For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogelbased systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.
Correlative Light and Electron Microscopy (CLEM) combines two imaging techniques at different scales to figure out the precise localization of rare elements in a well‐defined biological context. Light microscopy (LM) allows positional mapping of the sample labeled with a fluorochrome. Electron microscopy (EM) provides the nanometer resolution of the mapped area. The general principle of CLEM is to collect different informations from a single region in a sample. The data are then combined toward a global understanding of the sample ultrastructure. The precise localization of molecules of interest in their biological context serves to define their functional role. In the present study, we aimed to apply CLEM to tissue samples. We have chosen define regions of interest (ROI) by fiducial laser marks surrounding the ROI. For this purpose, we applied the “Near infrared branding” (NIRB) method (Bishop et al., 2011). This NIRB technique consists to create easily detectable fiducial marks in three dimensions in a fixed tissue sample. A specific region of the sample is imaged with LM, the fiducial marks are performed, and the same region is analysed with EM. The fiducial marks are detectable both in optical and electron microscopy. They are made with a femtosecond pulsed titanium‐sapphire laser. The laser is used to create a user defined pattern with line or points scans. The size of the marks depends on the fraction of laser power used or the number of laser swipes. The NIRB marks can be placed with a three‐dimensional micrometer precision in close proximity to the ROI. Laser‐made marks are visualized through tissue photo‐oxidation that induces autofluorescence in LM and is a characteristic marker for EM. When using Green Fluorescent Protein (GFP) labelling, no GFP photo‐oxidation is noticed when drawing NIRB. The present study focused on adjustments that are necessary to adapt the NIRB method to nervous tissue and skin. These two types of tissue present a very different composition and are therefore well suited to compare the conditions of NIRB implementation. The NIRB technique has been set using immunostaining of easily detectable and strongly represented antigens with a specific location in well‐defined regions. In nervous tissue, the constitutive extra‐membranous mitochondrial protein TOM20 was used as a target antigen in spinal cord sections. For the skin, the development relied on Langerin immunostaining (a type II transmembrane, C‐type lectin receptor on Langerhans cells). The goal is to locate the Langerhans cells in the epidermis. All immunostaining strategies are performed with a pre‐embedding method. It consists in immunostaining on chemically fixed samples before embedding in resin and ultra‐sectioning for TEM. First, we set the method to achieve the ROI localization. Then, we defined our own parameter settings using our multiphoton system. Finally, it is essential to adapt the marks to the size of the region of interest, and the type of sample. The delimited ROI can size up to 70μm3. Future perspectives are to develop three‐dimensional CLEM approach using electron tomography.
Understanding the biological role of molecules requires to know their precise localization and structural environment. Thanks to fluorescence microscopy and biotechnologies, the localization of proteins of interest has become relatively easy. But in more and more cases, this resolution is not enough and the lack of data on the molecule/cell environment does not allow the characterization of their biological function. Transmission Electron Microscopy (TEM) and immuno‐gold labelling can be used to characterize at the same time the cellular compartments associated with the protein of interest and the ultrastructure of the cellular environment at a high resolution. However, the antigenicity preservation as well as the antibody production are sometimes difficult. Moreover, TEM observations only give access to a static “snapshot” of a fixed, dead sample. For several years, many attempts have been made to correlate Fluorescent and Electron Microscopy in order to combine the advantages of both microscopy techniques on a unique sample. In this way, we develop on the BIC new protocols of sample preparation for animal and vegetal sample (culture cells and tissues), allowing to maintain simultaneously the fluorescence and the overall ultrastructure (Figure 1 and 2), using Quick Freeze Substitution (McDonald KL and Webb RI, J. Microsc, 2011). This technique will allow the correlation of both Light and Electron Microscopy data on the same section (CLEM: Correlative Light Electron Microscopy), works with classical fluorescent tags and improve the antigenicity for gold immunolabelling.
Electron microscopy (EM) and nano secondary ion mass spectrometry (NanoSIMS) aim to acquiring nanometric information, which also imply ultraresolution and therefore these techniques require the best preservation of samples. Analytical techniques such as X‐ray spectroscopy and NanoSIMS are able to identify, localize and quantify chemical elements both at the whole cell and at the intracellular level. These techniques can be coupled with biological structural analysis. The goal in sample preparation is to maintain chemical elements at their original localization site as well as at their physiological active site. Therefore, sample preparation has to prevent also delocalization of the biological molecules (e.g. proteins, lipids). In EM and NanoSIMS, samples are subjected to drastic conditions such as high vacuum and beam energy. Thus, due to the characteristics of these devices, analyses are incompatible with native biological systems. Moreover, these techniques require thin sections of samples (TEM/X‐EDS (70‐100nm) and NanoSIMS (200‐300nm)). The sample should not be destroyed too quickly under the beam or by vacuum sublimation and must be stable chemically. Diffusible elements are quickly lost during dehydration and embedding step used for the routine preparation of biological specimens and so these methods can only be used if it is already known that the elements of interest are tightly bound. Sample preparation for EM and NanoSIMS must immobilize the biological elements, must eliminate water and must allow sectioning of the sample. As generally accepted in the literature, cryofixation by high pressure freezing followed by cryosubstitution are the best methods to limit redistribution of metal ions. In general, the preparation of biological samples for TEM and NanoSIMS is rather similar. Therefore, both techniques can be easily applied together and in a complementary way for bioimaging. In this work, different biological sample preparation techniques will be presented. The goal is to compare several cryomethods like cryosubstitution with different resins or freeze‐drying. An advanced sample preparation protocol was developed basing on high pressure freezing cryofixation follow by cryosubstitution or freeze‐drying, in order to limit metal redistribution, and preparation of adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Best results were obtained by correlative imaging of a single cell by TEM and NanoSIMS combining the advantages of both techniques. As an illustration, the ultrastructure of a C. reinhardtii cell (Fig. 2) could be directly related to the spatial distribution of macro and trace elements present at basal levels in the cell (Figs. 1 and 3). Thus, metals could be localized in different cell organelles such as the pyrenoid and granules. For another example, epidermal cells on adherent culture (keratinocytes), localization of elements (e.g. Ni) demonstrates the importance of sample preparation. Correlative TEM and NanoSIMS shows potential for many future applications of subcellular imaging of trace elements in medicine and biology.
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