Cell composition and product comprising same, preparation method, storage method and use method

Abstract

The present application relates to a cell composition and a product comprising same, a preparation method, a storage method and a use method. Provided is a cell composition, comprising mesenchymal stem cells and natural killer cells, wherein the mesenchymal stem cells are one or more of bone marrow-derived, adipose tissue-derived, umbilical cord-derived, placenta-derived and dental pulp-derived mesenchymal stem cells, and the natural killer cells are peripheral blood and / or umbilical cord blood-derived natural killer cells. The cell composition has broad application prospects, and brings new hope for future clinical treatment of diseases such as cancer, trauma, and / or ulcers.

Description

Cell compositions and their products, preparation, storage and use methods Technical Field

[0001] This invention relates to the fields of tissue engineering and regenerative medicine, specifically to cell compositions of mesenchymal stem cells and natural killer cells, as well as their products, preparation, storage, and use methods. Background Technology

[0002] Mesenchymal stem cells (MSCs) can be isolated from tissues such as the umbilical cord, bone marrow, adipose tissue, and amnion. They possess immunosuppressive and immunomodulatory functions, inhibiting abnormal proliferation of immune cells and exhibiting anti-inflammatory immunomodulatory effects. MSCs mediate immunosuppression through the secretion of soluble factors, primarily hepatocyte growth factor (HGF), IL-4, IL-10, sHLA-G, and TGF-β1. HGF has biological activities that promote cell mitosis and inhibit apoptosis, and can create an immunosuppressive microenvironment. IL-4 can reduce Th1-dependent delayed-type hypersensitivity responses and resist the biological effects of Th1-secreted INF-γ, thereby exerting an immunosuppressive effect. IL-10 is a recognized Treg growth factor that antagonizes IL-12 in the immune response and is a negative regulator of immunity, partially antagonizing the immunosuppressive effects of MSCs. sHLA-G is an important immune tolerance molecule in the body; its abnormal expression can lead to loss of autoimmune tolerance or inflammatory dysregulation, thereby inducing immunosuppression. TGF-β1 is an important negative regulator of immunity that can suppress cells other than lymphocytes. In addition to the above cytokines, mesenchymal stem cells also secrete some small molecule chemicals that participate in mediating local immunosuppression, such as cyclooxygenase and BT-H1.

[0003] Mesenchymal stem cells can also secrete exosomes. Exosomes are miniature plasma membrane vesicles, approximately 30-120 nm in diameter, actively synthesized and secreted into the extracellular environment by the endoplasmic reticulum and Golgi complex, and are an important paracrine form besides soluble cytokines. Exosomes derived from mesenchymal stem cells contain various cytokines, heat shock proteins, and microRNAs. Exosomes can protect cytokines, heat shock proteins, and microRNAs from degradation and prolong their half-life. Exosomes have been shown to have a clear protective effect against multiple organ dysfunctions, including kidney damage, myocardial infarction, and nervous system diseases. They can reverse apoptosis, promote tissue cell repair, effectively promote ulcer repair, and accelerate wound healing.

[0004] Natural killer cells (NK cells) are the main cells of the body's innate immunity. Morphologically, NK cells are large granular lymphocytes, originating from the bone marrow. They are the third largest type of lymphocyte after T cells and B cells, accounting for approximately 15% of all immune cells (white blood cells) in the blood. NK cells exhibit non-specificity in recognizing target cells, participating in the recognition process through intercellular adhesion molecule-1 (ICAM-1) on the surface of target cells. In addition, NK cells kill target cells by secreting perforin, NK cell cytotoxic factors, and TNF. NK cells are characterized by being unrestricted by MHC, requiring no prior sensitization, and capable of non-specifically killing tumor cells and virus-infected cells. They participate in anti-tumor, anti-viral infection, and immune regulation processes, and are important members of the body's immune surveillance. Currently, NK cells used in immunotherapy are mainly derived from donor peripheral blood, allogeneic umbilical cord blood, and NK cell lines.

[0005] Using adult peripheral blood as a source of NK cells allows for the use of the recipient or other healthy adult donors, offering a lower cost and greater flexibility. While the use of NK cells is not restricted by MHC, they still possess some immunogenicity and may cause immune rejection when used in allogeneic form.

[0006] Currently, there are two main methods for NK cell culture. The first method primarily utilizes cytokine stimulation for expansion. For example, interleukin-2 (IL-2) and IL-15, either alone or in combination, are used to induce NK cell expansion. However, this method suffers from low expansion efficiency, often making it difficult to obtain a sufficient number of highly active NK cells for clinical treatment in a short period. Furthermore, NK cell therapy alone is not ideal in treating solid tumors. The second method primarily uses irradiated human leukemia tumor cells, specifically K562, as feeder cells for NK cell culture. K562 cells are a relatively common cell line with relatively mature and stable culture conditions; they also secrete various cytokines, such as interleukin-15 (IL-15). These cytokines can stimulate the growth and proliferation of NK cells, thereby increasing NK cell expansion efficiency. However, K562 cells themselves pose a certain tumorigenic risk. Although strict control measures are taken during NK cell culture, the potential harm to patients cannot be completely ruled out.

[0007] In existing technologies, there are also attempts to combine different types of cells with NK cells for culture, but usually a simple mixing is performed at the end of the culture, which cannot give full play to the synergistic effect between different cells, and cannot achieve efficient NK cell expansion and enhance its anti-tumor activity.

[0008] There is a need in this field for cell compositions of mesenchymal stem cells and NK cells. Summary of the Invention

[0009] Most existing wound repair products use mesenchymal stem cell (MSC) culture supernatant, which does not contain live cells. Compared to products containing live cells, their ability to promote wound healing is relatively weak. The supernatant refers to the culture medium used during the in vitro culture of MSCs. Its main components are cytokines, growth factors, exosomes secreted by the stem cells, as well as residual components from the culture medium. Functionally, the supernatant contains a large number of cellular active substances, which can promote cell activation and repair tissue damage, but the effect is often temporary. MSCs, as living organisms, continuously secrete growth factors and other active substances, resulting in a more lasting effect. From a safety perspective, the residual culture medium in the supernatant may cause allergic reactions due to the presence of animal-derived components (such as bovine serum). MSCs undergo a washing process to remove culture medium components and are low in immunogenicity, generally not causing allergic reactions or immune rejection. Therefore, live MSCs are more effective than the supernatant in promoting wound healing.

[0010] This invention significantly improves the expansion efficiency and cytotoxic effect of NK cells by co-culturing MSCs with NK cells at different time points, utilizing the immunomodulatory properties of MSCs, without any potential safety risks. The method of this invention yields a cell composition containing mesenchymal stem cells and NK cells.

[0011] In one aspect, a cell composition comprising mesenchymal stem cells and natural killer cells is provided.

[0012] In one embodiment, the mesenchymal stem cells are one or more of bone marrow-derived, adipose-derived, umbilical cord-derived, placental-derived, and dental pulp-derived mesenchymal stem cells. In one embodiment, the natural killer cells are natural killer cells derived from peripheral blood and / or umbilical cord blood.

[0013] In one implementation, the natural killer cell is an activated natural killer cell.

[0014] In one implementation, the natural killer cells are IL-15 activated natural killer cells.

[0015] In one implementation, the natural killer cells are natural killer cells activated by IL-15 for 2-5 hours (e.g., 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours).

[0016] In one embodiment, mesenchymal stem cells are mesenchymal stem cells obtained by passage primary mesenchymal stem cells 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 generations).

[0017] In one implementation, the mesenchymal stem cells are positive for IL-12, IL-15, IL-18, IL-21 and / or IL-2.

[0018] In one embodiment, the cell composition is produced by co-incubating mesenchymal stem cells and natural killer cells in a suspension.

[0019] In one embodiment, the mesenchymal stem cells are added on one or more of the following days: day 0, day 3, day 4, day 5, day 7, day 10, day 14, and day 21 of the natural killer cell culture.

[0020] In one embodiment, the mesenchymal stem cells and natural killer cells are co-incubated for 18-25 days (e.g., 18, 19, 20, 21, 22, 23, 24 or 25 days).

[0021] In one embodiment, the initial culture volume for co-incubation is 50-150 ml, preferably 100 ml.

[0022] In one embodiment, the ratio of mesenchymal stem cells to natural killer cells is 1:20-20:1 (e.g., 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1).

[0023] In one implementation scheme, the cells are expanded to 2 x 10⁻⁶. 8 Up to 4x 10 8 3x10 is preferred 8 When the volume of cells reaches 250-350 ml, preferably 300 ml, the cells are injected into a cell culture bag, preferably a 2 L cell culture bag, for culture. In one embodiment, complete co-culture medium is added until the cell density is 1 x 10⁻⁶ cells / mL. 6 cells / ml to 1.5 x 10⁻⁶ 6 Between cells / ml. In one embodiment, a substance that promotes natural killer cell proliferation and inhibits T cell and / or B cell proliferation is added during co-incubation. In one embodiment, said substance is one or more of IL-2, IL-7, IL-15, IL-18, IL-21, and FLT3L.

[0024] In one embodiment, mesenchymal stem cells are genetically modified to secrete or overexpress one or more of the following cytokines: IL-2, IL-7, IL-15, IL-18, IL-21, and FLT3L.

[0025] In one embodiment, mesenchymal stem cells and natural killer cells are co-incubated in a co-culture medium. In one embodiment, the co-culture medium is based on StemRD Mgro-500 MesenGro human mesenchymal stem cell serum-free medium and GIBCO AIM-V medium CTS serum-free medium. In one embodiment, the co-culture medium is supplemented with one or more of FLT-3L, SCF, IL-3, IL-7, TNF-α, GM-CSF, TPO, IL-6, IL-2, and IL-15.

[0026] In one embodiment, the ratio of StemRD Mgro-500 MesenGro serum-free human mesenchymal stem cell culture medium to GIBCO AIM-V medium CTS serum-free culture medium is 1:2. In one embodiment, the co-culture medium is supplemented with one or more of the following: 300 ng / ml FLT-3L, 300 ng / ml SCF, 10 ng / ml IL-3, 5 ng / ml IL-7, 50 IU / ml TNF-α, 100 ng / ml GM-CSF, 5 ng / ml TPO, 100 ng / ml IL-6, 40 ng / ml IL-2, and 20 ng / ml IL-15.

[0027] In one embodiment, the cell composition is an aggregate of natural killer cells and mesenchymal stem cells.

[0028] In one embodiment, the cell aggregates are generated by co-incubating mesenchymal stem cells and natural killer cells in an aggregate culture medium comprising basal medium, serum albumin, and insulin. In one embodiment, the basal medium is any one of DMEM high-glucose medium, DMEM low-glucose medium, α-MEM medium, DMEM / F12 medium, and F12 medium.

[0029] In one embodiment, the aggregate culture medium also contains non-essential amino acids and / or L-glutamine.

[0030] In one embodiment, the serum albumin concentration is 0.5-10% by mass (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), preferably 0.5-5%, and most preferably 2%. In one embodiment, the insulin concentration is 0.5-50 μg / mL, preferably 1-20 μg / mL, and most preferably 5 μg / mL.

[0031] In one implementation, the initial cell concentration is adjusted to 1 x 10⁻⁶. 5 / ml to 1x 10 7 Between / ml. In one embodiment, mesenchymal stem cells are mixed with said natural killer cells in a ratio of 1:1 to 10:1 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1). In one embodiment, the cells are seeded by hanging drop method and cultured for 8-48 hours (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours) to form cell aggregates.

[0032] In one embodiment, the cell composition is a cell sheet of natural killer cells and mesenchymal stem cells.

[0033] In one embodiment, the cell membrane sheet is circular in size with a diameter of 3-30 mm and a thickness of 50-1000 μm.

[0034] In one embodiment, the cell sheet is obtained by co-culturing natural killer cells and mesenchymal stem cells on a substrate coated with a matrix that facilitates the attachment of mesenchymal stem cells.

[0035] In one embodiment, the matrix is ​​one or more of collagen, gelatin, fibronectin, fibrinogen, hyalin, laminin, polyornithine, polylysine, and ornithine-lysine copolymer.

[0036] In one implementation, mesenchymal stem cells are mixed with natural killer cells in a ratio of 1:1 to 10:1 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1) and cultured for 2-48 hours.

[0037] In one implementation, the amount of cells added is 1.8 x 10⁻⁶. 5 -1.8x 10 7 Preferably 1 x 10 7The amount of culture medium added is 1-4 ml, preferably 3 ml.

[0038] In another aspect, a product is provided that comprises the cell composition described herein.

[0039] In one implementation, the product is one or more of a biological scaffold, a dressing such as a wound healing patch, or a pre-filled syringe.

[0040] In one embodiment, the bioscaffold further comprises one or more of the following: extracellular matrix components, such as collagen, proteoglycans / glycosaminoglycans, and glycoproteins, such as fibronectin, laminin, platelet-reactive proteins, and proteoglycan attachment peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid; and / or the scaffold is made of a biocompatible polymer, including one or more of the following: resins and naturally occurring polymers, such as collagen, fibroin, hyaluronic acid, agarose, and laminin-rich gels, alginates, or modified alginate materials.

[0041] In another aspect, a method for preparing the cell product described herein is provided, comprising any of the following:

[0042] (1) Mesenchymal stem cells and natural killer cells were co-incubated in a suspension;

[0043] (2) Mesenchymal stem cells and natural killer cells were co-incubated in aggregate culture medium containing basal medium, serum albumin, and insulin, wherein the basal medium was any one of DMEM high-glucose medium, DMEM low-glucose medium, α-MEM medium, DMEM / F12 medium, and F12 medium; and

[0044] (3) Natural killer cells and mesenchymal stem cells were co-cultured on a substrate coated with a matrix that facilitates the attachment of mesenchymal stem cells.

[0045] In one implementation, the method further includes one or more of the following steps: primary culture of umbilical cord mesenchymal stem cells, passage of umbilical cord mesenchymal stem cells, and culture of natural killer cells.

[0046] In one embodiment, the initial culture volume for co-culturing in step (1) is 50-150 ml, preferably 100 ml. In one embodiment, the ratio of the amount of mesenchymal stem cells to the amount of natural killer cells added in step (1) is 1:20-20:1 (e.g., 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20:1).

[0047] In one implementation scheme, the cells are expanded to 2 x 10⁻⁶. 8 Up to 4x 10 8 3x10 is preferred 8 When the volume of cells reaches 250-350ml, preferably 300ml, inject the cells into a cell culture bag, preferably a 2L cell culture bag, for culture.

[0048] In one implementation, co-culture complete medium is then added until the cell density is 1 x 10⁻⁶ cells / year. 6 cells / ml to 1.5 x 10⁻⁶ 6 Between cells / ml.

[0049] In one implementation, step (1) includes adding the mesenchymal stem cells on one or more of the following days: day 0, day 3, day 4, day 5, day 7, day 10, day 14, and day 21 of the natural killer cell culture.

[0050] In one implementation, step (1) includes co-incubating the mesenchymal stem cells and natural killer cells for 18-25 days.

[0051] In one implementation, step (1) includes adding a substance that promotes the expansion of natural killer cells and inhibits the expansion of T cells and / or B cells during co-incubation.

[0052] In one embodiment, the substance is one or more of IL-2, IL-7, IL-15, IL-18, IL-21, and FLT3L.

[0053] In one embodiment, mesenchymal stem cells and natural killer cells are co-incubated in a co-culture medium based on StemRD Mgro-500 MesenGro human mesenchymal stem cell serum-free medium and GIBCO AIM-V medium CTS serum-free medium, supplemented with one or more of FLT-3L, SCF, IL-3, IL-7, TNF-α, GM-CSF, TPO, IL-6, IL-2 and IL-15.

[0054] In one embodiment, the ratio of StemRD Mgro-500 MesenGro human mesenchymal stem cell serum-free medium to GIBCO AIM-V medium CTS serum-free medium is 1:2, and / or the co-culture medium is supplemented with one or more of the following: 300 ng / ml FLT-3L, 300 ng / ml SCF, 10 ng / ml IL-3, 5 ng / ml IL-7, 50 IU / ml TNF-α, 100 ng / ml GM-CSF, 5 ng / ml TPO, 100 ng / ml IL-6, 40 ng / ml IL-2, and 20 ng / ml IL-15.

[0055] In one embodiment, step (2) includes generating mesenchymal stem cells and natural killer cells by co-incubation in aggregate culture medium containing basal medium, serum albumin and insulin, wherein the basal medium is any one of DMEM high glucose medium, DMEM low glucose medium, α-MEM medium, DMEM / F12 medium and F12 medium.

[0056] In one embodiment, the aggregate culture medium also contains non-essential amino acids and / or L-glutamine.

[0057] In one embodiment, the serum albumin concentration is 0.5-10% by mass (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), preferably 0.5-5%, most preferably 2%, and / or the insulin concentration is 0.5-50 μg / mL, preferably 1-20 μg / mL, most preferably 5 μg / mL.

[0058] In one embodiment, the cell concentrations of the mesenchymal stem cells and the natural killer cells are adjusted to 1 x 10⁻⁶. 5 / ml to 1x10 7 / ml. In one embodiment, the mesenchymal stem cells and natural killer cells are mixed evenly at a ratio of 1:1 to 10:1, seeded by hanging drop method, and cultured for 8-48 hours to form cell aggregates. In one embodiment, the ratio of mesenchymal stem cells to natural killer cells is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In one embodiment, the culture time is, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0059] In one implementation, the ratio of mesenchymal stem cells to natural killer cells is 5:1 to 10:1, preferably 8:1.

[0060] In one implementation, step (3) includes co-culturing natural killer cells and mesenchymal stem cells on a substrate coated with a matrix that facilitates the attachment of mesenchymal stem cells.

[0061] In one embodiment, the matrix is ​​one or more of collagen, gelatin, fibronectin, fibrinogen, hyalin, laminin, polyornithine, polylysine, and ornithine-lysine copolymer.

[0062] In one embodiment, mesenchymal stem cells and natural killer cells are mixed homogeneously at a ratio of 1:1 to 10:19 and cultured for 2-48 hours. In one embodiment, the ratio of mesenchymal stem cells to natural killer cells is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In one embodiment, the culture time is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0063] In one implementation, the amount of cells added is 1.8 x 10⁻⁶. 5 -1.8x 10 7 Preferably 1 x 10 7 The amount of culture medium added is 1-4 ml, preferably 3 ml.

[0064] In another aspect, a method for treating a disease in a subject is provided, comprising administering the cell composition described herein to the subject.

[0065] In one implementation, the disease is cancer, trauma, and / or ulcer.

[0066] In one implementation, the cancer is one or more of liver cancer, ovarian cancer, lung cancer, mesenchymal stem cell carcinoma, and B-cell lymphoma.

[0067] In one implementation, trauma is burns, scalds, and / or mechanical injury.

[0068] In one implementation, the ulcer is an oral ulcer, such as a recurrent oral ulcer.

[0069] In one implementation, the subjects had diabetic foot.

[0070] In one implementation, the subject is a mammal, preferably a human.

[0071] In one embodiment, the method includes applying a wound healing patch containing the cellular composition described herein to an ulcer or wound.

[0072] In one implementation, the wound healing patch is applied 1-5 times daily, and / or the application continues for 1-10 days. In one implementation, the wound healing patch is applied 1, 2, 3, 4, or 5 times daily. In one implementation, the application continues for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0073] In another aspect, the use of the cell compositions described herein in the preparation of medicaments or kits for treating diseases is provided.

[0074] In one implementation, the disease is cancer, trauma, and / or ulcer.

[0075] In one implementation, the cancer is one or more of liver cancer, ovarian cancer, lung cancer, mesenchymal stem cell carcinoma, and B-cell lymphoma.

[0076] In one implementation, trauma is burns, scalds, and / or mechanical injury.

[0077] In one implementation, the ulcer is an oral ulcer, such as a recurrent oral ulcer.

[0078] In another aspect, a method for storing a dressing is provided, comprising immersing the dressing in a preservation solution at 2-8°C or storing it in a liquid nitrogen tank at -183°C, said dressing comprising the cellular composition described herein.

[0079] In one implementation, the dressing also includes a pharmaceutically acceptable carrier, such as maltodextrin.

[0080] In another aspect, a method for storing a pre-filled syringe is provided, comprising storing the pre-filled syringe in a liquid nitrogen tank at -183°C, the pre-filled syringe containing the cell composition and cryoprotectant described herein.

[0081] The beneficial technical effects of this invention include:

[0082] (1) A method for co-culturing MSCs and NK cells was provided;

[0083] (2) The therapeutic effects of hybrid cells obtained by co-culturing MSCs and NK cells were provided;

[0084] (3) A cell composition is provided that can continuously secrete active substances such as growth factors, resulting in a more lasting effect. This cell composition has low immunogenicity and generally does not cause allergic reactions or immune rejection. This cell composition is more effective than the supernatant in promoting wound healing. Attached Figure Description

[0085] Figure 1 shows the morphology of cell aggregates.

[0086] Figure 2 shows a photograph of a cell membrane sheet. Detailed Implementation

[0087] To facilitate understanding of this disclosure, certain terms are defined below. Further definitions of these terms, and others, are set forth throughout the specification.

[0088] Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators.

[0089] Unless otherwise specified in the context, the term “or” as used herein should be understood as inclusive and encompasses both “or” and “and”.

[0090] The term “and / or” should be considered as a specific disclosure of each of the two specified features or components, including or excluding the other. Thus, as used herein in phrases such as “A and / or B”, the term “and / or” is intended to include A and B, A or B, A (alone), and B (alone). Similarly, as used in phrases such as “A, B, and / or C”, the term “and / or” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0091] As used in this article, when referring to a range of values, it includes any integer within that range and any range thereof. For example, when referring to 1:20-20:1, it includes the individual values ​​within that range and any range between those values.

[0092] The term "multiple" should be understood to include, but is not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. It also includes any larger numerical values ​​or fractions in between.

[0093] Throughout this specification, the word “comprising” or variations such as “containing” or “including” should be understood to imply inclusion of the stated elements, integers, or steps, or groups of elements, integers, or steps, but does not exclude any other elements, integers, or steps, or groups of elements, integers, or steps. It should be understood that any aspect described herein using the language “comprising” is also provided for similar aspects described as “consisting of” and / or “substantially consisting of”.

[0094] As used herein, a “cell composition” refers to a composition formed from two or more different types of cells. Preferably, the cell composition is a composition of MSCs and NK cells. MSCs can promote tissue repair and regeneration by secreting cytokines and growth factors, making them suitable for treating various tissue injuries or diseases. NK cells, on the other hand, have the natural ability to monitor and kill tumor cells, clearing pathogens and tumor cells from the body and maintaining the stability of the immune system. In the field of wound repair, the combined application of MSCs and NK cells can exert a synergistic therapeutic effect. NK cells are responsible for clearing pathogens and microorganisms that infect wounds and maintaining local immune integrity, while MSCs reach the damaged site to play a repairing role and accelerate wound healing. One of the unresolved issues in cell-based therapies is cell migration and integration into the site of injury. This process is called “homing.” NK cells facilitate the migration, homing, and implantation of MSCs. As used herein, “administration” means introducing the cell composition described herein into a subject using any of the various methods and delivery systems known to those skilled in the art, such as intravenous injection, intraperitoneal injection, subcutaneous injection, or topical application.

[0095] Co-culture method of MSC cells and NK cells

[0096] This protocol relates to a method for efficiently expanding NK cells using MSC feeder cells. Specifically, the method includes the preparation of MSC cells, the preparation of primary MSC cells, the assessment of factor secretion by MSC cells, and the assessment of the co-culture period of MSCs and NK cells.

[0097] MSCs from different tissues

[0098] - Bone marrow: Bone marrow fluid is usually extracted from the patient's bone marrow, and the mononuclear cell layer containing mesenchymal stem cells (MSCs) is separated by methods such as density gradient centrifugation.

[0099] -Adipose tissue: Adipose tissue obtained during procedures such as liposuction, and MSCs are separated after digestion and processing.

[0100] - Perinatal tissues such as umbilical cord and placenta: These tissues are also rich in MSCs and can be collected and separated after delivery.

[0101] Preparation of primary MSC cells

[0102] - The isolated MSC-containing cell suspension is seeded into a culture vessel using MSC culture medium, which typically includes basal medium (such as DMEM, α-MEM, etc.) and a certain proportion of serum (fetal bovine serum, etc.) and growth factors. The cells are then cultured in a constant temperature incubator at 37°C and 5% CO2. MSCs will gradually adhere to the culture vessel and grow, forming a monolayer. When the primary cultured MSCs reach a certain density (usually 80%-90% confluence), cell morphology and growth status are observed under a microscope, and the cells are passaged. Cells are cryopreserved after passages 3-10.

[0103] Factor secretion status of MSC cells

[0104] During MSC cell passage, the culture supernatant of MSC cells is collected, and the secretion of certain factors in the MSC cell supernatant is detected by ELISA to determine whether the MSC cells meet the requirements for NK cell expansion and their impact on NK cell growth and functional characteristics. The cytokines detected may include, but are not limited to, IL-12, IL-15, IL-18, IL-21, and IL-2.

[0105] Co-culture period of MSC and NK cells

[0106] 1) Exploration of the effects of cytokines secreted by MSCs of different generations on NK cell expansion and function.

[0107] MSC cells from each passage were co-cultured with NK cells until day 21, and the expansion rate, purity, and functional characteristics of NK cells were assessed to determine the optimal MSC cell passage.

[0108] 2) The timing of MSC cell addition affects NK cell expansion and functional exploration.

[0109] MSCs of a specific passage were added on one or more individual days (days 0, 3, 4, 5, 7, 10, 14, and 21) of NK cell culture, and co-cultured until day 21. The expansion effect, purity, and functional characteristics of NK cells were then assessed to determine the optimal timing for adding MSCs.

[0110] 3) The effect of MSC cell addition on NK cell expansion and functional exploration.

[0111] Under the conditions of selected MSC cell passages and addition timing, the effects of different amounts of added MSC cells (MSC cell to NK cell ratios of 20:1, 15:1, 10:1, 5:1, 1:1, 1:5, 1:10, 1:15, 1:20, etc.) on NK cell expansion and function were explored.

[0112] 4) In addition to adding MSC cells, additional cytokines / small molecule peptides that promote NK cell proliferation and inhibit T cell / B cell proliferation can be added. By adjusting the proportion of cytokines, the proliferation and functional characteristics of NK cells can be improved.

[0113] For example, commonly used cytokines such as IL-2, IL-7, IL-15, IL-18, IL-21, and FLT3L can enhance the expansion and functional characteristics of NK cells.

[0114] 5) Genetically modify MSC cells to enable them to secrete certain cytokines, thereby promoting the expansion and functional characteristics of NK cells.

[0115] Against the backdrop of increasing demand for cancer treatment, this method addresses the problems of low NK cell expansion efficiency and poor efficacy of traditional NK cell therapy for solid tumors. The preparation method has clearly defined steps and includes specific culture conditions and process control. By using MSCs as feeder cells and co-culturing them with NK cells at different time points, the immunomodulatory properties of MSCs are utilized to significantly improve the expansion efficiency and killing effect of NK cells. In terms of application, this provides a new strategy and approach for cancer immunotherapy, and is expected to improve the efficacy of cancer treatment and patient survival rates. Compared to a simple mixture of MSCs and NK cells at the culture endpoint, co-cultured NK cells and MSCs exhibit better therapeutic effects. For different cancer patients, different culture protocols can be used in the early stages to precisely control the ratio of mesenchymal stem cells to NK cells, thereby achieving precise and effective treatment. This innovative co-culture method has broad application prospects and brings new hope to future clinical treatment. NK cells can be derived from peripheral blood, umbilical cord blood, etc., without restriction. In existing technologies, some attempts have been made to combine different types of cells with NK cells for culture. However, these methods typically involve simple mixing at the end of the culture period, failing to fully leverage the synergistic effects between different cell types and thus hindering efficient NK cell expansion and enhanced anti-tumor activity. This invention utilizes MSCs as feeder cells, co-culturing them with NK cells at different time points. By leveraging the immunomodulatory properties of MSCs, it significantly improves the expansion efficiency and NK cell killing effect, while eliminating potential safety risks.

[0116] Cell microspheres

[0117] The characteristics of cell microspheres (such as the umbilical cord mesenchymal stem cell microspheres described herein) may include: (1) being spherical or ellipsoidal cell aggregates with a diameter of approximately 30-300 μm; (2) having intact extracellular matrix; (3) containing injectable human serum albumin in the cell gaps; (4) secreting large amounts of pro-angiogenic factors such as HGF, VEGF, and IL-8; (5) secreting large amounts of IL-6 and IL-8 anti-inflammatory factors; (6) being able to inhibit the Th1 subtype of lymphocytes, inhibit lymphocyte proliferation, and inhibit lymphocyte TNFα secretion; (7) being able to promote microvascular formation; (8) being either a cryopreserved preparation or a fresh preparation; (9) each aggregate containing 10-5000 umbilical cord mesenchymal stem cells, preferably 100-3000, and most preferably 500-2000.

[0118] The features and functions of the aggregates in this application are as follows:

[0119] (1) High safety: The culture medium used in the aggregate preparation process has a simple formulation and does not contain animal-derived components or exogenous growth factors; and

[0120] (2) Superior efficacy: Compared with single-cell suspensions, aggregates have a longer survival time and higher survival rate in vivo, and can perform their functions more persistently and efficiently.

[0121] Cell aggregates are a more efficient method of stem cell transplantation. Compared with the previous method of stem cell suspension injection, cell aggregates can effectively avoid the loss of stem cells during the transplantation process and improve the utilization rate of stem cells.

[0122] The preparation process of umbilical cord mesenchymal stem cell aggregates is as follows: passaged umbilical cord mesenchymal stem cells are digested into single cells using biological enzymes, then washed to remove residual umbilical cord mesenchymal stem cell culture medium, and then resuspended in aggregate culture medium. After that, the cells are seeded at a certain cell density by hanging drop seeding, low adherent microplate seeding, centrifuge tube seeding, shake flask seeding, stirred flask seeding, or rotating screen seeding.

[0123] Umbilical cord mesenchymal stem cell aggregates can be preserved in a preservation solution at 2-8℃, room temperature, or 37℃ for immediate use after preparation. Alternatively, they can be cryopreserved in liquid nitrogen for immediate use.

[0124] Human umbilical cord mesenchymal stem cell aggregates can promote microvascular regeneration, regulate inflammatory responses, and delay tissue fibrosis through paracrine effects. This allows them to repair ischemic injuries caused by diseases or trauma to the myocardium, liver, kidneys, skin, uterus, and other organs.

[0125] Product Form

[0126] The cell compositions of this application can be prepared into various products. The cell compositions of this invention can be prepared and used immediately before product commercialization or use. Products may include biological scaffolds, dressings, and pre-filled syringes, etc.

[0127] 1) Biological scaffold

[0128] The scaffold composition has varying permeability, allowing cells to flow out only in certain physical areas of the scaffold. Cell outflow can be modulated by altering the composition of the scaffold, for example, by using adhesion molecules to attract or delay cell migration. The scaffold is made from a variety of synthetic biocompatible polymers, including resins and naturally occurring polymers such as, but not limited to, collagen, fibroin, hyaluronic acid, agarose, and laminin-rich gels. A preferred hydrogel material is alginate or modified alginate. Calcium-crosslinked alginate hydrogels can be used in dental applications, wound dressings, chondrocyte transplantation, and as a matrix for other cell types. Relatively low molecular weight alginate or other polysaccharides are used, preferably with a size at the renal clearance threshold after dissolution, preferably with a molecular weight of 1000 to 60,000 Daltons, particularly preferably 1000 to 50,000 Daltons.

[0129] The scaffold is nanoporous with a diameter of less than about 10 nm. At least one cell adhesion molecule is incorporated into or onto the polymer matrix. Examples of cell adhesion molecules include, but are not limited to, peptides, proteins, and polysaccharides. More specifically, cell adhesion molecules include fibronectin, laminin, etc. Particularly preferred cell adhesion molecules are peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD).

[0130] The composition is assembled into a cell scaffold structure using methods known in the art, such as injection molding, freeze-drying of pre-formed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber formation / processing, particle leaching, or combinations thereof.

[0131] Stem cell populations are encapsulated in a hydrogel using standard encapsulation techniques, such as alginate bead formation. The first hydrogel contains factors necessary to maintain stem cell pluripotency. These factors are released slowly and continuously from the gel via covalent coupling to the polymer forming the gel or through their presence. A second layer of gel (e.g., a bilayer of alginate beads) is then coated with factors that do not retain stem cells but promote their rapid proliferation and the generation of numerous more specialized daughter cells.

[0132] The scaffold includes at least one cell growth factor that prevents premature terminal differentiation of transplanted cells in the polymer matrix and induces the migration of transplanted cells and their progeny out of the polymer matrix. The cell growth factor is incorporated into the polymer matrix before polymerization or may be coupled to the polymer matrix after polymerization.

[0133] 2) Applying plaster

[0134] The cell composition of this application can be formulated into a dressing. Since the agent uses live mesenchymal stem cells, the prepared dressing needs to be immersed in a special preservation solution to maintain cell viability and stored at 2-8°C. Before use, it should be removed from the preservation solution and allowed to warm to room temperature for 10-30 minutes. The dressing may contain maltodextrin. Maltodextrin can form a protective film on the wound surface, effectively preventing contact and friction between the wound and other areas, reducing pain, and providing a sustained-release effect for the drug, resulting in a longer-lasting efficacy than conventional drugs.

[0135] 3) Pre-filled syringe

[0136] The cell composition can also be mixed with cryoprotectant and loaded into a pre-filled syringe for long-term storage in liquid nitrogen. The pre-filled syringe is made of materials resistant to liquid nitrogen, such as glass, plastic, or stainless steel. The pre-filled syringe is an assembly, mainly composed of a tube, piston, plunger, cone, and cap.

[0137] Product preservation

[0138] The cell composition of this application can be refrigerated at 4°C for 7 days in culture medium. Before use, it can be brought to room temperature. If necessary, it can be stored long-term in liquid nitrogen. For example, after the wound repair patch is manufactured, it can be stored in a liquid nitrogen tank at -183°C for a long time. When needed, it is revived using a certain method. The revived wound repair patch is stored in culture medium and can be refrigerated at 4°C for 7 days. Before use, it can be brought to room temperature. The dressing preparation can be stored at 2-8°C and has a shelf life of about 1 week. The pre-filled preparation can be placed in liquid nitrogen and can be stored for a long time.

[0139] Treatment course

[0140] The products in this application, such as wound healing patches, can be refrigerated in a household refrigerator for 7 days, so the treatment course is designed to last 1-7 days.

[0141] Indications

[0142] 1) Complex and refractory oral ulcers

[0143] The main causes of recurrent oral ulcers include autoimmune disorders, local injury, psychological stress, and diet. One of the causes of oral ulcers is an autoimmune reaction. Studies have found that recurrent oral ulcers are due to abnormal cellular immune function and an imbalance in immune regulation. This disorder of normal immune regulation constitutes an autoimmune disease. Its pathological process involves not only innate immunity but also a series of immune disorders, including Th1 / Th2 imbalance, reduced function and number of CD4+CD25+Foxp3+ Treg cells, disordered cytokine secretion, mucosal antibody formation, low expression of sIgA in the oral mucosa, and allergic reactions. Furthermore, changes in local microcirculation and oxygen free radical-mediated immune disorders have also been found to contribute to the pathogenesis of recurrent oral ulcers.

[0144] Before applying the product of this application (e.g., wound healing patch), clean the oral ulcer by brushing your teeth and rinsing your mouth (using mouthwash will enhance the effect). Remove the product from the container and allow it to return to room temperature. Then, apply the patch to the oral ulcer, spreading it evenly. Do not eat or drink for 2 hours. Apply the medication 3-5 times daily until the ulcer heals.

[0145] 2) Diabetic foot

[0146] Diabetic foot is caused by a combination of factors, including long-term hyperglycemia leading to neuropathy, vascular disease, and abnormal immune function. Specifically, chronic hyperglycemia causes microvascular disease, restricting blood supply and resulting in symptoms such as dry skin, thickened keratin, hardening of the soles, and cracking. Simultaneously, hyperglycemia can damage nerves, causing loss of sensation or abnormal sensation in the feet, making patients prone to ignoring foot pain and injuries, leading to wound aggravation and infection. Furthermore, abnormal immune function also makes diabetic foot patients more susceptible to infection.

[0147] Before applying the product described in this application, clean the wound thoroughly, removing any necrotic tissue, ulcers, and pus. Remove the wound repair patch and allow it to return to room temperature. Then apply it to the foot ulcer, spreading it evenly, and secure it with gauze or a bandage. Apply the medication once daily, changing the dressing every other day. Significant improvement should be observed after one week of continuous treatment.

[0148] 3) Burns and scalds

[0149] Burns and scalds are generally tissue damage caused by high-temperature liquids, steam, or hot metals, primarily affecting the skin or mucous membranes. The product described in this application has a certain therapeutic effect during the tissue repair phase after the acute phase of a burn or scald. Remove the product from the wound and allow it to return to room temperature before applying it to the wound, spreading it evenly, and securing it with gauze or a bandage. Apply the medication once daily, changing the dressing every other day. Significant improvement can be seen after one week of continuous treatment.

[0150] Example

[0151] Example 1: Co-incubation of mesenchymal stem cell suspension with NK cells

[0152] 1) Resuscitate the cryopreserved NK cells and umbilical cord mesenchymal stem cells respectively. After thawing the corresponding cell cryopreservation tubes in a 37°C water bath, quickly add them to 10 times the volume of DMEM basal medium for dilution and centrifuge at 1500 rpm for 5 min. Remove the supernatant and resuspend them in co-culture complete medium.

[0153] 2) The co-culture complete culture medium formula is based on a 1:2 mixture of StemRD Mgro-500 MesenGro human mesenchymal stem cell serum-free medium and GIBCO AIM-V medium CTS serum-free medium, with the addition of 300 ng / ml FLT-3L, 300 ng / ml SCF, 10 ng / ml IL-3, 5 ng / ml IL-7, 50 IU / ml TNF-α, 100 ng / ml GM-CSF, 5 ng / ml TPO, 100 ng / ml IL-6, 40 ng / ml IL-2, and 20 ng / ml IL-15;

[0154] 3) The initial culture volume for co-culture inoculation is 100 ml. Resuscitated NK cells and P5 generation umbilical cord mesenchymal stem cells are directly suspended and cultured together at a ratio of 1:10. The density of nucleated cells in the umbilical cord blood is controlled to be greater than 1 x 10⁻⁶. 6 cells / ml;

[0155] 4) Wait until the cells expand to 3 x 10⁻⁶ 8 When the volume of cells reaches 300ml, the cells are injected into a 2L cell culture bag for culture. The culture conditions are 37℃, saturated humidity, and 5% CO2. Samples are taken and counted every 2 days during the culture process.

[0156] 5) After completing the sampling and counting, replenish fresh co-culture complete culture medium until the cell density is 1 x 10⁻⁶. 6 cells / ml to 1.5 x 10⁻⁶ 6 Between cells / ml;

[0157] 6) After replenishing the culture medium to 2L based on the cell density, culture for another 2 days. Harvest all the cell suspension by centrifuging at 1500rpm for 5min to collect all cells. Wash the cells 2-3 times with physiological saline. Finally, collect all cells and take samples for counting.

[0158] Preferably, the NK cells are activated NK cells. Preferably, the NK cells have been activated with IL-15, such as by IL-15 activation for 4 hours. Preferably, the NK cells are peripheral blood natural killer cells (PB-NK).

[0159] Example 2: Co-incubation of mesenchymal stem cell microspheres with NK cells

[0160] The fabrication process of mesenchymal stem cell microspheres and NK cell aggregates is as follows:

[0161] 1) Umbilical cord mesenchymal stem cell culture

[0162] Primary culture and passage culture

[0163] The culture medium used during the culture process can be any medium that supports the growth of umbilical cord mesenchymal stem cells, including serum-containing and serum-free media.

[0164] Primary culture of umbilical cord mesenchymal stem cells

[0165] After cleaning the collected neonatal umbilical cord with physiological solution, the arteries, veins, and adventitia were removed. Wharton's jelly was isolated, and the tissue was cut into 0.1–2 mm pieces. These tissue pieces were then evenly spread in a culture container coated with matrix, with a spacing of 2–30 mm between each piece. The culture container was then placed in a cell culture incubator. After 2–7 days, an appropriate amount of complete culture medium was added to cover the tissue pieces. After 8–21 days, umbilical cord mesenchymal stem cells could be observed emerging. When the cells reached 70–100% confluence, the tissue pieces were removed, and the cells were passaged.

[0166] Umbilical cord mesenchymal stem cell culture:

[0167] When the cells reach 70-100% confluence, they are passaged: the cells are separated from the culture vessel by methods including trypsin digestion and cell scraping. Then, the cells are dispersed in the culture medium by methods such as pipetting, stirring, and vortexing (including but not limited to), at a rate of 500-100,000 / cm³. 2 Cells were seeded into culture containers at a suitable density. An appropriate amount of culture medium was added, and the medium was replaced with fresh medium every 1-5 days depending on the cell growth status. When the cells reached 70-100% confluence, the passage process was repeated. Umbilical cord mesenchymal stem cells exhibited fibroblast-like adherent growth and uniform morphology.

[0168] 2) NK cell culture

[0169] (1) On day -1, the cell culture plate was coated with activator.

[0170] The activators include the following antibodies: anti-human CD16 monoclonal antibody with a final concentration of 2-10 μg / ml, anti-human HER-2 mAb with a final concentration of 0.1-1 μg / ml, and retrotronectin with a final concentration of 5-20 μg / ml. The three activators are mixed with PBS, and the mixture is pipetted 10 times. The mixture is then added to cell culture plates or culture flasks and incubated overnight at 4°C for later use.

[0171] (2) On day 0, PBMC cells were revived.

[0172] (a) Remove the frozen PBMC cells from the liquid nitrogen container and thaw them rapidly in a 37°C water bath for approximately 2 minutes. Remove the cells when they contain a small number of still-frozen cells (no need to wait for complete thawing). Use a 1ml pipette to pipette the cell suspension once in each cryovial. Transfer 1ml of the cell suspension to a 50ml centrifuge tube and slowly add 9ml of IMDM basal medium. Add 1ml of the suspension to the cryovial, pipette once (to wash away any remaining cells), and transfer to a 50ml centrifuge tube. Pipette twice with a 10ml pipette to mix. At this point, the PBMC cells should be singly distributed without any flocculent cell clumps. Centrifuge at 500g for 5 minutes and discard the supernatant.

[0173] (b) Resuspend PBMC cells in 3 ml of IMDM medium containing 10% autologous plasma, and perform two replicate cell counts using 20 μl of the medium; follow the 6 x 10⁻⁶ regimen. 6 The cell count ratio was set at 6 / ml, and PBMC cells were incubated at 37°C for 6-8 hours.

[0174] (3) On day 0, PBMC cells were inoculated.

[0175] Remove PBMC cells from the 37℃ incubator after 6-8 hours of static incubation. Add activators (including 0.25 μg / ml recombinant human IL3, 0.25 μg / ml recombinant human IL7, 0.25 μg / ml recombinant human IL18, 0.25 μg / ml recombinant human IL21, 0.05 μg / ml recombinant human IL1β, 0.25 μg / ml recombinant human 4-1BB protein, 0.25 μg / ml recombinant human IL-15, and 1000 IU / ml human interleukin-2 for injection, etc., antibodies or cytokines), pipette three times, mix well and resuspend the PBMC cells. Take 20 μL for two replicate cell counts; according to 1.5 x 10⁻⁶ 6 PBMC cells were seeded into day-1 coated cell culture plates at a cell count ratio of / ml. The plates were then incubated statically at 37°C for 4 days.

[0176] (4) On the 4th day, add activation medium (including IMDM medium, 5% autologous plasma and activator) at a ratio of 1:1 and incubate at 37°C.

[0177] (5) From day 5 to day 21, every 1-2 days, add fresh amplification medium at a 1:1 ratio. The amplification medium includes IMDM medium, 2.5% autologous plasma, and amplification reagent, which includes 1000 IU / ml IL-2 and 0.25 μg / ml recombinant human 1L-15. Rotate the cells into different culture bottles as the volume of the culture medium increases. Observe the color of the culture medium, the size of the cell colonies, and the cell density daily, and replenish the medium as needed. Before each replenishment, gently tap the culture bottle to suspend and disperse the adherent colonies.

[0178] 3) Preparation of cell aggregates

[0179] (1) Cell digestion

[0180] Remove the old culture medium from the umbilical cord mesenchymal stem cells, then wash with PBS 1-3 times, add digestive enzymes (TryPLE or trypsin) to digest until the cells are in a single-cell state, add PBS, complete cell culture medium, physiological saline, DPBS, Hanks, etc. to stop or reduce the digestion of the enzymes, centrifuge, discard the supernatant, and obtain the cell pellet.

[0181] (2) Cell washing

[0182] Digested umbilical cord mesenchymal stem cells were washed with PBS, physiological saline, DPBS, and Hanks buffer, at a concentration of 5 x 10⁻⁶ mcg / mL. 5 Wash cells / mL at a density of at least 2 times.

[0183] The collected NK cells were washed with PBS, physiological saline, DPBS, and Hanks buffer, at a concentration of 5 x 10⁻⁶ cells / mL. 6 Wash cells / mL at a density of at least 2 times.

[0184] (3) Aggregate preparation

[0185] Stem cells and NK cells were washed to remove residual culture medium, then resuspended in aggregate medium and adjusted to a cell concentration of 1 x 10⁻⁶. 5 / ml to 1x 10 7 Stem cells and NK cells are mixed evenly at a ratio of 1:1 to 10:1 between 1 / ml and seeded into culture dishes or flasks, or into cell culture shake flasks, or into cell culture rotating flasks, or into low-absorption microplates, or into centrifuge tubes using the hanging drop method. After 8-48 hours of culture, cell aggregates are formed.

[0186] The preferred ratio of stem cells to NK cells is 5:1 to 10:1, with 8:1 being the most preferred. The aggregate culture medium consists of basal medium + human serum albumin + insulin + non-essential amino acids (optional) + L-glutamine (optional). The basal medium can be DMEM (high glucose), DMEM (low glucose), α-MEM, DMEM / F12, F12, etc., with α-MEM and DMEM / F12 being preferred, and DMEM / F12 being the most preferred. The concentration (mass fraction) of human serum albumin is 0.5-10%, preferably 0.5-5%, and most preferably 2%. The addition of human serum albumin promotes the secretion of extracellular matrix during aggregate formation, thus promoting aggregate formation. The insulin concentration is 0.5-50 μg / mL, preferably 1-20 μg / mL, and most preferably 5 μg / mL.

[0187] (4) Aggregate harvesting

[0188] Cell aggregates along with the culture medium were collected into centrifuge tubes. The culture supernatant was removed by centrifugation, and the aggregates were washed 1-6 times with PBS, physiological saline, DPBS, and Hanks buffer to minimize residual culture supernatant. Figure 1 shows the morphology of the cell aggregates.

[0189] Example 3: Co-incubation of mesenchymal stem cell sheets with NK cells

[0190] 1. Primary culture of umbilical cord mesenchymal stem cells

[0191] After washing the obtained neonatal umbilical cord with a physiological solution of equal osmotic pressure to human body fluids, the arteries, veins, and adventitia are removed. Wharton's jelly is isolated, and the tissue is cut into small pieces of 0.1-2 mm. These tissue pieces are evenly spread in a culture container coated with a substrate, with a spacing of 2-30 mm between each piece. The culture container is placed in a cell culture incubator. After 2-7 days, an appropriate amount of complete culture medium is added to cover the tissue pieces. Umbilical cord mesenchymal stem cells emerge from the culture container after 8-21 days. These emerging cells adhere to the culture vessel and grow in a fibrous, uniform morphology. When the emerging cells reach 70-100% confluence, the tissue pieces are removed, and the cells are passaged.

[0192] 2. Passaging of umbilical cord mesenchymal stem cells

[0193] When the cells reach 70-100% confluence, passage them: Separate the cells from the culture vessel using methods including but not limited to trypsin digestion and cell scraping. Disperse the cells in the culture medium using methods such as stirring and vortexing (including but not limited to these), at a rate of 500-100,000 / cm³. 2 Cells were seeded into culture containers at a suitable density. An appropriate amount of culture medium was added, and the medium was replaced with fresh medium every 1-5 days depending on the cell growth status. When the cells reached 70-100% confluence, the passage process was repeated. The cultured umbilical cord mesenchymal stem cells adhered to the culture vessel, exhibiting a fibroblast-like, uniform morphology.

[0194] 3. Temperature-sensitive intelligent culture dish coating

[0195] Before preparing membranes using a serum-free culture system and mesenchymal stem cells, a matrix that facilitates mesenchymal stem cell adhesion should be pre-coated onto the surface of a temperature-sensitive smart culture dish. This matrix includes, but is not limited to, collagen, gelatin, fibronectin, fibrinogen, fibronectin, laminin, polyornithine, polylysine, and ornithine-lysine copolymers. The coating matrix should be diluted with physiological buffers such as saline or PBS before being added to the temperature-sensitive smart culture dish, and the dish should be capped. The dish should then be placed in a 37°C, saturated humidity, 5% CO2 incubator for 0.5–48 hours.

[0196] 4. Cell membrane preparation

[0197] After coating, the coating matrix is ​​removed from the smart culture dish. Stem cells and NK cells are mixed evenly at a ratio of 1:1 to 10:1, and then added to the temperature-sensitive smart culture dish. In this embodiment, a 35mm diameter temperature-sensitive smart culture dish is used, and the cell quantity is 1.8 x 10⁻⁶ cells / day. 5 -1.8x 10 7(Optimal choice is 1x10) 7 Add 1-4 ml of culture medium (preferably 3 ml). Then, place the temperature-sensitive smart culture dish in a 37℃, saturated humidity, 5% CO2 incubator for 2-48 hours. After culture, transfer the temperature-sensitive smart culture dish to a 4-32℃ environment. The cells will detach from the bottom of the temperature-sensitive smart culture dish in sheet-like layers without the addition of any additional reagents or materials, forming cell sheets. Collect the liquid in the culture dish to obtain the cell sheet supernatant. The cell sheets are grayish-white, dense in structure, and have a smooth and flat surface. They are circular in diameter (3-30 mm) and 50-1000 μm thick. Tissue section observation shows that the cell sheets contain multilayered cellular structures and extracellular matrix components secreted by the cells (see Figure 2).

[0198] It should be understood that although the present invention has been described in conjunction with specific embodiments, the foregoing description is intended to be illustrative and not to limit the scope of the invention. The scope of this application is defined by the appended claims.

Claims

1. A cell composition comprising mesenchymal stem cells and natural killer cells, wherein the mesenchymal stem cells are one or more of bone marrow-derived, adipose-derived, umbilical cord-derived, placental-derived, and dental pulp-derived mesenchymal stem cells, wherein the natural killer cells are peripheral blood and / or umbilical cord blood-derived natural killer cells, wherein the natural killer cells are activated natural killer cells, and wherein the mesenchymal stem cells are mesenchymal stem cells obtained by passage primary mesenchymal stem cells 1-10 times.

2. The cell composition according to claim 1, wherein the natural killer cells are IL-15 activated natural killer cells; preferably, wherein the natural killer cells are IL-15 activated for 2-5 hours.

3. The cell composition according to claim 1 or 2, wherein the cell composition is produced by co-incubating mesenchymal stem cells and natural killer cells in a suspension; Preferably, the mesenchymal stem cells are added on one or more of the following days: day 0, day 3, day 4, day 5, day 7, day 10, day 14, and day 21 of the natural killer cell culture. Preferably, the mesenchymal stem cells and natural killer cells are co-incubated for 18-25 days.

4. The cell composition according to any one of claims 1-3, wherein the initial culture volume for co-incubation is 50-150 ml, preferably 100 ml; Preferably, the ratio of the amount of mesenchymal stem cells to the amount of natural killer cells is 1:20-20:

1.

5. The cell composition according to claim 4, wherein the cells are to be expanded to 2 x 10⁻⁶. 8 Up to 4 x 10 8 Preferred 3 x 10 8 When the volume of cells reaches 250-350ml, preferably 300ml, inject the cells into a cell culture bag, preferably a 2L cell culture bag, for culture.

6. The cell composition according to claim 5, wherein after culturing in a cell culture bag, co-culture complete medium is added until the cell density is 1 x 10⁻⁶. 6 cells / ml to 1.5 x 10 6 Between cells / ml.

7. The cell composition according to claim 3, wherein a substance that promotes the expansion of natural killer cells and inhibits the expansion of T cells and / or B cells is added during co-incubation; Preferably, the substance is one or more of IL-2, IL-7, IL-15, IL-18, IL-21 and FLT3L.

8. The cell composition according to any one of claims 1-7, wherein the mesenchymal stem cells are genetically modified to secrete or overexpress one or more of the following cytokines: IL-2, IL-7, IL-15, IL-18, IL-21 and FLT3L.

9. The cell composition according to any one of claims 1-8, wherein mesenchymal stem cells and natural killer cells are co-incubated in a co-culture medium, the co-culture medium being based on StemRD Mgro-500 MesenGro human mesenchymal stem cell serum-free medium and GIBCO AIM-V medium CTS serum-free medium, and supplemented with one or more of FLT-3L, SCF, IL-3, IL-7, TNF-α, GM-CSF, TPO, IL-6, IL-2 and IL-15; Preferably, the ratio of StemRD Mgro-500 MesenGro serum-free culture medium and GIBCO AIM-V medium CTS serum-free culture medium is 1:2, and / or the co-culture medium is supplemented with one or more of the following: 300 ng / ml FLT-3L, 300 ng / ml SCF, 10 ng / ml IL-3, 5 ng / ml IL-7, 50 IU / ml TNF-α, 100 ng / ml GM-CSF, 5 ng / ml TPO, 100 ng / ml IL-6, 40 ng / ml IL-2 and 20 ng / ml IL-15.

10. The cell composition according to claim 1 or 2, wherein the cell composition is a cell aggregate of natural killer cells and mesenchymal stem cells; Preferably, the cell aggregates are generated by co-incubating mesenchymal stem cells and natural killer cells in an aggregate culture medium containing basal medium, serum albumin and insulin, wherein the basal medium is any one of DMEM high glucose medium, DMEM low glucose medium, α-MEM medium, DMEM / F12 medium and F12 medium; Preferably, the aggregate culture medium further comprises non-essential amino acids and / or L-glutamine; Preferably, the serum albumin concentration is 0.5-10% by mass, more preferably 0.5-5%, and most preferably 2%, and / or the insulin concentration is 0.5-50 μg / ml, more preferably 1-20 μg / ml, and most preferably 5 μg / ml; Preferably, the initial cell concentration is adjusted to 1 x 10⁻⁶. 5 / ml to 1 x 10 7 Between / ml Preferably, the mesenchymal stem cells and the natural killer cells are mixed evenly in a ratio of 1:1 to 10:1, seeded by the hanging drop method, and cultured for 8-48 hours to form cell aggregates; Preferably, the ratio of mesenchymal stem cells to natural killer cells is 5:1 to 10:1, and more preferably 8:

1.

11. The cell composition according to claim 1 or 2, wherein the cell composition is a cell sheet of natural killer cells and mesenchymal stem cells; Preferably, the cell membrane sheet is a circle with a diameter of 3-30 mm and a thickness of 50-1000 μm; Preferably, the cell sheet is obtained by co-culturing natural killer cells and mesenchymal stem cells on a substrate coated with a matrix that facilitates the attachment of mesenchymal stem cells.

12. The cell composition according to claim 11, wherein mesenchymal stem cells and natural killer cells are mixed evenly in a ratio of 1:1 to 10:1 and cultured for 2-48 hours; Preferably, the amount of cells added is 1.8 x 10⁻⁶. 5 -1.8 x 10 7 Preferably 1 x 10 7 The amount of culture medium added is 1-4 ml, preferably 3 ml.

13. A product comprising the cell composition according to any one of claims 1-12; Preferably, the product is one or more of a biological scaffold, a dressing such as a wound healing patch, and a pre-filled syringe; Preferably, the bioscaffold further comprises an extracellular matrix component, such as one or more of collagen, proteoglycans / glycosaminoglycans, and glycoproteins, such as fibronectin, laminin, platelet-reactive protein, and proteoglycan attachment peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid, and / or the scaffold is made of a biocompatible polymer, including one or more of the following: resins and naturally occurring polymers, such as collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels, alginates, or modified alginate materials.

14. A method for preparing the cell composition according to any one of claims 1-12, comprising any one of the following: (1) Mesenchymal stem cells and natural killer cells were co-incubated in a suspension; (2) Mesenchymal stem cells and natural killer cells were co-incubated in aggregate culture medium containing basal medium, serum albumin, and insulin, wherein the basal medium was any one of DMEM high-glucose medium, DMEM low-glucose medium, α-MEM medium, DMEM / F12 medium, and F12 medium; and (3) Natural killer cells and mesenchymal stem cells were co-cultured on a substrate coated with a matrix that facilitates the attachment of mesenchymal stem cells.

15. The method according to claim 14, wherein the initial culture volume for co-culturing in step (1) is 50-150 ml, preferably 100 ml. Preferably, the ratio of mesenchymal stem cells to natural killer cells is 1:20-20:1; Preferably, step (1) includes adding the mesenchymal stem cells on one or more of the following days: day 0, day 3, day 4, day 5, day 7, day 10, day 14, and day 21 of the natural killer cell culture. Preferably, step (1) includes co-incubating the mesenchymal stem cells and natural killer cells for 18-25 days; Preferably, step (1) includes adding a substance that promotes the expansion of natural killer cells and inhibits the expansion of T cells and / or B cells during co-incubation; Preferably, the substance is one or more of IL-2, IL-7, IL-15, IL-18, IL-21 and FLT3L.

16. The method of claim 15, wherein the cells are to be expanded to 2 x 10⁻⁶. 8 Up to 4 x 10 8 Preferred 3 x 10 8 When the volume of cells reaches 250-350ml, preferably 300ml, inject the cells into a cell culture bag, preferably a 2L cell culture bag, for culture.

17. The method of claim 16, wherein after culturing in the cell culture bag, co-culture complete medium is added until the cell density is 1 x 10⁻⁶. 6 cells / ml to 1.5 x 10 6 Between cells / ml.

18. The method of claim 15, wherein mesenchymal stem cells and natural killer cells are co-incubated in a co-culture medium, the co-culture medium being based on StemRD Mgro-500 MesenGro human mesenchymal stem cell serum-free medium and GIBCO AIM-V medium CTS serum-free medium, and supplemented with one or more of FLT-3L, SCF, IL-3, IL-7, TNF-α, GM-CSF, TPO, IL-6, IL-2 and IL-15; Preferably, the ratio of StemRD Mgro-500 MesenGro serum-free culture medium and GIBCO AIM-V medium CTS serum-free culture medium is 1:2, and / or the co-culture medium is supplemented with one or more of the following: 300 ng / ml FLT-3L, 300 ng / ml SCF, 10 ng / ml IL-3, 5 ng / ml IL-7, 50 IU / ml TNF-α, 100 ng / ml GM-CSF, 5 ng / ml TPO, 100 ng / ml IL-6, 40 ng / ml IL-2 and 20 ng / ml IL-15.

19. The method of claim 14, wherein step (2) comprises generating mesenchymal stem cells and natural killer cells by co-incubation in aggregate culture medium, said aggregate culture medium comprising basal culture medium, serum albumin and insulin, said basal culture medium being any one of DMEM high glucose medium, DMEM low glucose medium, α-MEM medium, DMEM / F12 medium and F12 medium; Preferably, the aggregate culture medium further comprises non-essential amino acids and / or L-glutamine; Preferably, the serum albumin concentration is 0.5-10% by mass, more preferably 0.5-5%, and most preferably 2%, and / or the insulin concentration is 0.5-50 μg / mL, more preferably 1-20 μg / mL, and most preferably 5 μg / mL; Preferably, the initial cell concentrations of the mesenchymal stem cells and the natural killer cells are adjusted to 1 x 10⁻⁶. 5 / mL to 1 x 10 7 / mL, Preferably, the cells are mixed evenly in a ratio of 1:1 to 10:1, inoculated by the hanging drop method, and cultured for 8-48 hours to form cell aggregates; preferably, the ratio of mesenchymal stem cells to natural killer cells is 5:1 to 10:1, and more preferably 8:

1.

20. The method of claim 14, wherein step (3) comprises co-culturing natural killer cells and mesenchymal stem cells on a substrate coated with a matrix that facilitates the attachment of mesenchymal stem cells; Preferably, mesenchymal stem cells and natural killer cells are mixed evenly in a ratio of 1:1 to 10:1 and cultured for 2-48 hours. Preferably, the amount of cells added is 1.8 x 10⁻⁶. 5 -1.8 x 10 7 Preferably 1 x 10 7 The amount of culture medium added is 1-4 ml, preferably 3 ml.

21. Use of the cell composition according to any one of claims 1-12 in the preparation of a medicament or kit for treating a disease; Preferably, the disease is cancer, trauma, and / or ulcer; Preferably, the cancer is one or more of liver cancer, ovarian cancer, lung cancer, mesenchymal stem cell carcinoma, and B-cell lymphoma; Preferably, the trauma is a burn, scald, and / or mechanical injury; Preferably, the ulcer is an oral ulcer, such as a recurrent oral ulcer.

22. A method of storing a dressing, comprising immersing the dressing in a preservation solution at 2-8°C or storing it in a liquid nitrogen tank at -183°C, said dressing comprising the cell composition of any one of claims 1-12.

23. A method of storing a pre-filled syringe, comprising storing the pre-filled syringe in a liquid nitrogen tank at -183°C, said pre-filled syringe comprising the cell composition and cryoprotectant of any one of claims 1-12.

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