High-Throughput Screening Techniques

  • Rico Brendtke
  • Bart De WeverEmail author
  • Florian Groeber
  • Jan Hansmann
  • Freia Schmid
  • Heike Walles


The use of laboratory animals has been the ‘standard’ procedure for the dermal safety and efficacy evaluation of consumer, chemical and/or pharmaceutical products for many decades. However, both scientific and ethical considerations have driven the development of alternative methods aiming to reduce, replace and refine animal experimentation (Russell WMS, Burch RL, Hume CW. The principles of humane experimental technique. 1959). Especially, human tissue-engineered skin models demonstrate to be very valuable alternatives for dermal toxicity and efficacy testing. Since human skin models are available in quality ensured commercial companies, they are routinely used by industry for in-house screening and more recently also for regulatory toxicity testing applications. Whereas today only skin corrosion and skin irritation test methods are listed in regulatory guidelines, many new non-animal tissue-based dermal toxicity testing applications are currently in development and validation, including skin sensitization, genotoxicity and phototoxicity. In addition, the adoption of skin model-based tests in regulatory guidelines has stimulated regulatory bodies in other parts of the world such as Brazil, China, India and South Korea to harmonize with the global trend to accept validated alternative methods, indicating that the demand for skin models in the future will be largely increased. To ensure the availability of high-quality skin models in very large quantities, automated production is the ultimate solution. Although the field of high-throughput testing has expanded massively over the last years in pharmacological research, it is rarely used in the assessment of adverse health effects. The reason for that might be the higher complexity of three-dimensional (3D) reconstructed tissues employed in toxicology compared to two-dimensional (2D) systems routinely used for drug discovery applications. Furthermore, there is a lack for standardized test methods that can be implemented in a high-throughput approach. This chapter will focus on new production technologies to generate skin models in sufficient numbers and emerging nondestructive methods to assess tissues.


  1. 1.
    Russell WMS, Burch RL, Hume CW. The principles of humane experimental technique. 1959 [cited 2012 Sep 28].
  2. 2.
    Mayr LM, Bojanic D. Novel trends in high-throughput screening. Curr Opin Pharmacol. 2009;9(5):580–8.CrossRefPubMedGoogle Scholar
  3. 3.
    Smith A. Screening for drug discovery: the leading question. Nature. 2002;418:453–9.PubMedGoogle Scholar
  4. 4.
    Sundberg SA. High-throughput and ultra-high-throughput screening: solution- and cell-based approaches. Curr Opin Biotechnol. 2000;11(1):47–53.CrossRefPubMedGoogle Scholar
  5. 5.
    Martis E. High-throughput screening: the hits and leads of drug discovery- an overview. J Appl Pharm Sci. 2011;01(01):02–10.Google Scholar
  6. 6.
    Mayr LM, Fuerst P. The future of high-throughput screening. Off J Soc Biomol Screen. 2008;13(6):443–8.CrossRefGoogle Scholar
  7. 7.
    Rimann M, Angres B, Patocchi-Tenzer I, Braum S, Graf-Hausner U. Automation of 3D cell culture using chemically defined hydrogels. J Lab Autom. 2013;19(2):1–7.Google Scholar
  8. 8.
    Schmidt CW. TOX 21: new dimensions of toxicity testing. Environ Health Perspect. 2009;117(8):A348–53.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Shukla SJ, Huang R, Austin CP, Xia M. The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform. Drug Discov Today. 2010;15(23–24):997–1007.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Borg DJ, Dawson RA, Leavesley DI, Hutmacher DW, Upton Z, Malda J. Functional and phenotypic characterization of human keratinocytes expanded in microcarrier culture. J Biomed Mater Res A. 2009;88:184–94.CrossRefPubMedGoogle Scholar
  11. 11.
    Kalyanaraman B, Boyce S. Assessment of an automated bioreactor to propagate and harvest keratinocytes for fabrication of engineered skin substitutes. Tissue Eng. 2007;13(5):983–93.CrossRefPubMedGoogle Scholar
  12. 12.
    Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev. 2008;14(1):61–86.CrossRefPubMedGoogle Scholar
  13. 13.
    Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science. 2001;294(1998):1708–12.CrossRefPubMedGoogle Scholar
  14. 14.
    Gieseck RL, Hannan NRF, Bort R, Hanley NA, Drake RA, Cameron GW, et al. Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One. 2014;9(1):e86372.CrossRefPubMedGoogle Scholar
  15. 15.
    Kempner M. A review of cell culture automation. J Assoc Lab Autom. 2002;7(2):56–62.CrossRefGoogle Scholar
  16. 16.
    Graeve T, Noll M. Three-dimensional skin model. Google Patents. 2002.
  17. 17.
    Lemper M, De Paepe K, Rogiers V. Practical problems encountered during the cultivation of an open-source reconstructed human epidermis model on a polycarbonate membrane and protein quantification. Skin Pharmacol Physiol. 2013;27(2):106–12.CrossRefPubMedGoogle Scholar
  18. 18.
    Poumay Y, Dupont F, Marcoux S, Leclercq-Smekens M, Hérin M, Coquette A. A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies. Arch Dermatol Res. 2004;296(5):203–11.CrossRefPubMedGoogle Scholar
  19. 19.
    Williams JA. Keys to bioreactor selections. Chem Eng Prog. 2002;98(3):34–41.Google Scholar
  20. 20.
    Hambor JE. Bioreactor design and bioprocess controls for industrialized cell processing. BioProcess Int. 2012;10(6):22–33.Google Scholar
  21. 21.
    Placzek MR, Chung I-M, Macedo HM, Ismail S, Blanco TM, Lim M, et al. Stem cell bioprocessing: fundamentals and principles. J R Soc Interface. 2009;6(32):209–32.CrossRefPubMedGoogle Scholar
  22. 22.
    Baudoin R, Griscom L, Prot JM, Legallais C, Leclerc E. Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor. Biochem Eng J. 2011;53(2):172–81.CrossRefGoogle Scholar
  23. 23.
    Krishnan V, Vogler EA, Sosnoski DM, Mastro AM. In vitro mimics of bone remodeling and the vicious cycle of cancer in bone. J Cell Physiol. 2014;229(4):453–62. doi: 10.1002/jcp.24464.CrossRefPubMedGoogle Scholar
  24. 24.
    Chaudhuri J. Special issue: design of bioreactor systems for tissue engineering. PRO. 2015;3(1):46–9.Google Scholar
  25. 25.
    Groeber F, Kahlig A, Loff S, Walles H, Hansmann J. A bioreactor system for interfacial culture and physiological perfusion of vascularized tissue equivalents. Biotechnol J. 2013;8(3):308–16. doi: 10.1002/biot.201200160.CrossRefPubMedGoogle Scholar
  26. 26.
    Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. Trends Biotechnol. 2004;22(2):80–6.CrossRefGoogle Scholar
  27. 27.
    Chen H-C, Hu Y-C. Bioreactors for tissue engineering. Biotechnol Lett. 2006;28(18):1415–23. doi: 10.1007/s10529-006-9111-x.CrossRefPubMedGoogle Scholar
  28. 28.
    Korossis SA, Bolland F, Kearney JN, Fisher J, Ingham E. Bioreactors in tissue engineering. Top Tissue Eng. 2005;2(8):1–23.Google Scholar
  29. 29.
    Plunkett N, O’Brien FJ. Bioreactors in tissue engineering. Technol Health Care. 2011;19(1):55–69. doi: 10.3233/THC-2011-0605.CrossRefPubMedGoogle Scholar
  30. 30.
    Hansmann J, Groeber F, Kahlig A, Kleinhans C, Walles H. Bioreactors in tissue engineering—principles, applications and commercial constraints. Biotechnol J. 2013;8(3):298–307. doi: 10.1002/biot.201200162.CrossRefPubMedGoogle Scholar
  31. 31.
    Tocchio A, Tamplenizza M, Martello F, Gerges I, Rossi E, Argentiere S, et al. Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor. Biomaterials. 2015;45:124–31.CrossRefPubMedGoogle Scholar
  32. 32.
    Ebrahimkhani MR, Young CL, Lauffenburger DA, Griffith LG, Borenstein JT. Approaches to in vitro tissue regeneration with application for human disease modeling and drug development. Drug Discov Today. 2014;19(6):754–62.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Bowcock AM, Krueger JG. Getting under the skin: the immunogenetics of psoriasis. Nat Rev Immunol. 2005;5(9):699–711. doi: 10.1038/nri1689.CrossRefPubMedGoogle Scholar
  34. 34.
    Mahabeleshwar GH, Byzova TV. Angiogenesis in melanoma. Semin Oncol. 2007;34(6):555–65.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Baker SC, Shabir S, Southgate J. Biomimetic urothelial tissue models for the in vitro evaluation of barrier physiology and bladder drug efficacy. Mol Pharm. 2014;11(7):1964–70. doi: 10.1021/mp500065m.CrossRefPubMedGoogle Scholar
  36. 36.
    Grabinger T, Luks L, Kostadinova F, Zimberlin C, Medema JP, Leist M, et al. Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy. Cell Death Dis. 2014;5:e1228. doi: 10.1038/cddis.2014.183.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Osorio-Lozada A, Surapaneni S, Skiles GL, Subramanian R. Biosynthesis of drug metabolites using microbes in hollow fiber cartridge reactors: case study of diclofenac metabolism by Actinoplanes species. Drug Metab Dispos. 2008;36(2):234–40.CrossRefPubMedGoogle Scholar
  38. 38.
    Lister PD. The role of pharmacodynamic research in the assessment and development of new antibacterial drugs. Biochem Pharmacol. 2006;71(7):1057–65.CrossRefPubMedGoogle Scholar
  39. 39.
    Shah P, Vedarethinam I, Kwasny D, Andresen L, Dimaki M, Skov S, et al. Microfluidic bioreactors for culture of non-adherent cells. Sensors Actuators B Chem. 2011;156(2):1002–8.CrossRefGoogle Scholar
  40. 40.
    Leclerc E, Sakai Y, Fujii T. Microfluidic PDMS (Polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes. Biotechnol Prog. 2004;20(3):750–5. doi: 10.1021/bp0300568.CrossRefPubMedGoogle Scholar
  41. 41.
    Altmann B, Löchner A, Swain M, Kohal R-J, Giselbrecht S, Gottwald E, et al. Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions. Biomaterials. 2014;35(10):3208–19.CrossRefPubMedGoogle Scholar
  42. 42.
    Marx U, Walles H, Hoffmann S, Lindner G, Horland R, Sonntag F, et al. “human-on-a-chip” developments: a translational cutting-edge alternative to systemic safety assessment and efficiency evaluation of substances in laboratory animals and man? Altern Lab Anim. 2012;40(5):235–57.PubMedGoogle Scholar
  43. 43.
    Esch MB, Smith AST, Prot J-M, Oleaga C, Hickman JJ, Shuler ML. How multi-organ microdevices can help foster drug development. Adv Drug Deliv Rev. 2014;69–70:158–69.CrossRefPubMedGoogle Scholar
  44. 44.
    Bhatia SN, Ingber DE. Microfluidic organs-on-chips. Nat Biotechnol. 2014;32(8):760–72. doi: 10.1038/nbt.2989.CrossRefPubMedGoogle Scholar
  45. 45.
    Bhise NS, Ribas J, Manoharan V, Zhang YS, Polini A, Massa S, et al. Organ-on-a-chip platforms for studying drug delivery systems. J Control Release. 2014;190:82–93.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Mansbridge J. Commercial considerations in tissue engineering. J Anat. 2006;209(4):527–32. doi: 10.1111/j.1469-7580.2006.00631.x.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Amanullah A, Otero JM, Mikola M, Hsu A, Zhang J, Aunins J, et al. Novel micro-bioreactor high throughput technology for cell culture process development: reproducibility and scalability assessment of fed-batch CHO cultures. Biotechnol Bioeng. 2010;106(1):57–67. doi: 10.1002/bit.22664.CrossRefPubMedGoogle Scholar
  48. 48.
    Spencer TJ, Hidalgo-Bastida LA, Cartmell SH, Halliday I, Care CM. In silico multi-scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor. Biotechnol Bioeng. 2013;110(4):1221–30. doi: 10.1002/bit.24777.CrossRefPubMedGoogle Scholar
  49. 49.
    Liu N, Zang R, Yang S-T, Li Y. Stem cell engineering in bioreactors for large-scale bioprocessing. Eng Life Sci. 2014;14(1):4–15. doi: 10.1002/elsc.201300013.CrossRefGoogle Scholar
  50. 50.
    Betts JPJ, Warr SRC, Finka GB, Uden M, Town M, Janda JM, et al. Impact of aeration strategies on fed-batch cell culture kinetics in a single-use 24-well miniature bioreactor. Biochem Eng J. 2014;82(0):105–16.CrossRefGoogle Scholar
  51. 51.
    De Napoli IE, Scaglione S, Giannoni P, Quarto R, Catapano G. Mesenchymal stem cell culture in convection-enhanced hollow fibre membrane bioreactors for bone tissue engineering. J Membr Sci. 2011;379(1-2):341–52.CrossRefGoogle Scholar
  52. 52.
    Misener R, Fuentes Garí M, Rende M, Velliou E, Panoskaltsis N, Pistikopoulos EN, et al. Global superstructure optimisation of red blood cell production in a parallelised hollow fibre bioreactor. Comput Chem Eng. 2014;71:532–53.CrossRefGoogle Scholar
  53. 53.
    Leite SB, Teixeira AP, Miranda JP, Tostões RM, Clemente JJ, Sousa MF, et al. Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: improved in vitro models for toxicological applications. Toxicol In Vitro. 2011;25(4):825–32.CrossRefPubMedGoogle Scholar
  54. 54.
    Gunness P, Mueller D, Shevchenko V, Heinzle E, Ingelman-Sundberg M, Noor F. 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. Toxicol Sci. 2013;133(1):67–78.CrossRefPubMedGoogle Scholar
  55. 55.
    National Research Council. Toxicity testing in the 21st century: a vision and a strategy: Washington, DC, National Academies Press; 2007.Google Scholar
  56. 56.
    Xia M, Huang R, Witt KL, Southall N, Fostel J, Cho MH, et al. Compound cytotoxicity profiling using quantitative high-throughput screening. Environ Health Perspect. 2008;116(3):284–91.CrossRefPubMedGoogle Scholar
  57. 57.
    Cotovio J, Grandidier M, Portes P, Roguet R, Rubinstenn G. The in vitro skin irritation of chemicals: optimisation of the EPISKIN prediction model within the framework of the ECVAM validation process. Altern Lab Anim. 2005;33(4):329–49.PubMedGoogle Scholar
  58. 58.
    Bouhifd M, Bories G, Casado J, Coecke S, Norlén H, Parissis N, et al. Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests. Food Chem Toxicol. 2012;50(6):2084–96.CrossRefPubMedGoogle Scholar
  59. 59.
    Mahmud FA, Hastings GW, Martini M. Model to characterize strain generated potentials in bone. J Biomed Eng. 1988;10(1):54–6.CrossRefPubMedGoogle Scholar
  60. 60.
    Schenke-Layland K, Riemann I, Damour O, Stock UA, König K. Two-photon microscopes and in vivo multiphoton tomographs - powerful diagnostic tools for tissue engineering and drug delivery. Adv Drug Deliv Rev. 2006;58(7):878–96.CrossRefPubMedGoogle Scholar
  61. 61.
    Centonze V, White J. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J. 1998;75(4):2015–24.CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Skala MC, Riching KM, Bird DK, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, et al. In vivo multiphoton fluorescence lifetime imaging of protein-bound and free nicotinamide adenine dinucleotide in normal and precancerous epithelia. J Biomed Opt. 2007;12(2):024014.CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Schenke-Layland K, Madershahian N, Riemann I, Starcher B, Halbhuber KJ, König K, et al. Impact of cryopreservation on extracellular matrix structures of heart valve leaflets. Ann Thorac Surg. 2006;81:918–26.CrossRefPubMedGoogle Scholar
  64. 64.
    Brauchle E, Schenke-Layland K. Raman spectroscopy in biomedicine - non-invasive in vitro analysis of cells and extracellular matrix components in tissues. Biotechnol J. 2012;8(3):1–10.Google Scholar
  65. 65.
    Pudlas M, Koch S, Bolwien C, Thude S, Jenne N, Hirth T, et al. Raman spectroscopy: a noninvasive analysis tool for the discrimination of human skin cells. Tissue Eng Part C. 2011;17(10):1027–40.CrossRefGoogle Scholar
  66. 66.
    Votteler M, Carvajal Berrio DA, Pudlas M, Walles H, Stock UA, Schenke-Layland K. Raman spectroscopy for the non-contact and non-destructive monitoring of collagen damage within tissues. J Biophotonics. 2011;5(1):47–56.CrossRefPubMedGoogle Scholar
  67. 67.
    Tfayli A, Piot O, Draux F, Pitre F, Manfait M. Molecular characterization of reconstructed skin model by Raman microspectroscopy: comparison with excised human skin. Biopolymers. 2007;87(4):261–74.CrossRefPubMedGoogle Scholar
  68. 68.
    Thakoersing VS, van Smeden J, Mulder AA, Vreeken RJ, El Ghalbzouri A, Bouwstra JA. Increased presence of monounsaturated fatty acids in the stratum corneum of human skin equivalents. J Invest Dermatol. 2013;133(1):59–67.CrossRefPubMedGoogle Scholar
  69. 69.
    Pierce MC, Strasswimmer J, Park BH, Cense B, de Boer JF. Advances in optical coherence tomography imaging for dermatology. J Invest Dermatol. 2004;123(3):458–63.CrossRefPubMedGoogle Scholar
  70. 70.
    Yamamoto T, Yamamoto Y. Analysis for the change of skin impedance. Med Biol Eng Comput. 1977;15(3):219–27.CrossRefPubMedGoogle Scholar
  71. 71.
    Nyrén M, Kuzmina N, Emtestam L. Electrical impedance as a potential tool to distinguish between allergic and irritant contact dermatitis. J Am Acad Dermatol. 2003;48(3):394–400.CrossRefPubMedGoogle Scholar
  72. 72.
    Worth A. ECVAM protocol for rat skin transcutaneous electrical resistance: an in vitro assay for assessing dermal corrosivity. In Vitro. 2002;12(4):93–110.Google Scholar
  73. 73.
    Knott A, Koop U, Mielke H, Reuschlein K, Peters N, Muhr G, et al. A novel treatment option for photoaged skin. J Cosmet Dermatol. 2008;7(1):15–22.CrossRefPubMedGoogle Scholar
  74. 74.
    Kandárová H. Evaluation and validation of reconstructed human skin models as alternatives to animal tests in regulatory toxicology. Berlin: Freie Universität Berlin; 2006. Google Scholar
  75. 75.
    Millipore Corporation. ERS-2 User Guide. 2009.Google Scholar
  76. 76.
    Groeber F, Engelhardt L, Egger S, Werthmann H, Monaghan M, Walles H, et al. Impedance spectroscopy for the non-destructive evaluation of in vitro epidermal models. Pharm Res. 2014;32(5):1–10.Google Scholar
  77. 77.
    Yaskawa. Motoman dual arm robot in biomedical cell. 2013.
  78. 78.
    King RD, Rowland J, Oliver SG, Young M, Aubrey W, Byrne E, et al. The automation of science. Science. 2009;324(5923):85–9.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • Rico Brendtke
    • 1
    • 2
  • Bart De Wever
    • 3
    Email author
  • Florian Groeber
    • 4
  • Jan Hansmann
    • 1
    • 4
  • Freia Schmid
    • 4
  • Heike Walles
    • 1
    • 4
  1. 1.Department Tissue Engineering and Regenerative Medicine (TERM)University Hospital WuerzburgWuerzburgGermany
  2. 2.Senetics Healthcare Group GmbH & Co. KGErlangenGermany
  3. 3.ATERA SASNiceFrance
  4. 4.Translational Center Wuerzburg ‘Regenerative Therapies in Oncology and Musculoskeletal Diseases’Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB)WuerzburgGermany

Personalised recommendations