Indian Journal of Urology
: 2013  |  Volume : 29  |  Issue : 1  |  Page : 80--81

Long-term cryopreservation of vestigial muscle cells, as the source of stem cells for the treatment of stress urinary incontinence: Is it possible?

Saurabh Sudhir Chipde 

Correspondence Address:
Saurabh Sudhir Chipde

How to cite this article:
Chipde SS. Long-term cryopreservation of vestigial muscle cells, as the source of stem cells for the treatment of stress urinary incontinence: Is it possible?.Indian J Urol 2013;29:80-81

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Chipde SS. Long-term cryopreservation of vestigial muscle cells, as the source of stem cells for the treatment of stress urinary incontinence: Is it possible?. Indian J Urol [serial online] 2013 [cited 2020 Jan 21 ];29:80-81
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The use of stem-cell injection into the degenerated external urethral sphincter is an emerging treatment modality for stress urinary incontinence (SUI). Sumino et al. described the possibility of long-term cryopreserved pyramidalis muscle (PM) specimens as a source of striated muscle stem cells for the treatment of post-prostatectomy SUI.[1] They analyzed 5 male patients, aged 61-70 years, who underwent open radical retropubic prostatectomy for prostate cancer. PM specimens (volume 125 mm 3 ) were obtained and divided into two portions. One portion was used for direct primary culture. The other portion was stored at −80°C in a manufactured cell freezing medium. After 24-60 months, the preserved samples were thawed and directly incubated in Ham's F-10 medium with 20% fetal bovine serum. Selection of satellite cells was performed using a magnetic affinity cell sorting separator (MACS) and antibody to neural cellular adhesion molecule (NCAM), which is a marker of satellite cells. Both the cryopreserved and non-cryopreserved NCAM-positive cells were evaluated by Cell proliferation assays (CPA) as well as myogenic, osteogenic and adipogenic differentiation assays (DA). CPA was done by counting cell numbers. For myogenic DA, myotube formation was detected by immune-cytochemistry using anti-desmin and anti-myosin heavy chain (MHC) antibody. Osteogenic and adipogenic differentiation were induced by bone morphogenic protein-7 (BMP-7) and γ-linolenic acid, respectively. For detection of osteoblasts and accumulated oil droplets in adipocytes, alkaline phosphatase (ALP) staining and Sudan III staining were used, respectively.

The longest period for cryopreservation was 60 months. NCAM-positive cells were confirmed as striated muscle origin by the expression of desmin and MyoD, which are specific markers of striated muscle. The cells fused, became multinucleated, and differentiated into myotubes 7 days after incubation. These myotubes expressed MHC, which is a marker of myogenic differentiation. In contrast, NCAM negative cells neither expressed myogenic markers nor differentiated into myotubes. Cells were counted at 24, 48, 72, and 96 hours. At 48 hours, cryopreserved cells showed a numerically lower proliferation rate, but by 96 hours, they showed proliferation rate similar to that of non-cryopreserved cells. After addition of BMP-7 and ƴ-linolenic acid to the differentiation medium, NCAM-positive cells expressed ALP (a marker of osteoblasts) and stored lipid droplets within the cytoplasm (adipocyte characteristic), In contrast, NCAM negative cells did not express these features after the treatments. These results show that NCAM-positive cells in the PM were satellite cells, which are able to differentiate in multiple cell lines, i.e. stem cells. The authors concluded that the long-term cryopreservation of PM specimens can be used to culture muscle stem cells and this method may be utilized for the treatment of SUI when necessary.


It is known that rhabdosphincter (RS) is degenerated by the ageing or due to surgical injury in radical prostatectomy (RP), causing stress urinary incontinence (SUI).[2] There is a potential use of stem cells is in repair and regeneration.[3] Although most studies evaluating the use of stem cell in SUI are done in animal - models of female SUI, but there are few studies which evaluated its use in male SUI after RP.[2],[3],[4],[5]

The present study has addressed several important issues. 1) Cryopreserved specimens retained the ability to differentiate in various cell types and can be used as a 'cell banking' for future treatment in the same individual. 2) We know that unlike the embryonic stem cells, tissue derived stem cells (TDSCs), have no ethical issue and they don't trans-differentiate spontaneously into malignant phenotype. These TDSCs may be mesenchymal or myogenic origin. Mesenchymal cells are derived from the bone-marrow stroma and need painful procedures that require general or spinal anesthesia. Myogenic cells are derived from extremity muscle, which may cause pain and fear during muscle biopsy. On the other hand PM, which is considered vestigial, can be harvested at the time of radical prostatectomy, while the patient is sedated. 3) This study may have surgical impact also. It is known that the prostatic apex is the commonest site of positive surgical margin during RP. The possible cause of it may be an inadequate apical dissection, which is usually done because of the close proximity to the RS, the damage of which is directly linked to SUI. Thus, when stem cell therapy becomes more advanced, complete removal of the prostate without fear of injury to the RS in patients with apical cancer or T3 prostate cancer may be possible.

There are some weak points of study. 1) Authors have used only Cell Banker as the freezing medium, containing animal serum, which may cause the problem of antigenicity. To avoid it one must use the serum of each patient so that the sample is suitable for him in the case of clinical application. 2) The study has a very low power due to very small sample size of only 5 patients. 3) Authors have not described the cost of therapy. 4) Post-prostatectomy incontinence is usually identified at an early stage, so the sample may not need cryopreservation for such a prolonged period of five years. 5) Although open retropubic prostatectomy enables easy sampling of PM, the laparoscopic and robotic approaches, which are recently becoming more common, are not friendly for PM sampling. 6) If stem cell retrieval is to be done during anaesthesia only, than why only PM, but not any other tissue; even routine extremity muscle can be harvested. 7) At last, authors have not described any practical experience of the use of cryopreserved stem cells in patients.

Nevertheless, this is an important study giving insight on the cryopreservation of stem cells for their potential prolonged use.


1Sumino Y, Hirata Y, Hanada M, Akita Y, Sato F, Mimata H. Long-term cryopreservation of pyramidalis muscle specimens as a source of striated muscle stem cells for treatment of post-prostatectomy stress urinary incontinence. Prostate 2011;71:1225-30.
2Kim SO, Na HS, Kwon D, Joo SY, Kim HS, Ahn Y. Bone-marrow-derived mesenchymal stem cell transplantation enhances closing pressure and leak point pressure in a female urinary incontinence rat model. Urol Int 2011;86:110-6.
3Lin CS, Lue TF. Stem cell therapy for stress urinary incontinence: A critical review. Stem Cells Dev 2012;21:834-43.
4Sèbe P, Doucet C, Cornu JN, Ciofu C, Costa P, de Medina SG, et al. Intra-sphincteric injections of autologous muscular cells in women with refractory stress urinary incontinence: A prospective study. Int Urogynecol J 2011;22:183-9.
5Mitterberger M, Marksteiner R, Margreiter E, Pinggera GM, Frauscher F, Ulmer H, et al. Myoblast and fibroblast therapy for post-prostatectomy urinary incontinence: 1-year followup of 63 patients. J Urol 2008;179:226-31.