Method for proliferating cells

Abstract

Embodiments are described that relate to methods and systems for expanding cells in hollow fiber bioreactors. In embodiments, the cells are exposed to multiple growth factors, including combinations of recombinant growth factors. In other embodiments, the cells are grown in co-culture with other cells, e.g., hMSCs. In embodiments, the cells comprise CD34+ cells. The coated membrane comprises a membrane having a first coating configured to promote cell attachment to the membrane and a second coating comprising a soluble protein moiety.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit and priority of the following U.S. Provisional Patent Applications under 35 U.S.C. § 119(e): U.S. Provisional Patent Application No. 63 / 165,060, filed on March 23, 2021, with the title "Cell Proliferation"; U.S. Provisional Patent Application No. 63 / 169,173, filed on March 31, 2021, with the title "Cell Proliferation"; U.S. Provisional Patent Application No. 63 / 183,591, filed on May 3, 2021, with the title "Cell Proliferation"; U.S. Provisional Patent Application No. 63 / 227,293, filed on July 29, 2021, with the title "Cell Proliferation"; U.S. Provisional Patent Application No. 63 / 228,561, filed on August 2, 2021, with the title "Cell Proliferation"; U.S. Provisional Patent Application No. 63 / 275,389, filed on November 3, 2021, with the title "Methods and Systems for Separating Target Cells Using a Multipart Membrane Substrate"; U.S. Provisional Patent Application No. 63 / 275,793, filed on November 4, 2021, with the title "Methods and Systems for Separating Target Cells Using a Multipart Membrane Substrate"; U.S. Provisional Patent Application No. 63 / 304,467, filed on January 28, 2022, with the title "Methods and Systems for Separating Target Cells Using a Multipart Membrane Substrate". The entire disclosures of the above applications are hereby incorporated by reference into this application.

[0002] The present disclosure generally relates to separating and growing live cells, and more particularly to separating target cells using a membrane and growing the separated cells.

Background Art

[0003] The cell processing system includes a cell collection system and a cell expansion system (CES). The cell collection system collects cells from a source, and the CES is used to expand and differentiate various cell types. The expanded and / or differentiated cells can be used for both research and / or therapeutic purposes. As an example, hematopoietic stem cells (HSCs) are pluripotent, self-renewing, and capable of producing mature blood cells such as red blood cells, white blood cells, platelets, and lymphocytes. CD34 is a marker for human HSCs, and all colony-forming activity of human bone marrow (BM) cells is found in the fraction of cells that express CD34 (i.e., the "CD34+ HSCs" or "CD34+ cells" or "CD34+ fraction"). HSCs can be collected from bone marrow, umbilical cord blood, or peripheral blood, and CD34+ HSCs are recognized as a potential treatment for diseases such as blood cancers (e.g., lymphoma, leukemia, myeloma). Umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable CD34+ HSCs.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Effective treatment or transplantation with CD34+ HSCs requires a minimum dose of HSCs. Therefore, after separating CD34+ HSCs from a suitable source such as CB, the CD34+ HSCs must be grown (i.e., "expanded") from an initial amount to an amount that is considered to be at least effective for treatment or transplantation.

Means for Solving the Problems

[0005] The present disclosure provides procedures, devices, and compositions useful in the separation, expansion, and administration of CD34+ HSCs.

[0006] This section is for the purpose of explaining the aspects of the present invention in a simplified form and is not intended to identify important or essential elements of the present invention, nor is it intended to limit the scope of the claims.

[0007] The present disclosure provides a cell capture and expansion system, and a method for expanding target cells that can be collected from a mixed cell population. Examples include membranes useful for capturing, collecting, and / or retaining target cells, particularly CD34+ HSCs. Using the methods of the present disclosure, HSCs are collected and rapidly and efficiently expanded significantly while minimizing or eliminating HSC differentiation. In the systems and methods of the present disclosure, HSCs are expanded at least 50-fold. The cells may be target cells collected from a donor fluid (e.g., one or more blood components). These target cells include, but are not limited to, stem cells, CD34+ HSCs, T cells, natural killer (NK) cells, monocytes, etc. The membrane may comprise one or more layers or coatings (i.e., membranes) configured to attract and collect target cells. The membrane may include a substrate that promotes cell adhesion to at least one of its own surfaces. The substrate has a first surface and a second surface, and may further have at least one coating on the first surface and / or the second surface. The at least one coating may correspond to any molecule or material that promotes cell adhesion to the first surface and / or the second surface of the substrate. The at least one coating may include a first coating material and a second coating material. The first coating material may be fibronectin or a fibronectin equivalent, and the second coating material may be a soluble protein moiety. The second coating material may target specific target cells from a mixed cell population. For example, the second coating material may be a chemokine such as stromal cell-derived factor-1 (SDF-1) used to enhance the collection power of CD34+ HSCs. Additional coating materials may be used to collect the same or different cells from a mixed cell population. The membrane can be provided in any form, such as a flat sheet, filter matrix, hollow fiber, any combination thereof, and / or some of them.

[0008] The present disclosure also provides methods for growing cells, particularly CD34+ HSCs, in a bioreactor such as a hollow fiber bioreactor (hollow fiber type bioreactor). These methods provide for introducing cells (e.g., hematopoietic stem cells (HSCs), e.g., including CD34+ HSCs) into the bioreactor and exposing the cells to growth conditions for growing a plurality of cells in the bioreactor. The growth conditions may include introducing one or a combination of growth factors into the bioreactor. Alternatively or additionally, the growth conditions may include the presence of co-culture cells in the bioreactor. After growing the cells in the bioreactor, the plurality of expanded cells may then be removed from the bioreactor for use in storage, transplantation, or treatment such as cancer treatment.

[0009] The present disclosure provides a method of growing cells having the step of introducing a plurality of cells including CD34+ hematopoietic stem cells (HSCs) into the hollow fibers of a hollow fiber bioreactor. Each of the hollow fibers of the bioreactor includes an inner lumen portion and an outer lumen portion. Further, the hollow fiber has a coating on at least one of the inner lumen portion surface and the outer lumen portion surface. The coating on the surface includes stromal cell-derived factor-1 (SDF-1) and fibronectin or an isoform, or functional equivalents thereof. In these methods, the plurality of cells in the hollow fiber are exposed to growth conditions and at least a portion of the plurality of cells are grown in the hollow fibers of the bioreactor to produce a plurality of expanded CD34+ HSCs. Using these methods, the plurality of cells introduced into the hollow fibers of the bioreactor are expanded at least 50-fold.

[0010] The present disclosure also provides methods for growing cells by perfusion in a cell growth system. These methods include coating a hollow fiber bioreactor with a first fluid containing a signaling factor and / or a coating factor. In these methods, a plurality of cells are introduced into the hollow fiber membrane of the hollow fiber bioreactor. In these methods, the plurality of cells within the hollow fiber membrane may be exposed to a second fluid containing a plurality of growth factors. In these methods, the plurality of cells in the hollow fiber bioreactor may be grown in a single culture or a co-culture.

[0011] The present disclosure also provides methods for capturing cells, including introducing a mixture of target cells and non-target cells into the hollow fibers of a hollow fiber bioreactor. Each of these hollow fibers includes an inner lumen portion and an outer lumen portion, and the hollow fiber further has a coating on at least one of the inner lumen surface and the outer lumen surface of the hollow fiber. The coating on the surface includes stromal cell-derived factor-1 (SDF-1) and fibronectin or an isoform, or a functional equivalent thereof. In these methods, the mixture of target cells and non-target cells in the hollow fiber is exposed to capture conditions to capture at least a portion of the target cells on at least one of the inner lumen surface and the outer lumen surface of the hollow fiber. At least a portion of the non-target cells can be washed out of the hollow fiber, leaving the target cells bound to the surface of the hollow fiber.

[0012] The present disclosure also provides methods for capturing a target species. In these methods, a mixture of a target species and a non-target species is introduced into a hollow fiber having an inner lumen portion and an outer lumen portion. Further, the hollow fiber may have a coating on at least one of the inner lumen surface and the outer lumen surface of the hollow fiber. The coating may include at least one of streptavidin, avidin, a biotinylated molecule, and an anti-biotin antibody or a functional fragment thereof. In these methods, the mixture of the target species and the non-target species in the hollow fiber is exposed to capture conditions to capture at least a portion of the target species on at least one of the inner lumen surface and the outer lumen surface of the hollow fiber. In these methods, at least a portion of the non-target species may be washed out of the hollow fiber.

[0013] The present disclosure also provides a coated hollow fiber membrane. These membranes are hollow fiber membranes having an inner capillary surface and an outer capillary surface. These membranes may include a coating on at least one of the inner capillary surface and the outer capillary surface of the hollow fiber. The coating may include stromal cell-derived factor-1 (SDF-1) and fibronectin or isoforms, or functional equivalents thereof.

[0014] The present disclosure also provides a method of forming a coated hollow fiber membrane. The method includes providing a hollow fiber membrane having an inner capillary surface and an outer capillary surface, and applying a first coating on the inner capillary surface of the hollow fiber membrane. In these methods, the first coating includes a material that promotes cell adhesion to at least one of the inner capillary surface of the hollow fiber membrane and the outer capillary surface of the hollow fiber membrane. In these methods, a second coating may be applied on the inner capillary surface of the hollow fiber membrane. The second coating may include a soluble protein moiety.

[0015] The present disclosure also provides a composition useful in the proliferation of CD34+ HSCs. These compositions include glial cell line-derived neurotrophic factor (GDNF) and an aryl hydrocarbon receptor (AHR) antagonist.

[0016] The foregoing is intended to provide a simplified overview of some aspects of the present disclosure. This overview is neither an extensive nor an exhaustive overview of the present disclosure and its various aspects, implementations, and configurations. It is not intended to identify the main or important elements of the present disclosure, nor to delineate the scope of the present disclosure. Instead, it is intended to present selected concepts of the present disclosure in a simplified form as an introduction to the more detailed description presented below. As will be understood, other aspects, implementations, and configurations of the present disclosure are possible using one or more of the features described above or detailed below, alone or in combination, and will be apparent to those skilled in the art in view of the following detailed description and with reference to the drawings.

[0017] The accompanying drawings are incorporated herein and constitute a part of this specification to illustrate some examples of the present disclosure. These drawings, together with the detailed description, explain the principles of the present disclosure. The drawings merely show preferred examples and other possible examples of how the present disclosure can be made and used, and should not be construed as limiting the present disclosure to only the illustrated and described examples.

Brief Description of the Drawings

[0018]

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[0019] The principle of the present invention will be further understood by referring to the following detailed description and the embodiments shown in the accompanying drawings. Although specific features are shown and described in the detailed embodiments, it should be understood that the present invention is not limited to the embodiments described below.

[0020] In the following, the embodiments shown in the accompanying drawings will be referred to and described in detail. As much as possible, the same reference numerals are used for the same or similar parts in the drawings and the following description.

[0021] FIG. 1 shows a front view of an example of a hollow fiber bioreactor 100 used with the present invention. The hollow fiber bioreactor 100 has a longitudinal axis LA-LA and includes a chamber housing 104. In at least one embodiment, the chamber housing 104 has four openings or four ports, namely, an in-capillary (IC) inlet port 108, an IC outlet port 120, an out-capillary (EC) inlet port 128, and an EC outlet port 132.

[0022] In an embodiment of the present disclosure, the fluid in the first circulation path enters the hollow fiber bioreactor 100 through the IC inlet port 108 at the first longitudinal end 112 of the hollow fiber bioreactor 100, enters and passes through the inside of the fine tubes of the plurality of hollow fibers 116 (in various embodiments, referred to as the lumen of the hollow fiber membrane, the inner side of the capillary (''IC''), or ''IC space''), and exits the hollow fiber bioreactor 100 through the IC outlet port 120 located at the second longitudinal end 124 of the hollow fiber bioreactor 100. The fluid path between the IC inlet port 108 and the IC outlet port 120 defines the IC portion 126 of the hollow fiber bioreactor 100. The fluid in the second circulation path enters the hollow fiber bioreactor 100 through the EC inlet port 128, contacts the outside or the outer side of the fine tubes of the hollow fibers 116 (referred to as the ''EC side'' or ''EC space'' of the membrane), and exits the hollow fiber bioreactor 100 through the EC outlet port 132. The fluid path between the EC inlet port 128 and the EC outlet port 132 constitutes the EC portion 136 of the hollow fiber bioreactor 100. The fluid entering the hollow fiber bioreactor 100 through the EC inlet port 128 contacts the outside of the hollow fibers 116. Small molecules (e.g., ions, water, oxygen, lactate) can diffuse through the hollow fibers 116 from the inside of the hollow fibers, i.e., the IC space, to the outside, i.e., the EC space, or from the EC space to the IC space. High molecular weight molecules such as growth factors are too large to pass through the hollow fibers and remain within the IC space of the hollow fibers 116. In an embodiment, the medium may be exchanged and replenished if necessary. Also, if necessary, the medium may be circulated through an oxygen supply device or a gas transfer module to exchange gases (see, for example, the cell growth system 500 (FIG. 5) and the cell growth system 600 (FIG. 6)). Cells can be contained in the first circulation path and / or the second circulation path and can be present on the IC side and / or the EC side of the membrane as described below.

[0023] The material used to fabricate the hollow fiber membrane may be any biocompatible polymer material that can be processed into hollow fibers and has appropriate permeability to small molecules such as ions, water, oxygen, glucose, lactic acid, etc. In embodiments of the present disclosure, one material that can be used is a synthetic polysulfone-based material. To attach cells to the surface of the hollow fiber, the surface may be modified by methods such as coating at least the cell growth surface with a protein (such as a glycoprotein like fibronectin or collagen) or irradiating the surface with radiation. By gamma-ray treating the membrane surface, it becomes possible to attach adherent cells without performing additional coating with fibronectin or the like on the membrane. In embodiments of the present disclosure, other coatings and / or other treatments for cell attachment may be used.

[0024] In FIG. 2, an embodiment of a cell growth system 200 having a pre-provided fluid transport assembly according to an embodiment of the present disclosure is shown. The CES 200 has a cell growth device 202. The cell growth device 202 has a hatch or an openable and closable door 204 that engages with a rear portion 206 of the cell growth device 202. The internal space 208 within the cell growth device 202 has features such that it can receive, engage, and fix a pre-provided fluid transport assembly 210 that includes a bioreactor 100. The pre-provided fluid transport assembly 210 is removably attached to the cell growth device 202, and in the cell growth device 202, the used fluid transport assembly 210 can be replaced relatively quickly with a new or unused fluid transport assembly 210. By the operation of a single cell growth device 202, a first set of cells can be grown or proliferated using the first fluid transport assembly 210, and then, without the need to sterilize during the replacement of the first fluid transport assembly 210 with the second fluid transport assembly 210, a second set of cells can be grown or proliferated using the second fluid transport assembly 210. The pre-provided fluid transport assembly has a bioreactor 100 and an oxygen supply or gas transfer module 212. In an embodiment, a piping guide slot 214 for receiving various medium pipes connected to the fluid transport assembly 210 is shown.

[0025] Figure 3 shows the rear portion 206 of the cell growth device 202 before removably attaching a pre-provided fluid transport assembly 210 (see FIG. 2) according to an embodiment of the present disclosure. The openable and closable door 204 (see FIG. 2) is omitted in FIG. 3. The rear portion 206 of the cell growth device 202 is provided with a plurality of different structures that operate in combination with the components of the fluid transport assembly 210. Specifically, the rear portion 206 of the cell growth device 202 has a plurality of peristaltic pumps (IC circulation pump 218, EC circulation pump 220, IC inlet pump 222, EC inlet pump 224) that cooperate with the pump loop in the fluid transport assembly 210. Also, the rear portion 206 of the cell growth device 202 has a plurality of valves (IC circulation valve 226, reagent valve 228, IC medium valve 230, air removal valve 232, cell inlet valve 234, washing valve 236, distribution valve 238, EC medium valve 240, IC waste valve 242, EC waste valve 244, harvest valve 246). Further, a plurality of sensors (IC outlet pressure sensor 248, IC inlet pressure / temperature sensor 250, EC inlet pressure / temperature sensor 252, EC outlet pressure sensor 254) are associated with the rear portion 206 of the cell growth device 202. Further, an optical sensor 256 for an air removal chamber is shown.

[0026] An axis or rocker control unit 258 for rotating the bioreactor 100 according to an embodiment is shown in FIG. 3. By an axis fitting portion 260 associated with the axis or rocker control unit 258, proper alignment of the axis access opening (for example, the opening 424 (see FIG. 4) of the pipe storage portion 300 (see FIG. 4)) of the fluid transport assembly 210 or the fluid transport assembly 400 can be performed with respect to the rear portion 206 of the cell growth device 202. By rotating the axis or rocker control unit 258, a rotational movement is imparted to the axis fitting portion 260 and the bioreactor 100. Therefore, when an operator or user of the CES200 attaches a new or unused fluid transport assembly 400 (see FIG. 4) to the cell growth device 202, the alignment becomes a relatively simple operation such as properly orienting the axis access opening 424 (see FIG. 4) of the fluid transport assembly 400 with respect to the axis fitting portion 260.

[0027] FIG. 4 is a perspective view of a removably pre-attached fluid transport assembly 400. The pre-attached fluid transport assembly 400 is removably attached to the cell growth device 202 such that in the cell growth device 202, the used fluid transport assembly 400 can be replaced relatively quickly with a new or unused fluid transport assembly 400. As shown in FIG. 4, the bioreactor 100 is attached to a bioreactor coupling including a shaft fitting portion 402. The shaft fitting portion 402 has one or more shaft fastening mechanisms (such as a biased arm or spring member 404) for engaging the shaft (such as shaft 258 shown in FIG. 3) of the cell growth device 202.

[0028] In an embodiment, a mechanism of a cell growth system for rotating the bioreactor 100 is connected to the shaft fitting portion 402 and the spring member 404. For example, in some embodiments, the cell growth system may be part of a rotatable QUANTUM® cell expansion system (CES) (manufactured by Terumo BCT, Lakewood, Colo.). Examples of cell growth systems for rotating a bioreactor are described at least in U.S. Patent No. 8,399,245, issued March 19, 2013 (Title: Rotation System for a Cell Growth Chamber of a Cell Expansion System and Method of Use Thereof), U.S. Patent No. 8,809,043, issued February 13, 2013 (Title: Rotation System for a Cell Growth Chamber of a Cell Expansion System and Method of Use Thereof), and U.S. Patent No. 9,057,045, issued June 16, 2015 (Title: Method of Loading and Dispersing Cells in a Bioreactor of a Cell Expansion System). The entire disclosures of the above U.S. patent applications are hereby expressly incorporated by reference into the present application.

[0029] According to the embodiment, the fluid transport assembly 400 has pipes 408A, 408B, 408C, 408D, 408E and various pipe joints. Thereby, as described below, fluid paths as shown in FIGS. 5 and 6 are provided. The pump loops 406A, 406B are also provided for the pumps. Although various culture media may be provided at the location where the cell growth device 202 is disposed, in the embodiment, the pre-mount type fluid transport assembly 400 has pipes long enough to extend outside the cell growth device 202, so that pipes associated with the culture medium bag may be joined to the pipes.

[0030] FIG. 5 is a schematic diagram of a cell growth system 500 in one embodiment. FIG. 6 shows a schematic diagram of a cell growth system 600 in another embodiment. In the embodiments shown in FIGS. 5 and 6, as described below, cells are grown in the IC space. In other embodiments, cells are grown in the EC space. In still other embodiments, in the case of co-culturing cells, the first cells may be grown in the EC space and the second cells may be grown in the IC space. Co-culture of cells may be performed by growing the first cells and the second cells in the EC space, or by growing the first cells and the second cells in the IC space.

[0031] FIG. 5 shows the CES500 according to an embodiment. The CES500 has a first fluid circulation path 502 (also referred to as a “capillary inner loop” or “IC loop”) and a second fluid circulation path 504 (also referred to as a “capillary outer loop” or “EC loop”). The first fluid flow path 506 is fluidly associated with the hollow fiber bioreactor 501 and at least partially constitutes the first fluid circulation path 502. Fluid flows into the hollow fiber bioreactor 501 through the IC inlet port 501A, passes through the hollow fibers in the hollow fiber bioreactor 501, and flows out through the IC outlet port 501B. The pressure measuring device 510 measures the pressure of the medium leaving the hollow fiber bioreactor 501. The medium flows through the IC circulation pump 512 used to control the medium flow rate / circulation flow rate. The IC circulation pump 512 can pump the fluid in a first direction (e.g., clockwise) or a second direction opposite to the first direction (e.g., counterclockwise). The outlet port 501B may be used as an inlet in the opposite direction. The medium flowing into the IC loop 502 flows in through the valve 514. As can be understood by those skilled in the art, by arranging additional valves and / or other devices at various positions, the medium can also be isolated and / or the characteristics of the medium can be measured along a specific portion of the fluid path. Therefore, the schematic diagram shown shows one possible configuration for a plurality of elements of the CES500, and it should be understood that the schematic diagram can be deformed within the scope of one or more embodiments.

[0032] For the IC loop 502, a sample of the medium is obtained from the sample port 516 or the sample coil 518 during operation. The pressure and temperature of the medium can be measured during operation by the pressure / temperature measuring device 520 disposed in the first fluid circulation path 502. Then, the medium returns to the IC inlet port 501A to complete the circulation in the fluid circulation path 502. The cells grown / proliferated in the hollow fiber bioreactor 501 flow out of the hollow fiber bioreactor 501, enter the harvest bag 599 through the valve 598, or are redistributed within the hollow fibers and further grown.

[0033] In the second fluid circulation path 504, the fluid enters the hollow fiber bioreactor 501 through the EC inlet port 501C and leaves the hollow fiber bioreactor 501 through the EC outlet port 501D. In the EC loop 504, the culture medium contacts the outside of the hollow fibers of the hollow fiber bioreactor 501, thereby enabling the diffusion of small molecules into and out of the hollow fibers.

[0034] Before the culture medium enters the EC space of the hollow fiber bioreactor 501, the pressure and temperature of the culture medium can be measured by the pressure / temperature measuring device 524 disposed in the second fluid circulation path 504. After the culture medium leaves the hollow fiber bioreactor 501, the pressure of the culture medium in the second fluid circulation path 504 can be measured by the pressure measuring device 526. For the EC loop, a sample of the culture medium can be obtained from the sample port 530 or the sample coil during operation.

[0035] In an embodiment, after leaving the EC outlet port 501D of the hollow fiber bioreactor 501, the fluid in the second fluid circulation path 504 reaches the oxygen supplier or gas transfer module 532 through the EC circulation pump 528. The EC circulation pump 528 can pump fluid in both directions. The second fluid flow path 522 is fluidly associated with the oxygen supplier or gas transfer module 532 via the oxygen supplier inlet port 534 and the oxygen supplier outlet port 536. During operation, the fluid medium flows into the oxygen supplier or gas transfer module 532 through the oxygen supplier inlet port 534 and flows out of the oxygen supplier or gas transfer module 532 through the oxygen supplier outlet port 536. The oxygen supplier or gas transfer module 532 adds oxygen to the medium in the CES500 and removes both carbon dioxide and bubbles. In various embodiments, the medium in the second fluid circulation path 504 is in equilibrium with the gas entering the oxygen supplier or gas transfer module 532. The oxygen supplier or gas transfer module 532 can be any oxygen supplier or gas transfer device of appropriate size. Air or gas flows into the oxygen supplier or gas transfer module 532 through the filter 538 and flows out of the oxygen supplier or gas transfer device 532 through the filter 540. The filters 538, 540 reduce or prevent contamination of the oxygen supplier or gas transfer module 532 and the associated medium. The air or gas purged from the CES500 when part of the priming step is being performed can be vented to the atmosphere through the oxygen supplier or gas transfer module 532.

[0036] In the configuration shown for the CES500, the fluid media in the first fluid circulation path 502 and the second fluid circulation path 504 flow through the hollow fiber bioreactor 501 in the same direction (in a co-current configuration). The CES500 may be configured to flow in a counter-current manner.

[0037] In at least one embodiment, the medium containing cells (from bag 562) and the fluid medium from bag 546 are introduced into the first fluid circulation path 502 via the first fluid flow path 506. The fluid container 562 (e.g., a cell input bag or a saline priming fluid for priming air out of the system) is fluidly associated with the first fluid flow path 506 and the first fluid circulation path 502 via valve 564.

[0038] The fluid container, i.e., the medium bag 544 (e.g., a reagent), is fluidly associated with the first fluid inlet path 542 via valve 548, the fluid container, i.e., the medium bag 546 (e.g., an IC medium), is fluidly associated with the first fluid inlet path 542 via valve 550, or bags 544, 546 are fluidly associated with the second fluid inlet path 574 via valves 548, 550, 570. Also, sterilizable and sealable first and second input priming paths 508, 509 are provided. An air removal chamber (ARC) 556 is fluidly associated with the first circulation path 502. The air removal chamber 556 may have one or more ultrasonic sensors. The sensors include an upper sensor and a lower sensor for detecting air, fluid deficiency, and / or gas / fluid boundaries, e.g., an air / fluid boundary, at specific measurement points within the air removal chamber 556. For example, near the bottom and / or near the top of the air removal chamber 556, ultrasonic sensors can be used to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors can be used without departing from the scope of the present disclosure. For example, according to embodiments of the present disclosure, optical sensors may be used. The air or gas purged from the CES 500 during part of the priming step or other protocols can be vented to the atmosphere from the air valve 560 through line 558 fluidly associated with the air removal chamber 556.

[0039] The EC medium (from bag 568) or the washing solution (from bag 566) is added to the first fluid flow path or the second fluid flow path. The fluid container 566 is fluidly associated with the valve 570. The valve 570 is fluidly associated with the first fluid circulation path 502 via the distribution valve 572 and the first fluid inlet path 542. Also, by opening the valve 570 and closing the distribution valve 572, the fluid container 566 can be fluidly associated with the second fluid circulation path 504 via the second fluid inlet path 574 and the EC inlet path 584. Similarly, the fluid container 568 is fluidly associated with the valve 576. The valve 576 is fluidly associated with the first fluid circulation path 502 via the first fluid inlet path 542 and the distribution valve 572. Also, by opening the valve 576 and closing the distribution valve 572, the fluid container 568 can be fluidly associated with the second fluid inlet path 574. Optionally, a heat exchanger 552 may be provided for introducing the medium reagent or the washing solution.

[0040] In the IC loop, the fluid is first sent by the IC inlet pump 554. In the EC loop, the fluid is first sent by the EC inlet pump 578. An air detector 580, such as an ultrasonic sensor, may be associated with the EC inlet path 584.

[0041] In at least one embodiment, the first and second fluid circulation paths 502, 504 are connected to the waste line 588. When the valve 590 is opened, the IC medium reaches the waste bag, i.e., the outlet bag 586, via the waste line 588. Similarly, when the valve 582 is opened, the EC medium flows to the waste bag, i.e., the outlet bag 586, via the waste line 588.

[0042] In an embodiment, the cells are harvested via the cell harvest path 596. The cells from the hollow fiber bioreactor 501 are harvested into the cell harvest bag 599 via the cell harvest path 596 and the valve 598 by pumping the IC medium containing the cells.

[0043] Each component of the CES500 is housed within a device or housing such as the cell growth device 202 (Figs. 2 and 3), which maintains the cells and media at a predetermined temperature.

[0044] FIG. 6 is a schematic diagram of another embodiment of a cell growth system 600. The CES600 has a first fluid circulation path 602 (also referred to as the “inner capillary loop” or “IC loop”) and a second fluid circulation path 604 (also referred to as the “outer capillary loop” or “EC loop”). The first fluid flow path 606 is fluidly associated with the hollow fiber bioreactor 601 to constitute the first fluid circulation path 602. Fluid flows into the hollow fiber bioreactor 601 through the IC inlet port 601A, passes through the hollow fibers within the hollow fiber bioreactor 601, and flows out through the IC outlet port 601B. The pressure sensor 610 measures the pressure of the media leaving the hollow fiber bioreactor 601. In addition to pressure, in an embodiment, the sensor 610 may be a temperature sensor that detects the media pressure and media temperature during operation.

[0045] The media flows through an IC circulation pump 612 that is used to control the media flow rate or circulation rate. The IC circulation pump 612 can pump the fluid in a first direction (e.g., counterclockwise) or a second direction opposite the first direction (clockwise). The outlet port 601B can be used as an inlet in the reverse direction. The media flowing into the IC loop can flow in through the valve 614. As will be understood by those skilled in the art, by placing additional valves and / or other devices at various locations, the media can also be isolated and / or the properties of the media can be measured along a particular portion of the fluid path. A sample of the media is obtained from the sample coil 618 during operation. Then, the media returns to the IC inlet port 601A to complete the fluid circulation path 602.

[0046] Cells grown / proliferated in the hollow fiber bioreactor 601 are flowed out of the hollow fiber bioreactor 601 and enter the harvest bag 699 via the valve 698 and the line 697. Alternatively, if the valve 698 is closed, the cells are redistributed to the hollow fiber bioreactor 601 and further grown. The schematic diagrams shown illustrate one possible configuration for the plurality of elements of the CES600, and it should be understood that changes to the schematic diagrams are possible within the scope of one or more embodiments.

[0047] In the second fluid circulation path 604, the fluid enters the hollow fiber bioreactor 601 via the EC inlet port 601C and leaves the hollow fiber bioreactor 601 via the EC outlet port 601D. In the EC loop, the medium contacts the outside of the hollow fibers of the hollow fiber bioreactor 601, thereby enabling, in one embodiment, the diffusion of small molecules into and out of the hollow fibers within the chamber 601.

[0048] Before the medium enters the EC space of the hollow fiber bioreactor 601, the pressure and temperature of the medium can be measured by the pressure / temperature sensor 624 disposed in the second fluid circulation path 604. After the medium leaves the hollow fiber bioreactor 601, the pressure and / or temperature of the medium in the second fluid circulation path 604 can be measured by the sensor 626. For the EC loop, a sample of the medium can be obtained from the sample port 630 or from the sample coil during operation.

[0049] After leaving the EC outlet port 601D of the hollow fiber bioreactor 601, the fluid in the second fluid circulation path 604 travels through the EC circulation pump 628 and reaches the oxygen supplier or gas transfer module 632. In an embodiment, the EC circulation pump 628 can also pump fluid in both directions. The second fluid flow path 622 is fluidly associated with the oxygen supplier or gas transfer module 632 via the inlet port 632A and the outlet port 632B of the oxygen supplier or gas transfer module 632. During operation, the fluid medium flows into the oxygen supplier or gas transfer module 632 through the inlet port 632A and flows out of the oxygen supplier or gas transfer module 632 through the outlet port 632B. The oxygen supplier or gas transfer module 632 adds oxygen to the medium in the CES600 and removes both carbon dioxide and bubbles.

[0050] In various embodiments, the medium in the second fluid circulation path 604 is in equilibrium with the gas entering the oxygen supplier or gas transfer module 632. The oxygen supplier or gas transfer module 632 can be any appropriately sized device useful for oxygen supply or gas transfer. Air or gas flows into the oxygen supplier or gas transfer module 632 through the filter 638 and flows out of the oxygen supplier or gas transfer device 632 through the filter 640. The filters 638, 640 reduce or prevent contamination of the oxygen supplier or gas transfer module 632 and the associated medium. The air or gas purged from the CES600 when performing a part of the priming step can be vented to the atmosphere through the oxygen supplier or gas transfer module 632.

[0051] In the configuration shown as the CES600, the fluid medium in the first fluid circulation path 602 and the second fluid circulation path 604 flows through the hollow fiber bioreactor 601 in the same direction (in a cocurrent configuration). In an embodiment, the CES600 may be configured to flow in a countercurrent manner.

[0052] In at least one embodiment, a medium containing cells (from a source such as a cell container, e.g., a bag) is attached to attachment point 662, and a fluid medium from a medium source is attached to attachment point 646. The cells and the medium are introduced into a first fluid circulation path 602 via a first fluid flow path 606. Attachment point 662 is fluidly associated with the first fluid flow path 606 via valve 664. Attachment point 646 is fluidly associated with the first fluid flow path 606 via valve 650. A reagent source may be fluidly connected to point 644 and associated with fluid inlet path 642 via valve 648, or may be associated with a second fluid inlet path 674 via valves 648 and 672.

[0053] An air removal chamber (ARC) 656 is fluidly associated with the first circulation path 602. The air removal chamber 656 may have one or more sensors. The sensors include upper and lower sensors for detecting air, fluid deficiency, and / or gas / fluid boundaries, e.g., air / fluid boundaries, at specific measurement points within the air removal chamber 656. For example, near the bottom and / or near the top of the air removal chamber 656, ultrasonic sensors can be used to detect air, fluid, and / or air / fluid boundaries at those locations. In embodiments, other types of sensors can be used without departing from the scope of the present disclosure. For example, according to embodiments of the present disclosure, optical sensors may be used. Air or gas purged from the CES 600 during part of a priming step or other protocol can be vented to the atmosphere from an air valve 660 through a line 658 fluidly associated with the air removal chamber 656.

[0054] The EC medium source is attached to the EC medium attachment point 668. The cleaning liquid source is attached to the cleaning liquid attachment point 666. Thereby, the EC medium and / or the cleaning liquid is added to the first fluid flow path or the second fluid flow path. The attachment point 666 is fluidly associated with the valve 670. The valve 670 is fluidly associated with the first fluid circulation path 602 via the valve 672 and the 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 circulation path 604 via the second fluid inlet path 674 and the second fluid flow path 684. Similarly, the attachment point 668 is fluidly associated with the valve 676. The valve 676 is fluidly associated with the first fluid circulation path 602 via the first fluid inlet path 642 and the valve 672. Also, by opening the valve 676 and closing the distribution valve 672, the fluid container 668 can be fluidly associated with the second fluid inlet path 674.

[0055] In the IC loop, the fluid is first sent by the IC inlet pump 654. In the EC loop, the fluid is first sent by the EC inlet pump 678. An air detector 680, such as an ultrasonic sensor, may be associated with the EC inlet path 684.

[0056] In at least one embodiment, the first and second fluid circulation paths 602, 604 are connected to the waste line 688. When the valve 690 is opened, the IC medium reaches the waste bag or the outlet bag 686 through the waste line 688. Similarly, when the valve 692 is opened, the EC medium flows into the waste bag or the outlet bag 686.

[0057] After the cells are grown in the hollow fiber type bioreactor 601, the cells are harvested via the cell harvest path 697. The cells from the hollow fiber type bioreactor 601 are harvested into the cell harvest bag 699 via the cell harvest path 697 by pumping the IC medium containing the cells with the valve 698 open.

[0058] Each component of the CES600 is housed within an apparatus or housing, such as the cell growth apparatus 202 (Figs. 2 and 3), which maintains cells and media at a predetermined temperature. Further, in embodiments, components of the CES600 and CES500 (Fig. 5) may be combined. In other embodiments, the CES may include fewer or more components than shown in the components of Figs. 5 and 6 within the scope of the present disclosure. In embodiments, certain portions of the CES500, CES600 are implemented by one or more features of the QUANTUM® cell expansion system (CES) manufactured by Terumo BCT, Lakewood, Colorado.

[0059] In one particular embodiment of using the CES600, hematopoietic stem cells (HSCs) (e.g., CD34+ HSCs) are expanded in an embodiment of the CES600. In this embodiment, HSCs (including CD34+ HSCs) collected using a leukapheresis process or a manual process (e.g., umbilical cord) are introduced into the bioreactor 601. HSCs (including CD34+ HSCs) can be introduced into the bioreactor 601 via the circulation path 602.

[0060] In some embodiments, the HSCs (including CD34+ HSCs) may be subjected to a selection step (e.g., a purification step) prior to introduction into the bioreactor 601. This step can involve the use of a centrifuge, a purification column, magnetic selection, or chemical selection. Some examples of cell selection / purification procedures include, for example, those performed using an isolation column manufactured by Miltenyi Biotec of Bergisch Gladbach, Germany. In one example, umbilical cord blood is first subjected to a cell selection process that selects for HSCs (including CD34+ HSCs) prior to introducing the cells into the bioreactor 601. In another example, an apheresis device is utilized to drastically reduce other cells that may be present in the HSCs (including CD34+ HSCs) when first collected. For example, the HSCs may be supplied from umbilical cord blood, bone marrow, or peripheral blood. After the initial collection and prior to introduction into the bioreactor 601, a volume of HSCs containing CD34+ HSCs can be processed to drastically reduce red blood cells, certain white blood cells, granulocytes, and / or other cells from that volume. These are merely exemplary and embodiments of the present invention are not limited thereto.

[0061] In other embodiments, the HSCs (including CD34+ HSCs) are added directly to the bioreactor 601 after collection without additional purification. For example, umbilical cord blood (including HSCs) is added to the bioreactor. In addition to many proteins and other bioactive molecules, umbilical cord blood contains HSCs (including CD34+ HSCs), red blood cells, platelets, granulocytes, and / or white blood cells.

[0062] In some embodiments, the HSCs are added to the bioreactor 601 after the priming step. As will be appreciated, the cells to be expanded are non - adherent and thus do not need to adhere to the hollow fiber wall of the bioreactor 601 for expansion. In these embodiments, there is no need to coat the inside of the hollow fibers with a coating agent (e.g., fibronectin) to promote adhesion. In these embodiments, the HSCs (purified or unpurified, including CD34 + HSCs) are introduced into the bioreactor 601 after the priming step without a bioreactor coating step. If the cells are adherent cells, a coating step is performed after the priming step and before the introduction of the HSCs.

[0063] Once inside the bioreactor 601, the cells are exposed to growth factors, activators, hormones, reagents, proteins, and / or other bioactive molecules that assist in cell proliferation. In one example, a co - culture cell line is grown / introduced in the bioreactor 601 in advance to optimize the conditions for growing HSCs (including CD34 + HSCs). In one particular embodiment, human mesenchymal stem cells (hMSCs) are co - cultured with HSCs (including CD34 + HSCs) to promote the proliferation of CD34 + HSCs. Without being bound by theory, it is thought that MSCs release factors (e.g., the SDF - 1 factor) that interact with HSCs (e.g., CD34 + HSCs) and promote the proliferation of these cells. In some embodiments, the use of co - cultured hMSCs may involve first performing a growth process under conditions optimized for hMSC growth before introducing the HSCs (including CD34 + HSCs) into the bioreactor 601. hMSCs are derived from bone marrow, peripheral blood, umbilical cord cells, adipose tissue, and / or teratogenic tissue in embodiments.

[0064] In addition to the co-cultured cells, to grow and expand the HSCs, supplements comprising one or more growth factors, activators, hormones, reagents, proteins, and / or other bioactive molecules may be added to the bioreactor 601. The supplements can be added as a single bolus addition or over a period of time (e.g., continuously, intermittently, or on a regular schedule). In one embodiment, a combination of cytokines and / or other proteins (e.g., recombinant cytokines, hormones) can be included as part of the supplement. As an example, the supplement can include one or more of recombinant human Flt3 ligand (rhFlt-3L), recombinant human stem cell factor (rhSCF), recombinant human thrombopoietin (rhTPO), recombinant human (rh) glial cell line-derived neurotrophic factor, and / or combinations thereof. An example of a supplement that can be used with the embodiments is the STEMCELL2MAX™ supplement (stemcell2MAX, Cantanide, Portugal).

[0065] Note that in some embodiments, the combination of factors may be included in the medium in which the cells are suspended. For example, the HSCs can be suspended in the medium and introduced into the bioreactor with the medium (1406). In an embodiment, the medium may include a combination of growth factors that assist in the growth of the HSCs.

[0066] After introducing the cells into the bioreactor with the supplement, co-cultured cells, and / or other materials for growing the cells, the cells are grown in the bioreactor (1410). During growth, there are several materials that can be added to or removed from the bioreactor. As an example, additional protein (e.g., cytokine) is added to the bioreactor 601. In some embodiments, two or more proteins or other bioactive agents can be used. The additional materials can be added individually, simultaneously, or at different times. Alternatively, they can be added in combination.

[0067] In some embodiments, for example, materials may be added more directly to bioreactor 501 via port 516 (FIG. 5). However, in other embodiments, materials may be added at a given location to bioreactor 601, for example, via flow path 606, to be perfused more slowly.

[0068] In addition to materials for assisting the growth of HSCs (including CD34+ HSCs), HSCs can also be fed by, for example, the addition of a medium containing some nutrients. In some embodiments, the medium is a commercially available medium containing serum. In other embodiments, the medium is serum-free and may contain other additives. The medium may be modified by the addition of some non-limiting examples including other materials, salts, serum, proteins, reagents, bioactive molecules, nutrients. An example of a medium used for feeding HSCs (including CD34+ HSCs) is CELLGRO® Serum-Free Medium (CellGenix, Freiburg, Germany).

[0069] In some embodiments, the co-culture cells are located within the IC space, but feeding can be performed within the EC space. In an embodiment, by performing feeding via the EC space, the amount of force sensed by the cells from the circulating fluid within the IC space can be reduced. The circulation of the medium within the EC space can, in an embodiment, provide sufficient nutrients for the growth of HSCs (including CD34+ HSCs).

[0070] As part of the growth of HSCs (including CD34+ HSCs), other conditions such as temperature, pH, oxygen concentration, carbon dioxide concentration, waste concentration, metabolite concentration are regulated within bioreactor 601. In some embodiments, various parameters are controlled using the flow rate on the EC side (e.g., path 604). For example, if it is desired to reduce the waste concentration or metabolite concentration on the IC side where cell growth is occurring, by increasing the flow rate on the EC side, waste and / or metabolites can be reliably removed from the IC side by movement from the IC side through the hollow fibers to the EC side.

[0071] After expanding the CD34+ HSCs, the cells are removed from the bioreactor 601. The CD34+ HSCs are collected in the container 699. In embodiments, the collected CD34+ HSCs are administered to a patient to reconstitute hematopoiesis. Such patients include, by way of non-limiting example, patients undergoing treatment for various cancers (such as leukemia, myelodysplasia, non-Hodgkin lymphoma, etc.) that can affect hematopoiesis. The cells may be administered together with other compounds or molecules.

[0072] In some embodiments, the use of the CES600 provides various advantages in the growth of HSCs (including CD34+ HSCs) compared to conventional processes. For example, the use of hollow fibers enables close cell-cell communication, as a result of which the growth of CD34+ HSCs is enhanced and proliferation can be initiated and continued. Also, the use of a hollow fiber type bioreactor such as the bioreactor 601 increases the surface area for cell growth, thereby allowing for a higher concentration or a higher amount of CD34+ HSCs to be obtained.

[0073] Furthermore, the conditions in the bioreactor 601 can be controlled using several different components of the CES600 (including the IC flow rate and the EC flow rate). Also, the CES600 can provide various locations for adding substances. This allows for direct or indirect introduction (such as perfusion) of cytokines into the bioreactor 601.

[0074] Furthermore, the CES600 provides a closed system. That is, the steps for growing CD34+HSCs can be performed without direct exposure to the surrounding environment. Thus, the cells are not contaminated by the surrounding environment, nor is the surrounding environment contaminated by the cells or the substances used for cell growth. In some embodiments, it is believed that the starting concentration of CD34+HSCs for proliferation can be made in a smaller amount compared to other methods / systems. In these embodiments, the harvest of CD34+HSC proliferation can be made larger compared to other methods / systems. Some embodiments are believed to be able to shorten the time for growing an effective amount of CD34+HSCs.

[0075] FIG. 7 shows an example of the components of a computer system 1100 in which an embodiment of the present invention is implemented. The computer system 1100 is used, for example, in embodiments where a cell growth system uses a processor to perform tasks (such as custom tasks or pre-programmed tasks performed as part of a process, as in the processes described above).

[0076] The computer system 1100 has a user interface 1102, a processing system 1104, and / or a storage device 1106. As would be understood by those skilled in the art, the user interface 1102 has an output device 1108 and / or an input device 1110. The output device 1108 may have 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 1110 capable of receiving and / or capturing physical contact from a user or operator, for example. The touchscreen may be a capacitive-structured liquid crystal display (LCD) that enables the processing system 1104 to estimate the contact position, as would be understood by those skilled in the art. In this case, the processing system 1104 can map the contact position to a user interface (UI) element displayed at a predetermined position in the application window. Also, in an embodiment, the touchscreen can receive contact via one or more other electronic structures. Other output devices 1108 include printers, speakers, and the like. Other input devices 1110 include keyboards, other touch input devices, mice, voice input devices, and the like, as would be understood by those skilled in the art.

[0077] In an embodiment of the present invention, the processing system 1104 may have a processing unit 1112 and / or a memory 1114. The processing unit 1112 may be a general-purpose processor operable to execute instructions stored in the memory 1114. The processing unit 1112 may include a single processor or multiple processors in an embodiment of the present invention. Further, in an embodiment, each processor may be a multi-core processor having one or more cores for individually reading and executing instructions. The processor may include a general-purpose processor, an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), and other integrated circuits, as would be understood by those skilled in the art.

[0078] In an embodiment of the present invention, the memory 1114 may include any storage device that stores data and / or processor-executable instructions in the short term or long term. As would be understood by those skilled in the art, the memory 1114 includes, for example, random access memory (RAM), read-only memory (ROM), or electrically erasable programmable read-only memory (EEPROM). Other storage media include, for example, CD-ROM, tape, digital versatile disk (DVD), or other optical storage devices, tape, magnetic disk storage devices, magnetic tape, and other magnetic storage devices, as would be understood by those skilled in the art.

[0079] The storage device 1106 is any long-term data storage device or component. In an embodiment of the present invention, the storage device 1106 may include one or more of the systems described in relation to the memory 1114. The storage device 1106 may be permanent or removable. In one embodiment, the storage device 806 stores data generated or provided by the processing system 1104.

[0080] The present disclosure provides methods for growing cells (i.e., increasing the number of cultured cells). In particular, these methods are useful for growing human hematopoietic stem cells (HSCs) that include HSCs expressing the CD34 protein (CD34-positive HSCs, or CD34+HSCs). In these methods, the CD34+HSCs may be CD45+ / CD34+HSCs and / or CD133+CD38− progenitor cells. Advantageously, these methods grow HSCs (e.g., at least 50-fold) rapidly and efficiently while minimizing the differentiation of these HSCs.

[0081] In an embodiment, by executing flow 1400, target cells such as CD34+HSCs can be grown in single culture or co-culture. Flow 1400 starts at step 1404 and proceeds to step 1412, where the grown cells (e.g., CD34+HSCs) are removed from the bioreactor.

[0082] Similarly, the cells grown in these methods can be cells collected from a donor fluid (e.g., one or more blood components) including stem cells, CD34+ HSCs, T cells, monocytes, and / or natural killer (NK) cells. In these methods, specific “target” cells (e.g., CD34+ HSCs) in the donor fluid can be grown and other cells present in the donor fluid can be removed or their numbers decreased.

[0083] These methods include growing the cells in a culture medium or on a membrane. In these methods of cell growth, such membranes are useful for capturing, collecting, and / or retaining cells. The membranes can be provided in any form such as flat sheets, filter matrices, hollow fibers, any combination thereof, and / or some of them. In these methods, the membrane may include a coating on at least one surface of the membrane. The coating includes stromal cell-derived factor-1 (SDF-1), and fibronectin or isoforms, or functional equivalents thereof. Particularly useful membranes in the methods of the present disclosure are hollow fibers or a plurality of hollow fibers as found within a hollow fiber bioreactor. Such hollow fibers include an inner portion (inner lumen) or inner surface within the lumen of the hollow fiber, and an outer surface (“extracapillary” side or “extracapillary” surface). The hollow fiber membrane may include a plurality of hollow fibers. An example of a hollow fiber may be as shown in the schematic of FIG. 8. FIG. 8 shows a length of hollow fiber 800 and an end of hollow fiber 802, having an inner lumen (inner capillary) 804 and an extracapillary 808 of hollow fiber 800. As shown in FIG. 8, the inner lumen surface (inner capillary surface) 806 has a coating 810.

[0084] In some examples of these methods, the membrane can be used in conjunction with a cell processing device. In one example, cell processing devices include the SPECTRA OPTIA® apheresis system, the COBE® Spectra apheresis system, and the TRIMA ACCEL® automated blood collection system, manufactured by Terumo BCT, Lakewood, Colorado. After collecting cells from a donor, the cells can be passed through the membrane to separate target cells therefrom.

[0085] In some examples of these methods, the membrane can be used in conjunction with a cell growth device. In one example, the cell growth device corresponds to the Quantum® cell growth system manufactured by Terumo BCT, Lakewood, Colorado. After the target cells are separated (e.g., as described above), the target cells are grown on the membrane, for example, the number of target cells contained in the membrane is increased.

[0086] The capture of small-sized species such as proteins or exosomes can be performed based on a continuous flow flowing on the membrane. Diffusion kinetics is effective in assisting the transport of these species to the membrane, and these species are captured by their conjugate chemicals deposited on the membrane. The transport to the hollow fiber wall is further assisted by moderate ultrafiltration from the inner lumen side 906 to the outside of the capillary of the hollow fiber 900, as shown in the schematic diagram of FIG. 9 (the arrow in FIG. 9 indicates the direction of the ultrafiltration flow). The ultrafiltration flow occurs through the coating 910 present on the inner lumen surface 906 of the hollow fiber 900. The flow is stopped to separate the mixed cell population, as shown in the schematic diagram of FIG. 10. Here, the particles 1002 in the inner lumen of the hollow fiber 1000 are floating in the inner lumen 1004, and the flow in the inner lumen 1004 is stopped. In the absence of flow, the particles 1002 (e.g., HSCs) fall to the bottom 1006 of the hollow fiber 1000, as shown in the schematic diagram of FIG. 11. At this point, the target species (such as cells) adhere to the surface of the membrane, and the non-target species (such as cells or cell debris) are washed away from the membrane, leaving the target species on the membrane. In one example, when the non-target species are removed, a release mechanism (including, but not limited to, pH change, temperature change, removal of the binding agent substance) can be used to release the association (such as binding) between the membrane component and the target species (e.g., between the aptamer and the cell membrane antigen), leaving the target species in its original unmodified state. For example, the aptamer can be cleaved by an appropriate nuclease to break the bond between the aptamer and the cell, releasing the cell from the binding.

[0087] The membrane may include one or more coatings configured to attract, collect, and / or retain target cells that will be grown later. When the membrane is the hollow fiber of a hollow fiber bioreactor, the hollow fiber may include a coating on one or both of the inner lumen (inside the capillary) surface and the extracapillary (outside the capillary) surface of the hollow fiber. The coatings provided in the present disclosure may be coatings that are chemically bonded to the membrane (e.g., via hydrophobic and hydrophilic interactions). In some examples, the base coating material may function as the first coating layer, and the second coating material may function as the second coating layer. These coating materials are applied to the membrane either sequentially or together. Examples of the first coating material include fibronectin, vitronectin, any extracellular matrix (ECM) glycoprotein, collagen, enzymes, their equivalents and / or combinations, and / or any molecule or material capable of enabling cell adhesion to the membrane or other surface. Examples of the second coating material include soluble protein moieties, biotinylated molecules, anti-biotin antibodies, biotin-binding and / or streptavidin-binding peptides, streptavidin, avidin, monoclonal antibodies, aptamers (e.g., aptamers targeting specific cell surface markers), cytokines (e.g., interleukin (IL)-6, IL-21), chemokines (e.g., stromal cell-derived factor (SDF)-1), their equivalents and / or combinations.

[0088] The coating may be applied in a single chemical operation. For example, a first molecule (e.g., the coating material of the first portion) and a second molecule (e.g., the coating material of the second portion) are conjugated away from the membrane and then coated onto the membrane simultaneously. When formed by the coating, the membrane of the present disclosure can be used (1) to create a selective bioreactor for growing cells and / or (2) to create a filter capable of capturing specific target cells or molecules (any biotinylated molecule or cell, etc.).

[0089] In one example, the membrane may include one or more materials that promote cell adhesion to at least one surface of the substrate. For example, the coating may include the dimeric glycoprotein fibronectin, or a functional equivalent of fibronectin (e.g., many known isoforms of fibronectin produced via alternative splicing of its pre-mRNA, or other proteins that include the integrin-binding sequence Arg-Gly-Asp (RGD) of the fibronectin protein that confers primary cell adhesion activity of fibronectin).

[0090] Further useful coatings may include one or more protein moieties. Such protein moieties may be selected to target specific target cells present in the donor fluid. For example, the protein moiety may be a chemokine such as stromal cell-derived factor-1 (SDF-1) that can be used to enhance the collection of CD34+ HSCs from the donor fluid (e.g., when compared to an uncoated membrane or a membrane coated only with fibronectin). Other useful protein moieties in these coatings are interleukin-21 (IL-21). Other useful protein moieties in these coatings are combinations of SDF-1 and IL-21. Other useful protein moieties in these coatings may be a combination of fibronectin and SDF-1, as shown in FIG. 12. In FIG. 12, a coating 1210 that includes a combination of fibronectin and SDF-1 is bound to the luminal surface of the hollow fiber 1200. Further coatings of the membrane may be selected to target, collect, and / or retain the same or different cells from the donor fluid.

[0091] In these methods, the membrane is coated with a mixture of fibronectin and a soluble protein moiety as shown in the schematic diagram of FIG. 12 In FIG. 12, a coating 1210 that includes a combination of fibronectin and SDF-1 is bound to the luminal surface of the hollow fiber 1200. Further coatings of the membrane may be selected to target, collect, and / or retain the same or different cells from the donor fluid.

[0092] As shown in FIG. 13, in these methods, a plurality of cells 1302 (such as a suspension of HSCs) are introduced into a hollow fiber membrane 1300 having a coating 1310 on its lumen surface 1306.

[0093] In some embodiments, the coated membrane is coated with a mixture of fibronectin and a soluble protein moiety to capture biotinylated molecules such as streptavidin, avidin, and / or anti-biotin antibodies and / or their functional equivalents.

[0094] As shown in the schematic diagram of FIG. 15, in some examples, the lumen surface 1506 of the hollow fiber 1500 may have a coating 1510 that is, for example, a coating containing a biotinylated molecule. In one example, this type of membrane coating allows the target species 1502 (such as HSCs) in a suspension of non-target species 1503 (such as red blood cells) to be captured. The coated membrane may be used to separate or capture target cells from a mixed population of cells. As shown in FIG. 16, the target cells 1502 are captured, for example, by a protein moiety present in the coating 1510 on the lumen surface 1506 of the hollow fiber 1500. After flushing 1550 of the hollow fiber 1500, the non-target species 1503 are removed from the lumen 1504 of the hollow fiber 1500, while the target species 1502 remain bound to the lumen surface 1506 of the hollow fiber 1500.

[0095] In these methods, a plurality of cells are oriented to contact a membrane that is the coated membrane of the present disclosure and are grown in contact with the membrane. When a hollow fiber membrane is used in these methods, a plurality of cells are introduced into the hollow fibers having an inner tube portion and an outer tube portion as described above of a hollow fiber bioreactor (1406). The plurality of cells are first purified by various means before being oriented to contact the membrane. Alternatively, the plurality of cells may be oriented to contact the membrane directly from, for example, collection from a cell donor source (e.g., collection of peripheral blood, or bone marrow, or cord blood (CB)) without first being purified. That is, it may include introducing a plurality of cells into a plurality of hollow fibers without any prior purification. The cells are oriented to contact the membrane prior to further circulation or movement of the membrane that is performed to “seed” additional cells onto the membrane or to remove residual cells and cell debris from the membrane and are then left in place to bind to the membrane. When the membrane includes the hollow fibers of a hollow fiber bioreactor, this procedure preferably involves circulating a plurality of cells within the lumen of the hollow fibers using a pump, then stopping the pump to attach a portion of the plurality of cells to a first portion of the lumen of the hollow fibers, then rotating the hollow fiber bioreactor 180 degrees from an initial position and then circulating the plurality of cells within the lumen of the hollow fibers again using the pump, and then stopping the pump to attach a portion of the plurality of cells to a second portion of the lumen of the hollow fibers.

[0096] Cells are grown (1410) by exposing the cells within the hollow fibers to growth conditions (1408). The growth conditions include exposing the cells to one or more cell growth media, for example, by circulating the cell growth media within the lumen of the hollow fibers of a hollow fiber bioreactor and / or outside of the capillaries of the hollow fibers. Alternatively, or additionally, the growth conditions may include exposing the cells to one or more growth factors. Useful growth factors can include FMS-like tyrosine kinase 3 ligand (Flt-3L), stem cell factor (SCF), thrombopoietin (TPO), glial cell line-derived neurotrophic factor (GDNF), interleukin-3 (IL-3), interleukin-6 (IL-6), IL-21, SDF-1, or combinations thereof. When GDNF is present in the growth media, a concentration of from 0.5% to 2% mass / volume percent concentration, for example, a concentration of about 10 ng / mL in the growth media, is particularly useful.

[0097] In these methods using the membranes of the hollow fibers of a hollow fiber bioreactor, a first media may be used within the lumen of the hollow fibers and a second media may be used in contact with the outside of the capillaries of the hollow fibers. In these methods, at least one component of the media in the lumen may be concentrated relative to the concentration of the same component outside of the capillaries of the hollow fibers. In these methods, the component to be concentrated (the concentrated component) may be GDNF, SR-1, SCF, TPO, Flt-3L, IL-3, IL-6, SDF-1, fibronectin, or combinations thereof. In these methods, the concentrated component is concentrated at least 5-fold, or at least 10-fold.

[0098] Other useful factors for cell proliferation can include aryl hydrocarbon receptor antagonists such as StemRegenin 1 (SR1) or UM171, developed at the University of Montreal and currently in clinical development for cell therapy by ExcellThera.

[0099] The coating can be used to provide a special environment for cell culture (for example, in the coating, when the base coating material is fibronectin or the like and the second layer coating material contains soluble protein moieties such as SDF-1, IL-21, etc.). Accordingly, the present disclosure provides compositions useful for growing CD34 + HSCs. These compositions can include at least one of glial cell line-derived neurotrophic factor (GDNF) and aryl hydrocarbon receptor (AHR) antagonists (such as SR-1, etc.). These compositions may also include at least one of SCF, TPO, Flt-3L, IL-3, IL-6, SDF-1, and fibronectin. In these compositions, GDNF can be present at a concentration of 0.5% to 2% by mass / volume percentage concentration, or at a concentration of at least 10 ng / mL. In these compositions, fibronectin and SDF-1 may be immobilized on the cell culture surface (such as a semipermeable membrane). These compositions increase the level of BCL2 and inhibit HSC differentiation.

[0100] The coated membranes of the present disclosure can be used to provide a special environment for capturing biotinylated molecules such as streptavidin, avidin, anti-biotin (for example, when the coating includes a first coating material such as fibronectin and a second coating material including a biotin capture moiety such as a biotinylated molecule, an aptamer targeting a specific cell surface marker, or a soluble moiety such as a cytokine (e.g., IL-6)).

[0101] At least one advantage of the chemical coatings described herein is the ability to manufacture membranes (such as hollow fiber membranes) with coatings in a sterile environment. A sterile package containing a chemically coated and sterilized coating membrane is in a state where it can be used immediately after being opened and the membrane is taken out of the package (for example, without the need for further treatment).

[0102] In one example, the hollow fiber membrane (HFM) of the Quantum® cell growth system bioreactor may be coated with a coating material containing streptavidin-fibronectin. This coating material can be used, for example, to select or isolate specific cell types when subsequently bound to biotinylated cell-specific monoclonal antibodies (mAbs).

[0103] In some examples, the fibronectin-streptavidin basis can be used as a coating material for the attachment of biotinylated molecules to functionalize the surface of polyethersulfone HFM bioreactors or pre-columns for cell selection. Fibronectin can bind to polyethersulfone HFM within the Quantum® cell growth system bioreactor via the adhesion and proliferation of adherent cells such as mesenchymal stromal / stem cells (MSCs), fibroblasts, and aortic endothelial cells. This fibronectin-streptavidin conjugation utilizes the high affinity of streptavidin binding to biotin. It is important to maintain the protocol directly and efficiently with minimal residues or reactants while adjusting their application in the manufacture of cell therapy products considering available protein-binding chemical structures. In one example, the fibronectin-streptavidin mixture or conjugate may be mixed and / or conjugated, thereby enabling HFM bioreactors or columns with biotinylated cytokines, chemokines, and / or other ligands to promote cell selection and / or proliferation. Other affinity separations of biomolecules may be used. In any case, this protein-protein conjugation can be regarded as a platform for affinity processes related to cell therapy using available techniques.

[0104] In these methods, a mixture of fibronectin and streptavidin can be used as a coating material for the coated membrane. This process may include reconstituting lyophilized fibronectin and streptavidin (e.g., at a mass ratio of 1:3.3) in water for about 30 minutes at ambient temperature. After fibronectin-streptavidin conjugation, the volume of the mixture is made up to 100 mL with Ca2+-Mg2+-free phosphate-buffered saline and can be introduced into the Quantum® cell growth system for a sufficient time (e.g., 8 hours) using the "Coat Bioreactor" task. After bioreactor coating, excess unbound conjugate protein may be washed away, and a selected biotinylated molecule, such as a cytokine (interleukin or growth factor), epitope, ligand, monoclonal antibody, stain, or aptamer, may be introduced into the Quantum® cell growth system bioreactor using the "Coat Bioreactor" task for coupling to the fibronectin-streptavidin coating. Upon completion, the resulting fibronectin-streptavidin-bio conjugate protein is ready for use in cell selection or cell signaling (including differentiation) applications. Other uses include coating a pre-HFM column or matrix that can be used for cell selection or differentiation before introducing cells into the Quantum® cell growth system.

[0105] In these methods, recombinant fibronectin or semi-synthetic fibronectin or fibrinogen can be used in place of plasma-derived fibronectin. Extracellular matrix proteins such as fibronectin can bind to a polyethersulfone hollow fiber membrane by polarity and hydrogen bonding. Fibronectin is inherently adhesive due to its glycoprotein structure and specific domains that allow fibronectin to bind to both polyethersulfone and cell membrane integrins.

[0106] In one example, the covalent attachment of fibronectin and streptavidin can be carried out at the same mass ratio as above using a streptavidin conjugation kit. This kit can utilize certain binding modifying factors and quenchers to generate covalent bonds between fibronectin and streptavidin over a period of 30 minutes to 24 hours, and in some embodiments, over a period of 3 hours to 15 hours. In some examples, the time for generating a covalent bond between fibronectin and streptavidin is about 4 hours ± 30 minutes. In the covalent coating method, the affinity of the selected biotinylated molecule for streptavidin is similar to the affinity of the biotinylated molecule in the fibronectin-streptavidin mixture coating method. One advantage of the covalent approach is the improved stability of the fibronectin-streptavidin bond.

[0107] For example, the attachment of streptavidin-biotinylated molecules to fibronectin using a molar ratio of 1:3 (fibronectin:streptavidin) is useful. In some examples, the attachment of fibronectin-streptavidin biotinylated molecules to the HFM bioreactor is a two-step process. The chemical structure of this conjugation coating is a platform for binding arrays of biotinylated molecules for cell selection, stimulation, proliferation, or differentiation.

[0108] The fibronectin-streptavidin protein conjugate can be selected as an adhesion molecule for the Quantum® cell growth system bioreactor. The binding of biotinylated cell-specific mAb or protein epitope to the fibronectin-streptavidin conjugate has a dissociation constant Kd = 10 -14 to 10 -15At a specific ratio of 1:4 or less with M, the high affinity of streptavidin for biotin can be elicited. Examples of biotinylated antibodies or epitopes that are cell-specific include anti-CD3 mAb against parental T cells, anti-CD4 / CD25 mAb against human T-reg cells, anti-CD8 mAb against human T-effector cells, anti-CD34 mAb against hematopoietic stem cells, or anti-CD56 mAb against NK cells. The streptavidin-biotin binding may include strong non-covalent bonds, and thus, by utilizing this functional specificity, substantially any cell type can be selected simply by changing the specificity of the biotinylated mAb conjugate. Furthermore, it is also possible to utilize the reverse approach in which biotinylated fibronectin binds to streptavidin-cell-specific mAb, and this can be used to select the cells of interest. When using the first approach, biotinylation of mAb using a small biotin molecule (m.w. 244.3 daltons) is less likely to affect mAb binding or cell antigen recognition. Second, the lack of glycosylated streptavidin and the net negative charge help minimize non-specific binding to cells. This concept can utilize the highly specific interaction and versatility of the streptavidin-biotin interaction to provide a better adhesion system. In some examples, cells are enzymatically separated from the streptavidin-fibronectin-biotin-mAb-cell complex by enzymatically cleaving a DNase-sensitive linker.

[0109] Accordingly, the present disclosure also provides coated membranes, as well as methods of making and using them. These coated membranes may be hollow fibers including the hollow fibers used in a hollow fiber bioreactor. These coated hollow fiber membranes may include a lumen surface (inner capillary surface) and a capillary outer surface (outer capillary surface), and may have a first coating on at least one of the lumen surface and the capillary outer surface. The first coating may include a material that promotes cell adhesion to at least one of the lumen surface and the capillary outer surface. The second coating on at least one of the lumen surface and the capillary outer surface may include a soluble protein moiety. In these coated hollow fiber membranes, the first coating may include fibronectin. In these coatings, the second coating may include at least one of a cytokine, an aptamer, a chemokine (e.g., SDF-1 or IL-21), a monoclonal antibody, streptavidin, avidin, a biotinylated molecule, and an anti-biotin antibody or a portion of its function. These membranes may be composed of a material including polysulfone or polyethersulfone.

[0110] In these coated hollow fiber membranes, the amount of fibronectin coating the hollow fiber may be 0.001 μg / cm 2 ~2 μg / cm 2 and may be 0.01 μg / cm 2 ~1.0 μg / cm 2 and may be 0.10 μg / cm 2 ~0.50 μg / cm 2 and may be 0.20 μg / cm 2 ~0.40 μg / cm 2 and may be 0.23 μg / cm 2 ~0.24 μg / cm 2 and may be 0.001 ng / cm 2 ~0.30 ng / cm 2 and may be 0.01 ng / cm 2 ~0.10 ng / cm2 may also be, 0.05 ng / cm 2 ~0.09 ng / cm 2 may also be, 0.075 ng / cm 2 may also be.

[0111] The present disclosure also provides a method for forming a coated hollow fiber membrane. These methods include preparing a hollow fiber membrane having an inner lumen surface and an outer capillary surface, applying a first coating on the inner lumen surface of the hollow fiber membrane, and applying a second coating on the inner lumen surface of the hollow fiber membrane. In these methods, the first coating can include a material (such as fibronectin) that promotes cell adhesion to at least one of the inner lumen of the hollow fiber membrane and the outer surface of the capillary of the hollow fiber membrane. The second coating can include a soluble protein moiety (e.g., one or more of a cytokine, an aptamer, a chemokine, a monoclonal antibody, streptavidin, avidin, a biotinylated molecule, and an anti-biotin antibody or a functional fragment thereof). In these methods, applying the first coating material and the second coating material may include conjugating the first coating material and the second coating material to a conjugate away from the hollow fiber membrane and coating the conjugate on the inner lumen surface of the hollow fiber membrane. These methods can include applying a first coating on the outer capillary surface of the hollow fiber membrane and / or applying a second coating on the outer capillary surface of the hollow fiber membrane. In these methods, the first coating may be fibronectin, and the second coating may be SDF-1 or interleukin-21 (IL-21).

[0112] As described herein, the bioreactor (e.g., HFM, hollow fiber device, and / or hollow fiber) may be continuously coated. By continuously coating the bioreactor, exposure to the SDF-1 moiety can be increased over time. For example, according to an exemplary protocol, on day -2 (e.g., 2 days prior to seeding), the bioreactor HFM may be coated with fibronectin (e.g., using the "Coat Bioreactor" task described above), on day -1 (e.g., 1 day prior to seeding), the bioreactor HFM may be coated with SDF-1 (e.g., using the "Coat Bioreactor" task described above), and on day 0 (e.g., the day of seeding), the bioreactor may be seeded with CB-derived CD34+ HSCs. In some examples, each coating may take 8 to 24 hours to complete.

[0113] In these methods, cells in contact with the membrane (e.g., cells within the lumen of a plurality of hollow fibers) are expanded by growing them in monoculture (i.e., substantially in the absence of other cell types) or co-culture (i.e., in the presence of other cell types). For example, in these methods, CD34+ HSCs expanded in the hollow fibers can be grown in a monoculture that is not co-cultured with other cell types in the hollow fibers. In these methods of expanding CD34+ HSCs in the hollow fibers in monoculture, the hollow fibers may have a coating comprising SDF-1 and fibronectin on at least one of the inner lumen surface and the outer capillary surface of the hollow fibers. In these methods, CD34+ HSCs are suitably expanded in the presence of SDF-1 and fibronectin without the need for other cells in co-culture.

[0114] In these methods, multiple cells (e.g., CD34+ HSCs) may be expanded by growing them in co-culture. CD34+ HSCs may be expanded in co-culture with mesenchymal stem cells. In these methods, the cells to be grown in co-culture (such as mesenchymal stem cells) may be introduced into the hollow fibers before introducing multiple expansion cells into the hollow fibers. The cells to be grown in co-culture (such as mesenchymal stem cells) may be expanded in single culture in the hollow fibers (such as by exposing the mesenchymal stem cells in the hollow fibers to growth conditions) before introducing the multiple cells (such as CD34+ HSCs) to be expanded. Alternatively or additionally, the multiple cells to be expanded (e.g., CD34+ HSCs) may first be expanded in co-culture (e.g., co-culture with mesenchymal stem cells) in a static growth chamber (e.g., a conventional cell culture well or flask), and all or part of the multiple cells to be expanded (e.g., CD34+ HSCs) may be removed from the static growth chamber and introduced into the hollow fibers.

[0115] In these methods, cell expansion may preferably be sufficient to expand a plurality of cells containing CD34+ HSCs obtained from 1 unit of blood or 1 unit of tissue to a number of expanded cells sufficient for at least one engraftment procedure for a human recipient. In these examples, 1 unit of blood may be umbilical cord blood, or 1 unit of tissue may be bone marrow.

[0116] In these methods, the expanded cells containing CD34+ HSCs may have a viability of at least 90% after expansion. In these methods, the expanded cells containing CD34+ HSCs may be expanded at least 50-fold.

[0117] In some examples, the present disclosure provides methods and apparatuses for separating a target species, such as a target cell or a target molecule, from a mixed population of non-target species. The methods and apparatuses are described using the separation of target cells from a mixed population of cells. A hollow fiber device similar to a hemodialyzer can be used in the cell separation procedure. As described above, the inner (lumen) wall of the hollow fiber may be coated so that a specific binding reagent (e.g., a coating material) adheres uniformly to the inner surface of the capillary of the hollow fiber device. In addition or alternatively, the outer wall of the hollow fiber capillary may be treated with a binding reagent to increase the surface area (e.g., for molecular capture).

[0118] The binding reagent corresponds to, for example, a monoclonal antibody (mAb) or a sequenced aptamer. The binding reagent can be selected to have specificity for a receptor molecule on the surface of the target cell that is the separation target. For example, when T cells are separated from a mononuclear cell (MNC) collection, a binding reagent having specificity for a combination of CD3, CD4, CD8, and / or T cell markers is attached (e.g., coated, deposited) to the inner surface and / or the outer surface of the capillary of the hollow fiber that constitutes the membrane. An example of a method for separating T cells from a mixed cell population involves attaching an antibody or aptamer to the cells prior to introduction into the membrane and then passing through a streptavidin-coated membrane such as a streptavidin-coated hollow fiber. In other examples, a counterflow confinement (CFC) approach is used, and a collection of cells is flowed into the lumen side of the hollow fiber membrane. Once the cells are contained on the lumen side of the membrane, the counterflow is minimized to a level sufficient to hold the cells within the hollow fiber. Once the target cells are bound to the lumen surface, both the longitudinal flow (from the lumen inlet header to the lumen outlet header) and the ultrafiltration flow can be utilized to remove unbound cells from the lumen of the hollow fiber.

[0119] In some examples, a releasing agent may be used to facilitate detachment of the target cells from their binding sites (e.g., to facilitate target cell harvesting). The releasing agent may be flowed longitudinally, flowed using ultrafiltration, or flowed using both.

[0120] Examples may be described herein in connection with a hollow fiber device (e.g., a bioreactor or other device including a coated membrane disposed as a hollow fiber), but it should be understood that any membrane can be used if it can be coated. For example, any one or more of the following devices can be used to receive various coatings and / or to perform the methods described herein: large surface area hollow fiber devices, dialysis devices (e.g., hemodialyzers), cell capture columns (e.g., magnetic cell sorting, magnetic column devices), polysulfone membrane filter devices, cell processing systems, and the like.

[0121] In these methods, at least a portion of the plurality of proliferating cells may be removed from a membrane (e.g., the hollow fiber of a hollow fiber bioreactor) (1412). The proliferated cells are then either stored or used for transplantation or administration in other treatment procedures for a patient (e.g., a cancer treatment protocol). In these methods, hematopoiesis in a patient can be reconstituted by administering a plurality of proliferating cells to the patient.

[0122] Human leukocyte antigen (HLA)-8 - allele - matched umbilical cord blood (CB) transplantation is an allogeneic treatment for the treatment of certain hematological malignancies, hemoglobinopathies, and autoimmune diseases. CB - derived CD34+ stem cells and progenitor cells can be selected for hematopoietic reconstitution. This is because, along with their ability to achieve rapid engraftment in a blood transplant recipient, their self - renewal ability and proliferation ability are improved, their telomeres are lengthened, and the incidence of graft - versus - host disease (GVHD) is reduced due to a decrease in the frequency of alloreactive T cells. However, one of the challenges in this situation is to provide a sufficient number of T - cell - depleted hematopoietic stem progenitor cells that are necessary to support a mixed allogeneic hematopoietic stem cell transplantation (HSCT). Among the umbilical cord blood units stored in a CB bank, for a 70 kg patient, one - unit transplantation (≧1.05×10 7 CD34+HSC) or two - unit transplantation (≧1.40×10 7Those containing a sufficient number of CD34+HSCs for (CD34+HSCs) are only about 4% to 5%.

[0123] Methods for expanding cord blood-derived CD34+HSCs in co-culture with either mesenchymal stromal cells or small molecules combined with various cytokine supplements often rely on the seeding of 4 - 6×10 6 or more CD34+HSCs. In some embodiments (e.g., to expand the range of stored CBUs), a primary cytokine cocktail consisting of recombinant human - stem cell factor (SCF), - thrombopoietin (TPO), - fms-like tyrosine kinase 3 ligand (Flt3L), - interleukin 3 (IL-3), and - interleukin 6 (IL-6) is used at one-tenth of the manufacturer's recommended concentration in a cell processing system (e.g., a perfusion-based two-chamber semi-permeable hollow fiber membrane (HFM) bioreactor such as the Quantum® cell expansion system) with 2×10 of pre-selected cord blood-derived CD34+HSCs 6A single culture expansion protocol for low initial seeding is provided. This cytokine cocktail is further supplemented with recombinant human glial cell line-derived neurotrophic factor (rhGDNF), for example, to maintain cell viability, and combined with the aryl hydrocarbon receptor (AHR) antagonist SR-1. GDNF can upregulate the expression of the anti-apoptotic gene BCL2 in human CB-CD34+ cell precursors, and SR-1 can limit the differentiation of HSCs during CD34+HSC expansion when implemented with other HSC cytokines. The proximity of mesenchymal stromal cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow sinusoids contributes to their inclusion in the hematopoietic co-culture process, in combination with perivascular support of HSPCs by SCF from CD146+ MSCs. Although attractive, co-culture of MSCs and HSPCs adds complexity, time, and potential variability to the stem and progenitor cell expansion process. Even so, MSC / HSPC co-culture may provide an alternative production strategy for CB-derived CD34+HSCs. By automating the hematopoietic cell and progenitor cell expansion process, cells selected for therapeutic applications can be provided in reliable quantities.

[0124] Furthermore, the Quantum® system supports the growth of both adherent MSCs and suspended CD3+ T cells and regulatory T cells using a perfusion-based HFM bioreactor. In the CB-derived CD34+ cell expansion method described herein, the inner capillary (IC) HFM lumen of the bioreactor is coated with a mixture of human fibronectin (Fn) and chemokine stromal-derived factor 1 (SDF-1) prior to seeding cells to mimic the stimulatory and homing effects of bone marrow-derived or Wharton's jelly-derived mesenchymal stromal cells. Pre-selected CB-derived CD34+ HSCs are subsequently expanded under suspension culture conditions and, during this process, adhere to the coated HFM IC surface and bind, for example, to the Fn-SDF-1 modified surface. In some cases, immobilized SDF-1 is required to develop integrin-mediated cell adhesion of CD34+ HSCs to murine endothelial cells by VLA-4 integrin. In this regard, hydrogel immobilization of SCF and SDF1α, along with the incorporation of a PEG-RGD integrin recognition sequence onto the cell culture surface, recapitulates certain aspects of the bone marrow microenvironment. The embodiments and examples described herein provide for the expansion of CB-derived CD34+ HSCs with modified extracellular matrix proteins.

[0125] In one example, a method and / or system for the automated monoculture expansion of CB-derived HSCs and progenitor cells is provided that begins with a positively selected CB-derived CD34+ HSC that is mixed. These cells are resuspended in serum-free media supplemented with a defined hematopoietic cytokine cocktail and grown for 8 days under a programmed but modifiable perfusion protocol, such as in a Fn-SDF-1-coated HFM bioreactor system, to minimize T cell differentiation. CB-derived CD34+ HSCs grown in a Quantum® system can generate a sufficient amount of cells to support both 1 unit and 2 unit minimum CD34+ dose equivalents while preserving the CD34+ phenotype and minimizing lymphocyte frequency. Additionally, these CB-derived expanded progenitor cells may demonstrate the ability to differentiate into mature hematopoietic colony-forming units (CFUs) under methylcellulose assay conditions.

[0126] In an exemplary embodiment, three master lots of preselected mixed CD34+ HSCs from umbilical cord blood are grown in a ~2.1 m 2 HFM bioreactor having a perfusion culture volume of approximately 124 mL and harvested using an automated suspension cell protocol. The cells are introduced into the inner capillary loop (e.g., IC loop) of the HFM bioreactor by a defined perfusion protocol and maintained within the lumen of the bioreactor using a custom countercurrent fluidics program.

[0127] As described above, using the membranes of the present disclosure, membranes can be effectively fabricated that can capture specific target cells or molecules. Accordingly, the present disclosure also provides a method of capturing cells. In an embodiment, Flow 2400 is performed to capture cells such as CD34+ HSCs. Flow 2400 starts at step 2404 and proceeds to step 2414, where the captured target cells (e.g., HSCs) are removed from the bioreactor. These methods include introducing a mixture of a target species (e.g., a cell or molecule) and a non-target species onto the membrane of the present disclosure (e.g., into the hollow fibers of a hollow fiber bioreactor) (2406). Here, each hollow fiber has a lumen and an extracapillary side. As detailed above, these membranes have a coating on at least one surface of the membrane that includes at least one of a material and a protein moiety that promote cell adhesion. When using a hollow fiber membrane, one or both of the inner lumen surface and the extracapillary surface of the hollow fiber are coated with a material and / or protein moiety that promote cell adhesion.

[0128] The mixture of species in contact with the membrane is exposed (2408) to conditions that enhance the association of the target species with the membrane (i.e., "capture conditions") (2410). Examples of capture conditions include changes in pH, temperature, tonicity, and / or addition or removal of a compound that enhances the association of the target species with the membrane. By implementing the capture conditions, at least a portion of the target cells can be effectively captured on the surface of the membrane (e.g., at least one of the inner lumen and the extracapillary surface of the hollow fiber). Thereafter, at least a portion of the non-target species is flushed from the membrane (e.g., from the inner lumen of the hollow fiber) (2412). In these capture methods, the target species may be, for example, CD34+ HSCs, and the non-target species may be, for example, other cell types or cell fragments or blood proteins.

[0129] In these capture methods, the coating material on the membrane that promotes cell adhesion to the membrane surface may include fibronectin. In these capture methods, the protein moiety may be at least one of stromal cell-derived factor-1 (SDF-1), interleukin-21 (IL-21), streptavidin, avidin, and anti-biotin antibody or functional fragments thereof. In these capture methods, the coating may include fibronectin and SDF-1. In these methods of capturing target cell populations (e.g., CD34+ HSCs), after at least a portion of the non-target cells are washed away from the membrane, the captured target cells may be expanded by altering other conditions in the medium and / or on the membrane to enhance the growth and proliferation of the captured cells such as CD34+ HSCs. These capture methods may include removing at least a portion of the captured target population (such as CD34+ HSCs) from the membrane. These captured populations are removed from the membrane as described above, after washing to remove non-target populations and capture the target population, or after the target cell population has been expanded following capture (2414).

[0130] The present disclosure also provides methods for capturing cells using the interaction between biotin and avidin. These methods include introducing a mixture of target and non-target species into a hollow fiber. In these methods, each hollow fiber includes an inner lumen and an extra-capillary side, and the hollow fiber may include a coating on at least one of the inner lumen surface and the extra-capillary side surface of the hollow fiber. In these methods, the coating may include at least one of streptavidin, avidin, a biotinylated molecule, and an anti-biotin antibody or a functional fragment thereof. In these methods, the target species and / or non-target species may be cells (e.g., HSCs) or molecules. In these methods, the mixture of target and non-target species can be exposed to capture conditions to capture at least a portion of the target species on at least one of the inner lumen and the extra-capillary side surface of the hollow fiber. And at least a portion of the non-target species is washed away from the hollow fiber. In these methods, the target cells introduced into the hollow fiber may include a biotinylated aptamer or a biotinylated antibody that binds to a coating on at least one of the inner lumen surface and the extra-capillary side surface of the hollow fiber.

[0131] These methods in which the target species is a cell may include, after washing away at least a portion of the non-target cells from the hollow fiber, exposing a portion of the target cells captured on the surface of the hollow fiber to growth conditions to proliferate a portion of the target cells captured within the hollow fiber, thereby generating a plurality of proliferated target cells. In these methods, after capture of the target cells and washing away of the non-target cells, at least a portion of the captured target cells is removed from the hollow fiber.

[0132] The present disclosure also provides methods for growing cells by perfusion in a cell growth system. These methods include coating a hollow fiber bioreactor with a first fluid containing a signaling factor and / or a coating factor. A plurality of cells are introduced into a hollow fiber bioreactor comprising a hollow fiber membrane. The plurality of cells are exposed to a second fluid containing a plurality of growth factors. The plurality of cells in the hollow fiber bioreactor may be grown in a single culture or a co-culture. In these methods, the first fluid may contain at least one of fibronectin and SDF-1. In these methods, fibronectin and SDF-1 may be mixed together before coating the hollow fiber bioreactor. In these methods, the hollow fiber bioreactor may be continuously coated by coating the hollow fiber bioreactor with fibronectin and then coating the hollow fiber bioreactor with SDF-1. In these methods, the hollow fiber bioreactor may be continuously coated by coating the hollow fiber bioreactor with SDF-1 and then coating the hollow fiber bioreactor with fibronectin. In these methods, the amount of fibronectin used to coat the hollow fiber bioreactor is 0.001 μg / cm 2 ~2 μg / cm 2 、or 0.01 μg / cm 2 ~1.0 μg / cm 2 、or 0.10 μg / cm 2 ~0.50 μg / cm 2 、or 0.20 μg / cm 2 ~0.40 μg / cm 2 、or 0.23 μg / cm 2 ~0.24 μg / cm 2 and may be. In these methods, the amount of SDF-1 used to coat the hollow fiber bioreactor is 0.001 ng / cm 2 ~0.30 ng / cm 2 、0.01 ng / cm 2 ~0.10 ng / cm 2 、or 0.05 ng / cm 2 ~0.09 ng / cm2 or 0.075 ng / cm 2 may also be used.

[0133] In these methods, the second fluid may contain GDNF. In these methods, the amount of GDNF in the second fluid may be 0.001 ng / mL to 40.0 ng / mL, or 0.01 ng / mL to 20 ng / mL, or 0.10 ng / mL to 15 ng / mL, or 1.0 ng / mL to 15 ng / mL, or 5.0 ng / mL to 15 ng / mL, or 10 ng / mL.

[0134] In these methods, the plurality of growth factors may include at least one of SCF, TPO, Flt-3L, IL-3, and IL-6. In these methods, the second fluid may contain stemregenin (SR-1). In these methods, the amount of SR-1 in the second fluid may be 0.001 μM to 3.0 μM, or 0.01 μM to 2.0 μM, or 0.10 μM to 1.0 μM, or 0.75 μM.

[0135] In these methods, before introducing the plurality of cells into the hollow fiber bioreactor, the hollow fiber bioreactor may be coated with a mixture of 5 mg of human plasma-derived fibronectin or 0.23 - 0.24 μg / cm 2 of fibronectin and 0.075 ng / cm 2 of recombinant human stem cell-derived factor 1 (SDF-1) for a predetermined period. In these methods, the predetermined period is 4.0 hours to 16.0 hours, or 8.0 hours to 12.0 hours.

[0136] Examples

[0137] Example 1 Short-term proliferation method of cord blood-derived CD34+ HSCs in the Quantum (registered trademark) system

[0138] Human cord blood-derived CD34+ hematopoietic stem cells (HSCs) expanded for 8 days or less engrafted better in a humanized immunodeficient mouse model than cells expanded for more than 8 days. As a result of expanding cord blood-derived CD34+ HSCs for 8 days or less, a BALB / C-RAG2 null IL-2r-gamma null mouse model humanized mouse (Clinical Immunology, 140:102-116, 2011) was obtained that showed more consistent human hematopoiesis and lymphoid engraftment.

[0139] Embodiments reduce or shorten the period for the expansion of cells (e.g., CD34+ HSCs and / or CB - CD34+ HSCs), for example, while improving cell yield, phenotype, and functionality. In one embodiment, it provides inoculum expansion and Quantum® system expansion of HSC CB - CD34+ HSCs for about 14 days in co - culture with mesenchymal stem cells (MSCs), which are an in situ source of SDF - 1. In a further embodiment, using a shortened single - culture protocol, for example, the yield, phenotype, and functionality are improved. Human umbilical cord blood - derived CD34+ HSCs can be expanded, for example, in the following two steps. (1) After the preparation expansion of an inoculum of about 1 million CB - CD34+ HSCs for about 3 days in a T25 flask, (2) to maintain the CD34+CD38 - CD133+ HSC phenotype and related engraftment function, viable CD34+ HSCs are expanded by perfusion in a Quantum® cell expansion system for about 5 days by using a single - culture technique with fibronectin - immobilized SDF - 1 and other growth factors / cytokines. For example, embodiments provide the following. (1) Use of a shortened timeline for about 8 days of cell expansion: for example, in a single - culture, (1) an immobilized SDF - 1 signaling factor is used in combination with (2) a novel growth factor cocktail, carried out for about 3 days in a flask and about 5 days in a Quantum® system. The cocktail utilizes, for example, one or more of SCF, TPO, Flt - 3L, IL - 3, IL - 6, GNDF±SR - 1, and combinations thereof. In one embodiment, a single - culture protocol (e.g., a shortened single - culture protocol) can use, for example, a bidirectional cell reseeding task in a Quantum® system.

[0140] CD34+ Mixed Cell Expansion

[0141] Flask Test

[0142] In one example, the flask test was carried out over 7 days. Cord blood - derived CD34+ HSCs were expanded at 37°C using CO2 CD34 complete medium without shaking. On day 1, the cells were placed at 1×10 in 7 mL5 They were seeded at cells / mL and harvested on the 7th day. A harvest of 5,700,000 was considered optimal. The CD34 medium produced 11,800,000 cells, while the CD34 medium diluted 1:10 with complete medium produced 9,200,000 cells, and the CD34 medium diluted 1:20 produced 5,900,000 cells. The cell viability was 86.2%, 90.3%, and 90.1% for undiluted, 1:10 diluted, and 1:20 diluted CD34 medium, respectively (n = 2 per arm, cell counting was performed 3 times).

[0143] Freeze - thaw test

[0144] The feasibility of the proliferation of thawed mixed umbilical cord blood - derived CD34 + HSC (Stem Cell Technologies, Lot 1907519003) was tested. Cells (1.1×10 6 cells and 2.1×10 6 cells of CB - derived CD34 + HSC were seeded respectively in two separate single - culture Quantum processes) were cultured in a T25 flask using a fibronectin - immobilized SDF - 1 - coated surface with SCGM medium (Cat.20802 - 0500, CellGenix GmbH, Freiburg, Germany) supplemented with a modified supplement cocktail (1 volume% StemSpan (Registered Trademark) CD34 + supplement with GDNF and SR - 1 added). Both the flask and the QuantumCES were coated overnight at 37°C with a fibronectin - SDF - 1 protein mixture before cell seeding. Hydrodynamically, the flask was in a static state. On the other hand, the Quantum system was perfused overnight (12 - 15 hours) using the Quantum CES "Coat Bioreactor" task (IC inlet rate: 0 mL / min, IC circulation rate: 20 mL / min with Fn / SDF - 1 added, EC inlet rate: 0.1 mL / min with PS added, and EC circulation rate: 30 mL / min, EC outlet). The cells were cultured for 3 days and then in the Quantum® system hollow fiber membrane (HFM) bioreactor for 5 days.

[0145] In the feasibility test, the performance of supporting the proliferation of CB-derived HSCs in single culture was evaluated for two media containing SCF, TPO, Flt-3L, IL-3, IL-6, and GNDF, with or without the SR-1 cocktail. In both experimental arms, 1×10 6 cells were seeded in T25 flasks. On day 3, cell inocula from each flask culture were seeded into the Q1893 (without SR-1) Quantum® system and the Q1894 (with SR-1) Quantum® system.

[0146] On day 8, the harvest yields were 4.49×10 7 cells (98.5% viability) without SR-1 and 5.57×10 7 cells (98.8% viability) with SR-1. Flow cytometry analysis of the phenotype of the cryopreserved hematopoietic stem cell Quantum® system harvests showed that the CD34+ cell fraction was 1.40×10 7 cells, i.e., 31.1% of the total harvest without SR-1, and 2.1×10 7 i.e., 37.7% of the total harvest with SR-1. The minimum and maximum CD34+ doses are 7,000,000 and 10,500,000 cells, respectively.

[0147] Example 2 Single culture proliferation method for cord blood-derived CD34+ HSCs in the Quantum® system

[0148] Embodiments provide an automated expansion protocol for CB-derived CD34+ HSCs in a Quantum® system's dynamic perfusion-based two-chamber semipermeable hollow fiber membrane (HFM) bioreactor using, for example, a novel cytokine cocktail composed of SCF, TPO, Flt-3L, IL-3, IL-6, and fibronectin-SDF-1 coated membranes. The cocktail is supplemented with GDNF and SR-1. Further, the inner capillary (IC) HFM lumen is coated with a mixture of human fibronectin and chemokine SDF-1 to mimic the stimulatory and homing effects of bone marrow-derived mesenchymal stromal cells.

[0149] In a series of tests of this automated expansion protocol, three master lots of preselected mixed CD34+ HSCs derived from thawed cord blood (CB) were grown in a ~2.1 m 6 HFM bioreactor with an IC volume of ~124 mL with an initial cell seeding of 2.0×10 2 CD34+ HSCs. First, the cells are resuspended in SCGM basal medium supplemented with the growth factor cocktail. The cells are thawed in a 37°C water bath, washed with 23 mL of complete medium, and resuspended in 50 mL of complete serum-free GMP SCGM medium (Cat.20802-0500, CellGenix GmbH, Freiburg, Germany). The complete serum-free GMP SCGM medium contains StemSpan (Registered Trademark)Supplemented with CD34 Supplement 10X (Cat. 2691, Stem Cell Technologies, Vancouver, BC, Canada) at 10 ng / mL, glial cell-derived neurotrophic factor (GDNF) (Cat. 212-GD-050, R&D Systems, Minneapolis, MN, USA) at 0.75 μM, StemRegenin 1 (SR-1) (Cat. 72342, Stem Cell Technologies, Vancouver, Canada), and penicillin-streptomycin-neomycin (PSN) antibiotic mixture 100X (Cat. 15640-055, ThermoFisher Scientific, Waltham, MA, USA) at a concentration of 1 volume %. StemSpan (Registered Trademark) CD34 Supplement 10X contains recombinant human FMS-like tyrosine kinase 3 ligand (Flt3l), stem cell factor (SCF), thrombopoietin (TPO), interleukin 3 (IL-3), and interleukin 6 (IL-6). The basal medium can be composed of serum-free GMP SCGM supplemented with SR-1 and PSN antibiotic mixture.

[0150] Prior to seeding the CD34+ HSC inoculum, the Quantum® system HFM bioreactor (21,000 cm 2 S.A.) was coated overnight at 37 °C with a mixture of 5 mg of human plasma-derived fibronectin (or 0.23 - 0.24 μg / cm 2+ -Mg 2+ free PBS (Cat. 17-516Q, Lonza Group, Walkersville, MD, USA)) and 0.075 ng / cm 2 of recombinant human stromal cell-derived factor 1 (SDF-1) (Cat. 6448-SD, R&D Systems, Minneapolis, MN, USA) in a mixed gas (5% CO2, 20% O2, balance N2). 2 ​

[0151] Next, the cells were introduced into the inner capillary loop (e.g., IC loop) of the HFM bioreactor according to a defined perfusion protocol and maintained within the lumen of the bioreactor using a custom countercurrent fluidics program. Using the automated tasks of the Quantum® system (the setup overview of which is shown in Table 1-3 below), CB-derived CD34+ HSCs were, after lumen and extra-capillary media exchange and conditioning, seeded in suspension in a 50 mL of complete medium (serum-free GMP SCGM basal medium containing the following cytokine cocktail: SCF, TPO, Flt-3L, IL-3, IL-6, GDNF, and SR-1) in a coated HFM bioreactor, grown in monoculture, and harvested on day 8 of cell culture. In the above conditioning, a Quantum®-embedded task entitled "Media Conditioning" was used, in which, by means of perfusion-based media circulation in the IE / EC loop of the bioreactor of the Quantum® system, conditioning was performed on the cell growth medium within the CES of Quantum® with the following circulation rates (IC circulation: 100 mL / min, EC circulation: 250 mL / min, EC inlet: 0.1 mL / min) set for at least 10 minutes. Thereby, the mixed gas (20% O2, 5% CO2, balance N2) in the bioreactor medium was equilibrated by gas exchange in the EC loop via the gas transfer module.

[0152]

Table 1

[0153]

Table 2

[0154]

Table 3

[0155]

Table 4

[0156]

Table 5

[0157] The default tasks are used for Quantum (registered trademark) system priming, IC medium / EC medium exchange, and medium adjustment tasks. During the process, glucose and lactate levels were monitored by an i-STAT Analyzer G and CG4+ cartridge (Abbott Point-of-Care, Princeton, NJ). During cell growth, the IC and EC inlet flows of the Quantum (registered trademark) system were adjusted according to the glucose consumption rate and lactate production rate and the type of automated task. This program uses a gas mixture of approximately 5% CO2, approximately 20% O2, and the remainder N2 at approximately 37 °C for only approximately 8 days to reduce (i.e., minimize) T cell differentiation during cell culture. Cells are harvested using an automated suspension cell protocol.

[0158] For example, the Quantum system inlet flow rate ranges from approximately +0.1 to approximately 100 mL / min, and the IC circulation flow rate ranges from approximately -40 to approximately 300 mL / min. During the cell culture process, the corresponding Quantum system EC inlet flow rate ranges from approximately 0 to approximately 148 mL / min, and the EC circulation flow rate ranges from approximately -1.7 mL / min to approximately 300 mL / min. During growth, glucose and / or lactate levels can be analyzed, for example, by an i-STAT analyzer (e.g., Abbott Point-of-Care, Princeton, NJ, USA) using G and CG4+ cartridges. At harvest, the cell count (e.g., using a Vi-CELL (Registered Trademark) XR cell analyzer, Beckman Coulter, Indianapolis, IN, USA) (Figure 17) (including quantification of cell viability by trypan blue) (Figure 18), cryopreserved in CryoStor CS10 cryopreservation medium (e.g., Biolife Solutions, Bothell, WA, USA), and stored in a liquid nitrogen gas phase until the next analysis.

[0159] Proliferation results

[0160] The average harvest was approximately 1.02×10 8 cells (ranging from approximately 4.02×10 7 to approximately 1.61×10 8 cells), and the average cell viability by trypan blue quantification was approximately 95.5% (ranging from approximately 93.3% to approximately 96.8%), determined by a cell viability counter (Vi-CELL™ XR, Beckman Coulter). The cell growth yield of 4.0×10 7 to 1.6×10 8 cells exceeded the minimum CD34+ cell dose of 1.5×10 5 cells / kg for a single unit transplant and the minimum CD34+ cell dose of 1.0×10 5 cells / kg for a two-unit transplant. This is equivalent to the minimum amounts of 1.1×10 7 CD34+ HSCs and 1.4×10 7 CD34+ HSCs for a 70 kg patient for a single unit transplant and a two-unit transplant, respectively.

[0161] The average cell population doubling was approximately 5.4, and the average cell population doubling time was approximately 34.9 hours. The average fold increase was 51.0-fold (ranging from approximately 20.1-fold to approximately 80.5-fold) over the course of the growth period. The IC medium input perfusion flow rate was adjusted in response to glucose and lactate metabolites and ranged from approximately 0.1 to approximately 0.2 mL / min.

[0162] The median cord blood unit (CBU) contained approximately 4.4×10 6 CD34+ HSCs and could contain up to approximately 2.0×10 7 CD34+ HSCs. When using the methods and systems described herein, the average growth yield from a single CBU was, for example, in the complete CBU CD34+ cell fraction, with the cell inoculum ranging from 2.0×10 6 cells to 4.4×10 6 to 2.0×10 7Using an automated 8-day monoculture cell growth protocol that simply increases cells up to the cellular level, it can become CB-derived stem cells or progenitor cells on the order of 2.2×10 8 ~1.0×10 9 This approach can increase the cell seeding density, for example, from 1.6×10 4 cells / mL to 3.6×10 4 ~1.6×10 5 cells / mL in a perfusion bioreactor in particular, and can result in a shorter growth time frame that can reduce the potential for cell differentiation.

[0163] Cryopreservation

[0164] By comparing such CD34 + HSC yields and pre-cryopreservation cell viability for various UCB donors, the relationship between growth yield and pre-cryopreservation cell viability was revealed (Figure 19). BC Vi-CELL (Registered Trademark) The QuantumCES CD34 + cell viability was measured at harvest by trypan blue dye exclusion using an XR Cell Analyzer. There is a wide range in pre-cryopreservation CD34 + cell viability. In the studies of the present inventors, the pre-cryopreservation cell viability ranged from 84% to 98%. The growth of CB-derived CD34 + HSCs in Quantum CES resulted in an average harvested cell viability of 95%.

[0165] Glycolytic metabolism

[0166] As shown by the monitoring results of glycolytic metabolism, the glucose consumption rate ranges from 0 mmol / day to a maximum value (day 5) of 0.596 mmol / day, and the lactate production rate ranges from 0 mmol / day to a maximum value (day 8) of 0.650 mmol / day (Figure 20). The difference in the days showing the maximum values of these two metabolites is thought to be due to the regulation of the medium flow rate, differences in the expression of enzymes controlling the flux of the glycolytic system, and the demand for central biosynthetic metabolites during cell growth.

[0167] Immunophenotype examination

[0168] For each of the three types of CB-derived CD34+ cells automatedly proliferated, 1x10 6 cells of each thawed cell harvest sample were resuspended and washed in complete medium, centrifuged at 500 g for 5 minutes, resuspended in 100 μL of BD Flow Stain Buffer, blocked with 5 μL of human BD Fc for 10 minutes, and then stained with the following conjugate staining solutions: BD Pharmingen anti-human CD45-APC-H7 (Cat.560178), anti-human CD34-APC (Cat.560940), anti-human CD133-PE (Cat.566593), anti-human CD38-BB515 (Cat.564499), anti-human CD41a-APC-H7 (Cat.561422), anti-human CD3-PE (Cat.555333), anti-human CD19-PE (Cat.555413), anti-human CD56 (555516), anti-human CD15-BB515 (Cat.565236), and 7-AAD (Cat.559925). The ISHAGE gating guidelines for counting CD34+ HSCs by flow cytometry may be referred to for the immunophenotypic examination of proliferating cells, and the CD34+ HSC population may be subordinate to the CD45+ parental cell population (Cytometry, 34:61-70, 1998). Furthermore, the CD34+ gating method was verified with a CD-Chex CD34 peripheral blood control (Streck, CD-Chex CD34, level 3). Cell sample data were acquired on a BD FACSCanto II flow cytometer equipped with BD FACSDiva v9.0 software (10,000 events / sample) and subsequently analyzed with FlowJo v10.7 software.

[0169] As shown in Table 1 and FIGS. 21A and 21B, flow cytometry showed that at the time of harvest on day 8 of automated culture, the mean frequency of the CD45+ / CD34+ immunophenotype was 54.3% (51.9 - 57.9%), and the more primitive CD133 + CD38 -It shows that the average frequency of the immunophenotype is 31.8% (25.9 - 39.0%). These results are comparable to other 7-day CD34+ HSC proliferation protocols using SR-1 (CD34+ HSC 10 - 25%) medium and the 21-day CD34+CD38- proliferation protocol using nicotinamide (CD34+ HSC 0.2 - 4.4%) medium for UCB-derived cell culture. The average frequencies of the differentiated cell lineages were 0.5% for lymphocytes (CD3+, CD19+, CD56+), 27.7% for neutrophils (CD15+), and 26.5% for platelets (CD41a+). The fact that biomarkers for both neutrophils and platelets are present in the expanded CB-derived CD34+ HSC population is thought to be partially due to the cytokine composition of the proliferation medium containing interleukin IL-3 and IL-6. Both cytokines are used to support CD34+ cell proliferation but are also involved in the development of myeloid cell lineages.

[0170]

Table 6

[0171] In vitro CB-CD34+ clone differentiation

[0172] The MethoCult™ CD34+ cell differentiation colony-forming unit (CFU) assay is performed using MethoCult™ H4034 Optimum medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with rh-cytokines SCF, GM-CSF, IL-3, G-CSF, and EPO. The cells generated hematopoietic progenitor cell lineages of GEMM, GM, BFU-E CFU.

[0173] Briefly, the UCB-derived CD34+ HSCs harvested with Quantum were washed, resuspended in IMDM containing 2% FBS, diluted in methylcellulose-based medium, vortexed, and seeded into a multiwell plate at seeding densities of 150, 500, and 1,000 cells / well in 1.1 mL / 35 mm well of medium. The CFU plates were incubated for 14 days in an incubator in a static state under conditions of 37 °C, 5% CO2, and humidity, and then the CFUs in each well were manually counted and scored using an Olympus CKX41 inverted microscope with CellSens 2.2 software at an objective magnification of ×4 (n = 6).

[0174] After performing methylcellulose-based cell culture for 14 days in MethoCult Optimum H4034 cytokine medium, the differentiated CFUs of CB-derived CD34+ cells were, among all the CFUs of the three expanded CB-derived CD34+ cell lines, on average 56% for the GM lineage, 23% for the GEMM lineage, and 21% for the BFU-E progenitor cell lineage (see, for example, FIGS. 22 and 23). The results of these CFU examples are that the majority of the lineages are GM-CFU (60%) clones, then BFU-E (36%) and GEMM-CFU (10%) clones, and / or that the majority of both recombinant and non-recombinant clones are GM-CFU (60%), then BFU-E (18-20%) and GEMM-CFU (5%) clones, which is equivalent to prior studies on the methylcellulose H4034 cytokine differentiation of electroporated non-recombinant CB-derived CD34+ HSCs. The differences in the relative distribution of CFU clones among these studies are thought to be due to differences in donor CBU cell source, stem cell selection method, recombinant in some cases, and cytokine cocktail formulations used in the expansion of CB-derived CD34+ HSCs prior to differentiation. Other small molecule supplements formulated with cytokines other than SR-1 may be options for hematopoietic stem cell culture for the purpose of increasing CB-derived CD34+ cell expansion and improving engraftment, and may include nicotinamide (a SIRT1 histone deacetylase and ribosylase inhibitor), valproic acid (a histone HDAC1 inhibitor), and UM171 (an inhibitor of histone HDAC1 deacetylation and LSD1 demethylation).

[0175] The MethoCult™ differentiation assay of the harvested cells can generate hematopoietic progenitor cell lineages of GEMM, GM, and BFU-E CFUs. Taking these results together, it was demonstrated that the automated Quantum® system single culture protocol can support the expansion of pre-selected CB-derived CD34+ hematopoietic stem cells for both single and double CBU dose equivalents while minimizing lymphocyte residual.

[0176] Those skilled in the art will understand that various modifications and changes can be made to the method and structure of the present invention without departing from the scope of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments or examples described above. Rather, the present invention is intended to include modifications and variations within the scope of the following claims and their equivalents.

[0177] Although embodiments and application examples of the present invention have been shown and described, it should be understood that the present invention is not limited to the configurations and resources described above. Various modifications, changes, etc. that are obvious to those skilled in the art can be made in the details of the configuration, operation, method, and system of the present invention without departing from the scope of the present invention.

Claims

1. A method for growing cells, the method comprising: A coating step of applying a coating to the inner side of the capillary tubes of a plurality of hollow fibers of a hollow fiber bioreactor, wherein the plurality of hollow fibers are semipermeable, and the coating is composed of fibronectin configured to adhere to the inner side of the capillary tubes and stromal cell-derived factor-1 (SDF-1) that adheres to the fibronectin and is configured to retain CD34+ hematopoietic stem cells (HSCs), the coating step; An introduction step of introducing a first plurality of cells containing CD34+ hematopoietic stem cells obtained from at least one of cord blood, bone marrow, and peripheral blood into the plurality of hollow fibers of the hollow fiber bioreactor; A concentration step of maintaining and concentrating the first plurality of cells in the plurality of hollow fibers by simultaneously introducing a fluid to both ends of the hollow fiber bioreactor; An exposure step of exposing the first plurality of cells in the hollow fibers to growth conditions; A growth step of introducing a soluble cytokine, an aryl receptor antagonist, and glial cell-derived neurotrophic factor (GDNF) that support growth into the plurality of hollow fibers and bringing them into contact with the first plurality of cells, and growing at least a part of the first plurality of cells in the plurality of hollow fibers of the hollow fiber bioreactor to generate a second plurality of growth target cells containing CD34+ hematopoietic stem cells that have been grown at least 50-fold; having The soluble cytokine that supports the growth includes at least one of stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (Flt-3L), interleukin-3 (IL-3), and interleukin-6 (IL-6). A method for growing cells.

2. In the method for growing cells according to Claim 1, the first plurality of cells are introduced into the hollow fibers without prior purification treatment. A method for growing cells.

3. In the method for growing cells according to Claim 1, the exposure step includes exposing the first plurality of cells to one or more growth factors including FMS-like tyrosine kinase 3 ligand (Flt-3L), stem cell factor (SCF), thrombopoietin (TPO), glial cell-derived neurotrophic factor (GDNF), and combinations thereof. A method for growing cells.

4. In the method for growing cells according to Claim 1, The exposing step includes circulating a cell growth medium inside the capillary of the hollow fiber. A method for growing cells. **Claim 5** In the method for growing cells according to claim 1, The exposing step includes circulating a cell growth medium outside the capillary of the hollow fiber. A method for growing cells. **Claim 6** In the method for growing cells according to claim 1, The introducing step includes: circulating a portion of the first plurality of cells inside the capillary of the hollow fiber; stopping the circulation and contacting one or more of the portion of the first plurality of cells with a first portion of the inside of the capillary of the hollow fiber; rotating the hollow fiber bioreactor 180 degrees from an initial position; circulating another portion of the first plurality of cells inside the capillary of the hollow fiber; stopping the circulation and contacting one or more of the other portion of the first plurality of cells with a second portion of the inside of the capillary of the hollow fiber; comprising A method for growing cells. **Claim 7** In the method for growing cells according to claim 1, The first plurality of cells are introduced simultaneously at both ends of the hollow fiber. A method for growing cells. **Claim 8** In the method for growing cells according to claim 1, The coating further includes a protein portion. A method for growing cells. **Claim 9** In the method for growing cells according to claim 1, The aryl receptor antagonist is at least one of StemRegenin 1 (SR1) and UM171. A method for growing cells. **Claim 10** In the method for growing cells according to claim 1, The soluble cytokine that supports the growth and includes at least one of stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (Flt-3L), interleukin-3 (IL-3), and interleukin-6 (IL-6) is included in a cytokine cocktail having a mass / volume percent concentration of 0.5% to 2%. A method for growing cells. **Claim 11** In the method for growing cells according to claim 1, The GDNF is at a concentration of at least 10 ng / mL. A method for growing cells. **Claim 12** In the method for growing cells according to claim 1, The hollow fiber contains a first culture medium inside the inner part of the capillary tube, and further contains a second culture medium that contacts the outer part of the capillary tube of the hollow fiber. A method for growing cells.

13. In the method for growing cells according to Claim 12, At least one component in the first culture medium inside the inner part of the capillary tube is denser than the concentration of the same component in the outer part of the capillary tube of the hollow fiber. A method for growing cells.

14. In the method for growing cells according to Claim 13, The component is GDNF. A method for growing cells.

15. In the method for growing cells according to Claim 13, The component contains at least one of SR-1, SCF, TPO, Flt-3L, IL-3, IL-6, SDF-1, and fibronectin. A method for growing cells.

16. In the method for growing cells according to Claim 1, By flowing the fluid simultaneously to both ends of the hollow fiber bioreactor, the second plurality of growth target cells are maintained and concentrated in the plurality of hollow fibers. A method for growing cells.

17. In the method for growing cells according to Claim 1, The first plurality of cells contain concentrated CD34+ cells. A method for growing cells.

18. In the method for growing cells according to Claim 1, The first plurality of cells contain at least 2 million cells. A method for growing cells.

19. In the method for growing cells according to Claim 1, The soluble cytokine that supports the growth and contains at least one of stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (Flt-3L), interleukin-3 (IL-3), and interleukin-6 (IL-6) has a volume concentration of 1% at a volume of 7 mL to 10,000 mL and multiples thereof. A method for growing cells.

20. In the method for growing cells according to Claim 1, The introduction step includes introducing 2×10^6 cord blood-derived CD34+ cells. A method for growing cells.

21. In the method for growing cells according to Claim 1, The introducing step includes introducing 1.6×10⁴ cord blood-derived CD34⁺ cells per mL of the complete medium in the inner part of the capillary tube, which contains at least one of SCF, TPO, Flt-3L, IL-3, IL-6, GDNF, and SR-1, into the hollow fiber bioreactor. A method for proliferating cells.

22. In the method for proliferating cells according to Claim 1, the second plurality of proliferating target cells includes at least 1.05×10⁷ CD34⁺ hematopoietic stem cells. A method for proliferating cells.

23. In the method for proliferating cells according to Claim 1, the introducing step includes introducing the first plurality of cells only into the first end of the plurality of hollow fibers and simultaneously introducing a medium in a countercurrent manner into the second end of the plurality of hollow fibers, wherein the first end is the end opposite to the second end. A method for proliferating cells.

24. In the method for proliferating cells according to Claim 1, the introducing step includes introducing the first plurality of cells only into the first end of the plurality of hollow fibers without introducing them in a countercurrent manner into the second end of the plurality of hollow fibers, wherein the first end is the end opposite to the second end. A method for proliferating cells.

25. In the method for proliferating cells according to Claim 1, the coating step performs the coating by ultrafiltration. A method for proliferating cells.

26. In the method for proliferating cells according to Claim 1, by simultaneously introducing a fluid into both ends of the hollow fiber bioreactor, the soluble cytokines that support the growth are maintained and concentrated in the plurality of hollow fibers. A method for proliferating cells.

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