Methods of analyzing soluble tumor necrosis factor receptor 2 (STNFR2) and uses thereof

Abstract

The present disclosure relates to methods of testing the immunomodulatory activity of cells, including, for example, mesenchymal stem cells, and the use of the cells determined to have immunomodulatory activity for the treatment of COVID-19 related acute respiratory distress syndrome (ARDS). Disclosed herein are in vitro methods of assessing the effective immunomodulatory effect of mesenchymal stem cells in vivo.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 303,585, filed on January 27, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to methods for determining the immunomodulatory activity of cells and their use in treating inflammatory diseases. Background Art

[0004] The safety and efficacy of mesenchymal stem cell (MSC)-based therapies are being investigated in multiple clinical trials for various conditions, including inflammatory, immune, autoimmune, musculoskeletal, cardiovascular, neurodegenerative, and gastrointestinal diseases. However, preliminary results from many such studies have revealed that these cell therapies have a high degree of variability, with cases of non-reproducibility in clinical observations.

[0005] What is needed is a novel in vitro method for assessing the effective immunomodulatory role of MSCs in vivo. Summary of the Invention

[0006] One embodiment of the present disclosure is a method for testing the immunomodulatory activity of multiple cells, the methods comprising

[0007] a. dividing the multiple cells into a first group of cells and a second group of cells;

[0008] b. culturing the first group of cells under basal conditions and culturing the second group of cells under inflammatory conditions;

[0009] c. collecting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells in the first group of cells, and the cells in the second group of cells;

[0010] d. determining the levels of soluble TNFR2 protein in the culture supernatant of the first group of cells and the culture supernatant of the second group of cells;

[0011] e. normalizing the levels of soluble TNFR2 protein in the first group of cells and the second group of cells with the total protein levels of the corresponding first group of cells and second group of cells collected in step c;

[0012] 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

[0013] g. If the ISI is higher than 1, it is determined that the plurality of cells are responsive to an inflammatory stimulus and have immunomodulatory activity.

[0014] In some embodiments, the plurality of cells include human mesenchymal stem cells or mesenchymal stromal cells or medical signaling cells. In some embodiments, the cells are derived from postpartum adipose tissue, infrapatellar fat pad, postpartum bone marrow, postpartum endometrium, perinatal umbilical cord, perinatal chorion, perinatal amnion, or perinatal placenta.

[0015] In some embodiments, the inflammatory condition includes the presence of TNFα and / or IFNγ. In some embodiments, the inflammatory condition further includes the presence of TNFβ, IL-1β, or connective tissue growth factor (CTGF).

[0016] One embodiment of the present disclosure is a method for treating COVID-19 related acute respiratory distress syndrome (ARDS) in a subject in need thereof, the method comprising administering to the subject a plurality of cells, which are determined to have immunomodulatory activity using the method of any of the foregoing aspects. In some embodiments, the plurality of cells having immunomodulatory activity are determined to have an inflammatory stimulus index (ISI) higher than 1.

[0017] One embodiment of the present disclosure is a method for treating an inflammatory disorder and / or fibrosis in a subject in need thereof, the method comprising administering to the subject a plurality of cells, which are determined to have immunomodulatory activity using the method of any of the foregoing aspects. In some embodiments, the plurality of cells having immunomodulatory activity are determined to have an inflammatory stimulus index (ISI) higher than 1.

[0018] One embodiment of the present disclosure is a method for treating an inflammatory condition in a subject in need thereof, the method comprising administering to the subject a plurality of cells, which are determined to have immunomodulatory activity using the method of any of the foregoing aspects. In some embodiments, the plurality of cells having immunomodulatory activity are determined to have an inflammatory stimulus index (ISI) higher than 1.

[0019] One embodiment of the present disclosure is a method for treating a fibrotic condition characterized in a subject in need thereof, the method comprising administering to the subject a plurality of cells, which are determined to have immunomodulatory activity using the method of any of the foregoing aspects. In some embodiments, the plurality of cells having immunomodulatory activity are determined to have an inflammatory stimulus index (ISI) higher than 1.

[0020] One embodiment of the present disclosure is a method of treating a condition in a subject in need thereof, characterized by an increase in TNF, the method comprising administering to the subject a plurality of cells, which are determined to have immunomodulatory activity using the method of any of the foregoing aspects. In some embodiments, the plurality of cells having immunomodulatory activity are determined to have an inflammatory stimulation index (ISI) greater than 1.

[0021] In some embodiments, the method of any of the foregoing aspects further comprises administering to the subject a therapeutically effective amount of an anti-COVID therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0023] Figure 1 Shows the steps for preparing a standard dilution.

[0024] Figure 2 Shows the layout for preparing samples and standards in a 96-well plate (see Table 5).

[0025] Figure 3 Shows a schematic diagram of measuring soluble TNFR2 release by normalized quantification and inflammatory stimulation index (ISI).

[0026] Figure 4 Shows the soluble TNFR2 (sTNFR2) release of UC-MSC under basal culture conditions for 3 days relative to inflammatory induction.

[0027] Figure 5 Shows the inflammatory stimulation index (ISI) of sTNFR2 release of UC-MSC, calculated as the ratio of sTNFR2 release under inflammatory conditions to sTNFR2 release under basal conditions.

[0028] Figure 6 Shows the observations of patients. Plasma concentrations of soluble tumor necrosis factor receptor 2 (sTNFR2), tumor necrosis factor α (TNFα), and tumor necrosis factor β (TNFβ) in subjects (n = 24) with COVID-19 acute respiratory distress syndrome (ARDS). On day 6, compared to the control, UC-MSC recipients had significantly elevated plasma sTNFR2 levels and significantly reduced TNFα and TNFβ levels. The data are presented as box plots indicating the median and minimum to maximum values and as scatter plots in which the lines indicate individual values.

[0029] Figure 7Shows the sTNFR2 inflammatory mimic index (ISI) of UC-MSC preparations from different donors and at different culture passages. The ISI values were obtained by a static sTNFR2 release assay. The histogram depicts the mean sTNFR2 ISI from triplicate tests; error bars indicate 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.

[0030] Figure 8 Shows a diagram for counting cells for the trypan blue viability test. Detailed Description

[0031] Reference will now be made in detail to embodiments of the invention, examples of which are illustrated in the accompanying drawings and examples. However, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0032] 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 disclosure belongs. The terms "comprising" and variations thereof as used herein are used synonymously with the terms "including" and variations thereof, and are open-ended and non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments and are also disclosed. The singular forms "a / an" and "the" as used in this disclosure and the appended claims include plural referents unless the context clearly indicates otherwise.

[0033] The following definitions are provided to facilitate a full understanding of the terms used in this specification.

[0034] The term

[0035] As used herein, the term "about", when referring to measurable values such as amounts, percentages, etc., is intended to cover variations of ±20%, ±10%, ±5% or ±1% of the measurable value.

[0036] "Administration" or "administering" to a subject encompasses any route that involves introducing or delivering an agent to the subject. Administration can be carried out via any suitable route, including oral, intravenous, intraperitoneal, intranasal, inhalation, etc. Administration includes self - administration and administration by others.

[0037] A "control" is an alternative subject or sample for comparison purposes in an experiment. The control can be "positive" or "negative".

[0038] As used herein, the terms "increased" or "increase" generally mean an increase in a statistically significant amount; for example, "increased" means an increase of at least 10% compared to a reference level, such as 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 up to and including 100% increase or any increase between 10 - 100%, 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 increase or any increase between 2 - fold and 10 - fold or more - fold compared to a reference level.

[0039] As used herein, the terms "reduced", "reduce", "reduction" or "decrease" generally mean a decrease in a statistically significant amount. However, for the sake of clarity, "reduced" means a decrease of at least 10% compared to a reference level, such as a decrease 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 up to and including 100% decrease or any decrease between 10 - 100%.

[0040] As used herein, the term "level" refers to the amount of a target molecule in a sample (e.g., a sample from a subject). The amount of the molecule can be determined by any method known in the art and will depend in part on the nature of the molecule (i.e., gene, mRNA, cDNA, protein, enzyme, etc.). Methods for quantifying nucleotides (e.g., genes, cDNA, mRNA, etc.) as well as proteins, polypeptides, enzymes, etc. are well - known in the art. It should be understood that the amount or level of the molecule in the sample does not need to be determined in absolute terms, but can be determined in relative terms (e.g., when compared to a control sample or a sham sample or an untreated sample).

[0041] As used herein, the terms "can", "optionally", and "optionally can" are used interchangeably and mean to include both the case where the condition occurs and the case where the condition does not occur. Thus, for example, the statement that a formulation "can contain an excipient" means to include both the case where the formulation contains an excipient and the case where the formulation does not contain an excipient.

[0042] As used herein, the term "subject" or "host" can 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 can be carried out in a dose and for a duration effective to treat the subject. In some embodiments, the subject is a human.

[0043] A "therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include both a therapeutic effect (e.g., treating a disorder or other undesirable physiological condition) and a prophylactic effect (e.g., preventing a disorder or other undesirable physiological condition). The term also encompasses pharmaceutically acceptable pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, etc. When the term "therapeutic agent" is used, or when a specific 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, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0044] As used herein, the terms "treat", "treating", "treatment" and their grammatical variants include partially or completely delaying, alleviating, reducing or decreasing the intensity of one or more accompanying infectious symptoms or conditions and / or alleviating, reducing or hindering one or more symptoms of COVID-19-related acute respiratory distress syndrome (ARDS). The treatment according to the present invention can be applied preventively, prophylactically, palliative or remedially. A prophylactic treatment is administered to a subject before the onset of infection (e.g., before the appearance of obvious signs of infection), during the early onset (e.g., when the initial signs and symptoms of infection appear) or after it is determined that the subject has an infection. Prophylactic administration can occur before the manifestation of the infection and continue for several minutes to several months.

[0045] Method

[0046] In some aspects, methods for testing the immunomodulatory activity of multiple cells are disclosed herein, the methods comprising

[0047] a. dividing the multiple cells into a first group of cells and a second group of cells;

[0048] b. Culture the first group of cells under basal conditions and the second group of cells under inflammatory conditions;

[0049] c. Collect the culture supernatants of the first group of cells, the culture supernatants of the second group of cells, the cells in the first group of cells, and the cells in the second group of cells;

[0050] d. Determine the levels of soluble TNFR2 protein in the culture supernatant of the first group and the culture supernatant of the second group;

[0051] e. Normalize the levels of soluble TNFR2 protein in the first group and the second group using the total protein levels of the cells of the corresponding groups collected in step c;

[0052] f. Calculate the inflammatory stimulation index (ISI) by dividing the normalized level of soluble TNFR2 protein in the second group by the normalized level of soluble TNFR2 protein in the first group; and

[0053] g. If the ISI is higher than 1, determine that the plurality of cells have immunomodulatory activity.

[0054] 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 "tumor necrosis factor receptor superfamily member 1B" or TNFRSF1B).

[0055] As used herein, the term "immunomodulatory activity" refers to the activity of reducing inflammatory activity, including, for example, the regulation of excessive inflammation (e.g., cytokine storm) or excessive immune responses. In some embodiments, the immunomodulatory activity is an anti-inflammatory effect.

[0056] In some embodiments, the plurality of cells include 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 postpartum islets or pancreatic tissue, postpartum adipose tissue, infrapatellar fat pad, postpartum bone marrow, postpartum endometrium, postpartum dental pulp, perinatal umbilical cord, perinatal chorion, perinatal amnion, or perinatal placenta. In some embodiments, the plurality of cells include mesenchymal stem cells.

[0057] As used herein, "mesenchymal stem cell" or "MSC" is a cell capable of differentiating into mesenchymal cell lineages (i.e., osteoblasts, chondrocytes, and adipocytes). These cells are identified using morphological and functional criteria well known to those of ordinary skill in the art. See Horwitz, supra; Dominici et al., supra; Trivedi P and Hematti P, "Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells", Exp. Hematol., January 5, 2008; Trivedi P and Hematti P, "Simultaneous generation of CD34+ primitive hematopoietic cells and CD56+ mesenchymal stem 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 herein by reference in its entirety as described. Mesenchymal stem cells or MSCs can be identified by their characteristic mononuclear ovoid, stellate, or spindle-shaped morphology, with a round to oval nucleus. The ovoid, elongated nucleus typically has a prominent nucleolus and is intermixed with heterochromatin and euchromatin. These cells have little cytoplasm but have many fine processes extending from the nucleus. Mesenchymal stem cells or MSCs can typically be stained for one, two, three, or more of the following markers: CD29, CD44, CD73, CD90, CD105, CD106 (VCAM), CD166 (ALCAM), and alkaline phosphatase, and are negative for hematopoietic lineage cell markers (e.g., CD14, CD34, or CD45) and endothelial lineage cell markers (e.g., CD31 and VE-cadherin). Mesenchymal stem cells or MSCs can also express STRO-1 and / or CD146 as markers.

[0058] In some embodiments, the inflammatory condition includes TNFα and / or IFNγ. In some embodiments, the inflammatory condition further includes TNFβ, IL-1β, connective tissue growth factor (CTGF). Thus, in some embodiments, the second group of cells is cultured in a cell culture medium comprising TNFα, IFNγ, TNFβ, IL-1β, or CTGF, or any combination thereof.

[0059] The cells can 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.

[0060] Also disclosed herein is a method of treating a subject in need of treatment for COVID-19 related acute respiratory distress syndrome (ARDS), 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.

[0061] Thus, in some aspects, disclosed herein is a method of treating a subject in need of treatment for COVID-19 related acute respiratory distress syndrome (ARDS), the method comprising administering to the subject a plurality of cells, wherein the cells are determined to have immunomodulatory activity using a method comprising:

[0062] a. dividing the plurality of cells into a first group of cells and a second group of cells;

[0063] b. culturing the first group of cells under basal conditions and culturing the second group of cells under inflammatory conditions;

[0064] c. collecting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells in the first group of cells, and the cells in the second group of cells;

[0065] d. determining the levels of soluble TNFR2 protein in the culture supernatant of the first group and the culture supernatant of the second group;

[0066] e. normalizing the levels of soluble TNFR2 protein in the first group and the second group with the total protein levels of the cells of the respective groups collected in step c;

[0067] f. calculating an inflammatory stimulation index (ISI) by dividing the normalized level of soluble TNFR2 protein in the second group by the normalized level of soluble TNFR2 protein in the first group; and

[0068] g. If the ISI is higher than 1, it is determined that the plurality of cells have immunomodulatory activity.

[0069] In some embodiments, the plurality of cells are determined to have an ISI higher than 1.

[0070] "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 breathing, shortness of breath, and / or low blood oxygen levels, and low blood oxygen levels can also produce a series of other symptoms, including confusion, dizziness, sweating, low blood pressure, and an increased heart rate. ARDS may be a consequence of inflammatory changes in the alveoli. In some embodiments, administering cells determined to have immunomodulatory activity using the methods disclosed herein can alleviate one or more symptoms, including, for example, extreme difficulty breathing, shortness of breath, and / or low blood oxygen levels. In some embodiments, administration of the cells reduces inflammation in the subject. In some embodiments, administration of the cells reduces the level 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 can refer to an increase or decrease compared to before treatment of the subject or compared to the incidence of this symptom in the general population or a study population.

[0071] Also disclosed herein 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 to have immunomodulatory activity using the methods disclosed herein.

[0072] Thus, in some aspects, disclosed herein 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 to have immunomodulatory activity using a method comprising:

[0073] a. Dividing the plurality of cells into a first group of cells and a second group of cells;

[0074] b. Culturing the first group of cells under basal conditions and culturing the second group of cells under inflammatory conditions;

[0075] c. Collecting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells in the first group of cells, and the cells in the second group of cells;

[0076] d. Determining the levels of soluble TNFR2 protein in the culture supernatant of the first group and the culture supernatant of the second group;

[0077] e. Normalizing the levels of soluble TNFR2 protein in the first group and the second group with the total protein level of the cells of the corresponding group collected in step c;

[0078] f. Calculate an Inflammatory Stimulation Index (ISI) by dividing the normalized level of the soluble TNFR2 protein of the second group by the normalized level of the soluble TNFR2 protein of the first group; and

[0079] g. If the ISI is higher than 1, determine that the plurality of cells has immunomodulatory activity.

[0080] In some embodiments, the plurality of cells is determined to have an ISI higher than 1.

[0081] In some embodiments, the method of any of the foregoing aspects further comprises administering to the subject a therapeutically effective amount of an anti-COVID therapeutic agent.

[0082] 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, sofosbuvir / daclatasvir, nirmatrelvir, budesonide, artesunate, type I interferon, telmisartan, nitazoxanide, niclosamide, bromhexine, dornasealfa), dexmedetomidine, fluoxetine, sabizabulin, ribavirin, molnupiravir, danoprevir, bemnifosbuvir, galidesivir, BCX-4430, opaganib, arbidol, chloroquine, dexamethasone, heparin, nitazoxanide, Tocilizumab, Sarilumab, Levilimab, Siltuximab, Clazakizumab, Sirukumab, Olokizumab, Anakinra, Canakinumab, Mavrilimumab, Lenzilumab, Gimsilumab, Otilimab, TJ003234, Emapalumab, Adalimumab, Infiximab, Secukinumab, Ixekizumab, Risankizumab, Lufotrelvir, Ensovibep, Fenretinide, Rintatolimod, Bemcentinib, Plitidepsin, Emetine hydrochloride, Stannous protoporphyrin, Antroquinonol, Apilimoddimesylate, Brequinar, Brilacidin, Sangivamycin, Tempol, RP-7214, PBI-0451, and Masitinib.

[0083] In one embodiment, in a static sTNFR2 release assay, cells are cultured in separate culture vessels and exposed to inflammatory or control (basal) medium. The sTNFR2 values are then measured in the respective medium, normalized, and the ratio between the sTNFR2 measured under these conditions is calculated.

[0084] Figure 7 Shown are the sTNFR2 inflammatory stimulation indices (ISIs) of UC-MSC preparations from different donors and at different culture passages. The ISI values were obtained by the static sTNFR2 release assay. The histograms depict the mean sTNFR2 ISI from triplicate tests; error bars indicate 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.

[0085] In one embodiment, in a dynamic sTNFR2 release assay, the same cell preparation is maintained fixed and the medium is flowed around the cells (‘perfusion’). In this embodiment, the same cells can be dynamically exposed to basal and inflammatory conditioned media by the flow of media with different compositions around the fixed cells during different time windows. Samples are collected at different time points throughout the perfusion assay, the sTNFR2 release values in the different samples can be measured, and the difference in the amount of sTNFR2 measured after exposure to inflammatory medium compared to basal medium can be calculated. Instruments are commercially available to automate the perfusion of medium around cells fixed in a chamber.

[0086] The difference in sTNFR2 release, referred to as ‘Delta sTNFR2’ or ‘ΔsTNFR2’, and the dynamic changes in sTNFR2 release can be used to compare multiple cell preparations.

[0087] Normalization of the sTNFR2 values can be based on the cell protein content at the end of the in vitro experiment, as presented in the preferred embodiment.

[0088] In other embodiments, normalization of sTNFR2 can be based on the cell number at initial seeding, or the cell seeding density or cell concentration in the medium.

[0089] In one embodiment, the culture medium contains 10% platelet lysate. In other embodiments, the culture medium contains 9%, or 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% platelet lysate. In other embodiments, the culture medium does not contain platelet lysate.

[0090] In one embodiment, the culture medium is a chemically defined medium.

[0091] In one embodiment, the inflammation induction is based on the addition of TNFα and IFNγ. In other embodiments, the inflammation induction is based on the addition of TNFα, or TNFβ, or IFNγ, or other inflammatory cytokines.

[0092] In certain embodiments, the concentration of TNFα is 0 - 15000 pg / ml. In certain embodiments, the concentration of TNFα is 0 - 12500 pg / ml. In certain embodiments, the concentration of TNFα is 0 - 10000 pg / ml. In certain embodiments, the concentration of TNFα is 0 - 7000 pg / ml. In certain embodiments, the concentration of TNFα is 0 - 4000 pg / ml. In one embodiment, the concentration of TNFα is 0 - 4000 pg / ml. In one embodiment, the concentration of TNFα is 0 - 3000 pg / ml. In one embodiment, the concentration of TNFα is 0 - 2500 pg / ml. In one embodiment, the concentration of TNFα is 0 - 2000 pg / ml. In one embodiment, the concentration of TNFα is 0 - 1500 pg / ml. In one embodiment, the concentration of TNFα is 0 - 1000 pg / ml. In one embodiment, the concentration of TNFα is 100 - 1500 pg / ml. In one embodiment, the concentration of TNFα is 500 - 2500 pg / ml. In one embodiment, the concentration of TNFα is 500 - 2000 pg / ml. In one embodiment, the concentration of TNFα is 500 - 1500 pg / ml. In one embodiment, the concentration of TNFα is 500 - 1000 pg / ml. In one embodiment, the concentration of TNFα is 500 - 4000 pg / ml. In one embodiment, the concentration of TNFα is 100 - 4000 pg / ml.

[0093] In certain embodiments, the concentration of TNFβ is 0 - 20000 pg / ml. In certain embodiments, the concentration of TNFβ is 0 - 15000 pg / ml. In certain embodiments, the concentration of TNFβ is 0 - 10000 pg / ml. In certain embodiments, the concentration of TNFβ is 0 - 5000 pg / ml. In certain embodiments, the concentration of TNFβ is 500 - 5000 pg / ml. In certain embodiments, the concentration of TNFβ is 500 - 10000 pg / ml. In certain embodiments, the concentration of TNFβ is 100 - 10000 pg / ml. In certain embodiments, the concentration of TNFβ is 100 - 7500 pg / ml. In certain embodiments, the concentration of TNFβ is 100 - 5000 pg / ml. In certain embodiments, the concentration of TNFβ is 100 - 20000 pg / ml. In certain embodiments, the concentration of TNFβ is 500 - 20000 pg / ml.

[0094] In certain embodiments, the concentration of IFNγ is 0 - 10000 pg / ml. In certain embodiments, the concentration of IFNγ is 0 - 5000 pg / ml. In certain embodiments, the concentration of IFNγ is 0 - 2000 pg / ml. In certain embodiments, the concentration of IFNγ is 0 - 1750 pg / ml. In certain embodiments, the concentration of IFNγ is 0 - 1500 pg / ml. In certain embodiments, the concentration of IFNγ is 0 - 1250 pg / ml. In certain embodiments, the concentration of IFNγ is 0 - 1000 pg / ml. In certain embodiments, the concentration of IFNγ is 500 - 1500 pg / ml. In certain embodiments, the concentration of IFNγ is 500 - 1250 pg / ml. In certain embodiments, the concentration of IFNγ is 100 - 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.

[0095] In other embodiments, the cells used for sTNFR2 release assays are cultured in suspension.

[0096] In other embodiments, the cells used for sTNFR2 release assays are cultured while adhered to microcarriers with or without agitation or in suspension.

[0097] In certain embodiments, the cells used for sTNFR2 release assays are adhered to a substrate.

[0098] In other embodiments, the cells used for sTNFR2 release assays are embedded in a material that enables the movement of sTNFR2 through pores in the material.

[0099] In certain embodiments, cells are immobilized by adhesion to a matrix or encapsulation in a container, and the culture medium flows around the cells (perfusion) in an in vitro system. A fluid, i.e., the culture medium, or a solution containing a drug, or a cell suspension, flows in the in vitro system to contact the cells. Using this method, the kinetics of sTNFR2 release from the immobilized cells can be analyzed in detail, and different samples can be collected over time and / or after exposure to a control culture medium or an inflammation-inducing culture medium.

[0100] When the goal is to increase the in vivo concentration of sTNFR2 and / or decrease the in vivo concentration of TNFα and / or TNFβ, the standardized in vitro methods presented herein can be used to screen cell preparations and select those with the greatest potential for clinical efficacy.

[0101] This method is capable of selecting cell populations based on in vitro potency related to sTNFR2 release and has the potential to predict the in vivo efficacy and effectiveness of cell populations.

[0102] Examples

[0103] The following examples are set forth below to illustrate the compositions, methods, and results in accordance with the disclosed subject matter. These examples are not intended to encompass all aspects of the disclosed subject matter, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the invention that are obvious to those skilled in the art.

[0104] Example 1. Measurement of sTNFR2 release from cultured UC-MSCs.

[0105] One embodiment of the present disclosure is a method for quantifying sTNFR2 release from cultured UC-MSCs by normalized ELISA and an inflammation stimulation index (ISI) in the presence or absence of inflammatory mediators. This is a representative method, and variations of this method are within the scope of the present disclosure.

[0106] Mesenchymal stem cells (MSCs) originate from human embryos and are considered adult pluripotent 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. The UC contains two umbilical arteries (UCA) and one umbilical vein (UCV), both of which are embedded in a specific mucous connective tissue (known as Wharton's jelly (WJ)) and covered by amniotic epithelium. The UC is considered medical waste and is collected in a non-invasive manner. In addition, obtaining the UC does not pose ethical issues. Similar to MSCs derived from other sources, UC-MSCs have unique self-renewal capabilities while maintaining their pluripotency, namely the ability to differentiate into adipocytes, osteocytes, chondrocytes, neurons, and hepatocytes, although some of the differentiation capabilities are known to be partial. In addition, due to their strong immunomodulatory, anti-inflammatory, and reparative properties, MSCs have been proposed as a treatment modality.

[0107] It has been observed that UC-MSC treatment has significant clinical benefits, including significantly improving the survival rate, survival rate without serious adverse events, and recovery time in patients with COVID-19 acute respiratory distress syndrome (ARDS). Studies have shown that on the 6th day after infusion, compared with the control, UC-MSC recipients had significantly elevated plasma soluble TNF receptor 2 (sTNFR2) levels and significantly reduced TNFα and TNFβ levels. These observations suggest that sTNFR2 plays a mechanistic role in mediating the effect of UC-MSCs on the plasma levels of TNFα and TNFβ, determining the reduction of inflammation in COVID-19 ARDS.

[0108] This study provides the quantification of sTNFR2 released by cultured UC-MSCs (a central mediator of the anti-inflammatory effect of UC-MSC treatment) by normalized ELISA and measurement of the inflammatory stimulation index (ISI) in the presence or absence of inflammatory mediators.

[0109] Definitions

[0110] A. GM: Growth medium

[0111] B. IIM: Inflammatory induction medium

[0112] C. ISI: Inflammatory stimulation index

[0113] D. MSC: Mesenchymal stem cell

[0114] E. Potency: The specific ability of a product to affect a given outcome.

[0115] F. SOP: Standard operating procedure

[0116] G. sTNFR2: Soluble TNF receptor 2

[0117] H. UC-MSC: Human umbilical cord-derived mesenchymal stem cells

[0118] I. WR: Working reagent

[0119] Equipment and Materials

[0120] A. Equipment:

[0121] 1. Biosafety cabinet (BSC)

[0122] 2. Centrifuge

[0123] 3. Hemocytometer with cover glass

[0124] 4. Incubator

[0125] 5. Micropipettes (2 - 20 μl, 20 - 200 μl, 100 - 1000 μl)

[0126] 6. Microplate reader ( iD3, Molecular Devices)

[0127] 7. Microscope

[0128] 8. Multichannel pipette

[0129] 9. Pipette aid

[0130] 10. Refrigerator

[0131] 11. Timer

[0132] B. Supplies:

[0133] 1. 1.5 ml microcentrifuge tubes

[0134] 2. 15 mL conical polypropylene tubes

[0135] 3. 25 cm 2 Rectangular neck cell culture flask with vented cap

[0136] 4. 96-well plates

[0137] 5. Face masks

[0138] 6. Gauze

[0139] 7. Gloves, non-sterile

[0140] 8. Gloves, sterile

[0141] 9. Micropipette tips, sterile: 2 - 200 μl and 100 - 1000 μl

[0142] 10. Serum Pipettes, Sterile: 5 ml, 10 ml, 25 ml, 50 ml

[0143] C. Reagents:

[0144] 1. CTS TM TrypLE TM Select Enzyme, cGMP grade, Thermo Fisher Scientific

[0145] 2. DMEM, low glucose, pyruvate, without glutamine, without phenol red, cGMP grade, Thermo Fisher Scientific

[0146] 3. GlutaMAX 100X, cGMP grade, Thermo Fisher Scientific

[0147] 4. IFNγ, for in vitro use, R&D Systems

[0148] 5. MEM-NEAA 100X, cGMP grade, Thermo Fisher Scientific

[0149] 6. Micro BCA Protein Assay Kit, for in vitro use, Thermo Fisher Scientific

[0150] 7. PLTGold, cGMP grade, Mill Creek

[0151] 8. RIPA Lysis and Extraction Buffer, for in vitro use, Thermo Fisher Scientific

[0152] 9. Human Soluble TNF Receptor 2, ELISA Kit, for in vitro use, Abcam

[0153] 10. TNFα, for in vitro use, R&D Systems

[0154] 11. Human TNF-RII (Soluble) Recombinant Protein, for in vitro use, Thermo Fisher Scientific D. Methods:

[0155] 1. UC-MSC Potency Assay: Batch Production Records.

[0156] 2. Medium and Reagent Preparation: Inflammatory Inducing Medium and Human Soluble TNF Receptor 2 Reagent.

[0157] 3. Standard Curve Preparation: Human Soluble TNF Receptor 2 Standard and Diluted Albumin (BSA) Standard.

[0158] Table 1. BSA

[0159]

[0160] Table 2. Layout BSA

[0161]

[0162]

[0163] Table 3. ELISA

[0164]

[0165] Additional Considerations

[0166] A. In one embodiment, at least two previously trained and qualified cGMP employees should be present throughout the procedure. In one embodiment, all calculation procedures should be verified by a second employee member present.

[0167] B. In one embodiment, the manufacturing process described herein consists of multiple steps and takes 6 days to complete. The cGMP facility personnel involved in product manufacturing should record each step of the process.

[0168] C. In one embodiment, aseptic / sterile techniques should be used for cell seeding and expansion. This involves working in a biosafety cabinet (BSC).

[0169] D. In one embodiment, the manufacturer, lot number, and expiration date of all supplies, media, and reagents used in this procedure should be recorded.

[0170] E. In one embodiment, all samples and reagents from the sTNFR2 human ELISA kit are placed at room temperature (18 - 25 °C) before use.

[0171] F. In one embodiment, open microplate wells or reagents can be stored at -20 °C for up to 1 month.

[0172] Procedure

[0173] A. Media, Reagent, and Standard Preparation

[0174] 1. Media Preparation

[0175] 1.1 Growth Medium (GM)

[0176] Prepare one bottle of medium and add the following reagents in the order indicated below:

[0177] a. 1000 ml DMEM

[0178] b. 54 ml PLTGoldTM (5% PLTGold)

[0179] c. 10.8 ml GlutaMAX supplement 200 mM

[0180] d. 10.8 ml MEM-NEAA, 10 mM, label the following on the culture medium:

[0181] · Name of the culture medium

[0182] · Date of preparation

[0183] · Expiration date (14 days after preparation)

[0184] · Initials of the person in the cGMP facility who prepared the culture medium

[0185] 1.2 Inflammatory Induction Medium (IIM)

[0186] Note: Prepare the culture medium 3 days before use.

[0187] Prepare 45 ml of IIM and add the following reagents in the indicated order.

[0188] a. 44.5 ml GM

[0189] b. 225 μl IFNγ (final concentration: 10 ng / ml)

[0190] c. 337.5 μl TNFα (final concentration: 15 ng / ml) Label the following on the culture medium: · Name of the culture medium

[0191] · Date of preparation

[0192] · For fresh use

[0193] · Initials of the person in the cGMP facility who prepared the culture medium

[0194] 2. Reagent preparation (Human Soluble TNF Receptor 2 ELISA Kit)

[0195] Table 4

[0196]

[0197] 3. Preparation of human soluble TNF receptor 2 standards

[0198] Note: Prepare serially diluted standards immediately before use according to the manufacturer's recommendations. Always prepare a fresh set of standards for each use.

[0199] 3.1 Reconstitute only 1 vial (1 vial / standard curve).

[0200] 3.2 Vial of short-term rotation sTNFR2 standard. Prepare a 50 ng / ml stock solution standard by adding 400 μl of 1X assay diluent B to the vial. Mix well and gently.

[0201] 3.3 Label tubes 1 - 7 with a dilution factor of 1 to 3 for serial standard dilutions.

[0202] 3.4 Prepare Standard 1 by adding 40 μl of the 50 ng / ml stock solution standard and 960 μl of 1X assay diluent B to Tube 1. Mix well and gently.

[0203] 3.5 Pipette 400 ul of 1X assay diluent B into the remaining tubes.

[0204] 3.6 Prepare Standard 2 by adding 200 μl of Standard 1 to Tube 2 and mixing well.

[0205] 3.7 Prepare Standard 3 by adding 200 μl of Standard 2 to Tube 3 and mixing well.

[0206] 3.8 Use Figure 2 as a guide to prepare more serial dilutions (see Table 5).

[0207] 3.9 Use 1X assay diluent B (Tube 8: negative control) as the zero standard (0 pg / ml) ( Figure 1 ).

[0208] Table 5. Preparation of Human Soluble TNF Receptor 2 Standards

[0209]

[0210] 4. Preparation of Diluted Albumin (BSA) Standards

[0211] Each 1 mL ampule of 2.0 mg / mL albumin standard is sufficient to prepare a set of diluted standards such that three replicates of each dilution can be included in the tube procedure. A fresh set of standards should be prepared for each use.

[0212] 4.1 Prepare a set of protein standards using distilled water. Use Table 6 as a guide.

[0213] Table 6. Preparation of Diluted Albumin (BSA) Standards

[0214]

[0215] 4.2 Determine the total volume of working reagent (WR) required using the following formula:

[0216] [(Number of standards × 2 replicates) + (Number of unknowns × 3 replicates)] x (150 μl of WR) = Total volume of WR required.

[0217] 4.3 Prepare the required volume of WR by mixing 25 parts of Micro BCA reagent A (MA) and 24 parts of reagent B (MB) with 1 part of Micro BCA reagent C (MC) (25:24:1).

[0218] Note: At room temperature, WR is stable when stored in a sealed container for one day.

[0219] B. Cell seeding and expansion

[0220] 1. Thaw the UC-MSC vial or fragment and perform cell expansion

[0221] 1.1 Remove 1 vial or 2 - 3 segments containing UC-MSC from cryogenic storage.

[0222] 1.2 Place the sample in a specimen bag and perform rapid thawing by gently swirling in a 37 °C water bath (until small ice crystals remain in the vial or segment). Handle the cells gently to avoid mechanical damage. This process should not exceed 3 minutes.

[0223] 1.3 Once thawed, wipe the sample with a pre-made sterile alcohol swab.

[0224] 1.4 Place the sample in the BSC and slowly transfer the cells to a sterile 50 ml conical tube using a syringe.

[0225] 1.5 Slowly add 10 ml of room temperature GM dropwise to the same 50 mL conical tube.

[0226] 1.6 Centrifuge the cells at 500 x g for 10 minutes at room temperature with the brake set to "high".

[0227] 1.7 Remove the cells from the centrifuge and gently aspirate the supernatant.

[0228] 1.8 Resuspend the pellet in 10 ml of GM and equilibrate to room temperature.

[0229] 1.9 Perform cell counting and viability ("Viability test using trypan blue or propidium iodide (PI) and thiazole orange (TO)"). Record the results. The trypan blue viability test is based on the ability of live (viable) cells to exclude the dye (trypan blue) from their cell membranes and the ability of non-viable cells to take up the dye. Evaluate the cells under a microscope and count both the viable and non-viable cells to determine the percentage of viability. Trypan blue should be filtered before use. The cells should be counted immediately after adding the dye. Viable cells will start to take up the dye within 2 - 3 minutes, which will result in a lower viability.

[0230] a. Reagents

[0231] · 0.4% Trypan Blue (filtered), Sigma or equivalent

[0232] · Phosphate Buffered Saline (PBS), MediaTech or equivalent

[0233] · 70% ethanol

[0234] b. Procedure

[0235] Viability of Fresh Cells

[0236] 1. The following procedure is used to prepare fresh cells in suspension.

[0237] 2. Ensure that the hemocytometer and coverslip are thoroughly cleaned and air-dried. Use 70% alcohol and lint-free cloth.

[0238] 3. Place the coverslip on the hemocytometer so that it partially covers two V-shaped slashes.

[0239] 4. Using a micropipette, mix the fresh cells well and place 100 μl in a small tube.

[0240] 5. Add 100 μl of 0.4% Trypan Blue. If the cell product is too concentrated, dilute it with PBS and mix well before adding Trypan Blue.

[0241] 6. Load the hemocytometer as follows:

[0242] a. Using a pipette, place a drop of the cell / Trypan Blue mixture on one of the V-shaped slashes between the coverslip and the counting chamber without disturbing the coverslip. Slowly fill the area completely and remove the pipette before the solution spills over the edge of the chamber section.

[0243] b. Do not overfill or underfill. If the chamber is overloaded or visible bubbles are present, clean the chamber and start over rather than trying to remove the excess liquid.

[0244] 7. Rotate to the 4x objective and carefully place the hemocytometer on the microscope stage.

[0245] 8. Count the cells after incubating for approximately 1 minute and 3 minutes before incubation. After incubating for 2 - 3 minutes, viable cells may start to absorb the dye, resulting in inaccurate counting.

[0246] 9. Viable cells will remain unstained, while dead cells will appear blue after absorbing the dye.

[0247] 10. Count the stained and unstained nucleated cells among a total of 100 random cells. These counts can be made on any of the quadrants numbered 1 - 5 in Figure 8 this figure.

[0248] 11. Record the data.

[0249] 12. Calculate the percentage of viability using the following formula:

[0250] % viable cells = number of viable cells × 100

[0251] total number of cells counted

[0252] Viability of thawed cells

[0253] 1. Rapidly thaw the cryopreserved cells.

[0254] 2. Follow steps 2 to 12 in the viability procedure for fresh cells.

[0255] 1.10 Seed the cells at a concentration of 500,000 viable cells per flask (20,000 cells / cm 2 ) in six 25 cm 2 flasks (3 flasks for each condition (‘basal’ and ‘inflammatory induction’ conditions), resuspended in 5 mL of GM per flask.

[0256] 1.11 Incubate the cells in GM for 72 hours (3 days) in an incubator at 37 °C, 5% CO2.

[0257] C. Inflammatory induction

[0258] 1. Inflammatory induction (TNFα / IFNγ)

[0259] 1.1 Prepare the IIM before use and preheat it at 37 °C.

[0260] 1.2 Remove all the medium from the flasks.

[0261] 1.3 Add 5 ml of preheated GM to one flask and label this flask ‘basal’.

[0262] 1.4 Add 5 ml of preheated IIM to another flask and label this flask ‘inflammatory induction’.

[0263] 1.5 Return the tissue culture flasks to the 37 °C incubator with 5% CO2 for 3 days.

[0264] D. Potency assay

[0265] On the 3rd day after induction:

[0266] 1. sTNFR2 Measurement

[0267] Note: It is recommended to measure all standards in duplicate and samples in triplicate. The 96-well strip plates included in the kit are ready for use at any time.

[0268] 1.1 Collect the culture supernatant in a 15 ml tube.

[0269] 1.2 Centrifuge the supernatant at 1,500 rpm for 5 minutes to remove debris.

[0270] 1.3 Gently pour the supernatant into a new 15 ml tube.

[0271] 1.4 Dilute the supernatant 5-fold with 1X Assay Diluent B (Reagent Preparation, Section V, A, 2) from the Human sTNFR2 ELISA Kit (Abcam).

[0272] 1.5 Add 100 μL of each standard in duplicate (see Standard Preparation, Section V, A, 3) and each sample (UC-MSC sample, TNFR2 standard, medium) in triplicate to the appropriate wells ( Figure 2 ). Cover the wells and incubate overnight with gentle shaking at 4 °C.

[0273] 1.5.1 TNFR2 Standard:

[0274] a) H is defined as high concentration (H)

[0275] b) M is defined as medium concentration (M)

[0276] c) L is defined as low concentration (L)

[0277] 1.5.2 Blank Medium:

[0278] a) GM

[0279] b) IIM

[0280] 1.5.3 Supernatant (Test Sample):

[0281] a) GM Supernatant (sGM)

[0282] b) IIM Supernatant (sIIM)

[0283] 1.6 The next day, discard the solution and wash 4 times with 1X Wash Solution. Wash by adding 1X Wash Solution (300 μL) to each well using a multi-channel pipette or an automated washer. After the last wash, remove any remaining wash buffer by decantation. Invert the plate and dry it with a clean paper towel.

[0284] 1.7 Add 100 μL of 1X biotinylated soluble TNF receptor 2 detection antibody to each well (Reagent Preparation, Section V, A, 2). Incubate with gentle shaking for 1 hour at room temperature.

[0285] 1.8 Discard the solution. Wash as in step 1.6.

[0286] 1.9 Add 100 μL of 1X HRP-streptavidin solution to each well. Incubate with gentle shaking for 45 minutes at room temperature.

[0287] 1.10 Discard the solution. Wash as in step 1.6.

[0288] 1.11 Add 100 μL of TMB one-step substrate reagent to each well. Incubate with gentle shaking for 30 minutes at room temperature in the dark.

[0289] 1.12 Add 50 μL of stop solution to each well.

[0290] 1.13 Load the plate into iD3 and immediately read the plate at 450 nm.

[0291] 2. Protein Quantification

[0292] Note: Normalize sTNFR2 secretion relative to the total protein content of the cell lysate. To this end, quantify the total protein content of the cell lysate using the 'MicroBCA Protein Assay Kit' (Thermo Fisher Scientific).

[0293] 2.1 Protein Lysates

[0294] a. Briefly, after removing the supernatant, wash the cell monolayer twice with 5 ml of cold DPBS.

[0295] b. Add 1 ml of cold RIPA buffer (4 °C) to each 25 cm 2 flask using a micropipette.

[0296] c. Incubate the flask at 4 °C for 5 minutes.

[0297] d. Collect the cell lysate and centrifuge at 10,000 rpm for 5 minutes to remove cell debris.

[0298] e. After centrifugation, transfer the cell lysate to a new microcentrifuge tube and further process for protein quantification.

[0299] 2.2 Protein Quantification Using Micro BCA Protein Assay

[0300] a. Preparation of bovine serum albumin standard curve (0 - 200 μg / ml) as described herein.

[0301] b. Dilute the cell lysate 1:100 with distilled water.

[0302] c. Add 150 μL of each standard in duplicate or the diluted cell lysate in triplicate to a 96 - well plate.

[0303] d. Add 150 μL of WR (25:24:1, reagent MA:MB:MC) to each well.

[0304] e. Mix well on a plate shaker for 30 seconds.

[0305] f. Incubate the plate at 37 °C for 2 hours.

[0306] g. Measure the absorbance at 562 nm on a microplate reader.

[0307] E. Analysis

[0308] 1. Perform the assay on three sets of induced and non - induced flasks and analyze the results using SoftMax Pro software.

[0309] 2. sTNFR2 ELISA:

[0310] 2.1 Calculate the sTNFR2 concentration of each sample using the assay standard curve (0 - 2,000 pg / ml) fitted to a 4 - parameter log - regression.

[0311] 2.2 Average the triplicate well values for two blank media samples and six test samples (3x induced and 3x non - induced) to obtain the average sTNFR2 concentration for each blank / flask.

[0312] 2.3 There should be a total of eight averages, one for each flask and one for each blank media.

[0313] 3. Total protein BCA assay:

[0314] 3.1 Calculate the total protein concentration of the cell lysate for each sample using the Micro BCA protein assay standard curve fitted to a linear regression.

[0315] 3.2 Average the triplicate well values for six test samples (3x induced and 3x non - induced) to obtain the average total protein for each flask.

[0316] 3.3 There should be a total of six averages, one for each flask.

[0317] 4. Normalized sTNFR2 Calculation:

[0318] 4.1 For all flasks (both UC-MSC basal group and UC-MSC inflammatory induction group), normalize the sTNFR2 concentration by dividing the sTNFR2 value of each flask by the corresponding total protein concentration value.

[0319] 4.2 Then calculate the normalized sTNFR2 under basal culture conditions by taking the average of the sTNFR2 concentrations normalized to UC-MSC basal. The resulting value should be ≥ 0.2 (pg / mL) / (μg of total protein).

[0320] 5. Inflammatory Stimulation Index (ISI) Calculation:

[0321] 5.1 Calculate the ISI for each group of flasks as the ratio of the induced normalized sTNFR2 concentration to the non-induced normalized sTNFR2 concentration.

[0322] 5.2 Take the average of the three ISIs, and the resulting value should be ≥ 1.5.

[0323] Note

[0324] A. Calculate the total cellular protein content based on the volume of cell lysate used (RIPA, 1.5 ml) and based on the dilution factor (1:100).

[0325] Quality Control

[0326] A. To evaluate the likelihood of false positive results in sTNFR2 quantification, analyze the media samples (growth media and inflammatory induction media).

[0327] B. Also run external sTNFR2 standards within the range of the standard curve to ensure the ELISA kit is working properly. These values should be within 20% of the theoretical concentration.

[0328] C. The %CV of induced and non-induced ELISA replicates should be ≤ 20% to ensure accurate results. If %CV > 20%, one value can be discarded as an outlier.

[0329] D. The standard curve should fit to a 4-parameter logarithmic regression, and an R2 value ≥ 0.98 is considered acceptable. If the R2 value < 0.98, as long as there is at least one value for each concentration of the standard, these values can be discarded as outliers.

[0330] E. All equipment used should be maintained.

[0331] Example 2. In vitro potency assay of immunomodulatory cells based on soluble tumor necrosis factor receptor 2 (sTNFR2) release assay.

[0332] In vitro potency assay of immunomodulatory cells based on soluble tumor necrosis factor receptor 2 (sTNFR2) release assay. This is a representative assay, and variations of this assay protocol are within the scope of the present disclosure.

[0333] The studies herein developed and interpreted a biologically relevant in vitro assay to determine the potency of immunomodulatory cells such as mesenchymal stem cells. The potency assay focused on measuring soluble tumor necrosis factor receptor 2 (sTNFR2) released by the cells. It was based on the quantification of sTNFR2 by ELISA, normalized by the cellular protein content, and the calculation of the inflammatory stimulation index (ISI) of sTNFR2 released by the cells. The ISI was calculated as the ratio of sTNFR2 release in inflammatory induction to sTNFR2 release under basal conditions. The assay was performed using cells cultured in vitro. In one embodiment, the cells analyzed by this assay were umbilical cord-derived mesenchymal stem cells (UC-MSCs). The basal conditions corresponded to culturing the cells in the same medium used for generating the final cell product. The inflammatory induction was sourced from the addition of TNFα (15 ng / mL) and IFNγ (10 ng / mL) to these cultured media. The cells were maintained for a specific amount of culture time under basal conditions or inflammatory induction, and in a preferred embodiment, for 3 days of culture. Then the supernatant was collected and tested using a commercially available kit (e.g., Abcam Human Soluble TNFR2 ELISA Kit, Abcam, catalog number ab100643) for sTNFR2 quantification. For normalization based on the total cellular protein content, the cells were lysed using RIPA lysis and extraction buffer (e.g., Thermo Fisher Scientific, catalog number 89900), and the protein content was obtained using the BCA method (e.g., using the Micro BCA Protein Assay Kit, Thermo Fisher Scientific, catalog number 23235). This assay is described in Figure 3 which. The SOP for this assay was provided in Example 1 above.

[0334] Data of the potency assay

[0335] The method described in Example 1 has been applied, where UC-MSCs were thawed from the "UC-MSC final product (batch, cryopreserved)" stage manufactured in the cGMP facility of the Institute of Diabetes. This experiment quantified the sTNFR2 release of UC-MSCs under basal culture conditions as well as under inflammatory (TNFα / IFNγ) induction for 3 days. The results are presented in Table 7, Figure 4 and Figure 5 which.

[0336] Table 7.

[0337]

[0338] Biological relevance of patient-related measurements

[0339] The excessive inflammatory response in COVID-19 patients with acute respiratory distress syndrome (ARDS) is characterized by high serum levels of pro-inflammatory mediators, including tumor necrosis factor (TNF) α and β. These two molecules, which are involved in the pathophysiology of ARDS, bind to TNFR2. It has been reported that the soluble form of TNFR2 has an inhibitory effect on TNF function. The study herein investigated the plasma levels of TNFα, TNFβ, and soluble TNFR2 (sTNFR2) in both the UC-MSC treatment group and the control group in a phase 1 / 2a clinical trial of COVID-19 ARDS. On day 6, sTNFR2 increased in the UC-MSC treatment group patients compared to those in the control group (see Figure 6 ). On day 6, TNFα and TNFβ were found to be decreased. The observations are presented in Figure 6 .

[0340] Methods (observations on patients)

[0341] Blood samples were obtained from randomized subjects in 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 on ice, and processed within 2 hours for plasma separation. The whole blood was centrifuged at 2,000 g for 15 minutes at 4°C, and the plasma was collected and stored at -80°C until processing. The plasma levels (pg / ml) of TNFR2, TNFα, and TNFβ in all samples were determined simultaneously using a quantitative multiplex protein array ( Q-Series, RayBiotech) according to the manufacturer's instructions. The fluorescence signals were visualized by a Cy3 wavelength laser scanner and converted into concentrations using the standard curves generated for each array.

[0342] Statistical analysis was performed using the two-sample t-test and the non-parametric Wilcoxon two-sample test. The paired comparison was performed using the signed rank test to examine the changes between the time points within the group. All tests were two-sided, and statistical significance was established as p < 0.05. The data are presented as the mean and the standard error of the mean.

[0343] Results (observations on patients)

[0344] There was no significant difference in the baseline protein levels of the patients in the UC-MSC group and the control group. In the control group, there was no significant difference in the levels of sTNFR2, TNFα, and TNFβ between day 0 and day 6. The levels of TNFα and TNFβ decreased significantly between day 0 and day 6 (p = 0.005 and p = 0.002, respectively). On day 6, the between-group comparison demonstrated that the levels of TNFα (319 ± 40 vs. 950 ± 226 pg / ml, p = 0.048) and TNFβ (810 ± 126 vs. 2,944 ± 735 pg / ml, p = 0.032) in the UC-MSC group were significantly lower than those in the control group. On day 6, the sTNFR2 level in the UC-MSC group was significantly higher than that in the control group (26,609 ± 846 pg / ml vs. 23,111 ± 760 pg / ml, p = 0.021). See Figure 6 .

[0345] In a recently completed phase 1 / 2a clinical trial, UC-MSC treatment was associated with accelerated clinical recovery in patients with COVID-19 ARDS. This article provides molecular evidence of key potential immune / inflammatory mediator axis differences that help to explain these results. On day 6, compared with the control, UC-MSC recipients had significantly elevated plasma sTNFR2 levels and significantly reduced TNFα and TNFβ levels. TNF receptor-based drugs have been tested for the treatment of chronic inflammatory diseases and may similarly be beneficial for reducing the excessive inflammation in severe COVID-19 patients. TNF blockade is clinically effective because it causes a rapid decrease in the levels of circulating interleukin (IL)-1 and IL-6 (<12 hours), as well as a decrease in adhesion molecules and vascular endothelial growth factor (VEGF) that strongly affect leukocyte transport and capillary permeability in inflamed tissues. Studies have shown that in anti-TNF therapy, the TNF concentration in inflamed tissues decreases as it enters the bloodstream and binds to anti-TNF antibodies.

[0346] In addition, sTNFR2 can bind to TNF and neutralize TNF-induced cytotoxicity and immunoreactivity, regulating the inflammatory response. For example, higher sTNFR2 levels lead to reduced T cell activation and the gradual generation of regulatory T cells (Tregs). On this basis, studies have shown that the expression of TNFR2 by MSCs is related to its higher ability to induce Foxp3+ Tregs. Therefore, the findings in this article demonstrate the key mechanism of the action of UC-MSC, and the plasma level of sTNFR2 can predict the progression of COVID-19 ARDS and the clinical outcome after treatment.

[0347] Based on the observations of the patients described above, and based on the potency assay observations described in the previous paragraphs, the potency assay developed in this article has biological relevance.

[0348] Potency Assurance

[0349] This potency assay (i.e., measuring soluble TNFR2 release by normalization quantification and inflammatory stimulation index (ISI)) is used to ensure the potency of the product intended for use in Phase 2b / 3 studies. The assurance criteria for the potency of each batch of UC-MSCs for Phase 2b / 3 studies are as follows:

[0350] · Soluble TNFR2 (sTNFR2) release, normalized by total cellular protein content, > 0.01 (pg / mL) / (μg) after 3 days of culture

[0351] · Inflammatory stimulation index (ISI) > 1

[0352] · In one embodiment, soluble TNFR2 (sTNFR2) release, normalized by total cellular protein content, > 0.02 (pg / mL) / (μg) after 3 days of culture under basal conditions

[0353] · Inflammatory stimulation index (ISI) > 1.5

[0354] · In one embodiment, soluble TNFR2 (sTNFR2) release, normalized by total cellular protein content, > 0.03 (pg / mL) / (μg) after 3 days of culture under inflammatory induction

[0355] · Inflammatory stimulation index (ISI) > 1.5

[0356] The present disclosure addresses the need for a potency assay of immunomodulatory cells (or their secretomes) that modify the tumor necrosis factor (TNF) inflammatory pathway in the human body. The types of immunomodulatory cells include mesenchymal stem cells, mesenchymal stromal cells, and mesenchymal stem cells (MSCs) for medical signal transduction.

[0357] This in vitro potency assay is based on the in vitro measurement of soluble tumor necrosis factor receptor 2 (sTNFR2) release under basal or inflammatory conditions to predict in vivo immunomodulatory function. Currently, the safety and efficacy of MSC-based therapies are being investigated in multiple clinical trials for various conditions, including inflammatory, immune, autoimmune, musculoskeletal, cardiovascular, neurodegenerative, and gastrointestinal diseases. However, preliminary results of many such studies have revealed that these cell therapies have a high degree of variability, with cases of non-reproducibility in clinical observations. Most importantly, the inconsistent evidence may be related to the inherent differences in the cell-based products used, including the lack of standardized characteristics in the formulations, which are reflected in potency differences. Therefore, a rapid and accurate method for a priori qualitative assessment of MSC batches in vitro is presented herein to determine effective in vivo immunomodulatory effects.

[0358] 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. The disclosures cited herein and the materials to which they are cited are specifically incorporated by reference.

[0359] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments of the present invention and that such changes and modifications can be made without departing from the spirit of the present invention. Accordingly, the appended claims are intended to cover all such equivalent variations that fall within the true spirit and scope of the present invention.

Claims

1. A method for testing the immunomodulatory activity of multiple cells, the method comprising: a. Dividing the multiple cells into a first group of cells and a second group of cells; b. Culturing the first group of cells under basal conditions and culturing the second group of cells under inflammatory conditions; c. Collecting the culture supernatant of the first group of cells, the culture supernatant of the second group of cells, the cells in the first group of cells, and the cells in the second group of cells; d. Determining the levels of soluble tumor necrosis factor receptor 2 (TNFR2) protein in the culture supernatant of the first group of cells and the culture supernatant of the second group of cells; e. Normalizing the levels of the soluble TNFR2 protein in the first group of cells and the second group of cells with the total protein levels of the corresponding first group of cells and second group of cells collected in step c; f. Calculating an inflammatory stimulation index (ISI) by dividing the normalized level of the soluble TNFR2 protein in the second group of cells by the normalized level of the soluble TNFR2 protein in the first group of cells; and g. If the ISI is higher than 1, determining that the multiple cells have immunomodulatory activity.

2. The method according to claim 1, wherein the multiple cells comprise human mesenchymal stem cells, mesenchymal stromal cells, or medical signaling cells.

3. The method according to claim 1 or 2, wherein the multiple cells are derived from postpartum adipose tissue, infrapatellar fat pad, postpartum bone marrow, postpartum endometrium, perinatal umbilical cord, perinatal chorion, perinatal amnion, or perinatal placenta.

4. The method according to any one of claims 1 to 3, wherein the inflammatory conditions comprise the presence of tumor necrosis factor α (TNFα) or interferon γ (IFNγ).

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

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

7. The method according to claim 6, wherein the multiple cells are determined to have an inflammatory stimulation index (ISI) higher than 1.

8. The method according to claim 6 or 7, further comprising administering a therapeutically effective amount of an anti-COVID therapeutic agent to the subject.

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

10. The method according to claim 9, wherein the multiple cells are determined to have an inflammatory stimulation index (ISI) higher than 1.

11. The method according to claim 9 or 10, further comprising administering to the subject a therapeutically effective amount of an anti-COVID therapeutic agent.

12. 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 according to any one of claims 1 to 5.

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

14. The method according to claim 12 or 13, further comprising administering to the subject a therapeutically effective amount of an anti-COVID therapeutic agent.

15. A method of treating a fibrotic condition characterized 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 according to any one of claims 1 to 5.

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

17. The method according to claim 15 or 16, further comprising administering to the subject a therapeutically effective amount of an anti-COVID therapeutic agent.

18. A method of treating a condition characterized by an increase in tumor necrosis factor (TNF) 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 according to any one of claims 1 to 5.

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

20. The method according to claim 18 or 19, further comprising administering to the subject a therapeutically effective amount of an anti-COVID therapeutic agent.

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