Methods of tissue stem cells extraction and culture

Abstract

The present invention relates to methods for extracting stem cells from a tissue, and culturing, expanding and / or recovering these stem cells, with at most one step of exposure to a protease. The present invention also relates to stem cells obtained by these methods, and compositions comprising thereof, and their applications.

Description

METHODS OF TISSUE STEM CELLS EXTRACTION AND CULTUREFIELD OF INVENTION

[0001] The present invention relates to methods for extracting stem cells from a tissue, and culturing, expanding and / or recovering these stem cells, with at most one step of exposure to a protease.BACKGROUND OF INVENTION

[0002] Stem cells are self-renewable cells and constitute powerful tools for many applications, in particular therapeutic applications such as regenerative medicine.

[0003] The production of stem cells, and in particular clinical grade stem cells, is thus a major challenge for the health industry. The steps of extracting stem cells from their tissue of origin, culturing / expanding them, and finally recovering / isolating them, must be optimized in order to yield high quantities of high-quality stem cells in a short amount of time.

[0004] Extraction of stem cells from tissues often rely on the use of proteases such as collagenase to digest the tissue and facilitate extraction. Besides, stem cells are often cultured according to classical cell culture procedure involving detaching the stem cells from their culture vessel with a protease such as trypsin in order to re-seed them an amplify them - repeatedly. However, these methods have a real impact on the purity and the growth capacity of isolated stem cells: while the enzymatic digestion helps to isolate a higher number of stem cells, it still damages the cellular viability and the cell size, as well as affect the expression of some surface markers.

[0005] While it is also possible to extract stem cells from a tissue without using an enzyme by relying on their migratory capabilities, this approach is not fully satisfactory because (i) the stem cells easily adhere to the plastic of culture supplies and invade theirwhole environment, which is difficult to adapt to large scale production, and (ii) precisely because they adhere to plastic, the culturing / amplification step will still require passaging, z.e., the repeated exposure to trypsin or the like.

[0006] There is thus an unmet need for a method that enables large scale production of stem cells while maintaining their viability, function, and surface proteome.SUMMARY

[0007] This invention thus relates to a non-protease-based method for extracting stem cells from a tissue, comprising contacting said tissue with at least one matrix suitable for cell adhesion in a culture vessel comprising a culture medium, wherein said culture vessel is a non-adherent culture vessel.

[0008] In some embodiments, the tissue is selected from the list (or group) comprising or consisting of umbilical cord, Wharton jelly, placenta, fetal tissue, bone marrow, peripheral blood, adipose tissue, dental pulp, thymus, and muscle.

[0009] In some embodiments, the tissue is Wharton jelly or placenta.

[0010] In some embodiments, the stem cells are selected from the group comprising or consisting of mesenchymal stem cells, neural stem cells, and epithelial stem cells, preferably mesenchymal stem cells (MSC).

[0011] In some embodiments, the non-adherent culture vessel is a low attachment culture vessel or an ultra-low attachment culture vessel.

[0012] In some embodiments, the at least one matrix suitable for cell adhesion is a microcarrier, preferably a dissolvable microcarrier.

[0013] In some embodiments, the method further comprises incubating said tissue with said at least one matrix suitable for cell adhesion for at least 168 hours.

[0014] In some embodiments, the method comprises the steps of:i. in a non-adherent culture vessel comprising at least one matrix suitable for cell adhesion, preferably at least one microcarrier, and a culture medium, placing the tissue; ii. incubating for at least 168 hours; and iii. obtaining the stem cells on the at least one matrix suitable for cell adhesion.

[0015] In some embodiments, step (i) is preceded by a step of placing at least one matrix suitable for cell adhesion, preferably at least one microcarrier, and a culture medium in a non-adherent culture vessel.

[0016] In some embodiments, the stem cells are not exposed to a protease selected from the group comprising or consisting of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, and any combination thereof.

[0017] The present invention further relates to a method of expanding stem cells extracted from a tissue, comprising or consisting of the steps of: i. extracting stem cells from a tissue with the method according to the present invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; v. optionally, recovering the stem cells from the matrixes suitable for cell adhesion comprising stem cells, preferably by trypsinization, and / or recovering the culture supernatant.

[0018] The present invention further relates to a method of obtaining or recovering mesenchymal stem cells extracted from a tissue selected from the group comprising orconsisting of placenta, umbilical cord, and Wharton jelly, the method comprising or consisting of the steps of: i. in a non-adherent culture vessel comprising a plurality of microcarriers and a culture medium, placing said tissue; ii. incubating for at least 168 hours; iii. obtaining a plurality of microcarriers comprising mesenchymal stem cells; iv. optionally, transferring the plurality of microcarriers comprising mesenchymal stem cells into one or more new non-adherent culture vessel comprising a plurality of microcarriers, and incubating for at least 24 hours; v. optionally, repeating steps (iii) to (iv) any number of times; vi. optionally, washing the plurality of microcarriers comprising mesenchymal stem cells; vii. contacting the plurality of microcarriers comprising mesenchymal stem cells with a composition comprising trypsin; and viii. recovering the mesenchymal stem cells; wherein the mesenchymal stem cells are exposed at most one time to a protease or to a cocktail of proteases, and optionally wherein the microcarriers are dissolvable microcarriers.

[0019] The present invention further relates to a stem cell or a population of stem cells obtained by the method according to the present invention.

[0020] The present invention further relates to a stem cell or a population of stem cells having increased expression of at least one gene selected form the group comprising or consisting of CXCR4, CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD44, Galectin, CXCR7, VLA-4, CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR5, CXCR6, MMP-1, MMP-2, MMP-3, TMP-1, TMP-2, integrins, PDGF-AB, CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD271, CD14, CD56, and CD146, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel.

[0021] The present invention further relates to a stem cell or a population of stem cells having increased expression of at least one gene selected form the group comprising or consisting of ALCAM, CXCR4, CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD44, Galectin, CXCR7, VLA-4, CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR5, CXCR6, IGF-1, IL-1B, IL-15, MMP-1, MMP-2, MMP-3, TLR3, TMP-1, TMP-2, integrins, PDGF-AB, VEGF, CD 13, CD29, CD44, CD73, CD90, CD105, CD106, CD271, CD14, CD56, and CD146, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time, preferably selected form the group comprising or consisting of TLR3, IL-15, CXCL8, ALCAM, VEGF, CD44, CXCL1, IL-1B, and IGF- 1.

[0022] The present invention further relates to a pharmaceutical composition comprising the stem cell or population of stem cells according to the present invention, and at least one pharmaceutically acceptable excipient.

[0023] The present invention further relates to a stem cell or population of stem cells, or pharmaceutical composition, according to the present invention, for use for treating a disease selected from the group comprising, inflammation, infection, cancer, autoimmune diseases, neurologic diseases, cardiologic diseases and genetic diseases.DEFINITIONS

[0024] In the present invention, the following terms have the following meanings:

[0025] “About”, when preceding a figure, means plus or less 10% of the value of said figure.

[0026] “And / Or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0027] “At least one” includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 103,104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015or more.

[0028] “Clinical grade”, when referring to a cell, means cells produced under culture conditions compatible with use in humans and conforming with the Good Practices of pharmaceutical production.

[0029] “Comprising", "comprises" and "comprised of" are used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. These terms also encompass “consisting of’.

[0030] “Culturing”, “cultivating”, “expanding”, and “amplifying”, when applied to stem cells, are used interchangeably to refer to procedures and conditions allowing for the increase or growth in the population of cells in vitro.

[0031] “Subject” or “individual” refers to an animal individual, preferably a mammalian individual, more preferably a human individual. In some embodiments, an individual may be a mammalian individual. Mammalians include, but are not limited to, all primates (human and non-human), cattle (including cows), horses, pigs, sheep, goats, dogs, cats, and any other mammal which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease or condition. In some embodiments, an individual may be a “patient”, ie., a warm-blooded animal, more preferably a human, who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease or condition. In some embodiments, the individual is an adult (e.g., an individual above the age of 18). In some embodiments, the individual is a child (e.g., an individual below the age of 18). In some embodiments, the individual is a male. In some embodiments, the individual is a female.

[0032] “Therapeutically effective amount” refers to the level or amount of an agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of the disease or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease or condition; (3) bringing about ameliorations of the symptoms of the disease or condition; (4) reducing the severity or incidence of the disease or condition; or (5) curing the disease or condition. A therapeutically effective amount may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after initiation of the disease or condition, for a therapeutic action.

[0033] “Treatment”, “alleviation”, “curation” and any declensions thereof refer to a therapeutic treatment, excluding prophylactic or preventative measures; wherein the object is to slow down, lessen, stop or even reverse (either partially or totally) the evolution of a targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well those suspected to have the disorder. A subject is successfully “treated” for the targeted pathologic condition or disorder if, after receiving treatment, they show observable and / or measurable reduction in or absence of one or more symptoms associated with the pathologic condition or disorder; relief to some extent; reduced morbidity and / or mortality; and / or improvement in quality-of-life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0034] “Wharton jelly” means the connective tissue of extra-embryonic mesoblast origin, enveloping the two arteries of the umbilical vein and thus protecting the umbilical cord.DETAILED DESCRIPTION

[0035] The present invention relates to a non-protease-based method for extracting stem cells from a tissue, comprising contacting said tissue with at least one matrix suitable forcell adhesion in a culture vessel comprising a culture medium, wherein said culture vessel is a non-adherent culture vessel.

[0036] The present invention relates to a method for extracting stem cells from a tissue without using a protease, comprising contacting said tissue with at least one matrix suitable for cell adhesion in a culture vessel comprising a culture medium, wherein said culture vessel is a non-adherent culture vessel.

[0037] It will be apparent to the person skilled in the art that the tissue may be a part or a fragment of one or more tissue or of one or more organ. In one embodiment, stem cells are extracted from one (z.e., a single) tissue. In another embodiment, stem cells are extracted from more than one (z.e., multiple) tissues, wherein the stem cells from a first tissue are the same type of stem cells than the stem cells from a second tissue. In certain embodiments, stem cells are extracted from 2, 3, or 4 different tissues, wherein the stem cells from a first tissue are the same type of stem cells than the stem cells from a second tissue.

[0038] In some embodiments, the tissue is selected from the list (or group) comprising or consisting of umbilical cord, Wharton jelly, placenta, fetal tissue, bone marrow, peripheral blood, adipose tissue, dental pulp, thymus, amnios, cord blood, and muscle. In some embodiments, the tissue is selected from the list (or group) comprising or consisting of umbilical cord, Wharton jelly, placenta, fetal tissue, bone marrow, peripheral blood, adipose tissue, dental pulp, thymus, and muscle. In some embodiments, the tissue is selected from the list (or group) comprising or consisting of umbilical cord, Wharton jelly, placenta, bone marrow, peripheral blood, adipose tissue, dental pulp, thymus, and muscle.

[0039] In some embodiments, the tissue is selected from the group comprising or consisting of umbilical cord, Wharton jelly, fetal tissue, cord blood, amnios, and placenta. In some embodiments, the tissue is selected from the group comprising or consisting of umbilical cord, Wharton jelly, fetal tissue and placenta. In some embodiments, the tissue is selected from the group comprising or consisting of umbilical cord, Wharton jelly, amnios, and placenta. In some embodiments, the tissue is selected from the groupcomprising or consisting of umbilical cord, Wharton jelly, and placenta. In some embodiments, tissue is Wharton jelly or placenta.

[0040] In some embodiments, the tissue is Wharton jelly. In some embodiments, the tissue is placenta. In some embodiments, the tissue is umbilical cord. In some embodiments, the tissue is a fetal tissue. In some embodiments, the tissue is bone marrow. In some embodiments, the tissue is peripheral blood. In some embodiments, the tissue is adipose tissue. In some embodiments, the tissue is dental pulp. In some embodiments, the tissue is thymus. In some embodiments, the tissue is muscle.

[0041] In one embodiment, stem cells are extracted only from Wharton jelly. In another embodiment, stem cells are extracted only from umbilical cord. In another embodiment, stem cells are extracted only from placenta. In another embodiment, stem cells are extracted from umbilical cord and placenta. In another embodiment, stem cells are extracted from Wharton jelly and placenta. In another embodiment, stem cells are extracted from umbilical cord and Wharton jelly. In another embodiment, stem cells are extracted from umbilical cord, Wharton jelly, and placenta.

[0042] It will be understood that, in the cases where stem cells are extracted from multiple tissue sources, the stem cells are all the same time of stem cells (e.g., all mesenchymal stem cells). Illustratively, stem cells may be extracted from umbilical cord and placenta, therefore obtaining umbilical mesenchymal stem cells and placental mesenchymal stem cells; or stem cells may be extracted from Wharton jelly and placenta, therefore obtaining Wharton jelly mesenchymal stem cells and placental mesenchymal stem cells.

[0043] In some embodiments, the tissue is an animal tissue, preferably a mammalian tissue, more preferably a human tissue. In some embodiments, the tissue is a human tissue. In some embodiments, the tissue is from a human donor. In some embodiments, the tissue is from an adult human donor.

[0044] In some embodiments, the tissue is extracted or collected shortly after birth. Illustratively, umbilical cord, Wharton jelly, and / or placenta, or fragment or parts thereof, may be extracted after birth. In some embodiments, isolation or extraction of the tissuedoes not involve the destruction of an embryo. In some embodiments, isolation or extraction of the tissue does not involve the destruction of a human embryo. In some embodiments, isolation or extraction of the tissue does not involve any invasive procedure in the human body.

[0045] While stem cells may be extracted from a tissue freshly isolated from a donor, it is also possible to extract stem cells from a frozen or cryopreserved tissue. Therefore, in some embodiments, the tissue is a fresh tissue (ie., freshly isolated or extracted tissue) or a frozen or cryopreserved tissue.

[0046] In one embodiment, the tissue is a fresh tissue. The person skilled in the art knows that if a fresh tissue is left untreated, its biological components start to decay after a certain period of time, thus the method of the invention is to be performed promptly after isolation or extraction of the fresh tissue.

[0047] In some embodiments, the method of the invention is performed within at most about 96 hours, about 72 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 8 hours, about 4 hours, about 2 hours, or less, after extraction or isolation of the fresh tissue from a donor. In some embodiments, the method of the invention is performed within at most about 24 hours after extraction or isolation of the fresh tissue from a donor. In some embodiments, the method of the invention is performed for a duration ranging from about 1 minute to about 48 hours after extraction or isolation of the fresh tissue from a donor, more preferably for a duration ranging from about 1 minute to about 24 hours after extraction or isolation of the fresh tissue from a donor. In some embodiments, the method of the invention is performed immediately after extraction or isolation of the fresh tissue from a donor.

[0048] In some embodiments, the fresh tissue may be stored at a temperature ranging from 2°C to 8°C, preferably about 4°C, for a duration of at most about 96 hours, about 72 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or less, after extraction or isolation of the fresh tissue from a donor. Storage as this temperature is meant to be temporary.

[0049] In another embodiment, the tissue is a frozen or cryopreserved tissue.

[0050] In some embodiments, after extraction or isolation from a donor, the tissue is frozen or cryopreserved.

[0051] In some embodiments, the tissue is contacted with at least one cryoprotectant or cryoprotective agent, typically a cryoprotectant solution, prior to being frozen. Non- limitative example of cryoprotective agents include glycerol, sucrose, Me2SO and DMSO.

[0052] In some embodiments, the tissue is frozen by liquid nitrogen and / or liquid nitrogen vapors.

[0053] In some embodiments, the frozen or cryopreserved tissue is stored at a temperature ranging from about -20°C to about -200°C, preferably ranging from about - 80°C and about -200°C. In some embodiments, the frozen or cryopreserved tissue is stored at a temperature of about -20°C, preferably about -80°C, more preferably about - 196°C.

[0054] In some embodiments, the tissue is frozen or cryopreserved within at most about 96 hours, about 72 hours, about 48hours, about 36 hours, about 24 hours, about 12 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, or less, after extraction or isolation of the tissue from a donor. In some embodiments, the tissue is frozen or cryopreserved within at most 24 hours after extraction or isolation of the tissue from a donor. In some embodiments, the tissue is frozen or cryopreserved for a duration ranging from about 1 minute to about 48 hours after extraction or isolation of the tissue from a donor, more preferably for a duration ranging from about 1 minute to about 24 hours after extraction or isolation of the tissue from a donor. In some embodiments, the tissue is frozen or cryopreserved immediately after extraction or isolation of the tissue from a donor.

[0055] In some embodiments, the frozen or cryopreserved tissue is stored for about 1 day, about 1 week, about 1 month, about 1 year, or more.

[0056] In some embodiments, the frozen tissue is thawed. Therefore, in certain embodiments, the tissue is a thawed tissue. Techniques to thaw a frozen tissue and tooptimize the recovery of stem cells from cryopreserved tissue are known in the art, such as, illustratively, in Munoz-Dominguez et al. (Cryobiology vol. 108 (2022): 34-41).

[0057] In some embodiments, the tissue is contacted with at least one antibiotic and / or antifungal agent prior to stem cell extraction. In some embodiments, the tissue is contacted with at least one antibiotic and / or antifungal agent for a duration from 10 minutes to 5 hours, preferably from 30 minutes to 2 hours, more preferably for about 1 hour, prior to stem cell extraction, optionally wherein the antibiotic is diluted or solubilized in a biological buffer (e.g., PBS). In some embodiments, the at least one antibiotic agent may any suitable antibiotic agent know in the art. In some embodiments, the at least one antibiotic agent is selected from the group comprising or consisting of amoxicillin, amphotericin, vancomycin, gentamycin, streptomycin, penicillin, ampicillin. In some embodiments, the at least one antibiotic agent is selected from the group comprising or consisting of amoxicillin, amphotericin, and vancomycin.

[0058] In some embodiments, the tissue is cut prior to stem cell extraction. In some embodiments, the tissue is cut in slices. In some embodiments, the tissue is cut in slices from 0.1 mm to 10 mm thick, preferably from 0.5 mm to 5 mm thick, more preferably from 1 mm to 4 mm thick, even more preferably from 2 mm to 3 mm thick. In some embodiments, the tissue is cut in slices from about 0.1 g to about 2 g, preferably about 0.6 g. In some embodiments, the tissue is cut in sterile conditions, preferably with a sterile scalpel or any sterile suitable tool. In some embodiments, the method of the invention is performed on tissue slices as described herein.

[0059] Within the scope of the present invention, the term “stem cells” encompasses a population of stem cells, a small group of stem cells, individual stem cells or isolated stem cells. As used herein, an “isolated cell” is a cell that is separated form a tissue or an organ, or a cell that is separated form cells of dissimilar phenotype or genotype, or a cell that has been removed from an organ or tissue for further use.

[0060] In some embodiments, the stem cells are selected from the group comprising or consisting of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cell. In some embodiments, the stem cells are selectedfrom the group comprising or consisting of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and epithelial stem cells.

[0061] In some embodiments, the stem cells are selected from the group comprising or consisting of mesenchymal stem cells, neural stem cells, and epithelial stem cells, preferably mesenchymal stem cells (MSC).

[0062] In some embodiments, the stem cells are animal stem cells, preferably mammals stem cells, more preferably human stem cells. In some embodiments, the stem cells are human stem cells.

[0063] In some embodiments, the stem cells are mesenchymal stem cells (MSC). In some embodiments, the stem cells are animal MSC, preferably mammals MSC, more preferably human MSC. In some embodiments, the stem cells are human MSC.

[0064] In some embodiments, the stem cells are neural stem cells. In some embodiments, the stem cells are animal neural stem cells, preferably mammals neural stem cells, more preferably human neural stem cells. In some embodiments, the stem cells are human neural stem cells.

[0065] In some embodiments, the stem cells are epithelial stem cells. In some embodiments, the stem cells are animal epithelial stem cells, preferably mammals epithelial stem cells, more preferably human epithelial stem cells. In some embodiments, the stem cells are human epithelial stem cells.

[0066] In some embodiments, the stem cells are hematopoietic stem cells. In some embodiments, the stem cells are animal hematopoietic stem cells, preferably mammals hematopoietic stem cells, more preferably human hematopoietic stem cells. In some embodiments, the stem cells are human hematopoietic stem cells.

[0067] In some embodiments, the stem cells are selected from the group comprising or consisting of fetal stem cells, placenta-derived stem cells, umbilical cord-derived stem cells, Wharton jelly-derived stem cells, amnios-derived stem cells, bone marrow-derived stem cells, peripheral blood-derived stem cells, adipose tissue-derived stem cells, dental pulp-derived stem cells, thymus-derived stem cells, and muscle-derived stem cells. Insome embodiments, the stem cells are selected from the group comprising or consisting of human fetal stem cells, human placenta-derived stem cells, human umbilical cord- derived stem cells, human Wharton jelly-derived stem cells, human amnios-derived stem cells, human bone marrow-derived stem cells, human peripheral blood-derived stem cells, human adipose tissue-derived stem cells, human dental pulp-derived stem cells, human thymus-derived stem cells, and human muscle-derived stem cells.

[0068] As used herein, the term “derived”, when used in the expression “tissue / organ- derived stem cell”, means that the stem cell originates from the tissue / organ. Illustratively, a “placenta-derived stem cell” originates strictly from placenta, and placenta only; an “umbilical cord-derived stem cell” originates strictly from umbilical cord, and umbilical cord only; or a “Wharton jelly -derived stem cell” originates strictly from Wharton jelly, and Wharton jelly only.

[0069] In some embodiments, the stem cells are selected from the group comprising or consisting of fetal stem cells, placenta-derived stem cells, umbilical cord-derived stem cells, and Wharton jelly-derived stem cells. In some embodiments, the stem cells are selected from the group comprising or consisting of placenta-derived stem cells, umbilical cord-derived stem cells, and Wharton jelly-derived stem cells. In some embodiments, the stem cells are placenta-derived stem cells or umbilical cord-derived stem cells. In some embodiments, the stem cells are selected from the group comprising or consisting of human fetal stem cells, human placenta-derived stem cells, human umbilical cord-derived stem cells, and human Wharton jelly-derived stem cells. In some embodiments, the stem cells are selected from the group comprising or consisting of human placenta-derived stem cells, human umbilical cord-derived stem cells, and human Wharton jelly-derived stem cells. In some embodiments, the stem cells are human placenta-derived stem cells or human umbilical cord-derived stem cells.

[0070] In some embodiments, the stem cells are fetal stem cells, preferably human fetal stem cells. In some embodiments, the stem cells are placenta-derived stem cells, preferably human placenta-derived stem cells. In some embodiments, the stem cells are umbilical cord-derived stem cells, preferably human umbilical cord-derived stem cells. In some embodiments, the stem cells are Wharton jelly-derived stem cells, preferablyhuman Wharton jelly-derived stem cells. In some embodiments, the stem cells are amnios-derived stem cells, preferably human amnios-derived stem cells. In some embodiments, the stem cells are bone marrow-derived stem cells, preferably human bone marrow-derived stem cells. In some embodiments, the stem cells are peripheral blood- derived stem cells, preferably human peripheral blood-derived stem cells. In some embodiments, the stem cells are adipose tissue-derived stem cells, preferably human adipose tissue-derived stem cells. In some embodiments, the stem cells are dental pulp- derived stem cells, preferably human dental pulp-derived stem cells. In some embodiments, the stem cells are thymus-derived stem cells, preferably human thymus- derived stem cells. In some embodiments, the stem cells are muscle-derived stem cells, preferably human muscle-derived stem cells.

[0071] In some embodiments, the stem cells are selected from the group comprising or consisting of fetal MSC, placenta-derived MSC, umbilical cord-derived MSC, Wharton jelly-derived MSC, amnios-derived MSC, bone marrow-derived MSC, peripheral blood- derived MSC, adipose tissue-derived MSC, dental pulp-derived MSC, thymus-derived MSC, and muscle-derived MSC. In some embodiments, the stem cells are selected from the group comprising or consisting of human fetal MSC, human placenta-derived MSC, human umbilical cord-derived MSC, human Wharton jelly-derived MSC, human amnios- derived MSC, human bone marrow-derived MSC, human peripheral blood-derived MSC, human adipose tissue-derived MSC, human dental pulp-derived MSC, human thymus- derived MSC, and human muscle-derived MSC.

[0072] In some embodiments, the stem cells are selected from the group comprising or consisting of fetal MSC, placenta-derived MSC, umbilical cord-derived MSC, and Wharton jelly-derived MSC. In some embodiments, the stem cells are selected from the group comprising or consisting of placenta-derived MSC, umbilical cord-derived MSC, and Wharton jelly-derived MSC. In some embodiments, the stem cells are placenta- derived MSC or umbilical cord-derived MSC. In some embodiments, the stem cells are selected from the group comprising or consisting of human fetal MSC, human placenta- derived MSC, human umbilical cord-derived MSC, and human Wharton jelly-derived MSC. In some embodiments, the stem cells are selected from the group comprising orconsisting of human placenta-derived MSC, human umbilical cord-derived MSC, and human Wharton jelly-derived MSC. In some embodiments, the stem cells are human placenta-derived MSC or human umbilical cord-derived MSC.

[0073] In some embodiments, the stem cells are fetal MSC, preferably human fetal MSC. In some embodiments, the stem cells are placenta-derived MSC, preferably human placenta-derived MSC. In some embodiments, the stem cells are umbilical cord-derived MSC, preferably human umbilical cord-derived MSC. In some embodiments, the stem cells are Wharton jelly-derived MSC, preferably human Wharton jelly-derived MSC. In some embodiments, the stem cells are amnios-derived MSC, preferably human amnios- derived MSC. In some embodiments, the stem cells are bone marrow-derived MSC, preferably human bone marrow-derived MSC. In some embodiments, the stem cells are peripheral blood-derived MSC, preferably human peripheral blood-derived MSC. In some embodiments, the stem cells are adipose tissue-derived MSC, preferably human adipose tissue-derived MSC. In some embodiments, the stem cells are dental pulp-derived MSC, preferably human dental pulp-derived MSC. In some embodiments, the stem cells are thymus-derived MSC, preferably human thymus-derived MSC. In some embodiments, the stem cells are muscle-derived MSC, preferably human muscle-derived MSC.

[0074] In some embodiments, isolation or extraction of the stem cells does not involve the destruction of an embryo. In some embodiments, isolation or extraction of the stem cells does not involve the destruction of a human embryo. In some embodiments, isolation or extraction of the stem cells does not involve any invasive procedure in the human body.

[0075] In some embodiments, the stem cells extracted by the method of the invention comprise or consist of one type of stem cells or a unique population of stem cells (z.e., the stem cells extracted by the method of the invention are homogeneous); or several types of stem cells or distinct populations of stem cells (z.e., the stem cells extracted by the method of the invention are heterogeneous).

[0076] In one embodiment, the stem cells extracted by the method of the invention comprise or consist of one type of stem cells. In one embodiment, the stem cells extractedby the method of the invention are homogeneous. In one embodiment, the stem cells extracted by the method of the invention comprise or consist of a unique population of stem cells.

[0077] In one embodiment, the stem cells extracted by the method of the invention are extracted from the same tissue. In another embodiment, the stem cells extracted by the method of the invention are extracted from two or more distinct tissues.

[0078] In one embodiment, the stem cells extracted by the method of the invention are extracted from the same tissue, and comprise or consist of one type of stem cells. In another embodiment, the stem cells extracted by the method of the invention are extracted from two or more distinct tissues, and comprise or consist of one type of stem cells.

[0079] In certain embodiments, the stem cells extracted by the method of the invention are substantially pure. As used herein, substantially pure means devoid of contaminants, in particular other cell types and / or other stem cell types. In some embodiments, the stem cells extracted by the method of the invention comprise less than 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or less, of one or more distinct cell type. In some embodiments, the stem cells extracted by the method of the invention are devoid of one or more distinct cell type.

[0080] In another, less preferred embodiment, the stem cells extracted by the method of the invention comprise or consist of several types of stem cells. In one embodiment, the stem cells extracted by the method of the invention are heterogeneous. In one embodiment, the stem cells extracted by the method of the invention comprise or consist of distinct populations of stem cells.

[0081] In some embodiments, the stem cells extracted by the method of the invention are devoid of chemical contaminants, and / or devoid of microorganisms (e.g., bacteria, fungi, viruses, amoeba, archaea, and the like).

[0082] Within the scope of the present invention, the general term “culture vessel” refers to any support or vessel suitable for maintaining and / or culturing cells, such as cell cultureplates, dishes, flasks, bioreactors, and the like. Such culture vessels are well-known in the art.

[0083] Within the scope of the present invention, the term “non-adherent culture vessel” refers to a culture vessel that is not conductive to the attachment of cells, preferably stem cells, more preferably mesenchymal stem cells, to said vessel. In other words, cells, preferably stem cells, more preferably mesenchymal stem cells, being cultured in a nonadherent culture vessel do not adhere nor attach, or very little (for example, 5%, 1%, 0.5%, 0.1% or less of adhesion), and do not spread to the inner surface of said culture vessel (z.e., to the walls and / or bottom of said culture vessel). It will be understood that matrixes suitable for cell adhesion, in particular microcarriers, are excluded from the definition of a “non-adherent culture vessel”.

[0084] It will be apparent to the person skilled in the art that stems cells, in particular MSC, are adherent cells - this property is also a prerequisite for stem cell self-renewal and often used as a quality control in the culture of stem cells.

[0085] It will be understood that the method of the invention comprises both a non- adherent culture vessel and at least one matrix suitable for cell adhesion. Therefore, in some embodiments, the stem cells adhere or attach to the at least one matrix suitable for cell adhesion and do not adhere nor attach to the non-adherent culture vessel. In some embodiments, less than 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or less, of the stem cells adhere or attach to the inner surface of the non-adherent culture vessel, and / or less than 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or less, of the stem cells spread to the inner surface of said culture vessel. In some embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, or less, of the stem cells adhere or attach to the inner surface of the non-adherent culture vessel, and / or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, or less, of the stem cells spread to the inner surface of said culture vessel. In some embodiments, stem cells’ attachment to the non-adherent culture vessel, and / or the spreading to the inner surface of the non- adherent culture vessel is almost zero. In some embodiments, the stem cells do not adherenor attach to the non-adherent culture vessel, and / or do not spread to the inner surface of the non-adherent culture vessel.

[0086] Examples of non-adherent culture vessels include, without being limited to, culture vessels made of glass, untreated culture vessels, low attachment culture vessels, and ultra-low attachment culture vessels. As used herein, the term “untreated culture vessel” refers to a culture vessel that did not undergo the chemical treatment or the coating commonly applied to culture vessels in order to enhance cell attachment to the culture vessels. In the field of cell culture, “treated culture vessel” commonly refers to a culture vessel, such as a plate or a dish, usually made of polystyrene, that underwent a chemical treatment or a coating in order to enhance the attachment of cells to the inner surface of the culture vessel.

[0087] In one embodiment, the non-adherent culture vessel is selected from the group comprising or consisting of low attachment culture vessels, ultra-low attachment culture vessels, culture vessels made of glass, and untreated culture vessels; preferably the non- adherent culture vessel is a low or ultra-low attachment culture vessel.

[0088] In some embodiments, the non-adherent culture vessel is a low attachment culture vessel or an ultra-low attachment culture vessel.

[0089] In some embodiments, the non-adherent culture vessel is selected from the group comprising or consisting of low and ultra-low attachment dishes, low and ultra-low attachment flasks and spinner flasks, low and ultra-low attachment plates such as low and ultra-low attachment multi-well plates or low and ultra-low attachment microplates, low and ultra-low attachment bioreactors, low and ultra-low attachment production platforms, and the like. In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment bioreactor, or a low or ultra-low attachment production platform. In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment bioreactor. In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment production platform. Typically, bioreactors may have a volume ranging from about 0.5 liter (L) to about 50 L, or from about 50 L to about 500 L, or from about 500 L to about 5,000 L, or more.

[0090] In some embodiments, the non-adherent culture vessel has a volume from 0.5 L to 5,000 L, from 0.5 L to 500 L, from 0.5 L to 50 L, from 0.5 L to 5 L, from 5 L to 500 L, or from 5 L to 5,000 L. In some embodiments, the non-adherent culture vessel has a volume from 0.1 L to 10,000 L, from 0.1 L to 1,000 L, from 0.1 L to 100 L, from 0.1 L to 10 L, from 0.1 L to 1 L, from 1 L to 100 L, from 1 L to 1,000 L, or from 1 L to 10,000 L.

[0091] In some embodiments, the non-adherent culture vessel has a volume of 0.100 liter (L), 0.250 L, 0.5 L, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 11 L, 12 L, 13 L, 14 L, 15 L, 16 L, 17 L, 18 L, 19 L, 20 L, 25 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 400 L, 500L, 1000 L, 2000 L, 3000 L, 4000 L, or 5000 L.

[0092] In some embodiments, the non-adherent culture vessel is a closed system or an open system. In one embodiment, the non-adherent culture vessel is a closed system. In another embodiment, the non-adherent culture vessel is an open system.

[0093] In some embodiments, the non-adherent culture vessel is a culture vessel designed for the culture of non-adherent cells.

[0094] In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment bioreactor, or a low or ultra-low attachment production platform, and is a closed system. In some embodiments, the non-adherent culture vessel is a low or ultralow attachment bioreactor, or a low or ultra-low attachment production platform, and is designed for the culture of non-adherent cells. In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment bioreactor, or a low or ultra-low attachment production platform, and is a closed system designed for the culture of non-adherent cells. In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment bioreactor, and is a closed system. In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment bioreactor designed for the culture of non-adherent cells. In some embodiments, the non-adherent culture vessel is a low or ultra-low attachment bioreactor and is a closed system designed for the culture of non-adherent cells.

[0095] It is known in the art that low attachment culture vessels and ultra-low attachment culture vessels may be characterized by the presence of a coating, typically a hydrophilicgel, that is covalently bound to the inner surface of the culture vessel. Said coating inhibits specific and nonspecific immobilization and thus prevents cell attachment to the inner surface of the culture vessel. Thus, in some embodiments, the non-adherent culture vessel is coated with a molecule or composition that inhibits specific and nonspecific attachment or adhesion of cells, preferably stem cells, to the inner surface of the non-adherent culture vessel. In some embodiments, the non-adherent culture vessel is coated with a hydrophilic gel that inhibits specific and nonspecific attachment or adhesion of cells, preferably stem cells, to the inner surface of the non-adherent culture vessel.

[0096] Low and ultra-low attachment culture vessels, in particular low attachment plates and ultra-low attachment plates, are readily available from suppliers and include, for example, low attachment culture vessels and ultra-low attachment culture vessels available from ThermoFisher (e.g., Nuclon Sphera®), SPL Life Sciences (e.g., SPL 3D Cell Floater®), Greiner Bio-One (e.g., CELLSTAR®), Wilson Wolf (e.g., G-Rex®), Corning (e.g., CellSTACK®, Costar® or Coming® Ultra-Low Attachment plates and flasks), S-Bio (e.g, PrimeSurface®), or Perkin Elmer (e.g, PhenoPlate®). Alternatively, methods to prepare low attachment culture vessels, in particular low attachment plates, are well known to those skilled in the art. Examples of such methods include, without being limited to, coating untreated culture vessels with, e.g., agarose or withpoly-2- hydroxyethyl methacrylate. In some embodiments, the non-adherent culture vessel is commercially available, or is prepared from a culture vessel by methods known in the art.

[0097] Within the scope of the present invention, the term “matrix” may be used interchangeably with the terms “support” or “substrate”; the matrix may be three- dimensional and / or porous.

[0098] Within the scope of the present invention, the term “at least one” matrix suitable for cell adhesion encompasses 1, 10, 100, 1,000, 10,000, 100,000, 1,000,000, or more matrixes suitable for cell adhesion, i.e., a plurality of matrixes suitable for cell adhesion.

[0099] In some embodiments, the at least one matrix suitable for cell adhesion is in suspension in the culture medium comprised in the non-adherent culture vessel, deposited in the non-adherent culture vessel, deposited at the bottom of the non-adherent culturevessel, or immobilized in the non-adherent culture vessel. In a preferred embodiment, the at least one matrix suitable for cell adhesion is in suspension in the culture medium comprised in the non-adherent culture vessel. In some embodiments, the at least one matrix suitable for cell adhesion is not attached to the non-adherent culture vessel.

[0100] In some embodiments, the at least one matrix suitable for cell adhesion is a three- dimensional (3D) matrix.

[0101] In some embodiments, the at least one matrix suitable for cell adhesion is spherical, ellipsoid, or irregularly shaped.

[0102] In some embodiments, the at least one matrix suitable for cell adhesion is sterile (before the start of the method). In some embodiments, the at least one matrix suitable for cell adhesion is cell-free (before the start of the method).

[0103] In some embodiments, the at least one matrix suitable for cell adhesion is solid, porous or non-porous. In some embodiments, the at least one matrix suitable for cell adhesion is solid. In some embodiments, the at least one matrix suitable for cell adhesion is non-porous. In some embodiments, the at least one matrix suitable for cell adhesion is porous. In some embodiments, the at least one matrix suitable for cell adhesion is macroporous, preferably the at least one macroporous matrix suitable for cell adhesion has a pore size of at least 1 pm, at least 10 pm, or at least 30 pm; or ranging from 30 pm to 400 pm. In some embodiments, the at least one matrix suitable for cell adhesion is microporous, preferably the at least one microporous matrix suitable for cell adhesion has a pore size of at most 10 pm, or at most 1 pm, or ranging from 0.1 pm to 10 pm.

[0104] In some embodiments, the at least one matrix suitable for cell adhesion is made of at least one material selected from the group comprising or consisting of polystyrene, cross-linked polystyrene, gelatin, cross-linked gelatin, dextran, cross-linked dextran, PGA, cross-linked PGA, plastic, cellulose, silica, alginate, collagen, glass, acrylamide, diethylaminoethyl-Sephadex, and chitin. In some embodiments, the at least one matrix suitable for cell adhesion is made of at least one material selected from the group comprising or consisting of polystyrene, cross-linked polystyrene, gelatin, cross-linked gelatin, dextran, and cross-linked dextran. In some embodiments, the at least one matrixsuitable for cell adhesion is made of polystyrene, gelatin, or dextran. In some embodiments, the at least one matrix suitable for cell adhesion is made of polystyrene. In some embodiments, the at least one matrix suitable for cell adhesion is made of gelatin. In some embodiments, the at least one matrix suitable for cell adhesion is made of dextran.

[0105] In some embodiments, the at least one matrix suitable for cell adhesion is coated with one or more agent selected from the group comprising or consisting of collagen, N,N-diethylaminoethyl (DEAE), positively or negatively charged molecules such as cationic molecules and anionic molecules, gelatin, extracellular matrix proteins, functional groups, recombinant proteins, peptides, poly-D-lysine, and any combination thereof. In some embodiments, the at least one matrix suitable for cell adhesion is coated with one or more agent selected from the group comprising or consisting of collagen, DEAE, positively or negatively charged molecules, and any combination thereof. In some embodiments, the at least one matrix suitable for cell adhesion is coated with extracellular matrix proteins. In some embodiments, the at least one matrix suitable for cell adhesion is coated with collagen. In some embodiments, the at least one matrix suitable for cell adhesion is coated with DEAE. The one or more agent may be charged, denatured, and / or modified. In some embodiments, the at least one matrix suitable for cell adhesion is positively charged. In some embodiments, the at least one matrix suitable for cell adhesion is negatively charged.

[0106] In some embodiments, the at least one matrix suitable for cell adhesion is biocompatible. In some embodiments, the at least one matrix suitable for cell adhesion is non-toxic for cells, preferably non-toxic for stem cells.

[0107] In some embodiments, the at least one matrix suitable for cell adhesion has a size or diameter from about 10 pm to about 600 pm, from about 50 pm to about 500 pm, from about 100 pm to about 400 pm. In some embodiments, the at least one matrix suitable for cell adhesion has a size or diameter of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 pm.

[0108] In some embodiments, the at least one matrix suitable for cell adhesion has a density from about 0.8 to about 1.2 g / cm3.

[0109] In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble, z.e., in some embodiments, the at least one matrix suitable for cell adhesion is a dissolvable or soluble matrix suitable for cell adhesion.

[0110] It will be understood that dissolvable matrixes suitable for cell adhesion enable release of the attached stem cells.

[0111] In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under certain conditions. Illustratively, a matrix may be dissolved under the action of one or more enzymes, above a certain temperature, under certain pH conditions, or in the presence of certain chemicals.

[0112] In a preferred embodiment, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of one or more enzymes. In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of one or more proteases. In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of one or more proteases selected from the group comprising or consisting of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, pectinase, pectolyase, and any combination thereof. In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of trypsin or pectinase. In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of pectinase. In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of trypsin.

[0113] In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of a chemical. In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of EDTA.

[0114] In some embodiments, the at least one matrix suitable for cell adhesion is dissolvable or soluble under the action of a composition comprising or consisting of trypsin, pectinase and / or EDTA.

[0115] In some embodiments, the at least one matrix suitable for cell adhesion is not dissolvable nor soluble under the action of temperature or heat. In some embodiments, the at least one matrix suitable for cell adhesion is not thermolabile. In some embodiments, the at least one matrix suitable for cell adhesion is not dissolvable nor soluble at a temperature superior or equal to 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 50°C, 60°C, 70°C, or more.

[0116] The person skilled in the art will know how to adapt the quantity of the at least one matrix suitable for cell adhesion depending of the volume of the non-adherent culture vessel and / or the quantity or concentration of stem cells. Typically, the quantity of such matrixes is expressed as a unit of surface, representing the surface available for cell attachment.

[0117] In some embodiments, the quantity of matrixes suitable for cell adhesion is from 1 cm2to 100,000,000 cm2, from 1 cm2to 10,000,000 cm2, from 1 cm2to 1,000,000 cm2, from 1 cm2to 100,000 cm2, from 1 cm2to 10,000 cm2, from 1 cm2to 1,000 cm2. In some embodiments, the quantity of matrixes suitable for cell adhesion is from 10 cm2to 100,000,000 cm2, from 100 cm2to 100,000,000 cm2, from 1,000 cm2to 100,000,000 cm2, from 10,000 cm2to 100,000,000 cm2, from 100,000 cm2to 100,000,000 cm2, or from 1,000,000 cm2to 100,000,000 cm2. In some embodiments, the quantity of matrixes suitable for cell adhesion is from 1 cm2to 1,000,000 cm2, from 10 cm2to 100,000 cm2, or from 100 cm2to 10,000 cm2. In some embodiments, the quantity of matrixes suitable for cell adhesion is about 500 cm2, 600 cm2, 700 cm2, 800 cm2, 900 cm2, 1,000 cm2, 2,000 cm2, 3,000 cm2, 4,000 cm2, 5,000 cm2, 10,000 cm2, 15,000 cm2, or 20,000 cm2.

[0118] In certain embodiments, a quantity of matrixes suitable for cell adhesion of 5,000 cm2represents about 1 gram (g) of matrixes suitable for cell adhesion.

[0119] In some embodiments, the quantity of matrixes suitable for cell adhesion is from 0,0002 g to 20000 g, from 0,0002 g to 2000 g, from 0,0002 g to 200 g, from 0,0002 g to20 g, from 0,0002 g to 2 g, from 0,0002 g to 0.2 g. In some embodiments, the quantity of matrixes suitable for cell adhesion is from 0.002 g to 20000 g, from 0.02 g to 20000 g, from 0.2 g to 20000 g, from 2 g to 20000 g, from 20 g to 20000 g, or from 200 g to 20000 g. In some embodiments, the quantity of matrixes suitable for cell adhesion is from 0,0002 g to 200 g, from 0.002 g to 20 g, or from 0.02 g to 2 g. In some embodiments, the quantity of matrixes suitable for cell adhesion is about 0.1 g, 0.12 g, 0.14 g, 0.16 g, 0.18 g, 0.2 g, 0.4 g, 0.6 g, 0.8 g, 1 g, 2 g, 3 g, or 4 g.

[0120] In some embodiments, the concentration of matrixes suitable for cell adhesion is from about 0.01 g / L to about 300 g / L, preferably from about 0.1 g / L to about 30 g / L, more preferably from about 1 g / L to about 3 g / L.

[0121] In some embodiments, the at least one matrix suitable for cell adhesion is selected from the group comprising or consisting of a microcarrier, a bead, a scaffold, a peptidic scaffold, an extracellular matrix or a derivative thereof, and / or any combination thereof. In some embodiments, the at least one matrix suitable for cell adhesion is a microcarrier or a scaffold.

[0122] In some embodiments, the at least one matrix suitable for cell adhesion is made of a material selected from the group comprising or consisting of polymers, polystyrene, gelatin, dextran, PGA, plastic, cellulose, silica, alginate, collagen, proteins, peptides, sugars, lipids, metal, glass, acrylamide, diethylaminoethyl-Sephadex, and chitin. In some embodiments, the at least one matrix suitable for cell adhesion is made of a material selected from the group comprising or consisting of polystyrene, gelatin, dextran, PGA, cellulose, collagen, and chitin.

[0123] In some embodiments, the at least one matrix suitable for cell adhesion is a microcarrier, preferably a dissolvable microcarrier. In a preferred embodiment, the at least one matrix suitable for cell adhesion a dissolvable microcarrier.

[0124] As used herein, the term “microcarrier” refers to a support matrix that allows for the growth of adherent cells, in particular stem cells, in suitable culture vessels, in particular bioreactors. Typically, in microcarrier-based cell culture, cells are cultured on the surface of a plurality of microcarriers such as spherical microcarriers, so that eachmicrocarrier carries one or more cells, typically several hundred cells. Microcarriers may be solid or porous as described hereinabove, may be optionally spherical, may have a diameter ranging from about 10 pm to about 600 pm, preferably from about 100 pm to about 300 pm, and may have a density of about 1.02 g / ml or more so that they remain in suspension in a stirred tank. Microcarriers may be made of any material and / or coated as described hereinabove.

[0125] In some embodiments, the at least one microcarrier is a three-dimensional (3D) microcarrier.

[0126] In some embodiments, the at least one microcarrier is spherical, ellipsoid, or irregularly shaped.

[0127] In some embodiments, the at least one microcarrier is sterile. In some embodiments, the at least one microcarrier is cell-free.

[0128] In some embodiments, the at least one microcarrier is solid, porous or non- porous. In some embodiments, the at least one microcarrier is solid. In some embodiments, the at least one microcarrier is non-porous. In some embodiments, the at least one microcarrier is porous. In some embodiments, the at least one microcarrier is macroporous. In some embodiments, the at least one microcarrier is microporous.

[0129] In some embodiments, the at least one microcarrier is made of at least one material selected from the group comprising or consisting of polystyrene, cross-linked polystyrene, gelatin, cross-linked gelatin, dextran, cross-linked dextran, PGA, crosslinked PGA, plastic, cellulose, silica, alginate, collagen, glass, acrylamide, diethylaminoethyl-Sephadex, and chitin. In some embodiments, the at least one microcarrier is made of at least one material selected from the group comprising or consisting of polystyrene, cross-linked polystyrene, gelatin, cross-linked gelatin, dextran, and cross-linked dextran. In some embodiments, the at least one microcarrier is made of polystyrene, gelatin, or dextran. In some embodiments, the at least one microcarrier is made of polystyrene. In some embodiments, the at least one microcarrier is made of gelatin. In some embodiments, the at least one microcarrier is made of dextran.

[0130] In some embodiments, the at least one microcarrier is coated with one or more agent selected from the group comprising or consisting of collagen, N,N- diethylaminoethyl (DEAE), positively or negatively charged molecules such as cationic molecules and anionic molecules, gelatin, extracellular matrix proteins, functional groups, recombinant proteins, peptides, poly-D-lysine, and any combination thereof. In some embodiments, the at least one microcarrier is coated with one or more agent selected from the group comprising or consisting of collagen, DEAE, positively or negatively charged molecules, and any combination thereof. In some embodiments, the at least one microcarrier is coated with extracellular matrix proteins. In some embodiments, the at least one microcarrier is coated with collagen. In some embodiments, the at least one microcarrier is coated with DEAE. The one or more agent may be charged, denatured, and / or modified. In some embodiments, the at least one microcarrier is positively charged. In some embodiments, the at least one microcarrier is negatively charged.

[0131] In some embodiments, the at least one microcarrier is biocompatible. In some embodiments, the at least one microcarrier is non-toxic for cells, preferably non-toxic for stem cells.

[0132] In some embodiments, the at least one microcarrier has a size or diameter from about 10 pm to about 600 pm, from about 50 pm to about 500 pm, from about 100 pm to about 400 pm. In some embodiments, the at least one microcarrier has a size or diameter of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 pm.

[0133] In some embodiments, the at least one microcarrier has a density from about 0.8 to about 1.2 g / cm3.

[0134] In some embodiments, the at least one microcarrier is dissolvable or soluble, z.e., in some embodiments, the at least one microcarrier is a dissolvable or soluble microcarrier.

[0135] In some embodiments, the at least one microcarrier is dissolvable or soluble under certain conditions. Illustratively, a microcarrier may be dissolved under the actionof one or more enzymes, above a certain temperature, under certain pH conditions, or in the presence of certain chemicals. In some embodiments, the at least one microcarrier is dissolvable or soluble under one or more conditions selected from the group consisting of action of one or more enzymes, pH, presence of certain chemicals, and temperature, preferably selected from the group consisting of action of one or more enzymes, pH, and presence of certain chemicals, more preferably selected from the group consisting of action of one or more enzymes and presence of certain chemicals, even more preferably action of one or more enzymes.

[0136] In a preferred embodiment, the at least one microcarrier is dissolvable or soluble under the action of one or more enzymes. In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of one or more proteases. In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of one or more proteases selected from the group comprising or consisting of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, pectinase, pectolyase, and any combination thereof. In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of trypsin or pectinase. In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of pectinase. In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of trypsin.

[0137] In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of a chemical. In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of EDTA.

[0138] In some embodiments, the at least one microcarrier is dissolvable or soluble under the action of a composition comprising or consisting of trypsin, pectinase and / or EDTA.

[0139] In some embodiments, the at least one microcarrier is not dissolvable nor soluble under the action of temperature or heat. In some embodiments, the at least one microcarrier is not thermolabile. In some embodiments, the at least one microcarrier is not dissolvable nor soluble at a temperature superior or equal to 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 50°C, 60°C, 70°C, or more.

[0140] The person skilled in the art will know how to adapt the quantity of the at least one microcarrier depending of the volume of the non-adherent culture vessel and / or the quantity or concentration of stem cells. Typically, the quantity of such microcarriers is expressed as a unit of surface, representing the surface available for cell attachment.

[0141] In some embodiments, the quantity of microcarriers is from 1 cm2to 100,000,000 cm2, from 1 cm2to 10,000,000 cm2, from 1 cm2to 1,000,000 cm2, from 1 cm2to 100,000 cm2, from 1 cm2to 10,000 cm2, from 1 cm2to 1,000 cm2. In some embodiments, the quantity of microcarriers is from 10 cm2to 100,000,000 cm2, from 100 cm2to 100,000,000 cm2, from 1,000 cm2to 100,000,000 cm2, from 10,000 cm2to 100,000,000 cm2, from 100,000 cm2to 100,000,000 cm2, or from 1,000,000 cm2to 100,000,000 cm2. In some embodiments, the quantity of microcarriers is from 1 cm2to 1,000,000 cm2, from 10 cm2to 100,000 cm2, or from 100 cm2to 10,000 cm2. In some embodiments, the quantity of microcarriers is about 500 cm2, 600 cm2, 700 cm2, 800 cm2, 900 cm2, 1,000 cm2, 2,000 cm2, 3,000 cm2, 4,000 cm2, 5,000 cm2, 10,000 cm2, 15,000 cm2, or 20,000 cm2.

[0142] In certain embodiments, a quantity of microcarriers of 5,000 cm2represents about 1 gram (g) of microcarriers.

[0143] In some embodiments, the quantity of microcarriers is from 0,0002 g to 20000 g, from 0,0002 g to 2000 g, from 0,0002 g to 200 g, from 0,0002 g to 20 g, from 0,0002 g to 2 g, from 0,0002 g to 0.2 g. In some embodiments, the quantity of microcarriers is from 0.002 g to 20000 g, from 0.02 g to 20000 g, from 0.2 g to 20000 g, from 2 g to 20000 g, from 20 g to 20000 g, or from 200 g to 20000 g. In some embodiments, the quantity of microcarriers is from 0,0002 g to 200 g, from 0.002 g to 20 g, or from 0.02 g to 2 g. In some embodiments, the quantity of microcarriers is about 0.1 g, 0.12 g, 0.14 g, 0.16 g, 0.18 g, 0.2 g, 0.4 g, 0.6 g, 0.8 g, 1 g, 2 g, 3 g, or 4 g.

[0144] In some embodiments, the concentration of microcarriers is from about 0.01 g / L to about 300 g / L, preferably from about 0.1 g / L to about 30 g / L, more preferably from about 1 g / L to about 3 g / L.

[0145] In some embodiments, the stem cells are maintained in a controlled atmosphere, ie., controlled temperature, pressure and composition. In some embodiments, the stemcells are maintained at a temperature of about 37°C. In some embodiments, the stem cells are maintained at an atmospheric pressure of about 1 atm. In some embodiments, the stem cells are maintained in an atmosphere comprising about 5% CO2. In some embodiments, the stem cells are maintained in an atmosphere comprising from about 5% O2 to about 20% O2. In some embodiments, the stem cells are maintained in an atmosphere comprising about 5 % CO2 and about 5% O2. In some embodiments, the stem cells are maintained in a closed system.

[0146] It will be understood that the non-adherent culture vessel used to perform the method of the invention comprises a culture medium. Therefore, in some embodiments, the stem cells are cultured in a culture medium sustaining their survival and / or proliferation, preferably a liquid culture medium. This culture medium both comprises the at least one matrix suitable for cell adhesion, and supports cell survival and growth.

[0147] In some embodiments, the at least one matrix suitable for cell adhesion is comprised in the culture medium. In some embodiments, the at least one matrix suitable for cell adhesion is added to the culture medium. In some embodiments, the culture medium is supplemented with the at least one matrix suitable for cell adhesion.

[0148] Culture media are known in the art. The terms “culture medium” or “cell culture medium” or “medium”, used herein interchangeably, refer to an aqueous liquid or gelatinous substance comprising nutrients which can be used for maintenance or growth of cells. Cell culture media can contain serum or be serum-free, and may be further supplemented with an appropriate mixture of organic or inorganic compounds may, providing for example nutrients and / or growth promoters.

[0149] In some embodiments, the culture medium comprises a basal medium formulation selected from the group comprising or consisting of Mesencult MSC Basal medium (StemCell technologies), MSCBMTM Basal Media (Lonza), StemPro MSC SFM (Gibco), alpha Minimum Essential Medium (a-MEM), Eagle's Minimum Essential Medium (MEM), OPTI-MEM, Dulbecco's Modified Eagle's Medium (DMEM), Basal Medium Essential (BME), Endothelial Cell Growth Medium-2 (EGM-2), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham),Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), GI-1640, Medium 199, Waymouth's MB 752 / 1 X-VIVO-15, and Williams Medium E, and modifications and / or combinations thereof. In some embodiments, the culture medium comprises Mesencult MSC Basal medium, or modifications and / or combinations thereof. In some embodiments, the culture medium comprises MSCBMTM Basal Media, or modifications and / or combinations thereof. In some embodiments, the culture medium comprises StemPro MSC SFM, or modifications and / or combinations thereof. In some embodiments, the culture medium comprises alpha Minimum Essential Medium (a- MEM), or modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for maintaining and / or culturing the stem cells.

[0150] In some embodiments, the culture medium comprises one or more molecule selected from the group comprising or consisting of inorganic salts (in particular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and / or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione) sources of carbon (e.g., glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodium acetate), and any combination thereof. In some embodiments, the culture medium is low glucose culture medium formulation. In one embodiment, the culture medium comprises sodium pyruvate. In one embodiment, the culture medium is devoid of sodium pyruvate.

[0151] In some embodiments, the culture medium is supplemented with one or more further components selected from the group comprising or consisting of transferrin, selenium salts, amino acids, sugar, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., ascorbate or P-mercaptoethanol. The person skilled in the art knows that some amino acids are known to be less stable when in solution and may thus be supplemented extemporaneously, e.g., L-glutamine.

[0152] In some embodiments, the culture medium is further supplemented with one or more antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amoxicillin, amphotericin, ampicillin, gentamycin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, vancomycin, zeocin, and combinations thereof. In some embodiments, the culture medium is further supplemented with amoxicillin, amphotericin, and / or vancomycin. In some embodiments, the culture medium is further supplemented with fungicide compounds.

[0153] In some embodiments, the culture medium is further supplemented with one or more hormones selected from the group comprising or consisting of D-aldosterone, diethylstilbestrol (DES), dexamethasone, estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / mammalian growth hormone (HGH), thyrotropin, thyroxine, L-thyronine, epithelial growth factor (EGF), mammalian recombinant epidermal growth factor, and combinations thereof. The final concentration of hormones may range from 0.01 to 1000 ng / mL, preferably from 0.1 to 100 ng / mL, more preferably from 1 to 10 ng / mL.

[0154] In some embodiments, the culture medium is further supplemented with one or more lipids and lipid carriers selected from the group comprising or consisting of cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, and the like, and combinations thereof.

[0155] In some embodiments, the culture medium is further supplemented with serum. In some embodiments, the concentration of the serum in the culture medium is from 0.01% to 20%, preferably from 0.1% to 20%, more preferably from 1% to 10%. In some embodiments, the concentration of the serum in the culture medium is about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In certain embodiments,the culture medium is serum-free. In some embodiments, the serum comprised in the culture medium as described herein is mammalian serum such as horse serum, goat serum, bovine serum, fetal bovine serum, or human serum. In some embodiments, the serum is bovine serum or fetal bovine serum. In some embodiments, the serum is human serum. In some embodiments, the serum is substantially free of extracellular vesicles and / or exosomes. In some embodiments, the culture medium is further supplemented with a serum replacement or analogue. The term "serum", as conventionally defined, is obtained from a sample of whole blood by first allowing clotting to take place in the sample and subsequently separating the so formed clot and cellular components of the blood sample from the liquid component (serum) by an appropriate technique, typically by centrifugation. An inert catalyst, e.g., glass beads or powder, can facilitate clotting. Serum often contains cellular factors and components that are necessary for viability and expansion.

[0156] In some embodiments, the culture medium is devoid of contamination by bacteria, fungi, protozoa, archaea, or any other microbial species.

[0157] In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion. In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion for a duration sufficient for part of the stem cells comprised in the tissue to migrate to, and adhere to, the at least one matrix suitable for cell adhesion. In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion for at least 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, 240 hours, 264 hours, 288 hours, 312 hours, 336 hours, 360 hours, or more. In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion for at least 168 hours. In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion for at least 216 hours. In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion for a duration from 168 hours to 360hours, preferably from 168 hours to 288 hours, more preferably from 168 hours to 216 hours. In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion for about 168 hours. In some embodiments, the method of the invention further comprises incubating the tissue with the at least one matrix suitable for cell adhesion for about 216 hours. It will be understood that this time corresponds to the time required for stem cells comprised in the tissue to migrate to, and adhere to, the matrixes suitable for cell adhesion, however the stem cells attached to the matrixes suitable for cell adhesion may be cultured for longer periods of time, e.g., a week, a month, or more.

[0158] In some embodiments, the method of the invention comprises the steps of: i. in a non-adherent culture vessel comprising at least one matrix suitable for cell adhesion, preferably at least one microcarrier, and a culture medium, placing the tissue; ii. incubating for at least 168 hours; and iii. obtaining the stem cells on the at least one matrix suitable for cell adhesion.

[0159] In some embodiments, the method of the invention comprises the steps of: i. in a non-adherent culture vessel comprising at least one matrix suitable for cell adhesion, preferably at least one microcarrier, and a culture medium, placing the tissue; ii. incubating for at least 168 hours; and iii. obtaining at least one matrix suitable for cell adhesion comprising stem cells.

[0160] In certain embodiments, step (i) is preceded by a step of placing at least one matrix suitable for cell adhesion, preferably at least one microcarrier, and a culture medium in a non-adherent culture vessel. Nature and quantity of the at least one matrix suitable for cell adhesion has been described above.

[0161] In some embodiments, at step (ii), a plurality of matrixes suitable for cell adhesion, preferably microcarriers, are further added. In some embodiments, step (ii) further comprises adding a plurality of matrixes suitable for cell adhesion, preferablymicrocarriers. It will be understood that providing additional matrixes suitable for cell adhesion, preferably microcarriers, further increases the total surface on which the stem cells from the tissue may attach / adhere to, and has a positive effect on viability and yield extraction, and limits confluency, as demonstrated by the Applicant (see Figures 4 and 6).

[0162] In some embodiments, step (ii) further comprises adding from 1 cm2to 100,000,000 cm2, from 1 cm2to 10,000,000 cm2, from 1 cm2to 1,000,000 cm2, from 1 cm2to 100,000 cm2, from 1 cm2to 10,000 cm2, from 1 cm2to 1,000 cm2of matrixes suitable for cell adhesion, preferably microcarriers; or from 10 cm2to 100,000,000 cm2, from 100 cm2to 100,000,000 cm2, from 1,000 cm2to 100,000,000 cm2, from 10,000 cm2to 100,000,000 cm2, from 100,000 cm2to 100,000,000 cm2, or from 1,000,000 cm2to 100,000,000 cm2of matrixes suitable for cell adhesion, preferably microcarriers; or from 1 cm2to 1,000,000 cm2, from 10 cm2to 100,000 cm2, or from 100 cm2to 10,000 cm2of matrixes suitable for cell adhesion, preferably microcarriers; or about 500 cm2, 600 cm2, 700 cm2, 800 cm2, 900 cm2, 1,000 cm2, 2,000 cm2, 3,000 cm2, 4,000 cm2, or 5,000 cm2of matrixes suitable for cell adhesion, preferably microcarriers.

[0163] The present invention further relates to a method of cultivating and / or expanding stem cells extracted from a tissue, comprising the steps of: i. extracting stem cells from a tissue with the method of the invention, as described hereinabove, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; and iv. optionally, repeating steps (ii) and (iii) any number of times.

[0164] In some embodiments, steps (ii) and (ii) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. Thus, in some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue comprises the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; and iv. repeating steps (ii) and (iii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.

[0165] As indicated above, at step (iii), a plurality of matrixes suitable for cell adhesion, preferably microcarriers, may be further added during incubation.

[0166] It will be understood that, when step (ii) is performed once, the at least one matrix suitable for cell adhesion comprising stem cells is obtained at step (i). However, when step (ii) is performed more than once, the at least one matrix suitable for cell adhesion comprising stem cells is obtained at step (iii). It is thus important to distinguish the “initial” (or “first”) non-adherent culture vessel, z.e., the non-adherent culture vessel from which the at least one matrix suitable for cell adhesion comprising stem cells is obtained, and the “new” (or “second”, “third” etc.) non-adherent culture vessel, z.e., the non- adherent culture vessel to which the at least one matrix suitable for cell adhesion comprising stem cells is transferred to.

[0167] In other words, in certain embodiments, the method of culturing and / or expanding stem cells extracted from a tissue comprises the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells in a first non-adherent culture vessel;ii. transferring at least one matrix suitable for cell adhesion comprising stem cells from the initial non-adherent culture vessel into one or more second non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; and iv. repeating steps (ii) and (iii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more, so that the matrixes from the one or more second non-adherent culture vessel are transferred into one or more third non-adherent culture vessel comprising additional matrixes suitable for cell adhesion, and so on.

[0168] In some embodiment, at step (ii), 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the at least one matrix suitable for cell adhesion comprising stem cells from the initial non-adherent culture vessel is transferred to the one or more new non-adherent culture vessel.

[0169] In some embodiment, at step (ii), the one or more new non-adherent culture vessel has the same volume or a higher volume, preferably a higher volume, than the non- adherent culture vessel of origin of the at least one matrix suitable for cell adhesion comprising stem cells. In some embodiment, at step (ii), the one or more new non- adherent culture vessel has the same volume or a higher volume, preferably a higher volume, than the initial non-adherent culture vessel. In some embodiment, at step (ii), the one or more new non-adherent culture vessel has the same volume or a higher volume, preferably a higher volume, than the non-adherent culture vessel used at step (i). Illustratively, and non-imitatively, the one or more new non-adherent culture vessel may have a volume of 3 L, 5 L or more, while the initial non-adherent culture vessel may have a volume of 1 L.

[0170] It will be understood that performing step (iv) enables culture and / or expansion of the stem cell sequentially, and / or enables scale-up of the culture volume. In some embodiments, the method is a sequential culture method.

[0171] In some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue as described herein is performed for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 30 days, 40 days, or more. In some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue as described herein is performed for at least 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 30 days, 40 days, or more. In some embodiments, the stem cells are cultured for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 30 days, 40 days, or more. In some embodiments, the stem cells are cultured for at least 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 30 days, 40 days, or more.

[0172] In some embodiment, the method of culturing and / or expanding stem cells extracted from a tissue comprises using from 1 to 600 slices of tissue, preferably from 1 to 60 slices of tissue. As used herein, “from 1 to 60” encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60. In some embodiment, the method of culturing and / or expanding stem cells extracted from a tissue comprises using from about 0.5 g to about 400 g of tissue, preferably from about 0.5 g to about 40 g of tissue.

[0173] In some embodiment, the method of culturing and / or expanding stem cells extracted from a tissue as described herein yields at least 1 million stem cells, at least 10 million stem cells, at least 100 million stem cells, at least 400 million stem cells, at least 500 million stem cells, or more. In some embodiment, the method of culturing and / or expanding stem cells extracted from a tissue as described herein yields at least 200 million stem cells after 6 days of culture, and / or at least 400 million stem cells after 8 days of culture.

[0174] In some embodiment, the method of culturing and / or expanding stem cells extracted from 1 to 60 tissue slices as described herein yields at least 1 million stem cells, at least 10 million stem cells, at least 100 million stem cells, at least 400 million stem cells, at least 500 million stem cells, or more. In some embodiment, the method of culturing and / or expanding stem cells extracted from a tissue as described herein yields at least 200 million stem cells after 6 days of culture, and / or at least 400 million stem cells after 8 days of culture. In some embodiment, the method of culturing and / or expanding stem cells extracted from 10 tissue slices as described herein yields at least from about 5 million stem cells to about 25 million stem cells. In some embodiment, the method of culturing and / or expanding stem cells extracted from about 0.5 g to about 40 g of tissue as described herein yields at least 1 million stem cells, at least 10 million stem cells, at least 100 million stem cells, at least 400 million stem cells, at least 500 million stem cells, or more. In some embodiment, the method of culturing and / or expanding stem cells extracted from a tissue as described herein yields at least 200 million stem cells after 6 days of culture, and / or at least 400 million stem cells after 8 days of culture.

[0175] In some embodiment, the method of culturing and / or expanding stem cells further comprises a step of counting cells or cell fragments in the culture medium to assess mortality.

[0176] In some embodiments, the stem cells are not exposed to a protease during extraction, culture and / or expansion. In some embodiments, the stem cells are not exposed to a protease selected from the group comprising or consisting of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, and any combination thereof. In some embodiments, extraction, culture and / or expansion of the stem cells from the tissue is performed without the use of a protease selected from the group comprising or consisting of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, and any combination thereof. In some embodiments, the method of extracting stem cells from a tissue as described herein, and the method of culturing and / or expanding stem cells extracted from a tissue as described herein, do not involve any exposure to a protease. In some embodiments, the method of extracting stem cells from a tissue as described herein, and the method of culturing and / or expanding stem cells extracted from a tissue asdescribed herein, do not involve any exposure to a protease selected from the group comprising or consisting of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, and any combination thereof.

[0177] It will be understood that: contrarily to classical stem cell extraction methods involving digesting a tissue with various enzymes, the method of the invention does not involve enzymatic digestion of the tissue; contrarily to classical stem cell culture methods involving passaging the cells (z. e. , recovering the cells attached to a first culture vessel by detaching them with the help of a protease, e.g., trypsin, then re-seeding the cells in a new culture vessel, and repeating these steps several times), the method of the invention does not involve any passaging and therefore does not involve the use of a protease such as trypsin during culture, therefore, the stem cells extracted and cultured by the method of the invention are not exposed to any protease such as trypsin. However, following extraction of the stem cells from the tissue and culture / expansion of the stem cells, the recovery of stem cells from the matrixes suitable for cell adhesion may comprise a single use of a protease such as trypsin; therefore, recovery or obtention of the stem cells extracted and cultured by the method may comprise at most one exposure to a protease such as trypsin.

[0178] In some embodiments, the stem cells are exposed at most once to one or more protease, from the step of extraction from the tissue to the step of recovery or obtention.

[0179] In some embodiments, the method of the invention further comprises obtaining at least one matrix suitable for cell adhesion comprising at least one stem cell from the tissue. In some embodiments, the method of the invention further comprises obtaining a plurality of matrixes suitable for cell adhesion comprising at least one stem cell from the tissue. In some embodiments, the method of the invention further comprises obtaining hundreds, thousands, or millions of matrixes suitable for cell adhesion comprising at least one stem cell from the tissue.

[0180] Thus, in some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue comprises the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; and v. optionally, recovering at least one matrix suitable for cell adhesion comprising stem cells.

[0181] In some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue as described herein further comprises obtaining at least one stem cell. In some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue as described herein further comprises obtaining a plurality of stem cells. In some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue as described herein further comprises obtaining hundreds, thousands, millions, or billions of stem cells. In some embodiments, the method further comprises a step of recovering the stem cells from the matrixes suitable for cell adhesion, preferably by trypsinization, and / or recovering the culture supernatant. Thus, in some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue comprises the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion;iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; and v. optionally, recovering the stem cells from the matrixes suitable for cell adhesion comprising stem cells, preferably by trypsinization, and / or recovering the culture supernatant.

[0182] As used herein, “by trypsinization” means by contacting the matrixes suitable for cell adhesion comprising stem cells with a composition comprising trypsin.

[0183] In some embodiments, the method of culturing and / or expanding stem cells extracted from a tissue comprises the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; v. optionally, recovering at least one matrix suitable for cell adhesion comprising at least one stem cell extracted from the tissue; and vi. optionally, recovering the stem cells from at least one matrix suitable for cell adhesion comprising stem cells, preferably by trypsinization, and / or recovering the culture supernatant.

[0184] The present invention further relates to a method of obtaining at least one matrix suitable for cell adhesion comprising at least one stem cell extracted from a tissue, comprising the steps of:i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; and v. recovering at least one matrix suitable for cell adhesion comprising stem cells.

[0185] The expression “matrix suitable for cell adhesion comprising stem cells” means that at least one stem cell, preferably a plurality of stem cells, more preferably hundreds or thousands of stem cells, are attached to the surface of the matrix suitable for cell adhesion, wherein attachment may occur on the external surface and / or inside the pore of the matrix suitable for cell adhesion (if said matrix suitable for cell adhesion is porous), optionally on the coating of said matrix suitable for cell adhesion.

[0186] The present invention further relates to a method of obtaining or recovering stem cells extracted from a tissue, comprising the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; and v. recovering the stem cells from the matrixes suitable for cell adhesion comprising stem cells, preferably by trypsinization.

[0187] In some embodiments, the method of obtaining or recovering stem cells extracted from a tissue comprises the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; v. recovering at least one matrix suitable for cell adhesion comprising stem cells; and vi. recovering the stem cells from the at least one matrix suitable for cell adhesion comprising stem cells, preferably by trypsinization.

[0188] In some embodiment, the method is a method of obtaining a population of stem cells. In some embodiment, the method is a method of obtaining a plurality of isolated stem cells. Stem cells have been described hereinabove.

[0189] In some embodiments, hundreds, thousands, millions, hundreds of millions, or more, of stem cells are obtained by the method described herein.

[0190] In some embodiments, the method of obtaining or recovering stem cells further comprises a step of washing the stem cells and / or at least one matrix suitable for cell adhesion comprising stem cells. In certain embodiments, the stem cells and / or at least one matrix suitable for cell adhesion comprising stem cells are washed 1 time, 2 times, 3 times, 4 times, 5 times, or more. In some embodiments, the stem cells and / or at least one matrix suitable for cell adhesion comprising stem cells are washed with a biological buffer or biocompatible buffer, and / or by centrifugation.

[0191] In some embodiments, the method of obtaining or recovering stem cells further comprises a step of counting cells. Methods to count cells are known in the art, such as use of a cell counter.

[0192] In some embodiments, the stem cells are recovered from the at least one matrix suitable for cell adhesion comprising stem cells by use of at least one protease, optionally administered in combination with a chemical and / or one or more additional enzyme. As used herein, “at least one protease” encompasses a single protease or a cocktail of two or more proteases. In some embodiments, the at least one protease is selected from the group comprising or consisting of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, and any combination thereof. In some embodiments, the at least one protease is trypsin. In some embodiments, the chemical is EDTA. In some embodiments, the one or more additional enzyme is pectinase and / or pectolyase. In some embodiments, the stem cells are recovered from the at least one matrix suitable for cell adhesion comprising stem cells by use of a composition comprising or consisting of trypsin supplemented with pectinase, pectolyase, and / or EDTA. In some embodiments, the stem cells are recovered from the at least one matrix suitable for cell adhesion comprising stem cells by use of a composition comprising or consisting of trypsin, pectinase and / or EDTA. In some embodiments, the one or more additional enzyme is pectinase and / or pectolyase. In some embodiments, the stem cells are recovered from the at least one matrix suitable for cell adhesion comprising stem cells by use of a composition comprising or consisting of trypsin supplemented with EDTA. In some embodiments, the stem cells are exposed to the at least one protease for at most 30 minutes, at most 20 minutes, at most 10 minutes, at most 5 minutes, at most 4 minutes, at most 3 minutes, at most 2 minutes, or at most 1 minute. In some embodiments, the stem cells are exposed to the at least one protease for a duration from 1 to 30 minutes, from 1 to 10 minutes, from 1 to 5 minutes, or from 1 to 3 minutes. The use of proteases such as trypsin is well known in the art, and the person skilled in the art readily know how to adjust the volumes or concentrations of the protease; it is also known in the art means to stop the action of trypsin, such as addition of serum or addition of a culture medium comprising serum.

[0193] In some embodiments, the method of obtaining or recovering stem cells from a tissue as described herein (z.e., after performing the method for extracting stem cells from a tissue according to the invention) comprises at most one exposure to at least one protease. In some embodiments, the method of obtaining or recovering stem cells from a tissue as described herein comprises at most one exposure to a single protease or to a cocktail of proteases.

[0194] In some embodiments, the method of obtaining or recovering stem cells from a tissue as described herein comprises at most one exposure to one or more of trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, pectinase, pectolyase, and EDTA.

[0195] The present invention further relates to a method of obtaining or isolating at least one molecule secreted by stem cells extracted from a tissue, comprising the steps of: i. extracting stem cells from a tissue with the method of the invention, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells; ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; v. recovering the culture supernatant; vi. optionally, recovering said at least one molecule secreted by stem cells from the supernatant; and vii. optionally, purifying the at least one molecule secreted by stem cells.

[0196] In some embodiments, the culture supernatant is centrifuged. As used herein, the term “centrifuged” encompasses both centrifugation and ultra-centrifugation

[0197] In some embodiments, the culture supernatant is centrifuged any number of times. In some embodiments, the culture supernatant is centrifuged from 1 time to 10 times, from 2 times to 5 times, or about 3 times.

[0198] In some embodiments, the culture supernatant is centrifuged at a speed ranging from 100 rpm to 100,000 rpm, or ranging from 1,000 to 50,000 rpm.

[0199] In some embodiments, the culture supernatant is differentially centrifugated (z. e. , subjected to differential centrifugation). The person skilled in the art knows how to perform differential centrifugation. Typically, differential centrifugation involves successive steps of centrifugation at increasing speeds to eliminate the heaviest components of the cells (membranes, organelles...) followed by one or more steps of ultra-centrifugation at high speeds; differential centrifugation by be combined with other techniques such as, e.g., density gradient purification.

[0200] In some embodiments, the at least one molecule secreted by stem cells is selected from the group comprising or consisting of exosomes, extracellular vesicles, secreted proteins, enzymes, growth factors, cytokines, hormones, and soluble mediators. In some embodiments, the at least one molecule secreted by stem cells is selected from the group comprising or consisting of exosomes, extracellular vesicles, and cytokines. In some embodiments, the at least one molecule secreted by stem cells is an exosome or extracellular vesicle. In some embodiments, the at least one molecule secreted by stem cells is an exosome. In some embodiments, the at least one molecule secreted by stem cells is an extracellular vesicle. In some embodiments, the at least one molecule secreted by stem cells is a secreted protein. In some embodiments, the at least one molecule secreted by stem cells is a cytokine. In some embodiments, the at least one molecule secreted by stem cells is an enzyme. In some embodiments, the at least one molecule secreted by stem cells is a growth factor. In some embodiments, the at least one molecule secreted by stem cells is a hormone. Methods to extract and optionally purify these molecules are known in the art.

[0201] In some embodiments, the at least one molecule secreted by stem cells is obtained, recovered, or otherwise isolated by one or more techniques, or combination oftechniques, known to the skilled in the art. In some embodiments, the at least one molecule secreted by stem cells is obtained, recovered, or otherwise isolated by one or more techniques selected from the group comprising centrifugation, differential centrifugation, density gradient, chromatography, affinity purification, size exclusion, immune-assays, and any combination thereof.

[0202] In some embodiments, the at least one molecule secreted by stem cells is obtained, recovered, or otherwise isolated by differential centrifugation as explained hereinabove. This technique is particularly adapted for embodiments wherein the at least one molecule secreted by stem cells is an exosome or an extracellular vesicle.

[0203] In some embodiments, the at least one molecule secreted by stem cells is obtained, recovered, or otherwise isolated by chromatography. In some embodiments, the at least one molecule secreted by stem cells is obtained, recovered, or otherwise isolated by affinity purification. In some embodiments, the at least one molecule secreted by stem cells is obtained, recovered, or otherwise isolated by size exclusion.

[0204] In some embodiments, the at least one molecule secreted by stem cells is purified. Illustratively and non-limitatively, the at least one molecule secreted by stem cells is pure when less than 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or less, of contaminants are present.

[0205] The at least one molecule obtained by the method may be placed in a suitable vial and stored in suitable conditions (for example frozen, etc.).

[0206] In certain embodiments, the method further comprises a step of analyzing the at least one molecule, by means known in the art such as LC-MS, detection of certain markers, and the like.

[0207] The present invention further relates to a method of obtaining or recovering stem cells extracted from a tissue, the method comprising the steps of: i. in a non-adherent culture vessel comprising a plurality of matrixes suitable for cell adhesion and a culture medium, placing the tissue;ii. incubating for at least 168 hours; iii. obtaining a plurality of matrixes suitable for cell adhesion comprising stem cells; iv. optionally, transferring the plurality of matrixes suitable for cell adhesion comprising stem cells into one or more new non-adherent culture vessel comprising a plurality of matrixes suitable for cell adhesion, and incubating for at least 24 hours; v. optionally, repeating steps (iii) to (iv) any number of times; vi. optionally, washing the plurality of matrixes suitable for cell adhesion comprising stem cells; vii. contacting the plurality of matrixes suitable for cell adhesion comprising stem cells with a composition comprising trypsin; and viii. recovering the stem cells; wherein the stem cells are exposed at most one time to a protease or to a cocktail of proteases, and optionally wherein the matrixes suitable for cell adhesion are dissolvable matrixes suitable for cell adhesion.

[0208] The present invention further relates to a method of obtaining or recovering mesenchymal stem cells extracted from a tissue selected from the group comprising or consisting of placenta, umbilical cord, and Wharton jelly, the method comprising the steps of: i. in a non-adherent culture vessel comprising a plurality of microcarriers and a culture medium, placing the tissue; ii. incubating for at least 168 hours; iii. obtaining a plurality of microcarriers comprising mesenchymal stem cells;iv. optionally, transferring the plurality of microcarriers comprising mesenchymal stem cells into one or more new non-adherent culture vessel comprising a plurality of microcarriers, and incubating for at least 24 hours; v. optionally, repeating steps (iii) to (iv) any number of times; vi. optionally, washing the plurality of microcarriers comprising mesenchymal stem cells; vii. contacting the plurality of microcarriers comprising mesenchymal stem cells with a composition comprising trypsin; and viii. recovering the mesenchymal stem cells; wherein the mesenchymal stem cells are exposed at most one time to a protease or to a cocktail of proteases, and optionally wherein the microcarriers are dissolvable microcarriers.

[0209] The present invention further relates to a method of obtaining or recovering stem cells extracted from a tissue, the method comprising the steps of: i. in a non-adherent culture vessel comprising a plurality of matrixes suitable for cell adhesion and a culture medium, placing the tissue; ii. incubating for at least 168 hours; iii. obtaining a plurality of matrixes suitable for cell adhesion comprising stem cells; iv. optionally, transferring the plurality of matrixes suitable for cell adhesion comprising stem cells obtained at step (iii) into one or more new non-adherent culture vessel comprising a plurality of matrixes suitable for cell adhesion, and incubating for at least 24 hours; v. optionally, repeating steps (iii) to (iv) any number of times;vi. optionally, washing the plurality of matrixes suitable for cell adhesion comprising stem cells obtained at step (iii) or (v); vii. contacting the plurality of matrixes suitable for cell adhesion comprising stem cells obtained at step (iii), (v) or (vi) with a composition comprising trypsin, thereby obtaining stem cells; and viii. recovering the stem cells obtained at step (vii); wherein the stem cells are exposed at most once to a protease or to a cocktail of proteases, and optionally wherein the matrixes suitable for cell adhesion are dissolvable matrixes suitable for cell adhesion.

[0210] The present invention further relates to a method of obtaining or recovering mesenchymal stem cells extracted from a tissue selected from the group comprising or consisting of placenta, umbilical cord, and Wharton jelly, the method comprising the steps of: i. in a non-adherent culture vessel comprising a plurality of microcarriers and a culture medium, placing the tissue; ii. incubating for at least 168 hours; iii. obtaining a plurality of microcarriers comprising mesenchymal stem cells; iv. optionally, transferring the plurality of microcarriers comprising mesenchymal stem cells obtained at step (iii) into one or more new non-adherent culture vessel comprising a plurality of microcarriers, and incubating for at least 24 hours; v. optionally, repeating steps (iii) to (iv) any number of times; vi. optionally, washing the plurality of microcarriers comprising mesenchymal stem cells obtained at step (iii) or (v);vii. contacting the plurality of microcarriers comprising mesenchymal stem cells obtained at step (iii), (v) or (vi) with a composition comprising trypsin, thereby obtaining mesenchymal stem cells; and viii. recovering the mesenchymal stem cells obtained at step (viii); wherein the mesenchymal stem cells are exposed at most once to a protease or to a cocktail of proteases, and optionally wherein the microcarriers are dissolvable microcarriers.

[0211] The present invention further relates to a stem cell or a population of stem cells obtained by the methods according to the invention.

[0212] In classical protocols for stem cell extraction from tissues and subsequent in vitro culture, the stem cells are repeatedly exposed to the action of proteases, for example during enzymatic digestion of the tissue, and during passages in cell culture wherein stem cells are detached from the culture flask, well or dish by the action of a protease, typically trypsin, that degrades attachment proteins on the stem cells. Repeated action of these proteases tends to degrade not only attachment protein, but also other surface proteins, markers or receptors, that may be of physiological and / or functional importance for the stem cells.

[0213] On the contrary, the methods of the invention enable extraction and expansion of stem cells without exposure to any protease, and recovery of these stem cells with at most one single exposure to a protease (or cocktail of proteases). The Applicant surprisingly demonstrates the methods of the invention allow for, inter alia, the obtention of a greater number of viable stem cells (see Figures 3 and 5), improved proliferation (see Figure 7), and that the resulting stem cells have improved migration capacity compared to stem cells undergoing several steps of trypsinization (see Figure 8A-8B).

[0214] Stem cells extracted with the method of the invention are described hereinabove.

[0215] In some embodiments, the stem cell or population of stem cells is a clinical grade stem cell.

[0216] It will be apparent to the person skilled in the art that stem cells or populations of stem cells extracted by the method of the invention are isolated and separated from any living organism. It will also be apparent to the person skilled in the art that stem cells or populations of stem cells extracted by the method of the invention are exposed to artificial conditions (culture medium, physico-chemical parameters of culture, etc.) and to an artificial environment (non-adherent culture vessel and matrixes suitable for cell adhesion), therefore the stem cells or populations of stem cells extracted by the method of the invention do not pertain to naturally occurring stem cells.

[0217] The present invention further relates to at least one molecule secreted by stem cells obtained by the methods according to the invention.

[0218] In some embodiments, the at least one molecule secreted by stem cells is selected from the group comprising or consisting of exosomes, extracellular vesicles, secreted proteins, enzymes, growth factors, cytokines, hormones, and soluble mediators. In some embodiments, the at least one molecule secreted by stem cells is selected from the group comprising or consisting of exosomes, extracellular vesicles, and cytokines. In some embodiments, the at least one molecule secreted by stem cells is an exosome or extracellular vesicle. In some embodiments, the at least one molecule secreted by stem cells is an exosome. In some embodiments, the at least one molecule secreted by stem cells is an extracellular vesicle. In some embodiments, the at least one molecule secreted by stem cells is a secreted protein. In some embodiments, the at least one molecule secreted by stem cells is a cytokine. In some embodiments, the at least one molecule secreted by stem cells is an enzyme. In some embodiments, the at least one molecule secreted by stem cells is a growth factor. In some embodiments, the at least one molecule secreted by stem cells is a hormone.

[0219] The present invention further relates to a stem cell or a population of stem cells having altered expression of at least one gene or protein compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has increased expression of at least one gene or protein comparedto a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0220] In some embodiments, the stem cell or population of stem cells has altered expression of 1, 2, 3, 4, 5, 6, 7,8 ,9, 10 or more genes or proteins.

[0221] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one gene or protein selected form the group comprising or consisting of ALCAM, ANKRD1, BDNF, CCL2, CCL5, CCND1, CCR1, CCR10, CCR2, CCR4, CCR7, CCR9, CD105, CD106, CD13, CD14, CD140a, CD142, CD146, CD202, CD209, CD271, CD29, CD29, CD40, CD44, CD47, CD49a, CD56, CD73, CD80, CD86, CD90, CDC20, CENPA, CITED2, CMKLR1, CTGF, CTTED2, CXCL1, CXCL16, CXCL2, CXCL5, CXCL6, CXCL8, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, cytokines, DAB2, DDAA1, DPP4, FJX1, Galectin, Galectin-9, GAS1, GREM1, HBEGF, HGF, HLA-G, IGFBP2, IL-1B, IL-15, IL-10, IL-12, IL-4, IL-6, IL-8, Indoleamine 2,3-dioxygenase (IDO), IGF-1, integrins, KGFBP2, LOX, MMP-1, MMP- 2, MMP-3, NP1, PDGFA, PDGF-AB, PDL-1, PDL-2, PGE2, PLK1, PLSCR4, Prostaglandin E2 (PGE2), RHOB, SERPINB2, SERPINE1, soluble HLA-G, SOX4, TGFP, THBS1, TLR agonists, TLR3, TLR4, TLR7, TLR8, TMP-1, TMP-2, TNFAIP6, TNFRSF11B, TNFa, IFNy, tolerogenic immune checkpoints, TSG-6, VCAM, VEGF, VLA-4, and WFDC1, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0222] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one gene or protein selected form the group comprising or consisting of ANKRD1, BDNF, CCL2, CCL5, CCND1, CCR1, CCR10, CCR2, CCR4, CCR7, CCR9, CD105, CD106, CD13, CD14, CD140a, CD142, CD146, CD202, CD209, CD271, CD29, CD29, CD40, CD44, CD47, CD49a, CD56, CD73, CD80, CD86, CD90, CDC20, CENPA, CITED2, CMKLR1, CTGF, CTTED2, CXCL1, CXCL16, CXCL2, CXCL5, CXCL6, CXCL8, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, cytokines, DAB2, DDAA1, DPP4, FJX1, Galectin, Galectin-9, GAS1, GREM1, HBEGF, HGF, HLA-G, IGFBP2, IL10, IL12, IL4, IL6, IL8, Indoleamine 2,3-dioxygenase (IDO), integrins, KGFBP2, LOX, MMP-1, MMP-2, MMP-3, NP1, PDGFA, PDGF-AB, PDL-1,PDL-2, PGE2, PLK1, PLSCR4, Prostaglandin E2 (PGE2), RHOB, SERPINB2, SERPINE1, soluble HLA-G, SOX4, TGFp, THBS1, TLR agonists, TLR4, TLR7, TLR8, TMP-1, TMP-2, TNFAIP6, TNFRSF1 IB, TNFa, ZFNy, tolerogenic immune checkpoints, TSG-6, VCAM, VEGF, VLA-4, and WFDC1, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0223] In some embodiments, the stem cell or population of stem cells has altered expression of at least one gene or protein selected form the group comprising or consisting of ALCAM, CXCR4, CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD44, Galectin, CXCR7, VLA-4, CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR5, CXCR6, IGF-1, IL-1B, IL-15, MMP-1, MMP-2, MMP-3, TLR3, TMP- 1, TMP-2, integrins, PDGF-AB, VEGF, CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD271, CD14, CD56, and CD146, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time, preferably selected form the group comprising or consisting of TLR3, IL-15, CXCL8, ALCAM, VEGF, CD44, CXCL1, IL-1B, and IGF-1.

[0224] In some embodiments, the stem cell or population of stem cells has altered expression of at least one gene or protein selected form the group comprising or consisting of CXCR4, CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD44, Galectin, CXCR7, VLA-4, CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR5, CXCR6, MMP-1, MMP-2, MMP-3, TMP-1, TMP-2, integrins, PDGF-AB, CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD271, CD14, CD56, and CD146, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has increased expression of at least one gene selected form the group comprising or consisting of CXCR4, CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD44, Galectin, CXCR7, VLA-4, CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR5, CXCR6, MMP-1, MMP-2, MMP-3, TMP-1, TMP-2, integrins, PDGF-AB, CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD271, CD14,CD56, and CD146, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0225] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one marker selected form the group comprising or consisting of CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD29, CD44, Galectin, CXCR4, VLA-4, CCR1, CCR2, CCR4, CCR7 , CCR9, CCR10, CXCR5, CXCR6, MMP-1, MMP-2, MMP-3, TMP-1, TMP-2, integrins, and PDGF-AB, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one marker selected form the group comprising or consisting of CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD29, and CD44, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one receptor selected form the group comprising or consisting of CD44, Galectin, CXCR4, CXCR3, CXCR7, VLA-4, CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR5, and CXCR6, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one marker selected form the group comprising or consisting of MMP-1, MMP-2, MMP-3, TMP-1, TMP-2, integrins, and PDGF-AB, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0226] In some embodiments, the stem cell or population of stem cells has altered expression of at least one gene or protein selected form the group comprising or consisting of CXCR4, CXCR3, CD142, IL-6, IL-10 and IL-IRA.

[0227] In some embodiments, the stem cell or population of stem cells has altered or increased expression of CXCR4 compared to a stem cell or a population of stem cellscultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0228] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one surface marker selected form the group comprising or consisting of CD73, CD105, CD106, CD56, CD146, and CD271, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one surface marker selected form the group comprising or consisting of CD73, CD105, CD106, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD56 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time, wherein the stem cell or population of stem cells are Wharton jelly MSC. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD106, CD146, CD271 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time, wherein the stem cell or population of stem cells are not Wharton jelly MSC.

[0229] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one surface marker selected form the group comprising or consisting of CD13, CD14, CD29, CD44, CD56, CD73, CD90, CD105, CD106, CD146, and CD271, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one surface marker selected form the group comprising or consisting of CD13, CD29, CD44, CD73, CD90, CD105, CD106, and CD271, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one surface markerselected form the group comprising or consisting of CD14, CD56, CD106, CD146, and CD271, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0230] In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD 13 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD29 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD44 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD73 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD90 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD 105 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD 106 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD 146 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD271 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0231] In some embodiments, the stem cell or population of stem cells has altered or increased expression of TSG-6 and / or TNFAIP6, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0232] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one immunomodulation marker selected form the group comprising or consisting of PD-L1, HLA-G, PGE2, cytokines, TGF[3, IL6, IL10, HGF, VEGF, and IDO, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0233] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one pro-inflammatory marker selected form the group comprising or consisting of TLR agonists (such as TLR4, TLR7 or TLR8), tolerogenic immune checkpoints (such as PD-L1, HLA-G or IDO), soluble HLA-G, PD-L1, IL6, IL8, IL 12, TNFa, TFNy, IL4, IL 10, CCL5, TGF[3, and PGE2, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one TLR agonist selected form the group comprising or consisting of TLR4, TLR7 and TLR8, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one tolerogenic immune checkpoint selected form the group comprising or consisting of PD- Ll, HLA-G and IDO, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one marker selected form the group comprising or consisting of soluble HLA-G, PD-L1, IL6, IL8, IL12, TNFa, IFNy, IL4, IL10, CCL5, TGF[3, and PGE2, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0234] In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one gene or protein selected form the group comprising or consisting of TLR3, IL-15, CXCL8, ALCAM, VEGF, CD44, CXCL1, IL-1B, and IGF- 1, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of at least one gene or protein selected form the group comprising or consisting of TLR3, IL- 15, ALCAM, CD44, CXCL1, IL- IB, and IGF-1, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0235] In some embodiments, the stem cell or population of stem cells has altered or increased expression of TLR3 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of IL-15 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CXCL8 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of ALCAM compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of VEGF compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CD44 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of CXCL1 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease morethan one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of IL-1B compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has altered or increased expression of IGF-1 compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0236] In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cell. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and epithelial stem cells.

[0237] In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of mesenchymal stem cells, neural stem cells, and epithelial stem cells, preferably mesenchymal stem cells (MSC).

[0238] In some embodiments, the stem cells or population of stem cells are animal stem cells, preferably mammal stem cells, more preferably human stem cells. In some embodiments, the stem cells or population of stem cells are human stem cells.

[0239] In some embodiments, the stem cells or population of stem cells are mesenchymal stem cells (MSC). In some embodiments, the stem cells or population of stem cells are animal MSC, preferably mammals MSC, more preferably human MSC. In some embodiments, the stem cells or population of stem cells are human MSC.

[0240] In some embodiments, the stem cells or population of stem cells are neural stem cells. In some embodiments, the stem cells or population of stem cells are animal neural stem cells, preferably mammals neural stem cells, more preferably human neural stem cells. In some embodiments, the stem cells or population of stem cells are human neural stem cells.

[0241] In some embodiments, the stem cells or population of stem cells are epithelial stem cells. In some embodiments, the stem cells or population of stem cells are animal epithelial stem cells, preferably mammals epithelial stem cells, more preferably human epithelial stem cells. In some embodiments, the stem cells or population of stem cells are human epithelial stem cells.

[0242] In some embodiments, the stem cells or population of stem cells are hematopoietic stem cells. In some embodiments, the stem cells or population of stem cells are animal hematopoietic stem cells, preferably mammals hematopoietic stem cells, more preferably human hematopoietic stem cells. In some embodiments, the stem cells or population of stem cells are human hematopoietic stem cells.

[0243] In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of fetal stem cells, placenta-derived stem cells, umbilical cord-derived stem cells, Wharton jelly-derived stem cells, amnios-derived stem cells, bone marrow-derived stem cells, peripheral blood-derived stem cells, adipose tissue-derived stem cells, dental pulp-derived stem cells, thymus-derived stem cells, and muscle-derived stem cells. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of human fetal stem cells, human placenta-derived stem cells, human umbilical cord-derived stem cells, human Wharton jelly-derived stem cells, human amnios-derived stem cells, human bone marrow-derived stem cells, human peripheral blood-derived stem cells, human adipose tissue-derived stem cells, human dental pulp-derived stem cells, human thymus-derived stem cells, and human muscle-derived stem cells.

[0244] In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of fetal stem cells, placenta-derived stem cells, umbilical cord-derived stem cells, and Wharton jelly-derived stem cells. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of placenta-derived stem cells, umbilical cord-derived stem cells, and Wharton jelly-derived stem cells. In some embodiments, the stem cells or population of stem cells are placenta-derived stem cells or umbilical cord-derived stem cells. In some embodiments, the stem cells or population of stem cells are selected fromthe group comprising or consisting of human fetal stem cells, human placenta-derived stem cells, human umbilical cord-derived stem cells, and human Wharton jelly-derived stem cells. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of human placenta-derived stem cells, human umbilical cord-derived stem cells, and human Wharton jelly-derived stem cells. In some embodiments, the stem cells or population of stem cells are human placenta-derived stem cells or human umbilical cord-derived stem cells.

[0245] In some embodiments, the stem cells or population of stem cells are fetal stem cells, preferably human fetal stem cells. In some embodiments, the stem cells or population of stem cells are placenta-derived stem cells, preferably human placenta- derived stem cells. In some embodiments, the stem cells or population of stem cells are umbilical cord-derived stem cells, preferably human umbilical cord-derived stem cells. In some embodiments, the stem cells or population of stem cells are Wharton jelly- derived stem cells, preferably human Wharton jelly-derived stem cells. In some embodiments, the stem cells or population of stem cells are amnios-derived stem cells, preferably human amnios-derived stem cells. In some embodiments, the stem cells or population of stem cells are bone marrow-derived stem cells, preferably human bone marrow-derived stem cells. In some embodiments, the stem cells or population of stem cells are peripheral blood-derived stem cells, preferably human peripheral blood-derived stem cells. In some embodiments, the stem cells or population of stem cells are adipose tissue-derived stem cells, preferably human adipose tissue-derived stem cells. In some embodiments, the stem cells or population of stem cells are dental pulp-derived stem cells, preferably human dental pulp-derived stem cells. In some embodiments, the stem cells or population of stem cells are thymus-derived stem cells, preferably human thymus-derived stem cells. In some embodiments, the stem cells or population of stem cells are muscle- derived stem cells, preferably human muscle-derived stem cells.

[0246] In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of fetal MSC, placenta-derived MSC, umbilical cord- derived MSC, Wharton jelly-derived MSC, amnios-derived MSC, bone marrow-derived MSC, peripheral blood-derived MSC, adipose tissue-derived MSC, dental pulp-derivedMSC, thymus-derived MSC, and muscle-derived MSC. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of human fetal MSC, human placenta-derived MSC, human umbilical cord-derived MSC, human Wharton jelly-derived MSC, human amnios-derived MSC, human bone marrow- derived MSC, human peripheral blood-derived MSC, human adipose tissue-derived MSC, human dental pulp-derived MSC, human thymus-derived MSC, and human muscle-derived MSC.

[0247] In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of fetal MSC, placenta-derived MSC, umbilical cord- derived MSC, and Wharton jelly-derived MSC. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of placenta- derived MSC, umbilical cord-derived MSC, and Wharton jelly-derived MSC. In some embodiments, the stem cells or population of stem cells are placenta-derived MSC or umbilical cord-derived MSC. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of human fetal MSC, human placenta-derived MSC, human umbilical cord-derived MSC, and human Wharton jelly- derived MSC. In some embodiments, the stem cells or population of stem cells are selected from the group comprising or consisting of human placenta-derived MSC, human umbilical cord-derived MSC, and human Wharton jelly-derived MSC. In some embodiments, the stem cells or population of stem cells are human placenta-derived MSC or human umbilical cord-derived MSC.

[0248] In some embodiments, the stem cells or population of stem cells are fetal MSC, preferably human fetal MSC. In some embodiments, the stem cells or population of stem cells are placenta-derived MSC, preferably human placenta-derived MSC. In some embodiments, the stem cells or population of stem cells are umbilical cord-derived MSC, preferably human umbilical cord-derived MSC. In some embodiments, the stem cells or population of stem cells are Wharton jelly-derived MSC, preferably human Wharton jelly-derived MSC. In some embodiments, the stem cells or population of stem cells are amnios-derived MSC, preferably human amnios-derived MSC. In some embodiments, the stem cells or population of stem cells are bone marrow-derived MSC, preferablyhuman bone marrow-derived MSC. In some embodiments, the stem cells or population of stem cells are peripheral blood-derived MSC, preferably human peripheral blood- derived MSC. In some embodiments, the stem cells or population of stem cells are adipose tissue-derived MSC, preferably human adipose tissue-derived MSC. In some embodiments, the stem cells or population of stem cells are dental pulp-derived MSC, preferably human dental pulp-derived MSC. In some embodiments, the stem cells or population of stem cells are thymus-derived MSC, preferably human thymus-derived MSC. In some embodiments, the stem cells or population of stem cells are muscle-derived MSC, preferably human muscle-derived MSC.

[0249] In some embodiments, the stem cell or population of stem cells has improved, enhanced, or otherwise increased proliferation, expansion, migration, viability, immune- modulatory and / or pro-inflammatory capabilities compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has improved, enhanced, or otherwise increased viability, proliferation, expansion, migration, capabilities compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has improved, enhanced, or otherwise increased proliferation capacity compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has improved, enhanced, or otherwise increased CFU potency compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has improved, enhanced, or otherwise increased migration capacity compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells has improved, enhanced, or otherwise increased viability compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time. In some embodiments, the stem cell or population of stem cells hasimproved, enhanced, or otherwise increased potency for inducing macrophage differentiation and / or reversion compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time.

[0250] In some embodiments, the stem cell or population of stem cells is a clinical grade stem cell.

[0251] It will be apparent to the person skilled in the art that the methods described herein are in vitro methods.

[0252] The present invention further relates to a composition comprising the stem cell or population of stem cells as described herein.

[0253] The present invention further relates to a pharmaceutical composition comprising the stem cell or population of stem cells as described herein, and at least one pharmaceutically acceptable excipient or vehicle.

[0254] In some embodiments, the pharmaceutically acceptable excipient or vehicle is selected in a group comprising or consisting of a solvent, a diluent, a carrier, an excipient, a dispersion medium, a coating, and any combinations thereof. The carrier, diluent, solvent or excipient must be “acceptable” in the sense of being compatible with the stem cell or stem cell population described herein, and not be deleterious upon being administered to an individual. Typically, the vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual.

[0255] For the particular purpose of human administration, the pharmaceutical compositions should meet general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).

[0256] The present invention further relates to a combination kit comprising (i) the stem cell or stem cell population described herein, or a pharmaceutical composition comprising the same and (ii) at least one other therapeutic agent. In some embodiments, the at leastone other therapeutic agent is for treating a disease selected from the group comprising or consisting of inflammation, infection, cancer, auto-immune diseases, neurologic diseases, cardiologic diseases and genetic diseases.

[0257] In some embodiments, the kit further comprises means to administer the stem cell or stem cell population described herein, or the pharmaceutical composition comprising the same, to a subject in need thereof.

[0258] The present invention further relates to a stem cell or population of stem cells as described herein, or a pharmaceutical composition as described herein, for use as a medicament.

[0259] The present invention further relates to a stem cell or population of stem cells as described herein, or a pharmaceutical composition as described herein, for use for treating a disease. The present invention further relates to a stem cell or population of stem cells as described herein, or a pharmaceutical composition as described herein, for use for treating a disease selected from the group comprising or consisting of inflammation, infection, cancer, auto-immune diseases, neurologic diseases, cardiologic diseases and genetic diseases. In some embodiment, the disease is inflammation. In some embodiment, the disease is an infection. In some embodiment, the disease is a cancer. In some embodiment, the disease is an auto-immune disease. In some embodiment, the disease is a neurologic disease. In some embodiment, the disease is a cardiologic disease. In some embodiment, the disease is a genetic disease.

[0260] The present invention further relates to a method for treating a disease selected from the group comprising or consisting of inflammation, infection, cancer, auto-immune diseases, neurologic diseases, cardiologic diseases and genetic diseases, in a subject in need thereof, comprising administering to said subject a therapeutically effective dose of the stem cell or population of stem cells as described herein, or the pharmaceutical composition as described herein.

[0261] The present invention further relates to a stem cell or population of stem cells as described herein, or a pharmaceutical composition as described herein, for use for the manufacture of a medicament for treating a disease selected from the group comprisingor consisting of inflammation, infection, cancer, auto-immune diseases, neurologic diseases, cardiologic diseases and genetic diseases.

[0262] The present invention further relates to the use of a stem cell or population of stem cells as described herein, or a pharmaceutical composition as described herein, for the manufacture of a medicament for treating a disease selected from the group comprising or consisting of inflammation, infection, cancer, auto-immune diseases, neurologic diseases, cardiologic diseases and genetic diseases.BRIEF DESCRIPTION OF THE DRAWINGS

[0263] Figure 1 is a histogram showing the yield of viable cell extraction in function of the method of cell extraction (classical 2D method, 3D method with fresh and frozen tissues).

[0264] Figure 2 is a histogram showing cellular viability after the cell extraction in function of the protocol tested (addition or not of microcarriers and time of extraction).

[0265] Figure 3 is a graph showing the total number of cells and cell viability in function of time.

[0266] Figure 4 is a graph showing cell expansion comparison between P3 cells classically extracted with 3 trypsinization steps and cultivated in bioreactor, and cells extracted with the method described herein (direct extraction) and cultivated in bioreactor.

[0267] Figure 5A-5B is a combination of a photograph and a histogram showing cell migration potential of stem cells following 3D-extraction and culture in bioreactor and stem cells undergoing three trypsinizations. (Fig. 5A) Visualization of the stained cells after 24H of migration trough a Boyden chamber. (Fig. 5B) Quantitative result of the cell migration after 24H of migration trough a Boyden chamber.

[0268] Figure 6 is a set of graphs showing cell expression of the membrane receptor CXCR4 by flow cytometry. Expression of the CXCR4 on the membranes of the cell in function of the culture condition. P0 cells are represented with the dark line, P3 cells are represented with the light grey line and bioreactor cells are represented with the grey lines.

[0269] Figure 7 is a histogram showing ISCT phenotypic marker expression after a production in bioreactor according to the invention and compared to a 2D production (P3 cells).

[0270] Figure 8 is a set of schemes showing the results of the digital PCR allowing to detect genetic abnormalities along the 23 hMSC chromosomes of the sample.

[0271] Figure 9A-9I is a set of histograms showing the transcriptomic analysis of certain markers in stem cells obtained with the method of the present invention: TLR3 (Fig. 9A), IL- 15 (Fig. 9B), CXCL8 (Fig. 9C), ALCAM (Fig. 9D), VEGF (Fig. 9E), CD44 (Fig. 9F), CXCL1 (Fig. 9G), IL- IB (Fig. 9H), and IGF-1 (Fig. 91).EXAMPLES

[0272] The present invention is further illustrated by the following examples.Example 1 : Culture in flasksMaterials and Methods

[0273] Stem cells derived from Wharton’s Jelly were extracted from fresh umbilical cords of just bom babies (UTCT - Centre Hospitalier Universitaire de Nancy, France) and were used during the 24 h following the birth.

[0274] The chosen method of extraction was non-enzymatic, based on the migration and plastic adhesion capabilities of the WJ-MSCs. Spontaneously, the cells migrate from the umbilical cord on plastic petri-dishes and multiply in suitable culture conditions. The cells were cultured in an alpha-Minimum Essential Medium (a-MEM medium) (Fisher Gibco12561-065, Grand Island, NY, USA), supplemented with Human Platelet Lysate (HPL) (PLT gold-GI, PLTGold500-GMP-GI, Mill Creek Life Sciences, Rochester, Minnesota, USA), and 2 mM of glutamine (Gibco A29168-01 lOOmL, lot 2302894, Grand Island, NY, USA). The petri-dishes were incubated at 37 °C with 5 % CO2.

[0275] Several slices of umbilical cord were placed by plate. The medium was completely replaced once a week. After 15 days, the umbilical cords slices were taken off the petri-dishes. The cells were still incubated for another 15 days, and the culture medium was changed twice a week. Once the cells have reached about 80 % confluency, they are being trypsinized with TrypLE Express (Gibco 12604-013, lot 2276862, Grand Island, NY, USA) and cryo-conserved in 90 % of HPL supplemented with 10 % in DMSO in cryotubes.Results

[0276] About 10 cords were treated without antibiotics and a mean number of cells extracted at P0 was about 14 million of total cells with a mean cell viability of 74 %.

[0277] The impact of the cord on the yield of cell extraction can also be measured, especially in the R&D extractions. The absence of antibiotics seemed to be beneficial for the yield cell extraction.

[0278] A mean of 40 slices of umbilical cord can be obtained when a 30 cm cord is used. Using Petri dishes of 90 mm of diameter it is possible to realize between 10 and 13 dishes. It certainly depends on the size of the plate. It is necessary to be careful about cell confluence and to not have too much aggregates. If this occurred a step of filtration after the trypsination would be required.

[0279] In conclusion, a more precise isolation of the Wharton’s jelly with biopsies patch could increase and improve the cell quality of explant.Example 2: Closed system for cell passaging

[0280] The use of closed systems with aseptic connections between unit operations greatly minimizes the exposure of process starting materials, process intermediates and the product to the environment. The use of custom single-use assemblies connecting medium preparation to the bioreactor, bioreactor to harvest systems and other downstream operations can ensure closed operations.Materials and Methods

[0281] Collection and screening of umbilical cord tissue

[0282] Human umbilical cord samples were collected from donors who gave informed consent. Each participant was thoroughly evaluated for donor eligibility based on medical history, and blood tests for transfusion-transmissible diseases were performed. Donors were 37 weeks or more gestation at the time of normal or cesarean delivery. Umbilical cords (minimum 20 cm long) were carefully collected by a licensed midwife, cleaned and stored in sterile containers with physiological serum. The transport to the laboratory was done by an approved transporter and at controlled temperature (2-8°C). The cords were processed after they been collected.

[0283] Cutting of the umbilical cords

[0284] After its reception, the cord was identified, measured, and then immersed in a D- PBS bath with antibiotics (amoxicillin, amphotericin, vancomycin) for 1 hour. Then, the entire umbilical cord was cut into 2 to 3 mm slices using a scalpel. All the slices were washed again with the D-PBS plus antibiotics solution. Then a part of the slices was used immediately and another part of the slices was frozen.

[0285] 3D method with fresh slices

[0286] For this method, a closed system was used with a solution of dissolvable microcarriers (DM) (Corning, 7290, 6,67 g / L). Then culture medium with a glucose supplementation was added. Several slices were deposited in the system. Then the system was put at 37°C 5% CO2 and 5% O2. Every week, concentration of glucose and lactate were determined.

[0287] 3D method with frozen slices

[0288] Several slices of umbilical cord were thawed, then the system was put at 37°C 5% CO2 and 5% O2. Every day, concentration of glucose and lactate were determined. Cultures lasted about 20 days.Results

[0289] At the end of the cell migration, the number of cells collected per slices of umbilical cord and their viability were determined either in 2D and 3D isolation (Figure 1). Thanks to the 3D extraction, the yield of viable cells extracted is more important than in a classical 2D extraction. About 1.5 million of viable cells can be extracted from frozen slices with the microcarriers, and a mean of 1 million of viable cells can be extracted from fresh slices. A higher variability can be noticed in the protocol of extraction from fresh slices because different protocols have been tested (variation of extraction time, addition of microcarriers, etc.).

[0290] The addition of microcarriers and the state of frozen tissues seemed to improve the cell viability after the extraction (Figure 2).

[0291] In conclusion, the closed system for the cell extraction is a good device thanks to the decrease of the manual operations (as the medium exchange or the trypsinization), but also, by the increase of the surface area bring by microcarriers it improves the yield of cell extraction. Thanks to this device, the process of cell extraction is close to an automated and closed system.3: Single Use cultures

[0292] Reproducible, scalable and robust manufacturing bioprocesses need to be developed before moving from basic research with initial preclinical evidence to cell therapies for clinical applications. There are no robust, reproducible manufacturing processes for the production of MSCs in the quantity and quality required for therapeutic applications (Garcia-Fernandez et al., 2020, Biochemical Engineering Journal 159;107601). In this part, a solution of standardized and high cell production was proposed in order to answer to this known problematic production of stem cells for clinical applications.Materials and Methods

[0293] Expansion in Stirred Tank Bioreactor with dissolvable microcarriers (DM)

[0294] IL bioreactor (Solaris) was pre-assembled, autoclaved, and calibrated for dissolved oxygen (20%), temperature (37°C), and pH status (7,4) with culture medium in it. After the 3D isolation of Wharton jelly mesenchymal stem cells (WJ-MSC) (see Example 2), the DM solution was transferred to the bioreactor. The agitation was set on 74 rpm.

[0295] Preparation of the BIOBLU 3c bioreactor

[0296] The tank bag was opened, removed from the bag and placed next to the controller. The controller was switched on. The temperature sensor was placed in the immersion sleeve, the O2 sensor was positioned so that it pushes against the membrane at the end of the immersion sleeve to expel air, the filter heater holding bar was placed, and the filter heater and exhaust tubing was slid onto it, the motor was placed, the gas addition tubing was connected to the overlay, and finally the heating blanket was placed on the tank. 1,250 mL of pre-warmed culture medium were transferred into a transfer bottle under the laminar flow cabinet. Using the pump, these 1,250 mL of culture medium were transferred into the tank.

[0297] Transfer inside 3L single use bioreactors

[0298] The transfer of the first bioreactor to the second one was performed by peristaltic pump. Then, fresh microcarriers were added in order to maintain a good cell confluence on microcarriers. With each addition of microcarriers, fresh cell culture medium was added.

[0299] Final harvest

[0300] After few days of culture, when the cells were ready to be used the detachment inside the vessel can be done. First, the agitation was stopped and the microcarriers were let to settle in the bottom of the bioreactor. Then, the supernatant was withdrawn without aspirating the microcarriers and the cells by using a peristaltic pump. When the cell culture medium was entirely collected, a cell counting was performed to assess the cell loss. Sterile D-PBS was added inside the tank and the agitation was turned on in order to rinse the microcarriers of medium’s leftover. The D-PBS was collected after having let the microcarriers to settle once again and realizing a cell count in the collected D-PBS. The solution of dissolution containing 100 U / mL pectinase and lOmM EDTA in TrypLE was added for 20 minutes. After the step of trypsinization, a volume of 600 mL of cell culture medium was added in order to inhibit the action of the enzyme, and all the harvest was collected inside a 2 L bottle; it was aseptically disconnected of the bioreactor and a cell count was performed under the safety cabinet.

[0301] Cell wash

[0302] The volume was transfered in 200 mL falcon tubes for a first centrifugation. The supernatant was discarded, the cells were re-suspended in 200 mL of D-PBS and centrifuged a second time. The supernatant was discarded, the cells were re suspended in 200 mL of D-PBS and centrifuged a third time. At each step of centrifugation, the cell count was monitored in order to follow the number of lost cells during the process.

[0303] Concentration and formulation

[0304] Finally, after the last centrifugation, the supernatant was discarded and the cells were re-suspended in 10 mL of cryoprotectant and counted. The volume of cryoprotectant was adjusted in order to obtain a cell concentration at 40 x 106cells / mL, and vials of 1 mL were prepared. The vials were freezed in a CoolCell in -80°C for 24 hours and then transferred to liquid nitrogen.Results

[0305] Culture for three days in the IL bioreactor allowed us to reach at least 60 million of cells with high cell viability. Then, after the transfer in the Bioblu3C the culture wasmaintained over 9 days with a maximal number of cells of 450 million produced with a high viability (> 80 %) (Figure 3).

[0306] According to data shown on Figure 4, the extraction method described herein (direct extraction) yields a greater number of cells after about one week compared to cells classically extracted and exposed to 3 trypsinization steps (P3 cells).

[0307] At the end of the downstream process, a yield of 83 % of recovery was obtained, leading to a formulation of 345 million of cells with a cell viability of 83 %.

[0308] A good reproducibility of the process developed in the inox vessels was demonstrated. Thanks to the BIOBLU3C a production of hMSC in a GMP compliant environment was possible, and able to produce at least 400 million of cells in less than 10 days. Moreover, the cells have preserved quality thanks to the innovative method of extraction and the use of only one step of trypsinization during the final phase of the process.Example 4: Quality AttributesMaterials and Methods

[0309] All manipulations were realized under sterile conditions with a type II laminar flow hood. Cells were cultivated in an incubator at 37°C with a humid atmosphere, 5 % CO2 and 5 % O2.

[0310] Boyden migration chamber

[0311] Under sterile conditions, the 24-well migration plate was allowed to warm-up at room temperature for 10 minutes. A cell solution containing IxlO6cells / mL in serum free media was prepared. 500 pL of media containing 10 % LPH was added to the lower well of the migration plate. 300 pL of the cell suspension solution was added to the inside of each insert, and incubated for 24 hours in a cell incubator.

[0312] The media was carefully aspired from the inside of the insert. The ends of 2-3cotton-tipped swabs were wetted with water, flattened by pressing them against a clean hard surface, and the interior of the inserts were gently swabbed to remove non-migratory cells, without puncturing the polycarbonate membrane. Cells on the inside perimeter of the insert were removed. The insert was transferred to a clean well containing 400 pL of Cell Stain Solution and incubated for 10 minutes at room temperature. The stained inserts were gently washed several times in a beaker of water, then dried. Each insert was transferred to an empty well, adding 200 pL of Extraction solution per well, then incubating 10 minutes on an orbital shaker.

[0313] 100 pL from each sample were transferred to a 96-well microtiter plate to measure the OD 560 nm in a plate reader.

[0314] Cell cytometry

[0315] Immune phenotypes (membrane level) were studied. Immunolabelling was performed under saturating conditions; antibodies were considered to bind to surface antigens through monovalent interaction. Cells were incubated with PBS / Bovine Serum Albumin 0.5% (PBS / BSA) (Sigma, France) and stained for 30 min at room temperature with direct membrane antibodies: anti-CXCR3-APC (Miltenyi, France) and anti-CXCR4- A700 (Miltenyi, France) antibodies. Afterwards, cells were washed with PBS- BSA and centrifuged before they were re-suspended in PBS- BSA. Negative controls were performed to detect cell auto-fluorescence. Cells were analyzed with the Facs SYMPHONY Al flow cytometer (BD Biosciences, USA) with an acquisition of 20,000 events. The cytometer was calibrated daily with Flow-Check Pro fluorospheres (Beckman Coulter, USA). Flow cytometry results were analyzed with FlowJo software (BD Biosciences, USA).Results

[0316] After the experiment in the Boyden chamber (Figure 5A-5B), it seems that we demonstrate a higher cell potential of migration after a 3D-extraction and a culture in bioreactor. Indeed, a 3.5 times higher cell migration was calculated in the well containing cells from the bioreactor, with only one step of trypsinization, than the cells having undergone three trypsinizations.

[0317] A study of the expression of the membrane receptor CXCR4 was also analyzed by flow cytometry and presented in the Figure 6-. It seems that the cells issued from bioreactors and that have undergone a single trypsinization express a greater number of membrane markers for CXCR4, leading to improved migration capacity.

[0318] More quality criteria were analyzed as presented in the next part. For two samples from the Inventors, production the activity of hTERT was not detected and the karyotype was normal (46 chromosomes) (data not shown). The immunophenotype markers were also studied and presented in Figure 7.

[0319] A more detailed study of the karyotype was carried out by Stem Genomics and the results are shown in Figure 8. Detailed research of possible genetic abnormalities (addition, deletion, mutation) was realized over the different cell chromosomes and compared between a production in bioreactor, initial P0 cells extracted from umbilical cords, and amplified P3 cells in 2D flasks. As showed by the figure 4.4, the mode of production (z.e., the bioreactor versus flasks) did not impact in a genetically way the umbilical cord stem cells. The cells remained similar to the cells just extracted from the umbilical cords (P0 cells).

[0320] The ISCT criteria (adherence to plastic, phenotype, differentiation) were respected after productions in bioreactors. Moreover, further studies showed no impact on the karyotype (no genetic abnormalities were found), and no expression of the hTERT telomerase. The cells produced by the Inventors remained stable and with high quality preserved.analysisMaterials and Methods

[0321] Cells from three different donors and derived from a bioreactor using the above method or a conventional culture method were compared with P0 umbilical cord cells.

[0322] The samples were thawed as described in order to extract RNA:- Preheat the water bath to 37°C.- Thaw the nine aliquots in the water bath until only a small ice cube remains. Transfer the entire volume of the cryotube to an Eppendorf tube.- Centrifuge at 200 g for 5 min.- Remove the supernatant.

[0323] After this first step, the RNA is extracted from the nine aliquots using the Qiagen Rneasy Mini Kit (Ref: 74104). The RNA elution was performed using 100 pL of elution buffer.

[0324] All samples were selected according to a minimum RNA concentration of 100 ng / pL and an RNA Integrity Number of at least 9. The purified RNAs were then stored in a -80 °C fridge until they were sent to the GENOM’IC platform at Cochin Hospital for sequencing. There, the library was prepared according to the Illumina® Stranded mRNA Prep Ligation protocol, with polyadenylated RNA captured using oligo (dT) magnetic beads. Finally, Illumina sequencing was performed to obtain an average of 2 * 51 million paired-end reads and about 25 Million reads per sample (Cortes Garcia E, et al., Antibody Mediated Rejection and T-cell Mediated Rejection Molecular Signatures Using Next-Generation Sequencing in Kidney Transplant Biopsies. Transpl Int. 2024 Jul 10;37: 13043).

[0325] Fastq files were then aligned using STAR algorithm (version 2.7.6a), on the Ensembl release 101 reference.

[0326] Reads were then count using RSEM (vl.3.1) and the statistical analyses on the read counts were performed with R (version 3.6.3) and the DESeq2 package (DESeq2_1.26.0) to determine the proportion of differentially expressed genes between two conditions.

[0327] The standard DESeq2 normalization method (DESeq2’s median of ratios with the DESeq function) was used, with a pre-filter of reads and genes (reads uniquely mapped on the genome, or up to 10 different loci with a count adjustment, and genes with at least 10 reads in at least 3 different samples).

[0328] Following the package recommendations, the Wald test with the contrast function and the Benjamini -Hochberg FDR control procedure were used to identify the differentially expressed genes. Results

[0329] As seen on Figure 9A-9I, the method of the invention (see “BIO” corresponding to “bioreactor” condition) preserves various markers of the MSCs present at P0 (z.e., cells extracted directly from tissue) compared to cells classically cultured (2D, with passages; see “P3” condition).

[0330] Some of these markers are involved in immunomodulation such as TLR3 (Fig.9A), CXCL1 (Fig. 9G), and IGF-1 (Fig. 91); pro-inflammation such as IL-15 (Fig. 9B), CXCL8 (Fig. 9C), and IL-1B (Fig. 9H); adhesion such as ALCAM (Fig. 9D) and CD44 (Fig. 9F); and growth such as VEGF (Fig. 9E).

[0331] These results show that the method of the invention yields MSCs with preserved expression of several markers involved in various biological functions, compared to MSCs that are passaged and exposed to trypsin multiple times.

Claims

CLAIMS1. A non-protease-based method for extracting stem cells from a tissue, comprising contacting said tissue with at least one matrix suitable for cell adhesion in a culture vessel comprising a culture medium, wherein said culture vessel is a non-adherent culture vessel.

2. The non-protease-based method according to claim 1, wherein said method further comprises incubating said tissue with said at least one matrix suitable for cell adhesion for at least 168 hours, preferably from 168 hours to 360 hours.

3. The non-protease-based method according to claim 1 or 2, wherein said method comprises the steps of: i. in a non-adherent culture vessel comprising at least one matrix suitable for cell adhesion, preferably at least one microcarrier, and a culture medium, placing the tissue; ii. incubating for at least 168 hours; and iii. obtaining the stem cells on the at least one matrix suitable for cell adhesion.

4. The non-protease-based method according to any one of claims 1 to 3, wherein said stem cells are not exposed to a protease selected from the group comprising trypsin, collagenase, dispase, papain, dextranase, hyaluronidase, and any combination thereof.

5. A method of expanding stem cells extracted from a tissue, comprising the steps of: i. extracting stem cells from a tissue by contacting said tissue with at least one matrix suitable for cell adhesion in a culture vessel comprising a culture medium, wherein said culture vessel is a non-adherent culture vessel, thereby obtaining at least one matrix suitable for cell adhesion comprising stem cells;ii. transferring said at least one matrix suitable for cell adhesion comprising stem cells in one or more new non-adherent culture vessel comprising additional matrixes suitable for cell adhesion; iii. incubating for at least 24 hours; iv. optionally, repeating steps (ii) and (iii) any number of times; v. optionally, recovering the stem cells from the matrixes suitable for cell adhesion comprising stem cells, preferably by trypsinization, and / or recovering the culture supernatant, wherein said method of expanding stem cells extracted from a tissue comprises at most one exposure to a single protease or to a cocktail of proteases.

6. The method according to any one of claims 1 to 5, wherein said tissue is selected from the list comprising umbilical cord, Wharton jelly, placenta, fetal tissue, bone marrow, peripheral blood, adipose tissue, dental pulp, thymus, and muscle, preferably wherein said tissue is Wharton jelly or placenta.

7. The method according to any one of claims 1 to 6, wherein said stem cells are selected from the group comprising mesenchymal stem cells, neural stem cells, and epithelial stem cells, preferably mesenchymal stem cells (MSC).

8. The method according to any one of claims 1 to 7, wherein said non-adherent culture vessel is a low attachment culture vessel or an ultra-low attachment culture vessel.

9. The method according to any one of claims 1 to 8, wherein said at least one matrix suitable for cell adhesion is a microcarrier, preferably a dissolvable microcarrier.

10. The method according to any one of claims 3 to 9, wherein step (i) is preceded by a step of placing at least one matrix suitable for cell adhesion, preferably at least one microcarrier, and a culture medium in a non-adherent culture vessel.

11. A method of obtaining or recovering mesenchymal stem cells extracted from a tissue selected from the group comprising placenta, umbilical cord, and Wharton jelly, the method comprising the steps of: i. in a non-adherent culture vessel comprising a plurality of microcarriers and a culture medium, placing said tissue; ii. incubating for at least 168 hours; iii. obtaining a plurality of microcarriers comprising mesenchymal stem cells; iv. optionally, transferring the plurality of microcarriers comprising mesenchymal stem cells into one or more new non-adherent culture vessel comprising a plurality of microcarriers, and incubating for at least 24 hours; v. optionally, repeating steps (iii) to (iv) any number of times; vi. optionally, washing the plurality of microcarriers comprising mesenchymal stem cells; vii. contacting the plurality of microcarriers comprising mesenchymal stem cells with a composition comprising trypsin; and viii. recovering the mesenchymal stem cells; wherein the mesenchymal stem cells are exposed at most one time to a protease or to a cocktail of proteases, and optionally wherein the microcarriers are dissolvable microcarriers.

12. A stem cell or a population of stem cells obtained by the method according to any one of claims 1 to 11.

13. A stem cell or a population of stem cells having increased expression of at least one gene selected form the group comprising ALCAM, CXCR4, CXCR3, CXCR7, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL16, CD44, Galectin, CXCR7, VLA-4, CCR1, CCR2, CCR4, CCR7, CCR9, CCR10, CXCR5, CXCR6, IGF-1, IL-IB, IL-15, MMP-1, MMP-2, MMP-3, TLR3, TMP-1, TMP-2, integrins, PDGF- AB, VEGF, CD13, CD29, CD44, CD73, CD90, CD105, CD106, CD271, CD14, CD56, and CD146, compared to a stem cell or a population of stem cells cultured on an adherent culture vessel and / or that has been exposed to a protease more than one time, preferably selected form the group comprising or consisting of TLR3, IL-15, CXCL8, ALCAM, VEGF, CD44, CXCL1, IL-1B, and IGF-1.

14. A pharmaceutical composition comprising the stem cell or population of stem cells according to claim 12 or 13, and at least one pharmaceutically acceptable excipient.

15. A stem cell or population of stem cells according to claim 12 or 13, or a pharmaceutical composition according to claim 14, for use for treating a disease selected from the group comprising inflammation, infection, cancer, auto-immune diseases, neurologic diseases, cardiologic diseases and genetic diseases.

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