Methods for cell expansion, differentiation and / or harvesting of natural killer cells using hollow fiber membranes

Abstract

A method for functionalizing a hollow fiber membrane for cell expansion of a target cell (e.g., a natural killer cell), the method comprising contacting a biotinylated molecule with a surface of a hollow fiber membrane comprising an extracellular matrix component, the biotinylated molecule binding to the extracellular matrix component and having an affinity for the target cell. The biotinylated molecule may be selected from the group consisting of cytokines, epitopes, ligands, monoclonal antibodies, stains, aptamers, and combinations thereof. The extracellular matrix component may be selected from the group consisting of fibronectin, vitronectin, fibrinogen, collagen, laminin, and combinations thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 18 / 234,470, filed on August 16, 2023; and also claims the benefit of U.S. Provisional Application No. 63 / 403,592, filed on September 2, 2022. The entire disclosure of the above application is hereby incorporated by reference herein. Technical Field

[0003] The present disclosure relates to methods of expanding cells using immobilized interleukin-21 (IL-21) and soluble interleukin-2 (IL-2). Background Art

[0004] This section provides background information related to the present disclosure which is not necessarily prior art.

[0005] Natural killer (NK) cells are innate lymphocytes that naturally attack certain cells, and are therefore candidates of interest for various cell therapies and treatments of various malignant diseases. For example, natural killer cells have recently been used as a cell type for engineered chimeric antigen receptor (CAR) cancer therapies. Although natural killer cell therapy is promising, its widespread clinical success is limited by the challenges of easily and effectively preparing large doses of natural killer cells. Current methods for natural killer cell expansion are typically based on flasks, which are time-consuming, expensive, and have relatively low success rates. Therefore, it is desirable to develop improved natural killer cell (and similar cell) expansion methods with improved success rates, less time consumption, and low costs. Summary of the invention

[0006] This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

[0007] In at least one exemplary embodiment, the present disclosure provides a method for functionalizing a hollow fiber membrane for cell expansion of a target cell. The method may include contacting a biotinylated molecule with a surface of a hollow fiber membrane including an extracellular matrix component. The biotinylated molecule may bind to the extracellular matrix component and may have affinity for the target cell.

[0008] In at least one exemplary embodiment, the biotinylated molecule may be selected from the group consisting of a cytokine, an epitope, a ligand, a monoclonal antibody, a dye, an aptamer, and combinations thereof.

[0009] In at least one exemplary embodiment, the cytokine may include interleukin-21.

[0010] In at least one exemplary embodiment, the extracellular matrix component may be selected from the group consisting of fibronectin, vitronectin, fibrinogen, collagen, laminin, and combinations thereof.

[0011] In at least one exemplary embodiment, the extracellular matrix component may include an extracellular matrix component-streptavidin conjugate, wherein the extracellular matrix component of the extracellular matrix component-streptavidin conjugate is bound to the surface of the hollow fiber membrane, and the streptavidin of the extracellular matrix component-streptavidin conjugate is bound to a biotinylated molecule.

[0012] In at least one exemplary embodiment, the extracellular matrix component-streptavidin conjugate may have a mass ratio of the extracellular matrix component to streptavidin of greater than or equal to about 1:3 to less than or equal to about 1:9.

[0013] In at least one exemplary embodiment, the extracellular matrix component-streptavidin conjugate may include a fibronectin-streptavidin conjugate, wherein the fibronectin has a molecular weight of greater than or equal to about 440 kDa to less than or equal to about 500 kDa, and the streptavidin has a molecular weight of greater than or equal to about 53 kDa to less than or equal to about 55 kDa.

[0014] In at least one exemplary embodiment, the method may further include preparing a fibronectin-streptavidin conjugate.

[0015] In at least one exemplary embodiment, preparing the fibronectin-streptavidin conjugate may include reconstituting the lyophilized fibronectin with streptavidin by soaking the lyophilized fibronectin and streptavidin in water.

[0016] In at least one exemplary embodiment, preparing a fibronectin-streptavidin conjugate may include covalently coupling fibronectin and streptavidin.

[0017] In at least one exemplary embodiment, the method may further include contacting an extracellular matrix component with a surface of the hollow fiber membrane.

[0018] In at least one exemplary embodiment, the extracellular matrix component can be contacted with the surface of the hollow fiber membrane for a period of greater than or equal to about 4 hours to less than or equal to about 24 hours prior to contacting the biotinylated molecule with the surface.

[0019] In at least one exemplary embodiment, after the period of time, and prior to contacting the biotinylated molecule with the surface, the method may further comprise washing the hollow fiber membrane to remove any unreacted and excess portions of the extracellular matrix components.

[0020] In at least one exemplary embodiment, the target cells may include natural killer cells.

[0021] In at least one exemplary embodiment, the surface may be an interior-facing surface.

[0022] In at least one exemplary embodiment, the surface may be an externally facing surface, or a combination of an internally facing surface and an externally facing surface.

[0023] In at least one exemplary embodiment, the present disclosure provides a method for functionalizing hollow fiber membranes for cell expansion of target cells. The method may include contacting an extracellular matrix component-streptavidin conjugate with a hollow fiber membrane, wherein the extracellular matrix component of the extracellular matrix component-streptavidin conjugate is combined with the hollow fiber membrane, and the streptavidin of the extracellular matrix component-streptavidin conjugate is combined with the extracellular matrix component. The method may also include contacting a biotinylated molecule with a hollow fiber membrane, wherein the biotinylated molecule is combined with the streptavidin of the extracellular matrix component-streptavidin conjugate. The biotinylated molecule may be selected from the group consisting of the following items: cytokines, epitopes, ligands, monoclonal antibodies, stains, aptamers, and combinations thereof.

[0024] In at least one exemplary embodiment, the extracellular matrix component of the extracellular matrix component-streptavidin conjugate may be selected from the group consisting of fibronectin, vitronectin, fibrinogen, collagen, laminin, and combinations thereof.

[0025] In at least one exemplary embodiment, the extracellular matrix component-streptavidin conjugate may include a fibronectin-streptavidin conjugate.

[0026] In at least one exemplary embodiment, the method may further include preparing a fibronectin-streptavidin conjugate. Preparing the fibronectin-streptavidin conjugate may include reconstituting the lyophilized fibronectin with streptavidin by immersing the lyophilized fibronectin and streptavidin in water, or covalently coupling the fibronectin and streptavidin.

[0027] In at least one exemplary embodiment, the extracellular matrix component-streptavidin conjugate can be contacted with the hollow fiber membrane for a period of greater than or equal to about 4 hours to less than or equal to about 24 hours prior to contacting the biotinylated molecule with the hollow fiber membrane.

[0028] In at least one exemplary embodiment, the method may further include: washing the hollow fiber membrane to remove any unreacted and excess portions of the extracellular matrix component-streptavidin conjugate prior to contacting the biotinylated molecule with the hollow fiber membrane.

[0029] Further areas of applicability will become apparent from the description provided herein.The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[0031] Figure 1 is a schematic diagram of an exemplary cell expansion system having a bioreactor according to at least one exemplary embodiment of the present disclosure;

[0032] Figure 2 is a schematic diagram of an exemplary bioreactor according to at least one exemplary embodiment of the present disclosure, which shows the circulation path through the bioreactor, and the exemplary bioreactor can be incorporated into Figure 1 In the cell expansion system of the cell expansion system shown;

[0033] Figure 3 is a schematic cross-sectional view of an exemplary hollow fiber membrane according to at least one exemplary embodiment of the present disclosure, which may be incorporated into Figure 1 The cell expansion system of the cell expansion system shown;

[0034] Figure 4 According to various aspects of the present disclosure, a bioreactor (e.g. Figure 2 A flow chart of an exemplary method for cell expansion, differentiation and / or harvesting of natural killer cells and other similar cells using a bioreactor (e.g., a bioreactor comprising a molecule of the invention); and

[0035] Figure 5 According to various aspects of the present disclosure, cytokines are used to prepare a bioreactor (e.g. Figure 2 Flowchart of an exemplary method for cell expansion, differentiation and / or harvesting of natural killer cells and other similar cells using a bioreactor (e.g., a bioreactor).

[0036] Figure 6 According to at least one exemplary embodiment of the present disclosure, a mobile bioreactor (such as Figure 2 Schematic diagram of an exemplary rocking device of a bioreactor;

[0037] Figure 7 An exemplary cell expansion system with pre-installed fluid delivery equipment (eg Figure 1 A perspective view of a cell expansion system shown;

[0038] Figure 8 Yes Figure 7A perspective view of an exemplary housing of an exemplary cell expansion system is shown;

[0039] Fig. 9 Yes Figure 7 A perspective view of a pre-installed fluid delivery device is shown;

[0040] Fig.10 is an exemplary cell expansion system (e.g. Figure 1 A schematic diagram of an exemplary flow path of a cell expansion system (shown in FIG. 1 );

[0041] Fig.11 is an exemplary cell expansion system (e.g. Figure 1 A schematic diagram of an exemplary flow path of a cell expansion system (shown in FIG. 1 );

[0042] Fig.12 According to at least one exemplary embodiment of the present disclosure, a cell expansion system (such as Figure 1 A flowchart of operational features of an exemplary process for performing cell expansion using a cell expansion system as shown; and

[0043] Fig.13 According to at least one exemplary embodiment of the present disclosure, in a cell expansion system (such as Figure 1 Block diagram of an exemplary processing system used in a cell expansion system (shown in FIG. 1 ).

[0044] Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings. DETAILED DESCRIPTION

[0045] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0046] Exemplary embodiments are provided to make the present disclosure comprehensive and to fully convey the scope to those skilled in the art. Many specific details, such as examples of specific components, devices, and methods, are described to provide a comprehensive understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, and that the exemplary embodiments may be implemented in a variety of different forms, and none of these forms should be construed as limiting the scope of the present disclosure. In certain exemplary embodiments, well-known procedures, well-known device structures, and well-known techniques are not described in detail.

[0047] The terms used herein are only used for the purpose of describing specific exemplary embodiments, and are not intended to be limited. As used herein, the singular forms "a", "an", and "the" are also intended to include plural forms, unless the context clearly indicates otherwise. The terms "comprises", "comprising", "including", and "having" are inclusive, and thus specify the presence of the features, integers, steps, operations, elements, and / or components described, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or their groups. Unless the order of performance is clearly indicated, the method steps, procedures, and operations described herein should not be interpreted as necessarily requiring them to be performed in the specific order discussed or shown. It should also be understood that additional or alternative steps may be adopted.

[0048] When an element or layer is referred to as being "located at," "engaged to," "connected to," or "coupled to" another element or layer, it may be directly located at, engaged to, connected to, or coupled to another element or layer, or there may be intermediate elements or layers. Conversely, when an element is referred to as being "directly located at," "directly engaged to," "directly connected to," or "directly coupled to" another element or layer, there may be no intermediate elements or layers. Other words used to describe the relationship between elements should be interpreted in a similar manner (e.g., "between" versus "directly between," "adjacent" versus "directly adjacent," etc.). As used herein, the term "and / or" includes any and all combinations of one or more associated listed items.

[0049] Although the terms "first", "second", "third", etc. can be used to describe various elements, components, regions, layers and / or parts in this article, these elements, components, regions, layers and / or parts should not be limited by these terms. These terms can only be used to distinguish an element, component, region, layer and / or part from another region, layer or part. When used herein, terms such as "first", "second" and other numerical terms do not mean order or sequence unless the context clearly indicates. Therefore, without departing from the teachings of the exemplary embodiments, the first element, component, region, layer and / or part discussed below may be referred to as the second element, component, region, layer and / or part.

[0050] For ease of description, spatially relative terms such as "inside", "outside", "beneath", "below", "lower", "above", "upper", etc. may be used herein to describe the relationship of one element or feature to another element or feature as shown in the figures. . In addition to the orientation depicted in the drawings, spatially relative terms may be intended to include different orientations of the device in use or operation. For example, if the device in the figure is turned over, elements described as "below" or "beneath" other elements or features will be oriented "above" the other elements or features. Therefore, the exemplary term "below" can cover both above and below orientations. The device can be oriented in other ways (rotated 90 degrees or in other orientations), and the spatially relative descriptors used herein are interpreted accordingly.

[0051] Various components are referred to herein as being "operably associated". As used herein, "operably associated" refers to components that are connected together in an operably manner, and encompasses embodiments in which components are directly connected, as well as embodiments in which additional components are placed between the connected components. Components that are "operably associated" may be "fluidically associated". "Fluidically associated" means that the components are connected together so that fluid can be transported between them. "Fluidically associated" encompasses embodiments in which additional components are disposed between two fluidically associated components, as well as between directly connected components. Fluidically associated components may include components that do not contact the fluid but contact other components to manipulate the system (e.g., a peristaltic pump that pumps fluid through a flexible tube by compressing the outside of the tube).

[0052] Example embodiments will now be described more fully with reference to the accompanying drawings.

[0053] The present disclosure relates to methods of performing cell expansion on cells (e.g., natural killer cells) using cell expansion systems, such as those described in U.S. patent application No. 8,309,347, entitled “Cell Expansion Systems and Methods of Use,” published on November 13, 2012, and / or U.S. patent application No. 9,677,042, entitled “Customizable Methods and Systems for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System,” published on June 13, 2017, and / or U.S. patent application No. 9,725,689, entitled “Configurable Methods and Systems for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System,” published on August 8, 2017, and / or U.S. application No. 15 / 943,536, entitled “Expanding Cells in a Bioreactor,” filed on April 2, 2018 and published on October 2, 2018, and / or U.S. patent application No. 10,577,585, entitled “Cell Expansion,” published on March 3, 2020. The entire disclosure of the above application is hereby incorporated by reference.

[0054] Cell expansion systems including hollow fiber bioreactors are cell culture systems used to expand and differentiate cells, including adherent and non-adherent cell types. Figure 1 As shown, the exemplary cell expansion system 10 includes a first fluid circulation path 12 and a second fluid circulation path 14. The first fluid circulation path 12 includes, for example, a first fluid flow path 16 having opposite ends 18 and 20. The first fluid flow path 16 can be in fluid communication with a hollow fiber cell growth chamber 24 (which can also be referred to as a "bioreactor"). For example, the first opposite end 18 of the first fluid flow path 16 can be in fluid communication with a first inlet 22 of the cell growth chamber 24, and the second opposite end 20 can be in fluid communication with a first outlet 28 of the cell growth chamber 24. The fluid in the first circulation path 12 can flow through the interior of a plurality of hollow fibers 116 of a hollow fiber membrane ("HFM") 117 disposed in the cell growth chamber 24 (see, for example, Figure 2 In at least one exemplary embodiment, the first fluid flow control device 30 may be operably coupled to the first fluid flow path 16 and may control the flow of the fluid in the first fluid circulation path 12 .

[0055] The second fluid circulation path 14 includes, for example, a second fluid flow path 34 and a second fluid flow control device 32. Like the first fluid flow path 16, the second fluid flow path 34 can have opposite ends 36 and 38. The opposite ends 36 and 38 of the second fluid flow path 34 can be in fluid communication with the inlet 40 and the outlet 42 of the cell growth chamber 24. For example, the first opposite end 36 of the second fluid flow path 34 can be in fluid communication with the inlet 40 of the cell growth chamber 24, and the second opposite end 38 of the second fluid flow path 34 can be in fluid communication with the outlet 42. The fluid in the second circulation path 14 can be in fluid communication with the hollow fiber membrane 117 (see, for example, Figure 2 ) external contact. In at least one exemplary embodiment, the second fluid flow control device 32 can be operably connected to the second fluid flow path 34 and can control the flow of the fluid in the second fluid circulation path 14.

[0056] The first fluid circulation path 12 and the second fluid circulation path 14 are maintained in the cell growth chamber 24 through the hollow fiber membrane 117, wherein the fluid in the first fluid circulation path 12 flows through the intracapillary ("IC") space of the hollow fiber membrane 117, and the fluid in the second circulation path 14 flows through the extracapillary ("EC") space of the cell growth chamber 24. The first circulation path 12 can be referred to as an "intracapillary loop" or "intracapillary space" or "IC loop". The second fluid circulation path 14 can be referred to as an "extracapillary loop" or "extracapillary space" or "EC loop". The fluid in the first fluid circulation path 12 can flow in a downstream or countercurrent direction relative to the flow of the fluid in the second fluid circulation path 14. As an example, Figure 3 A cross-section of an exemplary hollow fiber membrane 101 is shown, which includes a plurality of semipermeable hollow fibers (also referred to as hollow columns and / or hollow matrices) 121, wherein the space or voids 130 within the hollow fibers 121 define an intracapillary space, and the space outside the hollow fibers 121 defines an extracapillary space 110.

[0057] Typically, cells for expansion are seeded in the intracapillary space 130 (e.g., CD34+ hematopoietic stem cells / progenitor cells, monocytes, macrophages, hepatocytes, and / or endothelial cells derived from umbilical cord blood for expansion, differentiation, and / or harvesting), and cell culture medium is pumped through the extracapillary space to deliver nutrients to the cells via hollow fiber membrane perfusion during expansion. However, in other variations, cells for expansion may be seeded in the extracapillary space 110, and cell culture medium is pumped through the intracapillary space 130 to deliver nutrients to the cells via hollow fiber membrane perfusion during expansion. In further variations, cells for expansion may be seeded in the intracapillary space 130, and cell culture medium is pumped through the extracapillary space 110 and the intracapillary space 130. The movement of cell culture medium through the intracapillary space 130 can help remove excess cells that are not attached to the surface of the hollow fiber membrane 101. In each case, the material used to form the hollow fiber membrane 101 can be any biocompatible polymer material that can be made into a hollow fiber 121. For example, synthetic polysulfone-based materials such as polyethersulfone (PES) are commonly used to form hollow fibers.

[0058] In order to allow cells (such as natural killer cells (also known as NK cells)) to better attach to the hollow fibers 121 for cell expansion, differentiation, harvesting, etc., it may be beneficial to modify the surface of the hollow fibers 121 (e.g., the inner-facing surface (or hollow fiber membrane lumen) when the cells are expanded in the intracapillary space 130 or the outer-facing surface when the cells are expanded in the extracapillary space 110) in some way. For example, fibronectin (FN) and / or collagen can be used as surface modifiers, and / or the hollow fibers can be exposed to radiation. However, natural killer cells do not easily bind to fibronectin or collagen and / or gamma-treated surfaces. In various aspects, the present disclosure provides methods and materials for binding natural killer cells (isolated, such as buffy coated blood products, leukopaks and / or umbilical cord blood) and other similar cells to hollow fiber membranes (HFM) 101. The hollow fiber membrane 101 can be used as Figure 2 A hollow fiber membrane 117 is shown.

[0059] In at least one exemplary embodiment, the present disclosure provides for the use of a cell expansion system (e.g. Figure 1 The cell expansion system 10 and / or Figure 7 The cell expansion system 200 and / or Fig.10 The cell expansion system 500 and / or Fig.11 The method of the cell expansion system 600 shown in the figure includes forming a conjugated protein and biotinylating the conjugated protein to prepare (ie, functionalize) a bioreactor (eg, Figure 1 The bioreactor 24 and / or Figure 2 The bioreactor 100 and / or Fig.10 The hollow fiber membrane of the bioreactor 501 shown is used to attach natural killer cells and / or other similar cells. Using an automated perfusion-based cell expansion system, the biotinylated conjugated protein can be used to support the stimulation and / or monoculture of natural killer cells and / or other similar cells.

[0060] Figure 4 2 is a flow chart showing an exemplary method 201 for preparing a hollow fiber membrane of a cell expansion system for cell expansion, differentiation and / or harvesting of natural killer cells and other similar cells using a fibronectin-streptavidin (FN-SN) conjugate. The method 201 may include contacting 221 the fibronectin-streptavidin conjugate with the hollow fiber membrane, during which the fibronectin of the fibronectin-streptavidin conjugate contacts and attaches to (e.g., coats) at least a portion of the hollow fiber membrane and forms a modified hollow fiber membrane. In at least one exemplary embodiment, the contacting 221 may include introducing the fibronectin-streptavidin conjugate into the capillary intracapillary space of the hollow fiber membrane, wherein the fibronectin of the fibronectin-streptavidin conjugate contacts and attaches to (e.g., coats) at least a portion of the interior-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, contacting 221 may include introducing a fibronectin-streptavidin conjugate into the extracapillary space of the hollow fiber membrane, wherein the fibronectin of the fibronectin-streptavidin conjugate contacts and adheres to (e.g., coats) at least a portion of the exterior-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, contacting 221 may include introducing a fibronectin-streptavidin conjugate into the intracapillary space and the extracapillary space of the hollow fiber membrane, wherein the fibronectin of the fibronectin-streptavidin conjugate contacts and adheres to at least a portion of the interior-facing surface and at least a portion of the exterior-facing surface of the hollow fiber membrane.

[0061] In each case, the fibronectin of fibronectin-streptavidin conjugate can be attached to hollow fiber membrane to form one or more coatings. The coating can be a continuous coating, a discontinuous coating, a coating of variable thickness, a constant coating, etc. In at least one exemplary embodiment, the coating can be coated and / or cover the hole of the inner-facing surface defining the hollow fiber membrane. Fibronectin has a net positive charge and is attached to the hollow fiber membrane via polarity and hydrogen bonds. The hollow fiber membrane has a net negative charge at physiological pH (greater than or equal to about 7.2 to less than or equal to about 7.4). In addition, fibronectin has natural adhesion due to its glycoprotein structure and specific domains, which enables fibronectin to be combined with hollow fiber membrane (e.g., polyethersulfone (PES)) and cell membrane integrin. Although fibronectin is discussed, it should be recognized that other extracellular matrix (ECM) proteins with a net positive charge (such as vitronectin and / or fibrinogen and / or collagen and / or laminin, and their isoforms) can be conjugated with streptavidin and attached to one or more portions or regions of the hollow fiber membrane.

[0062] Reference again Figure 5 , the fibronectin-streptavidin conjugate can be contacted with the hollow fiber membrane 221 for a first time period. The first time period can be greater than or equal to about 4 hours to less than or equal to about 24 hours, and in some aspects, optionally about 12 hours. After contacting the fibronectin-streptavidin conjugate with the hollow fiber membrane 221, the method 201 can further include contacting the biotinylated molecule with the modified hollow fiber membrane 241. In at least one exemplary embodiment, the "coating bioreactor" setting of the cell expansion system can be used to contact the biotinylated molecule with the modified hollow fiber membrane 241. In at least one exemplary embodiment, for example, when the fibronectin-streptavidin conjugate is introduced into the capillary space of the hollow fiber membrane, the contact 241 may include introducing the biotinylated molecule into the capillary space. In at least one exemplary embodiment, for example, when the fibronectin-streptavidin conjugate is introduced into the extracapillary space of the hollow fiber membrane, the contact 241 may include introducing the biotinylated molecule into the extracapillary space. In at least one exemplary embodiment, for example, when a fibronectin-streptavidin conjugate is introduced into the intracapillary space and the extracapillary space of the hollow fiber membrane, contacting 241 may include introducing a biotinylated molecule into the intracapillary space and the extracapillary space. In each case, the biotinylated molecule may be selected from the group consisting of: a cytokine (including an interleukin or a growth factor), an epitope, a ligand, a monoclonal antibody, a stain, an aptamer, and a combination thereof.

[0063] Biotinylated molecules can be bound to fibronectin-streptavidin, and more specifically, can be bound to streptavidin to form biotinylated fibronectin-streptavidin conjugates that are ready for use in cell selection and / or cell signaling (including differentiation) applications, including enzyme-linked immunosorbent assays (ELISA) to quantify secreted cellular proteins. In at least one exemplary embodiment, streptavidin (having, for example, a molecular weight between about 52 kDa and about 55 kDa) can bind to up to four biotin molecules (having, for example, a molecular weight of about 244 Daltons) with high specificity and affinity (e.g., Kd=1E-14 to 1E-15) primarily through hydrogen bonds and van der Waals forces between amino acid residues that stabilize the polystreptavidin molecule.

[0064] In at least one exemplary embodiment, method 201 may include removing 231 excess unbound portions of conjugated proteins from the hollow fiber membrane prior to contacting 241 the biotinylated molecules with the modified hollow fiber membrane. For example, excess unbound portions of conjugated proteins may be removed using a washing process. In at least one exemplary embodiment, during the washing process, prior to contacting 241 the biotinylated molecules with the modified hollow fiber membrane, approximately 450 mL of phosphate buffered saline (PBS) or other buffer may be contacted with the hollow fiber membrane to remove unbound biotinylated proteins and / or other biotinylated molecules (e.g., aptamers).

[0065] In at least one exemplary embodiment, method 201 may include preparing 211 a fibronectin-streptavidin conjugate before contacting 221 the fibronectin-streptavidin conjugate with the hollow fiber membrane. In at least one exemplary embodiment, preparing 211 a fibronectin-streptavidin conjugate may include reconstituting the lyophilized fibronectin (having a molecular weight of, for example, greater than or equal to about 440 kDa to less than or equal to about 500 kDa) with streptavidin (having a molecular weight of, for example, greater than or equal to about 53 kDa to less than or equal to about 55 kDa) by contacting (e.g., immersing) the lyophilized fibronectin and streptavidin with deionized (DI) water (e.g., sterile deionized water (5 mg / 10 ml)) at room temperature (e.g., greater than or equal to about 20° C. to less than or equal to about 22° C.), and then diluting the mixture with phosphate buffered saline (PBS) (e.g., about 90 mL of phosphate buffered saline (PBS) without calcium ions and magnesium ions). In at least one exemplary embodiment, the preparation of 211 fibronectin-streptavidin conjugates may include a covalent coupling process that uses a connection modifier and a quencher chemistry to produce a covalent bond between fibronectin and streptavidin. For example, in at least one exemplary embodiment, the covalent coupling process may include the use of a Bio-Rad LYNX streptavidin conjugation kit. The covalent coupling process may occur within a time period of greater than or equal to about 3 hours to less than or equal to about 15 hours. In each variation, the fibronectin-streptavidin conjugate may have a mass ratio of fibronectin to streptavidin greater than or equal to about 1:2 to less than or equal to about 1:9, and in some aspects, optionally about 1:3.3. During cell selection and expansion, the selection of a specific mass ratio may be important for maintaining the function of fibronectin and streptavidin.

[0066] In at least one exemplary embodiment, the present disclosure provides for the use of a cell expansion system (e.g. Figure 1 The cell expansion system 10 and / or Fig.10 The method includes biotinylating one or more proteins to prepare (ie, functionalize) a bioreactor (eg, Figure 1 The bioreactor 24 and / or Figure 2 The bioreactor 100 and / or Fig.10 The hollow fiber membrane of the bioreactor 501 is shown for attaching natural killer cells and / or other similar cells. The biotinylated protein can support the stimulation and / or monoculture of natural killer cells and / or other similar cells using an automated perfusion-based cell expansion system.

[0067] Figure 5301 is a flow chart showing an exemplary method for preparing a hollow fiber membrane of a cell expansion system using cytokines for cell expansion, differentiation and / or harvesting natural killer cells and other similar cells. In at least one exemplary embodiment, the cytokine can be a Class I cytokine, including, for example, interleukin-21 (IL-21), interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), CD16, NKG2C / CD94, NKG2D, DNAM-1, 2B4 (CD48), Nkp30 and / or the like. Cytokines can be selected to enhance the in vitro expansion and cytotoxicity of natural killer cells. Method 301 may include contacting 311 fibronectin with the hollow fiber membrane, during which the fibronectin contacts and attaches to (e.g., coats) at least a portion of the hollow fiber membrane and forms a modified hollow fiber membrane. In at least one exemplary embodiment, contacting 311 may include introducing fibronectin into the intracapillary space of the hollow fiber membrane, wherein the fibronectin contacts and adheres to (e.g., coats) at least a portion of the inward-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, contacting 311 may include introducing fibronectin into the extracapillary space of the hollow fiber membrane, wherein the fibronectin contacts and adheres to (e.g., coats) at least a portion of the outward-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, contacting 311 may include introducing fibronectin into the intracapillary space and the extracapillary space of the hollow fiber membrane, wherein the fibronectin contacts and adheres to at least a portion of the inward-facing surface of the hollow fiber membrane and at least a portion of the outward-facing surface.

[0068] In each case, fibronectin can be attached to hollow fiber membrane to form one or more coatings.Coating can be a continuous coating, a discontinuous coating, a coating of variable thickness, a constant coating, etc. In at least one exemplary embodiment, coating can be coated and / or cover the hole of the inner-facing surface defining hollow fiber membrane.Fibronectin has a net positive charge and is attached to hollow fiber membrane via polarity and hydrogen bonds.Hollow fiber membrane has a net negative charge at physiological pH (greater than or equal to about 7.2 to less than or equal to about 7.4).In addition, fibronectin has natural adhesion due to its glycoprotein structure and specific domains, which enables fibronectin to be combined with hollow fiber membrane (such as polyethersulfone (PES)) and cell membrane integrin.Although fibronectin has been discussed, it should be appreciated that other extracellular matrix proteins (such as vitronectin and / or fibrinogen and / or collagen and / or laminin, and their isotypes) with a net positive charge can also be used, and they can be attached to one or more parts or regions of hollow fiber membrane.

[0069] Reference again Figure 5, fibronectin can contact 311 first time period with hollow fiber membrane.This first time period can be greater than or equal to about 4 hours to less than or equal to about 24 hours, and in some aspects, optionally be about 12 hours.After fibronectin is contacted 311 with hollow fiber membrane, method 301 can further include contacting 331 of cytokines with modified hollow fiber membrane.Cytokines can be combined with fibronectin to prepare hollow fiber membrane for cell selection and / or cell signaling (including differentiation) application.In at least one exemplary embodiment, for example, when fibronectin is introduced into the capillary space of hollow fiber membrane, contact 331 may include introducing cytokines into the capillary space.In at least one exemplary embodiment, for example, when fibronectin is introduced into the capillary extracapillary space of hollow fiber membrane, contact 331 may include introducing cytokines into the capillary extracapillary space. In at least one exemplary embodiment, for example, when fibronectin is introduced into the intracapillary space and the extracapillary space of the hollow fiber membrane, contacting 331 may include introducing cytokines into the intracapillary space and the extracapillary space.

[0070] In at least one exemplary embodiment, method 301 may include removing 321 excess unbound portions of fibronectin from the hollow fiber membrane prior to contacting 331 the cytokine with the modified hollow fiber membrane. For example, excess unbound portions of fibronectin may be removed using a washing process. In at least one exemplary embodiment, during the washing process, prior to contacting 331 the cytokine with the modified hollow fiber membrane, approximately 450 mL of phosphate buffered saline (PBS) or other buffer may be contacted with the hollow fiber membrane to remove unbound fibronectin and / or other molecules (e.g., aptamers).

[0071] Reference again Figure 1In at least one exemplary embodiment, the fluid inlet path 44 can be fluidically associated with the first fluid circulation path 12, and the fluid outlet path 46 can be fluidically associated with the second fluid circulation path 14. The fluid inlet path 44 allows fluid to enter the first fluid circulation path 12, while the fluid outlet path 46 allows fluid to leave the cell expansion system 10. In at least one exemplary embodiment, as shown, a third fluid flow control device 48 can be operably associated with the fluid inlet path 44. Although not shown, it should be recognized that in at least one exemplary embodiment, alternatively or additionally, a fourth fluid flow control device can be operably associated with the first outlet path 46. In at least one exemplary embodiment, the fluid flow control device (including the first fluid flow control device 30, the second fluid flow control device 32, the third fluid flow control device 48 and / or the fourth fluid flow control device) can include a pump, a valve, a clamp or any combination thereof. For example, a plurality of pumps, valves and clamps can be arranged in any combination. In at least one exemplary embodiment, the fluid flow control device can be a peristaltic pump, or include a peristaltic pump. The fluid circulation paths (including the first fluid circulation path 12 and / or the second fluid circulation path 14), inlet ports (including the fluid inlet port 44) and / or outlet ports (including the fluid outlet port 46) may include any known tubing materials, and any kind of fluid (including, for example, buffers, protein-containing fluids, and cell-containing fluids) may flow through the various circulation paths (including the first fluid circulation path 12 and / or the second fluid circulation path 14), inlet paths (including the fluid inlet port 44) and outlet paths (including the fluid outlet port 46). It should be appreciated that the terms "fluid," "medium," and "fluid medium" may be used interchangeably.

[0072] Figure 2 An exemplary hollow fiber cell growth chamber 100 (also referred to as a "bioreactor") is shown. The hollow fiber cell growth chamber 100 may be used as Figure 11 and 1 . The hollow fiber cell growth chamber 24 of the cell expansion system 10 is shown. The hollow fiber cell growth chamber 100 has a longitudinal axis (indicated by line LA-LA) and includes a cell growth chamber housing 104. The cell growth chamber housing 104 can have four openings or ports, including, for example, an intra-capillary inlet port 108, an intra-capillary outlet port 120, an extra-capillary inlet port 128, and an extra-capillary outlet port 132. A first fluid (also referred to as an intra-capillary fluid or medium) in a first circulation path (e.g., first fluid circulation path 12) can enter the cell growth chamber 100 through the intra-capillary inlet port 108 located at a first fluid manifold end 112 of the cell growth chamber 100, enter and pass through the intra-capillary space of a plurality of hollow fibers 116, and exit the cell growth chamber 100 through the extra-capillary outlet port 120 located at a second fluid manifold end 124 of the cell growth chamber 100. The fluid path between the intracapillary inlet port 108 and the intracapillary outlet port 120 defines an intracapillary portion 126 of the cell growth chamber 100. A second fluid (also referred to as an extracapillary medium or fluid) in a second circulation path (e.g., second fluid circulation path 14) can enter the cell growth chamber 100 through the extracapillary inlet port 128. The second fluid contacts the extracapillary space or the exterior of the hollow fiber membranes 117 and exits the cell growth chamber 100 via the extracapillary outlet port 132. The fluid path between the extracapillary inlet port 128 and the extracapillary outlet port 132 defines an extracapillary portion 136 of the cell growth chamber 100.

[0073] When the second fluid contacts the outside of the hollow fiber 116, small molecules (e.g., ions, water, oxygen, lactic acid, etc.) can diffuse from the interior or intracapillary space of the hollow fiber 116 to the exterior or extracapillary space through the hollow fiber 116, or alternatively or additionally, diffuse from the extracapillary space to the intracapillary space. Molecules of large molecular weight (e.g., growth factors and / or proteins) are generally too large to pass through the membrane wall of the hollow fiber 116 and remain in the intracapillary space of the hollow fiber 116 (or alternatively or additionally remain in the extracapillary space). The medium defining the first fluid and the second fluid can be replaced as needed, and alternatively or additionally, circulated through an oxygenator and / or a gas transfer module to exchange gases as needed. As discussed below, cells for expansion may be included in the first fluid circulation path 12 and / or the second fluid circulation path 14, and may enter the hollow fiber cell growth chamber 100 on one or both of the intracapillary space or the extracapillary space.

[0074] In at least one exemplary embodiment, cells may be seeded in the intracapillary space of the hollow fiber 116 (e.g., for expansion, differentiation and / or harvesting of cord blood-derived CD34+ hematopoietic stem / progenitor cells, monocytes, macrophages, hepatocytes and / or endothelial cells), and the cell culture medium is pumped through the extracapillary space of the hollow fiber 116 to deliver nutrients to the cells via perfusion of the hollow fiber membrane during expansion. However, in at least one other exemplary embodiment, cells for expansion may be seeded in the extracapillary space, and the cell culture medium is pumped through the intracapillary space to deliver nutrients to the cells via perfusion of the hollow fiber membrane during expansion. In at least one other exemplary embodiment, cells for expansion may be seeded in the intracapillary space, and the cell culture medium may be pumped through the extracapillary space and the intracapillary space. The movement of the cell culture medium through the intracapillary space and / or the extracapillary space can help remove excess cells, for example, those cells that are not attached to the surface of the hollow fiber membrane. In at least one exemplary embodiment, the material used to form the hollow fiber membrane 117 can be any biocompatible polymer material that can be made into a hollow fiber 121. For example, synthetic polysulfone-based materials such as polyethersulfone (PES) are commonly used to form hollow fibers.

[0075] In at least one exemplary embodiment, the cell expansion system 10 may also include a device configured to move or "rock" the cell growth chamber 100 relative to the other components of the cell expansion system 10. The device may be a rotating and / or lateral rocking device. For example, Figure 6 As shown, a cell growth chamber (also referred to as a bioreactor) 100 can be rotationally connected to one or more rotational rocking members 138 and lateral rocking members 140. The first rotational rocking member 138 can be rotationally associated with the bioreactor 100. For example, the first rotational rocking member 138 can be configured to rotate the bioreactor 100 around a first rotational axis or a central rotational axis 142. In at least one exemplary embodiment, the bioreactor 100 can be rotated in an alternating manner, for example, including, rotating in a first clockwise direction around the central axis 142 and then rotating in a second counterclockwise direction.

[0076] Although not shown, it should be appreciated that in at least one exemplary embodiment, the second rotational rocking member can be configured to move the bioreactor 100 about a second rotational axis 144 that passes through the center point of the bioreactor 100 and is perpendicular to the central axis 142. In at least one exemplary embodiment, the bioreactor 100 can be rotated in an alternating manner, for example including, for example, rotating about the second axis 144 in a first clockwise direction and then rotating in a second counterclockwise direction. In at least one exemplary embodiment, the bioreactor 100 can also be rotated about the second axis 144 and positioned in a horizontal or vertical orientation relative to gravity. The lateral rocking member 140 can be associated with the bioreactor 100 laterally. For example, the plane of the lateral rocking member 140 can be laterally movable in the x-direction and the y-direction.

[0077] Rotation and / or lateral movement of the bioreactor 100 can reduce the sedimentation of cells and the likelihood of cells being trapped within a portion of the bioreactor 100. In at least one exemplary embodiment, the rate at which cells settle in the cell growth chamber 100 can be proportional to the density difference between the cells and the suspension medium, based on Stokes' law. In at least one exemplary embodiment, repeated 180 degree rotations (rapid) with pauses as described above (e.g., with a total combined time of 30 seconds) can help keep non-adherent cells (e.g., T cells) suspended. A minimum rotation of about 180 degrees is preferred, however, various rotation angles can be used, including up to or greater than 360 degrees. Different rocking components can be used alone or in any combination. For example, a rocking component that rotates the bioreactor 100 around the central axis 142 can be combined with a rocking component that rotates the outside of the bioreactor 100 around the axis 144. Similarly, clockwise and counterclockwise rotations around different axes can also be performed independently in any combination.

[0078] In at least one exemplary embodiment, Figure 7 As shown, the cell expansion system 200 (similar to Figure 1The cell expansion system 10 shown in ) may include a pre-installed fluid delivery assembly 210. For example, the cell expansion system 200 may include a cell expansion machine 202 having a rear portion 206 and a hatch or closable door 204 engaged with the rear portion 206. The interior space 208 of the cell expansion machine 202 may be configured to receive the pre-installed fluid delivery assembly 210. The pre-installed fluid delivery assembly 210 may be removably attached to the cell expansion machine 202 to facilitate relatively quick replacement of a new or unused pre-installed fluid delivery assembly 210. For example, the cell expansion machine 202 may be operated to grow or expand a first group of cells using a first pre-installed fluid delivery assembly, and subsequently to grow or expand a second group of cells using a second pre-installed fluid delivery assembly without the need for sterilization between the exchange of the first pre-installed fluid delivery assembly 210 with the second pre-installed fluid delivery assembly. In each variation, the pre-installed fluid delivery assembly 210 includes a bioreactor (such as Figure 1 B and Figure 1 C) and an oxygenator or gas delivery module 212. The cell expansion system 200 includes a plurality of tubing guides 214 for receiving various media in fluid communication with the pre-installed fluid delivery assembly 210.

[0079] Figure 8 is a schematic diagram of the rear portion 206 of the cell expansion machine 202 prior to removably attaching a pre-installed fluid delivery assembly 210 . Figure 8 204 is omitted. As shown, the rear portion 206 of the cell expansion machine 202 may include a plurality of different structures for working in combination with the elements of the pre-installed fluid delivery assembly 210. For example, the rear portion 206 of the cell expansion machine 202 may include a plurality of peristaltic pumps for cooperating with the pump circuit of the pre-installed fluid delivery assembly 210, including, for example, an intra-capillary circulation pump 218, an extra-capillary circulation pump 220, an intra-capillary inlet pump 222, and / or an extra-capillary inlet pump 224. The rear portion 206 also includes a plurality of valves, including, for example, an intra-capillary circulation valve 226, a reagent valve 228, an intra-capillary medium valve 230, an air removal valve 232, a cell inlet valve 234, a wash valve 236, a dispensing valve 238, an extra-capillary medium valve 240, an intra-capillary waste valve 242, an extra-capillary waste valve 244, and / or a harvest valve 246. Several sensors may also be associated with the rear portion 206 of the cell expansion machine 202, including, for example, an intra-capillary outlet pressure sensor 248, a combined intra-capillary inlet pressure and temperature sensor 250, a combined extra-capillary inlet pressure and temperature sensor 252, and / or an extra-capillary outlet pressure sensor 254. In at least one exemplary embodiment, an optical sensor 256 for an air removal chamber may also be disposed in the rear portion 206.

[0080] The rear portion 206 may also include an axis or rocker control 258 for rotating the bioreactor 100. An axis fitting 260 may be associated with the axis or rocker control 258 to help ensure that the axis entry aperture 424 of the tubing organizer 300 of an exemplary pre-installed delivery assembly (e.g., pre-installed delivery assembly 400) is properly aligned with the rear portion 206 of the cell expansion machine 202. Rotation of the axis or rocker control 258 may cause the axis fitting 260 and the bioreactor 100 to rotate. Thus, when an operator or user of the cell expansion system 200 attaches a new or unused pre-installed fluid delivery assembly 400 to the cell expansion machine 202, alignment is a relatively simple matter of properly orienting the axis entry aperture 424 of the pre-installed fluid delivery assembly 400 with the axis fitting 260.

[0081] Fig. 9 4 is a perspective view of an exemplary removably-attachable pre-installed fluid delivery assembly 400. The pre-installed fluid delivery assembly 400 can be removably attached to the cell expansion machine 202 to facilitate quick placement of a new or unused pre-installed fluid delivery assembly 400 in the cell expansion machine 202. The bioreactor 100 can be attached to a bioreactor coupling that includes a shaft fitting 402. The shaft fitting 402 can include one or more shaft securing mechanisms, such as a biasing arm or spring member 404 for engaging the shaft 258 of the cell expansion machine 202.

[0082] The pre-installed fluid delivery assembly 400 may include a plurality of pipes (including, for example, pipes 408A, 408B, 408C, 408D, 408E) and various pipe fittings to form a Figure 8 and Fig. 9 4. The fluid paths shown are discussed below. Pump circuits 406A and 406B may also be provided. Although a variety of media may be provided in the cell expansion machine 202, in some variations, the pre-installed fluid delivery assembly 400 may include sufficient tubing length to extend to the exterior of the cell expansion machine 202 and be welded to tubing associated with a media bag or container.

[0083] Fig.10 is an exemplary cell expansion system 500 (which may be Figure 1Schematic diagram of a cell expansion system 100 shown in FIG. 1 , which shows an exemplary flow path. As shown, the cell expansion system 500 may include a first fluid circulation path 502 (also referred to as an "intracapillary loop" or "intracapillary space" or "IC loop") and a second fluid circulation path 504 (also referred to as an "extracapillary loop" or "extracapillary space" or "EC loop"). In at least one exemplary embodiment, cells may be positioned in the intracapillary space 502, and cell culture medium may be pumped through the extracapillary space 504 to deliver nutrients to the cells via hollow fiber membrane perfusion during expansion. However, it should be recognized that in at least one other exemplary embodiment, cells may be positioned in the extracapillary space 504, and cell culture medium may be pumped through the intracapillary space 502 to deliver nutrients to the cells via hollow fiber membrane perfusion during expansion. In at least one other exemplary embodiment, cells may be positioned in the intracapillary space 502, and cell culture medium may be pumped through the extracapillary space 504 and the intracapillary space 502. In at least one other exemplary embodiment, cells may be located in the extracapillary space 504 while cell culture medium is pumped through the extracapillary space 504 and the intracapillary space 502 .

[0084] The first fluid flow path 506 can be fluidly associated with a cell growth chamber (also referred to as a "bioreactor") 501 to form a first fluid circulation path 502. The cell growth chamber 501 can be used as Figure 1 The hollow fiber cell growth chamber 24 and / or Figure 2 The hollow fiber cell growth chamber 100 is shown. A first fluid can flow into the cell growth chamber 501 through the capillary inlet port 501A, which can be reversed to serve as an outlet. In a method of preparing a hollow fiber membrane for cell expansion of natural killer cells and other similar cells, a fibronectin-streptavidin conjugate can be introduced into the hollow fiber membrane through the first fluid flow path 506 and into the capillary inlet port 501A.

[0085] The first fluid can leave the cell growth chamber through the capillary outlet port 501B, which can be used as an inlet in reverse. For example, once entering the capillary space 502, the fibronectin of the fibronectin-streptavidin conjugate can contact and bind to (e.g., coat) the inner surface of the hollow fiber membrane, and any excess unbound conjugated protein in the capillary space 502 can be removed by the capillary outlet port 501B. After a period of time, the biotinylated molecule can be introduced into the hollow fiber membrane via the capillary inlet port 501A through the first fluid flow path 506. In the capillary space 502, the biotinylated molecule can be combined with the fibronectin-streptavidin conjugate, more specifically, combined with streptavidin to form a biotinylated fibronectin-streptavidin conjugate, which is ready for cell selection and / or cell signaling (including differentiation) applications. In at least one exemplary embodiment, a washing process may be used to flow a washing medium through the first fluid flow path 506, into the intra-capillary inlet port 501A, through the hollow fiber membranes, and out through the intra-capillary outlet port 501B.

[0086] In at least one exemplary embodiment, the first fluid circulation path 502 may include a pressure gauge 510 configured to measure the pressure of the first fluid leaving the cell growth chamber 501. In at least one exemplary embodiment, the first fluid circulation path 502 may include an intra-capillary circulation pump 512 configured to control the flow rate of the first fluid. For example, the intra-capillary circulation pump 512 may be configured to pump the first fluid in a first direction or a second direction opposite to the first direction. In the latter case, the intra-capillary outlet port 501B may be used as an inlet, and the intra-capillary inlet port 501A as an outlet. In at least one exemplary embodiment, the first fluid circulation path 502 may include a sample port 516 and / or a sample coil 518 configured for first fluid sample extraction. In at least one exemplary embodiment, the first fluid circulation path 502 may include a pressure gauge / thermometer 520 configured to detect the pressure and / or temperature of the first fluid during operation. In at least one exemplary embodiment, the first fluid may enter the intra-capillary loop 502 via a valve 514. In at least one exemplary embodiment, a portion of the cells can be flushed from the intra-capillary loop 502 into a harvest bag 599, for example, via valve 598. It should be appreciated that in at least one other exemplary embodiment, the first fluid circulation path 502 can include additional or fewer valves, pressure gauges, pressure sensors, temperature sensors, ports, and / or other devices configured to isolate and / or measure a property of the first fluid along a portion of the intra-capillary loop 502.

[0087] The second fluid may flow into the cell growth chamber 501 through the extracapillary inlet port 501C. The second fluid may exit the cell growth chamber 501 via the extracapillary outlet port 501D. In at least one exemplary embodiment, the second fluid in the extracapillary loop 504 may contact the exterior-facing surface of the hollow fibers disposed in the cell growth chamber 501, thereby allowing small molecules to diffuse into and out of the hollow fibers. In at least one exemplary embodiment, the extracapillary loop 504 may include a manometer / thermometer 524 configured to measure the pressure and / or temperature of the second fluid before the second fluid enters the cell growth chamber 501. In at least one exemplary embodiment, the extracapillary loop 504 may include a manometer 526 configured to measure, for example, the pressure of the second fluid as it exits the cell growth chamber 501. In at least one exemplary embodiment, the extracapillary loop 504 may include a sample port 530 configured for second fluid sample extraction.

[0088] In at least one exemplary embodiment, the extra-capillary loop 504 may include an extra-capillary circulation pump 528 and an oxygenator or gas delivery module 532. For example, after leaving the cell growth chamber 501, the second fluid may pass through the extra-capillary circulation pump 528 and enter and pass through the oxygenator or gas delivery module 532. In at least one exemplary embodiment, the extra-capillary circulation pump 528 may be configured to control the flow rate of the second fluid. For example, like the intra-capillary circulation pump 512, the extra-capillary circulation pump 528 may be configured to pump the second fluid in a first direction or in a second direction opposite to the first direction. In the latter case, the extra-capillary outlet port 501D may be used as an inlet, and the extra-capillary inlet port 501C may be used as an outlet.

[0089] In at least one exemplary embodiment, the second fluid flow path 522 can be fluidly associated with the oxygenator or gas delivery module 532 via an oxygenator inlet port 534 and an oxygenator outlet port 536. For example, the second fluid can flow into the oxygenator or gas delivery module 532 via the oxygenator inlet port 534 and can leave or flow out of the oxygenator or gas delivery module 532 via the oxygenator outlet port 536. In at least one exemplary embodiment, the oxygenator or gas delivery module 532 can be configured to add oxygen to the second fluid and / or remove bubbles from the second fluid. For example, air and / or gas can flow into the oxygenator or gas delivery module 532 via the first filter 538 and can leave or flow out (i.e., flow out) of the oxygenator or gas delivery device 532 via the second filter 540. The first filter 538 and the second filter 540 can be configured to reduce or prevent contaminants from entering the oxygenator or gas delivery module 532. The second fluid in the second fluid circulation path 504 can be in equilibrium with the gas entering the oxygenator or gas delivery module 532. In at least one exemplary embodiment, air and / or gas purged from the cell expansion system 500, such as during a priming sequence, can be exhausted to atmosphere via the oxygenator or gas delivery module 532. It should be appreciated that in at least one other exemplary embodiment, the second fluid circulation path 504 can include additional or fewer valves, pressure gauges, pressure sensors, temperature sensors, ports, and / or other devices configured to isolate and / or measure a property of the second fluid along a portion of the outer capillary loop 504.

[0090] In at least one exemplary embodiment, an air removal chamber (ARC) 556 can be fluidically associated with the first circulation path. The air removal chamber 556 can include one or more ultrasonic sensors. For example, the air removal chamber 556 can include an upper sensor and / or a lower sensor configured to detect a lack of air and / or fluid and / or an air-liquid interface at certain measurement locations within the air removal chamber 556. The upper sensor can be disposed near a first end (e.g., top) of the air removal chamber 556. The lower sensor can be disposed near a second end (e.g., bottom) of the air removal chamber 556. Although ultrasonic sensors are discussed, it should be understood that the air removal chamber 556 can additionally or alternatively include one or more other sensors, such as optical sensors. During portions of the pre-fill sequence and / or other protocols, air and / or gas purged from the cell expansion system 500 can be discharged to the atmosphere from the air valve 560 via a line 558 fluidically associated with the air removal chamber 556.

[0091] In at least one exemplary embodiment, the first fluid may include cells (e.g., fluid media (e.g., capillary media or fluid) from a first fluid container 562 (which may also be referred to as a first media bag or a first bag) and a second fluid container 546 (which may also be referred to as a second media bag or a second bag). The materials forming the first fluid container 562 and the second fluid container 546 (i.e., cells and / or capillary media) may enter the first fluid circulation path 502 via the first fluid flow path 506. The first fluid container 562 is connected to the first fluid flow path 506 and the first fluid circulation path 502 via a valve 564. In at least one exemplary embodiment, the second fluid container 546 and the third fluid container (also referred to as a third medium bag or a third bag) 544 can be fluidically associated with the first fluid inlet path 542, for example, via valves 548 and 550, respectively, or can be fluidically associated with the second fluid inlet path 574, for example, via valves 570 and 576, respectively. In at least one exemplary embodiment, the material from the second fluid container 546 and / or the third fluid container 544 can be in fluid communication with the first sterile sealable input pre-fill path 508 and / or the second sterile sealable input pre-fill path 509.

[0092] In at least one exemplary embodiment, the fourth fluid container 568 (which may also be referred to as a fourth media bag or a fourth bag) may include an extracapillary medium, and the fifth fluid container (which may also be referred to as a fifth media bag or a fifth bag) 566 may include a wash solution. Materials from the fourth fluid container 568 and the fifth fluid container 566 (i.e., the extracapillary medium and / or the wash solution) may enter the first fluid circulation path 502 and / or the second fluid circulation path 504. For example, in at least one exemplary embodiment, the fifth fluid container 566 may be fluidly associated with a valve 570, wherein the valve 570 is fluidly associated with the first fluid inlet path 542, for example, via a dispensing valve 572, and is fluidly associated with the first fluid circulation path 502. In at least one exemplary embodiment, the fifth fluid container 566 may be fluidly associated with the second fluid circulation path 504 via the second fluid inlet path 574 and the extracapillary inlet path 584, for example, by opening the valve 570 and closing the dispensing valve 572. The fourth fluid container 568 can be fluidly associated with a valve 576, wherein the valve 576 is fluidly associated with the first fluid circulation path 502, for example, via the first fluid inlet path 542 and the dispensing valve 572. In at least one exemplary embodiment, the fourth fluid container 568 can be fluidly associated with the second fluid inlet path 574 by opening the valve 576 and closing the dispensing valve 572. In at least one exemplary embodiment, the first fluid inlet path 542 and / or the second fluid inlet path 574 can be fluidly associated with the optional heat exchanger 552.

[0093] In at least one exemplary embodiment, the fluid may travel from the first fluid inlet path 542 and / or the second fluid inlet path 574 to the capillary inner loop 502 via the capillary inner inlet pump 554, and the fluid may travel to the capillary outer loop 504 via the capillary outer inlet pump 578. In at least one exemplary embodiment, an air detector 580 may also be associated with the capillary outer inlet path 584. The air detector 580 may include, for example, an ultrasonic sensor. In at least one exemplary embodiment, the first fluid circulation path 502 and the second fluid circulation path 504 may be fluidly associated with a waste line 588. For example, when the valve 590 is in an open state or position, the capillary inner medium may flow through the waste line 588 to the waste bag 586 (also referred to as the outlet bag). When the valve 582 is open, the capillary outer medium may flow to the waste bag 586 through the waste line 588. In at least one exemplary embodiment, cells may be harvested, for example, via a cell harvesting path 596. For example, cells from cell growth chamber 501 may be harvested by pumping capillary culture medium containing the cells through cell harvest path 596 and valve 598 to cell harvest bag 599 .

[0094] In at least one exemplary embodiment, as shown, the fluids in the first fluid circulation path 502 and the second fluid circulation path 504 flow through the cell growth chamber 501 in the same direction (i.e., a co-current configuration). Although not shown, it should be appreciated that in other exemplary embodiments, the cell expansion system 500 can also be configured to flow in a counter-current configuration. Fig.10 As shown, the fluid in the first fluid circulation path 502 can enter the bioreactor 501 at the capillary inlet port 501A and can leave or flow out of the bioreactor 501 at the capillary outlet port 501B. In at least one exemplary embodiment, the first fluid flow path 506 can be fluidically connected to the first fluid circulation path 502 via a connector 517, for example. The connector 517 can be a point or location where fluids can flow in opposite directions, for example, based on the direction and flow rate of the capillary inlet pump 554 and the fluid circulation pump 512. The connector 517 can include any type of fittings, couplings, fusions, passages, and / or pipes that allow the first fluid flow path 506 to be fluidically associated with the first fluid circulation path 502. In at least one exemplary embodiment, the connector 517 can include a T-shaped fitting or coupling and / or a Y-shaped fitting or coupling.

[0095] In at least one exemplary embodiment, one or more instruments (e.g., pressure gauge 510, pressure / temperature gauge 520, pressure / temperature gauge 524, and / or pressure gauge 526), ​​one or more valves (e.g., valve 514, valve 548, valve 550, valve 560, valve 564, valve 570, valve 572, valve 576, valve 582, valve 590, valve 596, and / or valve 598), one or more ports (e.g., capillary inlet port 501A, capillary outlet port 501B, extra-capillary inlet port 501C, extra-capillary outlet port 501D, sample port 516, The sample port 530, the oxygenator inlet port 534, and / or the oxygenator outlet port 536), one or more pumps (e.g., the intra-capillary circulation pump 512, the extra-capillary circulation pump 528, the intra-capillary inlet pump 554, and / or the extra-capillary inlet pump 578), one or more filters (e.g., the first filter 538 and / or the second filter 540), one or more coils (e.g., the sample coil 518), one or more modules (e.g., the oxygenator or gas delivery module 532), and / or one or more other components of the cell expansion system 500 can be in electrical communication with a control system (not shown). The control system may include a plurality of nodes, which may include various hardware, firmware, and / or software configured to control and / or communicate with mechanical, electromechanical, and electrical components of the cell expansion system 500, including, for example, controllers and memory.

[0096] The controller (also referred to as a processor) may be any type of microcontroller, microprocessor, field programmable gate array (FPGA), application specific integrated circuit (ASIC), etc. An exemplary controller may be a NK10DN512VOK10 microcontroller manufactured and sold by N9P USA, Incorporated, which is a microcontroller unit with a 32-bit architecture. Other exemplary controllers may include, for example, at least one of the following: 800 and 801, with 4G LTE integration and 64-bit computing 610 and 615, with 64-bit architecture A7 processor, M7 motion coprocessor, series, Core TM Series processors, Series processors, Atom TM Series processors, Intel Series processors, i5-4670K and i7-4770K 22nm Haswell, i5-3570K 22nm Ivy Bridge, FX TM Series processors, FX-4300, FX-6300 and FX-8350 32nm Vishera, Kaveri processors, Cortex TM -M processor, Cortex-A and ARM926EJ-S TM The processor, as well as other industry equivalent processors, may use any known or future developed standard, instruction set, library and / or architecture to perform computing functions. The memory may be any type of memory including random access memory (RAM), read-only memory (ROM), electrically erasable programmable ROM (EEPROM), portable compact disk read-only memory (CD-ROM), optical storage device, magnetic storage device, any suitable combination of the foregoing, or other type of storage device or storage device that stores and provides instructions to program and control the controller.

[0097] Fig.11 Another exemplary cell expansion system 600 (which may be similar to Figure 1 Schematic diagram of the cell expansion system 100 described above, showing exemplary flow paths. As shown, the cell expansion system 600 may include a first fluid circulation path 602 (also referred to as an "intracapillary loop" or "intracapillary space" or "IC loop") and a second fluid circulation path 604 (also referred to as an "extracapillary loop" or "extracapillary space" or "EC loop"). In at least one exemplary embodiment, cells may be positioned in the intracapillary space 602, while cell culture medium is pumped through the extracapillary space 604 to deliver nutrients to the cells via hollow fiber membrane perfusion during expansion. However, it should be recognized that in at least one other exemplary embodiment, cells may be positioned in the extracapillary space 604, while cell culture medium is pumped through the intracapillary space 602, to deliver nutrients to the cells via hollow fiber membrane perfusion during expansion. In at least one other exemplary embodiment, cells may be positioned in the intracapillary space 602, while cell culture medium is pumped through the extracapillary space 604 and the intracapillary space 602. In at least one other exemplary embodiment, cells may be located in the extracapillary space 604 , while cell culture medium is pumped through the extracapillary space 604 and the intracapillary space 602 .

[0098] The first fluid flow path 606 can be fluidically associated with the cell growth chamber 601 (also referred to as a "bioreactor") to form a first fluid circulation path 602. The cell growth chamber 601 can be used as Figure 1 The hollow fiber cell growth chamber 24 and / or Figure 1The hollow fiber cell growth chamber 100 is shown. A first fluid can flow into the cell growth chamber 601 through the capillary inlet port 601A. In a method of preparing a hollow fiber membrane for cell expansion of natural killer cells and / or other similar cells, a fibronectin-streptavidin conjugate can be introduced into the hollow fiber membrane through the first fluid flow path 606 and into the capillary inlet port 601A.

[0099] Fluid can leave the cell growth chamber 601 through the capillary outlet port 601B, which can be used as an inlet in reverse. For example, once entering the capillary space 602, the fibronectin of the fibronectin-streptavidin conjugate can contact and bind to (e.g., coat) the inner surface of the hollow fiber membrane, and any excess unbound conjugated protein in the capillary space can be removed through the capillary outlet port 601B. After a period of time, the biotinylated molecule can be introduced into the hollow fiber membrane through the first fluid flow path 606 and enter the capillary inlet port 601A. While in the capillary space 602, the biotinylated molecule can bind to fibronectin-streptavidin, more specifically, to streptavidin, to form a biotinylated fibronectin-streptavidin conjugate, thereby being ready for cell selection and / or cell signaling (including differentiation) applications. In at least one exemplary embodiment, a washing process may be used to flow a washing medium through the first fluid flow path 606, into the intra-capillary inlet port 601A, through the hollow fiber membranes, and out the intra-capillary outlet port 601B.

[0100] In at least one exemplary embodiment, first fluid circulation path 602 may include sensor 610. In at least one exemplary embodiment, sensor 610 may be configured to measure the pressure of the medium exiting cell growth chamber 601. In at least one exemplary embodiment, sensor 610 may be configured to measure the temperature of the medium exiting cell growth chamber 601. In at least one exemplary embodiment, sensor 610 may be configured to measure the pressure and temperature of the medium exiting cell growth chamber 601. In at least one exemplary embodiment, the medium may flow through an intra-capillary circulation pump 612 configured to control the flow rate of the medium. The intra-capillary circulation pump 612 may be configured to pump fluid in a first direction or a second direction opposite to the first direction.

[0101] In at least one exemplary embodiment, the (first) medium may enter the capillary back to 602 through valve 614. In at least one exemplary embodiment, a sample of the medium may be obtained from sample coil 618 during operation. The medium may then be returned to the capillary inlet port 601A to complete the fluid circulation path 602. In at least one exemplary embodiment, cells grown / expanded in the cell growth chamber 601 may be flushed from the cell growth chamber 601 to a harvest bag 699 through valve 698 and line 697. Alternatively, when valve 698 is closed, the cells may be redistributed into the chamber 601 for further growth.

[0102] Fluid in second fluid circulation path 604 may enter cell growth chamber 601 via extracapillary inlet port 601C and may exit cell growth chamber 601 via extracapillary outlet port 601D. In at least one exemplary embodiment, the (second) medium in extracapillary loop 604 may be in contact with the exterior of the hollow fibers in cell growth chamber 601, thereby allowing small molecules to diffuse into and out of the hollow fibers that may be located within chamber 601.

[0103] In at least one exemplary embodiment, second fluid circulation path 604 may include sensor 624. In at least one exemplary embodiment, sensor 624 may be configured to measure the pressure of the medium before the medium enters the extracapillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 624 may be configured to measure the temperature of the medium before the medium enters the extracapillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 624 may be configured to measure the pressure and temperature of the medium before the medium enters the extracapillary space of cell growth chamber 601.

[0104] In at least one exemplary embodiment, second fluid circulation path 604 may include sensor 626. In at least one exemplary embodiment, sensor 626 may be configured to measure the pressure of the medium after the medium leaves the extracapillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 626 may be configured to measure the temperature of the medium after the medium leaves the extracapillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 626 may be configured to measure both the pressure and the temperature of the medium after the medium leaves the extracapillary space of cell growth chamber 601.

[0105] After exiting the extracapillary outlet port 601D of the cell growth chamber 601, the fluid in the second fluid circulation path 604 can flow to the oxygenator or gas delivery module 632 through the extracapillary circulation pump 628. The extracapillary circulation pump 628 can also pump fluid in the opposite direction. The second fluid flow path 622 can be fluidically associated with the oxygenator or gas delivery module 632 via the inlet port 632A and the outlet port 632B of the oxygenator or gas delivery module 632. In operation, the fluid medium can flow into the oxygenator or gas delivery module 632 via the inlet port 632A and can leave the oxygenator or gas delivery module 632 via the outlet port 632B. The oxygenator or gas delivery module 632 can add oxygen to the medium in the cell expansion system 600 and remove bubbles therefrom. In at least one exemplary embodiment, the medium in the second fluid circulation path 604 can be in equilibrium with the gas entering the oxygenator or gas delivery module 632. The oxygenator or gas delivery module 632 can be any appropriately sized device for oxygenation or gas delivery. Air or gas flows into the oxygenator or gas delivery module 632 via filter 638 and flows out of the oxygenator or gas delivery device 632 through filter 640. Filter 638 and filter 640 can reduce or prevent contamination of the oxygenator or gas delivery module 632 and associated media. During portions of the priming sequence, air or gas purged from the cell expansion system 600 can be exhausted to the atmosphere via the oxygenator or gas delivery module 632.

[0106] Although the illustrated configuration of cell expansion system 600 shows the fluid media in first fluid circulation path 602 and second fluid circulation path 604 flowing through cell growth chamber 601 in the same direction (e.g., a co-current configuration), it should be appreciated that in at least one exemplary embodiment, cell expansion system 600 can also be configured for flow in a counter-current configuration.

[0107] In at least one exemplary embodiment, a medium, for example, including a cell container from a source (such as a bag of cells, can be attached at attachment point 662, and a fluid medium from the medium source can be attached at attachment point 646. The cells and the medium can be introduced into the first fluid circulation path 602 via the first fluid flow path 606. The attachment point 662 can be fluidly associated with the first fluid flow path 606 via valve 664, and the attachment point 646 can be fluidly associated with the first fluid flow path 606 via valve 650. A reagent source can be fluidly connected to point 644 and can be associated with the fluid inlet path 642 via valve 648, or associated with the second fluid inlet path 674 via valve 648 and valve 672.

[0108] An air removal chamber (ARC) 656 can be fluidically associated with the first circulation path 602. In at least one exemplary embodiment, the air removal chamber 656 can include one or more sensors (including an upper sensor and a lower sensor) for detecting air, lack of fluid, and / or a gas / fluid interface (e.g., an air / fluid interface) at certain measurement locations within the air removal chamber 656. For example, an ultrasonic sensor can be used near the bottom and / or top of the air removal chamber 656 to detect air, fluid, and / or air / fluid interfaces at these locations. It should be understood that many other types of sensors can be incorporated into the cell expansion system 600 without departing from the spirit and scope of the present disclosure. For example, in at least one exemplary embodiment, an optical sensor can be used according to an embodiment of the present disclosure. During portions of the priming sequence or other protocols, air or gas purged from the cell expansion system 600 can be discharged to the atmosphere from an air valve 660 via a line 658 fluidically associated with the air removal chamber 656.

[0109] A source of extracapillary medium may be attached to the extracapillary medium attachment point 668, and / or a source of wash liquid may be attached to the wash liquid attachment point 666 to add the extracapillary medium and / or wash liquid to the first or second fluid flow path. The attachment point 666 may be fluidically associated with a valve 670, which may be fluidically associated with the first fluid circulation path 602 via a valve 672 and the first fluid inlet path 642. Alternatively, the attachment point 666 may be fluidically associated with the second fluid circulation path 604 via a second fluid inlet path 674 and a second fluid flow path 684 by opening the valve 670 and closing the valve 672. Similarly, the attachment point 668 may be fluidically associated with a valve 676, which may be fluidically associated with the first fluid circulation path 602 via the first fluid inlet path 642 and the valve 672. Alternatively, the fluid container 668 may be fluidically associated with the second fluid inlet path 674 by opening the valve 676 and closing the valve 672.

[0110] In the intra-capillary loop, fluid may be initially propelled by an intra-capillary inlet pump 654. In the extra-capillary loop, fluid may be initially propelled by an extra-capillary inlet pump 678. An air detector 680, such as an ultrasonic sensor, may also be associated with the extra-capillary inlet path 684.

[0111] In at least one embodiment, the first fluid circulation path 602 and the second fluid circulation path 604 can be connected to a waste line 688. When the valve 690 is open, the intracapillary medium can flow through the waste line 688 and to the waste or outlet bag 686. Likewise, when the valve 692 is open, the extracapillary medium can flow to the waste or outlet bag 686.

[0112] After the cells are grown in the cell growth chamber 601, they can be harvested via the cell harvesting path 697. Here, when the valve 698 is open, the capillary medium containing the cells can be pumped through the cell harvesting path 697 into the cell harvesting bag 699 to harvest the cells from the cell growth chamber 601.

[0113] In at least one exemplary embodiment, the various components of cell expansion system 600 can be housed or positioned in a machine or housing, such as cell expansion machine 202, where the machine maintains the cells and media, for example, at a predetermined temperature. In at least one exemplary embodiment, the components of cell expansion system 600 and components of cell expansion system 500 can be combined. In at least one exemplary embodiment, the cell expansion system can include a plurality of components of cell expansion system 600, such as cell expansion machine 202, ... Figure 5 and Figure 6 The cell expansion systems shown may have fewer or additional components and still be within the scope of the present disclosure.

[0114] Fig.12 An exemplary process 800 for expanding cells is shown, which can be used with a cell expansion system, such as Figure 1 The cell expansion system 10 and / or Figure 7 The cell expansion system 200 and / or Fig.10 The cell expansion system 500 and / or Fig.11 802, the process 800 may include loading 804 a disposable tubing set onto the cell expansion system and pre-filling 806 the system. In at least one exemplary embodiment, a user or operator may provide a pre-filling instruction to the system by selecting a task for pre-filling. In at least one exemplary embodiment, the task for pre-filling may be a pre-programmed task. Then, the process 800 may proceed to coating 808 a bioreactor, wherein the bioreactor may be optionally coated with a coating agent. The coating step 808 is represented by a dotted line to indicate that the step is an optional step depending on the cell type being amplified, the operator's choice, other considerations or factors, etc. When the process 800 includes the coating step 808, reagents may be loaded into the capillary inner loop until the reagent container becomes empty. Reagents may be driven from the air removal chamber into the capillary inner loop, and then reagents may circulate in the capillary inner loop. In at least one exemplary embodiment, a coating reagent including fibronectin may be used. Once the bioreactor is coated 808 (or after the priming step 806 if the bioreactor is not coated), the procedure 800 may include a capillary / extra-capillary flush task 810, in which the fluids on the capillary and extra-capillary circulation loops are replaced. The replacement volume may be determined by the amount of capillary and extra-capillary volume replaced.

[0115] In order to maintain an appropriate or desired gas concentration in the fibers in the bioreactor membrane, an adjust medium task 812 may be performed so that the medium is balanced with the provided gas supply before the cells are loaded into the bioreactor. For example, rapid contact between the medium and the gas supply provided by the gas transfer module or oxygenator may be provided by using a high capillary extracellular circulation rate. The system may then remain in an appropriate or desired state until the user or operator is ready to load the cells into the bioreactor. In at least one exemplary embodiment, the system may be adjusted with a complete medium. The complete medium may be any source of medium for cell growth. In at least one exemplary embodiment, the complete medium may include, for example, alpha-MEM (α-MEM) and / or fetal bovine serum (FBS).

[0116] For example, process 800 may include loading 814 cells (e.g., natural killer cells), such as from a cell entry bag, into a bioreactor. In at least one exemplary embodiment, the cells may be loaded from the cell entry bag into the bioreactor until the bag becomes empty. The cells may then be driven from the air removal chamber to the bioreactor. In embodiments utilizing a larger chase volume, the cells may be spread and moved toward an intracapillary outlet. In at least one exemplary embodiment, the transmembrane distribution of cells may be promoted via intracapillary circulation, such as by an intracapillary circulation pump, in the absence of an intracapillary inlet.

[0117] In other embodiments, a "centrally load cells without circulation" task 814 may be used, wherein a first volume of fluid including a plurality of cells may be loaded into a cell expansion system at a first flow rate, wherein the cell expansion system includes a cell growth chamber. A second volume of fluid containing a medium may then be loaded into a portion of the first fluid circulation path at a second flow rate, for example to position the first volume of fluid in a first portion of the cell growth chamber. In at least one exemplary embodiment, the first portion of the cell growth chamber or bioreactor may include approximately a central region of the bioreactor. In at least one exemplary embodiment, the first volume may be the same as the second volume. In at least one exemplary embodiment, the first flow rate may be the same as the second flow rate. In at least one exemplary embodiment, the first volume may be different from the second volume. In at least one exemplary embodiment, the first flow rate is different from the second flow rate. In at least one exemplary embodiment, the sum of the first volume and the second volume may be equal to a percentage or ratio of the volume (e.g., total volume) of the first fluid circulation path. For example, the sum of the first volume and the second volume may be approximately 50% of the volume (e.g., total volume) of the first fluid circulation path. In at least one exemplary embodiment, the fluid in the first fluid circulation path flows through the intracapillary space of the bioreactor or cell growth chamber. In at least one exemplary embodiment, the fluid in the second fluid circulation path flows through the extracapillary space of, for example, a cell growth chamber or bioreactor. In at least one exemplary embodiment, the sum of the first volume and the second volume can be, for example, about 50% of the volume of the capillary loop, or, depending on the embodiment, can be another percentage or ratio of the volume of the capillary loop. In at least one exemplary embodiment, the sum of the first volume and the second volume can be about 50% of the volume of another fluid path, loop, etc., as applicable, or, depending on the embodiment, can be another percentage or ratio of its volume. Other percentages or ratios can be used, for example, including any percentage between and including about 1% and about 100%.

[0118] After loading 814 cells, process 800 may include feeding 816 cells. Cells may be expanded or grown 818. Although step 818 is shown after step 816, it should be appreciated that in at least one exemplary embodiment, step 818 may occur before step 816, or simultaneously with step 816. Next, process 800 proceeds to query 820 to determine whether any cell colonies, microcolonies, or clusters have been formed. A cell colony, microcolony, or cluster may be a group of one or more attached cells. If a cell colony, microcolony, or cluster has been formed, process 800 proceeds to "yes" to shear 822 any cell colony, microcolony, or cluster. For example, after amplifying a plurality of cells for a first time period, cells may be circulated at a first cycle rate in a second time period to reduce the number of cells in a cell colony, microcolony, or cluster. In at least one exemplary embodiment, circulating cells at a first cycle rate may cause a cell colony to generate shear stress, wherein one or more cells in a cell colony may detach from the cell colony. In at least one exemplary embodiment, for example, reducing the number of cells in a cell colony, microcolony, or cluster can provide a single cell suspension. For example, in at least one exemplary embodiment, during the cell culture period, the cells can be cycled every two days to shear 822 any colony, microcolony, or cluster to maintain uniform cell density and nutrient diffusion. In at least one exemplary embodiment, this shearing of any microcolony, colony, or cluster can begin on or after the 4th day. According to other exemplary embodiments, this shearing can begin on other dates or time periods. After shearing 822, process 800 can then return to the feeder cells 816.

[0119] For example, if query 820 determines not to shear any cell colonies or clusters, or if they do not exist, process 800 proceeds to "no" to resuspend 824 the cells. In at least one exemplary embodiment, circulating cells can be used to evenly resuspend those cells that may be loosely attached during culture. In at least one exemplary embodiment, step 824 may include circulating cells to evenly resuspend those cells that may be loosely attached before starting a harvesting task or other tasks to remove cells from the bioreactor. After the cells are resuspended 824, process 800 may then proceed to harvest 826 the cells. Optionally, at step 828, the removed cells may be further processed or otherwise analyzed, and process 800 may then terminate at end operation 830. If further processing / analysis is not desired, process 800 terminates at end operation 830.

[0120] It should be appreciated that, according to embodiments of the present disclosure, the described operational steps are provided for illustrative purposes and may be rearranged, combined into other steps, used in parallel with other steps, etc. Fewer or additional steps may be used in embodiments without departing from the spirit and scope of the present disclosure. Furthermore, in some embodiments, steps (and any sub-steps), such as priming, coating the bioreactor, loading cells may be performed automatically, such as by a processor executing pre-programmed tasks stored in a memory, wherein these steps are provided for illustrative purposes only.

[0121] Fig.13 2500 in which embodiments of the present disclosure may be implemented. Figure 1 The cell expansion system 10 and / or Figure 7 The cell expansion system 200 and / or Fig.10 The cell expansion system 500 and / or Fig.11 The cell expansion system 600 shown uses a processor to perform tasks, such as custom tasks or pre-programmed tasks performed as part of a process (such as the process shown and / or described herein). In some variations, the pre-programmed tasks may include, for example, based on "ready membrane", "IC / EC flushing" and / or "feeding cells".

[0122] As shown, computing system 2500 may include user interface 2502, processing system 2504 and / or storage device 2506. User interface 2502 may include output device 2508 and / or input device 2510. Output device 2508 may include one or more touch screens, wherein the touch screen may include a display area for providing one or more application windows. The touch screen may also be an input device 2510, which may, for example, receive and / or capture physical touch events from a user or operator. The touch screen may include a liquid crystal display (LCD) having a capacitive structure, thereby allowing processing system 2504 to infer the location of the touch event. Then, processing system 2504 may map the location of the touch event to a UI element presented at a predetermined position of the application window. According to an embodiment, the touch screen may also be configured to receive touch events through one or more other electronic structures. Other output devices 2508 may include printers, speakers, etc. Other input devices 2510 may include keyboards, other touch input devices, mice, voice input devices, etc.

[0123] The processing system 2504 may include a processing unit 2512 and / or a memory 2514. In at least one exemplary embodiment, the processing unit 2512 may be a general-purpose processor operable to execute instructions stored in the memory 2514. The processing unit 2512 may include a single processor or multiple processors. Further, each processor may be a multi-core processor having one or more cores to read and execute separate instructions. The processor may include a general-purpose processor, an application-specific integrated circuit (ASICs), a field programmable gate array (FPGA), other integrated circuits, etc.

[0124] Depending on the implementation, the memory 2514 may include any short-term or long-term storage device for data and / or processor executable instructions. The memory 1014 may include, for example, random access memory (RAM), read-only memory (ROM), or electrically erasable programmable read-only memory (EEPROM). Other storage media may include, for example, CD-ROM, magnetic tape, digital versatile disk (DVD) or other optical storage, tape, magnetic disk storage, magnetic tape, other magnetic storage devices, etc.

[0125] Storage 2506 may be any long-term data storage device or component. Depending on the implementation, storage 2506 may include one or more systems described in conjunction with memory 2514. Storage 2506 may be permanent or removable. Storage 2506 may be configured to store data generated or provided by processing system 2504.

[0126] Certain features of the current technology are further illustrated in the following non-limiting examples.

[0127] Example 1

[0128] Preparation of streptavidin coating

[0129] In some variations, the streptavidin coating can be prepared by contacting streptavidin with water (H2O) to form a 10 μg / mL solution. The solution (e.g., 100 μL) can be added to the wells and left to dry. After a period of time, a washing treatment can be performed. For example, 0.1 BSA can be washed and blocked at ambient temperature or room temperature (e.g., greater than or equal to about 20°C to less than or equal to about 22°C) for about 1 hour to prepare the hollow fiber.

[0130] In certain variations, the streptavidin coating process may include: on day -3, passing a phosphate buffered saline (PBS)-fibronectin solution, and coating the solution on a polyethersulfone (PES) membrane overnight at 37°C, wherein the solution is introduced at 0.1-20 mL / min and circulated in the capillary inner loop of the Quantum CES or similar platform at a rate of greater than or equal to about 0 mL / min to less than or equal to about 20 mL / min. This is followed by a phosphate buffered saline (PBS) wash step, which uses, for example, the QuantumCES IC fast rinse or IC / EC exchange task to remove unbound fibronectin. On day -2, a phosphate buffered saline (PBS)-streptavidin solution may be added after the washing step, which may be attached to the underlying fibronectin layer, may be circulated overnight in a similar manner, and then a Quantum rinse task may be performed. On day -1, a phosphate-buffered saline (PBS)-biotinylated IL-21 cytokine solution can be added and circulated in a similar manner within the capillary loop at 37°C, followed by a Quantum wash task prior to cell seeding.

[0131] Example 2

[0132] Bioreactor / column coating

[0133] There are several methods to create a fibronectin (FN)-streptavidin (SN) base for attaching biotinylated molecules to The surface of a system polyethersulfone (PES) hollow fiber membrane (HFM) bioreactor or a preparatory column for cell selection can be functionalized. For example, in certain variations, fibronectin may be functionalized by attaching and expanding adherent cells such as mesenchymal stromal / stem cells (MSCs), fibroblasts, and / or aortic endothelial cells. Polyethersulfone hollow fiber membrane binding in a cell expansion system (System) bioreactor. This process is likely based on the high affinity of streptavidin for biotin. While considering the available protein-conjugated biochemicals, it is important to keep the protocols direct and efficient, with minimal residues or reactants, to accommodate their adaptability in the manufacture of cell therapy products. Mixing and / or linking fibronectin-streptavidin mixtures or conjugates may support the functionalization of hollow fiber membrane bioreactors or columns with biotinylated cytokines, chemokines and other ligands to facilitate cell selection and expansion. Other affinity separations of biomolecules can be expected. In short, this protein-protein conjugation can be considered a platform for cell therapy-related affinity processes using existing technologies.

[0134] Two exemplary methods are described: (1) a simple mixture of fibronectin + streptavidin; (2) rapid covalent coupling of fibronectin and streptavidin using a modified Bio-Rad LYNX kit. As background, native and recombinant human dimeric fibronectin can have a molecular weight of greater than or equal to about 440 kDa to less than or equal to about 500 kDa, and tetrameric streptavidin can have a molecular weight of greater than or equal to about 53 kDa to less than or equal to about 55 kDa. In protein-protein coupling chemistry, the mass ratio of the reactants can be adjusted to optimize their molar ratios to maintain their functionality in cell selection and expansion. For example, the Bio-Rad LYNX kit can be designed to couple streptavidin and IgG class monoclonal antibodies (mAbs) at a mass ratio of approximately 1:1. Here, the Bio-Rad LYNX kit reagents can couple fibronectin and streptavidin at different mass ratios of about 1:3.3 in a reaction at an effective molar ratio of about 1:3 for a period of greater than or equal to about 3 hours to less than or equal to about 15 hours at ambient temperature. For example, see Table 1.

[0135] Table 1: FN:SN protein mass ratios used for mixing and conjugation

[0136]

[0137] Option 1: Coating with a mixture of fibronectin and streptavidin

[0138] This process may involve reconstituting lyophilized fibronectin and streptavidin (FN+SN) (e.g., 1:3.3 mass ratio) in deionized water (DI H2O) for about 30 minutes at room temperature. 2+ -Mg 2+ Increase the volume of the mixture to 100 mL with Longza PBS and introduce the mixture into After coating the bioreactor, excess unbound conjugated protein can be washed away and the biotinylated molecule of choice (e.g., cytokine (interleukin or growth factor), epitope, ligand, monoclonal antibody, dye, or aptamer) can be introduced into the system via the "Coat Bioreactor" task. The resulting fibronectin-streptavidin-bioconjugate protein is ready for cell selection or cell signaling (including differentiation) applications. Other applications could potentially include coating the prepared hollow fiber membrane columns or matrices, which can be used to introduce cells into The exact ratio of fibronectin to streptavidin and the method of conjugation may be modified during further development. For example, recombinant or semisynthetic fibronectin or fibrinogen may replace plasma-derived fibronectin. Extracellular matrix proteins such as fibronectin may bind to streptavidin by polarity and hydrogen bonding. The SurPASS binding layer of Anton Paar showed that under physiological pH conditions of 7.2-7.4, System polyethersulfone membranes may have a "net negative charge". Fibronectin may have a "net positive charge" due to the presence of positively charged amino acid residues (such as lysine). In addition, fibronectin may have natural adhesive properties due to its glycoprotein structure and specific domains that allow binding to polyethersulfone and cell membrane integrins.

[0139] Option 2: Coating with Rapid Covalent Coupling of Fibronectin and Streptavidin Using a Modified LYNX Kit

[0140] Covalent coupling of fibrinogen to streptavidin, using similar mass ratios as outlined in "Option 1", and may be another embodiment, can be accomplished using a modified commercially available Bio-Rad LYNX Rapid Streptavidin Conjugation Kit. The kit can use proprietary linker modifiers and quenching chemicals (LNK161STR, LNK162STR, LNK163STR) to produce a covalent link between fibronectin and streptavidin in a time period of greater than or equal to about 3 hours to less than or equal to about 15 hours. In the covalent coating method, the affinity of the selected biotinylated molecule for streptavidin may be similar to the affinity of the biotinylated molecule in the fibrinogen-streptavidin mixture coating method. The advantage of the covalent method may be the increased stability of the fibrinogen-streptavidin coupling.

[0141] The description of the above embodiments is provided for the purpose of illustration and description. It is not intended to be exhaustive or to limit the present disclosure. Single elements or features of a specific embodiment are generally not limited to that specific embodiment, but where applicable, they are interchangeable and can be used in selected embodiments even if not specifically shown or described. This can also be varied in many ways. These variations should not be considered as departing from the present disclosure, and all such modifications are intended to be included within the scope of the present disclosure.

Claims

1. A method for functionalizing a hollow fiber membrane for cell expansion of a target cell, the method comprising: A biotinylated molecule is brought into contact with the surface of the hollow fiber membrane including an extracellular matrix component to which it binds and has affinity for the target cell.

2. The method according to claim 1, wherein: The biotinylated molecule is selected from the group consisting of a cytokine, an epitope, a ligand, a monoclonal antibody, a dye, an aptamer, and combinations thereof.

3. The method according to claim 2, wherein: The cytokines include interleukin-21.

4. The method according to claim 1, wherein: The extracellular matrix component is selected from the group consisting of fibronectin, vitronectin, fibrinogen, collagen, laminin, and combinations thereof.

5. The method according to claim 1, wherein: The extracellular matrix component includes an extracellular matrix component-streptavidin conjugate, the extracellular matrix component of the extracellular matrix component-streptavidin conjugate is bound to the surface of the hollow fiber membrane, and the streptavidin of the extracellular matrix component-streptavidin conjugate is bound to the biotinylated molecule.

6. The method according to claim 5, wherein: The extracellular matrix component-streptavidin conjugate has a mass ratio of the extracellular matrix component to the streptavidin of greater than or equal to about 1:3 to less than or equal to about 1:

9.

7. The method according to claim 5, wherein: The extracellular matrix component-streptavidin conjugate includes a fibronectin-streptavidin conjugate, wherein the fibronectin has a molecular weight of greater than or equal to about 440 kDa to less than or equal to about 500 kDa, and the streptavidin has a molecular weight of greater than or equal to about 53 kDa to less than or equal to about 55 kDa.

8. The method according to claim 7, wherein: The method further comprises preparing the fibronectin-streptavidin conjugate.

9. The method according to claim 8, wherein: The preparation of the fibronectin-streptavidin conjugate includes reconstituting the lyophilized fibronectin with streptavidin by soaking the lyophilized fibronectin and streptavidin in water.

10. The method according to claim 8, wherein: Preparing the fibronectin-streptavidin conjugate includes covalently coupling the fibronectin and the streptavidin.

11. The method according to claim 1, wherein: The method further includes contacting the extracellular matrix component with a surface of the hollow fiber membrane.

12. The method according to claim 11, wherein: The extracellular matrix component is contacted with the surface of the hollow fiber membrane for a period of greater than or equal to about 4 hours to less than or equal to about 24 hours prior to contacting the biotinylated molecule with the surface.

13. The method according to claim 12, wherein: After the period of time, and prior to contacting the biotinylated molecule with the surface, the method further comprises washing the hollow fiber membrane to remove any unreacted and excess portions of the extracellular matrix components.

14. The method according to claim 1, wherein: The target cells include natural killer cells.

15. The method according to claim 1, wherein: The surface is the interior facing surface.

16. The method according to claim 1, wherein: The surface is an outwardly facing surface, or a combination of an inwardly facing surface and an outwardly facing surface.

17. A method for functionalizing a hollow fiber membrane for cell expansion of a target cell, the method comprising: contacting an extracellular matrix component-streptavidin conjugate with a hollow fiber membrane, wherein the extracellular matrix component of the extracellular matrix component-streptavidin conjugate binds to the hollow fiber membrane, and the streptavidin of the extracellular matrix component-streptavidin conjugate binds to the extracellular matrix component; and A biotinylated molecule is contacted with the hollow fiber membrane, the biotinylated molecule binds to the streptavidin of the extracellular matrix component-streptavidin conjugate, the biotinylated molecule is selected from the group consisting of: a cytokine, an epitope, a ligand, a monoclonal antibody, a dye, an aptamer, and a combination thereof.

18. The method according to claim 17, wherein: The extracellular matrix component of the extracellular matrix component-streptavidin conjugate is selected from the group consisting of fibronectin, vitronectin, fibrinogen, collagen, laminin and combinations thereof.

19. The method according to claim 17, wherein: The extracellular matrix component-streptavidin conjugate includes a fibronectin-streptavidin conjugate, and the method further includes preparing the fibronectin-streptavidin conjugate; The preparation of the fibronectin-streptavidin conjugate includes reconstituting the lyophilized fibronectin with streptavidin by immersing the lyophilized fibronectin and streptavidin in water, or covalently coupling the fibronectin and the streptavidin.

20. The method according to claim 17, wherein: Prior to contacting the biotinylated molecule with the hollow fiber membrane, contacting the extracellular matrix component-streptavidin conjugate with the hollow fiber membrane for a period of time greater than or equal to about 4 hours to less than or equal to about 24 hours, and the method further comprises, Prior to contacting the biotinylated molecule with the hollow fiber membrane, the hollow fiber membrane is washed to remove any unreacted and excess portions of the extracellular matrix component-streptavidin conjugate.

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