comprises a proximal portion and a distal portion, the proximal portion comprising a cross-sectional dimension that is greater than a cross-sectional dimension of the distal portion.

[0229] Embodiment 10. The centrifuge device of embodiment 9, wherein the input reservoir comprises a tapered portion that is tapered from the proximal portion toward the distal portion of the input reservoir.

[0230] Embodiment 11. The centrifuge device of any one of embodiments 1-10, comprising a force-regulated valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

[0231] Embodiment 12. The centrifuge device of embodiment 11, wherein the force- regulated valve comprises a burstable membrane configured to release the mixture comprising cells from the input reservoir when a threshold pressure in the centrifuge device is reached during centrifugation.

[0232] Embodiment 13. The centrifuge device of embodiment 12, wherein the force- regulated valve comprises a spring valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

[0233] Embodiment 14. The centrifuge device of embodiment 13, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when centrifuged to allow the mixture comprising cells to flow into the cell constricting component.

[0234] Embodiment 15. The centrifuge device of embodiment 13 or 14, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when a threshold centripetal force in the centrifuge device is reached.

[0235] Embodiment 16. The centrifuge device of any one of embodiments 11 or 13-15, wherein the housing comprises a first body and a second body, wherein the first body comprises at least a portion of the input reservoir and the second body is configured to block flow of the mixture comprising cells from the input reservoir before centrifugation.

[0236] Embodiment 17. The centrifuge device of embodiment 16, wherein the housing comprises a third body attachable to the first body to tune a release force of the spring valve.

[0237] Embodiment 18. The centrifuge device of any one of embodiments 1-17, wherein the housing comprises a first body and a second body, wherein the second body comprises the input reservoir and is at least partially disposed within the first body comprising the cell constricting component receiving portion.

[0238] Embodiment 19. The centrifuge device of embodiment 18, wherein an inner surface of the first body and an outer surface of the second body are helical, such that the second body is configured to translate within the first body during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

[0239] Embodiment 20. The centrifuge device of any one of embodiments 1-19, comprising a plunger body disposed within the input reservoir and configured to release the mixture comprising cells from the input reservoir during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

[0240] Embodiment 21. The centrifuge device of embodiment 20, wherein the plunger body is configured to release the mixture comprising cells from the input reservoir when a first threshold centripetal force in the centrifuge device is reached and prevent flow of the mixture comprising cells from the input reservoir when a second threshold centripetal force is reached.

[0241] Embodiment 22. The centrifuge device of any one of embodiments 1-21, comprising a magnet valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

[0242] Embodiment 23. The centrifuge device of embodiment 22, wherein the magnet valve is configured to release the mixture comprising cells from the input reservoir during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

[0243] Embodiment 24. The centrifuge device of embodiment 22 or 23, wherein the magnet valve comprises a fluid pathway fluidly connected to the input reservoir and the cell constricting component to allow the mixture comprising cells to flow into the cell constricting component during centrifugation.

[0244] Embodiment 25. The centrifuge device of any one of embodiments 22-24, wherein the magnet valve is configured to allow the mixture comprising cells to flow into the cell constricting component when a magnetic strength of the magnet valve is exceeded.

[0245] Embodiment 26. The centrifuge device of any one of embodiments 1-25, wherein the cell constricting component disposed in the cell constricting component receiving portion is configured to translate during centrifugation to allow passage of the mixture comprising cells through the cell constricting component and into the collection reservoir.

[0246] Embodiment 27. The centrifuge device of any one of embodiments 1-26, wherein the housing is configured to be disposed in centrifuge tubes having a volume capacity of at least 100 pL.

[0247] Embodiment 28. The centrifuge device of any one of embodiments 1-27, wherein the housing comprises polycarbonate, polypropylene, polyethylene, polytetrafluoroethylene (PTFE), and / or poly methyl methacrylate (PMMA).

[0248] Embodiment 29. The centrifuge device of any one of embodiments 1-28, wherein the cell constricting component comprises a cell perturbation filter that is a microsieved filter or a track-etched filter.

[0249] Embodiment 30. The centrifuge device of any one of embodiments 1-29, wherein the cell constricting component comprises a porous surface, each pore of the porous surface comprising a diameter between 1 pm and 15 pm.

[0250] Embodiment 31. The centrifuge device of embodiment 30, wherein the porous surface comprises uniformly sized pores.

[0251] Embodiment 32. The centrifuge device of embodiment 30 or 31, wherein the diameter of the pores is between 10% and 70% of a diameter of the cells.

[0252] Embodiment 33. The centrifuge device of any one of embodiments 1-32, wherein the cell constricting component comprises a thickness between 0.1 pm and 1 mm.

[0253] Embodiment 34. The centrifuge device of any one of embodiments 1-33, wherein the cell constricting component comprises silicon nitride, polycarbonate, silicon, glass, or another polymer.

[0254] Embodiment 35. The centrifuge device of any one of embodiments 1-34, wherein the cell constricting component comprises a leukocyte reduction filter or cell strainer.

[0255] Embodiment 36. A system for delivering a payload to cells, comprising: the centrifuge device of any one of embodiments 1-35; and a centrifuge tube.

[0256] Embodiment 37. A cell perturbation centrifuge tube for delivering a pay load to cells, comprising: a centrifuge tube comprising an input reservoir configured to receive a mixture comprising cells, a cell constricting component receiving portion disposed distal to the input reservoir, and a collection reservoir disposed distal to the cell constricting component receiving portion; and a cell constricting component disposed within the cell constricting component receiving portion of the centrifuge tube and fluidly connected to the input reservoir and the collection reservoir, wherein the cell constricting component is configured to perturb membranes of cells and to deposit a perturbed cell mixture into the collection reservoir during centrifugation.

[0257] Embodiment 38. The cell perturbation centrifuge tube of embodiment 37, wherein the cell constricting component comprises a cell perturbation filter or a microfluidic device comprising one or more channels.

[0258] Embodiment 39. The cell perturbation centrifuge tube of embodiment 37 or 38, wherein at least a portion of a wall of the centrifuge tube comprises at least a portion of a wall of the input reservoir.

[0259] Embodiment 40. The cell perturbation centrifuge tube of any one of embodiments 37-39, comprising a housing disposed within the centrifuge tube and comprising at least a portion of the input reservoir.

[0260] Embodiment 41. The cell perturbation centrifuge tube of any one of embodiments 37-40, wherein at least a portion of a wall of the centrifuge tube comprises at least a portion of a wall of the cell constricting component receiving portion.

[0261] Embodiment 42. The cell perturbation centrifuge tube of any one of embodiments 37-41, comprising a housing disposed within the centrifuge tube and containing at least a portion of the cell constricting component receiving portion.

[0262] Embodiment 43. The cell perturbation centrifuge tube of any one of embodiments 37-42, wherein at least a portion of a wall of the centrifuge tube comprises at least a portion of a wall of the collection reservoir.

[0263] Embodiment 44. The cell perturbation centrifuge tube of any one of embodiments 37-43, comprising a housing disposed within the centrifuge tube and containing at least a portion of the collection reservoir.

[0264] Embodiment 45. The cell perturbation centrifuge tube of any one of embodiments 37-44, wherein the collection reservoir contains a pay load.

[0265] Embodiment 46. The cell perturbation centrifuge tube of any one of embodiments 37-45, wherein the input reservoir is configured to receive a mixture comprising the cells and the payload.

[0266] Embodiment 47. The cell perturbation centrifuge tube of any one of embodiments 37-46, wherein the input reservoir comprises a proximal portion and a distal portion, the proximal portion comprising a cross-sectional dimension that is greater than a cross-sectional dimension of the distal portion.

[0267] Embodiment 48. The cell perturbation centrifuge tube of embodiment 47, wherein the input reservoir comprises a tapered portion that is tapered from the proximal portion toward the distal portion of the input reservoir.

[0268] Embodiment 49. The cell perturbation centrifuge tube of any one of embodiments 37-48, comprising a force-regulated valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

[0269] Embodiment 50. The cell perturbation centrifuge tube of embodiment 49, wherein the force-regulated valve comprises a burstable membrane configured to release the mixture comprising cells from the input reservoir when a threshold pressure in the cell perturbation centrifuge tube is reached during centrifugation.

[0270] Embodiment 51. The cell perturbation centrifuge tube of embodiment 49, wherein the force-regulated valve comprises a spring valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

[0271] Embodiment 52. The cell perturbation centrifuge tube of embodiment 51, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when centrifuged to allow the mixture comprising cells to flow into the cell constricting component.

[0272] Embodiment 53. The cell perturbation centrifuge tube of embodiment 51 or 52, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when a threshold centripetal force in the cell perturbation centrifuge tube is reached.

[0273] Embodiment 54. The cell perturbation centrifuge tube of any one of embodiments 37-53, comprising a magnet valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

[0274] Embodiment 55. The cell perturbation centrifuge tube of embodiment 54, wherein the magnet valve is configured to release the mixture comprising cells from the input reservoir during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

[0275] Embodiment 56. The cell perturbation centrifuge tube of embodiment 54 or 55, wherein the magnet valve comprises a fluid pathway fluidly connected to the input reservoir and the cell constricting component to allow the mixture comprising cells to flow into the cell constricting component during centrifugation.

[0276] Embodiment 57. The cell perturbation centrifuge tube of any one of embodiments 54-56, wherein the magnet valve is configured to allow the mixture comprising cells to flow into the cell constricting component when a magnetic strength of the magnet valve is exceeded.

[0277] Embodiment 58. The cell perturbation centrifuge tube of any one of embodiments 37-57, having a volume capacity of at least 100 pL.

[0278] Embodiment 59. The cell perturbation centrifuge tube of any one of embodiments 37-58, comprising one or more of polycarbonate, polypropylene, polyethylene, polytetrafluoroethylene (PTFE), and / or poly methyl methacrylate (PMMA).

[0279] Embodiment 60. The cell perturbation centrifuge tube of any one of embodiments 37-59, wherein the cell constricting component comprises a cell perturbation filter that is a micro-sieved filter or track-etched filter.

[0280] Embodiment 61. The cell perturbation centrifuge tube of any one of embodiments 37-60, wherein the cell constricting component comprises a porous surface, each pore of the porous surface comprising a diameter between 1 pm and 15 pm.

[0281] Embodiment 62. The cell perturbation centrifuge tube of embodiment 61, wherein the porous surface comprises uniformly sized pores.

[0282] Embodiment 63. The cell perturbation centrifuge tube of embodiment 61 or 62, wherein the diameter of the pores is between 10% and 70% of a diameter of the cells.

[0283] Embodiment 64. The cell perturbation centrifuge tube of any one of embodiments 37-63, wherein the cell constricting component comprises a thickness between 0.1 pm and 1 mm.

[0284] Embodiment 65. The cell perturbation centrifuge tube of any one of embodiments 37-64, wherein the cell constricting component comprises silicon nitride, polycarbonate, silicon, glass, or another polymer.

[0285] Embodiment 66. The cell perturbation centrifuge tube of any one of embodiments 37-65, wherein the cell constricting component comprises a leukocyte reduction filter or cell strainer.

[0286] Embodiment 67. A method for delivering a payload into cells, comprising: adding a mixture comprising cells to an input reservoir of a cell perturbation centrifuge tube; and centrifuging, using a centrifuge, the cell perturbation centrifuge tube to cause the mixture comprising cells to pass through a cell constricting component and deposit into a collection reservoir of the cell perturbation centrifuge tube as a perturbed cell mixture.

[0287] Embodiment 68. The method of embodiment 67, comprising preparing the mixture comprising cells by mixing a cell suspension and a payload.

[0288] Embodiment 69. The method of embodiment 67 or 68, comprising inserting a housing comprising at least the input reservoir into a centrifuge tube to assemble the cell perturbation centrifuge tube.

[0289] Embodiment 70. The method of embodiment 69, wherein inserting the housing into the centrifuge tube comprises inserting the housing into the centrifuge tube until a lip at a proximal end of the housing contacts a proximal end of the centrifuge tube.

[0290] Embodiment 71. The method of any one of embodiments 67-70, wherein the cell constricting component comprises a cell perturbation filter or a microfluidic device comprising one or more channels.

[0291] Embodiment 72. The method of any one of embodiments 69-71, wherein the cell constricting component is disposed in a cell constricting component receiving portion of the housing that is distal to the input reservoir.

[0292] Embodiment 73. The method of any one of embodiments 69-72, wherein the housing comprises the collection reservoir, and the collection reservoir is distal to the cell constricting component receiving portion of the housing.

[0293] Embodiment 74. The method of embodiment 72 or 73, wherein the housing comprises an opening configured to fluidly connect the cell constricting component to the collection reservoir.

[0294] Embodiment 75. The method of any one of embodiments 67-74, comprising sealing the input reservoir after the mixture is added to the input reservoir.

[0295] Embodiment 76. The method of embodiment 75, wherein sealing the input reservoir comprises securing a removable cap to a proximal end of the cell perturbation centrifuge tube.

[0296] Embodiment 77. The method of any one of embodiments 67-76, comprising loading the cell perturbation centrifuge tube into the centrifuge.

[0297] Embodiment 78. The method of any one of embodiments 67-77, wherein the centrifuge comprises a fixed angle rotor, a swing out rotor, or a vertical rotor.

[0298] Embodiment 79. The method of any one of embodiments 67-78, comprising providing operating parameters to the centrifuge that facilitate passage of the mixture through the cell constricting component.

[0299] Embodiment 80. The method of any one of embodiments 67-79, comprising unloading the cell perturbation centrifuge tube from the centrifuge.

[0300] Embodiment 81. The method of any one of embodiments 67-80, comprising removing the perturbed cell mixture from the collection reservoir of the cell perturbation centrifuge tube.

[0301] Embodiment 82. The method of any one of embodiments 67-81, wherein the cell constricting component is disposed within a cell constricting component receiving portion of the cell perturbation centrifuge tube, the cell constricting component receiving portion disposed distal to the input reservoir of the cell perturbation centrifuge tube.

[0302] Embodiment 83. The method of any one of embodiments 67-82, wherein the perturbed cell mixture has a cell viability of at least 50%.

[0303] Embodiment 84. The method of any one of embodiments 67-83, wherein the perturbed cell mixture has a pay load delivery of at least 30%.

[0304] Embodiment 85. The method of embodiment 79, wherein the operating parameters comprise a target relative centrifuge force (RCF) of the centrifuge between 50- 800 RCF.

[0305] Embodiment 86. The method of embodiment 85, wherein the target RCF of the centrifuge is between 50-200 RCF.

[0306] Embodiment 87. The method of embodiment 85 or 86, wherein centrifuging the cell perturbation centrifuge comprises centrifuging for a runtime of between 30-90 seconds.

[0307] Embodiment 88. The method of any one of embodiments 67-87, comprising setting a relative centrifuge force (RCF) of the cell perturbation centrifuge tube between 50- 200 RCF.

[0308] Embodiment 89. The method of embodiment 88, wherein setting the RCF of the cell perturbation centrifuge tube comprises adjusting a spring valve of the cell perturbation centrifuge tube.Definitions

[0309] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is 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 for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0310] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It is also to be understood that the term “and / or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It is further to be understood that the terms “includes, “including,” “comprises,” and / or “comprising,” when used herein, specify the presence of stated features, integers, steps, operations, elements, components, and / or units but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, units, and / or groups thereof.

[0311] The numerical ranges disclosed inherently support any range or value within the disclosed numerical ranges, including the endpoints, even though a precise range limitation is not stated verbatim in the specification because this disclosure can be practiced throughout the disclosed numerical ranges.Conclusion

[0312] The foregoing description, for the purpose of explanation, has been described with reference to specific embodiments. However, the illustrative discussions above are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments were chosen and described in order to best explain the principles of the techniques and their practical applications. Others skilled in the art are thereby enabled to best utilize the techniques and various embodiments with various modifications as are suited to the particular use contemplated.

[0313] Although the disclosure and examples have been fully described with reference to the accompanying figures, it is to be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications are to beunderstood as being included within the scope of the disclosure and examples as defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A centrifuge device for delivering a payload to cells, comprising: a housing configured to be disposed in a vessel, the housing comprising an input reservoir configured to receive a mixture comprising cells, and a cell constricting component receiving portion disposed distal to the input reservoir; and a cell constricting component disposed within the cell constricting component receiving portion of the housing and in fluid communication with the input reservoir and a collection reservoir, wherein the cell constricting component is configured to perturb membranes of cells and to deposit a perturbed cell mixture into the collection reservoir during centrifugation.

2. The centrifuge device of claim 1, wherein the cell constricting component comprises a cell perturbation filter or a microfluidic device comprising one or more channels.

3. The centrifuge device of claim 1 or 2, wherein the collection reservoir is connected to the cell constricting component through an opening in the housing.

4. The centrifuge device of claim 3, wherein in the opening is configured to deposit the perturbed cell mixture into an end of the centrifuge tube.

5. The centrifuge device of any one of claims 1-4, wherein the housing comprises the collection reservoir.

6. The centrifuge device of any one of claims 1-5, wherein the collection reservoir contains a payload.

7. The centrifuge device of any one of claims 1-6, wherein the input reservoir is configured to receive a mixture comprising the cells and the payload.

8. The centrifuge device of any one of claims 1-7, wherein the housing comprises a lip at a proximal end of the housing that is configured to contact a proximal end of the centrifuge tube when the housing is disposed in the centrifuge tube.

9. The centrifuge device of any one of claims 1-8, wherein the input reservoir comprises a proximal portion and a distal portion, the proximal portion comprising a cross-sectional dimension that is greater than a cross-sectional dimension of the distal portion.

10. The centrifuge device of claim 9, wherein the input reservoir comprises a tapered portion that is tapered from the proximal portion toward the distal portion of the input reservoir.

11. The centrifuge device of any one of claims 1-10, comprising a force-regulated valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

12. The centrifuge device of claim 11, wherein the force-regulated valve comprises a burstable membrane configured to release the mixture comprising cells from the input reservoir when a threshold pressure in the centrifuge device is reached during centrifugation.

13. The centrifuge device of claim 12, wherein the force-regulated valve comprises a spring valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

14. The centrifuge device of claim 13, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when centrifuged to allow the mixture comprising cells to flow into the cell constricting component.

15. The centrifuge device of claim 13 or 14, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when a threshold centripetal force in the centrifuge device is reached.

16. The centrifuge device of any one of claims 11 or 13-15, wherein the housing comprises a first body and a second body, wherein the first body comprises at least a portion of the input reservoir and the second body is configured to block flow of the mixture comprising cells from the input reservoir before centrifugation.

17. The centrifuge device of claim 16, wherein the housing comprises a third body attachable to the first body to tune a release force of the spring valve.

18. The centrifuge device of any one of claims 1-17, wherein the housing comprises a first body and a second body, wherein the second body comprises the input reservoir and is at least partially disposed within the first body comprising the cell constricting component receiving portion.

19. The centrifuge device of claim 18, wherein an inner surface of the first body and an outer surface of the second body are helical, such that the second body is configured to translate within the first body during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

20. The centrifuge device of any one of claims 1-19, comprising a plunger body disposed within the input reservoir and configured to release the mixture comprising cells from the input reservoir during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

21. The centrifuge device of claim 20, wherein the plunger body is configured to release the mixture comprising cells from the input reservoir when a first threshold centripetal force in the centrifuge device is reached and prevent flow of the mixture comprising cells from the input reservoir when a second threshold centripetal force is reached.

22. The centrifuge device of any one of claims 1-21, comprising a magnet valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

23. The centrifuge device of claim 22, wherein the magnet valve is configured to release the mixture comprising cells from the input reservoir during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

24. The centrifuge device of claim 22 or 23, wherein the magnet valve comprises a fluid pathway fluidly connected to the input reservoir and the cell constricting component to allowthe mixture comprising cells to flow into the cell constricting component during centrifugation.

25. The centrifuge device of any one of claims 22-24, wherein the magnet valve is configured to allow the mixture comprising cells to flow into the cell constricting component when a magnetic strength of the magnet valve is exceeded.

26. The centrifuge device of any one of claims 1-25, wherein the cell constricting component disposed in the cell constricting component receiving portion is configured to translate during centrifugation to allow passage of the mixture comprising cells through the cell constricting component and into the collection reservoir.

27. The centrifuge device of any one of claims 1-26, wherein the housing is configured to be disposed in centrifuge tubes having a volume capacity of at least 100 pL.

28. The centrifuge device of any one of claims 1-27, wherein the housing comprises polycarbonate, polypropylene, polyethylene, polytetrafluoroethylene (PTFE), and / or polymethyl methacrylate (PMMA).

29. The centrifuge device of any one of claims 1-28, wherein the cell constricting component comprises a cell perturbation filter that is a micro- sieved filter or a track-etched filter.

30. The centrifuge device of any one of claims 1-29, wherein the cell constricting component comprises a porous surface, each pore of the porous surface comprising a diameter between 1 pm and 15 pm.

31. The centrifuge device of claim 30, wherein the porous surface comprises uniformly sized pores.

32. The centrifuge device of claim 30 or 31, wherein the diameter of the pores is between 10% and 70% of a diameter of the cells.

33. The centrifuge device of any one of claims 1-32, wherein the cell constricting component comprises a thickness between 0.1 pm and 1 mm.

34. The centrifuge device of any one of claims 1-33, wherein the cell constricting component comprises silicon nitride, polycarbonate, silicon, glass, or another polymer.

35. The centrifuge device of any one of claims 1-34, wherein the cell constricting component comprises a leukocyte reduction filter or cell strainer.

36. A system for delivering a payload to cells, comprising: the centrifuge device of any one of claims 1-35; and a centrifuge tube.

37. A cell perturbation centrifuge tube for delivering a payload to cells, comprising: a centrifuge tube comprising an input reservoir configured to receive a mixture comprising cells, a cell constricting component receiving portion disposed distal to the input reservoir, and a collection reservoir disposed distal to the cell constricting component receiving portion; and a cell constricting component disposed within the cell constricting component receiving portion of the centrifuge tube and fluidly connected to the input reservoir and the collection reservoir, wherein the cell constricting component is configured to perturb membranes of cells and to deposit a perturbed cell mixture into the collection reservoir during centrifugation.

38. The cell perturbation centrifuge tube of claim 37, wherein the cell constricting component comprises a cell perturbation filter or a microfluidic device comprising one or more channels.

39. The cell perturbation centrifuge tube of claim 37 or 38, wherein at least a portion of a wall of the centrifuge tube comprises at least a portion of a wall of the input reservoir.

40. The cell perturbation centrifuge tube of any one of claims 37-39, comprising a housing disposed within the centrifuge tube and comprising at least a portion of the input reservoir.

41. The cell perturbation centrifuge tube of any one of claims 37-40, wherein at least a portion of a wall of the centrifuge tube comprises at least a portion of a wall of the cell constricting component receiving portion.

42. The cell perturbation centrifuge tube of any one of claims 37-41, comprising a housing disposed within the centrifuge tube and containing at least a portion of the cell constricting component receiving portion.

43. The cell perturbation centrifuge tube of any one of claims 37-42, wherein at least a portion of a wall of the centrifuge tube comprises at least a portion of a wall of the collection reservoir.

44. The cell perturbation centrifuge tube of any one of claims 37-43, comprising a housing disposed within the centrifuge tube and containing at least a portion of the collection reservoir.

45. The cell perturbation centrifuge tube of any one of claims 37-44, wherein the collection reservoir contains a payload.

46. The cell perturbation centrifuge tube of any one of claims 37-45, wherein the input reservoir is configured to receive a mixture comprising the cells and the payload.

47. The cell perturbation centrifuge tube of any one of claims 37-46, wherein the input reservoir comprises a proximal portion and a distal portion, the proximal portion comprising a cross-sectional dimension that is greater than a cross-sectional dimension of the distal portion.

48. The cell perturbation centrifuge tube of claim 47, wherein the input reservoir comprises a tapered portion that is tapered from the proximal portion toward the distal portion of the input reservoir.

49. The cell perturbation centrifuge tube of any one of claims 37-48, comprising a force- regulated valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

50. The cell perturbation centrifuge tube of claim 49, wherein the force-regulated valve comprises a burstable membrane configured to release the mixture comprising cells from the input reservoir when a threshold pressure in the cell perturbation centrifuge tube is reached during centrifugation.

51. The cell perturbation centrifuge tube of claim 49, wherein the force-regulated valve comprises a spring valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

52. The cell perturbation centrifuge tube of claim 51, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when centrifuged to allow the mixture comprising cells to flow into the cell constricting component.

53. The cell perturbation centrifuge tube of claim 51 or 52, wherein the spring valve is configured to release the mixture comprising cells from the input reservoir when a threshold centripetal force in the cell perturbation centrifuge tube is reached.

54. The cell perturbation centrifuge tube of any one of claims 37-53, comprising a magnet valve configured to control the flow of the mixture comprising cells from the input reservoir into the cell constricting component.

55. The cell perturbation centrifuge tube of claim 54, wherein the magnet valve is configured to release the mixture comprising cells from the input reservoir during centrifugation to allow the mixture comprising cells to flow into the cell constricting component.

56. The cell perturbation centrifuge tube of claim 54 or 55, wherein the magnet valve comprises a fluid pathway fluidly connected to the input reservoir and the cell constricting component to allow the mixture comprising cells to flow into the cell constricting component during centrifugation.

57. The cell perturbation centrifuge tube of any one of claims 54-56, wherein the magnet valve is configured to allow the mixture comprising cells to flow into the cell constricting component when a magnetic strength of the magnet valve is exceeded.

58. The cell perturbation centrifuge tube of any one of claims 37-57, having a volume capacity of at least 100 pL.

59. The cell perturbation centrifuge tube of any one of claims 37-58, comprising one or more of polycarbonate, polypropylene, polyethylene, polytetrafluoroethylene (PTFE), and / or polymethyl methacrylate (PMMA).

60. The cell perturbation centrifuge tube of any one of claims 37-59, wherein the cell constricting component comprises a cell perturbation filter that is a micro-sieved filter or track-etched filter.

61. The cell perturbation centrifuge tube of any one of claims 37-60, wherein the cell constricting component comprises a porous surface, each pore of the porous surface comprising a diameter between 1 pm and 15 pm.

62. The cell perturbation centrifuge tube of claim 61, wherein the porous surface comprises uniformly sized pores.

63. The cell perturbation centrifuge tube of claim 61 or 62, wherein the diameter of the pores is between 10% and 70% of a diameter of the cells.

64. The cell perturbation centrifuge tube of any one of claims 37-63, wherein the cell constricting component comprises a thickness between 0.1 pm and 1 mm.

65. The cell perturbation centrifuge tube of any one of claims 37-64, wherein the cell constricting component comprises silicon nitride, polycarbonate, silicon, glass, or another polymer.

66. The cell perturbation centrifuge tube of any one of claims 37-65, wherein the cell constricting component comprises a leukocyte reduction filter or cell strainer.

67. A method for delivering a payload into cells, comprising: adding a mixture comprising cells to an input reservoir of a cell perturbation centrifuge tube; and centrifuging, using a centrifuge, the cell perturbation centrifuge tube to cause the mixture comprising cells to pass through a cell constricting component and deposit into a collection reservoir of the cell perturbation centrifuge tube as a perturbed cell mixture.

68. The method of claim 67, comprising preparing the mixture comprising cells by mixing a cell suspension and a payload.

69. The method of claim 67 or 68, comprising inserting a housing comprising at least the input reservoir into a centrifuge tube to assemble the cell perturbation centrifuge tube.

70. The method of claim 69, wherein inserting the housing into the centrifuge tube comprises inserting the housing into the centrifuge tube until a lip at a proximal end of the housing contacts a proximal end of the centrifuge tube.

71. The method of any one of claims 67-70, wherein the cell constricting component comprises a cell perturbation filter or a microfluidic device comprising one or more channels.

72. The method of any one of claims 69-71, wherein the cell constricting component is disposed in a cell constricting component receiving portion of the housing that is distal to the input reservoir.

73. The method of any one of claims 69-72, wherein the housing comprises the collection reservoir, and the collection reservoir is distal to the cell constricting component receiving portion of the housing.

74. The method of claim 72 or 73, wherein the housing comprises an opening configured to fluidly connect the cell constricting component to the collection reservoir.

75. The method of any one of claims 67-74, comprising sealing the input reservoir after the mixture is added to the input reservoir.

76. The method of claim 75, wherein sealing the input reservoir comprises securing a removable cap to a proximal end of the cell perturbation centrifuge tube.

77. The method of any one of claims 67-76, comprising loading the cell perturbation centrifuge tube into the centrifuge.

78. The method of any one of claims 67-77, wherein the centrifuge comprises a fixed angle rotor, a swing out rotor, or a vertical rotor.

79. The method of any one of claims 67-78, comprising providing operating parameters to the centrifuge that facilitate passage of the mixture through the cell constricting component.

80. The method of any one of claims 67-79, comprising unloading the cell perturbation centrifuge tube from the centrifuge.

81. The method of any one of claims 67-80, comprising removing the perturbed cell mixture from the collection reservoir of the cell perturbation centrifuge tube.

82. The method of any one of claims 67-81, wherein the cell constricting component is disposed within a cell constricting component receiving portion of the cell perturbation centrifuge tube, the cell constricting component receiving portion disposed distal to the input reservoir of the cell perturbation centrifuge tube.

83. The method of any one of claims 67-82, wherein the perturbed cell mixture has a cell viability of at least 50%.

84. The method of any one of claims 67-83, wherein the perturbed cell mixture has a pay load delivery of at least 30%.

85. The method of claim 79, wherein the operating parameters comprise a target relative centrifuge force (RCF) of the centrifuge between 50-800 RCF.

86. The method of claim 85, wherein the target RCF of the centrifuge is between 50-200 RCF.

87. The method of claim 85 or 86, wherein centrifuging the cell perturbation centrifuge comprises centrifuging for a runtime of between 30-90 seconds.

88. The method of any one of claims 67-87, comprising setting a relative centrifuge force (RCF) of the cell perturbation centrifuge tube between 50-200 RCF.

89. The method of claim 88, wherein setting the RCF of the cell perturbation centrifuge tube comprises adjusting a spring valve of the cell perturbation centrifuge tube.

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