Patent Application
20210102171
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 8, 2020, is named SCH-5500-UT_SeqListing.txt and is 58 kilobytes in size.
The present invention relates to a mesenchymal stem cell storing or transport formulation, a method of preparing the mesenchymal stem cell storing or transport formulation as well as to methods of using the mesenchymal stem cell storing or transport formulation. Such methods include a method of transporting mesenchymal stem cells in this storing or transport formulation as well as a method of treating a subject having a disease, the method comprising topically administering mesenchymal stem cells that have been stored or transported in this storing or transport formulation. Also concerned is a unit dosage of the mesenchymal stem cells.
Mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord and their wound healing properties have been first reported in US patent application 2006/0078993 (leading to granted U.S. Pat. Nos. 9,085,755, 9,737,568 and 9,844,571) and the corresponding International patent application WO2006/019357. Since then, the umbilical cord tissue has gained attention as a source of multipotent cells; due to its widespread availability, the umbilical cord and in particular stem cells isolated from the amniotic membrane of the umbilical cord (also referred to as “cord lining stem cells”) have been considered as an excellent alternative source of cells for regenerative medicine. See, Jeschke et al. Umbilical Cord Lining Membrane and Wharton's Jelly-Derived Mesenchymal Stem Cells: the Similarities and Differences; The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 21-27. In the meantime, a population of such mesenchymal stem cells from the amniotic membrane of the umbilical cord has been described in US application 20181/27721 or the corresponding International Application WO 2018/067071.
The mesenchymal stem cell population described in the US application 2018/127721 or the corresponding International Application WO 2018/067071 has the advantage that 99% or more of the stem cells of this population express the three MSC markers CD73, CD90 and CD 105 while lacking expression of CD34, CD45 and HLA-DR. This extremely homogenous and well-defined cell population is thus an ideal candidate for clinical trials and cell based therapies as it, for example, fully meets the criteria generally accepted for human MSCs to be used for cellular therapy as defined, for example, by Dominici et al, “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement”, Cytotherapy (2006) Vol. 8, No. 4, 315-317, Sensebe et al., “Production of mesenchymal stromal/stem cells according to good manufacturing practices: a, review”, Stem Cell Research & Therapy 2013, 4:66), Vonk et al., Stem Cell Research & Therapy (2015) 6:94, or Kundrotas Acta Medica Lituanica. 2012. Vol. 19. No. 2. P. 75-79. As described in International Application WO 2018/067071, this mesenchymal stem cell population may, for example, be used in its undifferentiated state for wound healing purposes such as treatment of burns.
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 storage or transport formulations which keep cells viable and healthy for a period of time typically used for transport or storage of cells.
Accordingly, it is an object of the invention to provide a formulation suitable for storing and/or transporting of mesenchymal stem cells, that meets this need.
This object is accomplished by the methods, mesenchymal stem cell storing or transport formulation and the unit dosage having the features of the independent claims.
In a first aspect, the invention provides a method of preparing a mesenchymal stem cell storing or transport formulation, wherein the formulation comprises about 0.5 to about 10 million mesenchymal stem cells, the method comprising
i) Trolox;
ii) Na+;
iii) K+;
iv) Ca2+;
v) Mg2+;
vi) Cl−;
vii) H2PO4;
viii) HEPES;
ix) Lactobionate;
x) Sucrose;
xi) Mannitol;
xii) Glucose;
xiii) Dextran-40;
xiv) Adenosine, and
xv) Glutathione, thereby obtaining the mesenchymal stem cell storing or transport formulation comprising about 0.5 to about 10 million mesenchymal stem cells.
In a second aspect, the invention provides a mesenchymal stem cell storing or transport formulation obtained by a method as defined herein.
In a third aspect, the invention provides a mesenchymal stem cell storing or transport formulation obtainable by a method as defined herein.
In a fourth aspect, the invention provides a method of transporting mesenchymal stem cells, the method comprising transporting said mesenchymal stem cells in a mesenchymal stem cell storing or transport formulation as defined herein.
In a fifth aspect, the invention provides method of treating a subject having a disease, the method comprising topically administering mesenchymal stem cells that have been stored or transported in a mesenchymal stem cell storing or transport formulation as defined herein.
In a sixth aspect, the invention provides a unit dosage of mesenchymal stem cells obtainable by a method as defined herein.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings, in which:
As explained above, in a first aspect the invention is directed to a method of preparing a mesenchymal stem cell storing or transport formulation, wherein the formulation comprises about 0.5 to about 10 million mesenchymal stem cells, the method comprising
i) Trolox;
ii) Na+;
iii) K+;
iv) Ca2+;
v) Mg2+;
vi) Cl−;
vii) H2PO4;
viii) HEPES;
ix) Lactobionate;
x) Sucrose;
xi) Mannitol;
xii) Glucose;
xiii) Dextran-40;
xiv) Adenosine, and
xv) Glutathione.
thereby obtaining the mesenchymal stem cell storing or transport formulation comprising about 0.5 to about 10 million mesenchymal stem cells.
It has been surprisingly found in the present application that using a mesenchymal stem cell storing or transport formulation as described herein stabilizes the proliferation and metabolism of MSC during storage/transportation leading to an improved viability of MSC for up to 72 h. For example, after 3 days of storage of mesenchymal stem cells in the mesenchymal stem cell storing or transport formulation of the present invention about 90% of the cells were still viable (cf.
When used herein the term ‘transport’ or ‘transporting’ 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 mesenchymal stem cells (or the mesenchymal stem cell population as both terms are used herein interchangeably) of interest to the place of stem cell administration (for example, the GMP facility in which stem cells, respectively a stem cell population of interest is produced to the site of administration of the stem cells or 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 preparation of the mesenchymal stem cell storing or transport formulation comprises resuspending the MSCs in a pre-defined volume of the crystalloid solution. In the present invention, any volume of the crystalloid solution suitable to sufficiently resuspending MSCs can be used as the pre-defined volume. For example, the pre-defined volume may be in a range of about 0.5 ml to about 15 ml. In one example, the pre-defined volume may be in a range of about 1 ml to about 10 ml. In an illustrative example, the pre-defined volume of the crystalloid solution may be about 1 ml, about 2 ml, about 3 ml, about 4 ml or about 5 ml. By resuspending MSCs in the pre-defined volume of the crystalloid solution, a first cell suspension is generated. The resuspending is usually carried out after the mesenchymal stem cells/the mesenchymal stem cell population has been harvested after being cultivated for being pharmaceutically administered.
After determining the concentration of the MSCs in the first cell suspension and determining the volume of the first cell suspension needed to prepare a formulation comprising about 0.5 to about 10 million mesenchymal stem cells, the first cell suspension is mixed with a volume of a liquid carrier. The volume of the first cell suspension mixed with the liquid carrier may be abou 0.5 ml to about 10 ml. In an illustrative example, the determined volume of the first cell suspension with the volume of the liquid carrier, the total volume of the mesenchymal stem cell storing or transport formulation is about 1 ml. An amount of 0.5 to about 10 million mesenchymal stem cells is chosen to prepare a unit dosage that contain 0.5 to about 10 million mesenchymal stem cells preferably in a pre-defined volume such as 1 ml, 2 ml, etc. In the present invention, the pre-defined volume of the crystalloid solution comprises about 0.1 to about 15 million viable MSCs. In one example, the pre-defined volume of the crystalloid solution comprises about 0.5 to about 10 million MSCs. In an illustrative example, the mesenchymal stem cell storing or transport formulation comprises about 1 million MSCs, about 2 million MSCs, about 3 million MSCs, about 4 million MSCs, about 5 million MSCs or about 6 million MSCs. When used herein, the term “about” with respect to the number of mesenchymal stem cells may mean that the numerical value may vary by a specific percentage. For example, “about” may mean a numercical variation/deviation of ±1% to about ±15%. Thus, “about” may also mean ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9% or ±10%. It is evident for the person skilled in the art that such variations occur, in particular if the mesenchymal stem cell storing or transport formulation is prepared manually (which is still the usual approach for preparing such living cell based formulations) for subsequent storage and/or transport of the formulation to the administration site such as a wound healing clinic or a doctor's office.
In the present invention, MSCs may have been harvested directly from a culture of a MSCs containing tissue or from a culture of an isolated MSC or MSC population before being resuspended in the crystalloid solution. In either way, the cultivation of MSCs may have been carried out in a cell culture vessel. Consequently, the MSCs used in the present invention may have been harvested from the cell culture vessel prior to resuspending the MSCs in the pre-defined volume of the crystalloid solution.
The crystalloid solution and the liquid carrier of the present invention are both supplemented with serum albumin. Without wishing to be bound by theory, it is believed that serum albumin improves the viability of the mesenchymal stem cells/the mesenchymal stem cell population and may also improve the recovery of the stem cells from the vessel in which they are stored for transport of the stem cells to the site of administration. The concentration of the serum albumin may be the same or different in the crystalloid solution and the liquid carrier. Preferably, the concentrations of serum albumin are the same in both the crystalloid solution and the liquid carrier. In this context, any concentration of serum albumin suitable to, for example, improve MSC viability can be used. For instance, the crystalloid solution and the liquid carrier may each comprise about 0.5% (w/v), about 0.6% (w/v), about 0.7% (w/v), about 0.8 (w/v), about 0.9% (w/v), or about 1.0% (w/v) to about 5% (w/v) serum albumin. In one such example, the crystalloid solution and the liquid carrier may comprise about 1% (w/v) to about 3% (w/v) serum albumin. In an illustrative example, the crystalloid solution and the liquid carrier each comprise about 1% (w/v) serum albumin. Any pharmaceutically suitable serum albumin, for example, bovine or human serum albumin may be used herein. In an illustrative example, the crystalloid solution and the liquid carrier may both comprise human serum albumin (HSA). The serum albumin used herein is ideally obtained in a pharmaceutically acceptable quality. An example of such pharmaceutical grade serum albumin is the 25% solution (w/v) of human serum albumin commercially available under the tradename Plasbumin® from Grifols Therapeutics LLC, Clayton, N.C., USA.
The crystalloid solution may also comprise one or more components suitable for supporting the growth and/or proliferation of MSCs. Such a component may be a mineral such as sodium, potassium, iron, magnesium, zinc, selenium, chloride or a combination thereof. In one example, the crystalloid solution comprises sodium, potassium, magnesium and chloride. The crystalloid solution may be a commercially available solution including the further component suitable for supporting the growth and/or proliferation of MSCs. In an example, the crystalloid solution may be PlasmaLyte or Ringer's lactate. In the formulation of the present invention, the total amount of the the crystalloid solution may be limited to a specific percentage. For example, the mesenchymal stem cell storing or transport formulation may comprise not more than about 50%, not more than about 40%, not more than about 30%, not more than about 20%, not more than about 10% or not more than about 5% crystalloid solution. In an illustrative example, the mesenchymal stem cell storing or transport formulation may comprise not more than about 30% or about 20% or about 10% PlasmaLyte.
The transporting/storing can be performed for any period of time. For example, 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.
It is also contemplated that 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 15° 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 −10° C., more than about −15° 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 method of the present invention also envisions that the stem cell population (or the mesenchymal stem cells) stored or transported in any suitable concentration. As noted above the terms “mesenchymal stem cells” and “mesenchymal stem cell population” may be used interchangeably herein. It is also possible that, if reference is made herein to “mesenchymal stem cells” that these stem cells belong to the same mesenchymal stem cell population. For example, the mesenchymal stem cells may all belong to a mesenchymal stem cell population of which about 97% or more, about 98% or more, or about 99% or more of its cells express CD73, CD90 and CD105 while lacking expression of CD34, CD45 and HLA-DR. It is noted here that if the term “carrier” or “liquid carrier” may be used in context of a solution comprising MSCs, PlasmaLyte, HSA and Hyothermosol, the mesenchymal stem cell storing or transport formulation of the present invention may also be meant. Thus, the terms “carrier” or “liquid carrier” and “stem cell storing or transport formulation” may also be used interchangeably if the solution comprises MSCs, PlasmaLyte, HSA and Hyothermosol. The stem cell population as used herein 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. Therefore, 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. In principle, 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 cells 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.
In particular, 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.
As used herein 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. Notably, recent advances in embryonic stem cell research have led to the possibility of creating new embryonic stem cell lines without destroying embryos, for example by using a blastomere biopsy-based technique, which does not interfere with the embryo's developmental potential (Klimanskaya (2006) “Embryonic stem cells from blastomeres maintaining embryo viability.” Semin Reprod Med. 2013 January; 31(1):49-55). Furthermore, a large number of established embryonic stem cell lines are available in the art. Thus, it is possible to work with embryonic stem cells without the necessity to destroy an embryo. The pluripotent stem cells can be embryonic stem cells, which have not been obtained via the destruction of a human embryo. Thus, the pluripotent stem cells are embryonic stem cells obtained from an embryo, without the destruction of the embryo.
As used herein 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. For example, 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. For example, 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. In this context, it is noted that the culture medium described herein (e.g. PTT-6) allows the isolation of a mesenchymal stem cell population (also referred herein as “mesenchymal stem cells”) from the amniotic membrane under conditions that allow cell proliferation of the mesenchymal stem/progenitor cells without differentiation of the mesenchymal stem/progenitor cells. Thus, after isolation of the mesenchymal stem cells from the amniotic membrane as described herein the isolated mesenchymal stem/progenitor cell population has the capacity to differentiate into multiple cell types as described in US patent application 2006/0078993, U.S. Pat. No. 9,085,755, International patent application WO2006/019357, US patent 8,287,854 or WO2007/046775, for instance. As described in US patent application 2006/0078993, for example, the mesenchymal stem cells of the amniotic membrane of the umbilical cord have a spindle shape, express the following genes: POU5f1, Bmi-1, 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, β-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. An illustrative example of such an approach is the differentiation of the mesenchymal stem cells into insulin producing (3-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 WO2007/046775 in this respect). Alternatively, 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. In these therapeutic applications 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 WO2007/046775, for example).
In this context, it is noted that the MSCs may be derived from any mammalian tissue or compartment/body part known to contain MSCs. In illustrative examples, the MSCs may be MSCs of the umbilical cord, placental MSCs, MSCs of the cord-placenta junction, MSCs of the cord blood, MSCs of the bone marrow, or adipose-tissue derived MSCs. The MSCs of the umbilical cord may be (derived) from any compartment of umbilical cord tissue that contains MSCs such as the amnion, perivascular MSCs, MSCs of Wharton's jelly, MSCs of the amniotic membrane of umbilical cord but also mixed MSCs of the umbilical cord, meaning MSCs that includes stem cells of two or more of these compartments. 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. As shown, for example, in
Thus, in some embodiments 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 CD105. Additionally, or alternatively, 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). In further examples 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 MSCs express each of CD73, CD90 and CD105 while 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 MSCs may lack expression of CD34, CD45 and HLA-DR. In particular examples about 97% or more, about 98% or more, or about 99% or more of the MSCs express CD73, CD90 and CD105 while lacking expressing of CD34, CD45 and HLA-DR.
The marker CD73 is known to the skilled person. In this regard 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. In this regard CD90 refers to Cluster of Differentiation 90 also known as Thymocyte differentiation antigen 1 (Thy-1). The sequence of the human CD90 protein may have the sequence of SEQ ID NO: 2. The marker CD105 is known to the skilled person. CD105 is also known as Endoglin (ENG). The sequence of the human CD105 protein may have the sequence of SEQ ID NO: 3.
If 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. As explained, the mesenchymal stem cell population may lack expression of the following markers: CD34, CD45 and HLA-DR. In this context it is noted that 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.
As mentioned above, 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.
Thus, the stem cell population 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. Thus, 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. Such stem cell markers can include Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) as well as stem cell-specific antigen 3 and 4 (SSEA3/4). Also, 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).
In addition, 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 WO2009115295, WO2009144008 or EP2218778. Thus, the skilled artisan can obtain an iPSC by any method. In principle, 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.
The present invention is inter alia directed to a MSC storing or transporting formulation obtained by the method as described herein as well as to a MSC storing or transporting formulation obtainable by the method as described herein. Further, the present invention concers transporting MSCs comprising transporting said MSCs in a mesenchymal stem cell storing or transport formulation as defined herein. In this context, the present invention includes that the stem cell population as described herein is contacted with a liquid carrier. 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
By “Trolox” is meant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid of CAS Number 53188-07-1. It is a water-soluble analog of vitamin E and is suggested to reduce oxidative stress or damage.
Both of Na+ and Cl− are well known ions. The skilled person knows how to obtain these. For example, these ions may be added to the carrier as a NaCl salt. NaCl in GMP quality can be obtained from Sigma Aldrich.
Ca2+ and Mg2+ are also well-known ions. The skilled person knows how to obtain these. These ions may, for example, be added to the carrier as a CaCl2 or MgCl2 salt.
K+ and H2PO4− (dihydrogen phosphate) are also well known to the skilled person. It may be used e.g. as a KH2PO4 obtainable from SigmaAldrich.
HEPES also named 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS Number 7365-45-9) is commonly used as a zwitterionic organic chemical buffering agent. The person skilled in the art also knows where to obtain HEPES, which is commercially available. For example, she/he may obtain it from Sigma Aldrich; the corresponding data sheet shown in
Lactobionate is the carboxylate anion of lactobionic acid. Lactobionic acid (4-O-β-galactopyranosyl-D-gluconic acid) is a sugar acid. Lactobionate can be used in different ways. When used as potassium lactobionate it can e.g. provide osmotic support and prevent cell swelling and when combined with sodium it may have a preservative function. Alternatively, mineral salts of lactobionic acid can be used for mineral supplementation. For pharmaceutic applications, often the antibiotic erythromycin can inter alia be used as the salt erythromycin lactobionate. The skilled person also knows where to obtain lactobionate e.g. sodium lactobionate (Cas Number: 27297-39-8), namely from e.g. COMBI-BLOCKS, see product sheet in
Sucrose, also known as D-Glc-(1→2)-β-D-Fru, α-D-glucopyranosyl β-D-fructofuranoside, β-D-fructofuranosylαa-D-glucopyranoside, D(+)-saccharose or sugar (CAS Number 57-50-1) can as the other substances be commercially obtained and the skilled person knows where to buy it as well. The corresponding product sheet for sucrose from Sigma Aldrich is shown in
Mannitol is a type of sugar alcohol (CAS Registry Number: 69-65-8). The person skilled in the art knows how to obtain mannitol. For example, it may be obtained from Avantor. The respective product sheet is shown in
Glucose (CAS Number: 50-99-7) is also well known to the skilled person and commercially available. A respective product sheet from Sigma Aldrich is shown in
Dextran is a branched glucan composed of linear α (1→6) linked glucose units and α (1→3) link initiated branches. Dextran ranges in size from 10,000 to 150,000 Kd. Dextrans are used in many applications as volume extenders, stabilizers, matrix components, binding platforms, lubricants and physical structure components. Dextran 40 (CAS Number: 9004-54-0) as used in the carrier described herein is typically used in the development of new improved preservation solutions for organ transplantation. Dextran 40 may be used to determine cell tightness and flux parameters across cell layers. Dextran 40 can also be used as a colloidal plasma volume extender. Dextran-40 is commercially available and can inter alia be obtained from Sigma Aldrich (product sheet shown in
Adenosine (CAS Number 58-61-7) is a purine nucleoside composed of a molecule of adenine attached to a ribose sugar molecule (ribofuranose) moiety via a β-N9-glycosidic bond. Adenosine is commercially available inter alia from Sigma-Aldrich (the corresponding product sheet is shown in
Glutathione is also known as (2S)-2-Amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}butanoic acid. This component is commercially available and can inter alia be obtained from Sigma Aldrich (corresponding product sheet shown in
In principle any liquid carrier comprising the substances as listed in i)-xv) above can be used in the method of the present invention. The carrier is a liquid carrier. Thus, it is possible that the substances as listed in i)-xv) are dissolved in a liquid to form a solution/suspension. The liquid may be any suitable liquid. For example, the liquid can be a culture medium, water, buffer, or the like.
The carrier may additionally comprise further pH buffers, energy substrates, free radical scavengers, and osmotic/oncotic stabilizers—all known to the skilled person. Furthermore, the liquid carrier may be serum-free and/or protein-free. The liquid carrier may not comprise a dipolar aprotic solvent such as for example DMSO. In particular, the liquid carrier may be a carrier as described in WO 2010/064054. The carrier may be HypoThermosol™ or HypoThermosol™-FRS (HTS-FRS). HypoThermosol™-FRS (HTS-FRS) can be purchased from STEMCELL Technologies (according to the respective product sheet shown in
It is further envisioned that the carrier is 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)-xv) as described herein. The medium can also be one comprising substances as listed in i)-xv) as described herein and addition of (further) nutrients (both organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, and/or cofactors. In particular embodiments the medium is serum and/or protein free.
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)). Alternatively, 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.
In principle any amount of stem cells can be contacted with any amount of liquid carrier. In this regard 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. In some embodiments, the contacting is performed by suspending the stem cell population in a density of about 10 million/1 ml carrier.
After contacting the stem cell population with the mesenchymal stem cell storing or transport formulation, the stem cells contacted with the mesenchymal stem cell storing or transport formulation 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 mesenchymal stem cell storing or transport formulation. For example, the stem cells that have been contacted with the mesenchymal stem cell storing or transport formulation can be aliquoted into vials in a volume of about 1 ml.
It is further envisioned that 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.
In this regard, in the method of the present invention 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 μm and about 20 μm 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 μm and about 16 μm after transporting.
The stem cells transported/stored in the carrier as described herein secrete the same proteins/factors as viable stem cells. For example, the method of the present invention contemplates that after transport/storage the (mesenchymal) stem cell population may secrete about as much TGFbeta 1 as before transporting/storage. TGFbeta 1 (Transforming growth factor beta, TGF-β1) is known to the skilled person and may comprise the sequence as shown in SEQ ID NO. 7. Additionally, or alternatively, after transporting/storing the (mesenchymal) stem cell population may secrete about as much VEGF (Vascular endothelial growth factor), PDGF-AA (Platelet-derived growth factor subunit AA), Ang-1 (Angiogenin-1), and/or HGF (Hepatocyte growth factor) as before transporting/storing. All of VEGF, PDGF-AA, Ang-1, and/or HGF are known to the skilled person for their involvent in wound healing. In particular, VEGF may comprise a sequence as shown in SEQ ID NO. 8, PDGF-AA may have a sequence as shown in SEQ ID NO. 9, Ang-1 may have a sequence as shown in SEQ ID NO. 10 while 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-1, HGF or TGFβ1) that the stem cells secrete into the 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, Tex., USA). In this context, it is noted that involvement of the proteins Angiopoietin 1 (Ang-1), TGF-β1, VEGF, and HGF in the wound healing process is known to the person skilled in the art. For the involvement of Angiopoietin 1 in wound healing, see, for example, Li et al. Stem Cell Research & Therapy 2013, 4:113 “Mesenchymal stem cells modified with angiopoietin-1 gene promote wound healing”. For the involvement of Hepatocyte Growth Factor (HGF) in wound healing, in particular healing of chronic/non healing wounds see for example, Yoshida et al., “Neutralization of Hepatocyte Growth Factor Leads to Retarded CutaneousWound Healing Associated with Decreased Neovascularization and Granulation Tissue Formation. J. Invest. Dermatol. 120:335-343, 2003, Li, Jin-Feng et al. “HGF Accelerates Wound Healing by Promoting the Dedifferentiation of Epidermal Cells through β1-Integrin/ILK Pathway.” BioMed Research International 2013 (2013): 470418 or Conway et al, “Hepatocyte growth factor regulation: An integral part of why wounds become chronic”. Wound Rep Reg (2007) 15 683-692. For the involvement of Vascular Endothelial Growth Factor (VEGF) in wound healing, in particular healing of chronic/non-healing wounds, see for example Froget et al., Eur. Cytokine Netw., Vol. 14, Mar. 2003, 60-64 or Bao et al., “The Role of Vascular Endothelial Growth Factor in Wound Healing” J Surg Res. 2009 May 15; 153(2): 347-358.
For the involvement of Transforming Growth Factor Beta (including TGF-β1, TGF-β2, and TGF-β3) 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.
Turning now to the culture medium used in the present invention, the culture medium may comprise, for the isolation or cultivation 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 of “% (v/v)” as used herein refers to the volume of the individual component relative to the final volume of the culture medium. This means, if DMEM is, for example, present in the culture medium at a final concentration of about 55 to 65% (v/v), 1 liter of culture medium contains about 550 to 650 ml DMEM.
In other embodiments, the 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). In further embodiments, 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).
In addition to the above-mentioned components, the culture medium may comprise supplements that are advantageous for cultivation of the mesenchymal cord lining stem cells. The culture medium of the present invention may, for example, comprise Epidermal Growth Factor (EGF). If present, EGF may be present in the culture medium in a final concentration of about 1 ng/ml to about 20 ng/ml. In some of these embodiments, the culture medium may comprise EGF in a final concentration of about 10 ng/ml.
The culture medium may also comprise insulin. If present, insulin may be present in a final concentration of about 1 μg/m1 to 10 μg/ml. In some of these embodiments, the culture medium may comprise Insulin in a final concentration of about 5 μg/ml.
The culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyronine sodium salt (T3). In such embodiments, the culture medium may comprise all three of adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyronine sodium salt (T3). In these embodiments, the culture medium may comprise adenine in a final concentration of about 0.05 to about 0.1 μg/ml, hydrocortisone in a final concentration of about 1 to about 10 μg/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.
In one embodiment, the mesenchymal stem cells are cultured in PTT6 medium to obtain the highly purified mesenchymal stem cell population described and used herein. In this context it is noted that PTT6 medium as described herein is obtained by mixing to obtain a final volume of 500 ml culture medium:
i. 250 ml of DMEM
ii. 118 ml M171
iii. 118 ml DMEM/F12
iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of 2.5% (v/v)
v. EGF in a final concentration of 10 ng/ml
vi. Insulin in a final concentration of 5 μg/ml.
vii. Insulin 0.175 ml (final concentration of 5 μg/ml)
By “DMEM” is meant Dulbecco's modified eagle medium which was developed in 1969 and is a modification of basal medium eagle (BME) (cf.
By “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.
By “DMEM/F12” or “DMEM:F12” is meant a 1:1 mixture of DMEM with Ham's F12 culture medium (cf.
By “M171” is meant culture medium 171, which has been developed as basal medium for the culture and growth of normal human mammary epithelial cells (cf.
By “FBS” is meant fetal bovine serum (that is also referred to as “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. Suppliers of FBS that are ISIA members include Abattoir Basics Company, Animal Technologies Inc., Biomin Biotechnologia LTDA, GE Healthcare, Gibco by Thermo Fisher Scientific and Life Science Production, to mention only a few. In currently preferred embodiments, the FBS is obtained from GE Healthcare under catalogue number A15-151.
As mentioned above, a method of making a culture medium for isolating the mesenchymal stem cell population used in the invention comprises mixing to obtain a final volume of 500 ml culture medium:
i. 250 ml of DMEM
ii. 118 ml M171
iii. 118 ml DMEM/F12
iv. 12.5 ml Fetal Bovine Serum (FBS) to reach a final concentration of 2.5% (v/v).
As explained above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium. Thus, 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:
DMEM: 250 ml +59 ml=309 ml, corresponds to 309/500=61.8% (v/v)
M171: 118 ml, corresponds to 118/500=23.6% (v/v)
F12: 59 ml, corresponds to 59/500=11.8% (v/v).
Embodiments of this method of making a culture medium further comprise adding v. 1 ml EGF stock solution (5 μg/ml) to achieve a final EGF concentration of 10 ng/ml, and vi. Insulin 0.175 ml stock solution (14.28 mg/ml) to achieve a final insulin concentration of 5 μg/ml.
It is noted here that in these embodiments, the above-mentioned volumes of these components i. to vi. will result in a final volume of 499.675 ml 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 culture medium is 500 ml. In addition, it is also noted that components i. to iv. do not necessarily have to be added in the order in which they are listed but it is of course also possible to use any order to mix these components to arrive at the culture medium of the present invention. This means, that for example, 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).
In other embodiments, 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 culture medium. In this embodiment, the final concentration of these supplements in DMEM may be as follows: about 0.05 to 0.1 μg/ml adenine, for example about 0.025 μg/ml adenine, about 1 to 10 μg/ml hydrocortisone, about 0.5 to 5 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3), for example 1.36 ng/ml 3,3′,5-Triiodo-L-thyronine sodium salt (T3).
In line with the above disclosure, a cell culture medium used herein is obtainable or that is obtained by the method of making the medium as described here.
In addition, a method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, wherein this method comprises cultivating amniotic membrane tissue in the culture medium prepared by the method is described here.
Thus, the present invention is also directed to (the use of) a cell culture medium comprising:
In certain embodiments of the culture medium described here, 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). In other embodiments 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).
In addition, the culture medium may further comprise Epidermal Growth Factor (EGF) in a final concentration of about 1 ng/ml to about 20 ng/ml. In certain embodiments, the culture medium comprises EGF in a final concentration of about 10 ng/ml. The culture medium described herein may further comprise Insulin in a final concentration of about 1 μg/ml to 10 μg/ml. In such embodiments the culture medium may comprise Insulin in a final concentration of about 5 μg/ml.
The cell culture medium may further comprise at least one of the following supplements: adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyronine sodium salt (T3). In certain embodiments the culture medium comprises all three of adenine, hydrocortisone, and 3,3′,5-Triiodo-L-thyronine sodium salt (T3). If present, the culture medium may comprise adenine in a final concentration of about 0.01 to about 0.1 μg/ml adenine or of about 0.05 to about 0.1 μg/ml adenine, hydrocortisone in a final concentration of about 0.1 to about 10 μg/ml hydrocortisone or of about 1 to about 10 μg/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.
In embodiments of the cell culture medium, 500 ml of the cell culture medium of the present invention comprise:
In a particular example, the components i. to vi. of the culture medium used in the present invention are the components indicated in
In addition, the cell culture medium of the invention may comprise adenine in a final concentration of about 0.01 to about 0.1 μg/ml adenine or of about 0.05 to about 0.1 μg/ml adenine, hydrocortisone in a final concentration of about 0.1 to 10 μg/ml, of about 0.5 to about 10μg/ml, or of about 1 to about 10 μg/ml hydrocortisone 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.
To obtain the mesenchymal stem cell population as described herein the umbilical cord tissue may be cultured till a suitable number of (primary) mesenchymal cord lining stem cells have outgrown from the tissue. In typical embodiments, 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. It is noted here that 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.
Once a suitable number of primary cells (mesenchymal cord lining stem cells) have been obtained from the cord lining tissue by tissue explant, the mesenchymal stem cells are removed from the cultivation container used for the cultivation. By so doing, a master cell bank containing the (primary) isolated mesenchymal stem cells of the amniotic membrane can be obtained. Typically, since mesenchymal stem cells are adherent cells, removing is carried out using standard enzymatic treatment. For example, the enzymatic treatment may comprise trypsination as described in International US patent application 2006/0078993, International patent application WO2006/019357 or International patent application WO2007/046775, 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.
Once being harvested, 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. For subculturing, 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 0.5×106 cells/ml to about 5.0×106 cells/ml. In one embodiment the cells are suspended for subcultivation at a concentration of about 1.0×106 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, N.Y., USA) or Cellfactory (Nunc, part of Thermo Fisher Scientific Inc., Waltham, Mass., USA) that can be stacked in incubators. Alternatively, the subculturing can also be carried out in a closed self-contained system such as a bioreactor. Different designs of 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. “Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review”, supra. An illustrative example of a commercially available hollow-fiber bioreactor is the Quantum® Cell Expansion System (Terumo BCT, Inc). that has, for example, been used for the expansion of bone marrow mesenchymal stem cells for clinical trials (cf., Hanley et al, Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System, Cytotherapy. 2014 August; 16(8): 1048-1058). 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. Accordingly, 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. Also for the subcultivation, the mesenchymal stem cells can be cultured till a suitable number of cells have grown. In illustrative embodiments 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. Typically, 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. For example, human umbilical cord tissue and the mesenchymal cord lining stem cells, respectively, are usually cultivated at 37° C. in air atmosphere with 5% CO2. In this context, it is noted that the 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.
Once a desired/suitable number of mesenchymal cord lining stem cells have been obtained from the subculture, 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. The term “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 −196° 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.
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. In addition, in these embodiments 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. In particular embodiments, 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.
Thus, in line with the above disclosure 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. In preferred embodiments 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 isolate cell population express each of CD73, CD90 and CD105 (cf. the Experimental Section of the present application) can be used herein. In addition, 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. In particular embodiments about 97% or more, about 98% or more, or about 99% or more cells of the isolated mesenchymal stem cell population express CD73, CD90 and CD105 while lacking expressing of CD34, CD45 and HLA-DR. Such a highly homogenous population of mesenchymal stem cells derived from the amniotic membrane of the umbilical cord has been reported for the first time in US provisional application No. 62/404,582, filed Oct. 5, 2016 as well as in co-pending U.S. application Ser. No. 15/725,913, filed 5 Oct. 2017 as well as in co-pending PCT application PCT/SG2017/050500, also filed 5 Oct. 2017, 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 as described herein can comprise, mixing to obtain a final volume of 500 ml culture medium:
As explained above, DMEM/F12 medium is a 1:1 mixture of DMEM and Ham's F12 medium.
Thus, 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 cells that have been stored or transported in a mesenchymal stem cell storing or transport solution, or a population as described herein to the subject, wherein the mesenchymal stems are or the stem cell population is administered within about 96 hours from the time point the mesenchymal stem cell population has been harvested. The method of treating a subject may be carried out as described in International Patent Application WO2019/199229 “A Method Of Transporting Mesenchymal Stem Cells By Means Of A Transporting Solution And A Method Of Administering Stem Cells To Wounds” that has been published after the priority date of the present PCT application and is incorporated herewith in its entirety for all purposes.
Similarly, 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. Thus, in one embodiment, the subject is a vertebrate. The subject can also be a human subject. The subject therefore can be a subject in need of treatment. As such the subject may be afflicted with a disease as described elsewhere herein. In some embodiments the subject is afflicted with Type I or Type II diabetes with chronic foot ulcers. Preferably, 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. For example, 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. In one example, the mesenchymal stem cells may be applied in a dosage of about 3, about 5 or about 10 million cells. In a particular embodiment, 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, elven weeks or more.
Thus, 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.
It is also contemplated by the method of treatment of the present invention that the mesenchymal stem cells are or the mesenchymal stem cell population is applied in a dosage of about 1000 cells/cm2 to about 5 million cells/cm2. Here, the expression cm2 means the area of the wound/skin to which the stem cells are applied. It is also envisioned that the mesenchymal stem cell population is applied in a dosage of about 100,000 cells/cm2, 300,000 cells/cm2 or 500,000 cells/cm2. 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/cm2, about 300,000 cells/cm2 or about 500,000 cells/cm2.
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 cells or 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 in the mesenchymal stem cell storing or transport formulation as described in the present invention. Thus, aspects as described for the transporting/storing in the mesenchymal stem cell storing or transport formulation of the present application equally relate to the method of treating a subject comprising administering MCSs that have been stord in mesenchymal stem cell storing or transport formulation of the present invention mutatis mutandis.
The method of treating a subject of the present invention serves to alleviate a disease suffered by the subject. In principle, any disease that may be treated by the mesenchymal stem cell population as described herein is meant here. In particular, the disease may be a skin disease or a wound. The wound may be caused by any cause e.g. by a burn, 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. Notably, 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 WO2007/046775).
As described herein, between the harvesting of the mesenchymal stem cell population as described herein and their application to a subject the cells may be transported/stored in the carrier as defined herein. Therefore, 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. For example, the separating of the mesenchymal stem cell population from the carrier may comprise centrifugation. Additionally, or alternatively, separating the mesenchymal stem cell population from the carrier can comprise withdrawing the cell population from the vial by means of syringe.
After separating the stem cells from the mesenchymal stem cell storing or transport formulation or after harvesting the mesenchymal stem cells or after obtaining mesenchymal stem cell population as described herein by any other method these cells are topically applied to a subject. In principle any way of topical administration is meant herein. 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 by a film or bandage. An example for such a film or bandage may be 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 of mesenchymal stem cells obtained or obtainable by the method as described herein. For example, the unit dosage may comprise 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 in a volume of 1 ml.
It is also envisioned that 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. In one example, the unit dosage may comprise about 1 million, about 3 million, or about 5 million cells. Preferably the unit dosage comprises about 10 million cells. It is further envisioned that 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 cm2.
The unit dosage can be applied once, twice, three times or more a week. For example, 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 twice a week for 8 weeks, preferably onto 1 cm2.
The unit dosage can be contained in any suitable container. For example, 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 cm2. The unit dosage may alternatively be contained in a syringe.
The unit dosage of the present invention the cells can be in contact with a liquid carrier as defined herein. If this is the case then the mesenchymal stem cells are separated from the carrier before administration. For example, the cells can be centrifuged and isolated before administration to a subject. The carrier may comprise or be any carrieras described herein, such as HypoThermosol™ or Hypothermosol™-FRS.
The unit dosage of the present invention may comprise MSCs of the umbilical cord. As described above, MSCs of the umbilical cord may be (derived) from any compartment of umbilical cord tissue that contains MSCs. Thus, the unit dosage may comprise MSCs of the amnion, perivascular MSCs, MSCs of Wharton's jelly, MSCs of the amniotic membrane of umbilical cord. MSCs of the amniotic membrane of umbilical cord may be highly defined and homogenous. Thus, in one embodiment of the present invention, the unit dosage may comprise MSCs as described in International Application WO 2018/067071 is used. Thus, in typical examples of the method the unit dosage may comprise MSCs exhibiting 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 MSCs express each of the following markers: CD73, CD90 and CD105. Further, the unit dosage may comprise MSCs exhibiting 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 MSCs lacking expression of the following markers: CD34, CD45 and HLA-DR. In particular examples, the unit dosage comprises about 97% or more, about 98% or more, or about 99% or more of the MSCs express CD73, CD90 and CD105 while lacking expression of CD34, CD45 and HLA-DR. In further examples 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 MSCs express each of CD73, CD90 and CD105 while 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 MSCs may lack expression of CD34, CD45 and HLA-DR. In particular examples about 97% or more, about 98% or more, or about 99% or more of the MSCs express CD73, CD90 and CD105 while lacking expressing of CD34, CD45 and HLA-DR.
The method of treatment and the unit dosage of the present invention can comprise utilization of viable cells. How viability can be tested is described elsewhere herein.
The invention will be further illustrated by the following non-limiting Experimental Examples.
Sequences as used herein are depicted in below Table 1.
1. Cryopreservation of Umbilical Cord Tissue Prior to Isolation of Mesenchymal Stem Cells
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.
1.1 Washing of Umbilical Cord Tissue Sample:
NOTE: If the cord is not frozen right away, the umbilical cord tissue is kept in Plasmalyte A until ready to freeze.
1.2 Cryopreservation of Umbilical Cord Tissue:
2. Isolation of Mesenchymal Cord Lining Stem Cells from umbilical cord tissue
2.1. Preparing Media for Processing MSCs from Umbilical Cord Tissue:
The above-mentioned volumes of components i. to vi when result in a final volume of 499.675 ml 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 culture medium is 500 ml. Alternatively, a stock solution of an antibiotic such as Penicillin-Streptomycin-Amphotericin can be added to result in a final volume of 500 ml. It is also possible to add to the culture medium 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 culture medium.
2.2 Thawing of Umbilical Cord Tissue for MSC Harvesting:
2.3 Preparing for Tissue Processing:
2.4. Initiating MSC Outgrowth from Tissue:
With the size of the tissue explants being about 1-3mm, and the tissue explant/cell culture is performed in 175 mm squared culture dishes, 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 175 cm2 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 175 cm2 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.
2.5. Subculturing MSC in Cell Culture Dishes
1. “X” volume=Total viable cells/106 cells/ml
2. For example, if total viable cell number is 1.0×107;
3. “X”=107/106 cells/ml or 10 ml, therefore, you would bring your total cell volume up to 10 ml by adding 5 ml to your cell suspension (that is at 5 ml).
2.6 Cryopreserving MSC Cells
2.7. Subculturing (expansion) of MSC in a Quantum Bioreactor (Terumo BTC, Inc.) It is also possible to use a Quantum Bioreactor can 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.
Materials/reagents:
Equipment:
Procedure:
a) Priming the Quantum Bioreactor
b) Coating the bioreactor:
a) Loading and attaching the cells with Uniform Suspension:
b) Feeding and cultivation of the cells
3. Analysis of Stem Cell Marker Expression in Mesenchymal Cord Lining Stem Populations Isolated from Umbilical Cord Tissue, Using Different Culture Media
Flow cytometry experiments were carried out to analyse mesenchymal stem cells isolated from the umbilical cord for the expression of the mesenchymal stem cell markers CD73, CD90 and CD105.
For these experiments, 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.
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 US 2008/0248005 and the corresponding International patent application WO2007/046775 that consist of 90% (v/v) CMRL1066, and 10% (v/v) FBS (see paragraph [0183] of WO2007/046775) and c) the culture medium of the present invention PTT-6 the composition of which is described herein. In this flow cytometry analysis, two different samples of the cord lining mesenchymal stem cell (CLMC) population were analysed for each of the three used culture media.
The following protocol was used for the flow cytometry analysis.
Materials and Methods
Procedure
a) Cell isolation and cultivation from the umbilical cord lining membrane
b) Trypsinization of cells for experiments
The results of the flow cytometry analysis are shown in
The findings shown in
4. Experiments Showing that the Mesenchymal Stem Cell Population of the Invention Can Be Transported/Stored in HypoThermosol™:
To analyze health and viability of the mesenchymal stem cells as described herein in different storage or transport carrier, two different carriers were compared to each other. Namely, the carrier HypoThermosol™-FRS was compared to the carrier PlasmaLyte-A. Both are commercially available. HypoThermosol™ -FRS the product sheet of which is shown in
The experimental setup for comparison is shown in
In summary from the above data it can be concluded that storage of the mesenchymal stem cell population of the present invention in HypoThermosol™ outperforms storage in PlasmaLyte-A especially for the first 3 days of storage.
5. Experiments Showing that the Mesenchymal Stem Cell Population of the Invention Have Wound Healing Properties by Topical Treatment of Pigs:
Preclinical studies have also been performed using 10-week old female Yorkshire-Landrace pigs (50 kg). The treatments were performed at SingHealth Experimental Medicine Centre in Singapore. The pigs were rendered diabetic with 120 mg/kg streptozotocin and allowed to recover for 45 days prior to creating six 5 cm×5 cm full thickness wounds on their backs (see
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. Furthermore, there is clearly no sign of inflammation with the use of xenogeneic mesenchymal stem cells in either mice or pigs, and therefore the likelihood that allogeneic mesenchymal stem cells will have any serious adverse effect in humans is very low.
6. Experiments Showing that the Mesenchymal Stem Cells as Described Herein are Effective in Topical Treatments in Humans:
Experiments showing that the mesenchymal stem cells as described herein are effective in topical treatments in humans have been described in WO 2007/046775. In particular, as explained in Examples 23-26 of WO 2007/046775 mesenchymal stem cells of the amniotic membrane of the umbilical cord (UCMC) could alleviate full thickness burns (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). Notably, in accordance with Example 2 of WO 2007/046775 mesenchymal stem cells were resuspended in PTT-4 medium.
Notably, as depicted in
7. Experiments Showing that the Mesenchymal Stem Cells as Described Herein are Effective in Topical Treatments in Humans:
This is a planned study of escalating doses of the mesenchymal stem cell population obtained as described herein performed at the University of Colorado Anschutz Medical Campus in Aurora, Colo. The goal of this study is to determine a safe dose of mesenchymal stem cell population as described herein (human umbilical cord lining mesenchymal stem cells). This is a single-center, dose-escalation study where each of three dose levels will enroll five subjects for a total of fifteen subjects. The first group of 5 patients will receive 100,000 MSC/cm2 (skin/wound area) twice per week for 8 weeks. The second group of 5 patients will receive 300,000 MSC/cm2 twice per week for 8 weeks. The third group of 5 patients will receive 500,000 MSC/cm2 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. 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. A secondary endpoint of this study will be significant improvement in the condition of the wound. This endpoint will be based on the rate of wound closure, the percent of wound area successfully closed, and the percent of wounds fully closed, as measured using the Silhouette Wound Measurement and Documentation System. This device is approved by the FDA for this purpose.
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/cm2 skin/wound area, 300,000 cells/cm2 or 500,000 cells/cm2 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.
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/cm2 skin/wound area twice per week for 8 weeks. The second group of 5 patients will receive 300,000 MSC/cm2 twice per week for 8 weeks. The third group of 5 patients will receive 500,000 MSC/cm2 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.
Route of Administration. 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.
Dosing Procedure. Following suitable debridement, if needed, the patient is placed in the prone position and the affected leg bent at a 90° angle. This vial of the mesenchymal stem cell population as described herein is gently swirled to ensure equal distribution of the cells. The elevated foot is then treated by removing 100,000 (0.1 ml) to 500,000 (0.5 ml) cells per cm2 from the vial using a sterile syringe and placing it in the center of the wound. The wound is then sealed with a Tegaderm® membrane and gently massaged to distribute the cells evenly. The foot is maintained elevated for five minutes to allow the cells to settle and attach. The foot is then dressed with a crepe bandage to cover the Tegaderm® dressing.
8. Preparation of the Mesenchymal Stem Cell Storing or Transport Formulation (Comprising a Stem Cell Population of which more than 99% of the Cells Express Each of CD73, CD90 and CD105 and lack expression CD34 and HLA-DR).
Preparation for processing after cell passaging for the fourth time (Stage 4 processing):
Stage 4 processing is typically performed in an environmentally monitored (EM) clean room. The required solutions and utensils should be prepared for use beforehand.
Transfer cells from cryovial to a labeled 50m1 centrifuge tube.
Use the complete PTT6 medium within 5 minutes of removing from the refrigerator (record time of PTT6 media removal from refrigerator). Slowly add 9 ml of complete PTT6 medium to the cells while gently swirling to facilitate mixing.
Centrifuge at 1200rpm at room temperature (15-25° C.) for 5 minutes to pellet the cells. Record centrifuge use on Form Centrifuge Periodic Preventative Maintenance—CR and verify performance following SOP Centrifuge Preventative Maintenance.
Remove supernatant and re-suspend the cells in enough complete PTT6 medium for counting.
Counting:
To determine cell concentration (with or without trypan blue), perform cell count on TC20. In most cases, no sample dilution is necessary, as the TC20 accommodates a wide range of cell concentrations (5×104 to 1×107 cells/ml).
To determine viability, count on the hemocytometer following the same SOP. Make sure that at least 200 cells in total are counted.
If both viability and cell concentration are desired via hemocytometer, the suspension may have to be diluted to accommodate for the hemocytometer range (20-100 cells per outer square). If an estimated 10 million thawed cells are being re-suspended, a volume of 6m1 should yield that range.
Therapeutic Culture (P4):
Seed 300,000 live cells per 175cm2 flask in 30 ml complete PTT6 medium and incubate at 35-39° C. with 4-6% CO2. Make sure the incubator preventative maintenance is up-to-date, as per SOP Incubator Preventative Maintenance and General Use. Label flasks with P1-P4 MSC Processing Label.
Once most cells have become adherent (preferably overnight), perform a cursory examination of a sentinel flask under the inverted Nikon microscope located in the clean room to determine if there is an area of the flask that contains a noticeably greater density of cells. If so, use that area for continuous monitoring by the CytoSmart. If multiple flasks are seeded, a single flask may be used as a representative “sentinel” flask. As an option, CytoSmart the email alert notification is set to 60%, 70%, and 80% confluence for the “sentinel” flask.
Change the media with pre-warmed fresh 30m1 complete PTT6 per flask every 2-3 days and continue to incubate.
When cell outgrowth reaches 80% ±10% confluence, harvest the MSCs as follows:
Rinse each flask with 10m1 HBSS without Ca2+or Mg2+.
Add 5 ml 1× TrypLE per flask. Tilt the flask to coat the entire surface and immediately aspirate off the TrypLE by tilting the flask and removing the majority of TrypLE with a sterile serological pipette, leaving only enough TrypLE to cover the surface. Discard aspirated TrypLE.
Allow the cells to detach (10-20 minutes at 15-25° C.). Tilting the flask 30-45° allows cells to shift down by gravitational flow. Gentle tapping on the side of the flask expedites detachment. The flask will be monitored under the inverted microscope to ensure all cells have detached.
Add 5 ml HBSS without Ca2+ or Mg2+ to the first flask. Pipette up and down gently, then transfer cell suspension to the next flask. Repeat until cells are harvested from all the flasks and transfer to 50 ml centrifuge tube labeled with the processing label.
Repeat with fresh 5 ml of HBSS without Ca2+ or Mg2+ and combine with the suspension.
Confirm under the microscope that all cells are removed and if needed repeat for the third time to harvest the cells in all flasks.
Centrifuge the combined cell suspension for 5 minutes at 1200 rpm at 15-25° C. Record centrifuge use on Form Centrifuge Periodic Preventative Maintenance—CR and verify performance.
Prepare the Harvested Cells Suspension:
Remove supernatant without disturbing the pellet and re-suspend cells in 1.0 ml complete PTT6 medium per harvested flask with a serological pipette of suitable size. The medium need not be pre-warmed.
Spin down cells re-suspended in complete PPT6 medium at 1200 rpm for 5 minutes at room temperature.
Remove the complete PTT6 supernatant without disturbing the pellet and gently re-suspend the pellet in 1.0 ml “1% HSA in Plasmalyte” per harvested flask a serological pipette of suitable size. This is the Harvested Cells Suspension. Keep the harvested cells suspension in the cooling block from this point forward.
Count the harvested cells suspension.
Prior to each sampling for counting from the harvested cells suspension, ensure cells are mixed well.
To determine cell concentration (with or without trypan blue), count on the TC20 following SOP Cell Count and Viability Assay. In most cases, no sample dilution is necessary, as the TC20 accommodates a wide range of cell concentrations (5×104 to 1×107 cells/ml).
To determine viability, count on the hemocytometer following the same SOP. Make sure that at least 200 cells in total are counted.
Prepare the Vial Load Suspension (keep chilled in 50m1 conical in cooling block):
Based on previous count of the Harvested Cell Suspension, determine the volume of harvested cell suspension and “1% HSA in HypoThermosol” needed to prepare the required patient dose. Label conical tube with appropriate label. Keep conical containing vial load suspension in its own pre-chilled cooling block.
HypoThermosol and the prepared “1% HSA in HypoThermosol” are stored and used at refrigeration temperature range (2-8° C.), so keep the vial load suspension in the cooling block.
Record volume of each component (HSA, Plasmalyte-A, and HypoThermosol-FRS) used to prepare this final suspension. Based on this volume, also record volumes of HSA, Plasmalyte, and HypoThermosol that will be present in each AT-Closed Vial.
Count the vial load suspension:
Prior to each sampling for counting from the vial load suspension, ensure cells are mixed well.
To determine cell concentration (with or without trypan blue), count on the TC20 following SOP Cell Count and Viability Assay.
To determine viability, count on the hemocytometer following the same SOP. Make sure that at least 200 cells in total are counted. Viability may be performed only once on the VLS.
Load AT-Closed Vials as follows:
Remove the previously placed syringes+needles from the refrigerator and place into the biosafety cabinet (BSC).
Remove the AT-Closed Vials previously placed in the CoolRackSV10/XT Cooling Core assembly from the fridge and place the setup in the BSC cabinet. Start a timer to ensure loading completion within 30 minutes.
Wipe the injection port with the alcohol swab.
Before loading the vial, insert a sterile 22G needle in the stopper, near the center, to vent the vial (this is to avoid pressurizing the vial during filling).
Swirl the vial load suspension to mix, then slowly draw it into the syringe without introducing bubbles. Pierce the center of the stopper with the syringe and inject 1.0 ml into each AT-Closed Vial (reading from meniscus-to-meniscus on the syringe), being careful not to introduce bubbles.
Remove the loading syringe, then remove the pressure venting needle.
Cover the vial port with the accompanying cap and firmly press it all around. Return to the CoolRackSV10 and store at 2-8° C. for shipping to destination.
Sample Collection for post-P4 Cytokine Assessment (the cytokine assay is performed at least once per each lot number, i.e. identical CBU # and lot # of donor tissue):
Based on the vial load suspension concentration from above, dispense enough volume of vial load suspension into at least one well of a 6-well plate so that 100,000 total (live+dead) cells are dispensed per well. Add the vial load suspension directly into enough complete PTT6 medium already added into each well so that the total volume in each well is 2 ml. Mark incubation start time.
Incubate for 48 hours±1 hour. At the conclusion of incubation:
Take a single representative CytoSmart image placed randomly in approximate center of each well.
Measure lactate from each well and report on Lactate Test Result Form.
Collect the medium from each well and centrifuge at 1200 rpm at room temperature for 5 minutes. Record centrifuge use on Form Centrifuge Periodic Preventative Maintenance—CR and verify performance.
Dispense the medium supernatants into cryotubes and freeze within 1 hour of collection. Mark storage location on the Batch Record.
9. Stability Study of MSC Proliferation and Metabolism During Storage/Transportation
The umbilical cord tissues and cell from early passages are stored at −195° C. and have been tested for stability.
Original stability testing for the mesenchymal cord lining stem cells (MSCs) described herein having a purity of more than 99% with respect to the posive and negative markers (cf.
To mitigate cell loss due to the MSCs being put into HypoThermosol® alone at distribution, adhesion of MSCs to plastics at various steps of Stage 4 processing and to maximize recovery of the drug product from the vial used at distribution, Plasmalyte and human serum albumin (HSA), two pharmaceutically inactive ingredients, were found, when added, to optimize the quality of final drug product.
A new stability study was performed to support that the addition of these two pharmaceutcally inactive ingredients does not adversely affect the stability of the final drug product. The results are shown in
Viability Analysis
The mesenchymal stem cells were seeded into AT-Closed Vials® at 106 cells per vial in 1 mL of Plasmalyte/HSA/HypoThermosol®. Individual vials were sampled at various time points, with viability assessed manually with trypan blue (hemocytometer) and total cell number tallied by an automated system (TC20).
The MSCs were stored at 2 to 8° C. for 1 to 3 days to mimic shipping and storage of the product prior to application on the wounds. As shown in
Appearance Analysis
The MSCs were photographed after removal from the AT-Closed Vials and cultured for 24 hours at 37° C. As seen below, cells obtained up to 2 days in cold storage were capable of adhering to the tissue culture plates and forming the typical spindle structures. After storage for 2.5 days at 2-8° C., the cells exhibited increasingly spheroid shapes, suggestive of dying cells. The results are shown in
Analysis of the Proliferation and Metabolism
MSCs from the same cultures shown in
Individual vials have further been tested on Days 0, 1, 1.5, 2, 2.5 and 3 based on an established 3-day cell viability threshold. Trypan blue viability was performed immediately upon removing the cells from the sealed vials, and there was no appreciable loss of viability over 2.5 days (range 92-98%). The cells were also plated at 105 cell/cm2 in standard PTT6 medium, and lactate production was measured 24 and 48 hours later. Lactate is a product of glucose metabolism, which we have validated to be directly proportional to the rate of MSC cell growth.
Analysis of the Cytokine Production
Cytokine production was measured from the same cultures at 24 hours at 37° C. In alignment with the metabolism data, the ability of MSCs to produce Ang-1, TGF β, VEGF and HGF were within 10-20% of the controls (Day 0) when the cells were stored at 2-8° C. for 24 hours. The results shown in
Another analysis of cytokine production on MSCs stored for 0, 1, 1.5, 2, 2.5 or 3 days in Plasmalyte/HSA/HypoThermosol® verified the results obtained by the first cytokine analysis production (
In summary, based on the viability, appearance, metabolism and cytokine production demonstrated by the cells during these studies, an expiration of 72 hours from product vial closure may be set. Thus, since in principle any place in the (developed) word can be reached by air travel within 72 hours, the storage and transport formulation of the invention essentially allows transporting living MSC from the MSC production facility to basically any place in the world, where the MSC are administered to a subject. Thus, the storage and/or transport formulation of the present invention signifcantly reduces the complexity of GMP manufacturing and supply chain of pharmaceutically suitable mesenchymal stem cells/stem cell populations, thereby making therapies based on mesenchymal stem cells easily available for the greater public.
The invention is further characterized by the following items:
i) Trolox;
ii) Na+;
iii) K+;
iv) Ca2+,
v) Mg2+
vi) Cl—;
vii) H2PO4—;
viii) HEPES;
ix) Lactobionate;
x) Sucrose;
xi) Mannitol;
xii) Glucose;
xiii) Dextran-40;
xiv) Adenosine, and
xv) Glutathione,
thereby obtaining the mesenchymal stem cell storing or transport formulation comprising about 0.5 to about 10 million mesenchymal stem cells.
It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments of the invention will become apparent from the following claims.
When used herein, 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. For example, if 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. As used herein, 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.
The present application claims the benefit of priority of U.S. Provisional Application No. 62/912,368 filed Oct. 8, 2019, the content of which is hereby incorporated by reference it its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62912368 | Oct 2019 | US |
