Imaging mass spectrometry for in situ lipidomics: from cell structures to cardiac tissue

Imaging of cells and tissues is important for studying different processes within biological systems due to the spatial information provided for different molecular species during imaging. One powerful imaging technique is mass spectrometry imaging (MSI). It is a label free technique that provides chemical information of a sample at the same time as it allows for imaging at high spatial resolution. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) uses a focused primary ion beam to ablate and ionise molecules from the top layers of the sample surface which makes it a very surface sensitive technique. Recent developments in high energy gas cluster ion beam (GCIB) technology for ToF-SIMS has greatly improved the imaging of higher mass species, such as intact lipids. Lipids are important molecules found in all living organisms. They are used as building blocks for cells and are involved in a variety of important cellular processes such as energy storage and acting as important mediators in many signalling pathways, making them an interesting target for imaging studies. In this thesis, the ToF-SIMS imaging technique has been applied to both tissues and cells in order to perform in situ lipidomics analysis of various samples. Development of sample treatment methods that provides easier data interpretation and other method development for improving secondary ion yields have also been implemented in this work. In paper I, enhancement of negative secondary ion yields was induced by a combination of ion bombardment using a GCIB with simultaneous caesium flooding, for both inorganic and organic substrates. In paper II, ToF-SIMS imaging with a GCIB was used together with LC-MS to elucidate changes in lipid composition 6 hours after an induced myocardial infarction in mouse heart. The spatial information from the MSI allowed correlation of specific lipid species to infarcted and non-infarcted regions of the heart. Localised lipid accumulation was discovered in specific regions of the heart. In paper IV, these lipid changes were tracked over longer periods of time, 24 hours and 48 hours after infarction, and progression of the infarcted area was observed. In paper III, a simple method was developed in order to aid interpretation of the complex mass spectra collected from ToF-SIMS experiments of complex tissue sample such as heart tissue. Salt adduct formation was demonstrated as a means to discriminate between diacylglyceride and triacylglyceride, which are usually impossible to distinguish during ToF-SIMS analysis. In paper V, lipid changes in PC12 cell membranes were analysed after incubation with the essential fatty acids, omega-3 and omega-6. Using deuterium labelled fatty acids made it possible to track incorporation into phospholipids as well as the relative amount of each

Correlated fluorescence microscopy and multi-ion beam secondary ion mass spectrometry imaging reveals phosphatidylethanolamine increases in the membrane of cancer cells over-expressing the molecular chaperone subunit CCTδ

Changes in the membrane composition of sub-populations of cells can influence different properties with importance to tumour growth, metastasis and treatment efficacy. In this study, we use correlated fluorescence microscopy and ToF-SIMS with C60+ and (CO2)6k+ ion beams to identify and characterise sub-populations of cells based on successful transfection leading to over-expression of CCTδ, a component of the multi-subunit molecular chaperone named chaperonin-containing tailless complex polypeptide 1 (CCT). CCT has been linked to increased cell growth and proliferation and is known to affect cell morphology but corresponding changes in lipid composition of the membrane have not been measured until now. Multivariate analysis of the surface mass spectra from single cells, focused on the intact lipid ions, indicates an enrichment of phosphatidylethanolamine species in the transfected cells. While the lipid changes in this case are driven by the structural changes in the protein cytoskeleton, the consequence of phosphatidylethanolamine enrichment may have additional implications in cancer such as increased membrane fluidity, increased motility and an ability to adapt to a depletion of unsaturated lipids during cancer cell proliferation. This study demonstrates a successful fluorescence microscopy-guided cell by cell membrane lipid analysis with broad application to biological investigation. Graphical abstrac

Design and biofabrication of a leaf-inspired vascularized cell-delivery device

We designed and biofabricated a channeled construct as a possible cell-delivery device that can be endothelialized to overcome size limitations due to oxygen diffusion. The channeled device mimicking a leaf was designed using computer-aided design software, with fluid flow through the channels visualized using simulation studies. The device was fabricated either by form casting using a custom 3D-printed plastic mold or by 3D-bioprinting using Pluronic F-127 as sacrificial ink to print the channels. The actual leaf was cast or bioprinted using hydrogel made from a mixture of tunicate cellulose nanofibers and alginate that was cross-linked in calcium chloride solution to allow a stable device. The resulting device was a 20 7 8 7 3 mm or 35 7 18 7 3 mm (length 7 width 7 height) leaf with one main channel connected to several side channels. Surface modification using periodate oxidation, followed by laminin bioconjugation, was performed to enhance endothelial cell adhesion in the channels. We subsequently used human umbilical vein endothelial cells to demonstrate the efficacy of the device for promoting endothelialization. These results indicated that the biofabricated device has great potential for use in tissue-engineering for various applications associated with the need of perfusable vasculature

Biomaterial and biocompatibility evaluation of tunicate nanocellulose for tissue engineering

Extracellular matrix fibril components, such as collagen, are crucial for the structural properties of several tissues and organs. Tunicate-derived cellulose nanofibrils (TNC) combined with living cells could become the next gold standard for cartilage and soft-tissue repair, as TNC fibrils present similar dimensions to collagen, feasible industrial production, and chemically straightforward and cost-efficient extraction procedures. In this study, we characterized the physical properties of TNC derived from aquaculture production in Norwegian fjords and evaluated its biocompatibility regarding induction of an inflammatory response and foreign-body reactions in a Wistar rat model. Additionally, histologic and immunohistochemical analyses were performed for comparison with expanded polytetrafluoroethylene (ePTFE) as a control. The average length of the TNC as determined by atomic force microscopy was tunable from 3 mu m to 2.4 mu m via selection of a various number of passages through a microfluidizer, and rheologic analysis showed that the TNC hydrogels were highly shear-thinning and with a viscosity dependent on fibril length and concentration. As a bioink, TNC exhibited excellent rheological and printability properties, with constructs capable of being printed with high resolution and fidelity. We found that post-print cross-linking with alginate stabilized the construct shape and texture, which increased its ease of handling during surgery. Moreover, after 30 days in vivo, the constructs showed a highly-preserved shape and fidelity of the grid holes, with these characteristics preserved after 90 days and with no signs of necrosis, infection, acute inflammation, invasion of neutrophil granulocytes, or extensive fibrosis. Furthermore, we observed a moderate foreign-body reaction involving macrophages, lymphocytes, and giant cells in both the TNC constructs and PTFE controls, although TNC was considered a nonirritant biomaterial according to ISO 10993-6 as compared with ePTFE. These findings represent a milestone for future clinical application of TNC scaffolds for tissue repair. One sentence summary: In this study, the mechanical properties of tunicate nanocellulose are superior to nanocellulose extracted from other sources, and the biocompatibility is comparable to that of ePTFE

Autologous endothelialisation by the stromal vascular fraction on laminin-bioconjugated nanocellulose-alginate scaffolds

Establishing a vascular network in biofabricated tissue grafts is essential for ensuring graft survival. Such networks are dependent on the ability of the scaffold material to facilitate endothelial cell adhesion; however, the clinical translation potential of tissue-engineered scaffolds is hindered by the lack of available autologous sources of vascular cells. Here, we present a novel approach to achieving autologous endothelialisation in nanocellulose-based scaffolds by using adipose tissue-derived vascular cells on nanocellulose-based scaffolds. We used sodium periodate-mediated bioconjugation to covalently bind laminin to the scaffold surface and isolated the stromal vascular fraction and endothelial progenitor cells (EPCs; CD31+CD45−) from human lipoaspirate. Additionally, we assessed the adhesive capacity of scaffold bioconjugation in vitro using both adipose tissue-derived cell populations and human umbilical vein endothelial cells. The results showed that the bioconjugated scaffold exhibited remarkably higher cell viability and scaffold surface coverage by adhesion regardless of cell type, whereas control groups comprising cells on non-bioconjugated scaffolds exhibited minimal cell adhesion across all cell types. Furthermore, on culture day 3, EPCs seeded on laminin-bioconjugated scaffolds showed positive immunofluorescence staining for the endothelial markers CD31 and CD34, suggesting that the scaffolds promoted progenitor differentiation into mature endothelial cells. These findings present a possible strategy for generating autologous vasculature and thereby increase the clinical relevance of 3D-bioprinted nanocellulose-based constructs

Lipid Diversity in Cells and Tissue Using Imaging SIMS

Lipids are an important class of biomolecules with many roles within cells and tissue. As targets for study, they present several challenges. They are difficult to label, as many labels lack the specificity to the many different lipid species or the labels maybe larger than the lipids themselves, thus severely perturbing the natural chemical environment. Mass spectrometry provides exceptional specificity and is often used to examine lipid extracts from different samples. However, spatial information is lost during extraction. Of the different imaging mass spectrometry methods available, secondary ion mass spectrometry (SIMS) is unique in its ability to analyze very small features, with probe sizes <50 nm available. It also offers high surface sensitivity and 3D imaging capability on a subcellular scale. This article reviews the current capabilities and some remaining challenges associated with imaging the diverse lipids present in cell and tissue samples. We show how the technique has moved beyond show-and-tell, proof-of-principle analysis and is now being used to address real biological challenges. These include imaging the microenvironment of cancer tumors, probing the pathophysiology of traumatic brain injury, or tracking the lipid composition through bacterial membranes

BioArchitecture: New Futures of Sustainable Living

Public exhibition and lecture at the International Science Festival, Vetenskapsfestivalen 2022, in Gothenburg, Sweden. The presentation provides glimpses of an unknown future of living surrounded by architectural structures made from sustainable biomaterials. Audience is encouraged to reflect and create their own imaginings of such a future by experiencing physical samples representing fragments of architectural objects from such materials, 3D printed using digital machines and industrial robots.\ua0In the lecture, an unusual research collaboration between seemingly unrelated disciplines - architecture and chemistry - is discussed. The aim is to demonstrate how a crossover between artistic design, digital technology and natural sciences creates unprecedented opportunities for innovation. Such innovation relates to a sustainable future in which waste from the Swedish forestry industry is transformed into a novel material with great potentials for new applications in architecture and built environment

RePrint: Digital workflow for aesthetic retrofitting of deteriorated architectural elements with new biomaterial finishes

Digital fabrication offers new opportunities for revitalizing aged buildings in the time of craft expertise decline and higher demands regarding the sustainability of employed materials. Precise reproduction of architectural elements with digital 3D reconstruction methods such as photo-grammetry, and their repair using agile robotic 3D printing involving new environmentally friendly materials can save time and resources, leading to more circular design and manufacturing. This study presents digital workflows for architectural restoration, based on the concept of aesthetic retrofitting of deteriorated wooden architectural elements through the application of surface finishes from a novel biomaterial – nanocellulose hydrogel, upcycled from forestry waste. The workflows were established through experimental digital design and reproduction of wooden architectural details in an existing historical building, and executed within an integrated digital framework combining photogrammetry, 2D graphics processing, computational design and robotic 3D printing. Overall, the investigation has sought to demonstrate the potential of nanocellulose as a material suitable for applications in renovation and conservation. Further, the intention was to elucidate the role of digital tools as new media of restoration that enable to uplift cultural assets in an alterna-tive way - by allowing to embed aesthetic features conveying the contemporaneity of remedial interventions. Aiming to contribute to current work in experimental preservation, the study offers a novel approach in which deteriorated architectural elements are endowed with a new materiality that follows the new logic of circularity in contemporary design and construction

Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion

Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose

Relative quantification of deuterated omega-3 and -6 fatty acids and their lipid turnover in PC12 cell membranes using TOF-SIMS

Understanding FA metabolism and lipid synthesis requires a lot of information about which FAs and lipids are formed within the cells. We focused on the use of deuterated substrates of 100 μM α-linolenic acid and linoleic acid to determine the relative amounts of their converted PUFAs and specific phospholipids that are incorporated into cell plasma membranes. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to image and analyze lipids in model cell membranes with and without FA treatment. Because of its high spatial resolution, TOF-SIMS can be used to simultaneously provide both chemical information and distribution of various molecules in the sample surface down to the subcellular scale. Data obtained from this analysis of isotopes in the cell samples were used to calculate the relative amounts of long-chain PUFAs and phospholipids from their precursors, α-linolenic acid and linoleic acid. Our results show that the FA treatments induced an increase in the amounts of α-linolenic acid and linoleic acid and their long-chain conversion products. Moreover, an enhanced level of phospholipid turnover of these FAs in lipids such as phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols was also observed in the cell plasma membrane