Skip to main content

Advertisement

SpringerLink
Log in

Manufacturing autologous myoblast for regenerative medicine applications

  • Methods Paper
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Background: Autologous myoblasts have been tested in the treatment of muscle-related diseases. However, the standardization of manufacturing myoblasts is still not established. Here we report a flask and animal-free medium-based method of manufacturing clinical-grade myoblast together with establishing releasing criteria for myoblast products under Good Manufacturing Practice (GMP). Methods: Quadriceps muscle biopsy samples were donated from three patients with myogenic ptosis. After biopsy samples were digested through enzymatic dissociation, the cells were grown in T175 flasks (passage 0) and hyperflasks (passage 1) in the animal-free SkGMTM-2 skeletal muscle cell growth medium containing 5% human platelet lysate for 15–17 days. The harvested cells were released based on cell morphology, cell dose, viability, sterility, endotoxin, mycoplasma and immunophenotype. Myotube differentiation was also evaluated. Results: 400 to 500 million myoblast cells were manufactured within 15 to 17 days by the end of passage 1, which met pre-determined releasing criteria. The manufactured myoblast cells could differentiate and fuse into myotubes in vitro, with the possible trend that the donor age may impact the differentiation ability of myoblasts. Conclusions: The present study establishes a flask-based method of manufacturing myoblast in the animal-free medium together with releasing criteria, which is simple, robust, inexpensive and easily reproducible. This study will serve as the validation for a planned phase 1 clinical trial to assess the use of autologous myoblast transplants for the treatment of myogenic ptosis and other myogenic diseases.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Baj A, Bettaccini AA, Casalone R, Sala A, Cherubino P, Toniolo AQ (2005) Culture of skeletal myoblasts from human donors aged over 40 years: dynamics of cell growth and expression of differentiation markers. J Transl Med 3:21

    Article  Google Scholar 

  • Bentzinger, C.F., Wang, Y.X., and Rudnicki, M.A. (2012). Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol 4

  • Blau HM, Daley GQ (2019) Stem Cells in the Treatment of Disease. N Engl J Med 380:1748–1760

    Article  CAS  Google Scholar 

  • Bonavaud S, Thibert P, Gherardi RK, Barlovatz-Meimon G (1997) Primary human muscle satellite cell culture: variations of cell yield, proliferation and differentiation rates according to age and sex of donors, site of muscle biopsy, and delay before processing. Biol Cell 89:233–240

    Article  CAS  Google Scholar 

  • Boyer O, Bridoux V, Giverne C, Bisson A, Koning E, Leroi AM, Chambon P, Dehayes J, Le Corre S, Jacquot S et al (2018) Autologous Myoblasts for the Treatment of Fecal Incontinence: results of a Phase 2 Randomized Placebo-controlled Study (MIAS). Ann Surg 267:443–450

    Article  Google Scholar 

  • Corbu A, Scaramozza A, Badiali-DeGiorgi L, Tarantino L, Papa V, Rinaldi R, D’Alessandro R, Zavatta M, Laus M, Lattanzi G et al (2010) Satellite cell characterization from aging human muscle. Neurol Res 32:63–72

    Article  CAS  Google Scholar 

  • Endo T (2015) Molecular mechanisms of skeletal muscle development, regeneration, and osteogenic conversion. Bone 80:2–13

    Article  CAS  Google Scholar 

  • Guan Q, Ezzati P, Spicer V, Krokhin O, Wall D, Wilkins JA (2017) Interferon gamma induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells. Clin Proteomics 14:26

    Article  Google Scholar 

  • Guan Q, Li Y, Shpiruk T, Bhagwat S, Wall DA (2018) Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells. Cytotherapy 20:639–649

    Article  CAS  Google Scholar 

  • Jarocha D, Stangel-Wojcikiewicz K, Basta A, Majka M (2014) Efficient myoblast expansion for regenerative medicine use. Int J Mol Med 34:83–91

    Article  CAS  Google Scholar 

  • Menasche P, Alfieri O, Janssens S, McKenna W, Reichenspurner H, Trinquart L, Vilquin JT, Marolleau JP, Seymour B, Larghero J et al (2008) The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized placebo-controlled study of myoblast transplantation. Circulation 117:1189–1200

    Article  Google Scholar 

  • Miyagawa, S., Domae, K., Yoshikawa, Y., Fukushima, S., Nakamura, T., Saito, A., Sakata, Y., Hamada, S., Toda, K., Pak, K., et al. (2017). Phase I Clinical Trial of Autologous Stem Cell-Sheet Transplantation Therapy for Treating Cardiomyopathy. J Am Heart Assoc 6

  • Negroni E, Gidaro T, Bigot A, Butler-Browne GS, Mouly V, Trollet C (2015) Invited review: stem cells and muscle diseases: advances in cell therapy strategies. Neuropathol Appl Neurobiol 41:270–287

    Article  CAS  Google Scholar 

  • Perie S, Trollet C, Mouly V, Vanneaux V, Mamchaoui K, Bouazza B, Marolleau JP, Laforet P, Chapon F, Eymard B et al (2014) Autologous myoblast transplantation for oculopharyngeal muscular dystrophy: a phase I/IIa clinical study. Mol Ther 22:219–225

    Article  CAS  Google Scholar 

  • Peters KM, Dmochowski RR, Carr LK, Robert M, Kaufman MR, Sirls LT, Herschorn S, Birch C, Kultgen PL, Chancellor MB (2014) Autologous muscle derived cells for treatment of stress urinary incontinence in women. J Urol 192:469–476

    Article  Google Scholar 

  • Price DM, Lane FL, Craig JB, Nistor G, Motakef S, Pham QA, Keirstead H (2014) The effect of age and medical comorbidities on in vitro myoblast expansion in women with and without pelvic organ prolapse. Female Pelvic Med Reconstr Surg 20:281–286

    Article  Google Scholar 

  • Sawa Y, Yoshikawa Y, Toda K, Fukushima S, Yamazaki K, Ono M, Sakata Y, Hagiwara N, Kinugawa K, Miyagawa S (2015) Safety and efficacy of autologous skeletal myoblast sheets (TCD-51073) for the treatment of severe chronic heart failure due to ischemic heart disease. Circ J 79:991–999

    Article  Google Scholar 

  • Spinazzola, J.M., and Gussoni, E. (2017). Isolation of Primary Human Skeletal Muscle Cells. Bio Protoc 7

Download references

Acknowledgements

This study was supported by Misericordia Health Centre Foundation.

Author information

Authors and Affiliations

  1. Dept of Ophthalmology, University of Manitoba, Winnipeg, Canada

    Matthew Lee-Wing & Alanna Flynn

  2. Manitoba Centre for Advanced Cell & Tissue Therapy, Winnipeg, Canada

    David Szwajcer, Karla Anjos, Marie Tulloch, Angeline Giftakis & Qingdong Guan

  3. Cellular Therapy Laboratory, Manitoba Blood and Marrow Transplant Program, CancerCare Manitoba, MS773M, 820 Sherbrook St, Winnipeg, R3A 1R9, MB, Canada

    David Szwajcer, Karla Anjos, Marie Tulloch, Angeline Giftakis & Qingdong Guan

  4. Dept of Internal Medicine, University of Manitoba, Winnipeg, Canada

    David Szwajcer & Qingdong Guan

  5. Dept of Plastic Surgery, University of Manitoba, Winnipeg, Canada

    Anthony Lockwood

  6. Dept of Immunology, University of Manitoba, Winnipeg, Canada

    Qingdong Guan

Authors
  1. Matthew Lee-Wing
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Szwajcer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anthony Lockwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alanna Flynn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karla Anjos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marie Tulloch
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Angeline Giftakis
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Qingdong Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MLW, DS and QG conceived and designed the project; MLW recruited donors; QG developed the cell manufacturing protocol; AL, MLW and AF performed muscle biopsy; KA, MT, QG and AG manufactured cell products; QG analyzed the data; MLW, DS and QG wrote the manuscript; AL, AF, KA, MT and AG critically reviewed it. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Qingdong Guan.

Ethics declarations

Conflict of interest

The authors declare no competing financial interests.

Ethics approval and consent to participate

The study was approved by the University of Manitoba Research Ethics Board. Animal study was not used in this study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 23 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee-Wing, M., Szwajcer, D., Lockwood, A. et al. Manufacturing autologous myoblast for regenerative medicine applications. Cytotechnology 72, 605–614 (2020). https://doi.org/10.1007/s10616-020-00420-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-020-00420-9

Keywords

Search

Navigation