Applied Microbiology and Biotechnology

, Volume 68, Issue 4, pp 456–466 | Cite as

Microbiological control in stem cell banks: approaches to standardisation

  • Fernando Cobo
  • Glyn N. Stacey
  • Charles Hunt
  • Carmen Cabrera
  • Ana Nieto
  • Rosa Montes
  • José Luis Cortés
  • Purificación Catalina
  • Angela Barnie
  • Ángel Concha


The transplant of cells of human origin is an increasingly complex sector of medicine which entails great opportunities for the treatment of a range of diseases. Stem cell banks should assure the quality, traceability and safety of cultures for transplantation and must implement an effective programme to prevent contamination of the final product. In donors, the presence of infectious micro-organisms, like human immunodeficiency virus, hepatitis B virus, hepatitis C virus and human T cell lymphotrophic virus, should be evaluated in addition to the possibility of other new infectious agents (e.g. transmissible spongiform encephalopathies and severe acute respiratory syndrome). The introduction of the nucleic acid amplification can avoid the window period of these viral infections. Contamination from the laboratory environment can be achieved by routine screening for bacteria, fungi, yeast and mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an adventitious or endogenous virus, is a well-known fact that will also have to be considered for processes involving in vitro culture of stem cells. It is also a standard part of current good practice in stem cell banks to carry out routine environmental microbiological monitoring of the cleanrooms where the cell cultures and their products are prepared. The risk of viral contamination from products of animal origin, like bovine serum and mouse fibroblasts as a “feeder layer” for the development of embryonic cell lines, should also be considered. Stem cell lines should be tested for prion particles and a virus of animal origin that assure an acceptable quality.


  1. American Association of Tissue Banks (2002) Standards for tissue banking, 10th edn. American Association of Tissue Banks, McLean, VAGoogle Scholar
  2. Arnold DM, Neame PB, Meyer RM, Soamboonsrup P, Luinstra KE, O'hoski P, Garner J, Foley R (2005) Autologous peripheral blood progenitor cells are a potential source of parvovirus B19 infection. Transfusion 45:394–398CrossRefPubMedGoogle Scholar
  3. Barry T, Powell R, Gannon F (1990) A general method to generate DNA probes for microorganisms. Biotechnology 8:233–236CrossRefPubMedGoogle Scholar
  4. Cassinotti P, Burtonboy G, Fopp M, Siegl C (1997) Evidence for persistence of human parvovirus B19 DNA in bone marrow. J Med Virol 53:229–232CrossRefPubMedGoogle Scholar
  5. Centers for Disease Control and Prevention (2004) West Nile virus screening of blood donations and transfusion associated transmission—United States, 2003. MMWR Morb Mortal Wkly Rep 53:281–284Google Scholar
  6. Consensus Document (1999) Selection criteria in the organ donors respect to transmission of infections. Grupo GESITRA (SEIMC), SpainGoogle Scholar
  7. Dellepiane N, Griffiths E, Milstien JB (2000) New challenges in assuring vaccine quality. Bull World Health Organ 78:155–162PubMedGoogle Scholar
  8. Department of Health (2000) Guidance on the microbiological safety of human organs, tissues and cells used in transplantation. Advisory committee on the Microbiological Safety of Blood and Tissues for Transplantation, MSBT. Department of Health, London, UKGoogle Scholar
  9. Department of Health (2001) A code of practice for tissue banks. Department of Health, London, UKGoogle Scholar
  10. Directive 2004/23/CE of the European parliament and the council of March 31st, relating to the establishment of quality and safety norms to donate, to obtain, to assess, to process, to preserve, to store and to distribute cells and human tissuesGoogle Scholar
  11. Doblhoff-Dier O, Bachmayer H, Bennett A, Brunius G, Burki K, Cantley M, Collins C, Crooy P, Elmqvist A, Frontali-Botti C, Havenacer R, Haymerle H, Lelieveld H, Lex M, Mahler JL, Martinez L, Mosgaard C, Olsen L, Pazlarova J, Rudan F, Sarvas M, Stepankova H, Tzotzus G, Wagner K, Werner R (1999) Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The safety in Biotechnology Working Party of the European Federation of Biotechnology. Trends Biotechnol 17:307–311CrossRefPubMedGoogle Scholar
  12. Dodd RY, Stramer JL, Aberle-Grasse J, Notari E IV (2000) Risk of hepatitis and retroviral infections among blood donors and introduction of nucleic acid testing (NAT). Dev Biol Stand 102:19–27Google Scholar
  13. Dodd RY, Notari EP IV, Stramer SL (2002) Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population. Transfusion 42:975–979CrossRefPubMedGoogle Scholar
  14. Doyle A, Griffiths JB (1998) Elimination of contamination. In: Cell and tissue culture: laboratory procedures in biotechnology. Wiley, New YorkGoogle Scholar
  15. Drexler HG, Uphoff CC (2002) Mycoplasma contamination of cell cultures. Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39:75–90CrossRefGoogle Scholar
  16. EC Guide to Good Manufacturing Practice (2003) Manufacture of sterile medicinal products, revision to annex 1. EC, BrusselsGoogle Scholar
  17. European Medicines Evaluation Agency (1997) ICH Consensus guideline on quality of biotechnology products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin, publication no CPMP/ICH/295/95. European Medicines Evaluation Agency, Canary Wharf, LondonGoogle Scholar
  18. European Medicines Evaluation Agency (1999) EMEA workshop on application of pharmaceutical assays for markers of TSE: report to CPMP from the BIOT-WP (CPMP/BWP/257/99). EMEA, LondonGoogle Scholar
  19. European Medicines Evaluation Agency (2003) Note for guidance on the use of bovine serum in the manufacture on human biological medicinal products. EMEA, LondonGoogle Scholar
  20. European Pharmacopeia (2002) Pharmaceutical inspection convention. Recommendation on sterility testing, PIC/sGoogle Scholar
  21. European Pharmacopeia (2004a) European Pharmacopeia section 2.6.1 (Sterility). Maisonneuve SA, Sainte RuffineGoogle Scholar
  22. European Pharmacopeia (2004b) European Pharmacopeia section 2.6.7 (Mycoplasma). Maisonneuve SA, Sainte RuffineGoogle Scholar
  23. Fanci R, De Santis R, Zakrzewska K, Paci C, Azzi A (2004) Presence of TT virus DNA in bone marrow cells from hematologic patients. New Microbiol 27:113–117PubMedGoogle Scholar
  24. Frommer W, Archer L, Boon B, Brunius G, Collins CH, Crooy P, Doblhoff-Dier O, Donikian R, Economidis J, Frontali C (1993) Safe biotechnology (5). Recommendations for safe work with animal and human cell cultures concerning potential human pathogens. Appl Microbiol Biotechnol 39:141–147CrossRefPubMedGoogle Scholar
  25. Garbuglia AR, Iiezzi T, Capobianchi MR, Pignoloni P, Pulsoni A, Sourdis J, Pescarmona E, Vitolo D, Mandelli F (2003) Detection of TT virus in lymph node biopsies of B-cell lymphoma and Hodgkin's disease, and its association with EBV infection. Int J Immunopathol Pharmacol 16:109–118PubMedGoogle Scholar
  26. GPMD (2002) Rules and guidance for pharmaceutical manufacturers and distributors (EEC Orange Guide). The Stationery Office, LondonGoogle Scholar
  27. Hartung T, Balls M, Bardouille C, Blank O, Coecke S, Gstramthaler G, Lewis D (2002) Good cell culture practice. ECVAM Good Cell Culture Practice Task Force Report 1. ATLA 30:407–411PubMedGoogle Scholar
  28. Hitzler WE, Runkel S (2001) Screening of blood donations by hepatitis C virus polymerase chain reaction (HCV-PCR) improves safety of blood products by window period reduction. Clin Lab 47:219–222PubMedGoogle Scholar
  29. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1997) ICH harmonised tripartite guideline. Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. ICH, GenevaGoogle Scholar
  30. International Organization for Standarization (1999) ISO 14644. Cleanrooms and associated controlled environments. Part 1: Classification of air cleanliness. International Organization for StandarizationGoogle Scholar
  31. Ironside JW (1996) Review: Creutzfeldt–Jakob disease. Brain Pathol 6:379–388PubMedGoogle Scholar
  32. Janssen RS, Satten GA, Stramer SL, Rawal BD, O'Brien TR, Weiblen BJ, Hecht FM, Jack N, Cleghorn FR, Kahn JO, Chesney MA, Busch MP (1998) New testing strategy to detect early HIV-1 infection for use in incidence estimates and for clinical and prevention purposes. JAMA 280:42–48 (Erratum, JAMA 1999; 281: 1893)CrossRefPubMedGoogle Scholar
  33. Juang JL, Chen TC, Jiang SS, Hsiong CA, Chen WC, Cheng GW, Lin SM, Lin JH, Chiu SC, Lai YK (2004) Coupling multiplex RT-PCR to a gene chip assay for sensitive and semiquantitative detection of severe acute respiratory syndrome coronavirus. Lab Invest 84:1085–1091CrossRefPubMedGoogle Scholar
  34. Laassri M, Chizhikov V, Mikheev M, Shchelkunov S, Chumakov K (2003) Detection and discrimination of orthopoxviruses using microarrays of immobilized oligonucleotides. J Virol Methods 112:67–78CrossRefPubMedGoogle Scholar
  35. Lee DC, Stenland CJ, Hartwell RC, Ford EK, Cai K, Miller JL, Gilligan KJ, Rubenstein R, Fournel M, Petteway SR (2000) Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein. J Virol Methods 84:77–89CrossRefPubMedGoogle Scholar
  36. Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, MacKenzie J, Will RG (2004) Possible transmission of variant Creutzfeldt–Jakob disease by blood transfusion. Lancet 363:417–421CrossRefPubMedGoogle Scholar
  37. MacLeod RA, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG (1999) Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int J Cancer 83:555–563CrossRefPubMedGoogle Scholar
  38. Mayor S (2004) First stem cell bank in the world is opened in UK. BMJ 328:1277CrossRefPubMedGoogle Scholar
  39. McGarrity GJ (1982) Detection of mycoplasmal infection of cell cultures. In: Maramorosch K (ed) Advances in cell cultures, vol. 2. Academic, New York, pp 99–131Google Scholar
  40. McGarrity GJ, Kotani H, Butler GH (1992) Mycoplasmas and tissue culture cells. In: Maniloff J, McElhaney RN, Fich LR, Baseman JB (eds) Mycoplasmas—molecular biology and pathogenesis. American Society for Microbiology, Washington, D.C., pp 445–454Google Scholar
  41. Microbiological Control Methods (2003) In the European Pharmacopeia: present and future. European Directorate for the Quality of Medicines Meeting, Copenhagen, pp 5–7 and 29–31Google Scholar
  42. Nelson NC, Reynolds MA, Arnold LJ Jr (1995) Detection of acridinium esters by chemiluminescence. In: Kricka LJ (ed) Non isotopic probing, plotting and sequencing. Academic, San Diego, CA, pp 391–428Google Scholar
  43. Nelson-Rees WA, Daniels DW, Flandermeyer RR (1981) Cross-contamination of cells in culture. Science 212:446–452PubMedCrossRefGoogle Scholar
  44. Nolte FS, Caliendo AM (2003) Molecular detection and identification of microorganisms. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (eds) Manual of clinical microbiology. ASM, Washington, D.C., pp 234–256Google Scholar
  45. O'Hara CM, Weinstein MP, Miller JM (2003) Manual and automated systems for detection and identification of microorganisms. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH (eds) Manual of clinical microbiology. ASM, Washington, D.C., pp 185–207Google Scholar
  46. Parenteral Society (2002) Parenteral Society Technical Monograph No 2. Environmental contamination control practice. Parenteral Society, LondonGoogle Scholar
  47. Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW (2004) Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 264:527–529CrossRefGoogle Scholar
  48. Pincock S (2004) UK opens embryonic stem cell bank. Lancet 363:1778CrossRefPubMedGoogle Scholar
  49. Razin S (1994) DNA probes and PCR in diagnosis of mycoplasma infections. Mol Cell Probes 8:497–511CrossRefPubMedGoogle Scholar
  50. Rijsewijk F, Maris-Veldhuis MA, Oei HL, Claassen I (2002) Semi-quantitative detection of BVDV RNA in vaccine samples using RT-PCR with internal control RNA molecules. ID-lelystad/CIDCGoogle Scholar
  51. Saldanha J (2001) Validation and standardisation of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products. J Clin Virol 20:7–13CrossRefPubMedGoogle Scholar
  52. Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ (1996) The risk of transfusion-transmitted viral infections. The Retrovirus Epidemiology Donor Study. N Engl J Med 334:1685–1690CrossRefPubMedGoogle Scholar
  53. Stacey GN (2000) Detection of mycoplasma by DNA amplification. In: Doyle A, Griffitths JB (eds) Cell and tissue culture for medical research, chapter 2. Wiley, New York, pp 58–61Google Scholar
  54. Stacey GN, Phillips P (1999) Quality assurance for cell substrates. Dev Biol Stand 98:141–151PubMedGoogle Scholar
  55. Stacey GN, Masters JRW, Hay RJ, Drexler HG, MacLeod RAF, Freshney RI (2000) Cell contamination leads to inaccurate data: we must take action now. Nature 403:356CrossRefGoogle Scholar
  56. Stramer SL, Glynn SA, Kleinman SH, Strong M, Caglioti S, Wright DJ, Dodd RY, Busch MP (2004) Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid amplification testing. N Engl J Med 351:760–768CrossRefPubMedGoogle Scholar
  57. Takeuchi H, Kobayashi R, Hasegawa M, Hirai K (1996) Detection of latent infection by Epstein–Barr virus in peripheral blood cells of healthy individuals and in non-neoplastic tonsillar tissue from patients by reverse transcription-polymerase chain reaction. J Virol Methods 58:81–89CrossRefPubMedGoogle Scholar
  58. Toji LH, Lenchitz TC, Kwiatkowski VA, Sarama JA, Mulivor RA (1998) Validation of routine mycoplasma testing by PCR. In Vitro Cell Dev Biol, Anim 34:356–358CrossRefGoogle Scholar
  59. UK Code of Practice for Tissue Banks (2001) Providing tissues of human origin for therapeutic purposes, Department of Health, CrownGoogle Scholar
  60. UK MSBT (2000) Guidelines on the microbiological safety of human organs, tissues and cells used in transplantation. Advisory Committee on the Microbiological Safety of Blood and Tissues for Transplantation, MSBTGoogle Scholar
  61. UKBTS/NIBSC (2002) Guidelines for the blood transfusion services in the United Kingdom (Red Book), 6th edn. UKBTS/NIBSC, LondonGoogle Scholar
  62. Uphoff CC, Drexler HG (2005) Detection of mycoplasma contaminations. Methods Mol Biol 290:13–23PubMedGoogle Scholar
  63. US Food and Drugs Administration (1999) CFR (Code of Federal Regulations) section 1271 Subpart C-suitability determination for donors of human cellular and tissue based products, proposed rule 64 FR 189. US Food and Drugs Administration, Rockville, MDGoogle Scholar
  64. Wadsworth JD, Joiner S, Hill AF, Campbell TA, Desbruslais M, Luthert PJ, Collinge J (2001) Tissue distribution of protease resistant prion protein in variant Creutzfeldt–Jakob disease using a highly sensitive immunoblotting assay. Lancet 358:171–180CrossRefPubMedGoogle Scholar
  65. Weisburg WG, Tully JG, Rose DL, Petzel P, Oyaizu H, Yang D, Mandelco L, Sechrest J, Lawrence TG, Van Etten J, Maniloff J, Woese CR (1989) A phylogenetic analysis of the mycoplasmas: basis of their classification. J Bacteriol 171:6455–6467PubMedGoogle Scholar
  66. Zou S, Dodd RY, Stramer SL, Strong M (2004) Probability of viremia with HBV, HCV, HIV and HTLV among tissue donors in the United States. N Eng J Med 351:751–759CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Fernando Cobo
    • 1
  • Glyn N. Stacey
    • 2
  • Charles Hunt
    • 2
  • Carmen Cabrera
    • 1
  • Ana Nieto
    • 1
  • Rosa Montes
    • 1
  • José Luis Cortés
    • 1
  • Purificación Catalina
    • 1
  • Angela Barnie
    • 1
  • Ángel Concha
    • 1
  1. 1.Stem Cell Bank of Andalucía (Spanish Central Node)Hospital Universitario Virgen de las NievesGranadaSpain
  2. 2.UK Stem Cell BankNational Institute for Biological Standards and ControlHertfordshireUK

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