Viable Single Cells Research Articles

Factors Affecting Cell Viability during the Enzymatic Dissociation of Human Endocrine Tumor Tissues.

The enzymatic dissociation of human solid tissues is a critical process for disaggregating extracellular matrix and the isolation of individual cells for various applications, including the immortalizing primary cells, creating novel cell lines, and performing flow cytometry and its specialized type, FACS, as well as conducting scRNA-seq studies. Tissue dissociation procedures should yield intact, highly viable single cells that preserve morphology and cell surface markers. However, endocrine tissues, such as adrenal gland tumors, thyroid carcinomas, and pituitary neuroendocrine tumors, present unique challenges due to their complex tissue organization and morphological features. Our study conducted a morphological examination of these tissues, highlighting the intricate structures and secondary degenerative changes that complicate the dissociation process. We investigated the effects of various dissociation parameters, including the types of enzymes, incubation duration, and post-dissociation purification procedures, such as debris removal and nontarget blood cell lysis, on the viability of cells derived from different tumor types. The findings emphasize the importance of optimizing tissue digestion protocols to preserve cell viability and integrity, ensuring reliable outcomes for downstream analyses.

A simple method for the preparation of single cells and regeneration of colonies of Botryococcus braunii NIES836

Botryococcus braunii, a colonial alga, is known for notably slow growth; however, the growth rate and hydrocarbon productivity are expected to be improved using genetic modification techniques. Nevertheless, B. braunii has a hydrocarbon-rich extracellular matrix (ECM), and the ECM is a major barrier to DNA transformation. To analyse and utilize genetically modified B. braunii, it is essential to regenerate genetically homogeneous colonies derived from single cells. In this study, we developed a novel, simple method for harvesting viable single cells of B. braunii by centrifugation of the culture and subsequent filtration alone. The harvest of single cells was made possible by culturing B. braunii colonies in AF6 medium until the depletion of nitrogen and phosphorus sources and then releasing the single cells in colonies into the medium. Twenty-day culture of single cells in a 96-well plate resulted in 96% regeneration of colonies, and the regeneration of colonies was also confirmed on agar medium. This is the first report of colony regeneration from single cells of B. braunii. We believe that our method developed in this study will contribute greatly to the advancement of genetic modification techniques for B. braunii.

Isolation of planarian viable cells using fluorescence-activated cell sorting for advancing single-cell transcriptome analysis.

Preparing viable single cells is critical for conducting single-cell RNA sequencing (scRNA-seq) because the presence of ambient RNA from dead or damaged cells can interfere with data analysis. Here, we developed a method for isolating viable single cells from adult planarian bodies using fluorescence-activated cell sorting (FACS). This method was then applied to both adult pluripotent stem cells (aPSCs) and differentiating/differentiated cells. Initially, we employed a violet instead of ultraviolet (UV) laser to excite Hoechst 33342 to reduce cellular damage. After optimization of cell staining conditions and FACS compensation, we generated FACS profiles similar to those created using a previous method that employed a UV laser. Despite successfully obtaining high-quality RNA sequencing data for aPSCs, non-aPSCs produced low-quality RNA reads (i.e., <60% of cells possessing barcoding mRNAs). Subsequently, we identified an effective FACS gating condition that excluded low-quality cells and tissue debris without staining. This non-staining isolation strategy not only reduced post-dissociation time but also enabled high-quality scRNA-seq results for all cell types (i.e., >80%). Taken together, these findings imply that the non-staining FACS strategy may be beneficial for isolating viable cells not only from planarians but also from other organisms and tissues for scRNA-seq studies.

SpinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

O-060 Single-cell DNA sequencing reveals that current bulk DNA sequencing methods lead to an underestimation of chromosomal mosaicism in human blastocysts

Abstract Study question What is the incidence of chromosomal mosaicism in trophectoderm and inner cell mass from good quality human blastocysts after NGS-based single-cell molecular karyotyping? Summary answer Single-cell analysis showed that almost all human blastocysts are mosaic, involving low level mosaicism and/or abnormal cells resulting from reciprocal error events. What is known already Chromosome segregation in early human embryos is considered to be error prone. While meiotic errors affect all cells within embryos, mitotic errors lead to chromosomal mosaicism, the presence of cytogenetically different cells within an embryo. The reported incidence of mosaicism in blastocysts after bulk DNA sequencing of multicellular trophectoderm (TE) biopsies performed for preimplantation genetic testing for aneuploidy (PGT-A) varies between 2-19%. However, bulk DNA analysis can only assess net average chromosome gains or losses with a detection limit of 20-30%, and it makes distinguishing true mosaicism from technical artefact problematic. Study design, size, duration Observational study in human good quality surplus embryos which were donated for research purposes (CCMO, NL82597.000.22). The embryos were thawed and cultured until the blastocyst stage. From the blastocysts with at least a morphology grade 3BB, the ICM was dissociated from the TE. From 55 embryos, both samples were successfully disaggregated into single cells and manually placed in 384 well plates. For the cytogenetic analysis, a validated method for single-cell molecular karyotyping was used (scKaryo-seq). Participants/materials, setting, methods Embryos with at least two cytogenetically different cells were considered mosaic. To investigate if bulk DNA sequencing would have detected this mosaicism, we performed an in-silico reanalysis on our single-cell data. For each embryo, TE cells with the same mitotic abnormality were quantified. Next, we determined the number of mosaic embryos in which these abnormalities were present in at least 20% of TE cells, as a cut-off for what PGT-A methods are expected to detect. Main results and the role of chance On average 42% of the total number of cells that could be isolated per embryo were successfully karyotyped, giving a total of 1057 karyotyped cells (522 normal, 535 abnormal) from 55 embryos. Six embryos (11%) were normal, four (7%) were uniformly abnormal and 45 (82%) were mosaic. From these, 14 (26%) embryos were aneuploid mosaic with cytogenetically different abnormal cells and 31 (56%) were diploid-aneuploid mosaic with normal and abnormal cells. Here cytogenetically different abnormal cells were also frequently observed, with products of reciprocal events detected in 20 embryos. The in-silico reanalysis was performed on 38 embryos with a mosaic TE. It predicted that only 11% of the mitotic abnormalities observed in TE cells through scKaryo-seq would have been detected by bulk DNA sequencing. In 19 out of 38 embryos at least one mitotic abnormality affected more than 20% of the TE cells, meaning that only 50% of the embryos would have been recognized as mosaic with bulk DNA analysis. Bulk DNA sequencing methods used for PGT-A lead to an underestimation of mosaicism, since low-level mosaicism that affects a few cells within an embryo and products of reciprocal events without a net gain or loss remain undetected. Limitations, reasons for caution Isolation and cytogenetic analysis of viable single cells at the blastocyst stage is technically challenging and only 42% of the cells per embryo could be successfully karyotyped. Furthermore, it is unknown to what extent in vitro culture conditions and the freezing-thawing process influenced our findings. Wider implications of the findings These single cell observations support the notion that mosaicism is a common biological phenomenon in human blastocysts. Any selective mechanism eliminating embryos or cells with chromosomal abnormalities is still not fully active at this stage. The underestimation of mosaicism by bulk DNA analysis has potential implications for current PGT-A practices Trial registration number not applicable

Pancreatic Tissue Dissection to Isolate Viable Single Cells.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Abstract LB179: Automated viable circulating tumor cell (CTC) isolation enables efficient single-cell multi-omics analysis in a clinical setting

Abstract Circulating tumor cells (CTCs) are rare cells found in peripheral blood or other body fluids of cancer patients. Single-cell multi-omics analysis of CTCs can provide critical information and insights for tumor heterogeneity, early detection, residual disease, recurrence, and response and resistance to therapies. However, the adoption of single cell analysis in a clinical setting has been challenging due to complicated and lengthy workflows, lack of automation, low throughput, cell loss, inefficient cell picking, and nucleic acid degradation. To overcome these challenges, we have developed the fully automated LiquidScan liquid biopsy platform as a solution for CTC enrichment and single cell isolation. Whole blood samples were collected from breast, prostate, and lung cancer patients using BioFluidica blood collection tubes. CTCs were enriched with LiquidScan platform coupled with microfluidic affinity selection of rare cells with antibodies against cancer specific surface markers. Cell eluates released from microfluidic chips were further sorted and individual cells were placed into individual wells of PCR plates. The isolated single cells were processed with either whole genome amplification for DNA analysis or pre-amplification of RNA for transcriptome profiling. Cell viability and RNA integrity were assessed for enriched cells post LiquidScan processing. Whole blood samples were directly loaded to microfluidic chips and processed (capture, wash, release and elute) using Hamilton robot for automated processing with no require to red blood cell lysis. The sample processing time was approximately 3 hours with up to 8 samples processed simultaneously. Enriched cells were 70% viable on average post LiquidScan sample processing. The RNA RIN score for the enriched cells was 6 on average. Success rate of DNA amplifications and RNA pre-Amplifications were both over 90% across all samples processed. The LiquidScan platform provides a solution for fast, automated single viable cell isolation making downstream molecular analysis possible in a clinical setting. Protocols for RNA pre-amplification and DNA sequencing including copy number analysis and targeted resequencing have been developed. Single cell DNA methylation and proteomics assay development is in progress. Citation Format: Janet Dickerson, Dylan Dufek, Robbie Huff, Stephenie Jones, Jennifer Barber-Singh, Christopher Brandt, Judy Muller-Cohn, Rolf Muller, Yipeng Wang. Automated viable circulating tumor cell (CTC) isolation enables efficient single-cell multi-omics analysis in a clinical setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB179.

Abstract 5461: Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores

Abstract Single cell RNA-sequencing (scRNA-seq) yields valuable insights into the molecular heterogeneity of multiple cell types in normal and cancer tissues. scRNA-seq approach has the potential to not only answer basic research questions but also enhance clinical studies and clinical trials and accelerate drug development and testing. However, a consistent and reproducible method for isolating viable single cells and generating high quality single cell data from needle biopsy cores has been challenging to develop. Based on our experience isolating single cells from a large number of surgical tumor tissues, we set out to optimize a dissociation and processing protocol to generate viable single cell suspensions in adequate quantity for scRNA-seq from needle biopsy cores. A total of 17 de-identified 18-gauge needle biopsy samples were collected in collaboration with MD Anderson Cancer Center investigators. Samples were from liver, lymph node, pelvis, abdominal, neck, and lung tissues and included matched pre- and post-treatment samples. Patient biopsies were kept in RPMI medium on ice and transferred to EMPIRI (time to capture ranged from 3 to 6.5 hours after biopsy collection). Samples were dissociated using mechanical and enzymatic methods to achieve a single cell suspension. Cells were washed and viability and cell counts were verified before capture via 10x Chromium Connect. Captured cells were processed through the 10x Genomics Chromium Next GEM Single Cell workflow before sequencing on Illumina NovaSeq 6000 sequencer. Raw sequencing data were processed through EMPIRI’s computational pipeline that includes QC steps, doublet removal, and Seurat and other downstream analyses. Recovery of viable cells from individual 18-gauge biopsy cores ranged from 2,000-300,000 cells per core, depending on the tissue type and cellularity. Cell viability was strictly monitored and was maintained above 80% for 16 of 17 samples (most samples &amp;gt;90%). Cell counts after sequencing and pipeline analysis averaged 6,466 cells per sample. The median number of UMI counts per cell was 5,580, and an average of 1,699 genes were identified per cell. Average sequencing saturation was 73%. Here, we demonstrate the feasibility of integrating scRNA-seq analysis into clinical trials or clinical studies to obtain rapid insights into on-target drug effects and anticipated cellular responses to therapies, including immunotherapies, within 3 weeks of biopsy collection. We have established a method for isolating high quality single cells for scRNA-seq from tissue biopsy core, as small as a single 18-gauge needle core. We tested our protocol on 17 human samples and showed successful isolation of highly viable cells from lung, lymph node, pelvis, abdominal, neck, and lung tissues. We were able to generate scRNA-seq data sets from all samples for analysis. Acknowledgements: We thank our MDACC collaborators, particularly Drs. Scott Kopetz and Van Morris. Citation Format: Thomas Gallup, Sang Yun, Dave Gallup, Kyuson Yun. Gain early insights from single cell RNA-sequencing of clinical trial needle biopsy cores. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5461.

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation. Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes. The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Examination of resistance to nivolumab monotherapy through single-cell analysis of tumors from patients enrolled in the HCRN GU16-260 study of nivolumab monotherapy.

698 Background: Nivolumab (nivo) monotherapy demonstrated anti-tumor activity in treatment-naïve renal cell carcinoma (RCC) in Part A of HCRN GU16-260 across all IMDC groups and in multiple histologies. Patient tumor samples were collected to characterize the tumor-immune microenvironmental (TME) determinants of effective anti-tumor immunity with nivo. Methods: Eligible patients (with clear cell or non-clear cell histology) underwent tumor biopsy prior to and/or at resistance to nivo monotherapy. Fresh tissue fragments were cryopreserved locally and centrally processed to extract viable single-cells from the RCC TME. Single-cell RNA-sequencing (scRNA-seq) and T cell receptor (TCR)-sequencing (scTCR-seq) was performed on all tumor samples. Gene expression signatures discovered through scRNA-seq were used to interrogate previously published bulk transcriptomic data from the CheckMate-009/010/025 trials of nivo in the treatment-refractory setting. Results: In total, 72,730 viable single-cells (56,900 immune and 15,830 tumor or stromal cells) were sequenced from 17 patients (8 with baseline only, 7 with progression only, and 2 with paired baseline and progression samples) across 7 trial sites. Trajectory inference analysis of tumor-infiltrating T cells revealed a bifurcating trajectory, starting with naïve T cells and ending either in PMCH+ terminally exhausted CD8+ T cells or SLAMF7+ CD8+ T cells. Notably, the SLAMF7+ T cell population expressed high levels of cytotoxic genes (including GZMA, GZMB, GNLY) and markers of tissue residency ( ZNF683/HOBIT and ITGAE/CD103), and had a restricted TCR diversity (normalized Shannon index = 0.57). Among patients with at least 100 sequenced T cells (n = 14), a higher percentage of SLAMF7+ CD8+ T cells (relative to total T cells) was associated with primary resistance (progressive disease [PD] as best response) to nivo (mean percentage in PD [n = 4] patients 32.7%; stable disease [n = 4] patients, 9.1%; complete or partial response [CR/PR; n = 6] patients, 2.2%; p = 0.019 for CR/PR vs PD). A signature derived using genes expressed in the T cell trajectory branch containing the SLAMF7+ CD8+ T cell population was used to interrogate bulk RNA-seq data from 172 pre-treatment tumors from the nivo arms of the CheckMate-009/010/025 trials. The signature score was enriched in patients with PD compared to CR/PR as best response (p = 0.032). Analysis of bulk whole exome sequencing and RNA-seq from patients enrolled in the HCRN GU16-260 is pending. Conclusions: Single-cell transcriptomic analysis uncovered a SLAMF7+ CD8 + T cell population with markers of cytotoxicity and tissue residency that was associated with resistance to nivo monotherapy in RCC. Further, the study highlights that scRNA-seq is a viable scientific strategy for deep correlative analysis in multicenter clinical trials.

Dissociation of Mouse Olfactory Mucosae for Fluorescence-Activated Cell Sorting of Olfactory Sensory Neurons.

An increasing diversity of techniques investigating the biology of specific cell types and individual cells have elevated the importance of dissociation of viable cells from living tissues. Here we describe a method for the dissociation of single cells from samples of adult mouse olfactory mucosae, with an emphasis on maximizing yield of viable single cells from fluorescence-activated cell sorting. Yields are typically in the range of 80,000-150,000 viable cells per adult mouse.

Enhanced absorbance detection system for online bacterial monitoring in digital microfluidics.

We report a fully integrated digital microfluidic absorbance detection system with an enhanced sensitivity for online bacterial monitoring. Through a 100 μm gap in the chip, our optical detection system has a detection sensitivity for a BCA protein concentration of 0.1 mg mL-1. The absorbance detection limit of our system is 1.4 × 10-3 OD units, which is one order of magnitude better than that of the existing studies. The system's linear region is 0.1-7 mg mL-1, and the dynamic range is 0-25 mg mL-1. We measured the growth curves of wild-type and E. coli transformed with resistance plasmids and mixed at different ratios on chip. We sorted out the bacterial species including highly viable single cells based on the difference in absorbance data of growth curves. We explored the changes in the growth curves of E. coli under different concentrations of resistant media. In addition, we successfully screened for the optimal growth environment of the bacteria, in which the growth rate of PET30a-DH5α (in a medium with 33 μg mL-1 kanamycin resistance) was significantly higher than that of a 1 mg mL-1 resistance medium. In conclusion, the enhanced digital microfluidic absorbance detection system exhibits exceptional sensitivity, enabling precise bacterial monitoring and growth curve analysis, while also laying the foundation for DMF-based automated bioresearch platforms, thus advancing research in the life sciences.

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ Tcell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

A two-stage digestion of whole murine knee joints for single-cell RNA sequencing

ObjectiveSingle-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a technical challenge in preparing a viable and representative cell suspension suitable for use in scRNA-seq in minimal time, where under-digestion may exclude cells in hard tissues, over-digestion may damage soft tissue cells, and prolonged digestion may induce phenotypic drift. We developed a rapid two-stage digestion protocol to overcome these difficulties. DesignA two-stage digest consisting of first collagenase IV, an intermediate cell recovery, then collagenase II on the remaining hard tissue. Cells were sequenced on the 10x Genomics platform. ResultsWe observed consistent cell numbers and viable single cell suspensions suitable for scRNA-seq analysis. Comparison of contralateral knees and separate mice showed reproducible cell yields and gene expression patterns by similar cell-types. A diverse collection of structural and immune cells were captured with a majority from immune origins. Two digestions were necessary to capture all cell-types. ConclusionsThe knee contains a diverse mixture of stromal and immune cells that may be crucial for the study of osteoarthritis. The two-stage digestion presented here reproducibly generated highly viable and representative single-cell suspension for sequencing from the whole knee. This protocol facilitates transcriptomic studies of the joint as a complete organ.

EXTH-96. BIOPROCESSING OF SURGICAL PEDIATRIC BRAIN TUMOR SPECIMENS FOR GENOME-GUIDED PERSONALIZED DRUG TESTING

Abstract Novel treatment approaches for pediatric central nervous system (CNS) tumors are urgently needed. A lack of patient-derived tumor cells impedes progress towards developing such new therapies. We intended to overcome this challenge by establishing methods to create a biorepository of viable single cell suspensions of pediatric brain tumor surgical specimens. Quantitative and qualitative comparisons of tissue processing strategies were performed to preserve viability of heterogeneous tumor and immune cells. Novel drug targets were identified by analyzing pathways affected by RNA transcripts that are highly expressed (outliers) in a patients’ tumor; outliers for each patient were determined by RNA-seq data from individual patients’ tumors compared with a compendium of 12,747 pediatric and adult samples harmonized by the Treehouse Childhood Cancer Initiative at the UCSC Genomics Institute. The predicted anti-tumor efficacy of small molecule inhibitors of the outlier pathways was tested in cell viability assays against short-term cultured cells from matched patients. Successful tissue collection required obtaining informed consent, standard operating procedures, and sample recording using a Laboratory Inventory Management Software (LIMS). Since 2020 we have banked 67 pediatric CNS brain tumor specimens at Stanford. Amongst those, 51 cases yielded sufficient tissue for RNA-seq and cryoprotection. The most common tumor histology was low-grade glioma (LGG, 26 of 67), the majority of which were pilocytic astrocytoma (18 of 26). The second and third most common tumor types are embryonal tumors (6 medulloblastoma, 3 AT/RT) and ependymoma (4), respectively. We identified significant differences in cell viability with different preservation media. An outlier pathway previously not implicated in LGG was identified and sensitivity to a small molecule inhibitor of this outlier pathway was demonstrated. Taken together, we established feasibility for validating therapeutic vulnerabilities identified by a genome-guided approach in short-term cultures from surgical specimens. This works facilitates the rapid development of personalized CNS tumor treatment.

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters.

Electric-field facilitated rapid and efficient dissociation of tissues Into viable single cells

Single-Cell Analysis is a growing field that endeavors to obtain genetic profiles of individual cells. Disruption of cell–cell junctions and digestion of extracellular matrix in tissues requires tissue-specific mechanical and chemical dissociation protocols. Here, a new approach for dissociating tissues into constituent cells is described. Placing a tissue biopsy core within a liquid-filled cavity and applying an electric field between two parallel plate electrodes facilitates rapid dissociation of complex tissues into single cells. Different solution compositions, electric field strengths, and oscillation frequencies are investigated experimentally and with COMSOL Multiphysics. The method is compared with standard chemical and mechanical approaches for tissue dissociation. Treatment of tissue samples at 100 V/cm 1 kHz facilitated dissociation of 95 ± 4% of biopsy tissue sections in as little as 5 min, threefold faster than conventional chemical–mechanical techniques. The approach affords good dissociation of tissues into single cells while preserving cell viability, morphology, and cell cycle progression, suggesting utility for sample preparation of tissue specimens for direct Single-Cell Analysis.

Single-Fluorescence ATP Sensor Based on Fluorescence Resonance Energy Transfer Reveals Role of Antibiotic-Induced ATP Perturbation in Mycobacterial Killing.

ABSTRACTThe rapid emergence of multidrug-resistant/extensively drug-resistant tuberculosis (TB) is responsible for treatment failure in patients with TB and significantly endangers global public health. Recently, bioenergetics has become a new paradigm for anti-TB drug discovery and is based on the link between bacterial ATP levels and drug efficacy. A better understanding of the role of ATP fluctuations during antibiotic treatment may provide insight into antibiotic-mediated killing of mycobacteria. Here, we employed an advanced single-fluorescence FRET (fluorescence resonance energy transfer)-based ATP biosensor, ATPser, for the stable and convenient detection of intracellular ATP fluctuations in mycobacteria. This strategy correlated closely with the results obtained from conventional luminescence ATP assays, indicating the reliability of the system for bioenergetics analysis in mycobacteria. Moreover, the reporter strains expressing ATPser displayed obvious ATP changes when subjected to different stresses, such as starvation and ATP depletion. Interestingly, we observed that different antibiotics induced fluctuations in cellular ATP levels in individual cells of various magnitudes, revealing a strong connection between ATP fluctuations and drug efficacy. Furthermore, drug combinations accelerated ATP perturbation, resulting in increased cell death. We concluded that ATPser enabled real-time measurement of ATP at the single-cell level in mycobacteria, and monitoring ATP dynamics in drug-treated bacteria may shed light on novel treatment strategies.IMPORTANCE Bioenergetics has emerged as a new paradigm for antituberculosis (anti-TB) drug discovery, and the cellular ATP level is the core indicator reflecting bacterial metabolic homeostasis. Although several bulk assays have been designed for the measurement of cellular ATP content, a more convenient strategy is required for real-time ATP measurement of single viable cells. In this study, by combining the ε-subunit of Bacillus subtilis FoF1-ATP synthase with a circularly permuted green fluorescent protein [(cp)GFP], we constructed a FRET-based single-fluorescence ATP sensor, ATPser, for real-time single-cell ATP detection among a mycobacterial population. Using the ATPser, we designed different drug combinations containing components that have similar/opposite effects on ATP alternation. Our results demonstrated that increased cellular ATP fluctuations were associated with depletion of mycobacterial viability, while counteracting ATP fluctuations weakened the killing effect of the drug regime. Thus, potentially efficient drug combinations can be considered based on their similar effects on mycobacterial ATP levels, and ATPser may be a useful tool to study mycobacterial bioenergetics and to guide drug regime design.

Isolation of Viable Single Cells With High Yield and Purity Using a Small Amount of Human Kidney Tissue Biopsy.

Objective: Establishment of an efficient method of preparing human kidney single cell suspension, using a very small amount of tissue puncture. Methods: Samples of human kidney tissue puncture were cut into pieces, and then 80μL of the digestive enzyme were added to each punctured tissue to induce enzymatic digestion. The enzyme combination is composed of collagenases, DNase and hyaluronidase and the sample was incubated 20min at 37°C. The obtained cell suspension was filtered through a 70μm cell strainer, centrifuged at 300g for 5min and the supernatant was removed, then the pellet was resuspended in 3ml of DMEM (Dulbecco's Modified Eagle's Medium). Cell suspension was sorted and purified by flow sorting to remove dead cells and obtain a cell suspension with higher viability rate. Results: We found that 1) diverse single cells of human kidney can be obtained by the digestive enzyme, as observed under the light microscope, with different sizes, normal cell morphology and good dispersion. 2) (2-3) × 106 single cells can be extracted from one fresh punctured kidney tissue of about 10mg, with a cell viability rate of more than 80%. Conclusion: In this work we generated a comprehensive and high-resolution single-cell method, which is simple and efficient for preparing single cell suspension from a minimal amount of human kidney tissue. This method can facilitate the study of renal cell biology and the pathogenesis of kidney diseases.

An Optimized Tissue Dissociation Protocol for Single-Cell RNA Sequencing Analysis of Fresh and Cultured Human Skin Biopsies.

We present an optimized dissociation protocol for preparing high-quality skin cell suspensions for in-depth single-cell RNA-sequencing (scRNA-seq) analysis of fresh and cultured human skin. Our protocol enabled the isolation of a consistently high number of highly viable skin cells from small freshly dissociated punch skin biopsies, which we use for scRNA-seq studies. We recapitulated not only the main cell populations of existing single-cell skin atlases, but also identified rare cell populations, such as mast cells. Furthermore, we effectively isolated highly viable single cells from ex vivo cultured skin biopsy fragments and generated a global single-cell map of the explanted human skin. The quality metrics of the generated scRNA-seq datasets were comparable between freshly dissociated and cultured skin. Overall, by enabling efficient cell isolation and comprehensive cell mapping, our skin dissociation-scRNA-seq workflow can greatly facilitate scRNA-seq discoveries across diverse human skin pathologies and ex vivo skin explant experimentations.