Single Cell Culture Research Articles

A single-cell 3D dynamic volume control system for chondrocytes.

In articular cartilage, zone-specific cellular morphology is a typical characteristic of cartilage tissue, which is related with chondrocyte function, inflammation and osteoarthritis (OA). Chondrocyte hypertrophic phenotype is a criticle physiological process which indicates a hallmark of chondrocyte terminal differentiation and bone formation. Thus, developing a in vitro cell culture system for dynamic regulation of single chondrocyte volume at a three-dimensional (3D) level is particularly necessary for understanding how physical cues of matrix microenvironment regulate chondrocyte fate and the degeneration of articular cartilage. Here, based on the soft lithography techniques, we have constructed well-defined single-cell 3D dynamic volume control system to recapitulate the physiological matrix microenvironment of single chondrocyte niche. The results of finite element analysis indicated that the stress and strain distribution in the cell culture region is homogeneous during the stretching process. Additionally, 3D dynamic volume expansion and compression of single cells in physiological or hyperphysiological can be realized in this cell culture system. Our device for single-cell 3D dynamic culture provides a microphysiological culture system for chondrocytes to explore the mechanisms of cartilage hypertrophy, as well as develops a new paradigm for functional cartilage tissue engineering and regenerative medicine.

Glucotoxicity suppresses function of pancreatic beta and duct cells via miR-335-targeted Runx2 and insulin-mediated mechanism.

Pancreatic cell dynamics have important contributions to the development of type 2 diabetes and related diseases such as nonalcoholic fatty pancreas disease. The aim of this study was to investigate the effects of prolonged excessive glucose exposure on the functions of pancreatic beta cells and duct cells in single and co-culture conditions. In this study, we focused on the effects of glucotoxicity on insulin secretion which is the main function of beta cells and on progenitor functions of duct cells. Rat primary INS1 beta cells and ARIP duct cells were exposed to glucose (25mM) for 72h under single or indirect co-culture conditions. Glucotoxicity stimuli increased insulin secretion and decreased insulin expression in single beta cells while stimulating beta-cell differentiation and adipogenesis in single duct cells. On the other hand, glucotoxicity caused functional loss and increased proliferation and apoptosis in beta cells while increasing proliferation but suppressed beta-cell differentiation and adipogenesis in duct cells under co-culture conditions. The expression level of miR-335, a microRNA known to be upregulated by leptin and target Runx2, was measured. As a result, unlike single-cell culture, glucotoxicity upregulated miR-335, downregulated Runx2, and decreased insulin signaling in beta cells while downregulating miR-335 and upregulating Runx2, and decreased insulin signaling in duct cells under co-culture conditions. When the results of single and co-culture experiments are compared, insulin and miR-335 may be seen as important mediators for setting up the relation between beta and duct cells. Our findings are important for preventing the development of type 2 diabetes and nonalcoholic fatty pancreas disease, even developing new diagnosis and treatment strategies.

A novel method for the preparation of reproducible, stable, and non-infectious quality control materials for Chlamydia trachomatis nucleic acid detection.

Chlamydia trachomatis (CT) is a significant sexually transmitted pathogen known to evoke severe complications, including infertility. Nucleic acid amplification tests (NAATs) are recommended by the World Health Organization to detect CT infection. Furthermore, the establishment of methods, performance validation, internal quality control, and external quality assessment for CT NAATs necessitate the utilization of quality control materials (QCs). QCs are specimens or solutions that are analyzed for quality control purposes in a test system. In this study, we established a novel cell line that stably integrates CT amplification target sequences for producing QCs for CT NAATs. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, we integrated the CT plasmid-mediated sequence (comprising the full length of the cryptic plasmid and the major outer membrane protein gene, 9,136 bp) into the MUC4 gene of HEK293T cells. Positive clones were screened through flow cytometric sorting, single-cell culture, and PCR-based identification, followed by the establishment of stable cell lines. These cells were then processed using optimized cell preservation procedures to prepare QCs. The sequence insertion copy number was confirmed by real-time quantitative PCR. This novel CT QCs demonstrate excellent clinical applicability, non-infectiousness, quantifiability, and stability. With an integrated sequence exceeding 9 kb in length, it offers exceptional flexibility for adapting to new kit developments. Furthermore, maintaining a well-defined copy number and stable shelf life, the QCs closely aligns with the quality control requirements of CT NAATs. This study presents an innovative method for preparing QCs for CT nucleic acid detection, making a valuable contribution to improving the performance of CT NAATs.IMPORTANCEUntreated CT infections impose significant burdens on individuals and communities, underscoring the importance of early and accurate testing via CT NAATs for disease control. QCs are instrumental in identifying testing process issues. Hence, we developed a cell line integrating CT-amplified target sequences as readily accessible non-infectious QCs. These QCs boast several advantages: the integration of over 9 kb of CT sequence allows for broad applicability, allowing flexible adaptation to the development of new kits. Confirming the CT sequence copy number provides a reliable basis for QC concentration preparation and kit detection limit evaluation. Optimized preservation protocol enhances QC stability during storage, facilitating convenient shipment to clinical laboratories at ambient temperatures. In summary, our novel CT QCs offer a powerful tool for improving CT NAAT performance and present a fresh perspective on QC preparation for detecting nucleic acids from intracellular parasitic pathogens.

In-house polydimethylsiloxane microfluidic device on single cell trapping and culturing of Leukemia cell line: comparison between conventional and conventional with centrifugal methods

In-house polydimethylsiloxane microfluidic device on single cell trapping and culturing of Leukemia cell line: comparison between conventional and conventional with centrifugal methods

Forming Single-Cell-Derived Colon Cancer Organoid Arrays on a Microfluidic Chip for High Throughput Tumor Heterogeneity Analysis.

Single-cell-derived tumor organoids (STOs) possess a distinct genetic background, making them valuable tools for demonstrating tumor heterogeneity. In order to fulfill the high throughput demands of STO assays, we have developed a microfluidic chip containing 30 000 microwells, which is dedicated to a single cell culture approach for selective expansion and differential induction of cancer stem cells. The microwells are coated with a hydrophilic copolymer to eliminate cell adhesion, and the cell culture is supported by poly(ethylene glycol) (PEG) to establish a nonadhesive culture environment. By utilizing an input cell density of 7 × 103·mL-1, it is possible to construct a 4000 single cell culture system through stochastic cell occupation. We demonstrate that the addition of 15% PEG10000 in the cell culture medium effectively prevents cell loss while facilitating tumor stem cell expansion. As were demonstrated by HCT116, HT29, and SW480 colon cancer cells, the microfluidic approach achieved a STO formation rate of ∼20%, resulting in over 800 STOs generated from a single culture. Comprehensive analysis through histomorphology, immunohistochemistry, drug response evaluation, assessment of cell invasion, and biomarker detection reveals the heterogeneity among individual STOs. Specifically, the smaller STOs exhibited higher invasion and drug resistance capabilities compared with the larger ones. The developed microfluidic approach effectively facilitates STO formation and offers promising prospects for investigating tumor heterogeneity, as well as conducting personalized therapy-focused drug screening.

Synoviocytes assist in modulating the effect of Ross River virus infection in micromass-cultured primary human chondrocytes.

Introduction. Ross River virus (RRV) is a mosquito-borne virus prevalent in Australia and the islands of the South Pacific, where it causes an arthritogenic illness with a hallmark feature of severe joint pain. The joint space is a unique microenvironment that contains cartilage and synovial fluid. Chondrocytes and synoviocytes are crucial components of the joint space and are known targets of RRV infection.Hypothesis/Gap statement. Understanding the relationship between synoviocytes and chondrocytes during RRV infection will provide further insights into RRV-induced joint pathology.Methodology. To better understand the unique dynamics of these cells during RRV infection, we used primary chondrocytes cultured in physiologically relevant micromasses. We then directly infected micromass chondrocytes or infected primary fibroblast-like synoviocytes (FLS), co-cultured with micromass chondrocytes. Micromass cultures and supernatants were collected and analysed for viral load with a PCR array of target genes known to play a role in arthritis.Results. We show that RRV through direct or secondary infection in micromass chondrocytes modulates the expression of cellular factors that likely contribute to joint inflammation and disease pathology, as well as symptoms such as pain. More importantly, while we show that RRV can infect micromass-cultured chondrocytes via FLS infection, FLS themselves affect the regulation of cellular genes known to contribute to arthritis.Conclusion. Single-cell culture systems lack the complexity of in vivo systems, and understanding the interaction between cell populations is crucial for deciphering disease pathology, including for the development of effective therapeutic strategies.

Targeted mutagenesis of specific genomic DNA sequences in animals for the in vivo generation of variant libraries.

Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AIDevoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.

Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.)

BackgroundIsolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration.ResultsLow-temperature pre-treatment at 4 ℃ for 10–15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61–211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency.ConclusionsThis study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

Design and proof-of-concept of a micropillar-based microfluidic chip for trapping and culture of single cells

Design and proof-of-concept of a micropillar-based microfluidic chip for trapping and culture of single cells

Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor

BackgroundSingle-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides.ResultsThe water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists.ConclusionsWe established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

Abstract 5906: A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells

Abstract Despite advances in immune and targeted therapies, chemotherapy remains the primary treatment for ovarian cancer (OvCa) patients. Unfortunately, most OvCa patients develop chemotherapy-resistant disease that is inevitably fatal. Quiescence is an important yet poorly understood mechanism underlying chemotherapy resistance. Therefore, we aimed to characterize quiescent ovarian cancer (qOvCa) cells to identify novel therapeutic targets to improve OvCa patient outcomes. Primary patient-derived qOvCa cells and proliferating cells were isolated using a single-cell microfluidics culture device and collected for single-cell RNA-seq. scRNA-seq of qOvCa cells identified (i) downregulation of MBD3 and CHD4, which are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, and (ii) upregulation of proteasome-associated genes. Knockdown of either MBD3 or CHD4 by shRNA, or NuRD complex pharmacologic inhibition with the HDAC inhibitors such as Vorinostat resulted in significant cell growth arrest without cell death. Standard and Fucci reporter-based cell cycle analysis further confirmed that inhibition of the NuRD complex with Vorinostat induced 94.47% cells in the G0 phase (p<0.0001), suggesting a dense quiescent state. Through cell cycle phase maps produced by iterative immunofluorescence of 30 core cell cycle regulators and manifold learning, we found Vorinostat-treated OvCa cells demonstrated a completely different cell cycle structure from control cells, with most of the cells in an extreme G0 phase. Bulk RNA-seq of Vorinostat-treated cells in three OvCa cell lines validated the downregulation of pathways relating to cell cycle, cell division, and DNA replication (LFC<-1, p<0.05), which is consistent with stem cells. Upregulation of several pathways involved in proteostasis were also noted. We therefore performed a targeted drug screen for combinatorial therapy approaches to eradicate qOvCa. We found that Vorinostat combined with the proteasome inhibitor Carfilzomib demonstrated the most profound synergistic cell death (Loewe index synergy score 23-73). Increased proteasome activity was also found in both primary and Vorinostat-induced qOvCa, suggesting the essential role of the proteasome in qOvCa. Tumor xenograft studies confirmed that Vorinostat alone can delay tumor growth (p<0.05) and combinatorial therapy of Vorinostat and Carfilzomib is more efficacious (p=0.0049). Taken together, NuRD complex inhibition induces quiescence. Combination therapy with Vorinostat to induce quiescence combined with proteasome inhibitors resulted in the profound death of therapy-resistant quiescent cells. Our work suggests this is a novel therapeutic approach to eradicate chemoresistant qOvCa cells and increase cure rates. Citation Format: Qi Jiang, Michelle Ertel, Santiago Panesso- Gómez, Sara Sannino, April Sagan, Alexander J. Cole, Stacy C. McGonigal, Betsy Ann Varghese, Wayne Stallaert, Jeffrey L. Brodsky, Hatice U. Osmanbeyoglu, Ronald J. Buckanovich. A new combinatorial therapy that synergistically targets the NuRD complex and proteostasis to eradicate quiescent ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5906.

Abstract 2895: ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts

Abstract Introduction: Pancreatic cancer (PDAC) patients have poor prognosis partly due to excessive activity of cancer-associated fibroblasts (CAFs). CAFs drive the fibrosis that causes excessive type III collagen and extracellular matrix deposition that in turn reduces drug response resulting in poor survival. In support, high levels of the type III collagen serum biomarker PRO-C3 correlates with poor survival in PDAC. TGF-β is thought to be the main driver of PRO-C3 and tumor fibrosis. Cytokines such as Interleukin 1 (IL-1) play a key role in the pancreatic tumor microenvironment and may play a role in tumor fibrosis as well. In this study, we first investigated the potential of IL-1 in activating fibroblasts to drive fibrosis and produce PRO-C3. Subsequently, we established a co-culture of pancreatic cancer cells and pancreatic CAFs to investigate the anti-fibrotic properties of nadunolimab, an antibody that blocks IL-1 signaling by targeting IL-1 Receptor Accessory Protein (IL1RAP). Nadunolimab is currently in phase I/IIa clinical development for treatment of pancreatic cancer (CANFOUR, NCT03267316). Methods: Human primary pancreatic cancer-associated fibroblasts (CAFs) were cultured in Ficoll-media (Scar-in-a-jar, SiaJ) supplemented with TGF-β, IL-1α or IL-1β. The fibrotic activity of the fibroblasts was investigated by measuring the formation of type III collagen (PRO-C3) at days 3, 6, 9 and 12. Then, pancreatic cancer cells (BxPc3) and pancreatic CAFs were cultured either alone or in a co-culture. Nadunolimab or isotype control were added at the start of these cultures and supernatants were collected after three days. The level of PRO-C3 was measured by ELISA. Results: Both IL-1α or IL-1β were equipotent to TGF-β in inducing PRO-C3 in the SiaJ monoculture, indicating that IL-1 is pro-fibrotic. In addition, when cancer cells and CAFs were co-cultured, PRO-C3 levels increased compared to single-cell cultures (1.5-6-fold). When co-cultures were treated with nadunolimab, the induction of PRO-C3 was blocked to levels similar to monocultures, whereas the isotype control had no effect on PRO-C3 levels. Conclusion: IL-1 activated fibroblasts and induced type III collagen formation (PRO-C3), suggesting that IL-1 is a driver of tumor fibrosis. In support, pancreatic tumor cells induced collagen type III formation (PRO-C3) in pancreatic CAFs and blockade of IL1RAP with nadunolimab inhibited this collagen formation. Thus, nadunolimab may have anti-fibrotic properties and PRO-C3 could potentially be used for prognostic/predictive enrichment and as a pharmacodynamic marker in future studies evaluating anti-IL-1 modalities in PDAC. Citation Format: Neel I. Nissen, Nils Hansen, Elin Jaensson Gyllenbäck, Camilla R. Millrud, Marcus Järås, David Liberg, Morten A. Karsdal, Nicholas Willumsen. ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2895.

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

Single Cell Microgels for High‐Throughput Magnetic Sorting and Sequencing of Antigen‐Specific Antibodies

AbstractProgress in the discovery of potent antibodies for therapeutic and research purposes has been accelerated by mining the antibody repertoire of plasma cells and plasmablasts. Magnetic activated cell sorting (MACS) is a well recognized method for cell sorting, however, secreted IgGs rapidly diffuse away from cells, therefore requiring specialized technique to compartmentalize individual cells and capture their secretions for MACS isolation. To this end, a scalable method to prepare hydrogel‐based microcontainers for single cell culture and subsequent sorting based on their secreted IgG products at high‐throughput level are described. In situ crosslinkable microgels featuring tunable viscosity are fabricated to act as soft compartments that accommodate delicate primary cells and capture secreted proteins, while providing solid supports after solgel transition to enable immune recognition with chemically functionalized magnetic nanorods in aqueous medium for MACS separation. Splenocytes are sorted based on antigen‐specific IgG production from mice immunized with an antibiotic target ceftriaxone sodium, enriching a population of 220 000 000 to 800 000 prevalence following sorting. This enrichment ratio is substantially higher than conventional fluorescence activated cell sorting (FACS) methods, likely due to the multivalency of the antigen‐conjugated magnetic nanorods.

Radiation evaluation assay using a human three-dimensional oral cancer model for clinical radiation therapy.

With the development of various radiation-based cancer therapies, radiobiological evaluation methods instead of traditional clonogenic assays with monolayer single cell culture are required to bridge gaps in clinical data. Heterogeneity within cancer tissues is the reason for bridging the gap between basic and clinical research in cancer radiotherapy. To solve this problem, we investigated an evaluation assay using a three-dimensional (3D) model of cancer tissue. In this study, a 3D model consisting of tumor and stromal layers was used to compare and verify radiobiological effects with conventional two-dimensional (2D) methods. A significant difference in the response to radiation was observed between the 2D and 3D models. The relative number of cancer cells decreased with X-ray dose escalations in the 2D and 3D models. In contrast, the relative number of normal cells was quite different between the 2D and 3D models. Considering the ability of cells to recover from radiation-induced damage, the histological results of the 3D model were reflected in the clinical data. Histopathological analysis using a 3D model is a potential method for evaluating radiobiological effects on the tumor and tumor margins.

Regulation of headache response and transcriptomic network by the trigeminal ganglion clock.

To characterize the circadian features of the trigeminal ganglion in a mouse model of headache. Several headache disorders, such as migraine and cluster headache, are known to exhibit distinct circadian rhythms of attacks. The circadian basis for these rhythmic pain responses, however, remains poorly understood. We examined trigeminal ganglion exvivo and single-cell cultures from Per2::LucSV reporter mice and performed immunohistochemistry. Circadian behavior and transcriptomics were investigated using a novel combination of trigeminovascular and circadian models: a nitroglycerin mouse headache model with mechanical thresholds measured every 6 h, and trigeminal ganglion RNA sequencing measured every 4 h for 24 h. Finally, we performed pharmacogenomic analysis of gene targets for migraine, cluster headache, and trigeminal neuralgia treatments as well as trigeminal ganglion neuropeptides; this information was cross-referenced with our cycling genes from RNA sequencing data to identify potential targets for chronotherapy. The trigeminal ganglion demonstrates strong circadian rhythms in both exvivo and single-cell cultures, with core circadian proteins found in both neuronal and non-neuronal cells. Using our novel behavioral model, we showed that nitroglycerin-treated mice display circadian rhythms of pain sensitivity which were abolished in arrhythmic Per1/2 double knockout mice. Furthermore, RNA-sequencing analysis of the trigeminal ganglion revealed 466 genes that displayed circadian oscillations in the control group, including core clock genes and clock-regulated pain neurotransmitters. In the nitroglycerin group, we observed a profound circadian reprogramming of gene expression, as 331 of circadian genes in the control group lost rhythm and another 584 genes gained rhythm. Finally, pharmacogenetics analysis identified 10 genes in our trigeminal ganglion circadian transcriptome that encode target proteins of current medications used to treat migraine, cluster headache, or trigeminal neuralgia. Our study unveiled robust circadian rhythms in the trigeminal ganglion at the behavioral, transcriptomic, and pharmacogenetic levels. These results support a fundamental role of the clock in pain pathophysiology. Several headache diseases, such as migraine and cluster headache, have headaches that occur at the same time each day. We learned that the trigeminal ganglion, an important pain structure in several headache diseases, has a 24-hour cycle that might be related to this daily cycle of headaches. Our genetic analysis suggests that some medications may be more effective in treating migraine and cluster headache when taken at specific times of the day.

A multilayer microfluidic system for studies of the dynamic responses of cellular proteins to oxygen switches at the single-cell level.

Oxygen levels vary in the environment. Oxygen availability has a major effect on almost all organisms, and oxygen is far more than a substrate for energy production. However, less is known about related biological processes under hypoxic conditions and about the adaptations to changing oxygen concentrations. The yeast Saccharomyces cerevisiae can adapt its metabolism for growth under different oxygen concentrations and can grow even under anaerobic conditions. Therefore, we developed a microfluidic device that can generate serial, accurately controlled oxygen concentrations for single-cell studies of multiple yeast strains. This device can construct a broad range of oxygen concentrations, [O2] through on-chip gas-mixing channels from two gases fed to the inlets. Gas diffusion through thin polydimethylsiloxane (PDMS) can lead to the equilibration of [O2] in the medium in the cell culture layer under gas cover regions within 2min. Here, we established six different and stable [O2] varying between ~0.1 and 20.9% in the corresponding layers of the device designed for multiple parallel single-cell culture of four different yeast strains. Using this device, the dynamic responses of different yeast transcription factors and metabolism-related proteins were studied when the [O2] decreased from 20.9% to serial hypoxic concentrations. We showed that different hypoxic conditions induced varying degrees of transcription factor responses and changes in respiratory metabolism levels. This device can also be used in studies of the aging and physiology of yeast under different oxygen conditions and can provide new insights into the relationship between oxygen and organisms. Integration, innovation and insight: Most living cells are sensitive to the oxygen concentration because they depend on oxygen for survival and proper cellular functions. Here, a composite microfluidic device was designed for yeast single-cell studies at a series of accurately controlled oxygen concentrations. Using this device, we studied the dynamic responses of various transcription factors and proteins to changes in the oxygen concentration. This study is the first to examine protein dynamics and temporal behaviors under different hypoxic conditions at the single yeast cell level, which may provide insights into the processes involved in yeast and even mammalian cells. This device also provides a base model that can be extended to oxygen-related biology and can acquire more information about the complex networks of organisms.

Characterization of the osteogenic differentiation capacity of human bone cells on hybrid β-TCP/ZrO2 structures

This study deals with synthetic biomaterials that are biocompatible and mechanically stable to serve as a potential bone graft for improved mandibular reconstruction. To fabricate novel hybrid scaffolds containing beta-tricalcium phosphate (β-TCP) and zirconia (ZrO2), 3D printing was combined with Freeze Foaming to achieve a certain porosity, mimicking cancellous bone. The aim was to characterize the differentiation capacity of human pre-osteoblasts on the hybrid scaffolds. Although a good biocompatibility was demonstrated for the tested components of the hybrid scaffold, pure β-TCP foams showed the best results regarding osteogenic differentiation and pro-inflammatory processes. To enhance the osteoinductive properties of the β-TCP foam, the structures were also biofunctionalized with bone morphogenic protein 2 (BMP–2) and its effect was analyzed either on single cell cultures of pre-osteoblasts or pre-osteoblasts directly co-cultured with human peripheral blood mononuclear cells (PBMCs). The latter served to analyze the induction of bone remodelling processes. The immobilization of BMP-2 on scaffolds and its biological accessibility could be demonstrated, however, the biofunctionalization did not result in an enhanced differentiation capacity and bone remodeling processes of either pre-osteoblasts or directly co-cultured pre-osteoblasts and PBMCs.

Microbial consortium assembly and functional analysis via isotope labelling and single-cell manipulation of polycyclic aromatic hydrocarbon degraders.

Soil microbial flora constitutes a highly diverse and complex microbiome on Earth, often challenging to cultivation, with unclear metabolic mechanisms in situ. Here, we present a pioneering concept for the in situ construction of functional microbial consortia (FMCs) and introduce an innovative method for creating FMCs by utilizing phenanthrene as a model compound to elucidate their in situ biodegradation mechanisms. Our methodology involves single-cell identification, sorting, and culture of functional microorganisms, resulting in the formation of a precise in situ FMC. Through Raman-activated cell sorting-stable-isotope probing, we identified and isolated phenanthrene-degrading bacterial cells from Achromobacter sp. and Pseudomonas sp., achieving precise and controllable in situ consortia based on genome-guided cultivation. Our in situ FMC outperformed conventionally designed functional flora when tested in real soil, indicating its superior phenanthrene degradation capacity. We revealed that microorganisms with high degradation efficiency isolated through conventional methods may exhibit pollutant tolerance but lack actual degradation ability in natural environments. This finding highlights the potential to construct FMCs based on thorough elucidation of in situ functional degraders, thereby achieving sustained and efficient pollutant degradation. Single-cell sequencing linked degraders with their genes and metabolic pathways, providing insights regarding the construction of in situ FMCs. The consortium in situ comprising microorganisms with diverse phenanthrene metabolic pathways might offer distinct advantages for enhancing phenanthrene degradation efficiency, such as the division of labour and cooperation or communication among microbial species. Our approach underscores the importance of in situ, single-cell precision identification, isolation, and cultivation for comprehensive bacterial functional analysis and resource exploration, which can extend to investigate MFCs in archaea and fungi, clarifying FMC construction methods for element recycling and pollutant transformation in complex real-world ecosystems.

Cellular Thermal Biology Using Fluorescent Nanothermometers.

It has been known that cells have mechanisms to sense and respond to environmental noxiousness and mild temperature changes, such as heat shock response and thermosensitive TRP channels. Meanwhile, new methods of measuring temperature at the cellular level has recently been developed using fluorescent nanothermometers. Among these thermometers, fluorescent polymeric thermometers and fluorescent nanodiamonds excel in the properties required for intracellular thermometry. By using these novel methods to measure the temperature of single cells in cultures and tissues, it was revealed that spontaneous spatiotemporal temperature fluctuations occur within cells. Furthermore, the temperature fluctuations were related to organelles such as mitochondria and cellular and physiological functions, revealing a close relationship between intracellular temperature and cellular functions.