Abstract Accurate cell counting is essential in tissue engineering and cancer research. The ongoing transition towards advanced 3D in vitro tumour models raises a question about the validity of the standard cell counting protocols, particularly in the systems containing extracellular matrix-based scaffolds. Here, we provide a quantitative analysis of the performance of three popular plate reader-based cell counting/viability assays, such as the Alamar Blue, MTT, CellTiter Glo 3D assays, in 2D monolayer and 3D scaffold-based cultures of U251 human glioblastoma cells, including cell-laden Matrigel plugs, and original tissue engineering constructs based on the decellularised sheep brain scaffolds. We quantitatively characterized the assays’ linearity, precision, biological and technical reproducibility, proportionality, and inter-assay agreement. The study revealed that assays’ performance is highly platform-dependent, with 2D cultures allowing significantly more precise and reliable measurements than in 3D ECM scaffold-based cultures. The numerical results provided in this study can help researchers make informed decisions when working with 3D scaffold-based in vitro tumour models and for other tissue engineering purposes where precise cell counting is essential. ToC

This chapter presents a segmentation-free, cell ensemble-level workflow for detecting treatment-induced responses in confluent microglial cultures in vitro, using standard phase-contrast microscopy and Python-based imaging data analysis. Entire fields of view are analyzed after minimal preprocessing, avoiding fixation, staining, and single-cell segmentation. We implement multifractal, lacunarity, and texture analysis (MFTA) to characterize the microglial monolayers. In parallel, we introduce cell radiomics as the adaptation of radiomics principles to label-free microscopy, extracting a high-dimensional panel of intensity- and texture-based features directly from grayscale images of live cells. Together, MFTA and cell radiomics provide complementary, quantitative readouts that are sensitive to subtle changes in ensemble organization. Statistical comparisons employ false discovery rate control to identify robust discriminative features between conditions, with optional validation by conventional morphometry on a subset of segmented cells. The workflow is demonstrated on an experimental dataset by the successful reveal of the effect of a moderate static magnetic field on human C20 microglia cell line.

Microglia are the primary resident immune cells of the central nervous system (CNS) that respond to injury and infections. Being critical to CNS homeostasis, microglia also have been shown to contribute to neurodegenerative diseases and brain cancer. Hence, microglia are regarded as a potential therapeutic target in CNS diseases, resulting in an increased demand for reliable in vitro models. Two human microglia cell lines (HMC3 and C20) are being used in multiple in vitro studies, however, the knowledge of their biological and immunological characteristics remains limited. Our aim was to identify and compare the biological changes in these immortalised immune cells under normal physiological and immunologically challenged conditions. Using high-resolution quantitative mass spectrometry, we have examined in-depth proteomic profiles of non-stimulated and LPS or IFN-γ challenged HMC3 and C20 cells. Our findings reveal that HMC3 cells responded to both treatments through upregulation of immune, metabolic, and antiviral pathways, while C20 cells showed a response associated with mitochondrial and immune activities. Additionally, the secretome analysis demonstrated that both cell lines release IL-6 in response to LPS, while IFN-γ treatment resulted in altered kynurenine pathway activity, highlighting distinct immune and metabolic adaptations.

INTRODUCTION:Metastasis remains the major cause of death in breast cancer (BrCa) and lacks specific treatment strategies. The kynurenine pathway (KP) has been suggested as a key mechanism facilitating progression of BrCa. While KP activity has been explored in primary BrCa, its role in metastasis remains unclear. To better understand this, we examined changes in the KP of BrCa with no metastasis compared to BCa that produced local or distant metastases. Given that the cancer cell secretome plays a role in metastasis, we also investigated the relationship between changes in KP activity and serum proteins of patients with local or distant metastases. METHODS:To investigate changes in the KP in BrCa, with and without metastasis, we quantified KP metabolites in blood sera collected from patients with stage 1 BrCa (n = 34), BrCa with local metastases (n = 46), BrCa with distant metastases (n = 20) and healthy controls (n = 39). The serum protein profile of the BrCa patients with local or distant metastasis was determined before correlation analyses were carried out to examine the relationship between changes in the KP and cancer serum proteins using SPSS. RESULTS:We found that the KP was elevated in BrCa patients with local and distant metastasis compared to healthy controls and stage 1 BrCa patients. The activity of kynurenine monooxygenase (KMO) and kynureninase (KYNU) A was positively associated with disease stage and was higher compared to healthy controls. Proteome analysis in patients with local or distant metastasis revealed the dysregulation of 14 proteins, 9 of which were up-regulated and 5 down-regulated at the distant metastasis stage. Importantly, three of these proteins have not been previously linked to BrCa metastasis. In the correlation studies between the KP profile, cancer serum proteins and metastasis status, KYNU A had the greatest number of significant associations with cancer serum protein, followed by KMO. CONCLUSION:Our findings reveal that the KP was regulated differently at various stages of BrCa and was more dysregulated in patients with local or distant metastasis. These KP activity changes showed a significant association with cancer serum proteins in BrCa patients with local or distant metastasis, highlighting the potential role of KP in BrCa metastasis.

BACKGROUND:HE is a neuropsychiatric complication of liver disease characterized by systemic elevation in ammonia and proinflammatory cytokines. These neurotoxins cross the blood-brain barrier and cause neuroinflammation, which can activate the kynurenine pathway (KP). This results in dysregulated production of neuroactive KP metabolites, such as quinolinic acid, which is known to cause astrocyte and neuronal death. Our aim was to compare KP activity between patients with covert HE (CHE), patients without encephalopathic cirrhosis (NHE), and healthy controls (HCs). METHODS:This was a single-center prospective cohort study conducted between 2018 and 2021 at St Vincent's Hospital, Sydney. Overall, 13 patients with CHE, 10 patients with NHE, and 12 with HC were recruited. Patients with cirrhosis were diagnosed with CHE if they scored ≤-4 on the Psychometric Hepatic Encephalopathy Score. KP metabolite levels were quantified on plasma samples via HPLC and gas chromatography/mass spectrometry. One-way Kruskal-Wallis test was used to compare the expression levels of KP enzymes. RESULTS:KP was highly activated in patients with cirrhosis, demonstrated by higher levels of activity in the rate-limiting enzymes, indoleamine 2,3-dioxygenase, and tryptophan-2,3-dioxygenase in both CHE (65.04±20.72, p=0.003) and patients with NHE (64.85±22.10, p=0.015) compared to HC (40.95±7.301). Higher quinolinic acid concentrations were demonstrated in CHE (3726 nM±3385, p<0.001) and patients with NHE (1788 nM±632.3, p=0.032) compared to HC (624 nM±457). KP activation was positively correlated with inflammatory marker C-reactive protein in patients with CHE (Rs=0.721, p≤0.01). CONCLUSIONS:KP is highly activated in patients with CHE, resulting in heightened production of neurotoxic metabolites. Dysregulation of the pathway is demonstrable in patients who do not yet show clinical signs of neurocognitive impairment. Therapeutic agents that modulate KP activity may be able to alleviate symptoms of patients with CHE.

The overall goal of regenerative medicine is to restore the functional performance of the tissues and organs that have been severely damaged or lost due to traumas and diseases [...].

Background Defining the presence of acute and chronic brain inflammation remains a challenge to clinicians due to the heterogeneity of clinical presentations and aetiologies. However, defining the presence of neuroinflammation, and monitoring the effects of therapy is important given its reversible and potentially damaging nature. We inves-tigated the utility of CSF metabolites in the diagnosis of primary neuroinflammatory disorders such as encephalitis and explored the potential pathogenic role of inflammation in epilepsy.Methods Cerebrospinal fluid (CSF) collected from 341 paediatric patients (169 males, median age 5.8 years, range 0.1-17.1) were examined. The patients were separated into a primary inflammatory disorder group (n = 90) and epilepsy group (n = 80), who were compared with three control groups including neurogenetic and structural (n = 76), neurodevelopmental disorders, psychiatric and functional neurological disorders (n = 63), and headache (n = 32).Findings There were statistically significant increases of CSF neopterin, kynurenine, quinolinic acid and kynurenine/ tryptophan ratio (KYN/TRP) in the inflammation group compared to all control groups (all p < 0.0003). As bio-markers, at thresholds with 95% specificity, CSF neopterin had the best sensitivity for defining neuroinflammation (82%, CI 73-89), then quinolinic acid (57%, CI 47-67), KYN/TRP ratio (47%, CI 36-56) and kynurenine (37%, CI 28-48). CSF pleocytosis had sensitivity of 53%, CI 42-64). The area under the receiver operating characteristic curve (ROC AUC) of CSF neopterin (94.4% CI 91.0-97.7%) was superior to that of CSF pleocytosis (84.9% CI 79.5-90.4%) (p = 0.005). CSF kynurenic acid/kynurenine ratio (KYNA/KYN) was statistically decreased in the epilepsy group compared to all control groups (all p <= 0.0003), which was evident in most epilepsy subgroups.Interpretation Here we show that CSF neopterin, kynurenine, quinolinic acid and KYN/TRP are useful diagnostic and monitoring biomarkers of neuroinflammation. These findings provide biological insights into the role of in-flammatory metabolism in neurological disorders and provide diagnostic and therapeutic opportunities for improved management of neurological diseases.Funding Financial support for the study was granted by Dale NHMRC Investigator grant APP1193648, University of Sydney, Petre Foundation, Cerebral Palsy Alliance and Department of Biochemistry at the Children's Hospital at Westmead. Prof Guillemin is funded by NHMRC Investigator grant APP 1176660 and Macquarie University. Copyright (c) 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Precise characterization of a tissue’s extracellular matrix (ECM) protein composition (matrisome) is essential for biomedicine. However, ECM protein extraction that requires organ-specific optimization is still a major limiting factor in matrisome studies. In particular, the matrisome of mouse kidneys is still understudied, despite mouse models being crucial for renal research. Here, we comprehensively characterized the matrisome of kidneys in healthy C57BL/6 mice using two ECM extraction methods in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS), protein identification, and label-free quantification (LFQ) using MaxQuant. We identified 113 matrisome proteins, including 22 proteins that have not been previously listed in the Matrisome Database. Depending on the extraction approach, the core matrisome (structural proteins) comprised 45% or 73% of kidney ECM proteins, and was dominated by glycoproteins, followed by collagens and proteoglycans. Among matrisome-associated proteins, ECM regulators had the highest LFQ intensities, followed by ECM-affiliated proteins and secreted factors. The identified kidney ECM proteins were primarily involved in cellular, developmental and metabolic processes, as well as in molecular binding and regulation of catalytic and structural molecules’ activity. We also performed in silico comparative analysis of the kidney matrisome composition in humans and mice based on publicly available data. These results contribute to the first reference database for the mouse renal matrisome.

Renal fibrosis is the final common pathophysiological pathway in chronic kidney disease (CKD) regardless of the underlying cause of kidney injury. Tubulointerstitial fibrosis (TIF) is considered to be the key pathological predictor of CKD progression. Currently, the gold-standard tool to identify TIF is kidney biopsy, an invasive method that carries risks. Non-invasive diagnostics rely on an estimation of glomerular filtration rate and albuminuria to assess kidney function, but these fail to diagnose early CKD accurately or to predict progressive decline in kidney function. In this review, we summarize the current and emerging molecular biomarkers that have been studied in various clinical settings and in animal models of kidney disease and that are correlated with the degree of TIF. We examine the potential of these biomarkers to diagnose TIF non-invasively and to predict disease progression. We also examine the potential of new technologies and non-invasive diagnostic approaches in assessing TIF. Limitations of current and potential biomarkers are discussed and knowledge gaps identified.

Combination drug therapy addresses the auxiliary cancer pathways of the tumor progression unaffected by the standard adjuvant treatments such as radio- and chemotherapy. It is a particularly attractive strategy to improve the treatment outcomes and the quality of life in patients with the deadliest brain cancer, glioblastoma (GB). Testing of combination drug treatment protocols requires reliable, efficient, and biologically accurate preclinical testbeds applicable before the transition to clinical trials. The 3D in vitro models of GB are a promising platform for pharmacological research. However, there is notable methodological uncertainty and a highly scattered data landscape regarding drug testing in 3D in vitro models of GB. In particular, it is not completely clear how to mimic clinically relevant dozing and schedule of the main chemotherapy drug for GB, temozolomide (TMZ) in 3D in vitro GB models. Here, the authors carefully explore the available literature on the application of TMZ in 3D in vitro models of GB, both as a sole agent and in combination with other medications. The joint analysis of the tumor modeling approaches, the employed assays, and the obtained treatment responses provided in this review may be used as a roadmap for future research in combination treatments of GB.

Additive manufacturing (3D printing) and computer-aided design (CAD) still have limited uptake in biomedical and bioengineering research and education, despite the significant potential of these technologies. The utility of organ-scale 3D-printed models of living structures is widely appreciated, while the workflows for microscopy data translation into tactile accessible replicas are not well developed yet. Here, we demonstrate an accessible and reproducible CAD-based methodology for generating 3D-printed scalable models of human cells cultured in vitro and imaged using conventional scanning confocal microscopy with fused deposition modeling (FDM) 3D printing. We termed this technology CiTo-3DP (Cells-in-Touch for 3D Printing). As a proof-of-concept, we created dismountable CiTo-3DP models of human epithelial, mesenchymal, and neural cells by using selectively stained nuclei and cytoskeletal components. We also provide educational and research context for the presented cellular models. In the future, the CiTo-3DP approach can be adapted to different imaging and 3D printing modalities and comprehensively present various cell types, subcellular structures, and extracellular matrices. The resulting CAD and 3D printed models could be used for a broad spectrum of education and research applications.

Background Epileptic (previously infantile) spasms is the most common epileptic encephalopathy occurring during infancy and is frequently associated with abnormal neurodevelopmental outcomes. Epileptic spasms have a diverse range of known (genetic, structural) and unknown aetiologies. High dose corticosteroid treatment for 4 weeks often induces remission of spasms, although the mechanism of action of corticosteroid is unclear. Animal models of epi-leptic spasms have shown decreased brain kynurenic acid, which is increased after treatment with the ketogenic diet. We quantified kynurenine pathway metabolites in the cerebrospinal fluid (CSF) of infants with epileptic spasms and explored clinical correlations.Methods A panel of nine metabolites in the kynurenine pathway (tryptophan, kynurenine, kynurenic acid, 3-hydrox-ykynurenine, xanthurenic acid, anthranilic acid, 3-hydroxyanthranilic acid, quinolinic acid, and picolinic acid) were measured using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). CSF collected from paediatric patients less than 3 years of age with epileptic spasms (n=34, 19 males, mean age 0.85, median 0.6, range 0.3-3 yrs) were compared with other epilepsy syndromes (n=26, 9 males, mean age 1.44, median 1.45, range 0.3-3 yrs), other non-inflammatory neurological diseases (OND) (n=29, 18 males, mean age 1.47, median 1.6, range 0.1-2.9 yrs) and inflammatory neurological controls (n=12, 4 males, mean age 1.80, median 1.80, range 0.8-2.5 yrs).Findings There was a statistically significant decrease of CSF kynurenic acid in patients with epileptic spasms com-pared to OND (p<0.0001). In addition, the kynurenic acid/kynurenine (KYNA/KYN) ratio was lower in the epileptic spasms subgroup compared to OND (p<0.0001). Epileptic spasms patients who were steroid responders or partial steroid responders had lower KYNA/KYN ratio compared to patients who were refractory to steroids (p<0.005, p<0.05 respectively).Interpretation This study demonstrates decreased CSF kynurenic acid and KYNA/KYN in epileptic spasms, which may also represent a biomarker for steroid responsiveness. Given the anti-inflammatory and neuroprotective proper-ties of kynurenic acid, further therapeutics able to increase kynurenic acid should be explored. eBioMedicine ebiom.2022.104280 Funding Financial support for the study was granted by Dale NHMRC Investigator grant APP1193648, Petre Foun-dation, Cerebral Palsy Alliance and Department of Biochemistry at the Children's Hospital at Westmead. Prof Guil-lemin is funded by NHMRC Investigator grant APP1176660 and Macquarie University.Copyright (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Transcranial magnetic stimulation (TMS) is a widely available technology employing relatively strong (up to 4 T) pulsed (up to 300 Hz) magnetic fields for diagnostics of brain functions and treatment of various brain disorders. The current paradigm implies that the magnetic induction of Eddy currents in the brain neurons is the leading biological mechanism of TMS. At the same time, it is almost unknown how the TMS-like magnetic fields act on nonneuronal cells. Here, we explored the effects of TMS-like repetitive magnetic stimulation (RMS) on metabolic activity of human colorectal cancer (CRC) and hepatocellular carcinoma (HCC) cells. The HCT116 (CRC) and HuH7 (HCC) in vitro cell cultures were treated using a TMS device “Magstim Rapid 2 ” with an air-cooled “Magstim” figure-eight coil (AFC70). Five intermittent RMS (iRMS) and two burst RMS (bRMS) experimental protocols were applied to the monolayers of the cells as a single treatment session. The activity of succinate dehydrogenase, an enzyme essential for energy production in mitochondria, was measured by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric viability assay in 24 h post-stimulation. Compared to untreated control, in HCT116 cells, a low-frequency iRMS treatment ( $f $ = 1 Hz, 600 pulses, $B $ = 0.8 T) induced a statistically significant decrease in metabolic activity (viability), while the high-frequency iRMS and bRMS increased it. In HuH7 cells, all tested RMS protocols either did not change or stimulated the metabolic activity (viability) of the cells, in comparison to the untreated control. The analysis of correlations revealed the almost opposite trends in CRC and HCC cells response to the frequency, temporal patterns, and magnetic field flux density considered as the parameters of the experimental RMS. Our findings demonstrate the tumor type- and stimulation protocol-specific effects of the repurposed TMS technology on colorectal and liver cancer cells. The underlying mechanism of these differences requires further study. The current results indicate that TMS-like magnetic fields may have new potential applications as an adjuvant anticancer treatment modality.