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

Abstract

A method for functionalizing a hollow fiber membrane for cell proliferation of target cells (e.g., natural killer cells) includes contacting the surface of the hollow fiber membrane containing an extracellular matrix component with a biotinylated molecule that binds to the extracellular matrix component and has affinity for the target cells. The biotinylated molecule is selected from the group consisting of cytokines, epitopes, ligands, monoclonal antibodies, stains, aptamers, and combinations thereof. The extracellular matrix component is selected from the group consisting of fibronectin, vitronectin, fibrinogen, collagen, laminin, and combinations thereof.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Patent Application No. 18 / 234,470, filed August 16, 2023, and the benefit of U.S. Provisional Patent Application No. 63 / 403,592, filed September 2, 2022, the entire disclosures of which are incorporated herein by reference.

[0002] The present disclosure relates to a method for expanding cells using immobilized interleukin-21 (IL-21) and soluble interleukin-2 (IL-2). [Background technology]

[0003] This section provides background information related to the present disclosure that is not necessarily prior art.

[0004] Natural killer (NK) cells are innate lymphocytes that naturally attack certain cells and are therefore attractive candidates for various cell therapies and the treatment of various malignant diseases. For example, natural killer cells have recently been used as a cell type for cancer therapy using artificial chimeric antigen receptors (CARs). Summary of the Invention [Problem to be solved by the invention]

[0005] Despite their promise, the widespread clinical success of natural killer cell therapy has been limited in part by challenges in easily and efficiently producing large quantities of natural killer cells. Current methods for expanding natural killer cells are often flask-based, time-consuming, expensive, and have relatively low success rates. Therefore, it is desirable to develop improved methods for expanding natural killer cells (and similar cells) that have improved success rates and are less time-consuming and expensive. [Means for solving the problem]

[0006] This section provides a general overview of the disclosure and is not an exhaustive disclosure of the entire scope or all features.

[0007] In at least one exemplary embodiment, the present disclosure provides a method for functionalizing a hollow fiber membrane for cell proliferation of target cells, the method comprising contacting a surface of the hollow fiber membrane containing an extracellular matrix component with a biotinylated molecule, the biotinylated molecule binding to the extracellular matrix component and having affinity for the target cells.

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

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

[0010] In at least one exemplary embodiment, the extracellular matrix component is 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 comprises an extracellular matrix component-streptavidin conjugate, the extracellular matrix component of the extracellular matrix component-streptavidin conjugate binding to the surface of the hollow fiber membrane, and the streptavidin of the extracellular matrix component-streptavidin conjugate binding to the biotinylated molecule.

[0012] In at least one exemplary embodiment, the mass ratio of the extracellular matrix component to the streptavidin in the extracellular matrix component-streptavidin complex is about 1:3 or more and about 1:9 or less.

[0013] In at least one exemplary embodiment, the extracellular matrix component-streptavidin complex comprises a fibronectin-streptavidin complex, wherein the fibronectin in the fibronectin-streptavidin complex has a molecular weight of about 440 kDa or more and about 500 kDa or less, and the streptavidin in the fibronectin-streptavidin complex has a molecular weight of about 53 kDa or more and about 55 kDa or less.

[0014] In at least one exemplary embodiment, the method further comprises preparing a fibronectin-streptavidin complex.

[0015] In at least one exemplary embodiment, the step of preparing the fibronectin-streptavidin complex comprises reconstituting the lyophilized fibronectin with the streptavidin by soaking the lyophilized fibronectin and streptavidin in water.

[0016] In at least one exemplary embodiment, the step of preparing the fibronectin-streptavidin complex comprises covalently binding the fibronectin to the streptavidin.

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

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

[0019] In at least one exemplary embodiment, after the period of time and before the step of contacting the biotinylated molecule, the method further comprises the step of washing the hollow fiber membrane to remove unreacted and excess extracellular matrix components.

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

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

[0022] In at least one exemplary embodiment, the surface is an outward facing surface or a combination of an inward facing surface and the outward facing surface.

[0023] In at least one exemplary embodiment, the present disclosure provides a method for functionalizing a hollow fiber membrane for cell proliferation of target cells. The method includes contacting the hollow fiber membrane with an extracellular matrix component-streptavidin complex, wherein the extracellular matrix component of the extracellular matrix component-streptavidin complex binds to the hollow fiber membrane and the streptavidin of the extracellular matrix component-streptavidin complex binds to the extracellular matrix component. The method also includes contacting the hollow fiber membrane with a biotinylated molecule, wherein the biotinylated molecule binds to the streptavidin of the extracellular matrix component-streptavidin complex. The biotinylated molecule is selected from the group consisting of a cytokine, an epitope, a ligand, a monoclonal antibody, a stain, an aptamer, and combinations thereof.

[0024] In at least one exemplary embodiment, the extracellular matrix component of the extracellular matrix component streptavidin complex is 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 comprises a fibronectin-streptavidin conjugate.

[0026] In at least one exemplary embodiment, the method further comprises preparing a fibronectin-streptavidin complex, wherein the step of preparing the fibronectin-streptavidin complex comprises reconstituting the lyophilized fibronectin with streptavidin by immersing the lyophilized fibronectin and streptavidin in water, or covalently binding the fibronectin and streptavidin.

[0027] In at least one exemplary embodiment, prior to the step of contacting the biotinylated molecule, the extracellular matrix component-streptavidin complex is contacted with the hollow fiber membrane for a period of about 4 hours or more to about 24 hours or less.

[0028] In at least one exemplary embodiment, the method further comprises washing the hollow fiber membrane to remove unreacted and excess extracellular matrix component-streptavidin complexes prior to the step of contacting the biotinylated molecule.

[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.

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

[0031] [Figure 1]FIG. 1 is a diagram of an example of a cell growth system having a bioreactor, according to at least one exemplary embodiment of the present disclosure. [Figure 2] FIG. 2 is a diagram of an example bioreactor showing the circulation path through the bioreactor that may be incorporated into a cell growth system similar to the cell growth system illustrated in FIG. 1 , according to at least one exemplary embodiment of the present disclosure. [Figure 3] FIG. 3 is a cross-sectional schematic diagram of an example of a hollow fiber membrane incorporated into a cell growth system similar to the cell growth system shown in FIG. 1, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 4] FIG. 4 is a flow chart illustrating an example of a method for preparing a bioreactor similar to that of FIG. 2 for cell expansion, differentiation, and / or harvesting of natural killer and other similar cells using biotinylated protein conjugates according to various embodiments of the present disclosure. [Figure 5] FIG. 5 is a flow chart illustrating an example of a method for preparing a bioreactor similar to that of FIG. 2 for cell expansion, differentiation, and / or harvesting of natural killer and other similar cells using cytokines, according to various embodiments of the present disclosure. [Figure 6] FIG. 6 is a diagram of an example of a rocking device configured to move a bioreactor similar to the bioreactor of FIG. 2, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 7] FIG. 7 is a perspective view of an example of a cell growth system similar to that shown in FIG. 1, having a pre-mounted fluid transfer device. [Figure 8] FIG. 8 is a perspective view of an example of a housing for the cell growth system shown in FIG. [Figure 9] 9 is a perspective view of the pre-mounted fluid transfer device shown in FIG. [Figure 10] FIG. 10 is a schematic diagram illustrating an example of a flow path of a cell growth system similar to the cell growth system shown in FIG. 1, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 11] FIG. 11 is a schematic diagram illustrating an example of a flow path of a cell growth system similar to the cell growth system shown in FIG. 1, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 12] FIG. 12 is a flow diagram illustrating operational features of an example process for growing cells using a cell growth system similar to the cell growth system shown in FIG. 1, in accordance with at least one exemplary embodiment of the present disclosure. [Figure 13] FIG. 13 is a block diagram of an example of a processing system for use in a cell growth system similar to the cell growth system shown in FIG. 1, in accordance with at least one exemplary embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0032] Corresponding reference characters indicate corresponding parts shown in the several views of the drawings.

[0033] Exemplary embodiments will be described in more detail with reference to the accompanying drawings.

[0034] The exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope to those skilled in the art. Numerous specific details are set forth, such as examples of specific components, devices, and methods, to provide a thorough understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details are not necessary, that the exemplary embodiments may be embodied in many different forms, and that none of these should be construed as limiting the scope of the present disclosure. In some exemplary embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0035] The terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. The terms "comprises," "comprising," "including," and "having" are inclusive and thus specify the presence of stated features, entities, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The method steps, processes, and operations described herein should not be construed as necessarily requiring performance in the particular order described or illustrated, unless specifically identified as such. It should also be understood that additional or alternative steps may be employed.

[0036] When an element or layer is referred to as "on," "engaged to," "connected to," or "coupled to" another element or layer, it may be directly on, directly engaged with, directly connected to, or directly coupled to the other element or layer, or intervening elements or layers may be present. Conversely, when an element is referred to as "directly on," "directly engaged with," "directly connected to," or "directly coupled to" another element or layer, no intervening elements or layers are present. Other terms used to describe relationships between elements should be interpreted similarly (e.g., "between" vs. "directly between," "adjacent" vs. "directly adjacent," etc.). As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0037] Terms such as "first," "second," and "third" may be used herein to describe various elements, components, regions, layers, and / or sections, but these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are used only to distinguish one element, component, region, layer, or section from another region, layer, or section. Terms such as "first," "second," and other numerical terms, when used herein, do not imply a sequence or order unless clearly indicated by context. Thus, a first element, first component, first region, first layer, or first section described below could also be referred to as a second element, second component, second region, second layer, or second section without departing from the teachings of the exemplary embodiments.

[0038] Spatial terms such as "inside," "outside," "beneath," "below," "lower," "upper," and "above" are used herein for ease of description when describing the relationship of one element or feature to another element(s) or feature(s), as shown in the figures. Spatial terms may be intended to encompass different orientations of the device during use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures were inverted, elements described as being "beneath" or "beneath" other elements or features would change accordingly to be "above" them. Thus, for example, the term "below" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatial descriptions used herein should be interpreted accordingly.

[0039] Various components are referred to herein as "operably associated." As used herein, "operably associated" refers to components that are linked together in an operable manner and includes embodiments in which components are directly coupled as well as embodiments in which other components are disposed between the coupled components. "Operably associated" components can be "fluidically associated." "Fluidly associated" refers to components that are linked together so that fluid can be transported between them. The term "fluidly associated" includes embodiments in which another member is disposed between two fluidly associated members, embodiments in which members are directly connected, and the like. Fluidly associated members may include members that do not contact the fluid but that operate the system by contacting other members (e.g., a peristaltic pump that pumps fluid through a flexible tube by compressing the outside of the tube).

[0040] Exemplary embodiments will be described in more detail with reference to the accompanying drawings.

[0041] This disclosure relates to methods for cell expansion of cells, such as natural killer cells, using cell expansion systems such as those described in U.S. Patent No. 8,309,347, issued November 13, 2012, entitled "Cell Expansion Systems and Methods of Use," and / or U.S. Patent No. 9,677,042, issued June 13, 2017, entitled "Customizable Methods and Systems for Growing and Harvesting Cells in Hollow Fiber Bioreactor Systems," and / or No. 9,725,689, issued August 8, 2017 ("Configurable Method and System for Growing and Harvesting Cells in a Hollow Fiber Bioreactor System"), and / or U.S. Patent Application No. 15 / 943,536, filed April 2, 2018, published October 2, 2018 ("Cell Growth in Bioreactors"), and / or U.S. Patent No. 10,577,585, issued March 3, 2020 ("Cell Growth"), the entire disclosures of which are incorporated herein by reference.

[0042] A cell growth system including a hollow fiber bioreactor is a cell culture system used to grow and differentiate cells, including both adherent and non-adherent cell types. For example, as shown in FIG. 1, an exemplary cell growth system 10 includes a first fluid circuit 12 and a second fluid circuit 14. The first fluid circuit 12 includes, for example, a first fluid flow path 16 having opposing ends 18, 20. The first fluid flow path 16 may be in fluid communication with a hollow fiber cell growth chamber 24 (also referred to as a "bioreactor"). For example, the first end 18 of the first fluid flow path 16 is in fluid communication with a first inlet 22 of the cell growth chamber 24, and the second end 20 is in fluid communication with a first outlet 28 of the cell growth chamber 24. Fluid in the first fluid circuit 12 can flow through the interior of a plurality of hollow fibers 116 of a hollow fiber membrane ("HFM") 117 (see, for example, FIG. 2) disposed within the cell growth chamber 24. In at least one exemplary embodiment, a first flow control device 30 may be operably coupled to the first fluid flow path 16 to control the flow of fluid within the first fluid circuit 12 .

[0043] The second fluid circuit 14 includes, for example, a second fluid flow path 34 and a second flow controller 32. Similar to the first fluid flow path 16, the second fluid flow path 34 has opposing ends 36, 38. The opposing ends 36, 38 of the second fluid flow path 34 may be in fluid communication with an inlet port 40 and an outlet port 42 of the cell growth chamber 24. For example, the first end 36 of the second fluid flow path 34 is in fluid communication with the inlet port 40 of the cell growth chamber 24, and the second end 38 of the second fluid flow path 34 is in fluid communication with the outlet port 42. The fluid in the second fluid circuit 14 contacts the exterior of a hollow fiber membrane 117 (see, e.g., FIG. 2 ) disposed within the cell growth chamber 24. In at least one exemplary embodiment, the second flow controller 32 may be operably coupled to the second fluid flow path 34 to control the flow of fluid within the second fluid circuit 14.

[0044] The first and second fluid circuits 12, 14 are maintained within the cell growth chamber 24 by hollow fiber membranes 117. In this case, fluid in the first fluid circuit 12 flows through the inner capillary ("IC") space of the hollow fiber membranes 117, and fluid in the second fluid circuit 14 flows through the extra-capillary ("EC") space of the cell growth chamber 24. The first fluid circuit 12 is also referred to as the "inner capillary loop" or "inner capillary space" or "IC loop." The second fluid circuit 14 is also referred to as the "outer capillary loop" or "extra-capillary space" or "EC loop." The fluid in the first fluid circuit 12 may flow either co-currently or counter-currently with respect to the fluid flow in the second fluid circuit 14. By way of example, FIG. 3 shows a cross section of an exemplary hollow fiber membrane 101 comprising a plurality of semi-permeable hollow fibers (also referred to as a hollow column and / or hollow matrix) 121. Here, the space or void 130 within the hollow fibers 121 defines the intra-capillary space, and the space outside the hollow fibers 121 defines the extra-capillary space 110 .

[0045] In many cases, cells for expansion are seeded in the capillary inner space 130 (e.g., for expansion, differentiation, and / or harvesting of cord blood-derived CD34+ hematopoietic stem / progenitor cells, monocytes, macrophages, hepatocytes, and / or endothelial cells) while cell culture medium is pumped through the capillary outer space 110, delivering nutrients to the cells via hollow fiber membrane perfusion during expansion. However, in other variations, cells for expansion are seeded in the capillary outer space 110 while cell culture medium is pumped through the capillary inner space 130, delivering nutrients to the cells via hollow fiber membrane perfusion during expansion. In yet other variations, cells for expansion are seeded in the capillary inner space 130 while cell culture medium is pumped through both the capillary outer space 110 and the capillary inner space 130. The movement of cell culture medium in the capillary inner space 130 serves to remove excess cells that are not attached to the surface of the hollow fiber membrane 101. In each example, the material used to fabricate the hollow fiber membrane 101 can be any biocompatible polymeric material that can be made into hollow fibers 121. For example, synthetic polysulfone-based materials (e.g., polyethersulfone (PES)) are often used to form hollow fibers.

[0046] To allow cells (such as natural killer cells (NK cells)) to better attach to the hollow fibers 121 for cell growth, differentiation, harvesting, etc., it is beneficial to modify the surfaces of the hollow fibers 121 in some way (e.g., the inner-facing surface (i.e., hollow fiber membrane lumen) when cells are grown in the inner capillary space 130, or the outer-facing surface when cells are grown in the outer capillary space 110). 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 readily bind to fibronectin or collagen and / or gamma-treated surfaces. In various aspects, the present disclosure provides methods and materials for attaching natural killer cells (isolated, e.g., from buffy coat blood products, leukopaks, and / or umbilical cord blood) and other similar cells to hollow fiber membranes (HFMs) 101. The hollow fiber membrane 101 may be used as the hollow fiber membrane 117 shown in FIG.

[0047] In at least one exemplary embodiment, the present disclosure provides a method for using a cell expansion system (such as cell expansion system 10 shown in FIG. 1 and / or cell expansion system 200 shown in FIG. 7 and / or cell expansion system 500 shown in FIG. 10 and / or cell expansion system 600 shown in FIG. 11 ) comprising forming a conjugated protein and biotinylating the conjugated protein to prepare (i.e., functionalize) a hollow fiber membrane of a bioreactor (e.g., bioreactor 24 shown in FIG. 1 and / or bioreactor 100 shown in FIG. 2 and / or bioreactor 501 shown in FIG. 10 ) for adhesion with natural killer cells and / or other similar cells. The biotinylated conjugated protein can support stimulation and / or monoculture of natural killer cells and / or other similar cells using an automated perfusion-based cell expansion system.

[0048] FIG. 4 is a flowchart illustrating an exemplary method 201 for preparing hollow fiber membranes of a cell growth system for cell growth, differentiation, and / or harvesting of natural killer cells and other similar cells using a fibronectin-streptavidin (FN-SN) conjugate. Method 201 includes a step 221 of contacting the fibronectin-streptavidin conjugate with the hollow fiber membrane. During this step, the fibronectin of the fibronectin-streptavidin conjugate contacts and adheres to (e.g., coats) at least a portion of the hollow fiber membrane to form a modified hollow fiber membrane. In at least one exemplary embodiment, the contacting step 221 can include introducing the fibronectin-streptavidin conjugate into the capillary interior of the hollow fiber membrane. In this case, the fibronectin of the fibronectin-streptavidin conjugate contacts and adheres to (e.g., coats) at least a portion of the inner-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, contacting step 221 may include introducing a fibronectin-streptavidin complex into the extra-capillary space of the hollow fiber membrane. In this case, the fibronectin of the fibronectin-streptavidin complex contacts and adheres to (e.g., coats) at least a portion of the outer-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, contacting step 221 may include introducing a fibronectin-streptavidin complex into both the extra-capillary space and the extra-capillary space of the hollow fiber membrane. In this case, the fibronectin of the fibronectin-streptavidin complex contacts and adheres to at least a portion of the inner-facing surface and at least a portion of the outer-facing surface of the hollow fiber membrane.

[0049] In each case, the fibronectin in the fibronectin-streptavidin complex can adhere to the hollow fiber membrane to form one or more coating layers. The coating layer can be a continuous coating layer, a discontinuous coating layer, a coating layer with a variable thickness, a coating layer with a constant thickness, etc. In at least one exemplary embodiment, the coating layer coats and / or blocks the pores to define the inner-facing surface of the hollow fiber membrane. Fibronectin has a net positive charge and adheres to the hollow fiber membrane through polarity and hydrogen bonding. The hollow fiber membrane has a net negative charge at physiological pH, which is about 7.2 or higher to about 7.4 or lower. Furthermore, fibronectin has natural adhesive properties due to its glycoprotein structure and specific domains, which enable it to bind to both the hollow fiber membrane (e.g., polyethersulfone (PES)) and cell membrane integrins. Although fibronectin is described, it should be understood 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 also form conjugates with streptavidin and attach to one or more portions or regions of the hollow fiber membrane.

[0050] Referring again to FIG. 5 , the fibronectin-streptavidin complex is contacted with the hollow fiber membrane for a first period of time (221). The first period of time can be from about 4 hours to about 24 hours, and in certain embodiments, is optionally about 12 hours. After contacting the fibronectin-streptavidin complex with the hollow fiber membrane in step 221, method 201 further includes contacting a biotinylated molecule with the modified hollow fiber membrane in step 241. In at least one exemplary embodiment, the biotinylated molecule is contacted with the modified hollow fiber membrane using a "coat bioreactor" setting of a cell growth system in step 241. In at least one exemplary embodiment, for example, if the fibronectin-streptavidin complex is introduced into the capillary inner space of the hollow fiber membrane, contacting step 241 includes introducing the biotinylated molecule into the capillary inner space. In at least one exemplary embodiment, for example, when a fibronectin-streptavidin complex is introduced into the extra-capillary space of a hollow fiber membrane, contacting step 241 includes introducing a biotinylated molecule into the extra-capillary space. In at least one exemplary embodiment, for example, when a fibronectin-streptavidin complex is introduced into both the intra-capillary space and the extra-capillary space of a hollow fiber membrane, contacting step 241 includes introducing a biotinylated molecule into both the intra-capillary space and the extra-capillary space. In each case, the biotinylated molecule is selected from the group consisting of a cytokine (including an interleukin or growth factor), an epitope, a ligand, a monoclonal antibody, a stain, an aptamer, and combinations thereof.

[0051] The biotinylated molecule can bind to a fibronectin-streptavidin complex, more specifically, to streptavidin to form a biotinylated fibronectin-streptavidin complex ready for use in cell selection and / or cell signaling (including differentiation) applications, including enzyme-linked immunosorbent assays (ELISAs) for quantifying secreted cellular proteins. In at least one exemplary embodiment, streptavidin (e.g., having a molecular weight of about 52 kDa to about 55 kDa) can bind up to four molecules of biotin (e.g., having a molecular weight of about 244 daltons) with a high degree of specificity and affinity (e.g., Kd = 1E-14 to 1E-15) primarily through hydrogen bonding and van der Waals forces with amino acid residues that stabilize the multimeric streptavidin molecule.

[0052] In at least one exemplary embodiment, method 201 includes step 231 of removing excess unbound complex proteins from the hollow fiber membrane prior to step 241 of contacting the biotinylated molecules with the modified hollow fiber membrane. For example, excess unbound complex proteins are removed using a washing process. In at least one exemplary embodiment, the washing process involves contacting the hollow fiber membrane with approximately 450 mL of phosphate-buffered saline (PBS) or other buffer to remove unbound biotinylated proteins and / or other biotinylated molecules (e.g., aptamers), after which the biotinylated molecules can be contacted with the modified hollow fiber membrane (241).

[0053] In at least one exemplary embodiment, method 201 includes a step 211 of preparing a fibronectin-streptavidin complex prior to a step 221 of contacting the fibronectin-streptavidin complex with a hollow fiber membrane. In at least one exemplary embodiment, step 211 of preparing a fibronectin-streptavidin complex includes contacting (e.g., immersing) lyophilized fibronectin (e.g., having a molecular weight of about 440 kDa to about 500 kDa) and streptavidin (e.g., having a molecular weight of about 53 kDa to about 55 kDa) with deionized (DI) water (e.g., sterile deionized water (5 mg / 10 mL)) at ambient temperature (e.g., about 20°C to about 22°C) to reconstitute the lyophilized fibronectin with the streptavidin, 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, step 211 of preparing a fibronectin-streptavidin complex involves a covalent conjugation process using a binding modifier and a quencher to generate a covalent bond between fibronectin and streptavidin. For example, in at least one exemplary embodiment, the covalent conjugation process involves using a Bio-Rad LYNX Rapid Streptavidin Conjugation Kit. The covalent conjugation process is carried out for a period of about 3 hours or more and about 15 hours or less. In various variations, the fibronectin-streptavidin complex has a mass ratio of fibronectin:streptavidin of about 1:2 or more and about 1:9 or less, and in certain embodiments, optionally, about 1:3.3. Selecting a specific mass ratio is important for maintaining the functionality of fibronectin and streptavidin during cell selection and growth.

[0054] In at least one exemplary embodiment, the present disclosure provides a method for using a cell expansion system (such as cell expansion system 10 shown in FIG. 1 and / or cell expansion system 500 shown in FIG. 10) that includes biotinylating one or more proteins to prepare (i.e., functionalize) the hollow fiber membranes of the bioreactor (e.g., bioreactor 24 shown in FIG. 1 and / or bioreactor 100 shown in FIG. 2 and / or bioreactor 501 shown in FIG. 10) for adhesion with natural killer cells and / or other similar cells. The biotinylated proteins can support stimulation and / or monoculture of natural killer and / or other similar cells using the automated perfusion-based cell expansion system.

[0055] 5 is a flow chart illustrating an exemplary method 301 for preparing hollow fiber membranes of a cell expansion system for cell proliferation, differentiation, and / or harvesting of natural killer cells and other similar cells using cytokines. In at least one exemplary embodiment, the cytokines are Class I cytokines, 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. The cytokines are selected to enhance both the in vitro expansion potential and cytotoxicity of natural killer cells. Method 301 includes step 311 of contacting fibronectin with the hollow fiber membrane. During this step, the fibronectin contacts and adheres (e.g., coats) at least a portion of the hollow fiber membrane, forming a modified hollow fiber membrane. In at least one exemplary embodiment, the contacting step 311 may include introducing the fibronectin into the inner capillary space of the hollow fiber membrane. In this case, the fibronectin contacts and adheres (e.g., coats) at least a portion of the inner-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, the contacting step 311 may include introducing the fibronectin into the extra-capillary space of the hollow fiber membrane. In this case, the fibronectin contacts and adheres (e.g., coats) at least a portion of the outer-facing surface of the hollow fiber membrane. In at least one exemplary embodiment, the contacting step 311 may include introducing the fibronectin into both the inner and extra-capillary spaces of the hollow fiber membrane. In this case, the fibronectin contacts and adheres to at least a portion of the inner surface and at least a portion of the outer surface of the hollow fiber membrane.

[0056] In each case, fibronectin can adhere to the hollow fiber membrane to form one or more coating layers. The coating layer can be a continuous coating layer, a discontinuous coating layer, a coating layer with a variable thickness, a coating layer with a constant thickness, etc. In at least one exemplary embodiment, the coating layer coats and / or blocks the pores to define the inner-facing surface of the hollow fiber membrane. Fibronectin has a net positive charge and adheres to the hollow fiber membrane through polarity and hydrogen bonding. The hollow fiber membrane has a net negative charge at physiological pH, which is about 7.2 or higher to about 7.4 or lower. Furthermore, fibronectin has natural adhesive properties due to its glycoprotein structure and specific domains, which enable it to bind to both the hollow fiber membrane (e.g., polyethersulfone (PES)) and cell membrane integrins. Although fibronectin is described, it should be understood that other extracellular matrix proteins having a net positive charge (such as vitronectin and / or fibrinogen and / or collagen and / or laminin, and their isoforms) may also be used and may be attached to one or more portions or regions of the hollow fiber membrane.

[0057] Referring again to FIG. 5 , fibronectin is contacted with the hollow fiber membrane for a first period of time (311). The first period of time may be from about 4 hours to about 24 hours, and in certain embodiments, optionally, may be about 12 hours. After contacting fibronectin with the hollow fiber membrane in step 311, method 301 further comprises contacting a cytokine with the modified hollow fiber membrane in step 331. The cytokine binds to the fibronectin to prepare the hollow fiber membrane for use in cell selection and / or cell signaling (including differentiation) applications. In at least one exemplary embodiment, for example, if fibronectin is introduced into the inner capillary space of the hollow fiber membrane, contacting step 331 includes introducing a cytokine into the inner capillary space. In at least one exemplary embodiment, for example, if fibronectin is introduced into the extracapillary space of the hollow fiber membrane, contacting step 331 includes introducing a cytokine into the extracapillary space. In at least one example embodiment, for example, when fibronectin is introduced into the intra- and extra-capillary spaces of the hollow fiber membrane, the contacting step 331 includes introducing a cytokine into the intra- and extra-capillary spaces.

[0058] In at least one exemplary embodiment, method 301 includes step 321 of removing excess unbound fibronectin from the hollow fiber membrane prior to step 331 of contacting the cytokine with the modified hollow fiber membrane. For example, excess unbound fibronectin is removed using a washing process. In at least one exemplary embodiment, during the washing process, approximately 450 mL of phosphate buffered saline (PBS) or other buffer solution can be contacted with the hollow fiber membrane to remove unbound fibronectin and / or other molecules (e.g., aptamers) prior to contacting the cytokine with the modified hollow fiber membrane (331).

[0059] 1 , in at least one exemplary embodiment, a fluid inlet passage 44 is fluidly associated with the first fluid circuit 12, and a fluid outlet passage 46 is fluidly associated with the second fluid circuit 14. The fluid inlet passage 44 allows fluid to enter the first fluid circuit 12, while the fluid outlet passage 46 allows fluid to exit the cell growth system 10. In at least one exemplary embodiment, as shown, a third flow controller 48 is operably associated with the fluid inlet passage 44. Although not shown, it should be appreciated that in at least one exemplary embodiment, a fourth flow controller may alternatively or additionally be operably associated with the first outlet passage 46. In at least one exemplary embodiment, the flow controllers (including the first flow controller 30, the second flow controller 32, the third flow controller 48, and / or the fourth flow controller) include pumps, valves, clamps, or any combination thereof. For example, multiple pumps, multiple valves, and multiple clamps may be arranged in any combination. In at least one exemplary embodiment, the flow control device may be or may include a peristaltic pump. The fluid circuits (including first fluid circuit 12 and / or second fluid circuit 14), inlet ports (including fluid inlet port 44), and / or outlet ports (including fluid outlet port 46) may comprise any known tubing material, and any type of fluid, including, for example, buffers, protein-containing fluids, and cell-containing fluids, may flow through the various circuits (including first fluid circuit 12 and / or second fluid circuit 14), inlet channels (including fluid inlet port 44), and outlet channels (including fluid outlet port 46). It should be appreciated that the terms "fluid," "media," and "fluid media" are used interchangeably.

[0060] An exemplary hollow fiber cell growth chamber 100 (also referred to as a "bioreactor") is shown in Figure 2. Hollow fiber cell growth chamber 100 may be used as hollow fiber cell growth chamber 24 in cell growth system 10 shown in Figure 1. Hollow fiber cell growth chamber 100 has a longitudinal axis (represented by line LA-LA) and includes a cell growth chamber housing 104. Cell growth chamber housing 104 has four openings or ports, including, for example, capillary inner inlet port 108, capillary inner outlet port 120, capillary outer inlet port 128, and capillary outer outlet port 132. A first fluid (also referred to as intra-capillary fluid or intra-capillary medium) in a first circuit (such as first fluid circuit 12) enters cell growth chamber 100 through capillary inner inlet port 108 at first fluid manifold end 112 of cell growth chamber 100, enters and passes through the intra-capillary spaces of a plurality of hollow fibers 116, and exits cell growth chamber 100 through capillary inner outlet port 120 located at second fluid manifold end 124 of cell growth chamber 100. The flow path between capillary inner inlet port 108 and capillary inner outlet port 120 constitutes an intra-capillary portion 126 of cell growth chamber 100. A second fluid (also referred to as extra-capillary medium or extra-capillary fluid) in a second circuit (such as second fluid circuit 14) can enter cell growth chamber 100 through capillary outer inlet port 128. This second fluid contacts the extra-capillary space or the outside of hollow fiber membrane 117 and exits cell growth chamber 100 through extra-capillary outlet port 132. The fluid path between extra-capillary inlet port 128 and extra-capillary outlet port 132 constitutes extra-capillary portion 136 of cell growth chamber 100.

[0061] When the second fluid contacts the outside of the hollow fibers 116, small molecules (e.g., ions, water, oxygen, lactate, etc.) can diffuse through the hollow fibers 116 from the interior, i.e., intra-capillary space, of the hollow fibers 116 to the exterior, i.e., extra-capillary space, or from the extra-capillary space to the intra-capillary space. Larger molecular weight molecules (e.g., growth factors and / or proteins) are often too large to pass through the membrane walls of the hollow fibers 116 and remain within the intra-capillary space (or alternatively, or additionally, the extra-capillary space) of the hollow fibers 116. The media comprising the first and second fluids may be exchanged as needed, or alternatively, or additionally, may be circulated through an oxygenator and / or gas transfer module to exchange gases as needed. As described below, cells for growth are contained within first fluid circuit 12 and / or second fluid circuit 14 and enter hollow fiber cell growth chamber 100 at one or both of the intra-capillary space or the extra-capillary space.

[0062] In at least one exemplary embodiment, cells are seeded in the inner capillary space of hollow fibers 116 (e.g., for proliferation, differentiation, and / or harvesting of cord blood-derived CD34+ hematopoietic stem / progenitor cells, monocytes, macrophages, hepatocytes, and / or endothelial cells), while cell culture medium is pumped through the extra-capillary space of hollow fibers 116, with nutrients being delivered to the cells via hollow fiber membrane perfusion during proliferation. However, in at least one other exemplary embodiment, cells for proliferation are seeded in the extra-capillary space, while cell culture medium is pumped through the inner capillary space, with nutrients being delivered to the cells via hollow fiber membrane perfusion during proliferation. In at least one other exemplary embodiment, cells for proliferation are seeded in the inner capillary space, while cell culture medium is pumped through both the extra-capillary and inner capillary spaces. The movement of cell culture medium in the inner and / or outer capillary spaces helps remove excess cells, e.g., cells that are not attached to the surface of the hollow fiber membrane. In at least one exemplary embodiment, the material used to fabricate the hollow fiber membrane 117 can be any biocompatible polymeric material that can be made into hollow fibers 121. For example, synthetic polysulfone-based materials (e.g., polyethersulfone (PES)) are often used to form hollow fibers.

[0063] In at least one exemplary embodiment, the cell growth system 10 includes a device configured to move, or "rock," the cell growth chamber 100 relative to other components of the cell growth system 10. The device may be a rotational and / or lateral rocking device. For example, as shown in FIG. 6, the cell growth chamber (also referred to as a bioreactor) 100 may be rotatably connected to one or more rotational rocking components 138 and lateral rocking components 140. A first rotational rocking component 138 may be rotatably associated with the bioreactor 100. For example, the first rotational rocking component 138 is configured to rotate the bioreactor 100 about a first or central rotational axis 142. In at least one exemplary embodiment, the bioreactor 100 may be rotated in an alternating manner, including, for example, a first direction, a clockwise direction, and then a second direction, a counterclockwise direction, about the central axis 142.

[0064] Although not shown, it should be appreciated that in at least one exemplary embodiment, the second rotational rocking component may be configured to move the bioreactor 100 about a second rotational axis 144 that is perpendicular to the central axis 142 and passes through a center point of the bioreactor 100. In at least one exemplary embodiment, the bioreactor 100 may be rotated in an alternating manner, e.g., a first direction, a clockwise direction, and then a second direction, a counterclockwise direction, about the second rotational axis 144. In at least one exemplary embodiment, the bioreactor 100 may be rotated about the second rotational axis 144 to position it in a horizontal or vertical orientation relative to gravity. The lateral rocking component 140 is laterally associated with the bioreactor 100. For example, the plane of the lateral rocking component 140 may move laterally in the x and y directions.

[0065] Rotation and / or lateral movement of the bioreactor 100 reduces cell settling and the likelihood of cells becoming trapped in certain areas of the bioreactor 100. In at least one exemplary embodiment, the settling rate of cells in the cell growth chamber 100 is proportional to the density difference between the cells and the suspension medium, according to Stokes' equation. In at least one exemplary embodiment, repeated 180-degree rotations (fast) with pauses (e.g., a total pause and rotation time of 30 seconds), as described above, help maintain suspension of non-adherent cells (e.g., T-cells). A minimum rotation of approximately 180 degrees is preferred, but various rotation angles, including up to 360 degrees or more, can be used. Different rocking components may be used separately or in combination. For example, a rocking component that rotates the bioreactor 100 about central axis 142 can be combined with a rocking component that rotates the bioreactor 100 about axis 144. Similarly, clockwise and counterclockwise rotations about different axes can be independently combined.

[0066] In at least one exemplary embodiment, as shown in Figure 7, a cell growth system 200 (similar to cell growth system 10 shown in Figure 1) may include a pre-mounted fluid transfer assembly 210. For example, cell growth system 200 includes a cell growth apparatus 202 having a rear portion 206 and a hatch or closeable door 204 that engages with rear portion 206. An interior space 208 of cell growth apparatus 202 is configured to receive pre-mounted fluid transfer assembly 210. Pre-mounted fluid transfer assembly 210 may be removably attached to cell growth apparatus 202, thus allowing for relatively quick and easy replacement of a new or unused pre-mounted fluid transfer assembly 210. For example, in operation of one cell growth apparatus 202, a first set of cells can be grown or expanded using a first pre-mounted fluid transfer assembly 210, and then a second set of cells can be grown or expanded using a second pre-mounted fluid transfer assembly 210 without the need for sterilization when replacing the first pre-mounted fluid transfer assembly 210 with the second pre-mounted fluid transfer assembly 210. In each variation, the pre-mounted fluid transfer assembly 210 includes a bioreactor (such as the bioreactor 100 illustrated in FIG. 2) and an oxygenator or gas transfer module 212. The cell growth system 200 includes a plurality of tube guide slots 214 disposed in fluid communication with the pre-mounted fluid transfer assemblies 210 for receiving various media.

[0067] Figure 8 is a view of the rear portion 206 of the cell growth device 202 prior to the removably mounting of the pre-mounted fluid transfer assembly 210. The closable door 204 is omitted from Figure 8. As shown, the rear portion 206 of the cell growth device 202 includes several different components that operate in conjunction with the components of the pre-mounted fluid transfer assembly 210. For example, the rear portion 206 of the cell growth device 202 includes several peristaltic pumps (inner capillary circulation pump 218, outer capillary circulation pump 220, inner capillary inlet pump 222, and / or outer capillary inlet pump 224) that cooperate with pump loops in the pre-mounted fluid transfer assembly 210. The rear section 206 also includes a plurality of valves (e.g., capillary inner circulation valve 226, reagent valve 228, capillary inner medium valve 230, air removal valve 232, cell inlet valve 234, wash valve 236, dispense valve 238, capillary outer medium valve 240, capillary inner waste valve 242, capillary outer waste valve 244, and / or harvest valve 246). Additionally, a plurality of sensors (e.g., capillary inner outlet pressure sensor 248, combined capillary inner inlet pressure / temperature sensor 250, combined capillary outer inlet pressure / temperature sensor 252, and / or capillary outer outlet pressure sensor 254) are associated with the rear section 206 of the cell growth device 202. In at least one exemplary embodiment, an optical sensor 256 for the air removal chamber may also be located in the rear section 206.

[0068] The rear section 206 may also include a shaft or swing control 258 for rotating the bioreactor 100. The shaft mount 260 is associated with the shaft or swing control 258 to ensure proper alignment of the shaft access opening 424 of the tubing housing 300 of a pre-mounted fluid transfer assembly (e.g., pre-mounted fluid transfer assembly 400) relative to the rear section 206 of the cell growth device 202. Rotation of the shaft or swing control 258 imparts rotational motion to the shaft mount 260 and the bioreactor 100. Thus, when an operator or user of the cell growth system 200 installs a new or unused pre-mounted fluid transfer assembly 400 on the cell growth device 202, alignment is a simple matter of properly orienting the shaft access opening 424 of the pre-mounted fluid transfer assembly 400 relative to the shaft mount 260.

[0069] 9 is a perspective view of a removable pre-mounted fluid transfer assembly 400. The pre-mounted fluid transfer assembly 400 can be removably attached to the cell growth apparatus 202, allowing for quick and easy replacement of the cell growth apparatus 202 with a new or unused pre-mounted fluid transfer assembly 400. The bioreactor 100 is attached to a bioreactor coupling that includes a shaft mount 402. The shaft mount 402 has one or more shaft fastening mechanisms (biased arms or spring members 404) for engaging the shaft 258 of the cell growth apparatus 202.

[0070] The pre-mounted fluid transfer assembly 400 includes multiple tubes (e.g., tubes 408A, 408B, 408C, 408D, 408E, etc.) and various tube fittings that form the fluid paths shown in Figures 8 and 9, as described below. Pump loops 406A and 406B are also provided. While various media can be provided to the cell growth device 202, in certain variations, the pre-mounted fluid transfer assembly 400 includes sufficient tubing to extend outside the cell growth device 202 to allow for tubing associated with media bags or media containers to be welded together.

[0071] FIG. 10 is a schematic diagram of an exemplary cell growth system 500, similar to cell growth system 100 shown in FIG. 1, illustrating exemplary fluid paths. As shown, cell growth system 500 includes a first fluid circuit 502 (also referred to as an "inner capillary loop" or "inner capillary space" or "IC loop") and a second fluid circuit 504 (also referred to as an "outer capillary loop" or "extra capillary space" or "EC loop"). In at least one exemplary embodiment, cells are disposed within inner capillary space 502, while cell culture medium is pumped in extra capillary space 504 to deliver nutrients to the cells via hollow fiber membrane perfusion during growth. However, it should be appreciated that in at least one other exemplary embodiment, cells may be disposed within extra capillary space 504, while cell culture medium is pumped in inner capillary space 502 to deliver nutrients to the cells via hollow fiber membrane perfusion during growth. In at least one other exemplary embodiment, cells are disposed in the inner capillary space 502, while cell culture medium is pumped in both the outer capillary space 504 and the inner capillary space 502. In at least one other exemplary embodiment, cells are disposed in the outer capillary space 504, while cell culture medium is pumped in both the outer capillary space 504 and the inner capillary space 502.

[0072] First fluid flow path 506 is fluidly associated with cell growth chamber (also called a "bioreactor") 501 to form first fluid circuit 502. Cell growth chamber 501 may be used as hollow fiber cell growth chamber 24 shown in FIG. 1 and / or hollow fiber cell growth chamber 100 shown in FIG. 2. A first fluid enters cell growth chamber 501 through capillary inner inlet port 501A (which may be used as an outlet in the reverse direction). In a method of preparing hollow fiber membranes for cell growth of natural killer cells and other similar cells, a fibronectin-streptavidin complex enters capillary inner inlet port 501A through first fluid flow path 506 and is introduced into the hollow fiber membrane.

[0073] The first fluid exits the cell growth chamber through the capillary inner outlet port 501B (which can also be used as an inlet in the reverse direction). For example, upon entering the capillary inner space 502, the fibronectin of the fibronectin-streptavidin complex can contact and bind (e.g., coat) the inner-facing surface of the hollow fiber membrane, and excess unbound complex protein can be removed from the capillary inner space 502 through the capillary inner outlet port 501B. After a period of time, biotinylated molecules are introduced into the hollow fiber membrane through the capillary inner inlet port 501A via the first fluid flow path 506. While in the capillary inner space 502, the biotinylated molecules can bind to the fibronectin-streptavidin complex, more specifically, the streptavidin, to form a biotinylated fibronectin-streptavidin complex ready for use in cell selection and / or cell signaling (including differentiation) applications. In at least one exemplary embodiment, a wash process can be used to flow wash medium through the first fluid flow path 506 into the capillary inner inlet port 501A, through the hollow fiber membrane, and out the capillary inner outlet port 501B.

[0074] In at least one exemplary embodiment, the first fluid circuit 502 is provided with a pressure gauge 510 configured to measure the pressure of the first fluid exiting the cell growth chamber 501. In at least one exemplary embodiment, the first fluid circuit 502 is provided with an inner capillary circulation pump 512 configured to control the flow rate of the first fluid. For example, the inner capillary circulation pump 512 is configured to pump the fluid in a first direction or a second direction opposite the first direction. In the latter case, the inner capillary outlet port 501B is used as an inlet and the inner capillary inlet port 501A is used as an outlet. In at least one exemplary embodiment, the first fluid circuit 502 includes 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 circuit 502 is provided with a pressure / 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 can enter the capillary inner loop 502 via valve 514. In at least one exemplary embodiment, a portion of the cells are flushed from the capillary inner loop 502, for example, via valve 598, into a harvest bag 599. It should be understood that in at least one other exemplary embodiment, the first fluid circuit 502 can include more or fewer valves, manometers, pressure sensors, temperature sensors, ports, and / or other devices positioned to isolate the first fluid along portions of the capillary inner loop 502 and / or measure properties of the first fluid at portions thereof.

[0075] A second fluid can enter cell growth chamber 501 through capillary outer inlet port 501C. The second fluid can exit cell growth chamber 501 via capillary outer outlet port 501D. In at least one exemplary embodiment, the second fluid in capillary outer loop 504 contacts the exterior-facing surfaces of hollow fibers disposed within cell growth chamber 501, thereby allowing diffusion of small molecules into and out of the hollow fibers. In at least one exemplary embodiment, capillary outer loop 504 is provided with a pressure / thermometer 524 configured to measure the pressure and / or temperature of the second fluid before it enters cell growth chamber 501. In at least one exemplary embodiment, capillary outer loop 504 is provided with a pressure gauge 526 configured to measure the pressure of the second fluid, for example, as it exits cell growth chamber 501. In at least one exemplary embodiment, the capillary outer loop 504 is provided with a sample port 530 configured for the extraction of a second fluid sample.

[0076] In at least one exemplary embodiment, the capillary outer loop 504 is provided with a capillary outer circulation pump 528 and an oxygenator or gas transfer module 532. For example, after exiting the cell growth chamber 501, the second fluid passes through the capillary outer circulation pump 528 to and through the oxygenator or gas transfer module 532. In at least one exemplary embodiment, the capillary outer circulation pump 528 is configured to control the flow rate of the second fluid. For example, the capillary outer circulation pump 528, similar to the capillary inner circulation pump 512, is configured to pump the second fluid in a first direction or a second direction opposite the first direction. In the latter case, the capillary outer outlet port 501D is used as an inlet and the capillary outer inlet port 501C is used as an outlet.

[0077] In at least one exemplary embodiment, the second fluid flow path 522 is fluidly associated with an oxygenator or gas transfer module 532 via an oxygenator inlet port 534 and an oxygenator outlet port 536. For example, the second fluid flows into the oxygenator or gas transfer module 532 via the oxygenator inlet port 534 and leaves or flows out of the oxygenator or gas transfer module 532 via the oxygenator outlet port 536. In at least one exemplary embodiment, the oxygenator or gas transfer module 532 is configured to add oxygen to the second fluid and / or remove air bubbles from the second fluid. For example, air and / or gas flows into the oxygenator or gas transfer module 532 via a first filter 538 and leaves or flows out of the oxygenator or gas transfer module 532 (i.e., flows out) through a second filter 540. The first and second filters 538, 540 are configured to reduce or prevent contaminants from entering the oxygenator or gas transfer module 532. The second fluid in second fluid circuit 504 is in equilibrium with gas entering oxygenator or gas transfer module 532. In at least one exemplary embodiment, air and / or gas is purged from cell growth system 500, for example, during a priming sequence, and the air and / or gas is vented to atmosphere via oxygenator or gas transfer module 532. It should be understood that in at least one other exemplary embodiment, second fluid circuit 504 can include more or fewer valves, manometers, pressure sensors, temperature sensors, ports, and / or other devices positioned to isolate the second fluid and / or measure properties of the second fluid along portions of capillary outer loop 504.

[0078] In at least one exemplary embodiment, an air removal chamber (ARC) 556 is fluidly associated with the first fluid circuit 502. The air removal chamber 556 may include one or more ultrasonic sensors. For example, the air removal chamber 556 may include an upper sensor and / or a lower sensor configured to detect a lack of air and / or fluid and / or a gas-fluid interface at a specific measurement location within the air removal chamber 556. The upper sensor may be located near a first end (e.g., the top) of the air removal chamber 556. The lower sensor may be located near a second end (e.g., the bottom) of the air removal chamber 556. While an ultrasonic sensor is described, it should be understood that the air removal chamber 556 may additionally or alternatively include one or more other sensors, including, for example, an optical sensor. Air and / or gas purged from the cell growth system 500 during parts of the priming step and / or other protocols can be vented to the atmosphere through a line 558 fluidly associated with the air removal chamber 556 and through an air valve 560.

[0079] In at least one exemplary embodiment, the first fluid includes, for example, cells from a first fluid container (also referred to as a first medium bag or first bag) 562 and fluid medium (e.g., inner capillary medium or fluid) from a second fluid container (also referred to as a second medium bag or second bag) 546. Material (i.e., cells and / or inner capillary medium) from the first and second fluid containers 562, 546 can enter the first fluid circuit 502 via a first fluid flow path 506. The first fluid container 562 is fluidly associated with the first fluid flow path 506 and the first fluid circuit 502 via a valve 564. In at least one exemplary embodiment, second fluid container 546 and third fluid container (also referred to as third media bag or third bag) 544 are associated with first fluid inlet line 542, e.g., via valves 548 and 550, respectively, or are fluidly associated with second fluid inlet line 574, e.g., via valves 570 and 576, respectively. In at least one exemplary embodiment, material from second fluid container 546 and / or third fluid container 544 may be in fluid communication with first sterile sealable input priming line 508 and / or second sterile sealable input priming line 509.

[0080] In at least one exemplary embodiment, a fourth fluid container (also referred to as a fourth medium bag or fourth bag) 568 contains extra-capillary medium, and a fifth fluid container (also referred to as a fifth medium bag or fifth bag) 566 contains a wash solution. Materials (i.e., extra-capillary medium and / or wash solution) from the fourth and fifth fluid containers 568, 566 can enter the first fluid circuit 502 and / or the second fluid circuit 504. For example, in at least one exemplary embodiment, the fifth fluid container 566 is fluidly associated with a valve 570. The valve 570 is fluidly associated with the first fluid circuit 502, for example, via a distribution valve 572 and the first fluid inlet path 542. In at least one exemplary embodiment, the fifth fluid container 566 can be fluidly associated with the second fluid circuit 504 via the second fluid inlet passage 574 and the capillary outer inlet passage 584, for example, by opening valve 570 and closing distribution valve 572. The fourth fluid container 568 is fluidly associated with a valve 576. The valve 576 is fluidly associated with the first fluid circuit 502, for example, via the first fluid inlet passage 542 and distribution valve 572. In at least one exemplary embodiment, the fourth fluid container 568 can be fluidly associated with the second fluid inlet passage 574 by opening valve 576 and closing distribution valve 572. In at least one exemplary embodiment, the first fluid inlet passage 542 and / or the second fluid inlet passage 574 may optionally be fluidly associated with the heat exchanger 552.

[0081] In at least one exemplary embodiment, fluid is transferred from first fluid inlet passage 542 and / or second fluid inlet passage 574 to inner capillary loop 502 via inner capillary inlet pump 554, and fluid is transferred to outer capillary loop 504 via outer capillary inlet pump 578. In at least one exemplary embodiment, air detector 580 may also be associated with outer capillary inlet passage 584. Air detector 580 may include, for example, an ultrasonic sensor. In at least one exemplary embodiment, first fluid circuit 502 and second fluid circuit 504 may be fluidly associated with waste line 588. For example, when valve 590 is in an open state or position, inner capillary medium flows through waste line 588 to waste bag (also referred to as outlet bag) 586. When valve 582 is open, outer capillary medium flows through waste line 588 to waste bag 586. In at least one exemplary embodiment, cells are harvested, for example, via cell harvest channel 596. For example, cells can be harvested from cell growth chamber 501 by pumping the cell-containing inner capillary medium through cell harvest channel 596 and valve 598 into cell harvest bag 599.

[0082] In at least one exemplary embodiment, as shown, fluids in the first fluid circuit 502 and the second fluid circuit 504 flow in the same direction through the cell growth chamber 501 (i.e., a co-current configuration). Although not shown, it should be understood that in other exemplary embodiments, the cell growth system 500 may be configured to flow in a counter-current configuration. As shown in FIG. 10 , fluid in the first fluid circuit 502 enters the bioreactor 501 at a capillary inner inlet port 501A and leaves or exits the bioreactor 501 at a capillary inner outlet port 501B. In at least one exemplary embodiment, the first fluid flow path 506 may be fluidly connected to the first fluid circuit 502, for example, via a connection 517. The connection 517 may be a point or location where fluids flow in opposite directions, based, for example, on the direction and flow rate of the capillary inner inlet pump 554 and the direction and flow rate of the fluid circulation pump 512. Connection 517 may be any type of fitting, coupling, weld, passage, tubing, etc. that allows first fluid flow path 506 to be fluidly associated with first fluid circuit 502. In at least one exemplary embodiment, connection 517 may include a T-joint or T-junction and / or a Y-joint or Y-junction.

[0083] In at least one exemplary embodiment, the system includes one or more measuring instruments (e.g., pressure gauge 510, pressure / thermometer 520, pressure / thermometer 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 inner inlet port 501A, capillary inner outlet port 501B, capillary outer inlet port 501C, capillary outer outlet port 501D, sample port 516, sample port 518, sample port 519, sample port 520, sample port 521, sample port 522, sample port 523, sample port 524, sample port 525, sample port 526, sample port 527, sample port 528, sample port 529, sample port 530, sample port 531, sample port 532, sample port 533, sample port 534, sample port 535, sample port 536, sample port 537, sample port 538, sample port 539, sample port 540, sample port 541, sample port 542, sample port 543, sample port 544, sample port 545, sample port 546, sample port 547, sample port 548, sample port 550, sample port 551, sample port 552, sample port 553, sample port 554, sample port 555, sample port 556, sample port 557, sample port 558, sample port 559, sample port 560, sample port 561 30, oxygenator inlet port 534, and / or oxygenator outlet port 536), one or more pumps (e.g., inner capillary circulation pump 512, outer capillary circulation pump 528, inner capillary inlet pump 554, and / or outer capillary inlet pump 578), one or more filters (e.g., first filter 538 and / or second filter 540), one or more coils (e.g., sample coil 518), one or more modules (e.g., oxygenator or gas transfer module 532), and / or one or more other components of cell growth system 500 may be in electrical communication with a control system (not shown). The control system may include multiple nodes, which may include various hardware, firmware, and / or software (e.g., including controllers and memory) configured to control and / or communicate with mechanical, electromechanical, and electrical components of cell growth system 500.

[0084] The controller (also called a processor) may be any type of microcontroller, microprocessor, field programmable gate array (FPGA), application specific integrated circuit (ASIC), etc. An example of a controller may be the NK10DN512VOK10 microcontroller manufactured and sold by N9P USA, Incorporated, which is a microcontroller unit with a 32-bit architecture. Other examples of controllers include the Qualcomm® Snapdragon® 800 and 801, the Qualcomm® Snapdragon® 610 and 615 with 4G LTE Integration and 64-bit computing, the Apple® A7 processor with 64-bit architecture, the Apple® M7 motion coprocessor, the Samsung® Exynos® series, the Intel® Core™ processor family, the Intel® Xeon® processor family, the Intel® Atom™ processor family, the Intel Itanium® processor family, the Intel® Core™ i5-4670K and i7-4770K 22nm Haswell, the Intel® Core™ i5-3570K 22nm IvyBridge, the AMD® FX™ processor family, the AMD® FX-4300, FX-6300, and FX-8350 32nm It may include at least one of a Vishera, AMD® Kaveri processor, ARM® Cortex™-M processor, ARM® Cortex-A and ARM926EJ-S™ processor, other industry equivalent processor, and may perform computer functions using any known or future-developed standard instruction set, library, and / or architecture.The memory may be any type of memory including random access memory (RAM), read-only memory (ROM), electrically erasable programmable ROM (EEPROM), portable compact disc read-only memory (CD-ROM), optical storage, magnetic storage, any suitable combination of these, or any other type of storage or memory device that stores and provides instructions for programming and controlling the controller.

[0085] FIG. 11 is a schematic diagram of an exemplary cell growth system 600, similar to cell growth system 100 shown in FIG. 1, illustrating exemplary fluid paths. As shown, cell growth system 600 includes a first fluid circuit 602 (also referred to as an "inner capillary loop" or "inner capillary space" or "IC loop") and a second fluid circuit 604 (also referred to as an "outer capillary loop" or "extra capillary space" or "EC loop"). In at least one exemplary embodiment, cells are disposed within inner capillary space 602, while cell culture medium is pumped in extra capillary space 604 to deliver nutrients to the cells via hollow fiber membrane perfusion during growth. However, it should be appreciated that in at least one other exemplary embodiment, cells may be disposed within extra capillary space 604, while cell culture medium may be pumped in inner capillary space 602 to deliver nutrients to the cells via hollow fiber membrane perfusion during growth. In at least one other exemplary embodiment, cells are disposed in the inner capillary space 602, while cell culture medium is pumped in both the outer capillary space 604 and the inner capillary space 602. In at least one other exemplary embodiment, cells are disposed in the outer capillary space 604, while cell culture medium is pumped in both the outer capillary space 604 and the inner capillary space 602.

[0086] First fluid flow path 606 is fluidly associated with cell growth chamber (also referred to as a "bioreactor") 601 to form first fluid circuit 602. Cell growth chamber 601 may be used as hollow fiber cell growth chamber 24 of cell growth system 10 shown in FIG. 1 and / or hollow fiber cell growth chamber 100 shown in FIG. 1. A first fluid enters cell growth chamber 601 through capillary inner inlet port 601A. In a method of preparing hollow fiber membranes for cell growth of natural killer cells and / or other similar cells, a fibronectin-streptavidin complex is introduced into the hollow fiber membrane through first fluid flow path 606, into capillary inner inlet port 601A, and into the hollow fiber membrane.

[0087] Fluid exits cell growth chamber 601 through capillary inner outlet port 601B (which can also be used as an inlet in the reverse direction). For example, upon entering capillary inner space 602, the fibronectin of the fibronectin-streptavidin complex can contact and bind (e.g., coat) the inner-facing surface of the hollow fiber membrane, and excess unbound complex protein can be removed from the capillary inner space through capillary inner outlet port 601B. After a period of time, biotinylated molecules are introduced into the hollow fiber membrane through first fluid flow channel 606 via capillary inner inlet port 601A. While in capillary inner space 602, the biotinylated molecules can bind to fibronectin-streptavidin complexes, more specifically, streptavidin, to form biotinylated fibronectin-streptavidin complexes ready for use in cell selection and / or cell signaling (including differentiation) applications. In at least one exemplary embodiment, a wash process can be used to flow wash medium through the first fluid flow path 606 into the capillary inner inlet port 601A, through the hollow fiber membrane, and out the capillary inner outlet port 601B.

[0088] In at least one exemplary embodiment, first fluid circuit 602 includes 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 both the pressure and the temperature of the medium exiting cell growth chamber 601. In at least one exemplary embodiment, the medium flows through an inner capillary circulation pump 612 configured to control the flow rate of the medium flow. The inner capillary circulation pump 612 is configured to pump the fluid in a first direction or in a second direction opposite the first direction.

[0089] In at least one exemplary embodiment, the (first) culture medium can enter capillary inner loop 602 through valve 614. In at least one exemplary embodiment, a sample of the culture medium is taken from sample coil 618 during operation. The culture medium is then returned to capillary inner inlet port 601A, completing fluid circuit 602. In at least one exemplary embodiment, cells grown / expanded in cell growth chamber 601 are flushed out of cell growth chamber 601 and into harvest bag 699 via valve 698 and line 697. Alternatively, if valve 698 is closed, the cells can be redistributed back into chamber 601 for further growth.

[0090] In second fluid circuit 604, fluid enters cell growth chamber 601 via capillary outer inlet port 601C and leaves cell growth chamber 601 via capillary outer outlet port 601D. In at least one exemplary embodiment, in capillary outer loop 604, the (second) culture medium contacts the outside of the hollow fibers in cell growth chamber 601, thereby allowing diffusion of small molecules present in chamber 601 into and out of the hollow fibers.

[0091] In at least one exemplary embodiment, second fluid circuit 604 includes sensor 624. In at least one exemplary embodiment, sensor 624 is configured to measure the pressure of the culture medium before it enters the extra-capillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 624 is configured to measure the temperature of the culture medium before it enters the extra-capillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 624 is configured to measure the pressure and temperature of the culture medium before it enters the extra-capillary space of cell growth chamber 601.

[0092] In at least one exemplary embodiment, second fluid circuit 604 includes sensor 626. In at least one exemplary embodiment, sensor 626 is configured to measure the pressure of the culture medium after it exits the extra-capillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 626 is configured to measure the temperature of the culture medium after it exits the extra-capillary space of cell growth chamber 601. In at least one exemplary embodiment, sensor 626 is configured to measure both the pressure and the temperature of the culture medium after it exits the extra-capillary space of cell growth chamber 601.

[0093] After fluid in the second fluid circuit 604 exits the capillary outer outlet port 601D of the cell growth chamber 601, it travels through the capillary outer circulation pump 628 to the oxygenator or gas transfer module 632. The capillary outer circulation pump 628 is also capable of pumping fluid in both directions. The second fluid flow path 622 is fluidly associated with the oxygenator or gas transfer module 632 via the inlet port 632A and outlet port 632B of the oxygenator or gas transfer module 632. During operation, the fluid medium enters the oxygenator or gas transfer module 632 via the inlet port 632A and exits the oxygenator or gas transfer module 632 via the outlet port 632B. The oxygenator or gas transfer module 632 supplies oxygen to the medium in the cell growth system 600 and removes air bubbles from the medium. In at least one exemplary embodiment, the medium in second fluid circuit 604 is in equilibrium with gas entering oxygenator or gas transfer module 632. Oxygenator or gas transfer module 632 can be any suitably sized device useful for oxygenation or gas transfer. Air or gas enters oxygenator or gas transfer module 632 through filter 638 and exits oxygenator or gas transfer module 632 through filter 640. Filters 638, 640 reduce or prevent contamination of oxygenator or gas transfer module 632 and associated medium. Air or gas purged from cell growth system 600 during part of the priming step can be vented to atmosphere via oxygenator or gas transfer module 632.

[0094] The illustrated configuration of cell growth system 600 shows the fluid media in first fluid circuit 602 and second fluid circuit 604 flow in the same direction through cell growth chamber 601 (e.g., a co-current configuration). However, it should be understood that in at least one exemplary embodiment, cell growth system 600 may be configured to flow in a counter-current configuration.

[0095] In at least one exemplary embodiment, for example, media containing cells from a source (such as a cell container, e.g., a bag) is attached to attachment point 662, and fluid media from a media source is attached to attachment point 646. The cells and media are introduced into first fluid circuit 602 via first fluid flow path 606. Attachment point 662 is fluidly associated with first fluid flow path 606 via valve 664. Attachment point 646 is fluidly associated with first fluid flow path 606 via valve 650. A reagent source may be fluidly connected to point 644 and associated with first fluid inlet path 642 via valve 648, or with second fluid inlet path 674 via valves 648, 672.

[0096] An air removal chamber (ARC) 656 is fluidly associated with the first fluid circuit 602. In at least one exemplary embodiment, the air removal chamber 656 may include one or more sensors, including upper and lower sensors for detecting air, a lack of fluid, and / or a gas / fluid boundary (e.g., an air / fluid boundary) at specific measurement points within the air removal chamber 656. For example, ultrasonic sensors may be used near the bottom and / or top of the air removal chamber 656 to detect air, fluid, and / or an air / fluid boundary at those locations. It should be understood that numerous other types of sensors may be incorporated into the cell growth system 600 without departing from the spirit and scope of the present disclosure. For example, in at least one exemplary embodiment, optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from the cell growth system 600 during portions of the priming process or other protocols may be vented to the atmosphere through a line 658 fluidly associated with the air removal chamber 656 and through an air valve 660.

[0097] An extra-capillary medium source is attached to the extra-capillary medium attachment point 668. And / or a wash fluid source is attached to the wash fluid attachment point 666. This allows extra-capillary medium and / or wash fluid to be added to the first or second fluid flow path. The attachment point 666 is fluidly associated with a valve 670. The valve 670 is fluidly associated with the first fluid circuit 602 via a valve 672 and a first fluid inlet path 642. Also, by opening the valve 670 and closing the valve 672, the attachment point 666 can be fluidly associated with the second fluid circuit 604 via a second fluid inlet path 674 and a second fluid flow path 684. Similarly, the attachment point 668 is fluidly associated with a valve 676. The valve 676 is fluidly associated with the first fluid circuit 602 via the first fluid inlet path 642 and a valve 672. Additionally, fluid container 668 can be fluidly associated with second fluid inlet passage 674 by opening valve 676 and closing dispensing valve 672 .

[0098] In the inner capillary loop, fluid is first pumped by the inner capillary inlet pump 654. In the outer capillary loop, fluid is first pumped by the outer capillary inlet pump 678. An air detector 680, such as an ultrasonic sensor, may be associated with the outer capillary inlet channel 684.

[0099] In at least one embodiment, the first and second fluid circuits 602, 604 are connected to a waste line 688. When valve 690 is opened, the inside capillary medium flows through the waste line 688 to the waste or outlet bag 686. Similarly, when valve 692 is opened, the outside capillary medium flows to the waste or outlet bag 686.

[0100] After the cells have grown in the cell growth chamber 601, the cells are harvested via cell harvest line 697. In this case, cells from the cell growth chamber 601 are harvested into a cell harvest bag 699 via cell harvest line 697 by pumping the cell-containing media inside the capillary with valve 698 open.

[0101] In at least one exemplary embodiment, the various components of cell growth system 600 are housed or contained within a device or housing, such as cell growth device 202, which maintains the cells and culture medium, for example, at a predetermined temperature. In at least one exemplary embodiment, the components of cell growth system 600 and cell growth system 500 may be combined. In at least one exemplary embodiment, the cell growth system may include fewer or additional components than those shown in Figures 5 and 6 and still be within the scope of the present disclosure.

[0102] FIG. 12 illustrates an exemplary process 800 for growing cells for use in a cell growth system, such as cell growth system 10 shown in FIG. 1 , cell growth system 200 shown in FIG. 7 , cell growth system 500 shown in FIG. 10 , and / or cell growth system 600 shown in FIG. 11 . The process begins at step 802. Process 800 includes steps 804 of loading a disposable tubing set into the cell growth system and 806 of priming the system. In at least one exemplary embodiment, a user or operator instructs the system to prime by selecting a task for priming. In at least one exemplary embodiment, the task for priming is a pre-programmed task. Process 800 then proceeds to step 808 of coating a bioreactor. This step is optional, but in this step, the bioreactor is coated with a coating agent. Coating step 808 is shown with a dashed line to indicate that this step is optional, depending on the cell type being grown, operator preference, other considerations, or factors. If process 800 includes coating step 808, reagent may be introduced into the inner capillary loop until the reagent container is empty. Reagent is flowed from the air removal chamber into the inner capillary loop, and the reagent then circulates within the inner capillary loop. In at least one exemplary embodiment, a coating reagent containing fibronectin is used. Once the bioreactor is coated in step 808 (or following priming step 806 if the bioreactor is not coated), process 800 proceeds to inner capillary / outer capillary washing step 810, where the fluid in the inner capillary circulation loop and the outer capillary circulation loop are replaced. The replacement volume is determined by the number of inner capillary volumes and outer capillary volumes being exchanged.

[0103] To maintain the appropriate or desired gas concentration across the hollow fibers of the bioreactor membrane, a medium conditioning step 812 is performed to allow the medium to reach equilibrium with the gas supply before cells are added to the bioreactor. For example, a high outer capillary circulation flow rate can be used to rapidly achieve contact between the medium and the gas supply provided by a gas transfer module or oxygenator. The system is then maintained in the appropriate or desired state until the user or operator is ready to add cells to the bioreactor. In at least one exemplary embodiment, the system may be conditioned with complete medium. The complete medium may be any medium source used for cell growth. In at least one exemplary embodiment, the complete medium includes, for example, alpha MEM (α-MEM) and / or fetal bovine serum (FBS).

[0104] Process 800 includes step 814 of introducing cells (e.g., natural killer cells) into a bioreactor, for example, through a cell inlet bag. In at least one exemplary embodiment, the cells are introduced into the bioreactor from the cell inlet bag until the bag is empty. The cells are then pumped through the air removal chamber into the bioreactor. In embodiments utilizing a larger pumping volume, the cells spread and migrate toward the inner capillary outlet. In at least one exemplary embodiment, cell distribution is facilitated across the membrane via inner capillary circulation, such as by an inner capillary circulation pump without an inner capillary input.

[0105] In other embodiments, a "Central Loading of Cells Without Circulation" step 814 can be used, in which a first volume of fluid containing a plurality of cells is loaded at a first flow rate into a cell growth system, where the cell growth system includes a cell growth chamber. A second volume of fluid containing medium is then loaded at a second flow rate into a portion of the first fluid circuit, e.g., disposing 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 is a generally central region of the bioreactor. In at least one exemplary embodiment, the first volume is the same as the second volume. In at least one exemplary embodiment, the first flow rate is the same as the second flow rate. In at least one exemplary embodiment, the first volume is 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 is equal to a percentage or fraction of the volume (e.g., total volume) of the first fluid circuit. For example, the sum of the first volume and the second volume is, for example, about 50% of the volume (e.g., total volume) of the first fluid circuit. In at least one exemplary embodiment, the fluid in the first fluid circuit flows through an inner capillary space of a bioreactor or cell growth chamber. In at least one exemplary embodiment, the fluid in the second fluid circuit flows through, for example, an outer capillary space of 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 inner capillary loop, or some other percentage or fraction. In at least one exemplary embodiment, the sum of the first volume and the second volume, if applicable, may be about 50% or some other percentage or fraction of the volume of other fluid paths, other loops, etc. Other percentages or fractions may also be used, such as any percentage between about 1% and about 100%.

[0106] Following cell input step 814, process 800 proceeds to cell feeding step 816. In step 818, the cells are expanded and grown. While step 818 is shown after step 816, it should be appreciated that in at least one exemplary embodiment, step 818 may occur before or simultaneously with step 816. Process 800 then proceeds to query step 820 to determine whether cell colonies, microcolonies, or clusters have formed. A cell colony, microcolony, or cluster is a group of one or more adherent cells. If a cell colony, microcolony, or cluster has formed, process 800 proceeds to step 822, where the cell colony, microcolony, or cluster is sheared. For example, after expanding a plurality of cells for a first period of time, the cells are circulated at a first circulation flow rate for a second period of time to reduce the number of cells in the cell colony, microcolony, or cluster. In at least one exemplary embodiment, circulating the cells at a first circulation flow rate creates shear stress on the cell colony, causing one or more cells within the cell colony to split off from the cell colony. In at least one exemplary embodiment, for example, reducing the number of cells within a cell colony, microcolony, or cluster can provide a suspension of single cells. In at least one exemplary embodiment, step 822 of circulating the cells to shear colonies, microcolonies, or clusters can be used during cell culture, for example, every two days, to maintain uniform cell density and nutrient diffusion. In at least one exemplary embodiment, such shearing of any microcolony, colony, or cluster can begin, for example, on day four or later. According to other exemplary embodiments, such shearing can be initiated and performed on other days or time periods. After shearing step 822, process 800 then returns to cell feeding step 816.

[0107] If query step 820 determines not to shear cell colonies or clusters, or if, for example, there are no sheared colonies or clusters, process 800 proceeds to a "no" and resuspends the cells in step 824. In at least one exemplary embodiment, cell circulation is performed to uniformly resuspend cells that may be loosely attached during culture. In at least one exemplary embodiment, step 824 includes circulating the cells to uniformly resuspend cells that may be loosely attached before initiating a harvesting task or other task to remove the cells from the bioreactor. Following cell resuspension step 824, process 800 then proceeds to cell harvesting step 826. Further processing or other analysis of the removed cells may optionally be performed in step 828, and process 800 ends in end step 830. If no further processing / analysis is desired, process 800 ends in end step 830.

[0108] It should be understood that, according to embodiments of the present disclosure, the operational steps depicted in the above figures are for illustrative purposes only and may be rearranged, combined with, or performed in parallel with other steps. In embodiments, steps may be reduced or additional steps may be added without departing from the spirit and scope of the present disclosure. Additionally, steps (and substeps), such as priming, coating the bioreactor, and loading cells, may, in some embodiments, be performed automatically, e.g., by a processor executing pre-programmed tasks stored in memory. Such steps are presented herein for illustrative purposes only.

[0109] 13 illustrates an example of components of a computing system 2500 for implementing embodiments of the present disclosure. Computing system 2500 may be used, for example, in embodiments in which a cell growth system (such as cell growth system 10 shown in FIG. 1 , and / or cell growth system 200 shown in FIG. 7 , and / or cell growth system 500 shown in FIG. 10 , and / or cell growth system 600 shown in FIG. 11 ) employs a processor to execute tasks (such as custom tasks or pre-programmed tasks) performed as part of a process (such as a process shown and / or described herein). In variations, pre-programmed tasks may include, for example, "membrane preparation," "IC / EC wash," and / or "cell feeding."

[0110] As shown, computing system 2500 includes a user interface 2502, a processing system 2504, and / or a storage device 2506. User interface 2502 includes output device(s) 2508 and / or input device(s) 2510. Output device(s) 2508 may include one or more touchscreens. The touchscreen may have a display area for providing one or more application windows. The touchscreen may also be an input device 2510 capable of receiving and / or capturing physical touch from a user or operator, for example. The touchscreen may be a liquid crystal display (LCD) with a capacitive structure that allows processing system 2504 to estimate the location of the touch. In this case, processing system 2504 can map the location of the touch to a UI element displayed at a predetermined location in the application window. The touchscreen may also be configured to receive touch events via one or more other electronic structures in accordance with the present invention. Other output devices 2508 may include a printer, a speaker, etc. The other input devices 2510 include a keyboard, other touch input devices, a mouse, a voice input device, and the like.

[0111] 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 for independently loading and executing instructions. The processor may include a general-purpose processor, an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), other integrated circuits, etc.

[0112] In embodiments of the present invention, memory 2514 may include any storage device for short-term or long-term storage of data and / or processor-executable instructions. Memory 1014 may include, for example, random access memory (RAM), read-only memory (ROM), or electrically erasable programmable read-only memory (EEPROM). Other storage media may include, for example, CD-ROM, tape, digital versatile disk (DVD), or other optical storage devices, tape, magnetic disk storage devices, magnetic tape, or other magnetic storage devices.

[0113] Storage device 2506 is any long-term data storage device or component. In embodiments of the invention, storage device 2506 may include one or more of the systems described in connection with memory 2514. Storage device 2506 may be permanent or removable. Storage device 2506 is configured to store data generated by or provided to processing system 2504.

[0114] Features of the present technology are further illustrated in the following non-limiting examples.

[0115] Example 1 Streptavidin coating preparation

[0116] In a specific variation, streptavidin coating is prepared by contacting streptavidin with water (HO) to form a 10 μg / mL solution. The solution (e.g., 100 μL) is added to the well and allowed to dry. After a certain period of time, a washing process is applied. For example, 0.1% BSA is used for washing, and the hollow fibers are blocked at ambient or room temperature (e.g., about 20°C to about 22°C) for about 1 hour to prepare the hollow fibers.

[0117] In one variation, the streptavidin coating process involves coating a polyethersulfone (PES) membrane with a phosphate-buffered saline (PBS)-fibronectin solution overnight at 37°C on day -3 by introducing the solution at 0.1 mL / min to 20 mL / min and circulating it through the capillary inner loop of a Quantum CES or similar platform at about 0 mL / min to about 20 mL / min. This may be followed by a phosphate-buffered saline (PBS) wash step to remove unbound fibronectin, for example, using the Quantum CES IC Rapid Washout Task or IC / EC Exchange Task. On day -2, after the wash step, a phosphate-buffered saline (PBS)-streptavidin solution may be added, which adheres to the basal fibronectin layer and is circulated overnight in a similar manner, followed by the Quantum Washout Task. On day -1, a phosphate-buffered saline (PBS)-biotinylated IL-21 cytokine solution is added and circulated through the inner capillary loop at 37°C in a similar manner, followed by a Quantum Washout Task before cell seeding.

[0118] Example 2 Bioreactor / Column Coating

[0119] Several approaches exist for creating fibronectin (FN)-streptavidin (SN) substrata for attaching biotinylated molecules to functionalize the surface of Quantum® System polyethersulfone (PES) hollow fiber membrane (HFM) bioreactors or preparatory columns for cell selection. For example, in one variation, fibronectin is bound to the polyethersulfone hollow fiber membrane in the Quantum® Cell Expansion System bioreactor, mediated by the attachment and proliferation of adherent cells such as mesenchymal stromal / stem cells (MSCs), fibroblasts, and / or aortic endothelial cells. This process is based on the established high affinity of streptavidin binding to biotin. Given the biochemical nature of available protein conjugations, it can be important to keep protocols straightforward and efficient, minimizing residual materials and reactants, so that they can be adapted for the production of cell therapy products. Mixing and / or binding of fibronectin-streptavidin mixtures or complexes supports functionalization of hollow fiber membrane bioreactors or columns with biotinylated cytokines, chemokines, and other ligands to promote cell selection and proliferation. Other affinity separations of biomolecules are also anticipated. In either case, this protein-protein complex can be considered a platform for affinity processes related to cell therapy using available technologies.

[0120] Two exemplary approaches are described: (1) simple mixing of fibronectin and streptavidin, and (2) rapid covalent conjugation of fibronectin and streptavidin using a modified Bio-Rad LYNX kit. As background, native and recombinant human dimeric fibronectin has a molecular weight of approximately 440 kDa to approximately 500 kDa, and tetrameric streptavidin has a molecular weight of approximately 53 kDa to approximately 55 kDa. In protein-protein conjugation components, the mass ratio of reactants is adjusted to optimize their molar ratio to maintain their functionality in cell selection and proliferation. For example, the Bio-Rad LYNX kit is designed to conjugate streptavidin and an IgG class monoclonal antibody (mAb) at a mass ratio of approximately 1:1. The Bio-Rad LYNX kit reagents combine fibronectin and streptavidin at a different mass ratio of about 1:3.3, achieving an effective molar ratio of about 1:3 upon subsequent reaction at room temperature for a period of about 3 hours to about 15 hours (see, e.g., Table 1).

[0121] [Table 1]

[0122] Option 1: Coating with a mixture of fibronectin and streptavidin

[0123] This process involves reconstituting lyophilized fibronectin and streptavidin (FN+SN) (e.g., 1:3.3 by mass) with deionized water (DI H2O) at ambient temperature for approximately 30 minutes. After fibronectin-streptavidin binding, the volume of the mixture is reduced to 0.1% by volume with Ca. 2+ -Mg 2+The resulting fibronectin-streptavidin bioconjugate protein is ready for use in cell selection or cell signaling (including differentiation) applications. Other applications include coating pre-tube hollow fiber membrane columns or matrices used for cell selection or differentiation prior to introducing cells into the Quantum® System. The exact ratio of fibronectin to streptavidin and the method of conjugation may be modified in further development. For example, recombinant or semisynthetic fibronectin or fibrinogen may be used in place of plasma-derived fibronectin. Extracellular matrix proteins such as fibronectin bind to the polyethersulfone hollow fiber membrane of the Quantum® System bioreactor through polarity and hydrogen bonding. Studies of the SurPASS binding layer by Anton Paar have shown that the Quantum® System polyethersulfone membrane has a net negative charge under physiological pH conditions of 7.2 to 7.4. Fibronectin has a "net positive" charge due to the presence of positively charged amino acid residues, such as lysine. Furthermore, fibronectin possesses natural adhesive properties due to its glycoprotein structure and specific domains that allow it to bind to both polyethersulfone and cell membrane integrins.

[0124] Option 2: Rapid covalent coating of fibronectin and streptavidin using a modified LYNX kit

[0125] Another example is the covalent conjugation of fibrinogen to streptavidin using a mass ratio similar to that outlined in "Option 1," performed using a modified commercially available Bio-Rad LYNX Rapid Streptavidin Conjugation Kit. This kit uses proprietary binding modifiers and quencher substances (LNK161STR, LNK162STR, LNK163STR) to generate covalent bonds between fibronectin and streptavidin over a period of approximately 3 hours to approximately 15 hours. In the covalent coating method, the affinity of the selected biotinylated molecule for streptavidin is similar to that of the biotinylated molecule in the fibrinogen-streptavidin mixture coating method. The advantage of the covalent conjugation approach is the improved stability of the fibrinogen-streptavidin bond.

[0126] The foregoing description of the embodiments has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the present disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment and, even if not specifically shown or described, may be interchangeable and used in selected embodiments, where applicable. The same may be modified in many ways. Such modifications should not be considered a departure from the present disclosure, and all such variations are intended to be included within the scope of the present disclosure.

Claims

1. 1. A method for functionalizing a hollow fiber membrane for cell growth of a target cell, the method comprising: contacting a surface of the hollow fiber membrane containing extracellular matrix components with a biotinylated molecule that binds to the extracellular matrix components and has affinity for the target cells; method.

2. 10. The method of claim 1, The biotinylated molecule is selected from the group consisting of a cytokine, an epitope, a ligand, a monoclonal antibody, a stain, an aptamer, and combinations thereof. method.

3. 3. The method of claim 2, The cytokines include interleukin-21. method.

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

5. 10. The method of claim 1, the extracellular matrix component comprises an extracellular matrix component-streptavidin complex, the extracellular matrix component of the extracellular matrix component-streptavidin complex binds to the surface of the hollow fiber membrane, and the streptavidin of the extracellular matrix component-streptavidin complex binds to the biotinylated molecule; method.

6. 6. The method of claim 5, In the extracellular matrix component-streptavidin complex, the mass ratio of the extracellular matrix component to the streptavidin is about 1:3 or more and about 1:9 or less. method.

7. 6. The method of claim 5, The extracellular matrix component-streptavidin complex comprises a fibronectin-streptavidin complex, wherein the fibronectin in the fibronectin-streptavidin complex has a molecular weight of about 440 kDa or more to about 500 kDa or less, and the streptavidin in the fibronectin-streptavidin complex has a molecular weight of about 53 kDa or more to about 55 kDa or less. method.

8. 8. The method of claim 7, The method further comprises the step of preparing the fibronectin-streptavidin complex. method.

9. 9. The method of claim 8, the step of preparing the fibronectin-streptavidin complex comprises reconstituting the lyophilized fibronectin with the streptavidin by immersing the lyophilized fibronectin and streptavidin in water; method.

10. 9. The method of claim 8, The step of preparing the fibronectin-streptavidin complex includes a step of covalently binding the fibronectin and the streptavidin. method.

11. 10. The method of claim 1, The method further comprises contacting the extracellular matrix component with the surface of the hollow fiber membrane. method.

12. 12. The method of claim 11, contacting the extracellular matrix component with the surface of the hollow fiber membrane for a period of about 4 hours or more to about 24 hours or less prior to the step of contacting the biotinylated molecule; method.

13. 13. The method of claim 12, After the period of time and before the step of contacting the biotinylated molecule, the method further comprises the step of washing the hollow fiber membrane to remove unreacted and excess extracellular matrix components. method.

14. 10. The method of claim 1, The target cells include natural killer cells. method.

15. 10. The method of claim 1, the surface is an inward-facing surface; method.

16. 10. The method of claim 1, the surface is an outward-facing surface or a combination of an inward-facing surface and the outward-facing surface; method.

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

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

19. 18. The method of claim 17, the extracellular matrix component-streptavidin complex comprises a fibronectin-streptavidin complex; The method further comprises preparing the fibronectin-streptavidin complex; The step of preparing the fibronectin-streptavidin complex comprises reconstituting the freeze-dried fibronectin with streptavidin by immersing the freeze-dried fibronectin and streptavidin in water, or covalently binding the fibronectin and streptavidin. method.

20. 18. The method of claim 17, contacting the extracellular matrix component-streptavidin complex with the hollow fiber membrane for a period of about 4 hours or more to about 24 hours or less prior to the step of contacting the biotinylated molecule; The method further comprises a step of washing the hollow fiber membrane to remove unreacted and excess extracellular matrix component-streptavidin complexes prior to the step of contacting the biotinylated molecule. method.

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