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GMP Tiered Cell Banking of Non-enzymatically Isolated Dermal Progenitor Fibroblasts for Allogenic Regenerative Medicine

  • Alexis Laurent
  • Corinne Scaletta
  • Murielle Michetti
  • Nathalie Hirt-Burri
  • Anthony S. de Buys Roessingh
  • Wassim Raffoul
  • Lee Ann ApplegateEmail author
Part of the Methods in Molecular Biology book series


Non-enzymatically isolated primary dermal progenitor fibroblasts derived from fetal organ donations are ideal cell types for allogenic musculoskeletal regenerative therapeutic applications. These cell types are differentiated, highly proliferative in standard in vitro culture conditions and extremely stable throughout their defined lifespans. Technical simplicity, robustness of bioprocessing and relatively small therapeutic dose requirements enable pragmatic and efficient production of clinical progenitor fibroblast lots under cGMP standards. Herein we describe optimized and standardized monolayer culture expansion protocols using dermal progenitor fibroblasts isolated under a Fetal Transplantation Program for the establishment of GMP tiered Master, Working and End of Production cryopreserved Cell Banks. Safety, stability and quality parameters are assessed through stringent testing of progeny biological materials, in view of clinical application to human patients suffering from diverse cutaneous chronic and acute affections. These methods and approaches, coupled to adequate cell source optimization, enable the obtention of a virtually limitless source of highly consistent and safe biological therapeutic material to be used for innovative regenerative medicine applications.


Cell therapy Clinical cell banking Dermal fibroblasts GMP manufacturing Optimized protocols Organ donation Progenitor cells Safety testing 



Advanced therapy medicinal product


Bovine polyomavirus


Cluster of differentiation


Current good manufacturing practices




European Union Committee for Proprietary Medicinal Products


Dulbecco’s modified Eagle medium




Deoxyribonucleic acid


Dulbecco’s phosphate-buffered saline


Epstein-Barr virus


End of production


End of Production Cell Bank


Fluorescence-activated cell sorting


Fetal bovine serum


US Food and Drug Administration


Good laboratory practices


Good manufacturing practices


Hepatitis A virus


Human bocavirus


Hepatitis B virus


Human cytomegalovirus


Hepatitis C virus


Human herpes viruses types 6, 7 and 8


Human immunodeficiency viruses types 1 and 2


Human leukocyte antigen


Human platelet lysate


Human papilloma virus


Human serum albumin


Human T-cell leukemia-lymphoma viruses types 1 and 2


Human polyomavirus


In-process control


KI polyomavirus


Master Cell Bank


Parental Cell Bank


Polymerase chain reaction


Population doubling time


Population doubling value


Pilot Working Cell Bank


Quantitative fluorescent product enhanced reverse transcriptase


Quality risk management


Standard operating procedure


Transmission electron microscopy


Working Cell Bank


WU polyomavirus



We would like to thank the S.A.N.T.E and Sandoz Foundations for their commitments to the Fetal Biobanking Program through the years. We would like to thank Mrs. Judith Applegate for her reviewing of spelling and grammar of the manuscript.


  1. 1.
    Doyle A, Griffiths JB (1998) Cell and tissue culture: laboratory procedures in biotechnology. Wiley, New YorkGoogle Scholar
  2. 2.
    Vacanti JP, Langer R (1999) Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet 354(Suppl 1):SI32–SI34Google Scholar
  3. 3.
    Monti M, Perotti C, Del Fante C et al (2012) Stem cells: sources and therapies. Biol Res 45:207–214Google Scholar
  4. 4.
    Li Z, Maitz P (2018) Cell therapy for severe burn wound healing. Burns Trauma 6:13Google Scholar
  5. 5.
    Loebel C, Burdick JA (2018) Engineering stem and stromal cell therapies for musculoskeletal tissue repair. Cell Stem Cell 22:325–339Google Scholar
  6. 6.
    Marks P, Gottlieb S (2018) Balancing safety and innovation for cell-based regenerative medicine. N Engl J Med 378:954–959Google Scholar
  7. 7.
    Abbasalizadeh S, Baharvand H (2013) Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies. Biotechnol Adv 31:1600–1623Google Scholar
  8. 8.
    Heathman TR, Nienow AW, McCall MJ et al (2015) The translation of cell-based therapies: clinical landscape and manufacturing challenges. Regen Med 10:49–64Google Scholar
  9. 9.
    Hunsberger J, Harrysson O, Shirwaiker R et al (2015) Manufacturing road map for tissue engineering and regenerative medicine technologies. Stem Cells Transl Med 4:130–135Google Scholar
  10. 10.
    Abbasalizadeh S, Pakzad M, Cabral JMS et al (2017) Allogeneic cell therapy manufacturing: process development technologies and facility design options. Expert Opin Biol Ther 17:1201–1219Google Scholar
  11. 11.
    Pigeau GM, Csaszar E, Dulgar-Tulloch A (2018) Commercial scale manufacturing of allogeneic cell therapy. Front Med 5:233Google Scholar
  12. 12.
    Cass DL, Meuli M, Adzick NS (1997) Scar wars: implications of fetal wound healing for the pediatric burn patient. Pediatr Surg Int 12:484–489Google Scholar
  13. 13.
    De Buys Roessingh AS, Hohlfeld J, Scaletta C et al (2006) Development, characterization, and use of a fetal skin cell bank for tissue engineering in wound healing. Cell Transplant 15:823–834Google Scholar
  14. 14.
    Werner S, Krieg T, Smola H (2007) Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol 127:998–1008Google Scholar
  15. 15.
    Zuliani T, Saiagh S, Knol AC et al (2013) Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy. PLoS One 8:e70408Google Scholar
  16. 16.
    Larijani B, Ghahari A, Warnock GL et al (2015) Human fetal skin fibroblasts: extremely potent and allogenic candidates for treatment of diabetic wounds. Med Hypotheses 84:577–579Google Scholar
  17. 17.
    Varkey M, Ding J, Tredget EE (2015) Advances in skin substitutes-potential of tissue engineered skin for facilitating anti-fibrotic healing. J Funct Biomater 6:547–563Google Scholar
  18. 18.
    Akita S, Akino K, Imaizumi T et al (2008) Basic fibroblast growth factor accelerates and improves second-degree burn wound healing. Wound Repair Regen 16:635–641Google Scholar
  19. 19.
    De Buys Roessingh AS, Hirt-Burri N, Raffoul W et al (2015) A decade after foetal skin progenitor cell therapy in pediatric burn treatment. J Regen Med 4:1Google Scholar
  20. 20.
    Hayflick L, Plotkin SA, Norton TW et al (1962) Preparation of poliovirus vaccines in a human fetal diploid cell strain. Am J Hyg 75:240–258Google Scholar
  21. 21.
    Hebda PA, Dohar JE (1999) Transplanted fetal fibroblasts: survival and distribution over time in normal adult dermis compared with autogenic, allogenic, and xenogenic adult fibroblasts. Otolaryngol Head Neck Surg 121:245–251Google Scholar
  22. 22.
    Abdel-Sayed P, Hirt-Burri N, De Buys Roessingh AS et al (2019) Evolution of biological bandages as first cover for burn patients. Adv Wound Care (New Rochelle) 8:555–564Google Scholar
  23. 23.
    Hohlfeld J, De Buys Roessingh AS, Hirt-Burri N et al (2005) Tissue engineered fetal skin constructs for paediatric burns. Lancet 366:840–842Google Scholar
  24. 24.
    Quintin A, Hirt-Burri N, Scaletta C et al (2007) Consistency and safety of cell banks for research and clinical use: preliminary analysis of fetal skin banks. Cell Transplant 16:675–684Google Scholar
  25. 25.
    Applegate LA, Scaletta C, Hirt-Burri N et al (2009) Whole-cell bioprocessing of human fetal cells for tissue engineering of skin. Skin Pharmacol Physiol 22:63–73Google Scholar
  26. 26.
    Applegate LA, Weber D, Simon JP et al (2013) Organ donation and whole-cell bioprocessing in the Swiss fetal progenitor cell transplantation platform. In: Saidi RF (ed) Organ donation and organ donors. Nova Science Publishers, New YorkGoogle Scholar
  27. 27.
    Metcalfe AD, Ferguson MW (2008) Skin stem and progenitor cells: using regeneration as a tissue-engineering strategy. Cell Mol Life Sci 65:24–32Google Scholar
  28. 28.
    Ramelet AA, Hirt-Burri N, Raffoul W et al (2009) Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells. Exp Gerontol 44:208–218Google Scholar
  29. 29.
    Rayment EA, Williams DJ (2010) Concise review: mind the gap: challenges in characterizing and quantifying cell- and tissue-based therapies for clinical translation. Stem Cells 28:996–1004Google Scholar
  30. 30.
    Haack-Sørensen M, Kastrup J (2011) Cryopreservation and revival of mesenchymal stromal cells. Methods Mol Biol 698:161–174Google Scholar
  31. 31.
    Ratcliffe E, Thomas RJ, Williams DJ (2011) Current understanding and challenges in bioprocessing of stem cell-based therapies for regenerative medicine. Br Med Bull 100:137–155Google Scholar
  32. 32.
    Mount NM, Ward SJ, Kefalas P et al (2015) Cell-based therapy technology classifications and translational challenges. Philos Trans R Soc Lond B Biol Sci 370:20150017Google Scholar
  33. 33.
    Hunt CJ (2019) Technical considerations in the freezing, low-temperature storage and thawing of stem cells for cellular therapies. Transfus Med Hemother 46:134–150Google Scholar
  34. 34.
    Laurent-Applegate LA (2012) Preparation of parental cell bank from foetal tissue. European Patent No 2 732 030 B1, 10 July 2012Google Scholar
  35. 35.
    Abdel-Sayed P, Kaeppeli A, Siriwardena T et al (2016) Anti-microbial dendrimers against multidrug-resistant P. aeruginosa enhance the angiogenic effect of biological burn-wound bandages. Sci Rep 6:22020Google Scholar

Copyright information

© Springer Science+Business Media New York 2020

Authors and Affiliations

  • Alexis Laurent
    • 1
    • 2
  • Corinne Scaletta
    • 1
    • 2
  • Murielle Michetti
    • 1
    • 2
  • Nathalie Hirt-Burri
    • 1
    • 2
  • Anthony S. de Buys Roessingh
    • 3
  • Wassim Raffoul
    • 2
  • Lee Ann Applegate
    • 1
    • 2
    • 4
    • 5
    Email author
  1. 1.Regenerative Therapy Unit, Musculoskeletal Medicine DepartmentLausanne University Hospital, University of LausanneEpalingesSwitzerland
  2. 2.Plastic, Reconstructive & Hand Surgery Service, Lausanne University Hospital, University of LausanneLausanneSwitzerland
  3. 3.Pediatric Surgery Service, Lausanne University Hospital, University of LausanneLausanneSwitzerland
  4. 4.Oxford Suzhou Center for Advanced Research, Science and Technology Co. Ltd., Oxford UniversitySuzhouPeople’s Republic of China
  5. 5.Competence Center for Applied Biotechnology and Molecular Medicine, University of ZurichZurichSwitzerland

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