Treatment of organ dysfunction using a combination of mesenchymal stem cells and exosomes

Abstract

Embodiments relate to a method of treating a disease or condition in a subject in need thereof, the method comprising: administering a therapeutic effective amount of a pharmaceutical composition comprising mesenchymal cells and exosomes to the subject; wherein the mesenchymal cells and exosomes are in a ratio of about 1: 2500 to 1: 250000; wherein the mesenchymal cells have a stemness quality more than 90% and viability more than 95%, and the exosomes are free of a cellular contaminant as detected by immunoblotting technique, wherein the cellular contaminant comprises calnexin, and treating said disease or condition.

Description

Attorney Docket No. SASC-OOl-OIWOTREATMENT OF ORGAN DYSFUNCTION USING A COMBINATION OFMESENCHYMAE STEM CEELS AND EXOSOMESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C §119 of U.S. Provisional Application No. 63 / 714,195, filed on October 31, 2024, titled “Human Umbilical Cord-Derived Mesenchymal Stem Cells in combination with Small Extracellular Vesicles Improve Survival and Dramatically Reduce Fibrosis and Cirrhosis in Wistar Rats receiving Ccl4”, which is / arc hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to a pharmaceutical composition having mesenchymal cells and exosomes. In particular, this disclosure relates to a pharmaceutical composition having mesenchymal cells and exosomes derived from it. In some embodiment, the disclosure also relates to use of the composition in treatment of a disease in a mammal. In some embodiment, the disclosure also relates to method of isolating of mesenchymal stem cells and exosomes.BACKGROUND

[0003] Currently, there are numerous medical conditions that have limited treatment options. Innovative biological products including stem cells have the potential to fill this therapeutic void. Stem cells possess the remarkable capability to repair and regenerate damaged tissues and organs, offering new hope for patients with previously untreatable conditions.

[0004] Stem cells are capable of regenerating damaged tissues as they possess immunomodulatory, anti-inflammatory, anti-apoptotic, antifibrotic and neovascularization capabilities. By leveraging the powers bestowed by nature on stem cells, stem cell therapy can be seen as a “natural” way of treating diseases. A single stem cell product is a broad-spectrum drug that is capable of treating various medical conditions. Introducing stem cells into the body could be treated akin to infusing intelligence as stem cells are known to diagnose the medical condition and “custom” secrete different growth factors, cytokines and chemokines that repair the affected organ. Most importantly, numerous clinical trials involving several thousands of patients have established the safety of stem cells.

[0005] Extensive animal studies and preliminary data suggest that stem cells can treat numerous conditions not limited to lower back pain, joint degeneration, stroke injury, heart failure, kidney failure, various degenerative neurological conditions, chronic obstructive pulmonary disease, CO VID infection, Crohn’s disease, peripheral vascular disease and autoimmune diseases.

[0006] However, unlike small chemical entity drugs, stem cells are very complex. The large-scaleAttorney Docket No. SASC-OOl-OIWO production of stem cells can be challenging, and the method adopted for producing stem cells affects not only stem cell yield but also their quality. There is a need in the art for improved stem cells as well as for improved methods for preparing stem cells that achieve a high yield of stem cells and are cost-effective. There is also a need in the art for methods that yield stem cells with improved properties.

[0007] Mesenchymal stromal cells, also called mesenchymal stem cells (MSCs), are a type of adult stem cells that can be easily manipulated in vitro conditions. Stem cells present in early embryonic stages are pluripotent, whereas MSCs exhibit more limited plasticity, differentiating mainly into osteoblasts, adipocytes and chondrocytes (mesodermal cells) and to some extent also to skeletal muscle and endothelial cells. MSCs are present in the bone marrow and adipose tissue, and are also present in peripheral blood, placenta, umbilical cord blood, dental pulp, and other tissues.

[0008] MSCs secrete a plethora of biologically active proteins (Id., citing Tremain N, et al. Stem Cells 2001 ; 19: 408-418; Phinney D G, et al. Stem Cells 2006; 24: 186-198; Ren J, et al. Cytotherapy 2011; 13: 661-674).

[0009] High performance liquid chromatography (HPTC) and dynamic light scatter (DLS) analyses revealed that MSCs secrete cardioprotective microparticles with a hydrodynamic radius ranging from 30-150nm (Id., citing Chen et al., 2011 ; Tai et al. J. Mol. Cell. Cardiol. (2010) 48: 1215-1224).

[0010] MSC-derived EVs, which include exosomes and microvesicles (MV), are involved in cell- to-cell communication, cell signaling, and altering cell or tissue metabolism at short or long distances in the body, and can influence tissue responses to injury, infection, and disease (Phinney, D G and Pittenger, M F. Stem Cells (2017) 35: 851-58). Their content includes cytokines and growth factors, signaling lipids, mRNAs, and regulatory miRNAs (Id.). The content of MSC EVs is not static; they are a product of the MSC tissue origin, its activities, and the immediate intercellular neighbors of the MSCs (Id.).

[0011] Although MSC-derived EVs recapitulate to a large extent the immensely broad therapeutic effects previously attributed to MSCs, most studies fall short of rigorously validating this hypothesis (Id.) For example, various groups have compared the potency of MSCs versus MSC- derived EVs, and in some cases MSC-conditioned media, in animal models of myocardial infarction (Id., citing Bian S, et al. J Mol Med (Berlin) 2014; 92: 387-397), focal cerebral ischemia (Doeppner T R, et al. Stem Cells Transl Med 2015; 4: 1131-1143), gentamicin-induced kidney injury (Reis L A, et al. PLoS One 2012; 7: e44092), and silicosis (Choi M, et al. Mol Cells 2014; 37: 133-1394). While most studies report that MSC-derived EVs are equally effective as MSCs in sparing tissue and / or promoting functional recovery from injury, this desired outcome is compromised by lack of appropriate controls, comparable dosing, evaluation of the differentAttorney Docket No. SASC-OOl-OIWO disease endpoints, variations in frequency and timing of dosage, and absence of dose-dependent effects, thereby making it difficult to draw reliable conclusions about comparable efficacy and potency (Id.)SUMMARY

[0012] The successful translation of stem cell technology from the bench to bedside hinges on two important parameters. Firstly, the ease of large-scale manufacturing is critical as each patient dose comprises approximately 100 million cells. Secondly, maintaining the quality of the stem cells despite scaling up production is critical for good patient outcomes.

[0013] There is long felt need to understand the efficacy of a composition having mesenchymal cells and Exosomes derived from the mesenchymal cells, and ease of manufacturing large scale MSCs with high quality.

[0014] The present invention achieves the above goals through the methods described herein which improvise multiple steps involved in production of stem cells to achieve the target of obtaining large quantities of highly potent MSCs and exosomes. Thus, an aspect of the invention includes methods for producing mesenchymal stem cells (MSCs) under ideal growth conditions. While the principles of the invention are also applicable to other cell types, such as stem cells and chondrocytes, the embodiments described herein focus on MSCs for simplicity.

[0015] In an aspect, the disclosure relates to a method of treating a disease or condition in a subject in need thereof, the method comprising: administering a therapeutic effective amount of a pharmaceutical composition comprising mesenchymal cells and exosomes to the subject; wherein the mesenchymal cells and exosomes are in a ratio of about 1: 2500 to 1: 250000, wherein the mesenchymal cells have a sternness quality more than 90% and viability more than 95%, and the exosomes are free of a cellular contaminant as detected by immunoblotting technique, wherein the cellular contaminant comprises calnexin, and treating said disease or condition.

[0016] In an embodiment, the cellular contaminant further comprises GM130.

[0017] In an embodiment, the exosomes have a zeta potential in a range of -10 mV to -50 mV, and a size range between 85 nm to 130 nm as measured using the ZetaView system by Particle Metrix.

[0018] In an embodiment, a ratio of the mesenchymal cells to exosomes is 1 :75,000. In an embodiment, a ratio of the mesenchymal cells to exosomes is 1 :10,000. In an embodiment, a ratio of the mesenchymal cells to exosomes is 1:5000.

[0019] In an embodiment, method of treatment further includes administering peripheral blood cells along with the mesenchymal cells and the exosomes.

[0020] In an embodiment, a disease or condition is selected from a neurological disease, a muscular disease, an autoimmune disease, an inflammatory disease, a digestive disease, an energy homeostasis disease, aging, radiation induced injury, cell transplant rejection and a proliferativeAttorney Docket No. SASC-OOl-OIWO disease. In a particular embodiment, the disease is a fibrotic disease. In a more particular embodiment, the fibrotic disease comprises liver fibrosis.

[0021] In an embodiment, the mesenchymal cells are derived from umbilical cord of a mammal. The mammal includes human.

[0022] In an embodiment, the exosomes are derived from the mesenchymal cells.

[0023] In an embodiment, the composition can be administered at least one of intravenous administration, intramuscular administration, intranasal administration, intrathecal administration, intrastriatal administration, intracranial administration, intraarterial administration, and subcutaneous administration.

[0024] In an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, excipient or carrier.

[0025] In an embodiment, the pharmaceutical composition farther comprises at least one additional therapeutic agent. In an embodiment, the pharmaceutical composition is configured to inhibit a lymphocyte proliferation by more than 85% in the subject having a fibrotic disease.

[0026] In an embodiment, the mesenchymal cells have population doubling time (PDT) about 23 hours or less without any addition of a growth stimulant.

[0027] In an embodiment, the exosomes have biomarkers selected from CD9, TSG101, and / or HSP70, and encapsulated by miRNA selected from miR-125a-3p, miR-146a, and / or miR-133.

[0028] An aspect of the disclosure relates to a) taking an explant comprising an umbilical cord; b) allowing release of cells from the explant; c) cryopreserving and thawing the cells at a temperature about 30°C to 40°C; and Ij harvesting mesenchymal cells; wherein the mesenchymal cells have a sternness quality more than 95% and viability more than 90%, wherein the method is configured to give 1.1 million mesenchymal cells per cm of the umbilical cord.

[0029] In an embodiment, the mesenchymal cells have cell size less than 15 micron.

[0030] In an embodiment, the mesenchymal cells have PDT about 25 hours or less without any addition of a growth stimulant.

[0031] In an embodiment, the method farther comprising deriving exosomes from the mesenchymal cells.

[0032] In an embodiment, the exosomes have biomarkers selected from CD9, TSG101, and / or HSP70, and are free of a cellular contaminant comprising Calnexin and / or GM130.

[0033] In an embodiment, the method further includes adding a pharmaceutical acceptable excipient in the mesenchymal cells and exosomes to form a phamraceutical composition.

[0034] In an embodiment, the exosomes have a size range between about 85 nm to about 130 nm as measured using the ZetaView system by Particle Metrix.

[0035] In an embodiment, the method provides about 1.0 x 1010exosomes per milliliter to 1.0 xAttorney Docket No. SASC-OOl-OIWO 1012exosomes per milliliter (which is equivalent to 5.8 x 106exosomes per microgram to 5.8 x 108exosomes per microgram of protein).

[0036] In an embodiment, the mesenchymal cells have an expression level of immune lineage markers comprising CD34, CDl lb, CD19, CD45, and HLA-DR less than 1.5%.

[0037] In an aspect, the present disclosure relates to a pharmaceutical composition comprising mesenchymal cells and exosomes in a ratio of about 1 : 2500 to 1: 250000, wherein the mesenchymal cells have a sternness quality more than 95% and viability more than 90%, and the exosomes are free of a cellular contaminant as detected by immunoblotting technique, wherein the cellular contaminant comprises calnexin.

[0038] In an embodiment, the mesenchymal cells have an expression level of immune lineage markers comprising CD34, CD1 lb, CD19, CD45, and HLA-DR less than 1.5%.

[0039] In an embodiment, the mesenchymal cells have PDT about 25 hours or less without any addition of a growth stimulant.

[0040] In an embodiment, the exosomes have biomarkers selected from CD9, TSG101, and / or HSP70, and encapsulated by miRNA selected from miR-125a-3p, miR-146a, and / or miR-133.

[0041] In an embodiment, mesenchymal cells have cell size less than 15 micron, and the exosomes have a size in range of about 85 nm to about 130 nm as measured using the ZetaView system by Particle Metrix.

[0042] In an embodiment, the composition further comprises a pharmaceutical acceptable excipient.

[0043] In an embodiment, the composition has a ration of mesenchymal cells to exosomes in a ratio of about 1:5000 to 1 :100000.

[0044] In an embodiment, the mesenchymal cell is derived from an umbilical cord, and the exosomes are derived from the mesenchymal cells.

[0045] In an embodiment, the pharmaceutical composition is configured to treat a fibrotic disease.

[0046] In an embodiment, the pharmaceutical composition is configured to inhibit a lymphocyte proliferation by more than 85% in the subject having a fibrotic disease. In an embodiment, the composition is configured to inhibit a lymphocyte proliferation by more than 90% in the subject having a fibrotic disease. In an embodiment, the composition is configured to inhibit a lymphocyte proliferation by more than 95% in the subject having a fibrotic disease. In an embodiment, the composition is configured to inhibit a lymphocyte proliferation by more than 99% in the subject having a fibrotic diseaseBRIEF DESCRIPTION OF THE FIGURES

[0047] Fig. 1 shows expression of MSCs cell surface protein markers.

[0048] Fig. 2A shows representative images of expression of exosomal protein analysis earnedAttorney Docket No. SASC-OOl-OIWO out Exo-Check exosome antibody array.

[0049] Fig. 2B shows representative images of expression of exosomal protein analysis carried out immunoblotting techniques.

[0050] Fig. 3A shows images of exosome size analysed by ZetaView, Particle Matrix.

[0051] Fig. 3B shows morphology of exosomes by TEM.

[0052] Fig. 4A shows images of functional assays to establish effect of exosomes, MSCs and combination of exosomes & MSCs on immunomodulatory functions such as inhibition of lymphocyte proliferation.

[0053] Fig. 4B shows % inhibition of lymphocyte proliferation by exosomes, MSCs and combination of exosomes & MSCs.

[0054] Fig. 4C shows images of functional assays to establish effect of exosomes, MSCs and combination of exosomes & MSCs on immunomodulatory functions such as increase of regulatory T cells.

[0055] Fig. 4D shows % change in T regulatory cells by exosomes, MSCs and combination of exosomes & MSCs.

[0056] Fig. 4E shows images of functional assays to establish effect of exosomes, MSCs and combination of exosomes & MSCs on angiogenic functions such as tube formation assays.

[0057] Fig. 4F shows images of functional assays to establish effect of exosomes, MSCs and combination of exosomes & MSCs on angiogenic functions such as wound scratch assay.

[0058] Fig. 5A shows images of functional assays to establish effect of different ratios of MSCs to exosomes on immunomodulatory functions such as inhibition of lymphocyte proliferation.

[0059] Fig. 5B shows % inhibition of lymphocyte proliferation by different ratio different ratios of MSCs to exosomes on immunomodulatory functions such as

[0060] Fig. 5C shows images of functional assays to establish effect of different ratios of MSCs to exosomes on angiogenic functions such as tube formation assays

[0061] Fig. 5D shows images of functional assays to establish effect of different ratios of MSCs to exosomes on angiogenic functions such as wound scratch assay.

[0062] Fig. 6 Masson's Trichrome staining of liver tissues from disease control and treatment groups. Liver fibrotic histopathological scores are expressed as mean ± SEM. Statistical significance for the treatment group (n=14) compared to the disease control group (n=8) is indicated by ***p<0.001.

[0063] Fig. 7 Picrosirius Red (PSR) staining of liver tissues from disease control and treatment groups Liver fibrotic histopathological scores are expressed as mean ± SEM. Statistical significance for the treatment group (n=14) compared to the disease control group (n=8) is indicated by ***p<0.001.Attorney Docket No. SASC-OOl-OIWO

[0064] Fig. 8 Immunohistochemical staining with a-SMA in liver tissues from disease control and treatment animal groups. The IHC scores representing the percentage of positive area were determined using Fiji Software. The results are expressed as mean ± SEM. Statistical significance for the treatment group (n=14) compared to the disease control group (n=8) is indicated by ***p<0.001.

[0065] Fig. 9 Immunohistochemical staining with collagen (COL1 Al) in liver tissues from disease control and treatment animal groups. The IHC scores representing the percentage of positive area were determined using Fiji Software. The results are expressed as mean ± SEM. Statistical significance for the treatment group (n=14) compared to the disease control group (n=8) is indicated by ***p<0.001.

[0066] Fig. 10 Immunohistochemical staining with Cytokeratin 18 in liver tissues from disease control and treatment animal groups. The IHC scores representing the percentage of positive area were determined using Fiji Software. The results are expressed as mean ± SEM. Statistical significance for the treatment group (n=14) compared to the disease control group (n=8) is indicated by ***p<0.001.

[0067] Fig. 11 Immunohistochemical staining with Cytokeratin 19 in liver tissues from disease control and treatment animal groups. The IHC scores representing the percentage of positive area were determined using Fiji Software. The results are expressed as mean ± SEM. Statistical significance for the treatment group (n=14) compared to the disease control group (n=8) is indicated by ***p<0.001.

[0068] Fig. 12 shows effect of MSCs, exosomes, and combined MSCs + exosomes on liver morphology following acetaminophen-induced acute liver failure in rats.

[0069] Fig. 13 shows % cell distribution in two sets of cultured cells source from two independent raw materials, evaluated by single cell RNA- sequencing technology.

[0070] Fig. 14 shows Heat map of gene expression across biological pathways in mesenchymal stromal cells (MSCs) population evaluated by single cell RNA- sequencing technology.DETAILED DESCRIPTIONDefinitions and General Techniques

[0071] For simplicity and clarity of illustration, the drawing figures illustrate the general manner of construction, and descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the present disclosure. Additionally, elements in the drawing figures are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of embodiments of the present disclosure. The same reference numerals in different figures denotes the same elements.Attorney Docket No. SASC-OOl-OIWO

[0072] The terms “first," “second," “third," “fourth," and the like in the description and in the claims, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms “include,” and “have,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, device, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such process, method, system, article, device, or apparatus.

[0073] The terms “left,” “right,” “front,” “back,” “top,” “bottom,” “over,” “under,” and the like in the description and in the claims, if any, are used for descriptive purposes and not necessarily for describing permanent relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments of the apparatus, methods, and / or articles of manufacture described herein are, for example, capable of operation in other orientations than those illustrated or otherwise described herein.

[0074] No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include items, and may be used interchangeably with “one or more.” Furthermore, as used herein, the term “set” is intended to include items (e.g., related items, unrelated items, a combination of related items, and unrelated items, etc.), and may be used interchangeably with “one or more.” Where only one item is intended, the term “one” or similar language is used. Also, as used herein, the terms “has,” “have,” “having," or the like are intended to be open-ended terms. Further, the phrase “based on” is intended to mean “based, at least in part, on” unless explicitly stated otherwise.

[0075] The present disclosure may be embodied in other specific forms without departing from its spirit or characteristics. The embodiments described are to be considered in all respects only as illustrative and not restrictive. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

[0076] As defined herein, “approximately” or “about” can, in some embodiments, mean within plus or minus ten percent of the stated value. In other embodiments, “approximately” or “about” can mean within plus or minus five percent of the stated value. In further embodiments, “approximately” or “about” can mean within plus or minus three percent of the stated value. In yet other embodiments, “approximately” or “about” can mean within plus or minus one percent of the stated value.Attorney Docket No. SASC-OOl-OIWO

[0077] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, health monitoring described herein are those well-known and commonly used in the art.

[0078] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. The nomenclatures used in connection with, and the procedures and techniques of embodiments herein, and other related fields described herein are those well-known and commonly used in the art.

[0079] The following terms and phrases, unless otherwise indicated, shall be understood to have the following meanings.

[0080] Mesenchymal Stem Cells (MSCs)

[0081] Mesenchymal stem cells (MSCs) (also known as stromal stem cells) are non-blood adult stem cells found in a variety of tissues. They are characterized by their spindle-shape morphologically, by the expression of specific markers on their cell surface, and by their ability, under appropriate conditions, to differentiate along a minimum of three lineages (osteogenic, chondrogenic, and adipogenic).

[0082] In an embodiment, MSCs are derived from mammals. Mammals include humans, dogs, cats, mice, rats, and transgenic species thereof. In an embodiment, MSCs are derived from humans.

[0083] In an embodiment, MSCs are derived from tissues such as but not limited to bone marrow, adipose tissue, umbilical cord, placenta, dental tissue, and etc. In an embodiment, MSCs are derived from umbilical cord. In a specific embodiment, MSCs are derived from human umbilical cords.

[0084] In an embodiment, MSCs are derived from a desired tissue in an invitro condition. The in vitro condition refers to a sterile condition optimal to allow culturing of tissue, so that cells are free from contamination of microbes such as bacteria, fungi, mycoplasma, etc.. The in vitro condition involves providing adequate culture medium / nutrients (e.g., DMEM, RPMI, MEM) containing amino acids, salts, vitamins, glucose, buffering system (e.g., bicarbonate), physical environment such as culturing temperature, oxygen level, CO2 incubator, etc. while maintaining safety and regulatory standards.

[0085] In an embodiment, size of MSCs is in a range of about 8 pm to 12 pm, with minimum size selected from 8 pm, 9 pm, 10 pm and maximum size selected from 11 pm, 12 pm.Attorney Docket No. SASC-OOl-OIWO

[0086] In an embodiment, Umbilical Cord-derived Mesenchymal Stromal Cells (UC-MSCs) exhibit high-quality characteristics, including a notably small average cell size of 9.8 gm, significantly smaller than what has been reported in existing literature. Previous studies have shown that smaller-sized MSCs often possess enhanced functionality across various biological activities.

[0087] In an embodiment, human Umbilical Cord-derived Mesenchymal Stromal Cells (hUC- MSCs) exhibit high-quality characteristics, including a notably small average cell size of 9.8 pm, significantly smaller than what has been reported in existing literature. Previous studies have shown that smaller-sized MSCs often possess enhanced functionality across various biological activities.

[0088] The population doubling time (PDT) for MSCs refers to the time required for the cell population to double in number under specific culture conditions. It varies significantly depending on several biological and environmental factors. For example: bone marrow-derived MSCs (BM- MSCs) have PDT about 40-60 hours. It leads to slower proliferation: increases with donor age or passage number; adipose-derived MSCs (AD-MSCs) have PDT about 20-40 hours, generally faster growth than BM-MSCs; umbilical cord-derived MSCs (UC-MSCs) have about 18-36 hours , very high proliferative capacity, especially at early passages, and dental pulp-derived MSCs (DP- MSCs) have about 24-48 hours, which provides intermediate growth rate.

[0089] UC-MSCs of the present disclosure demonstrate robust proliferative capacity, as evidenced by a shorter population doubling time (PDT) of 24 + 2 hours and a high number of colony -forming units (CFUs), both of which are indicative of strong proliferative potential and cellular vitality.

[0090] In an embodiment, MSCs described in one or more embodiments of the present disclosure has PDT of about 18± 2 hours 19± 2 hours, 20± 2 hours, 21± 2 hours, 22+ 2 hours, 22+ 2 hours, 23+ 2 hours, 24+ 2 hours, 25+ 2 hours, 26+ 2 hours, 27+ 2 hours, 28+ 2 hours.

[0091] The viability of mesenchymal stem cells (MSCs) refers to the percentage of living, metabolically active cells in a given population after isolation, culture, cryopreservation, or experimental treatment.

[0092] In an embodiment, MSCs described in one or more embodiments of the present disclosure have viability more than 90%, 92%, 94%, 96%, 98%, 99%, 99.99% or more, as assessed by trypan blue exclusion.

[0093] In an embodiment, UC-MSCs consistently demonstrate over 90% viability as assessed by trypan blue exclusion. In addition to membrane integrity-based staining, IC-1 dye, which indicates cell metabolic fitness, and also indicates that more than 90% of the cells are metabolically active and healthy.

[0094] Membrane integrity-based staining is a class of cell viability assays that distinguishes liveAttorney Docket No. SASC-OOl-OIWO and dead cells based on the integrity of the plasma membrane. It’s widely used for MSCs and other primary cells because loss of membrane integrity is one of the earliest and most reliable indicators of cell death (necrosis or late apoptosis). In an embodiment, MSCs described in one or more embodiments of the present disclosure have membrane integrity-based staining more than 90%, 92%, 94%, 96%, 98%, 99%, 99.99% or more, as assessed by IC-1 dye.

[0095] According to some embodiments, the isolating MSCs comprises isolation of cells expressing a plurality of surface marker selected from CD9, CD10, CD13, CD26, CD29, CD44, CD36, CD46, CD47, CD49a, CD49b, CD49c, CD49d, CD49e, CD50, CD51 / 61, CD54, CD55, CD58, CD59, CD61, CD63, CD71, CD73, CD81, CD83, CD87, CD90, CD91, CD95, CD97, CD98, CD99, CD105, CD108, CD109, CD140b, CD142, CD146, CD147, CD151. CD164, CD165, CD166, CD273, 02-microglobulin, HLA-A,B,C, HLA-A2, and STRO1.

[0096] No single marker that delineates MSCs in vivo has been identified due to a lack of consensus regarding the MSC phenotype, but it generally is considered that MSCs are positive for cell surface markers such as CD105, CD166, CD90, and CD44, and that MSCs are negative for typical hematopoietic antigens, such as CD45, CD34, and CD14. As for the differentiation potential of MSCs, studies have reported that populations of bone marrow-derived MSCs have the capacity to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic-supporting stroma. Studies using transgenic and knockout mice and human musculoskeletal disorders have reported that MSCs differentiate into multiple lineages during embryonic development and adult homeostasis.

[0097] In an embodiment, MSCs described in one or more embodiments of the present disclosure have expression levels of the positive MSC markers selected from such as CD105, CD166, CD90, CD73 and CD44, more than 95%, 96%, 97%, 98%, 99% or more. In an embodiment, expression level of collective positive MSCs markers selected from CD105, CD166, CD90, CD73 and CD44 in MSCs described in one or more embodiments of the present disclosure is more than 95%, 96%, 97%, 98%, 99% or more. In an embodiment, expression level of different combinations of MSCs markers selected from CD105, CD166, CD90, CD73 and CD44 in MSCs described in one or more embodiments of the present disclosure is more than 95%, 96%, 97%, 98%, 99% or more.

[0098] In an embodiment, MSCs described in one or more embodiments of the present disclosure have expression levels of the negative MSCs markers selected from CD 14, CD34, CD1 lb, CD 19, CD45, and HLA-DR are less than 2%, 1%, 1.5%, 0.5%, 0.4%, 0.3%, 0.2% or less. In an embodiment, expression level of different combinations of negative MSCs markers is less than 2%, 1%, 1.5%, 0.5%, 0.4%, 0.3%, 0.2% or less. In an embodiment, expression level of different collective negative MSCs markers selected from CD 14, CD34, CDllb, CD19, CD45, and HLA-Attorney Docket No. SASC-OOl-OIWO DR is less than 2%, 1%, 1.5%, 0.5%, 0.4%, 0.3%, 0.2% or less.

[0099] In an embodiment, UC-MSCs of the present invention had expression levels of the positive MSC markers CD73 (99.6%), CD90 (99.8%), and CD105 (99.3%), all exceeding the >95% threshold established by the International Society for Cell & Gene Therapy (ISCT). In an embodiment, the expression level of positive MSC markers is more than 95%, 96%, 97%, 98%, 99%, 99.9% or more.

[0100] Conversely, the cells exhibited minimal expression of hematopoietic and immune lineage markers such as CD34, CD1 lb, CD19, CD45, and HLA-DR, with a combined expression level of 1.43%, well within the ISCT-recommended limit of <2%.

[0101] Sternness quality in mesenchymal stem (or stromal) cells (MSCs) refers to the extent to which MSCs retain their fundamental stem cell properties, such as their self-renewal ability, multi potency, and expression of characteristic surface markers and genes. In addition to common MSC markers, 9 additional functional protein markers denoting the “sternness” quality also demonstrate >90% expression consistently.

[0102] Analyses of the in vitro differentiation of MSCs under appropriate conditions that recapitulate the in vivo process have led to the identification of various factors essential for stem cell commitment. Among them, secreted molecules and their receptors (e.g., transforming growth factor-(3), extracellular matrix molecules (e.g., collagens and proteoglycans), the actin cytoskeleton, and intracellular transcription factors (e.g., Cbfal / Runx2, PPARy, Sox9, and MEF2) have been shown to play important roles in driving the commitment of multipotent MSCs into specific lineages, and maintaining their differentiated phenotypes.

[0103] The term “high expression” refers to the upregulation of a gene’s expression or function relative to a comparable product or control. High expressions could be at least one fold, 2 folds, 4 folds, 5 folds, 10 folds, 15 folds, 20 folds or more increase in the expression compared to the control.

[0104] In an embodiment, functionality of MSCs as described in one or more embodiments of present disclosure has high expression of at least one of anti-apoptotic and anti-oxidant genes, such as BCL2 (B-cell lymphoma 2), BCL-XL (BCL2L1) similar to BCL-2; MCL-1 (Myeloid cell leukemia 1), XIAP (X-linked inhibitor of apoptosis protein), BIRC5, FOXO3 (Forkhead box 03), SOD1, SOD2, SOD3 (Superoxide dismutases), GPX1, GPX3 (Glutathione peroxidases), CAT (Catalase), PRDXs (Peroxiredoxins 1-6), PRDX1 and PRDX2, GSR (Glutathione reductase), Nrf2 (Nuclear factor erythroid 2-related factor 2), antioxidant response elements (AREs), HO-1 (Heme oxygenase- 1).

[0105] In an embodiment, functionality of MSCs as described in one or more embodiments of present disclosure has high expression of at least one of TIMP1, VEGFA, EDIL3, DKK3, IGFBP3,Attorney Docket No. SASC-OOl-OIWO LIF, TGFB1, ALCAM, PTGS2, KLF4, NFKBIA. In an embodiment, umbilical cord derived MSC has high expression of at least one of TIMP1, VEGFA, EDIL3, DKK3, IGFBP3, LIF, TGFB 1, ALCAM, PTGS2, KLF4, NFKBIA.

[0106] Mesenchymal cells derived Exosomes

[0107] MSC-derived EVs, which include exosomes and microvesicles (MV), are involved in cell- to-cell communication, cell signaling, and altering cell or tissue metabolism at short or long distances in the body, and can influence tissue responses to injury, infection, and disease (Phinney, D G and Pittenger, M F. Stem Cells (2017) 35: 851-58). Their content includes cytokines and growth factors, signaling lipids, mRNAs, and regulatory miRNAs (Id.). The content of MSC EVs is not static; they are a product of the MSC tissue origin, its activities, and the immediate intercellular neighbors of the MSCs (Id.).

[0108] Exosomes are often regarded as superior to other extracellular particles, such as microvesicles and apoptotic bodies, due to their unique biogenesis, size, and functional properties. Unlike microvesicles, which bud directly from the plasma membrane, exosomes originate from endosomal compartments, resulting in a more consistent size (30-150 nm) and a more controlled molecular cargo. This endosomal origin allows for selective packaging of proteins, lipids, and RNAs, making exosomes highly effective in cell-to-cell communication. In contrast, apoptotic bodies are generally larger and result from cell death, containing random cellular debris rather than functional signaling molecules. Furthermore, exosomes are more stable in circulation and less immunogenic, which makes them especially attractive for use in regenerative medicine.

[0109] In an embodiment, exosomes derived from mesenchymal stem cells (MSCs) were isolated using the filtration method.

[0110] “Yield" means how many exosomes are produced or can be collected from a defined amount of substrate (protein). In an embodiment, exosomes are produced in an amount about 4x 107exosomes per microgram of protein, about 5x 107exosomes per microgram of protein, about 10 x 107exosomes per microgram of protein, about 20x 107exosomes per microgram of protein, about 30x 107exosomes per microgram of protein, about 50x 107exosomes per microgram of protein, 70x 107exosomes per microgram of protein, about lOOx 107exosomes per microgram of protein. In a particular embodiment, the exosome isolation technology used in the present disclosure consistently giving high yield, producing approximately 1.0 x 1011exosomes per milliliter, which is equivalent to 5.8 x 107exosomes per microgram of protein.

[0111] Zeta potential is a measure of the electrostatic charge on the surface of particles suspended in a liquid. Exosomes have a lipid bilayer membrane containing charged molecules (like phospholipids, proteins, and glycoproteins). Because of these surface molecules, exosomes typically carry a negative charge in physiological buffers. In an embodiment, the exosomes of oneAttorney Docket No. SASC-OOl-OIWO or more embodiments have a zeta potential of about 25 mV, about 30 mV, about 35 mV, about 40 mV, about 45 mV. In an embodiment, zeta potential of exosomes is in a range of about 30 mV to 40 mV. In an embodiment, the exosomes have zeta potential of about -37.4 ± 6.6 mV. This indicates strong colloidal stability and high bioactive functionality.

[0112] In an embodiment, exosomes are enriched with a range of beneficial microRNAs (miRNAs) and several functional proteins. Enriched means that a particular miRNA is about 2- 50x or more abundant in exosomes than in the donor cell or in control exosomes. In some cases, miRNA may be about 5 times, 10 times, 15 times, 20 times, 25 times, 50 times, 100 times or more abundant in exosomes than in the donor cell or in control exosomes

[0113] In an embodiment, the exosomes comprising one or more proteins selected from the group consisting of: CASK, COL3A1, B2M, CDH2, CTNNA1, DLG1, EGFR, F3, FARP1, GPC1, CDH2, CTNNA1, HAPLN1, LAMB1, LAMB2, LAMPCI, LGALS3BP, LOXL2, MCAM, NIDI, OLXNB2, S100A6, TNC, WNT5A, and PLXNB2.

[0114] In an embodiment, the exosomes comprising one or more miRNA selected from miR 378a 3p, miR 339 5p, miR 1273h 5p, miR-125a-3p, miR-146a, miR-133, miR-672-5p, miR 6724 5p, anti-inflammatory miRNAs, including miR-125a-3p, which is known to promote the expansion of regulatory T cells (Tregs); miR- 146a, which plays a key role in suppressing the NF-KB signalling pathway; and miR-133, which is involved in cellular repair mechanisms, highly scored miRNAs listed below along with beneficial roles reported in research papers.

[0115] In an embodiment, an average size of exosomes of one or more embodiments of present disclosure are about 60 nm, about 70 nm, 80 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, as measured according to ZetaView system by Particle Metrix.

[0116] In an embodiment, shape of exosomes of one or more embodiments of present disclosure are spherical to cup-shaped morphology.

[0117] In an embodiment, exosomes of the present disclosure have high purity. Purity refers to freeing exosome from contaminants, such as proteins, lipoproteins, or other extracellular vesicles (micro vesicles, apoptotic bodies, etc.). In an embodiment, the purity of exosomes is more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.99% ormore. High purity and identity of the exosome preparation, help in downstream therapeutic applications. In an embodiment, exosomes have contaminants less than 2%, 1.5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less. In an embodiment, the contaminant cannot be detected in a blotting gel such as western blotting. Negative markers: Calnexin, GM130, ApoAl can be used to confirm purity. In an em

[0118] In an embodiment, the exosomes as described in one or more embodiments of the present disclosure have at least one of a protein markers selected from CD63, EpCAM, ANXA5, TSG101,Attorney Docket No. SASC-OOl-OIWO Flotillin- 1 (Flotl), GM130, ICAM, Alix, and CD81. The expression level of one of these protein markers were more than 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In an embodiment, the exosomes have exosomal markers such as CD9, TSG101, and HSP70, wherein the expression of individual positive biomarkers or combinations of the positive biomarkers in the exosomes are more than 95%, 96%, 97%, 98%, 99%, or more.

[0119] In an embodiment, exosomes as described in one or more embodiments of the present disclosure have no detectable levels of cellular contaminants or negative markers selected from Calnexin, GM130 and ApoAl . No detectable means that the expression level of the proteins are less than 2%, 1.5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less that cannot be detected via an immunoblotting technique.

[0120] Absence of cellular contaminants in exosomes represents the integrity and specificity of the exosome preparation. No detectable means that protein band could not be seen in an immunoblotting technique.

[0121] Method of isolation of MSCs

[0122] In an embodiment, the present disclosure relates to high-potency adult mesenchymal stem cells (MSC) and high-potency adult mesenchymal stem cell population and to a method of preparing high-potency adult mesenchymal stem cells or high-potency adult mesenchymal stem cell population through a cost-effective and high-yield method. The method of the present application provides a high yield of stem cells capable of supplying one or more therapeutic doses and result in stem cells and populations having stable characteristics and properties.

[0123] An embodiment relates to method of isolation of MSCs from a tissue.

[0124] A “population”, as used herein, refers to a cell culture wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or all cells of the culture have a similar profile. In some embodiments, a population is an enriched population.

[0125] “MSC population”, as used herein, refers to a population of MCSs having a unique profile, particularly an expression profile, including but not limited to, one or more unique proteins (e.g., surface markers and secreted proteins), genes, and one or more coding and non-coding RNAs (e.g., miRNA and IncRNA). An MSC population may also be characterized by extracellular vesicles having a unique profile, secreted from said MSCs such as exosomes and extracellular vesicles.

[0126] MSC population may be isolated from a particular source or alternatively can be manipulated by various factors, known to a skilled artisan, to encompass and be defined by the unique profile as specified herein below. Tissue could be selected form bone marrow, adipose tissue, umbilical cord (Wharton’s jelly), Placenta / amniotic membrane, non-invasive (post-birth tissue), dental pulp / gingiva, synovial membrane / fluid, endometrial tissue, skin dermis. DifferentAttorney Docket No. SASC-OOl-OIWO MSC populations having a unique expression profile can be isolated from different tissues.

[0127] Optionally, tissue could be fragmented or sliced into small pieces, such as 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 10 mm, as desired.

[0128] The tissue as desired in one of the embodiments, then sterically allowed to incubate in a culture condition with optimal medium and physical condition. Optionally, to facilitate binding of released cells, the culturing of the tissue can be performed in a culture vessel coated with cell binding reagents.

[0129] After the population of cells in culturing tissue has reached about 95%, 90%, or 80% confluence, the cells are harvested and cryopreserved. For harvesting, protease enzyme could be used that would help to remove the cell bound on the culturing vessel.

[0130] The cryopreserved cells are thawed at a temperature of about 30-40°C, more specifically around 35 °C, and re-cultured into a suitable culture medium and condition. The cells are harvested after certain time or when the cells have reached a desired confluence of about 95%, 90%, or 80% in the culture. This step may help in selecting a subpopulation of MSCs that have similar profile compared to rest of cells in the population.

[0131] A “subpopulation”, as used herein, refers to one of the MSC populations defined herein, being further manipulated by various growth conditions so as to endow a further unique expression profile to said MSCs. Each population consists of additional subpopulations exhibiting different combinations of expression profiles or different levels of expression. As used herein, “various growth conditions” includes but is not limited to enrichment by one or more protein (e.g., growth factors), one or more coding or non-coding RNAs, and growth in selective conditions and / or media (e.g., scaffold, hypoxia, glucose concentrations, co-cultures).

[0132] The cells are checked with specific surface markers, micro RNA and etc. can be used to identify MSCs in the MSC population. “Surface marker”, as used herein, refers to any type of antigenic determinant on the surface of the plasma membrane of an MSC.

[0133] “Non-coding RNA”, as used herein, refers to an RNA molecule that is not translated into a protein. Non-coding RNAs include microRNAs (miRs) and long non-coding RNAs (IncRNAs). Both of these non-coding RNAs are known to regulate mRNA stability and translation and thus protein expression. Sequences for the miRs presented herein can be found in resources such as the miR databases “miRbase” (www.mirbase.org) and miRDB (www.mirdb.org). Sequences of IncRNAs presented herein can be found in IncRNA databases such as IncRNAdb (www.lncmadb.org) and LNCipedia (http: / / lncipedia.org).

[0134] As used herein, the term “P0 cells” or “seed cells” refer to the cells directly retrieved from the organ tissue.

[0135] As used herein, the term “Pl” refers to passage 1 which is the subculture of the P0 cells orAttorney Docket No. SASC-OOl-OIWO seed cells and Pl cells are the cells obtained from passage 1 or Pl.

[0136] As used herein, the term “P2” refers to passage 2 which is the subculture of the Pl cells and P2 cells are the cells obtained from passage 2 or P2. Similarly, as used herein the term “P3” refers to passage 3 which is the subculture of the P2 cells and P3 cells are the cells obtained from passage 3 or P3.

[0137] As used herein, the term “Pn” refers to passage n which is the subculture of the Pn-1 cells and Pn cells are the cells obtained from passage n or Pn. Here ‘n’ is any positive integer.

[0138] As used herein, the terms “media” or “medium” or “cell culture media” or “cell culture medium” are used interchangeably and refer to culture media, growth media or nutrient media in solid, semi-solid or liquid form, that comprises nutrients suitable for the growth of stem cells.

[0139] In an embodiment, platelets obtained from the blood bank are pooled with subsequent subjection to 3 freeze-thaw cycles. Centrifugation is performed to remove cell debris from the ruptured platelets. The collected supernatant is incubated with 20mM calcium chloride at 37C for 4 hours followed by 4C for 24 hours. Further centrifugation is performed at 10000 or more RPM. The supernatant is isolated and stored at -20C for future use.

[0140] In another aspect, the present invention provides novel methods of producing HPL that increase the efficiency of fibrinogen free HPL production. The method comprises the steps of: a. Subjecting platelets to three freeze thaw cycles; b. Centrifuging the platelets of step a. to remove cell debris and collecting the supernatant: c. Incubating the supernatant collected in step b. with 5mM-20mM calcium chloride at 37°C for 4 hours followed by 4°C for 20 to 30 hours; d. Centrifuging the supernatant of step c. and e. Collecting the supernatant.

[0141] In preferred aspects related to producing fibrinogen free HPL, the centrifugation in step d. is carried out at a high speed of 6000 RPM to 20,000 RPM or more. In even more preferred aspects, the centrifugation in step d. is carried out at a high speed of 10000 RPM.

[0142] In preferred embodiments, the fine pieces of organ tissue remaining after harvesting the P0 stem cells are again seeded in an altered culture vessel, In further preferred embodiments the process of re-seeding the fine pieces of organ tissue remaining after harvesting the P0 stem cells is repeated 2-10 times.

[0143] As used herein the term “culture vessel” refers to any vessel used for seeding, growth and / or culture of cells and / or tissues. The culture vessel could include a variety of cell or tissue culture flasks, plates, multi-well plates, dishes, bottles, tubes or bioreactors.

[0144] As used herein the term “altered culture vessel” refers to any culture vessel whose surface has been coated or treated to increase the adhesion of biological cells and tissues to the culture vessel. The altered culture vessels include but are not limited to culture vessels coated or treated with substances that increase the adhesion of biological cells and tissues to the culture vessel; orAttorney Docket No. SASC-OOl-OIWO with substances which alter the surface charge or oxygen level or hydrophilicity of the culture vessel; or with substances which increase the culture vessel surface oxygen level thereby increasing negative charge and making the culture vessel surface more hydrophilic.

[0145] In some embodiments, the altered culture vessel used in one or more steps of the method of the present invention is a culture vessel that is coated with one or more extracellular matrix (ECM) proteins such as collagen, fibronectin, laminin, gelatin, vitronectin, tenascin C, aggrecan, Matrigel ® (solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), cellbind or any other surface treatment that enhances tissue and cell attachment, etc. In further embodiments, the altered culture vessel used in one or more steps of the methods of the present invention is a culture vessel that is coated with synthetic polymers or peptides such as Synthemax®!! (a synthetic vitronectin peptide-copolymer), poly-L-lysine, etc.

[0146] In a preferred aspect, expanding the P0 stem cells by serial passage comprises seeding and culturing the P0 stem cells or the stem cells obtained from the previous passage in cell culture media present in roller bottles maintained at a low rotational speed of about 0.5 RPM to 9 RPM during the stages of both seeding and culturing, in each passage. The rotational speed of the roller bottles is increased to 5 to 80 RPM for harvesting the cells in each passage. The inventor of the present application found that maintaining a low rotational speed of about 0.5 RPM to 9 RPM during both the seeding and culturing stages in each passage. During harvest the RPM is increased to 80 for 30 mins.

[0147] In an embodiment, a method for reducing cell aggregation during harvesting of mesenchymal stem cells cultured in roller bottles, comprising performing periodic medium exchanges and harvesting the cells at a confluence level below 90%. Cell strainer can exclude large aggregates.

[0148] During the process, glucose and pH to be monitored daily or every other day throughout culturing of cells during each passage, to determine the optimum time for medium exchange. In preferred aspects, medium is exchanged at pH of 7-7.5 and the lowest glucose concentration of 30 mg / dl. By monitoring both glucose and pH during culturing, the inventor of the present invention found that medium exchange should be carried out at a much shorter duration than was being done conventionally. The medium exchange at the optimum time is important for optimal cell culturing.

[0149] At 90% confluence, cells are harvested with TrypLE, a gentle protease and further cryopreserved. The cell detachment agent or cell dissociation reagent suitable for use in the methods of the present invention include but are not limited to a gentle protease, such as a gentle trypsin; a trypsin-like protease, such as TrypEE™; a trypsin alternate, such as Accutase™ which is a mixture of proteolytic and collagenolytic enzymes from crustaceans; a neutral protease, such as dispase; or mixtures thereof. Gentle enzymes are less disruptive to cell morphology and function.Attorney Docket No. SASC-OOl-OIWO

[0150] In an embodiment, culturing mesenchymal stem cells (MSCs) under reduced oxygen tension (approximately 2-5% O2).

[0151] In one aspect, the present invention relates to a novel method for producing mesenchymal stem cells, that achieves large quantities of highly potent MSCs and provides a therapeutic dose or several therapeutic doses. The method comprises: i. Mincing an organ tissue to obtain fine pieces of organ tissue; ii. Seeding the fine pieces of organ tissue in an altered culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells; iii. Harvesting the P0 stem cells using a mild cell detachment agent; and iv. Expanding the P0 stern cells obtained in step c. by serial passage. v. Optionally, re-seeding the residual explants.

[0152] In an embodiment, the altered culture vessel is selected from the group consisting of:• culture vessel coated with extracellular matrix proteins such as collagen, fibronectin, laminin, gelatin, vitronectin, tenascin C, aggrecan, Matrigel®;• culture vessel coated with synthetic polymers or peptides like Synthemax®II, poly-L- lysine or with glycosaminoglycans such as heparan sulfate proteoglycan;• culture vessel with altered surface charge; and• culture vessel with altered surface oxygen levels or with altered surface hydrophilicity, such as, Corning® CellBIND® Surface.

[0153] In a further aspect, the organ tissue is minced into pieces of size 1 mm to 1 cm. In another aspect, the organ tissue is a tissue derived from adipose tissue, umbilical cord, or placenta. In preferred aspects, the organ tissue is a tissue derived from umbilical cord.

[0154] Further aspects of the present invention require glucose and pH to be monitored throughout culturing of cells during each passage, to determine the optimum time for medium exchange. In preferred aspects, glucose and pH are monitored daily or on alternate days. In preferred aspects, medium is exchanged at pH of 7-7.5 and a glucose concentration of 30-120 mg / dl.

[0155] In an aspect expanding the P0 stem cells by serial passage comprises seeding the P0 stem cells or the stem cells obtained from the previous passage into cell culture media, culturing the cells and harvesting the cells, in each passage. In a preferred aspect, expanding the P0 stem cells by serial passage comprises seeding and culturing the P0 stem cells or the stem cells obtained from the previous passage in cell culture media present in roller bottles maintained at a low rotational speed of about 0.5 RPM to 9 RPM during the stages of both seeding and culturing, in each passage. The rotational speed of the roller bottles is increased to 5 to 80 RPM for harvesting the cells in each passage.Attorney Docket No. SASC-OOl-OIWO

[0156] In yet another aspect, the present invention relates to a novel method for producing mesenchymal stem cells, that achieves large quantities of highly potent MSCs and provides a therapeutic dose or several therapeutic doses. The method comprises i. Mincing an organ tissue to obtain fine pieces of organ tissue; ii. Seeding the fine pieces of organ tissue in a culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells; iii. Monitoring glucose and pH every day or every alternate day, to determine the optimum time for medium exchange; iv. Harvesting the P0 stem cells using a mild cell detachment agent; and v. Expanding the P0 stem cells obtained in step c. by serial passage. vi. Optional re-seeding the residual explants.

[0157] In a further aspect, the present invention relates to a novel method for producing mesenchymal stem cells, that achieves large quantities of highly potent MSCs and provides a therapeutic dose or several therapeutic doses. The method comprises: i. Mincing an organ tissue to obtain fine pieces of organ tissue; ii. Seeding the fine pieces of organ tissue in an altered culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells; iii. Monitoring glucose and pH every day or every alternate day, to determine the optimum time for medium exchange; iv. Harvesting the P0 stem cells using a mild cell detachment agent; and v. Expanding the P0 stem cells obtained in step c. by serial passage. vi. Optional Re-seeding the residual explants.

[0158] The methods for producing mesenchymal stem cells of the present invention achieves the following surprising technical features: i. a very high MSC yield of about 384 million P0 cells, 6 billion Pl cells and about 98 billion P2 cells and up to 4000 trillion P9 cells from a single umbilical cord. ii. a very high MSC density of 1.1 million cells per cm of umbilical cord. iii. a high cell viability of more than 98%; iv. spindle-shaped morphology of nearly all cells; v. a cell diameter of about 9-11 pm; vi. a population doubling time (PDT) of about 22 hours at passage 0, 1 and 2; and vii. a CFU-F value of 25-40 per 100 cells.

[0159] In another aspect, the present invention relates to a novel method of producing human platelet lysate (HPL) that increases the efficiency of fibrinogen free HPL production. The method comprises the steps of:Attorney Docket No. SASC-OOl-OIWO a. Subjecting platelets to three freeze-thaw cycles; b. Centrifuging the platelets of step a. and collecting the supernatant; c. Incubating the supernatant collected in step b., with 5 to 20 mM calcium chloride. d. Centrifuging the supernatant of step c. at a high speed. e. Collecting the supernatant.

[0160] In preferred aspects related to producing fibrinogen free HPL, the centrifugation in step d. is carried out at a high speed of 10,000 RPM or more. In even more preferred aspects, the centrifugation in step d. is carried out at a high speed of 10,000-12,000 RPM. This high centrifugation speed efficiently eliminates fibrinogen from human platelet lysate and captures more than 90% HPL in volume.

[0161] In addition, preferred embodiments, in step c. the supernatant is incubated with 5 to 20 mM calcium chloride at about 37°C for about 4 hours and then at about 4°C for about 20-30 hours. In even more preferred embodiments, in step c. the supernatant is incubated with 5 to 20 mM calcium chloride at about 37°C for about 4 hours and then at about 4°C for about 24 hours.

[0162] Additionally, the present invention also relates to MSCs and MSC populations with improved properties including but not limited to improved viability, reduced cell size, maintenance of elongated spindle shape despite several population doublings, short population doubling time, high colony-forming units- fibroblast (CFU-F) counts and high expression of MSC markers and specific additional markers.

[0163] In one aspect, the present invention relates to MSCs that have unexpected technical features such as expression of multiple stem cell markers, namely, CD73, CD90 and CD105, CD9, CD10, CD13, CD29, CD44, CD54 / ICAM-1, CD106 / VCAM-1, CD 108, CD 146, CD 166, HLA-ABC, EPHA 2, S SEA-4 and NESTIN. Other useful features of the MSCs of the present invention include viability of more than 98%, spindle-shaped morphology and a cell diameter of about 9-11pm.

[0164] In one aspect, the present invention relates to MSC populations of that have unexpected technical features including an antigen profile comprising more than 99% cells positive for the CD73, CD90 and CD105 markers and more than 90% cells positive for the CD9, CD10, CD13, CD29, CD44, CD 106 / V CAM- 1, CD 108, CD 146, CD 166, HLA-ABC, EPHA 2 markers and more than 50% cells positive for the SSEA-4, CD54 / ICAM-1, and NESTIN markers. The MSC populations may additionally demonstrate one or more of the following technical features:1) a high cell viability of more than 98%;2) spindle-shaped morphology of nearly all cells;3) a cell diameter of about 9-10 pm;4) a population doubling time (PDT) of about 22 hours for passage Pl, P2 and P3 cells and up to 31 hours for passage P8 cells; andAttorney Docket No. SASC-OOl-OIWO5) a CFU-F value of 25-40 per 100 cells.

[0165] In one aspect, the present invention relates to a novel method for producing mesenchymal stem cells, that achieves large quantities of highly potent MSCs and provides a therapeutic dose or several therapeutic doses. The method comprises: i. Mincing an organ tissue to obtain fine pieces of organ tissue; ii. Seeding the fine pieces of organ tissue in an altered culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells; iii. Harvesting the P0 stem cells using a mild cell detachment agent; and iv. Expanding the P0 stem cells obtained in step c. by serial passage. v. Re-seeding the residual explants after step iv multiple times.

[0166] Further aspects of the present invention require glucose and pH to be monitored throughout culturing of cells during each passage, to determine the optimum time for medium exchange. In preferred aspects, medium is exchanged at pH of 7-7.5 and a glucose concentration of 30-120 mg / dl. By monitoring both glucose and pH during culturing, the inventor of the present invention found that medium exchange should be carried out at a much shorter duration than was being done conventionally. The medium exchange at the optimum time is important for optimal cell culturing.

[0167] In another aspect expanding the P0 stem cells by serial passage comprises seeding the P0 stem cells or the stem cells obtained from the previous passage into cell culture media, culturing the cells and harvesting the cells, in each passage. In a preferred aspect, expanding the P0 stem cells by serial passage comprises seeding and culturing the P0 stem cells or the stem cells obtained from the previous passage in cell culture media present in roller bottles maintained at a low rotational speed of about 0.5 RPM to 9 RPM during the stages of both seeding and culturing, in each passage. The rotational speed of the roller bottles is increased to 5 to 80 RPM for harvesting the cells in each passage. The inventor of the present application found that maintaining a low rotational speed of about 0.5 RPM to 9 RPM during both the seeding and culturing stages in each passage, resulted in more robust cell growth.

[0168] The source of the MSCs or the organ tissue from which the MSCs are obtained is selected from the group consisting of adipose tissue, umbilical cord, and placenta. In preferred embodiments, the source of the MSCs is the umbilical cord.

[0169] In one embodiment, the media is selected from alpha-minimum essential medium (aMEM) and Dulbecco’s Modified Eagle Medium (DMEM). In another embodiment, the media is alphaminimum essential medium (aMEM) supplemented with 5%-10% HPL. In yet another embodiment, the media is Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% %-10% HPL.Attorney Docket No. SASC-OOl-OIWO

[0170] In some embodiments, the altered culture vessel used in one or more steps of the method of the present invention is a culture vessel that is coated with one or more extracellular matrix (ECM) proteins such as collagen, fibronectin, laminin, gelatin, vitronectin, tenascin C, aggrecan, Matrigel® (solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), etc.

[0171] In further embodiments, the altered culture vessel used in one or more steps of the methods of the present invention is a culture vessel that is coated with synthetic polymers or peptides such as SynthemaxSII (a synthetic vitronectin peptide-copolymer), poly-L-lysine, etc.

[0172] In some embodiments the altered culture vessel used in one or more steps of the methods of the present invention is a culture vessel that is coated with glycosaminoglycans such as heparan sulfate proteoglycan.

[0173] In additional embodiments, the altered culture vessel used in one or more steps of the methods of the present invention is a positively / negatively charged culture vessel or is a culture vessel with increased surface oxygen level and therefore increased negative charge and increased hydrophilicity. In specific embodiments, the altered culture vessel used in one or more steps of the methods of the present invention is a culture vessel treated with a substance that increases the surface oxygen level of the culture vessel thereby increasing its negative charge and making the culture vessel surface more hydrophilic.

[0174] In preferred embodiments, an altered culture vessel is used for seeding of fine pieces of organ tissues and culturing to obtain P0 stem cells.

[0175] In some embodiments, the altered culture vessel used for seeding of fine pieces of organ tissues and culturing to obtain P0 stem cells is a culture vessel coated with one or more substances selected from the group consisting of extracellular matrix proteins, synthetic polymers or peptides or glycosaminoglycans.

[0176] In some embodiments, the altered culture vessel used for seeding of fine pieces of organ tissues and culturing to obtain P0 stem cells is a culture vessel that manipulates surface charge by increasing oxygen levels thereby increasing hydrophilicity. In further preferred embodiments, the altered culture vessel is a culture vessel treated with CellBIND® Surface.

[0177] As used herein, the term “cell detachment agent” or “cell dissociation reagent” refers to compounds, substances, solutions or mixtures that facilitate detachment or dissociation of adherent cells from the culture vessel. The cell detachment agent or cell dissociation reagent suitable for use in the methods of the present invention include but are not limited to a gentle protease, such as a gentle trypsin; a trypsin-like protease, such as TiypLE™; a trypsin alternate, such as Accutase™ which is a mixture of proteolytic and collagenol ytic enzymes from crustaceans; a neutral protease, such as dispase; or mixtures thereof.Attorney Docket No. SASC-OOl-OIWO

[0178] In one embodiment, the present invention relates to a novel method for producing stem cells that achieves large quantities of highly potent MSCs, comprising the steps of: i. Mincing an organ tissue to obtain fine pieces of organ tissue; ii. Seeding the fine pieces of organ tissue in an altered culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells, wherein the altered culture vessel is selected from the following: a. a culture vessel coated with extracellular matrix proteins or synthetic polymers or peptides or glycosaminoglycans; or b. a culture vessel with altered surface charge; or c. a culture vessel with altered surface oxygen levels or with altered surface hydrophilicity; iii. Harvesting the P0 stem cells using a cell detachment agent.

[0179] In another embodiment, the present invention relates to a novel method for producing stem cells that achieves large quantities of highly potent MSCs, comprising the steps of: i. Mincing an organ tissue to obtain fine pieces of organ tissue; ii. Seeding the fine pieces of organ tissue in an altered culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells, wherein the altered culture vessel is selected from following: a. a culture vessel coated with extracellular matrix proteins or synthetic polymers or peptides or glycosaminoglycans; or b. a culture vessel with altered surface charge; or c. a culture vessel with altered surface oxygen levels or with altered surface hydrophilicity; iii. Harvesting the P0 stem cells using a mild cell detachment agent; iv. Seeding the P0 stem cells into roller bottles comprising cell culture media and stirring the roller bottles at a low rotational speed during both seeding and culturing of stem cells; v. Assessing baseline glucose and pH of the cell culture medium followed by monitoring glucose and pH at regular intervals to determine the optimum time for medium exchange; vi. Harvesting Pl cells at 80-90% confluence using a protease; vii. Seeding the Pl cells into roller bottles comprising cell culture media and stirring the roller bottles at a low rotational speed during both seeding and culturing of stem cells; viii. Assessing baseline glucose and pH of the cell culture medium followed by monitoring glucose and pH at regular intervals to determine the time point for medium exchange; ix. Harvesting P2 cells at 90% confluence using a protease.Attorney Docket No. SASC-OOl-OIWO

[0180] In preferred embodiments of the method, the glucose and pH are monitored in in step v. every day or every alternate day.

[0181] In optional embodiments the Pl cells harvested in step vi. may be cryopreserved. In these embodiments, the cryopreserved Pl cells are prepared for seeding and recovered by the following steps: a. rapidly thawing in a water bath at 37°C; b. centrifuging to remove the cryoprotectant by discarding the supernatant comprising the cryoprotectant; c. collecting the pelleted cells and suspending them in cell culture medium; d. repeating steps b. and c. several times to ensure complete removal of cryoprotectant.

[0182] In preferred embodiments, the cryopreserved Pl cells are rapidly thawed in a water bath at 37°C for 30 to 120 seconds until only a small ice ball is seen.

[0183] In further preferred embodiments the process of re-seeding the fine pieces of organ tissue remaining after harvesting the P0 stem cells is optionally repeated 2-10 times.

[0184] In specific embodiments the organ tissue is umbilical cord tissue.

[0185] In further embodiments the umbilical cord is minced into pieces of size 1 mm to 1 cm.

[0186] In preferred embodiments, cell culture media used is alpha-minimum essential medium (aMEM) supplemented with 5%-10% HPL or Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% HPL.

[0187] In further preferred embodiments, the cell detachment agent is a trypsin-like protease. In preferred embodiments, the trypsin-like protease is TrypLE™.

[0188] In preferred embodiments the altered culture vessel used for seeding the fine pieces of organ tissue and the fine pieces of organ tissue remaining after harvesting P0 cells is a culture vessel with altered surface oxygen levels and therefore, increased hydrophilicity. In further preferred embodiments the altered culture vessel is a culture vessel treated with CellBIND® Surface.

[0189] In preferred embodiments, a glucose concentration of less than 30 mg / dl and a pH of 7.0 to 7.5 indicates the optimum time to exchange media during each passage.

[0190] In preferred embodiments, the low rotational speed at which the roller bottles are stirred during stem cell seeding and culturing is about 0.5 RPM to 9 RPM , in each passage.

[0191] In specifically preferred embodiments the low rotational speed at which the roller bottles are stirred during stem cell seeding is 0.5 RPM and the low rotational speed at which the roller bottles are stirred during stem cell culturing is 0.5 RPM to 9 RPM, in each passage.

[0192] The method for producing stem cells of the present invention can provide around 384 million P0 cells and around 98 billion P2 cells and up to 4000 trillion P9 cells from a single umbilical cord.Attorney Docket No. SASC-OOl-OIWO

[0193] In yet another aspect, the present invention relates to a novel method for producing mesenchymal stem cells, that achieves large quantities of highly potent MSCs and provides a therapeutic dose or several therapeutic doses. The method comprises vii. Mincing an organ tissue to obtain fine pieces of organ tissue; viii. Seeding the fine pieces of organ tissue in a culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells; ix. Monitoring glucose and pH every day or every alternate day, to determine the optimum time for medium exchange; x. Harvesting the P0 stem cells using a mild cell detachment agent; and xi. Expanding the P0 stem cells obtained in step c. by serial passage. xii. Optional re-seeding the residual explants after step iv. multiple times

[0194] In a further aspect, the present invention relates to a novel method for producing mesenchymal stem cells, that achieves large quantities of highly potent MSCs and provides a therapeutic dose or several therapeutic doses. The method comprises i. Mincing an organ tissue to obtain fine pieces of organ tissue; ii. Seeding the fine pieces of organ tissue in an altered culture vessel comprising a cell culture medium and culturing to obtain P0 stem cells; iii. Monitoring glucose and pH every day or every alternate day, to determine the optimum time for medium exchange; iv. Harvesting the P0 stem cells using a mild cell detachment agent; and v. Expanding the P0 stem cells obtained in step c. by serial passage. vi. Optional re-seeding the residual explants after step iv. multiple times

[0195] In another aspect, the present invention provides novel methods of producing HPL that increase the efficiency of fibrinogen free HPL production. The method comprises the steps of: a. Subjecting platelets to three freeze thaw cycles; b. Centrifuging the platelets of step a. to remove cell debris and collecting the supernatant; c. Incubating the supernatant collected in step b. with 5mM-20mM calcium chloride at 37°C for 4 hours followed by 4°C for 20 to 30 hours; d. Centrifuging the supernatant of step c. at a high speed; e. Collecting the supernatant.

[0196] In preferred aspects related to producing fibrinogen free HPL, the centrifugation in step d. is carried out at a high speed of 10,000 RPM or more. In even more preferred aspects, the centrifugation in step d. is carried out at a high speed of 10,000-12,000 RPM. This high centrifugation speed efficiently eliminates fibrinogen from human platelet lysate and captures more than 90% HPL in volume.Attorney Docket No. SASC-OOl-OIWO

[0197] In preferred embodiments of the above method, the platelets are subjected to three freeze thaw cycles, wherein each cycle comprises incubating at -80°C for 24 hours.

[0198] Additionally, the present invention achieves MSCs with improved properties including but not limited to improved viability, reduced cell size, maintenance of elongated spindle shape despite several population doublings, short population doubling time, high CFU-F counts and high expression of MSC markers and additional markers.

[0199] The novel MSCs and MSC populations of the present invention possess the following characteristics: i. at least 98% viability; ii. spindle-shaped morphology; iii. maintenance of the elongated spindle shape even after 8 population doublings (PDs); iv. diameter of about 9-11 pm; v. population doubling time (PDT) of about 22 hours for passage Pl , P2 and P3 cells . . .cells and up to 31 hours for passage 8 (P8) cells; vi. no significant increase in PDT or increase in cell size and no reduction in viability up to passage 9 (P9); vii. CFU-F of 25-40 colonies per 100 cells; viii. more than 99% of the cells are positive for the classic MSC markers, namely, CD73, CD90 and CD 105; ix. more than 90% of the cells are positive for additional stem cell markers CD9, CD10, CD13, CD29, CD44, CD106 (VCAM-1), CD108, CD146, CD166, HLA-ABC, EPHA 2; and x. more than 50% of the cells are positive for the markers CD54 (ICAM-1), SSEA-4 and NESTIN.

[0200] In an aspect, the novel MSCs and MSC populations of the present invention are obtained by the novel method for producing MSCs as described herein.The improved method for producing MSCs of the present invention is based on the following.Altering Culture Vessel for Superior Organ Tissue Attachment for Obtaining PO / Seed Cells

[0201] Organ tissue is finely minced and seeded into culture vessels. Attachment of the fragments of tissue to the culture vessel is fundamental for the migration of MSCs from the explants to the floor of the culture vessel. The attachment of tissue fragments to the culture surface is a complex process that involves several factors, including the properties of the culture substrate, the presence of specific extracellular matrix (ECM) proteins, the expression of cell adhesion molecules on the MSC surface, etc. The composition and topography of the culture substrate can significantly influence explant attachment. The inventor of the present application found that the following typesAttorney Docket No. SASC-OOl-OIWO of altered culture vessels enhance the quality and quantity of passage 0 (PO) cells, which are critical for subsequent passaging:1. culture vessel coated with extracellular matrix proteins such as collagen, fibronectin, laminin, gelatin, vitronectin, tenascin C, aggrecan, Matrigel® (solubilized basement membrane matrix secreted by Engelbreth-IIolm-Swarm (EIIS) mouse sarcoma cells);2. culture vessel coated with synthetic polymers or peptides like Synthemax®!! (a synthetic vitronectin pep tide-copolymer), poly-L-lysine or with glycosaminoglycans such as heparan sulfate proteoglycan;3. culture vessels with altered surface charge;4. culture vessels with altered surface oxygen levels or with altered surface hydrophilicity, such as culture vessels treated with Corning® CellBIND® Surface.

[0202] Another unexpected observation made by the inventor of the present application was that the use of the altered culture vessel for incubating the minced organ tissue increased MSC attachment and growth not just from the attached organ tissue pieces but from some of the nonattached organ pieces as well.

[0203] Re-culturing Residual Organ Tissue Pieces in Conjunction with Altering CultureVessel Substrate

[0204] By using altered culture vessels to enhance organ tissue attachment for every step of the repeated organ tissue culture, the process of the present application significantly enhances the number of MSCs that can be extracted from donor tissue.

[0205] Monitoring Both Glucose and pH to Determine the Optimum Time For Media Exchange

[0206] During traditional 2D cell culture glucose and pH are not monitored. However, additional glucose and pH vigilance during 2D culture ensures optimal culture conditions for cell growth and viability and serves as a guide for media exchange. During the experimentation in relation to the present invention, based on monitoring glucose and pH throughout the culture process, the inventor identified the need for earlier media exchange (due to significant drops in glucose and pH) than the usual media exchanges which were typically done every 3 days. This ensures that the rapidly proliferating cells have access to all the necessary nutrients. Glucose levels during culturing determine the regenerative properties of MSCs. Based on the studies conducted in relation to the present invention, the inventor of the present application identified the need to monitor glucose and pH levels throughout culturing in each passage, preferably every day or every alternate day to determine the right time for medium exchange.Attorney Docket No. SASC-OOl-OIWO

[0207] 2D Dynamic Culture

[0208] Conventionally, roller bottle cell culture requires a low rotational speed during the seeding phase, a higher rotational speed while culturing and a very high rotational speed during harvesting. However, the inventor of the present application found that maintaining a low rotational speed during both seeding and culturing of cells in each passage resulted in more robust growth compared to using a high rotational speed, such as 10 RPM, during culturing. According to the present method a low rotational speed of 0.5 RPM during seeding and 0.5-2 RPM during culturing and a high rotational speed of 5 to 80 RPM during harvesting in each passage gave robust growth.

[0209] The yield obtained by maintaining a low rotational speed during both seeding and culturing is typically 6 times more than that reported in the literature.

[0210] Based on the foregoing observations, the inventor of the present application arrived at novel methods for producing MSCs which not only yielded mesenchymal stem cells of high quality but also helped to achieve a high MSC yield.

[0211] The processes for producing stem cells of the present invention achieve the following advantages which are unprecedented.1. A significant increase in yield - About 1000 doses (100 million cells in a dose) of highly proliferative and potent, low population doubling cells can be obtained from a single donation of an umbilical cord by harvesting the cells at the end of passage 2.2. Maintenance of high regenerative capacity- It is well understood that regenerative capacity of stem cells may decline with in vitro aging from excessive culturing (passaging) due to factors such as telomere shortening, DNA damage, genetic alterations and changes in the stem cell niche. Since the method of the present invention can help to achieve a high number of MSCs from very limited population doublings and, therefore, requires limited cell passaging, the regenerative capacity of the MSCs is maintained.3. Production of unique stem cells which demonstrate the following desirable properties- i. Smaller size of stem cells- Smaller sized stem cells retain stem cell attributes and are better than larger sized cells. Larger sized MSCs are not as functional as smaller sized MSCs. The MSC diameter of approximately 10 microns achieved by the method of the present application is one of the smallest MSC sizes reported in literature. The MSCs of the present invention have a diameter of 9-1 1 pm and were able to retain a diameter of less than 10 pm even after eight passages (P8). ii. Fast growing cells- Proliferation of stem cells is indicative of stem cell potency. The population doubling time of the MSCs of the present invention is about 22 hours at passages Pl, P2 and P3, making the present MSCs one of the fastest growing cells. The population doubling time increases from less than 24 hour to 31 hours by P8.Attorney Docket No. SASC-OOl-OIWO iii. Colony -Forming Unit-Fibroblast (CFU-f)- CFU(f) is a measure that demonstrates the clonogenic capacity of stem cells, that is the ability of a single stem cell to grow into a colony. The MSCs of the present application have exceptionally high CFU-f counts compared to other MSCs reported in the literature. The MSCs of the present application were able to achieve CFU-f of 25-40 / 100 cells seeded. iv. The International Society for Cell and Gene Therapy (ISCT) requires only 3 positive cluster of differentiation (CD) surface markers to identify MSCs, namely, CD73, CD90 and CD105. The MSCs of the present invention not only express CD73, CD90, CD105, they have a consistently high expression of additional stem cell markers such as CD9, CD10, CD13, CD29, CD44, CD54 (ICAM-1), CD106 (VCAM-1), CD108, CD146, CD166, HLA-ABC, EPHA 2, SSEA-4, NESTIN. v. Source of viable exosomes- Exosomes secreted by excessively passaged MSCs are impaired compared to exosomes that are obtained from cells that have undergone fewer passages. Since the MSCs of the present application are obtained through a very small number of population doublings (less than 10 PD), the exosomes obtained from said cells are viable.

[0212] Stem cell populations are often heterogeneous and consist of a mixture of cells. Ensuring the purity, identity, and quality of stem cell preparations is essential for their safe and effective use in clinical settings, but achieving consistent quality control can be challenging. However, extensive characterization shows consistency in the functionality and homogenicity of the MSCs obtained by the methods of the present applications.

[0213] Size from automated cell counter, spindle shape morphology from microscope observation. Viability is checked with trypan blue test and JC dye flow cytometer test assessing mitochondrial potential. CD markers thro flow Cytometry. Population doubling is by the PD formula. Exosomes by NTA

[0214] In addition to these inventions, the inventor also developed an improved method of preparing fibrinogen-depleted human platelet lysate (Fd-hPL).

[0215] High RPM Centrifugation along with Calcium Chloride to Increase the Efficiency of Fibrinogen-Depleted hPL (Fd-hPL) Production

[0216] Fetal bovine serum (FBS) has been traditionally utilized to provide growth factors for cell growth. However, it has several drawbacks including batch-to-batch variations, presence of yet many unidentified components, potential transmission of zoonotic diseases, ethical considerations, supply uncertainties, immunogenicity, departure from xenofree culture practices, cellular incorporation of animal -derived components, and the potential for epigenetic alterations in culturedAttorney Docket No. SASC-OOl-OIWO cells. It has been estimated that approximately 7-30 mg of bovine protein are transferred per 108MSCs.

[0217] Human platelet lysate (hPL) not only makes the process of the present invention xenofree but also is exempt from the problems with FBS described above. Since hPL has significantly higher levels of growth factors and cytokines, studies have shown that cells cultured with hPL exhibit higher proliferation rates, multipotency, superior phenotypic and genomic stability. Furthermore, hPL derived cells are smaller in size and exhibit lesser senescence with higher viability. (Crespo- Diaz et al., Cell Transplantation. 2011;20(6):797-812. doi: 10.3727 / 096368910X543376; Riordan et al., J Transl Med. 2015 Jul 17;13:232. doi: 10.1186 / sl2967 -015-0561-6. PMID: 26183703; PMCID: PMC4504159; Blazquez-Prunera et al. Stem Cell Research & Therapy (2017) 8:103 )

[0218] In addition, use of HPL results in superior passage doubling time, is more angiogenic, and increases exosome secretion. (Palombella et al. Stem Cell Research & Therapy (2022) 13: 142 https: / / doi.org / 10.1186 / sl3287-022-02815-l) Further, HPL has more growth factors than FBS. (Mohamed et al., Blood Res. 2020 Mar;55(l):35-43. doi: 10.5045 / br.2020.55.1.35)

[0219] Eliminating fibrinogen from HPL enables HPL to remain in aqueous form when added to the cell culture media. Avoidance of clot generation ensures proper growth of MSCs. Typically, to produce fibrinogen-depleted HPL, platelet lysate is incubated with 20mM of calcium chloride for 24 hours to induce clot formation followed by centrifugation at 4000-5000 RPM. However, at these centrifuge speeds, minimal clot retraction is noted entrapping most of the HPL. In some experiments a loss of almost 50% HPL has been observed. By increasing the centrifuge speed to higher RPM, for example, 10000 RPM or more, the inventor of the present application could efficiently capture more of HPL in volume that drastically decreases the platelet donations required.

[0220] Composition having exosomes derived from mesenchymal cells, and mesenchymal cells.

[0221] The term “pharmaceutical composition” is used herein to refer to a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease. The terms “formulation” and “composition” are used interchangeably herein to refer to a product of the described invention that comprises all active and inert ingredients.

[0222] In an embodiment, the composition of the present disclosure comprises a combination of exosomes derived from mesenchymal cells, and mesenchymal cells. In an embodiment, the composition of the present disclosure comprises a combination of exosomes derived from mesenchymal cells, and mesenchymal cells, wherein the mesenchymal cells are derived from umbilical cord.Attorney Docket No. SASC-OOl-OIWO

[0223] In an embodiment, the composition of the present disclosure comprises a combination of exosomes derived from mesenchymal cells, and mesenchymal cells, wherein the combination is in a particular ratio. The ratio of the combination is about 1: 2000, about 1 : 1 :2500, about 1 : 3000, about 1: 3500, about 1: 5000, about 1: 7000, about 1 : 10000, about 1: 25000, about 1 : 50000, about 1: 75000, about 1:1000000, about 1 :1250000, about 1:1500000, about 1 :1750000, about 1:2000000, about 1 : 5000000, about 1: 7500000, about 1: 10000000,

[0224] In an embodiment, the composition has an effective amount of a combination of mesenchymal cells and exosomes. In an embodiment, the composition has an effective amount of combination of mesenchymal cells and exosomes from the mesenchymal cells. In an embodiment, an effective therapeutic amount of the composition of one or more embodiments is configured to treat a disease.

[0225] An “effective amount” of therapeutic compound or agent is an amount sufficient to prevent, treat, reduce and / or ameliorate the symptoms, neuronal damage and / or underlying causes of any of the referenced disorders or diseases. In some instances, an “effective amount” is sufficient to eliminate the symptoms of those diseases and, perhaps, overcome the disease itself.

[0226] The terms “disease” or “disorder” as used herein refer to an impairment of health or a condition of abnormal functioning. The disease or condition is selected from a neurological disease, cardiac / pulmonary / hepatic / metabolic disease, a muscular disease, an autoimmune disease, an inflammatory disease, a digestive disease, an energy homeostasis disease, aging, radiation induced injury, cell transplant rejection and a proliferative disease. According to some embodiments, the neurological disease is selected from brain cancer, cancer metastasis to the brain, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, neurological injury, radiation induced injury to the brain, hypoxic injury to the brain and Rett syndrome.

[0227] According to some embodiments, the brain cancer is one of an astrocytic tumor, a glioma, a medulloblastoma, a neuroblastoma and a meningioma. According to some embodiments, the glioma is glioblastoma. According to some embodiments, the muscular disease is selected from MS, a muscular dystrophy, muscle injury, muscle inflammation, cachexia and sarcopenia. According to some embodiments, the muscular dystrophy is Duchenne's muscular dystrophy (DMD), or Baker muscular dystrophy. According to some embodiments, the autoimmune disease is selected from MS, diabetes, colitis, and Chron's disease. According to some embodiments, the energy homeostasis disease is diabetes. According to some embodiments, the digestive disease is selected from irritable bowel syndrome (IBD), Chron's disease, and colitis. According to some embodiments, aging comprises at least one of skin aging, muscle aging, and brain aging. According to some embodiments, the proliferative disease is cancer.Attorney Docket No. SASC-OOl-OIWO

[0228] In an embodiment, MSCc and exosomes help counteract ageing primarily through secretion of growth factors (e.g., VEGF, TGF-p, HGF, IGF-1) and cytokines that enhance tissue repair and reduce inflammation, and down regulating pro-inflammatory cytokines (TNF-a, IL-6) and upregulate anti-inflammatory ones (IL-10). The formulation could further improve blood flow and nutrient delivery to ageing tissues. Stimulates collagen and elastin production, reducing wrinkles and improving skin elasticity.

[0229] The term “fibrotic disease” as used herein refers to a condition marked by an increase of interstitial fibrous tissue. According to some embodiments, the fibrotic disease is selected from one or more of a fibrotic lung disease, a fibrotic cardiac disease, a fibrotic renal disease, a fibrotic hepatic disease, a fibrotic skin disease, a fibrotic pancreatic disease, a fibrotic eye disease, a fibrotic joint disease, a fibrotic bone marrow disease, a fibrotic brain disease, a fibrotic intestinal disease, a fibrotic peritoneum disease, a fibrotic retroperitoneum disease, a fibrotic condition of the nerves or nervous system (e.g, CNS, PNS, ANS), a nerve compression, or an injury due to fibrosis. According to some embodiments, the fibrotic disease is a fibrotic lung disease.

[0230] In an embodiment, the composition comprises one or more adjuvant. As used herein, the term “carrier,” “excipient,” or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.

[0231] Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloringAttorney Docket No. SASC-OOl-OIWO agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present.

[0232] Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO.

[0233] These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety.

[0234] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0235] In an embodiment, the composition may have additional active agent in addition to the combination of exosomes and mesenchymal cells. Examples of such additional active therapeutic agents include one or more immunomodulators, analgesics, anti-inflammatory agents, anti-fibrotic agents, proton pump inhibitors, or oxygen therapy. Examples of immunomodulators include corticosteroids, for example, prednisone, azathioprine, mycophenolate, mycophenolate mofetil, colchicine, and interferon-gamma lb. Examples of analgesics include capsaisin, codeine, hydrocodone, lidocaine, oxycodone, methadone, resiniferatoxin, hydromorphone, morphine, and fentanyl. Examples of anti-inflammatory agents include aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sulindac, and tolmetin. Examples of anti-fibrotic agents are nintedanib and pirfenidone. Examples of proton pump inhibitors are omeprazole, lansoprazole.

[0236] A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants,Attorney Docket No. SASC-OOl-OIWO additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include: U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0237] In an embodiment, pharmaceutical composition as described in one or more embodiments, is configured to improve the liver morphology by more than 70%, more than 80%, more than 90%, more than 95%, more than 99% or more as detected by H&E staining.

[0238] In an embodiment, pharmaceutical composition as described in one or more embodiments, is configured to increase Tregulatory cells (Treg cells) by more than 20%, more than 25%, more than 30%, more than 40%, more than 50% or more.

[0239] In an embodiment, pharmaceutical composition as described in one or more embodiments, is configured to

[0240] As used herein, the terms “administering”, “administration” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for oral administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include parenteral, subcutaneous, intravenous, intramuscular, or intraperitoneal. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. In some embodiments, administering comprises at least one of intravenous administration, intramuscular administration, intranasal administration, intrathecal administration, intrastriatal administration, intracranial administration, intraarterial administration, and subcutaneous administration.

[0241] In an embodiment, the composition may be administered once, for a limited period of time or as a maintenance therapy over an extended period of time, for example until the condition is ameliorated, cured or for the life of the subject. A limited period of time may be for 1 week, 2 weeks, 3 weeks, 4 weeks and up to one year, including any period of time between such values, including endpoints. According to some embodiments, the pharmaceutical composition may be administered for about 1 day, for about 3 days, for about 1 week, for about 10 days, for about 2 weeks, for about 18 days, for about 3 weeks, or for any range between any of these values, including endpoints. According to some embodiments, the pharmaceutical composition may beAttorney Docket No. SASC-OOl-OIWO administered for more than one year, for about 2 years, for about 3 years, for about 4 years, or longer.

[0242] According to the foregoing embodiments, the composition or pharmaceutical composition may be administered once daily, twice daily, three times daily, four times daily or more.

[0243] All referenced journal articles, patents, and other publications are incorporated by reference herein in their entirety.

[0244] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0245] WORKING EXAMPLES

[0246] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.METHODOLOGY

[0244] Example 1 : Human Platelet Lysate (HPL) Production

[0245] Platelets obtained from the blood bank were pooled and subsequently subjected to 3 freezethaw cycles. Each freeze-thaw cycle required freezing at -80°C for 24 hours and thawing. Centrifugation was performed at 10,000 RPM to remove cell debris of the ruptured platelets. The collected supernatant was incubated with 20mM calcium chloride at 37°C for 4 hours followed by 4°C for 24 hours. Further centrifugation was performed at 10,000-4-34)00 or more RPM. The supernatant was collected and stored at -20°C for future use.

[0246] Example 2: Culturing Stem Cells

[0247] P0 Culture

[0248] Umbilical cord was acquired under strict sterile conditions from cesarean section. Prior donor screening was done and approvals were duly taken. The cord was minced into pieces of length of about 5 mm and washed two to three times with a mixture of phosphate buffered saline (PBS) and antibiotic solution (PCN 10000 U / ml, S TREP 10000 ug / ml) to remove the blood and any other contaminants. The washed explants were seeded into multiple Corning® CellBIND® Surface cell culture flasks containing media such as alpha-minimum essential medium (aMEM)Attorney Docket No. SASC-OOl-OIWO supplemented with 5% HPL or Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% to 10% HPL.

[0249] Within 7 days of seeding of the minced umbilical cord pieces, MSCs commenced migrating out of the explants. Around day 21, when MSCs got confluent around the explants, they were harvested by using a gentle protease, TrypLE™. The harvested cells were the P0 or seed cells.

[0250] After harvesting the MSCs, the residual umbilical cord pieces were re-seeded and cultured in cell culture flasks coated with CellBIND®.

[0251] Pl Culture

[0252] The harvested P0 cells were seeded into the roller bottles (2D dynamic culture) immediately for Pl culture. The RPM was set at 2, which remained unchanged throughout the Pl culture process. Baseline glucose and pH were checked for the media (aMEM with 5%-10% HPL or DMEM with 5 %-10% HPL) and then reassessed every day. Medium exchange was determined by these measured parameters. Culture media was exchanged when the pH was 7-7.5 and the glucose concentration fell to 30-120 mg / dl. Typically, one medium exchange was performed and a second exchange was incorporated only if necessary. At 90% confluence, cells were harvested with TrypLE™, a gentle protease and if needed, cryopreserved until further use.

[0253] Baseline Glucose and PH are checked for the media (aMEM or DMEM with 2.5-10% HPL) and then reassessed every day. Medium exchange is determined by these measured parameters. Typically one medium exchange is performed and only if necessary a second exchange is incorporated.

[0254] P2 Culture

[0255] If the Pl cells were cryopreserved, then they were made to undergo rapid thaw in a water bath at 37°C for 30 to 120 seconds until only a small ice ball is seen. The thawed cells were centrifuged. Following centrifugation, the supernatant was discarded and the cell pellet was suspended in cell culture medium (aMEM supplemented with 5% HPL or DMEM supplemented with 5% to 10% HPL). The cells suspended in cell culture medium were again centrifuged and the steps of centrifuging the cells suspended in cell culture medium, discarding the supernatant and resuspending the cell pellet in cell culture media were repeated until the cryoprotectant was completely removed.

[0256] The thawed Pl cells were then seeded into the roller bottles immediately for P2 culture. The RPM for culturing was set at 2 which remained unchanged throughout the P2 culture process.

[0257] Baseline glucose and pH were checked for the media (aMEM with 5% -10% HPL or DMEM with 5% -10% HPL) and then reassessed every day for determining the optimum time for medium exchange. Medium exchange was performed when the pH of the culture media changedAttorney Docket No. SASC-OOl-OIWO to 7-7.5 and the glucose level fell to 30 mg / dl. Typically, one medium exchange was performed, and a second exchange was incorporated only if necessary. At 90% confluence, cells were harvested with TrypLE™, a gentle protease.

[0258] For further passage of cells (P3, P4, P5, ....Pn) the same steps as mentioned above were followed.

[0259] It was observed that culturing mesenchymal stem cells (MSCs) under reduced oxygen tension (approximately 2-5% O2) enhances their immunomodulatory and regenerative properties. Exposure of MSCs to such hypoxic conditions leads to upregulation of hypoxia-inducible factor-1 alpha (HIE- la) and associated downstream pathways. This results in increased cell survival, proliferation, angiogenic factor secretion, and anti-inflammatory activity compared to cells maintained under normoxic (-20% O2) conditions.

[0260] The combination of these various above techniques resulted in a highly potent, high yield, homogenous (based on single cell RNA sequencing) MSC population and by extension superior quality exosomes

[0261] Example 3: Characterization of the MSCs Produced

[0262] The MSCs of the present application were evaluated for cell size, cell morphology, cell proliferation, fibroblastic colony forming units (CFU-F), cell surface markers and secretion of immunomodulatory cytokines using the methods described below.

[0263] METHODOLOGY FOR IN VITRO ANALYSIS1. CELL SIZE: Corning automated cell counter.2. CELL VIABILITY: Trypan blue for cell membrane integrity and for metabolic function, IC-1 dye is used to assess mitochondrial potential. High mitochondrial membrane potential represents robust metabolic activity and the intracellular permeable IC-1 dye emits red fluorescence while poor potential emits green.3. CELL MORPHOLOGY: Microscope.4. CELL PROLIFERATION: Calculating PDT using the traditional formula.5. CFU-F: 6 well plate is seeded with 100 cells. Number of colonies (>50 cells) assessed by crystal violet staining after 14 days. Ratio of colonies / 100 cells is measured.6. CELL SURFACE MARKERS: Flow cytometry7. CYTOKINES- ELISA8. CYTOKINES- RT-PCR9. INDUCTION TEST: IDO is not natively expressed by MSCs. However upon stimulation with inflammatory cytokines, significant secretion of IDO is noted. This burst of IDO secretion upon stimulation is regarded as one of the potency assays.10. MIXED LYMPHOCYTE REACTIONSAttorney Docket No. SASC-OOl-OIWO11. GENOMIC ANALYSIS INCLUDING SINGLE CELL RNA SEQUENCING

[0264] 3.1 CELL COUNT

[0265] Cell count and cell viability were determined using an automatic cell counter, Coming® automated cell counter, and the Trypan blue exclusion method. Trypan Blue dye was mixed with the cell suspension and the stained cell suspension was placed in the cell counter. In addition to evaluating the cell membrane integrity using the trypan blue test, metabolic health of the cells was also recorded by assessing the mitochondrial membrane potential using the JC-1 dye method with a flow cytometer.

[0266] Using the method of the present application, around 8 million cells per cell culture flask from the first explant culture (P0), were obtained. The inventor could plate 8 flasks from one cord resulting in 64 million cells. The residual explants after the first explant culture could be re-plated at least 6 times yielding 384 million P0 cells from a single cord.

[0267] The 384 million cells obtained from P0 were further cultured to create 6 billion cells which further underwent P2 culturing for a final yield of 98 billion cells from a single cord. Viability of more than 98% was reported for the cells obtained by the method of the present invention. In fact, using the method of the present invention, a good number of cells could be obtained in less than 10 passages.

[0268] This cell yield is much improved over the yields achieved by known methods. For example, Otte et al. (Otte A, Bucan V, Reimers K, Hass R. Mesenchymal stem cells maintain long-term in vitro sternness during explant culture. Tissue Eng Part C Methods. 2013 Dec;19(12):937-48. doi: 10.1089 / ten.TEC.2013.0007. Epub 2013 May 15. PMID: 23560527), reported the generation of 200 trillion UC-MSC cells from a single umbilical cord utilizing a multiple explant method (5 times) for 9 passages. In contrast, the process of the present invention can generate at least 4000 trillion cells from 9 passages.

[0269] Tozetti et al. (Tozetti PA, Caruso SR, Mizukami A, Fernandes TR, da Silva FB, Traina F, Covas DT, Orellana MD, Swiech K. Expansion strategies for human mesenchymal stromal cells culture under xeno-free conditions. Biotechnol Prog. 2017 Sep;33(5):1358-1367. doi: 10.1002 / btpr.2494. Epub 2017 May 30. PMID: 28486779.), used a 2150 cm roller bottle and seeded UC-MSCs at a density of 2000 cells / cm2. After 6 days they could achieve only 30 million cells (population doublings <3) with a viability of 90%. Through the process of the present application, the inventor was able to obtain 80-100 million cells after 5 days (4 population doublings) with a viability of more than 98% using a smaller roller bottle of 850 cm.

[0270] Hua et al. (Hua J, Gong J, Meng H, Xu B, Yao L, Qian M, He Z, Zou S, Zhou B, Song Z. Comparison of different methods for the isolation of mesenchymal stem cells from umbilical cord matrix: proliferation and multilineage differentiation as compared to mesenchymal stem cells fromAttorney Docket No. SASC-OOl-OIWO umbilical cord blood and bone marrow. Cell Biol Int. 2013 Oct 7. doi: 10.1002 / cbin.l0188. Epub ahead of print. PMID: 24123669) could obtain only a maximum of 67000 MSCs per cm of the umbilical cord using different explant protocols. In contrast, with the method of the present application, 1.1 million cells / cm could be obtained.

[0271] Majorie et al. (Majore, I., Moretti, P., Stahl, F. et al. Growth and Differentiation Properties of Mesenchymal Stromal Cell Populations Derived from Whole Human Umbilical Cord. Stem Cell Rev and Rep 7, 17-31 (2011). https: / / doi.org / 10.1007 / s l2015-010-9165-y), could obtain only 28 million MSCs from the entire cord whereas Xu et al. (Xu Y, Meng H, Li C, Hao M, Wang Y, Yu Z, Li Q, Han J, Zhai Q, Qiu L. Umbilical cord-derived mesenchymal stem cells isolated by a novel explantation technique can differentiate into functional endothelial cells and promote revascularization. Stem Cells Dev. 2010 Oct;19(10): 1511-22. doi: 10.1089 / scd.2009.0321. PMID: 20170363) and Yoon et al. (Yoon JH, Roh EY, Shin S, Jung NH, Song EY, Chang JY, Kim BJ, Jeon HW. Comparison of explant-derived and enzymatic digestion-derived MSCs and the growth factors from Wharton's jelly. Biomed Res Int. 2013;2013:428726. doi: 10.1155 / 2013 / 428726. Epub 2013 Apr 9. PMID: 23653895: PMCID: PMC3638666) extracted 30 million and 1.8 million passage P0 cells, respectively.

[0272] While Mori et al. (Mori Y, Ohshimo J, Shimazu T, He H, Takahashi A, Yamamoto Y, Tsunoda H, Tojo A, Nagamura-Inoue T. Improved explant method to isolate umbilical cord- derived mesenchymal stem cells and their immunosuppressive properties. Tissue Eng Part C Methods. 2015 Apr;21(4):367-72. doi: 10.1089 / ten.TEC.2014.0385. Epub 2015 Jan 7. PMID: 25220032) reported cell counts of 104 million from one cord, the same was obtained by utilizing a mesh to enhance explant attachment which increases contamination risk and is challenging to use for large scale manufacturing. However, when the mesh was not incorporated the yield decreased to 23.7 million. In contrast, the method of the present application gave about 98 billion P2 cells using one cord.

[0273] Although another study had significantly higher P0 numbers from explant culture (152 million) per cord, the cell viability was as low as 91% with cell size as high as 15 microns at Pl and P2.

[0274] Further, while Chu et al. (Chu, W., Zhang, F., Zeng, X. et al. A GMP-compliant manufacturing method for Wharton’s jelly-derived mesenchymal stromal cells. Stem Cell Res Ther 15, 131 (2024). https: / / doi.org / 10.1186 / sl3287-024-03725-0) reported 152 million P0 cells per cord, the cell viability was as low as 91% with cell size as high as 15 microns at Pl and P2.

[0275] 3.2 CELL QUALITY / VIABILITY

[0276] Maintaining quality during large scale manufacturing has been challenging. Although small batch production in academic trials resulted in favourable outcomes, most trials from large scaleAttorney Docket No. SASC-OOl-OIWO manufacturers failed to meet endpoints required for drug approvals. Poor quality of cells is resulting in inadequate performance of cells by the industry.

[0277] One Indian company that obtained regulatory approval has reported that only 80% of their cells display the stem cell canonical markers in contrast to our cells (>98%). Cell viability was only 85% compared to our viability of 95%. Their CFU-f numbers ranged from 2-17 per 100 cells which is significantly lower than our cells. 1.5% of their cells are senescent and our number is less than 1%.

[0278] The cell line from Mesoblast has only 70% viability and only 80% of cells are spindle shaped (typical for MSCs).

[0279] In contrast our cells have more than 98% viability and 100% of cells are spindle shaped. Moreover, they report only one of the 3 canonical stem cell markers.

[0280] The cells obtained by the method of the present invention with remarkable viability of over 98% is much improved over cells described in the prior art. For example, Yuanyuan Xie et al. (Xie, Y., Liu, W., Liu, S. et al. The quality evaluation system establishment of mesenchymal stromal cells for cell-based therapy products. Stem Cell Res Ther 11, 176 (2020). https: / / doi.Org / 10.l 186 / s 13287-020-01696-6) report a human umbilical cord mesenchymal stromal cell (HUCMSC) viability of at least 90%. Matthay et al. (Lancet Respir Med. 2018 Nov 16;7(2): 154-162. doi: 10.1016 / S2213-2600(18)30418-1) reported an MSC viability ranging from 36% to 85%. Matthay et al. further reported that the criteria of release of MSCs from the University of Minnesota was at least 70% viability. Similarly, US 11708560 also reported a stem cell viability of at least 70%.

[0281] Commercial products have shown viability of only 70%. Another experimental study regarding GMP manufacturing protocols showed that the viability of their cell line was as low as 85%.

[0282] 3.3 CELL SIZE AND MORPHOLOGY

[0283] Cell size is also an important parameter for determining the metabolic fitness and proliferative capability of MSCs. Stem cell health is inversely proportional to the cell size. Progressive passaging and culturing increase the cell size. Smaller cells are associated with rapid growth and enhanced sternness, making them particularly valuable for therapeutic applications. Cell size plays a crucial role in intra-arterial applications, as small increases in cell diameter can significantly increase the likelihood of embolism and micro-occlusions in the feeding artery, which can lead to severe and potentially disastrous consequences. With large size cells, such as MSCs, there is an obvious risk that the cells, rather than rolling and adhering to the postcapillary venule walls, clog up the entire capillary lumen, eliminating its function as a nutrient supplier and gas exchanger.Attorney Docket No. SASC-OOl-OIWO

[0284] Cell morphology, including cell shape and cell size, was studied by visualizing the roller bottles and flasks in different positions under an inverted microscope multiple times during culturing starting from day 0 until harvesting.

[0285] The diameter of cells obtained by the method of the present invention was around 9-11 pm for P0, Pl and P2 cells. Further passage up to P8 resulted in P8 cells which also had a dimeter less than 11pm. This is one of the smallest reported MSC cell sizes in literature that shows MSCs up to 25 microns (Guzman et al., Stroke. 2018 May; 49(5): 1075-1082. doi: 10.1161 / STROKEAHA.117.018288. Epub 2018 Apr 18. PMID: 29669876; PMCID: PMC6027638). In contrast to the MSCs obtained by the method of the present invention, the manufacturing protocol described in Chu et al.(>S em Cell Res Ther 15, 131 (2024). https: / / doi.Org / 10.l 186 / s 13287 -024-03725-0) generated umbilical cord MSCs of diameter of about 14.5 pm to more than 15 pm at P0, Pl and P2 and the size increased to 16 pm for the cells at P5.

[0286] A GMP manufacturing protocol study generated UC MSCs at a size of more than 15 microns at P0, Pl and P2 and increased to 16 for their product at P5.

[0287] Human Umbilical Cord-derived Mesenchymal Stromal Cells (hUC-MSCs) exhibit high- quality characteristics, including a notably small average cell size of 9.8 pm, significantly smaller than what has been reported in existing literature. Previous studies have shown that smaller-sized MSCs often possess enhanced functionality across various biological activities.

[0288] Our final cell size for patient infusion is around 10 microns. Smaller cells are associated with rapid growth and enhanced sternness properties, making them particularly valuable for therapeutic applications.

[0289] All cells obtained by the method of the present invention were spindle shaped. The elongated spindle shape was maintained even after 8 population doublings (PDs). This is a desirable feature as it has been seen in the prior art that prolonged culturing leads to alteration of the cell shape into wider, flattened phenotype that reflects poor performance.

[0290] The hUC-MSCs consistently demonstrate over 90% viability as assessed by trypan blue exclusion. In addition to membrane integrity-based staining, JC-1 dye which indicates cell metabolic fitness — also indicates that more than 90% of the cells are metabolically active and healthy.Table 1: Cell quality measures over serial passagingAttorney Docket No. SASC-OOl-OIWO

[0291] 3.4 CELL PROLIFERATION ANALYSIS

[0292] Cell proliferation analysis included determining population doubling time (PDT) which was calculated using the formula PDT= log2 (NH / N1). In the formula, N1 is the number of cells seeded and NH is the number of cells harvested.

[0293] Cell proliferation is known to be a surrogate marker of cell potency. Literature shows that despite addition of various growth stimulants like recombinant insulin, transferrin and TGFP to the cell culture media for industrial production, the population doubling time (PDT) of UC-MSCs was as high as 27 hours. In comparison, the population doubling time in the method of the present invention is around 22 hours without any additional growth stimulants for cells of passage Pl, P2 and P3. This is amongst the least PDT for MSCs. PDT increased from less than 24 hours to about 31 hours from Pl to P8.

[0294] In a separate study involving GMP manufacturing, the population doubling time demonstrated an increase at Passage 6 and increasing cell size / viability (87%) from passage 5 reflecting diminishing cell fitness. However, our cell line did not display these decreases until passage 9. Their final product had a viability of 87% and PDT of 28.9 hours compared to >98% and 23 hours of our product.

[0295] In Chu et al. (Stem Cell Res Ther 15, 131 (2024). https: / / doi.org / 10.1 186 / sl3287-024- 03725-0), the population doubling time increased at Passage 6 while an increase in cell size and reduction in viability (around 87%) was seen from passage 5 onwards which is indicative of diminished cell fitness.

[0296] However, the cell line obtained by the method of the present invention did not display the increase in PDT until passage 8. Chu et al.’s patient product had a PDT of 28.9 hours for passage Pl cells compared to a PDT of 23 hours for passage Pl cells obtained by the method of the present invention.

[0297] our hUC-MSCs demonstrate robust proliferative capacity, as evidenced by a shorter population doubling time (PDT) of 24 ± 2 hours and a high number of colony-forming units (CFUs), both of which are indicative of strong proliferative potential and cellular vitality.

[0298] 3.5 FIBROBLASTIC COLONY FORMING UNITS (CFU-F)

[0299] 100 MSCs per well were seeded in 6-well plates in triplicate. Colonies or aggregates comprising 50 cells or more were treated as fibroblastic colony forming units (CFU-F) and were assessed by crystal violet staining. After culturing the MSCs in triplicates in 6-well plates for 11 days, the cells were washed with 1XPBS and then fixed with a 1 :7 mixture of glacial acetic acid and methanol for 10 minutes. The cells were stained with 0.1% crystal violet for 30 minutesAttorney Docket No. SASC-OOl-OIWO followed by 4-5 washes with 1XPBS. The CFU-F values were reported as a ratio of colonies per 100 cells seeded.

[0300] The CFU-F assay is a measure of the clonogenic progenitor capacity of MSCs. The CFIJ- F counts of the MSCs of the present invention is typically high when measured against those reported in the literature.

[0301] A CFU-F of 25-40 per 100 cells seeded was recorded for the cells of the present invention.

[0302] This demonstrates an improvement over the prior art such as Chu et al. which reported a CFU-F of only about 17 colonies per 100 cells seeded. Kannan et al. (Kannan, S., Gokul Krishna, S., Gupta, P.K. et al. Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs. Sei Rep 14, 12654 (2024). https: / / doi.org / 10.1038 / s41598-024-62544-8) reported a CFU-F of 2 to 17.

[0303] Additionally, their CFU-f number was only 17 / 100 cells plated compared to 30 / 100 of our line. Furthermore, a significant increase in senescence was noted from Pl to P5 (final product) from 0.23% to 0.56%. The senescence for our cells are consistently reported 0%.

[0304] One study had a CFU-f of 10 among 100 cells plated.

[0305] 3.6 CELL SURFACE MARKERS

[0306] Cell surface markers were determined using flow cytometry. Fluorescently labelled antibodies from BD Biosciences were used. One million cells were incubated with the respective fluorescently labelled antibody. The samples were analysed using BD Biosciences and FlowJo™ and FACSDiva™ softwares.

[0307] The International Society for Cell and Gene Therapy (ISCT) enlisted a series of criteria to define MSCs. One of the criteria requires more than 95% of the cellular population to express the markers CD73, CD90, CD105. FACS analysis has revealed that more than 99% of the MSC population of the present invention expresses these classic MSC markers (CD73, CD90 and CD105). Additionally, more than 90% of the MSCs of the present invention express the additional stem cell markers CD9, CD10, CD13, CD29, CD44, CD54 (ICAM-1), CD106 (VCAM-1), CD108, CD146, CD166, HLA-ABC, EPHA 2, SSEA-4 and NESTIN.

[0308] Kannan et al. has reported that over 85% of their cells display the stem cell canonical markers in contrast to the cells obtained by the method of the present invention where >99% of stem cells display the canonical markers. Further, US 11,708,560 reported the expression of only CD105.

[0309] Our hUC-MSCs were extensively characterized using flow cytometry to assess their identity and quality based on surface marker expression. Our hUC-MSCs consistently demonstrated high expression levels of the positive MSC markers CD73 (99.6%), CD90 (99.8%), and CD105 (99.3%), all exceeding the >95% threshold established by the International Society forAttorney Docket No. SASC-OOl-OIWO Cell & Gene Therapy (IS CT). Conversely, the cells exhibited minimal expression of hematopoietic and immune lineage markers — CD34, CD 11b, CD 19, CD45, and HLA-DR — with a combined expression level of 1.43%, well within the ISCT-recommended limit of <2%. Significantly, in addition to common MSC markers, 9 additional functional protein markers denoting the “sternness” quality also demonstrate >90% expression consistently, as depicted in Fig. 1.

[0310] The functionality of our mesenchymal stem cells (MSCs) has been validated through a series of functional assays, including angiogenesis and immunomodulatory activity. Each of these assays has consistently demonstrated robust therapeutic potential, further RNA sequencing showed high expression of anti-apoptotic and anti-oxidant genes.

[0311] 3.6 INDUCTION TEST

[0312] Indoleamine-2, 3 -dioxygenase (IDO) is not natively expressed by MSCs. However, MSCs respond to inflammatory cues and can be induced to secrete immunomodulatory cytokines such as IDO, TSG-6, PGE2 etc, upon stimulation with inflammatory cytokines such as IFNy and TNFa. The immunomodulatory cytokines in turn impact immune cells to produce regenerative cytokines culminating in tissue repair and functional restoration. Thus, induction of IDO secretion by MSCs is an indicator of in vivo potency of MSCs.

[0313] For conducting the induction test, 10 million MSCs were induced with IFNy and TNFa at 37°C for 48 hours, in T75 flasks for the test samples (test and control). This sample was designated as “MSC Treatment” group. For uninduced control 10 million MSCs (MSCs that were not incubated with IFNy and TNFa) were incubated at 37°C for 48 hours in T75 flasks. This sample was designated as “MSC Control” group. Following incubation, the test and control samples were centrifuged at . . .RPM for . . . minutes. The supernatant was evaluated by ELISA while the pelleted MSCs were subjected to RNA sequencing.

[0314] ELISA of the supernatant demonstrated secretion of a higher concentration of IDO by the MSCs of the present invention, that is 223 pg / ml, as compared to 187pg / ml IDO secreted by the control.

[0315] The number of RNA transcript reads of IDO obtained by RNA sequencing of the cells was 58,635 in the induced MSCs of the present invention while the number of transcript reads of IDO for the control was zero. In addition, the following gene expression results were obtained:Table 2: List of genes with more than 1000 RNA transcript readsAttorney Docket No. SASC-OOl-OIWOTable 3: List of genes with less than 1000 RNA transcript reads

[0316] Further highly expressed genes listed in Table 4 represent efficacy of our hUC-MSCs.Table 4: Genes and their respective rolesAttorney Docket No. SASC-OOl-OIWO

[0317] Our MSC manufacturing process has yielded highly consistent results across five different production batches, underscoring the robustness and reproducibility of our quality control measures. The quality of the MSCs was further confirmed through karyotyping and single-cell RNA sequencing analyses. Remarkably, the single-cell RNA sequencing data revealed that 93 - 95% of the cells exhibited gene expression profiles characteristic of MSCs, affirming the purity and identity of our cell populations (Fig.13). Importantly, this confirmed the homogeneous population resulting from our culturing process which is unprecedented. The gene expression also revealed the immunomodulatory, anti-inflammatory, anti-apoptotic, angiogenic, anti-oxidant, and anti-fibrotic properties of our MSCs (Fig.14).

[0318] EXAMPLE 4: CHARACTERIZATION OF EXOSOMES

[0319] Exosomes derived from mesenchymal stem cells (MSCs) were isolated using the filtration method. Exosome isolation technology consistently giving high yield, producing approximately 1.0 x 1011exosomes per milliliter, which is equivalent to 5.8 x 107exosomes per microgram of protein. Our exosomes consistently exhibit a zeta potential of -37.4 ± 6.6 mV, indicating strong colloidal stability and high bioactive functionality.

[0320] These exosomes are highly encapsulated with a range of beneficial microRNAs (miRNAs) and several functional proteins. Preliminary miRNA profiling has revealed a strong presence of anti-inflammatory miRNAs, including miR-125a-3p, which is known to promote the expansion of regulatory T cells (Tregs); miR-146a, which plays a key role in suppressing the NF-KB signalling pathway; and miR-133, which is involved in cellular repair mechanisms, highly scored miRNAs listed below along with beneficial roles reported in research papers.

[0321] These exosomes are highly encapsulated with a range of beneficial microRNAs (miRNAs) and several functional proteins. Preliminary miRNA profiling has revealed a strong presence of anti-inflammatory miRNAs, including miR-125a-3p, which is known to promote the expansion of regulatory T cells (Tregs); miR-146a, which plays a key role in suppressing the NF-KB signalling pathway; and miR-133, which is involved in cellular repair mechanisms. Effects of miRNA are listed in Table 5.Table 5: Effects of miRNAAttorney Docket No. SASC-OOl-OIWO

[0322] In addition to our miRNA analysis, we are currently conducting in-depth proteomic studies to further characterize the functional properties of exosomal proteins and to better understand their therapeutic potential.

[0323] Critical quality attributes were evaluated through a Quality by Design (QbD) approach to ensure the consistency, safety, and efficacy of the final exosome product. Comprehensive characterization was performed using the Exo-Check exosome antibody array (System Biosciences, LLC, USA), targeting a panel of key exosomal protein markers, including CD63, EpCAM, ANXA5, TSG101, Flotillin-1 (Flotl), GM130, ICAM, Alix, and CD81. Further validation of selected markers — CD9, HSP70, TSG101, and the negative marker Calnexin — was conducted using immunoblotting techniques.

[0324] Our results demonstrated that the exosome product consistently expressed hallmark exosomal markers such as CD9, TSG101, and HSP70, while showing no detectable levels of cellular contaminants like Calnexin and GM 130 (Fig. 2A and Fig. 2B). These findings confirm the high purity and identity of the exosome preparation, supporting its potential for downstream therapeutic applications.

[0325] Our exosomal products, derived from human umbilical cord mesenchymal stem cells (hUC-MSCs), exhibit distinct and consistent characteristics. The average particle size was measured at 107.4 ± 18.0 nm using the ZetaView system by Particle Metrix, which falls within the commonly reported size range for exosomes (30-150 nm). (Fig. 3)

[0326] Transmission electron microscopy (TEM) data revealed that the exosomes consistently displayed a spherical to cup-shaped morphology across multiple batches, indicating the reproducibility and stability of our isolation and preparation methods. Result is shown in Fig.3

[0327] Example 5: Comparative efficacy results demonstrating treatment outcomes for (i) EVs alone, (ii) mesenchymal cells alone, and (iii) a combination of EVs and mesenchymal cells

[0328] The therapeutic potential of mesenchymal stem cells (MSCs) and their derived exosomes was systematically evaluated through a series of in vitro functional assays, including lymphocyte proliferation inhibition, angiogenic tube formation, and wound scratch assays. These assays were designed to assess the immunomodulatory, angiogenic, and regenerative / anti-apoptotic properties of the treatments.Attorney Docket No. SASC-OOl-OIWO

[0329] When tested individually, MSC-derived exosomes at an optimized concentration of 100 pg exhibited a significant inhibitory effect on lymphocyte proliferation, achieving 55.3% inhibition.

[0330] In comparison, MSCs alone demonstrated a higher inhibition rate of 70.5%, highlighting their strong immunosuppressive capacity.

[0331] Remarkably, the combination of MSCs with their exosomes produced a synergistic effect, enhancing the inhibition of lymphocyte proliferation to 89.9%. Further analysis of regulatory T cell (Treg) proliferation revealed significant increases with treatment by exosomes (100 pg), MSCs, and their combination, resulting in Treg populations of 17.2%, 16.4%, and 22.7%, respectively. This substantial increase suggests that co-administration of MSCs and exosomes may enhance immunomodulation and tissue repair outcomes. Result is shown in Fig. 4.

[0332] In angiogenic functional assays, individual treatments with exosomes or secretome showed moderate formation of capillary-like structures, indicating some degree of angiogenic potential. The combination of exosomes and secretome significantly enhanced the formation of interconnected tubular networks compared to either treatment alone. This suggests a synergistic effect where the combination promotes better endothelial cell organization, migration, and angiogenesis.

[0333] Individual treatments with exosomes or secretome showed moderate cell migration and partial wound closure over the observed time period. The combination of exosomes and secretome significantly enhanced cell migration and accelerated wound closure compared to either treatment alone. This suggests a synergistic effect, where exosomes and secretome together provide a more potent regenerative environment, promoting faster and more effective wound healing. Result is illustrated in Fig. 4.

[0334] Example 6: Combinations of mesenchymal cells and EVs in different ratios.

[0335] Based on previous research data, we conducted efficacy studies in an animal model using 1 :5000 MSCs-to-exosome ratio to evaluate the therapeutic potential of our combination dose. Surprisingly the results demonstrated a remarkable 0% mortality rate and 100% recovery from liver disease in the treated group, compared to the placebo group.

[0336] We investigated further with various ratios of mesenchymal stromal cells (MSCs) to exosomes to determine the optimal combination that would yield the most effective synergistic therapeutic effects under cell culture conditions. These different ratios were evaluated utilizing key functional assays that reflect the therapeutic potential of the combination. For assessing angiogenic capacity, we employed both tube formation and wound scratch assays, which measure the ability of the cells and exosomes to promote blood vessel formation and wound healing, respectively. Additionally, we examined immunomodulatory functions by evaluating the inhibition ofAttorney Docket No. SASC-OOl-OIWO lymphocyte proliferation, a critical aspect of the anti-inflammatory and immune -regulating potential of MSCs and their secreted exosomes.

[0337] Based on a concentration of MSCs at 1.0 x 107cells per milliliter of secretome and an estimated 1 x 1011exosomes per microgram of protein, tube formation and wound scratch assays were performed using various ratios of MSCs to exosomes, as outlined below in Table 6.Table 6: Ratio of Mesenchymal cells to exosomes in different samples.

[0338] All tested ratios demonstrated an incremental protective effect. Notably, the 1 :66,667 ratio (Exo 100 pg + Sec- 15) showed a synergistic response, outperforming the individual effects of exosomes or secretome alone.

[0339] Similarly, peripheral blood mononuclear cells (PBMCs) were co-cultured with MSCs along with varying concentrations of exosomes (25 pg, 50 pg, 100 pg, and 200 pg) to investigate the immunomodulatory effects at different MSC-to-exosome ratios. Under co-culture conditions, the 1:250,000 ratio (Exo 100 pg + MSCs) exhibited the strongest inhibition of lymphocyte proliferation and a notable increase in regulatory T cell (Treg) population, compared to other tested ratios as shown in Table 7.Table 7: Ratio of Mesenchymal cells to exosomes in different samples.

[0340] The optimal mesenchymal stem cell (MSC) to exosome ratio identified under in vitro conditions (1 :100,000) did not directly translate to in vivo efficacy, where a ratio of 1 :5,000 demonstrated superior therapeutic outcomes. Result is illustrated in Fig. 5.

[0341] This discrepancy is attributable to the fundamental biological differences between simplified in vitro systems and the complex physiological environment of living organisms. In vivo, exosome biodistribution, cellular uptake, clearance kinetics, immune modulation, and paracrine feedback loops significantly influence therapeutic dynamics. Moreover, stem cells exhibit context-dependent secretory and homing behaviours that are not fully replicated in vitro. Consequently, the observed shift in optimal ratio reflects the integrated effects of host biology,Attorney Docket No. SASC-OOl-OIWO tissue microenvironment, and systemic pharmacokinetics, rather than a methodological inconsistency.

[0342] Example 7: COMPARATIVE IN VIVO ANALYSIS

[0343] Human UC-MSCs were cultured via a xenofree, explant process with modifications to passage 3 and SEVs were obtained from supernatant of the cultured UC-MSCs. Two groups, each of 14. Wistar male rats, aged 7-8 weeks, received oral CCL4 with olive oil (Iml / kg) twice weekly for a total of 6 weeks from week 1 to week 6. Starting at week 4, after all animals in both groups received 6 induction doses of CCL4, one group of 14 animals received three weekly IV doses of UC-MSC + SEV at a dose of 1 million MSCs and 5 billion SEV each. All animals alive at week 7 were sacrificed. 14 animals who received CCL4 alone from weeks4-7 were control animals. The primary objectives were to examine the survival differences between two groups of animals and the effect of UC-MSC + SEV on fibrosis stage by Trichrome and Sirius Red. In our animal trial, 28 rats were given oral CCL4 (twice weekly) for 6 weeks to induce chronic liver failure for 6 wks.

[0344] Six animals in the control group died before week 6 whereas all 14 animals in the US-MSC + SEV were alive at week 6. The survival difference at 6 weeks was significant between two groups (100% with UC-MSC + SEV vs 57%, p=0.0066). The necropsy of 6 dead animals in the control group showed cirrhosis in all 6 animals. The comparison between 8 animals in the control group and 14 animals receiving US-MSC + SEV is shown in Table 4. Notably, liver fibrosis stage by both Trichrome and Sirius Red was significantly lower in the UC-MSC + SEV group. While there were no animals in the UC-MSC + SEV group had cirrhosis, there were 12 animals in the control group with cirrhosis. There were corresponding favorable liver biochemistry and liver immunohistochemistry changes in the UC-MSC +SEV group (Table 8 and Figs. 6 to 11).

[0345] After week 4, 14 animals received a total of 3 IV infusions of 1 million UC-MSCs and 5 billion exosomes weekly and the remaining 14 were considered as control groups. At week 7, 40% of the control group died whereas no animal perished in the treatment group. Furthermore, all the rats in the treatment group demonstrated reversal of fibrosis and cirrhosis compared to only 2 animals in the control group. Hepatomegaly, serum ALT and ALP, sirius red and masson trichrome staining for cirrhosis and fibrosis, Immunohistochemistry for liver collagen, a-SMA, CK-18 and CK-19 showed significant improvement in the treatment group.Table 8: Selected characteristics of CCL4 fed animals with and without US-MSC +SEV treatment

[0346] In a mice model, CCL4 was injected subcutaneously twice weekly for 8 weeks and GMP grade UC-MSCs were administered IV once in 2 weeks for 6 weeks. Low, medium and high MSC doses of 2.5 million, 5 million and 10 million respectively were infused into different groups. Survival rate for high, medium and low doses was 100%, 92.3% and 90.7% whereas it was only 55% for the control group. Although survival benefit was demonstrated in this study, large doses were required compared to our study with the lowest dose and high dose being 2.5 times and 10 times more than our doses. Moreover, biochemical parameters, liver co-efficient, gross liver appearance, histopathology, and favorable liver tissue expression of HGF, TGFb and IL- lb were more significant in the high dose when compared to low dose groups.

[0347] In a rat model of CCL4 induced liver failure (every other day IP injection of CCL4 for 16 weeks), portal vein infusion and intrahepatic injection of 1 million autologous adipose MSCs without exosomes failed to provide any survival benefit. Although, 5 weeks after MSC treatment, improvement in total bilirubin levels, sonographic appearance and histopathology changes were noted, no improvement in survival was reported despite the invasive mode of MSC administration.

[0348] Human UC-MSCs cultured to passage 3 in combination with SEV significantly improved the survival of the animals receiving low dose CCL4. UC-MSC + SEV dramatically reduced the development of fibrosis and cirrhosis induced by CCL4. Further, studies are needed to validate our observations and to test the combination of UC-MSC + SEV in other animal models and in humans with fibrotic liver diseases and liver failure.Attorney Docket No. SASC-OOl-OIWO

[0349] Example 9: Effect of effect of MSCs, exosomes, and combined MSCs + exosomes in rats

[0350] An acute liver failure model was induced in rats using a single intraperitoneal dose of 1000 mg acetaminophen. Animals were divided into five groups: Healthy Control (no acetaminophen, n=3), Vehicle Control (acetaminophen + vehicle, n=6), MSCs only (acetaminophen + mesenchymal stem cells, IxlO6cells / dose, n=6), Exosomes only (acetaminophen + exosomes, 5xl09particles / dose, n=6), and MSCs + Exosomes (combination therapy, n=6). All four treatments were given 3 hours after the acetaminophen administration in two doses with a 3-hour interval.

[0351] All animals survived the 48-hour study period. Early biochemical parameters (BUN, ALT, AST, Albumin) showed no significant differences among groups. However, histological analysis (H&E staining) revealed notable improvement in liver morphology in treatment groups, particularly in MSC + Exo, which showed near-normal architecture similar to healthy controls. In conclusion, combined MSC + Exosome therapy provided the highest hepatoprotective effect, nearly restoring normal liver morphology compared to controls.

[0352] Fig. 12 shows effect of MSCs, exosomes, and combined MSCs + exosomes on liver morphology following acetaminophen -induced acute liver failure in rats. Rats were sacrificed after 48 hours of treatment; liver sections were analyzed using hematoxylin and eosin (H&E) staining. The bar graph represents the percentage improvement in liver morphology compared to healthy controls. The Vehicle Control group showed minimal recovery (17%), while MSC only and Exosome only treatments significantly improved liver architecture (67% and 83%, respectively). The combination of MSCs and Exosomes exhibited the highest improvement (100%), comparable to the Healthy Control group, indicating synergistic hepatoprotective and regenerative effects.

[0353] From the foregoing, a person skilled in the art will appreciate that the disclosure provided herein is susceptible to variations and modifications and can be implemented in other forms without changing the technical spirit or essential features of the invention. The present disclosure includes all such variations, modifications and equivalent concepts. The specific embodiments described herein are only illustrative and the present disclosure is not to be limited in scope by the specific embodiments.

[0354] INCORPORATION BY REFERENCE

[0355] All references, including granted patents and patent application publications, referred in this document are incorporated herein by reference in their entirety.List of references are:US20210052657A1US20190269739A1Attorney Docket No. SASC-OOl-OIWOABBREVIATIONS USED:MSC- mesenchymal stem cellCCL4- Carbon tetrachlorideCFU-F- colony forming unit-fibroblastECM- extracellular matrixFBS- fetal bovine serumFd-hPL- Fibrinogen-Depleted hPL hPL- human platelet lysateIDO- Indoleamine-2,3-dioxygenaseISCT- International Society for Cell and Gene TherapyPD- population doublingPDT- population doubling timeRPM- revolutions per minuteUC- umbilical cord

Claims

Attorney Docket No. SASC-001-01WOWHAT IS CLAIMED IS:

1. A method of treating a disease or a condition in a subject in need thereof, the method comprising: administering a therapeutic effective amount of a pharmaceutical composition comprising mesenchymal cells and exosomes to the subject; wherein the mesenchymal cells and exosomes are in a ratio of about 1 : 2500 to 1: 250000 wherein the mesenchymal cells have a sternness quality more than 90% and viability more than 95%, and the exosomes are free of a cellular contaminant as detected by immunoblotting technique, wherein the cellular contaminant comprises calnexin, and treating said disease or condition.

2. The method of claim 1, wherein the cellular contaminant further comprises GM130.

3. The method of claim 1, wherein the exosomes have a zeta potential in a range of -30mV to -50 mV, and a size range between 85 nm to 130 nm as measured using the ZetaView system by Particle Metrix.

4. The method of claim 1, wherein a ratio of the mesenchymal cells to exosomes is 1: 150,000.

5. The method of claim 1, wherein a ratio of the mesenchymal cells to exosomes is 1:75,000.

6. The method of claim 1, wherein a ratio of the mesenchymal cells to exosomes is 1: 10,000.

7. The method of claim 1, wherein a ratio of the mesenchymal cells to exosomes is 1:5000.

8. The method of claim 1, further comprising administering peripheral blood mononuclear cells along with the mesenchymal cells and the exosomes.

9. The method of claim 1, wherein said disease or condition is selected from a neurological disease, a muscular disease, an autoimmune disease, an inflammatory disease, a digestive disease, an energy homeostasis disease, a fibrotic disease, aging, radiation induced injury, cell transplant rejection and a proliferative disease.

10. The method of claim 1 or 9, wherein the disease is a fibrotic disease.

11. The method of claim 10, wherein the fibrotic disease comprises a liver fibrosis.

12. The method of claim 1, wherein the mesenchymal cells are derived from umbilical cord of a mammal.Attorney Docket No. SASC-OOl-OIWO13. The method of claim 1 to 3, wherein the pharmaceutical composition is configured to improve the liver morphology by more than 80% as detected by H&E staining.

14. The method of claim 1 or 3, wherein the exosomes are derived from the mesenchymal cells.

15. The method of claim 1, wherein said administering comprises at least one of intravenous administration, intramuscular administration, intranasal administration, intrathecal administration, intrastriatal administration, intracranial administration, intraarterial administration, and subcutaneous administration.

16. The method of claim 1 to 3, wherein the pharmaceutical composition is configured to improve the liver morphology by more than 80% as detected by H&E staining.

17. The pharmaceutical composition of claim 1 to 3, wherein the pharmaceutical composition is configured to increase Treg cells by more than 20%.

18. The method of claim 1 to 3, wherein the pharmaceutical composition is configured to inhibit a lymphocyte proliferation by more than 85% in the subject having a fibrotic disease.

19. The method of claim 1, wherein the mesenchymal cells have population doubling time (PDT) about 23 hours or less without any addition of a growth stimulant.

20. The method of claim 1 to 3, wherein the exosomes have biomarkers selected from CD9, TSG101, and / or HSP70 having expression level more than 95%, and enriched by miRNA selected from miR-125a-3p, miR-146a, and / or miR-133.

21. A method, comprising: a) taking an explant comprising an umbilical cord; b) allowing release of cells from the explant; c) cryopreserving and thawing the cells at a temperature about 30°C to 40°C; and f) harvesting mesenchymal cells in an in vitro condition; wherein the mesenchymal cells have a sternness quality more than 90% and viability more than 95%, wherein the method is configured to give 1.1 million mesenchymal cells per cm of the umbilical cord.

22. The method of claim 21, wherein the mesenchymal cells have cell size less than 15 micron.

23. The method of claim 21 or 22, wherein the mesenchymal cells have PDT about 25 hours or less without any addition of a growth stimulant.Attorney Docket No. SASC-OOl-OIWO24. The method of claim 21, further comprising deriving exosomes from the mesenchymal cells.

25. The method of claim 24, wherein the exosomes have biomarkers selected from CD9, TSG101, and / or HSP70 having expression level more than 95%; and are free of a cellular contaminant comprising Calnexin and / or GM 130.

26. The method of claim 24 or 25, wherein the exosomes are enriched by miRNA selected from miR-125a-3p, miR-146a, and / or miR- 133.

27. The method of claim 21, further adding a pharmaceutical acceptable excipient in the mesenchymal cells and exosomes to form a pharmaceutical composition.

28. The method of claim 24, wherein the exosomes have a size range between about 85 nm to about 130 nm as measured using the ZetaView system by Particle Metrix.

29. The method of claim 24, wherein the method provides about 1 .0 x 1010exosomes per milliliter to 1.0 x 1012exosomes per milliliter (which is equivalent to 5.8 x 106exosomes per microgram to 5.8 x 108exosomes per microgram of protein).

30. The method of claim 21, wherein the mesenchymal cells have an expression level of immune lineage markers comprising CD34, CDl lb, CD19, CD45, HLA-DR or combinations thereof less than 1.5%.

31. A pharmaceutical composition comprising mesenchymal cells and exosomes in a ratio of about 1 : 2500 to 1 : 250000, wherein the mesenchymal cells have a sternness quality more than 90% and viability more than 95%, and the exosomes are free of a cellular contaminant as detected by immunoblotting technique, wherein the cellular contaminant comprises calnexin.

32. The pharmaceutical composition of claim 31, wherein the mesenchymal cells have an expression level of immune lineage markers comprising CD34, CDl lb, CD19, CD45, HLA-DR or combination thereof less than 1.5%.

33. The pharmaceutical composition of claim 31, wherein the mesenchymal cells have PDT about 25 hours or less without any addition of a growth stimulant.

34. The pharmaceutical composition of claim 31, wherein the exosomes have biomarkers selected from CD9, TSG101, and / or HSP70, and enriched by miRNA selected from miR-125a-3p, miR- 146a, and / or miR- 133.Attorney Docket No. SASC-OOl-OIWO35. The pharmaceutical composition of claim 31, wherein mesenchymal cells have cell size less than 15 micron, and the exosomes have a size in range of about 85 nm to about 130 nm as measured using the ZetaView system by Particle Metrix.

36. The pharmaceutical composition of claim 31, further comprises a pharmaceutical acceptable adjuvant, excipient or carrier.

37. The pharmaceutical composition of claim 31, wherein the ratio is about 1:5000 to 1 : 100000.

38. The pharmaceutical composition of claim 31, wherein the mesenchymal cell is derived from an umbilical cord, and the exosomes are derived from the mesenchymal cells.

39. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is configured to treat a fibrotic disease comprising a liver fibrosis.

40. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition is configured to inhibit a lymphocyte proliferation by more than 85% in the subject having a fibrotic disease.

41. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition further comprises at least one additional therapeutic agent.

42. The pharmaceutical composition of claim 31, wherein the cellular contaminant further comprises GM130.

43. The pharmaceutical composition of claim 31, wherein a ratio of the mesenchymal cells to exosomes is about 1 :75,000 to 1:125000.

44. The pharmaceutical composition of claim 31, wherein the exosomes have a zeta potential in a range of -30mV to -50 mV, as measured using the ZetaView system.

45. The pharmaceutical composition of claim 31, wherein said composition is configured to treat a disease or a condition selected from a neurological disease, a muscular disease, an autoimmune disease, an inflammatory disease, a digestive disease, an energy homeostasis disease, a fibrotic disease, aging, radiation induced injury, cell transplant rejection and a proliferative disease.

46. The pharmaceutical composition of claim 31, wherein the exosomes are derived from the mesenchymal cells.Attorney Docket No. SASC-OOl-OIWO47. The pharmaceutical composition of claim 31, wherein the mesenchymal cells are derived from umbilical cord of a mammal.

48. The pharmaceutical composition of claim 31, wherein the mesenchymal cells have more than 95% expression level of positive markers selected from CD73, CD90, and CD105.

49. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is configured to improve the liver morphology by more than 80% as detected by H&E staining.

50. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is configured to increase Treg cells by more than 20%.

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