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3D bioprinting is an emerging biofabrication strategy using bioinks, comprising cells and biocompatible materials, to produce functional tissue models. Despite progress in building increasingly complex objects, biological analyses in printed constructs remain challenging. Especially, methods that allow non-invasive and non-destructive evaluation of embedded cells are largely missing. Here, we implemented Raman imaging for molecular-sensitive investigations on bioprinted objects. Different aspects such as culture formats (2D, 3D-cast, and 3D-printed), cell types (endothelial cells and fibroblasts), and the selection of the biopolymer (alginate, alginate/nanofibrillated cellulose, alginate/gelatin) were considered and evaluated. Raman imaging allowed for marker-independent identification and localization of subcellular components against the surrounding biomaterial background. Furthermore, single-cell analysis of spectral signatures, performed by multivariate analysis, demonstrated discrimination between endothelial cells and fibroblasts and identified cellular features influenced by the bioprinting process. In summary, Raman imaging was successfully established to analyze cells in 3D culture in situ and evaluate them with regard to the localization of different cell types and their molecular phenotype as a valuable tool for quality control of bioprinted objects.

Two of the greatest challenges for successful application of small-diameter in situ tissue-engineered vascular grafts are 1) preventing thrombus formation and 2) harnessing the inflammatory response to the graft to guide functional tissue regeneration. This study evaluates the in vivo performance of electrospun resorbable elastomeric vascular grafts, dual-functionalized with anti-thrombogenic heparin (hep) and anti-inflammatory interleukin 4 (IL-4) using a supramolecular approach. The regenerative capacity of IL-4/hep, hep-only, and bare grafts is investigated as interposition graft in the rat abdominal aorta, with follow-up at key timepoints in the healing cascade (1, 3, 7 days, and 3 months). Routine analyses are augmented with Raman microspectroscopy, in order to acquire the local molecular fingerprints of the resorbing scaffold and developing tissue. Thrombosis is found not to be a confounding factor in any of the groups. Hep-only-functionalized grafts resulted in adverse tissue remodeling, with cases of local intimal hyperplasia. This is negated with the addition of IL-4, which promoted M2 macrophage polarization and more mature neotissue formation. This study shows that with bioactive functionalization, the early inflammatory response can be modulated and affect the composition of neotissue. Nevertheless, variability between graft outcomes is observed within each group, warranting further evaluation in light of clinical translation.

Smooth muscle cell (SMC) diversity and plasticity are limiting factors in their characterization and application in cardiovascular tissue engineering. This work aimed to evaluate the potential of Raman microspectroscopy and Raman imaging to distinguish SMCs of different tissue origins and phenotypes. Cultured human SMCs isolated from different vascular and non-vascular tissues as well as fixed human SMC-containing tissues were analyzed. In addition, Raman spectra and images of tissue-engineered SMC constructs were acquired. Routine techniques such as qPCR, histochemistry, histological and immunocytological staining were performed for comparative gene and protein expression analysis. We identified that SMCs of different tissue origins exhibited unique spectral information that allowed a separation of all groups of origin by multivariate data analysis (MVA). We were further able to non-invasively monitor phenotypic switching in cultured SMCs and assess the impact of different culture conditions on extracellular matrix remodeling in the tissue-engineered ring constructs. Interestingly, we identified that the Raman signature of the human SMC-based ring constructs was similar to the one obtained from native aortic tissue. We conclude that Raman microspectroscopic methods are promising tools to characterize cells and define cellular and extracellular matrix components on a molecular level. In this study, in situ measurements were marker-independent, fast, and identified cellular differences that were not detectable by established routine techniques. Perspectively, Raman microspectroscopy and MVA in combination with artificial intelligence can be suitable for automated quality monitoring of (stem) cell and cell-based tissue engineering products. STATEMENT OF SIGNIFICANCE: The accessibility of autologous blood vessels for surgery is limited. Tissue engineering (TE) aims to develop functional vascular replacements; however, no commercially available TE vascular graft (TEVG) exists to date. One limiting factor is the availability of a well-characterized and safe cell source. Smooth muscle cells (SMCs) are generally used for TEVGs. To engineer a TEVG, proliferating SMCs of the synthesizing phenotype are essential, whereas functional, sustainable TEVGs require SMCs of the contractile phenotype. SMC diversity and plasticity are therefore limiting factors, also for their quality monitoring and application in TE. In this study, Raman microspectroscopy and imaging combined with machine learning tools allowed the non-destructive, marker-independent characterization of SMCs, smooth muscle tissues and TE SMC-constructs. The spectral information was specific enough to distinguish for the first time the phenotypic switching in SMCs in real-time, and monitor the impact of culture conditions on ECM remodeling in the TE SMC-constructs.

Cryomedium toxicity is a major safety concern when transplanting cryopreserved organs. Therefore, thorough removal of potentially toxic cryoprotective agents (CPAs) is required before transplantation. CPAs such as dimethyl-sulfoxide (DMSO), propylene glycol (PG), and formamide (FMD), routinely employed in ice-free cryopreservation (IFC), have advantages in long-term preservation of tissue structures compared with conventional cryopreservation employing lower CPA concentrations. This study evaluated the impact of potential residual CPAs on human cardiac valves. Raman microspectroscopy and Raman imaging were established as nondestructive marker-independent techniques for in situ quantitative assessment of CPA residues in IFC valve tissues. In detail, IFC valve leaflets and supernatants of the washing solutions were analyzed to determine the washing efficiency. A calibration model was developed according to the CPA's characteristic Raman signals to quantify DMSO, PG and FMD concentrations in the supernatants. Single point Raman measurements were performed on the intact tissues to analyze penetration properties. In addition, Raman imaging was utilized to visualize potential CPA residues. Our data showed that washing decreased the CPA concentration in the final washing solution by 99%, and no residues could be detected in the washed tissues, validating the multistep CPA removal protocol routinely used for IFC valves. Raman analysis of unwashed tissues showed different permeation characteristics depending on each CPA and their concentration. Our results demonstrate a great potential of Raman microspectroscopy and Raman imaging as marker-independent in situ tissue quality control tools with the ability to assess the presence and concentration of different chemical agents or drugs in preimplantation tissues.

Summary: Aortic rupture and dissection are life-threatening complications of ascending thoracic aortic aneurysms (aTAAs), and risk assessment has been largely based on the monitoring of lumen size enlargement. Temporal changes in the extracellular matrix (ECM), which has a critical impact on aortic remodeling, are not routinely evaluated, and cardiovascular biomarkers do not exist to predict aTAA formation. Here, Raman microspectroscopy and Raman imaging are used to identify spectral biomarkers specific for aTAAs in mice and humans by multivariate data analysis (MVA). Multivariate curve resolution-alternating least-squares (MCR-ALS) combined with Lasso regression reveals elastic fiber-derived (Ce1) and collagen fiber-derived (Cc6) components that are significantly increased in aTAA lesions of murine and human aortic tissues. In particular, Cc6 detects changes in amino acid residues, including phenylalanine, tyrosine, tryptophan, cysteine, aspartate, and glutamate. Ce1 and Cc6 may serve as diagnostic Raman biomarkers that detect alterations of amino acids derived from aneurysm lesions.

A hallmark of Parkinson’s disease (PD) is the formation of Lewy bodies in the brain. Lewy bodies are rich in the aggregated form of misfolded α-Synuclein (α-Syn). The brain from PD patients can only be analysed after post-mortem, limiting the diagnosis of PD to the manifestation of motor symptoms. In PD patients and animal models phosphorylated α-Syn was detected in the gut, thus, raising the hypothesizes that early-stage PD could be diagnosed based on colon tissues biopsies. Non-invasive marker-free technologies represent an ideal method to potentially detect aggregated α-Syn in vivo. Raman microspectroscopy has been established for the detection of molecular changes such as alterations of protein structures. Here, the olfactory bulb in the brain and the muscularis mucosae of colon tissue sections of a human BAC-SNCA transgenic (TG) rat model was analysed using Raman imaging and microspectroscopy. Raman images from TG and WT rats were investigated using spectral, principal component and true component analysis. Spectral components indicated protein aggregates (spheroidal oligomers) in TG rat brain and colon tissues even at a young age but not in WT. In summary, we have demonstrated that Raman imaging is capable to detect α-Syn aggregates in colon tissues of a PD rat model and making it a promising tool for future use in PD pathology.

Hybrid scaffolds composed of synthetic polymers and naturally occurring components have become more relevant in the field of tissue engineering and regenerative medicine. Synthetic polymers are responsible for scaffold durability, strength and structural integrity; however, often do not provide biological signals. Introducing a biological component leads to more advanced and biocompatible scaffolds. In order to use these scaffolds as implants, a deeper knowledge of material characteristics and the impact of the biological component on the scaffold mechanical properties are required. Furthermore, it is necessary to implement fast, easy and non-invasive methods to determine material characteristics. In this work, we aimed to generate gelatin-poly-L-lactide (PLA) hybrids via electrospinning with defined, controllable and tunable scaffold characteristics. Using Raman microspectroscopy, we demonstrated the effectiveness of the cross-linking reaction and evaluated the increasing PLA content in the hybrid scaffolds with a non-invasive approach. Using multiphoton microscopy, we showed that gelatin fibers electrospun from a fluorinated solvent exhibit a second harmonic generation (SHG) signal typical for collagen-like structures. Compared to pure gelatin, where the SHG signal vanishes after cross-linking, the signal could be preserved in the hybrid scaffolds even after cross-linking. Furthermore, we non-invasively imaged cellular growth of human dermal fibroblasts on the hybrid electrospun scaffolds and performed fluorescence lifetime imaging microscopy on the cell-seeded hybrids, where we were able to discriminate between cells and scaffolds. Here, we successfully employed non-invasive methods to evaluate scaffold characteristics and investigate cell–material interactions.

Mammalian cells have become the predominant expression system for the production of biopharmaceuticals due to their capabilities in posttranslational modifications. In recent years, the efficacy of these production processes has increased significantly through technical improvements. However, the state of the art in the development of producer cell lines includes many manual steps and is as such very time and cost consuming. In this study we developed a process combination of Raman micro-spectroscopy, laser-induced forward transfer (LIFT) and surface-enhanced Raman spectroscopy (SERS) as an automated machine system for the identification, separation and characterization of single cell-clones for biopharmaceutical production. Raman spectra showed clear differences between individual antibody-producing and non-producing chinese hamster ovary (CHO) cells after their stable transfection with a plasmid coding for an immunoglobulin G (IgG) antibody. Spectra of producing CHO cells exhibited Raman signals characteristic for human IgG. Individual producing CHO cells were successfully separated and transferred into a multiwell plate via LIFT. Besides, changes in concentration of human IgG in solution were detected via SERS. SERS spectra showed the same peak patterns but differed in their peak intensity. Overall, our results show that identification of individual antibody-producing CHO cells via Raman micro-spectroscopy, cell separation via LIFT and determination of changes in concentrations of overexpressed protein via SERS are suitable and versatile tools for assembling a fully automated system for biopharmaceuticals manufacturing.

Aortic rupture and dissection are life-threatening complications of ascending thoracic aortic aneurysms (aTAAs) and the risk assessment has been largely based on the monitoring of a lumen size enlargement. Temporal changes of the extracellular matrix (ECM), which has a critical influence on aortic remodeling, are not routinely evaluated nor cardiovascular biomarkers exist to predict aTAA formation. Here, we performed Raman microspectroscopy and imaging and identified marker spectra specific for aTAAs in mice and humans by multivariate data analysis (MVA). Multivariate curve resolution-alternating least squares (MCR-ALS) combined with Lasso regression successfully identified elastic fiber-derived Ce1 and collagen fiber-derived Cc6 components, which were significantly increased in aTAA lesions of murine and human aortic tissues. Cc6 detected the changes in amino acid residues, including phenylalanine, tyrosine, tryptophan, cysteine, aspartate, and glutamate. Ce1 and Cc6 may serve as a diagnostic Raman spectral marker that detects alterations of amino acids derived from aneurysm lesions.

Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood. Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune-suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c+ myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin α9β1 and binding to CCL21, tenascin-C immobilized CD11c+ cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines, and matrisomal components were hallmarks of the tenascin-C–instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune-suppressive stromal properties, reducing tumor growth, progression, and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors, as high tenascin-C expression and an immune-suppressive stroma correlate to poor patient survival.

Summary Cardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.48 mL/min), cyclic strain (5%), and extended culture time to improve the maturation of murine and human ESC-CMs. Dynamically-cultured ESC-CMs showed an increased expression of cardiac-associated proteins and genes, cardiac ion channel genes, as well as increased SERCA activity and a Raman fingerprint with the presence of maturation-associated peaks similar to primary CMs. We present a bioreactor platform that can serve as a foundation for the development of human-based cardiac in vitro models to verify drug candidates, and facilitates the study of cardiovascular development and disease., Graphical Abstract, Highlights • Custom-made bioreactor exposes ESC-CMs to defined shear stress and cyclic stretch • Physical signals and extended culture significantly improve maturation of ESC-CMs • Biochemical fingerprint of dynamically cultured ESC-CMs is similar to primary CMs, In this study, we induce differentiation and maturation of mouse and human embryonic stem cell (ESC)-derived cardiomyocytes (CMs) by the application of defined pulsatile flow and cyclic strain in a custom-made bioreactor. The ESC-CMs cultured under dynamic conditions and extended culture time showed a significantly faster calcium decay, increased SERCA activity and sarcomeric length, as well as protein and gene expression patterns, and biochemical fingerprints comparable with primary CMs.

Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI). However, age-related differences in pancreatic matrix digestion have not been studied in detail at the molecular level. To address this, we investigated ECM digestion in purified ECM proteins and in pancreatic tissue sections from younger (≤30 years; n = 5) and older (>55 years; n = 5) BMI matched donors, using Raman microspectroscopy (RMS). The Raman spectral profiles for purified collagens I, IV, VI and laminins were significantly altered following controlled enzyme treatment. Pancreatic cryosections were treated with Serva collagenase, NP, or the two enzymes combined, at clinically relevant concentrations. RMS demonstrated that the ECM at the islet-exocrine interface was differentially digested with respect to donor age. The action of collagenase was affected to a greater extent than NP. RMS is a powerful, marker-independent technology for characterising the human pancreatic ECM and demonstrating differences between donor types. Ongoing detailed studies using RMS will assist the development of donor-specific enzyme blends, increasing the overall success of human islet isolation and benefiting many people with type 1 diabetes worldwide. Statement of Significance Pancreatic islet transplantation is a minimally invasive treatment, which can reverse Type 1 Diabetes Mellitus (T1DM) in selected patients. Islets of Langerhans are extracted (isolated) from the exocrine tissue of human donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current enzymes are ineffective at digesting pancreases from younger donors. Using Raman microspectroscopy we demonstrate that donor age affects the enzymatic digestion of the pancreatic ECM at the molecular level. Collagenase activity is affected to a greater extent than NP. These findings will assist the development of donor-specific enzymes, thereby increasing the overall success of islet isolation and benefiting many people with T1DM worldwide.

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1−/− cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.

Aggregates of misfolded α-Synuclein in the brain represent a hallmark of Parkinson’s disease (PD). In patients and animal models, phosphorylated α-Synuclein was detected in the gut, hence, raising the hypothesis that early-stage PD could be diagnosed based on colon tissues. Marker-independent technologies represent an ideal method to monitor disease progression and potentially detect early-stage aggregated α-Synuclein in vivo. Here, formalin-fixed, paraffinembedded colon tissues of a transgenic rat model were analyzed using Raman imaging. Detailed spectral and imagebased analysis was performed indicating the major spectral shifts that alter in PD rat tissues in the amide I region. Peak fitting and multivariate analysis specified an increase of β-sheet proteins in transgenic rat colon compared with wild-type colon. In summary, Raman imaging is capable to detect α-Synuclein aggregates in colon tissues of a PD rat model, indicating that it could be a useful tool to support diagnosis in PD pathology.

OBJECTIVES Frozen cryopreservation (FC) with the vapour phase of liquid nitrogen storage (-135°C) is a standard biobank technique to preserve allogeneic heart valves to enable a preferable allograft valve replacement in clinical settings. However, their long-term function is limited by immune responses, inflammation and structural degeneration. Ice-free cryopreserved (IFC) valves with warmer storage possibilities at -80°C showed better matrix preservation and decreased immunological response in preliminary short-term in vivo studies. Our study aimed to assess the prolonged performance of IFC allografts in an orthotopic pulmonary sheep model. METHODS FC (n = 6) and IFC (n = 6) allografts were transplanted into juvenile Merino sheep. After 12 months of implantation, functionality testing via 2-dimensional echocardiography and histological analyses was performed. In addition, multiphoton autofluorescence imaging and Raman microspectroscopy analysis were applied to qualitatively and quantitatively assess the matrix integrity of the leaflets. RESULTS Six animals from the FC group and 5 animals from the IFC group were included in the analysis. Histological explant analysis showed early inflammation in the FC valves, whereas sustainable, fully functional, devitalized acellular IFC grafts were obtained. IFC valves showed excellent haemodynamic data with fewer gradients, no pulmonary regurgitation, no calcification and acellularity. Structural remodelling of the leaflet matrix structure was only detected in FC-treated tissue, whereas IFC valves maintained matrix integrity comparable to that of native controls. The collagen crimp period and amplitude and elastin structure were significantly different in the FC valve cusps compared to IFC and native cusps. Collagen fibres in the FC valves were less aligned and straightened. CONCLUSIONS IFC heart valves with good haemodynamic function, reduced immunogenicity and preserved matrix structures have the potential to overcome the known limitations of the clinically applied FC valve.

Myocardial infarction (MI) results in debilitating remodeling of the myocardial extracellular matrix (ECM). In this proof-of-principle study it was sought to modulate this aggressive remodeling by injecting a hyaluronic acid-based reservoir delivering exogenous microRNA-29B (miR-29B). This proof-of-principal study was executed whereby myocardial ischemia/reperfusion was performed on C57BL/6 mice for 45 min after which five 10 μL boluses of a hydrogel composed of thiolated hyaluronic acid cross-linked with poly (ethylene glycol) diacrylate, containing exogenous miR-29B as an active therapy, were injected into the border zone of the infarcted myocardium. Following surgery, the myocardial function of the animals was monitored up to 5 weeks. Delivering miR-29B locally using an injectable hyaluronan-based hydrogel resulted in the maintenance of myocardial function at 2 and 5 weeks following MI in this proof-of-principle study. In addition, while animals treated with the control of a nontargeting miR delivered using the hyaluronan-based hydrogel had a significant deterioration of myocardial function, those treated with miR-29B did not. Histological analysis revealed a significantly decreased presence of elastin and significantly less immature/newly deposited collagen fibers at the border zone of the infarct. Increased vascularity of the myocardial scar was also detected and Raman microspectroscopy discovered significantly altered ECM-specific biochemical signals at the border zone of the infarct. This preclinical proof-of-principle study demonstrates that an injectable hyaluronic acid hydrogel system could be capable of delivering miR-29B toward maintaining cardiac function following MI. In addition, Raman microspectroscopy revealed subtle, yet significant changes in ECM organization and maturity. These findings have great potential with regard to using injectable biomaterials as a local treatment for ischemic tissue and exogenous miRs to modulate tissue remodeling.

The extracellular matrix (ECM) is extensively remodeled in tumor tissues. Overproduction of collagens, pathological collagen crosslinking and alignment of fibers are major processes that ultimately result in an increased tissue stiffness. Although it is known that glycosaminoglycans (GAGs) play an important role in tumor signaling, their contribution to the biomechanical properties of tumor ECM is unknown. In this study, ECM structures of human colon carcinoma and normal (control) colon tissues were histologically identified. Using atomic force microscopy (AFM) nanoindentation, we show that the collagen-rich regions within the ECM of colon carcinoma tissues were significantly stiffer than the submucosal collagen-rich layer of control tissues. Screening of these regions with Raman microspectroscopy revealed significantly different molecular fingerprints for collagen fibers in colon carcinoma tissues compared to control tissues. We further showed an increased alignment of collagen fibers and elevated levels of GAG immuno-reactivity within the collagen network of colon carcinoma tissues. GAGs such as heparan sulfate and chondroitin sulfate were detected in significantly elevated levels in collagen fibers of carcinoma tissues. Moreover, immunodetection of the collagen-associated proteoglycan decorin was significantly decreased in carcinomas tissues of individual patients when compared with the corresponding control tissues. Overall a strong patient-to-patient variability was evident in the ECM composition, structure and biomechanics of individual colon carcinoma tissues. Although, biomechanical characteristics of tumor ECM were not directly impacted by GAG content, GAGs might play an important role during the mechanical and structural remodeling of pathological tumor ECM. To manipulate GAG expression and deposition in tumor microenvironments could represent a novel potential therapeutic strategy.

Objectives Allogeneic frozen cryopreserved heart valves (allografts or homografts) are commonly used in clinical practice. A major obstacle for their application is the limited availability in particular for paediatrics. Allogeneic large animal studies revealed that alternative ice-free cryopreservation (IFC) results in better matrix preservation and reduced immunogenicity. The objective of this study was to evaluate xenogeneic (porcine) compared with allogeneic (ovine) IFC heart valves in a large animal study. Methods IFC xenografts and allografts were transplanted in 12 juvenile merino sheep for 1-12 weeks. Immunohistochemistry, ex vivo computed tomography scans and transforming growth factor-β release profiles were analysed to evaluate postimplantation immunopathology. In addition, near-infrared multiphoton imaging and Raman spectroscopy were employed to evaluate matrix integrity of the leaflets. Results Acellular leaflets were observed in both groups 1 week after implantation. Allogeneic leaflets remained acellular throughout the entire study. In contrast, xenogeneic valves were infiltrated with abundant T-cells and severely thickened over time. No collagen or elastin changes could be detected in either group using multiphoton imaging. Raman spectroscopy with principal component analysis focusing on matrix-specific peaks confirmed no significant differences for explanted allografts. However, xenografts demonstrated clear matrix changes, enabling detection of distinct inflammatory-driven changes but without variations in the level of transforming growth factor-β. Conclusions Despite short-term success, mid-term failure of xenogeneic IFC grafts due to a T-cell-mediated extracellular matrix-triggered immune response was shown.

To achieve safer patient treatments, serum-free cell culture conditions have to be established for cell therapies. In previous studies, we demonstrated that serum-free culture favored the proliferation of MSCA-1+ osteoprogenitors derived from the jaw periosteum. In this study, the in vitro formation of bone-specific matrix by MSCA-1+ jaw periosteal cells (JPCs, 3 donors) was assessed and compared under serum-free and serum-containing media conditions using the marker-free Raman spectroscopy. Based on a standard fluorescence assay, JPCs from one patient were not able to mineralize under serum-containing culture conditions, whereas the other cells showed similar mineralization levels under both conditions. Raman spectra from mineralizing MSCA-1+ JPCs revealed higher levels of hydroxyapatite formation and higher mineral to matrix ratios under serum-free culture conditions. Higher carbonate to phosphate ratios and higher crystallinity in JPCs cultured under serum-containing conditions indicated immature bone formation. Due to reduced collagen production under serum-free conditions, we obtained significant differences in collagen maturity and proline to hydroxyproline ratios compared to serum-free conditions. We conclude that Raman spectroscopy is a useful tool for the assessment and noninvasive monitoring of in vitro mineralization of osteoprogenitor cells. Further studies should extend this knowledge and improve JPC mineralization by optimizing culture conditions.

Tissue-engineered skin equivalents based on primary isolated fibroblasts and keratinocytes have been shown to be useful tools for functional in vitro tests, including toxicological screenings and drug development. In this study, a commercially available squamous cell carcinoma (SCC) cell line SCC-25 was introduced into epidermal and full-thickness skin equivalents to generate human-based disease-in-a-dish model systems. Interestingly, when cultured either in the epidermis or dermis of full-thickness skin equivalents, SCC-25 cells formed hyper-keratinized tumor cell nests, a phenomenon that is frequently seen in the skin of patients afflicted with SCC. Raman spectroscopy was employed for the label-free cell phenotype characterization within the engineered skin equivalents and revealed the presence of differential protein patterns in keratinocytes and SCC-25 cells. To conclude, the here presented SSC disease-in-a-dish approaches offer the unique opportunity to model SSC in human skin in vitro, which will allow further insight into SSC disease progression, and the development of therapeutic strategies.

Fibrillin microfibrils and elastic fibers are critical determinants of elastic tissues where they define as tissue-specific architectures vital mechanical properties such as pliability and elastic recoil. Fibrillin microfibrils also facilitate elastic fiber formation and support the association of epithelial cells with the interstitial matrix. Mutations in fibrillin-1 (FBN1) are causative for the Marfan syndrome, a congenital multisystem disorder characterized by progressive deterioration of the fibrillin microfibril/ elastic fiber architecture in the cardiovascular, musculoskeletal, ocular, and dermal system. In this study, we utilized Raman microspectroscopy in combination with principal component analysis (PCA) to analyze the molecular consequences of fibrillin-1 deficiency in skin of a mouse model (GT8) of Marfan syndrome. In addition, full-thickness skin models incorporating murine wild-type and Fbn1GT8/GT8 fibroblasts as well as human HaCaT keratinocytes were generated and analyzed. Skin models containing GT8 fibroblasts showed an altered epidermal morphology when compared to wild-type models indicating a new role for fibrillin-1 in dermal-epidermal crosstalk. Obtained Raman spectra together with PCA allowed to discriminate between healthy and deficient microfibrillar networks in murine dermis and skin models. Interestingly, results obtained from GT8 dermis and skin models showed similar alterations in molecular signatures triggered by fibrillin-1 deficiency such as amide III vibrations and decreased levels of glycan vibrations. Overall, this study indicates that Raman microspectroscopy has the potential to analyze subtle changes in fibrillin-1 microfibrils and elastic fiber networks. Therefore Raman microspectroscopy may be utilized as a non-invasive and sensitive diagnostic tool to identify connective tissue disorders and monitor their disease progression. Statement of Significance Mutations in building blocks of the fibrillin microfibril/ elastic fiber network manifest in disease conditions such as aneurysms, emphysema or lax skin. Understanding how structural changes induced by fibrillin-1 mutation impact the architecture of fibrillin microfibrils, which then translates into an altered activation state of targeted growth factors, represents a huge challenge in elucidating the genotype-phenotype correlations in connective tissue disorders such as Marfan syndrome. This study shows that Raman microspectroscopy is able to reveal structural changes in fibrillin-1 microfibrils and elastic fiber networks and to discriminate between normal and diseased networks in vivo and in vitro. Therefore Raman microspectroscopy may be utilized as a non-invasive and sensitive diagnostic tool to identify connective tissue disorders and monitor their disease progression.

Electroconductive substrates are emerging as promising functional materials for biomedical applications. Here, the development of biohybrids of collagen and pristine graphene that effectively harness both the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching native cardiac tissue) obtainable with pristine graphene is reported. As well as improving substrate physical properties, the addition of pristine graphene also enhances human cardiac fibroblast growth while simultaneously inhibiting bacterial attachment (Staphylococcus aureus). When embryonic-stem-cell-derived cardiomyocytes (ESC-CMs) are cultured on the substrates, biohybrids containing 32 wt% graphene significantly increase metabolic activity and cross-striated sarcomeric structures, indicative of the improved substrate suitability. By then applying electrical stimulation to these conductive biohybrid substrates, an enhancement of the alignment and maturation of the ESC-CMs is achieved. While this in vitro work has clearly shown the potential of these materials to be translated for cardiac applications, it is proposed that these graphene-based biohybrid platforms have potential for a myriad of other applications-particularly in electrically sensitive tissues, such as neural and neural and musculoskeletal tissues.

Tissue-engineered organs and implants hold promise for the replacement of damaged and diseased organs. However, the foreign body response (FBR) is a major obstacle that compromises the function of tissue-engineered constructs, typically causing them to fail. Two components of FBR are an inflammatory response and a lack of vascularization. To overcome these limitations, a collagen system was developed to release interleukin-6 (IL-6) siRNA and endothelial nitric oxide synthase (eNOS) pDNA in a staggered manner. Hollow collagen microspheres were assembled into a collagen sphere-in-hydrogel system that displayed a staggered release profile in vitro. This system was assessed in vivo in a subcutaneous rat model. The doses of IL-6 siRNA and eNOS pDNA were first individually optimized for their ability to reduce the volume fraction of inflammatory cells (7 days) and increase the length density of blood vessels (14 days), respectively. The identified optimal doses were combined, and the ability of the system to decrease the volume fraction of inflammatory cells and increase the length density of blood vessels was confirmed at both 7 and 14 days. Analysis of the tissue using Raman microspectroscopy revealed that in addition to changes in inflammation and angiogenesis, there were also changes in the extracellular matrix (ECM) at seven days. While changes in sulfated glycosaminoglycan (sGAG) content of the ECM were not detected, changes in the binding of sGAG of the ECM to growth factors were observed. Two growth factors tested, VEGF165 and bFGF showed increased binding to sGAG extracted from eNOS pDNA-treated samples at seven days, increasing the angiogenic potential of the ECM. Thus, we observe that changes in the tissue in terms of the balance of inflammation and angiogenesis as well changes in the activity of sGAG of the ECM occurs following dual delivery of nucleic acids from the collagen sphere-in-hydrogel system.

Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes.

Proteoglycans (PGs) are crucial extracellular matrix (ECM) components that are present in all tissues and organs. Pathological remodeling of these macromolecules can lead to severe diseases such as osteoarthritis or rheumatoid arthritis. To date, PG-associated ECM alterations are routinely diagnosed by invasive analytical methods. Here, we employed Raman microspectroscopy, a laser-based, marker-free and non-destructive technique that allows the generation of spectra with peaks originating from molecular vibrations within a sample, to identify specific Raman bands that can be assigned to PGs within human and porcine cartilage samples and chondrocytes. Based on the non-invasively acquired Raman spectra, we further revealed that a prolonged in vitro culture leads to phenotypic alterations of chondrocytes, resulting in a decreased PG synthesis rate and loss of lipid contents. Our results are the first to demonstrate the applicability of Raman microspectroscopy as an analytical and potential diagnostic tool for non-invasive cell and tissue state monitoring of cartilage in biomedical research. ((c) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

As a possible viable and non‐invasive method to identify high producing cells, Confocal Raman Microscopy was shown to be able to differentiate CHO host cell lines and derivative production clones. Cluster analysis of spectra and their derivatives was able to differentiate between different producer cell lines and a host, and also distinguished between an intracellular region of high lipid and protein content that in structure resembles the Endoplasmic Reticulum. This ability to identify the ER may be a major contributor to the identification of high producers. PCA enabled the discrimination even of host cell lines and their subclones with inherently higher production capacity. The method is thus a promising option that may contribute to early, non‐invasive identification of high potential candidates during cell line development and possibly could also be used for proof of identity of established production clones.

Raman spectroscopy is an established laser-based technology for the quality assurance of pharmaceutical products. Over the past few years, Raman spectroscopy has become a powerful diagnostic tool in the life sciences. Raman spectra allow assessment of the overall molecular constitution of biological samples, based on specific signals from proteins, nucleic acids, lipids, carbohydrates, and inorganic crystals. Measurements are non-invasive and do not require sample processing, making Raman spectroscopy a reliable and robust method with numerous applications in biomedicine. Moreover, Raman spectroscopy allows the highly sensitive discrimination of bacteria. Rama spectra retain information on continuous metabolic processes and kinetics such as lipid storage and recombinant protein production. Raman spectra are specific for each cell type and provide additional information on cell viability, differentiation status, and tumorigenicity. In tissues, Raman spectroscopy can detect major extracellular matrix components and their secondary structures. Furthermore, the non-invasive characterization of healthy and pathological tissues as well as quality control and process monitoring of in vitro-engineered matrix is possible. This review provides comprehensive insight to the current progress in expanding the applicability of Raman spectroscopy for the characterization of living cells and tissues, and serves as a good reference point for those starting in the field.

Summary One major obstacle to the application of stem cell-derived cardiomyocytes (CMs) for disease modeling and clinical therapies is the inability to identify the developmental stage of these cells without the need for genetic manipulation or utilization of exogenous markers. In this study, we demonstrate that Raman microspectroscopy can non-invasively identify embryonic stem cell (ESC)-derived chamber-specific CMs and monitor cell maturation. Using this marker-free approach, Raman peaks were identified for atrial and ventricular CMs, ESCs were successfully discriminated from their cardiac derivatives, a distinct phenotypic spectrum for ESC-derived CMs was confirmed, and unique spectral differences between fetal versus adult CMs were detected. The real-time identification and characterization of CMs, their progenitors, and subpopulations by Raman microspectroscopy strongly correlated to the phenotypical features of these cells. Due to its high molecular resolution, Raman microspectroscopy offers distinct analytical characterization for differentiating cardiovascular cell populations., Graphical Abstract, Highlights • Raman microspectroscopy identifies intrinsic molecular signatures of individual cells • Cardiovascular cell fate decisions can be assessed non-invasively • Chamber specificity is monitored marker-free and without genetic manipulation • Cardiac cell maturation and cell quality can be screened online in real time, Application of stem cell-derived cardiomyocytes (CMs) for disease modeling and clinical therapies is limited by the inability to identify these cells without genetic manipulation or exogenous markers. In this article, Schenke-Layland and colleagues demonstrate that Raman microspectroscopy is a suitable tool for the real-time marker-free identification and characterization of chamber-specific primary and pluripotent stem cell-derived CMs, their progenitors, and subpopulations, and to monitor CM maturation.

The increasing prevalence of diabetes, its heterogeneity, and the limited number of treatment options drive the need for physiologically relevant assay platforms with human genetic background that have the potential to improve mechanistic understanding and e\xpedite diabetes-related research and treatment. In this study, we developed an endocrine pancreas-on-a-chip model based on a tailored microfluidic platform, which enables self-guided trapping of single human pseudo-islets. Continuous, low-shear perfusion provides a physiologically relevant microenvironment especially important for modeling and monitoring of the endocrine function as well as sufficient supply with nutrients and oxygen. Human pseudo-islets, generated from the conditionally immortalized EndoC-βH3 cell line, were successfully injected by hydrostatic pressure-driven flow without altered viability. To track insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, dynamic sampling of the supernatant as well as non-invasive real-time monitoring using Raman microspectroscopy was established on-chip. Dynamic sampling indicated a biphasic glucose-stimulated insulin response. Raman microspectroscopy allowed to trace glucose responsiveness in situ and to visualize different molecular structures such as lipids, mitochondria and nuclei. In-depth spectral analyses demonstrated a glucose stimulation-dependent, increased mitochondrial activity, and a switch in lipid composition of insulin secreting vesicles, supporting the high performance of our pancreas-on-a-chip model.