State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve ...lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest.
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•Quiescent muscle stem cells undergo major transcriptomic alterations during isolation•Isolation induces histone H3 modifications but leaves intact DNA methylation•In situ fixation allows isolation of quiescent cells in their native, in vivo state•In situ fixation detects quiescence and early activation factors
Machado et al. demonstrate that muscle stem cells undergo changes in transcripts and histone modifications during isolation. The authors develop an in situ fixation-based methodology, which allows capture of cells in their native state. In light of these findings, some high-throughput analyses of tissue extracted cells may need to be revisited.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro ...microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.
Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model ...the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D) cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not ...been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner.
Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation.
Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes.
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•Immortalized cardiac cell lines show low resemblance to mature cardiac cells.•Organized sarcomeric structure is detectable in HL-1 cells but not in H9C2 or AC16.•Differentiation of AC16 or H9C2 induces minor changes towards cardiac phenotype.•Functional tests reveal the restricted responsiveness of all the cardiac cell lines.•This is the first systematic study showing major limitations of cardiac cell lines.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Interactions between neurons, astrocytes, and microglia critically influence neuroinflammatory responses to insult in the central nervous system. In vitro astrocyte and microglia cultures are ...powerful tools to study specific molecular pathways involved in neuroinflammation; however, in order to better understand the influence of cellular crosstalk on neuroinflammation, new multicellular culture models are required.
Primary cortical cells taken from neonatal rats were cultured in a serum-free "tri-culture" medium formulated to support neurons, astrocytes, and microglia, or a "co-culture" medium formulated to support only neurons and astrocytes. Caspase 3/7 activity and morphological changes were used to quantify the response of the two culture types to different neuroinflammatory stimuli mimicking sterile bacterial infection (lipopolysaccharide (LPS) exposure), mechanical injury (scratch), and seizure activity (glutamate-induced excitotoxicity). The secreted cytokine profile of control and LPS-exposed co- and tri-cultures were also compared.
The tri-culture maintained a physiologically relevant representation of neurons, astrocytes, and microglia for 14 days in vitro, while the co-cultures maintained a similar population of neurons and astrocytes, but lacked microglia. The continuous presence of microglia did not negatively impact the overall health of the neurons in the tri-culture, which showed reduced caspase 3/7 activity and similar neurite outgrowth as the co-cultures, along with an increase in the microglia-secreted neurotrophic factor IGF-1 and a significantly reduced concentration of CX3CL1 in the conditioned media. LPS-exposed tri-cultures showed significant astrocyte hypertrophy, increase in caspase 3/7 activity, and the secretion of a number of pro-inflammatory cytokines (e.g., TNF, IL-1α, IL-1β, and IL-6), none of which were observed in LPS-exposed co-cultures. Following mechanical trauma, the tri-culture showed increased caspase 3/7 activity, as compared to the co-culture, along with increased astrocyte migration towards the source of injury. Finally, the microglia in the tri-culture played a significant neuroprotective role during glutamate-induced excitotoxicity, with significantly reduced neuron loss and astrocyte hypertrophy in the tri-culture.
The tri-culture consisting of neurons, astrocytes, and microglia more faithfully mimics in vivo neuroinflammatory responses than standard mono- and co-cultures. This tri-culture can be a useful tool to study neuroinflammation in vitro with improved accuracy in predicting in vivo neuroinflammatory phenomena.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
BACKGROUND
Platelets have the highest bacterial contamination risk of all blood components, and septic transfusion reactions remain a problem. A good estimate of contamination rates could provide ...information about residual risk and inform optimal testing strategies. We performed a systematic review and meta‐analysis of platelet contamination rates by primary culture.
STUDY DESIGN AND METHODS
A literature search in December 2019 identified articles on platelet contamination rates using primary culture. We used meta‐analysis to estimate the overall rate of contamination and meta‐regression to identify heterogeneity. We studied the following sources of heterogeneity: collection method, sample volume, positivity criteria, and study date. Contamination rate estimates were obtained for apheresis (AP), platelet rich plasma (PRP), and buffy coat (BC) collection methods.
RESULTS
The search identified 6102 studies, and 22 were included for meta‐analysis. Among these 22 studies, there were 21 AP cohorts (4,072,022 components), 4 PRP cohorts (138,869 components), and 15 BC cohorts (1,474,679 components). The overall mean contamination rate per 1000 components was 0.51 (95% CI: 0.38‐0.67) including AP (0.23, 95% CI: 0.18‐0.28), PRP, (0.38, 95% CI: 0.15‐0.70), and BC (1.12, 95% CI: 0.51‐1.96). There was considerable variability within each collection method. Sample volume, positivity criteria, and publication year were significant sources of heterogeneity.
CONCLUSION
The bacterial contamination rate of platelets by primary culture is 1 in 1961. AP and PRP components showed a lower contamination rate than BC components. There is clinically significant between‐study variability for each method. Larger sample volumes increased sensitivity, and bacterial contamination rates have decreased over time.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS ...membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.
BACKGROUND.Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further ...collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries.
METHODS.A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan.
RESULTS.Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities.
CONCLUSIONS.The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.
Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, ...the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes.
Tendon and ligament (T/L) function is intrinsically related with their unique hierarchically and anisotropically organized extracellular matrix. Their natural healing capacity is, however, limited. ...Here, continuous and aligned electrospun nanofiber threads (CANT) based on synthetic/natural polymer blends mechanically reinforced with cellulose nanocrystals are produced to replicate the nanoscale collagen fibrils grouped into microscale collagen fibers that compose the native T/L. CANT are then incrementally assembled into 3D hierarchical scaffolds, resulting in woven constructions, which simultaneously mimic T/L nano‐to‐macro architecture, nanotopography, and nonlinear biomechanical behavior. Biological performance is assessed using human‐tendon‐derived cells (hTDCs) and human adipose stem cells (hASCs). Scaffolds nanotopography and microstructure induce a high cytoskeleton elongation and anisotropic organization typical of tendon tissues. Moreover, the expression of tendon‐related markers (Collagen types I and III, Tenascin‐C, and Scleraxis) by both cell types, and the similarities observed on their expression patterns over time suggest that the developed scaffolds not only prevent the phenotypic drift of hTDCs, but also trigger tenogenic differentiation of hASCs. Overall, these results demonstrate a feasible approach for the scalable production of 3D hierarchical scaffolds that exhibit key structural and biomechanical properties, which can be advantageously explored in acellular and cellular T/L TE strategies.
Advanced 3D textile scaffolds are fabricated by applying rational construction designs to mimic key functional features of native tendons/ligaments. Their unique nano‐to‐macro 3D hierarchical architecture prevents the phenotypic drift of human‐tendon‐derived cells, and triggers the tenogenic differentiation of human adipose stem cells without biochemical supplementation, demonstrated to be a noble solution for engineering these highly specialized, load‐bearing, anisotropic tissues.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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