Methods for obtaining muscle derived cells
The present invention relates to methods for obtaining skeletal muscle derived cells (SMDC), and the use of SMDCs in a method of preventing and/or treating neuromyopathies and/or myopathies, wherein the neuromyopathy and/or myopathy is incontinence, in particular a urinary and/or an anal or fecal incontinence.
1. A method of treating incontinence comprising administering a skeletal muscle derived cell population comprising at least 60% CD56 positive and 60% A2B5 positive cells to a subject in need thereof, wherein the skeletal muscle derived cell (SMDC) population is obtained according to the method comprising the steps of:
(a) cooling of a sample obtained from skeletal muscle tissue in a buffer;
(b) processing followed by cooling of the sample, wherein cooling is conducted at a temperature of 1 to 16° C. for a time in the range of 2 to 48 hours;
(c) resuspending the sample of step (b) in medium with serum comprising at least one enzyme and heating up to 38° C. for 1 to 20 hours; pelleting the sample and
(d) resuspending the pellet of the sample of step (c) to provide a single cell suspension from the sample of step (c), thereby obtaining SMDCs.
2. The method according to claim 1 , wherein the incontinence is fecal incontinence and/or urinary incontinence.
3. The method according to claim 1 , wherein the incontinence is anal incontinence.
4. The method according to claim 1 , wherein the SMDC population is characterized by the expression of distinct marker combinations selected from the positive expression of at least one or more of the markers selected from CD56, A2B5, CD105, Myf5, and Pax7, and the negative expression of at least one or more of the markers selected from CD34, and MyoD.
5. The method according to claim 1 , wherein step (a) is conducted at a temperature lower than 16° C., and for a time in the range of up to 96 hours.
6. The method according to claim 1 , wherein step (b) comprises the use of scissors, scalpel, tweezers, filter, or ball mill.
7. The method according to claim 1 , wherein the cooling in step (b) is conducted at a temperature in the range of 4 to 8° C., and for a time in the range of 2 hours to 48 hours.
8. The method according to claim 1 , step (c) comprises conducting the enzymatically treating with a solution comprising any one or more selected from the group consisting of trypsin, papain, elastase, hyaluronidase, collagenase, deoxyribonuclease, and DNAse.
9. The method according to claim 1 , wherein step (c) is conducted at a temperature in the range of 25 to 38° C.
10. The method according to claim 1 , wherein step (d) further comprises sorting the SMDCs according to a method selected from at least one of FACS sorting, centrifugation, electrokinetic sorting, acoustophoresis sorting, bead-based cell sorting, and optical sorting.
11. The method according to claim 1 , wherein a further step (e) is conducted after step (d) including incubating of the single cell suspension obtained in step (d), wherein the incubation in step (e) is conducted at a temperature in the range of 25 to 38° C., thereby obtaining adherent SMDCs.
12. The method according to claim 1 , wherein after step (e) a further step (f) is conducted comprising discarding of non-adherent cells of step (e), wherein step (f) is preferably conducted after at least 6 hours to 4 days.
13. The method according to claim 1 , wherein after step (f) a further step (g) is conducted of propagating of the adherent cells of step (e), the propagating in step (h) comprises culturing of the adherent cells for 1 to 5 passages to 70 to 80% confluency.