Surfaces for Cell Cultivation

We develop coatings made of thermoresponsive polymers for cell culture applications to allow the user to control cell adhesion effectively and non-invasively. The cells adhere and proliferate at typical cultivation temperatures in the same way as they would on a standard cell culture substrate. Reducing the temperature by a few degrees allows the cells to be detached from these coatings simply by rinsing them. Eliminating the use of invasive proteases ensures that cell vitality and membrane proteins are not adversely affected during this critical process step. The polymers are inexpensive to apply to conventional cell culture substrates homogeneously or in defined patterns, using straightforward methods such as spin coating, spray coating, spotting or printing. Along with its use as a cell culture substrate, the polymer coating is also suitable for cell tests that permit the cell migration to be examined (e.g. wound healing test), or for establishing co-cultures with defined geometric relationships.

Thermoresponsive polymer coatings for controlling cell adhesion

Thermoresponsive polymer coatings
© Fraunhofer IZI-BB

Controlling cell adhesion on thermoresponsive surfaces. The cells are adherent and spread across the surfaces at 37 °C. When the surface temperature is reduced to 25 °C, the cells detach from their substrate and can be removed straightforwardly by rinsing them.

Thermoresponsive polymer coatings
© Fraunhofer IZI-BB

Controlling cell adhesion on structured thermoresponsive surfaces. Thermoresponsive microgels can be applied locally to many different surfaces (in this case, to circular islands). Homogeneous cells develop at 37 °C (1). When the surface temperature is reduced to 25 °C, the cells detach from their substrate selectively (2 & 3). Once the temperature is increased again, the cells repopulate the thermoresponsive areas (4).

The institute is developing thermoresponsive polymer coatings that allow good cell adhesion at typical cultivation temperatures. Reducing the temperature by a few degrees allows the cells to be detached from these coatings simply by rinsing them, without requiring the use of invasive enzyme cocktails. This ensures that the vitality of the cells is not adversely affected during what is often a critical process step. The polymers are inexpensive to apply to almost all conventional cell culture substrates homogeneously or in defined patterns using straightforward methods such as spin coating, spotting or printing. This allows the range of applications for which thermoresponsive coatings are suitable to be extended considerably beyond conducting non-invasive standard protocols. Co-cultures with defined geometric relationships can be produced straightforwardly using this approach. Tests have shown that the polymer coatings allow considerable improvements to the way cell assays, for example for wound healing or cell migration, are handled, while also increasing reliability and precision. Establishing innovative cell assay formats is one of the other activities pursued by the Working Group.

Methods

 

  • Polyelectrolyte layers (layer-by-layer (LbL) application) and reservoirs for biomolecules for controlling adherent cells, layers (self-assembled monolayers (SAM)) made of polymers, and biomolecules for improving the biocompatibility of synthetic surfaces
  • Techniques for producing homogeneous and structured coatings: Spin coating, dip coating, spraying, spotting, printing (µ-contact printing)
  • Extensive range of methods for non-invasive examination and characterization of surfaces and coatings: Contact angle identification, ellipsometry, surface plasmon resonance spectroscopy (SPR), fluorescence microscopy techniques, fluorescence recovery after photobleaching (FRAP), atomic force microscopy (AFM)
  • Time-resolved examination of cell adhesion on functionalized surfaces by means of total internal reflection microscopy (TIRFM)
  • Characterization of mechanical properties of surfaces and coatings by means of microindentation
  • Storage and cultivation of eukaryotic cells at S1 biosafety level (mammalian cells, insect cells, primary cells, cell lines)

 

Equipment

 

  • Transmitted and reflected light microscopy with bright field, phase contrast, fluorescence, polarization and total reflection modes (TIRFM), super-resolution structured illumination microscopy (SIM), each equipped with computer-controlled and temperature-controlled specimen stages and cell culture chambers
  • Confocal scanning laser microscope with 3D image processing
  • Fully automated fluorescence microscope for producing images of living cells under physiological conditions (time-lapse microscopy) (Olympus CellR)
  • TIRF microscopy (Olympus)
  • Laser tweezers with laser microdissection (Palm/Zeiss)
  • Atomic force microscopy for biological applications/bio-AFM (JPK)
  • Variable microfluidics setup
  • Multiscope for imaging ellipsometry, surface plasmon resonance/SPR (Optrel)
  • Vapor deposition system for producing thin metallic layers (Edwards)
  • Microcontact printer (GeSiM)
  • Contact angle measuring device
  • Flow cytometer (Becton D.)
  • Micromanipulation, microinjection, microdissection (Eppendorf)

  • GeSiM mbH, Grosserkmannsdorf
  • Mikrofluidik ChipShop, Jena
  • BST Bio Sensor Technology GmbH
  • University of Jerusalem, Israel
  • École Polytechnique Fédéral de Lausanne, Switzerland
  • Centre Suisse d’Electronique et Microtechnique Neuchâtel, Switzerland
  • University of Bielefeld
  • Nottingham Trent University

Publications

  • Uhlig K, Wegener T, He J, Zeiser M, Bookhold J, Dewald I, Godino N, Jaeger MS, Hellweg T, Fery A, Duschl C. Patterned thermoresponsive microgel coatings for noninvasive processing of adherent cells. Biomacromolecules (2016),  17, S. 1110-1116.
  • Velk N, Uhlig K, Duschl C, Volodkin D. Mobility of Lysozyme in Poly(L-lysine)/Hyaluronic Acid Multilayer Films. Colloids Surfaces B (2016), 47, S. 343-350.
  • Vikulina AS, Anissimov YG, Singh P, Prokopović VZ, Uhlig U, Jaeger MS, von Klitzing R, Duschl C, Volodkin D. Temperature effect on build-up of exponentially growing polyelectrolyte multilayers. Exponential-to-linear transition point. Phys. Chem. Chem. Phys. (2016), 18, S. 7866-7874.
  • Prokopovic VZ, Duschl C, Volodkin D. Hyaluronic acid/poly-L-lysine Multilayers as Reservoirs for Storage and Release of Small Charged Molecules. Macromo. Biosci. (2015), 15, S. 1357-1363.
  • Vikulina AS, Aleed ST, Paulraj T, Vladimirov YA, von Klitzing R, Duschl C, Volodkin D. Temperature-induced molecular transport through polymer multilayers coated with pNIPAM microgels. Phys. Chem. Chem. Phys. (2015), 17, S. 12771-12777.
  • Paulraj T, Feoktistova N, Velk N, Uhlig K, Duschl C, Volodkin D. Microporous polymeric 3D scaffolds templated by the Layer-by-Layer self-assembly. Macromol. Rapid Comm. (2014) 35, S. 1408-1413.
  • Schmidt S, Uhlig K, Duschl C, Volodkin D. Stability and Cell Uptake of Calcium Carbonate Templated Insulin Microparticles. Acta Biomat. (2014), 10, S. 1423-1430.
  • Uhlig K, Boerner HG, Wischerhoff E, Lutz JF, Jaeger MS, Laschewsky A, Duschl C. On the interaction of adherent cells with thermoresponsive polymer coatings. Polymers. (2014), 6, 1164-1177.
  • Madaboosi N, Uhlig K, Jäger MS, Möhwald H, Duschl C, Volodkin D. Microfluidics as A Tool to Understand the Build-Up Mechanism of Exponential-Like Growing Films. Macromol Rapid Comm. (2012), 33(20), 1775-1779.
  • Madaboosi N, Uhlig K, Schmidt S, Jaeger MS, Möhwald H, Duschl C, Volodkin D. Microfluidics meets soft layer-by-layer films: selective cell growth in 3D polymer architectures. Lab Chip. (2012), 12, S. 1434-1436.
  • Schmidt S, Behra M, Uhlig K, Madaboosi N, Hartmann L, Duschl C, Volodkin D. Mesoporous Protein Particles through Colloidal CaCO3 Templates. Adv. Funct. Mat. (2012) 23, S. 116-123.
  • Uhlig K, Boysen B, Lankenau A, Jaeger MS, Wischerhoff E, Lutz JF, Laschewsky A, Duschl C. On the influence of the architecture of poly(ethylene glycol)-based thermoresponsive polymers on cell adhesion. Biomicrofluidics (2012), 6, S. 024129.
  • Uhlig K, Madaboosi N, Schmidt S, Jäger MS, Rose J, Duschl C, Volodkin D. 3D localization and diffusion of proteins in polyelectrolyte multilayers. Soft Matter (2012),  8, S. 11786-11789.
  • Volodkin VS, Schmidt S, Fernandes P, Larionova NI, Sukhorukov GB, Duschl C, Möhwald H, von Klitzing R. One-step formulation of protein microparticles with tailored properties: hard templating at soft conditions. Adv. Funct. Mat. (2012), 22, S. 1914-1922.
  • Schmidt S, Zeiser M, Hellweg T, Duschl C, Fery A, Möhwald H. Adhesion and Mechanical Properties of PNIPAM Microgel Films and their Potential Use as Switchable Cell Culture Substrates. Adv. Funct. Mat. (2010), 20, S. 3235-3244.
  • Uhlig K, Wischerhoff E, Lutz JF, Laschewsky A, Jaeger MS, Lankenau A, Duschl C. Monitoring cell detachment on PEG-based thermoresponsive surfaces using TIRF microscopy. Soft Matter. (2010), 6, 4262-4267.
  • Kessel S, Müller R, Schmidt S, Wischerhoff E, Laschewsky A, Lutz JF, Uhlig K, Lankenau A, Duschl C and Fery A. Thermoresponsive, PEG-based Polymer Layers: Surface Characterization with AFM Force Measurements. Langmuir (2009), 26, S. 3462–3467.
  • Ernst O, Lieske A, Holländer A, Lankenau A, Duschl C. Tailoring of Thermo-Responsive Self-Assembled Monolayers for Cell Type Specific Control of Adhesion. Langmuir (2008), 24, S. 10259.
  • Wischerhoff E, Uhlig K, Lankenau A, Börner HG, Laschewsky A, Duschl C, Lutz JF. Controlled Cell Adhesion on PEG-based Switchable Surfaces. Angew. Chem. (2008), 47, S. 5666-5668.
  • Ernst O, Lieske A, Jaeger M, Lankenau A, Duschl C. Control of cell detachment in a microfluidic device using a thermoresponsive copolymer on a gold substrate. Lab Chip. (2007), 7, 1322–1329.

 

Patents

  • Duschl C, Lankenau A, Lutz J-F, Laschewsky A, Wischerhoff E, Fuhr GR, Bier F. Substrat, Kultivierungseinrichtung und Kultivierungsverfahren für biologische Zellen. DE 10 2010 012 254 A1. 22. Sept. 2011.
  • Duschl C, Hellweg T, Lankenau A, Laschewsky A, Lutz J-F, Schmidt S, Wischerhoff E. Thermoresponsives Substrat mit Mikrogelen, Verfahren zu dessen Herstellung und Kultivierungsverfahren für biologische Zellen. EP 2 550 352 B1. 22. Sept. 2011.

Lowering the cultivation temperature from 37 °C to 28 °C causes a thermoresponsive cell cultivation substrate to switch from a cell-attractive to a cell-repellent state. This results in a non-invasive detachment of the cells from the substrate. 

© Video Fraunhofer IZI-BB

Image showing the contact surface of fibroblast cells on a thermoresponsive cell cultivation substrate during cell detachment.

© Video Fraunhofer IZI-BB