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Type 2 diabetes (T2D) constitutes a major public health problem, and despite prevention efforts, this pandemic disease is one of the deadliest diseases in the world. In 2022, 6.7 million patients with T2D died prematurely from vascular complications. Indeed, diabetes increases the risk of myocardial infarction or stroke eightfold. The identification of the molecular factors involved in the occurrence of cardiovascular complications and their prevention are therefore major axes. Our hypothesis is that factors brought into play during physiological aging appear prematurely with diabetes progression. Our study focused on the aging of the extracellular matrix (ECM), a major element in the maintenance of vascular homeostasis. We characterized the morphological and functional aspects of aorta, with a focus on the collagen and elastic fibers of diabetic mice aged from 6 mo to nondiabetic mice aged 6 mo and 20 mo. The comparison with the two nondiabetic models (young and old) highlighted an exacerbated activity of proteases, which could explain a disturbance in the collagen accumulation and an excessive degradation of elastic fibers. Moreover, the generation of circulating elastin-derived peptides reflects premature aging of the ECM. These extracellular elements contribute to the appearance of vascular rigidity, often the origin of pathologies such as hypertension and atherosclerosis. In conclusion, we show that diabetic mice aged 6 mo present the same characteristics of ECM wear as those observed in mice aged 20 mo. This accelerated aortic wall remodeling could then explain the early onset of cardiovascular diseases and, therefore, the premature death of patients with T2D. NEW & NOTEWORTHY Aortic elastic fibers of young (6-mo old) individuals with diabetes degrade prematurely and exhibit an appearance like that found in aged (20-mo old) nondiabetic mice. Exacerbated elastolysis and elastin-derived peptide production are characteristic elements, contributing to early aortic wall rigidity and hypertension development. Therefore, limiting this early aging could be a judicious therapeutic approach to reduce cardiovascular complications and premature death in patients with diabetes.

Transplantation of stem cell-derived β-cells is a promising therapeutic advancement in the treatment of type 1 diabetes mellitus. A current limitation of this approach is the long differentiation timeline that generates a heterogeneous population of pancreatic endocrine cells. To address this limitation, an inducible lentiviral overexpression system of mature β-cell markers was introduced into human induced-pluripotent stem cells (hiPSCs). Following the selection of the successfully transduced hiPSCs, the cells were treated with doxycycline in the pancreatic progenitor induction medium to support their transition toward the pancreatic lineage. Cells cultured with doxycycline presented the markers of interest, NGN3, PDX1, and MAFA, after five days of culture, and glucose-stimulated insulin secretion assays demonstrated that the cells were glucose-responsive in a monolayer culture. When cultured as a spheroid, the markers of interest and insulin secretion in a static glucose-stimulated insulin secretion assay were maintained; however, insulin secretion upon consecutive glucose challenges was limited. Comparison to human fetal and adult donor tissues identified that although the hiPSC-derived spheroids present similar markers to adult insulin-producing cells, they are functionally representative of fetal development. Together, these results suggest that with optimization of the temporal expression of these markers, forward programming of hiPSCs towards insulin-producing cells could be a possible alternative for islet transplantation., (© 2024. The Author(s).)

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs 1-3 . Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion., (© 2024. The Author(s).)

Pancreatic in vitro research is of major importance to advance mechanistic understanding and development of treatment options for diseases such as diabetes mellitus. We present a thermoplastic-based microphysiological system aiming to model the complex microphysiological structure and function of the endocrine pancreas with concurrent real-time read-out capabilities. The specifically tailored platform enables self-guided trapping of single islets at defined locations: β-cells are assembled to pseudo-islets and injected into the tissue chamber using hydrostatic pressure-driven flow. The pseudo-islets can further be embedded in an ECM-like hydrogel mimicking the native microenvironment of pancreatic islets in vivo . Non-invasive real-time monitoring of the oxygen levels on-chip is realized by the integration of luminescence-based optical sensors to the platform. To monitor insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, an automated cycling of different glucose conditions is implemented. The model's response to glucose stimulation can be monitored via offline analysis of insulin secretion and via specific changes in oxygen consumption due to higher metabolic activity of pseudo-islets at high glucose levels. To demonstrate applicability for drug testing, the effects of antidiabetic medications are assessed and changes in dynamic insulin secretion are observed in line with the respective mechanism of action. Finally, by integrating human pancreatic islet microtissues, we highlight the flexibility of the platform and demonstrate the preservation of long-term functionality of human endocrine pancreatic tissue.

Background: The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma., Methods: We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo., Results: ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo., Conclusion: In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials., (© 2024. The Author(s).)

Background: The prevalence of COVID-19 breakthrough infections in healthcare workers (HCWs) remains an issue of concern. This study examines the different characteristics associated with breakthrough infections in HCWs. Methods: From the total participants in the TüSeRe:exact study (n = 1046), we specifically included study participants who had received three vaccinations and were not infected prior to the third vaccination. Participants were invited to complete an online questionnaire, which included inquiries about any breakthrough infections they might have experienced. Univariate Cox regression analysis was used to investigate the association between participant characteristics and breakthrough infections. Results: Among 629 HCWs (497 female and 132 male), 241 (38%) experienced breakthrough infections during the follow-up period. The frequency of breakthrough infections was 39.2% (195/497) among female participants and 34.8% (46/132) among male participants ( p = 0.357). The Cox regression model adjusted for age and sex showed that participants with cardiovascular disease (hazard ratio (95%CI) = 0.621 (0.392-0.985); p = 0.043) and those taking antihypertensives (hazard ratio (95%CI) = 0.551 (0.331-0.915); p = 0.021) had a significantly lower hazard ratio for breakthrough infections. The use of analgesics after the first vaccine (hazard ratio (95%CI) = 1.343 (1.025-1.759); p = 0.032) was associated with an increased risk of breakthrough infections. Conclusions: These findings can inform targeted preventive measures and risk management strategies to protect frontline workers and maintain a resilient healthcare system during the ongoing pandemic.

In the context of multimodal treatments for abdominal cancer, including procedures such as cytoreductive surgery and intraperitoneal chemotherapy, recurrence rates remain high, and long-term survival benefits are uncertain due to post-operative complications. Notably, treatment-limiting side effects often arise from an uncontrolled activation of the immune system, particularly peritoneally localized macrophages, leading to massive cytokine secretion and phenotype changes. Exploring alternatives, an increasing number of studies investigated the potential of plasma-activated liquids (PAL) for adjuvant peritoneal cancer treatment, aiming to mitigate side effects, preserve healthy tissue, and reduce cytotoxicity towards non-cancer cells. To assess the non-toxicity of PAL, we isolated primary human macrophages from the peritoneum and subjected them to PAL exposure. Employing an extensive methodological spectrum, including flow cytometry, Raman microspectroscopy, and DigiWest protein analysis, we observed a pronounced resistance of macrophages towards PAL. This resistance was characterized by an upregulation of proliferation and anti-oxidative pathways, countering PAL-derived oxidative stress-induced cell death. The observed cellular effects of PAL treatment on human tissue-resident peritoneal macrophages unveil a potential avenue for PAL-derived immunomodulatory effects within the human peritoneal cavity. Our findings contribute to understanding the intricate interplay between PAL and macrophages, shedding light on the promising prospects for PAL in the adjuvant treatment of peritoneal cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Schultze-Rhonhof, Marzi, Carvajal Berrio, Holl, Braun, Schäfer-Ruoff, Andress, Bachmann, Templin, Brucker, Schenke-Layland and Weiss.)

The extracellular matrix (ECM) is essential for cell support during homeostasis and plays a critical role in cancer. Although research often concentrates on the tumor's cellular aspect, attention is growing for the importance of the cancer-associated ECM. Biochemical and physical ECM signals affect tumor formation, invasion, metastasis, and therapy resistance. Examining the tumor microenvironment uncovers intricate ECM dysregulation and interactions with cancer and stromal cells. Anticancer therapies targeting ECM sensors and remodelers, including integrins and matrix metalloproteinases, and ECM-remodeling cells, have seen limited success. This review explores the ECM's role in cancer and discusses potential therapeutic strategies for cell-ECM interactions.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established., (Creative Commons Attribution license.)

Spatial transcriptomics of histological sections have revolutionized research in life sciences and enabled unprecedented insights into genetic processes involved in tissue reorganization. However, in contrast to genomic analysis, the actual biomolecular composition of the sample has fallen behind, leaving a gap of potentially highly valuable information. Raman microspectroscopy provides untargeted spatiomolecular information at high resolution, capable of filling this gap. In this study we demonstrate spatially resolved Raman "spectromics" to reveal homogeneity, heterogeneity and dynamics of cell matrix on molecular levels by repurposing state-of-the-art bioinformatic analysis tools commonly used for transcriptomic analyses. By exploring sections of murine myocardial infarction and cardiac hypertrophy, we identify myocardial subclusters when spatially approaching the pathology, and define the surrounding metabolic and cellular (immune-) landscape. Our innovative, label-free, non-invasive "spectromics" approach could therefore open perspectives for a profound characterization of histological samples, while additionally allowing the combination with consecutive downstream analyses of the very same specimen., (© 2023. Springer Nature Limited.)

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Cardiomyopathies are associated with fibrotic remodeling of the heart, which is characterized by the excessive accumulation of collagen type I (COL I) due to chronic inflammation and suspected epigenetic influences. Despite the severity and high mortality rate of cardiac fibrosis, current treatment options are often inadequate, underscoring the importance of gaining a deeper understanding of the disease's underlying molecular and cellular mechanisms. In this study, the extracellular matrix (ECM) and nuclei in fibrotic areas of different cardiomyopathies were molecularly characterized by Raman microspectroscopy and imaging and compared with the control myocardium. Patient samples were obtained from heart tissue affected by ischemia, hypertrophy, and dilated cardiomyopathy and analyzed for fibrosis through conventional histology and marker-independent Raman microspectroscopy (RMS). Prominent differences between control myocardium and cardiomyopathies were revealed by spectral deconvolution of COL I Raman spectra. Statistically significant differences were identified in the amide I region of spectral subpeak at 1,608 cm -1 , which is a representative endogenous marker for alterations in the structural conformation of COL I fibers. Moreover, epigenetic 5mC DNA modification was identified within cell nuclei by multivariate analysis. A statistically significant increase in signal intensities of spectral features indicative of DNA methylation was detected in cardiomyopathies in accordance with immunofluorescence 5mC staining. Overall, RMS is a versatile technology in the discrimination of cardiomyopathies based on molecular evaluation of COL I and nuclei while providing insights into the pathogenesis of the diseases. NEW & NOTEWORTHY Cardiomyopathies are associated with severe fibrotic remodeling of the heart, which is characterized by the excessive accumulation of collagen type I (COL I). In this study, we used marker-independent Raman microspectroscopy (RMS) to gain a deeper understanding of the disease's underlying molecular and cellular mechanisms.

Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Katja Schenke-Layland reports financial support was provided by German Research Foundation. Katja Schenke-Layland & Garry Duffy reports financial support was provided by 10.13039/501100000780European Union. Chuan-En Lu reports equipment, drugs, or supplies was provided by Boston Scientific Ireland Ltd. Katja Schenke-Layland reports financial support was provided by State of Baden-Wurttemberg Ministry for Science Research and Art. Giulio Ghersi reports a relationship with Abiel Sr that includes: employment., (© 2023 The Authors.)

Fibrosis is a consequence of the pathological remodeling of extracellular matrix (ECM) structures in the connective tissue of an organ. It is often caused by chronic inflammation, which over time, progressively leads to an excess deposition of collagen type I (COL I) that replaces healthy tissue structures, in many cases leaving a stiff scar. Increasing fibrosis can lead to organ failure and death; therefore, developing methods that potentially allow real-time monitoring of early onset or progression of fibrosis are highly valuable. In this study, the ECM structures of diseased and healthy human tissue from multiple organs were investigated for the presence of fibrosis using routine histology and marker-independent Raman microspectroscopy and Raman imaging. Spectral deconvolution of COL I Raman spectra allowed the discrimination of fibrotic and non-fibrotic COL I fibers. Statistically significant differences were identified in the amide I region of the spectral subpeak at 1608 cm -1 , which was deemed to be representative for structural changes in COL I fibers in all examined fibrotic tissues. Raman spectroscopy-based methods in combination with this newly discovered spectroscopic biomarker potentially offer a diagnostic approach to non-invasively track and monitor the progression of fibrosis. STATEMENT OF SIGNIFICANCE: Current diagnosis of fibrosis still relies on histopathological examination with invasive biopsy procedures. Although, several non-invasive imaging techniques such as positron emission tomography, single-photon emission computed tomography and second harmonic generation are gradually employed in preclinical or clinical studies, these techniques are limited in spatial resolution and the morphological interpretation highly relies on individual experience and knowledge. In this study, we propose a non-destructive technique, Raman microspectroscopy, to discriminate fibrotic changes of collagen type I based on a molecular biomarker. The changes of the secondary structure of collagen type I can be identified by spectral deconvolution, which potentially can provide an automatic diagnosis for fibrotic tissues in the clinical applicaion., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Electrospinning has become a well-established method for creating nanofibrous meshes for tissue-engineering applications. The incorporation of natural extracellular components, such as electrospun pure collagen nanofibers, has proven to be particularly challenging, as electrospun collagen nanofibers do not constitute native collagen fibers anymore. In this study, we show that this detrimental effect is not only limited to fluorinated solvents, as previously thought. Rat tail collagen was dissolved in acetic acid and ethanol and electrospun at various temperatures. Electrospun collagen mats were analyzed using circular dichroism, enzymatic digestion, SDS-PAGE, western blotting, and Raman spectroscopy and compared to heat-denaturated and electrospun collagen in HFIP. Our data suggest that even non-fluorinated electrospinning solvents, such as acid-based solvents, do not yield structurally intact fibers. Interestingly, neither epithelial cells nor fibroblasts displayed a different cellular response to electrospun collagen compared to collagen-coated polyurethane scaffolds in F-actin staining and metabolic analysis using fluorescent lifetime imaging. The disruption of the structural integrity of collagen might therefore be underestimated based on the cell-material interactions alone. These observations expose the larger than anticipated vulnerability of collagen in the electrospinning process. Based on these findings, the influence of the electrospinning process on the distinct biochemical properties of collagen should always be considered, when ECM-mimicking fibrous constructs are manufactured.

Synthetic bone substitute materials (BSMs) are becoming the general trend, replacing autologous grafting for bone tissue engineering (BTE) in orthopedic research and clinical practice. As the main component of bone matrix, collagen type I has played a critical role in the construction of ideal synthetic BSMs for decades. Significant strides have been made in the field of collagen research, including the exploration of various collagen types, structures, and sources, the optimization of preparation techniques, modification technologies, and the manufacture of various collagen-based materials. However, the poor mechanical properties, fast degradation, and lack of osteoconductive activity of collagen-based materials caused inefficient bone replacement and limited their translation into clinical reality. In the area of BTE, so far, attempts have focused on the preparation of collagen-based biomimetic BSMs, along with other inorganic materials and bioactive substances. By reviewing the approved products on the market, this manuscript updates the latest applications of collagen-based materials in bone regeneration and highlights the potential for further development in the field of BTE over the next ten years.

Background: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction., Methods: We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals., Results: While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose., Conclusions: Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies., Competing Interests: Potential conflicts of interest. N. S. M. was a speaker at previous Luminex user meetings. The NMI is involved in applied research projects as a fee for services with the Luminex Corporation. M. Bi. reports payment or honoraria from MSD Sharp & Dohme GmbH for symposia; and also reports participation on advisory boards for Roche Pharma AG, Incyte Biosciences Germany GmbH, Bayer Vital GmbH, Bristol-Myers Squibb GmbH & Co KgaA, and MSD Sharp & Dohme GmbH. C. E. reports support for the present manuscript from MWK Sonderfördermaßnahme Kinderstudie (Kap. 1499 TG 93). B. L. reports receiving funding for the present manuscript from NaFOUniMedCovid19 (FKZ: 01KX2021) supported by the German Federal Ministry of Education and Research; and reports a leadership or fiduciary role for the German Center for Infection Research (TI BBD, DZIF), Transplant Cohort, and Steering Committee TBNet. A. Z. reports state technology funding for device infrastructure (7-4332.62-NMI/55), outside the conduct of this study. N. S.-M. reports support for the present manuscript from LAND BW (MULTICOV-AB and LAND BW, Automation in SARS-CoV-2) and payment or honoraria from Luminex Corporation for being a speaker at previous user meetings (the NMI is also involved in applied research projects as a fee for services with the Luminex Corporation). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Invasive lobular carcinoma (ILC) is a distinct type of breast cancer and is accounting up to 10-15 % of all mammary carcinomas showing a pronounced increase in incidence rates over the last two decades. We generated three induced pluripotent stem cell (iPSC) lines from CD34+ progenitor cells isolated from a mammary carcinoma patient diagnosed with ILC. Here, we describe the characterization of the iPSCs by array-based comparative genomic hybridization (array CGH), immunocytochemistry, flow cytometry, reverse transcriptase polymerase chain reaction and directed in vitro differentiation. The iPSC lines will find application in the field of breast cancer research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Transplantation of islets of Langerhans is a promising alternative treatment strategy in severe cases of type 1 diabetes mellitus; however, the success rate is limited by the survival rate of the cells post-transplantation. Restoration of the native pancreatic niche during transplantation potentially can help to improve cell viability and function. Here, we assessed for the first time the regulatory role of the small leucine-rich proteoglycan decorin (DCN) in insulin secretion in human β-cells, and its impact on pancreatic extracellular matrix (ECM) protein expression in vitro. In depth analyses utilizing next-generation sequencing as well as Raman microspectroscopy and Raman imaging identified pathways related to glucose metabolism to be upregulated in DCN-treated cells, including oxidative phosphorylation within the mitochondria as well as proteins and lipids of the endoplasmic reticulum. We further showed the effectiveness of DCN in a transplantation setting by treating collagen type 1-encapsulated β-cell-containing pseudo-islets with DCN. Taken together, in this study, we demonstrate the potential of DCN to improve the function of insulin-secreting β-cells while reducing the expression of ECM proteins affiliated with fibrotic capsule formation, making DCN a highly promising therapeutic agent for islet transplantation., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

As the Corona Disease 2019 (COVID-19) caused by SARS-CoV-2 persists, vaccination is one of the key measures to contain the spread. Side effects (SE) from vaccination are one of the reasons for reluctance to vaccinate. We systematically investigated self-reported SE after the first, second, and booster vaccinations. The data were collected during the TüSeRe: exact study (Tübinger Monitoring Studie zur exakten Analyse der Immunantwort nach Vakzinierung). Employees of health and research institutions were invited to participate. Study participants were asked to fill out an online questionnaire and report their SE after each dose of SARS-CoV-2 vaccination. A total of 1046 participants (mean age: 44 ± 12.9 years; female, n = 815 (78%); male, n = 231 (22%)) were included in the analysis. Local and systemic SE were more frequent after receiving the vector-based vaccine ChAdOx1 nCoV-19 in the first vaccination. However, local and systemic SE were more common after receiving mRNA vaccines (BNT162b2, mRNA-1273) in the second vaccination. Compared to the BNT162b2 vaccine, more SE have been observed after receiving the mRNA-1273 vaccine in the booster vaccination. In multivariate analysis, local and systemic side effects were associated with vaccine type, age and gender. Local and systemic SE are common after SARS-CoV-2 vaccines. The frequency of self-reported local and systemic SE differ significantly between mRNA and vector-based vaccines.

Conventional synthesis routes for thermoplastic polyurethanes (TPUs) still require the use of isocyanates and tin-based catalysts, which pose considerable safety and environmental hazards. To reduce both the ecological footprint and human health dangers for nonwoven TPU scaffolds, it is key to establish a green synthesis route, which eliminates the use of these toxic compounds and results in biocompatible TPUs with facile processability. In this study, we developed high-molecular-weight nonisocyanate polyurethanes (NIPUs) through transurethanization of 1,6-hexanedicarbamate with polycarbonate diols (PCDLs). Various molecular weights of PCDL were employed to maximize the molecular weight of NIPUs and consequently facilitate their electrospinnability. The synthesized NIPUs were characterized by nuclear magnetic resonance, Fourier-transform infrared spectroscopy, gel permeation chromatography, and differential scanning calorimetry. The highest achieved molecular weight ( M w ) was 58,600 g/mol. The NIPUs were consecutively electrospun into fibrous scaffolds with fiber diameters in the submicron range, as shown by scanning electron microscopy (SEM). To assess the suitability of electrospun NIPU mats as a possible biomimetic load-bearing pericardial substitute in cardiac tissue engineering, their cytotoxicity was investigated in vitro using primary human fibroblasts and a human epithelial cell line. The bare NIPU mats did not need further biofunctionalization to enhance cell adhesion, as it was not outperformed by collagen-functionalized NIPU mats and hence showed that the NIPU mats possess a great potential for use in biomimetic scaffolds., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

CD34+ cells were isolated from peripheral blood of a breast cancer patient. By the introduction of five integration-free episomal vectors, the CD34+ cells were successfully reprogrammed and resulted in four iPSC clones. Flow Cytometry, reverse transcriptase PCR and immunocytochemistry confirm a robust expression of pluripotency factors and the concomitant loss of exogenous reprogramming plasmids. The maintenance of genomic integrity was confirmed by array-based comparative genomic hybridization and iPSCs harbored the capacity to differentiate into all three germ layers. Here, we present the generation and characterization of four iPSC lines that will find application in the field of breast cancer research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

In cancer, the complex interplay between tumor cells and the tumor microenvironment results in the modulation of signaling processes. By assessing the expression of a multitude of proteins and protein variants in cancer tissue, wide-ranging information on signaling pathway activation and the status of the immunological landscape is obtainable and may provide viable information on the treatment response. Archived breast cancer tissues from a cohort of 84 patients (no adjuvant therapy) were analyzed by high-throughput Western blotting, and the expression of 150 proteins covering central cancer pathways and immune cell markers was examined. By assessing CD8α, CD11c, CD16 and CD68 expression, immune cell infiltration was determined and revealed a strong correlation between event-free patient survival and the infiltration of immune cells. The presence of tumor-infiltrating lymphocytes was linked to the pronounced activation of the Jak/Stat signaling pathway and apoptotic processes. The elevated phosphorylation of PPARγ (pS112) in non-immune-infiltrated tumors suggests a novel immune evasion mechanism in breast cancer characterized by increased PPARγ phosphorylation. Multiplexed immune cell marker assessment and the protein profiling of tumor tissue provide functional signaling data facilitating breast cancer patient stratification., Competing Interests: The authors declare no conflict of interest.

Endothelial cells (ECs) are a key cellular component of the vascular system as they form the inner lining of the blood vessels. Recent findings highlight that ECs express extensive phenotypic heterogenicity when following the vascular tree from the major vasculature down to the organ capillaries. However, in vitro models, used for drug development and testing, or to study the role of ECs in health and disease, rarely acknowledge this EC heterogenicity. In this review, we highlight the main differences between different EC types, briefly summarize their different characteristics and focus on the use of ECs in in vitro models. We introduce different approaches on how ECs can be utilized in co-culture test systems in the field of brain, pancreas, and liver research to study the role of the endothelium in health and disease. Finally, we discuss potential improvements to current state-of-the-art in vitro models and future directions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Three-dimensional (3D) organoid culture recapitulating patient-specific histopathological and molecular diversity offers great promise for precision medicine in cancer. In this study, we established label-free imaging procedures, including Raman microspectroscopy (RMS) and fluorescence lifetime imaging microscopy (FLIM), for in situ cellular analysis and metabolic monitoring of drug treatment efficacy. Primary tumor and urine specimens were utilized to generate bladder cancer organoids, which were further treated with various concentrations of pharmaceutical agents relevant for the treatment of bladder cancer (i.e., cisplatin, venetoclax). Direct cellular response upon drug treatment was monitored by RMS. Raman spectra of treated and untreated bladder cancer organoids were compared using multivariate data analysis to monitor the impact of drugs on subcellular structures such as nuclei and mitochondria based on shifts and intensity changes of specific molecular vibrations. The effects of different drugs on cell metabolism were assessed by the local autofluorophore environment of NADH and FAD, determined by multiexponential fitting of lifetime decays. Data-driven neural network and data validation analyses (k-means clustering) were performed to retrieve additional and non-biased biomarkers for the classification of drug-specific responsiveness. Together, FLIM and RMS allowed for non-invasive and molecular-sensitive monitoring of tumor-drug interactions, providing the potential to determine and optimize patient-specific treatment efficacy.

In light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease, we developed a 3D preclinical tumor model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) to identify individual treatment vulnerabilities and validate chemo-, immuno- and targeted therapy efficacies. Enzymatic digestion of primary ovarian cancer tissue and cultivation in defined serum-free media allowed rapid and efficient recovery of PDM, while preserving histopathological features of corresponding patient tumor tissue. Reverse-phase protein array (RPPA)-analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Co-cultures of PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Combining protein pathway analysis and drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision making.

Obesity and associated diseases, such as diabetes, have reached epidemic proportions globally. In this era of "diabesity", white adipose tissue (WAT) has become a target of high interest for therapeutic strategies. To gain insights into mechanisms of adipose (patho-)physiology, researchers traditionally relied on animal models. Leveraging Organ-on-Chip technology, a microphysiological in vitro model of human WAT is introduced: a tailored microfluidic platform featuring vasculature-like perfusion that integrates 3D tissues comprising all major WAT-associated cellular components (mature adipocytes, organotypic endothelial barriers, stromovascular cells including adipose tissue macrophages) in an autologous manner and recapitulates pivotal WAT functions, such as energy storage and mobilization as well as endocrine and immunomodulatory activities. A precisely controllable bottom-up approach enables the generation of a multitude of replicates per donor circumventing inter-donor variability issues and paving the way for personalized medicine. Moreover, it allows to adjust the model's degree of complexity via a flexible mix-and-match approach. This WAT-on-Chip system constitutes the first human-based, autologous, and immunocompetent in vitro adipose tissue model that recapitulates almost full tissue heterogeneity and can become a powerful tool for human-relevant research in the field of metabolism and its associated diseases as well as for compound testing and personalized- and precision medicine applications., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The equilibrium between scaffold degradation and neotissue formation, is highly essential for in situ tissue engineering. Herein, biodegradable grafts function as temporal roadmap to guide regeneration. The ability to monitor and understand the dynamics of degradation and tissue deposition in in situ cardiovascular graft materials is therefore of great value to accelerate the implementation of safe and sustainable tissue-engineered vascular grafts (TEVGs) as a substitute for conventional prosthetic grafts. In this study, we investigated the potential of Raman microspectroscopy and Raman imaging to monitor degradation kinetics of supramolecular polymers, which are employed as degradable scaffolds in in situ tissue engineering. Raman imaging was applied on in vitro degraded polymers, investigating two different polymer materials, subjected to oxidative and enzymatically-induced degradation. Furthermore, the method was transferred to analyze in vivo degradation of tissue-engineered carotid grafts after 6 and 12 months in a sheep model. Multivariate data analysis allowed to trace degradation and to compare the data from in vitro and in vivo degradation, indicating similar molecular observations in spectral signatures between implants and oxidative in vitro degradation. In vivo degradation appeared to be dominated by oxidative pathways. Furthermore, information on collagen deposition and composition could simultaneously be obtained from the same image scans. Our results demonstrate the sensitivity of Raman microspectroscopy to determine degradation stages and the assigned molecular changes non-destructively, encouraging future exploration of this techniques for time-resolved quality assessment of in situ tissue engineering processes., Competing Interests: The research labs from KS-L and AIS performed independent scientific contract work for the company Xeltis and received for this work financial compensation. MC, HB, and AS are employees of Xeltis. MC is shareholder of Xeltis and AB is a financially compensated scientific advisor to Xeltis. SS is an employee of SyMO-Chem BV. AB is an employee and shareholder in SupraPolix BV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marzi, Munnig Schmidt, Brauchle, Wissing, Bauer, Serrero, Söntjens, Bosman, Cox, Smits and Schenke-Layland.)

(1) The monocytic leukemia cell line THP-1 and primary monocyte-derived macrophages (MDMs) are popular in vitro model systems to study human innate immunity, wound healing, and tissue regeneration. However, both cell types differ significantly in their origin and response to activation stimuli. (2) Resting THP-1 and MDMs were stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ) and analyzed by Raman microspectroscopy (RM) before and 48 h after activation. Raman data were subsequently analyzed using principal component analysis. (3) We were able to resolve and analyze the spatial distribution and molecular composition of proteins, nucleic acids, and lipids in resting and activated THP-1 and MDMs. Our findings reveal that proinflammatory activation-induced significant spectral alterations at protein and phospholipid levels in THP-1. In MDMs, we identified that nucleic acid and non-membrane-associated intracellular lipid composition were also affected. (4) Our results show that it is crucial to carefully choose the right cell type for an in vitro model as the nature of the cells itself may impact immune cell polarization or activation results. Moreover, we demonstrated that RM is a sensitive tool for investigating cell-specific responses to activation stimuli and monitoring molecular changes in subcellular structures.

Postoperative abdominal adhesions are responsible for serious clinical disorders. Administration of plasma-activated media (PAM) to cell type-specific modulated proliferation and protein biosynthesis is a promising therapeutic strategy to prevent pathological cell responses in the context of wound healing disorders. We analyzed PAM as a therapeutic option based on cell type-specific anti-adhesive responses. Primary human peritoneal fibroblasts and mesothelial cells were isolated, characterized and exposed to different PAM dosages. Cell type-specific PAM effects on different cell components were identified by contact- and marker-independent Raman imaging, followed by thorough validation by specific molecular biological methods. The investigation revealed cell type-specific molecular responses after PAM treatment, including significant cell growth retardation in peritoneal fibroblasts due to transient DNA damage, cell cycle arrest and apoptosis. We identified a therapeutic dose window wherein specifically pro-adhesive peritoneal fibroblasts were targeted, whereas peritoneal mesothelial cells retained their anti-adhesive potential of epithelial wound closure. Finally, we demonstrate that PAM treatment of peritoneal fibroblasts reduced the expression and secretion of pro-adhesive cytokines and extracellular matrix proteins. Altogether, we provide insights into biochemical PAM mechanisms which lead to cell type-specific pro-therapeutic cell responses. This may open the door for the prevention of pro-adhesive clinical disorders.

(1) Background: Cervical intraepithelial neoplasia (CIN) of long-term persistence or associated with individual treatment indications often requires highly invasive treatments. These are associated with risks of bleeding, infertility, and pregnancy complications. For low- and middle-income countries (LMICs), standard treatment procedures are difficult to implement and manage. We characterized the application of the highly energized gas "noninvasive physical plasma" (NIPP) for tissue devitalization and the treatment of CIN. (2) Methods: We report the establishment of a promising tissue devitalization procedure by NIPP application. The procedure was characterized at the in vitro, ex vivo and in vivo levels. We performed the first prospective, single-armed phase-IIb trial in 20 CIN1/2 patients (NCT03218436). (3) Results: NIPP-treated cervical cancer cells used as dysplastic in vitro model exhibited significant cell growth retardation due to DNA damage, cell cycle arrest and apoptosis. Ex vivo and in vivo tissue assessments showed a highly noninvasive and tissue-preserving treatment procedure which induces transmucosal tissue devitalization. Twenty participants were treated with NIPP and attended a 24-week follow-up. Treatment success was achieved in 19 (95%) participants without postinterventional complications other than mild to moderate discomfort during application. (4) Conclusions: The results from this study preliminarily suggest that NIPP could be used for an effective and tissue-preserving treatment for CIN without the disadvantages of standard treatments. However, randomized controlled trials must confirm the efficacy and noninferiority of NIPP compared to standard treatments.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites 1 . Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1 + HOXA9 + MLLT3 + MECOM + HLF + SPINK2 + distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1 + KCNK17 + haemogenic endothelial cells, which arose from an IL33 + ALDH1A1 + arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Calcified aortic valve disease (CAVD) is the most prevalent valve disease in the elderly. Targeted pharmacological therapies are limited since the underlying mechanisms of CAVD are not well understood. Appropriate 3D in vitro models could potentially improve our knowledge of the disease. Here, we developed a 3D in vitro aortic heart valve model that resembles the morphology of the valvular extracellular matrix and mimics the mechanical and physiological behavior of the native aortic valve fibrosa and spongiosa. We employed cryogenic electrospinning to engineer a bi-layered cryogenic electrospun scaffold (BCES) with defined morphologies that allowed valvular endothelial cell (VEC) adherence and valvular interstitial cell (VIC) ingrowth into the scaffold. Using a self-designed cell culture insert allowed us to establish the valvular co-culture simultaneously by seeding VICs on one side and VECs on the other side of the electrospun scaffold. Proof-of-principle calcification studies were successfully performed using an established osteogenic culture protocol and the here designed 3D in vitro aortic heart valve model. STATEMENT OF SIGNIFICANCE: Three-dimensional (3D) electrospun scaffolds are widely used for soft tissue engineering since they mimic the morphology of the native extracellular matrix. Several studies have shown that cells behave more naturally on 3D materials than on the commonly used stiff two-dimensional (2D) cell culture substrates, which have no biological properties. As appropriate 3D models for the study of aortic valve diseases are limited, we developed a novel bi-layered 3D in vitro test system by using the versatile technique of cryogenic electrospinning in combination with the influence of different solvents to mimic the morphology, mechanical, and cellular distribution of a native aortic heart valve leaflet. This 3D in vitro model can be used to study valve biology and heart valve-impacting diseases such as calcification to elucidate therapeutic targets., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Growing evidence indicates that perivascular tissue is critical to modulate vessel function. We hypothesized that the arachnoid membrane surrounding middle cerebral artery (MCA) regulates its function via sphingosine-1-phosphate (S1P)-induced vasoconstriction. The MCA from 3- to 9-month-old male and female wild-type (Oncine France 1 and C57BL/6) mice and sphingosine kinase 2 knockout (SphK2-/-) mice in the C57BL/6 background was mounted in pressure myographs with and without arachnoid membrane. Raman microspectroscopy and imaging were used for in situ detection of S1P. The presence of arachnoid tissue was associated with reduced external and lumen MCA diameters, and with an increase in basal tone regardless of sex and strain background. Strong S1P-positive signals were detected in the arachnoid surrounding the MCA wall in both mice models, as well as in a human post-mortem specimen. Selective S1P receptor 3 antagonist TY 52156 markedly reduced both MCA vasoconstriction induced by exogenous S1P and arachnoid-dependent basal tone increase. Compared to 3-month-old mice, the arachnoid-mediated contractile influence persisted in 9-month-old mice despite a decline in arachnoid S1P deposits. Genetic deletion of SphK2 decreased arachnoid S1P content and vasoconstriction. This is the first experimental evidence that arachnoid membrane regulates the MCA tone mediated by S1P.

Enzymatic digestion of the pancreas during islet isolation is associated with disintegration of the islet basement membrane (IBM) that can cause reduction of functional and morphological islet integrity. Attempts to re-establish IBM by coating the surface of culture vessels with various IBM proteins (IBMP) have resulted in loss of islet phenotype and function. This study investigated the capability of Collagen-IV, Laminin-521 and Nidogen-1, utilised as single or combined media supplements, to protect human islets cultured in hypoxia. When individually supplemented to media, all IBMP significantly improved islet survival and in-vitro function, finally resulting in as much as a two-fold increase of islet overall survival. In contrast, combining IBMP enhanced the production of chemokines and reactive oxygen species diminishing all positive effects of individually added IBMP. This impact was concentration-dependent and concerned nearly all parameters of islet integrity. Predictive extrapolation of these findings to data from 116 processed human pancreases suggests that more than 90% of suboptimal pancreases could be rescued for clinical islet transplantation increasing the number of transplantable preparations from actual 25 to 40 when adding Nidogen-1 to pretransplant culture. This study suggests that media supplementation with essential IBMP protects human islets from hypoxia. Amongst those, certain IBMP may be incompatible when combined or applied at higher concentrations. STATEMENT OF SIGNIFICANCE: Pancreatic islet transplantation is a minimally-invasive treatment that can reverse type 1 diabetes in certain patients. It involves infusing of insulin-producing cell-clusters (islets) from donor pancreases. Unfortunately, islet extraction is associated with damage of the islet basement membrane (IBM) causing reduced islet function and cell death. Attempts to re-establish the IBM by coating the surface of culture vessels with IBM proteins (IBMP) have been unsuccessful. Instead, we dissolved the most relevant IBM components Collagen-IV, Laminin-521 and Nidogen-1 in media routinely used for clinical islet culture and transplantation. We found human islet survival and function was substantially improved by IBMP, particularly Nidogen-1, when exposed to a hypoxic environment as found in vivo. We also investigated IBMP combinations. Our present findings have important clinical implications., Competing Interests: Declaration of Competing Interest The authors of this manuscript have no conflict of interest to disclose as defined by Acta Biomaterialia., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing. In the present study, we analyzed human monocyte-derived macrophages (MDMs) using RM, revealing that macrophage activation by lipopolysaccharides (LPS), interferons (IFN), or cytokines can be identified by lipid composition, which significantly differs in M0 (resting), M1 (IFN-γ/LPS), M2a (IL-4/IL-13), and M2c (IL-10) MDMs. To identify the impact of a biomaterial on MDM phenotype and polarization, we cultured macrophages on titanium disks with varying surface topographies and analyzed the adherent MDMs with RM. We detected surface topography-induced changes in MDM biochemistry and lipid composition that were not shown by less sensitive standard methods such as cytokine expression or surface antigen analysis. Our data suggest that RM may enable a more precise classification of macrophage activation and biomaterial-macrophage interaction., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Water restriction is commonly used to motivate rodents to perform behavioral tasks; however, its effects on hydration and stress hormone levels are unknown. Here, we report daily body weight and bi-weekly packed red blood cell volume and corticosterone (CORT) in adult male rats across 80 days for three commonly used water restriction schedules. We also assessed renal adaptation to water restriction using postmortem histologic evaluation of renal medulla. A control group received ad libitum water. After one week of water restriction, rats on all restriction schedules resumed similar levels of growth relative to the control group. Normal hydration was observed, and water restriction did not drive renal adaptation. An intermittent restriction schedule was associated with an increase in CORT relative to the control group. However, intermittent restriction evokes a stress response which could affect behavioral and neurobiological results. Our results also suggest that stable motivation in behavioral tasks may only be achieved after one week of restriction., (Copyright © 2021 Vasilev et al.)

Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA., (© 2021. The Author(s).)

Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and lifestyle risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on the Complement Factor H ( CFH ) gene, leading to an amino acid substitution in the Factor H (FH) protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability toward oxidative stress. In this study, we established a novel co-culture model comprising CFH silenced RPE cells and porcine retinal explants derived from the visual streak of porcine eyes, which closely resemble the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observed changes involving the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE-derived FH for retinal homeostasis and provide a valuable model for AMD research.

In this study, we describe the production of hybrid gelatin-poly-L-lactide electrospun scaffolds whose hydrophilicity was controlled by binding increasing concentrations of hyaluronic acid (HA). We show that cross-linking has advantages over coating when aiming to functionalize the scaffolds with HA. The here described scaffolds structurely mimicked the complexity of the extracellular matrix, and when excited by second harmonic generation, they produced a signal that is typical of collagen-containing biological fibers. Fluorescence lifetime imaging microscopy (FLIM) was used to marker-independently monitor the growth of human dermal fibroblasts on the electrospun scaffolds using reduced (phosphorylated) nicotinamide adenine dinucleotide as target. Benefitting from the different fluorescence lifetimes of the polymer and the endogenous cellular fluorophore, we were able to distinguish and separate the signals produced by the cells from the signals generated by the electrospun scaffolds. FLIM further allowed the detection of metabolic differences in the cells seeded on the HA-functionalized scaffolds compared with cells that were cultured on nonfunctionalized control scaffolds.

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., (© 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Advancements in type 1 diabetes mellitus treatments have vastly improved in recent years. The move toward a bioartificial pancreas and other fully implantable systems could help restore patient's glycemic control. However, the long-term success of implantable medical devices is often hindered by the foreign body response. Fibrous encapsulation "walls off" the implant to the surrounding tissue, impairing its functionality. In this study we aim to examine how streptozotocin-induced diabetes affects fibrous capsule formation and composition surrounding implantable drug delivery devices following subcutaneous implantation in a rodent model. After 2 weeks of implantation, the fibrous capsule surrounding the devices were examined by means of Raman spectroscopy, micro-computed tomography (μCT), and histological analysis. Results revealed no change in mean fibrotic capsule thickness between diabetic and healthy animals as measured by μCT. Macrophage numbers (CCR7 and CD163 positive) remained similar across all groups. True component analysis also showed no quantitative difference in the alpha-smooth muscle actin and extracellular matrix proteins. Although principal component analysis revealed significant secondary structural difference in collagen I in the diabetic group, no evidence indicates an influence on fibrous capsule composition surrounding the device. This study confirms that diabetes did not have an effect on the fibrous capsule thickness or composition surrounding our implantable drug delivery device. Impact Statement Understanding the impact diabetes has on the foreign body response (FBR) to our implanted material is essential for developing an effective drug delivery device. We used several approaches (Raman spectroscopy and micro-computed tomography imaging) to demonstrate a well-rounded understanding of the diabetic impact on the FBR to our devices, which is imperative for its clinical translation.

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 analyzed after postmortem, therefore, limiting the diagnosis of PD to the manifestation of motor symptoms. In PD patients and animal models, phosphorylated α-Syn was detected in the peripheral tissues including the gut, thus, raising the hypothesis that early-stage PD could be diagnosed based on colon tissue biopsies. Non-invasive marker-free technologies represent ideal methods 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. Using Raman imaging and microspectroscopy, we analyzed the olfactory bulb in the brain and the muscularis mucosae of colon tissue sections of a human BAC-SNCA transgenic (TG) rat model. Raman images from TG and WT rats were investigated using principal component analysis (PCA) and true component analysis (TCA). Spectral components indicated protein aggregates (spheroidal oligomers) in the TG rat brain and in the colon tissues even at a young age but not in WT. In summary, we have demonstrated that Raman imaging is capable of detecting α-Syn aggregates in colon tissues of a PD rat model and making it a promising tool for future use in PD pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sevgi, Brauchle, Carvajal Berrio, Schenke-Layland, Casadei, Salker, Riess and Singh.)

In recent years, organ-on-chip (OoC) systems have provoked increasing interest among researchers from different disciplines. OoCs enable the recreation of in vivo -like microenvironments and the generation of a wide range of different tissues or organs in a miniaturized way. Most commonly, OoC platforms are based on microfluidic modules made of polydimethylsiloxane (PDMS). While advantageous in terms of biocompatibility, oxygen permeability, and fast prototyping amenability, PDMS features a major limitation as it absorbs small hydrophobic molecules, including many types of test compounds, hormones, and cytokines. Another common feature of OoC systems is the integration of membranes (i) to separate different tissue compartments, (ii) to confine convective perfusion to media channels, and/or (iii) to provide mechanical support for cell monolayers. Typically, porous polymer membranes are microstructured using track-etching (e.g., polyethylene terephthalate; PET) or lithography (e.g., PDMS). Although membranes of different biomechanical properties (rigid PET to elastic PDMS) have been utilized, the membrane structure and material remain mostly artificial and do not resemble in vivo conditions (extracellular matrix). Here, we report a method for the reliable fabrication and integration of electrospun membranes in OoC modules, which are made of laser-structured poly(methyl methacrylate) (PMMA). The choice of PMMA as base material provides optical parameters and biocompatibility similar to PDMS while avoiding the absorption problem. Using electrospinning for the generation of 3D membranes, microenvironments resembling the native extracellular matrix (ECM) can be generated. We tested two different kinds of electrospun membranes and established processes for a tight integration into PMMA modules. Human (microvasculature) endothelial as well as (retinal pigment) epithelial cell layers could be successfully cultured inside the systems for up to 7 days, while being either directly exposed to (endothelial cells) or protected (epithelial cells) from the shear flow. Our novel method enables the versatile fabrication of OoC platforms that can be tailored to the native environment of tissues of interest and at the same time are applicable for the testing of compounds or chemicals without constraints.

Peripheral-blood derived CD34 + hematopoietic stem and progenitor cells were isolated from a 49-year old male donor and were successfully reprogrammed into human induced pluripotent stem cells (hiPSCs) using integration-free episomal vectors. The hiPSC line exhibited a typical stem cell-like morphology and endogenously expressed several pluripotency markers by concomitant loss of exogenous reprogramming vectors. Genomic integrity was confirmed by microarray-based comparative genomic hybridization (array CGH). Further analysis affirmed the ability of this hiPSC line to differentiate into all three germ layers. Thus, the reported cell line may serve as a healthy control for disease modeling., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The use of biomaterials and biomaterial functionalization is a promising approach to support pancreatic islet viability posttransplantation in an effort to reduce insulin dependence for patients afflicted with diabetes mellitus type 1. Extracellular matrix (ECM) proteins are known to impact numerous reparative functions in the body. Assessing how endogenously expressed pancreatic ECM proteins are affected by posttransplant-like hypoxic conditions may provide significant insights toward the development of tissue-engineered therapeutic strategies to positively influence β-cell survival, proliferation, and functionality. Here, we investigated the expression of three relevant groups of pancreatic ECM proteins in human native tissue, including basement membrane (BM) proteins (collagen type 4 [COL4], laminins [LAM]), proteoglycans (decorin [DCN], nidogen-1 [NID1]), and fibril-forming proteins (fibronectin [FN], collagen type 1 [COL1]). In an in vitro hypoxia model, we identified that ECM proteins were differently affected by hypoxic conditions, contributing to an overall loss of β-cell functionality. The use of a COL1 hydrogel as carrier material demonstrated a protective effect on β-cells mitigating the effect of hypoxia on proteoglycans as well as fibril-forming protein expression, supporting β-cell functionality in hypoxia. We further showed that providing endothelial cells (ECs) into the COL1 hydrogel improves β-cell response as well as the expression of relevant BM proteins. Our data show that β-cells benefit from a microenvironment composed of structure-providing COL1 with the incorporation of ECs to withstand the harsh conditions of hypoxia. Such hydrogels support β-cell survival and can serve as an initial source of ECM proteins to allow cell engraftment while preserving cell functionality posttransplantation. Impact statement Expression analysis identifies hypoxia-induced pathological changes in extracellular matrix (ECM) homeostasis as potential targets to support β-cell transplants by encapsulation in biomaterials for the treatment of diabetes mellitus. A collagen-1 hydrogel is shown to attenuate the effect of hypoxia on β-cells and their ECM expression. The functionalization of the hydrogel with endothelial cells increases the β-cell response to glucose and rescues essential basement membrane proteins.

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen's κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.