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1.
Biotechnol Bioeng ; 119(7): 1728-1739, 2022 07.
Article in English | MEDLINE | ID: mdl-35355251

ABSTRACT

Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for nonspecific cell detachment. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 37°C, but become repulsive upon a cell-compatible temperature drop to, for example, 32°C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond. On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells and cells in serum-free culture. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for, for example, stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines.


Subject(s)
Microgels , Cell Adhesion , Cell Proliferation , Peptide Hydrolases , Polymers/chemistry , Surface Properties , Temperature
2.
Sci Rep ; 10(1): 13700, 2020 08 13.
Article in English | MEDLINE | ID: mdl-32792676

ABSTRACT

Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity.


Subject(s)
Bioreactors , Biosensing Techniques/methods , Cellular Microenvironment , Hepatocytes/metabolism , Oxygen Consumption/drug effects , Oxygen/metabolism , Acetaminophen/pharmacology , Animals , Biomarkers/analysis , Cytochrome P-450 CYP3A/metabolism , Hep G2 Cells , Hepatocytes/cytology , Hepatocytes/drug effects , Humans , Mice , Mice, Inbred C57BL
3.
ACS Appl Bio Mater ; 2(7): 2853-2861, 2019 Jul 15.
Article in English | MEDLINE | ID: mdl-35030819

ABSTRACT

In vitro cultured neuronal networks with defined connectivity are required to improve neuronal cell culture models. However, most protocols for their formation do not provide sufficient control of the direction and timing of neurite outgrowth with simultaneous access for analytical tools such as immunocytochemistry or patch-clamp recordings. Here, we present a proof-of-concept for the dynamic (i.e., time-gated) control of neurite outgrowth on a cell culture substrate based on 2D-micropatterned coatings of thermoresponsive polymers (TRP). The pattern consists of uncoated microstructures where neurons can readily adhere and neurites can extend along defined pathways. The surrounding regions are coated with TRP that does not facilitate cell or neurite growth at 33 °C. Increasing the ambient temperature to 37 °C renders the TRP coating cell adhesive and enables the crossing of gaps coated with TRP by neurites to contact neighboring cells. Here, we demonstrate the realization of this approach employing human neuronal SH-SY5Y cells and human induced neuronal cells. Our results suggest that this approach may help to establish a spatiotemporal control over the connectivity of multinodal neuronal networks.

4.
Polymers (Basel) ; 10(6)2018 Jun 12.
Article in English | MEDLINE | ID: mdl-30966690

ABSTRACT

For the effective use of live cells in biomedicine as in vitro test systems or in biotechnology, non-invasive cell processing and characterisation are key elements. Thermoresponsive polymer coatings have been demonstrated to be highly beneficial for controlling the interaction of adherent cells through their cultivation support. However, the widespread application of these coatings is hampered by limitations in their adaptability to different cell types and because the full range of applications has not yet been fully explored. In the work presented here, we address these issues by focusing on three different aspects. With regard to the first aspect, by using well-defined laminar flow in a microchannel, a highly controllable and reproducible shear force can be applied to adherent cells. Employing this tool, we demonstrate that cells can be non-invasively detached from a support using a defined shear flow. The second aspect relates to the recent development of simple methods for patterning thermoresponsive coatings. Here, we show how such patterned coatings can be used for improving the handling and reliability of a wound-healing assay. Two pattern geometries are tested using mouse fibroblasts and CHO cells. In terms of the third aspect, the adhesiveness of cells depends on the cell type. Standard thermoresponsive coatings are not functional for all types of cells. By coadsorbing charged nanoparticles and thermoresponsive microgels, it is demonstrated that the adhesion and detachment behaviour of cells on such coatings can be modulated.

5.
Macromol Biosci ; 18(2)2018 02.
Article in English | MEDLINE | ID: mdl-29231289

ABSTRACT

Polyelectrolyte multilayers assembled from hyaluronic acid (HA) and poly-l-lysine (PLL) are most widely studied showing excellent reservoir characteristics to host molecules of diverse nature; however, thick (HA/PLL)n films are often found cell repellent. By a systematic study of the adhesion and proliferation of various cells as a function of bilayer number "n" a correlation with the mechanical and chemical properties of films is developed. The following cell lines have been studied: mouse 3T3 and L929 fibroblasts, human foreskin primary fibroblasts VH-Fib, human embryonic kidney HEK-293, human bone cell line U-2-OS, Chinese hamster ovary CHO-K and mouse embryonic stem cells. All cells adhere and spread well in a narrow "cell-friendly" window identify in the range of n = 12-15. At n < 12, the film is inhomogeneous and at n > 15, the film is cell repellent for all cell lines. Cellular adhesion correlates with the mechanical properties of the films showing that softer films at higher "n" number exhibiting a significant decrease of the Young's modulus below 100 kPa are weakly adherent to cells. This trend cannot be reversed even by coating a strong cell-adhesive protein fibronectin onto the film. This indicates that mechanical cues plays a major role for cell behavior, also in respect to biochemical ones.


Subject(s)
Cell Communication , Hyaluronic Acid/chemistry , Polylysine/chemistry , 3T3 Cells , Animals , CHO Cells , Cell Communication/drug effects , Cell Proliferation/drug effects , Cell Shape/drug effects , Cricetinae , Cricetulus , Elastic Modulus , Epithelial Cells/cytology , Epithelial Cells/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Fibronectins/pharmacology , HEK293 Cells , Humans , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/drug effects
6.
Colloids Surf B Biointerfaces ; 147: 343-350, 2016 Nov 01.
Article in English | MEDLINE | ID: mdl-27552029

ABSTRACT

The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein-film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand.


Subject(s)
Hyaluronic Acid/chemistry , Microfluidics , Muramidase/chemistry , Polylysine/chemistry , Adsorption , Diffusion , Fluorescence , Humans , Hyaluronic Acid/metabolism , Microscopy, Confocal , Muramidase/metabolism , Polylysine/metabolism
7.
Phys Chem Chem Phys ; 18(11): 7866-74, 2016 Mar 21.
Article in English | MEDLINE | ID: mdl-26911320

ABSTRACT

In this study, the effect of temperature on the build-up of exponentially growing polyelectrolyte multilayer films was investigated. It aims at understanding the multilayer growth mechanism as crucially important for the fabrication of tailor-made multilayer films. Model poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers were assembled in the temperature range of 25-85 °C by layer-by-layer deposition using a dipping method. The film growth switches from the exponential to the linear regime at the transition point as a result of limited polymer diffusion into the film. With the increase of the build-up temperature the film growth rate is enhanced in both regimes; the position of the transition point shifts to a higher number of deposition steps confirming the diffusion-mediated growth mechanism. Not only the faster polymer diffusion into the film but also more porous/permeable film structure are responsible for faster film growth at higher preparation temperature. The latter mechanism is assumed from analysis of the film growth rate upon switching of the preparation temperature during the film growth. Interestingly, the as-prepared films are equilibrated and remain intact (no swelling or shrinking) during temperature variation in the range of 25-45 °C. The average activation energy for complexation between PLL and HA in the multilayers calculated from the Arrhenius plot has been found to be about 0.3 kJ mol(-1) for monomers of PLL. Finally, the following processes known to be dependent on temperature are discussed with respect to the multilayer growth: (i) polymer diffusion, (ii) polymer conformational changes, and (iii) inter-polymer interactions.

8.
Biomacromolecules ; 17(3): 1110-6, 2016 Mar 14.
Article in English | MEDLINE | ID: mdl-26879608

ABSTRACT

Cultivation of adherently growing cells in artificial environments is of utmost importance in medicine and biotechnology to accomplish in vitro drug screening or to investigate disease mechanisms. Precise cell manipulation, like localized control over adhesion, is required to expand cells, to establish cell models for novel therapies and to perform noninvasive cell experiments. To this end, we developed a method of gentle, local lift-off of mammalian cells using polymer surfaces, which are reversibly and repeatedly switchable between a cell-attractive and a cell-repellent state. This property was introduced through micropatterned thermoresponsive polymer coatings formed from colloidal microgels. Patterning was obtained through automated nanodispensing or microcontact printing, making use of unspecific electrostatic interactions between microgels and substrates. This process is much more robust against ambient conditions than covalent coupling, thus lending itself to up-scaling. As an example, wound healing assays were accomplished at 37 °C with highly increased precision in microfluidic environments.


Subject(s)
Cell Adhesion , Coated Materials, Biocompatible/chemistry , Hydrogels/chemistry , Animals , Cell Line , Cell Separation/methods , Coated Materials, Biocompatible/pharmacology , Fibroblasts/cytology , Fibroblasts/drug effects , Hydrogels/pharmacology , Mice , Microfluidics/methods , Temperature
9.
Macromol Rapid Commun ; 35(16): 1408-13, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25042776

ABSTRACT

Polymeric scaffolds serve as valuable supports for biological cells since they offer essential features for guiding cellular organization and tissue development. The main challenges for scaffold fabrication are i) to tune an internal structure and ii) to load bio-molecules such as growth factors and control their local concentration and distribution. Here, a new approach for the design of hollow polymeric scaffolds using porous CaCO3 particles (cores) as templates is presented. The cores packed into a microfluidic channel are coated with polymers employing the layer-by-layer (LbL) technique. Subsequent core elimination at mild conditions results in formation of the scaffold composed of interconnected hollow polymer microspheres. The size of the cores determines the feature dimensions and, as a consequence, governs cellular adhesion: for 3T3 fibroblasts an optimal microsphere size is 12 µm. By making use of the carrier properties of the porous CaCO3 cores, the microspheres are loaded with BSA as a model protein. The scaffolds developed here may also be well suited for the localized release of bio-molecules using external triggers such as IR-light.


Subject(s)
Polymers/chemistry , 3T3 Cells , Animals , Calcium Carbonate/chemical synthesis , Calcium Carbonate/chemistry , Cattle , Cell Adhesion/drug effects , Hydrogen-Ion Concentration , Infrared Rays , Mice , Microscopy, Confocal , Microspheres , Osmolar Concentration , Polymers/metabolism , Polymers/pharmacology , Porosity , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism
10.
Macromol Rapid Commun ; 33(20): 1775-9, 2012 Oct 26.
Article in English | MEDLINE | ID: mdl-22806940

ABSTRACT

Microfluidics is used here for the first time to efficiently tune the growth conditions for understanding the build-up mechanism of exponentially growing polyelectrolyte (PE) films. The velocity of PE supply and time of interaction can be successfully altered during the layer-by-layer assembly. Another advantage of this method is that the deposition of poly-L-lysine/hyaluronic acid (PLL/HA) films in microchannels can be monitored online by fluorescence microscopy. The study demonstrates that PE mass transport to the film surface and diffusion in the film are key parameters affecting PLL/HA film build-up. Increase of PE supply rate results in a change in the "transition" (exponential-to-linear growth) towards higher number of deposition steps, thus indicating a mass transport-mediated growth mechanism.


Subject(s)
Microfluidics , Electrolytes , Hyaluronic Acid/chemistry , Microscopy, Fluorescence , Polylysine/chemistry
11.
Langmuir ; 28(18): 7249-57, 2012 May 08.
Article in English | MEDLINE | ID: mdl-22509757

ABSTRACT

Chemical cross-linking is the standard approach to tune the mechanical properties of polymer coatings for cell culture applications. Here we show that the elastic modulus of highly swollen polyelectrolyte films composed of poly(L-lysine) (PLL) and hyaluronic acid (HA) can be changed by more than 1 order of magnitude by addition of gold nanoparticles (AuNPs) in a one-step procedure. This hydrogel-nanoparticle architecture has great potential as a platform for advanced cell engineering application, for example remote release of drugs. As a first step toward utilization of such films for biomedical applications we identify the most favorable polymer/nanoparticle composition for optimized cell adhesion on the films. Using atomic force microscopy (AFM) we determine the following surface parameters that are relevant for cell adhesion, i.e., stiffness, roughness, and protein interactions. Optimized cell adhesion is observed for films with an elastic modulus of about 1 MPa and a surface roughness on the order of 30 nm. The analysis further shows that AuNPs are not incorporated in the HA/PLL bulk but form clusters on the film surface. Combined studies of the elastic modulus and surface topography indicate a cluster percolation threshold at a critical surface coverage above which the film stiffness drastically increases. In this context we also discuss changes in film thickness, material density and swelling ratio due to nanoparticle treatment.


Subject(s)
Fibroblasts/cytology , Gold/chemistry , Membranes, Artificial , Metal Nanoparticles/chemistry , Polylysine/chemistry , Animals , Cell Adhesion , Electrolytes/chemistry , Hyaluronic Acid/chemistry , Mice , Microscopy, Atomic Force , Particle Size , Surface Properties
12.
Lab Chip ; 12(8): 1434-6, 2012 Apr 21.
Article in English | MEDLINE | ID: mdl-22382798

ABSTRACT

We present here the micropatterns of layer-by-layer (LbL) assembled soft films generated using microfluidic platform that can be exploited for selective cell growth. Using this method, the issue of cell adhesion and spreading on soft LbL-derived films, and simultaneous utilisation of such unmodified soft films to exploit their reservoir properties are addressed. This also paves the way for extending the culture of cells to soft films and other demanding applications like triggered release of biomolecules.


Subject(s)
Fibroblasts/cytology , Hyaluronic Acid/chemistry , Microfluidic Analytical Techniques/instrumentation , Polylysine/chemistry , Tissue Scaffolds/chemistry , Animals , Cell Adhesion , Cell Line , Cell Proliferation , Equipment Design , Mice , Microscopy, Atomic Force
13.
Biomicrofluidics ; 6(2): 24129, 2012 Jun.
Article in English | MEDLINE | ID: mdl-23761842

ABSTRACT

Thermoresponsive polymer surface coatings are a promising tool for cell culture applications. They allow for a mild way of cell detachment that preserves the activity of membrane proteins-a prerequisite for reliable cell analysis. To enlarge the application range of these coatings to cells with different adhesion properties, we synthesized various novel poly(ethylene glycol)-based thermoresponsive polymers and describe how (i) their chemical structure and (ii) their surface density affect their efficiency. In order to quantify the influence of both factors, the time for cell spreading and rounding efficiency were observed. As a result, efficiency of cell rounding, which is closely correlated to cell detachment, is less affected by both factors than the time needed for cell spreading. This time can effectively be adjusted by the molecular architecture which includes the length of the polymer backbone and the side chains. Based on this work, recommendations are given for future optimization of functionality of thermoresponsive polymer coatings for cell culture applications.

14.
Langmuir ; 26(5): 3462-7, 2010 Mar 02.
Article in English | MEDLINE | ID: mdl-19891449

ABSTRACT

Thermoresponsive polymer-coated surfaces based on poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol) methacrylate) [P(MEO(2)MA-co-OEGMA)] allow switching between cell attachment and detachment. Here, we investigate the temperature-dependent surface interactions between the polymer coating and a colloidal probe in an aqueous medium by means of atomic force microscopy (AFM) force-distance measurements. The analysis of the adhesion forces from AFM retraction curves identifies two kinds of regimes for the copolymer at temperatures below and above the lower critical solution temperature (LCST). Whereas at 25 degrees C the surface interactions with the polymer in the swollen state are dominated by repulsive forces, at 37 degrees C the surface interactions switch to attractive forces and a stronger adhesion is detected by AFM. Running several heating/cooling cycles repeatedly shows that switching the surface properties provides reproducible adhesion force values. Time-dependent measurements give insight into the switching kinetics, demonstrating that the cell response is coupled to the polymer kinetics but probably limited by the cellular rearrangements.


Subject(s)
Biocompatible Materials/chemistry , Methacrylates/chemistry , Microscopy, Atomic Force , Polyethylene Glycols/chemistry , Temperature , Animals , Biocompatible Materials/metabolism , Cell Adhesion , Cell Line , Colloids , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Gold/chemistry , Kinetics , Methacrylates/metabolism , Mice , Polyethylene Glycols/metabolism , Surface Properties
15.
Langmuir ; 25(10): 5949-56, 2009 May 19.
Article in English | MEDLINE | ID: mdl-19358594

ABSTRACT

A new versatile method for tuning the thickness of surface-tethered polymer brushes is introduced. It is based on the combination of polyelectrolyte multilayer deposition and surface-initiated atom transfer radical polymerization. To control the thickness of the brushes, the nonlinear growth of certain polyelectrolyte multilayer systems is exploited. The method is demonstrated to work with different polyelectrolytes and different monomers. The relevance for applications is demonstrated by cell adhesion experiments on grafted thermoresponsive polymer layers with varying thickness.


Subject(s)
Electrolytes/chemistry , Electrolytes/chemical synthesis , Polymers/chemistry , Polymers/chemical synthesis , Animals , Cell Adhesion , Cell Line , Mice , Surface Properties
17.
Biomaterials ; 29(2): 247-56, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17920675

ABSTRACT

Although they have been discovered decades ago, only in the last years forisome protein aggregates received broader attention due to their ability to convert chemical into mechanical energy. In contrast to most other motor proteins, these proteins from Fabaceae plants are independent of high-energy chemical compounds, like e.g. ATP, but undergo an anisotropic shape transition (longitudinally expanded to contracted) in response to ion concentration changes (Ca(2+), H(+), etc.), instead. We present morphological and functional data on forisomes obtained using atomic force microscopy (AFM). High-aspect ratio AFM tips allow the detailed elucidation of structural characteristics that are inaccessible with standard AFM tips. Microindentation measurements were employed to calculate the elasticity of the forisome material. Young's moduli were found to be approximately 32.7 kPa in the expanded state and approximately 2.748 kPa in the contracted state of the polymer. These results are compared to investigations where a tipless AFM cantilever was utilized to exert a load against the shape transition. In the latter experiments, an energy conversion of approximately 2.29 pJ per stroke was detected. Energetical considerations support the hypothesis that the switching process is accompanied by a change in cross-linking of the constituent subunits and allow estimating the extent of cooperativity during the pH-induced transition. Finally, useful parameters were identified and characterized that are crucial for the application of forisomes as functional elements in microfluidic chips.


Subject(s)
Contractile Proteins/metabolism , Contractile Proteins/ultrastructure , Hydrogen-Ion Concentration , Microscopy, Atomic Force , Vicia faba/metabolism
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