Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting

Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1 s)and type 2 cells(AEC2s)are the key cells in the repair of injured lungs as well as fetal lung development.Therefore,the purification and culture of AECls and AEC2s play an important role in the research of repair and regeneration of lung tissue.Methods:Sprague-Dawley rats(3-4 weeks,120-150 g)were purchased for experiment.Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells,and then magnetic bead cell sorting was performed to isolate Tla positive cells as AECls from the single-cell suspension by using polyclonal rabbit anti-Tla(a specific AECls membrane protein)antibodies combined with anti-rabbit IgG microbeads.Afterwards,alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining Tla-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads.Cell purity was identified by immunofluorescence staining and flow cytometry.Resii沾••The purity of AECls and AEC2s was 88.3%±3.8%and 92.6%±2.7%,respectively.The cell growth was observed as follows:AECls stretched within the 12-16 h,but the cells proliferated slowly;while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.Conclusion:AECls and AEC2s sorted by this method have high purity and good viability.Therefore,our method provides a new approach for the isolation and culture of AECls and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Microfluidic platform for circulating tumor cells isolation and detection

Circulating tumor cells(CTCs)are essential biomarkers for liquid biopsies,which are important in the early screening,prognosis,and real-time monitoring of cancer.However,CTCs are less abundant in the peripheral blood of patients,therefore,their isolation is necessary.Recently,the use of microfluidics for CTC sorting has become a research hotspot owing to its low cost,ease of integration,low sample consumption,and unique advantages in the manipulation of micron-sized particles.Herein,we review the latest research on microfluidics-based CTC sorting.Specifically,we consider active sorting using external fields(electric,magnetic,acoustic,and optical tweezers)and passive sorting using the flow effects of cells in specific channel structures(microfiltration sorting,deterministic lateral displacement sorting,and inertial sorting).The advantages and limitations of each method and their recent applications are summarized here.To conclude,a forward-looking perspective is presented on future research on the microfluidic sorting of CTCs.

AI-assisted cell identification and optical sorting[Invited]

Cell identification and sorting have been hot topics recently.However,most conventional approaches can only predict the category of a single target,and lack the ability to perform multitarget tasks to provide coordinate information of the targets.This limits the development of high-throughput cell screening technologies.Fortunately,artificial intelligence(AI)systems based on deep-learning algorithms provide the possibility to extract hidden features of cells from original image information.Here,we demonstrate an AI-assisted multitarget processing system for cell identification and sorting.With this system,each target cell can be swiftly and accurately identified in a mixture by extracting cell morphological features,whereafter accurate cell sorting is achieved through noninvasive manipulation by optical tweezers.The AI-assisted model shows promise in guiding the precise manipulation and intelligent detection of high-flux cells,thereby realizing semiautomatic cell research.

Whole-blood sorting,enrichment and in situ immunolabeling of cellular subsets using acoustic microstreaming

Analyzing undiluted whole human blood is a challenge due to its complex composition of hematopoietic cellular populations,nucleic acids,metabolites,and proteins.We present a novel multi-functional microfluidic acoustic streaming platform that enables sorting,enrichment and in situ identification of cellular subsets from whole blood.This single device platform,based on lateral cavity acoustic transducers(LCAT),enables(1)the sorting of undiluted donor whole blood into its cellular subsets(platelets,RBCs,and WBCs),(2)the enrichment and retrieval of breast cancer cells(MCF-7)spiked in donor whole blood at rare cell relevant concentrations(10 mL^(−1)),and(3)on-chip immunofluorescent labeling for the detection of specific target cellular populations by their known marker expression patterns.Our approach thus demonstrates a compact system that integrates upstream sample processing with downstream separation/enrichment,to carry out multi-parametric cell analysis for blood-based diagnosis and liquid biopsy blood sampling.

Numerical Study on Separation of Circulating Tumor Cell Using Dielectrophoresis in a Four-Electrode Microfluidic Device

This numerical study proposes a cell sorting technique based on dielectrophoresis(DEP)in a microfluidic chip.Under the joint effect of DEP and fluid drag,white blood cells and circulating tumor cells are separated because of different dielectric properties.First,the mathematical models of device geometry,single cell,DEP force,electric field,and flow field are established to simulate the cell motion.Based on the simulation model,important boundary parameters are discussed to optimize the cell sorting ability of the device.A proper matching relationship between voltage and flow rate is then provided.The inlet and outlet conditions are also investigated to control the particle motion in the flow field.The significance of this study is to verify the cell separating ability of the microfluidic chip,and to provide a logistic design for the separation of rare diseased cells.

Single cell metabolic phenome and genome via the ramanome technology platform:Precision medicine of infectious diseases at the ultimate precision?

Due to the limitations of existing approaches,a rapid,sensitive,accurate,comprehensive,and generally applicable strategy to diagnose and treat bacterial and fungal infections remains a major challenge.Here,based on the ramanome technology platform,we propose a culture‐free,one cell resolution,phenome‐genome‐combined strategy called single‐cell identification,viability and vitality tests and source tracking(SCIVVS).For each cell directly extracted from a clinical specimen,the fingerprint region of the D2O‐probed single cell Raman spectrum(SCRS)enables species‐level identification based on a reference SCRS database of pathogen species,whereas the C‐D band accurately quantifies viability,metabolic vitality,phenotypic susceptibility to antimicrobials,and their intercellular heterogeneity.Moreover,to source track a cell,Raman‐activated cell sorting followed by sequencing or cultivation proceeds,producinging an indexed,high coverage genome assembly or a pure culture from precisely one pathogenic cell.Finally,an integrated SCIVVS workflow that features automated profiling and sorting of metabolic and morphological phenomes can complete the entire process in only a few hours.Because it resolves heterogeneity for both the metabolic phenome and genome,targets functions,can be automated,and is orders‐of‐magnitude faster while cost‐effective,SCIVVS is a new technological and data framework to diagnose and treat bacterial and fungal infections in various clinical and disease control settings.

FACS-iChip:a high-efficiency iChip system for microbial‘dark matter’mining

The isolation chip method(iChip)provides a novel approach for culturing previously uncultivable microorganisms;this method is currently limited by the user being unable to ensure single-cell loading within individual wells.To address this limitation,we integrated flow cytometry-based fluorescence-activated cell sorting with a modified iChip(FACS-iChip)to effectively mine microbial dark matter in soils.This method was used for paddy soils with the aim of mining uncultivable microorganisms and making preliminary comparisons between the cultured microorganisms and the bulk soil via 16S rRNA gene sequencing.Results showed that the FACS-iChip achieved a culture recovery rate of almost 40%and a culture retrieval rate of 25%.Although nearly 500 strains were cultured from 19 genera with 8 FACS-iChip plates,only six genera could be identified via 16S rRNA gene amplification.This result suggests that the FACS-iChip is capable of detecting strains in the currently dead spaces of PCR-based sequencing technology.We,therefore,conclude that the FACS-iChip system provides a highly efficient and readily available approach for microbial‘dark matter’mining.

Viscoelastic microfluidics: progress and challenges

The manipulation of cells and particles suspended in viscoelastic fluids in microchannels has drawn increasing attention,in part due to the ability for single-stream three-dimensional focusing in simple channel geometries.Improvement in the understanding of non-Newtonian effects on particle dynamics has led to expanding exploration of focusing and sorting particles and cells using viscoelastic microfluidics.Multiple factors,such as the driving forces arising from fluid elasticity and inertia,the effect of fluid rheology,the physical properties of particles and cells,and channel geometry,actively interact and compete together to govern the intricate migration behavior of particles and cells in microchannels.Here,we review the viscoelastic fluid physics and the hydrodynamic forces in such flows and identify three pairs of competing forces/effects that collectively govern viscoelastic migration.We discuss migration dynamics,focusing positions,numerical simulations,and recent progress in viscoelastic microfluidic applications as well as the remaining challenges.Finally,we hope that an improved understanding of viscoelastic flows in microfluidics can lead to increased sophistication of microfluidic platforms in clinical diagnostics and biomedical research.