Condited media and uses thereof

Abstract

The present disclosure relates to conditioned media and methods of making the same. Such methods may be particularly useful for providing therapeutic compositions.

Description

Technical Field

[0001] The present disclosure relates to conditioned medium and methods for preparing the same. Such methods may be particularly useful for providing therapeutic compositions. Background Art

[0002] MLPSCs (e.g., multipotent mesenchymal stem cells (MSCs)) have been proposed as attractive candidates for therapeutic applications due to their high proliferation and differentiation potential, as well as immunomodulatory / anti-inflammatory and other beneficial properties (Caplan AI (2007) J. Cell Physiol., 213, 341-347; Prockop DJ (2007) Clin Pharmacol Ther., 82, 241-243). In vitro proliferation of sparse mesenchymal stem cell (MSC) populations is often necessary to generate quantities suitable for therapeutic applications.

[0003] Despite various advances, the in vitro expansion of MLPSCs, while significant, remains limited. Furthermore, recent studies have shown that MLPSCs may exert their biological effects through secreted factors.

[0004] Thus, there remains an unmet need for compositions and methods for the sustained production of therapeutically effective compositions comprising MLPSCs and / or factors derived therefrom. Summary of the Invention

[0005] The present inventors have identified that MLPSC cultures supplemented with neonatal serum unexpectedly produce conditioned medium with increased angiogenic potential. For example, newborn calf serum (NBCS) is typically sold as an equivalent / acceptable substitute for fetal bovine serum (FBS). However, the present inventors unexpectedly found that this was not the case because, as described herein, NBCS supplementation induced angiogenesis marker levels to increase. In addition, conditioned medium obtained from MLPSC cultured in a culture medium supplemented with NBCS enhanced angiogenesis. Analysis of neonatal serum for culturing MLPSC surprisingly revealed increased levels of cytokines, particularly cytokines for MLPSC expressing corresponding receptors. These findings lay the foundation for producing novel compositions by culturing and amplifying MLPSC with certain proinflammatory cytokines and / or in neonatal serum.

[0006] Thus, in one aspect, the present disclosure relates to a composition comprising a conditioned medium or an extracellular vesicle obtained therefrom, wherein the conditioned medium is obtained from a population of MLPSCs cultured and expanded in a medium containing interferon (IFN) -γ and / or tumor necrosis factor (TNF) -α, wherein the level of IFN-γ and / or TNF-α in the medium is <1 ng / ml. For example, the level of IFN-γ can be <500 pg / ml. In one example, the level of IFN-γ is <100 pg / ml. In one example, the level of TNF-α is <750 pg / ml. In another example, the level of TNF-α is <500 pg / ml. In one example, the levels of IFN-γ and TNF-α are <500 pg / ml. In these examples, the conditioned medium can be characterized by an increased level of angiogenesis markers, wherein the increased level of angiogenesis markers is determined relative to the conditioned medium obtained from a control population.

[0007] In another aspect, the present disclosure relates to a composition comprising conditioned medium or extracellular vesicles obtained therefrom, wherein the conditioned medium is characterized by increased levels of angiogenic markers, wherein the conditioned medium is obtained from a population of MLPSCs that has been expanded by culture in a medium containing:

[0008] -IFN-γ and / or TNF-α; and / or,

[0009] - one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, IP-10;

[0010] And, wherein the increased level of the angiogenic marker is determined relative to conditioned medium obtained from a control population.

[0011] In one example, the angiogenesis marker is increased levels of angiogenin relative to conditioned medium obtained from a control population.

[0012] In another example, the angiogenic marker is increased endothelial network formation relative to conditioned medium obtained from a control population.

[0013] In another example, the angiogenesis marker is increased endothelial length relative to conditioned medium obtained from a control population.

[0014] In another example, the angiogenesis marker is increased endothelial branch length relative to conditioned medium obtained from a control population.

[0015] In one example, the angiogenesis marker is one or more or all of the following:

[0016] - Increased levels of angiogenin relative to conditioned medium obtained from a control population;

[0017] - Increased endothelial network formation relative to conditioned medium obtained from control populations;

[0018] - Increased endothelial length relative to conditioned medium obtained from control populations;

[0019] - Increased endothelial branch length relative to conditioned medium obtained from control populations.

[0020] In one example, the control population is a population of MLPSCs that has been expanded in cell culture medium containing 10% fetal serum.

[0021] In another example, the conditioned medium contains an angiogenin level greater than about 1200 pg / ml. In another example, the conditioned medium contains an SDF-1 level greater than about 3000 pg / ml. In another example, the conditioned medium contains a VEGF level greater than about 3200 pg / ml. In another example, the conditioned medium induces a tumor at a level greater than about 0.12 mm Hg. 2 / mm 2 In another example, the conditioned medium induces endothelial network formation greater than about 5 mm 2 / mm 2 In another example, the conditioned medium induces an endothelial network length greater than about 15 1 / mm 2 Length of endothelial branches.

[0022] In another example, the culture medium contains three or more proinflammatory cytokines. In another example, the culture medium contains two or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. In another example, the culture medium contains IL-6. In another example, the culture medium contains IL-8 and / or IL-17A.

[0023] In another example, the culture medium contains IFN-γ and TNF-α. In another example, the level of IFN-γ is <1 ng / ml, preferably <500 pg / ml, more preferably <100 pg / ml. In another example, the level of TNF-α is <1 ng / ml, preferably <750 pg / ml, more preferably <400 pg / ml.

[0024] In another example, the culture medium contains serum containing pro-inflammatory cytokines.

[0025] In another example, the serum is newborn mammalian serum. For example, the newborn mammalian serum can be newborn calf serum. In one example, the serum is obtained no more than 21 days after birth.

[0026] In one example, the culture medium is characterized by one or more or all of the following:

[0027] i. IFN-γ level greater than 1 pg / ml;

[0028] ii. TNF-α level greater than 2 pg / ml;

[0029] iii. IL-6 level greater than 3 pg / ml;

[0030] iv. IL-8 level greater than 500 pg / ml;

[0031] v.IL-17A level greater than 0.2 pg / ml;

[0032] vi. MCP-1 level greater than 3 pg / ml;

[0033] vii. MIP-1-α level greater than 0.5 pg / ml;

[0034] viii. MIP-1-β level greater than 3 pg / ml;

[0035] ix. IP-10 levels were greater than 500 pg / ml.

[0036] In one example, the culture medium comprises at least 5% (v / v) newborn mammalian serum. In another example, the culture medium does not contain serum.

[0037] In one example, the extracellular vesicles obtained from the conditioned medium are exosomes.

[0038] In another embodiment, the present disclosure relates to conditioned medium produced by culturing a MLPSC population in neonatal serum and extracellular vesicles obtained therefrom. Thus, in one example, the present disclosure relates to a conditioned medium or extracellular vesicles obtained therefrom, wherein the conditioned medium is obtained from a MLPSC population that has been cultured and expanded in a medium containing neonatal mammalian serum. In one example, the serum is neonatal calf serum. In one example, the neonatal serum is obtained no more than 21 days after birth.

[0039] In another example, the conditioned medium comprises an increased level of angiogenesis marker relative to the conditioned medium obtained from the control population. In one example, the angiogenesis marker is an increased level of angiogenin relative to the conditioned medium obtained from the control population. In another example, the angiogenesis marker is increased endothelial network formation relative to the conditioned medium obtained from the control population. In another example, the angiogenesis marker is increased endothelial length relative to the conditioned medium obtained from the control population. In another example, the angiogenesis marker is increased endothelial branch length relative to the conditioned medium obtained from the control population. In one example, the angiogenesis marker is one or more or all of the following:

[0040] - Increased levels of angiogenin relative to conditioned medium obtained from a control population;

[0041] - Increased endothelial network formation relative to conditioned medium obtained from control populations;

[0042] - Increased endothelial length relative to conditioned medium obtained from control populations;

[0043] - Increased endothelial branch length relative to conditioned medium obtained from control populations.

[0044] In one example, the control population is a population of MLPSCs that have been cultured and expanded in a cell culture medium containing 10% fetal serum. In one example, the conditioned medium contains an angiogenin level greater than about 1200 pg / ml. In another example, the conditioned medium contains an SDF-1 level greater than about 3000 pg / ml. In another example, the conditioned medium contains a VEGF level greater than about 3200 pg / ml. In another example, the conditioned medium induces a tumor necrosis factor (TNF) level greater than about 0.12 mm Hg. 2 / mm 2 In another example, the conditioned medium induces endothelial network formation greater than about 5 mm 2 / mm 2 In another example, the conditioned medium induces an endothelial network length greater than about 15 1 / mm 2 Length of endothelial branches.

[0045] In another example, the neonatal serum is neonatal serum from day 1 to day 7 after birth. In one example, the concentration of the neonatal serum is about 2% (v / v) to about 12% (v / v). In another example, the concentration of the neonatal serum is about 5% (v / v).

[0046] In another example, the culture medium comprises fetal serum and neonatal serum from the same species, and the ratio of the concentration of fetal serum to the concentration of neonatal serum is 1:1 or less. For example, the concentration of fetal serum and the concentration of neonatal serum can each be 5% (v / v).

[0047] In another example, the concentration of fetal serum is lower than the concentration of neonatal serum.

[0048] In another example, the extracellular vesicles obtained from the conditioned medium are exosomes.

[0049] In one example, the MLPSC is a human mesenchymal stem cell (hMSC). In another example, the MLPSC is derived from STRO-1 + Pluripotent cell populations are expanded in culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1: Evaluation and comparison of serum cytokine levels. 1:1 FCS / NBCS (Serum A); fetal bovine serum (Serum B); and FBS from different suppliers (Serum C).

[0051] Figure 2: Usage The 96-well Kinetic Angiogenesis PrimeKit assay quantitatively measures in vitro angiogenesis induced by MLPSC-conditioned medium.

[0052] Figure 3 : Luminex assay results showed increased angiogenin production by MLPSCs cultured in the presence or absence of neonatal serum.

[0053] Figure 4 : Angiogenic marker levels in conditioned medium from cGMP batches of MLPSCs cultured in the presence or absence of neonatal serum. DETAILED DESCRIPTION

[0054] General techniques and definitions

[0055] Unless specifically defined otherwise, all technical and scientific terms used herein should be interpreted as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology, stem cell biology, and biochemistry).

[0056] Unless otherwise indicated, the cell culture techniques and assays used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (eds.), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory Press, (1988); and J. E. Colligan et al. (eds.) Current Protocols in Immunology, John Wiley & Son (including all updates up to now).

[0057] The term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be considered to provide clear support for both or either meanings.

[0058] Unless stated to the contrary, the term "about" as used herein means + / - 10%, more preferably + / - 5% of the specified value.

[0059] The term "level" is used to define the amount of a particular substance present in a sample, cell culture medium, serum preparation, or composition of the present disclosure. For example, a specific concentration, weight, percentage (e.g., v / v%), or ratio can be used to define the level of a particular substance.

[0060] The term "conditioned medium" is used in the context of the present disclosure to refer to a culture medium obtained from MLPSC under culture conditions. This culture medium contains MLPSC secretory protein groups, proteins and other particles shed from the MLPSC surface, such as extracellular vesicles. The conditioned medium disclosed herein contains pro-angiogenic factors, such as extracellular vesicles, angiogenin or secreted metabolites, such as prostaglandin E2. If necessary, the pro-angiogenic ability of the conditioned medium disclosed herein and / or the factors obtained therefrom can be confirmed using one or more angiogenesis assays disclosed herein (e.g., endothelial network formation, endothelial length, endothelial branch length). In certain examples, the present disclosure relates to extracellular vesicles, such as exosomes, obtained from the conditioned medium of MLPSC under culture conditions. In one example, the conditioned medium is obtained when MLPSC is in the exponential growth phase. In one example, the conditioned medium is obtained after culturing for at least two or three days. In another example, the conditioned medium is obtained after culturing for about 30 to 84 hours.

[0061] In one example, the level of a specific marker (such as an angiogenic factor) is measured under culture conditions. The term "culture conditions" is used to refer to cells grown in culture. In one example, culture conditions refer to a population of actively dividing cells. In one example, such cells can be in an exponential growth phase. Alternatively, such cells can be in a quiescent phase.

[0062] In one example, in the case of measuring the IL2-RA inhibition level, the culture conditions may include co-culturing of an MLPSC population disclosed herein and a second cell population, such as a population comprising peripheral blood mononuclear cells (PBMC). In one example, co-culturing includes culturing an MLPSC population disclosed herein and an activated PBMC population. For example, PBMC can be activated using anti-CD3 and anti-CD28 antibodies before co-culturing with an MLPSC population disclosed herein. In this example, "culture conditions" may include co-culturing MLPSC and T cells at a ratio of about 1 MLPSC: 2 T cells or less. For example, co-culturing MLPSC and T cells at a ratio of 1: 3, 1: 4, 1: 5, 1: 10, 1: 20, 1: 30, 1: 40, 1: 50, 1: 60, 1: 70, 1: 80, 1: 90 or 1 MLPSC: 100 T cells or less. In this example, the level of IL2-RA inhibition is determined after cell culture for about 30 to 84 hours under culture conditions.

[0063] In another example, the level of a specific marker can be determined by obtaining a sample of conditioned medium and measuring the level of the marker in the sample. In another example, the level of a specific marker can be determined by obtaining a sample of cells and measuring the level of the marker in a cell lysate. It will be understood by those skilled in the art that secreted markers can be measured by sampling the culture medium, while markers expressed on the cell surface can be measured by evaluating a sample of cell lysate. In one example, the sample is obtained when the cells are in the exponential growth phase. In one example, the sample is obtained after culturing for at least two or three days. In another example, the sample is obtained after culturing for about 30 to 84 hours. In one example, the sample is obtained when the cells are in the stationary phase.

[0064] In one example, a sample is obtained from a co-culture of MLPSC and activated PBMC. In this example, the cell sample can be lysed and the level of the marker can be determined. For example, the level of IL2-RA can be determined. In this example, the level of IL2-RA can be determined using a variety of methods, such as enzyme-linked immunosorbent assay (ELISA)-based methods. In one example, the ELISA includes:

[0065] (i) adding sample diluent to each well of a microplate pre-coated with a monoclonal antibody specific for IL2-RA;

[0066] (ii) adding the co-cultured samples to the wells of a microplate pre-coated with a monoclonal antibody specific for IL2-RA;

[0067] (iii) incubating the microplate for a time sufficient to allow the monoclonal antibody specific for IL2-RA to specifically bind to any IL2-RA in the sample;

[0068] (iv) washing the microplate;

[0069] (v) adding IL2-RA conjugate to the wells;

[0070] (vi) incubating the microplate for a time sufficient to allow the conjugate to bind specifically to any captured IL2-RA;

[0071] (vii) washing the microplate;

[0072] (viii) adding substrate solution to the wells;

[0073] (ix) incubating the microplate for a sufficient time to allow color development;

[0074] (x) Add stop solution to the wells;

[0075] (xi) reading the optical density on a microplate reader set to 450 nm and wavelength calibrated at 570 nm;

[0076] (xii) Determining the level of IL2-RA.

[0077] In another example, the level of IL2-RA is determined using fluorescence activated cell sorting (FACS) with an appropriate antibody (such as anti-CD25). Additional antibodies may be used if needed to differentiate CD25+ cell types. Although the above reference examples relate to IL-2RA, it should be understood that the levels of other markers disclosed herein (such as angiopoietin) can also be determined using similar methods. In these examples, the levels may not require co-culture to determine.

[0078] In another example, the level is measured based on an assessment of conditioned medium (or properties thereof) obtained from a MLPSC population under culture conditions. For example, conditioned medium can be obtained from a MLPSC population disclosed herein under culture conditions prior to use in one or more angiogenesis assays disclosed below.

[0079] In the context of this disclosure, the term "sufficient" is used to define an amount that provides a specific concentration when dissolved in a stem cell culture medium. The "sufficient amount" is determined by the volume of culture medium required.

[0080] As used herein, the term "angiogenesis marker" refers to an indicator of angiogenesis. As used herein, "angiogenesis marker" includes pro-angiogenic factors, such as angiopoietin, SDF-1α and VEGF. In another example, the angiogenesis marker is a cell indicator of angiogenesis, for example, endothelial network formation, endothelial network length and endothelial branch length. In this example, the cell indicator of angiogenesis is determined in an in vitro angiogenesis assay as disclosed herein. In one example, angiogenesis marker characterization can be used to characterize the MLPSC population disclosed herein (e.g., a cryopreserved intermediate or drug product disclosed herein).

[0081] In one example, the compositions of the present disclosure comprise MLPSCs that have not been genetically modified. As used herein, the term "non-genetically modified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, MLPSCs transfected with a nucleic acid encoding a protein will be considered genetically modified.

[0082] As used herein, the term "sample" refers to an extract from a cell culture in which the level of a particular marker can be measured. The term "sample" includes extracts and / or derivatives and / or fractions of a sample. In one example, a "sample" is a cell population, such as a cell population under culture conditions. In one example, a sample is a supernatant obtained after cell culture, such as cell-conditioned medium. In these examples, the sample is any extract from a cell culture in which the level of angiogenesis markers can be measured. In one example, the sample is contacted with another cell population to determine the level of angiogenesis markers.

[0083] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0084] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter shall be considered to encompass both one and more of those steps, compositions of matter, groups of steps, or groups of compositions of matter (i.e., one or more).

[0085] It will be understood by those skilled in the art that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the disclosure includes all such variations and modifications. The disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, whether individually or collectively, and any and all combinations or any two or more of said steps or features.

[0086] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods, as described herein, are clearly within the scope of this disclosure.

[0087] Unless expressly stated otherwise, any example disclosed herein should be considered applicable mutatis mutandis to any other example.

[0088] Mesenchymal lineage precursor or stem cells (MLPSC)

[0089] As used herein, the term "mesenchymal lineage precursor or stem cell (MLPSC)" refers to an undifferentiated multipotent cell that has the ability to self-renew while maintaining multipotency and the ability to differentiate into a number of cell types of mesenchymal origin (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesoderm origin (e.g., hepatocytes, neural cells, and epithelial cells). For the avoidance of doubt, "mesenchymal lineage precursor cell" refers to a cell that can differentiate into mesenchymal cells (such as bone, cartilage, muscle, and adipocytes) as well as fibrous connective tissue.

[0090] The term "mesenchymal lineage precursor or stem cell" includes both parental cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSC), perivascular mesenchymal precursor cells and their undifferentiated progeny.

[0091] Mesenchymal lineage precursors or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or isogenic. Autologous cells are isolated from the same individual into which they will be re-implanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0092] In one example, the mesenchymal lineage precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal lineage precursors or stem cells are culture-expanded and cryopreserved.

[0093] Mesenchymal lineage precursors or stem cells are mainly located in the bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. They are also present in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum; and can be differentiated into reproductive system such as mesoderm and / or endoderm and / or ectoderm. Therefore, mesenchymal lineage precursors or stem cells can be differentiated into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue, and fibrous connective tissue. The specific lineage-stereotype and differentiation pathways that these cells enter depend on the various effects of mechanical influences and / or endogenous bioactive factors (such as growth factors, cytokines, and / or the local microenvironmental conditions established by host tissues).

[0094] The term "enriched", "enrichment" or variations thereof are used herein to describe such cell colonies, in which the ratio of a particular cell type or the ratio of many particular cell types increases when compared to an untreated cell colony (e.g., cells in their native environment). In one example, the colony enriched for mesenchymal lineage precursors or stem cells comprises at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% mesenchymal lineage precursors or stem cells. In this regard, the term "cell colony enriched for mesenchymal lineage precursors or stem cells" will be deemed to provide clear support for the term "cell colony comprising X% mesenchymal lineage precursors or stem cells", where X% is a percentage as enumerated herein. In some examples, mesenchymal lineage precursors or stem cells can form clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (e.g., 50%, or 60%, or 70%, or 70%, or 90%, or 95%) can have such activity.

[0095] In one example of the present disclosure, mesenchymal lineage precursors or stem cells are mesenchymal stem cells (MSCs). MSCs can be homogeneous compositions or can be mixed cell colonies enriched with MSCs. Homogeneous MSC compositions can be obtained by culturing adhered bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified with unique monoclonal antibodies. Methods for obtaining cell colonies enriched with MSCs are described in, for example, U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, MSCs are allogenic. In one example, MSCs are cryopreserved. In one example, MSCs are cultured, expanded, and cryopreserved.

[0096] In another example, the mesenchymal lineage precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC.

[0097] The isolated or enriched mesenchymal lineage precursors or stem cells can be expanded in vitro by culture.The isolated or enriched mesenchymal lineage precursors or stem cells can be cryopreserved, thawed and subsequently expanded in vitro by culture.

[0098] In one example, isolated or enriched mesenchymal lineage precursors or stem cells are plated at 50,000 viable cells / cm 2Cells are plated in culture medium (serum-free or serum-supplemented), for example, alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessels overnight at 37° C., 20% O 2 . The culture medium is then replaced and / or changed as needed, and the cells are cultured at 37° C., 5% O 2 for an additional 68 to 72 hours.

[0099] As will be understood by those skilled in the art, cultured mesenchymal lineage precursors or stem cells are phenotypically different from cells in vivo. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal lineage precursors or stem cells are also biologically different from cells in vivo, having a higher proliferation rate than most non-circulating (quiescent) cells in vivo.

[0100] In one example, a cell population is enriched from a cell preparation comprising STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cells express markers (e.g., cell surface markers) that allow the selection of STRO-1+ cells. The marker can be STRO-1, but not necessarily. For example, as described and / or exemplified herein, cells (e.g., mesenchymal precursor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1bright). Therefore, the indication that a cell is STRO-1+ does not mean that the cell is selected only by STRO-1 expression. In one example, cells are selected based at least on STRO-3 expression, for example, they are STRO-3+ (TNAP+). For example, MPCs can be isolated from bone mononuclear cells using anti-STRO-3 antibodies.

[0101] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected from, isolated from, or enriched from a variety of sources. That is, in some examples, these terms provide support for selection from any tissue or vascularized tissue containing STRO-1+ cells (e.g., mesenchymal precursor cells) or tissue containing pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues listed herein.

[0102] In one example, the cells used in the present disclosure express one or more markers, alone or collectively, selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0103] "Individually" means that the disclosure individually encompasses the recited marker or marker groups, and although individual markers or marker groups may not be individually listed herein, the appended claims may define such markers or marker groups individually and separately from each other.

[0104] "Collectively" means that the present disclosure encompasses any number or combination of the listed markers or marker groups, and although such number or combination of markers or marker groups may not be specifically listed herein, the appended claims may define such combinations or subcombinations individually and separately from any other combination of markers or marker groups.

[0105] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, the TNAP is BAP. In one example, TNAP, as used herein, refers to a molecule that can bind to the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC on December 19, 2005, under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0106] Furthermore, in one example, STRO-1+ cells were able to generate clonogenic CFU-F.

[0107] In one example, a significant proportion of STRO-1+ cells are capable of differentiating into at least two different germ lineages. Non-limiting examples of lineages to which STRO-1+ cells can be committed include bone progenitor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural-restricted cells, which can give rise to glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; myocardial and cardiomyocyte precursors; and pancreatic beta cell lineages that secrete glucose-responsive insulin. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors of retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0108] In one example, mesenchymal lineage precursors or stem cells are obtained from a single donor or multiple donors, wherein the donor samples or mesenchymal lineage precursors or stem cells are subsequently pooled and then expanded in culture.

[0109] Mesenchymal lineage precursors or stem cells encompassed by the present disclosure can also be cryopreserved prior to administration to a subject. In one example, mesenchymal lineage precursors or stem cells are culture-expanded and cryopreserved prior to administration to a subject.

[0110] In one example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and their offspring, soluble factors derived therefrom and / or extracellular vesicles separated therefrom. In another example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and conditioned medium obtained therefrom under culture conditions. In another example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and extracellular vesicles separated therefrom. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be cultured and expanded for a period of time under conditions suitable for secretion of extracellular vesicles into cell culture medium. Secreted extracellular vesicles can then be obtained from the culture medium for use in therapy. If desired, such extracellular vesicles can be characterized using one or more angiogenesis assays disclosed herein (e.g., endothelial network formation, endothelial length, endothelial branch length).

[0111] As used herein, the term "extracellular vesicle" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to as large as 10 microns, but the size of the lipid particles is generally less than 200 nm. The lipid particles may contain extracellular vesicles from releasing cells (e.g., mesenchymal stem cells; STRO-1 +proteins, nucleic acids, lipids, metabolites or organelles of cells).

[0112] As used herein, the term "exosome" refers to a type of extracellular vesicle that is typically sized in the range of about 30 nm to about 150 nm and originates from the endosomal compartment of mammalian cells, from which it is transported to the cell membrane and released. They may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and play a role in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0113] As used herein, the term "pre-licensing" or "licensing" refers to the process by which MLPSCs achieve functional maturation such that, when administered to a subject, pre-licensed or licensed MLPSCs reduce the release of inflammatory cytokines to a greater extent than non-pre-licensed MLPSCs.

[0114] The terms "enriched," "enriched," or variations thereof are used herein to describe cell populations in which the proportion of a particular cell type or the proportion of a number of particular cell types is increased when compared to an untreated cell population (e.g., cells in their native environment). In one example, a population enriched for STRO-1+ cells comprises at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% STRO-1+ cells. In this regard, the term "cell population enriched for STRO-1+ cells" will be considered to provide clear support for the term "cell population comprising X% STRO-1+ cells," where X% is a percentage as recited herein. In some examples, STRO-1+ cells can form clonogenic colonies, such as CFU-F (fibroblasts) or a subpopulation thereof (e.g., 50%, or 60%, or 70%, or 80%, or 90%, or 95%) can have such activity.

[0115] In one example, a cell population is enriched from a cell preparation comprising STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cells express markers (e.g., cell surface markers) that allow the selection of STRO-1+ cells. The marker can be STRO-1, but not necessarily. For example, cells expressing STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 (e.g., mesenchymal precursor cells) also express STRO-1 (and may be STRO-1 bright). Therefore, the indication that a cell is STRO-1+ does not mean that the cell is selected by STRO-1 expression. In one example, cells are selected based at least on STRO-3 expression, for example, they are STRO-3+ (TNAP+).

[0116] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected from, isolated from, or enriched from a variety of sources. That is, in some examples, these terms provide support for selection from any tissue or vascularized tissue containing STRO-1+ cells (e.g., mesenchymal precursor cells) or tissue containing pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues listed herein.

[0117] In one example, the mesenchymal lineage precursors or stem cells used in the present disclosure express one or more markers selected individually or collectively from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0118] Use of the term "individually" means that the disclosure individually encompasses the recited marker or marker groups, and that even though individual markers or marker groups may not be individually listed herein, the appended claims may define such markers or marker groups individually and separately from each other.

[0119] Use of the term "collectively" means that the present disclosure encompasses any number or combination of the listed markers or marker groups, and that although such number or combination of markers or marker groups may not be specifically listed herein, the appended claims may define such combinations or subcombinations individually and separately from any other combination of markers or marker groups.

[0120] In one example, STRO-1+ cells are STRO-1 bright (syn.STRO-1 bri ). In another example, STRO-1 bricells relative to STRO-1 dim or STRO-1 中间 In another example, STRO-1 bri The cells are additionally one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β) and / or CD146+. For example, cells are selected for one or more of the aforementioned markers and / or are shown to express one or more of the aforementioned markers. In this regard, cells shown to express a marker do not need to be specifically tested, but rather previously enriched or isolated cells can be tested and subsequently used, and it is reasonable to assume that the isolated or enriched cells also express the same marker.

[0121] In one example, the mesenchymal precursor cells are perivascular mesenchymal precursor cells as defined in WO 2004 / 85630, characterized by the presence of the perivascular marker 3G5.

[0122] Cells that are referred to as "positive" for a given marker can express low (lo or dim) or high (bright, bri) levels of the marker, depending on the extent to which the marker exists on the cell surface, where these terms relate to fluorescence intensity or other markers used in cell sorting processes. The difference between lo (or dim or dull) and bri will be understood in the context of the marker used on the specific cell population being sorted. Cells that are referred to as "negative" for a given marker are not necessarily completely absent from the cell. The term means that the marker is expressed at a relatively very low level by the cell, and when detectably labeled, it produces a very weak signal, or is undetectable above background levels (e.g., levels detected using an isotype control antibody).

[0123] As used herein, the term "bright" or "bri" refers to a marker on the surface of a cell that produces a relatively strong signal when detectably labeled. While not wishing to be bound by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., an antigen recognized by STRO-1) than other cells in the sample. For example, when labeled with a FITC-conjugated STRO-1 antibody, STRO-1 is expressed as detected by fluorescence activated cell sorting (FACS) analysis. bri cells than non-bright cells (STRO-1 dull / dim ) produces a stronger fluorescent signal. In one example, the "bright" cells comprise at least about 0.1% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In other examples, the "bright" cells comprise at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In one example, STRO-1 bright cells have a relative high fluorescence intensity relative to "background" (i.e., STRO-1- In contrast, STRO-1 dim and / or STRO-1 中间 The cells have surface expression of STRO-1 that is less than 2 logs above "background," typically about 1 log or less.

[0124] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, the TNAP is BAP. In one example, TNAP, as used herein, refers to a molecule that can bind to the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC on December 19, 2005, under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0125] Furthermore, in one example, STRO-1+ cells were able to generate clonogenic CFU-F.

[0126] In one example, a significant proportion of STRO-1+ multipotent cells are able to differentiate into at least two different germ lineages. Non-limiting examples of lineages to which multipotent cells can commit include bone progenitor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells, which can generate glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; myocardium and precursors of cardiomyocytes; and pancreatic beta cell lineages that secrete glucose-responsive insulin. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors of retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0127] In one aspect of the present disclosure, the mesenchymal lineage precursors or stem cells currently described are MSCs. MSCs can be homogeneous compositions or can be mixed cell populations enriched in MSCs. Homogeneous MSC cell compositions can be obtained by culturing adhered bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified with unique monoclonal antibodies. Methods for obtaining cell populations enriched in MSCs are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0128] In another example, the mesenchymal lineage precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC (e.g., remestemcel-L).

[0129] As will be understood by those skilled in the art, cultured mesenchymal lineage precursors or stem cells are phenotypically different from cells in vivo. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal lineage precursors or stem cells are also biologically different from cells in vivo, having a higher proliferation rate than most non-circulating (quiescent) cells in vivo.

[0130] Mesenchymal lineage precursors or stem cells cultured using the methods of the present disclosure can also be cryopreserved.

[0131] Conditioned medium

[0132] In one example, the conditioned medium or the extracellular vesicles obtained therefrom are characterized by the expression of angiogenic markers. For example, the conditioned medium or the extracellular vesicles obtained therefrom can be characterized by increased levels of VEGF, angiopoietin and / or SDF-1α under culture conditions. In another example, the conditioned medium or the extracellular vesicles obtained therefrom can be characterized based on one or more functional criteria. In one example, when endothelial cells are treated with conditioned medium obtained from culture-expanded MLPSCs or extracellular vesicles obtained therefrom, the conditioned medium or the extracellular vesicles obtained therefrom increase the level of endothelial network formation, endothelial network length and / or endothelial branch length in the cell population. In one example, the increase is determined relative to the conditioned medium from a control MLPSC population or the extracellular vesicles obtained therefrom. In one example, the control population is an MLPSC population that has been cultured and expanded in a cell culture medium containing 10% fetal serum.

[0133] In one example, the conditioned medium is characterized by a level of VEGF greater than about 3 ng / ml. In one example, the level of VEGF is between about 3 ng / ml and 4 ng / ml. In one example, the level of VEGF is greater than about 3.1 ng / ml. In one example, the level of VEGF is greater than about 3.2 ng / ml. In one example, the level of VEGF is greater than about 3.3 ng / ml. In one example, the level of VEGF is greater than about 3.4 ng / ml. In one example, the level of VEGF is greater than about 3.5 ng / ml. In one example, the level of VEGF is between about 3.2 ng / mL and 3.6 ng / mL. In one example, the level of VEGF is about 3.45 ng / mL.

[0134] In one example, the conditioned medium or extracellular vesicles obtained therefrom contain increased levels of angiogenin relative to a control population. In one example, the conditioned medium is characterized by angiogenin levels greater than about 1000 pg / ml. In one example, the angiogenin levels are greater than about 1100 pg / ml. In one example, the angiogenin levels are between about 1000 pg / ml and 1200 pg / ml. In one example, the angiogenin levels are between about 1100 pg / ml and 1150 pg / ml. In one example, the angiogenin levels are about 1114 pg / ml.

[0135] In one example, the conditioned medium is characterized by a level of SDF-1α greater than about 3000 ng / ml. In one example, the level of SDF-1α is greater than about 3100 ng / ml. In one example, the level of SDF-1α is greater than about 3200 ng / ml. In one example, the level of SDF-1α is greater than about 3300 ng / ml. In one example, the level of SDF-1α is greater than about 3400 ng / ml. In one example, the level of SDF-1α is greater than about 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3400 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3300 ng / ml. In one example, the level of SDF-1α is between about 3100 ng / ml and 3400 ng / ml. In one example, the level of SDF-1α is between about 3100 ng / ml and 3300 ng / ml.

[0136] In one example, the conditioned medium stimulated the formation of endothelial networks larger than about 0.1 mm 2 / mm 2In one example, the endothelial network is formed at about 0.1 mm 2 / mm 2 to 0.2mm 2 / mm 2 In another example, the endothelial network is formed to be approximately 0.12 mm 2 / mm 2 .

[0137] In one example, the conditioned medium stimulated endothelial network length to be greater than about 4 mm. 2 / mm 2 In one example, the endothelial network length is about 4 mm. 2 / mm 2 to about 6mm 2 / mm 2 In one example, the endothelial network length is about 5 mm. 2 / mm 2 In one example, the conditioned medium stimulates endothelial branch length to be greater than about 12 1 / mm 2 In one example, the endothelial branch length was approximately 12 1 / mm. 2 About 17 1 / mm 2 In one example, the endothelial branch length is about 15 1 / mm 2 .

[0138] In one example, the conditioned medium or the extracellular vesicles obtained therefrom are characterized in that the level of one or more angiogenesis markers is increased relative to the conditioned medium obtained from the MLPSC population that has been cultured and expanded in a cell culture medium containing 10% fetal serum or the extracellular vesicles obtained therefrom. In one example, the level of the angiogenesis marker is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70%. In one example, the level of the angiogenesis marker is increased by about 5% to about 60%. In one example, the level of the angiogenesis marker is increased by about 5% to about 40%. In one example, the level of the angiogenesis marker is increased by about 40%. In one example, the level of the angiogenesis marker is increased by at least about 5%. In one example, the level of the angiogenesis marker is increased by at least about 10%. In one example, the level of the angiogenesis marker is increased relative to the conditioned medium obtained from the MLPSC population that has been cultured and expanded in a cell culture medium without IFN-γ or TNF-α or the extracellular vesicles obtained therefrom.

[0139] In one example, the conditioned medium or the extracellular vesicles obtained therefrom are characterized in that the level of one or more angiogenesis markers is increased relative to the conditioned medium obtained from the MLPSC population that has been cultured and expanded in a cell culture medium without neonatal serum or the extracellular vesicles obtained therefrom. In one example, the level of the angiogenesis marker is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70%. In one example, the level of the angiogenesis marker is increased by about 5% to about 60%. In one example, the level of the angiogenesis marker is increased by about 5% to about 40%. In one example, the level of the angiogenesis marker is increased by about 40%. In one example, the level of the angiogenesis marker is increased by at least about 5%. In one example, the level of the angiogenesis marker is increased by at least about 10%.

[0140] "Culture-expanded" MLPSCs are distinguished from freshly isolated cells in that they have been cultured in cell culture medium and passaged (ie, subcultured).

[0141] In one example, the freshly isolated cells are cultured and expanded for about 1 or 2 passages to provide an intermediate population. In one example, the freshly isolated cells are cultured and expanded for 2 passages to provide an intermediate population. In another example, the freshly isolated cells are cultured and expanded for about 1 to 3 passages to provide an intermediate population. In one example, the freshly isolated cells are STRO-1+.

[0142] Therefore, in one example, the relevant cells are isolated and the relevant cell cultures are expanded 2 times to provide an intermediate MLPSC population. In certain examples, the intermediate MLPSC population is then cultured and expanded to provide a drug product (DP). In one example, conditioned medium or extracellular vesicles obtained therefrom are produced by culturing cells from an intermediate cryopreserved MLPSC population or in other words, a cryopreserved intermediate. In one example, the intermediate cell population can be cultured for another three generations (i.e., a total of 5 generations) to provide DP. In another example, the conditioned medium or the extracellular vesicles obtained therefrom are obtained from DP MLPSC.

[0143] In one example, the MLPSC culture is expanded for about 4-10 generations to provide conditioned medium or extracellular vesicles obtained therefrom. In one example, the MLPSC culture is expanded for at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations to provide conditioned medium or extracellular vesicles obtained therefrom. For example, the MLPSC culture can be expanded for at least 5 generations to provide conditioned medium or extracellular vesicles obtained therefrom. In one example, the MLPSC culture can be expanded for at least 5-10 generations to provide conditioned medium or extracellular vesicles obtained therefrom. In one example, the MLPSC culture can be expanded for at least 5-8 generations to provide conditioned medium or extracellular vesicles obtained therefrom. In one example, the MLPSC culture can be expanded for at least 5-7 generations to provide conditioned medium or extracellular vesicles obtained therefrom. In one example, the MLPSC culture can be expanded for more than 7 generations to provide conditioned medium or extracellular vesicles obtained therefrom. In these examples, MLPSCs can be expanded in culture prior to cryopreservation to provide an intermediate cryopreserved MLPSC population, which can then be further expanded in culture to provide conditioned medium or extracellular vesicles derived therefrom.

[0144] In one example, the conditioned medium or the extracellular vesicles obtained therefrom are obtained from MLPSC cultured and expanded from a cryopreserved intermediate. In one example, the cell culture expanded from a cryopreserved intermediate is expanded for at least 3 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations. For example, MLPSC can be cultured and expanded for at least 3 generations. In one example, MLPSC can be cultured and expanded for at least 3-10 generations. In one example, MLPSC can be cultured and expanded for at least 3-8 generations. In one example, MLPSC can be cultured and expanded for at least 3-7 generations. In one example, MLPSC expanded from a cryopreserved intermediate is cultured and expanded in a culture medium disclosed herein (e.g., a culture medium containing newborn calf serum).

[0145] In one example, MLPSCs can be obtained from a single donor or multiple donors, wherein the donor samples or MLPSCs are subsequently pooled and then cultured and expanded as needed. In one example, the culture expansion process includes:

[0146] i. expanding a number of viable cells by passage expansion to provide a preparation of at least about 1 billion viable cells, wherein the passage expansion comprises establishing a primary culture of the isolated MLPSCs and then serially establishing a first non-primary (P1) culture of the isolated MLPSCs from the previous culture;

[0147] ii. expanding the isolated P1 culture of MLPSCs into a second non-primary (P2) culture of MLPSCs by subculture expansion; and,

[0148] iii. preparing and cryopreserving an in-process intermediate MLPSC preparation obtained from a P2 culture of MLPSC; and, optionally

[0149] iv. Thawing the cryopreserved in-process intermediate MLPSC preparation and expanding the in-process intermediate MLPSC preparation by passaging.

[0150] In one example, the methods of the present disclosure include selecting an intermediate population (e.g., a cryopreserved intermediate) for further culture expansion based on certain criteria (e.g., the level of one or more angiogenesis markers). The selection process is not particularly limited as long as it is capable of selecting a cell population characterized by the relevant criteria (such as the level of angiogenesis markers). In one example, the levels of angiogenesis markers of a series of intermediate MLPSC populations are assessed, and those populations expressing angiogenesis markers as described herein that exceed a threshold level are selected for further expansion.

[0151] It should be understood that the selection process does not require immediate culture expansion. Instead, the "selected" population can be cryopreserved and culture expanded at a later time. In one example, a portion of the intermediate cell population is culture expanded, and the remainder of the population is cryopreserved for culture expansion at a later time.

[0152] In one example, the selected cell population is immediately cultured and expanded. In another example, the selected cell population is cryopreserved for culture expansion at a later time.

[0153] In one example, the conditioned medium or extracellular vesicles obtained therefrom are obtained from a culture-expanded MLPSC population having an antigenic profile and an activity profile comprising:

[0154] i. Less than about 0.75% CD45+ cells;

[0155] ii. at least about 95% CD105+ cells;

[0156] iii. at least about 95% CD166+ cells.

[0157] The process of MLPSC isolation and ex vivo expansion can be carried out using any equipment and cell processing methods known in the art. Various culture expansion embodiments disclosed herein employ steps that require manipulation of cells, such as inoculation, feeding, dissociation of adherent cultures, or washing steps. Any step in manipulating cells is likely to damage the cells. Although MLPSCs can generally tolerate a certain amount of damage during preparation, it is preferred to manipulate the cells by a processing procedure and / or equipment that fully performs a given step while minimizing damage to the cells.

[0158] In one example, MLPSCs are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a rotating membrane filter having an inlet end and an outlet end, a filter bag, a mixing zone, a final product bag for washing the cells, and appropriate tubing, e.g., as described in US 6,251,295, which is hereby incorporated by reference.

[0159] In one example, a composition of MLPSCs cultured according to the present disclosure is 95% homogeneous with respect to being CD105 positive and CD166 positive and CD45 negative. In one example, this homogeneity persists through ex vivo expansion (ie, through multiple population doublings).

[0160] In one example, the MLPSC of the present disclosure is cultured and expanded in 2D culture. For example, the MLPSC of the present disclosure can be cultured and expanded in a cell factory. In certain examples, the 3D culture of the intermediate disclosed herein can be carried out using, for example, a bioreactor. In one example, the MLPSC of the present disclosure is initially cultured and expanded in 2D culture and then further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure has not yet been cultured and expanded in 3D culture. In one example, the level of one or more angiogenesis markers is assessed before subsequent culture expansion in a cell factory or 3D culture.

[0161] In one example, the MLPSCs of the present disclosure are expanded from an intermediate population culture. In one example, the MLPSCs of the present disclosure are expanded from an intermediate culture in 2D culture and then seeded in 3D culture.

[0162] In the case of both the intermediate population and the therapeutic composition expanded therefrom, in one example, the MLPSC of the present disclosure is cultured and expanded in 2D culture for at least 3 days and then seeded in another culture system (such as 3D culture in a cell factory or bioreactor). In one example, the MLPSC of the present disclosure is cultured and expanded in 2D culture for at least 4 days and then seeded in another culture system. In one example, the MLPSC of the present disclosure is cultured and expanded in 2D culture for 3 to 5 days and then seeded in another culture system. In these examples, 2D culture can be carried out in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma, Corning). In one example, the cell factory has at least 5 layers. In one example, the cell factory has at least 10 layers. In one example, the cell factory has at least 20 layers. 3D culture can be carried out in various types of bioreactors (such as stirred tanks, wave bags, and vertical wheels).

[0163] In one example, CO2 is provided during the culture expansion of MLPSC. In one example, MLPSC is cultured and expanded in less than 9% CO2. In one example, MLPSC is cultured and expanded in less than 8% CO2. In one example, MLPSC is cultured and expanded in 5% CO2. For example, MLPSC can be cultured and expanded in 5% + / - 2% CO2. In one example, MLPSC is cultured and expanded under passive preconditioning of CO2. For example, the cell factory can be passively preconditioned with 5% CO2.

[0164] Pretreatment of the cell factory maintains CO2 tension between the cell factory and the incubator and stabilizes the pH level of the growth medium. Active pretreatment involves actively passing CO2 gas through a bacterial ventilation filter into each culture dish (e.g., a cell factory) for a specified period of time (e.g., about 10 minutes). However, active pretreatment may introduce contamination into the culture because it requires an open port to provide gas. Passive pretreatment involves placing the closed culture system in an incubator with an appropriate CO2 concentration and then inoculating the cells (e.g., for about 12 to 72 hours). In one example, the cells of the present disclosure are STRO-3+ before being cultured and expanded to provide an intermediate cell population.

[0165] MLPSC culture medium

[0166] In one embodiment, the present disclosure encompasses culture expansion of MLPSCs in a culture medium supplemented with proinflammatory cytokines. In one example, the culture medium comprises IFN-γ and / or TNF-α. In one example, the culture medium comprises IFN-γ. For example, the level of IFN-γ may be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the culture medium comprises TNF-α. For example, the level of TNF-α may be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the culture medium comprises IFN-γ and TNF-α, and the levels of both are less than 1 ng / ml.

[0167] In one example, the culture medium contains one or more pro-inflammatory cytokines that are capable of binding to receptors on the surface of MLPSCs.

[0168] In one example, the culture medium comprises one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP- 10. For example, the culture medium may comprise IL-8.

[0169] In one example, the culture medium comprises IFN-γ and / or TNF-α and one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0170] In one example, the culture medium is characterized by one or more or all of the following:

[0171] i. IFN-γ level greater than 1 pg / ml;

[0172] ii. TNF-α level greater than 2 pg / ml;

[0173] iii. IL-6 level greater than 3 pg / ml;

[0174] iv. IL-8 level greater than 500 pg / ml;

[0175] v.IL-17A level greater than 0.2 pg / ml;

[0176] vi. MCP-1 level greater than 3 pg / ml;

[0177] vii. MIP-1-α level greater than 0.5 pg / ml;

[0178] viii. MIP-1-β level greater than 3 pg / ml;

[0179] ix. IP-10 levels were greater than 500 pg / ml.

[0180] In another example, the culture medium comprises serum characterized by one or more or all of the following:

[0181] i. IFN-γ level greater than 10 pg / ml;

[0182] ii. TNF-α level greater than 20 pg / ml;

[0183] iii. IL-6 level greater than 30 pg / ml;

[0184] iv. IL-8 levels greater than 5,000 pg / ml;

[0185] v.IL-17A level greater than 2 pg / ml;

[0186] vi. MCP-1 level greater than 30 pg / ml;

[0187] vii. MIP-1-α level greater than 50 pg / ml;

[0188] viii. MIP-1-β level greater than 30 pg / ml;

[0189] ix. IP-10 levels greater than 5,000 pg / ml.

[0190] In one example, the culture medium comprises IL-10. In another example, the culture medium comprises IL-36RA. In another example, the culture medium comprises IL-10 and IL-36RA. In one example, the level of IL-10 is greater than 0.3 pg / ml. For example, the level of IL-10 can be greater than 30 pg / ml. In one example, the level of IL-10 is greater than 400 pg / ml. In one example, the level of IL-36RA is greater than 50 pg / ml.

[0191] In one example, the culture medium does not contain serum.

[0192] In one example, the culture medium is serum-free and supplemented with PDGF and FGF2. In one example, the culture medium is serum-free and supplemented with PDGF, FGF2, and EGF. In one example, the PDGF is PDGF-BB. In one example, the serum-free culture medium is supplemented with 10 ng / ml PDGF-BB, 5 ng / ml EGF, and 1 ng / ml FGF2.

[0193] In one example, the above cytokines can each be provided at a concentration of <1 ng / ml. For example, the culture medium can be characterized by one or more or all of the following, each provided at <1 ng / ml: IFN-γ, TNF-α, IL-6, IL-17A, MCP-1, MIP-1-α, MIP-1-β, IP-10.

[0194] method

[0195] The cell culture methods of the present disclosure and their use for producing the conditioned medium disclosed herein involve culturing a cell population enriched for MLPSCs (eg, human MSCs) in a cell culture medium suitable for the maintenance and proliferation of MLPSCs.

[0196] In one example, the culture medium is the composition or culture medium mentioned above. In one example, the culture medium is supplemented with serum comprising one or more proinflammatory cytokines as described herein. In some preferred embodiments, the culture medium used is supplemented with neonatal serum. In some preferred embodiments, the culture medium to be used is supplemented with fetal serum and neonatal serum at the same concentration so that the total serum concentration in the culture medium is about 10% (v / v). In some preferred embodiments, MLPSC is pre-licensed in a cell culture medium containing 5% (v / v) neonatal serum and 5% (v / v) fetal serum.

[0197] In some embodiments, the methods disclosed herein include an additional step of determining or having determined the level of one or more proinflammatory cytokines in serum that is contained in the culture medium for pre-permitting MLPSCs. Methods for determining cytokine levels are well known in the art, such as ELISA.

[0198] In some embodiments, the methods disclosed herein also include determining or having determined in an in vitro assay that a culture medium (e.g., a culture medium supplemented with neonatal serum) stimulates MLPSC to produce a conditioned medium that promotes angiogenesis, for example, by analysis of tube formation and network length, network area, and branch point formation by human umbilical vein endothelial cells (HUVEC). In some embodiments, such an assay includes collecting MLPSC conditioned medium cultured in a culture medium supplemented with neonatal serum disclosed herein, and quantifying the effect of such conditioned medium in the above-mentioned angiogenesis assay or similar assay.

[0199] In some embodiments, the methods disclosed herein further comprise determining or having determined the level of one or more of angiogenin, angiopoietin (Ang1 / ANGPT1), SDF-1α, and VEGF in the conditioned medium described above.

[0200] In some embodiments, when the first batch of neonatal serum is used in the conditioned medium, the conditioned medium promotes angiogenesis or the release of angiogenic factors more than the conditioned medium using the second batch of neonatal serum. It can be concluded that using the first batch of neonatal serum for pre-licensure and culture expansion of MLPSCs will result in the production of MLPSCs with relatively greater therapeutic efficacy, particularly for the treatment of conditions where angiogenesis or anti-inflammatory therapeutic modes of action are useful.

[0201] In certain examples, the methods and cell culture media of the present disclosure maintain MLPSC in an undifferentiated state. When MLPSC have not yet been stereotyped into a specific differentiation lineage, they are considered to be undifferentiated. As discussed above, MLPSC exhibit morphological characteristics that distinguish them from differentiated cells. In addition, undifferentiated MLPSC express genes that can be used as markers for detecting a state of differentiation. Polypeptide products can also be used as markers for detecting a state of differentiation. Therefore, those skilled in the art can easily determine whether the methods of the present disclosure maintain MLPSC in an undifferentiated state using conventional morphological analysis, genetic analysis, and / or proteomic analysis. Methods for monitoring / confirming cell proliferation are also known in the art, and in certain examples, can be as preliminary as regular visual inspection of cell cultures to confirm an increase in cell number. Other methods may involve the use of cell viability dyes and / or live cell imaging and counting using commercially available products.

[0202] The term "medium" or "media" as used in the context of the present disclosure includes components of the environment surrounding cells. A medium contributes to and / or provides conditions suitable for allowing cell growth. A medium can be solid, liquid, gaseous, or a mixture of phases and materials. A medium can include liquid growth media as well as liquid media that do not sustain cell growth. Exemplary gaseous media include the gaseous phase to which cells grown on a culture dish or other solid or semisolid support are exposed.

[0203] In one example, the methods of the present disclosure encompass culturing and expanding in a cell culture medium comprising one or more proinflammatory cytokines. In one example, the cell culture medium comprises IFN-γ and / or TNF-α. In one example, the cell culture medium comprises IFN-γ. For example, the level of IFN-γ may be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the cell culture medium comprises TNF-α. For example, the level of TNF-α may be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the cell culture medium comprises IFN-γ and TNF-α, and the levels of both are less than 1 ng / ml.

[0204] In one example, the cell culture medium contains one or more pro-inflammatory cytokines that are capable of binding to receptors on the surface of MLPSCs.

[0205] In one example, the cell culture medium comprises one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP- 10. For example, the cell culture medium may comprise IL-8.

[0206] In one example, the cell culture medium comprises IFN-γ and / or TNF-α and one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0207] In one example, the cell culture medium is characterized by one or more or all of the following:

[0208] i. IFN-γ level greater than 1 pg / ml;

[0209] ii. TNF-α level greater than 2 pg / ml;

[0210] iii. IL-6 level greater than 3 pg / ml;

[0211] iv. IL-8 level greater than 500 pg / ml;

[0212] v.IL-17A level greater than 0.2 pg / ml;

[0213] vi. MCP-1 level greater than 3 pg / ml;

[0214] vii. MIP-1-α level greater than 0.5 pg / ml;

[0215] viii. MIP-1-β level greater than 3 pg / ml;

[0216] ix. IP-10 levels were greater than 500 pg / ml.

[0217] In another example, the culture medium comprises serum characterized by one or more or all of the following:

[0218] i. IFN-γ level greater than 10 pg / ml;

[0219] ii. TNF-α level greater than 20 pg / ml;

[0220] iii. IL-6 level greater than 30 pg / ml;

[0221] iv. IL-8 levels greater than 5,000 pg / ml;

[0222] v.IL-17A level greater than 2 pg / ml;

[0223] vi. MCP-1 level greater than 30 pg / ml;

[0224] vii. MIP-1-α level greater than 50 pg / ml;

[0225] viii. MIP-1-β level greater than 30 pg / ml;

[0226] ix. IP-10 levels greater than 5,000 pg / ml.

[0227] In one example, the culture medium comprises IL-10. In another example, the culture medium comprises IL-36RA. In another example, the culture medium comprises IL-10 and IL-36RA. In one example, the level of IL-10 is greater than 0.3 pg / ml. For example, the level of IL-10 can be greater than 30 pg / ml. In one example, the level of IL-10 is greater than 400 pg / ml. In one example, the level of IL-36RA is greater than 50 pg / ml.

[0228] In another example, the methods of the present disclosure encompass culture expansion in a cell culture medium comprising neonatal serum. Various examples of suitable serum (and levels thereof) are disclosed herein.

[0229] Cell culture media used for cell culture and expansion contain all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are classified into essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro).

[0230] Those skilled in the art will appreciate that, in order to obtain optimal results, the basal medium must be appropriate for the cell line of interest. For example, if it is found that the glucose (or other energy source) in the basal medium is depleted and therefore restricts growth, it may be necessary to increase the glucose (or other energy source) level in the basal medium or to add glucose (or other energy source) during the culture process. In one example, the dissolved oxygen (DO) level may also be controlled.

[0231] serum

[0232] "Neonatal serum" refers to serum obtained after childbirth. For example, the culture medium can be supplemented with mammalian neonatal serum (e.g., bovine). In one example, the culture medium can be supplemented with animal neonatal serum. In another example, the culture medium can be supplemented with human neonatal serum.

[0233] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25% v / v of neonatal serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of neonatal serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 10% v / v of neonatal serum. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v neonatal serum. In one example, the cell culture medium is supplemented with about 5% v / v neonatal serum.

[0234] In one example, the neonatal serum contains at least one inflammatory cytokine. As used herein, the term "inflammatory cytokine" refers to a signaling molecule that promotes inflammation. In an example, the one or more cytokines are selected from the group consisting of IL-1β, IL-6, TNF-α, IFN-γ, and / or IL-1ra.

[0235] In one example, the neonatal serum contains IFN-γ. In another example, the neonatal serum contains TNF-α. In another example, the neonatal serum contains IFN-γ and TNF-α. In another example, the neonatal serum contains one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the neonatal serum may contain IL-8. In one example, the neonatal serum contains IFN-γ and / or TNF-α and one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. In another example, the neonatal serum contains IFN-γ and TNF-α and one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. In one example, the level of IFN-γ is less than 1 ng / ml. In one example, the level of TNF-α is less than 1 ng / ml. In one example, the levels of IFN-γ and TNF-α are both less than 1 ng / ml. For example, the level of IFN-γ can be less than 500 pg / ml or less than 100 pg / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml.

[0236] Methods for detecting the presence of cytokines in serum are known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA). In another example, the presence of cytokines in serum is detected by measuring cytokine mRNA, for example, by polymerase chain reaction (PCR) techniques, such as reverse transcription PCR.

[0237] In one example, the neonatal serum can be newborn calf serum (NBCS). In one example, NBCS is obtained from a newborn calf that has been fed colostrum. In one example, the NBCS comprises elevated levels of at least one inflammatory cytokine relative to NBCS obtained from a calf that has not been fed colostrum. In one example, the NBCS comprises elevated levels of at least one inflammatory cytokine relative to fetal serum (such as FCS).

[0238] In one example, the NBCS is obtained within 4 weeks of the calf's birth. In one example, the NBCS is obtained within 21 days of the calf's birth. For example, the NBCS is obtained ≤ 21 days after the calf's birth. In one example, the NBCS is obtained between the day the calf is born and 21 days after birth. In one example, the NBCS is obtained between the day the calf is born and 14 days after birth. In one example, the NBCS is obtained between the day the calf is born and 10 days after birth. In one example, the NBCS is obtained between the day the calf is born and 7 days after birth. In one example, the NBCS is obtained between 6 hours after birth and 72 hours after birth. In one example, the NBCS is obtained between 6 hours after birth and 48 hours after birth. In one example, the NBCS is obtained between 6 hours after birth and 24 hours after birth. In one example, the NBCS is obtained between 12 hours after birth and 24 hours after birth.

[0239] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25% v / v of NBCS. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of NBCS. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v of NBCS. In one example, the cell culture medium is supplemented with at least about 5% v / v of NBCS.

[0240] In one example, the culture medium is further supplemented with fetal serum. In one example, the fetal serum is fetal calf serum (FCS). It is contemplated that the terms fetal calf serum (FCS) and fetal bovine serum (FBS) can be used interchangeably in the context of this disclosure. In one example, the cell culture medium is supplemented with less than 10% v / v FCS. In one example, the cell culture medium is supplemented with about 5% v / v FCS.

[0241] In one example, the cell culture medium does not contain fetal serum.

[0242] In one example, the cell culture medium does not contain FCS.

[0243] In one example, the culture medium is supplemented with a mixture of FCS and NBCS. In one example, the cell culture medium is supplemented with about 5% v / v FCS and about 5% v / v NBCS (i.e., a 1:1 ratio of FCS to NBCS). In one example, the culture medium can be supplemented with a mixture of FCS and NBCS such that the FCS:NBCS ratio is at least about 0.4:1, at least about 0.5:1, at least about 0.6:1, at least about 0.7:1, at least about 0.8:1, at least about 0.9:1, at least about 1:1, at least about 1.5:1, or at least about 2:1. In one example, the FCS:NBCS ratio is between about 0.5:1 and about 2:1. In one example, the FCS:NBCS ratio is between about 0.8:1 and about 1.5:1. In one example, the FCS:NBCS ratio is between about 0.8:1 and about 1.2:1. In one example, the FCS:NBCS ratio is about 1:1.

[0244] In one example, the mixture of FCS and NBCS can comprise at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 1% v / v to about 15% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 2% v / v to about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise between about 5% v / v and about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise between about 8% v / v and about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise between about 10% v / v of the cell culture medium. However, in this example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9% v / v, but less than 10% v / v of FCS. In one example, the cell culture medium is supplemented with between about 1% v / v and about 9% v / v of FCS. In one example, the cell culture medium is supplemented with between about 3% v / v and about 8% v / v of FCS. In one example, the cell culture medium is supplemented with between about 3% v / v and about 6% v / v of FCS. In one example, the cell culture medium is supplemented with about 5% v / v FCS.

[0245] ascorbic acid

[0246] In one example, the cell culture medium is supplemented with a short-acting ascorbic acid derivative. The term "short-acting" encompasses ascorbic acid derivatives that are approximately 80%-90% oxidized after 24 hours of cell culture at neutral pH and 37°C. In one example, the short-acting L-ascorbic acid derivative is an L-ascorbate salt, such as sodium L-ascorbate. In one example, the cell culture medium may contain at least about 0.005 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.01 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.02 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.03 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.04 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.05 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium can contain at least about 0.06 g / L of a fugitive ascorbic acid derivative.

[0247] In another example, the cell culture medium contains a short-acting ascorbic acid derivative but does not contain a significant amount of a long-acting ascorbic acid derivative. For example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.04 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.03 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.02 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.01 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.005 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not a significant amount of a long-acting ascorbic acid derivative. In another example, the cell culture medium contains L-ascorbic acid sodium salt but does not contain a significant amount of L-ascorbic acid-2-phosphate.

[0248] Other additives

[0249] In one example, the cell culture medium contains additives of human origin. For example, human serum and human platelet cell lysate can be added to the cell culture medium. In other examples, additional factors can be added to the cell culture medium. For example, the cell culture medium can be supplemented with one or more stimulatory factors selected from the group consisting of platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), lα, 25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and stromal-derived factor 1α (SDF-1α). In another embodiment, cells can also be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth. In another embodiment, cells can be cultured in the presence of heparin or its derivatives.

[0250] In the above examples, basal culture media such as αMEM or StemSpan TM The serum may be supplemented with a reference amount and, in some cases, with other additives. Other examples of suitable culture media for culturing stem cells may be found in, for example, WO2016139340.

[0251] It will be appreciated by those skilled in the art that many changes and / or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the embodiments of the present invention are considered to be illustrative and not restrictive in all aspects.

[0252] This application claims priority to US63 / 386,876 filed on December 9, 2022 and US63 / 507,013 filed on June 8, 2023, the disclosures of which are incorporated herein by reference.

[0253] All publications discussed and / or cited herein are incorporated herein in their entirety.

[0254] Any discussion of documents, acts, materials, devices, articles and the like has been included in this specification solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0255] Example:

[0256] Example 1: Serum analysis

[0257] Mesenchymal precursor lineage or stem cell populations were cultured and expanded in 5% FCS / 5% NBCS (Serum A) or 10% fetal bovine serum (Serum B). These MLPSCs were used in Examples 4-6.

[0258] Cytokine levels were assessed in 5% FCS / 5% NBCS (Serum A) and 10% fetal bovine serum (Serum B). To provide external controls, cytokine levels were also assessed in FBS from different suppliers (Serum C). In each case, cytokine levels were assessed in pure serum.

[0259] Surprisingly, serum preparations containing newborn calf serum showed higher levels of proinflammatory cytokines (Figure 1). Notably, there was an increase in proinflammatory cytokines known to bind to receptors expressed on the surface of MLPSCs, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukins. For example, the following was observed in serum preparations containing newborn calf serum relative to fetal bovine serum:

[0260] IFNγ increased at least 2-fold;

[0261] TNFα increased at least 13-fold;

[0262] IL-6 increased at least 8-fold;

[0263] IL-8 increased by at least 2-fold;

[0264] IL-17A increased at least 2-fold.

[0265] Example 2: MLPSC Composition Derived Using Culture Medium Containing Fetal Serum

[0266] Eagle's α-modification of Minimum Essential Medium (MEM) with Earle's Balanced Salts (commonly referred to as Eagle αMEM) contains nonessential amino acids, sodium pyruvate, and additional vitamins. These modifications were originally described for use in growing hybrid mouse and hamster cells (Stanners et al. 1971).

[0267] Eagle αMEM medium suitable for culturing primary stem cells is available from a variety of sources, including Life Technologies and Sigma.

[0268] Detailed methods for establishing primary stem cell cultures, including the required growth factors used in the exemplified procedures, are described in Gronthos and Simmons 1995.

[0269] The cells were supplemented with 10% fetal bovine serum, L-ascorbic acid-2-phosphate (100 μM), dexamethasone (10 -7MLPSCs were cultured in Eagle αMEM medium supplemented with 1% (400 μM) and / or inorganic phosphate (3 mM).

[0270] Example 3: MLPSC compositions derived using culture medium containing neonatal serum

[0271] For MLPSC culture medium containing neonatal serum, the serum component of Eagle αMEM medium described in Example 2 was modified by supplementing with 5% (volume / volume) neonatal serum (the difference between fetal serum medium and neonatal serum medium is shown in Table 1). The neonatal serum used was newborn calf serum (NBCS). NBCS is 100% bovine serum obtained from animals that meet standard fetal bovine serum specifications but are less than 20 days old.

[0272] NBCS was obtained from a commercial supplier where it is marketed as an FCS alternative. It is highly similar to FCS and can be used interchangeably with FCS and is expected to have the same effect on cell lines.

[0273] Table 1: Summary of differences between fetal serum media and licensed media

[0274]

[0275]

[0276] Example 4: Enhanced angiogenesis by culturing and expanding MLPSCs in a medium supplemented with neonatal serum

[0277] To characterize novel MLPSC populations generated by culture expansion in medium supplemented with neonatal serum and / or proinflammatory cytokines, we evaluated the angiogenic potential of conditioned medium obtained from MLPSCs cultured under different conditions.

[0278] Cell Culture: MPCs were cultured in 10% FCS or 5% NBCS / 5% FCS to produce MPC-conditioned medium. To control for donor variability, MPCs were obtained from the same donor and cultured under different conditions. In some experiments, MPCs from the same donor but cultured during different manufacturing expansion periods were represented by separate "lot" numbers.

[0279] Conditioned medium was obtained by separating cells from culture supernatant. Briefly, cryopreserved MPCs were thawed and plated at 50,000 / cm 2 The cells were seeded in αMEM and 10% FBS or 5% NBCS / 5% FCS. Conditioned medium (CM) was collected after incubation at 37°C under 5% CO2 for 72 hours. VEGF, SDF-1, and angiopoietin levels in CM were measured using Luminex (R&D Systems). 3k protein concentration filter columns ( CM was concentrated using a Sigma-Aldrich Ultra-15 and reconstituted back to 1x or 0.25x in assay medium.

[0280] Angiogenesis Potency Assay: In vitro angiogenesis was measured using a kinetic, quantitative 96-well co-culture angiogenesis model. Human umbilical vein endothelial cells (HUVECs) transduced with lentivirus expressing CytoLight Green (a GFP variant) were seeded into 96-well plates and incubated with Live cell analysis system imaging. This system can fluorescently identify HUVEC (CytoLight Green + ) cells and allows visualization of tube formation over time through time-lapse image acquisition. The acquired images are analyzed using an integrated angiogenesis algorithm to measure network length, network area, and branch point formation to quantify the stage and extent of angiogenesis throughout the assay.

[0281] Results: Surprisingly, conditioned medium from MPCs cultured in medium supplemented with newborn calf serum was found to increase angiogenesis. As shown in Figure 2, in the co-culture angiogenesis model, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS increased network area (Figure 2A), network length (Figure 2B), and branch points (Figure 2C). In addition, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS contained higher levels of VEGF compared to conditioned medium from cells cultured in 10% FCS (Figure 2A). Angiopoietin levels were also increased in conditioned medium from MPCs cultured in 5% NBCS / 5% FCS when compared to 10% FCS (Figure 2B). Figure 3 ). Figure 4 Shown is a further analysis of the levels of angiogenic factors SDF-1α, VEGF, and Ang1 (ANGPT1) present in MPCs cultured in 10% FCS ("Serum B medium") or 5% FCS / 5% NBCS ("Serum A medium"). These data demonstrate that both VEGF and SDF-1α are elevated in MPCs cultured in neonatal serum medium. These data demonstrate that both VEGF and SDF-1α are elevated in MPCs cultured in neonatal serum medium.

[0282] In view of the data provided in Example 1, these data indicate that culturing and expanding MLPSCs in a medium supplemented with neonatal serum and / or proinflammatory cytokines provides a novel conditioned medium with enhanced angiogenic potential. If desired, this enhanced potential can be characterized in a variety of ways (e.g., defining the novel conditioned medium composition identified by the inventors), including, for example:

[0283] Conditioned medium derived from MLPSCs can increase network area, network length, and / or branching points when in contact with HUVECs;

[0284] - Angiopoietin, VEGF and / or SDF-1 levels in conditioned medium.

[0285] Example 5: MLPSC isolation and expansion

[0286] MLPSCs can be isolated using techniques such as STRO-3+ immunoselection of MPCs or density gradient separation of MSCs.

[0287] Typically, bone marrow-derived MLPSCs are collected from healthy normal adult volunteers (20-35 years old). Briefly, 40 ml of BM is aspirated from the posterior iliac crest into a tube containing lithium heparin anticoagulant.

[0288] BMMNCs were prepared by density gradient separation using Lymphoprep (Nycomed Pharma, Oslo, Norway) as previously described (Zannettino et al. 1998). After centrifugation at 400 x g for 30 minutes at 4°C, the buffy coat was removed with a pipette and washed three times in "HHF" consisting of Hank's balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0289] In conjunction with immune selection, STRO-3+ (or TNAP+) cells were then isolated by magnetic activated cell sorting as previously described (Gronthos et al. 2003; Gronthos and Simmons 1995). Briefly, approximately 1-3×108 BMMNCs were incubated on ice for 20 minutes in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. The cells were incubated on ice for 1 hour with 200 ul of a 10 ug / ml solution of STRO-3 mAb in blocking buffer. The cells were then washed twice in HHF by centrifugation at 400 x g. A 1 / 50 dilution of goat anti-mouse biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer was added and the cells were incubated on ice for 1 hour. Cells were washed twice in MACS buffer (Ca2+- and Mn2+-free PBS supplemented with 1% BSA, 5 mM EDTA, and 0.01% sodium azide) as described above and resuspended in a final volume of 0.9 ml of MACS buffer.

[0290] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice and resuspended in 0.5 ml MACS buffer, then loaded onto a micro MACS column (MS column, Miltenyi Biotec) and washed three times with 0.5 ml MACS buffer to recover cells that were not bound to STRO-3 mAb (deposited with the American Type Culture Collection (ATCC) under the accession number PTA-7282 on December 19, 2005—see International Publication No. WO 2006 / 108229). After adding 1 ml of MACS buffer, the column was removed from the magnet and TNAP+ cells were separated by positive pressure. An aliquot of cells from each fraction can be stained with streptavidin-FITC and the purity assessed by flow cytometry.

[0291] Alternatively, MSCs can be expanded from BMMNCs using plastic adherence techniques. For example, bone marrow mononuclear cells can be isolated using ficoll-hypaque and placed in two T175 flasks, each containing 50 ml of culture expansion medium containing α-modified MEM (αMEM) containing gentamicin, glutamine (2 mM), and 10% (v / v) fetal bovine serum (FBS).

[0292] The cells were cultured at 37°C, 5% CO2 for 2-3 days, at which time the non-adherent cells were removed; the remaining adherent cells were cultured until the cell confluence reached 70% or greater (7-10 days), and then the cells were trypsinized and transferred to six T175 flasks containing expansion medium.

Claims

1. A composition comprising conditioned medium or extracellular vesicles obtained therefrom, wherein the conditioned medium is characterized by increased levels of angiogenic markers, wherein the conditioned medium is obtained from a population of MLPSCs that has been expanded by culture in a medium containing: -IFN-γ and / or TNF-α; and / or, - one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, IP-10; and wherein the increased levels of the angiogenesis markers are determined relative to conditioned medium obtained from a control population.

2. The composition of claim 1, wherein the angiogenesis marker is one or more or all of the following: i. angiogenin levels are increased relative to conditioned medium obtained from a control population; ii. increased endothelial network formation relative to conditioned medium obtained from control populations; iii. increased endothelial length relative to conditioned medium obtained from control populations; iv. Endothelial branch length is increased relative to conditioned medium obtained from control populations.

3. The composition of claim 1 or claim 2, wherein the control population is a population of MLPSCs that has been expanded by culture in a cell culture medium comprising 10% fetal serum.

4. The composition of any one of claims 1 to 3, wherein the conditioned medium contains angiogenin levels greater than about 1200 pg / ml.

5. The composition of any one of claims 1 to 4, wherein the conditioned medium contains a level of SDF-1 greater than about 3000 pg / ml.

6. The composition of any one of claims 1 to 5, wherein the conditioned medium contains a level of VEGF greater than about 3200 pg / ml.

7. The composition of any one of claims 1 to 6, wherein the conditioned medium induces a cytokine secretion greater than about 0.12 mm 2 / mm 2 endothelial network formation.

8. The composition of any one of claims 1 to 7, wherein the conditioned medium induces a cytokine secretion greater than about 5 mm 2 / mm 2 Length of the endothelial network.

9. The composition of any one of claims 1 to 8, wherein the conditioned medium induces an increase in the expression of greater than about 15 1 / mm 2 Length of endothelial branches.

10. The composition according to any one of claims 1 to 9, wherein the culture medium contains three or more pro-inflammatory cytokines.

11. The composition according to any one of claims 1 to 10, wherein the culture medium contains two or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, IP-10.

12. The composition according to any one of claims 1 to 11, wherein the culture medium contains IL-6.

13. The composition according to any one of claims 1 to 12, wherein the culture medium contains IL-8 and / or IL-17A.

14. The composition according to any one of claims 1 to 13, wherein the culture medium contains IFN-γ and TNF-α.

15. The composition according to any one of claims 1 to 14, wherein the level of IFN-γ is <1 ng / ml, preferably <500 pg / ml, more preferably <100 pg / ml.

16. The composition according to any one of claims 1 to 15, wherein the level of TNF-alpha is <1 ng / ml, preferably <750 pg / ml, more preferably <400 pg / ml.

17. The composition according to any one of claims 1 to 16, wherein the culture medium contains serum comprising the proinflammatory cytokines.

18. The composition of claim 17, wherein the serum is newborn mammalian serum, preferably newborn calf serum.

19. The composition of claim 17, wherein the serum is obtained no more than 21 days after birth.

20. The composition of any one of claims 1 to 19, wherein the culture medium is characterized by one or more or all of the following: i. IFN-γ level greater than 1 pg / ml; ii. TNF-α level greater than 2 pg / ml; iii. IL-6 level greater than 3 pg / ml; iv. IL-8 level greater than 500 pg / ml; v.IL-17A level greater than 0.2 pg / ml; vi. MCP-1 level greater than 3 pg / ml; vii. MIP-1-α level greater than 0.5 pg / ml; viii. MIP-1-β level greater than 3 pg / ml; ix. IP-10 levels were greater than 500 pg / ml.

21. The composition of any one of claims 1 to 20, wherein the culture medium comprises at least 5% (v / v) newborn mammalian serum.

22. The composition of any one of claims 1 to 16 or 20, wherein the culture medium is serum-free.

23. The composition of any one of claims 1 to 22, wherein the extracellular vesicles obtained from the conditioned medium are exosomes.

24. A composition comprising conditioned medium or extracellular vesicles obtained therefrom, wherein the conditioned medium is obtained from a population of MLPSCs that has been expanded by culture in a medium containing newborn mammalian serum.

25. The composition of claim 24, wherein the serum is newborn calf serum.

26. A composition according to claim 24 or claim 25, wherein the serum is obtained no more than 21 days after birth.

27. The composition of any one of claims 24 to 26, wherein the conditioned medium comprises increased levels of angiogenesis markers relative to conditioned medium obtained from a control population.

28. The composition of claim 27, wherein the angiogenesis marker is one or more or all of the following: i. angiogenin levels are increased relative to conditioned medium obtained from a control population; ii. increased endothelial network formation relative to conditioned medium obtained from control populations; iii. increased endothelial length relative to conditioned medium obtained from control populations; iv. Endothelial branch length is increased relative to conditioned medium obtained from control populations.

29. The composition of claim 27 or claim 28, wherein the control population is a population of MLPSCs that has been expanded by culture in a cell culture medium comprising 10% fetal serum.

30. The composition of any one of claims 24 to 29, wherein the conditioned medium contains angiogenin levels greater than about 1200 pg / ml.

31. The composition of any one of claims 24 to 30, wherein the conditioned medium contains a level of SDF-1 greater than about 3000 pg / ml.

32. The composition of any one of claims 24 to 31, wherein the conditioned medium contains a level of VEGF greater than about 3200 pg / ml.

33. The composition of any one of claims 24 to 32, wherein the conditioned medium induces greater than about 0.12 mm 2 / mm 2 endothelial network formation.

34. The composition of any one of claims 24 to 33, wherein the conditioned medium induces a cytokine secretion greater than about 5 mm 2 / mm 2 Length of the endothelial network.

35. The composition of any one of claims 24 to 34, wherein the conditioned medium induces a cytokine secretion greater than about 15 l / mm 2 Length of endothelial branches.

36. The composition of any one of claims 18 to 35, wherein the neonatal serum is neonatal serum from day 1 to day 7 after birth.

37. The composition of any one of claims 18 to 36, wherein the concentration of the neonatal serum is about 2% (v / v) to about 12% (v / v).

38. The composition of any one of claims 18 to 36, wherein the concentration of the neonatal serum is about 5% (v / v).

39. The composition of any one of claims 18 to 36, wherein the culture medium comprises fetal serum and neonatal serum from the same species, and the ratio of the concentration of the fetal serum to the concentration of the neonatal serum is 1:1 or less.

40. The composition of claim 39, wherein the concentration of the fetal serum and the concentration of the neonatal serum are each 5% (v / v).

41. The composition of claim 39, wherein the concentration of the fetal serum is lower than the concentration of the neonatal serum.

42. The composition of any one of claims 24 to 41, wherein the extracellular vesicles obtained from the conditioned medium are exosomes.

43. The composition of any one of claims 1 to 42, wherein the MLPSC is a human mesenchymal stem cell (hMSC).

44. The composition of any one of claims 1 to 43, wherein the MLPSCs are derived from STRO-1 + Pluripotent cell populations are expanded in culture.

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