Methods for preparing large-scale cultures of muscle progenitor cells (MPCs) and uses thereof

Abstract

Provided are methods for obtaining large-scale cultures of muscle-derived myogenic progenitor cells (MPCs) using microcarriers as growth substrates, methods for obtaining therapeutically effective amounts of these cells, cell populations obtained by the methods, as well as compositions comprising the expanded cells, and methods for preparing medicaments for use in, for example, treating skeletal muscle dysfunction.

Description

[Technical Field]

[0001] The present invention relates to a method for obtaining large-scale cultures of muscle precursor cells (MPCs), as well as cell populations comprising MPCs obtained by said method and compositions comprising said MPCs. Furthermore, the present invention relates to a method for preparing a medicament based on the obtained MPCs for use in treating skeletal muscle dysfunction. [Background technology]

[0002] Skeletal muscles damaged by injury or degenerative diseases such as muscular dystrophy can regenerate new muscle fibers, a process that primarily relies on myogenic progenitor cells. Therefore, transplantation of muscle progenitor cells (MPCs) has been investigated as a treatment for various inherited and acquired muscle disorders. Satellite cells are quiescent adult stem cells located beneath the membrane surrounding muscle fibers. After trauma or injury, satellite cells are activated as MPCs, proliferate, and participate in tissue regeneration by differentiating into myoblasts, which then fuse to form new muscle fibers. Because the majority of MPCs are committed to the myogenic lineage, they are ideally suited for muscle tissue engineering (Eberli et al., Cell Transplant 21 (2012), 2089-98).

[0003] MPCs, which are more differentiated than stem cells and more committed toward the muscle lineage, are a promising therapeutic option for damaged, diseased, and aging muscle tissue, and their potential is being widely explored. Stress urinary incontinence (SUI), the involuntary loss of urine due to coughing, laughing, sneezing, exercise, and other movements that increase intra-abdominal pressure on the bladder, is one example of a muscle dysfunction that could benefit from cell therapy.

[0004] For example, as reviewed by Schmid et al., cell-based therapeutic approaches for sphincter regeneration have been developed, in which progenitor cells are isolated from living human tissue biopsies, then expanded in vitro and reimplanted to repair or replace damaged or diseased tissue (Schmid et al., International Journal of Molecular Sciences 22 (2021), 3981). A particular option is the transplantation of autologous muscle progenitor cells into the sphincter region to strengthen and restore external urethral sphincter function, as also disclosed in WO 2019 / 215090.

[0005] However, one of the limiting steps in such cell-based therapies, especially for the treatment of muscle dysfunction, is the lack of an efficient method to generate sufficiently high quantities of MPCs for therapeutic use. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2019 / 215090 [Non-patent literature]

[0007] [Non-Patent Document 1] Eberli et al.,Cell Transplant 21(2012),2089-98 [Non-patent document 2] Schmid et al.,International Journal of Molecular Sciences 22(2021),3981 Summary of the Invention

[0008] The present invention generally relates to a method for obtaining large-scale cultures of muscle-derived progenitor cells (MPCs), the culture preferably being carried out in a 3D culture system. In particular, the method of the present invention comprises culturing MPCs in a vessel containing culture medium and microcarriers under conditions that allow the MPCs to attach to the microcarriers, the MPCs growing at 500-1500 cells / cm of the growth surface provided by the microcarriers. 2 The method of the present invention further comprises a step of increasing the growth surface area within the culture environment when the cell number from the initial seeding has increased, preferably by about 8-25 fold. In one embodiment, the step of increasing the growth surface area of ​​the method of the present invention is performed when the cell number increases to about 1.3 x 10 4 ~1.8×10 4 cells / cm 2 and / or when more than 80%, preferably more than 90%, of the microcarriers are occupied. 4 cells / cm 2 , i.e., at least 5-7.5 x 10 4 cells / cm 2 Or 5 to 7.5 x 10 4 cells / cm 2 or less, and / or 4 to 6.5 × 10 5 In a preferred embodiment, the method further comprises culturing the MPCs until a cell density of about 1.5 to 2.75 x 10 cells / ml is reached. 8 The culture medium is expanded and the cells are then further cultured until a cell number of Preferably, the MPCs are obtained from a patient, preferably a human patient, as described below.

[0009] Thus, with the help of the present invention, a culture system has been established that allows for the generation of large quantities of MPCs. MPCs are anchorage-dependent cells, commonly referred to as adherent cells. These cells need to adhere to a surface to remain viable and proliferate. The method of the present invention has certain advantages over previously applied methods that rely on MPC culture as a monolayer on a plate. In particular, high cell yields can be obtained using the growth surface provided by the method of the present invention without the need for a replating step. Therefore, the method of the present invention is less labor-intensive and time-consuming than conventional monolayer culture systems on plates, and the risk of contamination is also lower when applying the method of the present invention due to the closed system. It should also be emphasized that, because the method achieves large quantities of MPCs much more quickly than, for example, conventional 2D culture systems, the resulting population of MPCs is advantageously "closer" to the patient, for example, with respect to undesirable mutations known to accumulate during culture, i.e., the short culture time reduces the likelihood of undesirable mutations arising during culture.

[0010] A further advantage of the method of the present invention is the low seeding density required for efficient cell expansion. For example, as can be derived from WO 2019 / 215090, a seeding density of 5000 cells / cm is required to expand cells. 2 In the method of the present invention, a seeding density of 500-1500 cells / cm is used. 2 is already sufficient to provide cell proliferation and result in a high yield after the culturing process.

[0011] As shown in Example 2 and Figures 1 and 2, MPCs can be efficiently cultured and expanded on microcarriers to obtain sufficient cell numbers for downstream applications, e.g., therapeutic procedures. The concentration of cells in the culture medium and their ratio to the growth surface provided by the microcarriers is important to provide appropriate growth conditions. Thus, in a preferred embodiment, the method of the present invention allows for the cultivation of 800-1200 cells / cm. 2In a more preferred embodiment, the MPCs are seeded at a density of 800 to 1200 cells / cm in a culture volume as described below, preferably 130 ml of culture medium. 2 The seeds are sown at a density of

[0012] As described above, the methods of the present invention include a step of increasing the growth surface area within the culture environment upon reaching a specific cell density, cell number, and / or expansion fold for the cells, as defined elsewhere herein. In a preferred embodiment, the growth surface area, and optionally the volume of the culture medium, is increased by 2-4 fold, preferably 3 fold. In one embodiment, the starting volume used in the methods of the present invention is approximately 100-150 ml, and the volume of the culture medium is increased to approximately 400 ml. To obtain even higher cell numbers, the step of increasing the growth surface area can be repeated one or more times; i.e., upon reaching a specific cell density, cell number, and / or expansion fold for the cells, as defined elsewhere herein, the growth surface can be increased again by 2-4 fold. Thus, in one embodiment, the present invention includes one or more steps of increasing the growth surface area of ​​the culture environment.

[0013] As shown in Examples 1 and 2, a bioreactor system can be used in accordance with the methods of the present invention, i.e., MPCs can be cultured in a bioreactor to obtain large-scale cultures of MPCs. Thus, in one embodiment, the container used in accordance with the present invention is a closed bioreactor. In a preferred embodiment, the container is a bioreactor bag. In a further embodiment, the container used in accordance with the methods of the present invention is an expandable container. In a preferred embodiment, the container is an expandable bioreactor bag.

[0014] As shown by flow cytometry analysis in Example 3, the methods of the present invention result in populations containing MPCs, i.e., MPCs expressing myogenic markers. As further explained below, such populations also contain other cells adjacent to the MPCs at various stages of muscle differentiation, e.g., cells of earlier lineages, resulting in a heterogeneous population. Furthermore, the cultured cells exhibit similar characteristics to MPCs generated using the methods disclosed in WO 2019 / 215090, particularly with respect to characteristics essential for therapeutic utility, e.g., high expression of Pax7 and α-actinin, and low expression of CD34, confirming that the cells are therapeutically useful, as described below.

[0015] According to the method of the present invention, microcarriers are used that allow the cultivation of adherent cells in suspension and provide a large growth area available for cell proliferation. As explained above, this is advantageous over the traditional cultivation of MPCs in monolayer culture, since the yield limiting factor for large-scale adherent cell cultures is the limited growth area in 2D culture systems. Thus, in one embodiment, the microcarriers are coated microcarriers, preferably collagen-coated microcarriers. In one embodiment, the microcarriers are dissolvable, and in a preferred embodiment, the microcarriers used according to the present invention are collagen-coated and dissolvable.

[0016] In one embodiment, the culture medium used in accordance with the methods of the invention comprises human platelet lysate (hPL).

[0017] In one embodiment, the culture medium used in accordance with the methods of the invention comprises human platelet lysate (hPL), preferably fibrinogen-depleted hPL, and lacks heparin, and therefore lacks potentially allergenic components, such as serum or heparin, used in conventional growth media.

[0018] In one embodiment, the culture medium used in accordance with the methods of the invention comprises human platelet lysate (hPL) and lacks heparin, and therefore lacks potentially allergenic components, such as serum or heparin, used in conventional growth media.

[0019] For downstream applications, it is important that the cells themselves are available without adhering to the microcarriers. Therefore, in one embodiment, the method of the present invention includes a step of separating the MPCs from the microcarriers at the end of the culture. In a preferred embodiment, the separation includes complete dissolution of the microcarriers, which is preferably carried out by enzymatic digestion, preferably by adding TrypLE® and pectinase.

[0020] The present invention further relates to cell populations comprising MPCs obtainable by the methods of the invention disclosed herein.

[0021] As shown by flow cytometry analysis in Example 3, culture yields a population containing MPCs, i.e., MPCs expressing myogenic markers. Such a population is heterogeneous and includes other cells at various stages of muscle differentiation, e.g., cells of earlier lineages, adjacent to MPCs. Thus, the presence of cells at various stages of muscle differentiation contributes to the proportion of cells expressing specific marker genes shown in Example 3 and Figures 4 and 5. As seen in Figures 3 and 4, culture in a bioreactor yielded MPCs expressing myogenic markers, with approximately 99% of the cells being positive for Pax7, α-actinin, and A2B5, and negative for CD34 expression.

[0022] In one embodiment, more than 40% of the cells of the population express α-actinin, preferably more than 50%, preferably more than 60%, preferably more than 65%, preferably more than 70%, preferably more than 75%, preferably more than 80%, preferably more than 85%, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99% express α-actinin, and / or more than 60% of the cells of the population express Pax7, preferably more than 65%, preferably more than 70%, preferably more than 75%, preferably more than 80%, preferably More than 85%, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99% express Pax7 and / or less than 20% of the cells in the population express CD34, preferably less than 15%, preferably less than 10%, preferably less than 8%, preferably less than 7.5%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1.5%, preferably less than 1%, preferably less than 0.5%, preferably less than 0.25% of the cells express CD34.

[0023] In one embodiment, more than 40% of the cells of the population express α-actinin, preferably more than 50%, preferably more than 60%, preferably more than 65%, preferably more than 70%, preferably more than 75%, preferably more than 80%, preferably more than 85%, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99% express α-actinin, and more than 60% of the cells of the population express Pax7, preferably more than 65%, preferably more than 70%, preferably more than 75%, preferably more than 80%, preferably More than 85%, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99% express Pax7 and less than 20% of the cells in the population express CD34, preferably less than 15%, preferably less than 10%, preferably less than 8%, preferably less than 7.5%, preferably less than 5%, preferably less than 4%, preferably less than 3%, preferably less than 2%, preferably less than 1.5%, preferably less than 1%, preferably less than 0.5%, preferably less than 0.25% of the cells express CD34.

[0024] In a preferred embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, and ≦15% of the cells in the population express CD34, i.e., the population comprises ≧50% α-actinin-positive cells, ≧60% Pax7-positive cells, and ≦15% CD34-positive cells (≦15% of the population expresses CD34).

[0025] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, and ≦5% of the cells in the population express CD34, i.e., the population comprises ≧80% α-actinin-positive cells, ≧80% Pax7-positive cells, and ≦5% CD34-positive cells.

[0026] The populations of the invention may be further characterized by expression of A2B2. Thus, in one embodiment, more than 50%, preferably more than 60%, preferably more than 65%, preferably more than 70%, preferably more than 75%, preferably more than 80%, preferably more than 85%, preferably more than 90%, preferably more than 95%, preferably more than 98%, preferably more than 99% of the cells of the population express A2B5.

[0027] Thus, in one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≧60% of the cells in the population express A2B5, and ≦15% of the cells in the population express CD34, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≧60% A2B5 positive cells, and ≦15% CD34 positive cells (≦15% of the population expresses CD34).

[0028] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≧80% of the cells in the population express A2B5, and ≦5% of the cells in the population express CD34, i.e., the population comprises ≧80% α-actinin-positive cells, ≧80% Pax7-positive cells, ≧80% A2B5-positive cells, and ≦5% CD34-positive cells.

[0029] Additionally or alternatively, the populations of the present invention are further characterized by comprising cells that express desmin, and preferably 1% to 99%, preferably 10% to 90%, or 20% to 80%, preferably up to 75%, preferably up to 70%, preferably 20% to 70%, or up to 60% of the cells in the population express desmin.

[0030] Thus, in one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≦15% of the cells in the population express CD34, and the cells express desmin, preferably ≧10%, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, and ≦15% CD34 positive cells (≦15% of the population expresses CD34), and the population comprises cells that express desmin, preferably ≧10%.

[0031] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≦5% of the cells in the population express CD34, and the cells express desmin, preferably ≧10% desmin, i.e., the population comprises ≧80% α-actinin positive cells, ≧80% Pax7 positive cells, and ≦5% CD34 positive cells, and the population comprises cells expressing desmin, preferably ≧10%.

[0032] In one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≧60% of the cells in the population express A2B5, ≦15% of the cells in the population express CD34, and the cells express desmin, preferably ≧10%, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≧60% A2B5 positive cells, and ≦15% CD34 positive cells (≦15% of the population expresses CD34), and the population comprises cells that express desmin, preferably ≧10%.

[0033] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≧80% of the cells in the population express A2B5, ≦5% of the cells in the population express CD34, and the cells express desmin, preferably ≧10% desmin, i.e., the population comprises ≧80% α-actinin positive cells, ≧80% Pax7 positive cells, ≧80% A2B5 positive cells, and ≦5% CD34 positive cells, and the population comprises cells expressing desmin, preferably ≧10%.

[0034] Furthermore, as can be seen in Figure 5 , in addition to showing typical myogenic markers α-actinin and A2B5 (99.9% and 99.7%, respectively) and negative (0.1%) CD34, cells cultured in bioreactors were also positive for the expression of Myf5, myHC, and MyoD (67.6%, 8.7%, and 19.6%, respectively), and showed very low expression of CD56 (3.3%).

[0035] Thus, the populations of the present invention may be further characterized by comprising greater than 15% CD56-positive cells, 50% Myf5-positive cells, 30% MyHC-positive cells, and / or 10-40% MyoD-positive cells; preferably, the cell population comprises 10% CD56-positive cells, 60% Myf5-positive cells, 20% MyHC-positive cells, and / or 10-30% MyoD-positive cells; and most preferably, the cell population comprises 5% CD56-positive cells, 60-90% Myf5-positive cells, 15% MyHC-positive cells, and / or 15-25% MyoD-positive cells.

[0036] More specifically: The populations of the invention may be further characterized by expression of MyHC. In particular, in one embodiment, between 0% and 29% of the cells of the population express MyHC, and thus in one embodiment, <29% of the cells of the population express MyHC, preferably <25%, preferably <20%, preferably <15%, and more preferably <10% of the cells express MyHC.

[0037] Thus, in one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≦15% of the cells in the population express CD34, and ≦29% of the cells in the population express MyHC, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≦15% CD34 positive cells, and ≦29% MyHC positive cells.

[0038] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≦5% of the cells in the population express CD34, and ≦15% of the cells express MyHC, i.e., the population comprises ≧80% α-actinin positive cells, ≧80% Pax7 positive cells, ≦5% CD34 positive cells, and ≦15% MyHC positive cells.

[0039] In one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≧60% of the cells in the population express A2B5, ≦15% of the cells in the population express CD34, and ≦29% of the cells in the population express MyHC, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≧60% A2B5 positive cells, ≦15% CD34 positive cells, and ≦29% MyHC positive cells.

[0040] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≧80% of the cells in the population express A2B5, ≦5% of the cells in the population express CD34, and ≦15% of the cells express MyHC, i.e., the population comprises ≧80% α-actinin positive cells, ≧80% Pax7 positive cells, ≧80% A2B5 positive cells, ≦5% CD34 positive cells, and ≦15% MyHC positive cells.

[0041] Preferably, the cells of the populations of the present invention further express desmin, as set out above.

[0042] The populations of the present invention may be further characterized by expression of MyoD. In particular, in one embodiment, 10% to 40%, preferably 10% to 30%, preferably 15% to 30%, and more preferably 15% to 25% of the cells in the population express MyoD.

[0043] Thus, in one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≦15% of the cells in the population express CD34, and 10%-40% of the cells in the population express MyoD, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≦15% CD34 positive cells, and 10%-40% MyoD positive cells.

[0044] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≦5% of the cells in the population express CD34, and 10%-30% of the cells in the population express MyoD, i.e., the population comprises ≧80% α-actinin-positive cells, ≧80% Pax7-positive cells, ≦5% CD34-positive cells, and 10%-30% MyoD-positive cells.

[0045] In one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≧60% of the cells in the population express A2B5, ≦15% of the cells in the population express CD34, and 10%-40% of the cells in the population express MyoD, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≧60% A2B5 positive cells, ≦15% CD34 positive cells, and 10%-34% MyoD positive cells.

[0046] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≧80% of the cells in the population express A2B5, ≦5% of the cells in the population express CD34, and 10%-30% of the cells in the population express MyoD, i.e., the population comprises ≧80% α-actinin-positive cells, ≧80% Pax7-positive cells, ≧80% A2B5-positive cells, ≦5% CD34-positive cells, and 10%-30% MyoD-positive cells.

[0047] In one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≦15% of the cells in the population express CD34, ≦29% of the cells in the population express MyHC, and 10%-40% of the cells in the population express MyoD, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≦15% CD34 positive cells, ≦29% MyHC positive cells, and 10%-40% MyoD positive cells.

[0048] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≦5% of the cells in the population express CD34, ≦15% of the cells express MyHC, and 10%-30% of the cells in the population express MyoD, i.e., the population contains ≧80% α-actinin positive cells, ≧80% Pax7 positive cells, ≦5% CD34 positive cells, ≦15% MyHC positive cells, and 10%-30% MyoD positive cells.

[0049] In one embodiment, ≧50% of the cells in the population express α-actinin, ≧60% of the cells in the population express Pax7, ≧60% of the cells in the population express A2B5, ≦15% of the cells in the population express CD34, ≦29% of the cells in the population express MyHC, and 10%-40% of the cells in the population express MyoD, i.e., the population comprises ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≧60% A2B5 positive cells, ≦15% CD34 positive cells, ≦29% MyHC positive cells, and 10%-40% MyoD positive cells.

[0050] More preferably, ≧80% of the cells in the population express α-actinin, ≧80% of the cells in the population express Pax7, ≧80% of the cells in the population express A2B5, ≦5% of the cells in the population express CD34, ≦15% of the cells express MyHC, and 10%-30% of the cells in the population express MyoD, i.e., the population contains ≧80% α-actinin-positive cells, ≧80% Pax7-positive cells, ≧80% A2B5-positive cells, ≦5% CD34-positive cells, ≦15% MyHC-positive cells, and 10%-30% MyoD-positive cells.

[0051] Preferably, the cells of the populations of the present invention further express desmin, as set out above.

[0052] The populations of the invention may be further characterized by expression of CD56. In particular, in one embodiment, 0% to 15%, preferably 5% to 15%, of the cells of the population express CD56, and thus in one embodiment, ≦15% of the cells of the population express CD56, preferably ≦10%, more preferably ≦5% of the cells express CD56.

[0053] Thus, in one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, and ≦15% of the cells in the population express CD56.

[0054] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, ≧60%, preferably ≧80%, of the cells in the population express A2B5, and ≦15% of the cells in the population express CD56.

[0055] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, 10%-40%, preferably 10%-30%, of the cells in the population express MyoD, and ≦15% of the cells in the population express CD56.

[0056] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≧60%, preferably ≧80%, of the cells in the population express A2B5, ≦15%, preferably ≦5%, of the cells in the population express CD34, 10% to 40%, preferably 10% to 30%, of the cells in the population express MyoD, and ≦15% of the cells in the population express CD56.

[0057] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, ≦29%, preferably ≦15%, of the cells in the population express MyHC, and ≦15% of the cells in the population express CD56.

[0058] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, ≧60%, preferably ≧80%, of the cells in the population express A2B5, ≦29%, preferably ≦15%, of the cells in the population express MyHC, and ≦15% of the cells in the population express CD56.

[0059] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, 10% to 40%, preferably 10% to 30%, of the cells in the population express MyoD, ≦29%, preferably ≦15%, of the cells in the population express MyHC, and ≦15% of the cells in the population express CD56.

[0060] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≧60%, preferably ≧80%, of the cells in the population express A2B5, ≦15%, preferably ≦5%, of the cells in the population express CD34, 10% to 40%, preferably 10% to 30%, of the cells in the population express MyoD, ≦29%, preferably ≦15%, of the cells in the population express MyHC, and ≦15% of the cells in the population express CD56.

[0061] Preferably, the cells of the populations of the present invention further express desmin, as set out above.

[0062] The populations of the present invention may be further characterized by expression of Myf5. In particular, in one embodiment, ≥ 50% of the cells of the population express Myf5, preferably ≥ 60% of the cells of the population express Myf5, preferably 60%-89%, more preferably 65%-89% of the cells of the population express Myf5.

[0063] Thus, in one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0064] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, ≧60%, preferably ≧80%, of the cells in the population express A2B5, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0065] In one embodiment, ≧50%, preferably ≧80% of the cells in the population express α-actinin, ≧60%, preferably ≧80% of the cells in the population express Pax7, ≦15%, preferably ≦5% of the cells in the population express CD34, 10%-40%, preferably 10%-30% of the cells in the population express MyoD, and ≦50% of the cells in the population express Myf5, preferably 60%-89% of the cells in the population express Myf5.

[0066] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, ≦29%, preferably ≦15%, of the cells in the population express MyHC, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0067] In one embodiment, ≧50% of the cells in the population, preferably ≧80% express α-actinin, ≧60% of the cells in the population, preferably ≧80% express Pax7, ≦15% of the cells in the population, preferably ≦5% express CD34, ≦15% of the cells in the population express CD56, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0068] In one embodiment, ≧50%, preferably ≧80% of the cells in the population express α-actinin, ≧60%, preferably ≧80% of the cells in the population express Pax7, ≧60%, preferably ≧80% of the cells in the population express A2B5, ≦15%, preferably ≦5% of the cells in the population express CD34, 10% to 40%, preferably 10% to 30% of the cells in the population express MyoD, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0069] In one embodiment, ≧50%, preferably ≧80% of the cells in the population express α-actinin, ≧60%, preferably ≧80% of the cells in the population express Pax7, ≧60%, preferably ≧80% of the cells in the population express A2B5, ≦15%, preferably ≦5% of the cells in the population express CD34, ≦29%, preferably ≦15% of the cells in the population express MyHC, ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0070] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≧60%, preferably ≧80%, of the cells in the population express A2B5, ≦15%, preferably ≦5%, of the cells in the population express CD34, ≦15% of the cells in the population express CD56, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0071] In one embodiment, ≧50%, preferably ≧80%, of the cells of the population express α-actinin, ≧60%, preferably ≧80%, of the cells of the population express Pax7, ≦15%, preferably ≦5%, of the cells of the population express CD34, 10% to 40%, preferably 10% to 30%, of the cells of the population express MyoD, ≦29%, preferably ≦15%, of the cells of the population express MyHC, and ≦50% of the cells of the population express Myf5, preferably 60% to 89% of the cells of the population express Myf5.

[0072] In one embodiment, ≧50%, preferably ≧80% of the cells of the population express α-actinin, ≧60%, preferably ≧80% of the cells of the population express Pax7, ≧60%, preferably ≧80% of the cells of the population express A2B5, ≦15%, preferably ≦5% of the cells of the population express CD34, 10% to 40%, preferably 10% to 30% of the cells of the population express MyoD, ≦29%, preferably ≦15% of the cells of the population express MyHC, and ≦50% of the cells of the population express Myf5, preferably 60% to 89% of the cells of the population express Myf5.

[0073] In one embodiment, ≧50%, preferably ≧80%, of the cells in the population express α-actinin, ≧60%, preferably ≧80%, of the cells in the population express Pax7, ≦15%, preferably ≦5%, of the cells in the population express CD34, 10% to 40%, preferably 10% to 30%, of the cells in the population express MyoD, ≦15% of the cells in the population express CD56, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0074] In one embodiment, ≧50%, preferably ≧80% of the cells in the population express α-actinin, ≧60%, preferably ≧80% of the cells in the population express Pax7, ≧60%, preferably ≧80% of the cells in the population express A2B5, ≦15%, preferably ≦5% of the cells in the population express CD34, 10% to 40%, preferably 10% to 30% of the cells in the population express MyoD, ≦15% of the cells in the population express CD56, and ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0075] In one embodiment, ≧50%, preferably ≧80% of the cells in the population express α-actinin, ≧60%, preferably ≧80% of the cells in the population express Pax7, ≧60%, preferably ≧80% of the cells in the population express A2B5, ≦15%, preferably ≦5% of the cells in the population express CD34, ≦29%, preferably ≦15% of the cells in the population express MyHC, ≦15% of the cells in the population express CD56, ≦50% of the cells in the population express Myf5, preferably 60% to 89% of the cells in the population express Myf5.

[0076] In one embodiment, ≧50%, preferably ≧80%, of the cells of the population express α-actinin, ≧60%, preferably ≧80%, of the cells of the population express Pax7, ≦15%, preferably ≦5%, of the cells of the population express CD34, 10% to 40%, preferably 10% to 30%, of the cells of the population express MyoD, ≦15% of the cells of the population express CD56, ≦29%, preferably ≦15% of the cells of the population express MyHC, and ≦50% of the cells of the population express Myf5, preferably 60% to 89% of the cells of the population express Myf5.

[0077] In one embodiment, ≧50%, preferably ≧80% of the cells of the population express α-actinin, ≧60%, preferably ≧80% of the cells of the population express Pax7, ≧60%, preferably ≧80% of the cells of the population express A2B5, ≦15%, preferably ≦5% of the cells of the population express CD34, 10% to 40%, preferably 10% to 30% of the cells of the population express MyoD, ≦15% of the cells of the population express CD56, ≦29%, preferably ≦15% of the cells of the population express MyHC, and ≦50% of the cells of the population express Myf5, preferably 60% to 89% of the cells of the population express Myf5.

[0078] Preferably, the cells of the populations of the present invention further express desmin, as set out above.

[0079] As described above, the populations of the present invention may be characterized by comprising ≥50%, preferably ≥80%, of the cells expressing α-actinin, ≥60%, preferably ≥80%, of the cells expressing Pax7, and ≤15%, preferably ≤5%, of the cells expressing CD34.

[0080] In one embodiment, the population may be further characterized as comprising ≦29%, preferably ≦15% of cells that express MyHC.

[0081] Low expression of MyHC, a contractile protein marker that is expressed in more differentiated cell populations, indicates that the majority of cells are in an early stage, i.e., not differentiated.

[0082] Additionally or alternatively, the population may be further characterized by comprising cells that express MyoD, preferably comprising 10% to 40%, preferably 10% to 30%, of the cells that express MyoD. The population may be further characterized by comprising 60% to 89% of the cells that express Myf5.

[0083] Quiescent satellite cells are characterized by the expression of Pax7 and the absence of MyoD expression, whereas activated satellite cells express MyoD and / or Myf5. Thus, as can be derived from marker expression data, e.g., the presence of Pax7 and MyoD / Myf5 positive cells, the populations of the present invention contain a mixture of activated satellite cells, which can still dedifferentiate into dormant satellite cells to replenish the pool for potential future muscle injury.

[0084] Additionally or alternatively, the population is further characterized by comprising <15% cells expressing CD56. Low expression of CD56, a pure myoblast marker, indicates that the population is in an early differentiation state.

[0085] The population may be further characterized as comprising >60%, preferably >80% cells that express A2B5. Optionally, but preferably, the population further comprises cells that express desmin.

[0086] In a preferred embodiment, the cell population of the present invention preferably comprises at least 1 x 10 6 MPCs, preferably at least 1 x 10 7 In a preferred embodiment, the population comprises at least 1-3 x 10 MPCs. 8 Includes MPCs.

[0087] The invention also encompasses a method of preparing a pharmaceutical product, comprising the steps of the method for obtaining large scale cultures of MPCs of the invention disclosed herein, and optionally adding a biomaterial solution, preferably a hydrogel solution, more preferably a collagen solution, to the harvested MPCs, most preferably at a final concentration of 1-4 mg / mL, preferably 2 mg / mL. In one embodiment, the method further comprises filling the MPCs into a pharmaceutical container, preferably a syringe or vial.

[0088] Furthermore, the present invention relates to a composition comprising MPCs obtainable by the method for obtaining large-scale cultures of MPCs of the present invention disclosed herein. According to the present invention, the composition is used as a pharmaceutical. In a preferred embodiment, the present invention relates to a composition for use in treating muscle dysfunction, preferably, the muscle dysfunction is skeletal muscle dysfunction, more preferably, the skeletal muscle dysfunction is an abnormality of a sphincter, preferably the external urethral sphincter. Optionally, the composition of the present invention further comprises a biomaterial solution, preferably a hydrogel solution, more preferably a collagen solution, which is preferably mixed with MPCs, most preferably at the concentrations indicated above.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0090] Further embodiments of the present invention will become apparent from the following description and examples.

[0091] To avoid any doubt, expressions such as "in some embodiments," "in particular embodiments," "in particular cases," "optionally," "in a further embodiment," "in one embodiment," etc. are used, and it is emphasized that any of the embodiments described therein should be read with the combination of each of the features of those embodiments in mind, and that the present disclosure should be treated in the same manner as if that combination of features of those embodiments were recited in one embodiment. The same applies to any combination of embodiments and features in the appended claims, as well as any combination of embodiments and features shown in the examples, which are also intended to be combined with features from the corresponding embodiments disclosed in the description; only for the sake of consistency and conciseness, the embodiments are characterized by dependencies, but in fact, each embodiment and combination of features that can be interpreted due to dependency(s) must be considered as literally disclosed and not as a selection among various alternatives.

[0092] The term "between" is inclusive of the endpoints. [Brief explanation of the drawings]

[0093] [Figure 1]Quantification of cultured MPCs (passage 3). Growth curves for the cell numbers shown in Table 1, i.e., adjusted to account for biomass loss due to sampling, are shown in Figure 1. [Figure 2] Visual inspection of cultured MPCs at 40x and 100x magnification. [Figure 3] Flow cytometry analysis of MPCs cultured according to the invention compared to cultures in monolayer flasks where cells were cultured in the presence of 5% (A) and 10% (B) hPL. Expression of the markers Pax7, α-actinin and CD34 was analyzed. [Figure 4] Flow cytometry analysis of MPCs cultured according to the invention compared to culture in monolayer flasks. Expression of the markers Pax7, α-actinin, CD34 and A2B5 was analyzed. [Figure 5] Flow cytometry analysis of MPCs cultured according to the invention compared to culture in monolayer flasks. Expression of the markers α-actinin, A2B5, CD34, CD56, Myf5, MyHC and MyoD was analyzed. DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention relates to a method for obtaining muscle precursor cells (MPCs), preferably comprising culturing and expanding MPCs in a 3D culture system. More specifically, the method of the present invention relates to a method for obtaining a population comprising a therapeutically effective amount of MPCs, as defined below. In particular, the present invention relates to a method for obtaining MPCs, comprising expanding MPCs on microcarriers in suspension culture. Microcarriers are particles with a high surface-to-volume ratio. The surface provided by the microcarriers serves as a culture support for adherent cells, allowing them to be efficiently expanded in a small culture volume. According to the present invention, MPCs are cultured in a growth medium under conditions that allow the MPCs to attach to the microcarriers and expand thereon. When a certain density of cells attached to the microcarriers is reached, the growth surface provided by the microcarriers is increased. The cells are cultured until a desired cell number is reached.

[0095] Each type of cell requires different culture conditions, and a process established for one cell type usually cannot be used for another cell type; a new process must be established. Furthermore, the culture conditions for obtaining large-scale cultures are unpredictable. For example, spinner flask cultures have been used to grow MPCs in culture media containing microcarriers. In microcarrier-based expansion, cells must be transferred from one microcarrier to another, and this process requires maintaining a homogeneous distribution of cells on the microcarriers to achieve a high cell yield. In this context, a cell density of approximately 5,000 cells / cm is required. 2 A seeding density of only 500-1500 cells / cm was found to be optimal for reaching high cell yields. See the poster by Burer et al., "Optimization of Microcarrier-based Culture of Muscle Precursor Cells," Scinus Cell Expansion. Therefore, only 500-1500 cells / cm are used according to the present invention. 2 It is surprising that a seeding density as low as 1000 μg / ml is already sufficient to provide sufficient cell proliferation, resulting in high yields.

[0096] The culture systems used in accordance with the present invention, such as those described in Example 1, are specifically designed and adapted to grow and expand MPCs. In particular, the seeding density, i.e., the initial concentration of cells in the culture medium and their ratio to the growth surface provided by the microcarriers, has been found to be critical for successful culture. Thus, seeding is typically performed at 500-1500 cells / cm. 2 at a density of preferably 800-1200 cells / cm 2 and most preferably at a density of about 900 cells / cm 2 Therefore, in the most preferred embodiment, the cell concentration for inoculation is about 7500 cells / mL and the microcarrier concentration is 1.7 g / L. This seeding density is about 10 5 ~106 cells, preferably 10 6 This is particularly suitable when 1 x 10 cells are used to inoculate 130 mL of culture medium. The seeding density is specifically adapted to work well with the intended MPC protocol, taking into account the low donor cell number. Another important factor is expansion, and the cell density at which cells can be successfully expanded varies for each cell type. Therefore, a range of cell densities at which MPCs can be successfully expanded on microcarriers has been established, preferably 1.3 x 10 cells, which corresponds to an approximately 8- to 25-fold increase in cell number. 4 ~1.8×10 4 cells / cm 2 (i.e., 1.1×10 5 ~1.5×10 5 The surface area preferably increases by about three times when a density of 1000 cells / mL is reached and / or when more than 80%, preferably 90%, of the microcarriers are occupied. This occurs after about three days. Termination of the culture is a further important parameter. In particular, the culture is terminated when a cell density is reached, which provides sufficient cells for further applications, such as therapeutic applications, as described below. Preferably, a maximum of 5-7.5 x 10 4 cells / cm 2 (4~6.5×10 5 cells / ml), preferably up to 6.7 × 10 4 cells / cm 2 (5.7×10 5 A cell density of 1.3 x 10 cells / mL has been reached. This occurs after approximately 6 days of culture. More than 80%, preferably 90%, of the microcarriers are occupied, or a cell density of 1.3 x 10 cells / mL has been reached. 4 ~1.8×10 4 cells / cm 2 (i.e., 1.1×10 5 ~1.5×10 5 Upon reaching a cell density of about 1000 cells / mL, more cells can be obtained by a further expansion step, i.e., by further increasing the growth area, preferably by about 3-fold. Those skilled in the art will understand that such expansion steps can be further repeated each time such occupancy and / or cell density is reached.

[0097] muscle progenitor cells As used herein, the term "muscle progenitor cells" or "MPCs" or simply "cells" (unless otherwise indicated) refers to a pool of any muscle-derived progenitor cells that express muscle-specific markers and can give rise to new muscle fibers, as defined, for example, by Eberli et al., Methods 47 (2009), 98-103. MPCs are also called proliferating satellite cells. The terms "population of MPCs" or "population containing MPCs" or "MPCs" mean that MPCs are the predominant cell type in the population. However, a population of MPCs may contain other cell types besides MPCs; i.e., a population of MPCs preferably contains at least 60% MPCs, at least 65% MPCs, at least 70% MPCs, at least 75% MPCs, at least 80% MPCs, at least 85% MPCs, at least 90% MPCs, at least 95% MPCs, at least 98% MPCs, at least 99% MPCs, or about 100% MPCs. For example, a population of MPCs may contain myofibroblasts in addition to MPCs and still be considered a population of MPCs according to this definition. In addition, if myogenic markers can be detected as described herein, i.e., for example, if α-actinin, Pax7, A2B5 are present and CD34 is absent, the cell population is considered to be a population of MPCs. For more information regarding marker expression, see the section "Cell Populations and Therapeutic Aspects" herein.

[0098] The MPCs cultured according to and obtained using the methods of the present invention can be derived from muscle tissue of any species, preferably mammals, more preferably domestic animals, i.e., pets or livestock (livestock), or humans. Pets include, but are not limited to, dogs, cats, rabbits, guinea pigs, hamsters, and horses. Livestock include, but are not limited to, cattle, cows, pigs, sheep, goats, donkeys, camels, water buffalo, and elephants. Most preferably, the methods of the present invention are used to obtain human MPCs (hMPCs). Accordingly, the present invention relates to a method for obtaining large-scale cultures of MPCs, wherein the MPCs are preferably mammalian MPCs, more preferably domestic animal MPCs, such as pet MPCs or livestock MPCs as defined above, most preferably human MPCs (hMPCs). The MPCs are preferably derived from skeletal muscle, and are preferably obtained from healthy muscle tissue, preferably from tissue selected from the group consisting of soleus, rectus abdominis, quadriceps, vastus lateralis, and vastus intermedius. If the MPCs obtained by the methods of the present invention are intended to be used for the treatment of skeletal muscle dysfunction as outlined in the section "Cell Populations and Therapeutic Aspects" below, those skilled in the art will readily recall that biopsies are taken from healthy muscles with a similar structure to the target muscle, i.e., the injured muscle being treated. For example, slow-twitch muscle fibers are similar to sphincter muscles, and the soleus muscle primarily contains such slow-twitch muscle fibers. Thus, in one embodiment of the present invention, MPCs are obtained from slow-twitch muscle fibers, preferably from the soleus muscle (of the left or right leg), which is similar in composition to sphincter muscles and easily accessible. Alternatively, vastus lateralis can be used. Thus, the MPCs obtained by the methods of the present invention are preferably slow-twitch muscle fiber-derived MPCs, preferably soleus muscle-derived MPCs, rectus abdominis muscle-derived MPCs, quadriceps muscle-derived MPCs, vastus lateralis muscle-derived MPCs, or vastus intermedius muscle-derived MPCs, and most preferably soleus muscle-derived MPCs or vastus lateralis muscle-derived MPCs, particularly soleus muscle-derived MPCs. Depending on the muscle targeted by the MPCs of the present invention, for example, if the target muscle is a fast-twitch muscle, the biopsy may be taken from a fast-twitch muscle.

[0099] The MPCs cultured according to the methods of the present invention to obtain corresponding mass cultures can be obtained by various methods. A preferred method for isolating MPCs is described in WO 2019 / 115790, and in the methods of the present invention, these cells can be used as inoculation material, and thus the methods of the present invention can be used to obtain mass cultures of these cells. In a preferred embodiment, the MPCs cultured according to the methods of the present invention are isolated as described in WO 2019 / 215090, the contents of which are incorporated herein by reference, in particular Example 1.

[0100] Thus, in a preferred embodiment, to isolate MPCs, a muscle biopsy is taken from muscle tissue, preferably skeletal muscle, more preferably from predominantly slow-twitch or fast-twitch tissue, preferably slow-twitch, most preferably selected from the non-limiting group consisting of soleus, rectus abdominis, quadriceps, vastus lateralis, and vastus intermedius. In a further preferred embodiment, the biopsy is taken from the soleus or vastus lateralis, most preferably from the soleus.

[0101] In one specific embodiment, adipose tissue, tendon tissue, and / or connective tissue are removed from a human tissue sample, the biopsy material is cut into small pieces, preferably using scissors, to obtain a viscous mixture, which is then digested with one or more enzymes, preferably a mixture containing collagenase and dispase, to break down the tissue. Preferably, a mixture of about 0.05% to 2%, more preferably about 0.2%, collagenase type I (w / v) and about 0.1% to 2%, more preferably about 0.4% to 1.6%, dispase (w / v) is used. The enzymatic reaction is preferably carried out at 36-38°C for 15-75 minutes, preferably 45-75 minutes. Once the desired degree of digestion is reached, digestion is preferably terminated by the addition of cell culture medium, i.e., growth medium, as defined herein. See the section "Culture Medium" below.

[0102] In one embodiment, the step of cutting the biopsy is preceded by the steps of disinfecting the biopsy using a disinfectant and washing with PBS.

[0103] After adding the growth medium, the digest is mixed, preferably by pipetting, and centrifuged. After centrifugation, the pellet is resuspended in the growth medium, preferably by pipetting up and down. In one embodiment, the growth medium contains 1% penicillin / streptomycin, preferably 1% (supplemented only for this passage 0 step). In one embodiment, the growth medium does not contain penicillin / streptomycin. In an alternative embodiment, the growth medium contains one or more antibiotics other than penicillin or streptomycin, such as gentamicin. The cell suspension is filtered, preferably through a strainer with a pore size of 100 μm. The cells are then seeded onto a coated dish, in particular, the cell suspension is transferred to a culture plate, such as a 35 mm dish (6 wells), coated with an extracellular matrix protein, such as collagen, fibronectin, or laminin, preferably collagen, most preferably type I collagen.

[0104] In one embodiment of the present invention, a plate for culturing cells obtained from muscle biopsies is coated with a collagen solution, preferably type I collagen, at a concentration of about 0.03 to 1.5 mg / ml, preferably about 0.05 to 1 mg / ml, and more preferably 0.05 mg / ml. The collagen solution is transferred to the culture plate so that the bottom of the well is covered with the solution. The collagen solution is then removed, and the coated plate is washed three times with PBS.

[0105] As used herein, the term "collagen-coated plate" or "plate" is not limited to a culture plate, but also generally includes a culture dish suitable for culturing cells as a monolayer, such as a cell culture flask. Alternatively, in a further embodiment of the present invention, cells expanded from a biopsy are cultured as a multilayer, for example, in a multilayer flask or any other 2D culture system, or in any 3D culture system, for example, on microcarriers in a spinner flask.

[0106] As outlined above, cells are seeded onto plates coated with extracellular matrix proteins such as fibronectin or collagen. The cells are then incubated under appropriate culture conditions, preferably at 36-38°C and 5% CO2, for approximately 20-28 hours, preferably 24 hours. The supernatant containing non-adherent cells, primarily MPCs, is then replated onto dishes coated with extracellular matrix proteins, such as collagen or fibronectin, preferably collagen, most preferably type I collagen, to reduce the number of myofibroblasts. The plates are coated as outlined above. The MPCs are allowed to settle onto the coated dishes, thereby obtaining a population containing MPCs, preferably human MPCs. These cells are considered passage 0 (P0) MPCs.

[0107] The growth medium is preferably changed first after 2-4 days, and then every 2-4 days. Sufficient cells to seed a large-scale culture are obtained at P0, i.e., approximately 10 5 ~10 6 cells, preferably 10 6 If the cells have already been obtained, the MPCs, i.e., the population comprising MPCs, are transferred directly to a vessel for mass culture. Thus, in one embodiment, the MPCs cultured according to the methods of the present invention are preferably passage 0 (P0) cells obtained as described herein above.

[0108] Otherwise, the MPCs are split. To split, the MPCs are washed with PBS and enzymatically detached from the plate, preferably using an enzyme such as trypsin or TrypLE®, according to standard protocols. Growth medium is then added, and the MPCs are centrifuged to a density of 3,000-7,000 cells / cm. 2In one embodiment of the present invention, MPCs are seeded onto plates coated with extracellular matrix proteins, such as collagen or fibronectin. In an alternative embodiment, MPCs are seeded onto plates not coated with extracellular matrix proteins. These P1 cells are cultured with growth medium changes every 2-3 days. The cells are then split again or used to seed the larger-scale culture system defined herein. When the cells are detached from the plate after centrifugation and resuspension in growth medium, they are typically counted, including determining cell viability. If necessary, cells can be frozen according to standard protocols before larger-scale culture, e.g., for storage. If frozen, they are typically cultured as a monolayer for one passage before larger-scale culture.

[0109] Thus, in one embodiment, the MPCs cultured according to the methods of the present invention are first passage (P1) cells, preferably obtained as described herein above.

[0110] Optionally, the MPCs are replated for further cell expansion; preferably, the cells are replated onto coated, particularly collagen-coated, dishes and cultured in growth medium. In an alternative preferred embodiment, the MPCs are replated onto uncoated dishes. Thus, in another embodiment of the present invention, the MPCs cultured in the methods of the present invention are second passage (P2) or third passage (P3) MPCs, preferably obtained as described herein above. In a preferred embodiment, the MPCs are P1 or P2 cells, more preferably P1 MPCs.

[0111] When MPCs are not directly transferred to a mass culture system but are frozen and stored, for example, in liquid nitrogen, particularly in the vapor phase of liquid nitrogen, the MPCs are typically cultured as a monolayer for one passage on a culture dish before being seeded in a mass culture system. Thus, in one embodiment of the present invention, cells obtained from a biopsy material are, for example, frozen for storage, and then thawed and cultured as a monolayer for one passage. In an alternative embodiment, the thawed cells are seeded directly into a mass culture system.

[0112] Mass culture The terms "mass culture" or "mass cultivation" refer to the expansion of cells to obtain a sufficient quantity of cells for a desired downstream application. Of course, the number of cells required for a particular downstream application will vary and will be known or readily apparent to those skilled in the art.

[0113] The method according to the invention is illustratively carried out as described in Examples 1 and 2, for example, to obtain a total of about 2.8×10 8 The method of the present invention results in MPCs with a specific myogenic marker profile, including the presence of Pax7, α-actinin, and desmin, and the absence of CD34, as shown in Example 3. This means that sufficient cells with the necessary marker expression can be obtained for cell therapy procedures, such as the preparation of the pharmaceuticals and / or compositions of the present invention, as defined below. As a specific example, the target cell number for injection into each patient for the treatment of skeletal muscle dysfunction, such as urinary incontinence, preferably ranges from 80 million to 150 million cells in total. However, as outlined below, the therapeutic dose will largely depend on the indication being treated.

[0114] An exemplary culture method of the present invention is shown in Example 1. In the first step of the method of the present invention, a cell culture medium is inoculated with MPCs as defined hereinabove, and the MPCs are cultured in the culture medium containing microcarriers, the microcarriers providing a growth surface for the MPCs. In one embodiment, the method of the present invention includes pre-equilibration of the microcarriers with the culture medium under the desired culture conditions, i.e., the microcarriers are added to the culture medium before inoculating the culture medium with cells and are maintained in the vessel in which the MPCs are cultured. The culture medium containing the microcarriers defined below, and in particular its volume, are referred to herein as the "starting culture medium" and the "starting volume," respectively.

[0115] In one embodiment, the method of the present invention comprises culturing at least 10 5 pieces, preferably 10 5 ~10 6 This involves inoculating cells into a cell culture medium. Needless to say, the cell number is adapted depending on the culture volume so that approximately 750 to 8000 cells / ml are inoculated.

[0116] According to the methods of the present invention, MPCs are grown on a microcarrier-provided growth surface area of ​​1 cm. 2 The cells are seeded into a vessel containing culture medium and microcarriers at a density of 500-1500 cells per cm. The cells are incubated in the vessel, thereby allowing the cells to attach to the microcarriers. In a preferred embodiment, the cells grow over 1 cm of the growth surface area provided by the microcarriers. 2 In one embodiment, the concentration of cells seeded according to the method of the present invention is 750 to 8000 cells / ml, preferably about 7700 cells / ml. In one embodiment, the concentration of cells seeded according to the method of the present invention is 750 to 8000 cells / ml, preferably about 7700 cells / ml. 5 MPCs, preferably 10 5 ~10 6 MPCs, more preferably about 10 6 MPCs are seeded in a volume of 130 ml of culture medium.

[0117] In particular, in one embodiment of the present invention, the method for obtaining a large scale culture of MPCs comprises at least the following steps: (a) seeding MPCs into a vessel containing culture medium containing microcarriers and allowing the MPCs to attach to the microcarriers, wherein the MPCs do not grow over a growth surface area of ​​1 cm provided by the microcarriers; 2 The density should be 500-1500 cells per cm, preferably 800-1200 cells / cm. 2 Preferably, the microcarriers are seeded at a density of 5,000 to 10,000 cm 2 / L, and more preferably 8,500 cm 2 / L of growth surface area; and (b) culturing the MPCs in the container; and (c) increasing the growth surface area of ​​the culture medium when the cell number increases by about 8-fold to 25-fold; and (d) Preferably 5 to 7.5 × 10 4 cells / cm 2 and / or 4 to 6.5 × 10 5 further culturing the MPCs until a cell density of up to 1000 cells / ml is reached; and optionally (e) Harvesting the MPC.

[0118] In one embodiment of the present invention, a method for obtaining a large scale culture of MPCs comprises at least the following steps: (a) seeding MPCs into a vessel containing culture medium containing microcarriers and allowing the MPCs to attach to the microcarriers, wherein the MPCs do not grow over a growth surface area of ​​1 cm provided by the microcarriers; 2 The density should be 500-1500 cells per cm, preferably 800-1200 cells / cm. 2 Preferably, the microcarriers are seeded at a density of 5,000 to 10,000 cm 2 / L, and more preferably 8,500 cm 2 / L of growth surface area; and (b) culturing the MPCs in the container; and (c) Cell density is 1.3 × 10 4 ~1.8×10 4cells / cm 2 increasing the growth surface area of ​​the culture medium if (d) Preferably 5 to 7.5 × 10 4 cells / cm 2 and / or 4 to 6.5 × 10 5 further culturing the MPCs until a cell density of up to 1000 cells / ml is reached; and optionally (e) Harvesting the MPC.

[0119] In one embodiment of the present invention, a method for obtaining a large scale culture of MPCs comprises at least the following steps: (a) seeding MPCs into a vessel containing culture medium containing microcarriers and allowing the MPCs to attach to the microcarriers, wherein the MPCs grow within 1 cm of the growth surface area provided by the microcarriers; 2 The density should be 500-1500 cells per cm, preferably 800-1200 cells / cm. 2 Preferably, the microcarriers are seeded at a density of 5,000 to 10,000 cm 2 / L, and more preferably 8,500 cm 2 / L of growth surface area; and (b) culturing the MPCs in the container; and (c) increasing the growth surface area of ​​the culture medium when at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, or 95%, and most preferably at least 90% of the microcarriers are occupied; and (d) Preferably 5 to 7.5 × 10 4 cells / cm 2 and / or 4 to 6.5 × 10 5 further culturing the MPCs until a cell density of up to 1000 cells / ml is reached; and optionally (e) Harvesting the MPC.

[0120] In one embodiment of the present invention, a method for obtaining a large scale culture of MPCs comprises at least the following steps: (a) seeding MPCs in a vessel containing a culture medium containing microcarriers, and attaching the MPCs to the microcarriers, wherein the MPCs are seeded at an amount of 750 to 8000 cells / ml, preferably about 7700 cells / ml, and preferably the microcarriers are cultured at a density of 5,000 to 10,000 cm 2 / L, and more preferably 8,500 cm 2 / L of growth surface area; and (b) culturing the MPCs in the container; and (c) increasing the growth surface area of ​​the culture medium when the cell number increases by about 8-fold to 25-fold; and (d) Preferably 5 to 7.5 × 10 4 cells / cm 2 and / or 4 to 6.5 × 10 5 further culturing the MPCs until a cell density of up to 1000 cells / ml is reached; and optionally (e) Harvesting the MPC.

[0121] In one embodiment of the present invention, a method for obtaining a large scale culture of MPCs comprises at least the following steps: (a) seeding MPCs in a vessel containing a culture medium containing microcarriers, and attaching the MPCs to the microcarriers, wherein the MPCs are seeded at an amount of 750 to 8000 cells / ml, preferably about 7700 cells / ml, and preferably the microcarriers are cultured at a density of 5,000 to 10,000 cm 2 / L, and more preferably 8,500 cm 2 / L of growth surface area; and (b) culturing the MPCs in the container; and (c) increasing the growth surface area of ​​the culture medium when at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, or 95%, and most preferably at least 90% of the microcarriers are occupied; and (d) Preferably 5 to 7.5 × 10 4 cells / cm 2 and / or 4 to 6.5 × 10 5further culturing the MPCs until a cell density of up to 1000 cells / ml is reached; and optionally (e) Harvesting the MPC.

[0122] In a preferred embodiment, the growth surface area, and optionally the volume of culture medium, is increased by 2-4 fold, preferably 3 fold (step (c)). To obtain even higher cell numbers, the step of increasing the growth surface can be repeated one or more times, i.e., upon reaching a certain cell density, cell number, and / or growth fold of cells as defined elsewhere herein, the growth surface can be increased again by 2-4 fold. Thus, in one embodiment, the invention comprises one or more steps of increasing the growth area of ​​the culture environment, preferably 1-10 steps of increasing the growth area, preferably 1-8 steps of increasing the growth area, more preferably 1-4 steps, even more preferably 1 or 2 steps, and most preferably 2 steps.

[0123] The term "cultivation" refers to conditions for maintaining and growing cells in cell culture. During culturing according to the methods of the present invention, the vessel and culture medium are each kept in intermittent motion to keep the microcarriers in suspension. Cells attached to the microcarriers grow optimally when the microcarriers are kept in homogeneous suspension and do not settle or precipitate except as necessary to promote cell movement on the microcarriers. In particular, maintaining the microcarriers in suspension and motion avoids substantial cell aggregation, which can lead to MPC differentiation or senescence. The force applied to keep the microcarriers in suspension should be such that the microcarriers do not settle or precipitate, but the force should not be so great as to damage the cells or the microcarriers. Various possibilities exist for keeping the microcarriers in suspension / motion, including, but not limited to, agitation of the culture medium and rocking of the cell culture system, particularly the bioreactor described in Example 1.

[0124] Typical cell culture environments for mammalian cells are known to those skilled in the art. For example, the pH range for mammalian cell culture is typically 7.2-7.6, and the temperature is typically 36-37°C. This was confirmed by experiments performed according to the present invention, in which growth was observed at pH 7.3 and 37°C. Thus, in one embodiment, the pH is 7.2-7.6, preferably 7.3-7.4, and most preferably 7.3, the temperature set point is 36-37°C, and the temperature is preferably maintained at 37°C. The dissolved oxygen (DO) concentration is typically maintained between 20% and 80%, preferably between 30% and 75%. In particular, the DO set point in the method of the present invention is set at 75% and should not drop below 30%.

[0125] Experiments performed in accordance with the present invention have shown that it is beneficial to increase the concentration of cultured cells by seeding them in a small volume (starting volume). After a period of time, more growth area is required during cell expansion, and the growth surface is therefore increased by adding additional microcarriers. Therefore, the method of the present invention involves adding a cell culture medium containing microcarriers to a cell culture to increase the growth surface. The expanded culture medium, and in particular its volume, are referred to herein as the "expansion culture medium" and "expansion volume," respectively. In a preferred embodiment, the concentration of microcarriers in the expansion culture medium is approximately the same as that of the starting culture medium, preferably 1-2 g / L, more preferably about 1.7 g / L. According to the present invention, the volume of the culture medium is increased when the cell number has increased approximately 8-fold to 25-fold and / or when more than 80%, preferably more than 90%, of the microcarriers are occupied. In particular, increasing the volume of the culture medium while maintaining the microcarrier concentration provides more growth area for further cell expansion. In an alternative embodiment, the growth surface is increased by the addition of microcarriers to the vessel, but the culture medium is not increased to the same extent, ie, the resulting concentration of microcarriers is higher or lower than before.

[0126] A convenient marker for increasing the size of the expansion growth volume is after more than 50%, preferably at least 75%, 80% or 85%, most preferably at least 90% of the microcarriers are occupied.

[0127] In a preferred embodiment, the method of the present invention comprises: 4 ~1.8×10 4 cells / cm 2 This includes increasing the volume of culture medium and / or the growth surface area when a cell density of

[0128] In one embodiment, the method of the present invention involves increasing the growth area by 2-4 fold, preferably by about 3 fold. For example, the culture volume is increased by about 3 fold, while maintaining approximately the same microcarrier concentration in the expansion culture medium, i.e., adding culture medium containing approximately the same concentration of microcarriers as during seeding.

[0129] In one embodiment of the invention, the starting volume, i.e., the volume of culture medium into which the MPCs are inoculated, is about 100-150 ml, preferably about 130 ml. In a preferred embodiment, the starting volume is increased to an expansion growth volume of 400 ml.

[0130] In one embodiment, the method of the invention further comprises refreshing the culture medium after increasing the growth surface during further culturing of the cells, for example every other day, to provide sufficient nutrients to the MPCs.

[0131] Culture times can vary and depend on cell density as specified above. Preferably, MPCs are cultured for 5 to 21 days, more preferably 7 to 14 days, and even more preferably 8 to 10 days. However, longer or shorter culture periods can be envisioned depending on the number of cells seeded, and / or their proliferation rate, and / or the desired final cell number.

[0132] According to the method of the present invention, the culture is terminated when the desired cell number is reached, preferably when the cell number has increased by about 100- to 1000-fold and / or when the cell density has increased by about 10- to 200-fold, preferably about 13- to 130-fold. In particular, MPCs are cultured until they reach a number suitable for further subsequent therapeutic procedures, i.e., in particular up to 5-7.5 x 10 4 cells / cm 2 and / or 4 to 6.5 × 10 5 The cells are further cultured until a cell density of 5.7 x 10 cells / ml is reached. In a preferred embodiment, the cells are 5 cells / ml and cultured to a density of 5.7 x 10 5 This density of cells / ml has been found to be an optimal density that supports the maintenance of MPC characteristics, i.e., the absence of, for example, fibrogenesis. In one embodiment according to the invention, MPCs are cultured at a density of about 1.5 x 10 cells / ml. 8 ~2.75×10 8 Total cell number, preferably about 2.3 x 10 8 However, if the required number of cells cannot be obtained by a single expansion step, i.e., a single increase in culture volume, further expansion steps may be performed, whereby the culture volume and growth area are further increased, preferably three-fold, in each expansion step, while the concentration of microcarriers is preferably maintained the same.

[0133] In one embodiment, the MPCs are harvested after reaching the desired cell number and cell density, respectively. In particular, in one embodiment, the method of the present invention includes a step of detaching the MPCs from the microcarriers at the end of the culture by cleaving the scaffold protein via enzymatic or mechanical means (see above), and a step of recovering the MPCs from the bioreactor. In a preferred embodiment, the detachment includes complete dissolution of the microcarriers. If non-dissolving microcarriers are used, the cells are quickly separated from the microcarriers before they begin to reattach.

[0134] Since MPCs are grown on microcarriers in a suspension culture setting similar to that of non-adherent cells, it can be envisioned that in further expansion steps the volume and / or growth surface of the culture system can be further increased to obtain even larger numbers of cells.

[0135] Microcarriers According to the present invention, microcarriers generally refer to supports for culturing anchorage-dependent cells. "Microcarriers" or "carrier particles" are defined as small bead-like materials derived from silica, glass, dextran, or similar materials used for immobilizing biocatalysts or as supports for culturing anchorage-dependent animal cell lines (IUPAC Compendium of Chemical Terminology (2nd Edition, 1992, Vol. 64, p. 160)). Microcarriers increase the growth surface area in tissue culture for the attachment and yield of anchorage-dependent cells. The terms "growth surface," "growth area," and "surface area" are used interchangeably herein and refer to the surface area provided by the microcarrier to which anchorage-dependent cells attach for culturing. The microcarriers used according to the present invention are preferably spherical carrier materials and are suitable for culturing adherent growing cells, particularly animal cells, in suspension.

[0136] Microcarriers can be made from a wide variety of materials, including plastic, glass, ceramic, silicone, gelatin, dextran, cellulose, etc. In addition, microcarriers can be pretreated in various ways, including plasma treatment of plastic surfaces to create hydrophilic surfaces, or carriers can be coated (e.g., with gelatin, fibronectin, laminin, polyomithine, Matrigel, or with the binding motif of the RGD-binding domain of fibronectin). In a preferred embodiment, microcarriers used in accordance with the present invention are made from polygalacturonic acid (PGA) polymer chains cross-linked via calcium ions and coated with denatured collagen. Suitable commercially available microcarriers include Cytodex™ 1, Cytodex™ 3, Cytopore™ (Amersham Biosciences), Cultispher® G, Cultispher® S (Perbio), Pronectin®, FACT (Sigma), Biosilon®, Microhex™ (Nunc), ImmobasiSil™ (Dunn) and collagen-coated (dissolvable) microcarriers (Corning™).

[0137] In one embodiment, the microcarriers used in accordance with the methods of the present invention are collagen-coated microcarriers. The microcarriers may be dissolvable or non-dissolvable. In a preferred embodiment, the microcarriers are dissolvable, and thus the microcarriers are preferably collagen-coated dissolvable microcarriers. When the microcarriers are dissolvable, in one embodiment of the present invention, the microcarriers are dissolved by enzymatic digestion, preferably by adding a harvesting solution containing a peptidase, preferably an endopeptidase that cleaves proteins at specific sites, most preferably trypsin, or a corresponding trypsin substitute, such as TrypLE® or Accutase®, and pectinase. TrypLE® cleaves peptide bonds at the C-terminus of lysine and arginine and can be used as a direct replacement for trypsin. It is of animal-free origin. Accutase® is a natural enzyme mixture with proteolytic and collagenolytic enzyme activity. This means that it simultaneously mimics the actions of trypsin and collagenase. Thus, in a preferred embodiment, the harvest solution comprises either trypsin and pectinase, or TrypLE® and pectinase, or Accutase® and pectinase, most preferably TrypLE® and pectinase. In one embodiment, the above-mentioned harvest solution further comprises EDTA, which aids in complete dissolution of the microcarriers.

[0138] In one embodiment, the concentration of the microcarriers used according to the method of the present invention in the culture medium is about 0.5-3 g / L, preferably about 1-2 g / L, more preferably 1.7 g / L, the bead size is preferably 100-400 μm, preferably 200-300 μm fully hydrated, and the surface is preferably 1000-10,000 cm 2 / g dry weight, more preferably 3000 to 8000 cm 2 / g dry weight, more preferably 4000 to 7000 cm 2 / gram dry weight, most preferably 5000 cm2 / gram dry weight. Generally, the concentration of microcarriers in the culture medium depends on the specific microcarriers used. For example, 5,000-10,000 cm per liter of culture medium. 2 In a preferred embodiment, the microcarriers provide a growth surface of approximately 8500 cm per liter of culture medium. 2 This provides a growth surface of 5,000 cm 2 This corresponds to the use of 1.7 g / L of microcarriers with a surface of 1.7 g / L.

[0139] The purpose of the microcarriers is to provide an increased growth surface for adherent cells. Thus, in another preferred embodiment, the microcarriers are sized to have a density of 100-60,000 cm 2 growth surface area of ​​500 to 40,000 cm 2 growth surface area of ​​1,000 to 20,000 cm 2 As the number of cells increases, it may be apparent that additional microcarriers may be added to provide sufficient growth surface area. Depending on the amount of surface area occupied by the adherent cells, microcarriers may be added during the culture, for example, during the expansion step.

[0140] After the cells are grown until the desired amount of cells, i.e., the amount of cells defined herein above, is achieved, the MPCs are harvested, i.e., recovered from the culture system. In one embodiment of the present invention, the adherent cells, i.e., MPCs, are detached from the microcarriers. Detachment can be performed using a suitable detachment agent. Suitable detachment agents can be enzymes, thermo-responsive agents, and / or pH-responsive agents. In one embodiment of the present invention, the MPCs are detached with a digestive enzyme that cleaves the cells from the microcarriers. Preferably, the enzyme is an endopeptidase, more preferably selected from trypsin, TrypLE®, or Accutase®. When non-dissolvable microcarriers are used, the detached adherent cells can be removed by passing them through a 50-100 μm filter. The detached adherent cells pass through the filter, while the microcarriers are retained in the container. In the case of dissolvable microcarriers, the above-mentioned harvesting solution can be used.

[0141] In an alternative embodiment of the invention, MPCs are harvested without being detached from the microcarriers, i.e., the MPCs recovered from the culture system include the microcarriers. Thus, the cells can be directly prepared for injection without detachment, particularly if the carrier is biocompatible and / or biodegradable, such as a collagen carrier. Thus, in the case of biocompatible / biodegradable microcarriers, the cells are not detached and are injected directly while still attached to the microcarriers.

[0142] Culture medium As used herein, the terms "growth medium" and "culture medium" are used interchangeably and refer to a solution containing components and nutrients that support the viability and growth of cells cultured according to the present invention. Suitable culture media for growing MPCs are known to those skilled in the art and are described, for example, in International Publication Nos. WO 1999 / 056785, WO 2001 / 078754, WO 2008 / 066886, WO 2008 / 086040, WO 2009 / 045506 and WO 2019 / 115790.

[0143] Growth medium supplements derived from animal sources, such as fetal bovine serum (FBS), are still widely used in cell culture to promote cell attachment, proliferation, and maintenance. However, these reagents should be avoided for clinical use due to safety concerns. In cell therapy, xeno-free and serum-free reagents are highly desirable to enhance safety and quality. Possible alternatives to FBS are media supplemented with human serum, human platelet derivatives, allogeneic umbilical cord serum, or chemically defined media.

[0144] Thus, in one embodiment, the culture medium used in accordance with the methods of the present invention is a substantially xeno-free and / or serum-free medium, such as, for example, the culture medium disclosed in WO 2019 / 215090, which is incorporated herein by reference. Xeno-free and / or serum-free refers to the replacement of serum, such as FBS, with hPL.

[0145] In particular, in one embodiment, a growth medium containing preferably filtered human platelet lysate (hPL), preferably pooled human platelet lysate (phPL), is used in accordance with the present invention. In one embodiment, the final concentration of phPL in the growth medium used in accordance with the present invention is at least 5%, preferably about 5-20%, more preferably 7-12%, and most preferably about 10% or about 5% (volume percent). This 5% concentration of hPL has been shown to minimize microcarrier aggregation. In a specific embodiment, the culture medium contains an anticoagulant, preferably heparin. For this purpose, for example, heparin-Na (heparin sodium) (2500 IU / 5 ml) can be used. After adding heparin to the filtered phPL to form a mixture, the mixture is added to the nutrient solution of the growth medium to a preferred final concentration of 1-10 IU per ml of growth medium, 2-6 IU / ml, or about 2 IU / ml. Alternatively, other substances that prevent coagulation (e.g., EDTA) can be used. When fibrinogen-depleted phPL is used, there is no need to add an anticoagulant, since active clotting factors are no longer present.

[0146] Thus, in a preferred embodiment, the cell culture medium used in accordance with the present invention comprises fibrinogen-depleted human platelet lysate (hPL) and lacks heparin.

[0147] The cell culture medium may further comprise the following components: · A nutrient solution, preferably Dulbecco's Modified Eagle's Medium (DMEM), more preferably a 1:1 DMEM / F12 nutrient mixture (a 1:1 mixture of DMEM and Flam's F-12); human epidermal growth factor (hEGF), preferably added to the nutrient solution to give a final concentration of 2 to 20 ng / ml, more preferably about 10 ng / ml; human basic fibroblast growth factor (hbFGF), preferably added to the nutrient solution to give a final concentration of 0.5 to 2 ng / ml, more preferably about 1 ng / ml; Insulin, preferably human insulin, preferably added to the nutrient solution to give a final concentration of 5-20 pg / ml, more preferably about 10 μg / ml Dexamethasone preferably added to the nutrient solution to give a final concentration of 0.2-0.8 μg / ml, more preferably about 0.4 μg / ml.

[0148] According to one embodiment, the cell growth medium further comprises a solution containing an antibiotic, preferably penicillin and streptomycin, preferably at a final concentration of about 1% (Penicillin / Streptomycin: 10,000 units / ml penicillin and 10,000 μg / ml streptomycin in 10 mM citrate buffer (for pH stability) at 20° C.). In an alternative embodiment, the growth medium does not contain penicillin or streptomycin. In an alternative embodiment, the growth medium comprises an antibiotic that is not penicillin and / or streptomycin, but another antibiotic. Additional antibiotics and their use in cell culture media are well known to those skilled in the art and may be used in accordance with the present invention.

[0149] bioreactor To maintain optimal growth conditions, i.e., optimal growth conditions with respect to oxygen, carbon dioxide, and nutrient supply, any of the above-disclosed embodiments of the method of the present invention can be advantageously carried out in a bioreactor system, as exemplarily shown in Examples 1 and 2. A bioreactor is generally understood as a vessel suitable for culturing biological materials such as cells. One form of bioreactor is a stirred tank. In one embodiment of the method of the present invention, the vessel is a stirred tank reactor. A bioreactor is known to those skilled in the art as a closed system for culturing cells, in particular, in which growth parameters such as dissolved oxygen concentration, temperature, and pH can be controlled.

[0150] Thus, in one embodiment, the vessel used in the method of the invention is a closed bioreactor. Preferably, the closed bioreactor is a bioreactor bag. Bioreactor bags suitable for carrying out the method of the invention are disclosed in International Application WO 2011 / 142667, e.g., in the Examples section "Expansion in culture bags," the teachings of which are incorporated herein by reference. Advantageously, in one embodiment of the invention, the bioreactor bag is expandable, i.e., the culture volume can be expanded, e.g., to achieve an increased growth surface area, as described above. The MPCs are transferred aseptically to the container. Preferably, the MPCs are transferred through a bag having a connectable tube by first aspirating a cell suspension, preferably containing the MPCs, into a syringe under sterile conditions, then injecting the cell suspension into the bag under sterile conditions, and subsequently connecting the connectable tube to the container of the bioreactor system, thereby transferring the cell suspension to the container of the bioreactor system.

[0151] If there is a gas volume or headspace in the container, the movement of the container will cause excessive turbulence in the culture medium.Excessive turbulence may kill cells or have a negative effect on cell growth, especially on sensitive cells.Therefore, in a preferred embodiment, there is less than 20% headspace in the container, preferably less than 10%, and even more preferably there is no headspace.In the present invention, headspace refers to the proportion of the volume of the container that contains gas.20% headspace means that 80% of the container is made up of medium containing cells and microcarriers.

[0152] Depending on the needs of the cells, additional nutrients and / or supplements may be added during the culture. This may be done by perfusion. Sensors may be added to the bioreactor to measure nutrient and / or waste levels, pH, DO (dissolved oxygen), the amount of cells in the system, and / or other parameters. Preferably, these sensors operate automatically, and more preferably, the addition of nutrients and / or supplements is also automatic, and most preferably, the sensors direct the addition of nutrients and / or supplements.

[0153] To provide sufficient nutrients to the cells, fresh medium is preferably passed through the vessel while simultaneously removing medium to maintain a constant volume and / or pressure. By removing medium from the expansion vessel through a filter with a pore size larger than the cells but smaller than the microcarriers, e.g., about 100 μm, unattached cells and cellular debris are removed and the adherent cells are retained in the vessel.

[0154] In one embodiment, the method of the invention comprises replacing the culture medium with fresh medium every other day after increasing the growth surface, preferably 50% of the culture medium is replaced.

[0155] In specific embodiments, the methods of the invention include, for example (but not necessarily), the following steps for culturing MPCs: (i) Before culturing the MPC, the vessel was pre-equilibrated, with the dissolved oxygen concentration (DO) set to 75%, the temperature set to 37°C, and the pH set to 7.3; (ii) culturing the MPCs in the vessel without perfusion, pH control, and DO control for the first 24 hours to allow the MPCs to attach to the carrier particles, or until the DO falls below about 30% (whichever comes first) (pH and DO are not controlled but measured); (iii) 24 hours after the start of the culture or when the DO drops below about 30% (whichever occurs first), start DO control, pH control, and perfusion, setting the DO at 75%, the pH at 7.3, perfusion at 3 mL / min, and preferably maintaining a pressure in the bag between 80 and 120 mbarg; and / or (iv) The vessel is preferably agitated at the following set points: · Rocker speed 90° / sec; ·Maximum tilt angle 180°; ·Acceleration 90° / s 2 ; ·Deceleration 90° / s 2 ; ·Vertical holding time 10 seconds; Four 1-hour static intervals over 24 hours (holding time: 3600 seconds (horizontal rest)); number of mixing cycles: 1000 (horizontal rest)).

[0156] In this embodiment, if the DO drops below 40% or the cell density drops below 5.0 x 10 5 The static interval may be stopped (up to 0 seconds horizontal hold time and 0 mixing cycles during horizontal rest) when cells / ml is reached or when at least 70%, preferably 80%, and more preferably 90% of the microcarriers are occupied. Additionally or alternatively, if the DO and / or pH set points can no longer be achieved, perfusion may be increased up to 10 ml / min. Additionally or alternatively, if inhomogeneous mixing is observed, acceleration and deceleration rates may be increased to 210° / s. 2 However, the oscillation scheme can be adapted as also described in WO 2011 / 142667.

[0157] Cell Populations and Treatment Aspects Those skilled in the art can verify the presence of MPC characteristic markers. For example, WO 2019 / 215090 discloses in vitro and in vivo analyses, such as the presence of marker proteins by flow cytometry, fibrogenesis by Giemsa staining, and transplantation experiments.

[0158] MPCs obtainable by the methods of the present invention, i.e., the cell populations that contain MPCs and are harvested, exhibit the following myogenic marker expression: the population comprises at least 40% α-actinin positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin positive cells; and / or the population comprises at least 60% Pax7 positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax7 positive cells; and / or the population comprises at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5-positive cells; and / or the population comprises less than 20% CD34 positive cells, preferably less than 15%, more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34 positive cells; and optionally, The population preferably comprises 5% to 95%, more preferably 10% to 99%, even more preferably 20% to 95%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 90%, and most preferably 70% to 95% desmin-positive cells.

[0159] Thus, in one embodiment, the cell population comprises ≧40% α-actinin positive cells, ≧60% Pax7 positive cells, ≦20% CD34 positive cells, and optionally desmin positive cells, preferably ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≦15% CD34 positive cells, and optionally desmin positive cells.

[0160] Preferably, the cell population comprises more than 80% alpha-actinin positive cells, more than 80% Pax7 positive cells, less than 5% CD34 positive cells, and optionally desmin positive cells, most preferably more than 10% desmin positive cells.

[0161] In one embodiment, the cell population comprises ≧40% α-actinin positive cells, ≧60% Pax7 positive cells, ≧50% A2B5 positive cells, ≦20% CD34 positive cells, and optionally desmin positive cells, preferably ≧50% α-actinin positive cells, ≧60% Pax7 positive cells, ≧50% A2B5 positive cells, ≦15% CD34 positive cells, and optionally desmin positive cells.

[0162] Preferably, the cell population comprises greater than 80% alpha-actinin positive cells, greater than 80% Pax7 positive cells, greater than 80% A2B5 positive cells, less than 5% CD34 positive cells, and optionally desmin positive cells, most preferably greater than 10% desmin positive cells.

[0163] Generally, the cells of the populations of the invention also express early myogenic markers such that when administered to a patient the cells are capable of effecting muscle regeneration, i.e., inducing myogenesis. In one embodiment of the invention, the MPCs express MyoD and / or MyHC, preferably adjacent to other markers mentioned above.

[0164] Expressions regarding the expression rate of a marker reflect the percentage of cells that are positive for the marker in the total population, such as that a population contains a certain percentage of positive cells. If a cell expresses a marker, that is, if the marker can be detected at the protein level or mRNA level by any suitable detection method, such as flow cytometry, Western blot, immunostaining or qPCR, the cell is considered to be positive for that marker.

[0165] Thus, in one embodiment of the present invention, the cell population of the present invention comprises at least 40% α-actinin-positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin-positive cells. In a further embodiment of the present invention, the cell population of the present invention comprises at least 60% Pax7-positive cells, preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax7-positive cells. In a further embodiment of the invention, the cell population of the invention comprises at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5-positive cells. In a further embodiment of the invention, the cell population of the invention comprises less than 20% CD34-positive cells, preferably less than 15%, even more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34-positive cells.

[0166] In a further embodiment of the invention, the cell population of the invention comprises at least 40% α-actinin positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin positive cells, and at least 60% Pax7 positive cells, preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax 7 positive cells. In a further embodiment of the invention, the cell population of the invention comprises at least 40% α-actinin-positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin-positive cells, and at least 60% A2B5-positive cells, preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5-positive cells.In a further embodiment of the invention, the cell population of the invention comprises at least 40% α-actinin positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin positive cells, and less than 20% CD34 positive cells, preferably less than 15%, even more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34 positive cells. In a further embodiment of the invention, the cell population of the invention comprises at least 60% Pax7 positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax7 positive cells and at least 60% A2B5 positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5 positive cells.In a further embodiment of the invention, the cell population of the invention comprises at least 60% Pax7 positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax 7 positive cells, and less than 20% CD34 positive cells, preferably less than 15%, even more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34 positive cells. In a further embodiment of the invention, the cell population of the invention comprises at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5-positive cells, and less than 20% CD34-positive cells, preferably less than 15%, even more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34-positive cells.In a further embodiment of the invention, the cell population of the invention comprises at least 40% α-actinin positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin positive cells and at least 60% Pax7 positive cells, preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax 7-positive cells and at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5-positive cells.In a further embodiment of the invention, the cell population of the invention comprises at least 40% α-actinin positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin positive cells and at least 60% Pax7 positive cells, preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax 7-positive cells and less than 20% CD34-positive cells, preferably less than 15%, more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34-positive cells.In a further embodiment of the invention, the cell population of the invention comprises at least 60% Pax7 positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax 7-positive cells, at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5-positive cells, and less than 20% CD34-positive cells, preferably less than 15%, even more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34-positive cells.

[0167] In the most preferred embodiment of the invention, the cell population of the invention comprises at least 40% α-actinin positive cells, preferably at least 50%, more preferably at least 60%, even more preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% α-actinin positive cells, at least 60% Pax7 positive cells, preferably at least 65%, even more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% Pax 7-positive cells, at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% A2B5-positive cells, and less than 20% CD34-positive cells, preferably less than 15%, even more preferably less than 10%, even more preferably less than 8%, even more preferably less than 7.5%, even more preferably less than 5%, even more preferably less than 4%, even more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1.5%, even more preferably less than 1%, even more preferably less than 0.5%, and most preferably less than 0.25% CD34-positive cells.

[0168] In preferred embodiments, the cell populations of the present invention are further defined by the markers CD56, Myf5, MyHC, and / or MyoD (preferably in addition to the markers specified above) having the following percentages: In one embodiment, the cell populations of the present invention comprise less than 15% CD56-positive cells, preferably less than 10%, more preferably less than 5%, and most preferably about 3% CD56-positive cells. In a further embodiment, the cell populations of the present invention comprise at least 50% Myf5-positive cells, preferably more than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells. In a further embodiment, the cell populations of the present invention comprise less than 30% MyHC-positive cells, preferably less than 20%, more preferably less than 15%, and most preferably less than 10% MyHC-positive cells. In a further embodiment, the cell populations of the present invention comprise 10-40% MyoD-positive cells, preferably 10-30%, more preferably 15-25%, and most preferably about 20% MyoD-positive cells. In a further embodiment, the cell population of the present invention comprises less than 15% CD56-positive cells, preferably less than 10%, more preferably less than 5%, and most preferably about 3% CD56-positive cells, and at least 50% Myf5-positive cells, preferably greater than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells. In a further embodiment, the cell population of the present invention comprises less than 15% CD56-positive cells, preferably less than 10%, more preferably less than 5%, and most preferably about 3% CD56-positive cells, and less than 30% MyHC-positive cells, preferably less than 20%, more preferably less than 15%, and most preferably less than 10% MyHC-positive cells. In a further embodiment, the cell population of the present invention comprises less than 15% CD56-positive cells, preferably less than 10%, more preferably less than 5%, and most preferably about 3% CD56-positive cells, and 10-40% MyoD-positive cells, preferably 10-30%, more preferably 15-25%, and most preferably about 20% MyoD-positive cells.In a further embodiment, the cell population of the present invention comprises less than 15% CD56-positive cells, preferably less than 10%, more preferably less than 5%, and most preferably about 3% CD56-positive cells, at least 50% Myf5-positive cells, preferably more than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells, and less than 30% MyHC-positive cells, preferably less than 20%, more preferably less than 15%, and most preferably less than 10% MyHC-positive cells. In a further embodiment, the cell population of the present invention comprises less than 15% CD56-positive cells, preferably less than 10%, more preferably less than 5%, and most preferably about 3% CD56-positive cells, at least 50% Myf5-positive cells, preferably more than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells, and 10-40% MyoD-positive cells, preferably 10-30%, more preferably 15-25%, and most preferably about 20% MyoD-positive cells. In a further embodiment, the cell population of the present invention comprises less than 15% CD56-positive cells, preferably less than 10%, more preferably less than 5%, and most preferably about 3% CD56-positive cells, at least 50% Myf5-positive cells, preferably more than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells, less than 30% MyHC-positive cells, preferably less than 20%, more preferably less than 15%, and most preferably less than 10% MyHC-positive cells, and 10-40% MyoD-positive cells, preferably 10-30%, more preferably 15-25%, and most preferably about 20% MyoD-positive cells. In a further embodiment, the cell population of the present invention comprises at least 50% Myf5-positive cells, preferably more than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells, and less than 30% MyHC-positive cells, preferably less than 20%, more preferably less than 15%, and most preferably less than 10% MyHC-positive cells.In a further embodiment, the cell population of the present invention comprises at least 50% Myf5-positive cells, preferably greater than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells, and 10-40% MyoD-positive cells, preferably 10-30%, more preferably 15-25%, and most preferably about 20% MyoD-positive cells. In a further embodiment, the cell population of the present invention comprises at least 50% Myf5-positive cells, preferably greater than 60%, more preferably 60-90%, and most preferably about 65% Myf5-positive cells, less than 30% MyHC-positive cells, preferably less than 20%, more preferably less than 15%, and most preferably less than 10% MyHC-positive cells, and 10-40% MyoD-positive cells, preferably 10-30%, more preferably 15-25%, and most preferably about 20% MyoD-positive cells. In a further embodiment, the cell population of the present invention comprises less than 30% MyHC-positive cells, preferably less than 20%, more preferably less than 15%, and most preferably less than 10% MyHC-positive cells, and 10-40% MyoD-positive cells, preferably 10-30%, more preferably 15-25%, and most preferably about 20% MyoD-positive cells.

[0169] The term "about," particularly with respect to the percentage of positive cells, is defined to include a variation of about 10% positive cells.

[0170] Additionally, cell viability is at least 80%, at least 85%, at least 90%, at least 95% or at least 98%, preferably at least 80%. Preferably, when the cells and the corresponding cell composition containing collagen are each stored at 2-8°C for at least 24 hours, preferably at least 48 hours, and up to 120 hours, at least 80% (or more) cell viability remains.

[0171] Experiments performed according to the present invention have shown that the amount of desmin-positive cells varies among various MPC isolates even before large-scale cell culture, i.e., within cell populations obtained from different patient biopsies, and increases during culture with each cell passage. Therefore, the amount of desmin-positive cells is not an important criterion for cells suitable for downstream clinical applications, as long as desmin-positive cells are present in the population.

[0172] Thus, in a further aspect, the present invention relates to a cell population comprising MPCs obtainable by the method of the present invention disclosed above. The population of MPCs according to the present invention can be used for the manufacture of a medicament. In particular, the population of MPCs according to the present invention can be used for the manufacture of a medicament for treating muscle dysfunction, particularly skeletal muscle dysfunction, in human patients. See below. Thus, in one embodiment, the population obtainable by the method of the present invention comprises a therapeutically effective amount of MPCs. By therapeutically effective amount, we mean an amount suitable for treating muscle dysfunction, such as urinary incontinence. The MPCs are administered to the site of damaged muscle, preferably by injection, to regenerate skeletal muscle tissue. In one embodiment of the present invention, the skeletal muscle dysfunction being treated is a sphincter abnormality. In a preferred embodiment, the sphincter is selected from the non-limiting group of the external urethral sphincter, the internal urethral sphincter, and the external anal sphincter, the internal anal sphincter. Therefore, the indications related to sphincter abnormalities that can be treated according to the present invention are the above-mentioned indications related to sphincter, and include, but are not limited to, female and male urinary incontinence and fecal incontinence, and pathological reflux in gastroesophageal reflux disorder.Preferably, urinary incontinence is selected from stress urinary incontinence, urge urinary incontinence, overflow urinary incontinence, total urinary incontinence, or a combination of stress urinary incontinence and urge urinary incontinence.In addition to targeting sphincter muscles, the cell population obtained by the method of the present invention can be used to target other skeletal muscle abnormalities.For example, it can be envisioned that after these muscle injuries, MPCs can be administered to the site of muscle injury or muscle damage to support, promote, or initiate regeneration.

[0173] In one embodiment, the MPCs administered, preferably injected according to the present invention, comprise microcarriers. For example, in the above-described embodiment in which the MPCs are not detached from the microcarriers, the population comprising MPCs obtainable by the method of the present invention further comprises microcarriers to which the MPCs are attached. In this embodiment, the MPCs are preferably injected together with the microcarriers. Therefore, the carrier is preferably biocompatible. Furthermore, it is generally known that the carriers used herein, for example as scaffolds, should degrade in a timely manner to ensure proper reconstruction of muscle tissue, and therefore, the carrier should preferably be biodegradable. Biocompatible microcarriers and biodegradable microcarriers are known to those skilled in the art. Examples of biocompatible and biodegradable microcarriers include natural polymers (polysaccharides and proteins) and synthetic polymers (poly(α-hydroxyesters)), such as poly-epsilon caprolactone (PCL), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and poly(lactic-co-glycolic acid) (PLGA), as reviewed in Elmowafy, et al., J. Pharm. Investig. 49 (2019), 347-380.

[0174] In an alternative embodiment, the MPCs are administered without microcarriers, ie, the MPCs are stripped from the microcarriers prior to administration.

[0175] Generally, the amount of cells that can be considered therapeutically effective depends largely on the indication to be treated and the severity, extent or size of the injury to be treated. For example, it can be imagined that in the case of mild stress urinary incontinence, fewer cells are injected than in the case of severe stress urinary incontinence. In a preferred embodiment, this amount is at least 1 × 10 7 pieces, preferably 6 x 10 7 ~3×10 8 pieces, most preferably 1 to 3 × 10 8The culture medium comprises MPCs. Even larger cell numbers can be obtained by performing multiple expansion steps, i.e., increasing the volume of culture medium preferably three-fold, while preferably maintaining the concentration of microcarriers in the medium. Thus, the present invention also relates to a method for obtaining a therapeutically effective amount of MPCs, which method comprises the steps of the method for obtaining large-scale cultures of MPCs of the present invention described herein above.

[0176] As outlined above, the amount of MPC generally considered therapeutically effective is highly variable and therefore not particularly limited. In the specific example of stress urinary incontinence, the target cell number for injection into a patient preferably ranges from 60 to 200 million total cells, more preferably from about 80 to 150 million cells. However, as noted above, these numbers depend on the severity of the disorder being treated. In a preferred embodiment, cell viability is at least 80%.

[0177] The present invention further relates to a method for preparing a pharmaceutical product, the method comprising the steps of the method for obtaining a large-scale culture of MPCs of the present invention described hereinabove, and optionally adding a biomaterial solution, preferably a hydrogel solution, and more preferably a collagen solution, to the harvested MPCs. "Biomaterial solution" or "biocompatible material" refers, inter alia, to a carrier solution that ensures that injected MPCs remain at the injection site. Thus, in one embodiment of the present invention, MPCs are suspended in a biomaterial solution, such as a hydrogel. Hydrogels are generally ECM proteins used in tissue engineering that allow for good engraftment. In preferred embodiments, the hydrogel is selected from, but is not limited to, collagen, alginate, hyaluronic acid, fibrin, poly(N-isopropylacrylamide) (PNIPAAm), poly(ethylene glycol) (PEG), recombinant protein polymers that form Mixing-Induced Two-Component Hydrogels (MITCH), Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery (SHIELD), preferably collagen. Without being bound by theory, it can be envisioned that when MPCs are administered together with microcarriers, i.e., while attached to the microcarriers, the microcarriers (as a biomaterial solution) serve as a support for the injected MPCs, for example, to ensure that the injected cells remain at the injection site.

[0178] The pharmaceutical product can be used to treat skeletal muscle dysfunction via injection. To deliver 80 million MPCs with at least 80% viability at a final concentration of 20-25 million cells / ml, in one embodiment of the present invention, cultured cells (80-100 million) are suspended in 4 ml of a biomaterial solution, such as a collagen solution, as described below. The final product is preferably shipped in vials or syringes in a box at 5°C (+ / -3°C) controlled by a temperature measurement device. A syringe is defined as a container suitable for injecting pharmaceutical products into patients. Alternatively, the MPCs and biomaterial solution can be kept separate within a syringe, e.g., a dual-chamber syringe, and mixed only during pharmaceutical injection.

[0179] The present invention further encompasses compositions comprising the above-described MPCs and cell populations obtained by the methods of the present invention, respectively. In a preferred embodiment, the MPCs are suspended in a collagen solution at a concentration of 10 to 30 million cells / ml, preferably with a viability of at least 80%. In a preferred embodiment, the collagen solution contains type I collagen, preferably of porcine, bovine, or preferably human origin, and the concentration of collagen in the composition is preferably 1 to 4 mg / ml, preferably about 2 mg / ml (e.g., 2.1 mg / ml). In one embodiment, the composition is contained in a pharmaceutical container, preferably a syringe or vial, as defined hereinabove.

[0180] The MPCs obtained by the methods of the present invention and the corresponding compositions of the present invention can be used for various therapeutic applications, particularly related to muscle dysfunction, including, but not limited to, the treatment of stress urinary incontinence, as described in WO 2019 / 215090, for example, the treatment of post-prostatectomy male stress urinary incontinence, as described in WO 2004 / 096245, and the treatment of anal incontinence, as described in WO 2008 / 104883. Thus, the present invention relates to the MPCs and compositions of the present invention, respectively, for use as pharmaceuticals in the treatment of muscle dysfunction, for example, skeletal muscle dysfunction, as defined hereinabove. In a preferred embodiment, the skeletal muscle dysfunction may be, for example, a dysfunction of the external urethral sphincter or a dysfunction of the external anal sphincter. In a preferred embodiment, the skeletal muscle dysfunction is an abnormality of the external urethral sphincter, and therefore, the MPCs and compositions, respectively, are preferably used to treat urinary incontinence, particularly female stress urinary incontinence.

[0181] Treatment is typically performed by injecting the composition or pharmaceutical preparation into a subject, preferably a mammal, more preferably a domestic animal, such as a pet or livestock animal as defined above, and most preferably a human. In a preferred embodiment, treatment is performed by injecting the composition or pharmaceutical preparation into the same subject from which the muscle biopsy was taken, and thus autologous cells are preferably used for treatment. Optionally, the composition further comprises a collagen solution as described hereinabove. After injection of the composition, the pelvic floor of the human patient can be subjected to neuromuscular electromagnetic stimulation (NMES) as described in WO 2019 / 215090. The induced electric field strength at maximum output was 120 V / m at the surface of the stimulation coil. The electric field measured 5 cm above the stimulation coil was 22 V / m. NMES treatment after injection of the cell suspension can support muscle and nerve regeneration by activating muscle-nerve interactions and inducing maturation of the neuromuscular junction. Injection of the composition can be performed using injection devices known in the art. In a preferred embodiment, the injection is carried out using the injection device described in PCT / EP2023 / 074044, filed September 1, 2023, which claims priority to EP22 193 690.9, the contents of which are incorporated herein by reference. Preferably, 8-12 or 12-18 aliquots of the hMPC-collagen composition are injected into the pelvic floor in a total volume not exceeding 6 ml of composition.

[0182] Throughout the text of this specification, several documents are cited. The contents of all cited references (including literature, issued patents, published patent applications cited throughout this application, including the background section, and manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference, without any admission that the cited documents are in fact prior art with respect to this invention.

[0183] A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. [Example]

[0184] Example 1: Protocol for automated MPC culture in a bioreactor system MPCs are obtained after explantation of muscle biopsies. A total of 1 million P0 or P1 MPCs are collected in an adhesive bag, a container with a connectable tube. Methods for explanting muscle biopsies and harvesting MPCs from tissue are described in WO 2019 / 215090 and in the "Muscle Progenitor Cells" section above. The adhesive bag is then connected to a pre-equilibrated SCINUS cell expansion and growth system by sterile connection. The MPCs are then expanded inside the SCINUS until sufficient cells are obtained (approximately 6 days). The SCINUS system is described in WO 2011 / 142667.

[0185] SCINUS preparation Prepare the SCINUS system using the following set points, volumes and concentrations: 130 mL MPC culture medium (DMEM:F12 (Gibco™, Thermo Fisher Scientific Inc, USA), 5% hPL (human platelet lysate; PLTGold from MillCreek, Rochester, Minnesota, USA), 1 U / μg penicillin / streptomycin (Gibco™, Thermo Fisher Scientific Inc, USA), 10 ng / mL hEGF (Sigma-Aldrich, Saint Louis, USA, and Merck KGaA, Darmstadt, Germany, respectively), 0.4 μg / mL dexamethasone (Sigma-Aldrich, Saint Louis, USA, and Merck KGaA, Darmstadt, Germany, respectively), 1 ng / mL βFGF (Sigma-Aldrich, Saint Louis, USA, and Merck KGaA, Darmstadt, Germany, respectively) KGaA, Darmstadt, Germany), 10 μg / mL insulin (Sigma-Aldrich, Saint Louis, USA, and Merck KGaA, Darmstadt, Germany, respectively). 1.7 grams / L Corning Life Sciences Collagen-Coated Dissolvable Microcarriers (Corning™ Denatured Collagen Dissolvable Microcarriers) ·DO set point 75% ·pH set point 7.3 ·Temperature set point 37℃.

[0186] After the set point is achieved, the pre-equilibration conditions are maintained until inoculation of cells on day 0.

[0187] SCINUS - Seeding (Day 0) 1×10 6 After harvesting the MPC (P1), the vessel containing the cell suspension was attached to the pre-equilibrated SCINUS bioreactor system. Perfusion and control parameters (DO and pH) were stopped. The following settings were used during the seeding and attachment phase: Perfusion set point: No control pH set point: No control DO set point: No control Volume set point: 130mL Locker Setpoint: Rocker speed: 90° / sec Maximum angle: 180° ·Acceleration, 90° / s 2 ·Deceleration, 90° / s 2 ·Vertical holding time: 10 seconds ·Horizontal holding time: 3600 seconds (horizontal pause) Number of mixing cycles: 1000 (horizontal pause).

[0188] The pressure in the bag was maintained at 80-120mbarg. If the DO dropped below 30%, the following settings were enabled: ·pH: Set point 7.3 ·DO: Set point 75% ·Perfusion: set point 3mL / min

[0189] SCINUS - Start process control (from day 1 onwards) After the seeding and attachment stage, the MPC culture settings were initiated. Generally, this stage begins 24 hours after inoculation or when the DO drops below 30%, whichever comes first. The following settings were used during this stage: ·Perfusion set point: 3mL / min ·pH set point: 7.3 ·DO set point: 75% Volume set point: 130mL Locker Setpoint: Rocker speed: 90° / sec Maximum angle: 180° ·Acceleration, 90° / s 2 ·Deceleration, 90° / s 2 ·Vertical holding time: 10 seconds ·Horizontal holding time: 3600 seconds (horizontal pause) Number of mixing cycles: 1000 (horizontal pause). The pressure should be maintained at 80-120mbarg.

[0190] As the proliferation rate and characteristics of MPCs can vary significantly between donors, the following parameters were observed during culture: When DO falls below 40% or the biomass sensor is >5.0 × 10 5 When a cell density of 100 cells / mL or >90% occupied microcarriers is observed, stop horizontal resting by adjusting the following settings: Horizontal hold time: 0 seconds (no horizontal pause) Number of mixing cycles: 0 (no horizontal pauses) If DO and pH set points can no longer be achieved, increase the perfusion settings up to a maximum of 10 mL / min. If an inhomogeneous mixture is observed, adjust the following rocker settings to maintain a homogeneous mixture: ·Acceleration: 210° / s 2 ·Deceleration: 210° / s 2

[0191] Volume expansion (density based) 1.3 × 10 cells 4 ~1.8×10 4 cells / cm 2 (i.e., 1.1×10 5 ~1.5×10 5 After reaching a density of 1.7 g / L (270 cells / mL), the volume of the adherent bag was increased while maintaining the microcarrier concentration at 1.7 g / L. The volume was increased approximately three-fold, from 130 mL to 400 mL. A suspension of microcarriers in MPC culture medium (1.7 g / L) was attached to the medium inlet of the SCINUS system and added to the bag by either gravity or pressure. To increase the microcarrier volume from 130 mL to 400 mL, the following was done: the volume set point was adjusted to 400 mL, and 270 mL of microcarriers was added to the system via the inlet. Pressure was maintained between 80 and 120 mbarg, and the rocker, perfusion, pH, and DO set points were unchanged.

[0192] Medium refreshment (timing-based) On the day after the volume expansion step, medium refreshment is required. Every other day, 50% of the cell culture medium was refreshed by transferring 200 mL of medium from the adhesive bag to a waste bag and adding 200 mL of fresh MPC culture medium to the bag via the addition inlet. The pressure was maintained at 80-120 mbarg, and the set points for the rocker, perfusion, pH, and DO were not changed.

[0193] Harvesting (density-based) After sufficient cells had been cultured in the adherent bags, the cells were harvested from the dissolvable microcarriers by complete lysis using harvesting solution. To maintain MPC cell characteristics, 6.7 × 10 4 cells / cm 2 (5.7×10 5 cells / mL, total 2.3×10 8 The cells were cultured to a maximum density of 1000 cells.

[0194] The cells were washed once with an equal volume of PBS, and 200 mL of PBS was transferred from the adhesion bag to a waste bag. Then, 200 mL of collection solution (74% PBS, TrypE 2.5X, pectinase 49 U / mL, EDTA 5 M) was added to the adhesion bag through the addition port. The cells were incubated at 37°C for 15-20 minutes, with rocking every 5 minutes using the following rocking settings: Locker Setpoint: Rocker speed: 90° / sec Maximum angle: 180° ·Acceleration, 90° / s 2 ·Deceleration, 90° / s 2 ·Vertical holding time: 10 seconds ·Horizontal holding time: 300 seconds Number of mixing cycles: 1.

[0195] Cells were collected from the bag into a bottle / sample bag.

[0196] Example 2: Cultivation of MPC in a bioreactor system MPCs were cultured according to the protocol detailed in Example 1. Cells at passage 3 were used. The MPC culture medium was changed to one supplemented with 10% hPL (Paracelsus). The remaining components of the culture medium were used as indicated in Example 1. The culture method included a second expansion step after 10 days. The volume of the culture medium containing the microcarriers was increased from 400 ml to 800 ml, resulting in a total surface area of ​​6800 cm, as shown in Table 1. 2 This is what happened.

[0197] Cells were counted and visualized at least every 2-3 days (Table 1, Figure 1, and Figure 2). Therefore, a small, homogenous sample was taken from the bioreactor bag. 1 mL was used for visual inspection using an optical microscope, and images were taken at 40x and 100x magnification. For cell counting, the remaining volume was collected by dissolving the microcarriers using a collection solution (PBS, TrypLE, EDTA, and pectinase). The single-cell suspension was then counted using an NC-250 NucleoCounter. The total cell count was adjusted to account for biomass loss due to sampling. Therefore, the expected total cell count is shown in the last row of Table 1.

[0198] [Table 1]

[0199] Example 3: Characteristics of MPCs obtained in a large-scale culture system MPCs harvested from the culture and bioreactor were analyzed to confirm their myogenic characteristics. For clinical purposes, MPCs should express ≥50% α-actinin, ≥60% Pax7, desmin, and ≤15% CD34. The presence and levels of these MPC markers were analyzed by flow cytometry and compared to MPCs cultured as monolayers ( Figures 3 and 4 ). Each of the different culture methods used the MPC culture medium described in Example 1, modified to contain 5% PLT Gold HPL ( Figure 3A ) and 10% PLT Gold HPL ( Figures 3B and 4 ), respectively.

[0200] Briefly, frozen MPCs were thawed and cultured as monolayers for one passage (P1). Cells were then seeded into bioreactors or T75 monolayer flasks. For bioreactor culture, cells were seeded (1 × 10 6 MPCs), as described in Example 2, i.e., about 160-200 x 10 6 A second expansion step was included to obtain 100 cells, which were then cultured.

[0201] In principle, the cultivation in monolayer flasks was carried out as previously described, for example, in WO 2019 / 215090.

[0202] Harvested cells were analyzed for marker expression by flow cytometry. MPCs were fixed with 2% PFA (Alfa Aesar) in PBS for 10 minutes at room temperature and permeabilized (0.5% Titron-X-100 (VWR) for 10 minutes at room temperature). Nonspecific binding sites were then blocked (5% FBS (Sigma) in 0.5% Titron-X-100 in PBS for 20-60 minutes at 2-8°C). Surface and intracellular staining with direct unlabeled antibodies / isotype controls for 30 minutes at 2-8°C: ·APC-CD34(TFS) / APC-isoCD34=APC-msIgG1(TFS) Anti-Pax7 (Sigma) / msIgG2a (TFS) FITC-anti-alpha-actinin (Miltenyi) / FITC-REA (Miltenyi) ·Anti-A2B5 (Sigma) ·PE-CD56 / PE-msIgG1(Beckman Coulter) ·PE-CD105 / PE-msIgG1(Beckman Coulter) Anti-desmin (Sigma) / msIgG1 (Santa Cruz Biotechnology) Human / mouse Myf-5 AlexaFlour488 (R&D Systems) / msIgG2a (TFS) MyHC anti-human / mouse / rat-APC / REA control (S), human IgG1-APC (Miltenyi Biotec) ·Anti-MyoD(BD Biosciences) / msIgG1(Santa Cruz Biotechnology)

[0203] Antibodies were diluted in autoMACS Running Buffer (Miltenyi Biotec). MPCs were stained with FITC-conjugated secondary antibodies (BD) for Pax7 and corresponding isotype controls for 30 min at 2–8°C. Data were acquired using MACSQuant by Miltenyi using the manufacturer's protocol, including MACSQuant Running Buffer, MACSQuant Washing solution, MACSQuant Storage Solution, MACSQuant Calibration Beads, and the manufacturer's software (MACS Quantify Software). Data were analyzed using Flow Jo software.

[0204] As can be seen in Figures 3 and 4, both culture methods, i.e., bioreactor and monolayer flask, resulted in MPCs that expressed myogenic markers, with approximately 99% of the cells being positive for Pax7, α-actinin, and A2B5, and negative for CD34 expression. In addition, the various culture configurations resulted in similar populations of MPCs, and for that reason, it can be concluded that large-scale culture in bioreactors is suitable for generating large numbers of MPCs suitable for clinical downstream applications. Furthermore, as can be seen in Figure 5, in addition to exhibiting typical myogenic markers α-actinin and A2B5 (99.9% and 99.7%, respectively), and negative (0.1%) for CD34, cells cultured in the bioreactor were also positive for expression of Myf5, myHC, and MyoD (67.6%, 8.7%, and 19.6%, respectively), and showed very low expression of CD56 (3.3%). Desmin expression varied between different bioreactor runs.

Claims

1. A method for obtaining a large-scale culture of muscle progenitor cells (MPCs) derived from skeletal muscle, comprising at least the following steps: (a) culturing MPCs in a vessel containing a culture medium comprising the microcarriers under conditions that allow the MPCs to adhere to the microcarriers, wherein the MPCs grow within 1 cm of the growth surface area provided by the microcarriers; ２ at a density of 500-1500 cells per cm, preferably 800-1200 cells / cm ２ and (b) increasing the growth surface area of ​​the culture medium when cell number increases by about 8-fold to 25-fold; and (c) preferably 5 to 7.5 × 10 ４ cells / cm ２ and / or 4 to 6.5 × 10 ５ further culturing the MPCs until a cell density of up to 1000 cells / ml is reached; and optionally (d) harvesting said MPCs.

2. 2. The method of claim 1, wherein the growth surface is increased by a factor of 2 to 4, preferably about 3, and preferably the volume of the culture medium is increased to the same extent as the growth area.

3. 3. The method of claim 1 or 2, wherein steps (b) and (c) are repeated one or more times, preferably once or twice, most preferably twice.

4. The starting volume of the culture medium is about 100-150 ml, and the volume of the culture medium is (i) in the first step (b) to 400 ml, and optionally (ii) in a second step (b) the volume is increased to 800-1000 ml, preferably 1000 ml; Preferably, the further culturing step in step (c) comprises culturing about 1.5 to 2.75 x 10 ８ The method according to any one of claims 1 to 3, wherein the method is carried out until a total cell count of

5. The method according to any one of claims 1 to 4, wherein the vessel is a closed bioreactor, preferably a bioreactor bag.

6. The method according to any one of claims 1 to 5, wherein the vessel is an expandable vessel, preferably an expandable bioreactor bag.

7. 7. The method of any one of claims 1 to 6, wherein the microcarriers are coated microcarriers, more preferably wherein the microcarriers are collagen-coated microcarriers, and / or wherein the microcarriers are dissolvable.

8. The method of any one of claims 1 to 7, wherein the culture medium comprises human platelet lysate (hPL).

9. 9. The method of any one of claims 1 to 8, wherein at the end of the culture, the MPCs are separated and harvested from the microcarriers, preferably wherein said separation comprises complete lysis of the microcarriers, preferably wherein said lysis of the microcarriers is carried out by enzymatic digestion, preferably by the addition of an endopeptidase which cleaves proteins at specific sites, most preferably by trypsin, or a corresponding trypsin substitute, and pectinase.

10. 10. A cell population comprising MPCs obtainable by the method of any one of claims 1 to 9, preferably comprising ≧40% α-actinin-positive cells, ≧60% Pax7-positive cells, and / or ≦20% CD34-positive cells, and optionally desmin-positive cells, preferably ≧50% α-actinin-positive cells, ≧60% Pax7-positive cells, and / or ≦15% CD34-positive cells, and optionally desmin-positive cells, and most preferably ≧80% α-actinin-positive cells, ≧80% Pax7-positive cells, and / or ≦5% CD34-positive cells, and optionally desmin-positive cells, preferably ≧10% desmin-positive cells.

11. The population according to claim 10, comprising ≧60% A2B5 positive cells, preferably ≧80% A2B5 positive cells.

12. 12. The population according to claim 10 or 11, comprising ≦15% CD56 positive cells, preferably ≦10% CD56 positive cells, more preferably ≦5% CD56 positive cells.

13. The population according to any one of claims 10 to 12, comprising ≧50% Myf5 positive cells, preferably ≧60% Myf5 positive cells, preferably ≧60-90% Myf5 positive cells.

14. The population according to any one of claims 10 to 13, comprising < 30% MyHC positive cells, preferably < 20% MyHC positive cells, preferably < 15% MyHC positive cells.

15. The population according to any one of claims 10 to 14, comprising 10 to 40% MyoD positive cells, preferably 10 to 30% MyoD positive cells, preferably 5 to 25% MyoD positive cells.

16. A therapeutically effective amount of said MPCs, preferably at least 1 x 10 ７ MPCs, preferably 6 x 10 ７ ~3 x 10 ８ MPCs, most preferably 1-3 x 10 ８ 11. The population of claim 10, comprising MPCs.

17. 10. A method for preparing a pharmaceutical product, comprising the steps of the method of any one of claims 1 to 9 and optionally adding a biomaterial solution, preferably a hydrogel solution, more preferably a collagen solution, to the harvested MPCs.

18. 13. The method of claim 12, further comprising the step of filling the MPC into a pharmaceutical container, preferably wherein the container is a syringe or a vial.

19. A composition comprising MPCs obtainable by the method of any one of claims 1 to 9 or a population of MPCs according to any one of claims 10 to 16.

20. 20. The composition of claim 19, further comprising a biomaterial solution, preferably a collagen solution, more preferably at a final concentration of 1-4 mg / mL, more preferably 2 mg / mL.

21. 20. The composition of claim 19 for use as a medicament, preferably for use in a method for treating skeletal muscle dysfunction, preferably wherein the skeletal muscle dysfunction is an abnormality of a sphincter, preferably the external urethral sphincter, and optionally wherein the composition further comprises a biomaterial solution, preferably a hydrogel solution, more preferably a collagen solution.

22. 22. A composition for use according to claim 21, wherein the method comprises obtaining MPCs from a patient, preparing a mass culture of said MPCs according to the method of any one of claims 1 to 9, preparing a composition according to any one of claims 19 to 21, and administering said composition to said patient, preferably by injection of the muscle to be treated.

23. 23. The composition for use according to claim 21 or 22, wherein the method comprises the steps of: a) cutting into small pieces tissue obtained from a muscle biopsy of a patient, preferably by using scissors, wherein said tissue is obtained from a skeletal muscle of said patient, more preferably from tissue selected from the group consisting of soleus, rectus abdominis, quadriceps, vastus lateralis and vastus intermedius, preferably from soleus muscle tissue; b) digesting said tissue biopsy, preferably with a mixture comprising one or more enzymes, preferably collagenase and dispase, to break down said tissue; c) preparing a cell suspension and seeding said cell suspension onto a coated dish, preferably a dish coated with an extracellular matrix protein, preferably collagen; d) incubating the rapidly adhering cells under appropriate culture conditions that allow the cells to attach to the dish, preferably at about 36-38°C for about 20-28 hours; e) replated the supernatant containing the non-adherent cells, mainly MPCs, onto a dish coated with extracellular matrix protein, preferably collagen, thereby obtaining a population containing MPCs; f) growing the cells until they reach a number suitable for use as inoculum in the method according to any one of claims 1 to 9; g) subjecting said population of MPCs to the method of any one of claims 1 to 9 to obtain a mass culture of MPCs; h) preparing a composition by mixing said MPC with a biomaterial solution, preferably a collagen solution; and i) administering said composition to said patient, preferably by injection into the muscle to be treated.

24. The composition for use according to any one of claims 21 to 13, wherein the final concentration of collagen in the composition is 1 to 4 mg / mL, preferably about 2 mg / mL.

25. 25. A composition for use according to any one of claims 21 to 24, comprising 10 to 30 million cells / ml with a viability of at least 80%.

26. 26. The composition for use according to any one of claims 21 to 25, wherein said skeletal muscle dysfunction is an abnormality of a sphincter, preferably the external urethral sphincter, preferably wherein said skeletal muscle dysfunction is urinary incontinence, preferably female urinary incontinence.

27. 27. The composition for use according to any one of claims 21 to 26, wherein the method further comprises subjecting the patient to neuromuscular electromagnetic stimulation (NMES), preferably wherein the strength of the induced electric field at maximum output is 120 V / m at the surface of the stimulation coil.

28. 21. Use of a composition according to claim 19 or 20 for the manufacture of a medicament for a method of treating skeletal muscle dysfunction, preferably said method being a method as defined in any one of claims 21 to 27.

29. 28. A method for treating skeletal muscle dysfunction in a patient, the method being as defined in any one of claims 21 to 27.

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