Three-dimensional stem cell expansion bioreactor

Abstract

A novel bioreactor to efficiently expand cells in space. A two-chamber system where the chambers are separated by a porous membrane that retains the cells in one chamber but permits nutrients and wastes to pass through freely between chambers. Media can be exchanged in the non-cell chamber without disturbing the cells in the cell chamber or subjecting them to fluid shear. The culture cassette includes a larger cell chamber, no sharp corners to prevent dead spaces, fittings to inject and manipulate fluids through the chambers without the need for needles. The cell chamber has been outfitted with a thin plastic film that permits gas exchange. The two-chamber system can be mounted into a tray that includes a fluidic loop to circulate fresh media through the non-cell chamber using a dedicated peristaltic pump. To monitor the cell expansion cycle, pH and OD sensors are integrated into each bioreactor tray.

Description

[0001] THREE-DIMENSIONAL STEM CELL EXPANSION

[0002] BIOREACTOR

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 539,227, filed September 19, 2023.

[0005] STATEMENT OF GOVERNMENT INTEREST

[0006] This invention was made with government support under grant number NNJ 13ZBG00 IN awarded by NASA. The government has certain rights in the invention.

[0007] FIELD OF INVENTION

[0008] This invention relates to methods and devices for cell expansion. More specifically, the invention relates to a low-shear, three-dimensional cell expansion bioreactor and methods for expanding cells using bioreactors.

[0009] BACKGROUND OF THE INVENTION

[0010] The transplantation of human stem cells for the treatment of human disease and regenerative medicine is an exciting area of research and development with enormous potential. To date, the Federal Drug Administration (FDA) has approved over 20 cellular-based products from human sources for clinical use, with most of these products based on hematopoietic stem cells or genetically modified T-cell applications. Extensive stem cell clinical trials are underway that would indicate that many more future FDA-approved cell therapy products are on the horizon. Stem cells can be derived from a variety of sources, including embryonic stem cells, induced pluripotent stem cells (iPSCs), and multipotent hematopoietic stem cells (HSCs) from bone marrow, mobilized peripheral blood (mPB) and umbilical cord blood (UCB). HSC transplantation has become the standard of care for numerous medical conditions and is being studied extensively for applications in regenerative medicine and the treatment of autoimmune diseases.

[0011] Sources of HSCs for allogeneic transplant are bone marrow (BM), mPB, and UCB, with each source having advantages. Unmanipulated umbilical cord blood (UCB) has been used in over 40,000 HSC transplants worldwide since the late 1980s. UCB has an advantage over other stem cell sources in that human leukocyte antigen (HLA) matching can be less stringent and graft versus host disease (GVHD) is a lower risk. However, UCB has a low total number of cells collected, and a double transplant is typically required for adults, adding cost, and making HLA matching more difficult. Cellular expansion of UCB could eliminate this disadvantage while enhancing clinical outcomes.

[0012] SUMMARY OF THE INVENTION

[0013] A novel bioreactor to efficiently expand cells in space. A two-chamber system where the chambers are separated by a porous membrane that retains the cells in one chamber, but permits nutrients and wastes to pass through freely between the chambers. With this system, cells can be cultured in one chamber and the other chamber kept cell-free. Media can be exchanged in the non-cell side without disturbing the cells in the cell chamber or subjecting them to fluid shear. The two-chamber system forms part of a culture cassette. The culture cassette includes a larger cell chamber, with no sharp corners to prevent dead spaces during media transfer, and fittings to inject and manipulate fluids through the chambers without the need for needles. Mesh supports can be utilized for the integrity of membranes and films for the chambers, if necessary, and the non-cell chamber can be smaller than the cell chamber (e.g., one-fourth the volume) to support more rapid molecular mass transport requirements. The cell chamber has two large surface area sides with one side having the porous membrane separating the cell chamber from the non-cell chamber and the opposing side of the cell chamber being outfitted with a thin plastic film (e.g., FEP Teflon) that permits gas exchange (O2 and CO2), while minimizing water loss and maintaining a completely sealed, sterile interior environment.

[0014] The two-chamber system has been designed to support non-invasive optical density (OD) measurement through the thin plastic film of cell density and pH and can be manufactured as a single-use, relatively low-cost bioreactor system. The two-chamber system can be mounted into a tray that includes a fluidic loop to circulate fresh media through the non-cell chamber using a dedicated peristaltic pump. To monitor the cell expansion cycle, pH and OD sensors are integrated into each bioreactor tray.

[0015] Also provided are methods of expanding a population of cells. The methods can include the steps of providing a population of cells from a subject; transporting the cells to a lower gravity environment; placing the cells in fresh media; incubating the cells in an environment providing media perfusion to the expanding cell population; and recovering cells following incubation. In a particularly advantageous embodiment the method(s) expands the cells in the novel bioreactor employing the two-chamber system as disclosed herein.

[0016] In a first aspect the present invention provides a two-chamber cell culture system. The system can include (1) a first chamber adapted to expand a plurality of cells, (2) a second chamber in fluid communication with the first chamber, and (3) a porous membrane separating the first chamber from the second chamber. The porous membrane has a pore size that allows for the exchange of media from one chamber to another chamber, but prevents the passage of cells from the first chamber to the second chamber.

[0017] In certain embodiments the two-chamber cell culture system can include a media reservoir to hold an additional supply of media. The media reservoir can be in fluid communication with the second chamber of the first aspect. The system can also include a pump, such as a peristaltic pump, to move media from the reservoir to the second chamber. In an advantageous embodiment the volume of the media reservoir is about two times to about five times (most preferably about five times) the volume of the first chamber.

[0018] The first chamber is preferentially devoid of sharp angles or corners. The lack of sharp corners protects the cells from damage during expansion.

[0019] In an advantageous embodiment the porous membrane has a pore size of about 3 pm or smaller. The pore size is designed to prevent the passage of cells from the first chamber to the second chamber, while allowing the exchange of media and waste between chambers.

[0020] The two-chamber cell culture system can include a mesh support system surrounding the two- chamber cell culture system. The mesh support provides support for the system and prevents rupture or breakage of the chambers. The two-chamber cell culture system can also include a thin plastic film (e.g., FEP Teflon) that permits gas exchange (O2 and CO2) on the side of the cell chamber opposite the permeable membrane and a noninvasive optical density (OD) measurement system to measure the pH and / or the cell density of the first chamber.

[0021] In an advantageous embodiment the volume of the first chamber is about 30 to about 200 mis, preferably about 45 to about 100 mis, more preferably about 50 to about 70 mis, or most preferably about 55 mis or greater. The volume of the second chamber can be about one-third to one-seventh the volume of the first chamber. Accordingly, the volume of the second chamber can be about 10- 20 mis where the volume of the first chamber is about 55 mis.

[0022] The two-chamber cell culture system according to the first aspect can further include one or more of the following; (1) a media supply bag to supply additional media to the two-chamber cell culture system; (2) a pump to pump media from the media bag to the second chamber of the the two- chamber cell culture system; (3) a sensor to monitor the pH or optical density of the media in the first chamber of the two-chamber cell culture system; and / or (4) a heater element to control the temperature of the media in the first chamber. In a second aspect the present invention provides a method of expanding a population of cells. The method can include the steps of (1) providing a population of cells from a subject; (2) transporting the cells to a lower gravity environment, wherein the lower gravity is relative to the standard gravity of earth; (3) placing the cells in fresh media; (4) incubating the cells in an environment providing media perfusion to the expanding cell population; and (5) recovering cells following incubation. The method of expanding a population of cells according to the second aspect is advantageously performed in a two-chamber cell culture system according to the first aspect. The lower gravity environment can be a low earth orbit or higher orbit.

[0023] In an advantageous embodiment the population of cells from the subject is cryopreserved prior to transport and the cells are washed to remove cryoprotectant prior to placing the cells in fresh media.

[0024] During the incubating step, the cells can be incubated in a system providing automated media perfusion. Automated media perfusion provides fresh media to the expanded cells and removes spent media and waste from the incubation environment of the first chamber. The method according to the second aspect can further include the steps of placing the recovered cells in a cryopreservative and freezing the cells for transport away from the lower gravity environment following the recovering step. In addition, about 70% to about 95% of the cells can be removed at the recovering step and the remaining cells can be placed in fresh media and subjected to a second round of incubation and expansion. This provides a second round of expansion and increases the yield of cells. Similarly, about 70% to about 95% if the cells can be removed at a second recovering step and the remaining cells can be placed in fresh media and subjected to a third incubation and expansion.

[0025] Alternatively, about 90% of the cells can be removed at the recovering step and the remaining 10% of the cells are placed in fresh media and subjected to a second round of incubation and expansion. Likewise, about 90% of the cells can be removed at a second recovering step and the remaining 10% of the cells can be placed in fresh media and subjected to a third incubation and expansion.

[0026] The lower gravity environment is reduced gravity, microgravity or lower than microgravity gravity environment.

[0027] In a particularly advantageous embodiment the expanded cells are hematopoietic stem cells. However, the methods taught herein can be used to expand a wider variety of cells, including induced pluripotent stem cells (iPSCs) and other stem cells as discussed herein. Similarly, this technology could apply to literally any suspended cell culture used for research or manufacturing (e.g. CHO cells).

[0028] In a third aspect the present invention provides additional embodiments of a two-chamber cell culture system. The system according to the third aspect can include (1) a first chamber adapted to expand a plurality of cells; (2) a second chamber in fluid communication with the first chamber; and (3) a porous membrane separating the first chamber from the second chamber. The second chamber has a volume about one-sixth to one-third of the volume of the first chamber and the second chamber is adapted to facilitate the exchange of media from the first chamber where the media is used to expand cells in the first chamber. The porous membrane has a pore size that allows for the exchange of media from one chamber to another chamber, but prevents the passage of cells from the first chamber to the second chamber.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

[0031] FIG. l is a drawing showing an isometric view of the bottom of a bioreactor. Perfusion ports are shown extended on media side for connection to the fluid loop of a tray. Cell side ports can be extended for extraction.

[0032] FIG. 2 is a drawing showing an exploded isometric view of a bioreactor seen from the media side. The bioreactor is a dual-chamber system divided vertically into cell and media sides, separated by a permeable membrane.

[0033] FIG. 3 is a drawing showing internal features of a bioreactor according to aspects of the invention.

[0034] FIG. 4 is a black and white line drawing of the bioreactor shown in FIG. 3.

[0035] FIG. 5 is a pair of drawings ((A) and (B)) showing an exploded view of a bioreactor with the center membrane. The upper image (A) is in gray scale, while the lower image (B) is a black and white line drawing.

[0036] FIG. 6 is a pair of drawings showing an isometric view of a BioServe In-Space Cell Expansion Platform (BICEP) tray. (A) The BICEP tray houses the cell expansion bioreactor along with temperature control system, media supply, peristaltic pump, sensors and control electronics. The tray also provides an additional level of containment while enabling gas exchange for oxygen and CO2. FIG. 6 (B) shows the BICEP tray of (A) in black and white line drawing.

[0037] FIG. 7 is a drawing in gray scale showing a top plan view of a BICEP tray show in in FIG. 6(A). FIG. 8 is a black and white line drawing showing a top plan view of a BICEP tray show in in FIG. 6 (B).

[0038] FIG. 9 is an illustration showing a Space Automated Bioproduct Laboratory (SABL) incubator. Four BICEP trays slide into each SABL incubator with atmosphere control module (ACM) CO2 control system in the rear of the incubator.

[0039] FIG. 10 is a schematic showing a top plan view of an open BICEP tray cell culture cassette, peristaltic pump and primary fluid lines and sensors.

[0040] FIG. 11 is an image demonstrating Jurkat cell expansion using a dual-chamber bioreactor setup with peristaltic pump to demonstrate proof-of-concept. Cell density started at 105cells / ml were grown in RPMI 1640 media with 10% FBS.

[0041] FIG. 12 is a graph showing the results of Jurkat cell expansion using a dual-chamber bioreactor setup with peristaltic pump to demonstrate proof-of-concept. Cell density started at 105cells / ml were grown in RPMI 1640 media with 10% FBS. The graph shows the total number of cells and viability results for BICEP configuration compared with standard flask culture.

[0042] FIG. 13 is a set of drawings of a bioreactor according to aspects of the invention. The bioreactor is seen from the top and the bottom.

[0043] FIG. 14 is a set of drawings showing internal features of the bioreactor. (A) An isometric view of the bottom of a bioreactor. Perfusion ports are shown extended on media side for connection to BICEP tray fluid loop. Cell side ports can be extended for extraction. (B) A cross-section view of the bioreactor.

[0044] FIG. 15 is a drawing showing an exploded view of a bioreactor with the center porous membrane.

[0045] FIG. 16 is a drawing depicting an exploded view of a BICEP tray.

[0046] FIG. 17 is a drawing showing the fluid loop of a BICEP tray including the bioreactor.

[0047] FIG. 18 is a drawing showing some of the components of the fluid loop of a BICEP tray including the bioreactor.

[0048] FIG. 19 is a set of drawings showing parts of a BICEP tray including the bioreactor interface and heater of the tray.

[0049] FIG. 20 is a drawing showing parts of a BICEP tray including the bioreactor interface and heater of the tray.

[0050] FIG. 21 is a drawing showing aspects of the bioreactor tray. FIG. 22 is a set of drawings showing the BICEP tray interface with Space Automated Bioproduct Laboratory (SABL). BICEP trays are housed in SABL via a drawer-style rack. BICEP trays can be individually installed / removed, with power & data mated / de-mated. Temperature is controlled by SABL. The CO2 environment is controlled by ACM (in back of SABL).

[0051] FIG. 23 is a drawing showing the BICEP tray interface with Space Automated Bioproduct Laboratory (SABL). BICEP trays are housed in SABL via a drawer-style rack. BICEP trays can be individually installed / removed, with power & data mated / de-mated. Temperature is controlled by SABL. The CO2 environment is controlled by ACM (in back of SABL).

[0052] FIG. 24 is a of drawing showing an exploded view of the BICEP interface with SABL. The exploded view shows the orientation of BICEP and SABL components.

[0053] FIG. 25 is a schematic showing the SABL - ACM - BICEP tray architecture.

[0054] FIG. 26 is a schematic showing the SABL - ACM - BICEP interfaces and thermal environment.

[0055] FIG. 27 is a schematic showing the SABL power / data hub architecture. Power and Data Distribution between SABL and each BICEP Science Tray as well as ACM (CO2 control) Power- Data Hub: (1) Power supply / switching each tray (reboot, power-down for tray removal); (2) USB hub / data routing;(3) Fan air circulation assist. Each TRAY independently controlled (pProcessor, sensors, actuators): (1) Data acquisition (p, rH, T, CO2, pH(media), pH / OD(culture), local storage and pass-through USB to SABL / Earth; (2) Ctrl: Local heater control at culture (37°C, adjustable); (3) Ctrl: Media circulation (pump on / off); (4) Each Science Tray usable individually in standard lab incubators (at »35°C) for ground controls using a dedicated interface “hub” (laptop optional). ACM: CO2 control using existing architecture (SABL).

[0056] FIG. 28 is a schematic showing the BICEP tray electrical operations and power / heat thermal control.

[0057] FIG. 29 is a schematic showing the science tray architecture.

[0058] FIG. 30 is a schematic showing tray actuators and sensors.

[0059] FIG. 31 is a set of graphs and images showing tray sensors for optical sensing of pH and optical density.

[0060] FIG. 32 is a set of graphs and images showing optical pH and optical density flow through a cell.

[0061] FIG. 33 shows two images. (A) BICEP components configured onto a flat sheet (see (B)) and placed in a cylinder for testing of clino-rotation as a means to simulate microgravity and to potentially provide a terrestrial alternative to microgravity cell expansion. The cylinder in set on a Thermo Fisher bottle roller, which rotates the cylinder at about 10 rpm. The bottle roller and the integrated cell expansion bioreactor are placed in a standard laboratory CO2 incubator for operation / expansion. (B) BICEP components for clino-rotation without the cylinder for clarity. The version shown in the picture includes a smaller bioreactor to the right side of the BICEP, with a media bag in the center of the BICEP. The non-cell chamber of the bioreactor is connected through fluid lines to the peristaltic pump on the left side and the media bag in the center. An electronics breadboard is included on the back side (not visible), along with a high-capacity battery to power the system while it is rotating.

[0062] FIG. 34 is a black and white line drawing of the embodiment of the bioreactor show in FIG. 2.

[0063] FIG. 35 is a black and white line drawing of the embodiment of the bioreactor show in FIG. 13.

[0064] FIG. 36 is an image a fully loaded bioreactor and media bag assembly (MBA).

[0065] FIG. 37 is an image a fully loaded bioreactor and media bag assembly (MBA).

[0066] FIG. 38 is a BICEP tray inside the ISS Life Sciences Glovebox (LSG), which is used to maintain sterility during assembly.

[0067] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0068] An in-space cell expansion platform is contemplated to perform a plurality of controlled cell expansions in multiple cell culture vessels, where each vessel, or bioreactor, employs a two- chamber cell culture system. The two-chamber cell culture system in the in-space cell expansion platform is a core piece of the technology that operationally enables the full system (i.e., the inspace cell expansion platform).

[0069] With reference to FIGS. 1, the in-space cell expansion platform is optimized through the features of the two-chamber bioreactor (10) as taught herein. The two-chamber bioreactor (10) has an upper clamp plate (12) and a lower clamp plate (22). A thin plastic film (20) (e.g., FEP Teflon) is affixed to the lower clamp plate (22). The film (20) forms a side of the cell chamber and permits gas exchange (O2 and CO2) while minimizing water loss and maintaining a completely sealed, sterile interior environment. The lower clamp plate (22) has an opening that exposes the surface of the film (20) to facilitate gas exchange and optical measurements, such as for measurements of optical density of the cells.

[0070] With reference to FIG. 2, a first chamber of the two-chamber bioreactor device houses cells, such as the hematopoietic stem cells. A second chamber of the two-chamber bioreactor is separated from the first chamber by a porous membrane (16). The first chamber (cell chamber) is formed by the space between the film (20), the cell side center plate (18), and the porous membrane (16). The second chamber (non-cell chamber) is formed by the space between the non-cell side (media side) center plate (14) and the porous membrane (16). A set of assembly fasteners (26) engages the upper clamp plate (12) and the lower clamp plate (22) to form an assembled two-chamber bioreactor (10).

[0071] With reference to FIG. 3, there is shown a disassembled two-chamber bioreactor (10). For the purposes of illustration, the porous membrane (16) and the assembly fasteners (26) are not shown. The cell chamber 18(a) can be seen in the lower half (10b) of the disassembled bioreactor (10) and the non-cell chamber 14(a) or media chamber can be seen in the upper half (10a) of the disassembled bioreactor (10).

[0072] FIG. 4 shows a black and white line drawing of the disassembled two-chamber bioreactor (10) shown in FIG. 3. The bioreactor (10) has a first cell chamber (18a) adapted to expand a population of cells, such as hematopoietic stem cells. The first chamber (18a) is separated from a second chamber (14a) by a porous membrane (16 - not shown). The porous membrane (16) has pores of sufficient size (e.g., 0.4pm to 3 pm) to allow media with nutrients and additives to diffuse between the chambers, but too small for cells to pass from the first chamber to the second chamber. The outer facing surfaces (20) of the cell chamber (i.e., the portion that is not co-extensive between the two chambers) can be constructed of an FEP teflon film. The outer walls of the chambers may further include a mesh support to guard against rupturing of the porous membrane during operation, removal or further storage. The bioreactor (10) also includes a pair of inlet Luer lock needleless ports (29) and a pair of outlet Luer lock needleless ports (27).

[0073] The second chamber is a small reservoir that holds media that is continuously recirculated from a larger media bag. This configuration leaves the hematopoietic stem cells undisturbed in the first chamber, but allows for the replenishment of fresh media and removal of spent media and other un-wanted by-products.

[0074] The present invention provides a novel bioreactor to efficiently expand cells in space. A modified version of the bioreactor, using similar principles, is also developed and disclosed herein for Earthbased cell expansion applications.

[0075] In certain aspects, the present invention provides a two-chamber version of the bioreactor space flight hardware. The two chambers are separated by a porous membrane that retains the cells in one chamber, but permits nutrients and wastes to pass through freely between chambers. With this culture device, cells can be cultured in one chamber and the other chamber kept cell-free. Media can be exchanged in the non-cell side of the bioreactor without disturbing the cells in the cell chamber or subjecting them to fluid shear. Key features of the design and culture cassette include a larger cell chamber, no sharp corners in the chambers to prevent dead spaces during media injection in the non-cell chamber (see e.g., FIG. 8(A)), fittings to inject and manipulate fluids through the chambers without the need for needles(see e.g., (24) in FIG. 3), mesh support of membranes and films, if necessary, and a smaller non-cell chamber to support more rapid molecular mass transport requirements. The cell chamber can be outfitted with a thin plastic film (20) (e.g., FEP Teflon) that permits gas exchange (O2 and CO2) while minimizing water loss and maintaining a completely sealed, sterile interior environment. The bioreactor has been designed to support non-invasive optical density (OD) measurement of cell density and pH. The bioreactor can be manufactured as a single-use, relatively low-cost bioreactor system. The two-chamber bioreactor can be mounted into a tray for containment and environmental control. The tray can include a fluidic loop to circulate fresh media through the non-cell chamber using a dedicated peristaltic pump. To monitor the cell expansion cycle, pH and OD sensors can be integrated into each bioreactor tray.

[0076] The trays will be inserted into another piece of hardware, BioServe’s Space Automated Bioproduct Laboratory (SABL), for use in microgravity or a standard cell culture incubator for use in a terrestrial lab. SABL or a similar piece of equipment can provide temperature and CO2 control, along with power and data interfaces to each tray. Multiple trays can be interfaced to a single SABL incubator through a common power and data breakout box located on the side of the SABL’s temperature and gas-controlled Science Research Module (SRM).

[0077] This present specification outlines the key features and design considerations for the BICEP system, including the cell culture chamber, media circulation, monitoring sensors, and integration with the BioServe Space Automated Bioproduct Laboratory (SABL) incubator. The main premise of BICEP is that suspended cells in microgravity (e.g., hematopoietic stem cells) can be suspended in a quiescent, extremely low-shear environment, where cells can grow and divide without mechanical load or perturbation. The system is designed to allow cultures to expand without the possible negative impacts of fluid shear that can cause cells to differentiate, or alter gene expression or DNA integrity.

[0078] The cell culture chamber in the BICEP system is a crucial element. The novel two-chamber bioreactor taught herein was developed to support the NTH ImmunoSenescence, NSF Cardiomyocyte Spheroid (Project EAGLE), and other previously flown payloads. One chamber is used for cell culture (the cell chamber) and the other for perfusing media (the non-cell chamber). The two chambers are separated by a porous membrane that retains the cells, but allows ready transport of media components (e.g., salts, nutrients, growth factors, dissolved oxygen, etc.) between chambers and removal of waste products (e.g., lactate, carbon dioxide, etc.) by diffusion from the cell chamber to the non-cell chamber. The porous membrane can be made of polycarbonate tracked etch (PCTE), polyethylene tracked etch (PETE), mixed cellulose esther (MCE) or other materials and have pore sizes up to 3pm.

[0079] To meet the volume requirements for cell expansion, the bioreactor’s cell chamber will be substantially larger (e.g., a minimum volume of 55mls). The chamber’s design will eliminate corners, ensuring easy addition and removal of media and cells with uniform laminar flow without creating “dead” spaces where media and nutrient exchange is compromised. Fittings for fluid manipulation will be upgraded to needleless ports using self-sealing, simple Luer-lock fittings, replacing pierceable septa that would otherwise require needles. The latter will be beneficial to minimize crew operation and potential contamination and greatly reduce shear on HSCs that are injected prior to expansion and removal of HSCs during harvest operations. Additionally, prototype testing will evaluate the need for mesh support on the chamber's exterior fluorinated ethylene propylene (FEP) Teflon films and / or inner porous membrane to prevent rupture during use.

[0080] The bioreactor system incorporates a non-cell chamber designed to hold a smaller volume (e.g., a volume of 10-20mls) than the cell chamber to support rapid media exchange and waste removal with predominantly diffusive molecular mass transport across the porous membrane into and out of the cell chamber. The media in the non-cell chamber can circulate continuously or periodically, as culture needs require, facilitating efficient nutrient supply and waste removal, while minimizing molecular gradients within the cell chamber. The system aims to maintain a high cell density of up to or in excess of 5 x 106cells / ml, while ensuring the media in both the cell and non-cell chambers remains optimal for cell growth and health.

[0081] Materials selection, cleaning protocols, and assembly processes for the bioreactor system align with anticipated current Good Manufacturing Practice (cGMP) requirements. The cell chamber can incorporate a non-invasive optical density (OD) measurement capability to monitor cell density and / or pH. The system can be manufactured for single use as a relatively low-cost bioreactor, with each bioreactor cell chamber system discarded after use. This eliminates risk of possible cross-donor HSC contamination.

[0082] The bioreactor cell culture cassette can be mounted into a tray. The tray stores and circulates media to support the expanding population of cells, such as hematopoietic stem cells (HSCs). The tray system provides a second level of containment, required for spaceflight application, in addition to the cell culture cassette, media bag, and fluid lines. Four bioreactor trays can fit into a single SABL incubator, which features temperature (37°C ± 0.5°C) and CO2 (5% ± 0.25%) control.

[0083] Each bioreactor tray can have a peristaltic pump for simultaneous media circulation in and out of the non-cell chamber. The pump can be configured to not contact the media directly. The pump maintains a neutral volume balance within the cell cassette. The media bag, with a volume of up to 5 times that of the cell chamber (e.g., 200mls), ensures an adequate supply of media throughout the expansion cycle. Using a large media supply, the system can operate autonomously over a full duration of cell growth and expansion without having to add or exchange additional media. The media bag can be made from FEP Teflon film to enable the entire media contents to equilibrate with the gas composition of the BICEP enclosure, including, most importantly, oxygen and carbon dioxide (typ. 5%). The bioreactor trays can include integrated OD and colorimetric sensors for monitoring cell density and pH levels. These sensors, calibrated to cell density and phenol red color shift, respectively, will provide critical feedback for determining the end of the growth cycle.

[0084] The bioreactor tray design includes built-in electronics to control the peristaltic pump, control the precise temperature of the bioreactor, and measure and record sensor values. A common power and data breakout box (power / data hub) located within the SABL Science Research Module (SRM) provides the interface between multiple bioreactor trays (up to four operating simultaneously) and the SABL incubator.

[0085] The bioreactor system represents a significant advancement in stem cell expansion technology in microgravity. With its larger cell culture chamber, improved fluid manipulation, advanced monitoring sensors, and integration with the SABL incubator, the bioreactor system aims to achieve high cell density, maintain optimal media conditions, and ensure the reliable and efficient expansion of hematopoietic stem cells. By meeting anticipated cGMP requirements and incorporating single-use, low-cost bioreactor technology, the BICEP system holds great promise for stem cell research and production applications in space exploration and manufacturing, respectively.

[0086] Example 1 - Earth-Based Microgravity Simulation System with Clino-Rotated Cylinder Integration

[0087] The Earth-Based Microgravity Simulation System with Clino-Rotated Cylinder Integration is an innovative technology designed to replicate certain aspects of microgravity conditions experienced in space for stem cell expansion and research and manufacturing purposes. This system utilizes a cylindrical configuration that undergoes clino-rotation, allowing the creation of a simulated microgravity environment on Earth. The system incorporates key design features from the space-based bioreactor system, including the cell culture chamber, fluid manipulation, monitoring sensors, and single-use bioreactor concept. A key to the success of the Earth-based system is that suspended cells (e.g., hematopoietic stem cells) remain suspended in a low-shear condition throughout growth and expansion. As with the microgravity configuration of the system, the clino-rotated version allows cells to grow without significant mechanical load. The system differs from the microgravity configuration in that clinostat conditions are not identical to microgravity, primarily because gravity is still present, suspended cells do move within the media, and the rotation does produce some shear.

[0088] Cell Culture Chamber: The cell culture chamber in the earth-based microgravity simulation system is exactly based on the two-chamber bioreactor design used in the BICEP system. To meet the volume requirements for cell expansion for possible clinical application, the chamber will be significantly larger than previously designs for other bioreactor payloads, with a minimum volume of 55mls, as specified in the space-based system. The chamber will feature a modified design with smooth curves and no sharp corners to facilitate easy addition and removal of media and cells. Fittings for fluid manipulation will also be upgraded to needleless ports with Luer-lock fittings.

[0089] Clino-Rotated Cylinder Integration: The core element of the Earth-based microgravity simulation system is the integration of a large cylinder, or set of cylindrical wheels, that undergoes clino- rotation. This clino-rotated configuration creates a controlled environment that emulates certain microgravity conditions on Earth. By subjecting the cell culture chamber to continuous rotation along the longitudinal axis, gravitational forces are averaged to zero, allowing cells to experience a simulated microgravity environment. Ideally, this is done with no gas bubbles in the cell chamber so that the cell cassette with cells and media can move in solid body rotation. Cells under this condition remain suspended, but still move through the media in a circular fashion dependent on their sedimentation rate and the rate of rotation.

[0090] Media Circulation and Support: Like the space-based system, the Earth-based microgravity simulation system incorporates a non-cell chamber designed to support rapid molecular mass transport requirements. The media in this chamber will circulate continuously through a media supply bag, ensuring efficient nutrient supply and waste removal. The system maintains a high cellular density while preventing the media from becoming spent and unhealthy for the cells. The specific volume ratios and circulation rates, or duty cycle, are optimized for the simulated microgravity conditions generated by the clino-rotated cylinder. Monitoring Sensors and Control: The Earth-based microgravity simulation system includes integrated sensors to monitor key parameters for cell growth and culture conditions. Optical density (OD) sensors, like those used in the space-based system, measure cell density. Additional sensors for pH and other relevant parameters can be integrated to provide comprehensive monitoring capabilities. The sensor data provides feedback on expansion kinetics and ensures optimal conditions for stem cell expansion are maintained within the simulated microgravity environment.

[0091] Power and Data Interfaces: The Earth-based microgravity simulation system is designed with power and data interfaces to enable continuous automated operation during rotation. Power is provided to the rotating system’s pump and electronics either by using (1) power interface slip ring, (2) integrated high-energy battery, or (3) inductive power transfer. Sensor and system data is transferred by wireless communication. Rotation of the system is achieved either using a direct drive motor or through a roller-bottle or roller-bottle-like device.

[0092] Single-Use bioreactor Concept and Cylindrical Design: Consistent with the space-based system, the Earth-Based Microgravity Simulation System is designed as a single-use bioreactor system. Each cell culture chamber, along with its associated wetted components, can be disposed of after use to maintain sterility and prevent cross-contamination. The system is designed to allow for efficient and cost-effective manufacturing, while ensuring compatibility with the simulated microgravity conditions generated by the clino-rotated cylinder or wheel approach.

[0093] Applications and Benefits: The Earth-based microgravity simulation system with clino-rotated cylinder configuration offers numerous applications and benefits. It provides researchers and scientists with a controlled terrestrial environment, similar to what can be attained during spaceflight, to efficiently expand stem cells under simulated microgravity conditions, enabling an improved physiological environment, and potential applications in cellular therapy, regenerative medicine and tissue engineering. The system’s compatibility with the single-use bioreactor concept ensures simplicity, sterility, and cost-effectiveness, making it an attractive option for various research and potential cellular clinical production applications.

[0094] Example 2 - Expansion of HSCs in Microgravity

[0095] The present invention provides methods and an HSC transplantation product that is expanded in space and returned to Earth to treat patients with a variety of hematological malignancies and disorders. The product is based on expanding CD34-enriched cell populations derived and isolated from UCB or mobilized peripheral blood (mPB) HSCs. The proposed system provides for in-space-enhanced cell therapy, starting with cells provided by a patient / subject, expanding the cells in space using an FDA-approved cGMP process, and then returning them to the customer. In addition, the goal of in-space or terrestrial clinostat cellular expansion would be to create a clinical cellular product that improves overall patient survival both in the short-term and long-term.

[0096] The number of allogeneic HSC transplants in the US and worldwide has increased by 7% per year between 2006 and 2016, with UCB transplants in decline. Companies and institutions are working to develop expanded HSC UCB products, which could become more favorable than other sources and allow more people to receive treatment. HSC treatments have been proven efficacious and can be used to treat multiple conditions. The global prevalence of rheumatoid arthritis and other autoimmune diseases is growing, with as many as 20 million people suffering. Gene-edited and subsequently expanded HSCs could provide a curative cellular-based therapy for rheumatoid arthritis and other inherited autoimmune diseases.

[0097] Expanding umbilical cord blood (UCB) cells either in space or on Earth with simulated microgravity offers several potential competitive advantages over traditional cell culture methods. Some of the key advantages include:

[0098] 1. Enhanced Stem Cell Expansion: Microgravity environments, whether in space or simulated on Earth, have shown the potential to enhance the expansion of UCB cells. Reduced gravitational forces and the absence of hydrostatic pressure have been shown to increase proliferation rates.

[0099] 2. Improved Differentiation Potential: Microgravity conditions have been observed to influence stem cell behavior and differentiation potential. UCB cells expanded in microgravity environments should exhibit greater multipotency and reduced differentiation compared to cells cultured under normal gravity conditions. This advantage would have significant implications for the development of advanced cell-based therapies and tissue engineering applications.

[0100] 3. Mimicking Native Tissue Environment: Microgravity or clinostat environments can provide a three-dimensional culture setting that better mimics the native tissue environment compared to traditional two-dimensional cell culture methods. This closer representation of the physiological conditions can improve the functionality and behavior of HSCs, making them more suitable for clinical applications.

[0101] 4. Increased Production Efficiency: Expanding UCB cells in space or simulated microgravity on Earth can potentially offer higher production efficiency compared to terrestrial cell culture methods. The optimized growth conditions and reduced differentiation potential can lead to a superior cell therapy product that can improve treatment outcomes for a wide variety of patients.

[0102] 5. Unique Research Opportunities: Conducting UCB cell expansion experiments in space or simulated microgravity environments opens unique research opportunities. Microgravity provides a novel setting to study cell behavior, gene expression, signaling pathways, and other cellular processes that can be difficult to replicate on Earth. These insights will contribute to a better understanding of stem cell biology and enable the development of more advanced and tailored cell therapies.

[0103] The unique microgravity environment, whether in space or simulated on Earth, offers several potential expanded applications for umbilical cord blood (UCB) stem cells. Some of the potential applications include:

[0104] 1. Regenerative Medicine: UCB stem cells have the potential to be manipulated to become pluripotent stem cells and subsequently differentiated into various cell types, including those found in different tissues and organs. UCB hematopoietic stem cells may be advantageous as a source for induced pluripotent stem cells. Microgravity environments can enhance the expansion and proliferation of UCB induced pluripotent stem cells, making them a valuable resource for regenerative medicine. These cells could be used to regenerate damaged tissues, repair injured organs, or replace malfunctioning cells.

[0105] 2. Tissue Engineering: Microgravity provides a more favorable culture environment for three- dimensional tissue engineering. UCB stem cells expanded in microgravity can be used to construct complex tissue structures, such as blood vessels, cartilage, or even whole organs. The unique conditions of microgravity allow for the creation of more functional and biomimetic tissue constructs.

[0106] 3. Drug Discovery and Toxicity Testing: UCB stem cells expanded in microgravity can serve as valuable tools for drug discovery and toxicity testing. The ability to culture cells in a more physiologically relevant environment can provide more accurate insights into drug effects and potential toxicities. Microgravity -based UCB stem cell models can help identify and develop safer and more effective drugs.

[0107] 4. Disease Modeling and Understanding: UCB stem cells, when exposed to microgravity conditions, can exhibit unique physiological and molecular characteristics. These features make them useful for studying diseases and gaining a deeper understanding of their underlying mechanisms. Microgravity-based UCB stem cell models can be employed to model diseases such as cancer, neurodegenerative disorders, or cardiovascular diseases, allowing researchers to investigate disease progression, test potential therapies, and identify novel therapeutic targets.

[0108] 5. Immunotherapy and Cell-Based Therapies: UCB stem cells have shown potential in immunotherapies, particularly in the field of cell-based cancer therapies. Expanding UCB stem cells in microgravity can enhance their proliferation and differentiation into immune cells, such as natural killer (NK) cells or T cells, which are crucial for immunotherapeutic approaches. Microgravity -based UCB stem cell-derived immune cells may offer enhanced therapeutic efficacy and improved patient outcomes.

[0109] 6. Space Medicine: The study of UCB stem cells in microgravity has relevance to space medicine. Understanding how UCB stem cells respond to microgravity environments can help address health concerns for astronauts on long-duration space missions. It can also provide insights into potential countermeasures against the detrimental effects of space travel, such as muscle and bone loss or immune system dysregulation.

[0110] The potential use for expanded HSCs in treating hematologic cancers and serious blood disorders is vast. In addition, new research is evaluating the potential for genetically modified, expanded HSCs to treat rheumatoid arthritis (5 million patients), multiple sclerosis (1 million patients), ankylosing spondylitis (8 million patients), and Type I diabetes (3 million patients). Improvements in the manufacturing of long-term engrafting HSC would benefit all HSC transplantation applications and would enhance commercial potential.

[0111] Microgravity offers further advancement in this arena. Biochemical cues are important, but the physical environment is also critical to mimic human physiology. We have flown a few investigations on the ISS to evaluate differentiated and induced pluripotent, mesenchymal, and hemopoietic stem cells. Our work has paved the way to explore and develop commercial production of HSCs in low-Earth orbit (LEO) and on Earth for clinical use.

[0112] The transplantation of human stem cells for treatment of human disease and regenerative medicine is an exciting area of research and development with enormous potential. To date, the Federal Drug Administration (FDA), through the Center for Biologies Evaluation and Research (CBER), has approved over 20 cellular-based products from human sources for clinical use with the majority of these products based in hematopoietic stem cell or genetically modified T-cell applications for the treatment of certain cancers, severe immune deficiencies and hematological conditions. Extensive stem cell clinical trials are underway that would imply that many more future FDA approved cell therapy products are on the horizon. Stem cells can be derived from a variety of sources. Human embryonic stem cells, derived from an embryo, are pluripotent, which means they can be grown theoretically indefinitely, without losing their pluripotency, and can be differentiated into essentially any cell type or tissue. However, embryonic stem cells carry significant ethical and policy considerations, and are not readily available for clinical use. As an alternative, induced pluripotent stem cells (iPSCs) are derived from adult cells and forced, through genetic manipulation, to revert to an embryonic stem cell-like state. Because iPSCs are pluripotent, potential future cell therapy applications are extensive. However, due to the genetic manipulation of these cell lines and associated safety concerns, manufacturing and scale-up challenges, requirements for human leukocyte antigen (HLA) matching, and infancy of R&D in this arena, FDA approval of any prospective iPSC-based product is likely many years away. Other sources of stem cells include multipotent hematopoietic stem cells (HSCs) derived from bone marrow (BM), mobilized peripheral blood (PB), and umbilical cord blood (UCB). These sources form the basis for most of the FDA-approved cell therapies and for the majority of cell therapy clinical trials being undertaken.

[0113] Hematopoietic stem cell transplantation (HSCT) has become the standard of care for numerous egregious medical conditions including various leukemias, lymphomas, multiple myeloma, aplastic and sickle cell anemia, certain autoimmune diseases and some solid tumors. Hematopoietic stem cells are also being studied extensively for applications in regenerative medicine and treatment of autoimmune disease such as rheumatoid arthritis. Hematopoietic stem cell transplantation can be either autologous, with stem cells derived from the patient, or allogeneic, with hematopoietic stem cells derived from a separate donor. Allogenic transplant is the only or preferred option when the stem cells from the patient may be diseased or if the disease being treated is a congenital or acquired hematological or immunological condition. The primary sources of hematopoietic stem cells for allogeneic transplant, depending on the disease being treated, are bone marrow, peripheral blood and umbilical cord blood, with each source having certain advantages when considering effectiveness (disease remission, no disease recurrence), graft vs. host disease (GVHD), non-recurring disease mortality, human leukocyte antigen (HLA) matching and overall disease recurrence and survival. According to the Center for International Blood and Marrow Transplant Research (CIBMTR), more than 24,000 hematopoietic stem cell transplantations were performed in the U.S. in 2020, with just over 14,000 being autologous and just under 10,000 being allogeneic. Worldwide, more than 1.5 million hematopoietic stem cell transplants have been performed as of 2019 with an annual increase in allogeneic hematopoietic stem cell transplantation procedures of 7.0%. Successful allogeneic hematopoietic stem cell transplantation depends on several factors including HLA matching (e.g., 5- 6 of 6 or 8-10 of 10 loci), readily available source of hematopoietic stem cells and the quantity (total number of cells) and quality (specific cell types) available for transplant. HLA matching is critical to graft success as either host vs graft rejection or GVHD can occur. Poor HLA matching also means that ongoing patient immunosuppression may be required, with concomitant compromised immunity and other side effects. The total quantity and quality of cells are also critical as these factors underly how quickly and effectively engraftment takes place in a patient. Most hematopoietic stem cell transplantation procedures follow myeloablative conditioning, to eradicate or greatly reduce cancerous or disease- causing cells, in which native bone marrow stem cells are largely destroyed to the point where the patient will not recover a hematopoietic system without a transplant. As a result, this procedure immediately leaves the patient vulnerable to infection, bleeding, and other life-threatening complications. The more quickly the hematopoietic stem cell transplantation engrafts, the sooner normal immunological and hematological function can be restored, and patient recovery can occur. This is also critical to total cost of care regarding treatment resources, hospital stay time, etc.

[0114] Unmanipulated umbilical cord blood has been used in over 40,000 hematopoietic stem cell transplantations worldwide since the first such transplant in the late 1980s. Umbilical cord blood has an advantage over other stem cell sources in that HLA matching can be less stringent. Graft vs. host disease is a lower risk with umbilical cord blood than with mobilized PB or BM allogeneic transplants. In addition, umbilical cord blood is readily available with over 800,000 umbilical cord blood units available in public cord banks worldwide, many millions of units available in private cord banks, and more than 100 million live births annually. Umbilical cord blood units can be harvested, HLA typed, cryogenically preserved for long-term storage, and can be transported relatively easily to the hospital where a transplant will be performed. Collection of umbilical cord blood is completely non-invasive and presents no risk either to the mother or the newborn. The biggest drawback to the use of umbilical cord blood in transplantation relates to the relatively low total number of cells collected even though umbilical cord blood has a higher proportion of proliferative hematopoietic stem cells compared to mobilized PB or BM hematopoietic stem cells. As a result, allogeneic hematopoietic stem cell transplantations are more typically done using mobilized PB or BM even though the risk of graft vs. host disease is greater. For hematopoietic stem cell transplantations, a single umbilical cord blood unit may be acceptable for children, but a double umbilical cord blood transplant is typically required for adults, adding cost, and making HLA matching more difficult. If done well, cellular expansion of umbilical cord blood could eliminate this disadvantage while enhancing clinical outcomes. Ex vivo expansion of unmanipulated umbilical cord blood or CD34+ cells isolated from umbilical cord blood (thus, enriched in multipotent stem cells) can be achieved with the use of various growth stimulating cytokines, with or without small molecules to inhibit differentiation or cell senescence, added to specialized serum-containing or serum-free culture media. Commercial development is taking place around the idea of expanding umbilical cord blood hematopoietic stem cells as an enhanced treatment option (product) for patients in need of hematopoietic stem cell transplant. Clinical trials have demonstrated that these expanded umbilical cord blood products can shorten the engraftment period and reduce the risk of post-transplantation infection compared with unmanipulated double umbilical cord blood unit transplantation. With positive results from a recent Phase III clinical trial with Omidubicel (expanded umbilical cord blood), the first expanded stem cell therapeutic product has now been approved by the FDA for clinical use. Even so, while the results with these various expanded umbilical cord blood products are encouraging, there is still significant potential for further improvement in production of cell therapy products and clinical outcomes.

[0115] Turning to FIG. 9, there is shown an in-space cell expansion platform with a partially removed BICEP tray and three additional trays inserted and stacked on top in the SABL incubator. FIG. 7 shows the BICEP tray (40) with a two-chamber bioreactor cell culture cassette (10). The BICEP tray (40) holds components in addition to the two-chamber bioreactor cell culture cassette (10), including the electronics, sensors, pump, media lines, needleless ports, heater plate, and the media bag. The cell culture cassette (10) houses cells in a first chamber and circulated media in a second chamber. Media for the cells is circulated / recirculated through the second non-cell chamber of bioreactor cell culture cassette (10) to the media bag (42) using the peristaltic pump (44). By adopting a two-chamber system, the cells in the first chamber of the bioreactor (20) are not disturbed as media is pumped by the peristaltic pump (40) from the media bag (30). The two chambers are separated by a membrane through which the cells cannot move, which ensures that the cells remain relatively undisturbed in the cell chamber. Further, having no sharp angles or corners in either chamber protects the integrity of the cells by enabling fresh media to access the whole volume of the non-cell chamber and exchange nutrients and wastes across the membrane. With this approach, all cells in the cell chamber will be maintained under optimum healthy conditions. FIG. 8 shows an embodiment of the cell culture cassette (20) according to aspects of the invention. The bioreactor (20) has a first cell chamber (22) adapted to expand a population of cells, such as hematopoietic stem cells. The first chamber (22) is separated from a second chamber (24) by a porous membrane (26). The porous membrane (26) has pores of sufficient size (e.g., 0.4pm to 3 pm) to allow media with nutrients and additives to diffuse between the chambers, but too small for cells to pass from the first chamber to the second chamber (e.g., a pore size of about 5 pm or less, but preferably about 3pm or less). The outer facing surfaces (28) (i.e., the portion that is not co-extensive between the two chambers) of the two chambers can be constructed of an FEP teflon film. The walls of the membranes may further include a mesh support to guard against rupturing of the porous membrane during operation, removal or further storage. The cell culture cassette (20) also includes a pair of inlet Luer lock needleless ports (29) and a pair of outlet Luer lock needleless ports (27).

[0116] When compared to prior test versions of the single chamber bioreactor, the first chamber of the two-chamber system is much larger (a minimum 55mls) to meet the volume requirements for the cell expansion. As shown in FIG. 8A, the chambers are designed without comers so that media and cells can be added and removed without chamber “dead” spaces. Fittings to install and then manipulate fluids through the chambers is changed from pierceable septa to needleless ports using simple Luer-lock fittings. The external surface of the chambers can be made of a film such as an FEP Teflon film. Other films could include polystyrene, polypropylene or expanded PTFE Teflon sheet. A large size chamber volume may benefit from some type of mesh support (e.g., biocomatible screen, 3-D printed mesh, etc.) on the outside of the FEP Teflon films and / or possibly on the inner porous membrane to prevent porous membrane rupture during use.

[0117] The non-cell chamber (i.e., the second chamber) is designed to hold a smaller volume (e.g., lOmls to a 55 ml first cell chamber or l / 5th the volume of the cell / first chamber) to support rapid molecular mass transport requirements (nutrients in and wastes out) through circulating media (i.e., rapid media turnover to minimize molecular gradients within the cell chamber). The smaller volume allows the non-cell chamber to be completely exchanged by the peristaltic pump quickly (e.g., 5-10 min). By exchanging this media on a frequent schedule (1-3 times per hour), fresh media will be in contact with the porous membrane continuously. This will enable steeper concentration gradients of fresh nutrients across the membrane from the non-cell to cell side and for waste products across the membrane from cell to non-cell side. This approach will improve the rate at which nutrients are supplied to the cells and waste products are removed. While a volume ratio of 1 :5 is envisioned to be particularly advantageous, it is contemplated that the range could advantageously be 1 :2 to 1 : 10 non-cell chambercell chamber volume. Materials selection, cleaning, sterilization and assembly of the bioreactor will be designed to be consistent with anticipated cGMP requirements and protocols.

[0118] The in-space cell expansion platform cell chamber (bioreactor) is designed to support non- invasive optical density (OD) measurement of cell density. The system is designed to be manufactured as a single-use relatively low-cost bioreactor system. Once used for stem cell expansion on orbit, each in-space cell expansion platform cell chamber system is discarded.

[0119] The in-space cell expansion platform cell culture cassette is mounted into a tray with additional components to store and circulate media to supply the needs of the expanding population of hematopoietic stem cells (FIG. 6). Trays will provide a second level of containment, the first level of containment being the cell culture cassette, media bag and fluid lines. Four in-space cell expansion platform trays will fit into a single BioServe Space Automated Bioproduct Laboratory (SABL) incubator (3 SABLs currently on-board the ISS) (See e.g., FIG. 9).

[0120] A peristaltic pump (44) is mounted to each in-space cell expansion platform tray (40) to pump media through the non-cell chamber of the cell culture cassette. (See FIG. 7) The low pressure drop through the circulation loop will allow a neutral volume balance to be maintained within the cell cassette. The media bag (42) will hold a total media volume of 2-5x the volume in the cell chamber (55mls). So, by way of example, if the first cell chamber holds about 55 mis, the media bag would hold 110 to 275 mis or more of media. Circulating this media through the non-cell chamber (the second chamber) of the cassette allows the cellular density to achieve a high number of cells (minimum of 5 x 106cells / ml) while ensuring that the media does not become spent (low glucose and / or high lactic acid) and harmful to the cells during the full expansion cycle duration.

[0121] To monitor the cell expansion cycle, pH and optical density (OD) sensors are integrated into each in-space cell expansion platform tray. OD is measured using a photo sensor / multi-spectral LED pair evaluating the media and cells inside the interior of the cell chamber. This approach will allow both the cell density and pH, based on phenol red color change, to be measured. The sensors are calibrated to cell density so that the cycle can be monitored and stopped before the cell density becomes too great, which would endanger the viability of the cells. pH is monitored based on the standard phenol red pH indicator additive in the culture media. The pH sensors may be placed to assess the cell chamber directly or may be placed across a separate inline photocell that will only measure the circulating media on the non-cell side of the system without interference from the expanding cell culture. Additional sensors are integrated into each tray to measure pressure, CO2, temperature and humidity. In addition, SABL’s Science Research Module (SRM) CO2 concentration is measured within the integrated Atmosphere Control Module (ACM).

[0122] The SABL incubator will provide temperature (37°C ± 0.5°C) and CO2 (5% ± 0.25%) control along with power and data interfaces to each in-space cell expansion platform tray. Each in-space cell expansion platform tray will also hold electronics that will control the peristaltic pump, bioreactor heater plate and measure and record sensor values. Multiple in-space cell expansion platform trays are interfaced to a single SABL incubator through a common power and data breakout box (hub) located on the left side of the SRM.

[0123] As a preliminary proof-of-concept, we tested one configuration of the hardware to demonstrate how this system would work (FIG. 11). We prepared a two-chamber bioreactor and inoculated the cell side with 105cells / ml of Jurkat immortalized human T-lymphocytes. The non-cell side of the bioreactor was connected through a peristaltic pump to a media bag containing approximately 3x the media in the cell side (FIG. 11 A). Cells were grown for a total of 7 days and compared to cells grown under standard T-flask static culture conditions (not shown). Results show that the two- chamber bioreactor was able to produce approximately 3-times more cells (5 vs 1.7xl06cells / ml) in the same period with a much higher viability (92% vs 61%) (FIG. 12) compared to standard T- flask culture. The color change of the circulating media is observed in the post-expansion media bag hanging in the cell culture incubator (FIG.1 IB), demonstrating the proposed use of colorimetric feedback, which along with cellular OD, is used to determine the end of the growth cycle. This level of cellular expansion was achieved with a non-cell media volume to cell volume ratio of only 3: 1. Expansion of hematopoietic stem cells in microgravity is performed with a ratio of 2-5: 1, which should easily support the required cell density.

[0124] The median total count of CD34+ cells in over 125,000 UCB units has been assessed, and has been shown to be 44 x 105, which means with this starting point, an expansion of 3x would theoretically be all that’s required to produce enough cells to treat an adult weighing 90kg based on the current UCBT recommendation of 1.5 x 105CD34+ cells / kg body weight. However, a recent clinical trial using expanded hematopoietic stem cells, demonstrating significant favorable clinical outcomes, administered a dose of 29 x 105CD34+cells / kg, expanding from the unmanipulated UCBs that were used in the study approximately 200x on average. We propose to select and expand UCB units starting with a total of IxlO6isolated and cryopreserved CD34+ cells. Once thawed on orbit or on the ground, cells are washed to remove cryoprotectant, resuspended in fresh media, and seeded into the in-space cell expansion platform cell chamber in a total volume of 55mls. The system will then perform automated media perfusion and cell expansion such that the total cell density increases 275-fold to 5xl06cells / ml. Once expanded, 90% of the cells are removed, a cryopreservative is added and the cells are frozen for return to Earth. A second expansion cycle is initiated in the bioreactor by adding back fresh media to replace the 90% volume removed with harvest. The 10% of cells left at harvest will seed the next cycle. Growth and harvest cycles could be repeated a total of 3 times. Cycles 2 and 3 will only need to expand cells lOx to achieve the same density as at the completion of the first cycle. However, the total expansion represented by these 3 cycles is 275x10x10=27, 500-fold, which if successful in regard to cellular quality and safety of the expansion products, would exceed UCB expansions reported in the literature by more than two orders of magnitude.

[0125] We hypothesize that hematopoietic stem cells expanded under microgravity are enriched with hematopoietic stem cell subsets that express ITGA3+ markers previously reported to be associated with long term repopulating capacity and robust multipotentiality. As such less than 200 million microgravity expanded hematopoietic stem cells may be needed for full adult hematopoietic stem cell transplant. Each harvest cycle should produce enough total cells (250 x 106cells total) to support an adult patient hematopoietic stem cell transplant. Taken to its fullest potential, the first cycle could theoretically seed 10 more parallel cycles which could in turn each seed 10 more to treat as many as 100 patients. While theoretically possible, this is not how on orbit production would generally be performed. Rather, CD34+ cells from UCB or mobilized PB would be expanded as much as would be needed to produce the inventory of cell transplant products that would be appropriately stored on Earth for patient use. For example, a minimum of 10 units expanded per UCB unit launched would enable the development of a high-quality, standardized expanded UCB cell bank on Earth. Based on expansion approaches that have been used terrestrially, the first expansion cycle (up to 300-fold) might require as many as 12 days to complete. Subsequent growth / harvest cycles would be expected to take 3-7 days; thus, expanding a single launch UCB unit into 10 hematopoietic stem cell transplantation units might take as few as 15-20 days.

[0126] Once cryopreserved properly, the hematopoietic stem cell units can be stored in low-Earth orbit until the next scheduled spacecraft transportation return. Return materials will include, for example, ten frozen 50-ml bags of cells per expanded unit. Once the full set of harvest cycles is complete, the in-space cell expansion platform cell chamber and all other wetted components is discarded and new disposable cell cassettes, tubing, syringes, and other components is installed into the system, enabling a new set of CD34+ cells to be expanded.

[0127] In certain aspects, the present invention proposes to produce an HSC transplantation product that is expanded in space and returned to Earth, or in clino-rotation simulated microgravity, to treat patients with a variety of hematological malignancies and disorders along with certain autoimmune diseases. The system achieves this goal by expanding CD34 enriched cell populations derived and isolated from UCB or mPB. It is further contemplated that the invention can employ gene-edited mPB as cells to start the expansion. Expanded UCB HSC - it is expected that up to 5 million CD34+ cells from either previously (cryopreserved) or freshly harvested UCB can be isolated. These cryopreserved cells will be transported to a cGMP space production facility and expanded with a single BICEP cassette 50- 300x to 250-500 million cells. These cells will then be split and loaded into 10 additional BICEP cassettes and expanded through 1 or 2 additional cycles another 10-fold per cycle to provide 10 or 20 units of 250 million cells per unit. These units will be packaged, cryopreserved, transported to Earth, and banked until needed for clinical transplantation.

[0128] Expanded gene-edited mPB - CD34+cells from mPB can be isolated from a given patient and gene edited with a selection marker (tNGFR). Since gene-editing is only 10% efficient, approximately 5 million isolated, gene-edited cells will be expanded with an emphasis on longterm repopulating HSCs. Cells will be cryopreserved, launched into LEO, expanded using BICEP to 250-500 million cells, cryopreserved and returned to Earth. Once returned to Earth, this cell therapy product will be infused into the patient (autologous transplant) in a single treatment.

[0129] Cells can be provided by a specific patient or subject (e.g., cell therapy partner, cord blood bank, hospital, individual doctor / patient), expanded in space using an FDA approved cGMP process, and returned to the patient or subject,

[0130] There are currently more than 10,000 allogeneic HSC transplants performed in the US annually (CIBMTR.org) and more than 40,000 worldwide with the number of transplants worldwide increasing on average by approximately 7% per year between 2006 and 2016, with this growth expected to continue. It should be noted that of the allogeneic transplants undertaken, the majority are performed using mPB collected through apheresis from haploidentical or unrelated donors. The number of UCB transplants is relatively small and in decline. The benefits of using UCB, such as proposed herein, will become more favorable than other sources of cells and may even allow more people to receive treatment, especially for racial and ethnic minorities where HLA matching to find a suitable donor is currently far more challenging. HSC treatments have been proven efficacious and have been used for many years to treat multiple conditions including acute lymphocytic leukemia, multiple myeloma, various lymphomas, acute myeloid leukemia, neuroblastoma, germ cell tumors, autoimmune disorders, and amyloidosis to name a few. Because HSCTs are already FDA approved, benefits of in-space produced HSCs, financially and clinically could be realized sooner than other stem cell products.

[0131] The markets for expanded gene-edited HSC transplants to treat autoimmune disorders is even greater than that for hematological malignancies and blood disorders. The global prevalence of RA was 246 cases per 100,000 in 2017 and growing, which means there are as many as 20 million people suffering from RA world-wide today. Prevalence and incidence rates of other autoimmune diseases, such as Type 1 diabetes, multiple sclerosis, Chron’s disease, ankylosing spondylitis, myasthenia gravis and celiac disease reveal many millions of people world-wide suffering from these diseases with an increase in incidence of nearly 20% per year. These conditions and diseases represent an enormous unmet need for the potentially curative treatment that gene-edited HSCs represent.

[0132] As more clinical trials in regenerative medicine with HSCs are undertaken, the number of indications for HSCTs will certainly increase. Finally, it should be noted that while the present invention disclosure focuses on expansion of UCB HSCs, the technology and spaceflight production facilities developed will be applicable to any cellular based therapy including iPSCs, mesenchymal stem cells and other sources of HSCs. Thus, the application of the technology goes well beyond that for expanded cellular UCB and mPB transplants.

[0133] MATERIALS AND METHODS

[0134] Prior to the ISS flight experiments, UCB or PB HSCs will be expanded using the BICEP (BioServe In-space Cell Expansion Platform) platform (prototypes, engineering units and eventually flight hardware). Isolation and characterization of umbilical cord blood derived CD34+cells will first be performed with cells derived from freshly obtained UCB obtained under an existing IRB approved protocol (Zubair Co- PI). CD34+cell isolation will be isolated using CliniMACS CD34 Reagent System according to manufacturer’s instructions. The CliniMACS system is an FDA- approved method for selecting hematopoietic stem cells for clinical application. If needed, we will also be able to utilize UCB CD34+cells obtained commercially.

[0135] The methods used for cell growth and expansion will be identical to those planned for flight. Validation and optimization of CD34+ cell culture conditions in BICEP will be performed. HSC will also be expanded using standard tissue culture flask to compare with HSC expanded in BICEP. Assays for pre-flight comparisons will include total number of cells (TNC), cell viability and flow cytometric analysis of cell subpopulations (CD34, CD133, ITGA3 and various lineage differentiation markers). See methods under Flight Experiment #1 and Post-Flight Experiments for details. [Kwee BI, Sung KE. Engineering microenvironments for manufacturing therapeutic cells. Exp Biol Med (Maywood). 2021;246(16):1845-1856. doi: 10.1177 / 15353702211026922; Choi YS, Noh SE, Lim SM, Kim D il. Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures. Biotechnol Lett. 2010;32(12): 1969-1975. doi: 10.1007 / sl0529- 010-0355-0], For the first flight study, CD34+cells from 4 distinct UCB donors will be expanded and evaluated post-flight for quality, safety and performance using a variety of standard metrics.

[0136] UCB CD34 HSC expansion. UCB CD34+cells (IxlO6total per vial) will be isolated utilizing the CliniMACS system or commercially sourced and stored cryogenically in our labs until they are packed for launch or used in ground controls. Vials will be transported to the ISS at -95°C and stored in MELFI of GLACIER until use. Expansion media (StemSpan SFEM II, StemCell Tech, Cat 09655) will be packaged in syringes (Becton Dickinson (BD) 60cc) and FEP Media Bags (330mls / bag), frozen on the ground and transported to the ISS at -20°C or lower. Growth promoting cytokines (all recombinant human, stem cell factor (SCF, lOOng / ml), thrombopoietin (TPO, 50ng / mI), Fms-like tyrosine kinase 3 (Flt3, 50ng / ml) ligand, and Interleukin-6 (IL-6, 50ng,ml)) with differentiation inhibitors (SB203580-protein kinase 38 (pK38) inhibitor [Bari S, Zhong Q, Fan X, et al. Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem and Progenitor Cells from Non-Enriched Umbilical Cord Blood with Azole Compounds. Stem Cells Translational Medicine. 2018;7(5):376-393. doi: 10.1002 / sctm, 17- 0251; Wang Y, Kellner J, Liu L, Zhou D. Inhibition of p38 Mitogen- Activated Protein Kinase Promotes Ex Vivo Hematopoietic Stem Cell Expansion. Stem Cells and Development. 2011;20(7): 1143-1152. doi: 10.1089 / scd.2010.0413; Peled T, Shoham H, Aschengrau D, et al. Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment. Experimental Hematology. 2012;40(4):342- 355. el. doi : 10.1016 / j . exphem.2011.12.005] and / or nicotinamide) will be prepared at lOOx concentration and frozen in lee syringes. To initiate the first growth expansion cycle, the BICEP tray will be set up, utilizing the Life Sciences Glovebox (LSG), and warmed to 37°C in SABL. Briefly, media will be added directly to the cell expansion cassette from the 60cc syringe and the input / output lines primed. The cassette and first media bag will be integrated into the BICEP tray, interconnected, and a waste syringe used to pull media from the media bag through the lines to purge any remaining air. The circulation tubing will then be connected to form a closed loop and threaded into the peristaltic pump. The cytokines and small molecule differentiation inhibitors will be diluted and added into the expansion cassette and media bag to final concentrations of 50pg / ml (cytokines) and 5pM (differentiation inhibitors). Once the BICEP tray is setup, a vial of CD34+cells will be rapidly thawed to room temperature (RT) using BioServe’ s flight-proven thaw system, drawn into a 5cc syringe and injected / mixed into the cell chamber of the expansion cassette. The BICEP tray will then be installed in SABL, power / data interfaces mated, and the peristaltic pump activated. SABL will provide precise temperature (37°C ± 0.5°C) and CO2 (5% ± 0.25%) control.

[0137] Cellular Product Harvest and Preservation. Following the completion of the expansion cycle, as determined by feedback monitoring of pH and OD, the BICEP tray will be transferred to the LSG. A 60cc syringe with pre-warmed media will be attached to the cell chamber inlet port and an empty 60cc syringe attached to the outlet port. A gentle cycle of pulling from the outlet and pushing in the inlet (5mls per stroke) will allow 90% of the cell chamber contents in 50ml volume to be drawn into the harvest syringe. Once complete, the harvest syringe will be detached, the contents transferred to a 50ml conical tube, the cells spun down, resuspended in a cGMP cryopreservative media containing 10% DMSO. Aliquots (1.5mls) of resuspended cells will be placed in 5 cryovials. The remaining cell suspension will be transferred to a cryobag to be frozen down. The harvested cells, both in the cryobag and in the cryotubes will be frozen down using the controlled rate capability of BioServe’s Space Automated Laboratory Incubator (SALI) to - 20°C [Mousavi S, Zarrabi M, Abroun S, Ahmadipanah M, Abbaspanah B. Umbilical cord blood quality and quantity: Collection up to transplantation. Asian J Transfus Sci. 2019;13(2):79. doi: 10.4103 / ajts.AJTS_124_18; Hunt CJ. Technical Considerations in the Freezing, Low- Temperature Storage and Thawing of Stem Cells for Cellular Therapies. Transfus Med Hemother. 2019;46(3): 134-150. doi: 10.1159 / 000497289] and then transferred to GLACIER to complete freezing to -135°C. Frozen cells will be stored on the ISS ideally at -135°C (-95°C acceptable for short durations of a few days) and subsequently returned to Earth at -95°C.

[0138] Cell count, viability, and biomarker phenotype (Characterization): One vial of expanded cells from each subject and each cycle will be thawed and processed directly for total number of mononuclear cells (MNCs) and viability (live / dead assay, ApexBio, K2081). Samples will be gently mixed, and 50pL acquired by a MACSQuant 16 Analyzer flow cytometer (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany). Cells will be stained using select antibodies (anti-ITGA3, anti-CD34, anti-CD45RA, anti-CD90, anti-EPCR, anti-CD133, anti- CD143, anti-GPR56, anti-CD318, anti-CD38) in the dark for 20 minutes at room temperature. Stained cells will be resuspended for flow cytometric analysis as described previously [Tomellini E, Fares I, Lehnertz B, et al. Integrin-a3 Is a Functional Marker of Ex Vivo Expanded Human Long-Term Hematopoietic Stem Cells. Cell Reports. 2019;28(4): 1063- 1073. e5. doi: 10.1016 / j.celrep.2019.06.084].

[0139] Chromosomal analysis (Safety): Chromosomal karyotype will be performed on expanded cell samples at Mayo Clinic Molecular Pathology Core Facility. Sample vials with cells will be rapidly thawed, washed and resuspended in growth media. Colcemid solution (37.5ml, CAT# 10295892001, Sigma-Aldrich) will be added and the cells incubated at 37 °C with 5% CO2, 5% O2 and 90% N2 for 16-22 hours. Cells in media will be transferred to microcentrifuge tubes and centrifuged at 1,200 rpm for 8 minutes. Medium will be removed and the pellet re-suspended in 10 ml of 50:50 hypotonic solution (potassium chloride 0.075M: sodium citrate 0.8%) and incubated at RT for 20-30 minutes. A fixative (methanol: glacial acetic acid, 3: 1) will be added and cells centrifuged. Pelleted cells will be re-suspended in fixative (methanol: glacial acetic acid, 3 : 1) a total of three times. Cells will be karyotyped using a standard G banding methods.

[0140] DNA damage assay (Safety): Samples of expanded cells (5 x 104cells / tube) will be thawed, washed, resuspended in PBS and stimulated with 100 pM Etoposide (Abeam) for 2 hours as appropriate. Following Etoposide treatment, cells will be washed 3 times with PBS and fixed with 4% paraformaldehyde (PF A) (37 °C, 15 minutes). After fixation, cells will be washed 3 times with PBS, permeabilized with 0.1% Triton-X 100 in PBS for 4 minutes and blocked with 3% Bovine Serum Albumin (BSA) and 0.05% Tween 20 in PBS (blocking solution) for 1 hour at RT. Samples will be incubated with pS.139-gH2A.X antibody (Cell Signaling Technology) in blocking solution at 1 :250 for 24 hours at 4 °C, washed 5 times with PBS and further incubated with Goat antiRabbit IgG (H+L) Secondary Antibody (Fisher Scientific) at 1 :800 and DAPI (Sigma- Aldrich) at 1 :5000 in blocking solution for 2 hours at RT. Subsequently, a cytospin prep will be prepared and washed 5x with PBS, mounting media will be added, and images captured.

[0141] Cell cycle analysis (Characterization): Cells will be fixed and permeabilized with BD Cytofix / Cytoperm kit, prior to staining with anti-Ki67 (FITC, BD Biosciences #556026) and propidium iodide. Flow cytometry data will be analyzed using Flow Jo software.

[0142] Long-term culture differentiation assay (Performance): MS5 stroma cells66will be seeded at an appropriate cell density per well in gelatin coated (0.1%) 96-well plates in a-MEM (GIBCO) supplemented with 12.5% horse serum and 12.5% FBS. Sorted cells (1000, 100, 10 and 1 cell / well, 24 wells per condition) will be plated the following day on irradiated (15 Gy gamma irradiation) stroma cells in a-MEM (GIBCO) supplemented with 12.5% horse serum, 12.5% FBS, SCF (100 ng / mL), IL-7 (20 ng / mL), TPO (50 ng / mL), IL-2 (10 ng / mL) and 2-mercaptoethanol. [Itoh K, Tezuka H, Sakoda H, et al. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp Hematol. 1989;17(2): 145-153.] Cultures will be maintained for 4 weeks with weekly half media changes. Cells will be stained with anti-CD45, anti-CD19, anti-CD14, anti-CD33 and anti-CD56, and analyzed by flow cytometry. Human (CD45+) lymphoid colonies will be identified as CD19+(B cells), CD56+(NK cells). Myeloid colonies will be identified as CD33+or CD14+in CD19_ / CD56_ cells.

[0143] Colony forming unit (CFU) cell assay (Performance): Thawed, washed and resuspended HSCs (3xl03) will be placed in sterile microtubes containing cold 2% FBS / IMDM. The volume of 2% FBS / IMDM will be preadjusted so that the final suspension volume will be approximately 0.3 ml. The cell suspension will be added to a 3 ml aliquot of thawed and supplemented Hematopoietic Progenitor Expansion Medium DXF (C-28021, Sigma-Aldrich). Cells will be mixed well and left static for 3 min and then spread evenly into two 30 mm non-treated. Cells be cultured for 14-17 days. Colonies will be characterized and scored according to their morphology with an inverted microscope at 40x magnification using a culture dish marked with a scoring grid and following manufacturer’s instruction.

[0144] Telomerase activity assay (Characterization): Telomerase activity will be measured by the TRAP assay as modified by Wright et al. [Wright WE, Shay JW, Piatyszek MA. Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity. Nucl Acids Res. 1995;23(18):3794-3795. doi: 10.1093 / nar / 23.18.3794.] Briefly, 105cells will be pelleted, lysed, and centrifuged at 14,000 x g for 20 min. The supernatants will be removed and flash frozen. Detection of telomerase activity in the extracts from cultured cells will be performed in a two-step process: (i) telomerase-mediated extension of an oligonucleotide and (ii) PCR amplification of the resultant product with forward and reverse primers. An appropriate amount, which is in the linear range of telomerase activity versus the internal standard (generally the equivalent of 100 to 1,000 cells), of each of the cell extracts will be incubated for 30 min at room temperature with the forward primer in a tube containing the reverse primer under a wax barrier. After telomerase synthesized TTAGGG repeats onto the forward primer, the reaction products will be amplified by PCR for 30 cycles in the presence of 5 attograms (ag) of an internal TRAP assay standard (ITAS); followed by electrophoresis on 10% polyacrylamide gels. Gels will be analyzed and products will be quantified by using the Phosphorimaging system from Molecular Dynamics. Relative telomerase activities will be quantified by taking the ratio of the ITAS to the entire telomerase ladder in each lane and comparing the ratio with that obtained from 0G versus 1G culture expanded HSCs.

[0145] NOD / SCID repopulating assay (Pre-Clinical Performance, Part A): To evaluate in vivo HSC repopulating potential of ex vivo expanded UCB HSC, NOD / SCID mice (Jackson Laboratories, Bar Harbor, ME, USA) will be used. The NOD / SCID assay will be performed as described previously [Araki H, Mahmud N, Milhem M, et al. Expansion of human umbilical cord blood SCID- repopulating cells using chromatin-modifying agents. Experimental Hematology. 2006;34(2): 140-149. doi: 10.1016 / j.exphem.2005.10.002; Milhem M, Mahmud N, Lavelle D, et al. Modification of hematopoietic stem cell fate by 5aza 2'deoxy cytidine and trichostatin A. Blood. 2004;103(l l):4102-4110. doi:10.1182 / blood-2003-07-2431]. Briefly, 3000 or 5000 CD34+cells from expanded CB samples will be transplanted in 10 mice per condition. Cells will be injected through the tail vein 12 to 16 hours after sublethal total body irradiation (300 cGy). Mice will be sacrificed after 8 weeks of transplantation. BM cells from each mouse will be harvested separately from femurs and tibias. BM cells will be analyzed using flow cytometry to detect human cell engraftment. To quantify the total number of human cells present, an aliquot of cells will be stained and analyzed by flow cytometry as outlined above. The engraftment of human CB cells in the bone marrow of mice will be monitored by flow cytometry at 3-, 12-, and 24-weeks post-transplantation. For each sample, a profile of 50,000 live cells will be acquired. NOD / SCID BM containing at least 0.1% human CD45+cells will be considered indicative of human hematopoietic cell engraftment.

[0146] Gene editing of human HSCs (Part B): CD34+cells isolated from patients, with known HLA typing for DRBl*04:01, will be obtained from Clinlmmune Cell and Gene Therapy. Briefly, stem cells from healthy donors will be mobilized using an injection of granulocyte colony stimulating factor and collected via apheresis. CD34+stem cells will then isolated using an automated, magnet-based separation system (CliniMACS Plus) that is programed to perform CD34+cell separation and enrichment of cells. CD34+cells will be nucleofected with the CAS9 / sgRNA ribonucleoprotein (RNP) made with guide RNAs. The adeno associated virus (AAV) repair virus will be added directly after nucleofection. CD34+cells will be grown in Iscove's Modified Dulbecco's Medium (IMDM) medium supplemented with 10% FBS, 50 pM 2- mercaptoethanol, 2 mM GlutaMAX, IL-6 (10 ng / mL), stem cell factor (20 ng / mL), and FLT3 (10 ng / mL), as described [Lang J, Zhang B, Kelly M, et al. Replacing mouse BAFF with human BAFF does not improve B-cell maturation in hematopoietic humanized mice. Blood Adv. 2017;l(27):2729-2741. doi: 10.1182 / bloodadvances.2017010090], Starting on day 2, cells will be analyzed by flow cytometry for HLA-DR expression and truncated nerve growth factor receptor (tNGFR) (CD271) expression. Once a timeline has been established for when tNGFR expression is detectable, double-positive cells will be sorted. Sorted cells will be injected the day of the sort into BALB / c- Rag2nullI12rgnull (BRG) strain in the Humanized Mouse Core facility at Clinlmmune Labs.

[0147] Characterization of CD34+gene-edited cells (Characterization, Part B): Flow cytometry will be performed on freshly isolated CD34+ cells and after each step in the process (expansion, editing, etc.). HSC markers will be utilized to confirm that expanded and / or gene edited CD34+ cells remain undifferentiated. Cells will be stained with antibodies against CD34, CD38, CD90, CD45RA, CD49f and CD117. In addition, CFU assays will be performed as a potency test and an important criterion for stem cell engraftment. The cells will be plated in a Methylcellulose-based medium formulated to support the optimal growth of erythroid progenitor cells (BFU-E and CFU- E), granulocyte-macrophage progenitor cells (CFU-GM, CFU-G and CFU- M), and multipotential granulocyte, erythroid, macrophage and megakaryocyte progenitor cells (CFU-GEMM). CFU enumeration will be performed after 14 days of incubation.

[0148] Gene-edited repopulation in humanized mice (Pre-Clinical Performance, Part B): The BALB / c-Rag2nullI12rgnull (BRG) strain, when transplanted with hHSCs, develop human B cells, T cells, and, with varying frequencies, other human hematopoietic cell types (See review57). This strain has been recently modified into BRGS with the introduction of the NOD-derived Sirpa allele (SirpaNOD) that further improves human chimerism. CRISPR gene-edited CD34+cells (approximately 100,000 to 400,000) will be injected IV into 1- to 3- day-old BRG(S) that were previously irradiated with 350 rad. Hu-mice will be analyzed 23 to 24 weeks after CD34+celltransplant as described previously [Lang J, Zhang B, Kelly M, et al. Replacing mouse BAFF with human BAFF does not improve B-cell maturation in hematopoietic humanized mice. Blood Adv. 2017;l(27):2729-2741. doi: 10.1182 / bloodadvances.2017010090], Mice will be checked for reconstitution and chimerism by flow cytometry by staining peripheral blood, which will be collected every 3-4 weeks. Once significant T cell chimerism is established (generally between 18 and 24 weeks), mice will be euthanized, and tissues collected for analysis. Flow cytometry will be used to measure the percentage of human hematopoietic (hCD45+) cells in both primary (bone marrow and thymus) and secondary (blood, lymph nodes and spleen) lymphoid tissues over time, as described in Lang, above. As a control, unedited DRBl*04:01+CD34+cells will be expanded and treated in an identical manner as the experimental group to demonstrate that DRBl*04:01 cells repopulate the mice.

[0149] GLOSSARY OF TERMS AND ACRONYMS

[0150] Acronyms:

[0151] 1. ACM - Atmosphere Control Module: A system within space incubators (such as SABL) responsible for controlling CO2 and atmosphere conditions during cell culture.

[0152] 2. BICEP - BioServe In-Space Cell Expansion Platform: A specialized platform developed by BioServe for expanding stem cells in microgravity environments, primarily used on the International Space Station (IS S) and other low Earth orbit facilities. 3. BM - Bone Marrow: One of the sources of hematopoietic stem cells (HSCs) used for transplantation.

[0153] 4. cGMP - Current Good Manufacturing Practice: Standards for ensuring products are consistently produced and controlled according to quality standards, important in biomanufacturing.

[0154] 5. FDA - Federal Drug Administration: U.S. agency responsible for approving medical products, including cell therapies.

[0155] 6. FEP - Fluorinated Ethylene Propylene: A type of Teflon film used in bioreactors to maintain sterile environments while permitting gas exchange.

[0156] 7. Flt3 - Fms-like tyrosine kinase 3: A growth factor ligand used in stem cell expansion media.

[0157] 8. GVHD - Graft-versus-Host Disease: A condition where donor cells attack the host’s body after stem cell transplantation.

[0158] 9. HL A - Human Leukocyte Antigen: Proteins on the surface of cells important for the immune system’s ability to recognize self from non-self, critical for matching in stem cell transplants.

[0159] 10. HSC - Hematopoietic Stem Cell: A type of stem cell that can develop into all types of blood cells and is commonly used in transplant therapies.

[0160] 11. IL-6 - Interleukin 6: A cytokine used to promote the growth of stem cells in culture.

[0161] 12. ISS - International Space Station: A space station where microgravity-based experiments like cell expansion take place.

[0162] 13. LEO - Low Earth Orbit: The area of space where the International Space Station operates, typically used for microgravity research.

[0163] 14. OD - Optical Density: A method of measuring cell density or media quality in a culture system.

[0164] 15. PB - Peripheral Blood: Blood circulating in the body, another source of hematopoietic stem cells used for transplantation.

[0165] 16. SABL - Space Automated Bioproduct Laboratory: A space incubator used to control the environment for bioreactors in microgravity research.

[0166] 17. SCF - Stem Cell Factor: A cytokine used in stem cell expansion media to promote cell growth.

[0167] 18. TPO - Thrombopoietin: A growth factor used to stimulate stem cell expansion, particularly in hematopoietic stem cells. 19. UCB - Umbilical Cord Blood: A rich source of hematopoietic stem cells used in transplantation therapies.

[0168] Key Terms:

[0169] 1. Automated - Systems or processes that operate without human intervention, often used in bioreactors to ensure controlled conditions for cell expansion.

[0170] 2. Bioreactor - A device or system that provides a controlled environment to grow and expand cells, such as stem cells, by regulating nutrients, gases, and waste.

[0171] 3. Cardiomyocyte - A type of heart muscle cell, often studied in regenerative medicine and space-based biological research.

[0172] 4. Cell Chamber - The section of a bioreactor where the cells are cultured, separated from the media by a porous membrane.

[0173] 5. Circulation - Refers to the movement of media through the non-cell chamber of a bioreactor to supply nutrients and remove waste from the cells.

[0174] 6. Clino-Rotated - A terrestrial simulation of microgravity by rotating a cell culture device on a clinostat, reducing the effects of Earth’s gravity.

[0175] 7. Diffusion - The movement of nutrients and waste products across the porous membrane in a bioreactor, driven by concentration gradients.

[0176] 8. Expansion - The process of increasing the number of cells, such as hematopoietic stem cells, through culture in a bioreactor.

[0177] 9. Hematopoietic - Referring to the formation of blood cells, often related to stem cells that can develop into various types of blood cells.

[0178] 10. Immunotherapy - A type of treatment that boosts or modifies the immune system to fight diseases, particularly cancer, using expanded stem cells or immune cells.

[0179] 11. Leukemia - A type of cancer affecting blood cells, commonly treated with hematopoietic stem cell transplantation.

[0180] 12. Lymphoma - A type of cancer that affects the lymphatic system, often treated with hematopoietic stem cell therapies.

[0181] 13. Media Exchange - The process of replacing the used media in a bioreactor with fresh media to maintain optimal conditions for cell growth.

[0182] 14. Microgravity - The condition of very weak gravity, experienced in space, which can affect cell growth and expansion in unique ways.

[0183] 15. Multipotency - The ability of a stem cell to develop into multiple, but not all, cell types (e.g., hematopoietic stem cells). 16. Peristaltic Pump - A type of pump used in bioreactors to move fluids, such as media, through the system without contamination.

[0184] 17. Pluripotent - A type of stem cell that can develop into any type of cell in the body, such as induced pluripotent stem cells (iPSCs).

[0185] 18. Regenerative Medicine - A field of medicine focused on repairing or regenerating damaged tissues and organs, often using stem cells.

[0186] 19. Stem Cell - A cell with the ability to divide for long periods and differentiate into various specialized cell types, such as hematopoietic stem cells.

[0187] 20. Teflon - A type of polymer used in bioreactor membranes to allow gas exchange while maintaining a sterile environment.

[0188] 21. Transplantation - The process of transferring cells, tissues, or organs from one site to another, often referring to hematopoietic stem cell transplants in medical treatments.

[0189] GLOSSARY OF CLAIM TERMS

[0190] As used throughout the entire application, the terms “a” and “an” are used in the sense that they mean “at least one”, “at least a first”, “one or more” or “a plurality” of the referenced components or steps, unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0191] The term “and / or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.

[0192] The term “about” or “approximately” as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0193] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used.

[0194] As used herein, the term “comprising” is intended to mean that the products, compositions and methods include the referenced components or steps, but not excluding others. “Consisting essentially of’ when used to define products, compositions and methods, shall mean excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers. “Consisting of’ shall mean excluding more than trace elements of other components or steps.

[0195] The advantages set forth above, and those made apparent from the foregoing description, are efficiently attained. Since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0196] All references cited in the present application are incorporated in their entirety herein byreference to the extent not inconsistent herewith.

[0197] It will be seen that the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0198] It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween. Now that the invention has been described,

[0199] Table 1 - Dimensions and other parameters for the bioreactor.

[0200] Table 2 - Materials for the bioreactor.

[0201] Table 3 - Dimensions and other parameters for the bioreactor tray. Table 4 - Materials for the bioreactor tray.

Claims

What is claimed is:

1. A two-chamber cell culture system comprising: a first chamber adapted to expand a plurality of cells; a second chamber in fluid communication with the first chamber; and a porous membrane separating the first chamber from the second chamber, wherein the membrane has a pore size that allows for the exchange of media from one chamber to another chamber, but prevents the passage of cells from the first chamber to the second chamber.

2. The two-chamber cell culture system according to claim 1 further comprising a media reservoir to hold an additional supply of media, wherein the media reservoir is in fluid communication with the second chamber.

3. The two-chamber cell culture system according to claim 2 further comprising a pump to move media from the reservoir to the second chamber.

4. The two-chamber cell culture system according to claim 3 wherein the pump is a peristaltic pump.

5. The two-chamber cell culture system according to claim 2 wherein the volume of the media reservoir is about two times to about five times (most preferably about five times) the volume of the first chamber.

6. The two-chamber cell culture system according to claim 1 wherein the first chamber is devoid of sharp angles or corners, whereby the lack of sharp corners protects the cells from damage during expansion.

7. The two-chamber cell culture system according to claim 1 wherein the porous membrane has a pore size of about 3 pm or smaller.

8. The two-chamber cell culture system according to claim 1 further comprising a mesh support system surrounding a portion of the two-chamber cell culture system to provide support for the system and to prevent rupture or breakage of the chambers.

9. The two-chamber cell culture system according to claim 1 wherein the first chamber has at least two sides and one side of the first chamber has the porous membrane and asecond side of the first chamber has thin plastic film (e.g., FEP Teflon) that permits gas exchange (O2 and CO2).

10. The two-chamber cell culture system according to claim 1 wherein the system further comprises a noninvasive optical density (OD) measurement system to measure the pH and / or the cell density of the first chamber.

11. The two-chamber cell culture system according to claim 1 wherein the volume of the first chamber is about 30 to about 200 mis, about 45 to about 100 mis, about 50 to about 70 mis, or about 55 mis or greater.

12. The two-chamber cell culture system according to claim 11 wherein the volume of the second chamber is about one-third to one-seventh the volume of the first chamber.

13. The two-chamber cell culture system according to claim 11 wherein the volume of the second chamber is about 10-20 mis.

14. A cell expansion system comprising the two-chamber cell culture system according to one of claims 1-13 and further comprising: a. a media supply bag to supply additional media to the two-chamber cell culture system; b. a pump to pump media from the media bag to the second chamber of the two- chamber cell culture system; c. a sensor to monitor the pH or optical density of the media in the first chamber of the two-chamber cell culture system; and d. a heater element to control the temperature of the media in the first chamber.

15. A method of expanding a population of cells comprising: providing a population of cells from a subject; transporting the cells to a lower gravity environment, wherein the lower gravity is relative to the standard gravity of earth; placing the cells in fresh media; incubating the cells in an environment providing media perfusion to the expanding cell population; and recovering cells following incubation.

16. The method of expanding a population of cells according to claim 15 wherein the cells are incubated in a two-chamber cell culture system according to one of claims 1-14.

17. The method of expanding a population of cells according to claim 16 wherein the lower gravity environment is a low earth orbit or higher orbit.

18. The method of expanding a population of cells according to claim 16 wherein a population of cells from the subject is cryopreserved prior to transport and the cells are washed to remove cryoprotectant prior to placing the cells in fresh media.

19. The method of expanding a population of cells according to claim 16 wherein the cells are incubated in a system providing automated media perfusion, whereby the media perfusion provides fresh media to the expanded cells and removes spent media and waste from the incubation environment of the first chamber.

20. The method of expanding a population of cells according to claim 16 further comprising the steps of placing the recovered cells in a cryopreservative and freezing the cells for transport away from the lower gravity environment.

21. The method of expanding a population of cells according to claim 16 wherein about 70% to about 95% of the cells are removed at the recovering step and the remaining cells are placed in fresh media and subjected to a second round of incubation and expansion.

22. The method of expanding a population of cells according to claim 21 wherein about 70% to about 95% if the cells are removed at a second recovering step and the remaining cells are placed in fresh media and subjected to a third incubation and expansion.

23. The method of expanding a population of cells according to claim 16 wherein about 90% of the cells are removed at the recovering step and the remaining 10% of the cells are placed in fresh media and subjected to a second round of incubation and expansion.

24. The method of expanding a population of cells according to claim 23 wherein about 90% of the cells are removed at a second recovering step and the remaining 10% of the cells are placed in fresh media and subjected to a third incubation and expansion.

25. The method of expanding a population of cells according to claim 16 wherein lower gravity environment is reduced gravity, microgravity or lower than microgravity gravity environment.

26. The method of expanding a population of cells according to claim 16 wherein the cells are hematopoietic stem cells.

27. A two-chamber cell culture system comprising: a first chamber adapted to expand a plurality of cells; a second chamber in fluid communication with the first chamber, wherein the second chamber has a volume about one-sixth to one-third of the volume of the first chamber and the second chamber is adapted to facilitate the exchange of media from the first chamber where the media is used to expand cells in the first chamber; and a porous membrane separating the first chamber from the second chamber, wherein the membrane has a pore size that allows for the exchange of media from one chamber to another chamber, but prevents the passage of cells from the first chamber to the second chamber.

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