Methods for assaying soluble tumor necrosis factor receptor 2 (STNFR2) and uses thereof

Abstract

The present disclosure relates to methods for testing the immunomodulatory activity of cells, including, for example, mesenchymal stem cells, and the use of such cells determined to have immunomodulatory activity to treat COVID-19-associated acute respiratory distress syndrome (ARDS). Disclosed herein are in vitro methods for evaluating mesenchymal stem cells for their effective immunomodulatory effects in vivo. In some embodiments, the plurality of cells comprises human mesenchymal stem cells, or mesenchymal stromal cells, or medical signaling cells. In some embodiments, the cells are derived from postnatal adipose tissue, infrapatellar fat pad, postnatal bone marrow, postnatal endometrium, perinatal umbilical cord, perinatal chorion, perinatal amnion, or perinatal placenta.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 303,585, filed January 27, 2022, which is incorporated by reference in its entirety.

[0002] Field The present disclosure relates to methods for determining the immunomodulatory activity of cells and uses for treating inflammatory diseases. [Background technology]

[0003] background The safety and efficacy of mesenchymal stem cell (MSC)-based therapies have been investigated in many clinical trials for a variety of disorders, including inflammatory, immune, autoimmune, musculoskeletal, cardiovascular, neurodegenerative, and gastrointestinal diseases. However, early results from many such trials have revealed that these cell therapies have a significant degree of variability and may not be reproducible in clinical observations.

[0004] Novel in vitro methods are needed to evaluate MSCs for their potential immunomodulatory effects in vivo. Summary of the Invention [Means for solving the problem]

[0005] Abstract One embodiment of the present disclosure is a method for testing an immunomodulatory activity of a plurality of cells, the method comprising: a. separating the plurality of cells into a first population of cells and a second population of cells; b. culturing a first population of said cells in basal conditions and culturing a second population of said cells in inflammatory conditions; c. harvesting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells of the first group of cells, and the cells of the second group of cells; d. determining the level of soluble TNFR2 protein in the culture supernatant of the first group of cells and in the culture supernatant of the second group of cells; e. normalizing the levels of soluble TNFR2 protein in said first group of cells and said second group of cells to the total protein levels in said first group of cells and said second group of cells, respectively, collected in step c; f. calculating an inflammatory stimulation index (ISI) by dividing the normalized level of soluble TNFR2 protein of the second group of cells by the normalized level of soluble TNFR2 protein of the first group of cells; and g. determining that the plurality of cells is responsive to an inflammatory stimulus and an immunomodulatory activity if the ISI is greater than 1; The method includes the steps of:

[0006] In some embodiments, the plurality of cells comprises human mesenchymal stem cells, or mesenchymal stromal cells, or medical signaling cells. In some embodiments, the cells are derived from postnatal adipose tissue, infrapatellar fat pad, postnatal bone marrow, postnatal endometrium, perinatal umbilical cord, perinatal chorion, perinatal amnion, or perinatal placenta.

[0007] In some embodiments, the inflammatory condition comprises the presence of TNFα and / or IFNγ, In some embodiments, the inflammatory condition further comprises the presence of TNFβ, IL-1β, or connective tissue growth factor (CTGF).

[0008] An embodiment of the present disclosure is a method of treating COVID-19 associated acute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are determined as having immunomodulatory activity using the method of any preceding aspect. In some embodiments, the plurality of cells having immunomodulatory activity are determined as having an inflammatory stimulation index (ISI) greater than 1.

[0009] One embodiment of the present disclosure is a method of treating an inflammatory disorder and / or fibrosis in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are determined as having immunomodulatory activity using the method of any preceding aspect. In some embodiments, the plurality of cells having immunomodulatory activity are determined as having an inflammatory stimulation index (ISI) greater than 1.

[0010] One embodiment of the present disclosure is a method of treating an inflammatory condition in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are determined to have immunomodulatory activity using the method of any preceding aspect. In some embodiments, the plurality of cells having immunomodulatory activity are determined to have an inflammatory stimulation index (ISI) greater than 1.

[0011] One embodiment of the present disclosure is a method of treating a characterized fibrotic condition in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are determined to have immunomodulatory activity using the method of any preceding aspect. In some embodiments, the plurality of cells having immunomodulatory activity are determined to have an inflammatory stimulation index (ISI) greater than 1.

[0012] An embodiment of the present disclosure is a method of treating a condition characterized by increased TNF in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are determined as having immunomodulatory activity using the method of any of the preceding aspects. In some embodiments, the plurality of cells having immunomodulatory activity are determined as having an inflammatory stimulation index (ISI) greater than 1. In some embodiments, the method of any of the preceding aspects further comprises administering to the subject a therapeutically effective amount of an anti-COVID therapeutic. [Brief description of the drawings]

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0014] [Figure 1] FIG. 1 shows the steps of standard dilution preparation.

[0015] [Diagram 2] FIG. 2 shows the layout for preparing samples and standards in a 96-well plate (see Table 5).

[0016] [Diagram 3] Figure 3 shows a schematic diagram of measuring soluble TNFR2 release via normalized quantification and the inflammatory stimulation index (ISI).

[0017] [Figure 4] FIG. 4 shows soluble TNFR2 (sTNFR2) release by UC-MSCs over a 3-day period in basal culture conditions upon induction of inflammation.

[0018] [Diagram 5] FIG. 5 shows the Inflammatory Stimulation Index (ISI) of sTNFR2 release by UC-MSCs, calculated as the ratio of sTNFR2 release in inflammatory conditions to basal conditions.

[0019] [Figure 6] Figure 6 shows observations in patients. Plasma concentrations of soluble tumor necrosis factor receptor 2 (sTNFR2), tumor necrosis factor alpha (TNFα), and tumor necrosis factor beta (TNFβ) in subjects (n=24) with COVID-19 acute respiratory distress syndrome (ARDS). At day 6, UC-MSC recipients had significantly elevated levels of plasma sTNFR2 and significantly decreased levels of TNFα and TNFβ compared to controls. Data are shown as median values ​​and box plots showing minimum to maximum values, as well as lined scatter plots showing individual values.

[0020] [Figure 7] Figure 7 shows the sTNFR2 inflammatory stimulation index (ISI) of UC-MSC preparations from different donors and at different culture passages. ISI values ​​were obtained via static sTNFR2 release assay. Histograms show the average sTNFR2 ISI from triplicate studies; error bars show the standard error of the mean. A: UC-MSC SCI-St passage 2; B: UC-MSC SCI-St passage 4; C: UC-MSC SCI-St passage 5; D: UC-MSC SCI-R01 passage 4; E: UC-MSC SCI-R01 passage 5; F: UC-MSC SCI-MCB-1 batch 1 passage 2; G: UC-MSC SCI-MCB-1 batch 2 passage 2; H: UC-MSC SCI-MCB-1 passage 4.

[0021] [Figure 8] FIG. 8 shows a diagram for cell counting in the trypan blue viability test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description Reference will now be made in detail to the embodiments of the present invention, examples of which are illustrated in the drawings and examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The term "comprising" and variations thereof, as used herein, are used synonymously with the term "including" and variations thereof, and are open and non-limiting terms. The terms "comprising" and "including" are used herein to describe various embodiments, and the terms "essentially consisting of" and "consisting of" can be used in place of "comprising" and "including" to provide more specific embodiments, and are similarly disclosed. As used in this disclosure and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0024] The following definitions are provided for a full understanding of terms used herein.

[0025] Terminology The term "about," when used herein in reference to a measurable value such as an amount, percentage, etc., is meant to encompass a variation of ±20%, ±10%, ±5%, or ±1% from that measurable value.

[0026] "Administration" or "administering" to a subject includes any route of introducing or delivering an agent to a subject. Administration may be by any suitable route, including oral, intravenous, intraperitoneal, intranasal, inhalation, etc. Administration includes self-administration and administration by another.

[0027] A "control" is a substitute subject or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0028] The term "increased" or "increase" as used herein generally refers to an increase by a statistically significant amount; for example, "increased" means an increase of at least 10% when compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase up to and including 100%, or any increase between 10-100% when compared to a reference level, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more than 10-fold when compared to a reference level.

[0029] The terms "reduced", "reduce", "reduction" or "decrease" as used herein generally refer to a decrease by a statistically significant amount. However, for the avoidance of doubt, "reduced" refers to a decrease of at least 10% when compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% decrease or a decrease up to and including 100%, or any decrease between 10-100% when compared to a reference level.

[0030] As used herein, the term "level" refers to the amount of a target molecule in a sample (e.g., a sample derived from a subject). The amount of the molecule can be determined by any method known in the art and depends in part on the nature of the molecule (i.e., gene, mRNA, cDNA, protein, enzyme, etc.). The art is familiar with quantitative methods for nucleotides (e.g., genes, cDNA, mRNA, etc.), as well as proteins, polypeptides, enzymes, etc. It is understood that the amount or level of a molecule in a sample need not be determined in absolute terms, but can be determined in relative terms (e.g., when compared to a control or sham or untreated sample).

[0031] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases where the condition occurs and cases where the condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include cases where the formulation includes an excipient and cases where the formulation does not include an excipient.

[0032] As used herein, the term "subject" or "host" may refer to a living organism, such as a mammal, including but not limited to humans, livestock, dogs, cats, and other mammals. Administration of a therapeutic agent may be at a dosage and for a period of time effective for treatment of the subject. In some embodiments, the subject is a human.

[0033] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects (e.g., treatment of a disorder or other undesirable physiological condition) and prophylactic effects (e.g., prevention of a disorder or other undesirable physiological condition). The term also encompasses pharmaceutically acceptable, pharmacologically active derivatives (including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, etc.) of the beneficial agents specifically mentioned herein. When the term "therapeutic agent" is used, then, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0034] The terms "treat", "treating", "treatment" and grammatical variations thereof, as used herein, include partially or completely delaying, alleviating, relieving, or reducing the intensity of one or more associated symptoms of an infection or condition, and / or alleviating, alleviating, or preventing one or more symptoms of COVID-19-associated acute respiratory distress syndrome (ARDS). Treatment according to the present invention may be applied preventatively, prophylactically, palliatively, or therapeutically. Prophylactic treatment is administered to a subject prior to onset (e.g., before overt signs of infection), during early onset (e.g., at early signs and symptoms of infection), or after the establishment of an outbreak of infection. Prophylactic administration may occur minutes to months prior to the appearance of infection.

[0035] method In some aspects, a method for testing an immunomodulatory activity of a plurality of cells, the method comprising: a. separating the plurality of cells into a first population of cells and a second population of cells; b. culturing a first population of said cells in basal conditions and culturing a second population of said cells in inflammatory conditions; c. harvesting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells of the first group of cells, and the cells of the second group of cells; d. determining the level of soluble TNFR2 protein in said first group culture supernatant and in said second group culture supernatant; e. normalizing the levels of soluble TNFR2 protein in said first and second groups to the total protein levels in each of said groups of cells collected in step c; f. calculating an inflammatory stimulation index (ISI) by dividing the normalized level of the second group of soluble TNFR2 protein by the normalized level of the first group of soluble TNFR2 protein; and g. determining that the plurality of cells has immunomodulatory activity if the ISI is greater than 1; Disclosed herein is a method comprising:

[0036] It should be understood, and is contemplated herein, that soluble tumor necrosis factor receptor 2 (soluble TNFR2) is also known as sTNFR2, sTNF-RII, TNFRSF1B, CD120b, TBPII, TNF-R-II, TNF-R75, TNFBR, TNFR1B, TNFR2, TNFR80, p75, p75TNFR, tumor necrosis factor receptor superfamily member 1B, TNF receptor superfamily member 1B (which is the product of the gene "TNF receptor superfamily member 1B" or TNFRSF1B).

[0037] As used herein, the term "immunomodulatory activity" refers to activity that reduces inflammatory activity, including, for example, modulation of a hyperinflammatory (e.g., cytokine storm) or hyperimmune response. In some embodiments, the immunomodulatory activity is an anti-inflammatory effect.

[0038] In some embodiments, the plurality of cells comprises human mesenchymal stem cells or mesenchymal stromal cells, medical signaling cells, or multipotent stromal cells. In some embodiments, the cells are derived from organs and tissues such as postnatal pancreatic islets or pancreatic tissue, postnatal adipose tissue, infrapatellar fat pad, postnatal bone marrow, postnatal endometrium, postnatal dental pulp, perinatal umbilical cord, perinatal chorion, perinatal amnion, or perinatal placenta. In some embodiments, the plurality of cells comprises mesenchymal stem cells.

[0039] As used herein, "mesenchymal stem cells" or "MSCs" are cells that can differentiate into mesenchymal cell lineages (i.e., osteoblasts, chondroblasts and adipocytes). Morphological and functional criteria well known to those skilled in the art are used to identify these cells. See Horwitz et al., supra; Dominici et al., supra; Trivedi P & Hematti P, "Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells", Exp. Hematol. Jan. 5, 2008; Trivedi P & Hematti P, "Simultaneous generation of CD34+ primitive hematopoietic cells and CD56+ mesenchymal stromal cells from human embryonic stem cells cocultured with murine OP9 stromal cells", Exp. Hematol. 35: 146-154(2007); and U.S. Published Patent Application No. 2006 / 0008902, each of which is incorporated by reference herein as if set forth in its entirety. Mesenchymal stem cells or MSCs can be recognized by their characteristic mononuclear ovoid, star-shaped or spindle-shaped, round to elliptical nuclei. The oval, elongated nuclei typically have prominent nucleoli and a mixture of heterochromatin and euchromatin. These cells have little cytoplasm, but many thin processes that appear to extend from the nucleus. Mesenchymal stem cells or MSCs are negative for hematopoietic lineage cell markers (e.g., CD14, CD34, or CD45) and endothelial lineage cell markers (e.g., CD31 and VE-cadherin), but may stain for one, two, three, or more of the following markers: CD29, CD44, CD73, CD90, CD105, CD106 (VCAM), CD166 (ALCAM), and alkaline phosphatase.Mesenchymal stem cells or MSCs may also express STRO-1 and / or CD146 as markers.

[0040] In some embodiments, the inflammatory condition comprises TNFα and / or IFNγ. In some embodiments, the inflammatory condition further comprises TNFβ, IL-1β, connective tissue growth factor (CTGF). Thus, in some examples, the second population of cells is cultured in a cell culture medium comprising TNFα, IFNγ, TNFβ, IL-1β, or CTGF, or any combination thereof.

[0041] The cells may be cultured for at least 1 minute, at least 1 hour, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 20 days, at least 30 days, or at least 60 days. In some embodiments, the cells are cultured for at least 1 day. In some embodiments, the cells are cultured for at least 2 days. In some embodiments, the cells are cultured for at least 3 days.

[0042] Also disclosed herein is a method of treating COVID-19 associated acute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are determined as having immunomodulatory activity using the methods disclosed herein.

[0043] Thus, in some aspects, there is provided a method of treating COVID-19 associated acute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising: administering to the subject a plurality of cells, wherein the cells comprise: a. separating the plurality of cells into a first population of cells and a second population of cells; b. culturing a first population of said cells in basal conditions and culturing a second population of said cells in inflammatory conditions; c. harvesting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells of the first group of cells, and the cells of the second group of cells; d. determining the level of soluble TNFR2 protein in said first group culture supernatant and in said second group culture supernatant; e. normalizing the levels of soluble TNFR2 protein in said first and second groups to the total protein levels in each of said groups of cells collected in step c; f. calculating an inflammatory stimulation index (ISI) by dividing the normalized level of the second group of soluble TNFR2 protein by the normalized level of the first group of soluble TNFR2 protein; and g. determining that the plurality of cells has immunomodulatory activity if the ISI is greater than 1; is determined to have immunomodulatory activity using a method comprising: A method is disclosed herein.

[0044] In some embodiments, the plurality of cells is determined as having an ISI higher than one.

[0045] "ARDS" or "acute respiratory distress syndrome" refers to a disease that occurs when fluid accumulates in the tiny elastic air sacs (alveoli) in the lungs. Symptoms of ARDS include, for example, extreme difficulty in breathing, shortness of breath, and / or low oxygen levels in the blood, which also result in a series of other symptoms including confusion, dizziness, excessive sweating, low blood pressure, and increased heart rate. ARDS can be the result of inflammatory changes in the alveoli. In some embodiments, administration of the cells determined to have immunomodulatory activity using the methods disclosed herein can alleviate one or more of the symptoms including, for example, extreme difficulty in breathing, shortness of breath, and / or low oxygen levels in the blood. In some embodiments, administration of the cells reduces inflammation in the subject. In some embodiments, administration of the cells reduces levels of SARS-CoV-2 virus in the subject. It should be understood, and is contemplated herein, that the terms "increase" and "decrease" as used herein may refer to an increase or decrease compared to before treatment of the subject, or compared to the incidence of such condition in a general or test population.

[0046] Also disclosed herein are methods of treating inflammatory disorders and / or fibrosis in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are determined to have immunomodulatory activity using the methods disclosed herein.

[0047] Thus, in some aspects, there is provided a method of treating an inflammatory disorder and / or fibrosis in a subject in need thereof, the method comprising administering to the subject a plurality of cells, wherein the cells are: a. separating the plurality of cells into a first population of cells and a second population of cells; b. culturing a first population of said cells in basal conditions and culturing a second population of said cells in inflammatory conditions; c. harvesting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells of the first group of cells, and the cells of the second group of cells; d. determining the level of soluble TNFR2 protein in said first group culture supernatant and in said second group culture supernatant; e. normalizing the levels of soluble TNFR2 protein in said first and second groups to the total protein levels in each of said groups of cells collected in step c; f. calculating an inflammatory stimulation index (ISI) by dividing the normalized level of the second group of soluble TNFR2 protein by the normalized level of the first group of soluble TNFR2 protein; and g. determining that the plurality of cells has immunomodulatory activity if the ISI is greater than 1; is determined to have immunomodulatory activity using a method comprising: A method is disclosed herein.

[0048] In some embodiments, the plurality of cells is determined as having an ISI higher than one.

[0049] In some embodiments, the method of any of the aforementioned aspects further comprises administering to the subject a therapeutically effective amount of an anti-COVID therapeutic.

[0050] In one embodiment, the anti-COVID therapeutic agent is one or more of the following: Baricitinib, Ruxolitinib, Tofacitinib, Imatinib, Fluvoxamine, Methylprednisolone, Lopinavir, Ritonavir, Darunavir, Favipiravir, Remdesivir, Sovosbuvir / Daclatasvir, Nilmatrelorvir, Budesonide, Artesunate, Type I Interferon, Telmisartan, Nitazoxanide, Niclosamide, Bromhexine, Dornase alfa, Dexmedetomidine, Fluoxetine, Sabizablin, Ribavirin, Molnupiravir, Danoprevir, Bemnifosbuvir, Galidesivir, BCX-4430, Opaganib, Arbidol, Chloroquine, Dexamethasone Heparin, Nitazoxanide, Tocilizumab, Sarilumab, Revilimab, Siltuximab, Clazakizumab, Sirukumab, Olokizumab, Anakinra, Canakinumab, Mavrilimumab, Lenzilumab, Gimcilumab, Otilimab, TJ003234, Emapalumab, Adalimumab, Infiximab, Secukinumab, Ixekizumab, Risankizumab, Lufotrevir, Ensovivep, Fenretinide, Lintatorimod, Bemcentinib, Plitidepsin, Emetine Hydrochloride, Tin Protoporphyrin, Antroquinonol, Apilimod Dimesylate dimesylate, brequinar, brilacidin, sangivamycin, Tempol, RP-7214, PBI-0451, and masitinib.

[0051] In one embodiment, in static sTNFR2 release assay, cells are cultured in separate culture vessels and exposed to inflammatory medium or control (basal) medium.Then, the value of sTNFR2 is measured in each medium, normalized, and the ratio between the sTNFR2 measured under the above conditions is calculated.

[0052] Figure 7 shows sTNFR2 inflammatory stimulation index (ISI) in UC-MSC preparations from different donors and at different culture passages. ISI values ​​were obtained via static sTNFR2 release assay. The histogram shows the average sTNFR2 ISI from triplicate studies; error bars show the standard error of the mean. A: UC-MSC SCI-St passage 2; B: UC-MSC SCI-St passage 4; C: UC-MSC SCI-St passage 5; D: UC-MSC SCI-R01 passage 4; E: UC-MSC SCI-R01 passage 5; F: UC-MSC SCI-MCB-1 batch 1 passage 2; G: UC-MSC SCI-MCB-1 batch 2 passage 2; H: UC-MSC SCI-MCB-1 passage 4.

[0053] In one embodiment, in a dynamic sTNFR2 release assay, the same cell preparation is kept fixed and culture medium flows around the cells ("perifusion"). In this embodiment, the same cells can be dynamically exposed to basal and inflammatory condition media through the flow of media with different compositions around the fixed cells for different time ranges. Samples can be collected at different time points throughout the perfusion assay, and values ​​of sTNFR2 release can be measured in the various samples, and the difference can be calculated in the amount of sTNFR2 measured after exposure to inflammatory media compared to basal media. Instruments for performing automated perfusion of media around cells fixed in a chamber are commercially available.

[0054] The difference in sTNFR2 release (termed "delta sTNFR2," or "ΔsTNFR2"), and the kinetics of change in sTNFR2 release, are useful when comparing multiple cell preparations.

[0055] Normalization of sTNFR2 values ​​may be based on cellular protein content at the conclusion of the in vitro experiments, as shown in a preferred embodiment.

[0056] In other embodiments, normalization of sTNFR2 may be based on cell number at initial seeding, or cell seeding density, or cell concentration in the culture medium.

[0057] In one embodiment, the culture medium comprises 10% platelet lysate. In other embodiments, the culture medium comprises 9%, or 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% platelet lysate. In other embodiments, the culture medium does not comprise platelet lysate.

[0058] In one embodiment, the culture medium is a chemically defined culture medium.

[0059] In one embodiment, the induction of inflammation is based on the addition of TNFα and IFNγ, hi other embodiments, the induction of inflammation is based on the addition of TNFα, or TNFβ, or IFNγ, or other inflammatory cytokines.

[0060] In certain embodiments, the concentration of TNFα is 0 to 15000 pg / ml. In certain embodiments, the concentration of TNFα is 0 to 12500 pg / ml. In certain embodiments, the concentration of TNFα is 0 to 10000 pg / ml. In certain embodiments, the concentration of TNFα is 0 to 7000 pg / ml. In certain embodiments, the concentration of TNFα is 0 to 4000 pg / ml. In one embodiment, the concentration of TNFα is 0 to 4000 pg / ml. In one embodiment, the concentration of TNFα is 0 to 3000 pg / ml. In one embodiment, the concentration of TNFα is 0 to 2500 pg / ml. In one embodiment, the concentration of TNFα is 0 to 2000 pg / ml. In one embodiment, the concentration of TNFα is 0 to 1500 pg / ml. In one embodiment, the concentration of TNFα is 0 to 1000 pg / ml. In one embodiment, the concentration of TNFα is 100 to 1500 pg / ml. In one embodiment, the concentration of TNFα is 500 to 2500 pg / ml. In one embodiment, the concentration of TNFα is 500 to 2000 pg / ml. In one embodiment, the concentration of TNFα is 500 to 1500 pg / ml. In one embodiment, the concentration of TNFα is 500 to 1000 pg / ml. In one embodiment, the concentration of TNFα is 500 to 4000 pg / ml. In one embodiment, the concentration of TNFα is 100 to 4000 pg / ml.

[0061] In certain embodiments, the concentration of TNFβ is 0 to 20000 pg / ml. In certain embodiments, the concentration of TNFβ is 0 to 15000 pg / ml. In certain embodiments, the concentration of TNFβ is 0 to 10000 pg / ml. In certain embodiments, the concentration of TNFβ is 0 to 5000 pg / ml. In certain embodiments, the concentration of TNFβ is 500 to 5000 pg / ml. In certain embodiments, the concentration of TNFβ is 500 to 10000 pg / ml. In certain embodiments, the concentration of TNFβ is 100 to 10000 pg / ml. In certain embodiments, the concentration of TNFβ is 100 to 7500 pg / ml. In certain embodiments, the concentration of TNFβ is 100 to 5000 pg / ml. In certain embodiments, the concentration of TNFβ is 100 to 20,000 pg / ml, In certain embodiments, the concentration of TNFβ is 500 to 20,000 pg / ml.

[0062] In certain embodiments, the concentration of IFNγ is 0 to 10000 pg / ml. In certain embodiments, the concentration of IFNγ is 0 to 5000 pg / ml. In certain embodiments, the concentration of IFNγ is 0 to 2000 pg / ml. In certain embodiments, the concentration of IFNγ is 0 to 1750 pg / ml. In certain embodiments, the concentration of IFNγ is 0 to 1500 pg / ml. In certain embodiments, the concentration of IFNγ is 0 to 1250 pg / ml. In certain embodiments, the concentration of IFNγ is 0 to 1000 pg / ml. In certain embodiments, the concentration of IFNγ is 500 to 1500 pg / ml. In certain embodiments, the concentration of IFNγ is 500 to 1250 pg / ml. In certain embodiments, the concentration of IFNγ is 100 to 1000 pg / ml. In certain embodiments, the concentration of IFNγ is 100-2000 pg / ml, In certain embodiments, the concentration of IFNγ is 500-2000 pg / ml.

[0063] In other embodiments, the cells to be analyzed for sTNFR2 release are grown in suspension culture.

[0064] In other embodiments, the cells to be analyzed for sTNFR2 release are cultured attached or in suspension on microcarriers, either without or with agitation.

[0065] In certain embodiments, the cells being analyzed for sTNFR2 release are adherent to a matrix.

[0066] In other embodiments, the cells to be analyzed for sTNFR2 release are embedded in a material that allows movement of sTNFR2 through pores in the material.

[0067] In certain embodiments, the cells are fixed by adhering onto a matrix or by being enclosed in a container, and culture medium is made to flow around the cells in an in vitro system (perfusion).The fluid, i.e., culture medium, or a solution containing a drug, or a suspension of cells, flows through the in vitro system and contacts the cells.Using this method, the kinetics of the release of sTNFR2 from the fixed cells can be analyzed in detail, and different samples can be collected over time and / or after exposure to control medium or inflammation-inducing medium.

[0068] The standardized in vitro methods presented herein can be used to screen cell preparations to select those with the greatest potential for clinical efficacy when the goal is to increase the in vivo concentration of sTNFR2 and / or decrease the in vivo concentration of TNFα and / or TNFβ.

[0069] This method allows for the selection of cell populations based on in vitro potency related to release of sTNFR2, with the potential to predict the in vivo efficacy and effectiveness of the cell population. EXAMPLES

[0070] The following examples are presented below to illustrate the compositions, methods, and results according to the subject matter of the present disclosure.These examples are not intended to be illustrative of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results.These examples are not intended to exclude the equivalents and variants of the present invention that are obvious to those skilled in the art.

[0071] Example 1. Measurement of sTNFR2 release by cultured UC-MSCs One embodiment of the present disclosure is a method for measuring sTNFR2 release by cultured UC-MSCs in the presence or absence of inflammatory mediators through normalized ELISA quantification and inflammatory stimulation index (ISI).This is a representative method, and variations in the above method are within the scope of the present disclosure.

[0072] Mesenchymal stem cells (MSCs) originate from human embryos and are considered adult multipotent stem cells. MSCs are a heterogeneous subset of stromal stem cells that can be isolated from bone marrow, mobilized peripheral blood, umbilical cord blood, umbilical cord (UC), placenta, adipose tissue, dental pulp, and even fetal liver and lung. UC contains two umbilical arteries (UCA) and one umbilical vein (UCV), both of which are embedded in a specific mucous-like connective tissue known as Wharton's jelly (WJ), which is covered by amniotic epithelium. UCs are considered medical waste and are collected in a non-invasive manner. Moreover, access to UCs has never been hindered by ethical issues. UC-MSCs are similar to MSCs derived from other sources and have a distinct capacity for self-renewal while maintaining their multipotency, i.e., the ability to differentiate into adipocytes, osteocytes, chondrocytes, neurons, and hepatocytes, although some differentiation capacity is known to be partial. Furthermore, MSCs have been proposed as a therapeutic modality due to their potent immunomodulatory, anti-inflammatory, and reparative properties.

[0073] UC-MSC treatment was observed to be associated with significant clinical benefits, including significantly improved survival, survival free of severe adverse events, and time to recovery in patients with COVID-19 acute respiratory distress syndrome (ARDS). Six days after infusion, UC-MSC recipients were shown to produce significantly increased levels of plasma soluble TNF receptor 2 (sTNFR2) and significantly decreased levels of TNFα and TNFβ compared to controls. These observations suggested that sTNFR2 plays a mechanistic role in mediating UC-MSC effects on TNFα and TNFβ plasma levels, which determine reduced inflammation in COVID-19 ARDS.

[0074] This study provides measurement of sTNFR2 (a central mediator of the anti-inflammatory effects of UC-MSC treatment) released by cultured UC-MSCs, via normalized ELISA quantification and the inflammatory stimulation index (ISI), in the presence or absence of inflammatory mediators.

[0075] definition A. GM: Growth medium B. IIM: Inflammation-inducing medium C. ISI: Inflammatory stimulation index D. MSC: Mesenchymal stem cell E. Potency: The specific ability or capacity of a product to produce a given result. F. SOP: Standard Operating Procedure G. sTNFR2: Soluble TNF receptor 2 H. UC-MSC: Umbilical Cord-derived Mesenchymal Stem Cells I. WR: working reagents.

[0076] Equipment and Materials A. Equipment: 1. Biological Safety Cabinet (BSC) 2. Centrifuge 3. Hemocytometer and coverslips 4. Incubator 5. Micropipettes (2-20 μl, 20-200 μl, 100-1000 μl) 6. Microplate reader (SpectraMax® iD3, Molecular Devices) 7. Microscope 8. Multichannel Pipettes 9. Pipette Aid 10. Refrigerator 11. Timer B. Supplies: 1. 1.5ml microcentrifuge tube 2. 15mL conical polypropylene tube 3. 25cm with vent cap 2 Rectangular Slanted Neck Cell Culture Flask 4. 96-well plates 5. Face masks 6. Gauze 7. Gloves (non-sterile) 8. Gloves (sterile) 9. Micropipette tips, sterile: 2-200μl and 100-1000μl 10. Serological pipettes, sterile: 5ml, 10ml, 25ml, 50ml C. Reagents: 1. CTS TM TrypLE TM Select Enzyme, cGMP Grade, ThermoFisher Scientific 2. DMEM, low glucose, pyruvate, no glutamine, no phenol red, cGMP grade, ThermoFisher Scientific 3. GlutaMAX 100X, cGMP grade, ThermoFisher Scientific 4. IFNγ, in vitro use, R&D Systems 5. MEM-NEAA 100×, cGMP grade, ThermoFisher Scientific 6. Micro BCA Protein Assay Kit for in vitro use, ThermoFisher Scientific 7. PLTGold, cGMP Grade, Mill Creek 8. RIPA Lysis and Extraction Buffer for In Vitro Use, ThermoFisher Scientific 9. Soluble TNF receptor 2 human, ELISA KIT, for in vitro use, Abcam 10. TNFα, in vitro use, R&D Systems 11. Human TNF-RII (soluble) recombinant protein, for in vitro use, ThermoFisher Scientific D. Method: 1. UC-MSC potency assay: batch production records. 2. Media and reagent preparation: Inflammation-inducing medium and soluble TNF receptor 2 human reagent 3. Standard curve preparation: Soluble TNF receptor 2 human standard and diluted albumin (BSA) standard [Table 1] [Table 2] [Table 3]

[0077] Further considerations A. In one embodiment, at least two trained and qualified cGMP personnel should be present throughout this procedure. In one embodiment, all calculation procedures should be verified by a second staff member present. B. In one embodiment, the manufacturing process described herein involves multiple steps and takes six days to complete. cGMP facility personnel involved in product manufacturing should provide documentation at each step of the process. C. In one embodiment, cell seeding and expansion should be performed using sterile / aseptic techniques, which includes working in a biosafety cabinet (BSC). D. In one embodiment, the manufacturer, lot number and expiration date of all supplies, media and reagents used in this procedure should be recorded. E. In one embodiment, all samples and reagents from the sTNFR2 Human ELISA KIT are allowed to come to room temperature (18-25° C.) prior to use. F. In one embodiment, opened microplate wells or reagents may be stored at -20°C for up to one month.

[0078] procedure A. Media, Reagents, and Standard Preparation 1. Medium preparation 1.1 Growth medium (GM) To prepare one bottle of medium, add the following reagents in the order shown: a. 1000ml DMEM b. 54ml PLTGoldTM (5% PLTGold) c. 10.8ml GlutaMAX Supplement 200mM d. 10.8 ml MEM-NEAA, with the following to make the medium 10 mM: Culture medium name · Preparation date Expiration date (14 days after preparation) Initials of the cGMP facility personnel who will prepare the media 1.2 Inflammation-inducing medium (IIM) NOTE: Prepare medium prior to use on day 3. To prepare 45 ml of IIM, add the following reagents in the order shown: a. 44.5ml GM b. 225μl IFNγ (final concentration: 10ng / ml) c. 337.5μl TNFα (final concentration: 15ng / ml) in medium with: Culture medium name · Preparation date Use fresh produce Initials of the cGMP facility personnel who will prepare the media 2. Reagent preparation (Soluble TNF receptor 2 human ELISA kit) [Table 4] 3. Preparation of soluble TNF receptor 2 human standard NOTE: Prepare serially diluted standards immediately prior to use according to the manufacturer's recommendations. Always prepare a fresh set of standards for each use. 3.1 Reconstitute only one vial (1 vial / standard curve) 3.2 Briefly centrifuge the vial of sTNFR2 standard. Prepare a 50 ng / ml stock standard by adding 400 μl of 1× Assay Diluent B to the vial. Mix thoroughly and gently. 3.3 Label tubes #1-7 for serial standard dilutions with dilution factors of 1-3. 3.4 Prepare Standard #1 by adding 40 μl of 50 ng / ml Stock Standard to 960 μl of 1× Assay Diluent B in tube #1. Mix thoroughly and gently. 3.5 Pipette 400 μl of 1× Assay Diluent B into the remaining tubes. 3.6 Prepare standard #2 by adding 200 μl standard #1 to tube #2 and mix thoroughly. 3.7 Prepare standard #3 by adding 200 μl standard #2 to tube #3 and mix thoroughly. 3.8 Using Figure 2 as a guide, prepare further serial dilutions (see Table 5). 3.9 1x Assay Diluent B (Tube 8: Negative Control) serves as the zero standard (0 μg / ml) (Figure 1). [Table 5] 4. Preparation of Dilute Albumin (BSA) Standards Each 1 mL ampoule of the 2.0 mg / mL albumin standard is sufficient to prepare a series of diluted standards so that three replicates of each dilution can be included in the test tube procedure. A fresh series of standards should be prepared for each use. 4.1 Prepare a series of protein standards using distilled water. Use Table 6 as a guide. [Table 6] 4.2 Determine the total volume of Working Reagent (WR) needed using the following formula: [(# standards x 2 replicates) + (# unknowns x 3 replicates)] x (150 μl WR) = total volume of WR required 4.3 Prepare the required WR volume by mixing 25 parts Micro BCA Reagent A (MA) and 24 parts Reagent B (MB) with 1 part Micro BCA Reagent C (MC) (25:24:1). Note: WR is stable for 1 day when stored in a closed container at room temperature. B. Cell Seeding and Expansion 1. Thawing of UC-MSC vial or segment(s) and cell expansion 1.1 Retrieve 1 vial or 2–3 segments containing UC-MSCs from frozen storage. 1.2 Place the sample in a specimen bag and thaw it quickly by gently swirling it in a 37°C water bath (until small ice crystals remain in the vial or segment(s). Handle the cells gently to avoid mechanical damage. This should take no more than 3 minutes. 1.3 Once the sample has thawed, wipe it down using a sterile alcohol prep pad. 1.4 Place the sample inside the BSC and slowly transfer the cells with a syringe into a sterile 50 ml conical tube. 1.5 Slowly, in a dropwise manner, add 10 ml of room temperature GM to the same 50 mL conical tube. 1.6 Centrifuge the cells at 500 x g for 10 minutes at room temperature with the brake set on "high". 1.7 Remove the cells from the centrifuge and gently aspirate off the supernatant. 1.8 Resuspend the pellet in 10 ml of GM and equilibrate to room temperature. 1.9 Cell count and viability (Perform a "Viability test using Trypan Blue or Propidium Iodide (PI) and Thiazole Orange (TO)" and record the results. The trypan blue viability test is based on the ability of the cell membrane of live (viable) cells to exclude the dye and for non-viable cells to take up the dye. Cells are evaluated under a microscope and a count of both viable and non-viable cells is performed to determine percent viability. Trypan blue should be filtered before use. Cells should be counted immediately after adding the dye. Viable cells will begin to take up the dye within 2-3 minutes, resulting in low viability. A. Reagents 0.4% Trypan Blue (filtered), Sigma or equivalent Phosphate Buffered Saline (PBS), MediaTech or equivalent 70% ethanol b. Procedure Viability in fresh cells 1. The following procedure is for freshly prepared cells in suspension. 2. Be sure to thoroughly clean the hemocytometer and cover slips and allow to air dry. Use 70% alcohol and lint-free wipes. 3. Place the coverslip onto the hemocytometer so that it partially covers both V-notches. 4. Using a micropipette, mix the fresh cells thoroughly and place 100 μl into a small tube. 5. Add 100 μl of 0.4% Trypan Blue. If the cell product is too thick, dilute with PBS and mix thoroughly before adding Trypan Blue. 6. Load the hemocytometer as follows: Using a pipette, place a drop of the cell / trypan blue mixture into one of the V-notches, between the cover slip and the counting chamber, without disturbing the cover slip. Slowly fill the area completely and remove the pipette before the solution spills over the edges of the chamber section. b. Don't overfill or underfill: If you have overloaded the chamber or air bubbles are visible, do not attempt to remove the excess liquid; clean the chamber and start again. 7. Rotate to a 4x objective and carefully place the hemocytometer on the microscope stage. 8. Count cells after approximately 1 minute of incubation but before 3 minutes. After 2-3 minutes of incubation, viable cells may begin to take up the dye, resulting in inappropriate counts. 9. Dead cells appear blue after taking up the dye while viable cells remain unstained. 10. Count the number of stained versus unstained nucleated cells in a total of 100 random cells. These counts can be done in any of the quadrants numbered 1-5 in Figure 8. 11. Record the data. 12. Calculate percent survival using the following formula: % Viable cells = Number of viable cells x 100 Total cell count Viability of thawed cells 1. Thaw the cryopreserved cells quickly. 2. Follow steps #2-#12 from the Viability in Fresh Cells procedure. 1.10 500,000 viable cells / flask (20.000 cells / cm) resuspended in 5 mL GM / flask 2) at a concentration of 6 x 25 cm 2 Seed in flasks (3 flasks per condition: "basal" and "inflammation-induced"). 1.11 The above cells are cultured in GM for 72 hours (3 days) in a 37°C, 5% CO2 incubator. C. Inflammation induction 1. Inflammation induction (TNFα / IFNγ) 1.1 Prepare IIM and warm it to 37°C before use. 1.2 Remove all medium from the flask. 1.3 Add 5 ml of pre-warmed GM to one flask and mark that flask as "Basal." 1.4 Add 5 ml of pre-warmed IIM to the other flask and mark that flask as "Inflammation Induced." 1.5 Return the tissue culture flasks to a 37°C incubator with 5% CO2 for 3 days. D. Potency Assays After day 3 of induction: 1. sTNFR2 Measurement NOTE: It is recommended that all standards be assayed in duplicate and samples in triplicate. The 96-well plate strips included in the kit are supplied ready to use. 1.1 Collect the culture supernatant into a 15 ml tube. 1.2 The supernatant is centrifuged at 1,500 rpm for 5 minutes to remove debris. 1.3 Gently pour off the supernatant into a new 15ml tube. 1.4 Dilute the supernatant 5-fold with 1x Assay Diluent B from the sTNFR2 Human ELISA Kit (Abcam) (Reagent Preparation, Section V.A.2). 1.5 Add 100 μL of each standard in duplicate (see Standard Preparation V, A, section 3) and each sample (UC-MSC samples, TNFR2 standards, media) in triplicate to the appropriate wells (Figure 2). Cover wells and incubate overnight at 4°C with gentle shaking. 1.5.1 TNFR2 standards: a) H is defined as high concentration (H b) M is defined as the medium concentration (M c) L is defined as the low concentration (L 1.5.2 Blank medium: a) GM b) IIM 1.5.3 Supernatant (Test Sample): a) Supernatant GM (sGM) b) Supernatant IIM (sIIM) 1.6 The next day, discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with 1x Wash Solution (300 μL) using a multichannel pipette or an automated washer. After the last wash, remove any remaining wash buffer by decanting. Invert the plate and blot on clean paper towels. 1.7 Add 100 μL of 1× Biotinylated Soluble TNF Receptor 2 Detection Antibody (Reagent Preparation, Section V.A.2) to each well. Incubate for 1 hour at room temperature with gentle shaking. 1.8 Discard the solution. Repeat the wash as in step 1.6. 1.9 Add 100 μL of 1× HRP-Streptavidin Solution to each well. Incubate for 45 minutes at room temperature with gentle shaking. 1.10 Discard the solution. Repeat the wash as in step 1.6. 1.11 Add 100 μL of TMB 1-Step Substrate Reagent to each well. Incubate for 30 minutes at room temperature with gentle shaking and protected from light. 1.12 Add 50 μL of Stop Solution to each well. 1.13 Load the plate into the SpectraMax® iD3 and immediately read the plate at 450 nm. 2. Protein quantification NOTE: sTNFR2 secretion is normalized to the cell lysate total protein content, which is quantified with the "MicroBCA Protein Assay Kit" (ThermoFisher Scientific). 2.1 Protein lysates a. Briefly, after removing the supernatant, the cell monolayer is washed twice with 5 ml of cold DPBS. b. Add 1 ml of cold RIPA buffer (4°C) to each 25 cm 2 Add to flask with a micropipette. c. Incubate the flask at 4° C. for 5 minutes. d. Cell lysates are collected and centrifuged at 10,000 rpm for 5 minutes to remove cell debris. e. After centrifugation, the cell lysate is transferred to a new microcentrifuge tube and further processed for protein quantification. 2.2 Protein quantification using the Micro BCA Protein Assay a. Prepare a bovine serum albumin standard curve (0-200 μg / ml) as described herein. b. Dilute the cell lysate 1:100 in distilled water. c. Add 150 μL of each standard in duplicate or 150 μL of diluted cell lysate in triplicate to a 96-well plate. d. Add 150 μL of WR (25:24:1, Reagents MA:MB:MC) to each well. e. Mix thoroughly on a plate shaker for 30 seconds. f. Incubate the plate at 37° C. for 2 hours. g. Measure the absorbance at 562 nm in a microplate reader. E. Analysis 1. Assays are performed in triplicate sets of induced and non-induced flasks and results are analyzed using SoftMax Pro Software. 2. sTNFR2 ELISA: 2.1 Calculate the sTNFR2 concentration of each sample using the assay standard curve (0-2,000 µg / ml) fitted to a four-parametric logistic regression. 2.2 Average the values ​​of triplicate wells of the two blank medium samples and the six test samples (3x induced and 3x uninduced) to obtain the average sTNFR2 concentration for each blank / flask. 2.3 There should be a total of eight averages (one for each flask and one for each blank medium). 3. Total Protein BCA Assay: 3.1 Calculate cell lysate total protein concentration for each sample using a linear regression fitted Micro BCA Protein Assay standard curve. 3.2 Average the values ​​of triplicate wells of the six test samples (3x induced and 3x uninduced) to obtain the average total protein for each flask. 3.3 There should be a total of 6 averages (one for each flask). 4. Normalized sTNFR2 calculation: 4.1 For all flasks (both UC-MSC basal and UC-MSC inflammation-induced groups), sTNFR2 concentrations are normalized by dividing each flask's sTNFR2 value by its corresponding total protein concentration value. 4.2 The normalized sTNFR2 in basal culture conditions is then calculated by averaging the UC-MSC basal normalized sTNFR2 concentrations. The resulting value should be ≧0.2 (pg / mL) / (μg of total protein). 5. Inflammatory stimulation index (ISI) calculation: 5.1 ISI is calculated for each set of flasks as the ratio of induced normalized sTNFR2 concentration to uninduced normalized sTNFR2 concentration. 5.2 Average the three ISIs and the resulting value should be ≧1.5.

[0079] Note A. Total cell protein content is calculated based on the volume of cell lysate used (RIPA, 1.5 ml) and the dilution factor (1:100).

[0080] quality control A. Media samples (growth medium and inflammation-inducing medium) are analyzed to assess the potential for false positive results for sTNFR2 quantification. B. External sTNFR2 standards with concentrations spanning the range across the standard curve are also run to ensure the ELISA kit is functioning properly. These values ​​should be within 20% of the theoretical concentration. C. The %CV of induced and uninduced ELISA replicates should be ≦20% to ensure accurate results. If the %CV is >20%, one value may be dismissed as an outlier. D. The standard curve should be fitted with a four-parameter logistic regression and should have an R2 value of > 0.98 to be considered acceptable. If the R2 value is < 0.98, the value can be dismissed as an outlier as long as there is at least one value for each concentration of the standard. E. All equipment used should be maintained.

[0081] Example 2. In vitro potency assay for immunomodulatory cells based on analysis of soluble tumor necrosis factor receptor 2 (sTNFR2) release The in vitro potency assay for immunomodulatory cells was based on analysis of soluble tumor necrosis factor receptor 2 (sTNFR2) release. This is a representative assay and variations in the assay protocol are within the scope of this disclosure.

[0082] The studies herein have been qualified by developing a biologically relevant in vitro assay to determine the potency of immune-modulating cells (e.g., mesenchymal stem cells). The potency assay focuses on the measurement of soluble tumor necrosis factor receptor 2 (sTNFR2) released by the cells. It is based on sTNFR2 quantification via ELISA, normalized to cellular protein content and calculation of the inflammatory stimulation index (ISI) of sTNFR2 released by the cells. ISI is calculated as the ratio of sTNFR2 release in inflammatory induction to basal conditions. The assay is performed on cells cultured in vitro. In one embodiment, the cells to be analyzed in the assay are umbilical cord-derived mesenchymal stem cells (UC-MSCs). The basal conditions correspond to culturing the cells in the same medium utilized for the production of the final cell product. The inflammatory induction is obtained by adding TNFα (15 ng / mL) and IFNγ (10 ng / mL) to the medium of these cultures. The cells are maintained under basal conditions or under inflammatory induction for a certain amount of time in culture, and in a preferred embodiment, for 3 days in culture.The supernatant is then collected and tested with a commercially available kit for sTNFR2 quantification (e.g., Abcam Soluble TNFR2 Human ELISA KIT, Abcam, catalog number ab100643).For normalization based on total cell protein content, the cells are lysed with RIPA lysis and extraction buffer (e.g., ThermoFisher Scientific, catalog number 89900), and protein content is obtained by BCA method (e.g., Micro BCA Protein Assay Kit (ThermoFisher Scientific, catalog number 23235)).The assay is described in Figure 3.The SOP for the assay is provided in Example 1 above herein.

[0083] Potency Assay Data The method described in Example 1 was applied using UC-MSCs thawed from the stage of "UC-MSC final product (batch, cryopreserved)" manufactured at the Diabetes Research Institute cGMP facility. From the experiment, sTNFR2 release by UC-MSCs over 3 days under basal culture conditions was quantified in response to inflammation (TNFα / IFNγ) induction. The results are shown in Table 7, Figure 4, and Figure 5. [Table 7]

[0084] Biological relevance of patient-related assays Hyperinflammatory response in COVID 19 patients with acute respiratory distress syndrome (ARDS) is characterized by high serum levels of proinflammatory mediators, including tumor necrosis factor (TNF) α and β. These two molecules, involved in ARDS pathophysiology, bind to TNFR2. TNFR2 soluble form was reported to have an inhibitory effect on TNF function. The study herein investigated the plasma levels of TNFα, TNFβ, and soluble TNFR2 (sTNFR2) in both UC-MSC-treated and control groups in a phase l / 2a clinical trial for COVID-19 ARDS. sTNFR2 was increased in UC-MSC-treated patients compared to control patients at day 6 (see Figure 6). TNFα and TNFβ were found to be decreased at day 6. The observations are shown in Figure 6.

[0085] Methods (observations in patients) Blood samples were obtained from randomized subjects of the clinical trial on day 0 (before infusion) and day 6 (3 days after the second infusion). Briefly, whole blood was collected into EDTA-treated tubes, transferred onto ice, and processed for plasma separation within 2 hours. Whole blood was centrifuged at 2,000g for 15 minutes at 4°C, and plasma was collected and stored at -80°C until processing. Quantitative multiplex protein arrays (RayBio® Q-Series, RayBiotech) were utilized to simultaneously determine the plasma levels (pg / ml) of TNFR2, TNFα, and TNFβ in all samples according to the manufacturer's instructions. Fluorescent signals were visualized via a Cy3 wavelength laser scanner and converted to concentrations using standard curves generated for each array.

[0086] Statistical analysis was performed using two-sample T-tests and non-parametric Wilcoxon two-sample tests. Matched rank tests were used for pairwise comparisons examining changes between time points within groups. All tests were two-sided, and statistical significance was established at p<0.05. Data are presented as mean and standard error of the mean.

[0087] Results (observations in patients) Patients in the UC-MSC and control groups showed no significant differences in baseline protein levels. In the control group, sTNFR2, TNFα and TNFβ levels were not significantly different between days 0 and 6. TNFα and TNFβ levels were significantly decreased between days 0 and 6 (p=0.005 and p=0.002, respectively). Comparison between groups on day 6 clearly showed significantly lower levels in the UC-MSC group compared to the control group for TNFα (319±40 vs. 950±226pg / ml, p=0.048) and TNFβ (810±126 vs. 2,944±735pg / ml, p=0.032). sTNFR2 showed significantly higher levels in the UC-MSC group compared to the control on day 6 (26,609±846 pg / ml vs. 23,111±760 pg / ml, p=0.021). See FIG. 6.

[0088] In a recently completed phase l / 2a clinical trial, UC-MSC treatment was associated with accelerated clinical recovery in patients with COVID-19 ARDS. Molecular evidence of differences in key underlying immune / inflammatory mediator axes that help explain these results is provided herein. On day 6, UC-MSC recipients had significantly elevated levels of plasma sTNFR2 and significantly decreased levels of TNFα and TNFβ compared to controls. TNF receptor-based drugs have been tested to treat chronic inflammatory diseases and may be similarly beneficial for hyperinflammation attenuation in severe COVID-19 patients. TNF blockade is clinically effective as it results in rapid reduction (<12 hours) of circulating interleukin (IL)-1 and IL-6 levels, as well as reduction of adhesion molecules and vascular endothelial growth factor (VEGF), which strongly affect leukocyte trafficking and capillary permeability in inflamed tissues. Studies have shown that upon anti-TNF treatment, TNF concentrations in inflamed tissues were reduced as it bound to anti-TNF antibodies and passed into the blood circulation.

[0089] In addition, sTNFR2 can bind TNF, neutralize TNF-induced cytotoxicity and immune reactivity, and modulate inflammatory responses. For example, higher sTNFR2 levels result in reduced T cell activation and progressive generation of regulatory T cells (Tregs). Based on this, studies have shown that the expression of TNFR2 by MSCs correlates with their higher Foxp3+ Treg induction ability. Thus, the findings herein indicate an important mechanism of UC-MSC effects, whereas sTNFR2 plasma levels may be a predictor of COVID-19 ARDS progression and clinical outcomes after treatment.

[0090] Now, based on the observations in patients described above, and based on the observations of the efficacy assays described in the previous paragraph, the efficacy assays developed herein are biologically meaningful.

[0091] Guaranteed Efficacy This potency assay, normalized quantification and measurement of soluble TNFR2 release via the Inflammatory Stimulation Index (ISI) will be utilized to ensure the potency of the product to be used in the proposed Phase 2b / 3 study. The criteria for ensuring the potency of each batch of UC-MSC utilized for Phase 2b / 3 study are as follows: Soluble TNFR2 (sTNFR2) release normalized by total cellular protein content >0.01 (pg / mL) / (μg) over 3 days of culture · Inflammatory irritation index (ISI)>1 In one embodiment, soluble TNFR2 (sTNFR2) release normalized by total cellular protein content >0.02 (pg / mL) / (μg) over a 3-day culture in basal conditions · Inflammatory irritation index (ISI)>1.5 In one embodiment, soluble TNFR2 (sTNFR2) release normalized by total cellular protein content >0.03 (pg / mL) / (μg) over a 3-day culture period under inflammatory induction · Inflammatory irritation index (ISI)>1.5

[0092] The present disclosure addresses the need for efficacy assays for immunomodulatory cells (or their secretomes) that modify tumor necrosis factor (TNF) inflammatory pathways in humans. Types of immunomodulatory cells include mesenchymal stem cells, mesenchymal stromal cells, and medicinal signaling cells (MSCs).

[0093] This in vitro potency assay is based on the measurement of soluble tumor necrosis factor receptor 2 (sTNFR2) release in vitro under basal or inflammatory conditions to estimate immune-modulatory function in vivo. Currently, the safety and efficacy of MSC-based therapies are being investigated in many clinical trials for a variety of disorders, including inflammatory, immune, autoimmune, musculoskeletal, cardiovascular, neurodegenerative, and gastrointestinal diseases. However, early results from many of these trials have revealed that these cell therapies have a significant degree of variability and may not be reproducible in clinical observations. Most importantly, the inconsistent evidence is potentially related to intrinsic differences in the cell-based products used, including the lack of standardized characteristics of the preparations that are reflected in the inconsistencies in efficacy. Thus, a rapid and accurate method to a priori qualitatively evaluate MSC batches in vitro for effective immune-modulatory effects in vivo is presented herein.

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated herein by reference.

[0095] Those skilled in the art will recognize that many changes and modifications may be made to the preferred embodiments of the present invention, and that such changes and modifications may be made without departing from the spirit of the present invention. It is therefore intended that the appended claims encompassing all such equivalent variations be within the true spirit and scope of the present invention.

Claims

1. 1. A method for testing the immunomodulatory activity of a plurality of cells, the method comprising: a. separating the plurality of cells into a first population of cells and a second population of cells; b. culturing a first population of said cells in basal conditions and a second population of said cells in inflammatory conditions; c. harvesting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells of the first group of cells, and the cells of the second group of cells; d. determining the level of soluble tumor necrosis factor receptor 2 (TNFR2) protein in the culture supernatant of the first group of cells and in the culture supernatant of the second group of cells; e. normalizing the levels of soluble TNFR2 protein in the first group of cells and the second group of cells to the total protein levels in the first group of cells and the second group of cells, respectively, collected in step c; f. calculating an inflammatory stimulation index (ISI) by dividing the normalized level of soluble TNFR2 protein in the second group of cells by the normalized level of soluble TNFR2 protein in the first group of cells; and g. determining that the plurality of cells has immunomodulatory activity if the ISI is greater than 1; A method that encompasses

2. The method of claim 1 , wherein the plurality of cells comprises human mesenchymal stem cells, mesenchymal stromal cells, or medical signaling cells.

3. 2. The cells of claim 1, wherein the plurality of cells are derived from postnatal adipose tissue, infrapatellar fat pad, postnatal bone marrow, postnatal endometrium, perinatal umbilical cord, perinatal chorion, perinatal amnion, or perinatal placenta.

4. 10. The method of claim 1, wherein the inflammatory condition comprises the presence of tumor necrosis factor alpha (TNFα) or interferon gamma (IFNγ).

5. 5. The method of claim 4, wherein the inflammatory condition further comprises the presence of TNFβ, IL-1β, or connective tissue growth factor (CTGF).

6. A composition for treating COVID-19-associated acute respiratory distress syndrome (ARDS) in a subject in need thereof, comprising a plurality of cells, wherein the plurality of cells have been determined to have immunomodulatory activity using a method according to any one of claims 1 to 5.

7. 7. The composition of claim 6, wherein the plurality of cells are determined to have an inflammatory stimulation index (ISI) greater than 1.

8. The composition described in claim 6, characterized in that the composition is administered to the subject in combination with an anti-COVID therapeutic agent.

9. A composition for treating at least one of an inflammatory disorder or a fibrotic condition in a subject in need thereof, comprising a plurality of cells, wherein the cells have been determined to have immunomodulatory activity using a method according to any one of claims 1 to 5.

10. 10. The composition of claim 9, wherein the plurality of cells are determined to have an inflammatory stimulation index (ISI) greater than 1.

11. The composition described in claim 9, characterized in that the composition is administered to the subject in combination with an anti-COVID therapeutic agent.

12. A composition for treating an inflammatory condition in a subject in need thereof, comprising a plurality of cells, wherein the cells have been determined to have immunomodulatory activity using a method according to any one of claims 1 to 5.

13. 13. The composition of claim 12, wherein the plurality of cells are determined to have an inflammatory stimulation index (ISI) greater than 1.

14. The composition of claim 12, wherein the composition is administered to the subject in combination with an anti-COVID therapeutic agent.

15. A composition for treating a fibrotic condition characterized in a subject in need thereof, comprising a plurality of cells, wherein the cells have been determined to have immunomodulatory activity using a method according to any one of claims 1 to 5.

16. 16. The composition of claim 15, wherein the plurality of cells are determined to have an inflammatory stimulation index (ISI) greater than 1.

17. The composition of claim 15, wherein the composition is administered to the subject in combination with an anti-COVID therapeutic agent.

18. A composition for treating a condition characterized by increased tumor necrosis factor (TNF) in a subject in need thereof, comprising a plurality of cells, wherein the cells have been determined to have immunomodulatory activity using a method according to any one of claims 1 to 5.

19. 20. The composition of claim 18, wherein the plurality of cells are determined to have an inflammatory stimulation index (ISI) greater than 1.

20. The composition of claim 18, wherein the composition is administered to the subject in combination with an anti-COVID therapeutic agent.

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