WO2024129945A2

WO2024129945A2 - Consumable for centrifugation of biological cells with a variable collection chamber for supernatant removal

Info

Publication number: WO2024129945A2
Application number: PCT/US2023/083977
Authority: WO
Inventors: Jeff Smith; Ivan RAZINKOV; Myo THU
Original assignee: Synthego Corporation
Priority date: 2022-12-16
Filing date: 2023-12-14
Publication date: 2024-06-20

Abstract

A cell centrifugation consumable and a method of using the cell centrifugation consumable is disclosed. The cell centrifugation consumable includes an inner fluid chamber comprising a tapered portion having a collection chamber at a narrow end of the tapered portion. The inner fluid chamber is configured to contain a cell solution. Upon centrifugation of the cell solution, a cell pellet forms within the collection chamber. The cell centrifugation consumable further includes a supernatant fluid port fluidly couplable to the collection chamber configured to allow transfer of supernatant from the collection chamber. The cell centrifugation consumable further includes a sensor detection line within the collection chamber configured so that a sensor can detect the presence of a supernatant or a cell pellet at the sensor detection line. The cell centrifugation consumable further includes a piston configured to translate the cell pellet within the collection chamber toward the sensor detection line.

Description

CONSUMABLE FOR CENTRIFUGATION OF BIOLOGICAL CELLS WITH A VARIABLE COLLECTION CHAMBER FOR SUPERNATANT REMOVAL

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C. §119(e), to U.S. Provisional Application No. 63/433,056, as filed December 16, 2022, the contents of which are incorporated herein by reference in its entirety.

BACKGROUND

Processing of biological samples such as cell-containing solutions often requires the sterile and cautious movement of liquids from one container (e.g., a beaker, flask, or test tube) to another. These containers are traditionally designed for manual holding and grasping. As the number of biological samples to be processed increases, automated solutions (i.e., robotics) are often used to perform repetitive tasks. However, the requirements of robotic handing of biological samples are different than the requirement for human handing of biological samples. For example, the act of decanting a solution (i.e., drawing off a liquid portion of a biological sample in a container) can be performed easily by a skilled technician using traditional containers. Nonetheless, decanting of biological samples, such as the removal of supernatant without disrupting a cell pellet, via traditional containers is not easily performed through robotic means. Therefore, there is a need for biological sample processing containers that can be easily and accurately manipulated via robotic means to facilitate procedures such as removing a supernatant from a cell pellet.

SUMMARY

Described herein is a cell centrifugation consumable. The cell centrifugation consumable includes an inner fluid chamber that includes a tapered portion having a collection chamber at a narrow end of the tapered portion, wherein the inner fluid chamber is configured to contain a cell solution, wherein upon centrifugation of the cell solution, a cell pellet forms within the collection chamber. The cell centrifugation consumable further includes a supernatant fluid port fluidly couplable to the collection chamber configured to allow transfer of supernatant from the collection chamber. The cell centrifugation consumable further includes a sensor detection line within the collection chamber configured so that a sensor can detect the presence of a supernatant or a cell

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SUBSTITUTE SHEET (RULE 26) pellet at the sensor detection line. The cell centrifugation consumable further includes a piston configured to translate the cell pellet within the collection chamber toward the sensor detection line.

Also described herein is a method for processing samples, such as cell-containing solutions, via the cell centrifugation consumable. The method may include providing the cell centrifugation consumable. The method may further include receiving a suspension of cells within the inner fluid chamber. The method may further include centrifuging the cell centrifugation consumable to concentrate the cells as a cell pellet in the collection chamber, with a supernatant remaining in the inner fluid chamber. The method may further include removing a first portion of the supernatant. The method may further include detecting the presence of a remaining supernatant at the sensor detection line. The method may further include translating the piston toward the sensor detection line until the presence of the cell pellet at the sensor detector line is detected. The method may further include removing a second portion of the remaining supernatant from the collection chamber via the supernatant fluid port.

DESCRIPTION OF THE FIGURES

The detailed description is described with reference to the accompanying figures. The use of the same reference numbers in different instances in the description and the figures may indicate similar or identical items. Various embodiments or examples of the present disclosure are disclosed in the following detailed description and the accompanying drawings. The drawings are not necessarily to scale. In general, operations of disclosed processes may be performed in an arbitrary order, unless otherwise provided in the claims.

FIG. 1 A is a drawing illustrating a perspective view of a cell centrifugation consumable, in accordance with one or more embodiments of the disclosure.

FIG. IB is a drawing illustrating a perspective cut-away view of a cell centrifugation consumable, in accordance with one or more embodiments of the disclosure.

FIG. 1C is a drawing illustrating a perspective cut-away view of a cell centrifugation consumable, in accordance with one or more embodiments of the disclosure.

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SUBSTITUTE SHEET (RULE 26) FIGS. 1D-1E are drawings illustrating a cutaway perspective views of a cell collection section 108, with a piston translated to different positions within a collection chamber, in accordance with one or more embodiments of the disclosure.

FIG. 2 is a drawing illustrating a cutaway perspective view of a cell centrifugation consumable with a top half of the liquid holding section removed, in accordance with one or more embodiments of the disclosure.

FIG. 3 is a block diagram illustrating a system for processing a cell sample, in accordance with one or more embodiments of the disclosure.

FIG. 4 is a flow diagram illustrating a method for centrifugation of a sample, in accordance with one or more embodiments of the disclosure.

FIG. 5 is a block diagram illustrating an automated robotic system for handling a cell centrifugation consumable, in accordance with one or more embodiments of the disclosure.

FIG. 6A is a drawing illustrating a side view of a cell centrifugation consumable, in accordance with one or more embodiments of the disclosure.

FIG. 6B is a drawing illustrating a side view of a cell centrifugation consumable placed within a centrifuge adapter, in accordance with one or more embodiments of the disclosure.

FIG. 6C is a drawing illustrating a portion of a liquid holding section and a cell collection section of a cell centrifugation consumable, in accordance with one or more embodiments of the disclosure.

FIG. 6D is a drawing illustrating the cell collection consumable inserted within a centrifuge adapter and having a reading taken by a sensor, in accordance with one or more embodiments of the disclosure.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

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SUBSTITUTE SHEET (RULE 26) As used herein a letter following a reference numeral is intended to reference an embodiment of the feature or element that may be similar, but not necessarily identical, to a previously described element or feature bearing the same reference numeral (e.g., 1, 1a, lb). Such shorthand notations are used for purposes of convenience only and should not be construed to limit the disclosure in any way unless expressly stated to the contrary.

Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by anyone of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

In addition, use of “a” or “an” may be employed to describe elements and components of embodiments disclosed herein. This is done merely for convenience and “a” and “an” are intended to include “one” or “at least one,” and the singular also includes the plural unless it is obvious that it is meant otherwise.

Finally, as used herein any reference to “one embodiment” or “some embodiments” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment disclosed herein. The appearances of the phrase “in some embodiments” in various places in the specification are not necessarily all referring to the same embodiment, and embodiments may include one or more of the features expressly described or inherently present herein, or any combination of sub-combination of two or more such features, along with any other features which may not necessarily be expressly described or inherently present in the instant disclosure.

Described herein is a container for a biological sample. The container is be configured for processing cell solutions, such as processing cell solutions during and after centrifugation, and may be disposable/consumable (i.e., a cell centrifugation consumable). The cell centrifugation consumable is configured for automated processing, such as automated removal of fluid, (e.g., supernatants) after centrifugation. The cell centrifugation consumable may also be configured for automatic detection, removal, and/or resuspension of a cell pellet. The cell centrifugation consumable may be used with various systems including automated cell processing systems such as those disclosed in Intemational/PCT Application PCT/US2022/012820, published as WO 2022/155610, which is incorporated herein by reference in its entirety. The cell centrifugation

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SUBSTITUTE SHEET (RULE 26) consumable may also include supernatant decanting and cell incubation as disclosed in US Provisional Patent Application No. 63/424,559, filed November 11, 2022, entitled "Consumable for Centrifugation of Biological Cells," which is incorporated herein by reference in its entirety.

FIG. 1A is a drawing illustrating a perspective view of a cell centrifugation consumable 100, in accordance with one or more embodiments of the disclosure. The cell centrifugation consumable 100 is configured as a container that can contain a volume of liquid, such as a volume of media containing cells (e.g., a cell solution). The cell centrifugation consumable 100 is disposable in that it may be discarded after a single use. However, the cell centrifugation consumable 100 may be discarded after any number of uses (i.e., biological samples processed) including but not limited to two uses, five uses, ten uses, one hundred uses, one thousand uses, or more. The cell centrifugation consumable 100 may be used for centrifugation processes. For example, a cell centrifugation consumable 100 containing a cell solution may be placed within the rotor of a centrifuge, whereupon the spinning of the rotor, cells within the cell solution may form a cell pellet due to gravitational (g) forces incurred by the cell centrifugation consumable 100. The cell centrifugation 100 may be constructed by injection molding or additive manufacturing and may include any material including but not limited to polycarbonate, polystyrene, polyester, and nylon.

In embodiments, the cell centrifugation consumable 100 includes a liquid holding section 104 and a cell collection section 108. The liquid holding section 104 is configured to hold a volume of fluid, such as the cell solution. The cell collection section 108 is configured to receive a concentrated cell mass as the cell centrifugation consumable 100 is centrifuged, and the applied centrifugal force (indicated by downward arrow 112) drives cells within the liquid holding section 104 to form a cell pellet within the cell collection section 108. The liquid holding section 104 may be formed from one or more sections (e.g., an upper section 116 and a lower section 120) that facilitate the ease of manufacture and/or disassembly. A seal 124 may be placed between each section to prevent leakage. The cell centrifugation consumable 100 further includes a housing 128 that protects the inner workings of the cell centrifugation consumable 100. The cell centrifugation consumable 100 may further include a lid 132 configured to cover an opening 134. The lid 132 may prevent spillage of media within the cell centrifugation consumable 100 and may be of any size or shape. Once attached to the cell centrifugation consumable 100, the lid may be removable, permitting access to the media and/or cell pellet, or nonremovable, with access to the media and cell

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SUBSTITUTE SHEET (RULE 26) pellet requiring other modes of access (e.g., with the lid attaching at an open end of the inner fluid chamber 136 opposite the collection chamber 148).

FIG. IB is a drawing illustrating a perspective cut-away view of the cell centrifugation consumable 100, in accordance with one or more embodiments of the disclosure. In embodiments, the cell centrifugation consumable 100 includes a media-holding inner fluid chamber 136 that is encapsulated on four or more sides by the housing 128. The inner fluid chamber 136 includes a high-volume portion 140 that includes a majority of the volume of the inner fluid chamber 136. The high-volume portion 140 may have any shape including but not limited to a spherical shape or a cuboidal shape. For example, the high-volume portion 140 may be configured as having a generally cuboidal shape with rounded side-edges, as shown in FIG. IB.

The inner fluid chamber 136 further includes a tapered portion 144 configured as a narrowing of the cross-sectional width of the inner fluid chamber 136 as compared to the high- volume portion 140. The tapered portion 144 may be configured with a shape of any tapered structure including but not limited to a conical shape. For example, the 112 may be configured as a conically shaped portion of the inner fluid chamber 136, similar to conical shapes seen in centrifuge tubes. The inner fluid chamber 136 further includes a collection chamber 148 positioned at a narrow end 152 of the tapered portion 144. The arrangement of the high-volume portion 140, the tapered portion 144, and the collection chamber 148 permit the pelleting of a cell pellet within the collection chamber 148 when a cell solution (e.g., a cell suspension) is transferred to the cell centrifugation consumable 100, and the cell centrifugation consumable 100 is subjected to a centrifugal force 112 that forces the cells from the cell solution to the collection chamber 148.

The inner fluid chamber 136 may be configured with any total volume (e.g., the total amount of fluid that can be contained by the inner fluid chamber) including but not limited to total volumes of 20 ml, 50 ml, 100 ml, 200 ml, 250 ml, 500 ml, 600 ml, 750 ml, 1.0 L, 1.5L, or 2 L, or approximate volumes thereof. The collection chamber 148 may be configured with any total volume including but not limited to total volumes of 0.1 ml, 0.5 ml, 1.0 ml. 2.5 ml, 5 ml, 10 ml, 13 ml or 15 ml, or approximate volumes thereof. For example, the collection chamber may be configured to contain a cell pellet 156 of Jurkat cells having a volume of 11 ml (e.g., approximately 10¹⁰ cells). The cell pellet 156 may include any of number of cells. For example, the cell pellet 156

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SUBSTITUTE SHEET (RULE 26) may include 60 million cells (e.g., a 0.04 ml cell pellet 156) and up to, or greater than, the aforementioned IO¹⁰ cells.

The cell centrifugation consumable 100 may be configured with any size or set of dimensions. For example, the cell centrifugation consumable 100 may be configured to any dimension/footprint standard as detailed by the Society for Laboratory Automation and Screening (SLAS). For example, the cell centrifugation consumable 100 may have a footprint dimension of approximately 85 mm x 128 mm. For instance, the footprint may be 84.48 mm x 127.76 mm. In some embodiments, the inner fluid chamber 136 is configured to include a total volume considerably smaller than the dimensions of the cell centrifugation consumable 100. For example, the dimension of the cell centrifugation consumable 100 may have a footprint of approximately 85 mm x 128 mm, and have an inner fluid chamber volume of approximately 20 ml.

The collection chamber 148 may include any shape including but not limited to a cylindrical shape or a conical shape. For example, a resultant cell pellet may form a cylindrical shape based on a cylindrical shape of the collection chamber 148. In another example, the resultant cell pellet may form a cylindrical shape based on the cylindrical shape of the collection chamber 148 as well as a conical form if the cell pellet 116 is large enough to overfill the collection chamber 148 and form a conical shape within the tapered portion 144. In another example, the resultant cell pellet may be a conical form based on a conical shape of the collection chamber 148.

FIG. 1C is a drawing illustrating a cutaway perspective view of the cell collection section 108, in accordance with one or more embodiments of the disclosure. The cell collection section 108 is shown post-centrifugation, with a cell pellet 156 formed within the collection chamber 148, and supernatant 160 (e.g., residual media) pooled above the cell pellet 156.

In some embodiments, the cell collection section 108 is interchangeable. For example, the cell centrifugation consumable 100 may include a liquid holding section 104 that is couplable to two or more of a set of cell collection sections 108 having different cell collection volumes. For instance, the liquid holding section 104 may be couplable to both a cell collection section 108 having a collection chamber 148 with a volume of 2.5 ml, and a cell collection section 108 having a collection chamber 148 with a volume of 13 ml.

In embodiments, the cell collection section 108 further includes a piston 164 disposed within the collection chamber 148 and configured to travel (e.g., translate) within collection chamber 148.

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SUBSTITUTE SHEET (RULE 26) The piston 164 may be coupled to a gasket 168 that makes contact with the cell pellet 156. Together, movement of the piston 164 and the gasket 168 along the collection chamber 148 translates the cell pellet 156 towards the narrow end 152 of the inner fluid chamber 136. Once the cell pellet 156 had been moved towards the narrow end 152 of the inner fluid chamber 136, the cell pellet 156 may be resuspended within the collection chamber 148. The piston 164 may be further configured with a hard-stop mechanism, such as an interference fit or block, that prevents loss of the piston 164 from the collection chamber 148 during centrifugation.

In embodiments, the piston 164 is biased to move, or translate, along the collection chamber 148 via pressure acting upon a contact surface 172 of the piston 164. The pressure applied to the contact surface 172 may be air pressure or fluid pressure (e.g., applied by pneumatic or hydraulic means) or may be pressure applied by mechanical means, such as pressure applied via an electric motor combined with a geared mechanism, or a spring-based mechanism. The cell collection section 108 may include a pressure fluid port 176 that delivers a pressurized medium (e.g., fluid, which may include air) to a reservoir 180 that contacts the contact surface 172 of the piston 164. The addition of air and/or fluid to the reservoir 180 results in an increased pressure within the reservoir 180, which is released upon the movement of the piston 164 along the collection chamber 148 (e.g., the fluid biases the piston 164). Fluid entering the pressure fluid port 176 may be sourced from a pressure delivery system that is operatively couplable to the cell centrifugation consumable 100.

In embodiments, the cell collection section 108 includes a supernatant fluid port 184 operatively coupled to the collection chamber 148. The supernatant fluid port 184 is configured to permit removal of supernatant from the cell centrifugation consumable 100 post centrifugation. The supernatant fluid port 184 may be sealable and/or resealable (e.g., via a resealable septum) such that a fluid removal apparatus, such as an aspirator, can be coupled to the supernatant fluid port 184 to remove the supernatant. For example, the aspirator may include a needle that penetrates a seal of the supernatant fluid port 184, and the supernatant is then removed via aspiration/suction. The fluid removal apparatus may include any type of fluid removing device, such as the aforementioned aspirator, or a peristaltic pump. In some embodiments, the supernatant fluid port 184 may be used to remove the majority of the supernatant 160 (e.g., a first portion of the supernatant 160). In some embodiments, the majority of the supernatant is removed by other means, such as by decanting or

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SUBSTITUTE SHEET (RULE 26) another fluid port, and the supernatant fluid port 184 is then used to remove a smaller portion of the supernatant 160 (e.g., a second portion of the supernatant).

In embodiments, the cell collection section 108 includes a collection chamber fluid port 188 operatively coupled to the collection chamber 148. The collection chamber fluid port 188 is configured to allow resuspension fluid to enter the collection chamber after the supernatant 160 has been effectively removed. The resuspension fluid may then at least partially resuspend the pellet. The resuspension fluid may be sourced from a fluid source, such as a container or flask. The fluid source may be couplable to the collection chamber fluid port 188 via a resuspension device (e.g., a pump). The fluid source may include the resuspension device. The collection chamber fluid port 188 may be sealable or resealable (e g., via a first resealable septum), and the resuspension device may include a needle or other instrument that can puncture the seal and allow resuspension fluid to enter the cell centrifugation consumable 100. In some embodiments, the resuspended cells are removed from the cell centrifugation consumable 100 via the collection chamber fluid port 188. For example, once the cells are resuspended, the collection chamber fluid port 188 is then coupled to an aspirator or other fluid removal device that removes the resuspended cells to a cell collection container, such as a test tube. The fluid removal apparatus may include any type of fluid removing device, such as the aforementioned aspirator, or a peristaltic pump. In some cases, the pump that pumps resuspension fluid into the collection chamber 148 is reversed so that the resuspended cells are removed by the same pump. The collection chamber fluid port 188 may be designed or positioned based on the estimated size/volume of the cell pellet 156.

FIG. ID- IE are drawings illustrating a cutaway perspective views of the cell collection section 108, with the piston 164 translated to different positions within the collection chamber 148, in accordance with one or more embodiments of the disclosure. In embodiments, the cell collection section 108 includes a sensor detection line 192 (e.g., a line of sight) that allows a sensor to determine the position of the cell pellet 156 and/or supernatant 160 within the collection chamber 148. For example, the housing 128 at the sensor detection line 192 may be translucent, allowing electromagnetic energy, such as light, to be transmitted on one side of the sensor detection line 192 (e.g., on one side of the cell collection section 108), which can be sensed by a sensor on the other side of the sensor detection line 192 (e.g., the opposite side of the cell collection section 108). For instance, and as shown in FIG. ID, the supernatant 160 may be positioned within the sensor

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SUBSTITUTE SHEET (RULE 26) detection line 192. The sensor 212 may then determine the presence of the supernatant 160 via the light transmitted through the line-of-sight 192, possibly through a change in absorbance. As the piston 164 is raised (e.g., in an upward direction 196) the cell pellet 156 moves into the sensor detection line 192, as shown in FIG. IE. The sensor 212 may then determine the presence of the cell pellet 156 based upon a change of absorbance or some other characteristic.

FIG. 2 is a drawing illustrating a cutaway perspective view of the cell centrifugation consumable 100 with the top half of the liquid holding section 104 removed, in accordance with one or more embodiments of the disclosure. The cell collection section 108 is shown as partially transparent and positioned adjacent to a transmitter 204 and a receiver 208 of the sensor 212. The sensor detection line 192 is shown passing through the cell collection section 108, just above the cell pellet 156. The sensor 212 may include any type of sensing technology and the use of any sensing medium. For example, the sensor 212 may utilize but not be limited to visible light, ultraviolet, infrared, laser, and sound technologies.

The sensor 212 may be operatively coupled to a controller 300 that provides processing functionality to at least the sensor 212. as shown in FIG. 3, in accordance with one or more embodiments of the disclosure. For example, controller 300 may cause the transmitter 204 to send a test signal and may determine if the cell pellet 156 is blocking the sensor detection line 192 by interpreting the results received from the receiver 208. The controller 300 includes one or more processors 304 (e.g., micro-controllers, circuitry, field programmable gate array (FPGA), central processing units (CPU), application-specific integrated circuit (ASIC), or other processing systems), and resident or external memory 308 for storing data, executable code, and other information. The controller 300, via the one or more processors 304, can execute one or more software programs embodied in a non-transitory computer readable medium (e.g., memory 308) that implement techniques described herein. The controller 300 is not limited by the materials from which it is formed or the processing mechanisms employed therein and, as such, can be implemented via semiconductor(s) and/or transistors (e.g., using electronic integrated circuit (IC) components), and so forth.

The memory 308 can be an example of tangible, computer-readable storage medium that provides storage functionality to store various data and/or program code associated with operation of the controller 300, such as software programs and/or code segments, or other data to instruct the io

SUBSTITUTE SHEET (RULE 26) controller 300, and possibly other components operatively coupled to the controller 300, to perform the functionality described herein. Thus, the memory 308 can store data, such as a program of instructions, and so forth (e.g., the instructions causing the one or more processors to implements the techniques described herein). It should be noted that while a single memory 308 is described, a wide variety of types and combinations of memory 308 (e.g., tangible, non-transitory memory) can be employed. The memory 308 may be integral with the controller 300, may include stand-alone memory, or may be a combination of both. Some examples of the memory 308 may include removable and non-removable memory components, such as random-access memory (RAM), readonly memory (ROM), flash memory (e.g., a secure digital (SD) memory card, a mini-SD memory card, and/or a micro-SD memory card), solid-state drive (SSD) memory, magnetic memory, optical memory, universal serial bus (USB) memory devices, hard disk memory, external memory, and so forth.

The communication interface 312 may be operatively configured to communicate with components of the controller 300. For example, the communication interface 312 can be configured to retrieve data from the controller 300 or other components, transmit data for storage in the memory 308, retrieve data from storage in the memory 308, and so forth. The communication interface 312 can also be communicatively coupled with the controller 300 to facilitate data transfer between components of the controller 300. The computation unit 104 can also include and/or connect to one or more input/output (I/O) devices. In embodiments, the communication interface 312 includes or is coupled to a transmitter, receiver, transceiver, physical connection interface, or any combination thereof.

The controller 300 may also be operatively linked to the pressure delivery system 316 and the supernatant delivery device 320. In this manner, the pressure delivery system 316 and the fluid removal apparatus 320 may act upon the cell centrifugation consumable 100 based on results obtained from the sensor 212. For example, and in referring to FIGS. 1D-E, the sensor 212 may detect supernatant 160 within the collection chamber 148 at the sensor detection line 192 (e.g., as shown in FIG, ID). To remove essentially all supernatant from the collection chamber 148, the controller 300 may cause the pressure delivery system 316 to apply air/fluid to the reservoir 180, causing the piston 164 to rise (e.g., in the upward direction 196) along with the cell pellet 156. As shown in FIG. IE, once the cell pellet 156 reaches the sensor detection line 192, the sensor 212

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SUBSTITUTE SHEET (RULE 26) detects the cell pellet 156 (e.g., as an increase in absorbance or occlusion) causing the controller 300 to pause the pressure delivery system 316. The controller 300 may then switch on the fluid removal apparatus 320, and the supernatant 160 is removed via the supernatant fluid port 184.

In embodiments, the controller 300 is operatively coupled to a cell resuspension device 324 that is itself fluidly couplable to the cell centrifugation consumable 100 via the collection chamber fluid port 188. For example, once the supernatant 160 has been thoroughly removed from the cell collection chamber 148, controller 300 may then cause the cell resuspension device 324 to add resuspension fluid to the cell pellet 156 via the collection chamber fluid port 188. The addition of the resuspension fluid may partially or totally resuspend the cell pellet 156. For example, the cell resuspension device 324 may be configured to consecutively add and remove the resuspension fluid (e.g., via reciprocating pumps or plungers within the cell resuspension device 324), effectively resuspending the cell pellet 156. Once the cell pellet 156 has been effectively resuspended, the cell resuspension device 324 may remove the resuspended cells from the cell concentration consumable 100 and transfer the resuspended cells to another container.

In some embodiments, a portion of the supernatant 160 (e.g., the first portion) is removed by decanting fluid through the top of the cell centrifugation consumable 100. For example, after centrifugation and the lid 132 partially or wholly removed to expose an opening 134 to the inner fluid chamber 136, the cell centrifugation consumable 100 may be tilted so as to dump out a portion of the supernatant. The cell centrifugation consumable 100 may then be titled into a position where the sensor 212 can determine if there is supernatant left in the collection chamber 148. The controller 300 may then operatively move the piston 164 to move the cell pellet 156 to a position immediately below the supernatant fluid port, based on the detection of the cell pellet 156 by the sensor 212, where the remaining supernatant 160 may be removed via the supernatant fluid port 184, as detailed above.

In some embodiments, the cell centrifugation consumable 100 is hermetically sealed so that there is no exchange of air during cell processing. Alternatively, the cell centrifugation consumable 100 may include one or more air ports The air may be configured to permit flow of air between the air within the inner fluid chamber 136 and air outside of the cell centrifugation consumable 100. For example, the cell centrifugation consumable 100 containing a cell solution may be place in an incubator, such as a CO2 cell culture incubator, wherein the air ports may allow the cells within the

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SUBSTITUTE SHEET (RULE 26) cell centrifugation consumable 100 to be regulated by the conditions of the incubator. The air ports may also be configured to allow pressurization of the cell centrifugation consumable 100 to transfer fluid. The air ports may include a filter, such as a 0.22 pm pore size filter that allows air flow without allowing the transmission of bacteria through the air port. In another example, the air ports may include port covers that prevent airflow, which can be subsequently removed to allow air flow.

In embodiments, the air ports, or other air flow regulators, may control the flow of air into and out of the inner fluid chamber 136. For example, during centrifugation and/or removal of supernatant 160, the air ports and/or air regulators may be configured to allow free air flow to and from the inner fluid chamber 136, thereby preventing a positive or negative pressure within the inner fluid chamber 136 from affecting the centrifugation or supernatant removal process. During cell pellet resuspension, the air ports or air regulators may be configured to prevent free air flow from occurring. Under these conditions, resuspension fluid entering the collection chamber 148 would be pressurized, facilitating the back and forth flow of resuspension fluid and the eventual removal of the cell pellet 156 via the collection chamber fluid port 188.

In some embodiments, the cell centrifugation consumable 100 includes an inner chamber fluid port operatively coupled to the inner fluid chamber 136 (e.g., a port similar to the supernatant fluid port 184, but located in the inner fluid chamber 136). The inner chamber fluid port is configured to permit removal supernatant from the cell centrifugation consumable 100 post centrifugation. The supernatant fluid port 184 may be sealable and/or resealable (e.g., via a second resealable septum) such that a fluid removal apparatus, such as an aspirator, can be coupled to the supernatant fluid port 184 to remove the supernatant. For example, the aspirator may include a needle that penetrates a seal of the supernatant fluid port 184, and the supernatant is then removed via aspiration/suction. The fluid removal apparatus may include any type of fluid removing device, such as the aforementioned aspirator, or a peristaltic pump. The cell centrifugation consumable 100 may also include supernatant fluid port tubing configured to couple to the inner chamber fluid port to the inner fluid chamber 136. The cell centrifugation consumable 100 may also be configured such that the supernatant 160 may be decanted from the inner fluid chamber 136 out of the opening 134 (e.g., the opening 134 reversibly sealed by the lid 132). In some embodiments, the supernatant 160 may be decanted into a decanting space located within the cell centrifugation consumable 100

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SUBSTITUTE SHEET (RULE 26) via a decanting port. Examples of the inner chamber fluid port, supernatant port tubing, decanting space, and decanting port are also shown in US Provisional Patent Application No. 63/424,559.

FIG. 4 is a flow diagram illustrating a method 400 for processing of a cell solution, in accordance with one or more embodiments of the disclosure. The method 400 may include the formation of a cell pellet and the removal of supernatant. The method 400 may be used to centrifuge any type of biological sample, such as a cell suspension containing human cells.

In embodiments, the method 400 includes a step 404 of providing the cell centrifugation consumable 100. The cell centrifugation consumable 100 may include the inner fluid chamber 136. The inner fluid chamber 136 includes the tapered portion 144, The tapered portion 144 is coupled to the collection chamber 148 at the narrow end 152 of the tapered portion 144. The collection chamber 148 is configured so that upon centrifugation of the cell centrifugation consumable 100 containing a cell solution, a cell pellet 156 forms within the collection chamber 148.

The cell centrifugation consumable 100 may further include the supernatant fluid port 184 fluidly couplable to the collection chamber 148 configured for removal of supernatant 160 from the collection chamber 148. For example, the supernatant fluid port 184 may include a resealable septum that, when accessed by needle or other sharp object, temporarily breaks the seal of the septum, allowing supernatant 160 to flow out off the collection chamber 148 via the supernatant fluid port 184.

The cell centrifugation consumable 100 may further include the sensor detection line 192 configured to so that the sensor 212 can detect the presence of the supernatant 160 or the cell pellet 156 within the cell chamber 148. The sensor detection line 192 may be any portion or section of the cell solution consumable 100 that can provide to the sensor 212 an unobstructive view of the level of the supernatant 160 and/or cell pellet 156 within the collection chamber 148.

The cell centrifugation consumable 100 may further include the piston 164 disposed within the collection chamber 148 and operatively coupled to the cell pellet 156 so as to be able to translate the cell pellet 156 toward the sensor detection line 192. The piston 164 may directly contact the cell pellet 192. Alternatively, the cell pellet 156 may form on a gasket 168 or another surface that is physically coupled to the piston 164. In some embodiments of the method, the cell centrifugation consumable 100 further includes the pressure fluid port 176 operatively coupled to the piston 164,

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SUBSTITUTE SHEET (RULE 26) wherein upon addition of fluid from, or through, the pressure fluid port 176, the piston 164 translates the cell pellet 156 toward the sensor detection line 192.

In embodiments, the method 400 includes a step 408 of receiving a suspension of cells within the inner fluid chamber 136. For example, the inner fluid chamber 136 may be partially filled (e.g., via the opening 134 at the top of the cell centrifugation consumable 100) with primary cells (e.g., T cells from a patient), or cells from a cell line, that have been suspended in cell media. The cell centrifugation consumable 100 may then be placed into a centrifuge. In some embodiments, the cell centrifugation consumable 100 is placed into an incubator for an incubation time (e.g., several minutes to several weeks) before being placed in the centrifuge.

In embodiments, the method 400 includes a step 412 of centrifuging the cell centrifugation consumable to concentrate the cells as a cell pellet 156 in the collection chamber 148, with a supernatant 160 remaining in the inner fluid chamber. The cell centrifugation consumable 100 is capable of sustaining g-forces (e.g., 10² to 10⁴ x g) that are required for pelleting the cells of the cellcontaining solution. The cell pellet 156 is then formed on or near the piston 168, such as on the gasket 168.

In embodiments, the method 400 further includes a step 416 of removing a first portion of the supernatant 160. The first portion of the supernatant 160 may be removed by one of several means. For example, the first portion of the supernatant 160 may be removed by decanting. In another example, the first portion of the supernatant 160 may be removed via an inner chamber port fluidly coupled to a fluid removal apparatus 320. In another example, the first portion of the supernatant 160 may be removed via the supernatant fluid port 184. For instance, a fluid removal apparatus 320 configured as a suction device may introduce a needle or pipette tip into a resealable septum of the supernatant fluid port and draw out the supernatant 160 via the supernatant fluid port 184

In embodiments, the method 400 includes a step 420 of detecting the presence of remaining supernatant 160. For example, the sensor 212 may be used to assess whether there is remaining supernatant at the sensor detector line 192 (e.g., via a change in absorbance). Data generated from the sensor 212 may then be sent to the controller 300.

In embodiments, the method 400 includes a step 424 of translating the piston 164 toward the sensor detection line 192 until the presence of the cell pellet 156 at the sensor detector line 192 is

15

SUBSTITUTE SHEET (RULE 26) detected. For example, the if the data sent to the controller 300 from the sensor 212 indicates the detection of only supernatant 160 at the sensor detection line 192, the controller 300 may then cause the pressure delivery system 316 to add fluid to the pressure fluid port 176. The increased fluid increases pressure at the contact surface 172 of the piston 164 biasing the piston, which moves the cell pellet 156 toward the sensor detection line 192. The sensor 212 continues to send detection data to the controller 300 as the piston 164 is translating, along with the cell pellet 156, within the collection chamber 148. When the cell pellet 156 reaches the sensor detection line 192, the sensor 212 detects the cell pellet 156 (e.g., as an increase in absorbance or other characteristic) and sends the corresponding data to the controller 300. The controller 300 then pauses the pressure delivery system 316, preventing the piston 156 from translating further.

In embodiments, the method includes a step 428 of removing a second portion of the supernatant 160 from the collection chamber 148 via the supernatant fluid port 184. For example, once the cell pellet 156 has been translated to the sensor detector line 192, the controller 300 causes the fluid removal apparatus 320 to access the supernatant fluid port 184 (if the fluid removal apparatus 320 is not already coupled to the supernatant fluid port 184) and begin removing the remaining supernatant 160. The rate of removal of the remaining supernatant 160 is controlled so that the cell pellet 156 remains relatively undisturbed. Removing the second portion of the supernatant 160 may include removing nearly all the supernatant from the collection chamber 148, with a small amount of supernatant 160 being left in the collection chamber 148. The amount of supernatant 160 left in the collection chamber 160 may vary. For example, the amount of supernatant 160 left in the collection chamber may be less than 2.0 ml, 1.5 ml, 1.0 ml, 0.75 ml, 0.5 ml, 0.25 ml, 0.1 ml, 0.05 ml, or 0.01 ml.

In embodiments, the cell concentration consumable 100 further includes the collection chamber fluid port 188, and the method 400 further includes a step of providing additional fluid to the collection chamber 148 via the collection chamber fluid port 204 and at least partially resuspending the cell pellet 156 using the additional fluid. For example, the cell resuspension device 324 may introduce cell resuspension fluid into the cell collection chamber 148 via the collection chamber fluid port 204. Once the additional fluid is added to the collection chamber, the method 400 progresses to the next step of at least partially resuspending the cell pellet 125 using the additional fluid. The resuspension of the cell pellet 156 may occur solely due to the addition of the

16

SUBSTITUTE SHEET (RULE 26) additional fluid or may also include a coordinated movement of the cell centrifugation consumable 100 (e.g., shaking, tilting, or rotating) along with the addition of the additional fluid.

In embodiments, the cell centrifugation consumable 100 is removably connected, or couplable, to an automated robotic system 500 as shown in FIG. 5, in accordance with one or more embodiments of the disclosure. For example, the automated robotic system 500 may include a robotic arm 504 configured to clamp onto, or otherwise couple to, the cell centrifugation consumable 100, and move the cell centrifugation consumable between different stations, such as stations within the automated robotic system 500, (e.g., a centrifuge 508). In another example, the automated robotic system may clamp onto, or otherwise secure to, the cell centrifugation consumable 100 within a rack 512 that is itself integrated within the automated robotic system 500. For instance, while the cell centrifugation consumable 100 is secured to the rack 512, the automated robotic system 500 may move various instruments, such as septum-piercing pipets, to the cell centrifugation consumable 100 in a coordinated matter. In another instance, the rack 512 itself may be mobile and configured to move the cell centrifugation consumable 100 to different stations (e.g., a pipetting station) within the automatic robotic system 500. The cell centrifugation consumable 100 may have one or more attachment points couplable to the robotic arm 504, rack 512, or other aspects of the automated robotic system 500.

In embodiments, the method 400 further includes a step of using the automated robotic system 500 to move the cell centrifugation consumable 100 to the centrifuge 508 for centrifugation. For example, the automated robotic system 500 may include the robotic arm 504 that couples to the attachment points of the cell centrifugation consumable 100 and places the cell centrifugation consumable 100 into the rotor assembly (e.g., a bucket) of the centrifuge 508. After the centrifugation is completed, the automated robotic system 500 may then remove the cell centrifugation consumable 100 from the rotor (e.g., via the robotic arm 504). The robotic arm 504 may also be programmed to decant the supernatant 160 from the cell concentration consumable 100 into another container. The robotic arm 504 may also be programmed to transfer the cell concentration consumable 100 to a mixer 516 (e.g., such as a vortex or other vibrating device) to resuspend the cell pellet 156.

As shown in FIG. 5, the components of the automated robotic system 500 are organized around an automated robotic system platform 520 that includes the robotic arm 504 and other

17

SUBSTITUTE SHEET (RULE 26) componentry, such as automatic pipettors and pumps. For example, the sensor 212, the pressure deliver system 316, the fluid removal apparatus 320, and/or the cell resuspension device 324 may be functionally couplable to the automatic robotic system platform 520. One of more aspects of the automated robotic system 500 may be operatively linked to one or more controllers 300.

EXAMPLES

The following examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

Example 1

Protocol for Processing of Cell Solution Using the Cell Centrifugation Consumable 100. The protocol includes steps performed by the automated robotic system 500.

(1) The cell centrifugation consumable 100 will be pulled from a storage rack by a robot arm 504 and loaded onto an automated robotic system platform 520.

(2) The cell centrifugation consumable 100 will be partially or wholly filled with a suspended cell culture or cell solution.

(3) The robot will move the cell centrifugation unit 100 to the centrifuge 508.

(4) The centrifuge 508 will concentrate the cells into a cell pellet 156 within the cell centrifugation consumable 100. For example, the centrifugation will concentrate the cells into a cell pellet ranging 156 from 0.1 ml to 13 ml.

(5) The robot arm 504 will move the cell centrifugation consumable 100 to the automated robotic system platform 520.

(6) The pressure delivery system 316 will then move the cell pellet 156 (e.g., via the piston 164) of unknown size upwards in the collection chamber 148 until the top surface of the cell pellet 156 is detected by the sensor 212. Movement of the cell pellet 156 is currently done by a piston 164 at the bottom of the collection chamber 148. A controller 300 operatively coupled to the automated robotic system 500 controls the movement of the piston 164 by a pneumatic interface or electrical actuator. The detection of the top surface of the cell pellet 156 is performed by the sensor 212 (e.g., optical sensor, or other type of sensor) operatively coupled to the automated robotic system 500.

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SUBSTITUTE SHEET (RULE 26) (8) Once the top of the cell pellet is raised to the proper level, the supernatant fluid port 184 positioned right above the cell pellet surface will pull the remaining supernatant 160 from the cell centrifugation consumable, leaving less than 0.5 ml of supernatant in the collection chamber 148.

(9) Buffer solution is added to the cell pellet 156 in order to resuspend the cell culture and allow removal of cells from the cell centrifugation consumable 100.

(12) The cell centrifugation consumable 100 is reused or discarded. For example, the cell centrifugation consumable 100 may be reused multiple times for a single patient.

Example 2

Processing of Cell Solutions Using the Cell Centrifugation Consumable 100.

A cell centrifugation consumable 100 was fabricated, as shown in FIG. 6A, in accordance with one or more embodiments of the disclosure. The liquid holding section 104, which included the high-volume portion 140 and the tapered portion 144, was constructed of a transparent resin. A lid 132 was attached. A cell collection section 108 was attached to the tapered portion 144 at the narrow end 152 (e.g., via a Luer taper). The cell collection section 108 includes the collection chamber 148, which further includes a combined piston 164 and gasket 168, as well as a supernatant fluid port 184 coupled to tubing 600. The combined piston 164 and gasket is arranged such that the pressure may be applied manually to the piston 164 (e.g., via a finger) in order to translate the piston 164 along the collection chamber 148.

FIG. 6B is a drawing illustrating the cell centrifugation consumable 100 inserted into a centrifuge adapter 604. The centrifuge adapter 604 stabilizes the cell centrifugation consumable 100 as it is placed within the bucket of a centrifuge rotor. The centrifuge adapter may itself be configured to attach directly to the centrifuge rotor, without a need for buckets. The cell centrifugation consumable 100 is shown with a portion of the tapered portion 144 filled with a cell solution 608.

FIG. 6C is a drawing illustrating the cell collection section 108 and a portion of the liquid holding section 104 from FIG. 6B after the cell centrifugation consumable has been centrifuged (400 g x 5 minutes) and removed from the centrifuge adapter 604. The cell solution 608 has now separated into a clear supernatant 160 and a small cell pellet 156 resting upon the combined piston 164 and gasket 168.

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SUBSTITUTE SHEET (RULE 26) FIG. 6D is a drawing illustrating the cell collection consumable 100 inserted within the centrifuge adapter 604 and containing a cell solution that has been centrifuged (400 g x 5 minutes). The cell collection consumable is shown having a reading taken by the sensor 212. In this example, the sensor 212 is a laser-based sensor that transmits a laser beam 612 (e.g., indicated by the dot) by a transmitter 204 across the collection chamber 148 along the sensor detection line 192 to a receiver 208 on the other side of the collection chamber 148 (receiver 208 not shown). The piston 164 is shown having been translated by the addition of fluid through the pressure fluid port 176 so that the cell pellet 156 is immediately below the laser beam 612. The cell pellet 156 is also immediately below the supernatant fluid port. With the cell pellet 156 at this position, the second portion of the supernatant may be removed via the supernatant fluid port 184. Once the supernatant 160 has been removed, the cell pellet 156 will be at least partially resuspended by resuspension fluid entering from the collection chamber fluid port 188 (not shown).

It is to be understood that embodiments of the methods disclosed herein may include one or more of the steps described herein. Further, such steps may be carried out in any desired order and two or more of the steps may be carried out simultaneously with one another. Two or more of the steps disclosed herein may be combined in a single step, and in some embodiments, one or more of the steps may be carried out as two or more sub-steps. Further, other steps or sub-steps may be carried in addition to, or as substitutes to one or more of the steps disclosed herein.

Although inventive concepts have been described with reference to the embodiments illustrated in the attached drawing figures, equivalents may be employed and substitutions made herein without departing from the scope of the claims. Components illustrated and described herein are merely examples of a system/device and components that may be used to implement embodiments of the inventive concepts and may be replaced with other devices and components without departing from the scope of the claims. Furthermore, any dimensions, degrees, and/or numerical ranges provided herein are to be understood as non-limiting examples unless otherwise specified in the claims.

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SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS What is claimed is:

1. A cell centrifugation consumable, comprising: an inner fluid chamber comprising a tapered portion having a collection chamber at a narrow end of the tapered portion, wherein the inner fluid chamber is configured to contain a cell solution, wherein upon centrifugation of the cell solution, a cell pellet forms within the collection chamber; a supernatant fluid port fluidly couplable to the collection chamber configured to allow transfer of supernatant from the collection chamber; a sensor detection line within the collection chamber configured so that a sensor can detect the presence of a supernatant or a cell pellet at the sensor detection line; and a piston configured to translate the cell pellet within the collection chamber toward the sensor detection line.

2. The cell centrifugation consumable of claim 1, further comprising a pressure fluid port operatively coupled to the piston, wherein an addition of fluid from the pressure fluid port translates the piston.

3. The cell centrifugation consumable of claim 2, further including a reservoir operatively coupled to the pressure fluid port and a contact surface of the piston, wherein the reservoir is configured to receive fluid from the pressure fluid port, wherein the received fluid is configured to bias the piston.

4. The cell centrifugation consumable of claim 1, wherein the tapered portion comprises a conical shape.

5. The cell centrifugation consumable of claim 1, wherein the collection chamber comprises a cylindrical shape.

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SUBSTITUTE SHEET (RULE 26)

6. The cell centrifugation consumable of claim 1, wherein the collection chamber includes a collection chamber fluid port.

7. The cell centrifugation consumable of claim 6, wherein the collection chamber fluid port is fluidly couplable to a fluid source, wherein the fluid source is configured to deliver fluid to the collection chamber fluid port.

8. The cell centrifugation consumable of claim 7, wherein a delivered fluid from the fluid source at least partially resuspends the cell pellet.

9. The cell centrifugation consumable of claim 7, wherein the collection chamber fluid port is couplable to the fluid source via a resealable septum.

10. The cell centrifugation consumable of claim 1, wherein the supernatant fluid port is couplable to a fluid removal apparatus, wherein the fluid removal apparatus is configured to remove fluid from the collection chamber, and wherein the fluid removal apparatus is configured to remove fluid from the tapered portion outside of the inner fluid chamber and leave fluid remaining in the collection chamber.

11. The cell centrifugation consumable of claim 10, wherein the supernatant fluid port is couplable to the fluid source via a resealable septum.

12. The cell centrifugation consumable of claim 1, further comprising an inner chamber fluid port couplable to a fluid removal apparatus.

13. The cell centrifugation consumable of claim 1, further including an opening, wherein a cell solution may be added to, or decanted from, the cell centrifugation consumable via the opening.

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SUBSTITUTE SHEET (RULE 26)

14. The cell centrifugation consumable of any of the preceding claims, further comprising a lid configured to reversibly seal the opening.

15. The cell centrifugation consumable of any of the preceding claims, wherein the inner fluid chamber comprises a volume of 500ml.

16. The cell centrifugation consumable of any of the preceding claims, wherein the piston is translated by an electrical actuator.

17. A method for centrifugation of a sample, comprising: providing a cell centrifugation consumable including: an inner fluid chamber comprising a tapered portion and having a collection chamber at a narrow end of the tapered portion configured to contain a cell solution; a supernatant fluid port fluidly coupled to the collection chamber configured for removal of supernatant from the collection chamber; a sensor detection line within the collection chamber configured so that a sensor can detect the presence of a supernatant or a cell pellet at the sensor detection line; and a piston configured to translate the cell pellet within the collection chamber toward the sensor detection line; receiving a cell solution within the inner fluid chamber; centrifuging the cell centrifugation consumable to concentrate the cells from the cell solution as a cell pellet in the collection chamber, with a supernatant remaining in the inner fluid chamber; removing a first portion of the supernatant; detecting the presence of a remaining supernatant at the sensor detection line; translating the piston toward the sensor detection line until the presence of the cell pellet at the sensor detector line is detected; and

23

SUBSTITUTE SHEET (RULE 26) removing a second portion of the remaining supernatant from the collection chamber via the supernatant fluid port.

18. The method of claim 17, wherein the cell centrifugation consumable further comprises a pressure fluid port operatively coupled to the piston, wherein upon addition of fluid from the pressure fluid port, the piston translates the cell pellet.

19. The method of claim 17, wherein the collection chamber of the cell centrifugation consumable includes a collection chamber fluid port, wherein the method further comprises: providing additional fluid to the collection chamber via the collection chamber fluid port, and at least partially resuspending the cell pellet using the additional fluid.

20. The method of claim 17, wherein the tapered portion further includes an inner chamber fluid port, and wherein the first portion of the supernatant is removed via the inner chamber fluid port.

21. The method of claim 17, wherein the first portion of the supernatant is removed by decanting fluid through an opening.

22. The method of claim 17, wherein the first portion of the supernatant is removed via the supernatant fluid port

23. The method of any one of claims 17-21, wherein the cell centrifugation consumable is removably connected to an automated robotic system.

24. The method of claim 22, wherein centrifuging the cell centrifugation consumable further comprises:

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SUBSTITUTE SHEET (RULE 26) using the automated robotic system to move the cell centrifugation consumable to a centrifuge for centrifugation, and using the automated robotic system to remove the cell centrifugation consumable from the centrifuge following centrifugation.

25

SUBSTITUTE SHEET (RULE 26)