Journal of Biosciences

, Volume 34, Issue 1, pp 59–69 | Cite as

Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

  • Axel Blau
  • Tanja Neumann
  • Christiane Ziegler
  • Fabio Benfenati


An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/day. Depending on the lid membrane thickness, pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm2 through PDMS lids as compared with 18.69 g/day/dm2 with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.


Hyperosmolarity long-term cell culture PDMS lids replica moulding pH stabilization 

Abbreviations used


days in vitro


fluorinated ethylene propylene


Hanks balanced salt solution


indium tin oxide


microelectrode array




polymethyl methacrylate






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  1. Arimochi H and Morita K 2005 High salt culture conditions suppress proliferation of rat C6 glioma cell by arresting cell-cycle progression at S-phase; J. Mol. Neurosci. 27 293–301PubMedCrossRefGoogle Scholar
  2. Bavister B D 1988 A minichamber device for maintaining a constant carbon dioxide in air atmosphere during prolonged culture of cells on the stage of an inverted microscope; In Vitro Cell. Dev. Biol. 24 759–763PubMedCrossRefGoogle Scholar
  3. Blau A, Weinl C, Mack J, Kienle S, Jung G and Ziegler C 2001 Promotion of neural cell adhesion by electrochemically generated and functionalized polymer films; J. Neurosci. Methods 112 65–73PubMedCrossRefGoogle Scholar
  4. Blau A W and Ziegler C M 2001 Prototype of a novel autonomous perfusion chamber for long-term culturing and in situ investigation of various cell types; J. Biochem. Biophys. Methods 50 15–27PubMedCrossRefGoogle Scholar
  5. Bruner S 2003 Avoiding cure inhibition and bubbles with Lightspan optical materials (application notes) ( Google Scholar
  6. Chen J, Wang W, Fang J and Varahramyan K 2004 Variable-focusing microlens with microfluidic chip; J. Micromech. Microeng. 14 675–680CrossRefGoogle Scholar
  7. Chen Z, Hothi S S, Xu W and Huang C L 2007 Conduction velocities in amphibian skeletal muscle fibres exposed to hyperosmotic extracellular solutions; J. Muscle Res. Cell Motil. 28 195–202PubMedCrossRefGoogle Scholar
  8. De Bartolo L, Salerno S, Morelli S, Giorno L, Rende M, Memoli B, Procino A, Andreucci V E, Bader A and Drioli E 2006 Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor; Biomaterials 27 4794–4803PubMedCrossRefGoogle Scholar
  9. Dong H W and Buonomano D V 2005 A technique for repeated recordings in cortical organotypic slices; J. Neurosci. Methods 146 69–75PubMedCrossRefGoogle Scholar
  10. Dow Corning Corporation 2005 SYLGARD® 184 silicone elastomer kit (product information) ( Google Scholar
  11. Forsythe I D and Coates R T 1988 A chamber for electrophysiological recording from cultured neurones allowing perfusion and temperature control; J. Neurosci. Methods 25 19–27PubMedCrossRefGoogle Scholar
  12. Goldman S A and Nedergaard M 1992 Newly generated neurons of the adult songbird brain become functionally active in long-term culture; Dev. Brain Res. 68 217–223CrossRefGoogle Scholar
  13. Gross G W and Schwalm F U 1994 A closed chamber for long-term electrophysiological and microscopical monitoring of monolayer neuronal networks; J. Neurosci. Methods 52 73–85PubMedCrossRefGoogle Scholar
  14. Hing W A, Poole C A, Jensen C G and Watson M 2000 An integrated environmental perfusion chamber and heating system for long-term, high resolution imaging of living cells; J. Microsc. 199(Pt 2) 90–95PubMedCrossRefGoogle Scholar
  15. Ho C L, Mou T Y, Chiang P S, Weng C L and Chow N H 2005 Mini chamber system for long-term maintenance and observation of cultured cells; Biotechniques 38 267–273PubMedCrossRefGoogle Scholar
  16. Hu S, Ren X, Bachman M, Sims C E, Li G P and Allbritton N 2002 Surface modification of poly(dimethylsiloxane) microfluidic devices by ultraviolet polymer grafting; Anal. Chem. 74 4117–4123PubMedCrossRefGoogle Scholar
  17. Ince C, van Dissel J T and Diesselhoff M M 1985 A teflon culture dish for high-magnification microscopy and measurements in single cells; Pflugers Arch. 403 240–244PubMedCrossRefGoogle Scholar
  18. Kallos M S, Behie L A and Vescovi A L 1999 Extended serial passaging of mammalian neural stem cells in suspension bioreactors; Biotechnol. Bioeng. 65 589–599PubMedCrossRefGoogle Scholar
  19. Kim S U, Osborne D N, Kim M W, Spigelman I, Puil E, Shin D H and Eisen A 1988 Long-term culture of human fetal spinal cord neurons: morphological, immunocytochemical and electrophysiological characteristics; Neuroscience 25 659–670PubMedCrossRefGoogle Scholar
  20. Klebe R J, Lyn S, Magnuson V L and Zardeneta G 1990 Cultivation of mammalian cells in heat-sealable pouches that are permeable to carbon dioxide; Exp. Cell Res. 188 316–319PubMedCrossRefGoogle Scholar
  21. Krause G, Lehmann S, Lehmann M, Freund I, Schreiber E and Baumann W 2006 Measurement of electrical activity of long-term mammalian neuronal networks on semiconductor neurosensor chips and comparison with conventional microelectrode arrays; Biosens. Bioelectron. 21 1272–1282PubMedCrossRefGoogle Scholar
  22. Leclerc E, Sakai Y and Fujii T 2004 Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes; Biotechnol. Prog. 20 750–755PubMedCrossRefGoogle Scholar
  23. Lesuisse C and Martin L J 2001 Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death; J. Neurobiol. 51 9–23CrossRefGoogle Scholar
  24. Luh E H, Shackford S R, Shatos M A and Pietropaoli J A 1996 The effects of hyperosmolarity on the viability and function of endothelial cells; J. Surg. Res. 60 122–128PubMedCrossRefGoogle Scholar
  25. Maher M P and McKinney S 1995 Careful control of osmolarity and pH enhances survival in hippocampal cultures (San Diego: Society for Neuroscience) 11–16 NovemberGoogle Scholar
  26. Martinoia S, Bonzano L, Chiappalone M, Tedesco M, Marcoli M and Maura G 2005 In vitro cortical neuronal networks as a new high-sensitive system for biosensing applications; Biosens. Bioelectron. 20 2071–2078PubMedCrossRefGoogle Scholar
  27. Morin F O, Takamura Y and Tamiya E 2005 Investigating neuronal activity with planar microelectrode arrays: achievements and new perspectives; J. Biosci. Bioeng. 100 131–143PubMedCrossRefGoogle Scholar
  28. Morita K 2007 High salt culture conditions inhibit serum- and NGF- but not PMA-induced Egr-1 gene transcription in rat C6 glioma cells; J. Mol. Neurosci. 33 216–223PubMedCrossRefGoogle Scholar
  29. Morton M 1987 Rubber technology (New York: Springer)Google Scholar
  30. Olsen M, Sarup A, Larsson O M and Schousboe A 2005 Effect of hyperosmotic conditions on the expression of the betaine-GABA-transporter (BGT-1) in cultured mouse astrocytes; Neurochem. Res. 30 855–865PubMedCrossRefGoogle Scholar
  31. Pentz S and Horler H 1992 A variable cell culture chamber for ‘open’ and ‘closed’ cultivation, perfusion and high microscopic resolution of living cells; J. Microsc. 167 97–103PubMedGoogle Scholar
  32. Petronis S, Stangegaard M, Christensen C B and Dufva M 2006 Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion; Biotechniques 40 368–376PubMedCrossRefGoogle Scholar
  33. Potter S M and DeMarse T B 2001 A new approach to neural cell culture for long-term studies; J. Neurosci. Methods 110 17–24PubMedCrossRefGoogle Scholar
  34. Prokop A, Prokop Z, Schaffer D, Kozlov E, Wikswo J, Cliffel D and Baudenbacher F 2004 NanoLiterBioReactor: long-term mammalian cell culture at nanofabricated scale; Biomed. Microdev. 6 325–339CrossRefGoogle Scholar
  35. Schmied B M, Ulrich A, Matsuzaki H, Ding X, Ricordi C, Moyer M P, Batra S K, Adrian T E and Pour P M 2000 Maintenance of human islets in long-term culture; Differentiation 66 173–180PubMedCrossRefGoogle Scholar
  36. Vicario-Abejón C 2004 Long-term culture of hippocampal neurons; in Current protocols in neuroscience (eds) J N Crawley, C R Gerfen, M A Rogawski, D R Sibley, P Skolnick and S Wray (New York: John Wiley) pp Unit 3.2Google Scholar
  37. Wacker 2006 Elastosil RT601 (product information) ( Google Scholar
  38. Walker G M, Ozers M S and Beebe D J 2002 Insect cell culture in microfluidic channels; Biomed. Microdev. 4 161–166CrossRefGoogle Scholar
  39. Wuertz K, Urban J P, Klasen J, Ignatius A, Wilke H J, Claes L and Neidlinger-Wilke C 2007 Influence of extracellular osmolarity and mechanical stimulation on gene expression of intervertebral disc cells; J. Orthop. Res. 25 1513–1522PubMedCrossRefGoogle Scholar
  40. Xiang Z, Hrabetova S, Moskowitz S I, Casaccia-Bonnefil P, Young S R, Nimmrich V C, Tiedge H, Einheber S, Karnup S, Bianchi R and Bergold P J 2000 Long-term maintenance of mature hippocampal slices in vitro; J. Neurosci. Methods 98 145–154PubMedCrossRefGoogle Scholar
  41. Yeung C K, Lauer L, Offenhausser A and Knoll W 2001 Modulation of the growth and guidance of rat brain stem neurons using patterned extracellular matrix proteins; Neurosci. Lett. 301 147–150PubMedCrossRefGoogle Scholar
  42. Zhang H, Zhao Y, Zhao C, Yu S, Duan D and Xu Q 2005 Long-term expansion of human neural progenitor cells by epigenetic stimulation in vitro; Neurosci. Res. 51 157–165PubMedCrossRefGoogle Scholar


  1. Arkles B 1997 Silicon esters; in Kirk-Othmer encyclopedia of chemical technology (New York: John Wiley) pp 69–81Google Scholar
  2. Chang W-J, Akin D, Sedlak M, Ladisch M R and Bashir R 2003 Polydimethylsiloxane (PDMS) and silicon hybrid biochip for bacterial culture; Biomed. Microdev. 5 281–290CrossRefGoogle Scholar
  3. Dow Corning Corporation 2005 SYLGARD® 184 silicone elastomer kit (product information) ( Google Scholar
  4. efunda 2007 Engineering fundamentals: polymers ( Google Scholar
  5. Ertel S I, Ratner B D, Kaul A, Schway M B and Horbett T A 1994 In vitro study of the intrinsic toxicity of synthetic surfaces to cells; J. Biomed. Mat. Res. 28 667–675CrossRefGoogle Scholar
  6. Favre E, Schaetzel P, Nguygen Q T, Clement R and Neel J 1994 Sorption, diffusion and vapor permeation of various penetrants through dense poly(dimethylsiloxane) membranes: a transport analysis; J. Membr. Sci. 92 169–184CrossRefGoogle Scholar
  7. Gelest Inc. 2006 Reference Scholar
  8. Goodfellow 2006 Material propertieswww.goodfellow.comGoogle Scholar
  9. Grzybowski B A, Brittain S T and Whitesides G M 1999 Thermally actuated interferometric sensors based on the thermal expansion of transparent elastomeric media; Rev. Sci. Instr. 70 2031–2037CrossRefGoogle Scholar
  10. Houston K S, Weinkauf D H and Stewart F F 2002 Gas transport characteristics of plasma treated poly(dimethyl siloxane) and polyphosphazene membrane materials; J. Membr. Sci. 205 103–112CrossRefGoogle Scholar
  11. Jo B H, Van Lerberghe L M, Motsegood K M and Beebe D J 2000 Three-dimensional micro-channel fabrication in polydimethylsiloxane (PDMS) elastomer; J. Micromech. Syst. 9 76–81CrossRefGoogle Scholar
  12. Kuo A C M 1999 Polymer data handbook (Oxford: Oxford University Press)Google Scholar
  13. Lee J N, Park C and Whitesides G M 2003 Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices; Anal. Chem. 75 6544–6554PubMedCrossRefGoogle Scholar
  14. Lynch D S and Lynch W 1978 Handbook of silicone rubber fabrication (New York: Van Nostrand Reinhold Company)Google Scholar
  15. Merkel T C, Bondar V I, Nagai K, Freeman B D and Pinnau I 2000 Gas sorption, diffusion, and permeation in poly(dimethylsiloxane); J. Polym. Sci., Part B: Polym. Phys. 38 415–434CrossRefGoogle Scholar
  16. Noll W 1968 Chemistry and technology of silicones (New York: Academic Press)Google Scholar
  17. NuSil Technology 2004 Silicone terms and definitions ( Google Scholar
  18. Parker S, Meththananda I, Braden M and Pearson G 2006 Characterisation of some experimental silicones; J. Mater. Sci. — Mater. Med. 17 1255–1258PubMedCrossRefGoogle Scholar
  19. Piruska A, Nikcevic I, Lee S H, Ahn C, Heineman W R, Limbach P A and Seliskar C J 2005 The autofluorescence of plastic materials and chips measured under laser irradiation; Lab. Chip 5 1348–1354PubMedCrossRefGoogle Scholar
  20. Randall G C and Doyle P S 2005 Permeation-driven flow in poly(dimethylsiloxane) microfluidic devices; Proc. Natl. Acad. Sci. USA 102 10813–10818PubMedCrossRefGoogle Scholar
  21. Reilly B and Bruner S 2004 Silicones as a material of choice for drug delivery applications (Honolulu, Hawaii: 31st Annual Meeting and Exposition of the Controlled Release Society) 12–16 JuneGoogle Scholar
  22. Riegler B, Bruner S and Thomaier R 2004 Optical silicones for use in harsh operating environments (Philadelphia: Optics East) 25–28 OctoberGoogle Scholar
  23. Robb W L 1968 Thin silicone membranes — their permeation properties and some applications; Ann. N.Y. Acad. Sci. 146 119–137PubMedCrossRefGoogle Scholar
  24. Schirrer R, Thepin P and Torres G 1992 Water absorption, swelling, rupture and salt release in salt-silicone rubber compounds; J. Mater. Sci. 27 3424–3434CrossRefGoogle Scholar
  25. Stern S A 1968 The ‘Barrer’ permeability unit; J. Polym. Sci. 6 1933–1934Google Scholar
  26. Tanimura M 1993 Handbook of silicone materials (Tokyo: Toray Dow Corning Silicone)Google Scholar
  27. Wacker 2006 Elastosil RT601 (product information) ( Google Scholar
  28. Watson J M and Baron M G 1996 The behaviour of water in poly(dimethylsiloxane); J. Membr. Sci. 110 47–57CrossRefGoogle Scholar
  29. Younan Xia G M W 1998 Soft lithography; Angew. Chem. Int. Ed. 37 550–575CrossRefGoogle Scholar


  1. Bruner S 2003 Avoiding cure inhibition and bubbles with Lightspan optical materials (application notes) ( Google Scholar

Copyright information

© Indian Academy of Sciences 2009

Authors and Affiliations

  • Axel Blau
    • 1
  • Tanja Neumann
    • 2
  • Christiane Ziegler
    • 2
  • Fabio Benfenati
    • 1
  1. 1.Department of Neuroscience and Brain TechnologiesThe Italian Institute of TechnologyGenoaItaly
  2. 2.Department of Physics and BiophysicsUniversity of KaiserslauternKaiserslauternGermany

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