Systems and devices for high throughput cell and vector co-localizations

Abstract

The systems and methods of the disclosure can include an insert that can form an enclosed chamber within a well so that a mixture of cells and vectors can be uniformly spread within the enclosed chamber to a constrained height. In some examples, the system may include at least one insert. Each insert configured to be disposed within a well of a culture device. Each insert may have a first end and a second end. The second end may be configured to contact a bottom surface of the culture device. Each insert may include a chamber within the second end or the culture device may include a chamber within the bottom surface so that an enclosed chamber is formed when the insert is disposed on the bottom surface of the well. The enclosed chamber may be configured to constrain a transduction mixture to a height defined by the chamber.

Description

SYSTEMS AND DEVICES FOR HIGH THROUGHPUT CELL AND VECTOR COLOCALIZATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 466,432 filed May 15, 2023. The entirety of this application is hereby incorporated by reference for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under HL129141 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Cell culture vessels such as plates, dishes, flasks, and bags, are commonly used for transduction of cells with viral vectors or other non-viral vectors, such as lipid nanoparticles. Due to the fixed surface area of these culture vessels, parameters such as cell surface density or cell volumetric density, can be difficult to maintain as the cell number is varied. Generally, transduction performed in a 96-well plate will not linearly scale up to a larger device, such as a cell culture bag . Thus, this can result in a lack of translatability during scale up of transduction for clinical manufacturing.

[0004] Small-scale transductions, such as platforms utilizing channel-based designs of fixed microscale heights, can overcome this issue while improving cell-vector contact and minimizing wasted vector due to dead space. However, small-scale transductions performed in channel-based platforms can be cumbersome and low throughput due to the microliter volumes that must be manually loaded into tiny channels. Collection can also be difficult and may result in loss of cells.SUMMARY

[0005] Thus, there is need for devices that can allow for easy loading and collection enabling efficient, small-scale transduction while maintaining translatability with larger-scale devices.

[0006] The devices and systems according to the disclosure can include an insert that can form an enclosed chamber within a well so that a mixture of cells and vectors can uniformly spread within the enclosed chamber to a constrained height. Thus, the co-localization of cells and vector can be increased. Additionally, the systems and devices can enable a high-throughput set-up for screening, optimization, and characterization of transduction parameters simultaneously.

[0007] In some examples, the devices and systems according to the disclosure can include a system that includes at least one insert. Each insert may be configured to be disposed within a well of a culture device. Each insert may have a first end and an opposing, second end. The second end may be configured to contact a bottom surface of the culture device. In some examples, each insert may include a chamber within the second end or the culture device may include a chamber within the bottom surface, so that an enclosed chamber is formed when the insert is disposed on the bottom surface of the well. The enclosed chamber may be configured to constrain a transduction mixture to a height defined by the chamber.

[0008] In some examples, each insert may include the chamber within the second end. The chamber may be a defined depression within the second end and may be surrounded by a wall defined by a periphery region and a top surface. The enclosed chamber may be defined by the wall, the top surface, and the bottom surface.

[0009] In some examples, the insert may include a first through hole extending between a first end and a second end. The second end may be disposed within the chamber and having an opening that is in fluid communication with the chamber.

[0010] In some examples, the insert may include a protruding region that is disposed within the defined space and next to the wall. The protruding region may surround the chamber. The first through hole may be disposed within the protruding region.

[0011] In some examples, the insert may further include a second through hole. In some examples, the insert may also include a reservoir disposed above the first end of the second through hole. The first end of the second through hole may be disposed within the reservoir. In some examples, the chamber and insert may each have a circular shape.

[0012] In some examples, each insert may include a first member and a second member disposed above the first member. The first member may include the chamber and the second member may include a handle. In some examples, at least a portion of the first member and thesecond member may have different stiffness properties. For example, the second member may have a stiffness higher than at least a portion of the first member.

[0013] In some example, the system may further include the culture device and a plurality of inserts. The culture device may include a plurality of wells. In some examples, the system may also include a lid. The lid may be configured to be disposed on the culture device. The lid may include the plurality of inserts extending therefrom so that each chamber of each insert faces a bottom surface of each well so that each insert is aligned with each well when the lid is disposed on the culture device.

[0014] In some examples, the culture device may include a chamber within the bottom surface. The chamber may be a defined depression within the bottom surface. The chamber may include a surface and a wall that surrounds the surface. Each insert may include an end having a flat surface and being configured to contact the bottom surface of the well. The enclosed chamber may be defined by the wall, the end, and the surface.

[0015] In some examples, the insert may further include a through hole extending between the end and a perpendicular side surface.

[0016] In some examples, the height of the chamber may be about 50-250 pm.

[0017] In some examples, the method may include loading a droplet of a transduction mixture into a well of a culture device. The method may also include lowering an insert into a well of a culture device forming a seal between the well and the culture device and forming an enclosed chamber between the well and the insert. In this example, the enclosed chamber may constrain the transduction mixture to a height of the enclosed chamber. The method may also include incubating for an incubation period. The method may also include lifting the insert from well after the incubation period.

[0018] In some examples, the well may include a chamber and the insert may include a flat surface that engages the well. In other examples, the insert may include the chamber

[0019] In some examples, the insert may be attached to a lid, and the lid may be lowered and lifted.

[0020] In some examples, the method may include providing an insert that includes a chamber, a first through hole in communication with the chamber, a reservoir disposed above the chamber, and a second through hole extending between the chamber and reservoir. In some examples, the method may include lowering the insert into a well of a culture device forming a seal between thewell and the culture device and forming an enclosed chamber between the well and the insert. The method may also include delivering a transduction mixture to the reservoir. The method may further include delivering suction to the first through hole causing the transduction mixture to dispense to the enclosed chamber. The enclosed chamber may constrain the transduction mixture to a height of the chamber. The method may also include incubating for an incubation period. The method may also include lifting the insert from well after the incubation period.

[0021] In some examples, the methods may be performed by an automated fluid handling system.

[0022] In some examples, the methods may further include delivering a washing solution to each well, and collecting the mixture from each well for cell processing.

[0023] Additional advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure. The advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The disclosure can be better understood with the reference to the following drawings and description. The components in the figures are not necessarily to scale, emphasis being placed upon illustrating the principles of the disclosure.

[0025] Figures 1 A-B show views of an example of a system according to embodiments. Figure1A shows a top view of an example of a system for according to embodiments. Figure IB shows a side view of the system shown in Figure 1 A.

[0026] Figures 2A-B show views of the insert shown in Figures 1 A-B. Figure 2A shows a side view of the insert. Figure 2B shows a top view of the insert shown in Figure 2A.

[0027] Figures 3 A-B show views of an example of an insert according to embodiments. Figure3A shows a side view of the insert. Figure 3B shows a top view of the insert shown in Figure 3A.

[0028] Figures 4A-B show views of an example of an insert according to embodiments. Figure 4A shows a side view of the insert. Figure 4B shows a top view of the insert shown in Figure 4A.

[0029] Figures 5 A-B show views of an example of an insert according to embodiments. Figure5A shows a side view of the insert. Figure 5B shows a top view of the insert shown in Figure 5A.

[0030] Figures 6A-E show illustrative examples of a transduction method steps performed using the insert shown in Figures 5A-B. Figure 6A shows an example of the inserts placed in wells of a culture plate. Figure 6B shows an illustrative example of dispensing fluid into the insert. Figure 6C shows an illustrative example of suction being performed to load the fluid through the insert. Figure 6D shows an illustrative example of a period of incubation. Figure 6E shows an illustrative example of ending the transduction.

[0031] Figure 7 shows an example of an insert according to embodiments.

[0032] Figures 8A-C show an illustrative examples of a transduction method steps performed using the insert shown in Figure 7. Figure 8A shows an illustrative example of loading a cell in wells of a culture plate. Figure 8B shows an illustrative example of lowering a lid including the inserts for transduction. Figure 8C shows an illustrative example of ending the transduction.

[0033] Figures 9A-B show views of an example of a system according to embodiments. Figure 9A shows a side view of the system according to embodiments. Figure 9B shows a side view of the system shown in Figure 9A.

[0034] Figures 10A-C show an illustrative examples of a transduction method steps performed using the system shown in Figures 9A-B. Figure 10A shows an illustrative example of loading a cell in wells of the culture plate. Figure 10B shows an illustrative example of a period of incubation. Figure 10C shows an illustrative example of ending the transduction.

[0035] Figure 11 shows an example of a system according to embodiments.

[0036] Figures 12A-C show an illustrative examples of a transduction method steps performed using the system shown in Figure 11. Figure 12A shows an illustrative example of loading a cell in wells of the culture plate. Figure 12B shows an illustrative example of a period of incubation. Figure 12C shows an illustrative example of ending the transduction.DESCRIPTION OF THE EMBODIMENTS

[0037] In the following description, numerous specific details are set forth such as examples of specific components, devices, methods, etc., in order to provide a thorough understanding of embodiments of the disclosure. It will be apparent, however, to one skilled in the art that these specific details need not be employed to practice embodiments of the disclosure. In other instances,well-known materials or methods have not been described in detail in order to avoid unnecessarily obscuring embodiments of the disclosure. While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that there is no intent to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure. Unless otherwise defined herein, scientific and technical terms used in connection with the disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. As used herein, the term“ one or more” such as one or more members of a group of members, can be understood to encompass inter alia a reference to any one of the members, or to any two or more of the members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of the members, and up to all of the members.

[0038] The systems and devices of the disclosure can provide a comparable transduction performance as a microfluidic channel device while enabling a more efficient and easier transduction process. For example, the devices can be more easily loaded and flushed due to the lack of a need for tubing or connection to syringes and other accessories needed to perform transduction in a microfluidic device. Additionally, the cells can be washed and collected at the end of transduction directly from the culture device. Therefore, the systems and devices can enable higher throughput transduction of multiple transduction conditions for different applications associated with cell products and processing, such as screening or testing of multiple donors / cell types.

[0039] Additionally, the systems and devices of the disclosure can enable a high throughput setup, including an automated high throughput set up, for evaluating multiple transduction conditions, such as screening, optimization, and characterization of several transduction parameters (e.g., transduction enhancers, vector dosage, transduction time, etc.), multiple vector constructs, and treatments (e.g., small molecule treatments), simultaneously.

[0040] The systems and devices of the disclosure can be used in a linearly scalable transduction platform, that can evaluate transduction conditions of a small number of cells (e.g.,104-l 05) to determine transduction conditions for a large-scale transduction of cells (e g., 106-l 09). Examples of a linearly scalable transduction platform are described in concurrently filed International Patent Application entitled “Systems and Methods for Optimizing Parameters For Target Cell Transduction Outcomes” with the docket number 22118 PCT, which is incorporated herein by reference. This way, the systems and methods of the disclosure can be used to more efficiently and effectively determine the best transduction conditions and treatments for large-scale transduction, such as clinical scale manufacturing, using a small number of cells. Additionally, the systems and devices of the disclosure can be used to standardize the evaluation of multiple transduction conditions and parameters; therefore, the systems and devices of the disclosure can enable patient-specific mapping of the transduction-parameter space.

[0041] In some examples, the systems and devices of the disclosure can include an insert (also can be referred to as a “piston”) when placed within a well of a culture device forms an enclosed chamber that can uniformly spread an amount of a cell and viral vector mixture, such as a droplet of the mixture, to a constrained height defined by a chamber disposed in the insert and / or well. In some examples, a droplet of the mixture may be dispensed to each well. In some examples, a droplet of the mixture may be about 10-100 pL of the mixture. In some examples, about 20 pL of the mixture, may be dispensed. In some examples, the height may be a microscale height. In some examples, the height may be about 50-250 pm. By spreading the mixture, the co-localization of cells and vectors can be increased. Therefore, the cells can be more efficiently transduced with a viral vector, such as retrovirus, lentivirus, adenovirus, adeno-associated and / or or simple herpesvirus vector.

[0042] For example, the cell and viral vector mixture can also be referred to as transduction mixture. In some examples, the transduction mixture may include a mixture of cells, viral vectors, and reagent(s) (e.g., activators, cytokines, enhancers, etc.) that have been prepared according to one or more protocols. In some examples, the transduction mixture may be prepared using an automatic fluid handling system.

[0043] In some examples, the insert may be made of one or more gas permeable materials. In some examples, the one or more gas permeable materials may include but is not limited to polydimethylsiloxane elastomer (PDMS), other polymeric materials, others, or any combination thereof. In some examples, the insert may be made with more than one PDMS composite having different properties, for example, by changing the amount of crosslinker. For example, the insertcan be made with two or more layers of PDMS that have different stiffness properties (e.g., Elastic Modulus properties).

[0044] In some examples, the insert and associated system may be used with an automated cell processing system, such as an automated fluid handling system.

[0045] Figures 1A-12C show examples of systems and devices, and illustrative examples of methods for performing transduction using the systems and devices according to the disclosure. In some examples, the systems according to the disclosure may include an insert and a culture device. It will be understood that the systems and devices are not limited to the configuration and / or combination of features of the inserts and / or culture devices as shown and described in the figures. The systems and devices may also include any combination of embodiments of the inserts and / or culture devices as shown and described.

[0046] In some examples, the insert may include a chamber. Figures 1A-8C show examples of systems and / or devices having an insert that includes the chamber and illustrated examples of associated methods. The inserts shown and described in Figures 1 A-8C can be used with a culture device and are not limited to the culture device as shown and described in Figures 1A-B and 6A- 8C. The inserts as shown and described in Figures 1A-8C may be used with any culture device and is not limited to the six well-plate as shown and described with respect to Figures 1A-B and 6A-8C. For example, the culture device may have any number of wells (e.g., 1, 12, 24, 48, 96, 384 etc.) and different-sized well(s). The culture device may also be a different culture device. For example, the inserts as shown and described in Figure 1 A-8C may be used with any commercially available, standard, well plate. By configuring the inserts to be used with any existing culture plates, it can enable a simple workflow and high throughput setup for screening, optimization, and characterization of several parameters simultaneously. The inserts as shown and described in Figure 1A-8C may also be used with a proprietary culture device specific to an automated cell processing system, such as any commercially available fluid handling systems. It will also be understood that the culture device as shown and described in Figures 1A-8C may also be omitted.

[0047] Figures 1A and B shows an example of a culture system 100 according to some embodiments. Figure 1A shows a top view of the system 100 and Figure IB shows a side view of the system 100. In some embodiments, the system 100 may include a culture device 110 in which the cellular processes, such as transduction, can be performed on a cell sample. In some examples, the culture device 110 may be a well-plate having a number of wells 120. Each well 120 mayinclude a flat, bottom surface 122. In some examples, the system 100 may include a lid 180 configured to be disposed on the culture device 110.

[0048] In some examples, the system 100 may include an insert 200 configured to be disposed within the respective well 120. In some examples, as shown in Figures 2A and 2B, the insert 200 may include a first end 202 and an opposing, second end 204. In some examples, the insert 200 may include a chamber 230 in the second end 204. In some examples, the chamber 230 may be a defined depression having a height h within the second end 204. In some examples, the surface area of the chamber 230 may be defined by a top surface 232 and an (interior) wall 234 of a periphery region 224 of the second end 204. As shown in Figure IB, each insert 200 may be configured to be disposed within each well 120 so that a surface 222 of the periphery region 224 of the second end 204 is disposed directly on the flat, bottom surface 122 of the well 120. The surface 222 may be flat such that a seal is formed when the insert 200 is disposed on the corresponding flat, bottom surface 122.

[0049] When the insert 200 is disposed in the well 120, an enclosed chamber 130 is formed by the top surface 230, the wall 234, and the bottom surface 122. As shown in Figure IB, a surface area of the enclosed chamber 130 can be defined by the chamber 230 so that any mixture disposed within the chamber is confined by the height of the chamber 230. Due to the elasticity of the material of the insert 200 and the flatness of the surface 122 and the surface 222, the chamber 130 can be enclosed and substantially sealed to form the enclosed chamber 230 when the insert 200 is disposed within the well 120.

[0050] In some examples, the insert 200 may include a first member 220 and a second member 250 disposed above or on the first member 220. In some examples, the first member 220 may include the chamber 230. In some examples, the first member 220 may have shape that corresponds to a shape of the respective well 120. For example, the first member 220 and the well 120 may both have a circular shape. In other examples, the first member 220 may have a different shape. In some examples, the chamber 230 may have a shape corresponding to the first member 220. In this example, the chamber 230 may have a circular shape. In other examples, the chamber 230 may have a different shape.

[0051] In some examples, the second member 250 may include a handle or knob configured to enable easy handling by the user. For example, the knob or handle can be used to move the insert 200 in and out of the well 120, for example, manually by hand and / or by a machine. In someexamples, the second member 250 may have a different shape. For example, the second member 250 may have a shape specific to the component of an automatic cell processing system configured to move the insert 200.

[0052] In some examples, the insert 200 may include more than one PDMS material having different stiffness properties. By way of example, the first member 220 and the second member 250 may include PDMS materials having different stiffness properties. For example, at least a portion of the first member 220 may have a stiffness lower than the second member 250. In some examples, the entire first member 220 may have a stiffness lower than the second member 250. In other examples, a portion of the first member 220, such as portion 212, may have a stiffness lower than another portion 210 of the first member and the second member 250. In some examples, the other portion 210 and the second member 250 may have the same stiffness. In other examples, the other portion 210 and the second member 250 may have different stiffness properties.

[0053] By incorporating different stiffness properties, the low stiffness of the first member 220 can enable the insert 200 to better adhere to the well surface 122, minimizing the chance of leakage and evaporation. The higher stiffness of the second member 250 can also enable easier handling.

[0054] In some examples, the system 100 may be used for cell transduction. For example, the method of using the system 100 for cell transduction may include loading an amount of a transduction mixture in the center of the surface 122 of the well 120. Next, the insert 200 may be aligned within the well 120 over the mixture. Next, the insert 200 may be lowered, by hand or by machine, into the well 120 so that the mixture spreads out along the surface while being constrained within the enclosed chamber 130 defined by the chamber 230. These steps may be repeated for the other wells, if desired. Next, the lid 180 may be provided onto the device 110, by hand or by machine, to cover each well 120 to prevent evaporation. The system 100 may then incubate for the desired incubation period. After the incubation period ends, the lid 180 may be removed. Next, each insert 200 may be removed from the respective well 120, and each well may be filled with a washing solution, such as phosphate buffered saline (PBS). Each insert 200 may also be dipped into the washing solution in the respective well 120 to remove any residual cells adhered or attached to the insert 200. Next, the cells may be collected from each respective well 120 and processed for downstream cell culture or analysis, for example, according to any known cell processing techniques.

[0055] In some examples, a system may include an insert that includes one or more through holes. The through holes may be pathways (whether straight, angled, curved, single, etc.) that allow for movement of liquids and / or gasses.

[0056] In some examples, as shown in Figures 3A, an insert 300 may include a through hole 310 that extends between a first end 312, an opposing, second end 314, and a length therebetween. Each end may have an opening. In some examples, the through hole 310 may be disposed within the first member 220 so that the second end 314 is disposed within the chamber 230. In some examples, the through hole 310 may be configured to provide a path for air to escape as the mixture spreads across the surface 122 of the well 120 with the enclosed chamber 130. In this example, the through hole 310 may be disposed next to the periphery region 224. However, it will be understood that the through hole 310 may be disposed at other locations within the chamber 230 and / or second member 210.

[0057] In some examples, an insert may further include an additional structure configured to restrain the droplet within the chamber to minimize leaking out of the chamber due to the increased resistance. For example, as shown in Figures 4A and B, an insert 400 may further include a protruding region 410 surrounding the chamber 230. The protruding region 410 may be configured to act as a barrier. In some examples, the height of the protruding region 410 may be less than the height (A) of the chamber 230. The shape of the protruding region 410 may correspond to the shape of the chamber 230. In this example, the protruding region 410 may have a circular shape so it may be shaped like an outer ring. In some examples, the width of the protruding region 410 should be approximately 0.5 mm or less,

[0058] In some examples, as shown in Figures 4A and B, the through hole 310 can be positioned within the region 410. In some examples, the through hole 310 may be disposed in a different area within the chamber 230. In some examples, the insert 400 may include one or more additional through holes 310 disposed at different areas within the chamber 230 in addition the through hole disposed within the region 410.

[0059] In some examples, the inserts 300 and 400 may be used in transduction processes. For example, a method for transduction using the inserts 300 and 400 may be similar to the method using the insert 200 described above.

[0060] In some examples, an insert may further include a reservoir configured to load the transduction mixture, such as a droplet of the mixture, into the enclosed chamber. Figures 5A andB shows an example of an insert 500 that further includes a reservoir 510 disposed above the chamber 230. By using a reservoir, the insert 500 can reduce user error by reducing the need for sensitive handling to spread the mixture within the enclosed chamber 130. It can also enable easier loading and flushing compared to comparable microfluidic channel-based device. By including a reservoir, proper placement of the insert within the plate, such as confirming a formation of a proper seal between the insert and the surface of the well plate, before loading the mixture, reducing the risk of leakage or evaporation.

[0061] In some examples, the insert 500 may include a second through hole 520 extending between the reservoir 510 and the chamber 230. In some examples, the through hole 520 may include a first end 522 disposed within the reservoir 510, an opposing, second end 522 disposed within the chamber 230, a length therebetween. Each end may have an opening. In some examples, the second through hole 520 may be configured to deliver the mixture dispensed into the reservoir 510 to the chamber 230 / enclosed chamber 130 when disposed within a well 120. Although shown, it will be understood that the insert 500 may omit the first through hole 310.

[0062] In some examples, the insert 500 may be used for cell transduction. Figures 6A-6E show illustrative examples of a method of transducing cells using the insert 500. The method may be performed manually or by an automated cell processing system, such as an automated fluid handling system.

[0063] For example, the insert 500 may be placed within one or more wells of a culture plate. For example, as shown in the illustrative example 610 in Figure 6A, the insert 500 may be placed within one or more wells 120 so that the wall 222 of the periphery region 224 is in good contact with the bottom surface 122 of the well 120 to substantially seal off the chamber 230 thereby forming the enclosed chamber 130. Next, as shown as in the illustrative example 620 in Figure 6B, a transduction mixture 602, for example, a droplet, may be dispensed into one or more reservoirs 510. In some examples, the mixture may be the same for one or more reservoirs 510 / wells 120. In some examples, different mixtures 602 may be disposed into the reservoirs 510 so that different mixing ratios or treatments can be analyzed. In some examples, an automated fluid handling system may deliver the mixture to each reservoir 510.

[0064] In some examples, for example, as shown in the illustrative example 630 in Figure 6C, suction 632 may be applied to the through hole 310, for example, by an automated fluid handling system, if transduction is automated, or a pipette, if transduction is performed manually. Thesuction may cause the transduction mixture 602 to move from the reservoir 510 to the respective enclosed chamber 130, 230 via the respective through hole 520. Like the method described with respect to the insert 200, when the mixture 602 reaches the chamber 130, 230, the droplet spreads out along the surface but remains constrained within the enclosed chamber 130 defined by the chamber 230.

[0065] Next, as shown in the illustrative example 640 in Figure 6D, the lid 180 may be provided onto the device 110 to prevent evaporation. The system may then incubate for the desired incubation period. After the incubation period ends, the lid 180 may be removed.

[0066] Next, as shown in the illustrative example 650 in Figure 6E, each insert 500 may be removed from the well 120, and each well may be filled with a washing solution 652, such as phosphate buffered saline (PBS), manually or using an automated fluid handling system. Each insert 500 may also be dipped into the washing solution of the respective well 120 to remove any residual cells form the insert 500. In some examples, although not shown, the washing solution 652 may be delivered through the one or more through holes, such as the through hole 310 and / or the reservoir 510 / through hole 520. In some examples, the washing solution 652 may disengage the seal formed between the insert 500 and the well 120.

[0067] Next, the cells may be collected from each well 120 and processed for downstream cell culture or analysis, for example, according to any known cell processing techniques.

[0068] In some examples, the one or more inserts may be fixedly or removably disposed on a lid for the culture device. For example, the second member 250 may be omitted. For example, as shown in Figure 7, an insert 720 may include the first member 220 and each insert 720 may be configured to be fixedly or removably disposed on a lid 780. For example, each insert 720 may be configured to be removably or fixedly attached to the lid 780 using known fasteners. In some examples, the lid 780 may be specific to the culture device, such as device 110.

[0069] In some examples, the insert 720 may be used in automated or manual transduction processes. Figures 8A-C show illustrative examples of a method of transducing cells using the insert 700. The method may be performed manually or by an automated cell processing system, such as an automated fluid handling system. For example, as shown in the illustrative example 810 in Figure 8A, the method may include loading a transductions mixture 802, such as a droplet of the mixture 802, of a transduction mixture in center of surface 122 of the well 120. Next, as shown in the illustrative example 820 in Figure 8B, the device 700 may be aligned to the culture device220 so that each insert 720 aligns with the respective well 120. Next, the device 700 may be lowered into the well 120, for example, by moving the lid 780 onto the culture device 110 using an automated fluid handling system, so that the mixture 802 spreads out along the surface but remains constrained within the enclosed chamber 130 defined by the chamber 230. Next, the system may then incubate for the desired incubation period. After the incubation period ends, as shown in the illustrative example 830 in Figure 8C, the device 700 may be removed, for example, by lifting the lid 780 using an automated fluid handling system. Next, each well 120 may be filled with a washing solution, such as phosphate buffered saline (PBS). The device 700 may be lowered so that each insert 720 may also be dipped into the washing solution of the respective well 120 to remove any residual cells adhered or attached to the member 220. Next, the cells may be collected from each well 120, for example, using an automated fluid handling system, and transferred for downstream processing, such as for downstream cell culture or analysis, for example, according to any known cell processing techniques.

[0070] In some examples, a culture device may include a chamber in each well. Figures 9A- 12C show examples of systems and / or devices including the culture device having the chamber and associated methods. The culture device as shown and described in Figures 9A-12C is not limited to the six well-plate and may have any number of wells (e.g., 12, 24, 48, 96, 384, etc.). The culture device may be modified to be used by an automated cell processing system, such as any commercially available fluid handling systems.

[0071] Figures 9A and B shows an example of a culture system 900 according to some embodiments. Figure 9A shows a top view of the system 900 and Figure 9B shows a side view of the system 900. In some embodiments, the system 900 may include a culture device 910 in which the cellular processes, such as transduction, can be performed on a cell sample. In some examples, the culture device 910 may be a well-plate having a number of wells 920. Each well 920 may include a bottom surface 922. In some examples, each well 920 may include a chamber 930. In some examples, similar the chamber 230, the chamber 930 may be a defined depression having a height h. Each chamber 930 may be formed within the bottom surface 922. In some examples, the surface area of the chamber 930 may be defined by a surface 932 and an (interior) wall 934 of the respective chamber 930. The height h of the wall 934 may define the height of the chamber 930

[0072] In some examples, the system 900 may include a lid 980 configured to be disposed on the culture device 910. In some examples, the system 900 may include an insert 940 configured tobe disposed within the respective well 920. Each insert 940 may be fixedly or removably attached to the lid 980.

[0073] In some examples, as shown in Figure 9B, the insert 940 may include a first end 952, an opposing, second end 954, and a side 946 therebetween. In some examples, the first end 952 is configured to be disposed on the lid 980. The second end 944 may have a flat surface 950 and configured to face the bottom surface 922 of each well 820. The flat surface 950 of the second end 944 may be configured to directly contact the bottom surface 922 of the culture device.

[0074] This way, a seal may form when the insert 940 is disposed in the well 920 so that an enclosed chamber (960), for example, as shown in Figure 10B, can be formed by the surface 932, the wall 934, and the surface 950. As shown in Figure 10B, a surface area of the enclosed chamber 960 can be defined by the chamber 930 so that any mixture disposed within the chamber is confined by the height of the chamber 930. Due to the elasticity of the material of the insert 940 and the flatness of the surface 950 and the surface 922, the chamber 930 can be enclosed and substantially sealed to form the enclosed chamber 930 when the insert 940 is disposed within the well 920.

[0075] Figures 10A-10C show illustrative examples of a method of transducing cells using the system 900. The method may be performed manually or by an automated cell processing system, such as an automated fluid handling system. If performed by an automated cell processing system, the delivering of the transduction mixture and any solutions, the movement (e.g., lifting and lowering) of the lid 980 and insert 940, among other processes, can be performed automatically by the automated fluid handling system.

[0076] To initiate the process, shown as in the illustrative example 1010 in Figure 10A, a transduction mixture 1002, such as a droplet of the mixture 1002, may be dispensed into one or more chambers 930. In some examples, the mixture 1002 may be the same for each well 920. In some examples, different mixtures 1002 may be disposed into the wells 920 to analyze different mixing ratios or treatments. In some examples, an automated fluid handling system may deliver the mixture to each well 920.

[0077] In some examples, for example, as shown in the illustrative example 1020 in Figure 10B, the lid 980 with the inserts 940 may be lowered into the wells 920 so to evenly spread each mixture 1002 along the surface 932 while being constrained within the enclosed chamber 960 defined by the chamber 930. Next, the system 900 may then incubate for the desired incubationperiod. After the incubation period ends, the lid 980 may be removed, as shown in the illustrative example 1030 in Figure 10C. After the lid 980 is removed, although not shown, each well 920 may be fdled with a washing solution, such as phosphate buffered saline (PBS), manually or using an automated fluid handling system. Next, the cells may be collected from each well 920 and processed for downstream cell culture or analysis, for example, according to any known cell processing techniques.

[0078] In some examples, similar to inserts 300 and 400, a system may include an insert that includes one or more through holes. The through holes may be pathways (whether straight, angled, curved, single, etc.) that allow for movement of liquids and / or gasses.

[0079] In some examples, as shown in Figure 11, a system 1100 may include an insert 1140 that includes one or more through holes 1110. In this example, the through hole 1110 may be angled so as to extend between two perpendicular sides of the insert 1140. As shown in Figure 11, the through hole 1110 may include (i) a first end 1162 disposed on the second 944,and(ii) a second end 1164 disposed on the side 946, so that the through hole 1110 extends at an angle between the second end 944 and the side 946. In other examples, the through hole 1110 may be disposed at other locations within the insert 1140. In some examples, the through hole 1110 may be configured to provide a path for any overflow to escape as the droplet spreads across the surface 932 of the well 120 with the enclosed chamber 930.

[0080] Figures 12A-12C show illustrative examples of a method of transducing cells using the system 1100. The method may be performed manually or by an automated cell processing system, such as an automated fluid handling system. If performed by an automated cell processing system, the delivering of the transduction mixture and any solutions, the movement (e.g., lifting and lowering) of the lid 980 and insert 1140, among other processes, can be performed automatically by the automated fluid handling system.

[0081] To initiate the process, like the examples shown in Figures 10A-C, a transduction mixture 1202, such as a droplet, may be dispensed into one or more chambers 930 as shown in the illustrative example 1210 shown in Figure 12A. In some examples, the mixture 1202 may be the same for each well 920. In some examples, different mixtures 1202 may be disposed into the wells 920 to analyze different mixing ratios or treatments. In some examples, an automated fluid handling system may deliver the mixture to each well 920.

[0082] In some examples, for example, as shown in the illustrative example 1220 in Figure 12B, the lid 980 with the inserts 1140 may be lowered into the wells 920 so to evenly spread each mixture 1202 along the surface 932 while remaining constrained within the enclosed chamber 960 defined by the chamber 930. Any overflow of the mixture 1202 can be routed from the enclosed chamber 960 through the through hole 1110. Next, the system 1100 may then incubate for the desired incubation period. After the incubation period ends, the lid 980 may be removed, as shown in the illustrative example 1230 in Figure 12C. After the lid 980 is removed, although not shown, each well may be filled with a washing solution, such as phosphate buffered saline (PBS), manually or using an automated fluid handling system. Next, the cells may be collected from each well 920 and processed for downstream cell culture or analysis, for example, according to any known cell processing techniques.

[0083] After the cells are collected, for example, as discussed with respect to Figures 6A-E, 8A-C, and Figures 12A-C, the cells may be processed to determine transduction outcomes, such as transduction efficiency and average vector copy number (VCN). The transduction outcomes can be processed, for example, using the systems and methods described in the above incorporated International Patent Application entitled “Systems and Methods for Optimizing Parameters For Target Cell Transduction Outcomes” with the Attorney Docket Number 22118 PCT, to optimize the transduction parameters for a larger-scale device.

[0084] While the disclosure has been described in detail with reference to exemplary embodiments, those skilled in the art will appreciate that various modifications and substitutions may be made thereto without departing from the spirit and scope of the disclosure as set forth in the appended claims. For example, elements and / or features of different exemplary embodiments may be combined with each other and / or substituted for each other within the scope of this disclosure and appended claims.

Claims

CLAIMSWhat is claimed:

1. A culture system, comprising: at least one insert; each insert configured to be disposed within a well of a culture device; and each insert having a first end and an opposing, second end, the second end being configured to contact a bottom surface of the culture device;(i) each insert including a chamber within the second end or (ii) the culture device including a chamber within the bottom surface, so that an enclosed chamber is formed when the insert is disposed on the bottom surface of the well, the enclosed chamber being configured to constrain a transduction mixture to a height defined by the chamber.

2. The system according to claim 1, wherein: each insert includes the chamber within the second end; the chamber being a defined depression within the second end and surrounded by a wall defined by a periphery region and a top surface; and the enclosed chamber is defined by the wall, the top surface, and the bottom surface.

3. The system according to claims 1 or 2, wherein the insert includes a first through hole extending between a first end and a second end, the second end disposed within the chamber and having an opening that is in fluid communication with the chamber.

4. The system according to claim 3, wherein: the insert includes a protruding region that is disposed within the defined space and next to the wall, the protruding region surrounding the chamber; and the first through hole is disposed within the protruding region.

5. The system according to any of claims 1-4, wherein the insert further includes: a second through hole;a reservoir is disposed above the first end of the second through hole, the first end of the second through hole being disposed within the reservoir.

6. The system according to any of claims 1-5, wherein: the chamber is circular; and the insert has a circular shape.

7. The system according to any of claims 1-6, wherein: each insert includes a first member and a second member disposed above the first member; and the first member includes the chamber and the second member includes a handle.

8. The system according to claim 7, wherein at least a portion of the first member and the second member has different stiffness properties.

9. The system according to claim 8, wherein the second member has a stiffness higher than at least a portion of the first member.

10. The system according to any of claims 1-6, further comprising: the culture device, the culture device including a plurality of wells; the plurality of inserts; and a lid configured to be disposed on the culture device, the lid including a plurality of inserts extending therefrom so that each chamber of each insert faces a bottom surface of each well so that each insert is aligned with each well when the lid is disposed on the culture device.

11. The system according to claim 10, wherein: the culture device including a chamber within the bottom surface; the chamber being a defined depression within the bottom surface, the chamber including a surface and a wall that surrounds the surface; each insert includes an end having a flat surface and being configured to contact the bottom surface of the well; andthe enclosed chamber is defined by the wall, the end, and the surface.

12. The system according to claim 11, wherein the insert further includes a through hole extending between the end and a perpendicular side surface.

13. The system according to any of claims 1-11, wherein the height is about 50-250 pm.

14. A method for transducing cells, comprising: loading a droplet of a transduction mixture into a well of a culture device; lowering an insert into a well of a culture device forming a seal between the well and the culture device and forming an enclosed chamber between the well and the insert; the enclosed chamber constraining the transduction mixture to a height of the enclosed chamber; incubating for an incubation period; and after the incubation period, lifting the insert from well.

15. The method according to claim 14, wherein the well includes a chamber and the insert includes a flat surface that engages the well.

16. The method according to any of claims 14 or 15, wherein: the insert is attached to a lid, and the lid is lowered and lifted.

17. The method according to claim 16, wherein the insert includes a chamber.

18. A method for transducing cells, comprising: providing an insert that includes a chamber, a first through hole in communication with the chamber, a reservoir disposed above the chamber, and a second through hole extending between the chamber and reservoir; lowering the insert into a well of a culture device forming a seal between the well and the culture device and forming an enclosed chamber between the well and the insert; delivering a transduction mixture to the reservoir;delivering suction to the first through hole causing the transduction mixture to dispense to the enclosed chamber, the enclosed chamber constraining the transduction mixture to a height of the chamber; incubating for an incubation period; and after the incubation period, lifting the insert from the well.

19. The method according to any of claims 12-17, wherein the method is performed by an automated fluid handling system.

20. The method according to any of claims 12-19, further comprising: delivering a washing solution to each well; and collecting the mixture from each well for cell processing.

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