WO2019199230A1 - Procédé de transport de cellules souches mésenchymateuses au moyen d'un milieu de culture cellulaire et procédé d'administration de cellules souches à des plaies - Google Patents

Procédé de transport de cellules souches mésenchymateuses au moyen d'un milieu de culture cellulaire et procédé d'administration de cellules souches à des plaies Download PDF

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WO2019199230A1
WO2019199230A1 PCT/SG2019/050198 SG2019050198W WO2019199230A1 WO 2019199230 A1 WO2019199230 A1 WO 2019199230A1 SG 2019050198 W SG2019050198 W SG 2019050198W WO 2019199230 A1 WO2019199230 A1 WO 2019199230A1
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carrier
cells
stem cell
million cells
cell population
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PCT/SG2019/050198
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English (en)
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Toan Thang Phan
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Cellresearch Corporation Pte. Ltd.
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

Definitions

  • the present invention relates to a method of transporting a stem cell population, the method comprising transporting the stem cell population contacted with a liquid carrier.
  • the present invention concerns a method of treating a subject having a disease, the method comprising topically administering a defined mesenchymal stem cell population to the subject, wherein the mesenchymal stem cell population is administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested.
  • a unit dosage comprising about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells of a defined mesenchymal stem cell population.
  • MSCs human mesenchymal stem cells derived from the amniotic membrane of the umbilical cord (umbilical cord lining (CL-MSCs), umbilical cord blood (CB-MSCs), placenta (P-MSCs), and Wharton’s jelly (WJ-MSCs)
  • CL-MSCs amniotic membrane of the umbilical cord
  • CB-MSCs umbilical cord blood
  • P-MSCs placenta
  • WJ-MSCs Wharton’s jelly
  • mesenchymal stem cells of the amniotic membrane can easily be obtained using the protocol as described in US patent application 2006/0078993 and International patent application W02006/019357, it would be of advantage for clinical trials with these cord lining MSC to have at hand a method that allows to isolate a population of these cord lining MSC’s that is highly homogenous and can thus be used for clinical trials.
  • Stem cells such as the mesenchymal stem cells as described above are however typically not applied/administered to patients at the site where they are produced. Often a substantial amount of time passes in between the harvesting of cells and their further utilization. There is thus a need for the provision of certain carriers which keep cells viable and healthy for a period of time typically used for transport or storage of cells.
  • the invention provides a method of transporting a stem cell population, the method comprising transporting said stem cell population contacted with a liquid carrier, liquid carrier, wherein the liquid carrier comprises a cell culture medium.
  • the invention provides a method of treating a subject having a disease, the method comprising topically administering a mesenchymal stem cell population as described herein to the subject, wherein the mesenchymal stem cell population is administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested.
  • the invention provides a unit dosage comprising about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells of a mesenchymal stem cell population as described herein.
  • the invention provides the use of a liquid carrier for transporting a stem cell population, wherein the liquid carrier comprises a cell culture medium.
  • Fig. 1 shows the technical information sheet of Lonza for Dulbecco’s modified eagle medium, including the catalogue number of the DMEM used for the making of the illustrative example of a medium of the invention (PTT-6) in the Experimental Section;
  • Fig. 2 shows the technical information sheet of Lonza for Ham’s F12 medium
  • FIG. 3 shows the technical information sheet of Lonza for DMEM:Fl2 (1: 1) medium, including the catalogue number of the DMEM:Fl2 (1: 1) medium used for the making of the illustrative example of a medium of the invention (PTT-6) in the Experimental Section;
  • Fig. 4 shows the technical information sheet of Life Technologies Corporation for M 171 medium, including the catalogue number of the M 171 medium used for the making of the illustrative example of a medium of the invention (PTT-6) in the Experimental Section;
  • Fig. 5 shows the list of ingredients, including their commercial supplier and the catalogue number that have been used in the Experimental Section for the making of the medium PTT-6.
  • Fig. 6 shows the results of flow cytometry experiments in which mesenchymal stem cells isolated from the umbilical cord have been analysed for the expression of the mesenchymal stem cell markers CD73, CD90 and CD 105.
  • mesenchymal stem cells were isolated from umbilical cord tissue by cultivation of the umbilical cord tissue in three different cultivation media, followed by subculturing of the mesenchymal stem cells in the respective medium.
  • the three following culture media were used in these experiments: a) 90% (v/v/ DMEM supplemented with 10 % FBS (v/v), b) the culture medium PTT-4 described in US patent application 2006/0078993 and the corresponding International patent application W02006/019357 that consist of 90% (v/v) CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of W02006/019357 and c) the culture medium of the present invention PTT-6 the composition of which is described herein.
  • PTT-6 the composition of which is described herein.
  • Fig. 6a shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD 105 after isolation from umbilical cord tissue and cultivation in DMEM/lO% FBS
  • Fig.6b shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in PTT-4
  • Fig. 6c shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in PTT-6.
  • Fig. 7 shows the results of flow cytometry experiments in which mesenchymal stem cells isolated from the umbilical cord have been analysed for their expression of stem cells markers (CD73, CD90 and CD105, CD34, CD45 and HLA-DR (Human Leukocyte Antigen - antigen D Related) that are used for defining the suitability of multipotent human mesenchymal stem cells for cellular therapy and compared to the expression of these markers by bone marrow mesenchymal stem cells.
  • stem cells markers CD73, CD90 and CD105, CD34, CD45 and HLA-DR (Human Leukocyte Antigen - antigen D Related) that are used for defining the suitability of multipotent human mesenchymal stem cells for cellular therapy and compared to the expression of these markers by bone marrow mesenchymal stem cells.
  • Fig. 7a shows the percentage of isolated mesenchymal cord lining stem cells that express the stem cell markers CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR after isolation from umbilical cord tissue and cultivation in PTT- 6 medium
  • Fig. 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA- DR.
  • FIG. 8 shows photographs obtained from a preclinical study with the mesenchymal stem cell population of the present invention in pigs.
  • the pigs were rendered diabetic with 120 mg/kg streptozotocin and allowed to recover for 45 days prior to creating six 5 cm x 5 cm full thickness wounds on their backs.
  • the two control pigs were treated with PBS. Wounds were photographed on postoperative day 0 (PODay 0) and every seven days until postoperative Day 35. The wounds were analyzed for surface area size by ImageJ.
  • the invention is directed to a method of transporting/storing a stem cell population, the method comprising transporting/storing said stem cell population contacted with a liquid carrier, wherein liquid carrier comprises or is a cell culture medium, in particular a cell culture medium that contains or is DMEM.
  • a liquid carrier such as a culture medium of PTT-6 as described herein provides a high number of viable stem cells and the possibility to isolate a highly homogenous population of mesenchymal stem cells (cf. Fig. 6).
  • cultivation of mesenchymal stem cells as described herein in PTT-6 (cell culture) medium provides for a mesenchymal stem cells population which is essentially 100% pure (with respect to the expression of the positive markers CD73, CD90 and CD105, and lack of expression of the negative markers CD34, CD45 and HLA-DR) and contains 97.5 % viable cells.
  • the so obtained stem cell population meets the criteria that mesenchymal stem cells are to fulfill in order to be used for cell therapy (95% or more cells of the stem cell population express CD73, CD90 and CD105, while, also 95 % or more cells of the stem cell population lack expression of CD34, CD45 and HLA-DR, see for example, Dominici et al, “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement”, supra, or Sensebe et al.’’Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra.
  • mesenchymal stem cells of the amniotic membrane adhere to plastic in standard culture conditions and differentiate in vitro into osteoblasts, adipocytes and chondroblasts, see US patents 9,085,755, US patent 8,287,854 or W02007/046775, for example, and thus meet the further criteria generally accepted for use of mesenchymal stem cells in cellular therapy.
  • the PTT-6 medium comprises different components. As described in the Experimental Section, for making 500 ml PTT6 (culture/growth media) the following ingredients were added:
  • DMEM is the most prominent component of the PTT-6 cell culture medium. It is therefore also clear that stem cells as described herein will also be healthy and viable in a medium/carrier that comprises DMEM or consists of DMEM. It is therefore also clear that cells will remain viable and healthy when kept in a carrier comprising or consisting of only DMEM for transport/storage of cells over a certain period of time.
  • transport any transport is meant. Such transport may be performed with any vehicle, such as car, train, and airplane or by a person carrying/transporting a container comprising the stem cells contacted with the liquid carrier from one place to another place. In one embodiment, transporting is carried out from the place of production of the stem cell population of interest to the place of stem cell administration (for example, the GMP facility in which a stem cell population of interest is produced to the site of administration of the stem cell population, for example, a clinic or a doctor’s office). It is however also envisioned that the term‘transporting relates to a storage of cells at the same place for a period of time. For example, stem cells may be stored after harvest until their application to a subject at one place.
  • the container in which the stem cells can be stored or transported can be any container suitable for the method of the present invention.
  • the transporting/storing can be performed for any period of time.
  • the transporting/storing can be performed for about 7 days or less. It is also envisioned that the transporting/storing can be performed for about 6, 5, 4, 3, 2, 1, day(s) or less. It can thus be that the transporting/storing is performed for about 48 hours or about 24 hours or less.
  • the transporting/storing is performed at any temperature suitable for the method of the present invention. For example, the transporting/storing can be performed at a temperature of about -5°C to about l5°C. It is therefore also envisioned that the transporting/storing can be performed at a temperature of about 2°C to about 8°C.
  • the transporting can also be carried out at a temperature of more than about -5°C, more than about -l0°C, more than about -l5°C , or more than about -20°C. Further it is envisioned that transporting/storing can be performed at a temperature of below 20°C, below 18 °C, below 15 °C, below 12 °C or below 10 °C.
  • the stem cell population is stored or transported in any suitable concentration.
  • the stem cell population can be transported/stored in a concentration of about 70 million cells per 1 ml carrier, of about 60 million cells million cells per 1 ml carrier, of about 50 million cells per 1 ml carrier, of about 40 million cells per 1 ml carrier, of about 30 million cells per 1 ml carrier, of about 20 million cells per 1 ml carrier, of about 10 million cells per 1 ml carrier, of about 5 million cells per 1 ml carrier, of about 4 million cells per 1 ml carrier, of about 3 million cells per 1 ml carrier, of about 2 million cells per 1 ml carrier, of about 1 million cells per 1 ml carrier, of about 0.5 million cells per 1 ml carrier, of about 0.1 million cells per 1 ml carrier or of less than 0.1 million cells per 1 ml carrier. Therefore, the stem cell population can be transported/stored in a concentration of about 10 million cells per ml carrier, of about 60 million cells million cells per 1 ml
  • the method of the present invention also envisions that the stem cell population is stored or transported in any suitable concentration.
  • the stem cell population may, for example, be transported/stored in a concentration of about 70 million cells per 1 ml carrier, of about 60 million cells million cells per 1 ml carrier, of about 50 million cells per 1 ml carrier, of about 40 million cells per 1 ml carrier, of about 30 million cells per 1 ml carrier, of about 20 million cells per 1 ml carrier, of about 10 million cells per 1 ml carrier, of about 5 million cells per 1 ml carrier, of about 4 million cells per 1 ml carrier, of about 3 million cells per 1 ml carrier, of about 2 million cells per 1 ml carrier, of about 1 million cells per 1 ml carrier, of about 0.5 million cells per 1 ml carrier, of about 0.1 million cells per 1 ml carrier or of less than 0.1 million cells per 1 ml carrier.
  • the stem cell population can be transported/stored in a concentration of about 10 million cells per ml carrier to about 1 million cells per 1 ml carrier.
  • the method of the present invention concerns the transporting/storing of stem cells.
  • any stem cell can be used in the method of the present invention.
  • One characterizing feature of stem cells is their ability to self-renew.‘Self-renewal’ is the ability to go through numerous cell cycles of cell division while maintaining the undifferentiated state. Methods for testing if a cell has the capacity to self-renew are known to the skilled artisan. For example, self-renewal may be tested by passaging the cells over more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more passages.
  • Passaging includes splitting of the cell population before re-plating them as a single cell suspension.
  • a further characteristic of stem cells is their multipotency or pluripotency as will also be described elsewhere herein. In principle, multipotency or pluripotency can be tested by differentiating said stem cells into different lineages.
  • the stem cell population used in the method of the present invention can be an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population or an induced pluripotent stem cell population.
  • an“embryonic stem cell population” is a "pluripotent stem cell population”.
  • a pluripotent cell when referred to herein relates to a cell type having the capacity for self-renewal, and the potential of differentiation into different cell types.
  • Pluripotent stem cells can differentiate into nearly all cells, i.e. cells derived from any of the three primary germ layers: ectoderm, endoderm, and mesoderm.
  • the term pluripotent stem cell also encompasses stem cells derived from the inner cell mass of an early stage embryo known as a blastocyst.
  • the pluripotent stem cells can be embryonic stem cells, which have not been obtained via the destruction of a human embryo.
  • the pluripotent stem cells are embryonic stem cells obtained from an embryo, without the destruction of the embryo.
  • an "adult stem cell population” is a multipotent stem cell population.
  • a multipotent stem cell population can give rise a restricted number of cell types, therefore they are somatic fate restricted.
  • a neural stem cell can give rise to both neuronal and glial cells.
  • Adult stem cells have the capability to self-renew and may be obtained from any suitable source.
  • adult stem cells may be obtained from bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelia of the skin and digestive system, cornea, retina, liver, or pancreas.
  • the stem cell population used in the method of the present invention may also be a mesenchymal stem cell population.
  • the culture medium described herein e.g. PTT-6
  • the isolated mesenchymal stem/progenitor cell population has the capacity to differentiate into multiple cell types as described in US patent application 2006/0078993, US patent 9,085,755, International patent application W02006/019357, US patent 8,287,854 or W02007/046775, for instance.
  • the mesenchymal stem cells of the amniotic membrane of the umbilical cord have a spindle shape, express the following genes: POU5fl, Bmi-l, leukemia inhibitory factor (LIF), and secrete Activin A and Follistatin.
  • the mesenchymal stem cells isolated in the present invention can, for example, be differentiated into any type of mesenchymal cell such as, but not limited to, adipocytes, skin fibroblasts, chondrocytes, osteoblasts, tenocytes, ligament fibroblasts, cardiomyocytes, smooth muscle cells, skeletal muscle cells, mucin producing cells, cells derived from endocrine glands such as insulin producing cells (for example, b- islet cells) or neurectodermal cells.
  • the stem cells isolated in accordance with the method described herein can be differentiated in vitro in order to subsequently use the differentiated cell for medical purposes.
  • mesenchymal stem cells are differentiated into insulin producing b-islet cells which can then be administered, for example by implantation, to a patient that suffers from an insulin deficiency such as diabetes mellitus (cf. also W02007/046775 in this respect).
  • the mesenchymal stem cells described herein can be used in their undifferentiated state for cell- based therapy, for example, for wound healing purposes such as treatment of burns or chronic diabetic wounds.
  • the mesenchymal stem cells of the invention can either serve to promote wound healing by interacting with the surrounding diseased tissue or can also differentiate into a respective skin cell (cf., again W02007/046775, for example).
  • the mesenchymal stem cell population described herein can be isolated and cultivated (i.e. are derived) from any umbilical cord tissue as long as the umbilical cord tissue contains the amniotic membrane (which is also referred to as “cord lining”). Accordingly, the mesenchymal stem cell population can be isolated from (pieces of) the entire umbilical cord as described in the Experimental section of the present application. This umbilical cord tissue may thus contain, in addition to the amniotic membrane, any other tissue/component of the umbilical cord.
  • the amniotic membrane of the umbilical cord is the outermost part of the umbilical cord, covering the cord.
  • the umbilical cord contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry deoxygenated, nutrient-depleted blood away from the fetus).
  • Wharton's jelly a gelatinous substance largely of mucopolysaccharides.
  • the umbilical cord tissue used herein can also comprise this one vein, the two arteries and the Wharton's jelly.
  • the use of such an entire (intact) section of the umbilical cord has the advantage that the amniotic membrane does not need to be separated from the other components of the umbilical cord. This reduces the isolation steps and thus makes the method described herein, simpler, faster, less error prone and more economical - which are all important aspects for the GMP production that is necessary for therapeutic application of the mesenchymal stem cells.
  • the isolation of the mesenchymal stem cells can thus start by tissue explant, which may be followed by subsequent subculturing (cultivation) of the isolated mesenchymal stem cells if greater amounts of the mesenchymal stem cells are desired, for example, for use in clinical trials.
  • tissue explant or“tissue explant method” is used in its regular meaning in the art to refer a method in which a tissue, once being harvested, or a piece of the tissue is being placed in a cell culture dish containing culture (growth) medium and by which over time, the stem cells migrate out of the tissue onto the surface of the dish.
  • the primary stem cells can then be further expanded and transferred into fresh dishes through micropropagation (subculturing) as also described here.
  • a master cell bank of the isolated mesenchymal stem cells is obtained, while with the subsequent subculturing, a working cell bank can be obtained.
  • the stem cell population thus is a mesenchymal stem cell population.
  • the mesenchymal stem cell population may be isolated from the amniotic membrane of the umbilical cord by a method comprising cultivating umbilical cord tissue in a culture medium comprising DMEM (Dulbecco’s modified eagle medium), F12 (Ham’s F12 Medium), M171 (Medium 171) and FBS (Fetal Bovine Serum), in particular using the medum PTT-6.
  • DMEM Denbecco’s modified eagle medium
  • F12 Ham’s F12 Medium
  • M171 Medium 171
  • FBS Fetal Bovine Serum
  • FTsing such a medium provides for the isolation of a mesenchymal stem cell population from the amniotic membrane of the umbilical cord of which more than 90 %, or even 99 % or more of the cells are positive for the three mesenchymal stem cell markers CD73, CD90 and CD105 while at the same these stem cells lack expression of CD34, CD45 and HFA-DR (see the Experimental Section), meaning 99 % or even more cells of this population express the stem cell markers CD73, CD90 and CD 105 while not expressing the markers CD34, CD45 and HFA-DR .
  • Such an extremely homogenous and well defined cell population as well as the medium PTT-6 has been reported for the first time in co-pending FTS application Serial No.
  • the present invention allows transporting/storing amounts of stem cells that are needed for therapeutic applications, such as their use in wound healing, in a cost efficient manner.
  • all components used for making the culture medium of the present invention are commercially available in GMP quality. Accordingly, the present invention opens the route to transport/store a GMP produced and highly homogenous mesenchymal stem cell population from the amniotic membrane of the umbilical cord.
  • the mesenchymal stem cell population is an isolated mesenchymal stem population of the amniotic membrane of the umbilical cord. It is further envisioned that at least about 90 % or more cells of the isolated mesenchymal stem cell population express each of the following markers: CD73, CD90 and CD 105. For example, at least about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more cells of the isolated mesenchymal stem cell population express each of CD73, CD90 and CD 105.
  • At least about 90 % or more, about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more of the isolated mesenchymal stem cells lack expression of the following markers: CD34, CD45 and HLA-DR (Human Leukocyte Antigen - antigen D Related).
  • the mesenchymal stem cell population is an isolated mesenchymal stem population of the amniotic membrane of the umbilical cord. It is further envisioned that at least about 90 % or more cells of the isolated mesenchymal stem cell population express each of the following markers: CD73, CD90 and CD105. For example, at least about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more cells of the isolated mesenchymal stem cell population express each of CD73, CD90 and CD 105.
  • the isolated mesenchymal stem cells lack expression of the following markers: CD34, CD45 and HLA-DR (Human Leukocyte Antigen - antigen D Related).
  • CD73 refers to cluster of differentiation 73 also known as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase.
  • the sequence of the human CD73 protein may have the sequence of SEQ ID NO. 1.
  • the marker CD90 is known to the skilled person.
  • CD90 refers to Cluster of Differentiation 90 also known as Thymocyte differentiation antigen 1 (Thy-l).
  • the sequence of the human CD90 protein may have the sequence of SEQ ID NO: 2.
  • the marker CD 105 is known to the skilled person.
  • CD 105 is also known as Endoglin (ENG).
  • the sequence of the human CD 105 protein may have the sequence of SEQ ID NO: 3.
  • a mesenchymal stem cell population of the invention (in particular a population of the mesenchymal stem cells of which at least about 98% or 99 % or express each of the markers CD73, CD90 and CD105 and lack expression of each of the markers: CD34, CD45 and HLA-DR) is used for clinical trials or as an approved therapeutic, a cell population of the working cell bank will typically be used for this purpose.
  • the mesenchymal stem cell population may lack expression of the following markers: CD34, CD45 and HLA-DR.
  • the marker CD34, CD45 and HLA-DR are known to the skilled person.
  • the human CD34 protein may have the sequence of SEQ ID NO. 4.
  • the human CD45 protein may have the sequence of SEQ ID NO: 5.
  • the human HLA-DR protein may have the sequence of SEQ ID NO: 6.
  • Both the stem cell population of the isolation step (which may make up the master cell bank) and the stem cell population of the subculturing step (which may make up the working cell bank) can, for example, be stored in cryo-preserved form.
  • the present method of isolating mesenchymal stem cells from the amniotic membrane of umbilical cord has the advantage that all components used in the culture medium of the invention are available in GMP quality and thus provide the possibility to isolate the mesenchymal stem cells under GMP conditions for subsequent therapeutic administration.
  • the liquid carrier used to transport the stem cell population of interest comprises or is a cell culture medium such as, but not limited to, DMEM or PTT-6 which are, as mentioned above, available in GMP quality.
  • the present invention does not make use of cryo-preserved cells but the stem cell population of interested is transported as viable cells in liquid carrier/cell culture medium (for example, at a temperature of about 2°C to about 8°C), administration of the stem cells according to the present invention is devoid of any freezing and thawing steps, thereby simplifying both the GMP manufacturing and the therapeutic administration.
  • the use of viable cells without intermediate freezing and thawing elimates the risk of damaging the cells and thus affecting their therapeutic efficacy.
  • any cell culture medium can be used as liquid carrier as long as the cell culture medium maintains, at least for a period of time, the viability and functionality such as the wound healing properties of the stem cell population of interest.
  • the term“cell culture medium” or also“culture medium”,“cultivation medium” or“growth medium” is used herein in its regular meaning in the art to refer to a liquid medium that contains all necessary ingredients to grow and maintain mammalian cells such as mesenchymal stem cells under controlled conditions outside their natural environment (cf., for example, Pollard, J.W. and Walker, J.M. (1997) Basic Cell Culture Protocols, Second Edition, Humana Press, Totowa, NJ; Freshney, R.I.
  • DMEM Dulbecco's Modified Eagle Medium
  • DMEM-F12 DMEM-F12
  • RPMI media Dulbecco's Modified Eagle Medium
  • EpiLife medium CMRL1066, or Medium 171 that are described in US patent application 2006/0078993 and the corresponding International patent application W02006/019357 for the cultivation of mesenchymal stem cells of the amniotic membrane of umbilical cord.
  • the media may be supplemented with fetal calf serum (FCS) or fetal bovine serum (FBS) as well as antibiotics, growth factors, amino acids, inhibitors or the like, which is well within the general knowledge of the skilled artisan.
  • FCS fetal calf serum
  • FBS fetal bovine serum
  • the cell culture medium may also comprise DMEM and further components. See in this context, for example, DMEM supplemented with 10 % FBS that has been used in US patent application 2008/0248005 and the corresponding International patent application W02007/046775 for cultivation of mesenchymal stem cells of the amniotic membrane of umbilical cord or the medium PTT-6 that is described and used herein for the isolation and cultivation of the mesenchymal stem cell population of the present invention.
  • a suitable cell culture medium is the culture medium PTT-4 that is also described in US patent application 2008/0248005 and the corresponding International patent application W02007/046775 and that consist of 90% (v/v) CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of W02007/046775, for example).
  • the stem cell population used herein can also be an induced pluripotent stem cell population.
  • “Induced pluripotent stem cells”, as used herein, refer to adult somatic cells that have been genetically reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells.
  • induced pluripotent stem cells can be derived/generated from a non-pluripotent cell.
  • Induced pluripotent stem cells are an important advancement in stem cell research, as they allow obtaining pluripotent stem cells without the use of embryos.
  • Mouse iPSCs were first reported in 2006 (Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”. Cell 126 (4): 663-76), and human iPSCs (hiPSCs) were first reported in 2007 (Takahashi et al. (2007) “Induction of pluripotent stem cells from adult human fibroblasts by defined factors.” Cell; 131(5 ):861-72).
  • Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expression of stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development.
  • Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers.
  • stem cell markers can include Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) as well as stem cell- specific antigen 3 and 4 (SSEA3/4).
  • ALP alkaline phosphatase
  • SSEA3/4 stem cell-specific antigen 3 and 4
  • the chromatin methylation patterns of iPSC are similar to that of embryonic stem cells (Tanabe, Takahashi, Yamanaka (2014)“Induction of pluripotency by defined factors.” Proc. Jpn. Acad., 2014, Ser. B 90).
  • iPSCs are able to self-renew in vitro and differentiate into all three germ layers.
  • the pluripotency or the potential to differentiate into different cell types of iPSC can tested, e.g., by in vitro differentiation into neural or glia cells or the production of germline chimeric animals through blastocyst injection.
  • Methods for the generation of human induced pluripotent stem cells are well known to the skilled person and for example described in W02009115295, W02009144008 or EP2218778.
  • induced pluripotent stem cells may be obtained from any adult somatic cell (of a subject).
  • Exemplary somatic cells include peripheral blood Mononuclear Cells (PBMCs) from blood or fibroblasts obtained from skin tissue biopsies.
  • PBMCs peripheral blood Mononuclear Cells
  • the method of the present invention includes that the stem cell population as described herein is contacted with a liquid carrier wherein the liquid carrier comprises or consists of a cell culture medium. It is envisioned that in the method of the present invention the stem cell population as described herein is contacted with the carrier before transporting/storing. Additionally or alternatively, the stem cell population is contacted with the carrier after its harvest. How harvesting can be performed is described in detail elsewhere herein as well as in the Experimental Section. For example, the stem cell population can be contacted with the carrier about 0 minutes, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 45 minutes, about 60 minutes or a longer time after its harvest.
  • Harvesting can comprise separating the stem cell population from culture medium e.g. from PTT-6. Suitable techniques for such separation are known to the skilled person. For example, separating can be performed by centrifuging the stem cells within a culture medium and decanting the culture medium.
  • the stem cell population is contacted with a liquid carrier, wherein the liquid carrier comprises DMEM.
  • any liquid carrier comprising DMEM can be used in the method of the present invention.
  • the carrier is a liquid carrier.
  • DMEM is contacted with further ingredients to provide for a solution/suspension.
  • the liquid may be any suitable liquid.
  • the liquid can be a culture medium, another buffer, or the like.
  • the liquid carrier comprising DMEM is a cell culture medium.
  • the carrier may be a transport/storage medium or an excipient.
  • a transport/storage medium may be a natural medium, which consists solely of naturally occurring biological fluids, which additionally comprise substances as listed in i)-xiii) as described herein.
  • the medium can also be a medium comprising substances as listed in i)-xiii) as described herein and addition of (further) nutrients (both organic and inorganic), vitamins, salts, 0 2 and C0 2 gas phases, serum proteins, carbohydrates, and/or cofactors.
  • the carrier may also be an excipient.
  • An“excipient” is a substance formulated alongside the active ingredient of a medication. In the present method the active ingredient is the stem cell population.
  • the carrier may further comprise biocompatible scaffolds or microcarriers.
  • the scaffolds or microcarriers can, for example, be biodegradable polymeric substances, most preferably poly(D,L lactic-co-glycolic acid) (PLGA)).
  • the scaffolds or micro carriers may be smooth, macroprorous or microporous structures comprising substances including poly-L-lactide (PLLA), collagen, fibronectin, glycosaminoglycans (GAGs), fibrin, starch, cellulose arabinogalactan (larch gum), alginic acid, agar, carrageenan, chitin, hyaluronic acid, dextran, gellan gum, pullulan, hydroxyapatite, polyhydroxyalkanoates (PHAs), hydrogels or other self-assembling materials such as peptide based nanostructured fibrous scaffolds.
  • PLLA poly-L-lactide
  • GAGs glycosaminoglycans
  • fibrin starch
  • cellulose arabinogalactan larageenan
  • alginic acid alginic acid
  • agar carrageenan
  • chitin chitin
  • hyaluronic acid alginic acid
  • dextran gellan
  • any amount of stem cells can be contacted with any amount of liquid carrier.
  • the contacting can be performed by suspending the stem cell population in a density of about 70 million/ml, of about 60 million/ml, of about 50 million/ml, of about 40 million/ml, of about 30 million/ml, of about 20 million/ml, of about 10 million/ml, of about 5 million/ml, of about 4 million/ml, of about 3 million/ml, of about 2 million/ml, of about 1 million/ml, of about 0.5 million/ml, of about 0.1 million/ml or of less than 0.1 million cells in 1 ml of the carrier.
  • the contacting is performed by suspending the stem cell population in a density of about 10 million/l ml carrier.
  • the stem cells contacted with the carrier can be aliquoted into vials in a volume of about 50 ml, of about 20 ml, of about 10 ml, of about 5 ml, of about 4 ml, of about 3 ml, of about 2 ml, of about 1 ml, of about 0.5 ml, of about 0.25 ml or of less than 0.25 ml carrier.
  • the stem cells that have been contacted with the carrier can be aliquoted into vials in a volume of about 1 ml.
  • the method of the present invention does not comprise a thawing or freezing step. This may include that after their harvest the stem cell population is transported/stored without the need to freeze and thaw the stem cell population.
  • the carrier used in the method of transporting/storing the stem cell population as described herein is particularly suited for this purpose.
  • One advantage of this carrier is that substantially all stem cells transported/stored therein remain viable.
  • A“viable cell” is a cell able to live. The person skilled in the art knows how to detect viable cells.
  • One such method is staining cells with the dye Trypan blue. Viable cells do not stain positive with Trypan blue.
  • At most about 50 %, about 40 %, about 30 %, about 20 %, about 10 % or less than about 10 % of the stem cells of the population may die during transporting/storing compared to the number/amount of viable stem cells before transporting/ storing.
  • the method of the present invention also contemplates that the stem cell population has any cell diameter after transporting/storage.
  • the person skilled in the art knows how to measure the diameter of a cell. For example, cell size/diameter may be determined by capturing a microscope image and using secondary software to measure the diameter of the cell. Most of the stem cells in the stem cell population can therefore have a cell diameter between about 9 pm and about 20 pm after transporting/storage. It is also envisioned that most of the stem cells in the stem cell population have a cell diameter between about 12 pm and about 16 pm after transporting.
  • TGFbeta 1 Transforming growth factor beta, TGFP 1
  • TGFP 1 Transforming growth factor beta
  • VEGF Vascular endothelial growth factor
  • PDGF-AA Plate-derived growth factor subunit AA
  • Ang-l Angiogenin-l
  • HGF Hepatocyte growth factor
  • All of VEGF, PDGF-AA, Ang-l, and/or HGF are known to the skilled person for their involvent in wound healing.
  • VEGF may comprise a sequence as shown in SEQ ID NO. 8
  • PDGF-AA may have a sequence as shown in SEQ ID NO.
  • Ang-l may have a sequence as shown in SEQ ID NO.
  • HGF may have a sequence as shown in SEQ ID NO. 11. Additionally or alternatively, essentially no PDGF-BB and/or IL-10 is detected before and/or after transporting. Both of PDGF-BB (Platelet-derived growth factor subunit BB) and/or IL-10 (interleukin- 10) are also known to the skilled person. PDGF-BB may comprise a sequence as shown in SEQ ID NO. 12 while IL-10 may comprise a sequence as shown in SEQ ID NO: 13.
  • the secretion of these factors can be determined with any suitable method, for example, by measuring the amount of protein (i.e., for example, PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-l, HGF or TGFp 1 ) that the stem cells secrete into the culture medium/carrier.
  • the amount of protein can be measured by commercially available antibodies/immunoassays in an automated fashion, using, for example a system such as the FLEXMAP 3D system (Luminex Corporation, Austin, Texas, USA).
  • FLEXMAP 3D system Luminex Corporation, Austin, Texas, USA.
  • involvement of the proteins Angiopoietin 1 (Ang- 1 ), TGF-b 1 , VEGF, and HGF in the wound healing process is known to the person skilled in the art.
  • HGF Hepatocyte Growth Factor
  • Transforming Growth Factor Beta (including TGF-b!, TGF ⁇ 2, and TGF ⁇ 3) in wound healing, in particular healing of chronic/non-healing wounds see for example, Ramirez et al.“The Role of TGFb Signaling in Wound Epithelialization” Advances In Wound Care, Volume 3, Number 7, 2013, 482-491 or Pakyari et al., Critical Role of Transforming Growth Factor Beta in Different Phases of Wound Healing, Advances In Wound Care, Volume 2, Number 5, 2012, 215-224.
  • the carrier or culture medium may comprise both for the isolation or cultivation or the transport of the mesenchymal cord lining stem cells DMEM in a final concentration of about 55 to 65 % (v/v), F12 in a final concentration of about 5 to 15 % (v/v), M171 in a final concentration of about 15 to 30 % (v/v) and FBS in a final concentration of about 1 to 8 % (v/v).
  • the value (v/v)” as used herein refers to the volume of the indivual component relative to the final volume of the carrier or culture medium. This means, if DMEM is, for example, present in the culture medium a final concentration of about 55 to 65 % (v/v), 1 liter of culture medium contains about 550 to 650 ml DMEM.
  • the carrier or culture medium may comprise DMEM in a final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v) .
  • the culture medium may comprise DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (v/v), M171 in a final concentration of about 23.6 % (v/v) and FBS in a final concentration of about 2.5 % (v/v).
  • the carrier or culture medium may comprise supplements that are advantageous for cultivation of the mesenchymal cord lining stem cells.
  • the carrier or culture medium used in the present invention may, for example, comprise Epidermal Growth Factor (EGF). If present, EGF may be present in the carrier or culture medium in a final concentration of about 1 ng/ml to about 20 ng/ml. In some of these embodiments, the carrier or culture medium may comprise EGF in a final concentration of about lOng/ml.
  • EGF Epidermal Growth Factor
  • the carrier or culture medium may also comprise insulin. If present, insulin may be present in a final concentration of about 1 pg/ml to 10 pg/ml. In some of these embodiments, the carrier or culture medium may comprise Insulin in a final concentration of about 5 pg/ml.
  • the carrier or culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3, 3', 5-Triiodo-L-thyronine sodium salt (T3).
  • the carrier or culture medium may comprise all three of adenine, hydrocortisone, and 3, 3', 5-Triiodo-L-thyronine sodium salt (T3).
  • the carrier or culture medium may comprises may comprise adenine in a final concentration of about 0.05 to about 0.1 pg/ml, hydrocortisone in a final concentration of about 1 to about 10 pg/ml and/or 3, 3', 5-Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • adenine in a final concentration of about 0.05 to about 0.1 pg/ml
  • hydrocortisone in a final concentration of about 1 to about 10 pg/ml and/or 3, 3', 5-Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • T3 5-Triiodo-L-thyronine sodium salt
  • the mesenchymal stem cells are cultured in PTT-6 medium to obtain the highly purified mesenchymal stem cell population described and used herein.
  • PTT-6 medium can also be used as liquid carrier for transporting the mesenchymal stem cell population.
  • PTT-6 carrier/medium as described herein is obtained by mixing to obtain a final volume of 500 ml culture medium: i. 250 ml of DMEM
  • Fetal Bovine Serum FBS
  • Fetal Bovine Serum FBS
  • EGF EGF
  • DMEM Dulbecco’s modified eagle medium which was developed in 1969 and is a modification of basal medium eagle (BME) (cf. Fig.1 showing the data sheet of DMEM available from Lonza).
  • BME basal medium eagle
  • the original DMEM formula contains 1000 mg/L of glucose and was first reported for culturing embryonic mouse cells.
  • DMEM has since then become a standard medium for cell culture that is commercially available from various sources such as ThermoFisher Scientific (catalogue number 11965-084), Sigma Aldrich (catalogue number D5546) or Lonza, to new only a few suppliers.
  • any commercially available DMEM can be used in the present invention.
  • the DMEM used herein is the DMEM medium available from Lonza under catalog number 12-604F. This medium is DMEM supplemented with 4.5 g/L glucose and L- glutamine). In another preferred embodiment the DMEM used herein is the DMEM medium of Sigma Aldrich catalogue number D5546 that contains 1000 mg/L glucose, and sodium bicarbonate but is without L-glutamine.
  • Ham Ham
  • F12 medium is meant Ham’s F12 medium.
  • This medium is also a standard cell culture medium and is a nutrient mixture initially designed to cultivate a wide variety of mammalian and hybridoma cells when used with serum in combination with hormones and transferrin (cf. Fig. 2, showing the data sheet of Ham’s F12 medium from Lonza).
  • Any commercially available Ham’s F12 medium for example, from ThermoFisher Scientific (catalogue number 11765-054), Sigma Aldrich (catalogue number N4888) or Lonza, to new only a few suppliers
  • Ham’s F12 medium from Lonza is used.
  • DMEM/F12 or“DMEM:Fl2” is meant a 1 : 1 mixture of DMEM with Ham’s F12 culture medium (cf. Fig. 3 showing the data sheet for DMEM: F12 (1: 1) medium from Lonza).
  • DMEM/F12 (1: 1) medium is a widely used basal medium for supporting the growth of many different mammalian cells and is commercially available from various supplier such as ThermoFisher Scientific (catalogue number 11330057), Sigma Aldrich (catalogue number D6421) or Lonza. Any commercially available DMEM:Fl2 medium can be used in the present invention.
  • the DMEM:Fl2 medium used herein is the DMEM/F12 (1: 1) medium available from Lonza under catalog number 12-719F (which is DMEM: F12 with L-glutamine, 15 mM HEPES, and 3.151 g/L glucose).
  • M171 is meant culture medium 171, which has been developed as basal medium for the culture of for the growth of normal human mammary epithelial cells (cf. Fig. 4 showing the data sheet for M171 medium from Life Technologies Corporation). Also this basal medium is widely used and is commercially available from suppliers such as ThermoFisher Scientific or Life Technologies Corporation (catalogue number M 171500), for example. Any commercially available M 171 medium can be used in the present invention. In preferred embodiments, the M171 medium used herein is the M171 medium available from Life Technologies Corporation under catalogue number M171500.
  • FBS fetal bovine serum
  • fetal calf serum i.e. the blood fraction that remains after the natural coagulation of blood, followed by centrifugation to remove any remaining red blood cells.
  • Fetal bovine serum is the most widely used serum- supplement for in vitro cell culture of eukaryotic cells because it has very low level of antibodies and contains more growth factors, allowing for versatility in many different cell culture applications.
  • the FBS is preferably obtained from a member of the International Serum Industry Association (ISIA) whose primary focus is the safety and safe use of serum and animal derived products through proper origin traceability, truth in labeling, and appropriate standardization and oversight.
  • ISIA International Serum Industry Association
  • FBS FBS
  • ISIA members include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTD A, GE Healthcare, Gibco by Thermo Fisher Scientific and Life Science Production, to mention only a few.
  • the FBS is obtained from GE Healthcare under catalogue number A15-151.
  • a method of making a culture medium for isolating the mesenchymal stem cell population used in the invention or a carrier used in the present invention comprises mixing to obtain a final volume of 500 ml culture medium:
  • DMEM/F12 medium is a 1:1 mixture of DMEM and Ham’s
  • 118 ml DMEM/F12 medium contain 59 ml DMEM and 59 ml F12. Accordingly, when using this method of making a culture medium, the final concentrations (v/v) with 500 ml total volume are as follows:
  • Embodiments of this method of making a culture medium further comprise adding
  • Insulin 0.175 ml stock solution 14.28 mg/ml to achieve a final insulin concentration of 5 pg/ml.
  • the above-mentioned volumes of these components i. to vi. will result in a final volume of 499.675 ml carrier or culture medium. If no further components are added to the culture medium, the remaining 0.325 ml (to add up to a volume of 500 ml) can, for example, be any of components i. to iv., that means either DMEM, M171, DMEM/F12 or FBS. Alternatively, the concentration of the stock solution of EGF or Insulin can of course be adjusted such that the total volume of the carrier or culture medium is 500 ml. In addition, it is also noted that compoents i. to iv.
  • M171 and DMEM/F12 can be mixed together and then combined with DMEM and FBS to reach final concentrations as described here, i.e. a final concentration of DMEM of about 55 to 65 % (v/v), a final concentration of F12 of about 5 to 15 % (v/v), a final concentration of M171 of about 15 to 30 % (v/v) and a final concentration of FBS of about 1 to 8 % (v/v).
  • the method further comprises adding to DMEM a volume of 0.325 ml of one or more of the following supplements: adenine, hydrocortisone, 3,3 ',5- Triiodo-L-thyronine sodium salt (T3), thereby reaching a total volume of 500 ml carrier or culture medium.
  • the final concentration of these supplements in DMEM may be as follows:
  • 0.05 to 0.1 pg/ml adenine for example about 0.025 pg/ml adenine, about 1 to 10 mg/ml hydrocortisone,
  • T3 3,3',5-Triiodo-L-thyronine sodium salt
  • T3 1,3',5-Triiodo-L-thyronine sodium salt
  • a carrier or cell culture medium used herein is obtainable or that is obtained by the method of making the carrier/ medium as described here.
  • the present invention is also directed to (the use of) a cell culture medium comprising:
  • the medium comprises DMEM in the final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v).
  • the culture medium may comprise DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (v/v), M171 in a final concentration of about 23.6 % (v/v) and FBS in a final concentration of about 2.5 % (v/v).
  • the culture medium or carrier may further comprise Epidermal Growth Factor (EGF) in a final concentration of about 1 ng/ml to about 20 ng/ml.
  • EGF Epidermal Growth Factor
  • the culture medium comprises EGF in a final concentration of about lOng/ml.
  • the culture medium described herein may further comprise Insulin in a final concentration of about 1 pg/ml to 10 pg/ml. In such embodiments the culture medium may comprise Insulin in a final concentration of about 5pg/ml.
  • the cell culture medium or carrier may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3, 3', 5-Triiodo-F-thyronine sodium salt (T3).
  • the culture medium comprises all three of adenine, hydrocortisone, and 3, 3', 5-Triiodo-F-thyronine sodium salt (T3).
  • the culture medium may comprise adenine in a final concentration of about 0.01 to about 0.1 pg/ml or of about 0.05 to about 0.1 pg/ml, hydrocortisone in a final concentration of about 0.1 to about 10 mg/ml or of about 1 to about 10 mg/ml and/or 3,3',5-Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • adenine in a final concentration of about 0.01 to about 0.1 pg/ml or of about 0.05 to about 0.1 pg/ml
  • hydrocortisone in a final concentration of about 0.1 to about 10 mg/ml or of about 1 to about 10 mg/ml
  • T3 3,3',5-Triiodo-L-thyronine sodium salt
  • 500 ml of the cell culture medium of the present invention comprise:
  • Fetal Bovine Serum (FBS) (final concentration of 2.5%)
  • the cell culture medium may further comprise
  • Both, insulin and and EGF can be added to to the culture medium using a stock solution of choice, such that the total volume of the culture medium does not exceed 500 ml.
  • the components i. to vi. of the culture medium used in the present invention or in the carrier are the components indicated in Figure 5, meaning they are obtained from the respective manufacturers using the catalogue number indicated in Figure 5.
  • the medium that is obtained from mixing the components i. to vi. as indicated in Figure 5 is also referred herein as“PTT-6”.
  • PTT-6 the constituents i. to vi. as well as any other ingredient such as an antibiotic of any other commercial supplier can be used in making the medium or carrier used in the invention.
  • This PTT-6 medium can also be used as a carrier as described herein.
  • the cell culture medium or carrier used in the invention may comprise adenine in a final concentration of about 0.01 to about 0.1 pg/ml or of about 0.05 to about 0.1 pg/ml, hydrocortisone in a final concentration of about 0.1 to 10 pg/ml, of about 0.5 to about l0pg/ml, or of about 1 to about 10 pg/ml and/or 3, 3', 5-Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.1 to about 5 ng/ml or of about 0.5 to about 5 ng/ml.
  • adenine in a final concentration of about 0.01 to about 0.1 pg/ml or of about 0.05 to about 0.1 pg/ml
  • hydrocortisone in a final concentration of about 0.1 to 10 pg/ml, of about 0.5 to about l0pg/ml, or of about 1 to about 10 pg/ml and/
  • the umbilical cord tissue may be cultured till a suitable number of (primary) mesenchymal cord lining stem cells have outgrown from the tissue.
  • the umbilical cord tissue is cultivated until cell outgrowth of the mesenchymal stem cells of the amniotic membrane reaches about 70 to about 80% confluency.
  • the term “confluency” or“confluence” is used in its regular meaning in the art of cell culture and is meant as an estimate/indicator of the number of adherent cells in a culture dish or a flask, referring to the proportion of the surface which is covered by cells. For example, 50 percent confluence means roughly half of the surface is covered and there is still room for cells to grow. 100 percent confluence means the surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer.
  • the mesenchymal stem cells are removed from the cultivation container used for the cultivation.
  • a master cell bank containing the (primary) isolated mesenchymal stem cells of the amniotic membrane can be obtained.
  • mesenchymal stem cells are adherent cells, removing is carried out using standard enzymatic treatment.
  • the enzymatic treatment may comprise trypsination as described in International US patent application 2006/0078993, International patent application W02006/019357 or International patent application W02007/046775, meaning outgrowing cells can be harvested by trypsinization (0.125% trypsin/0.05 % EDTA) for further expansion. If the harvested mesenchymal stem cells are, for example, used for generating a master cell bank, the cells can also be cryo-preserved and stored for further use as explained herein below.
  • the mesenchymal stem cells can be transferred to a cultivation container for subculturing.
  • the subculturing can also be started from frozen primary cells, i.e. from the master cell bank.
  • any suitable amount of cells can be seeded in a cultivation container such as cell culture plate.
  • the mesenchymal stem cells can, for this purpose, be suspended in a suitable medium (most conveniently, the culture medium PTT-6) for subculturing at a concentration of, for example, about 0.5 x 10 6 cells/ml to about 5.0 x 10 6 cells/ml. In one embodiment the cells are suspended for subcultivation at a concentration of about 1.0 x 10 6 cells/ml.
  • the subculturing can be carried out by cultivation either in simple culture flasks but also, for example, in a multilayer system such as CellStacks (Corning, Corning, NY, USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, MA, USA) that can be stacked in incubators.
  • the subculturing can also be carried out in a closed self-contained system such as a bioreactor.
  • bioreactors are known to the person skilled in the art, for example, parallel-plate, hollow-fiber, or micro-fluidic bioreactors. See, for example, Sensebe et al.
  • Another example of commercially available bioreactors that can be used for the subculturing of the mesenchymal stem cell population of the present invention is the Xuri Cell Expansion System available from GE Heathcare.
  • the cultivation of the mesenchymal stem cell population in an automated system such as the Quantum® Cell Expansion System is of particular benefit if a working cell bank for therapeutic application is to be produced under GMP conditions and a high number of cells is wanted.
  • the subculturing of the mesenchymal cord ling stem cells described herein takes place in a culture medium described herein such as the PTT-6 medium.
  • the culture medium such as PTT-6 can be used both for the isolation of the mesenchymal stem cells from the amniotic membrane and the subsequent cultivation of the isolated primary cells by subcultivation.
  • the mesenchymal stem cells can be cultured till a suitable number of cells have grown.
  • the mesenchymal stem cells are subcultured till the mesenchymal stem cells reach about 70 to about 80% confluency.
  • the isolation/cultivation of the population of mesenchymal cord lining stem cells can be carried out under standard conditions for the cultivation of mammalian cells.
  • the method of the invention of isolating the population of the mesenchymal cord lining stem cells is typically carried out at conditions (temperature, atmosphere) that are normally used for cultivation of cells of the species of which the cells are derived.
  • conditions temperature, atmosphere
  • human umbilical cord tissue and the mesenchymal cord lining stem cells, respectively, are usually cultivated at 37°C in air atmosphere with 5%C0 2 .
  • mesenchymal cells may be derived of any mammalian species, such as mouse, rat, guinea pig, rabbit, goat, horse, dog, cat, sheep, monkey or human, with mesenchymal stem cells of human origin being preferred in one embodiment.
  • the mesenchymal stem cells can be harvested by removing them from the cultivation container used for the subcultivation.
  • the harvesting of the mesenchymal stem cells is typically again carried out by enzymatic treatment, including trypsination of the cells.
  • the isolated mesenchymal stem cells are subsequently collected and are either directly used or preserved for further use. Typically, preserving is carried out by cryo-preservation.
  • cryo-preservation is used herein in its regular meaning to describe a process where the mesenchymal stem cells are preserved by cooling to low sub zero temperatures, such as (typically) -80°C or -l96°C (the boiling point of liquid nitrogen). Cryo-preservation can be carried out as known to the person skilled in the art and can include the use of cryo-protectors such as dimethylsulfoxide (DMSO) or glycerol, which slow down the formation of ice-crystals in the cells of the umbilical cord.
  • DMSO dimethylsulfoxide
  • glycerol glycerol
  • the isolated population of the mesenchymal cord lining stem cells that is obtained by the isolation method as described herein is highly defined and homogenous. In typical embodiments of the method at least about 90 % or more, about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more of the isolated mesenchymal stem cells express the following markers: CD73, CD90 and CD105.
  • At least about 90 % or more, about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more of the isolated mesenchymal stem cells may lack expression of the following markers: CD34, CD45 and HLA-DR.
  • about 97 % or more, about 98 % or more, or about 99 % or more of the isolated mesenchymal stem cell population express CD73, CD90 and CD105 while lacking expression of CD34, CD45 and HLA-DR.
  • a mesenchymal stem population isolated from the amniotic membrane of the umbilical cord wherein at least about 90 % or more cells of the stem cell population express each of the following markers: CD73, CD90 and CD105.
  • at least about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more cells of the isolated mesenchymal stem cell population are CD73+, CD90+ and CD105+, meaning that this percentage of the isolated cell population express each of CD73, CD90 and CD 105 (cf.
  • At least about 90 % or more, about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more of the isolated mesenchymal stem cells may lack expression of the lack expression of the following markers.
  • about 97 % or more, about 98 % or more, or about 99 % or more of the isolated mesenchymal stem cell population express CD73, CD90 and CD105 while lacking expressing of CD34, CD45 and HLA-DR.
  • mesenchymal stem cells derived from the amniotic membrane of the umbilical cord has been reported for the first time in co-pending US application Serial No. 15/725,913, filed 5 October as well as in co-pending PCT application PCT/SG2017/050500 and meets the criteria for mesenchymal stem cells to be used for cellular therapy (also cf. the Experimental Section and, for example, Sensebe et al.”Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra). It is noted in this context that this mesenchymal stem cell population can be obtained by either the isolating method of the present invention but also by a different method such as cell sorting, if needed.
  • a method of making a culture medium for isolating mesenchymal stem cells or a liquid carrier as described herein or a carrier as used in the present invention can comprise, mixing to obtain a final volume of 500 ml culture medium:
  • Fetal Bovine Serum iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of 2.5% (v/v).
  • DMEM/F 12 medium is a 1 : 1 mixture of DMEM and Ham’ s
  • 118 ml DMEM/F12 medium contain 59 ml DMEM and 59 ml F12. Accordingly, when using this method of making a culture medium, the final concentrations (v/v) with 500 ml total volume are as follows:
  • the present invention also relates to a method of treating a subject having a disease, the method comprising topically administering a mesenchymal stem cell population as described herein to the subject, wherein the mesenchymal stem cell population is administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested.
  • the present invention also relates to mesenchymal stem cell population as described herein for use in a method of treating a disease of a subject, wherein the mesenchymal stem cell population is topically administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested
  • the subject to be treated may be any suitable subject.
  • the subject can be a vertebrate, more preferably a mammal. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, dogs, horses, mice and rats.
  • a mammal can also be a human, dog, cat, cow, pig, mouse, rat etc.
  • the subject is a vertebrate.
  • the subject can also be a human subject.
  • the subject therefore can be a subject in need of treatment.
  • the subject may be afflicted with a disease as described elsewhere herein.
  • the subject is afflicted with Type I or Type II diabetes with chronic foot ulcers.
  • the subject is negative for HLA antibodies to the mesenchymal stem cell population.
  • the mesenchymal stem cell population may be applied in any dosage.
  • the dosage may be therapeutically effective.
  • The“therapeutically effective amount/dosage” can vary with factors including but not limited to the activity of the cells used, stability of the cells in the patient's body, the severity of the conditions to be alleviated, the age and sensitivity of the patient to be treated, adverse events, and the like, as will be apparent to a skilled artisan.
  • the amount of administration can be adjusted as the various factors change over time.
  • the dosage in which the mesenchymal stem cells are applied can also be a unit dosage.
  • the mesenchymal stem cell population can be applied in a unit dosage of about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells.
  • the mesenchymal stem cell population is applied in a unit dosage of about 10 million cells.
  • the mesenchymal stem cells may be applied several times to the same subject. For example, stem cells are applied once, twice, three times or more a week. In principle any unit dosage of mesenchymal stem cells may be applied for the number of times suitable to cure or alleviate the disease. For example, the mesenchymal stem cell population can be applied once, twice, three times or more a week. The mesenchymal stem cell population may also be applied for one, two, three, four, five, six, seven, eight, nine, ten, eleven weeks or more.
  • the unit dosage of about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells is administered once or twice a week.
  • the unit dosage of about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells can also be administered once or twice a week for a period of time of three weeks, of four weeks, or five weeks or of six weeks, or of seven weeks, or of eight weeks or of ten weeks or more weeks.
  • the mesenchymal stem cell population is applied in a dosage of about 1000 cells/cm 2 to about 5 million cells/cm 2 .
  • the expression cm 2 means the area of the wound/skin to which the stem cells are applied.
  • the mesenchymal stem cell population is applied in a dosage of about 100,000 cells/cm 2 , 300,000 cells/cm 2 or 500,000 cells/cm 2 .
  • the mesenchymal stem cell population can also be applied two times a week for about 8 weeks in a dosage of about 100,000 cells/cm 2 , about 300,000 cells/cm 2 or about 500,000 cells/cm 2 .
  • the mesenchymal stem cell population is administered within about 96 hours from the time point where the mesenchymal stem cell population has been harvested. How harvesting can take place is described elsewhere herein. It is also possible that the mesenchymal stem cell population is applied within about 72 hours, about 48 hours, about 24 hours, about 12 hours, about 6 hours or less from the time point where the mesenchymal stem cell population has been harvested. Between the time of harvesting and application, the mesenchymal stem cell population may be transported or stored by the method of transporting/storing of the present invention. Thus, aspects as described for the method of transporting/storing of the present application equally relate to the method of treatment of the present invention mutatis mutandis.
  • the method of treatment of the present invention serves to alleviate a disease suffered by the subject.
  • any disease that may be treated by the mesenchymal stem cell population as described herein is meant here.
  • the disease may be a skin disease or a wound.
  • the wound may be caused by any cause e.g. by a bum, a bite, a trauma, a surgery, or a disease.
  • the wound can also be caused by diabetic disease. Therefore, the wound can also be a diabetic wound.
  • the wound may also be a diabetic foot ulcer.
  • the mesenchymal stem cell population may, for example, be placed directly onto a wound such as a burn or a diabetic wound (see International patent application W02007/046775).
  • the method of treating a subject of the present invention may also comprise the step of separating the mesenchymal stem cell population from the carrier before administering the mesenchymal stem cell population to the subject.
  • the person skilled in the art knows how to perform the separation of cells from a carrier.
  • the separating of the mesenchymal stem cell population from the carrier may comprise centrifugation.
  • separating the mesenchymal stem cell population from the carrier can comprise withdrawing the cell population from the vial by means of syringe.
  • the administering the mesenchymal stem cell population may be performed by means of a syringe. It is however also possible, to contact the mesenchymal stem cells within a cream, ointment, gel, suspension or any other suitable substance before applying the mesenchymal stem cells to the subject.
  • the mesenchymal stem cell population after application to the subject may be held in place e.g. by a dressing such as Tegaderm® dressing and a crepe bandage to cover the Tegaderm® dressing. For a more even distribution of cells the application site may be gently massaged.
  • the present invention also relates to a unit dosage comprising about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells of a mesenchymal stem cell population as described herein.
  • the unit dosage comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, about 0.5, about 0.25, or about 0.1 million cells.
  • the unit dosage comprises about 10 million cells.
  • the unit dosage comprises about 1000 cells to about 5 million cells.
  • the unit dosage can be applied in a dosage of about 100,000 cells, 300,000 cells or 500,000 cells. As described herein the unit dosage may be applied topically. For example, the unit dosage may be applied topically per cm 2 .
  • the unit dosage can be applied once, twice, three times or more a week.
  • the unit dosage can be applied for one, two, three, four, five, six, seven, eight, nine, ten, elven weeks or more.
  • the unit dosage comprising of about 100,000 cells, about 300,000 cells or about 500,000 cells can be applied two times a week for 8 weeks, preferably onto 1 cm 2 .
  • the unit dosage can be contained in any suitable container.
  • the unit dosage can be contained in a 1 ml vial. In such cases, for example 0.1 ml of the vial can be applied onto the subject, preferably per cm 2 .
  • the unit dosage may alternatively be contained in a syringe.
  • the cells can be in contact with a liquid carrier as defined herein. If this is the case then before administration the mesehcnymal stem cells are separated from the carrier before administration.
  • the cells can be centrifuged and isolated before administration to a subject.
  • the carrier may be any carrier as described herein.
  • the carrier may be DMEM, PTT-6, PTT-4, DMEM-F12, RPMI media, EpiLife medium, CMRL1066, or Medium 171.
  • the method of treatment and the unit dosage of of the present invention can comprise utilization of viable cells. How viability can be tested is described elsewhere herein.
  • Umbilical cord tissue (the umbilical cords were donated with informed consent of the mother) was processed for the subsequent isolation of the mesenchymal stem cells from the amniotic membrane of the umbilical cord as follows.
  • the l50mm culture dish may be used in place of the cups.
  • c. Use the cover of the 150 mm culture dish as a resting place for forceps and scalpel.
  • d. Remove 25ml Wash Buffer 1 (Plasmalyte A with 50ug/ml Streptomycin, 50ug/ml Gentamycin, 10 pg/ml Ciprofloxacin) with a 30 ml syringe. Hold the syringe at a 45° angle using one hand and dispense the Wash Buffer 1 directly onto the umbilical cord tissue.
  • f. Hold the umbilical cord securely using the forceps and cut the cord into 1 cm thick pieces using a scalpel.
  • umbilical cord tissue can also be cryopreserved in CryoStor CS5 (a commercial cryoprotectant which contains 5% DMSO available in GMP grade from STEMCELL Technologies Inc.).
  • Preparing media for processing MSCs from umbilical cord tissue a. To make 500 ml PTT6 (culture/growth media) add the following in the order listed: i. DMEM, 250 ml
  • an antibiotic such as Penicillin-Streptomycin- Amphotericin can be added to result in a final volume of 500 ml.
  • Umbilical cord tissue processing should be performed in an environmentally monitored (EM) clean room:. At the end of each shift, full room and hood cleaning are performed b. Prepare/clean the biosafety cabinet.
  • EM environmentally monitored
  • the average number of mesenchymal stem cells harvested from an explant is typically about 4,000 - 6,000 cells/explant. Accordingly, when the mesenchymal stem cells are simultaneously grown out of 48 explants about 300,000 cells can be obtained at harvest. These 300,000 mesenchymal stem cells collected from explants can then be used for subculturing by seeding a l75cm 2 cell culture flask with such 300,000 cells as described in the following Example 2.5 (this can be referred to as Passage 1).
  • the mesenchymal stem cells obtained from this passage 1 can then be used to seed again l75cm 2 flasks (Passage 2) and expand the cells as described in the following Example 2.5.
  • the cells obtained from both Passage 1 and Passage 2 can be “banked” by cryo-preservation, with the mesenchymal stem cells obtained after Passage 2 being considered to represent the Master Cell Bank which will be for further expansion of the mesenchymal stem cells, for example, in a bioreactor as explained below in Example 2.7.
  • Count cells using a hemocytometer Expect to count 20-100 cells/square. If the count higher than 100, dilute the original sample 1:5 and repeat Trypan Blue method using a hemocytometer.
  • Viable cells/ml viable cell count x dilution factor x 10 4
  • Total viable cells viable cell count x dilution factor x total volume x 10 4
  • % viability viable cell count x 100 /(viable cell count + dead cell count)
  • total viable cell number is 1.0 x 10 7 ;
  • cell suspension is less than l06/ml, determine the volume required to seed 2 x 106 cells for each 150 mm petri dish or 175 cm2 flask.
  • viable cells/ml is 8 x 10 5 cells/ml
  • viii Seed 2 x 10 6 cells to each 150 mm petri dish or 175 cm 2 flask with 30 ml PTT6. ix. Observe cells for attachment, colony formation, and confluence every three days. When cells reach 40-50% confluence, observe cells every one-two days to prevent over-expansion. DO NOT allow cells to expand beyond 80% confluence. A real time cell culturing monitoring system can be used in place of the light microscope.
  • Quantum Bioreactor can be used to expand the MSC.
  • the starting cell number for the expansion in the Quantum Bioreactor should range between 20 to 30 million cells per run.
  • the typical yield per run is 300 to 700 million MSC at harvest.
  • the Bioreactor is operated following the protocol of the manufacturer.
  • the so obtained mesenchymal stem cells are typically cryo-preserved (see below) and serve as Working Cell Bank.
  • mesenchymal stem cells were isolated from umbilical cord tissue by cultivation of the umbilical cord tissue in three different cultivation media, followed by subculturing of the mesenchymal stem cells in the respective medium as set forth in Example 2.
  • Fig. 6a shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in DMEM/10% FBS
  • Fig. 6b shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD 105 after isolation from umbilical cord tissue and cultivation in PTT-4
  • Fig. 6c shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD 105 after isolation from umbilical cord tissue and cultivation in PTT-6.
  • Fig. 6a shows the percentage of isolated mesenchymal cord lining stem cells expressing stem cell markers CD73, CD90 and CD105 after isolation from umbilical cord tissue and cultivation in PTT-6.
  • the population isolated using DMEM/10 % FBS as culture medium cultivation has about 75% CD73+ cells, 78 % 90+ cells and 80 % CD105+ cells (average of two experiments), while after isolation/cultivation of umbilical cord tissue using PTT-4 culture medium (see Fig. 6b) the number of mesenchymal stem cells that are CD73-positive, CD90-positive and CDl05-positive are about 87 % (CD73+ cells), 93 % /CD90+ cells) and 86 % (CD 105+ cells) average of two experiments.
  • the purity of the mesenchymal stem cell population that was obtained by means of cultivation in the PTT-6 medium of the present invention is at least 99.0 % with respect to all three markers (CD73, CD90, CD105), meaning the purity of this cell population is significant higher than for cultivation using PTT-4 medium or DMEM/10 % FBS.
  • the mesenchymal stem cell population obtained by means of cultivation in PTT-6 is essentially a 100% pure and defined stem cell population. This makes the stem cell population of the present invention the ideal candidate for stem cell-based therapies. Thus, this population of mesenchymal cord lining stem cells may become the gold standard for such stem cell based therapeutic approaches.
  • Fig. 7a shows the percentage of isolated mesenchymal cord lining stem cells (mesenchymal stem cells of the amniotic membrane of umbilical cord) that express the stem cell markers CD73, CD90 and CD105 and lack expression of CD34, CD45 and HFA-DR after isolation from umbilical cord tissue and cultivation in PTT-6 medium.
  • stem cells mesenchymal stem cells of the amniotic membrane of umbilical cord
  • the mesenchymal stem cell population contained 97.5 % viable cells of which 100 % expressed each of CD73, CD90 and CD105 (see the rows“CD73+CD90+” and“CD73+CD105+”) while 99.2 % of the stem cell population did not express CD45 and 100 % of the stem cell population did not express CD34 and HFA-DR (see the rows “CD34-CD45- and“CD34-HFA-DR-).
  • the mesenchymal stem cells population obtained by cultivation in PTT-6 medium is essentially a 100% pure and defined stem cell population that meets the criteria that mesenchymal stem cells are to fulfill to be used for cell therapy (95% or more of the stem cell population express CD73, CD90 and CD105, while 98 % or more of the stem cell population lack expression of CD34, CD45 and HFA-DR, see Sensebe et al.’’Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra).
  • mesenchymal stem cells of the amniotic membrane are adhere to plastic in standard culture conditions and differentiate in vitro into osteoblasts, adipocytes and chondroblasts, see US patents 9,085,755, US patent 8,287,854 or W02007/046775 and thus meet the criteria generally accepted for use of mesenchymal stem cells in cellular therapy.
  • Fig. 7b shows the percentage of isolated bone marrow mesenchymal stem cells that express CD73, CD90 and CD105 and lack expression of CD34, CD45 and HLA-DR.
  • the bone marrow mesenchymal stem cell population contained 94.3 % viable cells of which 100 % expressed each of CD73, CD90 and CD105 (see the rows “CD73+CD90+” and“CD73+CD105+”) while only 62.8 % of the bone marrow stem cell population lacked expression of CD45 and 99.9 % of the stem cell population lacked expression CD34 and HLA-DR (see the rows“CD34-CD45- and“CD34-HLA-DR-).
  • the bone marrow mesenchymal stem cells that are considered to be gold standard of mesenchymal stem cells are by far less homogenous/pure in terms of stem cell marker than the mesenchymal stem cells population (of the amniotic membrane of the umbilical cord) of the present application.
  • This finding also shows that the stem cell population of the present invention may be the ideal candidate for stem cell based therapies and may become the gold standard for stem cell based therapeutic approaches.
  • the mesenchymal stem cell population grown in PTT-6 medium was also analysed for its secretion of tgrowth factors that are known to have a positive influence on wound healing.
  • concentration of PDGF-AA, PDGF-BB, VEGF, IL-10, Ang-l, HGF and TGFP 1 were measured in supernatant from the mesenchymal stem cell population using the respective Magnetic Luminex Performance Assay Kit by a FLEXMAP 3D system (Luminex Corporation). This experiment showed that the mesenchymal stem cells secrete high levels of PDGF-AA, VEGF, Ang-l, HGF and TGFP 1 while no secretion of PDGF-BB and IL-10 was detected (data not shown).
  • Mesenchymal stem cell population as described herein can be transported/stored in a carrier comprising or consisting of a cell culture medium
  • a liquid carrier such as the culture medium PTT-6 described herein (or a DMEM containing culture medium in general) provides a high number of viable cstem cells and the possibility to isolate a highly homogenous population of mesenchymal stem cells (Fig. 6).
  • cultivation of mesenchymal stem cells as described herein in PTT-6 medium provides for a mesenchymal stem cells population which is essentially 100% pure.
  • a so obtained stem cell population meets the criteria that mesenchymal stem cells are to fulfill to be used for cell therapy (95% or more of the stem cell population express CD73, CD90 and CD 105, while 98 % or more of the stem cell population lack expression of CD34, CD45 and HLA-DR, see Sensebe et al.’’Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra).
  • mesenchymal stem cells of the amniotic membrane adhere to plastic in standard culture conditions and differentiate in vitro into osteoblasts, adipocytes and chondroblasts, see US patents 9,085,755, US patent 8,287,854 or W02007/046775 and thus meet the criteria generally accepted for use of mesenchymal stem cells in cellular therapy.
  • this stem cell population secretes high levels of PDGF- AA, VEGF, Ang- 1 , HGF and TGFP 1 , i.e. of proteins that are associated in wound healing and tissue regeneration.
  • a mesenchymal stem cell population obtained by culturing the cells in PTT-6 cell culture medium is hightly suitable for therapy, in particular for wound healing.
  • Fig. 7a confirms that the mesenchymal stem cell population as described herein and isolated via PTT-6 medium contained 97.5 % viable cells. It can therefore be safely assumed that stem cells will also remain healthy and viable, when kept in a carrier such as PTT-6 for transport/storage of cells over a certain period of time.
  • the PTT-6 medium comprises different components. As described in the Experimental Section above, for making 500 ml PTT6 (culture/growth media) the following ingredients were added:
  • DMEM Insulin 0.175 ml (final concentration of 5pg/ml)
  • DMEM is the most prominent component of the PTT-6 cell culture medium. It is therefore clear that stem cells as described herein will also be healthy and viable in a medium/carrier comprising or consisting of DMEM. It is therefore also clear that cells will remain viable and healthy when kept in a carrier comprising only DMEM for transport/storage of cells over a certain period of time.
  • the pig model is not spontaneous, but the skin architecture most closely resembles humans.
  • the data suggest that umbilical cord lining mesenchymal stem cell population of the present invention will improve wound healing without the risk of serious adverse side effects.
  • These data thus strongly support the hypothesis that human umbilical cord lining mesenchymal stem cell population as described herein can promote chronic wound healing by suppressing inflammation and promoting angiogenesis.
  • mesenchymal stem cells of the amniotic membrane of the umbilical cord (UCMC) could alleviate full thickness bums (Example 23), partial-thickness wounds (Example 24), non-healing radiation wound (Example 25) as well as non-healing diabetic wound and non-healing diabetic foot wounds (Example 26).
  • mesenchymal stem cells were resuspended in PTT-4 medium.
  • the stem cell population obtained by cultivation when using PTT6 (as used herein) cultivation medium is significantly more homogenous than the population of cells obtained by using PTT4 medium (used in WO 2007/046775). Since PTT-4 was used as medium for mesenchymal stem cells in Examples 23-26 of WO 2007/046775 it is clear that the even more homogenous mesenchymal stem cell population isolated after cultivation in PTT-6 (as used herein) will have the same beneficial effects in wound healing applications, such as full thickness bums, partial-thickness wounds, non-healing radiation wound as well as non-healing diabetic wound and non-healing diabetic foot wounds.
  • wound healing applications such as full thickness bums, partial-thickness wounds, non-healing radiation wound as well as non-healing diabetic wound and non-healing diabetic foot wounds.
  • the third group of 5 patients will receive 500,000 MSC/cm 2 twice per week for 8 weeks. This schedule will continue until either the highest dose is reached, or until at least 2 subjects at a dose level have > Grade 2 allergic reaction that is suspected to be related to mesenchymal stem cell population as obtained herein or 2 or more subjects at a dose level experience an unexpected, treatment-related serious adverse event or dose limiting toxicity within 14 days following the initial dose of mesenchymal stem cell population as obtained as described herein. All of the patients will be evaluated 30 days posttreatment for the production of anti-HLA antibodies and for wound closure. At the present time, we do not consider production of HLA antibodies to be an absolute contraindication to a particular dose, but it will factor into our overall assessment of safety.
  • Subject Population Patients with Type I or Type II diabetes with chronic foot ulcers that have not healed after at least 30 days of conventional therapy and are negative for HLA antibodies to the mesenchymal stem cell population as described herein. Patients will continue with conventional wound treatment for the first 2 weeks commencing at the time of enrollment, at which time they will have already been screened for having a diabetic foot ulcer that has not healed in 30 days. Photodocumentation and measurement of wound parameters will start at this time. Conventional dressing changes will be performed twice a week for the first 2 weeks, after which mesenchymal stem cell population as described herein will be applied to the wound at the specified concentrations twice a week. The mesenchymal stem cell population as described herein -treated wounds will also be covered with Tegaderm® and a crepe dressing.
  • Dose Levels The goal of this study is to determine a safe dose of human umbilical cord lining mesenchymal stem cells as described herein for further study. Patients will be treated with one of three doses: 100,000 cells/cm 2 skin/wound area, 300,000 cells/cm 2 or 500,000 cells/cm 2 twice a week for 8 weeks. Each 100,000 cell dose represents 0.1 ml of the mesenchymal stem cell population as described herein from a vial containing 1 million cells/ml in HypoThermosol (however the use of PTT-6 or DMEM as carrier is also contemplated in this study).
  • Dosing Regimen This is a safety and tolerability study of escalating doses of mesenchymal stem cells as described herein. The goal of this study is to determine a safe dose of the human umbilical cord lining mesenchymal stem cells as described herein for further study. Each of three dose levels will enroll five subjects. The first group of 5 patients will receive 100,000 MSC/cm 2 skin/wound area twice per week for 8 weeks. The second group of 5 patients will receive 300,000 MSC/cm 2 twice per week for 8 weeks. The third group of 5 patients will receive 500,000 MSC/cm 2 twice per week for 8 weeks.
  • This schedule will continue until either the highest dose is reached, or until at least 2 subjects at a dose level have > Grade 2 allergic reaction that is suspected to be related to mesenchymal stem cells as described herein or 2 or more subjects at a dose level experience an unexpected, treatment- related serious adverse event or dose limiting toxicity within 30 days following the initial dose of a mesenchymal stem cell population as described herein. All of the patients will be evaluated 30 days posttreatment for the production of anti-HLA antibodies and for degree of wound closure. At the present time, we do not consider production of HLA antibodies to be an absolute contraindication to a particular dose, but it will factor into our overall assessment of safety. This is an open-label study where all subjects will be taking the study drug and all study personnel will know the dose each subject receives.
  • the mesenchymal stem cell population as described herein as described herein are applied topically to debrided diabetic foot ulcers and held in place by a Tegaderm® bandage.
  • the invention is further characterized by the following items.
  • a method of transporting a stem cell population comprising transporting said stem cell population contacted with a liquid carrier, said liquid carrier comprises a cell culture medium.
  • the stem cell population is transported in a concentration of about 70 million cells per 1 ml carrier, of about 60 million cells million cells per 1 ml carrier, of about 50 million cells per 1 ml carrier, of about 40 million cells per 1 ml carrier, of about 30 million cells per 1 ml carrier, of about 20 million cells per 1 ml carrier, of about 10 million cells per 1 ml carrier, of about 5 million cells per 1 ml carrier, of about 4 million cells per 1 ml carrier, of about 3 million cells per 1 ml carrier, of about 2 million cells per 1 ml carrier, of about 1 million cells per 1 ml carrier, of about 0.5 million cells per 1 ml carrier, of about 0.1 million cells per 1 ml carrier or of less than 0.1 million cells per 1 ml carrier.
  • the stem cell population is an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population or an induced pluripotent stem cell population.
  • the stem cell population is a mesenchymal stem cell population.
  • the mesenchymal stem cell population is an isolated mesenchymal stem population of the amniotic membrane of the umbilical cord.
  • separating is performed by centrifuging the stem cells within a culture medium and decanting the culture medium.
  • 21 The method of any one of the foregoing items, wherein the contacting is performed by suspending the stem cell population in a density of about 70 million/ml, of about 60 million/ml, of about 50 million/ml, of about 40 million/ml, of about 30 million/ml, of about 20 million/ml, of about 10 million/ml, of about 5 million/ml, of about 4 million/ml, of about 3 million/ml, of about 2 million/ml, of about 1 million/ml, of about 0.5 million/ml, of about 0.1 million/ml or of less than 0.1 million cells in the carrier.
  • the cell culture medium is selected from the group of Dulbecco's Modified Eagle Medium (DMEM), DMEM-F12, RPMI media, EpiLife medium, and Medium 171.
  • DMEM Dulbecco's Modified Eagle Medium
  • DMEM-F12 DMEM-F12
  • RPMI media RPMI media
  • EpiLife medium RPMI media
  • Medium 171 Dulbecco's Modified Eagle Medium
  • the carrier/cell culture medium comprises DMEM in the final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v).
  • the carrier/ cell culture medium comprises DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (n/n), M171 in a final concentration of about 23.6 % (v/v) and FBS in a final concentration of about 2.5 % (v/v).
  • the carrier/ cell culture medium further comprises Epidermal Growth Factor (EGF) in a final concentration of about 1 ng/ml to about 20 ng/ml.
  • EGF Epidermal Growth Factor
  • the carrier further comprises at least one of the following supplements: adenine, hydrocortisone, and 3,3',5-Triiodo-L- thyronine sodium salt (T3).
  • the carrier/cell culture medium comprises adenine in a final concentration of about 0.05 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 1 to about 10 pg/ml hydrocortisone and/or 3,3',5-Triiodo-L- thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • T3 3,3',5-Triiodo-L- thyronine sodium salt
  • 500 ml of the carrier/cell culture medium comprise: i. 250 ml of DMEM ii. 118 ml M171 iii. 118 ml DMEM/F12 iv. 12.5 ml Fetal Bovine Serum (FBS) (final concentration of 2.5%)
  • the carrier/cell culture medium further comprises v. EGF in a final concentration of lOng/ml vi. Insulin in a final concentration of 5pg/ml. vi. Insulin 0.175 ml (final concentration of 5pg/ml).
  • the carrier/ cell culture medium further comprises adenine in a final concentration of about 0.05 to about 0.1 pg/ml adenine, hydrocortisone in a final concentration of about 1 to about 10 pg/ml hydrocortisone and/or 3, 3', 5- Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • adenine in a final concentration of about 0.05 to about 0.1 pg/ml adenine
  • hydrocortisone in a final concentration of about 1 to about 10 pg/ml hydrocortisone and/or 3, 3', 5- Triiodo-L-thyronine sodium salt (T3) in a final concentration of about 0.5 to about 5 ng/ml.
  • T3 5- Triiodo-L-thyronine sodium salt
  • a method of treating a subject having a disease comprising topically administering a mesenchymal stem cell population as defined in any one of items 12-15 to the subject, wherein the mesenchymal stem cell population is administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested.
  • separating the mesenchymal stem cell population from the carrier comprises withdrawing the cell population from the vial by means of syringe.
  • a unit dosage comprising about 20 million cells, of about 15 million cells, of about 10 million cells, of about 5 million cells, of about 4 million cells, of about 3 million cells, of about 2 million cells, of about 1 million cells, of about 0.5 million cells, of about 0.25 million cells or of less than 0.25 million cells of a mesenchymal stem cell population as defined in any one of items 12-15.
  • the unit dosage of item 69, wherein the unit dosage comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, about 0.5, about 0.25, or about 0.1 million cells.
  • the unit dosage of item 69 or 70, wherein the unit dosage comprises about 10 million cells.
  • the unit dosage of any one of items 69 to 71, wherein the unit dosage comprises of about 100,000 cells, about 300,000 cells or about 500,000 cells of any one of items is applied two times a week for 8 weeks.
  • stem cell population is transported in a concentration of about 10 million cells per ml carrier to about 1 million cells per 1 ml carrier.
  • the stem cell population is an embryonic stem cell population, an adult stem cell population, a mesenchymal stem cell population or an induced pluripotent stem cell population.
  • mesenchymal stem cell population is an isolated mesenchymal stem population of the amniotic membrane of the umbilical cord.
  • any one of items 95 to 97 wherein at least about 90 % or more, about 91 % or more, about 92 % or more, about 93 % or more, about 94 % or more, about 95 % or more, about 96 % or more, about 97 % or more, about 98 % or more about 99 % or more of the isolated mesenchymal stem cells lack expression of the following markers: CD34, CD45 and HLA-DR (Human Leukocyte Antigen - antigen D Related).
  • DMEM Dulbecco's Modified Eagle Medium
  • DMEM-F12 DMEM-F12
  • RPMI media RPMI media
  • EpiLife medium fetal calf serum
  • the carrier/cell culture medium comprises DMEM in the final concentration of about 57.5 to 62.5 % (v/v), F12 in a final concentration of about 7.5 to 12.5 % (v/v), M171 in a final concentration of about 17.5 to 25.0 % (v/v) and FBS in a final concentration of about 1.75 to 3.5 % (v/v).
  • the carrier/ cell culture medium comprises DMEM in a final concentration of about 61.8 % (v/v), F12 in a final concentration of about 11.8 % (n/n), M171 in a final concentration of about 23.6 % (v/v) and FBS in a final concentration of about 2.5 % (v/v).
  • the term "about” is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value.
  • a formulation comprises about 5 mg/ml of a compound
  • this is understood to mean that a formulation can have between 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml.
  • an interval which is defined as“(from) X to Y” equates with an interval which is defined as“between X and Y”. Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of“5 mg/ml to 10 mg/ml” or“between 5 mg/ml and 10 mg/ml” includes a concentration of 5, 6, 7, 8, 9, and 10 mg/ml as well as any given intermediate value.

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Abstract

La présente invention concerne un procédé de transport d'une population de cellules souches, le procédé comprenant le transport de la population de cellules souches en contact avec un support liquide. Dans le mode de réalisation préféré, le support comprend du DMEM, du F12, du M171 et du FBS. De plus, la présente invention concerne un procédé de traitement d'un sujet souffrant d'une maladie telle qu'une plaie, le procédé comprenant l'administration topique d'une population de cellules souches mésenchymateuses définie au sujet, la population de cellules souches mésenchymateuses étant administrée pendant environ 96 heures à partir du moment où la population de cellules souches mésenchymateuses a été récoltée. L'invention concerne également un dosage unitaire comprenant la population de cellules souches mésenchymateuses définie provenant, par exemple, de la membrane amniotique du cordon ombilical, au moins 90 % des cellules exprimant chacun des marqueurs suivants : CD73, CD90 et CD105.
PCT/SG2019/050198 2018-04-09 2019-04-09 Procédé de transport de cellules souches mésenchymateuses au moyen d'un milieu de culture cellulaire et procédé d'administration de cellules souches à des plaies WO2019199230A1 (fr)

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WO2022019832A1 (fr) * 2020-07-20 2022-01-27 Cellresearch Corporation Pte Ltd Procédé de génération d'une cellule souche pluripotente induite, cellule souche pluripotente induite et procédés d'utilisation de la cellule souche pluripotente induite
EP3775163A4 (fr) * 2018-04-12 2022-03-09 CellResearch Corporation Pte. Ltd. Procédé d'induction ou d'amélioration des propriétés de cicatrisation de cellules souches mésenchymateuses

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3775163A4 (fr) * 2018-04-12 2022-03-09 CellResearch Corporation Pte. Ltd. Procédé d'induction ou d'amélioration des propriétés de cicatrisation de cellules souches mésenchymateuses
WO2022019832A1 (fr) * 2020-07-20 2022-01-27 Cellresearch Corporation Pte Ltd Procédé de génération d'une cellule souche pluripotente induite, cellule souche pluripotente induite et procédés d'utilisation de la cellule souche pluripotente induite

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