Automated production and collection

Abstract

To provide methods for producing, isolating, and / or collecting cellular products released or secreted from cells.SOLUTION: Cells are expanded in the intracapillary (or extracapillary) space of a bioreactor of a cell expansion system with media. Cells release cellular products into the fluid space of the bioreactor. Examples of the released cellular products include extracellular particles, such as extracellular vesicles (EVs). In order to collect the extracellular particles released from the cells expanded, rather than extracellular particles from other sources, serum proteins are eliminated in a washout step prior to collecting the released extracellular particles from the expanding cells. The released cellular products are collected or concentrated through the control of outlet parameters, and at the same time, nutrients reach the cells through the diffusion of media through a semi-permeable membrane. The released cellular products are harvested.SELECTED DRAWING: Figure 9

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 332,426, entitled "Automated Production and Harvesting," filed May 5, 2016, U.S. Provisional Patent Application No. 62 / 333,013, entitled "Automated Production and Harvesting," filed May 6, 2016, and U.S. Provisional Patent Application No. 62 / 500,962, entitled "Automated Production and Harvesting," filed May 3, 2017, the entireties of which are expressly incorporated herein by reference. [Background technology]

[0002] Cell expansion systems (CES) are used to expand and differentiate cells. A variety of adherent and suspension cells can be expanded (e.g., grown) using cell expansion systems. For example, cell expansion systems can be used to expand mesenchymal stem cells (MSCs) and other types of cells, such as bone marrow cells. Stem cells expanded from donor cells can be used to repair or replace damaged or defective tissues and have a wide range of clinical applications for a wide range of diseases. Growth of both adherent and non-adherent cell types occurs in the bioreactor of the cell expansion system. Summary of the Invention [Problem to be solved by the invention]

[0003] Embodiments of the present disclosure generally relate to the production, isolation, and / or collection of cell products released or secreted from cells. Such released or secreted cell products may be referred to as released or secreted agents, released or secreted constituents, cell produced substances, cell produced components, released or secreted particles, released or secreted molecules, extracellular particles, released or secreted proteins, transfer mechanisms, etc. Examples of extracellular particles include, but are not limited to, extracellular vesicles (EVs), viral vectors, etc. [Means for solving the problem]

[0004] Extracellular vesicles (EVs) are produced by cells and released during cell culture into the fluid or medium in which they are cultured or grown (often called conditioned medium due to the presence of important by-products produced during growth). EVs include, for example, exosomes and microvesicles. EVs contain proteins, RNA, and DNA that are essential for cell communication and other important cellular processes. EVs can be isolated from body fluids, for example, serum, plasma, urine, and cell culture supernatants.

[0005] In embodiments, intercellular communication plays an important function in cell biology. The ability of cells to communicate with other cells allows complex mechanisms such as protein synthesis to occur. There are many ways that cells can communicate with each other, for example, direct cell-cell contact or the transfer of secreted molecules. EVs, such as microvesicles and exosomes, have the ability to mediate intracellular communication and facilitate the transfer of genetic information. Microvesicles are direct buds from the plasma membrane and often contain surface markers similar to the membrane of origin. Exosomes are formed when vesicular endosomes fuse with the plasma membrane and bud into the extracellular space. Due to their active role in genetic information transfer, microvesicles and exosomes can be used for therapeutic applications. For example, EVs act as antigen-presenting cells to stimulate immune responses, and microvesicles transfer and activate chemokine receptors, resulting in anti-apoptotic effects.

[0006] In an embodiment, a cell growth system is used to grow cells. Such growth is achieved by the use of a bioreactor or cell growth chamber. In one embodiment, such a bioreactor or cell growth chamber includes, for example, a hollow fiber membrane. Such a hollow fiber membrane includes an outer capillary (EC) space and an inner capillary (IC) space. The cell growth system can grow various types of cells, such as mesenchymal stem cells, cancer cells, T cells, fibroblasts, and myoblasts. Each of these types of cells releases EVs into the fluid space of the bioreactor, which can then be collected via an outlet bag. The semi-permeable hollow fibers of the bioreactor allow essential nutrients (e.g., glucose) to reach the cells and metabolic waste products (e.g., lactate) to exit the system via diffusion. The cells are retained inside the capillaries of the hollow fibers, and the EVs are concentrated in the fluid space, and then the EVs are harvested from the system without harvesting the cells if harvesting of the cells is not desired.

[0007] Embodiments of the present disclosure further relate to the use of automated washing procedures to remove serum proteins used to culture or grow the cells prior to collection of released cell products (e.g., EVs or viral vectors, etc.) from the proliferating cells. Such washing procedures allow the system to purify the released cell products (e.g., EVs or viral vectors, etc.) by first removing them from the serum or other source used to grow the cells prior to commencing collection of the released cell products from the proliferating cells.

[0008] The embodiments of the present disclosure allow for the collection or concentration of released cell products by the use of a multi-compartment bioreactor. By controlling the outlet parameters, for example by closing the IC outlet valve (leaving the EC outlet open), the released cell products increase in concentration on the IC side, while nutrients (e.g. glucose) reach the cells on the IC side by the addition of medium to the EC side and diffusion through the membrane. Such collection is continued for a period of time, such as about 24 hours to about 72 hours. In one embodiment, such collection is continued for about 48 hours. After increasing the concentration of the released cell products, the released cell products are collected in a harvest bag or other container. According to one embodiment, the attached cells may remain in the bioreactor during the harvest process, until it is no longer desired to release and harvest such cells, if at all.

[0009] Embodiments of the present disclosure allow for such generation and / or collection of released cell products to be performed using one or more protocols or tasks for use with a cell growth system. Such protocols or tasks may include pre-programmed protocols or tasks. In other embodiments, such protocols or tasks may include custom or user-defined protocols or tasks. Custom or user-defined protocols or tasks may be created via user interface (UI) and graphical user interface (GUI) elements. A task may include one or more steps. In other embodiments, a pre-programmed task, a default task, or a previously saved task may be selected. In yet other embodiments, such generation and / or collection may be performed through the use of one or more manual protocols or tasks for use with a cell growth system.

[0010] This section is provided to present a selection of concepts in a simplified form that are described in more detail below in the Detailed Description. This section is not intended to be used to limit the scope of the subject matter defined by the claims. In addition to the features presented herein, there may be included features that include equivalents and modifications thereof.

[0011] In the following, embodiments of the present invention will be described with reference to the accompanying drawings, in which like reference numerals denote like elements, and in which: [Brief description of the drawings]

[0012] [Figure 1A] FIG. 1A is a diagram of one embodiment of a cell expansion system (CES). [Figure 1B] FIG. 1B is a front view of one embodiment of a bioreactor showing the circuit through the bioreactor. [Figure 1C] FIG. 1C is a diagram of a rocking device for rotationally or laterally moving a cell growth chamber during operation of a cell growth system according to an embodiment of the present disclosure. [Diagram 2] FIG. 2 is a perspective view of a cell growth system with a pre-attached fluid transfer device according to an embodiment of the present disclosure. [Diagram 3] FIG. 3 is a perspective view of a housing of a cell growth system according to an embodiment of the present disclosure. [Figure 4] FIG. 4 is a perspective view of a pre-attached fluid transfer device according to an embodiment of the present disclosure. [Diagram 5] FIG. 5 is a schematic diagram of a cell expansion system according to one embodiment of the present disclosure. [Figure 6] FIG. 6 is a schematic diagram of a cell expansion system according to another embodiment of the present disclosure. [Figure 7] FIG. 7 is a flow chart illustrating operational features of a process for generating and / or collecting releasable components according to an embodiment of the present disclosure. [Figure 8]FIG. 8 is a flow chart illustrating operational features of a process for generating and / or collecting released cell product according to an embodiment of the present disclosure. [Figure 9] FIG. 9 is a flow chart illustrating operational features of a process for generating and / or collecting an emission material according to an embodiment of the present disclosure. [Figure 10] FIG. 10 illustrates an example of a processing system of a cell growth system in which embodiments of the present disclosure may be implemented. [Figure 11] FIG. 11 is a diagram showing an example of the results of protein extraction from a culture medium in a cell growth system according to one embodiment of the present disclosure. [Figure 12] FIG. 12 shows example results of EV generation using a cell expansion system according to one embodiment of the present disclosure. [Figure 13A] FIG. 13A shows example results of EV generation using a cell expansion system according to one embodiment of the present disclosure. [Figure 13B] FIG. 13B shows example results of EV generation using a cell expansion system according to one embodiment of the present disclosure. [Figure 13C] FIG. 13C shows example results of EV generation using a cell expansion system according to one embodiment of the present disclosure. [Figure 14] FIG. 14 shows example results of EV generation using a cell expansion system according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the following detailed description, exemplary embodiments are described with reference to the accompanying drawings. Although certain embodiments will be included herein, they should not be construed as limiting or restricting the present disclosure. In addition, in describing the embodiments, for example, specific terms may be used for features, operations, and / or structures, but the scope of the claims of the present disclosure is not limited to these features, operations, and / or structures. Those skilled in the art will appreciate that other embodiments, including improvements, are within the spirit and scope of the present disclosure. Furthermore, any alternatives or additions, including any listed as other embodiments, may be used or incorporated with any other embodiments described herein.

[0014] Embodiments of the present disclosure generally relate to systems and methods for generating, isolating and / or harvesting released cellular products (e.g., extracellular vesicles (EVs), viral vectors, etc.) in cell growth systems. Embodiments of the present disclosure further provide for enabling the harvesting or concentration of released cellular products, for example, by the use of multi-compartment bioreactors.

[0015] In an embodiment, the permeability of the hollow fiber membrane allows cells to be separated from other components by retaining them in the capillary inner (IC) loop, while allowing soluble molecules to pass freely through the capillary outer (EC) loop. This can eliminate the need for an additional separation step. Media added to the EC side of the bioreactor can be used to meet the metabolic needs of the cells in culture (e.g., glucose, lactate, amino acids, vitamins). Such media can diffuse through the semi-permeable membrane of the bioreactor. Media components that are too large in molecular weight to diffuse through the membrane may be added to the IC side of the bioreactor using ultrafiltration (e.g., either continuous or intermittent bolus addition). In one embodiment, ultrafiltration (IC outlet valve closed) is used to maintain components that are too large to diffuse through the membrane in the IC side of the bioreactor. EVs produced by the cells may not be able to diffuse through the membrane, i.e., their molecular weight may be too large. Thus, EVs are maintained in the IC side of the bioreactor during growth (or a given harvest period) during which the EV concentration is continuously increased. The EVs are then harvested, for example, from the IC side of the bioreactor into a harvest container or harvest bag at the end of a predefined interval or the entire process. Without the benefit of the two fluid compartments of the hollow fiber membrane, EV concentration or collection is limited to the rate at which the cells produce EVs, and fresh medium is added to the culture environment to meet nutritional requirements.

[0016] For example, by controlling the outlet parameters, such as closing the IC outlet valve (leaving the EC outlet open), the released cell products will increase in concentration on the IC side, but nutrients (e.g., glucose) can still reach the cells on the IC side by adding medium to the EC side and diffusing through the membrane. In other embodiments, nutrients can be provided to the cells on the IC side by adding medium on the IC side. In yet other embodiments, cell growth can also occur on the EC side by adding medium to the IC side (or EC side) and diffusing through the membrane to reach the cells. Collection of EVs can continue for a period of about 24 hours or more. In other embodiments, such collection can continue for less than about 24 hours. In other embodiments, such collection can continue for, for example, about 48 hours to about 72 hours. After increasing the concentration of the released cell products, the released cell products can be harvested into a harvest bag or other container. In some embodiments, the attached cells may remain in the bioreactor during the harvest process until it is desired to release and harvest the cells, if at all.

[0017] The embodiments relate to a cell growth system, as described above. In an embodiment, the cell growth system is a closed system. The closed cell growth system includes contents that are not directly exposed to the atmosphere. Such a cell growth system may be automated. In an embodiment, both adherent or non-adherent types or suspension types of cells may be grown in the bioreactor of the cell growth system. According to an embodiment, the cell growth system may include a basal medium or other type of medium. A method of replenishing the medium is provided for cell growth performed in the bioreactor of the closed cell growth system. In an embodiment, the bioreactor used with such a system is a hollow fiber bioreactor. According to an embodiment of the invention, various bioreactors may be used.

[0018] In an embodiment, the system includes a bioreactor including a first fluid flow path having at least two ends, a first end of the first fluid flow path being fluidly associated with a first port of a hollow fiber membrane, a second end of the first fluid flow path being fluidly associated with a second port of the hollow fiber membrane, and the first fluid flow path including a capillary inner portion of the hollow fiber membrane. In an embodiment, the hollow fiber membrane includes a plurality of hollow fibers. The system includes a fluid inlet passage fluidly associated with the first fluid flow path, through which a plurality of cells are introduced into the first fluid flow path. Also included is a first pump for circulating a fluid in the first fluid flow path of the bioreactor. In an embodiment, the system includes a controller for controlling the operation of the first pump. In an embodiment, the controller is, for example, a computing system having a processor. In an embodiment, the controller controls the pump to circulate a fluid in the first fluid flow path at a first rate. In some embodiments, the system further includes a second pump for transferring an inner capillary input fluid from an inner capillary medium bag to the first fluid flow path, and a second controller for controlling the operation of the second pump. In embodiments, the second controller controls the second pump, for example, to transfer cells from a cell input bag to the first fluid flow path. In embodiments, additional controllers (e.g., a third controller, a fourth controller, a fifth controller, a sixth controller, etc.) may be used. In embodiments of the present disclosure, additional pumps (e.g., a third pump, a fourth pump, a fifth pump, a sixth pump, etc.) may be used. Furthermore, although the present disclosure refers to a medium bag, a cell input bag, etc., multiple bags may be used, e.g., a first medium bag, a second medium bag, a third medium bag, a first cell input bag, a second cell input bag, a third cell input bag, etc., and / or other types of containers. In other embodiments, a single medium bag, a single cell input bag, etc. is used. Also, in embodiments, additional or alternative flow paths (e.g., a second fluid flow path, a second fluid inlet path, etc.) may be provided.

[0019] In another embodiment, the system is controlled by a processor, e.g., coupled to the cell growth system, a display device in communication with the processor for displaying data, and a memory in communication with the processor and readable by the processor, storing a set of instructions. In an embodiment, instructions executed by the processor include, e.g., receiving an instruction directing coating of a bioreactor. In response to the instruction, the processor performs a set of steps to coat the bioreactor, and then receiving an instruction to, e.g., introduce cells into the bioreactor. In response to the instruction, the processor performs a set of steps to introduce cells into the bioreactor, e.g., from a cell input bag.

[0020] An example of a cell growth system (CES) according to an embodiment of the present invention is shown diagrammatically in FIG. 1A. The terms "CES" and "system" are used interchangeably. The CES 10 includes a first fluid circuit 12 and a second fluid circuit 14. According to an embodiment, the first fluid flow path 16 has at least two ends 18, 20 that are fluidly associated with a hollow fiber cell growth chamber 24 (also referred to as a "bioreactor"). In particular, the end 18 is fluidly associated with a first inlet 22 of the cell growth chamber 24, and the end 20 is fluidly associated with a first outlet 28 of the cell growth chamber 24. In the first circuit 12, fluid passes through the interior of hollow fibers 116 (see FIG. 1B) of a hollow fiber membrane 117 (see FIG. 1B) disposed in the cell growth chamber 24 (the cell growth chamber and hollow fiber membrane are described in more detail below). Additionally, a first flow controller 30 is operatively connected to the first fluid flow path 16 to control the flow of fluid in the first circuit 12.

[0021] The second fluid circuit 14 includes a second fluid flow path 34, a cell growth chamber 24, and a second flow controller 32. According to an embodiment, the second fluid flow path 34 has at least two ends 36, 38. The end 36 of the second fluid flow path 34 is fluidly associated with an inlet port 40 of the cell growth chamber 24, and the end 38 is fluidly associated with an outlet port 42 of the cell growth chamber 24. Fluid flowing through the cell growth chamber 24 contacts the exterior of a hollow fiber membrane 117 (see FIG. 1B) of the cell growth chamber 24. The hollow fiber membrane comprises a plurality of hollow fibers. The second fluid circuit 14 is operatively connected to the second flow controller 32.

[0022] Thus, the first and second fluid circuits 12, 14 are separated in the cell growth chamber 24 by a hollow fiber membrane 117 (see FIG. 1B). The fluid in the first fluid circuit 12 flows through the inner capillary (IC) space of the hollow fibers in the cell growth chamber 24. The first fluid circuit 12 is therefore referred to as the "IC loop." The fluid in the second fluid circuit 14 flows through the outer capillary (EC) space of the hollow fibers in the cell growth chamber 24. The second fluid circuit 14 is therefore referred to as the "EC loop." According to an embodiment, the fluid in the first fluid circuit 12 may flow either co-currently or counter-currently with respect to the flow of the fluid in the second fluid circuit 14.

[0023] The fluid inlet passage 44 is fluidly associated with the first fluid circuit 12. The fluid inlet passage 44 allows fluid to be introduced into the first fluid circuit 12, and the fluid outlet passage 46 allows fluid to be removed from the CES 10. A third flow control device 48 is operatively associated with the fluid inlet passage 44. Alternatively, the third flow control device 48 may be associated with the first outlet passage 46.

[0024] According to embodiments, the flow control device as used herein can be a pump, a valve, a clamp, or a combination thereof. Any combination of pumps, valves, and clamps may be arranged. In various embodiments, the flow control device is or includes a peristaltic pump. In other embodiments, the fluid circuit, inlet ports, and outlet ports may be comprised of tubing of any material.

[0025] Various members are described as being "operably associated," but as used herein, "operably associated" refers to members that are connected together in an operable manner, including embodiments in which the members are directly connected, or in which another member is disposed between two connected members, etc. "Operatively associated" members can be "fluidically associated." "Fluidly associated" refers to members that are connected together such that fluid can move between the members. The term "fluidly associated" includes embodiments in which another member is disposed between two fluidly associated members, or in which the members are directly connected, etc. Fluidly associated members may include members that do not contact the fluid but that operate the system by contacting other members (e.g., a peristaltic pump that compresses the outside of a flexible tube to pump fluid through the tube).

[0026] In general, any type of fluid can flow through the circuit, inlet and outlet channels, such as buffers, protein-containing fluids, cell-containing fluids, etc. As used herein, the terms "fluid," "media," and "fluid medium" are used interchangeably.

[0027] 1B is a front view of an example of a hollow fiber cell growth chamber 100 for use with the present invention. Cell growth chamber 100 has a longitudinal axis LA-LA and comprises a cell growth chamber housing 104. In at least one embodiment, cell growth chamber housing 104 has four openings or ports: IC inlet port 108, IC outlet port 120, EC inlet port 128, and EC outlet port 132.

[0028] According to an embodiment of the invention, fluid in a first circuit enters cell growth chamber 100 through IC inlet port 108 at first longitudinal end 112 of cell growth chamber 100, passes into and through the capillary inside of a plurality of hollow fibers 116 that make up hollow fiber membrane 117 (referred to in various embodiments as the intra-capillary ("IC") side or "IC space" of the hollow fiber membrane), and exits cell growth chamber 100 through IC outlet port 120 located at second longitudinal end 124 of cell growth chamber 100. The flow path between IC inlet port 108 and IC outlet port 120 constitutes IC portion 126 of cell growth chamber 100. Fluid in a second circuit enters cell growth chamber 100 through EC inlet port 128, contacts the capillary outside or exterior of hollow fibers 116 (referred to as the "EC side" or "EC space" of the membrane), and exits cell growth chamber 100 through EC outlet port 132. The flow path between the EC inlet port 128 and the EC outlet port 132 constitutes the EC portion 136 of the cell growth chamber 100. Fluid entering the cell growth chamber 100 through the EC inlet port 128 contacts the outside of the hollow fibers 116. Small molecules (e.g., ions, water, oxygen, lactate, etc.) can diffuse through the hollow fibers 116 from the interior or IC space of the hollow fibers to the exterior or EC space, or from the EC space to the IC space. High molecular weight molecules such as growth factors are typically too large to pass through the hollow fiber membrane and remain within the IC space of the hollow fibers 116. In embodiments, the medium may be replaced if necessary. Also, if necessary, the medium may be circulated through an oxygenator or gas transfer module to replace gas. In embodiments, cells may be contained within the first and / or second circuits and may be present on the IC and / or EC side of the membrane, as described below.

[0029] The material used to make the hollow fiber membrane 117 can be any biocompatible polymeric material that can be made into a hollow fiber. According to one embodiment of the present invention, one of the materials used is a synthetic polysulfone-based material. To allow cells to attach to the surface of the hollow fiber, according to an embodiment, the surface can be modified in some way. For example, the surface can be modified by coating at least the cell growth surface with a protein such as fibronectin (FN) or collagen, or by irradiating the surface. Gamma treatment of the membrane surface allows for the attachment of adherent cells without additional coatings such as fibronectin or cryoprecipitate. Bioreactors made with gamma irradiated membranes can be reused. Other coatings and / or treatments for cell attachment can be performed in embodiments of the present disclosure.

[0030] In embodiments, the CES (e.g., CES500 (FIG. 5) and / or CES600 (FIG. 6)) has apparatus configured to move or "rock" the cell growth chamber relative to other components of the cell growth system by attaching it to a rotational and / or lateral rocking device. FIG. 1C illustrates one such device. In one embodiment, in the device, the bioreactor 100 is rotationally connected to two rotational rocking components and one lateral rocking component.

[0031] The first rotational rocker part 138 rotates the bioreactor 100 about a central axis 142 of the bioreactor 100. The rotational rocker part 138 is rotationally associated with the bioreactor 100. In an embodiment, the bioreactor 100 can be rotated continuously in a single direction (clockwise or counterclockwise) about the central axis 142. Alternatively, the bioreactor 100 can be rotated alternately, for example, first clockwise and then counterclockwise about the central axis 142.

[0032] The CES also has a second rotational oscillating component that rotates the bioreactor 100 about the axis of rotation 144. The axis of rotation 144 passes through a center point of the bioreactor 100 and is perpendicular to the central axis 142. In embodiments, the bioreactor 100 can be continuously rotated in a single direction about the axis of rotation 144, either clockwise or counterclockwise. Alternatively, the bioreactor 100 can be alternately rotated about the axis of rotation 144, for example, first clockwise and then counterclockwise. In various embodiments, the bioreactor 100 can be rotated about the axis of rotation 144 and can be placed in a horizontal position or a vertical position relative to gravity.

[0033] In an embodiment, a lateral oscillating element 140 is associated with the bioreactor 100 for lateral movement. In an embodiment, the plane of the lateral oscillating element 140 moves in the x and y directions, such that settling of cells in the bioreactor is reduced by movement of cell-containing medium within the hollow fibers.

[0034] The rotational and / or lateral movement of the rocking device reduces settling of cells within the device and also reduces the possibility of cells becoming trapped in certain parts of the bioreactor. The settling rate of cells in the cell growth chamber is proportional to the density difference between the cells and the suspension medium according to Stokes' law. In one embodiment, non-adherent red blood cells are kept in suspension by alternating 180° rotations (fast) and pauses (e.g., total time 30 seconds) as described above. In one embodiment, a minimum rotation of about 180° is preferred. However, rotations of 360° or more can also be performed. Different rocking components may be used separately or in combination. For example, a rocking component that rotates the bioreactor 100 about the central axis 142 can be combined with a rocking component that rotates the bioreactor 100 about the axis 144. Similarly, clockwise and counterclockwise rotations about different axes can be independently combined.

[0035] 2, an embodiment of a cell growth system 200 having a pre-mounted fluid transport assembly is shown in accordance with an embodiment of the present invention. The CES 200 includes a cell growth apparatus 202. The cell growth apparatus 202 has a hatch or closeable door 204 that engages a rear portion 206 of the cell growth apparatus 202. An interior space 208 within the cell growth apparatus 202 is characterized to receive and securely engage the pre-mounted fluid transport assembly 210. The pre-mounted fluid transport assembly 210 is removably attached to the cell growth apparatus 202 to allow for relatively quick replacement of a used fluid transport assembly 210 with a new or unused fluid transport assembly 210 in the cell growth apparatus 202. A single cell growth device 202 can be operated to grow or expand a first set of cells using a first fluid transport assembly 210 and then grow or expand a second set of cells using a second fluid transport assembly 210 without sterilizing the cell growth device 202 when replacing the first fluid transport assembly 210 with the second fluid transport assembly 210. The pre-mounted fluid transport assembly 210 can include a bioreactor 100 and an oxygenator or gas transfer module 212 (see FIG. 4). An embodiment of a line guide slot is shown as 214 for receiving various media lines that are connected to the fluid transport assembly 210.

[0036] Figure 3 illustrates the rear portion 206 of the cell growth apparatus 202 prior to removable attachment of a pre-mounted fluid transport assembly 210 (see Figure 2) according to an embodiment of the present invention. The closeable door 204 (see Figure 2) has been omitted from Figure 3. The rear portion 206 of the cell growth apparatus 202 includes a number of different structures that operate in conjunction with the components of the fluid transport assembly 210. In particular, the rear portion 206 of the cell growth apparatus 202 includes a number of peristaltic pumps (IC circulation pump 218, EC circulation pump 220, IC inlet pump 222, EC inlet pump 224) that operate in conjunction with pump loops in the fluid transport assembly 210. The rear portion 206 of the cell growth apparatus 202 also includes a number of valves (IC circulation valve 226, reagent valve 228, IC media valve 230, air removal valve 232, cell inlet valve 234, wash valve 236, distribution valve 238, EC media valve 240, IC waste valve 242, EC waste valve 244, harvest valve 246). Additionally, a number of sensors (IC outlet pressure sensor 248, IC inlet pressure / temperature sensor 250, EC inlet pressure / temperature sensor 252, EC outlet pressure sensor 254) are associated with the rear portion 206 of the cell growth apparatus 202. Also shown is an optical sensor 256 for an air removal chamber (ARC), according to one embodiment.

[0037] According to an embodiment, a shaft or rocker control 258 is shown for rotating the bioreactor 100. A shaft fitting 260 associated with the shaft or rocker control 258 provides proper alignment of the shaft access opening of the fluid transport assembly 210 or the fluid transport assembly 400 (e.g., opening 424 (FIG. 4) in the tubing housing 300 (FIG. 4)) relative to the rear portion 206 of the cell growth device 202. Rotation of the shaft or rocker control 258 imparts rotational motion to the shaft fitting 260 and the bioreactor 100. Thus, when an operator or user of the CES 200 installs a new or unused fluid transport assembly 400 (FIG. 4) to the cell growth device 202, alignment is a relatively simple matter of properly orienting the shaft access opening of the fluid transport assembly 210 or the fluid transport assembly 400 (e.g., opening 424 in FIG. 4) relative to the shaft fitting 260.

[0038] FIG. 4 is a perspective view of a pre-mounted, removable fluid transport assembly 400. The pre-mounted fluid transport assembly 400 is removably mounted to the cell growth device 202 (FIGS. 2 and 3) to allow for relatively quick replacement of a used fluid transport assembly 400 with a new or unused fluid transport assembly 400 in the cell growth device 202. As shown in FIG. 4, the bioreactor 100 is mounted to a bioreactor coupling that includes a shaft fitting 402. The shaft fitting 402 has one or more shaft fastening mechanisms (biased arms or spring members 404) for engaging the shaft (258 in FIG. 3) of the cell growth device 202.

[0039] According to an embodiment, the fluid transport assembly 400 includes tubing 408A, 408B, 408C, 408D, 408E, etc. and fittings to provide the fluid paths shown in Figures 5 and 6, as described below. Pump loops 406A, 406B are also provided for the pumps. In an embodiment, various media may be provided where the cell growth device 202 is located, but according to an embodiment, the pre-mounted fluid transport assembly 400 includes sufficient tubing to extend outside the cell growth device 202 to allow tubing associated with a media bag or media container to be welded.

[0040] Next, Fig. 5 is a schematic diagram of an embodiment of a cell growth system 500. Fig. 6 is a schematic diagram of another embodiment of a cell growth system 600. In the embodiment shown in Figs. 5 and 6, the cells are grown in the IC space, as described below. However, the invention is not limited to such an example. In another embodiment, the cells may be grown in the EC space.

[0041] FIG. 5 shows a CES 500 according to an embodiment. The CES 500 has a first fluid circuit 502 (also called the "capillary inner loop" or "IC loop") and a second fluid circuit 504 (also called the "capillary outer loop" or "EC loop"). A first fluid flow path 506 is fluidly associated with a cell growth chamber 501 to form the first fluid circuit 502. Fluid enters the cell growth chamber 501 via an IC inlet port 501A, passes through hollow fibers in the cell growth chamber 501, and exits via an IC outlet port 501B. A pressure measuring device 510 measures the pressure of the medium leaving the cell growth chamber 501. The medium flows via an IC circulation pump 512, which is used to control the medium flow rate. The IC circulation pump 512 can pump the fluid in a first direction or a second direction opposite the first direction. The outlet port 501B can be used as an inlet in the reverse direction. Media entering the IC loop may enter through valve 514. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports and / or other devices may be placed in various locations to isolate media and / or measure properties of the media in certain portions of the fluid path. Thus, it should be understood that the schematic shown is one possible configuration for elements of CES 500 and that variations are possible within the scope of one or more embodiments.

[0042] For the IC loop 502, samples of medium are taken from sample port 516 or sample coil 518 during operation. A pressure / temperature measuring device 520 located in the first fluid circuit 502 allows medium pressure and temperature to be measured during operation. The medium then returns to the IC inlet port 501A completing the fluid circuit 502. Cells grown / expanded in the cell growth chamber 501 are flushed out of the cell growth chamber 501 and passed through valve 598 into a harvest bag 599 or redistributed into the hollow fibers for further growth.

[0043] In second fluid circuit 504, fluid enters cell growth chamber 501 through EC inlet port 501C and leaves cell growth chamber 501 through EC outlet port 501D. In EC loop 504, medium contacts the outside of the hollow fibers of cell growth chamber 501, thereby allowing diffusion of small molecules into and out of the hollow fibers.

[0044] In an embodiment, the pressure and temperature of the medium can be measured by a pressure / temperature measuring device 524 located in the second fluid circuit 504 before the medium enters the EC space of the cell growth chamber 501. After the medium leaves the cell growth chamber 501, the pressure of the medium in the second fluid circuit 504 can be measured by a pressure measuring device 526. For the EC loop, a sample of the medium is obtained from a sample port 530 or from a sample coil during operation.

[0045] In embodiments, after leaving the EC outlet port 501D of the cell growth chamber 501, the fluid in the second fluid circuit 504 passes through an EC circulation pump 528 to an oxygenator or gas transfer module 532. The EC circulation pump 528 is capable of pumping fluid in both directions. The second fluid flow path 522 is fluidly associated with the oxygenator or gas transfer module 532 via an oxygenator inlet port 534 and an oxygenator outlet port 536. In operation, the fluid medium enters the oxygenator or gas transfer module 532 via the oxygenator inlet port 534 and exits the oxygenator or gas transfer module 532 via the oxygenator outlet port 536. The oxygenator or gas transfer module 532 adds oxygen and removes air bubbles to the medium in the CES 500, for example. In various embodiments, the medium in the second fluid circuit 504 is in equilibrium with the gas entering the oxygenator or gas transfer module 532. Oxygenator or gas transfer module 532 may be any suitable sized oxygenator or gas transfer device. Air or gas enters oxygenator or gas transfer module 532 through filter 538 and exits oxygenator or gas transfer device 532 through filter 540. Filters 538, 540 reduce or prevent contamination of oxygenator or gas transfer module 532 and associated media. Air or gas purged from CES 500 during part of the priming process may be vented to atmosphere through oxygenator or gas transfer module 532.

[0046] In a configuration including a CES 500, the fluid media in the first fluid circuit 502 and the second fluid circuit 504 flow in the same direction (co-current configuration) through the cell growth chamber 501. The CES 500 may also be configured for counter-current flow.

[0047] In at least one embodiment, the cell-containing medium (from bag 562) and fluid medium from bag 546 are introduced into first fluid circuit 502 via first fluid flow path 506. A fluid reservoir 562 (e.g., a cell input bag or saline priming fluid for priming air out of the system) is fluidly associated with first fluid flow path 506 and first fluid circuit 502 via valve 564.

[0048] A fluid container (or media bag) 544 (e.g., a reagent) is fluidly associated with the first fluid inlet passage 542 via valve 548 or with the second fluid inlet passage 574 via valve 576, and a fluid container 546 (e.g., IC media) is fluidly associated with the first fluid inlet passage 542 via valve 550 or with the second fluid inlet passage 574 via valve 570. Also provided are first and second sterile and sealable input priming passages 508, 509. An air elimination chamber (ARC) 556 is fluidly associated with the first circuit 502. The air elimination chamber 556 may have one or more ultrasonic sensors, including upper and lower sensors for detecting air, lack of fluid, and / or gas / fluid boundaries, e.g., air / fluid boundaries, at specific measurement points within the air elimination chamber 556. For example, ultrasonic sensors may be used near the bottom and / or top of the air removal chamber 556 to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors may be used without departing from the scope of the present disclosure. For example, optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from the CES 500 during part of the priming process or other protocols may be vented to atmosphere through an air valve 560 via a line 558 in fluidic association with the air removal chamber 556.

[0049] EC medium (e.g., from bag 568) or wash solution (e.g., from bag 566) is added to the first or second fluid flow paths. Fluid container 566 is fluidly associated with valve 570. Valve 570 is fluidly associated with first fluid circuit 502 via distribution valve 572 and first fluid inlet path 542. By opening valve 570 and closing distribution valve 572, fluid container 566 can also be fluidly associated with second fluid circuit 504 via second fluid inlet path 574 and EC inlet path 584. Similarly, fluid container 568 is fluidly associated with valve 576. Valve 576 is fluidly associated with first fluid circuit 502 via first fluid inlet path 542 and distribution valve 572. By opening valve 576 and closing distribution valve 572, fluid container 568 can also be fluidly associated with second fluid inlet path 574.

[0050] An optional heat exchanger 552 may be provided for the introduction of media reagents or for the introduction of wash fluids.

[0051] In the IC loop, the fluid is first pumped by IC inlet pump 554. In the EC loop, the fluid is first pumped by EC inlet pump 578. An air detector 580, such as an ultrasonic sensor, may be associated with the EC inlet line 584.

[0052] In at least one embodiment, the first and second fluid circuits 502, 504 are connected to a waste line 588. When valve 590 is opened, IC medium flows through waste line 588 to waste or outlet bag 586. Similarly, when valve 582 is opened, EC medium flows through waste line 588 to waste or outlet bag 586.

[0053] In an embodiment, cells are harvested through cell harvest channel 596. Here, cells from the cell growth chamber 501 are harvested by pumping the IC medium containing the cells through cell harvest channel 596 and valve 598 into a cell harvest bag 599.

[0054] The various components of the CES 500 are housed within a device or housing, such as a cell growth device 202 (see FIGS. 2 and 3), which maintains the cells and culture medium, for example, at a predetermined temperature.

[0055] FIG. 6 is a schematic diagram of another embodiment of a cell growth system 600. The CES 600 has a first fluid circuit 602 (also referred to as the "capillary inner loop" or "IC loop") and a second fluid circuit 604 (also referred to as the "capillary outer loop" or "EC loop"). A first fluid flow path 606 is fluidly associated with a cell growth chamber 601 to form the first fluid circuit 602. Fluid enters the cell growth chamber 601 via an IC inlet port 601A, passes through hollow fibers in the cell growth chamber 601, and exits via an IC outlet port 601B. A pressure sensor 610 measures the pressure of the medium leaving the cell growth chamber 601. In addition to pressure, the sensor 610 may be a temperature sensor that senses medium pressure and medium temperature during operation. The medium flows through an IC circulation pump 612 that is used to control the medium flow rate. The IC circulation pump 612 can pump the fluid in a first direction or a second direction opposite the first direction. Outlet port 601B can be used as an inlet in the reverse direction. Media entering the IC loop can enter through valve 614. As will be appreciated by those skilled in the art, additional valves, pressure gauges, pressure / temperature sensors, ports and / or other devices can be placed in various locations to isolate media and / or measure properties of the media in certain portions of the fluid path. Thus, it should be understood that the schematic shown is one possible configuration for elements of CES 600 and that variations are possible within the scope of one or more embodiments.

[0056] For the IC loop, samples of media are taken from sample coil 618 during operation and the media returns to IC inlet port 601A completing the fluid circuit 602. Cells grown / expanded in cell growth chamber 601 are flushed out of cell growth chamber 601 and into harvest bag 699 via valve 698 and line 697. Alternatively, if valve 698 is closed the cells are redistributed back into chamber 601 for further growth.

[0057] In the second fluid circuit 604, fluid enters the cell growth chamber 601 through EC inlet port 601C and leaves the cell growth chamber 601 through EC outlet port 601D. In the EC loop, the medium contacts the outside of the hollow fibers of the cell growth chamber 601, thereby allowing diffusion of small molecules into and out of the fibers within the chamber 601.

[0058] Before the medium enters the EC space of the cell growth chamber 601, the pressure and temperature of the medium can be measured by a pressure / temperature sensor 624 located in the second fluid circuit 604. After the medium leaves the cell growth chamber 601, the pressure and / or temperature of the medium in the second fluid circuit 604 can be measured by a sensor 626. For the EC loop, a sample of the medium is obtained from a sample port 630 or from a sample coil during operation.

[0059] After leaving the EC outlet port 601D of the cell growth chamber 601, the fluid in the second fluid circuit 604 passes through an EC circulation pump 628 to an oxygenator or gas transfer module 632. In embodiments, the EC circulation pump 628 is also capable of pumping fluid in both directions. The second fluid flow path 622 is fluidly associated with the oxygenator or gas transfer module 632 via an inlet port 632A and an outlet port 632B of the oxygenator or gas transfer module 632. In operation, the fluid medium enters the oxygenator or gas transfer module 632 via an inlet port 632A and exits the oxygenator or gas transfer module 632 via an outlet port 632B. The oxygenator or gas transfer module 632 adds oxygen and removes air bubbles to the medium in the CES 600, for example. In various embodiments, the medium in the second fluid circuit 604 is in equilibrium with the gas entering the oxygenator or gas transfer module 632. The oxygenator or gas transfer module 632 may be any suitably sized device useful for oxygen supply or gas transfer. Air or gas enters the oxygenator or gas transfer module 632 through filter 638 and exits the oxygenator or gas transfer module 632 through filter 640. Filters 638, 640 reduce or prevent contamination of the oxygenator or gas transfer module 632 and associated media. Air or gas purged from the CES 600 during part of the priming process may be vented to atmosphere through the oxygenator or gas transfer module 632.

[0060] In the configuration shown as CES 600, the fluid media in the first fluid circuit 602 and the second fluid circuit 604 flow in the same direction (co-current configuration) through the cell growth chamber 601. Embodiments of the CES 600 may also be configured for counter-current flow.

[0061] In at least one embodiment, media containing cells (from a source such as a cell container, e.g., a bag) is attached to attachment point 662, and fluid media from a media source is attached to attachment point 646. The cells and media are introduced into the first fluid circuit 602 via a first fluid flow path 606. Attachment point 662 is fluidly associated with the first fluid flow path 606 via a valve 664. Attachment point 646 is fluidly associated with the first fluid flow path 606 via a valve 650. A reagent source may be fluidly connected to point 644 and associated with the fluid inlet path 642 via a valve 648, or associated with a second fluid inlet path 674 via valves 648, 672.

[0062] An air elimination chamber (ARC) 656 is fluidly associated with the first circuit 602. The air elimination chamber 656 may have one or more sensors, including upper and lower sensors for detecting air, lack of fluid, and / or gas / fluid boundaries, e.g., air / fluid boundaries, at specific measurement points within the air elimination chamber 656. For example, ultrasonic sensors may be used near the bottom and / or top of the air elimination chamber 656 to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors may be used without departing from the scope of the present disclosure. For example, optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from the CES 600 during part of the priming process or other protocols may be vented to atmosphere through a line 658 in fluid association with the air elimination chamber 656 and through an air valve 660.

[0063] An EC medium source is attached to EC medium attachment point 668. A wash fluid source is attached to wash fluid attachment point 666, which allows EC medium and / or wash fluid to be added to the first or second fluid flow paths. Attachment point 666 is fluidly associated with valve 670. Valve 670 is fluidly associated with first fluid circuit 602 via valve 672 and first fluid inlet path 642. Also, by opening valve 670 and closing valve 672, attachment point 666 can be fluidly associated with second fluid circuit 604 via second fluid inlet path 674 and second fluid flow path 684. Similarly, attachment point 668 is fluidly associated with valve 676. Valve 676 is fluidly associated with first fluid circuit 602 via first fluid inlet path 642 and valve 672. Additionally, fluid container 668 can be placed in fluid association with second fluid inlet passage 674 by opening valve 676 and closing dispensing valve 672 .

[0064] In the IC loop, the fluid is first pumped by the IC inlet pump 654. In the EC loop, the fluid is first pumped by the EC inlet pump 678. An air detector 680, such as an ultrasonic sensor, may be associated with the EC inlet line 684.

[0065] In at least one embodiment, the first and second fluid circuits 602, 604 are connected to a waste line 688. When valve 690 is opened, IC medium flows through the waste line 688 to the waste or outlet bag 686. Similarly, when valve 692 is opened, EC medium flows to the waste or outlet bag 686.

[0066] After the cells are grown in the cell growth chamber 601, the cells are harvested via cell harvest line 697. Cells from the cell growth chamber 601 are harvested into a cell harvest bag 699 via cell harvest line 697 by pumping the IC medium containing the cells with valve 698 open.

[0067] The various components of the CES 600 are housed within a device or housing, such as cell growth device 202 (see FIGS. 2 and 3), which maintains the cells and medium, for example, at a predetermined temperature. Additionally, the components of the CES 600 and CES 500 (see FIG. 5) may be combined. In other embodiments, a CES may include fewer or more components than those shown in FIGS. 5 and 6, while remaining within the scope of the present invention. One example of a cell growth system incorporating features of the present invention is the Quantum® Cell Growth System, manufactured by Terumo BCT, Inc. (Lakewood, Colorado).

[0068] Examples and detailed descriptions of cell expansion systems are provided in U.S. Patent No. 8,309,347 ("Cell Expansion Systems and Methods of Use" issued November 13, 2012), the entirety of which is expressly incorporated herein by reference.

[0069] Having described various embodiments of cell expansion systems and associated methods, FIG. 7 illustrates an example of operational steps 700 of a process for producing, purifying and / or harvesting a release component for use with a cell expansion system such as the CES500 (FIG. 5) or CES600 (FIG. 6) in accordance with embodiments of the present disclosure. Upon initiation at START step 702, the process 700 proceeds to step 704 of seeding a bioreactor with cells. In an embodiment, cells are seeded on Day 0. In one embodiment, MSCs are seeded. As will be appreciated by one of skill in the art, any cells may be used as long as they release the desired cell product (e.g., EVs, viral vectors, etc.).

[0070] Next, according to one embodiment, the cells are grown in medium until confluent (step 706). In other embodiments, the cells are grown until a desired number of cell doublings have occurred (e.g., 1 cell doubling, 2 cell doublings, 3 cell doublings, 4 cell doublings, 5 cell doublings, 6 or more cell doublings, etc.). In other embodiments, the cells may be grown for a particular period of time. For example, the cells may be grown for a period of about 24 hours to about 48 hours. In other embodiments, the cells may be grown for a period of about 48 hours to about 72 hours. In other embodiments, such growth occurs, for example, on days 1 to 3. According to other embodiments, the cells are grown for a period of less than about 24 hours. In other embodiments, the cells may be grown for a period of more than 72 hours. For example, in one embodiment, the cells may be grown for about 7 days to about 8 days before collecting the exosomes. Any period of time may be used in accordance with embodiments of the present disclosure.

[0071] The cells may be grown (step 706), for example, in complete medium. In one embodiment, the medium includes a serum-containing medium, such as alpha-MEM (α-MEM) and serum. In one embodiment, animal-derived serum may be used. In other embodiments, human-derived serum may be used. In other embodiments, synthetic serum may be used. In yet other embodiments, another type of serum may be used. An example of a serum-containing medium includes α-MEM+GlutaMAX+10% fetal bovine serum (FBS). In further embodiments, serum-free medium may be used. Any type of medium known to one of skill in the art may be used.

[0072] Returning to FIG. 7, process 700 then proceeds to optional step 708 to wash out the first medium, such as serum-containing medium. In one embodiment, the serum-containing medium is replaced with a second medium, such as a basal medium. Examples of basal medium include α-MEM+GlutaMAX. Any type of medium known to those skilled in the art can be used. In order to isolate or purify released components from the growing cells (as opposed to those also present in serum or other protein sources), a wash-and-wash procedure removes serum (e.g., serum proteins) according to an embodiment. In one embodiment, the wash procedure is performed on day 3. In one embodiment, execution of step 708 is optional, e.g., if no animal-derived serum is used, if only human-derived serum is used, if serum-free medium is used, and / or if there are no other additional protein sources to be removed, e.g.,

[0073] Process 700 then proceeds to step 710 of collecting the released components or substances (e.g., EVs or viral vectors, etc.) in a loop (e.g., IC loop). In one embodiment, such collection is performed by concentrating the released components or substances (e.g., EVs or viral vectors, etc.) in the IC loop by closing the IC outlet (e.g., closing the IC outlet valve). Such concentration is performed for a defined period of time. In one embodiment, such time includes concentrating the released components in the IC loop for about 48 hours. In other embodiments, such time includes concentrating the released components for about 24 hours to about 48 hours. In other embodiments, such time includes concentrating the released components for about 24 hours to about 72 hours. In other embodiments, such time includes concentrating the released components for about 48 hours to about 72 hours. In one embodiment, such time includes concentrating the released components for longer than about 72 hours. In one embodiment, such collection is performed, for example, on days 3 to 5. In yet other embodiments, such time includes concentrating the released components for less than about 24 hours. Any period can be used according to the embodiments of the present disclosure. During such harvesting period, cells are supplemented with protein-free medium. Such medium is added from the EC side and diffused through the semi-permeable membrane. In other embodiments, medium is added from the IC side, e.g., protein-free medium is supplemented to cells.

[0074] After collecting the released components in the IC (or EC) loop, process 700 proceeds to step 712 of harvesting the released components. In one embodiment, such harvesting occurs, for example, on day 5. In one embodiment, the released components (e.g., EVs or viral vectors, etc.) are transferred in suspension from the capillary inner circulation loop of the bioreactor to a harvest bag or harvest container. In one embodiment, a protein-free medium is used in such a harvesting task.

[0075] Process 700 then proceeds to optional step 714 of performing further processing, where, for example, the harvested released components are processed for analysis. In other embodiments, further processing step 714 can include further concentrating the released material from the medium collected in the harvest bag. In other embodiments, further processing step 714 can include separating the released material from other components in the bag (e.g., cells if suspension cells are used and harvested together with the released material). In one embodiment, such further processing step 714 can include further isolation and / or characterization of the released components. From optional further processing step 714, process 700 ends at end step 716. Alternatively, if optional further processing step 714 is not desired, process 700 can proceed directly from harvesting step 712 to end step 716 and end.

[0076] Next, Figure 8 is described with an example setup and media introduction example. However, the embodiments presented herein are not limited to this example and can be modified to meet other requirements or system designs or configurations. Starting at START step 802, process 800 proceeds to step 804 where a disposable tubing set is loaded into the cell growth system.

[0077] Next, the system is primed in step 806. In one embodiment, for example, a user or operator can provide an instruction to the system to prime by selecting a task for priming. In one embodiment, such a task for priming can be a pre-programmed task. The system 500 (FIG. 5) or the system 600 (FIG. 6) can be primed with, for example, phosphate buffered saline (PBS). To prime the bioreactor 501, 601, a bag (546) is attached to the system 500, 600 (e.g., at connection point 646). When referring to symbols in the figures, for example, when referring to "reference number, reference number" (e.g., 500, 600), such language means "reference number and / or reference number" (e.g., 500 and / or 600). Valves 550, 650 are opened. PBS is then directed into the first fluid circuit 502, 602 by setting the IC inlet pump 554, 654 to pump PBS into the first fluid circuit 502, 602. Valve 614 is opened and PBS enters the bioreactor 601 through the inlet 501A, 601A and exits through the outlet 501B, 601B. According to one embodiment, medium enters the bioreactor 501, 601 and / or the first fluid circuit 502, 602 while air is removed by the air removal chamber 556, 656, priming the bioreactor 501, 601.

[0078] To further prime the bioreactor 501, 601, a bag 586 is attached to the system 500, 600 (e.g., at connection point 668). Valve 576, 676 is opened. PBS can then be directed to the second fluid circuit 502, 602 by setting the IC inlet pump 554, 654 to pump PBS into the first fluid circuit 504, 604. Valve 692 is closed and PBS enters the bioreactor 601 through the inlet 501C, 601C and exits through the outlet 501D, 601D of the EC loop. According to one embodiment, the bioreactor 501, 601 is primed by entering medium into the bioreactor 501, 601 and / or the second fluid circuit 504, 604 while air is removed by the air removal chamber 580, 680.

[0079] The process 800 then proceeds to step 808 of coating the bioreactor. Here, the bioreactor 501, 601 is coated with a coating agent, for example, 5 mg of fibronectin (FN). For example, in one embodiment, a reagent can be dosed (e.g., from connection point 644) into the IC loop 502, 602 until the reagent bag 544 is empty. The reagent 544 is flowed into the IC loop 502, 602 from the air removal chamber 556, 656, and then the reagent 544 is circulated in the IC loop 502, 602 by operating the circulation pump 512, 612 and / or the inlet pump 554, 654. Any coating reagent (e.g., FN or cryoprecipitate) known to one of skill in the art can be used.

[0080] Examples of solutions that may be introduced into the system 500, 600 to coat the bioreactor are shown below.

[0081] [Table 1]

[0082] The coating of the bioreactor is carried out in three stages. An example of a setup for the system 500, 600 for the first stage of introducing the above solutions is shown below.

[0083] [Table 2]

[0084] An example of the setup of the system 500, 600 for the second stage of coating the bioreactor, flushing the reagents from the air removal chamber 556, 656, is shown below.

[0085] [Table 3]

[0086] An example of a setup of the system 500, 600 for the third stage of coating a bioreactor, circulating reagents in an IC loop 502, 602, is shown below.

[0087] [Table 4]

[0088] Once the bioreactor is coated, an IC / EC cleaning task is performed in step 810, where the fluids in the IC circulation loop 502, 602 and the EC circulation loop 504, 604 are replaced. The replacement volume is determined by the number of replacements of the IC volume and the EC volume. Examples of solutions introduced to the CES 500, 600 during the IC / EC cleaning task are shown below.

[0089] [Table 5]

[0090] An example of the IC / EC cleaning task settings for the system 500, 600 is shown below.

[0091] [Table 6]

[0092] Next, to maintain the correct or desired gas concentration across the fibers in the bioreactor membrane, a medium conditioning task 812 is performed to allow the medium to reach equilibrium with the provided gas supply before the cells are loaded into the bioreactor. Rapid contact between the medium and the gas supply provided by the gas transfer module or oxygenator 532, 632 is achieved, for example, by using a high EC circulation rate. The system 500, 600 is then maintained in the correct or desired state, for example, until a user or operator is ready to load cells into the bioreactor 501, 601. In one embodiment, the CES 500, 600 is conditioned with, for example, complete medium. Complete medium is any medium source used for cell growth. In one embodiment, complete medium includes, for example, alpha-MEM (α-MEM) and fetal bovine serum (FBS). Any type of medium known to those skilled in the art can be used.

[0093] The Condition Media task 812 is a two-step process. In the first step, the system 500, 600 rapidly contacts the media with the gas supply by using a high EC circulation rate. In the second step, the system 500, 600 maintains the bioreactor 501, 601 in the proper conditions until the operator is ready to load the cells. An example of a solution loaded into the CES 500, 600 during the Condition Media task 812 is shown below.

[0094] [Table 7]

[0095] An example of the settings for the first step of the Medium Conditioning task 812 is shown below.

[0096] [Table 8]

[0097] An example of the settings for the second step of the Medium Conditioning task 812 is shown below.

[0098] [Table 9]

[0099] The process 800 then proceeds to step 814 of loading cells from the cell loading bag 562 (at connection point 662) into the bioreactor 501, 601. The cell loading may be a three-stage process. In the first stage, cells are loaded in a uniform suspension from the cell loading bag 562 (at connection point 662) into the bioreactor 501, 601 until the bag 562 is empty (step 814). In other embodiments, the cells may be loaded by another type of loading procedure, such as a bulls-eye loading procedure (step 814). Any type of loading procedure may be used according to the embodiment. In the second stage, the cells are flushed from the air removal chamber 556, 656 towards the bioreactor 501, 601. In configurations utilizing a larger loading volume, the cells can spread and migrate towards the IC outlet 590, 690. In the third stage, the dispersion of cells across the membrane is promoted through IC circulation (eg, through IC circulation pumps 514, 614 in the absence of IC input).

[0100] Examples of solutions that may be introduced into the system 500, 600 in step 814 to input cells are shown below.

[0101] [Table 10]

[0102] As mentioned above, the cell input step 814 is performed in three stages. An example of the settings for the system 500, 600 for the first stage is shown below.

[0103] [Table 11]

[0104] An example of a configuration for the systems 500, 600 for the second stage is shown below.

[0105] [Table 12]

[0106] An example of a configuration for the systems 500, 600 for the third stage is shown below.

[0107] [Table 13]

[0108] Additionally, cells (e.g., adherent cells) can be attached, for example, to the hollow fibers (816). In one embodiment, during cell attachment (816), adherent cells can be attached to the bioreactor membrane while flow is occurring on the EC circulation loop 504, 604 with the pump 514, 614 flow rate to the IC loop 502, 602 at zero. Examples of solutions introduced to the CES 500, 600 in the process of attaching cells to the membrane 816 are shown below.

[0109] [Table 14]

[0110] An example setup for membrane attachment 816 in the system 500, 600 is shown below.

[0111] [Table 15]

[0112] The cells are fed in step 818. A flow rate (e.g., a low flow rate) is continuously added to the IC circulation loop 502, 602 and / or the EC circulation loop 504, 604. The outlet settings allow for the removal of the fluid added to the system 500, 600. Examples of solutions that may be introduced to the system 500, 600 in the feeding step 818 are shown below.

[0113] [Table 16]

[0114] An example setup for the feeding step 818 in the system 500, 600 is shown below.

[0115] [Table 17]

[0116] Process 800 then proceeds to optional step 820, where the serum-containing medium is washed off and replaced with basal or protein-free medium. If protein-free medium was used to load, feed, etc., the cells in the previous step, step 820 need not be performed. However, if serum-containing protein is used, the wash procedure 820 is initiated, for example, after the cells reach confluency, or after a defined period of time, or after a desired number of cell doublings, in anticipation of isolation and / or collection of released cell products (e.g., EVs or viral vectors).

[0117] For example, it may be desirable to begin purification and / or collection of the released cell product after a minimum of 2 cell doublings, 3 cell doublings, 4 cell doublings, 5 cell doublings, 6 or more cell doublings, etc. One or more processes may be performed to purify the medium in the system 500, 600 for collection of the released EV product. Such purification procedures may include a 5×IC·EC wash (5 IC·EC washes) 820, a negative pressure ultrafiltration wash 822, an IC·EC wash 824, and / or another type of wash procedure. First, the 5×IC·EC wash 820 involves replacing the fluid in both the IC circulation loop 502, 602 and the EC circulation loop 504, 604, where the replacement volume is specified by the number of IC volumes and EC volumes replaced.

[0118] Examples of solutions introduced into the system 500, 600 in the 5x IC / EC Clean task 820 are shown below.

[0119] [Table 18]

[0120] An example of the settings for the 5x IC / EC Clean task 820 of the system 500, 600 is shown below.

[0121] [Table 19]

[0122] Additionally, an optional negative pressure ultrafiltration wash 822 can use negative pressure ultrafiltration to clean the IC circulation loop 502, 602 to help lift off components that may have deposited on the IC side of the hollow fibers. Examples of solutions that may be introduced to the CES 500, 600 in negative pressure ultrafiltration 822 are shown below.

[0123] [Table 20]

[0124] An example of a setup for negative pressure ultrafiltration 822 of the system 500, 600 is shown below.

[0125] [Table 21]

[0126] Furthermore, the optional step IC·EC wash 824 can be used to replace the fluids in both the IC circulation loop 502, 602 and the EC circulation loop 504, 604. The replacement volume is specified by the number of replacements of the IC volume and the EC volume. In such a wash procedure, the medium used is, for example, a basal medium or a medium without protein. Examples of solutions introduced into the system 500, 600 in the optional step IC·EC wash 824 are shown below.

[0127] [Table 22]

[0128] An example of settings for IC·EC cleaning 824 of the systems 500, 600 is shown below.

[0129] [Table 23]

[0130] The tasks described above are custom or user-defined tasks. In other configurations, such tasks may be pre-programmed or default tasks. In other embodiments, such tasks may be performed manually, for example, by a user or operator.

[0131] In embodiments, to verify removal (e.g., complete removal) of serum or serum proteins, total protein in system 500, 600 is measured on the IC side (or EC side in other configurations) at different time points during the wash procedure (e.g., before 5x wash, after 5x wash 820, after negative pressure ultrafiltration 822, after basal medium replacement 824). Additionally, steps 820, 822, and / or step 824 are optional, for example, if no animal-derived serum is used, if only human-derived serum is used, if serum-free medium is used, and / or if there are no other such additional protein sources to be removed.

[0132] According to one embodiment, the effectiveness of the above method is represented by graph 1100 in FIG. 11. Graph 1100 represents protein measurements that can be obtained by a system such as system 500, 600 implementing the above-mentioned procedures. As shown, the amount of protein in system 500, 600 is represented by line 1104. Before the above-mentioned procedures 820, 822, 824, the amount of protein in system 500, 600 is at peak 1108 (e.g., above 7000 μg / mL). During the 5× wash 820, the amount of protein gradually decreases as represented by portion 1112 of line 1104. Indeed, at the end of the 5× wash 820, the protein concentration is approximately 0 μg / mL as represented by portion 1116 of line 1104. In such a case, for example, neither the negative pressure ultrafiltration wash 822 nor the basal medium replacement 824 need to be performed. However, steps 822, 824, according to one embodiment, may more effectively remove proteins in the system 500, 600.

[0133] After flushing the serum-containing medium from the bioreactor, the cells in the bioreactor are fed with protein-free medium for a defined period of time (e.g., about 48 hours) by adding medium to the EC side 504, 604 of the bioreactor 502, 602 and allowing it to diffuse through the semi-permeable membrane (826). The EC inlet 668 can supply EC medium (e.g., protein-free medium) to provide nutrients to the cells while the released cell products are concentrated. In such a configuration, no IC input is used. Instead, the semi-permeable hollow fibers of the bioreactor 502, 602 allow essential nutrients (e.g., glucose) to reach the cells through continuous perfusion, and metabolic waste products (e.g., lactate) are actively removed and exit the system 500, 600 through diffusion (EC outlets open 582, 692). Such feeding is performed for a defined period of time (e.g., about 48 hours). In other situations, a protein-free medium is used to feed the cells for about 24 hours to about 72 hours. In other embodiments, such feeding with a protein-free medium (e.g., a second medium) is performed for about 48 hours to about 72 hours to feed the cells. In other embodiments, such feeding with a second medium (e.g., a protein-free medium) is performed for about 24 hours to about 48 hours. In other embodiments, such feeding with a second medium is performed for about 24 hours to about 48 hours. In still other embodiments, such feeding with a protein-free medium is performed for less than about 24 hours. Any time period of feeding with a second medium may be used according to the embodiments. In another configuration, the IC inlet may provide IC medium (e.g., a protein-free medium) to provide nutrients to the cells. Such feeding may be performed for any time period according to the embodiments of the present disclosure. Such cell feeding 826 includes closing the IC outlet by closing the IC outlet valve 590, 690. Examples of solutions introduced into the system 500, 600 in cell feeding 826 are: , as shown below.

[0134] [Table 24]

[0135] An example setup for cell feeding 826 in the system 500, 600 is shown below.

[0136] [Table 25]

[0137] In other embodiments, closure of the IC outlet, allowing collection of released cell products, is performed, for example, in step 828. Such closure of the IC outlet (by closing valves 590, 598 or valves 690, 698) allows cell products released by the cells to be collected or concentrated in the bioreactor (828). By keeping the EC outlet open (valves 582, 692), waste products can be actively removed via the EC side 504, 604 by ultrafiltration. Furthermore, the semi-permeable membrane allows essential nutrients to reach the cells by continuous perfusion. For example, as shown in Tables 24, 25, according to one embodiment, the cells are fed by optionally adding medium (e.g., protein-free) to the IC side (826). In one embodiment, a medium bag 546 can be connected to the connection point 646. Medium (e.g., protein-free) from the bag 546 is sent to the IC inlet line 506, 606 via valves 550, 650. From the inlet line 506,606 the medium enters the IC loop 502,602 and feeds the cells in the IC part of the bioreactor 501,601.

[0138] Additionally or alternatively, step 826 and / or step 828 may also include an optional step 827, 829 of exchanging the outlet or waste bag 586, 686. In one embodiment, exchanging the outlet or waste bag 586, 686 allows the contents of the bag to be monitored or tested, such as monitoring glucose / lactate levels, determining if released cell products have crossed the membrane (since the IC outlet is closed), etc. As noted above, the cell products (e.g., EVs or viral vectors, etc.) produced by the cells are too large (e.g., large molecular weight) to diffuse through the membrane. When the IC outlet is closed, the released cell products (e.g., EVs) are maintained on the IC side of the bioreactor 501, 601 during growth (or during a defined harvest period) where the concentration of released cell products (e.g., EVs) is continually increasing. Cell feeding 826 may be performed simultaneously with the closing of the IC outlet. In other embodiments, step 826 may be performed after the closing of the IC outlet, for example, during harvest step 828. In yet other embodiments, step 826 is performed prior to closure of the IC outlet, for example, in collection step 828. In other embodiments, the outlet bag 586, 686 is not replaced if testing / monitoring is not desired after closure of the IC outlet. In yet other embodiments, an outlet container or waste container or outlet bag or waste bag is used to collect and harvest released material. It should be noted that while the IC outlet is described as being closed in process 800, in other embodiments where cell growth is performed on the EC side, for example, the EC outlet is closed.

[0139] When it is determined to begin harvesting the concentrated or collected cell product 828 in the loop (e.g., IC loop 502, 602) (e.g., after about 48 hours, or another period of time, etc.), in one embodiment, the harvest valve 598, 698 is opened and the cell product is harvested (830) from the IC side 502, 602 of the bioreactor 501, 601 into a harvest vessel 599, 699. In one embodiment, the IC valve 590, 690 at the IC outlet 586, 686 is opened to allow for harvesting. In other embodiments, the harvest valve 598, 698 is opened to allow for harvesting into a harvest bag 599, 699 or harvest vessel. In one embodiment, such harvesting occurs at the end of the entire process. In other embodiments, such harvesting occurs at predetermined intervals. In one embodiment, the cellular product (e.g., EVs or viral vectors, etc.) is transferred in suspension from the capillary inner circulation loop 502, 602 in the bioreactor 501, 601 to a harvest bag 599, 699 or harvest vessel. In one embodiment, protein-free media is used in such harvesting tasks. Examples of solutions that may be introduced into the system 500, 600 in the collection 828 of the concentrated or harvested cellular product are shown below.

[0140] [Table 26]

[0141] An example setup for collection of concentrated or harvested cell product 828 using system 500, 600 is provided below.

[0142] [Table 27]

[0143] After harvesting 830 of the released material, the process 800 can proceed directly from cell product harvesting 830 to an END step 838 and terminate if further processing 832 or cell harvesting 836 is not desired.

[0144] Alternatively, from the harvest step 830, the process 800 can optionally proceed to allow for further processing 832. Such further processing 832, in one embodiment, includes processing for analysis. In other embodiments, such further processing 832 includes further concentration of the cell product from the medium or fluid collected in the harvest bag. In other embodiments, such further processing 832 includes separation of the cell product from other components in the bag (such as suspension cells, if such cells are grown and harvested with the release). In one embodiment, such further processing 832 includes further isolation and / or characterization of the released cell product. If the cells remaining in the bioreactor (e.g., adherent cells, etc.) are not to be harvested, the process 800 can end from the optional further processing step 832 at an END step 838.

[0145] On the other hand, if it is desired to harvest the cells remaining in the bioreactor (e.g., adherent or attached cells), then process 800 proceeds to cell detachment step 834. Alternatively, if further processing 832 is not desired but detachment 834 of the cells from the bioreactor is desired, then process 800 proceeds directly from cell product harvest step 830 to cell detachment step 834. The attached cells are detached 834 from the membrane of the bioreactor and suspended, for example, in an IC loop. In one embodiment, an IC / EC wash task is performed as part of step 834 in preparation for adding reagents to detach the cells. For example, the IC / EC medium may be supplemented with nutrients such as protein, calcium (Ca), and / or phosphate buffer in preparation for the addition of trypsin or other chemical detachment agents to detach the adherent cells. 2+ ) and magnesium (Mg 2+For example, the IC / EC medium is replaced with phosphate buffered saline (PBS) to remove any residual erythrocytes. Reagents are added to the system until the reagent bags are empty. Reagents are flushed into the IC loop where they are mixed. After detachment of the adherent cells, in a harvest step 836, cells in suspension are transferred from the IC circulation loop, e.g., containing the cells remaining in the bioreactor, to a harvest bag or harvest container. The process 800 then ends at an END step 838.

[0146] Next, FIG. 9 illustrates exemplary operational steps 900 of a process for manufacturing, isolating, and / or collecting released material (e.g., EVs or viral vectors) that can be used with a cell growth system such as the CES500 (FIG. 5) or CES600 (FIG. 6) according to an embodiment of the present disclosure. The START step 902 begins and the process 900 proceeds to step 904 of loading a disposable tubing set into the cell growth system. The system is then primed in step 906. In one embodiment, for example, a user or operator can provide instructions to the system to prime by selecting a task for priming. In one embodiment, such a task for priming may be a pre-programmed task. The process 900 then proceeds to step 908 of coating the bioreactor. Here, the bioreactor is coated with a coating agent. For example, in one embodiment, reagents are pumped into the IC loop until the reagent container is empty. The reagents may be pumped into the IC loop from an air removal chamber, after which the reagents circulate in the IC loop. Any coating reagent known to those skilled in the art can be used, such as fibronectin or cryoprecipitate. Once the bioreactor is coated, an IC / EC wash task is performed (910), in which the fluids on the IC circulation loop and the EC circulation loop are replaced. According to one embodiment, the replacement volume is determined by the number of replacements of the IC volume and the EC volume.

[0147] Next, to maintain the proper or desired gas concentration across the yarns in the bioreactor membrane, a medium conditioning task 912 is performed to allow the medium to be in equilibrium with the provided gas supply before loading the cells into the bioreactor. Rapid contact between the medium and the gas supply provided by the gas transfer module or oxygenator is achieved, for example, by using a high EC circulation rate. The system is then maintained in the proper or desired state, for example, until the user or operator is ready to load the cells into the bioreactor. In one embodiment, the system is conditioned, for example, with complete medium. Complete medium is any medium source used for cell growth. In one embodiment, complete medium includes, for example, alpha-MEM (α-MEM) and fetal bovine serum (FBS). Any type of medium known to one of skill in the art can be used.

[0148] Next, the process 900 proceeds to step 914 with loading cells into the bioreactor, for example, from a cell input bag. In one embodiment, cells are loaded into the bioreactor from the cell input bag until the bag is empty. The cells are flushed into the bioreactor from the air removal chamber. In embodiments utilizing a larger flush volume, the cells can spread out and be directed to the IC outlet. The distribution of cells to the membrane can be facilitated by IC circulation, for example, via an IC circulation pump, rather than IC loading. Additionally, cells (e.g., adherent cells) are attached to the hollow fibers in step 916 and fed in step 918. In one embodiment, during cell attachment (916), the adherent cells can be attached to the bioreactor membrane while allowing flow on the EC circulation loop with the pump flow rate to the IC loop set to zero. The cells are grown / expanded in step 920. In one embodiment, the cells are expanded for 3-4 days. In other embodiments, the cells are grown for a period of time to achieve a desired number of cell doublings. In other embodiments, the cells are grown for a period of time to reach confluence. In one embodiment, the cells are grown for about 7 to about 8 days before collecting EVs (e.g., exosomes). Any period of time may be used according to embodiments of the present disclosure.

[0149] Next, the process 900 proceeds to a query step 922 to determine whether collection of cell products (such as EVs or viral vectors) released by the cells into the conditioned medium during the cell growth / expansion step 920 is desired. It may be desirable to begin isolation or purification of the released material for collection after a particular or defined number of cell doublings, e.g., 2 doublings, 3 doublings, 4 doublings, 5 doublings, 6 or more cell doublings, etc. For example, in one embodiment, it may be desirable to begin isolation and / or collection of the released material after a minimum of 2 cell doublings. In other embodiments, it may be desirable to begin purification and / or collection of the released material after a minimum of 5 or more cell doublings, for example. In one embodiment, there may not be a set number of cell doublings before beginning isolation and / or collection of the released material. As will be appreciated by one of skill in the art, any number of cell doublings, predefined time periods, and / or other indicators may be used.

[0150] Returning to query step 922, if it is desired to isolate and / or collect the released material, process 900 branches to "Yes" and proceeds to wash and replace step 924. In one embodiment, step 924 involves flushing out the serum-containing medium, where the medium is replaced with basal medium, for example. In one embodiment, the wash and replace step includes one or more of the following steps: (1) a 5x IC·EC wash, (2) a negative pressure ultrafiltration wash with phosphate buffered saline (PBS) to remove as much serum as possible from the bioreactor, and (3) a 2.5x IC·EC wash with basal medium to replenish the metabolic products lost in the PBS wash. In one embodiment, all of steps (1)-(3) above are performed. In other embodiments, only one of steps (1)-(3) above is performed. In other embodiments, two of steps (1)-(3) above are performed. Additionally, the steps may be in any order, provided that the embodiment is followed. In yet other embodiments, wash step 924 may not be performed. For example, step 924 may be optional if no serum of animal origin is used, if only serum of human origin is used, if serum-free media is used, and / or if there are no other such additional protein sources to be removed.

[0151] After the serum-containing medium is flushed from the bioreactor (924), the IC outlet is closed by closing the IC outlet valve (926) to allow the concentration of the substances released by the cells to increase in the bioreactor. As part of step 926, an outlet bag or waste bag may be optionally replaced (928). By replacing the outlet bag or waste bag, the contents of the bag can be monitored or tested, such as to monitor glucose / lactate levels, to determine if the released substances have crossed the membrane (since the IC outlet is closed), etc. As described above, the released substances (e.g., EVs or viral vectors, etc.) produced by the cells are too large (e.g., too large molecular weight) to diffuse through the membrane. When the IC outlet is closed, the released substances (e.g., EVs) are maintained on the IC side of the bioreactor during growth (or during a defined harvest period) where the concentration of the released substances (e.g., EVs or viral vectors, etc.) is continually increasing. In one embodiment, step 928 can be performed simultaneously with the closing of the IC outlet. In other embodiments, step 928 occurs after closing of the IC outlet. In yet other embodiments, step 928 occurs before closing of the IC outlet. In other embodiments, the outlet bag may not be replaced if testing / monitoring or repurposing of the outlet bag is not desired after closing of the IC outlet. It should be noted that while process 900 describes the IC outlet as being closed, in other embodiments where cell growth is performed on the EC side, for example, the EC outlet is closed.

[0152] Next, the process 900 proceeds to a cell feeding step 930, a release material collection step 932, where protein-free medium is added to the EC side of the bioreactor and diffused through the semi-permeable membrane to the IC side. In such an embodiment, the EC inlet is fed with EC medium (e.g., protein-free medium) to provide nutrients to the cells while the released materials are concentrated. In this case, for example, no IC input is performed. Instead, the semi-permeable hollow fibers of the bioreactor allow essential nutrients (e.g., glucose) to reach the cells through continuous perfusion, and metabolic waste products (e.g., lactate) are actively removed and leave the system through diffusion (opening the EC outlet). In other embodiments, the cells are fed by adding medium to the IC side. In one embodiment, a basal medium is used to feed the cells for about 48 hours. In other embodiments, a basal medium is used to feed the cells for about 24 hours to about 72 hours. In embodiments, collection or concentration of released material (e.g., EVs or viral vectors, etc.) in the IC loop (IC outlet closure 926) occurs for a defined period of time, such as about 48 hours, or in other embodiments, from about 24 hours to about 72 hours. In other embodiments, feeding with basal medium and collection of released particles occurs for a period longer than about 72 hours (e.g., from about 72 hours to about 96 hours). In other embodiments, feeding with second medium and collection of released particles occurs for a period of about 72 hours to about 120 hours. In yet other embodiments, such feeding and collection occurs for a period of less than 24 hours.

[0153] If it is decided to start harvesting the concentrated or collected releasable material (e.g., after about 48 hours or another time period), in one embodiment, a harvesting valve is opened (934) and the releasable material is harvested from the IC side of the bioreactor into a harvesting vessel (936). In one embodiment, the IC valve at the IC outlet is opened to allow for harvesting. In other embodiments, the harvesting valve is opened to allow for harvesting into a harvesting bag or harvesting vessel. In one embodiment, such harvesting occurs at the end of the entire process. In other embodiments, such harvesting occurs at predefined intervals. In one embodiment, the releasable material (e.g., EVs or viral vectors, etc.) is transferred in suspension from the capillary inner circulation loop in the bioreactor into a harvesting bag or harvesting vessel. In one embodiment, a protein-free medium is used in such a harvesting task.

[0154] After the releasable material harvesting step 936, the process 900 can optionally proceed to further processing 944. Such further processing 944, in one embodiment, includes processing for analysis. In other embodiments, such further processing 944 includes further concentration of the releasable material from the medium collected in the harvest bag. In other embodiments, such further processing 944 includes separation of the releasable material from other components in the bag (such as suspension cells, if such cells are grown and harvested with the releasable material). In one embodiment, such further processing 944 includes further isolation and / or characterization of the releasable material. If the cells remaining in the bioreactor (e.g., adherent cells, etc.) are not to be harvested, the process 900 can proceed from the optional further processing step 944 to an END step 946 and end. In other embodiments, the process 900 can proceed from the product harvesting step 936 directly to an END step 946 and end if neither further processing 944 nor cell harvesting 940 is desired.

[0155] Returning to step 944, if it is desired to harvest the cells remaining in the bioreactor (e.g., adherent or attached cells), the process 900 proceeds to an optional cell detachment step 938. Alternatively, if further processing 944 is not desired but detachment 938 of the cells from the bioreactor is desired, the process 900 proceeds directly from the cell product harvest step 936 to the optional cell detachment step 938. The attached cells are detached 938 from the membrane of the bioreactor and suspended, for example, in an IC loop. In one embodiment, an IC / EC wash task is performed as part of step 938 in preparation for adding reagents to detach the cells. For example, protein, calcium (Ca), or erythrocyte detachment (EC) may be performed in preparation for adding trypsin or other chemical detachment agents to detach the adherent cells. 2+ ) and magnesium (Mg 2+ ) the IC / EC medium is replaced with phosphate buffered saline (PBS). Reagents may be added to the system until the reagent bags are empty. The reagents are flushed into the IC loop and mixed therein. After detachment of the adherent cells, in a harvest step 940, the cells in suspension are transferred from the IC circulation loop, for example, containing the cells remaining in the bioreactor, to a harvest bag or harvest container. And as part of the process 900, in step 942, the disposable set is optionally removed from the cell expansion system and the process 900 ends at END step 946.

[0156] In accordance with embodiments of the present disclosure, the operational steps described in the processes depicted in Figures 7, 8, and 9 are for illustrative purposes only and may be modified, combined, or performed in parallel with other steps. In embodiments, steps may be reduced or additional steps may be added without departing from the spirit and scope of the present disclosure. Also, steps (and sub-steps), such as priming, coating the bioreactor, and loading the cells, may be performed automatically in some embodiments, e.g., by a processor executing pre-programmed tasks stored in a memory. Such steps are presented herein for illustrative purposes only.

[0157] Examples and detailed descriptions of tasks and protocols, including custom tasks and pre-programmed tasks, that may be used with the cell growth system are provided in U.S. Patent Application Publication No. 13 / 269,323, filed October 7, 2011, entitled "CONFIGURABLE METHOD AND SYSTEM FOR CELL GROWTH AND CELL HARVEST IN A HOLLOW FIBER BIOREACTOR SYSTEM," and U.S. Patent Application Publication No. 13 / 269,351, filed October 7, 2011, entitled "CUSTOMIZABLE METHOD AND SYSTEM FOR CELL GROWTH AND CELL HARVEST IN A HOLLOW FIBER BIOREACTOR SYSTEM," both of which are expressly incorporated herein by reference in their entireties.

[0158] 10 illustrates an example of components of a computing system 1000 for carrying out embodiments of the present invention. The computing system 1000 may be used in embodiments where the cell growth system employs a processor to perform tasks, such as custom tasks or pre-programmed tasks, for example as part of processes such as processes 700, 800, 900 described above. In embodiments, the pre-programmed tasks may include, for example, "IC / EC wash" and / or "cell feeding."

[0159] The computing system 1000 includes a user interface 1002, a processing system 1004, and / or a storage device 1006. The user interface 1002 includes an output device 1008 and / or an input device 1010, as will be appreciated by those skilled in the art. The output device 1008 may include one or more touch screens. The touch screen may have a display area for providing one or more application windows. The touch screen may also be an input device 1010 capable of accepting and / or capturing physical contact from a user or operator, for example. The touch screen may be a liquid crystal display (LCD) with a capacitive structure that allows the processing system 1004 to estimate the location of the touch, as will be appreciated by those skilled in the art. In this case, the processing system 1004 may map the location of the touch to a UI element that is displayed at a predetermined location in the application window. The touch screen may also accept touch via one or more other electronic structures in the present invention. Other output devices 1008 may include printers, speakers, etc. Other input devices 1010 may include a keyboard, other touch-based input device, a mouse, a voice input device, etc., as would be understood by one of ordinary skill in the art.

[0160] In embodiments of the invention, the processing system 1004 may include a processing unit 1012 and / or a memory 1014. The processing unit 1012 may be a general-purpose processor operable to execute instructions stored in the memory 1014. The processing unit 1012 may include a single processor or multiple processors in embodiments of the invention. Further, in embodiments, each processor may be a multi-core processor having one or more cores for separately reading and executing instructions. The processor may include a general-purpose processor, an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), or other integrated circuit, as would be understood by one of ordinary skill in the art.

[0161] In embodiments of the present invention, memory 1014 may include any storage device that provides short-term or long-term storage of data and / or processor-executable instructions. Memory 1014 may include, for example, random access memory (RAM), read-only memory (ROM), or electrically erasable programmable read-only memory (EEPROM), as would be understood by one of ordinary skill in the art. Other storage media may include, for example, CD-ROM, tape, digital versatile disk (DVD), or other optical storage devices, tape, magnetic disk storage devices, magnetic tape, or other magnetic storage devices, as would be understood by one of ordinary skill in the art.

[0162] Storage 1006 is any long-term data storage device or component. In embodiments of the invention, storage 1006 may include one or more of the systems described in connection with memory 1014. Storage 1006 may be permanent or removable. In one embodiment, storage 1006 stores data generated by or provided to processing system 1004.

[0163] Working Example Some examples of methods / processes / protocols / configurations that can implement aspects of the embodiments are described below. Although specific features may be described in these examples, they are provided for illustrative purposes only. The present invention is not limited to the following examples.

[0164] Example 1 Examples of cell expansion and extracellular particle collection results from implementation of the above systems and methods are shown in graph 1200 of FIG. 12. Graph 1200 shows a series of EV production runs using, for example, methods 700, 800, 900 and CES 500, 600 described above. For example, the production / collection can use the Quantum® Cell Expansion System (Quantum® System or Quantum® CES) manufactured by Terumo BCT, Inc. (Lakewood, Colorado). As shown, various concentrations of EVs can be produced and collected using the above processes and systems. Such production can result in various concentrations of EVs ranging from a high of over 2.5E+07 EVs / mL (1208) to a low of between 5.00E+06 EVs / mL and 1.00E+07 EVs / mL (1212).

[0165] Example 2 Examples of results of extracellular particle generation and / or collection using the above-described methods 700, 800, 900 and / or systems 500, 600 according to embodiments are shown in graphs 1300, 1304, 1308 of Figures 13A, 13B, and 13C. The EV concentrations 1312a, 1312c when generated by systems 500, 600, respectively, were 225 cm 2 The EV concentrations 1316a, 1316c are close to or higher than those generated in 225 cm tissue culture flasks (T225), which for comparison were seeded at substantially similar cell densities and similarly treated (e.g., with similar cell densities and similar feeding) as systems 500, 600. As shown in FIG. 13B, 2 The EV concentration 1316b when produced in a tissue culture flask (T225) is higher than the EV concentration 1312b when produced by systems 500, 600, where the flask was seeded at a substantially similar cell density and treated similarly as systems 500, 600 for comparison.

[0166] As an example, FIG. 13A shows the results of EV production harvest in an experiment numbered as “Q1468” 1312a in a Quantum® Cell Expansion System compared to EVs 1316a produced and / or harvested in T-flasks (Q1468T-flasks). The flasks are seeded at substantially similar cell densities and treated similarly for comparison. For example, Q1468 produces EVs in a Quantum® System (21,000 cm 2 The bioreactor in the Q1468T-flask (surface area of ​​225 cm) was charged with 1.25E+08 cultured cells. 2 surface area) was loaded with 9.24E+05 culture cells. This equates to loading substantially similar cell densities in both the Quantum® System and the T-Flask. The results in FIG. 13A show concentrations between 6.00E+07 EVs / mL and 7.00E+07 EVs / mL for both 1312a (Quantum® System) and 1316a (T-Flask), and graph 1300 shows a higher concentration of EVs 1312a collected from the Quantum® System compared to the concentration of EVs 1316a collected from the T-Flask (Q1468T-flask).

[0167] The results in Figures 13A and 13C show that the production / harvesting of the system (e.g., Quantum® Cell Expansion System, Terumo BCT, Lakewood, CO) provides a higher concentration of EVs than, for example, the concentration can be achieved by using a similar cell input (e.g., similar cell density) and feeding volume in a conventional T-flask. The CES500, 600 allows for automation of some functions, reducing labor and, in some embodiments, provides a larger surface area for growing cells, thus providing more EVs.

[0168] Example 3 For example, exemplary results of antigen-specific extracellular particle (e.g., EV) production and / or collection using the above-described methods 700, 800, 900 and / or systems 500, 600 according to embodiments are shown in FIG. 14. As shown in FIG. 14, exemplary results can be obtained from a representative sample of purified exosomes obtained from a conventional T-flask and a system 500, 600 (e.g., a Quantum® Cell Expansion System from Terumo BCT, Lakewood, Colorado). As shown in graph 1400, the types of antigen-specific exosomes include CD9, CD63, and / or CD81. The number of CD9 exosomes produced and collected from the system 500, 600 is shown in column 1404, the number of CD63 exosomes produced and collected from the system 500, 600 is shown in column 1408, and the number of CD81 exosomes produced and collected from the system 500, 600 is shown in column 1412. Additionally, cells were cultured at 225 cm 2 For comparison, tissue culture flasks (T225) are seeded at substantially the same cell density as systems 500, 600 (e.g., Quantum® systems) and similarly processed. As shown in graph 1400, the number of CD9 exosomes produced and collected from tissue culture flasks (T225) is shown in column 1416, the number of CD63 exosomes produced and collected from tissue culture flasks (T225) is shown in column 1420, and the number of CD81 exosomes produced and collected from tissue culture flasks (T225) is shown in column 1424. As shown in FIG. 14, for each antigen, the number of exosomes produced and collected by systems 500, 600 is higher than the number of exosomes produced and collected by tissue culture flasks (T225). For example, FIG. 14 shows that, according to one embodiment, the amount of each of the antigen-specific exosomes from the CES harvest is 2-3 times that of the T225 harvest.

[0169] Example 4 As described with respect to FIG. 8, the Quantum® system (e.g., CES500, 600) is primed with PBS and coated overnight with 5 mg of FN. The following day, the system undergoes a 2.5× IC·EC wash and is conditioned with complete medium (αMEM with GlutaMAX+10% FBS). Preselected MSCs are seeded into the bioreactor and allowed to grow for 4-5 days. At this point, the system undergoes a 5× IC·EC wash and a negative pressure ultrafiltration wash with PBS to remove as much serum as possible from the bioreactor. A 2.5× IC·EC wash with basal medium (αMEM with GlutaMAX only) is then performed to replenish metabolites lost during the PBS wash. The cells are fed with basal medium for 48 hours while the conditioned medium is collected into a harvest bag. Flasks are also seeded at a substantially similar seeding density as the bioreactor for comparison purposes and are treated similarly to the Quantum® system.

[0170] For example, results from the above methods 700, 800, 900 and / or systems 500, 600 used as part of Example 4 showed a 9.9×10 6 When cells were loaded into bioreactors 501 and 601, grown for 6 days, serum was removed from the system, and exosomes were collected by IC loop for 2 days, a total of 4.71 × 10 11 After the exosomes were collected, they were harvested from the IC loop, and in this case, 135 million MSCs were observed to be recovered from bioreactors 501 and 601.

[0171] Embodiments of the present disclosure may include, for example, one or more of the following aspects.

[0172] An embodiment and / or implementation of the invention includes a method of harvesting a cell product comprising the steps of: introducing cells into a bioreactor, feeding the cells with a medium, growing the cells whereby the cells release cell products, concentrating the released cell products, and harvesting the concentrated cell products from the bioreactor.

[0173] In one or more of the above embodiments and / or in any of the one or more of the above embodiments, the released cellular products include extracellular particles.

[0174] In any one or more of the above embodiments and / or one or more of the above implementations, the extracellular particles comprise extracellular vesicles.

[0175] In one or more of the above embodiments and / or any of the one or more of the above embodiments, the extracellular vesicles comprise exosomes.

[0176] In one or more of the above embodiments and / or any of the one or more of the above embodiments, the extracellular vesicles comprise microvesicles.

[0177] In one or more of the above embodiments and / or in any of one or more of the above embodiments, the extracellular particle comprises a viral vector.

[0178] In any one or more of the above embodiments and / or one or more of the above implementations, the harvested cell product is collected in a bag.

[0179] In one or more of the above embodiments and / or any of one or more of the above implementations, the method further comprises replacing the culture medium.

[0180] In one or more of the above embodiments and / or in any of one or more of the above embodiments, the medium comprises serum.

[0181] An embodiment and / or implementation of the present invention includes a cell growth system comprising a bioreactor having hollow fiber membranes, a first fluid flow path having at least two ends, a processor, and a memory in communication with and readable by the processor, the first fluid flow path being fluidly associated with an inner capillary portion of the hollow fiber membrane, the memory having a set of instructions that, when executed by the processor, cause the processor to perform operations such as, when selected, feeding cells, and, when selected, closing an outlet of the inner capillary portion to concentrate particles released from the cells in the inner capillary portion and transferring the particles to a harvest bag.

[0182] In any of one or more of the above embodiments and / or one or more of the above implementations, the processor further performs at least one of performing five flush washes, performing negative pressure ultrafiltration, and performing a flush wash.

[0183] In one or more of the above embodiments and / or in any of one or more of the above embodiments, the particles released from the cells comprise extracellular particles.

[0184] In one or more of the above embodiments and / or in any of the one or more of the above embodiments, the extracellular particles comprise exosomes.

[0185] In one or more of the above embodiments and / or any of the one or more of the above embodiments, the extracellular particles comprise microvesicles.

[0186] In any one or more of the above embodiments and / or one or more of the above implementations, the processor is further configured to, upon selection, replace fluid in the inner capillary portion and the outer capillary portion of the hollow fiber membrane.

[0187] In one or more of the above embodiments and / or in any of one or more of the above embodiments, when replacing the fluid, proteins are removed from the first medium used to feed the cells.

[0188] In one or more of the above embodiments and / or in any of one or more of the above embodiments, the first medium used for feeding the cells is replaced with a protein-free second medium.

[0189] In any one or more of the above embodiments and / or one or more of the above implementations, the processor further tests the first medium and / or the second medium in the bioreactor to determine whether the protein has been removed.

[0190] An embodiment and / or implementation of the present invention includes a cell growth system comprising a bioreactor having hollow fiber membranes, a first fluid flow path having a first inlet and a first outlet at at least opposite ends of the bioreactor and in fluid association with an inner capillary portion of the hollow fiber membrane, a second fluid flow path having a second inlet and a second outlet and in fluid association with an outer capillary portion of the hollow fiber membrane, a first connection port in fluid association with the first fluid flow path, a second connection port in fluid association with the first fluid flow path, a third connection port in fluid association with the first and second fluid flow paths, a fourth connection port in fluid association with the second fluid flow path, and a harvest bag in fluid association with the first fluid flow path. A first bag attached to the first connection port introduces cells into the bioreactor; a second bag containing a first medium containing a protein is connected to the second connection port, and the first medium is supplied from the second bag through the first fluid flow path to the bioreactor to feed the cells until a predetermined number of cell doublings occur; a third bag containing a second medium is connected to the third connection port, and the second medium is supplied from the third bag through the first fluid flow path and the second fluid flow path to the bioreactor; the first medium is flushed from the bioreactor to wash it; after the washing, a fourth bag containing a third medium not containing a protein is connected to the fourth connection port to feed the cells; and when feeding the cells with the third medium, the first outlet of the first fluid flow path is closed, and particles released from the cells are concentrated in the capillary inner portion of the bioreactor, and the concentrated particles are transferred to the harvest bag.

[0191] An embodiment and / or implementation of the invention includes a method of producing cell particles in a cell growth system comprising: a bioreactor comprising a hollow fiber membrane having an inner capillary portion and an outer capillary portion, a first fluid flow path having a first inlet and a first outlet at at least opposite ends of the bioreactor and in fluid association with the inner capillary portion of the hollow fiber membrane, a second fluid flow path having a second inlet and a second outlet and in fluid association with the outer capillary portion of the hollow fiber membrane, a first connection port in fluid association with the first fluid flow path, a second connection port in fluid association with the first fluid flow path, a third connection port in fluid association with the first and second fluid flow paths, a fourth connection port in fluid association with the second fluid flow path, and a harvest bag. The method includes the steps of priming the cell growth system, connecting a first bag to the first connection port to introduce cells into the bioreactor, connecting a second bag containing a first medium containing a protein to the second connection port and supplying the first medium to the bioreactor through the first fluid flow path to feed the cells until a predetermined number of cell doublings have occurred, and after the predetermined number of cell doublings have occurred, connecting a third bag containing a second medium to the third connection port to supply the second medium through the first fluid flow path and the second fluid flow path. and supplying the first medium to the bioreactor through the first fluid flow path to wash the first medium out of the bioreactor; after the washing, closing the first outlet of the first fluid flow path to concentrate particles released from the cells in the inside portion of the capillary of the bioreactor; connecting a fourth bag containing a third medium not containing protein to the fourth connection port to feed the cells; connecting the harvest bag to the first fluid flow path for harvesting; and harvesting the concentrated particles in the harvest bag.

[0192] Embodiments and implementations of the present invention include any one or more of the above embodiments and implementations or combinations thereof.

[0193] The embodiments and implementations of the present invention include means for carrying out one or more of the above-described embodiments and implementations.

[0194] While embodiments and applications of the present invention have been shown and described, it is to be understood that the present invention is not limited to the configurations set forth above. Various modifications, changes, and the like apparent to those skilled in the art may be made in the structure, operation, and details of the method and system of the present invention without departing from the scope of the invention.

[0195] As used herein, "at least one," "one or more," and "and / or" are open-ended expressions that function both conjunctively and disjunctively. For example, each of "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means either A only, B only, C only, A and B, A and C, B and C, or A, B, and C.

[0196] Those skilled in the art will appreciate that various modifications and variations can be made to the methods and structures of the present invention without departing from the scope of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments or examples described above. Rather, the present invention is intended to include modifications and variations within the scope of the following claims and their equivalents.

Claims

1. A method for collecting extracellular vesicles in a bioreactor, the method comprising: priming the hollow fiber membranes of the bioreactor with a buffer; a coating step of coating the hollow fiber membrane with a coating agent; a fluid washing step of washing the buffer-containing fluid from the bioreactor; introducing cells into an inner capillary portion of the hollow fiber membrane such that the cells are distributed across the inner capillary portion of the hollow fiber membrane by inner capillary circulation in the inner capillary portion of the hollow fiber membrane; A settling step of settling the cells on the inner part of the capillary of the hollow fiber membrane; a feeding step of feeding a protein-free medium to the outer capillary portion of the hollow fiber membrane, the protein-free medium providing nutrients to the cells settled in the inner capillary portion of the hollow fiber membrane through perfusion; A growth step of growing the cells that have settled on the inner capillary portion of the hollow fiber membrane, the growth step being for releasing the extracellular vesicles from the cells into a fluid medium contained in the hollow fiber membrane by growing the cells; closing the outlet of the inner capillary section and continuing to circulate the outer capillary section of the bioreactor to continue to supply the medium to the cells, thereby concentrating the extracellular vesicles released into the inner capillary section; A fluid medium washing step of washing the fluid medium containing the extracellular vesicles from the hollow fiber membrane; and collecting the extracellular vesicles from the fluid medium washed from the hollow fiber membrane. method.

2. In the method according to claim 1, the extracellular vesicles are exosomes. method.

3. In the method according to claim 1 or 2, the fixing step comprises: permitting flow to a capillary outer circulation loop of said bioreactor; allowing flow through the capillary outer circulation loop of the bioreactor while blocking flow through the capillary inner circulation loop of the bioreactor; method.

4. In the method according to any one of claims 1 to 3, the coating step includes introducing the coating agent into the inner part of the capillary of the bioreactor. method.

5. The method according to claim 1, wherein the coating agent is fibronectin. method.

6. The method according to any one of claims 1 to 5, further comprising the step of replacing the fluid medium washed from the hollow fiber membrane with a basal medium. method.

7. The method according to claim 1, further comprising, prior to the fluid medium washing step, determining to initiate isolation of the extracellular vesicles after a predetermined number of cell doublings have occurred; The isolation of the extracellular vesicles includes initiating the fluid medium washing step. method.

8. The method of claim 7, wherein the predetermined number of cell doublings is two. method.

9. The method of claim 7, wherein the predetermined number of cell doublings is 5. method.

10. The method according to claim 1, further comprising: releasing the extracellular vesicles from the cells into the fluid medium contained in the hollow fiber membrane over a predetermined period of time by growing the cells; The predetermined time is 48 hours. method.

11. A bioreactor having a hollow fiber membrane having an inner capillary portion and an outer capillary portion; A processor; a memory in communication with and readable by said processor; A cell proliferation system comprising: The memory has a series of instructions that, when executed by the processor, cause the processor to: Upon selection, priming the hollow fiber membrane with a buffer; After selection, the hollow fiber membrane is coated with a coating agent; Upon selection, flushing the buffer-containing fluid from the bioreactor; upon selection, introducing the cells into the inner capillary portion such that the cells are distributed throughout the inner capillary portion by inner capillary circulation in the inner capillary portion; subjecting the cells to selection to colonize the inner portion of the capillary; Proliferating the cells that have settled on the inner portion of the capillary upon selection, and releasing extracellular vesicles from the cells into a fluid medium contained within the hollow fiber membrane by propagating the cells; Upon selection, closing the outlet of the inner capillary section and continuing to circulate the outer capillary section of the bioreactor to continue to supply the cells with protein-free medium, thereby concentrating the extracellular vesicles released into the inner capillary section; Upon selection, washing the fluid medium containing the extracellular vesicles from the hollow fiber membrane; Upon selection, harvesting the extracellular vesicles from the fluid medium that has been washed through the hollow fiber membrane. Cell proliferation system.

12. In the cell proliferation system according to claim 11, the extracellular vesicles are exosomes. Cell proliferation system.

13. In the cell proliferation system of claim 11 or 12, when the cell is allowed to settle in the inner portion of the capillary upon receipt of the selection, the instructions are further executed by the processor, whereby the processor further permitting flow to a capillary outer circulation loop of said bioreactor; allowing flow through the capillary outer circulation loop of the bioreactor while blocking flow through the capillary inner circulation loop of the bioreactor; Cell proliferation system.

14. The cell proliferation system according to any one of claims 11 to 13, wherein, upon receipt of the selection, when coating the hollow fiber membrane with the coating agent, the instructions are further executed by the processor, whereby the processor further introduces the coating agent into an inner portion of the capillary of the bioreactor. Cell proliferation system.

15. The cell proliferation system according to claim 11, wherein the coating agent is fibronectin. Cell proliferation system.

16. The cell proliferation system of claim 11, wherein the set of instructions executed by the processor further selects to replace the fluid medium washed from the hollow fiber membrane with a basal medium. Cell proliferation system.

17. In the cell proliferation system according to any one of claims 11 to 16, the set of instructions executed by the processor upon said selection and before washing the fluid medium containing the extracellular vesicles from the hollow fiber membrane, the processor further determines to initiate isolation of the extracellular vesicles after a predetermined number of cell doublings have occurred, The isolation of the extracellular vesicles includes initiating the washing out of the fluid medium. Cell proliferation system.

18. The cell proliferation system of claim 17, wherein the predetermined number of cell doublings is two. Cell proliferation system.

19. The cell proliferation system of claim 17, wherein the predetermined number of cell doublings is 5. Cell proliferation system.

20. The cell proliferation system according to claim 11, further comprising: proliferating the cells to release the extracellular vesicles from the cells into the fluid medium contained in the hollow fiber membrane for a predetermined period of time; The predetermined time is 48 hours. Cell proliferation system.

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