RESUMO
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.
Assuntos
Microgéis , Adesão Celular , Proliferação de Células , Peptídeo Hidrolases , Polímeros/química , Propriedades de Superfície , TemperaturaRESUMO
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.
Assuntos
Adesão Celular , Materiais Revestidos Biocompatíveis/química , Hidrogéis/química , Animais , Linhagem Celular , Separação Celular/métodos , Materiais Revestidos Biocompatíveis/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Hidrogéis/farmacologia , Camundongos , Microfluídica/métodos , TemperaturaRESUMO
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.
RESUMO
Prediction of drug-induced toxicity is complicated by the failure of animal models to extrapolate human response, especially during assessment of repeated dose toxicity for cosmetic or chronic drug treatments. In this work, we present a 3D microreactor capable of maintaining metabolically active HepG2/C3A spheroids for over 28 days in vitro under stable oxygen gradients mimicking the in vivo microenvironment. Mitochondrial respiration was monitored using two-frequency phase modulation of phosphorescent microprobes embedded in the tissue. Phase modulation is focus independent and unaffected by cell death or migration. This sensitive measurement of oxygen dynamics revealed important information on the drug mechanism of action and transient subthreshold effects. Specifically, exposure to antiarrhythmic agent, amiodarone, showed that both respiration and the time to onset of mitochondrial damage were dose dependent showing a TC50 of 425 µm. Analysis showed significant induction of both phospholipidosis and microvesicular steatosis during long-term exposure. Importantly, exposure to widely used analgesic, acetaminophen, caused an immediate, reversible, dose-dependent loss of oxygen uptake followed by a slow, irreversible, dose-independent death, with a TC50 of 12.3 mM. Transient loss of mitochondrial respiration was also detected below the threshold of acetaminophen toxicity. The phenomenon was repeated in HeLa cells that lack CYP2E1 and 3A4, and was blocked by preincubation with ascorbate and TMPD. These results mark the importance of tracing toxicity effects over time, suggesting a NAPQI-independent targeting of mitochondrial complex III might be responsible for acetaminophen toxicity in extrahepatic tissues.
Assuntos
Acetaminofen/toxicidade , Amiodarona/toxicidade , Analgésicos não Narcóticos/toxicidade , Antiarrítmicos/toxicidade , Reatores Biológicos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Citocromo P-450 CYP2E1/metabolismo , Hepatócitos/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Mitocôndrias Hepáticas/efeitos dos fármacos , Consumo de Oxigênio , Acetaminofen/metabolismo , Ativação Metabólica , Amiodarona/metabolismo , Analgésicos não Narcóticos/metabolismo , Antiarrítmicos/metabolismo , Biomarcadores/metabolismo , Microambiente Celular , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Desenho de Equipamento , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/patologia , Esferoides Celulares , Fatores de TempoRESUMO
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.
Assuntos
Polímeros/química , Células 3T3 , Animais , Carbonato de Cálcio/síntese química , Carbonato de Cálcio/química , Bovinos , Adesão Celular/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Raios Infravermelhos , Camundongos , Microscopia Confocal , Microesferas , Concentração Osmolar , Polímeros/metabolismo , Polímeros/farmacologia , Porosidade , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismoRESUMO
Polyelectrolyte multilayers (PEM) loaded with bioactive molecules such as proteins serve as excellent mimics of an extracellular matrix and may find applications in fields such as biomedicine and cell biology. A question which is crucial to the successful employment of PEMs is whether conformation and bioactivity of the loaded proteins is preserved. In this work, the polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) technique is applied to investigate the conformation of the protein lysozyme (Lys) loaded into the poly(L-lysine)/hyaluronic acid (PLL/HA) multilayers. Spectra are taken from the protein in the PEMs coated onto an ATR crystal during protein adsorption and desorption. For comparison, a similar investigation is performed for the case of Lys in contact with the uncoated crystal. The study highlights the presence of both "tightly" and "poorly bound" Lys fractions in the PEM. These fractions differ in their conformation and release behavior from the PEM upon washing. Comparison of spectra recorded with different polarizations suggests preferential orientation of alpha helical structures, beta sheets and turns in the "tightly bound" Lys. In contrast, the "poorly bound" fraction shows isotropic orientation and its conformation is well preserved.
RESUMO
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.
Assuntos
Fibroblastos/citologia , Ouro/química , Membranas Artificiais , Nanopartículas Metálicas/química , Polilisina/química , Animais , Adesão Celular , Eletrólitos/química , Ácido Hialurônico/química , Camundongos , Microscopia de Força Atômica , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
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.
Assuntos
Microfluídica , Eletrólitos , Ácido Hialurônico/química , Microscopia de Fluorescência , Polilisina/químicaRESUMO
The mechanical behavior of biological cells is mainly determined by the cytoskeleton. Its properties are closely interlinked with many cellular events, including disease-related processes, and, thus, may be exploited as potent biomarkers. We have stretched two types of cells between microelectrodes through the application of dielectrophoretic forces. Small numbers of cells of cancerous origin (MCF-7) and from related noncancerous tissue (MCF-10A) were sufficient to obtain data that allowed us to unambiguously distinguish these cells. The Maxwell tension applied has been estimated to be 56 Pa. A detailed analysis of the cells showed that the differences in the stretching response are due to cell-specific mechanical properties. Through the addition of an actin- and a microtubule-specific toxin to the cells, differences in the microtubular structures of the two cell types have been identified as the major cause for the behavior observed. Our approach shows enormous potential for parallelization and automation. Hence, it should be suitable for achieving throughputs that make it attractive for many biomedical diagnostic purposes.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Eletrofisiologia/métodos , Microtúbulos/metabolismo , Resistência ao Cisalhamento , Estresse Mecânico , Adesão Celular/fisiologia , Linhagem Celular Tumoral/metabolismo , Elasticidade/fisiologia , Campos Eletromagnéticos , Células Epiteliais/metabolismo , Feminino , Humanos , Cinética , Microeletrodos , Processamento de Sinais Assistido por Computador/instrumentaçãoRESUMO
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.
Assuntos
Materiais Biocompatíveis/química , Metacrilatos/química , Microscopia de Força Atômica , Polietilenoglicóis/química , Temperatura , Animais , Materiais Biocompatíveis/metabolismo , Adesão Celular , Linhagem Celular , Coloides , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Ouro/química , Cinética , Metacrilatos/metabolismo , Camundongos , Polietilenoglicóis/metabolismo , Propriedades de SuperfícieRESUMO
We present a method for label-free imaging and sorting of cancer cells in blood, which is based on a dielectrophoretic microfluidic chip and label-free interferometric phase microscopy. The chip used for imaging has been embedded with dielectrophoretic electrodes, and therefore it can be used to sort the cells based on the decisions obtained during the cell flow by the label-free quantitative imaging method. Hence, we obtained a real-time, automatic, label-free imaging flow cytometry with the ability to sort the cells during flow. To validate our model, we combined into the label-free imaging interferometer a fluorescence imaging channel that indicated the correctness of the label-free sorting. We have achieved above 98% classification success and 69% sorting accuracy at flow rates of 4 to 7 µL hr-1 . In the future, this method is expected to help in label-free sorting of circulating tumor cells in blood following an initial state-of-the-art cell enrichment.
Assuntos
Holografia , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes , Contagem de Células , Separação Celular , Citometria de Fluxo , Humanos , MicrofluídicaRESUMO
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.
Assuntos
Reatores Biológicos , Técnicas Biossensoriais/métodos , Microambiente Celular , Hepatócitos/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Oxigênio/metabolismo , Acetaminofen/farmacologia , Animais , Biomarcadores/análise , Citocromo P-450 CYP3A/metabolismo , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In order to elucidate the dynamics of cellular processes that are induced in context with intercellular communication, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed on the level of individual cells and correlated with each other. Here we present an approach that allows the initiation of cell-cell or cell-particle interactions and the analysis of cellular reactions within various regimes while the identity of each individual cell is preserved. It utilizes dielectrophoresis (DEP) and microfluidics in a lab-on-chip system. With high spatial and temporal precision we contacted single T cells with functionalized microbeads and monitored their immediate cytosolic Ca(2+) response. After this, the cells were released from the chip system and cultivated further. Expression of the activation marker molecule CD69 was analyzed the next day and correlated with the previously recorded Ca(2+) signal for each individual cell. We found a significant difference in the patterns of Ca(2+) traces between activated and non-activated cells, which shows that Ca(2+) signals in T cells can provide early information about a later reaction of the cell. Although T cells are non-excitable cells, we also observed irregular Ca(2+) transients upon exposure to the DEP field only. These Ca(2+) signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, we could identify experimental conditions that caused the least interference with the physiology of the cell.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Cálcio/metabolismo , Regulação da Expressão Gênica , Ativação Linfocitária , Linfócitos T/imunologia , Anticorpos/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Sinalização do Cálcio/imunologia , Divisão Celular/imunologia , Sobrevivência Celular/imunologia , Citosol/metabolismo , Eletroforese , Humanos , Células Jurkat , Lectinas Tipo C , Potenciais da Membrana/imunologia , Técnicas Analíticas Microfluídicas , Microesferas , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de TempoRESUMO
With the advent of single-cell technologies comes the necessity for efficient protocols to process single cells. We combine dielectrophoresis with open source computer vision programming to automatically control the trajectories of single cells inside a microfluidic device. Using real-time image analysis, individual cells are automatically selected, isolated and spatially arranged.
Assuntos
Eletroforese , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Análise de Célula Única , Eletroforese/instrumentação , Eletroforese/métodos , Desenho de Equipamento , Análise de Célula Única/instrumentação , Análise de Célula Única/métodosRESUMO
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.
RESUMO
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.
Assuntos
Proteínas Contráteis/metabolismo , Proteínas Contráteis/ultraestrutura , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Vicia faba/metabolismoRESUMO
The gentle and careful in vitro processing of live cells is essential in order to make them available to future therapeutic applications. We present a protocol for the activation of single-T cells based on the contact formation with individual anti-CD3/anti-CD28 presenting microbeads in a lab-on-chip environment. The chips consist of microfluidic channels and microelectrodes for performing dielectrophoretic manipulation employing a.c. electric fields. The dielectrophoretic guiding elements allow the assembly of cell-bead pairs while avoiding ill-defined physical contacts with their environment. After overnight cultivation of the manipulated cells, 77% of the bead-associated T cells expressed the activation marker molecule CD69. Physiological stress on the cells was shown to be mainly due to the single-cell cultivation and not to the manipulation in the chips. The same approach could be useful for the in vitro regulation of stem cell differentiation.
Assuntos
Eletroforese em Microchip/métodos , Técnicas Analíticas Microfluídicas/métodos , Linfócitos T/metabolismo , Anticorpos/farmacologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD28/imunologia , Complexo CD3/imunologia , Células Cultivadas , Eletroforese em Microchip/instrumentação , Desenho de Equipamento , Humanos , Lectinas Tipo C , Ativação Linfocitária/efeitos dos fármacos , Técnicas Analíticas Microfluídicas/instrumentação , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
We present a simple lab-on-chip device for handling small samples of delicate cells, e.g. stem cells. It uses a combination of sedimentation and dielectrophoresis. The transport of cells is driven by gravitation. Dielectrophoresis uses radio-frequency electric fields for generating particle-selective forces dependent on size and polarisability. Electrodes along the channels hold particles and/or cells in a defined position and deflect them towards different outlets. The absence of external pumping and the integration of injection and sampling ports allow the processing of tiny sample volumes. Various functions are demonstrated, such as contact-free cell trapping and cell/particle sorting. Pairs of human cells and antibody-coated beads, as they are formed for T cell activation, are separated from unbound beads. The cells experience only low stress levels compared with the stress levels in dielectrophoresis systems, where transport depends on external pumping. Our device is a versatile yet simple tool that finds applications in cellular biotechnology, in particular when an economic solution is required.
Assuntos
Biotecnologia/métodos , Técnicas de Química Analítica/métodos , Eletroforese/instrumentação , Biotecnologia/instrumentação , Técnicas de Cultura de Células/métodos , Separação Celular , Células Cultivadas , Eletrodos , Campos Eletromagnéticos , Eletroforese/métodos , Desenho de Equipamento , Gravitação , Humanos , Ativação Linfocitária , Monócitos/citologia , Polimetil Metacrilato/química , Linfócitos T/metabolismoRESUMO
Comprehensive analysis of the multifractional molecular diffusion provides a deeper understanding of the diffusion phenomenon in the fields of material science, molecular and cell biology, advanced biomaterials, etc. Fluorescence recovery after photobleaching (FRAP) is commonly employed to probe the molecular diffusion. Despite FRAP being a very popular method, it is not easy to assess multifractional molecular diffusion due to limited possibilities of approaches for analysis. Here we present a novel simulation-optimization-based approach (S-approach) that significantly broadens possibilities of the analysis. In the S-approach, possible fluorescence recovery scenarios are primarily simulated and afterward compared with a real measurement while optimizing parameters of a model until a sufficient match is achieved. This makes it possible to reveal multifractional molecular diffusion. Fluorescent latex particles of different size and fluorescein isothiocyanate in an aqueous medium were utilized as test systems. Finally, the S-approach has been used to evaluate diffusion of cytochrome c loaded into multilayers made of hyaluronan and polylysine. Software for evaluation of multifractional molecular diffusion by S-approach has been developed aiming to offer maximal versatility and user-friendly way for analysis.
RESUMO
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.