Interactions between tumor cells and immune cells are sparsely explored in 3D models, although occasional studies with 3D cell culture technologies have confirmed that tumor architecture influences cancer cell-immune system interactions. The development of technologies enabling controlled analysis of tumor-immune system interactions especially in 3D is highly challenging. Two photon polymerization (2PP) as a method for fabrication of various microstructures suitable for preparation of 3D tumor models finds applications in this context. In the present study 2PP technology was used to fabricate polymeric microfibers in 3D microspace that mimic fibers of extracellular matrix. UV-curable polymer OrmoComp (Micro Resist Technology GmbH) was irradiated by Newport Spirit ultrafast amplified laser operating at 520 nm. The fibers were made with one side anchored to the substrate with the supporting structure, whereas the other side was freely movable in space. The shape and curliness of fibers were adjusted by alternating parameters of fabrication, namely energy intensities and speed of fabrication. Before exposing fabricated fibers to live cells, microstructures were processed with KOH based treatment to enhance adhesion of cancer cells. Arrangement of the cancer cells in the network of polymeric fibers was visualized by confocal scanning microscopy. Cancer cells were able to proliferate and form spatial cellular clusters among polymeric fibers after several days of cultivation. At the same time they were available to immune cells that could be supplemented to the culture at any time. The results of the present study document feasibility to use 2PP technology to develop in vitro 3D models suitable for studies of tumor-immune cell interactions.
Nanoparticles (NPs) from various metals (Zinc, Nickel, Cobalt, Copper) were designed and fabricated by direct synthesis using femtosecond laser ablation in liquids. Employing confocal microscopy with spectral detection and fluorescence lifetime imaging (FLIM), we have evaluated interaction of fabricated NPs with living Chlorella sp. algae by means of their naturally presented endogenous fluorescence. Live cell imaging was done in spectral region 500-550 nm and 650- 710 nm to evaluate the effect of NPs on both, the green and the red fluorescence that is derived from flavonoids/carotenoids and chlorophylls respectively. We observed fluorescence intensity decrease in the red spectral region by all but Ni NPs. The presence of NPs also lead to an increase in the blue fluorescence at 477-488 nm, possibly resulting from reflected light. Gathered observations constitute the first step towards creation of methodological approaches for fast natural biosensing of the effects of environmental pollution directly in live algae.
The last decade has witnessed a rapid growth of nanoscale-oriented biosensors that becomes one of the most promising and rapidly growing areas of modern research. Despite significant advancements in conception of such biosensors, most are based at evaluation of molecular, or protein interactions. It is however becoming increasingly evident that functionality of a living system does not reside in genome or in individual proteins, as no real biological functionality is expressed at these levels. Instead, to comprehend the true functioning of a biological system, it is essential to understand the integrative physiological behavior of the complex molecular interactions in their natural environment and precise spatio-temporal topology. In this contribution we therefore present a new concept for creation of biosensors, bio-inspired from true functioning of living cells, while monitoring their endogenous fluorescence, or autofluorescence.
Design and fabrication of appropriate biocompatible microstructures that ensure fixation and control of experimental conditions for live cell and bacteria observations is an important prerequisite for number of real time experiments. Our approach is to design engineered microfabricated 3D structures for growth of cells in culture without significant modification of their metabolic state. Presented approach is aimed at evaluation of the potential applicability of biocompatible constructs in the biomedical field and thus live cell monitoring in controlled conditions. Design and evaluation of properties of materials and structures with mesoscopic arrangement and their interaction with biological objects is a prerequisite for establishment of physiologically relevant in vitro models of pathologies as well as for development of a new generation of nano / micro / bio-sensors.
Fabricated micro- and nano-structured surfaces were evaluated for use with living cells. Metabolic state was tested by means of endogenous flavin fluorescence of living peripheral blood mononuclear cells (PBMC) positioned on a coverslip, non-covered, or covered with micro- or nano-structured surfaces (OrmoComp polymer structures produced by 2-photon photopolymerisation, or Zinc Oxide (ZnO) layer fabricated by pulsed laser deposition). Confocal microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) were employed to gather flavin fluorescence lifetime images of living PBMC on structured surfaces. Gathered data are the first step towards monitoring of the live cell interaction with different micro/nano-structured surfaces and thus evaluate their potential applicability in the biomedical field.