Reproducible 3D culture of multicellular tumor spheroids in supramolecular hydrogel from cancer stem cells sorted by sedimentation field-flow fractionation.

Three-dimensional (3D) cancer models, such as multicellular tumor spheroids (MCTS), are biological supports used for research in oncology, drug development and nanotoxicity assays. However, due to various analytical and biological challenges, the main recurring problem faced when developing this type of 3D model is the lack of reproducibility. When using a 3D support to assess the effect of biologics, small molecules or nanoparticles, it is essential that the support remains constant over time and multiples productions. This constancy ensures that any effect observed following molecule exposure can be attributed to the molecule itself and not to the heterogeneous properties of the 3D support. In this study, we address these analytical challenges by evaluating for the first time the 3D culture of a sub-population of cancer stem cells (CSCs) from a glioblastoma cancer cell line (U87-MG), produced by a SdFFF (sedimentation field-flow fractionation) cell sorting, in a supramolecular hydrogel composed of single, well-defined molecule (bis-amide bola amphiphile 0.25% w/v) with a stiffness of 0.4 kPa. CSCs were chosen for their ability of self-renewal and multipotency that allow them to generate fully-grown tumors from a small number of cells. The results demonstrate that CSCs cultured in the hydrogel formed spheroids with a mean diameter of 336.67 ± 38.70 µm by Day 35, indicating reproducible growth kinetics. This uniformity is in contrast with spheroids derived from unsorted cells, which displayed a more heterogeneous growth pattern, with a mean diameter of 203.20 ± 102.93 µm by Day 35. Statistical analysis using an unpaired t-test with unequal variances confirmed that this difference in spheroid size is significant, with a p-value of 0.0417 (p < 0.05). These findings demonstrate that CSC-derived spheroids, when cultured in a well-defined hydrogel, exhibit highly reproducible growth patterns compared to spheroids derived from unsorted cells, making them a more reliable 3D model for biological research and drug testing applications.

Targeting RSV-neutralizing B cell receptors with anti-idiotypic antibodies.

Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies.

Isolation and Selection of Protein-Rich Mutants of Chlorella vulgaris by Fluorescence-Activated Cell Sorting with Enhanced Biostimulant Activity to Germinate Garden Cress Seeds.

Microalgae are a promising feedstock with proven biostimulant activity that is enhanced by their biochemical components (e.g., amino acids and phytohormones), which turns them into an appealing feedstock to reduce the use of fertilisers in agriculture and improve crop productivity and resilience. Thus, this work aimed to isolate protein-rich microalgal mutants with increased biostimulant activity. Random mutagenesis was performed with Chlorella vulgaris, and a selection of protein-rich mutants were sorted through fluorescence-activated cell sorting (FACS), resulting in the isolation of 17 protein-rich mutant strains with protein contents 19-34% higher than that of the wildtype (WT). Furthermore, mutant F4 displayed a 38%, 22% and 62% higher biomass productivity, growth rate and chlorophyll content, respectively. This mutant was then scaled up to a 7 L benchtop reactor to produce biomass and evaluate the biostimulant potential of this novel strain towards garden cress seeds. Compared to water (control), the germination index and the relative total growth increased by 7% and 19%, respectively, after the application of 0.1 g L-1 of this bioproduct, which highlights its biostimulant potential.

Measurement of adhesion and traction of cells at high yield reveals an energetic ratchet operating during nephron condensation.

Developmental biology-inspired strategies for tissue-building have extraordinary promise for regenerative medicine, spurring interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here, we present multiplexed adhesion and traction of cells at high yield (MATCHY). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and a semiautomated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely on a priori knowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for rearranged during transfection (RET) tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We then use MATCHY to create a biophysical atlas of mouse embryonic kidney primary cells and identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an "energetic ratchet" that accounts for spatial trends in nephron progenitor cell condensation as they differentiate into early nephron structures, which we validate through agent-based computational simulation. MATCHY offers semiautomated cell biophysical characterization at >10,000-cell throughput, an advance benefiting fundamental studies and new synthetic tissue strategies for regenerative medicine.

Screening of Neutralizing Antibodies against FaeG Protein of Enterotoxigenic Escherichia coli.

The misuse of antibiotics in veterinary medicine presents significant challenges, highlighting the need for alternative therapeutic approaches such as antibody drugs. Therefore, it is necessary to explore the application of antibody drugs in veterinary settings to reduce economic losses and health risks. This study focused on targeting the F4ac subtype of the FaeG protein, a key adhesion factor in enterotoxigenic Escherichia coli (ETEC) infections in piglets. By utilizing formaldehyde-inactivated ETEC and a soluble recombinant FaeG (rFaeG) protein, an antibody library against the FaeG protein was established. The integration of fluorescence-activated cell sorting (FACS) and a eukaryotic expression vector containing murine IgG Fc fragments facilitated the screening of anti-rFaeG IgG monoclonal antibodies (mAbs). The results demonstrate that the variable regions of the screened antibodies could inhibit K88-type ETEC adhesion to IPEC-J2 cells. Furthermore, in vivo neutralization assays in mice showed a significant increase in survival rates and a reduction in intestinal inflammation. This research underscores the potential of antibody-based interventions in veterinary medicine, emphasizing the importance of further exploration in this field to address antibiotic resistance and improve animal health outcomes.

Identification of a Novel Subset of Human Airway Epithelial Basal Stem Cells.

The basal cell maintains the airway's respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell. To address functional heterogeneity amongst the basal cell population, we developed a novel monoclonal antibody, HLO1-6H5, that identifies a subset of KRT5+ (cytokeratin 5) basal cells. We used HLO1-6H5 and other known basal cell-reactive reagents to isolate viable airway subsets from primary human airway epithelium by Fluorescence Activated Cell Sorting. Isolated primary cell subsets were assessed for the stem cell capabilities of self-renewal and differentiation in the bronchosphere assay, which revealed that bipotent stem cells were, at minimum 3-fold enriched in the HLO1-6H5+ cell subset. Crosslinking-mass spectrometry identified the HLO1-6H5 target as a glycosylated TFRC/CD71 (transferrin receptor) proteoform. The HLO1-6H5 antibody provides a valuable new tool for identifying and isolating a subset of primary human airway basal cells that are substantially enriched for bipotent stem/progenitor cells and reveals TFRC as a defining surface marker for this novel cell subset.

Lab-on-Chip Systems for Cell Sorting: Main Features and Advantages of Inertial Focusing in Spiral Microchannels.

Inertial focusing-based Lab-on-Chip systems represent a promising technology for cell sorting in various applications, thanks to their alignment with the ASSURED criteria recommended by the World Health Organization: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Delivered. Inertial focusing techniques using spiral microchannels offer a rapid, portable, and easy-to-prototype solution for cell sorting. Various microfluidic devices have been investigated in the literature to understand how hydrodynamic forces influence particle focusing in spiral microchannels. This is crucial for the effective prototyping of devices that allow for high-throughput and efficient filtration of particles of different sizes. However, a clear, comprehensive, and organized overview of current research in this area is lacking. This review aims to fill this gap by offering a thorough summary of the existing literature, thereby guiding future experimentation and facilitating the selection of spiral geometries and materials for cell sorting in microchannels. To this end, we begin with a detailed theoretical introduction to the physical mechanisms underlying particle separation in spiral microfluidic channels. We also dedicate a section to the materials and prototyping techniques most commonly used for spiral microchannels, highlighting and discussing their respective advantages and disadvantages. Subsequently, we provide a critical examination of the key details of inertial focusing across various cross-sections (rectangular, trapezoidal, triangular, hybrid) in spiral devices as reported in the literature.

High-Throughput Single-Cell Metabolic Labeling, Sorting, and Sequencing of Active Antibiotic-Resistant Bacteria in the Environment.

Active antibiotic-resistant bacteria (ARB) play a major role in spreading antimicrobial resistance (AMR) in the environment; however, they have remained largely unexplored. Herein, we coupled bio-orthogonal noncanonical amino acid tagging with high-throughput fluorescence-activated single-cell sorting (FACS) and sequencing to characterize the phenome and genome of active ARB in complex environmental matrices. Active ARB, conferring resistance to six antibiotics throughout wastewater treatment, were distinguished and quantified. The percentage and concentration of active ARB ranged from 0.28% to 45.3% and from 1.1 × 104 to 2.09 × 107 cells/mL, respectively. Notably, the final effluents retained up to 4.79 × 104 cells/mL of active ARB. Targeted FACS and genomic sequencing revealed a distinct taxonomic composition of active ARB compared with that of the overall population. The coexistence of antibiotic resistome and mobilome in active ARB was also identified, including three high-quality metagenomic assembly genomes assigned to pathogenic bacteria, highlighting the substantial health risks due to their activity, phenotypic resistance, mobility, and pathogenicity. This study advances our understanding of previously overlooked active ARB in the environment by linking their resistance phenotype to their genotype. This high-throughput method will enable efficient quantitative surveillance of active AMR, providing valuable insights into risk control and management.

Different Biomechanical Cell Behaviors in an Epithelium Drive Collective Epithelial Cell Extrusion.

In vertebrates, many organs, such as the kidney and the mammary gland form ductal structures based on the folding of epithelial sheets. The development of these organs relies on coordinated sorting of different cell lineages in both time and space, through mechanisms that remain largely unclear. Tissues are composed of several cell types with distinct biomechanical properties, particularly at cell-cell and cell-substrate boundaries. One hypothesis is that adjacent epithelial layers work in a coordinated manner to shape the tissue. Using in vitro experiments on model epithelial cells, differential expression of atypical Protein Kinase C iota (aPKCi), a key junctional polarity protein, is shown to reinforce cell epithelialization and trigger sorting by tuning cell mechanical properties at the tissue level. In a broader perspective, it is shown that in a heterogeneous epithelial monolayer, in which cell sorting occurs, forces arising from epithelial cell growth under confinement by surrounding cells with different biomechanical properties are sufficient to promote collective cell extrusion and generate emerging 3D organization related to spheroids and buds. Overall, this research sheds light on the role of aPKCi and the biomechanical interplay between distinct epithelial cell lineages in shaping tissue organization, providing insights into the understanding of tissue and organ development.

Profiling of B cells and their subsets by whole blood gene expression analysis versus flow cytometry in multiple sclerosis.

We investigated if differentially expressed mRNA targets could be used as surrogate markers for circulating B cells and subsets. In paired blood samples from patients with untreated, anti-CD20-treated, fingolimod-treated, and natalizumab-treated multiple sclerosis, whole blood expression of CD19 correlated with B cell counts determined by flow cytometry, ROR1 with transitional B cells, TCL1A and ZNF727 with naïve B cells, NEXMIF with memory B cells and BCMA with plasmablasts. CD19 expression distinguished patients with B cell repletion and may be used as an alternative to flow cytometry, but NEXMIF was unsuitable for memory B cell monitoring in rituximab-treated patients.

In-vitro blood purification using tiny pinch holographic optical tweezers based on deep learning.

In-vitro blood purification is essential to a wide range of medical treatments, requiring fine-grained analysis and precise separation of blood components. Despite existing methods that can extract specific components from blood by size or by magnetism, there is not yet a general approach to efficiently filter blood components on demand. In this work, we introduce the first programmable non-contact blood purification system for accurate blood component detection and extraction. To accurately identify different cells and artificial particles in the blood, we collected and annotated a new blood component object detection dataset and trained a collection of deep-learning-based object detectors upon it. To precisely capture and extract desired blood components, we fabricated a microfluidic chip and set up a customized holographic optical tweezer to trap and move cells/particles in the blood. Empirically, we demonstrate that our proposed system can perform real-time blood fractionation with high precision reaching up to 96.89%, as well as high efficiency. Its scalability and flexibility open new research directions in blood treatment.

MitoTracker Red for isolation of zone-specific hepatocytes and characterization of hepatic sublobular metabolism.

BACKGROUND: The liver lobule is divided into three zones or regions: periportal (PP or Zone 1) that is highly oxidative and active in ureagenesis, pericentral (PC or Zone 3) that is more glycolytic, and midzonal (MZ or Zone 2) with intermediate characteristics. AIM: Our goal was to isolate and metabolically characterize hepatocytes from specific sublobular zones. METHODS: Mice were administered rhodamine123 (Rh123) or MitoTracker Red (MTR) prior to intravital imaging, liver fixation, or hepatocyte isolation. After in vivo MTR, hepatocytes were isolated and sorted based on MTR fluorescence intensity. Alternatively, E-cadherin (Ecad) and cytochrome P450 2E1 (CYP2E1) immunolabeling was performed in fixed liver slices. Ecad and CYP2E1 gene expression in sorted hepatocytes was assessed by qPCR. Oxygen consumption rates (OCR) of sorted hepatocytes were also assessed. RESULTS: Multiphoton microscopy showed Rh123 and MTR fluorescence distributed zonally, decreasing from PP to PC in a flow-dependent fashion. In liver cross-sections, Ecad was expressed periportally and CYP2E1 pericentrally in association with high and low MTR labeling, respectively. Based on MTR fluorescence, hepatocytes were sorted into PP, MZ, and PC populations with PP and PC hepatocytes enriched in Ecad and CYP2E1, respectively. OCR of PP hepatocytes was ∼4 times that of PC hepatocytes. CONCLUSIONS: MTR treatment in vivo delineates sublobular hepatic zones and can be used to sort hepatocytes zonally. PP hepatocytes have substantially greater OCR compared to PC and MZ. The results also indicate a sharp midzonal demarcation between hepatocytes with PP characteristics (Ecad) and those with PC features (CYP2E1). This new method to sort hepatocytes in a zone-specific fashion holds the potential to shed light on sublobular hepatocyte metabolism and regulatory pathways in health and disease.

Environmental community transcriptomics: strategies and struggles.

Transcriptomics is the study of RNA transcripts, the portion of the genome that is transcribed, in a specific cell, tissue, or organism. Transcriptomics provides insight into gene expression patterns, regulation, and the underlying mechanisms of cellular processes. Community transcriptomics takes this a step further by studying the RNA transcripts from environmental assemblies of organisms, with the intention of better understanding the interactions between members of the community. Community transcriptomics requires successful extraction of RNA from a diverse set of organisms and subsequent analysis via mapping those reads to a reference genome or de novo assembly of the reads. Both, extraction protocols and the analysis steps can pose hurdles for community transcriptomics. This review covers advances in transcriptomic techniques and assesses the viability of applying them to community transcriptomics.

Evaluation of single-cell sorting accuracy using antibody-derived tag-based qPCR.

Single-cell sorting (index sorting) is a widely used method to isolate one cell at a time using fluorescence-activated cell sorting (FACS) for downstream applications such as single-cell sequencing or single-cell expansion. Despite widespread use, few assays are available to evaluate the proteomic features of the sorted single cell and further confirm the accuracy of single-cell sorting. With this caveat, we developed a novel assay to confirm the protein expression of sorted single cells by co-staining cells with the same marker using both antibody-derived tags (ADTs) and fluorescent antibodies. After single-cell sorting, we amplified the oligo of the ADT reagent as a surrogate signal for the protein expression using multiplex TaqMan™ qPCR on sorted cells. This assay is not only useful for confirming the identity of a sorted single cell but also an efficient method to profile proteomic features at the single-cell level. Finally, we applied this assay to characterize protein expression on whole cell lysate. Because of the sensitivity of the TaqMan™ qPCR, we can detect protein expression from a small number of cells. In summary, the ADT-based qPCR assay developed here can be utilized to confirm single-cell sorting accuracy and characterizing protein expression on both single cells and whole cell lysate.

Detachable acoustofluidic droplet-sorter.

BACKGROUND: Cell sorting is crucial in isolating specific cell populations. It enables detailed analysis of their functions and characteristics and plays a vital role in disease diagnosis, drug discovery, and regenerative medicine. Fluorescence-activated cell sorting (FACS) is considered the gold standard for high-speed single-cell sorting. However, its high cost, complex instrumentation, and lack of portability are significant limitations. Additionally, the high pressure and electric fields used in FACS can harm cell integrity. In this work, an acoustofluidic device was developed in combination with surface acoustic wave (SAW) and droplet microfluidics to isolate single-cell droplets with high purity while maintaining high cell viability. RESULT: Human embryonic kidney cells, transfected with fluorescent reporter plasmids, were used to demonstrate the targeted droplet sorting containing single cells. The acoustofluidic sorter achieved a recovery rate of 81 % and an accuracy rate higher than 97 %. The device maintained a cell viability rate of 95 % and demonstrated repeatability over 20 consecutive trials without compromising efficiency, thus underscoring its reliability. Thermal image analysis revealed that the temperature of the interdigital transducer (IDT) during SAW operation remained within the permissible range for maintaining cell viability. SIGNIFICANCE: The findings highlighted the sensitivity and effectiveness of the developed acoustofluidic device as a tool for single-cell sorting. The detachable microfluidic chip design enables the reusability of the expensive IDT, making it cost-effective and reducing the risk of cross-contamination between different biological samples. The results underscore its capability to accurately isolate individual cells on the basis of specific criteria, showcasing its potential to advance research and clinical applications requiring precise cell sorting methodologies.

Enriching Cardiomyocytes Derived from hiPSCs by Magnetic-Activated Cell Sorting (MACS).

Cardiomyocytes (CMs) derived from human-induced pluripotent stem cells (hiPSCs) are considered a promising platform for multiple applications, including disease modeling, regenerative medicine, screening of drug toxicity and investigation of cardiomyogenesis. Despite remarkable improvement in methodology enabling differentiation of hiPSCs into CMs, applied protocols generate heterogeneous cell populations composed of CMs along with differentiated non-cardiac cell-types and undifferentiated hiPSCs. Here we describea procedure of automated Magnetic-Activated Cell Sorting (autoMACS) for the purification of hiPSCs-derived CMs under sterile culture conditions. We illustrate that this approach led to a robust depletion of non-cardiac cells and enrichment of CMs, a result particularly crucial for hiPSC lines with poor cardiac differentiation efficiencies.

Label-Free Continuous Cell Sorting Using Optofluidic Chip.

In the field of biomedicine, efficiently and non-invasively isolating target cells has always been one of the core challenges. Optical fiber tweezers offer precise and non-invasive manipulation of cells within a medium and can be easily integrated with microfluidic systems. Therefore, this paper investigated the mechanism of cell manipulation using scattering force with optical fiber tweezers. We employed flat-ended single-mode fiber to drive and sort cells and derived the corresponding scattering force formula based on the T-matrix model. A single-mode optical tweezers system for cell sorting was developed, and an optofluidic experimental platform was constructed that effectively integrates the optical system with microfluidic chips. The chip, featuring an expanded cross-channel design, successfully achieved continuous separation of yeast cells (8~10 µm in diameter) and polystyrene microspheres (15~20 µm in diameter), with a sorting efficiency of up to 86% and maintaining viability in approximately 90% of the yeast cells. Compared to other sorting systems, this system does not require labeling and can achieve continuous sorting with cell viability at a lower cost of instrumentation.

Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - Shedding light on spore superdormancy at 550 MPa.

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency. Bacillus subtilis spores were pressurized for 50 s - 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions. The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43-52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions. The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

Isolation of Rare Antigen-Specific Memory B Cells via Antigen Tetramers.

Immunological memory, which sets the foundation for the adaptive immune response, plays a key role in disease protection and prevention. Obtaining a deeper understanding of the mechanisms underlying this phenomenon can aide in research aimed to improve vaccines and therapies. Memory B cells (MBCs) are a fundamental component of immunological memory but can exist in rare populations that prove challenging to study. By combining fluorescent antigen tetramers with multiple enrichment processes, a highly streamlined method for identifying and sorting antigen-specific MBCs from human blood and lymphoid tissues can be achieved. With the output of this process being viable cells, there is a multitude of downstream operations that can be used in conjunction with the antigen-specific cell sorting outlined in this chapter. Single-cell RNA-sequencing paired with B cell repertoire sequencing, which can be linked to distinct antigens in a high-throughput fashion, is a downstream application widely used in disease and vaccination research. Incorporation of this protocol can lead to a variety of applications and a diversity of outcomes aiding in a deeper understanding of how immunological memory not only forms but is recalled and impacted by infection and vaccination.

Markers of Dermal Fibroblast Subpopulations for Viable Cell Isolation via Cell Sorting: A Comprehensive Review.

Fibroblasts are among the most abundant cell types in the human body, playing crucial roles in numerous physiological processes, including the structural maintenance of the dermis, production of extracellular matrix components, and mediation of inflammatory responses. Despite their importance, fibroblasts remain one of the least characterized cell populations. The advent of single-cell analysis techniques, particularly single-cell RNA sequencing (scRNA-seq) and fluorescence-activated cell sorting (FACS), has enabled detailed investigations into fibroblast biology. In this study, we present an extensive analysis of fibroblast surface markers suitable for cell sorting and subsequent functional studies. We reviewed over three thousand research articles describing fibroblast populations and their markers, characterizing and comparing subtypes based on their surface markers, as well as their intra- and extracellular proteins. Our detailed analysis identified a variety of distinct fibroblast subpopulations, each with unique markers, characteristics dependent on their location, and the physiological or pathophysiological environment. These findings underscore the diversity of fibroblasts as a cellular population and could lead to the development of novel diagnostic and therapeutic tools.