Process for preparation of large scale cultures of muscle precursor cells (mpc) and uses thereof

Abstract

Provided are methods of obtaining large scale cultures of muscle-derived muscle precursor cells (MPCs) using microcarriers as growth matrices, methods for obtaining therapeutically effective amounts of these cells, populations of cells obtained by the methods, and compositions comprising expanded cells, and a method for preparing a medicament, for example for the treatment of skeletal muscle dysfunction.

Description

Technical Field

[0001] The present invention relates to methods for obtaining large-scale cultures of muscle progenitor cells (MPCs), cell populations comprising MPCs obtained by said methods, and compositions comprising said MPCs. In addition, the present invention relates to methods for preparing a medicament for treating skeletal muscle dysfunction based on the obtained MPCs. Background Art

[0002] Skeletal muscle damaged by injury or by a degenerative disease such as muscular dystrophy is capable of regenerating new muscle fibers, wherein the regeneration mainly relies on myogenic progenitor cells. Therefore, muscle progenitor cell (MPC) transplantation has been investigated for treating various genetic and acquired muscle disorders. Satellite cells are quiescent adult stem cells and are located beneath the membrane surrounding muscle fibers. After trauma or injury, satellite cells are activated into MPCs and participate in tissue regeneration by proliferating and differentiating into myoblasts, which then fuse to form new muscle fibers. Most MPCs belong to the myogenic lineage and are thus most suitable for muscle tissue engineering (Eberli et al., Cell Transplant 21 (2012), 2089-98).

[0003] Given their regenerative capacity, MPCs are more differentiated than stem cells and are more prone to differentiate into the muscle lineage. They are a promising therapeutic option in injured, diseased, and aged muscle tissues, and their potential has been widely explored. Stress urinary incontinence (SUI), namely involuntary urine loss due to coughing, laughing, sneezing, exercise, and other actions that increase intravesical abdominal pressure, is an example of muscle dysfunction that can benefit from cell therapy.

[0004] For example, as commented by Schmid and his colleagues, cell-based therapeutic approaches have been developed to regenerate the sphincter, wherein progenitor cells are isolated from a living human tissue biopsy, then propagated in vitro and reimplanted to repair or replace injured or diseased tissues (Schmid et al., International Journal of Molecular Sciences 22 (2021), 3981). A particular option is to implant autologous muscle progenitor cells into the sphincter region to strengthen and restore external urethral sphincter function, as also disclosed in WO 2019 / 215090A1.

[0005] However, in such cell-based therapies for treating muscle dysfunction, one of the limiting steps is in particular the absence of an effective method to produce a sufficiently high amount of MPCs for therapeutic applications. Summary of the Invention

[0006] The present invention generally relates to a method for obtaining a large-scale culture of muscle-derived precursor cells (MPCs), wherein the culture is preferably carried out in a 3D culture system. In particular, the method of the present invention comprises culturing MPCs in a container comprising a culture medium and microcarriers under conditions that allow the MPCs to attach to the microcarriers, wherein the MPCs are preferably inoculated at a density between 500 and 1500 cells / cm 2 of the growth surface provided by the microcarriers. The method of the present invention further comprises the step of increasing the growth surface area in the culture environment when the number of cells from the initial inoculation preferably increases by about 8-fold to 25-fold. In one embodiment, when the number of cells increases to about 1.3x10 4 -1.8x10 4 cells / cm 2 and / or when more than 80%, preferably more than 90%, of the microcarriers are occupied, the step of increasing the growth area in the method of the present invention is carried out. The method of the present invention further comprises culturing the MPCs until the cell density reaches preferably 5 - 7.5x10 4 cells / cm 2 , i.e., at least or not more than 5 - 7.5x10 4 cells / cm 2 and / or 4 - 6.5x10 5 cells / ml. In a preferred embodiment, the cells are further cultured after the addition of the culture medium until a cell number of about 1.5 - 2.75x10 8 is obtained. Preferably, the MPCs are obtained from a patient, preferably a human patient as described below.

[0007] Thus, with the present invention, a culture system capable of generating a large amount of MPCs is established. MPCs are adherent-dependent cells, commonly referred to as adherent cells. These cells need to adhere to a surface to remain viable and proliferate. Compared with the application methods that have hitherto relied on monolayer MPC cultures on plates, the method of the present invention has unique advantages. In particular, using the growth area provided by the method of the present invention, a high cell yield can be obtained without using a replating step. Therefore, compared with the traditional monolayer culture system on plates, the method of the present invention is more labor-saving and time-saving, and due to the use of a closed system, the risk of contamination when applying the method of the present invention is also lower. It should also be emphasized that since this method can reach a large number of MPCs faster than the traditional 2D culture system, advantageously, the obtained MPC population is "closer" to the patient, for example, in terms of the adverse mutations known to accumulate during culture, i.e., due to the shorter culture time, the chance of adverse mutations occurring during culture is lower.

[0008] Another advantage of the method of the present invention is the lower seeding density required for efficient cell expansion. For example, from WO 2019 / 215090A1, it is known that using 5000 cells / cm2 Inoculation density to grow cells. In the method of the present invention, 500 - 1500 cells / cm 2 of inoculation density is sufficient to ensure cell growth and obtain a high yield after the culture step.

[0009] As shown in Example 2 and Figure 1 and Figure 2 as shown, MPC can be efficiently cultured and proliferated on microcarriers to obtain a large enough cell number for downstream applications, such as for therapeutic methods. To provide appropriate growth conditions, the cell concentration in the culture medium and its ratio to the growth surface provided by the microcarriers are crucial. Therefore, in a preferred embodiment, the method of the present invention includes inoculating MPC at a density between 800 - 1200 cells / cm 2 In another preferred embodiment, MPC is inoculated at a density between 800 - 1200 cells / cm 2 in the culture volume described below, preferably inoculated in 130 ml of culture medium.

[0010] As mentioned above, the method of the present invention includes the following steps: when a specific cell density, cell number, and / or cell fold increase as defined in other parts of this document are reached, the growth surface in the culture environment is increased. In a preferred embodiment, the growth surface area and optionally the culture medium volume are increased to between two and four times, preferably three times. In one embodiment, the starting volume used in the method of the present invention is about 100 to 150 ml, and the culture medium volume is increased to about 400 ml. To obtain a higher cell number, the step of increasing the growth surface can be repeated once or more, that is, when a specific cell density, cell number, and / or cell fold increase as defined in other parts of this document are reached, the growth surface can be increased again to between two and four times. Therefore, in one embodiment, the present invention includes one or more steps of increasing the growth area of the culture environment.

[0011] As shown in Examples 1 and 2, according to the method of the present invention, a bioreactor system can be used, that is, MPC can be cultured in a bioreactor to obtain a large-scale culture of MPC. Therefore, in one embodiment, the container used according to the present invention is a closed bioreactor. In a preferred embodiment, the container is a bioreactor bag. In another embodiment, the container used according to the method of the present invention is an expandable container. In a preferred embodiment, the container is an expandable bioreactor bag.

[0012] As shown by the flow cytometry analysis in Example 3, the method of the present invention generates MPCs, i.e., a population of MPCs that express myogenic markers. As further explained below, such a population includes, in addition to MPCs, other cells at different stages during muscle differentiation, such as cells of early lineages, and thus the population is a heterogeneous population. In addition, the cultured cells exhibit characteristics similar to those of MPCs generated by the method disclosed in WO2019 / 215090 A1, especially with respect to the characteristics necessary for therapeutic use, such as high expression of Pax7 and α-actinin and low expression of CD34, which confirms that these cells have the therapeutic use as described below.

[0013] According to the method of the present invention, microcarriers are used, which are capable of suspension culturing adherent cells and providing a large growth area available for cell growth. As explained above, this is more advantageous than conventional monolayer culturing of MPCs because the factor limiting the yield of large-scale cultures of adherent cells is the limited growth area in a 2D culture system. Thus, in one embodiment, the microcarriers are coated microcarriers, preferably collagen-coated microcarriers. In one embodiment, the microcarriers are soluble, and in a preferred embodiment, the microcarriers used according to the present invention are collagen-coated and soluble.

[0014] In one embodiment, the culture medium used according to the method of the present invention comprises human platelet lysate (hPL).

[0015] In one embodiment, the culture medium used according to the method of the present invention comprises human platelet lysate (hPL), preferably fibrinogen-depleted hPL, and is heparin-free, and thus does not contain allergy-prone components used in conventional growth media, such as serum or heparin.

[0016] In one embodiment, the culture medium used according to the method of the present invention comprises human platelet lysate (hPL) and is heparin-free, and thus does not contain allergy-prone components used in conventional growth media, such as serum or heparin.

[0017] For downstream applications, it is important that the cells themselves are available without being attached to the microcarriers. Thus, in one embodiment, the method of the present invention includes the 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, wherein the dissolution of the microcarriers is preferably carried out by enzymatic digestion, preferably by adding and pectinase.

[0018] The present invention further relates to a cell population comprising MPCs that can be obtained by the method of the present invention as disclosed herein.

[0019] As shown by the flow cytometry analysis in Example 3, culturing produced MPCs, i.e., a population comprising MPCs expressing myogenic markers. This population is a heterogeneous population and, in addition to MPCs, also includes other cells at different stages during muscle differentiation, such as cells of early lineages. Thus, the presence of cells at different stages during muscle differentiation explains the percentage of cells expressing specific marker genes shown in Example 3 as well as Figure 4 and Figure 5 . As Figure 3 and Figure 4 show, culturing in a bioreactor produced MPCs expressing myogenic markers, where approximately 99% of the cells were positive for Pax7, α - actinin, and A2B5 and negative for CD34 expression.

[0020] In one embodiment, more than 40% of the cells in 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 in 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.

[0021] In one embodiment, more than 40% of the cells in 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 in 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.

[0022] 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, that is, the population includes ≥50% α - actinin - positive cells, ≥60% Pax7 - positive cells and ≤15% CD34 - positive (≤15% of the cells in the population express CD34) cells.

[0023] 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, that is, the population includes ≥80% α - actinin - positive cells, ≥80% Pax7 - positive cells and ≤5% CD34 - positive cells.

[0024] The population of the present invention can be further characterized by its expression of A2B5. Thus, in one embodiment, more than 50% of the cells in the population express A2B5, 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%.

[0025] 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, that is, the population includes ≥50% α - actinin - positive cells, ≥60% Pax7 - positive cells, ≥60% A2B5 - positive cells and ≤15% CD34 - positive (≤15% of the cells in the population express CD34) cells.

[0026] 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.

[0027] Additionally, or alternatively, the population of the invention is further characterized in that it comprises cells that express desmin, preferably, wherein 1% to 99% of the cells in the population, preferably between 10% and 90%, or between 20% and 80%, preferably 75%, preferably up to 70%, preferably between 20% and 70%, or 60% of the cells express desmin.

[0028] 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 wherein the cells that express desmin are preferably ≥10%, i.e., the population comprises ≥50% α-actinin-positive cells, ≥60% Pax7-positive cells, and ≤15% CD34-positive (≤15% of the cells in the population express CD34) cells, and wherein the population comprises cells that express desmin, preferably ≥10%.

[0029] 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, and wherein 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 wherein the population comprises cells that express desmin, preferably ≥10%.

[0030] 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 wherein the cells that express desmin are preferably ≥10%, i.e., the population comprises ≥50% α-actinin-positive cells, ≥60% Pax7-positive cells, ≥60% A2B5-positive cells, and ≤15% CD34-positive (≤15% of the cells in the population express CD34) cells, and wherein 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, ≥80% of the cells in the population express A2B5 and ≤5% of the cells in the population express CD34, and wherein, 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 wherein, the population comprises cells expressing desmin, preferably ≥10%.

[0032] In addition, as Figure 5 visible in, in addition to showing typical myogenic markers α-actinin and A2B5 (99.9% and 99.7% respectively) and CD34 negativity (0.1%), the cells cultured in the bioreactor also showed positive expression of Myf5, myHC and MyoD (67.6%, 8.7% and 19.6% respectively), and showed very low CD56 expression (3.3%).

[0033] Therefore, the population of the present invention can be further characterized as: comprising ≤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; most preferably, the cell population comprises ≤5% CD56-positive cells, ≥60 - 90% Myf5-positive cells, ≤15% MyHC and / or 15 - 25% MyoD-positive cells.

[0034] More specifically:

[0035] The population of the present invention can be further characterized as: expressing MyHC. In particular, in one embodiment, 0% to 29% of the cells in the population express MyHC, and thus, in one embodiment, ≤29% of the cells in the population express MyHC, preferably ≤25%, preferably ≤20%, preferably ≤15%, more preferably ≤10% of the cells express MyHC.

[0036] Therefore, 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Preferably, the cells in the population of the present invention further express desmin as described above.

[0041] The population of the present invention can be further characterized in that it expresses MyoD. In particular, in one embodiment, 10% to 40% of the cells in the population, preferably 10% to 30% of the cells in the population, preferably 15% to 30% of the cells in the population, more preferably 15% to 25% of the cells in the population, express MyoD.

[0042] 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% to 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% to 40% MyoD-positive cells.

[0043] 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% to 30% of the cells in the population express MyoD, i.e., the population includes ≥80% α-actinin-positive cells, ≥80% Pax7-positive cells, ≤5% CD34-positive cells, and 10% to 30% MyoD-positive cells.

[0044] 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% to 40% of the cells in the population express MyoD, i.e., the population includes ≥50% α-actinin-positive cells, ≥60% Pax7-positive cells, ≥60% A2B5-positive cells, ≤15% CD34-positive cells, and 10% to 34% MyoD-positive cells.

[0045] 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% to 30% of the cells in the population express MyoD, i.e., the population includes ≥80% α-actinin-positive cells, ≥80% Pax7-positive cells, ≥80% A2B5-positive cells, ≤5% CD34-positive cells, and 10% to 30% MyoD-positive cells.

[0046] 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% to 40% of the cells in the population express MyoD, i.e., the population includes ≥50% α-actinin-positive cells, ≥60% Pax7-positive cells, ≤15% CD34-positive cells, ≤29% MyHC-positive cells, and 10% to 40% MyoD-positive cells.

[0047] 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 in the population express MyHC, and 10% to 30% of the cells in the population express MyoD, i.e., the population includes ≥80% α-actinin-positive cells, ≥80% Pax7-positive cells, ≤5% CD34-positive cells, ≤15% MyHC-positive cells, and 10% to 30% MyoD-positive cells.

[0048] 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% to 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% to 40% MyoD-positive cells.

[0049] 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% to 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, ≤15% MyHC-positive cells, and 10% to 30% MyoD-positive cells.

[0050] Preferably, the cells in the population of the present invention further express desmin as described above.

[0051] The population of the present invention can be further characterized by its expression of CD56. Specifically, in one embodiment, 0% to 15% of the cells in the population express CD56, preferably 5% to 15%, and thus, in one embodiment, ≤15% of the cells in the population express CD56, preferably ≤10%, more preferably ≤5% of the cells express CD56.

[0052] 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.

[0053] 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.

[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, 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.

[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, ≥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.

[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, ≤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.

[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, ≥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.

[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, 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.

[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, ≥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 cell population in the population expresses CD56.

[0060] Preferably, the cells in the population of the present invention further express desmin as described above.

[0061] The population of the present invention can be further characterized by its expression of Myf5. Specifically, in one embodiment, ≥50% of the cells in the population express Myf5, preferably ≥60% of the cells in the population express Myf5, and preferably 60% to 89%, more preferably 65% to 89% of the cells in the population express Myf5.

[0062] 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.

[0063] 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.

[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, 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.

[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, ≤29%, preferably ≤15% of the cells in the population express MyHC, and ≤50% of the cells in the population express Myf5, and preferably 60% to 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, ≤15% of the cells in the population express CD56, and ≤50% of the cells in the population express Myf5, and preferably 60% to 89% of the cells in the population express Myf5.

[0067] 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, and 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, ≤29%, preferably ≤15% of the cells in the population express MyHC, and ≤50% of the cells in the population express Myf5, and 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, ≤15% of the cells in the population express CD56, and ≤50% of the cells in the population express Myf5, and 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, ≤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 ≤50% of the cells in the population express Myf5, and preferably 60% to 89% of the cells in the population express Myf5.

[0071] 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 ≤50% of the cells in the population express Myf5, and preferably 60% to 89% of the cells in the population express Myf5.

[0072] 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, and preferably 60% to 89% of the cells in 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, ≥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, and 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, ≤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, and 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, ≤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, ≤29%, preferably ≤15% of the cells in the population express MyHC, ≤50% of the cells in the population express Myf5, and preferably 60% to 89% of the cells in the population express Myf5.

[0076] 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, ≤29%, preferably ≤15% of the cells in the population express MyHC, ≤50% of the cells in the population express Myf5, and preferably 60% to 89% of the cells in the population express Myf5.

[0077] Preferably, the cells in the population of the present invention further express desmin as described above.

[0078] As described above, the population of the present invention can be characterized in that it comprises ≥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.

[0079] In one embodiment, the population can be further characterized in that it comprises ≤29%, preferably ≤15% of the cells expressing MyHC.

[0080] Low expression of MyHC (which is a marker of contractile proteins and is expressed in more differentiated cell populations) indicates that most cells are in an early stage, i.e., undifferentiated.

[0081] Alternatively, or in addition, the population can be further characterized as comprising cells that express MyoD, and preferably, the population comprises between 10% and 40% (preferably between 10% and 30%) of cells that express MyoD. The population can be further characterized as comprising between 60% and 89% of cells that express Myf5.

[0082] 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, based on the marker expression data (e.g., the presence of Pax7 and MyoD / Myf5 positive cells), it can be concluded that the population of the present invention comprises a plurality of activated satellite cells that are still capable of redifferentiating back into quiescent cells to fill the gap for potential future muscle injury.

[0083] Alternatively, or in addition, the population is further characterized as comprising ≤15% of cells that express CD56. The low expression of CD56, which is a marker for pure myoblasts, indicates that the population is in an early differentiation state.

[0084] The population can be further characterized as comprising ≥60%, preferably ≥80%, of cells that express A2B5. Optionally, but preferably, the population further comprises cells that express desmin.

[0085] In a preferred embodiment, the cell population of the present invention comprises a therapeutically effective amount of MPCs, preferably at least 1x10 6 MPCs, preferably at least 1x10 7 MPCs. In a preferred embodiment, the population comprises at least 1 - 3x10 8 MPCs.

[0086] The present invention also encompasses a method for preparing a medicament, the method comprising the steps of: a method for obtaining a large-scale culture of MPCs of the present invention as disclosed herein; and optionally, adding a biomaterial solution, preferably a hydrogel solution, more preferably a collagen solution, most preferably at a final concentration of 1 - 4 mg / mL, preferably 2 mg / mL, to the harvested MPCs. In one embodiment, the method further comprises the step of filling the MPCs into a medicament container, which is preferably a syringe or a vial.

[0087] In addition, the present invention relates to a composition comprising MPCs obtainable by a method of obtaining a large-scale culture of the MPCs of the present invention as disclosed herein. According to the present invention, the composition is used as a medicament. In a preferred embodiment, the present invention relates to a composition for treating muscle dysfunction, preferably wherein the muscle dysfunction is skeletal muscle dysfunction, more preferably wherein the skeletal muscle dysfunction is a defect of the sphincter (preferably the external urethral sphincter). Optionally, the composition of the present invention further comprises a biological material solution, preferably a hydrogel solution, more preferably a collagen solution, which is preferably mixed with the MPCs, most preferably at the concentrations specified above.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein 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 not intended to be limiting.

[0089] Further embodiments of the present invention will be apparent from the following description and examples.

[0090] To avoid any doubt, it is emphasized that the phrases "in some embodiments", "in certain embodiments", "in certain cases", "in some cases", "in further embodiments", "in one embodiment", etc. are used, and it is meant that when reading any of the embodiments described therein, each feature of those embodiments is to be considered in combination, and the present disclosure must be treated in the same way as if the combination of the features of those embodiments were set forth in detail in one embodiment. The same is true for any combination of the embodiments and features exemplified in the appended claims and examples, which are also intended to be combined with the features of the corresponding embodiments disclosed in the specification, where, for reasons of consistency and brevity only, the features of the embodiments are in a subordinate relationship, but in fact, each combination of an embodiment and a feature, which can be construed due to the (multiple) subordinate relationships, must be considered to be explicitly disclosed rather than as an alternative among different options.

[0091] The term "between" includes the endpoints. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 : Quantification of cultured MPCs (passage 3). Figure 1 Growth curves of the cell counts presented in Table 1 are given, i.e., growth curves of the cell counts adjusted for biomass loss due to sampling.

[0093] Figure 2 : Visually inspect the cultured MPCs at magnifications of 40x and 100x.

[0094] Figure 3 : Flow cytometry analysis of MPCs cultured according to the present invention, compared with cells cultured in monolayer culture flasks in the presence of 5% (A) and 10% (B) hPL. The expression of markers Pax7, α - actinin, and CD34 was analyzed.

[0095] Figure 4 : Flow cytometry analysis of MPCs cultured according to the present invention, compared with culture in monolayer culture flasks. The expression of markers Pax7, α - actinin, CD34, and A2B5 was analyzed.

[0096] Figure 5 : Flow cytometry analysis of MPCs cultured according to the present invention, compared with culture in monolayer culture flasks. The expression of markers α - actinin, A2B5, CD34, CD56, Myf5, MyHC, and MyoD was analyzed. Detailed Description

[0097] The present invention relates to a method for obtaining muscle progenitor cells (MPCs), which includes culturing and expanding MPCs, preferably in a 3D culture system. More specifically, the method of the present invention relates to a method for obtaining a population of MPCs comprising a therapeutically effective amount as defined below. In particular, the present invention relates to a method for obtaining MPCs, which includes growing MPCs on microcarriers in a suspension culture. Microcarriers are particles with a high surface area to volume ratio. The surface provided by the microcarriers can serve as a culture substrate for adherent cells, and thus adherent cells can be effectively expanded in a smaller 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. Once the cells attached to the microcarriers reach a certain density, the growth surface provided by the microcarriers increases. The cells are cultured until the desired cell number is reached.

[0098] Each type of cell requires different culture conditions, and the process established for one cell type generally cannot be used for another cell type, and a new process must be established. In addition, the culture conditions for obtaining large - scale cultures are difficult to predict. For example, roller bottle cultures have been performed, in which MPCs grow in a medium comprising microcarriers. For microcarrier - based expansion, the cells need to migrate from one microcarrier to another, and during this process, the cells must be kept evenly distributed on the microcarriers to achieve a high cell yield. In this context, we found that about 5000 cells / cm 2The seeding density is the best choice to achieve a high cell yield; see the poster presentation of Burer et al., "Optimization of Microcarrier-based Culture of Muscle Precursor Cells", Scinus Cell Expansion. Thus, surprisingly, according to the present invention, a low seeding density of only 500 - 1500 cells / cm 2 is already sufficient to provide sufficient cell growth and achieve a high yield.

[0099] The culture system used according to the present invention, such as the culture system described in Example 1, is specifically designed and modified for the growth and expansion of MPC. In particular, it has been found that the seeding density, i.e., the initial concentration of cells in the culture medium and the ratio of cells to the growth surface provided by the microcarriers, is crucial for successful culture. Thus, the seeding density is between 500 - 1500 cells / cm 2 , preferably between 800 - 1200 cells / cm 2 , and most preferably about 900 cells / cm 2 . Thus, in the most preferred embodiment, the seeded cell concentration is about 7500 cells / mL and the microcarrier concentration is 1.7 g / L. When about 10 5 to 10 6 cells, preferably 10 6 cells, are seeded in 130 mL of culture medium, this seeding density is particularly suitable. Given the small number of donor cells, the seeding density has been specifically adjusted to fit perfectly with the expected MPC protocol. Another important factor is the expansion, where the cell density allowing successful expansion of cells varies for each cell type. Thus, a range of cell densities allowing successful expansion of MPC on microcarriers has been established, and when the cell number increases to about 8 to 25 times, preferably when the density reaches 1.3x10 4 -1.8x10 4 cells / cm 2 (i.e., 1.1x10 5 -1.5x10 5 cells / mL), and / or when more than 80% (preferably 90%) of the microcarriers are occupied, the surface area increases, preferably increasing to about 3 times. This occurs after about three days. The end of the culture is another key parameter. In particular, when a certain cell density is reached, the culture ends, which provides sufficient cells for further applications (such as the therapeutic applications mentioned below). Preferably, the following cell density has been reached: 5 - 7.5x10 4 cells / cm 2 (4 - 6.5x10 5cells / ml), preferably 6.7x10 4 cells / cm 2 (5.7x10 5 cells / mL). This is approximately after six days of culture. More cells can be obtained by additional expansion steps, i.e., by further increasing the growth area, preferably increased to about 3 times, when more than 80%, preferably 90%, of the microcarriers are occupied or when the cell density reaches 1.3x10 4 -1.8x10 4 cells / cm 2 (i.e., 1.1x10 5 -1.5x10 5 cells / mL). Those skilled in the art will understand that whenever such an occupancy rate and / or cell density is reached, such an expansion step can be repeated additionally.

[0100] Muscle precursor cells

[0101] As used herein, the term "muscle precursor cell" or "MPC" or simply "cell" (if not otherwise specified) refers to the collection of all muscle-derived precursor cells that express muscle-specific markers and are capable of generating new muscle fibers, such as those defined, for example, by Eberli et al., Methods 47(2009), 98-103. MPCs are also referred to as proliferating satellite cells. The term "MPC population" or "population comprising MPCs" or "MPC", etc., means that MPCs represent the main cell type of the population. However, an MPC population may include other cell types in addition to MPCs, i.e., an MPC population includes preferably 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 approximately 100% MPCs. For example, an MPC population may include myofibroblasts in addition to MPCs, and according to this definition, is still considered an MPC population. Additionally, when myogenic markers can be detected as described herein (i.e., for example, the presence of α-actinin, Pax7, A2B5 and the absence of CD34), the cell population is considered an MPC population; for detailed information on marker expression, see the "Cell Populations and Therapeutic Aspects" section herein.

[0102] MPCs cultured according to the method of the present invention and MPCs obtained by the method of the present invention can be derived from muscle tissues of any species, preferably mammals, more preferably domestic animals, namely pets or livestock (farm animals), or humans. Pets include, but are not limited to, dogs, cats, rabbits, guinea pigs, hamsters, and horses. Livestock include, but are not limited to, cows, dairy cows, pigs, sheep, goats, donkeys, camels, water buffalo, and elephants. Most preferably, the method of the present invention is used to obtain human MPCs (hMPCs). Accordingly, the present invention relates to a method for obtaining a large-scale culture of MPCs, wherein the MPCs are preferably mammalian MPCs, more preferably domestic animal MPCs, such as pet MPCs or livestock MPCs as defined above, and most preferably human MPCs (hMPCs). The MPCs are preferably derived from skeletal muscle and are preferably taken from healthy muscle, preferably from tissues selected from the group consisting of soleus, rectus abdominis, quadriceps femoris, vastus lateralis, and vastus intermedius. If the MPCs obtained by the method of the present invention are intended to be used for treating skeletal muscle dysfunction as outlined in the "Cell Populations and Therapeutic Aspects" section below, those skilled in the art can readily envision that, depending on the target muscle, i.e., the damaged muscle to be treated, the biopsy is taken from a healthy muscle with a similar structure. For example, slow twitch muscle fibers are similar to sphincter muscles, and the soleus mainly contains such slow twitch fibers. Thus, in one embodiment of the present invention, the MPCs are obtained from slow twitch muscle fibers and preferably from the soleus (left or right leg), which has a composition similar to that of the sphincter muscle and is easily accessible. Alternatively, the vastus lateralis can be used. Accordingly, the MPCs obtained by the method of the present invention are preferably MPCs derived from slow twitch muscle fibers, preferably MPCs derived from soleus, rectus abdominis, quadriceps femoris, vastus lateralis, or vastus intermedius, and most preferably MPCs derived from soleus or vastus lateralis, especially MPCs derived from soleus. Depending on the target muscle of the MPCs according to the present invention, for example, if the target muscle is a fast twitch muscle, the biopsy can be taken from a fast twitch muscle.

[0103] According to the method of the present invention, the MPCs to be cultured can be obtained in different ways to obtain the corresponding large-scale culture. WO 2019 / 115790 A1 describes a preferred method for isolating MPCs, and these cells can be used as an inoculum in the method of the present invention, and thus, the method of the present invention can be used to obtain a large-scale culture of those cells. In a preferred embodiment, the MPCs cultured according to the method of the present invention are isolated as described in WO 2019 / 215090 A1, particularly as described in Example 1, the content of which is incorporated herein by reference.

[0104] Thus, in a preferred embodiment, for the isolation of MPCs, muscle biopsies are taken from muscle tissue, preferably from skeletal muscle, and more preferably from muscle tissue dominated by slow-twitch or fast-twitch muscle, preferably from slow-twitch muscle, and most preferably from the non-limiting group consisting of: soleus muscle, rectus abdominis muscle, quadriceps femoris muscle, vastus lateralis muscle, vastus intermedius muscle. In another preferred embodiment, the biopsy is taken from the soleus muscle or the vastus lateralis muscle, and most preferably from the soleus muscle.

[0105] In a particular embodiment, fat and / or tendon, and / or connective tissue are removed from the human tissue sample, and the biopsy is minced, preferably by using scissors, to obtain a viscous mixture and digested, preferably by a mixture containing one or more enzymes, preferably collagenase and dispase. Preferably, a mixture of about 0.05% to 2%, more preferably about 0.2% type I collagenase (w / v) and about 0.1% to 2%, more preferably about 0.4% - 1.6% dispase (w / v) is used. The enzymatic reaction is preferably carried out at 36 - 38 °C for 15 to 75 min, preferably 45 to 75 min. Once the desired degree of digestion is reached, the digestion can be terminated, preferably by adding cell culture medium (i.e., the growth medium defined herein); see the "Medium" section below.

[0106] In one embodiment, prior to the step of mincing the biopsy, there is a step of disinfecting the biopsy with a disinfectant and washing it with PBS.

[0107] After adding the growth medium, the digested mixture is mixed, preferably by pipetting and centrifugation. After centrifugation, the pellet is resuspended in the growth medium, preferably by pipetting up and down. In one embodiment, the growth medium includes 1% penicillin / streptomycin, preferably 1% (supplemented only in the passage 0 step). In one embodiment, the growth medium is free of penicillin / streptomycin. In an alternative embodiment, the growth medium includes one or more antibiotic agents other than penicillin or streptomycin, such as gentamycin. The cell suspension is filtered through a filter (preferably a filter with a pore size of 100 μm). Thereafter, the cells are seeded onto a coated culture dish. In particular, the cell suspension is transferred to a culture plate, such as a 35 mm culture dish (6 wells), coated with an extracellular matrix protein, such as collagen, fibronectin or laminin, preferably collagen, and most preferably type I collagen.

[0108] In one embodiment of the present invention, the plate on which cells obtained from a muscle biopsy are to be cultured is coated with a collagen solution, preferably a type I collagen solution, at a concentration of about 0.03 - 1.5 mg / ml, preferably about 0.05 - 1 mg / ml, and more preferably 0.05 mg / ml. The collagen solution is transferred to the culture plate such that the bottom of the wells is covered with the solution. Subsequently, the collagen solution is removed, and the coated plate is washed 3 times with PBS.

[0109] As used herein, the terms "collagen-coated plate(s)" or "plate(s)" are not limited to culture plates and also include culture dishes generally suitable for monolayer cell culture, such as cell culture flasks. Alternatively, in other embodiments of the present invention, the cells to be amplified from the biopsy can be cultured in multiple layers, for example, in multi-layer culture flasks or any other 2D culture system, or in any 3D culture system, such as on microcarriers in spinner flasks.

[0110] After inoculating the cells onto a plate coated with an extracellular matrix protein such as fibronectin or collagen as described above, the cells are incubated under appropriate culture conditions, preferably at 36 - 38 °C and 5% CO2 for about 20 to 28 h, preferably for 24 h. Thereafter, the supernatant containing non-adherent cells (mainly MPCs) is re-plated onto a culture dish coated with an extracellular matrix protein (such as collagen or fibronectin, preferably collagen, and most preferably type I collagen) to reduce the number of myofibroblasts. As described above, the plate is coated. The MPCs are allowed to settle in the coated culture dish, thereby obtaining a population comprising MPCs (preferably human MPCs). These cells are regarded as passage 0 (P0) MPCs.

[0111] The growth medium is preferably changed for the first time after 2 to 4 days and then changed every 2 to 4 days. If sufficient cells for inoculation into a large-scale culture system have been obtained in P0, i.e., about 10 5 to 10 6 cells, preferably 10 6 cells, then the MPCs (i.e., the population comprising MPCs) are directly transferred to a container for large-scale culture. Thus, in one embodiment, the MPCs cultured according to the method of the present invention are passage 0 (P0) cells, which are preferably obtained as described above.

[0112] Otherwise, the MPCs are split. To split the MPCs, they are washed with PBS and enzymatically detached from the plate according to a standard protocol, preferably using an enzyme such as trypsin, TrypLE etc. Thereafter, growth medium is added, the MPCs are centrifuged, and plated at 3000 - 7000 cells / cm 2Inoculate onto the plate at a density. In one embodiment of the present invention, MPCs are inoculated onto a plate coated with extracellular matrix proteins such as collagen or fibronectin. In an alternative embodiment, MPCs are inoculated onto a plate not coated with extracellular matrix proteins. These P1 cells are cultured by replacing the growth medium every 2 to 3 days. Then, these cells can be passaged again or used for inoculation into the large-scale culture system defined herein. When the cells are separated from the plate after centrifugation and resuspended in the growth medium, they are typically counted, including determination of cell viability. If necessary, the cells can also be frozen according to standard protocols, for example, for storage, before large-scale culture. If the cells have been frozen, they are typically passaged once in monolayer culture before large-scale culture.

[0113] Thus, in one embodiment, the MPCs cultured according to the method of the present invention are first-generation (P1) cells, which are preferably obtained as described above.

[0114] Optionally, the MPCs are replated to further expand the cells. Preferably, the cells are replated onto a coated culture dish, particularly a collagen-coated culture dish, and cultured in the growth medium. In an alternative preferred embodiment, the MPCs are replated onto an uncoated culture dish. Thus, in another embodiment of the present invention, the MPCs to be cultured in the method of the present invention are second-generation (P2) or third-generation (P3) MPCs, which are preferably obtained as described above. In a preferred embodiment, the MPCs are P1 or P2 cells, more preferably P1 MPCs.

[0115] If the MPCs are not directly transferred to the large-scale culture system but are frozen, for example, stored in liquid nitrogen, particularly cryopreserved in the gas phase of liquid nitrogen, they are typically passaged once in monolayer culture on a culture dish and then inoculated into the large-scale culture system. Thus, in one embodiment of the present invention, the cells obtained from a biopsy are frozen, for example, for storage, and passaged once in monolayer culture after thawing. In an alternative embodiment, the thawed cells are directly inoculated into the large-scale culture system.

[0116] Large-scale culture

[0117] The terms "mass culture" or "mass cultivation" refer to the amplification of cells to obtain a large enough cell number for a desired downstream application. Of course, the cell numbers required for certain downstream applications vary, but are known or can be envisioned by those skilled in the art.

[0118] The method according to the present invention is exemplarily carried out as described in Examples 1 and 2. For example, a total of about 2.8x10 8An MPC. Additionally, as shown in Example 3, the MPCs produced by the method of the present invention have a specific myogenic marker profile, including the presence of Pax7, α-actinin, desmin and the absence of CD34. This means that cells with sufficient and desired marker expression are obtained for use in cell therapy methods, such as for preparing the medicaments and / or compositions of the present invention as defined below. As a specific example, the target cell count injected into each patient for treating skeletal muscle dysfunction (such as urinary incontinence) preferably totals in the range of 80 million to 150 million cells. However, as described below, the therapeutic dose depends to a large extent on the indication to be treated.

[0119] An exemplary culturing method of the present invention is as shown in Example 1. In the first step of the method of the present invention, the MPCs as defined above are inoculated into a cell culture medium, and the MPCs are cultured in a medium comprising microcarriers, wherein the microcarriers provide a growth surface for the MPCs. In one embodiment, the method of the present invention includes pre-equilibrating the microcarriers with the medium under the required culturing conditions, that is, adding the microcarriers to the medium before inoculating the cells into the medium and storing them in the container for MPC culturing. The medium comprising the microcarriers as defined below and especially its volume are herein referred to as "starting medium" and "starting volume", respectively.

[0120] In one embodiment, the method of the present invention includes inoculating at least 10 5 cells, preferably 10 5 to 10 6 cells into the cell culture medium. Of course, according to the culture volume, the cell number is adjusted so that it is inoculated at about 750 cells / ml to 8000 cells / ml.

[0121] According to the method of the present invention, the MPCs are inoculated into a container comprising a medium and microcarriers at a density of 500 - 1500 cells / cm 2 growth surface area provided by the microcarriers. The cells are incubated in the container so that the cells attach to the microcarriers. In a preferred embodiment, the cells are inoculated at a density between 800 - 1200 cells / cm 2 growth surface area provided by the microcarriers. In one embodiment, the cell concentration inoculated according to the method of the present invention is 750 - 8000 cells / ml, preferably about 7700 cells / ml. In one embodiment, at least 10 5 MPCs, preferably 10 5 to 10 6 MPCs, more preferably about 10 6 MPCs are inoculated into a medium with a volume of 130 ml.

[0122] 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:

[0123] (a) Inoculate MPCs into a container containing a culture medium, which includes microcarriers, and allow the MPCs to attach to the microcarriers, wherein the MPCs are inoculated at a density between 500 - 1500 cells / cm 2 of the growth surface area provided by the microcarriers, preferably inoculated at a density between 800 - 1200 cells / cm 2 ; preferably, the growth area provided by the microcarriers is 5,000 - 10,000 cm 2 / L, more preferably 8,500 cm 2 / L; and

[0124] (b) Cultivate the MPCs in the container; and

[0125] (c) When the cell number increases to about 8 to 25 times, increase the growth surface area of the culture medium; and

[0126] (d) Further cultivate the MPCs, preferably until the cell density reaches 5 - 7.5x10 4 cells / cm 2 and / or 4 - 6.5x10 5 cells / ml, and optionally

[0127] (e) Harvest the MPCs.

[0128] In one embodiment of the present invention, the method for obtaining a large-scale culture of MPCs comprises at least the following steps:

[0129] (a) Inoculate MPCs into a container containing a culture medium, which includes microcarriers, and allow the MPCs to attach to the microcarriers, wherein the MPCs are inoculated at a density between 500 - 1500 cells / cm 2 of the growth surface area provided by the microcarriers, preferably inoculated at a density between 800 - 1200 cells / cm 2 ; preferably, the growth area provided by the microcarriers is 5,000 - 10,000 cm 2 / L, more preferably 8,500 cm 2 / L; and

[0130] (b) Cultivate the MPCs in the container; and

[0131] (c) When the cell density is 1.3x10 4 - 1.8x10 4 cells / cm 2 , increase the growth surface area of the culture medium; and

[0132] (d) Further cultivate MPC, preferably until the cell density reaches 5 - 7.5x10 4 cells / cm 2 and / or 4 - 6.5x10 5 cells / ml, and optionally

[0133] (e) Harvest the MPC.

[0134] In one embodiment of the present invention, the method for obtaining a large-scale culture of MPC comprises at least the following steps:

[0135] (a) Inoculate MPC into a container containing a culture medium, which includes microcarriers, and allow the MPC to attach to the microcarriers, wherein the MPC is inoculated at a density between 500 - 1500 cells / cm 2 of the growth surface area provided by the microcarriers, preferably inoculated at a density between 800 - 1200 cells / cm 2 ; preferably, the growth area provided by the microcarriers is 5,000 - 10,000 cm 2 / L, more preferably 8,500 cm 2 / L; and

[0136] (b) Cultivate the MPC in the container; and

[0137] (c) When at least 70% of the microcarriers, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, or 95%, most preferably at least 90% are occupied, increase the growth surface area of the culture medium; and

[0138] (d) Further cultivate MPC, preferably until the cell density reaches 5 - 7.5x10 4 cells / cm 2 and / or 4 - 6.5x10 5 cells / ml, and optionally

[0139] (e) Harvest the MPC.

[0140] In one embodiment of the present invention, the method for obtaining a large-scale culture of MPC comprises at least the following steps:

[0141] (a) Inoculate MPC into a container containing a culture medium, which includes microcarriers, and allow the MPC to attach to the microcarriers, wherein the MPC is inoculated in an amount of 750 - 8000 cells / ml, preferably at an amount of about 7700 cells / ml, preferably, the growth area provided by the microcarriers is 5,000 - 10,000 cm 2 / L, more preferably 8,500 cm 2 / L; and

[0142] (b) Cultivate MPC in a container; and

[0143] (c) When the cell number increases to about 8 - fold to 25 - fold, increase the growth surface area of the culture medium; and

[0144] (d) Further cultivate MPC, preferably until the cell density reaches 5 - 7.5×10 4 cells / cm 2 and / or 4 - 6.5×10 5 cells / ml, and optionally

[0145] (e) Harvest MPC.

[0146] In one embodiment of the present invention, the method for obtaining a large - scale culture of MPC comprises at least the following steps:

[0147] (a) Inoculate MPC into a container containing a culture medium, which includes microcarriers, and allow MPC to attach to the microcarriers, wherein MPC is inoculated in an amount of 750 - 8000 cells / ml, preferably about 7700 cells / ml, and preferably wherein the growth area provided by the microcarriers is 5,000 - 10,000 cm 2 / L, more preferably 8,500 cm 2 / L; and

[0148] (b) Cultivate MPC in the container; and

[0149] (c) When at least 70% of the microcarriers, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, or 95%, most preferably at least 90% are occupied, increase the growth surface area of the culture medium; and

[0150] (d) Further cultivate MPC, preferably until the cell density reaches 5 - 7.5×10 4 cells / cm 2 and / or 4 - 6.5×10 5 cells / ml, and optionally

[0151] (e) Harvest MPC.

[0152] In a preferred embodiment, the growth surface area and optionally the culture volume are increased between two- to four-fold, preferably three-fold (step (c)). In order to obtain a higher cell number, the step of increasing the growth surface can be repeated one or more times, i.e., when a specific cell density, cell number, and / or cell fold increase as defined elsewhere herein is reached, the growth surface can be increased again between two- to four-fold. Thus, in one embodiment, the present invention includes one or more steps of increasing the growth area of the culture environment, preferably 1 to 10 steps of increasing the growth area, preferably 1 to 8 steps of increasing the growth area, more preferably 1 to 4 steps, more preferably 1 or 2 steps, and most preferably 2 steps.

[0153] The term "culturing" refers to the conditions for maintaining and growing cells in cell culture. During the culturing according to the method of the present invention, the container and the culture medium are each kept in intermittent motion to keep the microcarriers suspended. When the microcarriers are kept uniformly suspended and do not settle or deposit (unless required to promote cell migration on the microcarriers), the cells attached to the microcarriers can reach an optimal growth state. In particular, keeping the microcarriers suspended and in motion can avoid a large number of cell aggregates that may lead to MPC differentiation or senescence. The force applied to keep the microcarriers suspended should ensure that the microcarriers do not settle or deposit. However, the force should not be too large so as not to damage the cells or the microcarriers. There are different possibilities for keeping the microcarriers suspended / moving and include but are not limited to agitating the culture medium and shaking the cell culture system, especially shaking bioreactors, as described in Example 1.

[0154] A typical cell culture environment for mammalian cells is known to those skilled in the art. For example, the pH range for culturing mammalian cells is generally between 7.2 and 7.6, and the temperature is generally between 36°C and 37°C. Experiments conducted according to the present invention confirm this, where growth was observed at pH 7.3 and 37°C. Thus, in one embodiment, the pH is between 7.2 and 7.6, but preferably, the pH is maintained between 7.3 and 7.4, most preferably 7.3; and the temperature set point is between 36°C and 37°C, preferably, the temperature is maintained at 37°C. The dissolved oxygen (DO) concentration is generally 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 to 75% and should not drop below 30%.

[0155] Experiments conducted according to the present invention show that inoculating cells with a smaller volume (starting volume) is beneficial for increasing the concentration of cultured cells. After a period of time, during cell expansion, a larger growth area is required, and for this purpose, the growth surface is increased by adding additional microcarriers. Thus, the method of the present invention comprises adding a cell culture medium comprising microcarriers to the cell culture to increase the growth surface. The increased culture medium, especially its volume, is hereinafter referred to as "amplification medium" and "amplification volume" respectively. In a preferred embodiment, the concentration of microcarriers in the amplification medium is approximately the same as the concentration in the starting medium, preferably between 1 - 2 g / L, more preferably about 1.7 g / L. According to the present invention, once the cell number increases to about 8 to 25 times, and / or when more than 80% of the microcarriers, preferably more than 90% are occupied, the volume of the culture medium is increased. In particular, increasing the volume of the culture medium while maintaining the microcarrier concentration means providing more growth area for further expansion of the cells. In an alternative embodiment, the growth surface is increased by adding microcarriers to the container, while the culture medium is not increased to the same extent, i.e., the final microcarrier concentration is higher or lower than before.

[0156] A convenient indicator for increasing the size of the amplification volume is when more than 50% of the microcarriers are occupied, preferably at least 75%, 80% or 85%, most preferably at least 90%.

[0157] In a preferred embodiment, the method of the present invention comprises when the cell density reaches 1.3x10 4 -1.8x10 4 cells / cm 2 , increasing the volume of the culture medium and / or the growth surface area.

[0158] In one embodiment, the method of the present invention comprises increasing the growth area to two to four times, preferably to approximately three times. For example, increasing the culture volume to about three times and substantially maintaining the microcarrier concentration in the amplification medium, i.e., adding a culture medium containing microcarriers having a concentration approximately the same as that during inoculation.

[0159] In one embodiment of the present invention, the starting volume, i.e., the volume of the culture medium inoculating the MPC, is about 100 to 150 ml, preferably about 130 ml. In a preferred embodiment, the starting volume is increased to 400 ml amplification volume.

[0160] In one embodiment, the method of the present invention further comprises the step of renewing the culture medium after increasing the growth surface, for example, renewing it every other day during further culturing of the cells, in order to provide sufficient nutrients for the MPC.

[0161] The cultivation time can vary and depends on the cell density as described above. MPC is preferably cultivated for 5 - 21 days, more preferably for 7 - 14 days, and even more preferably for 8 - 10 days. However, longer or shorter cultivation periods can be envisioned depending on the number of cells inoculated and / or their growth rate and / or the desired final cell number.

[0162] According to the method of the present invention, once the desired cell number is reached, preferably once the cell number has increased by about 100 - fold to 1000 - fold, and / or once the cell density has increased by about 10 - fold to 200 - fold, preferably about 13 - fold to 130 - fold, the cultivation is ended. In particular, MPC is further cultivated until it reaches a number suitable for further subsequent treatment methods, that is, specifically, until the cell density reaches 5 - 7.5x10 4 cells / cm 2 and / or 4 - 6.5x10 5 cells / ml. In a preferred embodiment, the cells are cultivated until a density of 5.7x10 5 cells / ml, which density has been confirmed to be the optimal density for supporting the maintenance of MPC characteristics (i.e., for example, not forming fibers). In one embodiment according to the present invention, MPC is cultivated until a total cell number of about 1.5x10 8 - 2.75x10 8 is obtained, preferably a total of about 2.3x10 8 cells. However, if a single amplification step (i.e., increasing the culture volume once) does not yield the required cell number, additional amplification steps can be carried out, thereby further increasing the culture volume and growth area, preferably increasing it by a factor of 3 for each amplification step, while preferably keeping the concentration of the microcarriers constant.

[0163] In one embodiment, MPC is harvested after reaching the desired cell number and cell density, respectively. In particular, in one embodiment, the method of the present invention includes, at the end of cultivation, the step of separating MPC from the microcarriers by lysing the anchoring protein (see above) via enzymatic or mechanical means, and the step of removing MPC from the bioreactor. In a preferred embodiment, the separation includes complete dissolution of the microcarriers. If insoluble microcarriers are used, the cells will be quickly separated from the microcarriers before the cells start to attach again.

[0164] Since MPC grows on microcarriers in a suspension culture environment similar to non - adherent cells, it can be envisioned that in additional amplification steps, the volume and / or growth surface of the culture system can be further increased to obtain an even greater number of cells.

[0165] Microcarrier

[0166] According to the present invention, a microcarrier generally refers to a negative carrier for culturing adherent-dependent cells. A "microcarrier" or "carrier particle" is defined as a bead-like material derived from silica, glass, dextran or similar materials, used for immobilizing biocatalysts or for culturing as a negative carrier for adherent-dependent animal cell lines (IUPAC Compendium of Chemical Terminology (2nd Edition, 1992, Vol. 64, p. 160)). Microcarriers can increase the growth surface area in tissue culture for the attachment and yield of adherent-dependent cells. The terms "growth surface area", "growth area" and "surface area" are used interchangeably herein and refer to the surface area provided by the microcarrier for adherent-dependent cells to attach to the culture. The microcarriers used according to the present invention are carrier materials, preferably in spherical form, and are suitable for suspension culture of adherent-growing cells, especially animal cells.

[0167] Microcarriers can be produced from a variety of materials, including plastics, glass, ceramics, silicone, gelatin, dextran, cellulose, etc. Additionally, microcarriers can be pretreated in various ways, including plasma treatment of plastic surfaces to produce hydrophilic surfaces, or the carrier can be coated (e.g., with gelatin, fibronectin, laminin, polyornithine, matrigel or with the binding motif of the fibronectin RGD-binding domain). In a preferred embodiment, the microcarriers used according to the present invention are made of polygalacturonic acid (PGA) polymer chains crosslinked via calcium ions and are coated with denatured collagen. Suitable commercially available microcarriers include Cytodex TM 1, Cytodex TM 3, Cytopore TM (Amersham Biosciences), G, S (Perbio), FACT (Sigma), Microhex TM (Nunc), lmmobaSil TM (Dunn) and collagen-coated (soluble) microcarriers (Corning TM ).

[0168] In one embodiment, the microcarriers used according to the method of the present invention are collagen-coated microcarriers. The microcarriers can be soluble or insoluble microcarriers. In a preferred embodiment, the microcarriers are soluble, and thus, the microcarriers are preferably collagen-coated soluble microcarriers. If the microcarriers are soluble, in one embodiment of the present invention, the dissolution of the microcarriers is carried out by enzymatic digestion, preferably by adding a harvesting solution comprising a peptidase (preferably an endopeptidase that cleaves proteins at specific sites, most preferably trypsin) or a corresponding trypsin substitute (such as or ) and pectinase. TrypLE cleaves the peptide bonds at the C-terminus of lysine and arginine and can directly replace trypsin and is animal component-free. is a natural enzyme mixture with proteolytic and collagenase activities. This means that it can simultaneously mimic the actions of trypsin and collagenase. Thus, in a preferred embodiment, the harvesting solution comprises trypsin and pectinase, or and pectinase, or and pectinase, most preferably and pectinase. In one embodiment, the above harvesting solution further comprises EDTA, which helps in the complete dissolution of the microcarriers.

[0169] 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 to 3 g / L, preferably about 1 - 2 g / L, more preferably 1.7 g / L, wherein the bead size is preferably 100 to 400 μm, preferably 200 - 300 μm when fully hydrated, and the surface is preferably 1000 - 10,000 cm 2 / g dry weight, more preferably 3000 - 8000 cm 2 / g dry weight, more preferably 4000 - 7000 cm 2 / g dry weight and most preferably 5000 cm 2 / g dry weight. Generally, the concentration of the microcarriers in the culture medium depends on the specific microcarriers used. For example, the microcarriers should provide a growth surface of 5,000 - 10,000 cm 2 / L of the culture medium. In a preferred embodiment, the microcarriers provide a growth surface of about 8500 cm 2 / L of the culture medium. This corresponds to using microcarriers at a concentration of 1.7 g / L and a surface of 5,000 cm 2 / g.

[0170] The purpose of the microcarriers is to provide an increased growth surface for adherent cells. Thus, in another preferred embodiment, the growth surface area provided by the microcarriers is 100 to 60,000 cm 2, more preferably, the growth surface area is 500 to 40,000 cm 2 , most preferably, the growth surface area is 1,000 to 20,000 cm 2 . Obviously, as the number of cells increases, additional microcarriers can be added to provide sufficient growth surface area. According to the amount of surface area occupied by the adherent cells, microcarriers can be added during the culture (e.g., during the expansion step).

[0171] After the cells grow until the desired cell amount (i.e., the cell amount defined above) is reached, 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 separated from the microcarriers. The separation can be carried out using a suitable separating agent. Suitable separating agents can be enzymes, thermoresponsive agents, and / or pH-responsive agents. In one embodiment of the present invention, a digestive enzyme that cleaves cells is used to separate the MPCs from the microcarriers. Preferably, the enzyme is an endopeptidase, more preferably selected from trypsin, or If insoluble microcarriers are used, the adherent cells that have been separated from the microcarriers can be removed through a 50 - 100 μm filter. The separated adherent cells will pass through the filter while the microcarriers remain in the container. For soluble microcarriers, the above-mentioned harvesting solution can be used.

[0172] In an alternative embodiment of the present invention, the harvesting of MPCs does not require separation from the microcarriers, i.e., the MPCs taken out from the culture system include the microcarriers. Therefore, the cells can be directly prepared for injection without separation, especially when the carrier is biocompatible and / or biodegradable (such as a collagen carrier). Therefore, for biocompatible / biodegradable microcarriers, the cells do not need to be separated and can be directly injected when attached to the microcarriers.

[0173] Culture medium

[0174] As used herein, the terms "growth medium" and "medium" are used interchangeably and refer to a solution comprising components and nutrients that support the viability and proliferation of the cells to be cultured according to the present invention. Suitable media for growing MPCs are known to those skilled in the art, such as those described in WO 1999 / 056785A2, WO 2001 / 078754 A2, WO 2008 / 066886A2, WO 2008 / 086040A1, WO 2009 / 045506 A2, and WO 2019 / 115790 A1.

[0175] 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, due to safety concerns, the use of these reagents should be avoided in clinical settings. Animal-free and serum-free reagents are highly desirable for enhancing the safety and quality of cell therapy methods. Possible alternatives to FBS are media supplemented with human serum, human platelet derivatives, allogeneic cord blood serum, or chemically defined media. Thus, in one embodiment, the medium used in the method according to the present invention is a substantially animal-free and / or serum-free medium, for example, a medium disclosed in WO 2019 / 215090 A1, which is incorporated herein by reference. Animal-free and / or serum-free means replacing serum such as FBS with hPL.

[0176] In particular, in one embodiment, according to the present invention, a growth medium comprising human platelet lysate (hPL), preferably pooled human platelet lysate (phPL), which is preferably filtered, is used. In one embodiment, the final concentration of phPL in the growth medium used according to the present invention is at least 5%, preferably about 5 - 20%, more preferably 7 - 12%, most preferably about 10% or about 5% (volume percentage). A 5% hPL concentration has been shown to minimize microcarrier aggregation. In certain embodiments, the medium comprises an anticoagulant factor, preferably heparin. For this purpose, for example, heparin Na (sodium heparin) (2500 IU / 5 ml) can be used. Heparin is added to the filtered phPL to form a mixture, and then the mixture is added to the nutrient solution of the growth medium, preferably at a final concentration of 1 - 10 IU / ml, 2 - 6 IU / ml, or about 2 IU / ml of the growth medium. Alternatively, other substances that anticoagulate (e.g., EDTA) can be used. In the case of using fibrinogen-depleted phPL, no anti-aggregation agent needs to be added as there are no longer active clotting factors present.

[0177] Thus, in a preferred embodiment, the cell culture medium used according to the present invention comprises fibrinogen-depleted human platelet lysate (hPL) and does not contain heparin.

[0178] The cell culture medium can further comprise the following components:

[0179] - A nutrient solution, preferably Dulbecco's Modified Eagle Medium (DMEM), more preferably a 1:1 DMEM / F12 nutrient mixture (a 1:1 mixture of DMEM and Flam's F-12);

[0180] - Human epidermal growth factor (hEGF), preferably added to the nutrient solution to a final concentration of 2 - 20 ng / ml, more preferably about 10 ng / ml;

[0181] - Human basic fibroblast growth factor (hbFGF), preferably added to the nutrient solution to a final concentration of 0.5 - 2 ng / ml, more preferably about 1 ng / ml;

[0182] - Insulin, preferably human insulin, preferably added to the nutrient solution to a final concentration of 5 - 20 μg / ml, more preferably about 10 μg / ml

[0183] - Dexamethasone, preferably added to the nutrient solution to a final concentration of 0.2 - 0.8 μg / ml, more preferably about 0.4 μg / ml.

[0184] According to one embodiment, the cell growth medium further comprises a solution containing an antibiotic agent, preferably containing penicillin and streptomycin, preferably at a final concentration of about 1% (penicillin (Pen) / streptomycin (Strep): 10,000 units / ml of penicillin and 10,000 μg / ml of streptomycin in 10 mM citrate buffer (for pH stability), at 20 °C). In an alternative embodiment, the growth medium is free of penicillin and streptomycin. In an alternative embodiment, the growth medium comprises an antibiotic agent other than penicillin and / or streptomycin. Other antibiotics and their use in cell culture media are well known to those skilled in the art and can be used according to the present invention.

[0185] Bioreactor

[0186] To maintain optimal growth conditions, i.e., in terms of oxygen, carbon dioxide, and nutrient supply, all of the above-disclosed embodiments of the method of the present invention can be conveniently carried out in a bioreactor system as exemplified in Examples 1 and 2. Generally, a bioreactor is understood to be a container 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 container is a stirred tank reactor. Bioreactors are systems known to those skilled in the art, particularly closed systems for culturing cells, in which growth parameters such as dissolved oxygen concentration, temperature, and pH can be controlled.

[0187] Thus, in one embodiment, the container used in the method of the present invention is a closed bioreactor. Preferably, the closed bioreactor is a bioreactor bag. Bioreactor bags suitable for carrying out the method of the present invention have been disclosed in International Application WO 2011 / 142667 A1, for example, in the example section of "Amplification in Culture Bags"; the teachings of this application are incorporated herein by reference. Advantageously, in one embodiment of the present invention, the bioreactor bag is expandable, i.e., the culture volume can be increased, for example, to achieve an increase in the growth area as described above.

[0188] The MPC is transferred into a container in a sterile manner. Preferably, the MPC is transferred via a bag with a weldable tube. Preferably, first the cell suspension comprising the MPC is aspirated into a syringe under sterile conditions and then the cell suspension is injected into the bag under sterile conditions. Subsequently, the weldable tube is welded to the container of the bioreactor system, and thereby the cell suspension is transferred into the container of the bioreactor system.

[0189] When there is a gas volume or headspace in the container, due to the movement of the container, additional turbulence will be generated in the culture medium. The additional turbulence may cause cell death or have a negative impact on cell growth, especially for 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, the headspace means the volume percentage of gas contained in the container. 20% headspace means that 80% of the container is composed of the culture medium with cells and microcarriers.

[0190] During the culture, additional nutrients and / or supplements can be added according to the needs of the cells. This can be done by perfusion. Sensors can be added in the bioreactor to measure nutrient levels and / or waste levels, pH, DO (dissolved oxygen), the amount of cells in the system, and / or other parameters. Preferably, these sensors operate automatically. More preferably, the addition of nutrients and / or supplements is also automatic. Most preferably, the sensors direct the addition of nutrients and / or supplements.

[0191] To provide sufficient nutrients to the cells, preferably, fresh culture medium is passed through the container while removing the culture medium to maintain a constant volume and / or pressure. By removing the culture medium from the amplification container through a filter with a pore size larger than the cells but smaller than the microcarriers (for example, with a pore size of about 100 μm), unattached cells and cell debris can be removed, and the adherent cells can be retained in the container.

[0192] In one embodiment, the method of the present invention includes replacing the culture medium with fresh culture medium every other day after the growth surface is increased, preferably wherein 50% of the culture medium is replaced.

[0193] In a specific embodiment, the method of the present invention includes, for example (but not necessarily), the following steps to culture MPC:

[0194] (i) Before culturing MPC, pre-equilibrate the container, wherein the dissolved oxygen concentration (DO) is set to 75%, the temperature is set to 37 °C, and the pH is set to 7.3;

[0195] (ii) Culture the MPC in a container. For the first 24 hours or until the DO drops below about 30% (whichever occurs earlier), without perfusion, pH control, and DO control, to enable the MPC to adhere to the carrier particles (pH and DO are not controlled, but need to be measured);

[0196] (iii) Start DO control, pH control, and perfusion 24 hours after the start of the culture or when the DO drops below about 30% (whichever occurs earlier), where the DO is set to 75%, the pH is set to 7.3, and the perfusion is carried out at 3 mL / min. Preferably, the pressure inside the bag is maintained between 80 and 120 mbarg; and / or

[0197] (iv) Shake the container, preferably with the following set points:

[0198] - The shaker speed is 90° / s;

[0199] - The maximum tilt angle is 180°;

[0200] - The acceleration is 90° / s 2 ;

[0201] - The deceleration is 90° / s 2 ;

[0202] - The vertical hold time is 10 s;

[0203] - There are four 1-hour stationary intervals within a 24-hour period (horizontal hold time: 3600 s (horizontal pause); number of mixing cycles: 1000 times (horizontal pause)

[0204] In this embodiment, if the DO drops below 40%, or when the cell density reaches 5.0x10 5 cells / ml, or when at least 70%, preferably 80%, more preferably 90% of the microcarriers are occupied (the horizontal hold time is 0 s, and the mixing cycle during the horizontal pause is 0 times), then the static interval can be stopped. Additionally or alternatively, if the set points of DO and / or pH can no longer be achieved, the perfusion rate can be increased to a maximum of 10 ml / min. Additionally or alternatively, if uneven mixing is observed, the acceleration and deceleration are each adjusted to 210° / s 2 . However, the shaking protocol can also be adjusted as described in WO 2011 / 142667 A1.

[0205] Cell populations and therapeutic aspects

[0206] Those skilled in the art can verify the presence of MPC characteristic markers. For example, WO 2019 / 215090A1 discloses in vitro and in vivo assays, such as detecting the presence of marker proteins by flow cytometry, detecting fiber formation by Giemsa staining, and transplantation experiments.

[0207] The MPCs obtained by the method of the present invention, i.e., the cell population comprising MPCs and harvested, show the following myogenic marker expressions:

[0208] - The population comprises at least 40% α-actinin-positive cells, preferably at least 50%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% α-actinin-positive cells; and / or

[0209] - The population comprises at least 60% Pax7-positive cells, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% Pax 7-positive cells; and / or

[0210] - The population comprises at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99% A2B5-positive cells; and / or

[0211] - The population comprises less than 20% CD34-positive cells, preferably less than 15%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5%, and most preferably less than 0.25% CD34-positive cells; and optionally

[0212] - The population comprises desmin-positive cells, preferably between 5% and 95%, more preferably between 10% and 99%, more preferably between 20% and 95%, more preferably at least 70%, more preferably at least 75%, more preferably at least 90%, and most preferably between 70% and 95%.

[0213] 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.

[0214] Preferably, the cell population comprises greater than 80% α - actinin - positive cells, greater than 80% Pax7 - positive cells, less than 5% CD34 - positive cells and optionally desmin - positive cells, and most preferably greater than 10% desmin - positive cells.

[0215] 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, and preferably ≥50% α - actinin - positive cells, ≥60% Pax7 - positive cells, ≥50% A2B5 - positive cells, ≤15% CD34 - positive cells and optionally desmin - positive cells.

[0216] Preferably, the cell population comprises greater than 80% α - 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, and most preferably greater than 10% desmin - positive cells.

[0217] Generally, the cell population of the present invention also expresses early myogenic markers such that when these cells are administered to a patient, muscle regeneration can be induced, that is, muscle formation can be induced. In one embodiment of the present invention, the MPCs express MyoD and / or MyHC, preferably in addition to the other markers described above.

[0218] Regarding the expression percentage of markers, such as a cell population comprising a certain percentage of positive cells, it reflects the proportion of marker - positive cells in the total population. A cell is considered positive for a marker if the marker can be detected at the protein or mRNA level by any suitable detection method (such as flow cytometry, Western blotting, immunostaining or qPCR) when the cell expresses the marker.

[0219] Thus, in one embodiment of the present invention, the cell population of the present invention comprises at least 40% of α-actinin-positive cells, preferably at least 50%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% of α-actinin-positive cells. In a further embodiment of the present invention, the cell population of the present invention comprises at least 60% of Pax7-positive cells, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% of Pax7-positive cells. In a further embodiment of the present invention, the cell population of the present invention comprises at least 60% of A2B5-positive cells, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% of A2B5-positive cells. In a further embodiment of the present invention, the cell population of the present invention comprises less than 20% of CD34-positive cells, preferably less than 15%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5% and most preferably less than 0.25% of CD34-positive cells.

[0220] In a further 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%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% Pax7-positive cells. In a further 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%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% A2B5-positive cells. In a further 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%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5% and most preferably less than 0.25% CD34-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%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% A2B5-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%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% Pax7-positive cells, and less than 20% CD34-positive cells, preferably less than 15%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5% and most preferably less than 0.25% CD34-positive cells. In a further embodiment of the present invention, the cell population of the present invention comprises at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5% and most preferably less than 0.25% CD34-positive cells.In a further 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%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% A2B5-positive cells. In a further 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%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% α-actinin-positive cells; at least 60% positive cells of Pax7, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% Pax7-positive cells; and less than 20% CD34-positive cells, preferably less than 15%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5% and most preferably less than 0.25% CD34-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%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% Pax7-positive cells; at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5% and most preferably less than 0.25% CD34-positive cells.

[0221] In a most preferred 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%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, 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%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% Pax7-positive cells; at least 60% A2B5-positive cells, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98% and most preferably at least 99% A2B5-positive cells; less than 20% CD34-positive cells, preferably less than 15%, more preferably less than 10%, more preferably less than 8%, more preferably less than 7.5%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1.5%, more preferably less than 1%, more preferably less than 0.5% and most preferably less than 0.25% CD34-positive cells.

[0222] In a preferred embodiment, the cell population of the present invention is further defined by one or more markers CD56, Myf5, MyHC, and / or MyoD (preferably in addition to the specific markers mentioned above) in the following percentages: In one 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 approximately 3% CD56-positive cells. In another embodiment, the cell population comprises at least 50% Myf5-positive cells, preferably greater than 60%, more preferably 60 - 90%, and most preferably approximately 65% Myf5-positive cells. In another 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. In another embodiment, the cell population of the present invention comprises 10 - 40% MyoD-positive cells, preferably 10 - 30%, more preferably 15 - 25%, and most preferably approximately 20% MyoD-positive cells. In another 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 approximately 3% CD56-positive cells, and at least 50% Myf5-positive cells, preferably greater than 60%, more preferably 60 - 90%, and most preferably approximately 65% Myf5-positive cells. In another 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 approximately 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 another 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 approximately 3% CD56-positive cells and 10 - 40% MyoD-positive cells, preferably 10 - 30%, more preferably 15 - 25%, and most preferably approximately 20% MyoD-positive cells. In another 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 approximately 3% CD56-positive cells, at least 50% Myf5-positive cells, preferably more than 60%, more preferably 60 - 90%, and most preferably approximately 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 another 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 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 another 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 greater than 60%, more preferably 60 - 90%, 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 another 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 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 another 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 another 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 another 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.

[0223] The term "about" particularly refers to the percentage of positive cells, and the definition includes a variation of more or less than 10% of positive cells.

[0224] In addition, the 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 comprising collagen are stored at 2-8 °C for at least 24 hours, preferably at least 48 hours and up to 120 hours, the cell viability remains at least 80% (or higher).

[0225] Experiments conducted according to the present invention have shown that, prior to large-scale cell culture, the amount of desmin-positive cells among different MPC isolates already varies, i.e., there are differences in the cell populations obtained from biopsies of different patients, and increases with each cell passage during culture. Thus, as long as desmin-positive cells are present in the population, the amount of desmin-positive cells is not a critical criterion for whether the cells are suitable for downstream clinical applications.

[0226] 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 to manufacture a medicament. In particular, the population of MPCs according to the present invention can be used to manufacture a medicament for treating muscle dysfunction (especially skeletal muscle dysfunction) in a human patient; see below. Thus, in one embodiment, the population obtained by the method of the present invention comprises a therapeutically effective amount of MPCs. A therapeutically effective amount means an amount suitable for treating muscle dysfunction such as urinary incontinence. The MPCs are preferably administered by injection to the damaged muscle site to regenerate skeletal muscle tissue. In one embodiment of the present invention, the skeletal muscle dysfunction to be treated is a sphincter defect. In a preferred embodiment, the sphincter is selected from the non-limiting group consisting of the external urethral sphincter and the internal urethral sphincter and the external anal sphincter and the internal anal sphincter. Thus, the sphincter defect-related indications treated according to the present invention are indications related to the above-mentioned sphincters and are selected from, but not limited to, female and male urinary incontinence and fecal incontinence, and pathological reflux in gastroesophageal reflux disease. Preferably, the urinary incontinence is selected from stress urinary incontinence, urge urinary incontinence, overflow urinary incontinence, total urinary incontinence or a mixed form of stress urinary incontinence and urge urinary incontinence. In addition to targeting the sphincter, the cell population obtained by the method of the present invention is also used to target defects in other skeletal muscles. For example, after damage to these muscles, it is conceivable to support, promote or initiate regeneration by administering MPCs to the muscle damage or injury site.

[0227] In one embodiment, the MPC to be administered according to the present invention (preferably by injection) comprises microcarriers. For example, in the above-described embodiment where the MPC is not separated from the microcarrier, the population comprising the MPC obtainable by the method of the present invention further comprises the microcarrier to which the MPC is attached. In this embodiment, the MPC is preferably injected together with the microcarrier. Thus, the carrier is preferably biocompatible. In addition, as is well known, for example, the carrier used here as a scaffold should be degraded in a timely manner to ensure that the muscle tissue can undergo proper remodeling, and thus, these carriers should preferably be biodegradable. Biocompatible and biodegradable microcarriers are known to those skilled in the art. Examples of biocompatible and biodegradable microcarriers are natural polymers (polysaccharides and proteins) and synthetic polymers (poly(α-hydroxy esters), such as poly(ε-caprolactone) (PCL), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and poly(lactic-co-glycolic acid) (PLGA); reviewed in Elmowafy, et al., J. Pharm. Investig. 49 (2019), 347-380).

[0228] In an alternative embodiment, the MPC is administered without a microcarrier, i.e., the MPC is separated from the microcarrier before administration.

[0229] Generally, the amount of cells considered therapeutically effective is highly dependent on the indication to be treated and the severity, degree, or size of the lesion to be treated. For example, it can be envisaged that fewer cells are to be injected in the case of mild stress urinary incontinence compared to the severe form of stress urinary incontinence. In a preferred embodiment, the amount comprises at least 1x10 7 cells, preferably 6x10 7 to 3x10 8 cells, most preferably 1 - 3x10 8 MPC. By performing more than one amplification step, i.e., increasing the volume of the culture medium, preferably increasing it to 3-fold, while preferably maintaining the concentration of the microcarrier in the culture medium, an even greater number of cells can be obtained. Thus, the present invention also relates to a method for obtaining a therapeutically effective amount of MPC, which comprises the steps of the method for obtaining a large-scale culture of MPC of the present invention described above.

[0230] As described above, the amount of MPC generally considered therapeutically effective is highly variable and thus not particularly limited. In a specific example of stress urinary incontinence, the target cell count injected into the patient is preferably in the range of 60 million - 200 million cells in total, more preferably about 80 million - 150 million cells. However, as described above, these figures depend on the severity of the defect to be treated. In a preferred embodiment, the viability of the cells is at least 80%.

[0231] The present invention further relates to a method for preparing a medicament, the method comprising the steps of a method for obtaining a large-scale culture of the MPC of the present invention as described above, and optionally, adding a biocompatible material solution, preferably a hydrogel solution, more preferably a collagen solution, to the harvested MPC. A "biocompatible material solution" or "biocompatible material" refers to a carrier solution, especially for ensuring that the injected MPC remains at the injection site. Thus, in one embodiment of the present invention, the MPC is suspended in a biocompatible material solution (such as a hydrogel). Generally, hydrogels are ECM proteins used in tissue engineering, which can achieve better implantation. In a preferred embodiment, the hydrogel is selected from, but not limited to, collagen, alginate, hyaluronic acid, fibrin, poly(N-isopropylacrylamide) (PNIPAAm), poly(ethylene glycol) (PEG), recombinant protein polymers forming a mixed induction bicomponent hydrogel (MITCH), shear-thinning hydrogels for injectable encapsulation and long-term delivery (SHIELD), preferably collagen. Without being bound by theory, it is conceivable that when the MPC is administered together with the microcarrier, that is, when the MPC is attached to the microcarrier, the microcarrier (as a biocompatible material solution) can serve as a negative carrier for injecting the MPC, for example, to ensure that the injected cells remain at the injection site.

[0232] The medicament can be used for treating skeletal muscle dysfunction by injection. In order to deliver 80 million MPCs with at least 80% viability at a final concentration of 20 million - 25 million cells / ml, in one embodiment of the present invention, the cultured cells (80 million - 100 million) are suspended in 4 ml of a biocompatible material solution, such as a collagen solution as described below. The final product is preferably packaged in a vial or syringe, placed in a box, and transported at 5°C (+ / - 3°C) controlled by a temperature measuring device. A syringe is defined as a container suitable for injecting the medicament into a patient. Alternatively, the MPC and the biocompatible material solution are stored separately in the syringe, for example, in a dual-chamber syringe, and are only mixed during the injection of the medicament.

[0233] The present invention further includes a composition, the composition comprising the above-mentioned MPC and the above-mentioned cell population respectively obtained by the method of the present invention, wherein, in a preferred embodiment, the MPC is suspended in a collagen solution, preferably at a concentration of 10 million - 30 million cells / ml, with at least 80% viability. In a preferred embodiment, the collagen solution contains type I collagen, preferably porcine, bovine or preferably human origin, and wherein the concentration of collagen in the composition is preferably 1 - 4 mg / ml, preferably about 2 mg / ml (such as 2.1 mg / ml). In one embodiment, the composition is included in a medicament container as defined above, preferably included in a syringe or vial.

[0234] The MPCs obtained by the method of the present invention and the corresponding compositions of the present invention can be used in a variety of therapeutic applications, especially those related to muscle dysfunction, including but not limited to the treatment of stress urinary incontinence as described in WO 2019 / 215090 A1, the treatment of male stress urinary incontinence after prostatectomy as described in, for example, WO 2004 / 096245 A2, and the treatment of fecal incontinence as described in, for example, WO2008 / 104883 A1. Accordingly, the present invention relates to the MPCs and compositions of the present invention for use as medicaments, preferably for the treatment of muscle dysfunction, such as skeletal muscle dysfunction as defined above. In a preferred embodiment, the skeletal muscle dysfunction can be, for example, external urethral sphincter dysfunction or external anal sphincter dysfunction. In a preferred embodiment, the skeletal muscle dysfunction is an external urethral sphincter defect, and thus, the MPCs and compositions are preferably used for the treatment of urinary incontinence, especially female stress urinary incontinence.

[0235] Treatment is generally carried out by injecting the above composition or medicament into a subject, preferably a mammal, more preferably a domestic animal, such as a pet or livestock as defined above, and most preferably a human. In a preferred embodiment, the treatment is carried out by injecting the above composition or medicament into the same subject from whom the muscle biopsy was taken, and thus preferably using autologous cells for treatment. Optionally, the composition further comprises a collagen solution, which is described above. After injecting the composition, the pelvic floor of the human patient can be subjected to neuromuscular electrical stimulation (NMES) as described in WO 2019 / 215090A1. At the surface of the stimulation coil, the intensity of the induced electric field at maximum output is 120 V / m. At 5 cm above the stimulation coil, the measured field strength is 22 V / m. NMES treatment after injecting the cell suspension can support muscle and nerve regeneration by activating muscle-nerve cross-talk and induce the maturation of neuromuscular junctions. The injection of the composition can be carried out using an injection device known in the art. In a preferred embodiment, the injection is carried out using the injection device described in PCT / EP2023 / 074044, filed on September 1, 2023, claiming the priority of EP22 193 690.9, the content of which is incorporated herein by reference. Preferably, 8-12 or 12-18 equal portions of the hMPC-collagen composition are injected into the pelvic floor, and the total amount of the composition does not exceed 6 ml.

[0236] This specification incorporates by reference several documents in their entirety. The content of all the references cited in this application (including the literature references incorporated by reference in the present application in its entirety, issued patents, published patent applications, including the background section and manufacturer's instructions, specifications, etc.) is hereby incorporated by reference herein; however, it is not admitted that any of the cited documents is indeed prior art to the present invention.

[0237] A more complete understanding can be obtained by referring to the following specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0238] Example

[0239] Example 1: Automated MPC culture protocol in a bioreactor system

[0240] MPCs were obtained after explant muscle biopsy. A total of one million P0 or P1 MPCs were collected in a container (i.e., a cell culture flask) with a weldable tube. The methods for explant muscle biopsy and harvesting MPCs from tissues have been described in WO 2019 / 215090 A1 and in the "Muscle precursor cells" section above. Then, the cell culture flask was welded to a pre-balanced SCINUS cell expansion system by aseptic welding. Then the MPCs were grown in the SCINUS until sufficient cells were obtained (about 6 days). The SCINUS system has been described in WO 2011 / 142667 A1.

[0241] SCINUS preparation

[0242] The SCINUS system was prepared with the following set points, volumes, and concentrations:

[0243] - 130 mL of MPC medium (DMEM:F12 (Gibco TM , Thermo Fisher Scientific Inc, USA), 5% hPL (human platelet lysate; PLTGold from MillCreek (Rochester, Minnesota, USA), 1 U / μg penicillin / streptomycin (Gibco TM , Thermo Fisher Scientific Inc, USA), 10 ng / mL hEGF (Sigma-Aldrich, St. Louis, USA and Merck KGaA, Darmstadt, Germany respectively), 0.4 μg / mL dexamethasone (Sigma-Aldrich, St. Louis, USA and Merck KGaA, Darmstadt, Germany respectively), 1 ng / mL βFGF (Sigma-Aldrich, St. Louis, USA and Merck KGaA, Darmstadt, Germany respectively), 10 μg / mL insulin (Sigma-Aldrich, St. Louis, USA and Merck KGaA, Darmstadt, Germany respectively))

[0244] - 1.7 g / L of collagen-coated soluble microcarriers from Corning Life Sciences (CorningTM Denatured collagen soluble microcarrier)

[0245] - DO set point 75%

[0246] - pH set point 7.3

[0247] - Temperature set point 37 °C.

[0248] Once the set point is reached, maintain the pre - equilibrium state until cell seeding on day 0.

[0249] SCINUS-inoculation (day 0)

[0250] Once 1x10 6 MPCs (P1) are harvested, weld the container with the cell suspension to the pre - equilibrated SCINUS bioreactor system. Turn off perfusion and parameter (DO and pH) control. Use the following settings during the seeding and attachment phases:

[0251] - Perfusion set point: No control

[0252] - pH set point: No control

[0253] - DO set point: No control

[0254] - Volume set point: 130 mL

[0255] - Shaker set point:

[0256] - Shaker speed: 90 ° / s

[0257] - Maximum angle: 180 °

[0258] - Acceleration, 90 ° / s 2

[0259] - Deceleration, 90 ° / s 2

[0260] - Vertical hold time: 10 s

[0261] - Horizontal hold time: 3600 s (horizontal pause)

[0262] - Number of mixing cycles: 1000 times (horizontal pause).

[0263] The pressure inside the bag is maintained between 80 - 120 mbarg. If the DO drops below 30%, enable the following settings:

[0264] - pH: Set point 7.3

[0265] - DO: Set point 75%

[0266] - Perfusion: Set point 3 mL / min

[0267] SCINUS-start process control (from day 1)

[0268] After the seeding and attachment phases, start the MPC culture settings. Typically, this phase is started 24 hours after seeding or when the DO drops below 30% (whichever occurs earlier). The following settings are used during this phase:

[0269] - Perfusion set point: 3 mL / min

[0270] - pH set point: 7.3

[0271] - DO set point: 75%

[0272] - Volume set point: 130 mL

[0273] - Shaker set point:

[0274] - Shaker speed: 90° / s

[0275] - Maximum angle: 180°

[0276] - Acceleration, 90° / s 2

[0277] - Deceleration, 90° / s 2

[0278] - Vertical hold time: 10 s

[0279] - Horizontal hold time: 3600 s (horizontal pause)

[0280] - Number of mixing cycles: 1000 times (horizontal pause).

[0281] The pressure should be maintained between 80 - 120 mbarg.

[0282] Since the growth kinetics and characteristics of MPCs can vary significantly between donors, the following parameters are observed during culture.

[0283] - If the DO drops below 40%, or when the biomass sensor indicates a cell density > 5.0x10 5 cells / mL, or when > 90% of the microcarriers are occupied, then stop the horizontal pause by adjusting the following settings:

[0284] - Horizontal hold time: 0 s (no horizontal pause)

[0285] - Number of mixing cycles: 0 (no horizontal pause)

[0286] - If the DO and pH set points can no longer be achieved, increase the perfusion setting to a maximum of 10 mL / min.

[0287] - If uneven mixing is observed, adjust the following shaker settings to maintain even mixing:

[0288] - Acceleration: 210° / s 2

[0289] - Deceleration: 210° / s 2

[0290] Volume expansion (based on density)

[0291] Once the cells reach a density of 1.3x10 4 - 1.8x10 4 cells / cm 2 (i.e., 1.1x10 5 - 1.5x10 5 cells / ml), increase the volume of the adherent bag while maintaining the microcarrier concentration at 1.7 g / L. The volume is increased to approximately 3-fold, from 130 mL to 400 mL. Connect the suspension of microcarriers in MPC medium (1.7 g / L) to the medium inlet of the SCINUS system and add it to the bag by gravity or pumping. To increase the microcarrier volume from 130 mL to 400 mL, the following was done: adjust the volume set point to 400 mL and add 270 mL of microcarriers to the system via the inlet. The pressure is maintained between 80 - 120 mbarg, and the shaker, perfusion, pH, and DO set points remain unchanged.

[0292] Culture medium renewal (based on time)

[0293] The medium needs to be updated in the days following the volume expansion step. Every other day, update 50% of the cell medium by transferring 200 mL of medium from the adherent bag to the waste bag and adding 200 mL of fresh MPC medium to the bag via the feed inlet. The pressure is maintained between 80 - 120 mbarg, and the shaker, perfusion, pH, and DO set points remain unchanged.

[0294] Harvest (based on density)

[0295] Once sufficient cells have grown in the adherent bag, harvest the cells from the dissolvable microcarriers by completely dissolving them using the harvest solution. To maintain the characteristics of the MPC cells, culture the cells to 6.7x10 4 cells / cm 2 (5.7x10 5 cells / mL, for a total of 2.3x10 8The maximum density of (number of cells).

[0296] Wash the cells once with an equal volume of PBS, and transfer 200 mL of PBS from the adherent bag to the waste bag. Then, add 200 mL of the harvest solution (74% PBS, TryplE 2.5X, pectinase 49 U / mL, EDTA 5 M) to the adherent bag via the feeding inlet. Incubate the cells at 37 °C for 15 - 20 minutes, shaking the system every 5 minutes with the following shaking settings:

[0297] - Shaker set point:

[0298] - Shaker speed: 90° / s

[0299] - Maximum angle: 180°

[0300] - Acceleration, 90° / s 2

[0301] - Deceleration, 90° / s 2

[0302] - Vertical hold time: 10 s

[0303] - Horizontal hold time: 300 s

[0304] - Number of mixing cycles: 1.

[0305] Remove the cells from the bag and place them in a bottle / sample bag.

[0306] Example 2: Culturing MPC in a bioreactor system

[0307] Culture MPC according to the protocol detailed in Example 1. Use passage 3 cells. The MPC medium was changed, which was supplemented with 10% hPL (Paracelsus). The remaining components of the medium were used as described in Example 1. The culturing method included a second amplification step carried out after day 10. The volume of the medium including microcarriers has been increased from 400 ml to 800 ml, resulting in a total surface area of 6800 cm 2 , as shown in Table 1.

[0308] Perform cell counting and observation at least every 2 - 3 days (Table 1,[[]] Figure 1 and Figure 2)。Therefore, a small amount of homogeneous sample was obtained from the bioreactor bag. 1 mL was used for visual inspection using an optical microscope, and images were taken at magnifications of 40x and 100x. The remaining volume was harvested by dissolving the microcarriers with the harvest solution (PBS, TrypLE, EDTA, and pectinase) for cell counting. Then, a single-cell suspension was counted using a NC-250 NucleoCounter. The total cell number was adjusted according to the biomass loss caused by sampling. Therefore, the expected total cell number is listed in the last row of Table 1.

[0309]

[0310] Example 3: Characterization of MPCs Obtained in a Large-Scale Culture System

[0311] MPCs cultured and harvested from the bioreactor were analyzed to confirm that they have myogenic characteristics. Especially for clinical purposes, MPCs should express ≥50% α-actinin, ≥60% Pax7, desmin, and ≤15% CD34. The presence and levels of the MPC markers were analyzed by flow cytometry and compared with MPCs cultured in monolayers ( Figure 3 and Figure 4 ). In each of the different culture methods, the MPC medium described in Example 1 was used, but with the difference that the medium contained 5% PLT Gold HPL ( Figure 3 A) and 10% PLT Gold HPL ( Figure 3 B and Figure 4 ).

[0312] Briefly, cryopreserved MPCs were thawed and cultured in monolayer for one passage (P1). Then the cells were seeded into a bioreactor or a T75 monolayer culture flask. For bioreactor culture, the cells were seeded (1x10 6 MPCs) and cultured as described in Example 2, i.e., including a second amplification step to obtain approximately 160 - 200x10 6 cells.

[0313] In principle, the culture in the monolayer culture flask was carried out as previously described, for example, as described in WO 2019 / 215090A1.

[0314] The harvested cells were analyzed for marker expression by flow cytometry. The MPCs were fixed with 2% PFA (Alfa Aesar) in PBS for 10 min at RT and permeabilized (permeabilized with 0.5% Titron-X-100 (VWR) for 10 min at RT). Then, non-specific binding sites were blocked (blocked with 5% FBS (Sigma) in 0.5% Titron-X-100 in PBS for 20 - 60 min at 2 - 8 °C). At 2 - 8 °C, surface and intracellular staining was performed for 30 min using direct and unlabeled antibodies / isotype controls:

[0315] -APC-CD34 (TFS) / APC-isoCD34 = APC-msIgG1 (TFS)

[0316] -Anti-Pax7 (Sigma) / msIgG2a (TFS)

[0317] -FITC-anti-α-actinin (Miltenyi) / FITC-REA (Miltenyi)

[0318] -Anti-A2B5 (Sigma)

[0319] -PE-CD56 / PE-msIgG1 (Beckman Coulter)

[0320] -PE-CD105 / PE-msIgG1 (Beckman Coulter)

[0321] -Anti-desmin (Sigma) / msIgG1 (Santa Cruz Biotechnology)

[0322] -Human / mouse Myf-5 AlexaFlour488 (R&D Systems) / msIgG2a (TFS)

[0323] -MyHC anti-human / mouse / rat - APC / REA control (S), human IgG1-APC (Miltenyi Biotec)

[0324] -Anti-MyoD (BD Biosciences) / msIgG1 (Santa Cruz Biotechnology)

[0325] Antibodies were diluted with autoMACS running buffer (Miltenyi Biotec). MPCs were stained with FITC-labeled Pax7 secondary antibodies (BD) and corresponding isotype controls for 30 min at 2-8°C. Data were acquired using Miltenyi's MACSQuant using the manufacturer's protocol with MACSQuant running buffer, MACSQuant wash solution, MACSQuant storage solution, MACSQuant calibration beads, and the manufacturer's software (MACS QuantifySoftware). Data analysis was performed using Flow Jo software.

[0326] like Figure 3 and Figure 4 As can be seen in the results, both culture methods (i.e., bioreactor and monolayer culture flask) were able to generate MPCs expressing myogenic markers, with approximately 99% of the cells being positive for Pax7, α-actinin, and A2B5 and negative for CD34 expression. In addition, the different culture settings generated similar MPC populations, so it can be concluded that large-scale culture in bioreactors is suitable for the production of large numbers of MPCs suitable for clinical downstream applications.

[0327] In addition, if Figure 5 As can be seen in the Figure 3, in addition to showing typical myogenic markers α-actin and A2B5 (99.9% and 99.7%, respectively) and CD34 negativity (0.1%), cells cultured in the bioreactor 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%). There was variation in the expression of desmin between the different bioreactor runs.

Claims

1. A method for obtaining a large-scale culture of muscle precursor cells (MPCs) derived from skeletal muscle, comprising at least the following steps: (a) In a container comprising a culture medium, which includes microcarriers, MPCs are cultured under conditions that allow the MPCs to attach to the microcarriers, wherein the MPCs are seeded at a density between 500 - 1500 cells / cm 2 of the growth surface area provided by the microcarriers, preferably at a density between 800 - 1200 cells / cm 2 ; and (b) When the cell number increases to about 8-fold to 25-fold, increasing the growth surface area of the culture medium; and (c) Further cultivate the MPCs, preferably until the cell density reaches 5 - 7.5x10 4 cells / cm 2 and / or 4 - 6.5x10 5 cells / ml, and optionally (d) Harvesting the MPCs.

2. The method according to claim 1, wherein, The growth surface is increased to between two to four times, preferably increased to about three times, preferably wherein, The volume of the culture medium is increased to the same extent as the growth area.

3. The method according to claim 1 or 2, wherein Steps (b) and (c) are repeated once or more, preferably once or twice, most preferably twice.

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

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

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

7. The method according to any one of claims 1 to 6, wherein The microcarrier is a coated microcarrier, more preferably wherein, The microcarrier is a collagen-coated microcarrier, and / or wherein, the microcarrier is soluble.

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

9. The method according to any one of claims 1 to 8, wherein At the end of the culture, the MPCs are separated from the microcarriers and harvested. Preferably, wherein, the separation includes complete dissolution of the microcarriers. Preferably, wherein, the dissolution of the microcarriers is carried out by enzymatic digestion, preferably by adding an endopeptidase that cleaves proteins at specific sites, most preferably by trypsin or a corresponding trypsin substitute and pectinase.

10. A cell population comprising MPCs obtainable by the method according to any one of claims 1 to 9, preferably wherein the cell population comprises ≥40% alpha-actinin positive cells, ≥60% Pax7 positive cells and / or ≤20% CD34 positive cells, and optionally desmin positive cells; preferably wherein, The population includes ≥50% of α-actinin-positive cells, ≥60% of Pax7-positive cells and / or ≤15% of CD34-positive cells, and optionally desmin-positive cells; most preferably, wherein, the cell population includes ≥80% of α-actinin-positive cells, ≥80% of Pax7-positive cells and / or ≤5% of CD34-positive cells, and optionally desmin-positive cells, preferably ≥10% of desmin-positive cells.

11. The group according to claim 10, wherein, The population includes ≥60% of A2B5-positive cells, preferably ≥80% of A2B5-positive cells.

12. The group according to claim 10 or 11, wherein, The population includes ≤15% of CD56-positive cells, preferably ≤10% of CD56-positive cells, more preferably ≤5% of CD56-positive cells.

13. The group according to any one of claims 10 to 12, wherein The population includes ≥50% of Myf5-positive cells, preferably ≥60% of Myf5-positive cells, preferably ≥60 - 90% of Myf5-positive cells.

14. The group according to any one of claims 10 to 13, wherein The population includes ≤30% of MyHC-positive cells, preferably ≤20% of MyHC-positive cells, preferably ≤15% of MyHC-positive cells.

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

16. The group according to claim 10, said group comprising a therapeutically effective amount of MPC, preferably wherein said group comprises at least 1x10 7 MPCs, preferably 6x10 7 to 3x10 8 MPCs, most preferably 1 - 3x10 8 MPCs.

17. A method for preparing a drug, comprising the steps of the method according to 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. The method according to claim 12, the method further comprising the step of filling the MPC into a drug container, preferably wherein the container is a syringe or a vial.

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

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

21. Use of the composition according to claim 19 for use as a medicament, preferably for use in a method of treating skeletal muscle dysfunction, preferably wherein the skeletal muscle dysfunction is a sphincter defect, preferably an external urethral sphincter defect, optionally wherein, The composition further comprises a biological material solution, preferably a hydrogel solution, more preferably a collagen solution.

22. The composition for the use according to claim 21, wherein, The method comprises: obtaining MPC from a patient; preparing a large-scale culture of the MPC by the method according to any one of claims 1 to 9; preparing a composition according to any one of claims 19 to 21; and administering the composition to the patient, preferably by injecting the muscle to be treated.

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

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

25. A composition for use according to any one of claims 21 to 24, wherein, The composition comprises 10 million - 30 million cells / ml and has a viability of at least 80%.

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

27. A composition for use according to any one of claims 21 to 26, wherein, The method further comprises subjecting the patient to neuromuscular electrical magnetic stimulation (NMES), preferably wherein, The intensity of the maximum output induced electric field at the surface of the stimulating coil is 120 V / m. Use of the composition according to claim 19 or 20 in the manufacture of a medicament for a method of treating skeletal muscle dysfunction, preferably wherein the method is a method as defined in any one of claims 21 to 27.

29. A method for treating skeletal muscle dysfunction in a patient, wherein, The method is a method as defined in any one of claims 21 to 27.

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