, Volume 63, Issue 4, pp 325–335 | Cite as

Online- and offline- monitoring of stem cell expansion on microcarrier

  • C. Justice
  • J. Leber
  • D. Freimark
  • P. Pino Grace
  • M. Kraume
  • P. CzermakEmail author
Method in Cell Science


In the biopharmaceutical industry, adherent growing stem cell cultures gain worldwide importance as cell products. The cultivation process of these cells, such as in stirred tank reactors or in fixed bed reactors, is highly sophisticated. Cultivations need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time on-line monitoring tools are recommended. A tool meeting the new requirements may be the dielectric spectroscopy for online viable cell mass determination by measurement of the permittivity. To establish these tools, proper offline methods for data correlation are required. The cell number determination of adherent cells on microcarrier is difficult, as it requires cell detachment from the carrier, which highly increases the statistical error. As an offline method, a fluorescence assay based on SYBR®GreenI was developed allowing fast and easy total cell concentration determination without the need to detach the cells from the carrier. The assay is suitable for glass carriers used in stirred tank reactor systems or in fixed bed systems, may be suitable for different cell lines and can be applied to high sample numbers easily. The linear dependency of permittivity to cell concentration of suspended stem cells with the dielectric spectroscopy is shown for even very small cell concentrations. With this offline-method, a correlation of the cell concentration grown on carrier to the permittivity data measured by the dielectric spectroscopy was done successfully.


Microcarrier culture Process analytical technology Fluorescence Dielectric spectroscopy Biomass monitoring 



We would like to thank the Hessen State Ministry of Higher Education, Research and the Arts in Germany for the financial support within the Hessen initiative for scientific and economic excellence (LOEWE-Program). Moreover, the authors would like to thank the Federal Ministry of Economics and Technology of Germany (KF2268901UL9) for the financial support.


  1. Baldi L, Hacker DL, Adam M, Wurm FM (2007) Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol Lett 29:677–684CrossRefGoogle Scholar
  2. Butler M (2005) Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68:283–291CrossRefGoogle Scholar
  3. Butler M, Spearman M (2007) Cell counting and viability measurements. Methods Biotechnol 24:205CrossRefGoogle Scholar
  4. Carvell JP, Dowd JE (2006) On-line measurements and control of viable cell density in cell culture manufacturing processes using radio-frequency impedance. Cytotechnology 50:35–48CrossRefGoogle Scholar
  5. Clementschitsch F, Bayer K (2006) Improvement of bioprocess monitoring: development of novel concepts. Microb Cell Fact 5:19CrossRefGoogle Scholar
  6. Davey CI, Guan Y, Kemp RB, Kell DB (1997) Real-time monitoring of the biomass content of animal cell cultures using dielectric spectroscopy. Anim Cell Technol Basic Appl Asp 8:61–65Google Scholar
  7. Degouys V, Cerckel I, Garcia A, Harfield J, Dubois D, Fabry L, Miller AO (1993) Dielectric spectroscopy of mammalian cells. 2. Simultaneous in situ evaluation by aperture impedance pulse spectroscopy and low frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3. Cytotechnology 13:195–202CrossRefGoogle Scholar
  8. Ducommun P, Kadouri A, von Stockar U, Marison IW (2002a) On-line determination of animal cell concentration in two industrial high-density culture processes by dielectric spectroscopy. Biotechnol Bioeng 77:316–323CrossRefGoogle Scholar
  9. Ducommun P, Rueux PA, Kadouri A, Von Stockar U, Marison IW (2002b) Monitoring of temperature effects on animal cell metabolism in a packed bed process. Ferment Bioindustrial Chem 77:838–842Google Scholar
  10. Eibl R, Eibl D, Poertner R, Catapano G, Czermak P (2008) Cell and tissue reaction engineering. Springer, New YorkGoogle Scholar
  11. FDA (2004) Guidance for industry PAT- a framework for innovative pharmaceutical manufacturing and quality assurance. Accessed 19 Feb 2009
  12. Fernandes AM, Marinho PAN, Sartore RC, Paulsen BS, Mariante RM, Castilho LR, Rehen SK (2009) Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system. Braz J Med Biol Res 42:515–522Google Scholar
  13. Frauenschuh S, Reichmann E, Ibold Y, Goetz PM, Sittinger M, Ringe J (2007) A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Biotechnol Prog 23:187–193CrossRefGoogle Scholar
  14. Freimark D, Pino-Grace P, Pohl S, Weber C, Wallrapp C, Geigle P, Poertner R, Czermak P (2010) Use of encapsulated stem cells to overcome the bottleneck of cell availability for cell therapy approaches. Transfus Medicine Hemotherapy 37:66–73Google Scholar
  15. Guan Y, Kemp RB (2002) The viable cell monitor: a dielectric spectroscope for growth and metabolic studies of animal cells on macroporous beads. 15th ESACT Meeting: New Developments and New Applications in Animal Cell Technology, Part 7, pp 321–328. doi: 10.1007/0-306-46860-3_58
  16. Justice C, Pino-Grace P, Freimark D, Kraume M, Wallrapp C, Geigle P, Czermak P (2010) Process intensification of stem cell cultivation. Biomed Eng 55(Suppl. 1):Ref 163. doi: 10.1515/BMT.2010.706
  17. Justice C, Brix A, Freimark D, Kraume M, Pfromm P, Eichenmueller B, Czermak P (2011) Process control in cell culture technology using dielectric spectroscopy. Biotechnol Adv. doi: 10.1016/j.biotechadv.2011.03.002
  18. Levine DW, Wang DIC, Thilly WG (1979) Optimization of growth surface parameters in microcarrier cell culture. Biotechnol Bioeng 21:821–845CrossRefGoogle Scholar
  19. Myers MA (1998) Direct measurement of cell numbers in microtitre plate cultures using the fluorescent dye SYBR green I. J Immunol Methods 212:99–103CrossRefGoogle Scholar
  20. Noll T, Biselli M (1998) Dielectric spectroscopy in the cultivation of suspended and immobilized hybridoma cells. J Biotechnol 63:187–198CrossRefGoogle Scholar
  21. Oh SKW, Chen AK, Mok Y, Chen X, Lim U (2009) Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res 2:219–230CrossRefGoogle Scholar
  22. Rahman ARA, Register J, Vuppala G, Bhansali S (2008) Cell culture monitoring by impedance mapping using a multielectrode scanning impedance spectroscopy system (CellMap). Physiol Meas 29:227CrossRefGoogle Scholar
  23. Rengarajan K, Cristol SM, Mehta M, Nickerson JM (2002) Quantifying DNA concentrations using fluorometry: a comparison of fluorophores. Mol Vis 8:416–421Google Scholar
  24. Rudolph G, Brueckerhoff T, Bluma A, Korb G, Scheper T (2007) Optische Inline- Messverfahren zur Zellzahl- und Zellgrößenbestimmung in der Bioprozesstechnik. Chemie Ingenieur Technik 79:42–51CrossRefGoogle Scholar
  25. Schop D, Janssen FW, Borgart E, de Bruijn JD, van Dijkhuizen-Radersma R (2008) Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism. J Tissue Eng Regen Medicine 2:126–135CrossRefGoogle Scholar
  26. Shimizu S, Eguchi Y, Kamiike W, Itoh Y, Hasegawa J, Yamabe K, Otsuki Y, Matsuda H, Tsujimoto Y (1996) Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-XL. Cancer Res 56:2161Google Scholar
  27. Tavernarakis N (2006) Proteolytic pathways in necrotic cell death. Biotech Int. Accessed 12 Jan 2011.
  28. Teixeira AP, Oliveira R, Alves PM, Carrondo MJT (2009) Advances in on-line monitoring and control of mammalian cell cultures: supporting the PAT initiative. Biotechnol Adv 27:726–732CrossRefGoogle Scholar
  29. Velden-de Groot CAM (1995) Microcarrier technology, present status and perspective. Cytotechnology 18(1):51–56CrossRefGoogle Scholar
  30. Vojinovic V, Cabral JMS, Fonseca LP (2006) Real-time-bioprocess monitoring part I: in situ sensors. Sens Actuators B 114:1083–1091CrossRefGoogle Scholar
  31. Weber C, Pohl S, Poertner R, Wallrapp C, Kassem M, Geigle P, Czermak P (2007a) Expansion and Harvesting of hMSC-TERT. Open Biomed Eng J 1:38–46Google Scholar
  32. Weber C, Pohl S, Poertner R, Wallrapp C, Kassem M, Geigle P, Czermak P (2007b) Cultivation and differentiation of encapsulated hMSC-TERT in a disposable small-scale syringe-like fixed bed reactor. Open Biomed Eng J 1:64–70Google Scholar
  33. Weber C, Pohl S, Poertner R, Wallrapp C, Geigle P, Czermak P (2009) Development of a production process for stem cell based cell therapeutic implants using disposable bioreactor systems. In: Vander Sloten J, Verdonck P, Nyssen M, Haueisen J (eds) ECIFMBE 2008, IFMBE Proceedings 22, Springer-Verlag, Berlin, pp 2277–2280Google Scholar
  34. Weber C, Pohl S, Poertner R, Pino-Grace P, Freimark D, Wallrapp C, Geigle P, Czermak P (2010a) Production process for stem cell based therapeutic implants: expansion of the production cell line and cultivation of encapsulated cells. Adv Biochem Eng Biotechnol 123:143–162Google Scholar
  35. Weber C, Pohl S, Freimark D, Poertner R, Pino-Grace P, Wallrapp C, Geigle P, Czermak P (2010b) Expansion of human mesenchymal stem cells in a fixed-bed bioreactor system based on non-porous glass carrier—part A: inoculation, cultivation, and cell harvest procedures. Int J Bioartificial Organs 33:512–525Google Scholar
  36. Weber C, Pohl S, Poertner R, Pino-Grace P, Wallrapp C, Geigle P, Czermak P (2010c) Expansion of human mesenchymal stem cells in a fixed-bed bioreactor system based on non-porous glass carrier—part B: modeling and scale up. Int J Bioartificial Organs 33:782–795Google Scholar
  37. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398CrossRefGoogle Scholar
  38. Zipper H, Brunner H, Bernhagen J, Vitzthum F (2004) Investigations on DNA intercalation and surface binding by SYBR green I, its structure determination and methodological implications. Nucleic Acids Res 32:103CrossRefGoogle Scholar
  39. Zweigerdt R (2009) Large scale production of stem cells and their derivatives. Adv Biochem Eng Biotechnol 114:201–235Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • C. Justice
    • 1
  • J. Leber
    • 1
  • D. Freimark
    • 1
  • P. Pino Grace
    • 1
  • M. Kraume
    • 2
  • P. Czermak
    • 1
    • 3
    Email author
  1. 1.Institute of Bioprocess Engineering and Pharmaceutical TechnologyUniversity of Applied Sciences MittelhessenGiessenGermany
  2. 2.Department of Chemical EngineeringUniversity of Technology BerlinBerlinGermany
  3. 3.Department of Chemical EngineeringKansas State UniversityManhattanUSA

Personalised recommendations