Products for culturing or maintaining normal cells, precancerous cells, and cancer stem and progenitor cells

Abstract

In alternative embodiments, biosensing bioreactors are provided to detect normal cells, pre-cancerous cells and cancer stem cells. In alternative embodiments, bioreactors are provided for culturing, manipulating and maintaining primary human hematopoietic stem and progenitor cells, which may include a micro-peristaltic pump to mimic blood flow. In alternative embodiments, articles of manufacture in the form of bioreactors or nano-bioreactors or customized cell culture bags with defined three-dimensional (3D) stromal microenvironments, kits containing same, and methods of making and using same are provided. In alternative embodiments, articles of manufacture provided herein, including bioreactors, nano-bioreactors, bags, customized cell culture bags and kits provided herein, are used to culture, maintain or support the culture and / or differentiate hematopoietic stem cells, including human hematopoietic stem cells isolated from bone marrow or blood donors.

Description

[Technical field]

[0001] This PCT international application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 298,936, filed January 12, 2022. The foregoing application is expressly incorporated by reference in its entirety for all purposes. All publications, patents, and patent applications cited herein are expressly incorporated by reference for all purposes.

[0002] The present invention generally relates to biosensing bioreactors and cancer biology. In an alternative embodiment, a biosensing bioreactor is provided for detecting normal cells, pre-cancerous cells and cancer stem cells. In an alternative embodiment, a bioreactor, nano-bioreactor, bag or culture bag for culturing, manipulating, differentiating and maintaining primary human hematopoietic stem and progenitor cells is provided, which may include a micro-peristaltic pump for circulating fluids and simulating or reproducing blood flow. In an alternative embodiment, an article of manufacture in the form of a bioreactor or nano-bioreactor, bag or customized cell culture bag with a defined three-dimensional (3D) stromal microenvironment, and kits containing the same, as well as methods of making and using the same, are provided, and optionally also include bioluminescent and fluorescent lentiviral reporter vectors capable of quantifying the function of normal cells, pre-cancerous cells and cancer stem cells, including FUCCI2BL cell cycle reporter, ADAR1-nanoluciferase-GFP reporter self-replication reporter and RFP / GFP splicing reporter. In alternative embodiments, the articles of manufacture provided herein, including the bioreactors, nanobioreactors, customized cell culture bags and kits provided herein, can be used to culture, maintain or support the culture, and / or differentiate hematopoietic stem cells, including human hematopoietic stem cells isolated from bone marrow or blood donors. In alternative embodiments, the articles of manufacture provided herein, including the bioreactors, nanobioreactors, customized cell culture bags and kits provided herein, can be used to support or maintain human primary hematopoietic stem cell cultures. [Background technology]

[0003] While stem cells play a key role during embryonic and fetal development, they also maintain the integrity of adult tissues, mobilize in response to injury, and repair and regenerate tissues. Stem cells are functionally defined based on their ability to self-renew (divide without differentiating), differentiate into tissue-specific progenitor cells, and remain dormant in protected microenvironments. Stem cells normally reside in the body in a homeostatic, healthy state. These microenvironments remain highly specific and tightly regulated, and are often referred to as "niches." The hematopoietic stem cell niche is located within the highly vascularized cavernous bone marrow.

[0004] (Abstract) Summary of the Invention [Means for solving the problem]

[0005] In an alternative embodiment, an article of manufacture for cell culture, optionally wherein the cells are stem cells or human primary hematopoietic stem and / or progenitor cell cultures, and / or for the maintenance, proliferation and / or differentiation of these cell cultures (which may be in the form of a bioreactor, or a nanobioreactor, or a customized cell culture bag with a defined three-dimensional (3D) stromal microenvironment), comprising: a container, enclosure or bag having gas permeable (and optionally liquid impermeable or liquid impermeable) walls or sides, and a three-dimensional (3D) sponge matrix or sponge-like material contained within the container, enclosure or bag; Where: The sponge matrix or sponge-like material is infused with a mixture of cell culture medium and cells, including stem cells and / or bone marrow matrix cells; The interior of the container, enclosure or bag is sterile; An article of manufacture is provided, wherein the container, enclosure or bag includes at least one fluid or cell input port, or at least two fluid or cell input ports.

[0006] In an alternative embodiment of the article of manufacture provided herein, the article of manufacture (which may also be referred to as a bag or vessel and is in the form of a bioreactor, a nanobioreactor, a customized cell culture bag with a defined three-dimensional (3D) stromal microenvironment) is fabricated as a gas-permeable (and optionally liquid-impermeable or liquid-impermeable) cell culture bag or vessel, or equivalent; the container, enclosure or bag is constructed as a gas permeable (and optionally liquid impermeable or liquid impermeable) cell culture environment or bag; the vessel, enclosure or bag includes at least two liquid or cell input ports, one output port and one inflow port, and optionally a micro peristaltic pump is operably connected to the inflow and outflow ports for circulating liquid within the vessel, enclosure or bag; a mixture of cells comprising stem cells or human stem cells, bone marrow matrix cells or human bone marrow matrix cells, or cancer stem cells or human cancer stem cells, or organoid cells, or any combination thereof; Optionally, the mixture of cells comprises human hematopoietic cells (HSCs) from a CD34+ donor, or human hematopoietic cells (HSCs) from a human CD34+ donor, or pre-cancerous stem cells or cancer stem cells or cancer cell lines lentivirally transduced with a biosensing reporter of stem cell activity (including, for example, FUCCI2BL, ADAR1 and / or splicing reporters), or any combination thereof; The sponge or sponge-like material is an absorbable gelatin sponge (optionally an absorbable human or porcine gelatin sponge); solubilized or reconstituted basement membrane matrix; solubilized or reconstituted laminin / collagen IV rich basement membrane extracellular matrix, optionally Matrigel™ (Corning Life Sciences) or GELTREX™ (ThermoFisher Scientific); a compressed sponge comprising absorbable gelatin, optionally GELFOAM™ (Pfizer); Decalcified cancellous sponge (Berkeley Advanced Biomaterials (BAB), Berkeley CA), or VIASORB™ (Globus Medical, Audubon, PA), and / or A hydrogel-based macroporous sponge or a porous hydroxypropylcellulose (HPC) scaffold, optionally CelluSponge™, CelluSponge-GAL™, or CelluSponge-COL™ (Bio-Bybios), optionally a cancellous bone (or cancellous tissue) sponge, such as Cancellous Sponge™ (VMI Medical). Including, the sponge or sponge-like material further comprises a demineralized cancellous bone matrix sponge or a demineralized bone matrix (DBM) component, optionally comprising OSTEOSPONGE™ (Xtant Medical); and / or ·About 10 6 pieces ~ about 10 10 cells, or approximately 10 5 pieces ~ about 10 11 cells, approximately 10 5 pieces ~ about 10 12 The cells are placed in or cultured in an article of manufacture.

[0007] In alternative embodiments, the articles of manufacture provided herein further comprise a detectable vector or reporter, optionally the detectable vector or reporter is inserted (e.g., optionally transduced or transfected) into a cell (or if the vector is a viral vector or the reporter is comprised in a viral vector, the viral vector is capable of infecting a cell), or into a substantially majority of the cells in the article of manufacture, or into all of the cells in the article of manufacture, optionally comprising a bioluminescent vector and / or a fluorescent vector, optionally comprising a bioluminescent vector and / or a fluorescent lentiviral reporter vector, optionally the bioluminescent vector and / or the fluorescent vector is capable of quantifying a cell or a function of a cell, optionally capable of quantifying a function of a normal cell, a pre-cancerous cell, and / or a cancer stem cell, and optionally the detectable vector or reporter comprises a FUCCI2BL cell cycle reporter, an ADAR1-nanoluciferase-GFP reporter self-replication reporter, and / or an RFP / GFP splicing reporter.

[0008] In alternative embodiments, methods are provided for culturing, maintaining or differentiating a wide range of stem cells, including tissue-specific organoids and malignant organoids representative of solid tumors such as breast cancer, thereby promoting a plurality of stem cells in the articles of manufacture provided herein.

[0009] In an alternative embodiment, a method of producing an organoid is provided, comprising incubating a plurality of stem cells in a product provided herein, optionally further comprising cytokines capable of differentiating tissue-specific stem cells and / or pre-cancerous or cancer stem cells, and matrigel or bone marrow stromal cells.

[0010] In an alternative embodiment, kits are provided that contain the articles of manufacture provided herein and, optionally, further contain instructions for practicing the methods provided herein.

[0011] In alternative embodiments, there is provided a use of the articles of manufacture provided herein for human primary hematopoietic stem cell culture, or for the maintenance of stem cells, or for the differentiation of cells, or for the generation of organoids.

[0012] In alternative embodiments, an article of manufacture or kit for use in culturing human primary hematopoietic stem cells, or for maintaining stem cells, or for differentiating cells, or for generating organoids is provided, wherein the article of manufacture is an article of manufacture provided herein.

[0013] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0014] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0015] Description of the drawings The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] The drawings described herein are intended to illustrate exemplary embodiments provided herein and are not intended to limit the scope of the invention as encompassed by the claims. [Brief description of the drawings]

[0017] [Figure 1A-B]Figure 1A shows images of an exemplary article of manufacture for culturing and / or maintaining primary human hematopoietic stem and progenitor cells, also referred to as a bioreactor, nanobioreactor, customized cell culture bag, or bioreactor bag, including a sponge, where the bag is filled with cell culture medium, the left panel is an exemplary 30 ml cell culture bag with a two-port system, and the right panel is an exemplary 7 ml cell culture bag with a one-port system. Figure 1B shows representative images of A549 adherent cells (e.g., cells maintained and / or cultured therein) in an exemplary article of manufacture provided herein, where the cells have been transduced with a GFP fluorescent marker, and the images confirm the viability and imaging capability of the adherent cell line using bright field and fluorescence through an exemplary article of manufacture (which may be referred to as a "bag") including a sponge material, and the images were taken with a 4x objective. [Figure 1C] FIG. 1 shows representative images of TF1a suspension cells in an exemplary article of manufacture provided herein (e.g., cells are maintained and / or cultured therein), where the cells have been transduced with mVenus and mCherry fluorescent markers to confirm viability and imaging capabilities of the suspension cell line, where bright field and fluorescence through the bag and sponge were used, and images were taken with a 20x objective. [Figure 1D] FIG. 1 shows a representative image of human hematopoietic stem cells isolated from a donor's bone marrow two weeks after seeding, where the cells are in an exemplary article of manufacture provided herein (e.g., the cells are maintained and / or cultured therein), the image confirms the viability of the primary stem cells and their attachment to the sponge matrix and proliferation within the article of manufacture, thus making it a nanobioreactor, and the image was taken with a 20x objective. [Figure 1E]1 shows representative images of human hematopoietic stem cells isolated from donor bone marrow two weeks after seeding and transduced with mVenus fluorescent marker, where the cells are in an exemplary article of manufacture provided herein (e.g., the cells are maintained and / or cultured therein), and the images confirm the viability of the primary stem cells and the ability to image brightfield and fluorescent through the bag and sponge. Images were taken with a 20x objective. [Figure 1F] FIG. 1 shows representative images of human hematopoietic stem cells isolated from donor bone marrow 4 weeks after seeding and transduced with mVenus and mCherry fluorescent markers, where the cells are in an exemplary article of manufacture provided herein (e.g., the cells are maintained and / or cultured therein), and the images confirm primary stem cell viability in the article of manufacture (or bioreactor) after 4 weeks and the ability to image through the article of manufacture (or exemplary bags and sponges) using bright field and fluorescence, and the images were taken with a 10x objective. [Diagram 2] FIG. 1 is a schematic diagram of an exemplary modeling of a cell or culture niche or bone marrow niche for an exemplary article of manufacture provided herein (alternatively referred to as a bioreactor, nanobioreactor, customized cell culture bag or bioreactor bag), showing cancellous bone structure reproduced using gelatin sponge, and reproduction of blood circulation including arterioles and sinusoids, and also showing an exemplary ring pump with inflow and outflow ports, and in the left sub-image, press point 1, press point 2, press point 3 and tubes for outflow and inflow ports, as well as an eccentric motor with a rotating shaft, pump base and ring, where the press points of the tubes move from 1, 2 to 3 (press point 1, press point 2 and press point 3) (caused by the rotation of the eccentric rotor), and shown in the figure is an exemplary Takasago 2 ml / min peristaltic pump in a 2-port exemplary system. [Diagram 3]FIG. 1 is a schematic illustrating the processing and isolation of CD34+ cells from primary human bone marrow for use in an exemplary article of manufacture provided herein (alternatively referred to as a bioreactor, nanobioreactor, customized cell culture bag, or bioreactor bag), showing centrifugation of donor bone marrow to isolate mononuclear cells using a density gradient or isopycnic centrifuge, e.g., FICOLL-PAQUE PLUS™ (Cytiva, Fisher Scientific, Thermo Fisher), which separates whole blood or bone marrow into an upper layer containing plasma, a lower layer containing lymphocytes, monocytes, and platelets, a lower layer containing FICOLL-PAQUE PLUS™, and a bottom layer containing granulocytes and red blood cells, and magnetic labeling with microbeads to separate CD34+ stromal cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). [Figure 4] FIG. 1 is a schematic diagram illustrating the assembly of an exemplary article of manufacture (alternatively referred to as a bioreactor, nanobioreactor, customized cell culture bag or bioreactor bag) provided herein (in this figure, an exemplary two-port bag is used) to contain stromal cell support and HSPCs; the design of this exemplary system includes lentiviral transduction of targeted HSPCs with fluorescent reporters and demonstrates live cell imaging capabilities. [Diagram 5] FIG. 1 illustrates an exemplary article of manufacture provided herein (alternatively referred to as a bioreactor, nanobioreactor, customized cell culture bag, or bioreactor bag) for supporting long-term culture of primary hematopoietic stem and progenitor cells in space (in this figure, an exemplary two-port bag is used). [Figure 6]FIG. 1 is a graphical representation of flow cytometry analysis of cells returned from an orbital space mission (SpX-CRS24 mission); the data confirms the ability to culture and maintain human primary hematopoietic stem cell viability and progenitor cell viability in space in the exemplary articles of manufacture (or nanobioreactors) provided herein; 50,000 cells were analyzed using a flow cytometer Fortessa X-20™ (BD Biosciences) and DAPI (i.e., 4,6-diamidino-2-phenylindole) staining, a marker of membrane viability and therefore cell viability. [Figure 7] FIG. 1 is a graphical representation of flow cytometry analysis of CD34+ cells returned from an orbital space mission (the SpX-CSR24 mission); the data confirms the ability to culture human primary hematopoietic stem and progenitor cells in space in the exemplary articles of manufacture (or nanobioreactors) provided herein; 50,000 cells were analyzed using a flow cytometer Fortessa X-20™ (BD Biosciences) and DAPI staining was used. [Figure 8] FIG. 1 shows images of fluorescently labeled cells obtained during an orbital space mission (SpX-CRS24 mission). The data confirms the ability to image live cells in space in an exemplary article of manufacture (or nanobioreactor) provided herein. Hematopoietic stem and progenitor cells (HSPCs) were lentivirally transduced with a lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter or a Fucci2BL™ cell cycle reporter, and images were obtained at 20x using a high-resolution live cell imaging scope (i.e., Etaluma™, Carlsbad, Calif., scope), and images of cells cultured under normal laboratory conditions (top row of images, "ground") are compared to images of cells cultured during a space mission (bottom row of images, "flight"). [Figure 9]FIG. 13 graphically depicts fluorescence quantification of HSPCs labeled with a lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter (Fucci2BL™) during a 34-day space mission, measured using software capable of 3D analysis of fluorescent images (Volocity™, Ontario, Canada) and graphed with data analysis software (Prism™, GraphPad Software, San Diego, CA), also using mVenus, a basic, constitutively fluorescent yellow fluorescent protein, and MCherry, a basic, constitutively fluorescent red fluorescent protein, compared to cells cultured in a normal laboratory environment ("Ground" diagram) compared to cells cultured during a space mission ("Flight" diagram). [Figure 10A] FIG. 1 shows a schematic of a colony assay after a space mission where cells are seeded (at 10,000, or 10K cells / ml), incubated for 2 weeks, followed by a survival (viability) assay, cells are harvested, mixed, and re-seeded at approximately 1,000 cells / ml, followed by incubation for 2 weeks, followed by a self-renewal assay. [Figure 10B] 10A-10C are graphical representations of data from assays confirming the ability or effectiveness of using exemplary articles of manufacture (or nanobioreactors) provided herein to maintain long term cell cultures; in the left image of FIG. 10B, primary cell colonies are analyzed 2 weeks after seeding, and in the right image of FIG. 10B, secondary cell colonies are analyzed 4 weeks after seeding, with cells cultured in a normal laboratory environment ("Ground" image) compared to cells cultured during a space mission ("Flight" image) measuring granulocyte macrophage progenitor (CFU-GM), multi-lineage cells, and total cell viability. [Figure 11]FIG. 1 shows an assay that provides a cytokine profile for hematopoietic stem cell fitness following low Earth orbit exposure; analysis was performed on cell-free supernatants (from cell cultures grown in exemplary articles of manufacture (or nanobioreactors) provided herein) harvested following payload splashdown, with cells cultured in a normal laboratory environment ("ground" plate) compared to cells cultured during a space mission ("flight" plate); data shows that genes with increased expression in spaceflight are IGFBP-2, PDGF-BB, IL-6, IL1a, OPG (or TNFRSF118), while genes with increased expression during ground culture are TIMP-1, TIMP-2, and IL6-R. [Figure 12A] Figures 12A-C show the results of whole genome and whole transcriptome sequencing analysis, the data demonstrating the ability to culture HSPCs in space using the exemplary articles of manufacture (or nanobioreactors) provided herein and return viable cells for bulk DNA and RNA isolation. Figure 12A graphically illustrates telomere length of cells from flight (space mission) versus ground samples, measured using a computational tool that estimates the average telomere length (TL) of paired-end whole genome sequencing (WGS) samples, i.e., Telomerecat™ (see, e.g., Farmery et al., Scientific Reports, Vol. 8, Article No: 1300 (2018)), and software that specifically characterizes the footprint of telomere maintenance machinery in genomes, i.e., TelomereHunter™ (see, e.g., Feuerbach et al., BMC Bioinformatics, Vol. 20, Article No: 272 (2019)). [Figure 12B] FIG. 1 is a graphical representation of normalized singleton counts using different probes in flight (space mission) cell samples versus ground sample cells, with the nucleic acid probes being TTTGGG, TGAGGG, TTGGGG, TGAGGG, TAAGGG, CTAGGG, TCAGGG, TCCGGG, ATAGGG, and CATGGG. [Figure 12C] FIG. 13 graphically outlines the distribution of flight sample cells versus ground sample cells.

[0018] Like reference numbers in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In alternative embodiments, articles of manufacture in the form of bioreactors, nanobioreactors, bags, or customized cell culture bags, and kits containing same, as well as methods of making and using same, are provided. In alternative embodiments, articles of manufacture provided herein, including bioreactors, nanobioreactors, bags, customized cell culture bags, and kits provided herein, are used to culture, support culture, maintain culture, and / or differentiate cells, e.g., blood cells, stem cells, and / or hematopoietic stem cells, including human hematopoietic stem cells, which may be isolated from bone marrow or blood donors. In alternative embodiments, articles of manufacture provided herein, including bioreactors, nanobioreactors, bags, customized cell culture bags, and kits provided herein, are used to support or maintain (e.g., maintain cell viability of cell cultures) human primary hematopoietic stem cell cultures.

[0020] In an alternative embodiment, the articles of manufacture provided herein, including the bioreactors, nanobioreactors, bags, customized cell culture bags and kits provided herein, recreate or simulate or substantially simulate or substantially resemble a bone marrow niche, such as a human bone marrow niche, and in an alternative embodiment, this environment is generated or created by using or including a sponge matrix (or sponge-like material) or equivalent in the article of manufacture (or bioreactor, nanobioreactor, bag or customized cell culture bag) to simulate or mimic (or substantially simulate or mimic) cancellous bone structure, and the sponge or sponge-like material or equivalent is separated or disposed within a gas permeable container or gas permeable cell culture bag, thereby allowing for three-dimensional cell culture (e.g., including proliferation or differentiation or maintenance) of hematopoietic stem cells, including human hematopoietic stem cells, including, for example, CD34+ donor-derived human hematopoietic cells (HSC). In an alternative embodiment, the CD34+ fraction of the same donor bone marrow is seeded (or added) to the article of manufacture (or bioreactor, nanobioreactor, bag or customized cell culture bag) along with the HSCs to produce cytokines and growth factors to closely mimic the bone marrow niche and / or cancellous bone structural conditions.

[0021] In an alternative embodiment, the articles of manufacture provided herein, including the bioreactors, nanobioreactors, bags, customized cell culture bags and kits provided herein, replicate (or substantially replicate or replicate) the human bone marrow niche by including the use of a sponge or sponge-like matrix or equivalent to mimic cancellous bone structure, the sponge or sponge-like matrix or equivalent being inserted or placed within a gas permeable container, such as a gas permeable bag, such as a gas permeable cell culture bag, thereby allowing for three-dimensional cell culture of, for example, stem cells or hematopoietic cells (HSCs) or human hematopoietic cells (HSCs) from a CD34+ donor. ... -The fraction is seeded into an article of manufacture (eg, a bioreactor or bag) along with HSCs, thereby producing cytokines and growth factors to closely or substantially mimic or recreate niche conditions, eg, bone marrow niche conditions or cancellous bone structure.

[0022] In an alternative embodiment, the articles of manufacture provided herein, including the bioreactors, nanobioreactors, bags, customized cell culture bags and kits provided herein, are designed for the purpose of supporting or maintaining human primary hematopoietic stem cell cultures in a low gravity environment, e.g., a low (space) orbit environment, e.g., on orbit within the International Space Station (ISS). However, this design can be used to recreate and simulate the hematopoietic stem cell niche for any other purpose and in any other context.

[0023] In alternative embodiments, the articles of manufacture provided herein, including the bioreactors, nanobioreactors, bags, customized cell culture bags and kits provided herein, can support the maintenance, development, growth and / or differentiation of organoids, including organoids derived from (or grown from) stem cells (such as human hematopoietic cells (HSC)) and / or cancer stem cells. In alternative embodiments, this is facilitated by conditioning or augmenting the sponge or sponge-like matrix or equivalent, by the addition of a composition that promotes the support, maintenance, development, growth and / or differentiation of stem cells, or by the addition of cell culture medium, such as a cell culture medium that can promote the support, maintenance, development, growth and / or differentiation of stem cells. In alternative embodiments, the sponge or sponge-like matrix or equivalent can also include demineralized cancellous bone matrix sponge, or demineralized bone matrix components, including, for example, OSTEOSPONGE™ (Xtant Medical). In alternative embodiments, the sponge or sponge-like matrix or equivalent can also comprise an absorbable gelatin sponge, such as an absorbable human or porcine gelatin sponge, a solubilized or reconstituted basement membrane matrix or a solubilized or reconstituted laminin / collagen IV rich basement membrane extracellular matrix (e.g., a compressed sponge comprising absorbable gelatin, such as Matrigel™ (Corning Life Sciences) or GELTREX™ (ThermoFisher Scientific) or GELFOAM™ (Pfizer), or a hydrogel-based macroporous sponge or a porous hydroxypropylcellulose (HPC) scaffold, such as CelluSponge™, CelluSponge-GAL™, or CelluSponge-COL™ (Bio-Bybios).

[0024] In alternative embodiments, any cell culture medium capable of supporting hematopoietic stem cell culture can be used.

[0025] In alternative embodiments, any gas permeable bag or equivalent container can be used, or materials similar to known gas permeable containers can be used, for example, the articles of manufacture provided herein can include the use of gas permeable materials or membranes comprising fluorinated ethylene propylene (FEP) copolymers found in VueLife 2PF-0290™ (American Fluoroseal Corporation, Gaithersburg, Md) and PermaLife™ (OriGen Biomedical, Austin, Tex), and / or gas permeable materials or membranes comprising polyolefins, such as LIFECELL™ (3-L bag, Baxter, Deerfield, Ill.) culture bags or CultiLife™ (Takara) cell culture bags or MACS™ culture bags (Miltenyi Biotec, Bergisch Gladbach, Germany) made from polyolefin blends.

[0026] In alternative embodiments, the gas permeable materials or membranes used to manufacture the articles of manufacture provided herein include non-porous polystyrene, microporous polyolefins such as POLYFLEX® (Plastics Suppliers), microporous high density polyethylene (HDPE) such as TYVEK® or TYVEK® 1073 (DuPont), microporous polypropylene, microporous polyvinylidene fluoride, track etched polycarbonate (optionally small diameter and non-wettable in the liquid phase), hydrophobically treated nylon, polyurethane, microporous polyester with hydrophobic pores, microporous inorganic polymers and nonporous silicone rubber, any inorganic polymer, coextruded polystyrene and nonporous polyethylene or styrene such as butadiene-styrene or ethyl vinyl acetate or styrene-butadiene-styrene (SBS / EVA / SBS) three-layer coextruded film or styrene-butadiene-styrene / polyethylene (SBS / PE) two-layer coextruded film.

[0027] In an alternative embodiment, the gas permeable (and optionally liquid impermeable or liquid impermeable) membrane used is about 0.002 inches thick.

[0028] In an alternative embodiment, the gas-permeable (and optionally liquid-impermeable or liquid-impermeable) membrane used comprises a microporous membrane coated with a thin layer of silicone, which due to its permeability is particularly suitable for gas exchange; optionally the surface coating can be a thin layer of poly[1-(trimethylsilyl)-1-propyne] (PTMSP), which is known to be very permeable to gases.

[0029] In an alternative embodiment, the gas permeable (and optionally liquid impermeable or liquid impermeable) membrane used comprises a hydrophobic microporous hollow fiber membrane for degassing applications to remove oxygen, carbon dioxide and other gases from culture media, water and / or other liquids. In an alternative embodiment, a commercially available membrane module such as Liqui-Cel® membrane contactor (Membrana, Charlotte, NC) is used, which may comprise polypropylene (PP) X40 or X50 hollow fibers. In an alternative embodiment, a microporous PP hollow fiber membrane (e.g., CELGARD®) is used for oxygen delivery applications. In an alternative embodiment, a large membrane surface area (e.g., 220 m2) is used for degassing applications. 2 In this case, a membrane module suitable for large-scale industrial applications, for example Liqui-Cel®, having an active membrane surface area of ​​100 nm is used.

[0030] In alternative embodiments, the gas permeable (and optionally liquid impermeable or liquid impermeable) membrane used comprises poly(vinylidene fluoride) (PVDF), polyethylene (PE), PP, poly(vinyl chloride) (PVC) or other polymeric materials, optionally with a pore size in the range of 0.03-0.4 μm, and optionally with hollow fibers, e.g., with an outer diameter of 0.5-2.8 mm and an inner diameter of 0.3-1.2 mm.

[0031] Since low Earth orbit (LEO) can be used as a model to study inflammation, aging and malignant transformation in stem cells, the inventors designed and developed the bioreactor system provided herein to support cell culture of donor-derived human HSPCs in LEO. In an alternative embodiment, a sponge matrix, continuous flow pump and stromal cells are used, and this system or the article of manufacture provided herein can be used to model the bone marrow niche. Viability assessment by flow cytometry shows that our system (our article of manufacture provided herein) has the ability to maintain stem cell compatibility over a 6-week period. Cell cycle tracking shows a sharp decline in hematopoietic stem and progenitor cells in the resting phase (G0 / G1) of the cell cycle. In vitro colony assays during and after spaceflight compared to ground confirmed the reduced viability and loss of self-renewal capacity of stem cells returning from spaceflight. These data from the LEO SpX-24 mission clearly show a trend towards hematopoietic stem cell depletion and reduced "stemness" after 30 days of exposure to LEO.

[0032] Culture conditions In alternative embodiments, the articles of manufacture and kits provided herein comprising cells are cultured at cell culture growth compatible temperatures and environmental conditions, e.g., as described in Boudewijn Van Der Sanden, J Cell Biochem., 2010 November;111(4):801-807; or Borowski et al., Basic pluripotent stem cell culture protocols, StemBook, Cambridge (MA): Harvard Stem Cell Institute; 2008.

[0033] In alternative embodiments, the articles of manufacture and kits include the use of culture media compatible with stem cells, cultured cancer cells or organoids, which can be supplemented with growth factors or serum, see, e.g., Lee et al., Cell Biol Int., 2022 Jan;46(1):139-147, Epub 2021 Nov 27; Zhang J Tissue Eng 2020 Jan 24;11.

[0034] In an alternative embodiment, the article of manufacture further includes a pump, e.g., a peristaltic pump, e.g., a micro peristaltic pump, for circulating a liquid such as a culture medium within the container, enclosure or bag, the pump being operably connected to the inlet and outlet ports.

[0035] In alternative embodiments, the articles of manufacture and kits further include sensors for measuring the flow rate of liquid from one or more pumps, or for measuring incubation environment parameters, such as the temperature and / or pressure of the environment within the article of manufacture, or the amount of oxygen or carbon dioxide within the article of manufacture, or the pH of the article of manufacture, or a measurement of the viscosity or turbidity of the liquid within the article of manufacture. In alternative embodiments, the output of the sensor and / or pump is communicated to a remote device that can be read by a user, allowing the user to provide feedback or adjust the operation of the pump, or adjust another parameter, such as pH, temperature, gas volume (e.g., amount of oxygen), and / or pressure.

[0036] In an alternative embodiment, the rate of oxygen consumption by cells in the article of manufacture is balanced by adjusting the amount of oxygen transported through the membrane or into the article of manufacture by the flow of culture or biological fluid through the article of manufacture. The kinetics of oxygen consumption by cells is given by the oxygen uptake rate (OUR). The oxygen uptake rate can be expressed by Michaelis-Menten kinetics, and can be expressed as the number of moles of oxygen consumed per unit time per cell multiplied by the number of cells. The rate of oxygen transport through the membrane is the volumetric flow rate of oxygen per unit partial pressure difference (expressed as the volume of oxygen per unit area per unit time per unit partial pressure difference), where the partial pressure difference between the inside and outside of the membrane is used. It is given as the product of the projected areas of the film. The rate of oxygen transport by the culture or biological fluid depends on the diffusion rate of oxygen in the biological fluid, the oxygen concentration gradient along the direction perpendicular to the membrane at the membrane surface where the cells are cultured, and the membrane composition.

[0037] Cellular sensor and reporter vehicles or vectors In alternative embodiments, the cells in the articles of manufacture and kits further comprise a molecular sensor or reporter, e.g., a bioluminescent and / or fluorescent sensor or reporter comprising a vector, a plasmid or a viral vector, such as a bioluminescent and fluorescent lentiviral or adenoviral reporter vector, which can measure and / or quantitate one or more cellular functions, e.g., the function and / or viability of normal cells, pre-cancerous and / or cancerous cells or stem cells.

[0038] In alternative embodiments, any molecular sensor can be used, for example any vector, for example any viral vector, such as a lentiviral vector, that includes a cell cycle reporter, such as a FUCCI2BL cell cycle reporter, a self-replicating reporter, such as an ADAR1 (adenosine deaminase acting on RNA-1)-nanoluciferase-GFP (green fluorescent protein) self-replicating reporter, and / or a splicing reporter, such as a monomeric red fluorescent protein (mRFP) reporter or an RFP / GFP splicing reporter.

[0039] Manufactured Products and Kits Articles of manufacture and kits are provided for practicing the methods provided herein, and optionally, the articles of manufacture and kits can further include instructions for practicing the methods provided herein.

[0040] Any of the above aspects and embodiments may be combined with any other aspect or embodiment as disclosed in the Summary, Figures and / or Detailed Description sections.

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0042] The term "or" as used herein is understood to be inclusive, including both "or" and "and," unless otherwise stated or clear from the context.

[0043] The term "about" as used herein is understood to mean within the normal tolerance in the art, for example, within 2 standard deviations of the mean, unless otherwise specified or clear from the context. About (use of the term "about") can be understood to mean within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified with the term "about".

[0044] The terms "substantially all," "substantially a majority," "substantially all," or "majority" as used herein, unless otherwise specified or apparent from the context, include at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more of the referenced amount of the composition.

[0045] Each patent, patent application, publication, and document referred to herein is incorporated herein by reference in its entirety. Citation of the above patents, patent applications, publications, and documents is not an admission that any of the above is pertinent prior art, nor is it an admission as to the contents or dates of such publications or documents. The incorporation by reference of these documents should not, in and of itself, be construed as a claim or admission that any portion of the contents of any document shall be deemed essential material to satisfy national or local statutory disclosure requirements for patent applications. The right is nevertheless reserved to rely on any of such documents, where appropriate, to provide material deemed essential to the claimed subject matter by an examining authority or court.

[0046] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the present invention has been described in substantial detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications may be made to the embodiments specifically disclosed in this application, and that these modifications and improvements are within the scope and spirit of the present invention. The present invention illustratively described herein may also be suitably practiced in the absence of elements not specifically disclosed herein. Thus, for example, in each example of the present specification, any of the terms "comprise", "essentially consist of" and "consist of" may be replaced with any of the other two terms. Thus, the terms and expressions used are used as descriptive terms, and are not intended to be limiting, and do not exclude equivalents of the features shown and described, or portions thereof, and it is recognized that various modifications are possible within the scope of the present invention. Embodiments of the present invention are described in the following claims.

[0047] The present invention will be further described with reference to the embodiments described herein, but it will be understood that the invention is not limited to such embodiments. EXAMPLES

[0048] [Example 1] This example describes exemplary protocols for making and using the articles of manufacture, eg, bioreactors, provided herein.

[0049] material ULine Tabletop Impulse Heatsealer H-163™ OriGen PermaLife™ Gas Permeable Cell Culture Bags ETHICON SURGIFOAM(trademark) Absorbable porcine gelatin sponge Sterile scissors, tweezers, scalpel Sterile Luer Lock Syringe

[0050] Sterilize all non-sterile equipment by autoclaving. Work in a sterile environment such as a biosafety cabinet and spray and wipe down all materials with 70% EtOH before use.

[0051] 1) Using a scalpel, cut the sponge into the desired shape and size.

[0052] 2) Pre-soak the sponge in the medium you want to use for cell culture to remove any air bubbles inside the sponge. Consider degassing the medium depending on your downstream application.

[0053] 3) Using scissors, cut open the OriGen cell culture bag along the side opposite from the port.

[0054] 4) Using tweezers, place the pre-soaked sponge into the bag, being careful not to get the edges of the bag wet as this will prevent resealing.

[0055] 5) Using a heat sealer, seal the bag 9 times in succession at the highest temperature setting, waiting approximately 2 seconds between impulses.

[0056] 6) Leave the bag in the heat sealer for 5 minutes to cool before peeling it off to avoid the vinyl of the bag tearing and leaking.

[0057] 7) Carefully peel the bioreactor bag from the heat sealer and test for resealing using tweezers. If resealing is good and no holes are found, proceed to step 8. If not, repeat from step 5.

[0058] 8) Load the desired cell culture medium into a syringe and fill the bioreactor bag approximately halfway (test volume will depend on the size of cell culture bag used).

[0059] 9) Load the cells at the desired density into a syringe and seed the bioreactor bag. Load the second half of the medium into the syringe and load the bioreactor bag, allowing the cells to flow through the port into the bag and sponge.

[0060] 10) Allow the cells to settle for 8-24 hours before proceeding with downstream applications.

[0061] Since low Earth orbit (LEO) can be used as a model to study inflammation, aging and malignant transformation in stem cells, the inventors designed and developed the bioreactor system provided herein to support cell culture of donor-derived human HSPCs in LEO. In an alternative embodiment, a sponge matrix, continuous flow pump and stromal cells are used, and this system or the article of manufacture provided herein can be used to model the bone marrow niche. Viability assessment by flow cytometry shows that our system (our article of manufacture provided herein) has the ability to maintain stem cell compatibility over a 6-week period. Cell cycle tracking shows a sharp decline in hematopoietic stem and progenitor cells in the resting phase (G0 / G1) of the cell cycle. In vitro colony assays during and after spaceflight compared to ground confirmed the reduced viability and loss of self-renewal capacity of stem cells returning from spaceflight. These data from the LEO SpX-24 mission clearly show a trend towards hematopoietic stem cell depletion and reduced "stemness" after 30 days of exposure to LEO.

[0062] Although several embodiments of the invention have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. An article of manufacture for culturing and / or maintaining human primary hematopoietic stem cells, comprising: a container, enclosure or bag having gas permeable (and optionally liquid impermeable or liquid impervious) walls or sides, and a sponge matrix or sponge-like material contained within the container, enclosure or bag; The sponge matrix or sponge-like material is infused with a mixture of cell culture medium and cells, including stem cells and / or bone marrow matrix cells; and The interior of the container, enclosure or bag is sterile; and An article of manufacture wherein the container, enclosure or bag includes at least one fluid or cell input port.

2. 10. The article of manufacture of claim 1, wherein the container, enclosure, or bag is fabricated as a gas permeable cell culture environment or bag.

3. 10. The article of manufacture of claim 1, wherein the container, enclosure or bag comprises at least two liquid or cell input ports, one output port and one inlet port.

4. 4. The article of manufacture of claim 3, wherein a micro peristaltic pump for circulating a liquid within the container, enclosure, or bag is operably connected to the inlet and outlet ports.

5. 2. The article of manufacture of claim 1, wherein the mixture of cells comprises stem cells or human stem cells, bone marrow matrix cells or human bone marrow matrix cells, or cancer stem cells or human cancer stem cells, or organoid cells or organoid cells comprising cells from malignant organoids representing solid tumors.

6. 10. The article of manufacture of claim 1, wherein the mixture of cells comprises CD34+ donor-derived human hematopoietic cells (HSCs) or human CD34+ donor-derived human hematopoietic cells (HSCs).

7. The sponge matrix or sponge-like material is an absorbable gelatin sponge (optionally an absorbable human or porcine gelatin sponge); solubilized or reconstituted basement membrane matrix, solubilized or reconstituted laminin / collagen IV-rich basement membrane extracellular matrix, optionally Matrigel™ (Corning Life Sciences) or GELTREX™ (ThermoFisher Scientific); a compressed sponge comprising absorbable gelatin, optionally GELFOAM™ (Pfizer), and / or 10. The article of manufacture of claim 1, comprising a hydrogel-based macroporous sponge or porous hydroxypropyl cellulose (HPC) scaffold, optionally CelluSpongé™, CelluSpongé-GAL™, or CelluSpongé-COL™ (Bio-Bybio's).

8. 8. The article of manufacture of claim 7, wherein the sponge matrix or sponge-like material further comprises a demineralized cancellous bone matrix sponge or a demineralized bone matrix component, optionally OSTEOSPONGE™ (Xtant Medical).

9. about 10 ６ ~about 10 １０ 10. The article of manufacture of claim 1, wherein the cells are placed in or cultured in the article of manufacture.

10. 10. The article of manufacture of claim 1, further comprising a detectable vector or reporter, optionally comprising a bioluminescent and / or fluorescent vector, optionally comprising a bioluminescent and / or fluorescent lentiviral reporter vector, optionally wherein the bioluminescent and / or fluorescent vector is capable of quantitating a cell or a function of a cell, optionally a function of a normal cell, a precancerous cell, and / or a cancer stem cell, and optionally wherein the detectable vector or reporter comprises a FUCCI2BL cell cycle reporter, an ADAR1-nanoluciferase-GFP reporter self-renewal reporter, and / or an RFP / GFP splicing reporter.

11. A method for culturing, maintaining or differentiating a plurality of stem cells, the method comprising placing or culturing the plurality of stem cells in an article of manufacture according to any one of claims 1 to 10.

12. A method for generating organoids, optionally malignant organoids representing solid tumors, optionally breast cancer solid tumors, comprising incubating a plurality of stem cells in an article of manufacture according to any one of claims 1 to 10, optionally further comprising a cytokine capable of differentiating the stem cells.

13. A kit comprising the article of manufacture of any one of claims 1 to 8, optionally further comprising instructions for carrying out the method of claim 11.

14. Use of the product of any of claims 1 to 8 for culturing human primary hematopoietic stem cells, or for maintaining stem cells, or for differentiating cells, or for generating organoids.

15. 14. Use of the kit according to claim 13 for culturing human primary hematopoietic stem cells, or for maintaining stem cells, or for differentiating cells, or for generating organoids.

16. 14. An article of manufacture or kit for use in culturing human primary hematopoietic stem cells, or for use in maintaining stem cells, or for differentiating cells, or for generating organoids, wherein the article of manufacture is the article of manufacture described in any one of claims 1 to 7, or the kit is the kit described in claim 13.

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