Manipulation Of Cells Research Articles

Integrating Micro and Nano Technologies for Cell Engineering and Analysis: Toward the Next Generation of Cell Therapy Workflows.

The emerging field of cell therapy offers the potential to treat and even cure a diverse array of diseases for which existing interventions are inadequate. Recent advances in micro and nanotechnology have added a multitude of single cell analysis methods to our research repertoire. At the same time, techniques have been developed for the precise engineering and manipulation of cells. Together, these methods have aided the understanding of disease pathophysiology, helped formulate corrective interventions at the cellular level, and expanded the spectrum of available cell therapeutic options. This review discusses how micro and nanotechnology have catalyzed the development of cell sorting, cellular engineering, and single cell analysis technologies, which have become essential workflow components in developing cell-based therapeutics. The review focuses on the technologies adopted in research studies and explores the opportunities and challenges in combining the various elements of cell engineering and single cell analysis into the next generation of integrated and automated platforms that can accelerate preclinical studies and translational research.

Design of Optical Tweezers Manipulation Control System Based on Novel Self-Organizing Fuzzy Cerebellar Model Neural Network

Holographic optical tweezers have unique non-physical contact and can manipulate and control single or multiple cells in a non-invasive way. In this paper, the dynamics model of the cells captured by the optical trap is analyzed, and a control system based on a novel self-organizing fuzzy cerebellar model neural network (NSOFCMNN) is proposed and applied to the cell manipulation control of holographic optical tweezers. This control system consists of a main controller using the NSOFCMNN with a new self-organization mechanism, a robust compensation controller, and a higher order sliding mode. It can accurately move the captured cells to the expected position through the optical trap generated by the holographic optical tweezers system. Both the layers and blocks of the proposed NSOFCMNN can be adjusted online according to the new self-organization mechanism. The compensation controller is used to eliminate the approximation errors. The higher order sliding surface can enhance the performance of controllers. The distances between cells are considered in order to further realize multi-cell cooperative control. In addition, the stability and convergence of the proposed NSOFCMNN are proved by the Lyapunov function, and the learning law is updated online by the gradient descent method. The simulation results show that the control system based on the proposed NSOFCMNN can effectively complete the cell manipulation task of optical tweezers and has better control performance than other neural network controllers.

Characterization of the In Vitro Cultured Ovarian Cells in the Asian Yellow Pond Turtle (Mauremys mutica).

Simple SummaryGonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes’ functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells’ culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we isolated an ovarian cell line (fGSCs), from the juvenile Asian yellow pond turtle in vitro. The cultured fGSCs were characterized with germline stem cell specific markers’ expression using transcriptome sequencing on Illumina platform, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence (IF). Our study aims to provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle even in reptiles, and also, the established cell line has potential applications to enrich the conservation strategy of aquatic animal germplasm resources.Gonadal cell lines possess the abilities of self-renewal and differentiation, being used as an efficient tool to analyzing the genes’ functions involved in sex differentiation and gametogenesis. Although some significant achievements have been obtained in the gonadal cells’ culture or manipulation across multiple phyla including teleost and mammals, there is limited study on gonadal cell manipulation in turtles. In this study, we established a new ovarian cell line from the young Asian yellow pond turtle (Mauremys mutica), which exhibited a normal diploid karyotype with high alkaline phosphatase activity. The cell line, designated as YTO2, was then characterized through the analysis of gene expression profiles. The transcriptome analysis and the reverse transcription polymerase chain reaction (RT-PCR) showed that the cells expressed germline genes such as tdrd7, nanos1, klf5, igtb1, hsd17b4 and rad51. Moreover, the immunostaining showed that the germ cell markers, Tdrd7 and Rad51 proteins, were detected predominant in cytoplasm of perinuclear region, while proliferation marker, PCNA, was dominantly observed in the nuclei of cultured cells. Intriguingly, the cells could respond to the retinoic acid induction with significantly increasing the expression level of some meiosis genes, including vasa, dazl, figla, and dmc1. Furthermore, YTO2 cells could be efficiently transfected with the pHBAd-BHG-EGFP adenovirus and properly expressed the exogenous genes. To sum up, an ovarian cell line of the Asian yellow pond turtle had been established and could be stably propagated under in vitro culture condition, as well as being capable of efficiently expressing the exogenous gene tdrd7. This cell line would provide a valuable tool to elaborate the molecular mechanisms behind germ cells development, differentiation and oogenesis in the turtle, even in reptiles.

Continuously tuneable acoustic metasurface for high order transmitted acoustic vortices

Existed tuneable acoustic metasurfaces (AMSs) can only transfer high order orbital angular momentum by converting the incident wave into reflected acoustic vortices (AVs). Here, a continuously tuneable AMS is designed and fabricated to produce transmitted AVs with multiple orders. The proposed AMS is composed of multiple layers of reconfigurable sector-shaped resonator-based units. A new design mechanism of the tuneable AMS is proposed to ensure that the discrete precision of the phase shift is not affected by the number of the layer. The theoretical, numerical and experimental results prove that the proposed AMS is able to continuously generate high-order transmitted AVs, which is promising in applications of real-time acoustic rotational manipulations such as acoustic microfluidics, cell manipulation, acoustic tweezers and acoustic communication.

Magnetophoretic Cell Sorting: Comparison of Different 3D-Printed Millifluidic Devices

Cell sorting is a highly applicable technology for multiple biological, biotechnological, and medical applications. Magnetic cell sorting can be realized with microfluidic and millifluidic flow cells. Additive manufacturing and 3D printing allow for fast prototyping and validating separation processes on this small scale. Therefore, our novel approach is to use this technology to print millifluidic channels and to directly evaluate them on their magnetic separation performance and their handling for cell manipulation. In this study, two different flow cells manufactured with a 3D printer are compared in regard to their use for the magnetic cell sorting of algae. One linear flow cell geometry and one spiraling flow cell geometry have been investigated with perpendicular magnetic fields. Iron oxide nanoparticles have been synthesized and characterized prior to their use as a magnetic label for algae cells. Particle uptake by algae are investigated by a phenanthroline assay, and the particle/algae mixtures are studied by microscopy, dynamic light scattering, zeta potential, and magnetophoretic mobility measurements. Depending on magnetic susceptibility, the cells undergo different magnetophoretic forces. Interestingly, the spiraling geometry leads to a better fractionation of algae cells in accordance with their iron oxide load.

Genetic engineering of baculovirus-insect cell system to improve protein production.

The Baculovirus Expression Vector System (BEVS), a mature foreign protein expression platform, has been available for decades, and has been effectively used in vaccine production, gene therapy, and a host of other applications. To date, eleven BEVS-derived products have been approved for use, including four human vaccines [Cervarix against cervical cancer caused by human papillomavirus (HPV), Flublok and Flublok Quadrivalent against seasonal influenza, Nuvaxovid/Covovax against COVID-19], two human therapeutics [Provenge against prostate cancer and Glybera against hereditary lipoprotein lipase deficiency (LPLD)] and five veterinary vaccines (Porcilis Pesti, BAYOVAC CSF E2, Circumvent PCV, Ingelvac CircoFLEX and Porcilis PCV). The BEVS has many advantages, including high safety, ease of operation and adaptable for serum-free culture. It also produces properly folded proteins with correct post-translational modifications, and can accommodate multi-gene– or large gene insertions. However, there remain some challenges with this system, including unstable expression and reduced levels of protein glycosylation. As the demand for biotechnology increases, there has been a concomitant effort into optimizing yield, stability and protein glycosylation through genetic engineering and the manipulation of baculovirus vector and host cells. In this review, we summarize the strategies and technological advances of BEVS in recent years and explore how this will be used to inform the further development and application of this system.

Abstract PR010: Development of a pre-clinical metastatic model of human sarcoma to identify therapeutic targets

Abstract Targeted therapies have led to significant advances in the treatment of multiple tumor types resulting in more effective and often, less toxic therapeutic options. In sarcomas, the development of targeted therapies has been met limited success. There are more than 70 sarcoma subtypes that vary in histology, clinical course and patient demographics. Despite these clear differences, clinically, sarcomas are treated similarly with variable efficacy. Patients with localized disease are treated with surgery, radiation, and often chemotherapy. Even with this aggressive multimodality treatment, 35% of patients will develop incurable metastatic disease. This highlights the need for additional therapies targeting micrometastatic disease or cells within the primary tumor with a high propensity for metastasis. Understanding the pathways driving the formation of sarcoma metastasis would allow for the development of new therapies. Targeted treatments are limited due to the heterogeneity of the disease and the paucity of pre-clinical models that accurately reflect the human disease. We have focused on creating in vivo models of sarcoma development and metastasis that can be used as the basis of further studies and to test potential therapeutic targets. Mesenchymal stem cells (MSCs) are the presumed cell of origin for sarcomas and therefore, the starting cell for our investigations. We hypothesized that by recreating key genetic events in human MSCs, we could generate sarcomas in vivo that are reflective of the human disease. RB1 and P53, tumor suppressors that are often mutated or functionally inactive in sarcomas were first targeted using CRISPR-Cas9 technology in MSCs that have been immortalized by human telomerase (hTERT). Genes that are overexpressed or amplified in The Cancer Genome Atlas (TCGA) data were identified and a library of potential oncogenes was generated. This library was then added to RB1-/-P53+/- cells through lentiviral transduction. Targeting of key tumor suppressors and adding oncogenic drivers resulted in the formation of high-grade human sarcomas subcutaneously. We then sought to investigate the ability of these cells to metastasize. Injection of cells intramuscularly (into the thigh) in immunocompromised mice resulted in the formation of spontaneous lung metastasis without clear evidence of disease in other organs. This pattern clinically reflects that of human disease. Comparing oncogenic genes from metastatic outgrowths to primary tumors identified KLF4, DDIT3, JUN, and KRAS as being enriched in metastatic cells. This system allows for reproducible and robust genetic manipulation of tumor cells to characterize key drivers of metastatic growth. This will result in the identification and validation of new therapeutic targets to treat or prevent metastatic disease in our patients. Citation Format: Janai R. Carr-Ascher. Development of a pre-clinical metastatic model of human sarcoma to identify therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR010.

Confined Nanopipet as a Versatile Tool for Precise Single Cell Manipulation.

The precise manipulation of single cells plays a fundamental role for single cell measurement, which is crucial for understanding the diverse cellular mechanisms. Unusual single cell behavior could thus be identified by integrating with advanced analytical methods such as single cell omics, unraveling the intrinsic cellular heterogeneity hidden in ensemble measurements. Herein, this technical note reports a nanopipet-based versatile method for manipulation of an ultrasmall volume of liquid, which further enables the precise manipulation of single cells. Femtoliter volumes of cytoplasm were extracted from single living cells and analyzed by time-of-flight secondary ion mass spectrometry. Moreover, several kinds of exogenous components were injected simultaneously into a cell, offering a delicate tool for multi-imaging in single living cells.

Tissue-resident memory T cells in chronic liver diseases: Phenotype, development and function.

Tissue-resident memory (TRM) T cells are a unique subset of memory T cells that are critical for the first line of defense against pathogens or antigens in peripheral non-lymphoid tissues such as liver, gut, and skin. Generally, TRM cells are well adapted to the local environment in a tissue-specific manner and typically do not circulate but persist in tissues, distinguishing them from other memory T cell lineages. There is strong evidence that liver TRM cells provide a robust adaptive immune response to potential threats. Indeed, the potent effector function of hepatic TRM cells makes it essential for chronic liver diseases, including viral and parasite infection, autoimmune liver diseases (AILD), nonalcoholic fatty liver disease (NAFLD), hepatocellular carcinoma (HCC) and liver transplantation. Manipulation of hepatic TRM cells might provide novel promising strategies for precision immunotherapy of chronic liver diseases. Here, we provide insights into the phenotype of hepatic TRM cells through surface markers, transcriptional profiles and effector functions, discuss the development of hepatic TRM cells in terms of cellular origin and factors affecting their development, analyze the role of hepatic TRM cells in chronic liver diseases, as well as share our perspectives on the current status of hepatic TRM cell research.

Abstract IA26: Understanding CLL and Richter’s syndrome biology through mouse models of human genetics

Abstract Large-scale human next generation sequencing studies have progressively defined the complex genetic landscape of chronic lymphocytic leukemia (CLL) and Richter’s syndrome (RS), yet the difficulties in rapid manipulation of primary CLL cells and the limited availability of cell lines have challenged studies aimed at interrogating the function of the so-called disease “drivers”, defined in human studies through statistical inference. Mouse models represent powerful tools to study mechanisms of normal and malignant B cell biology. Conditional knock-in/knock-out systems have allowed B-cell restricted modeling of CLL genetic lesions, with MDR mice being the first example of a genetically-faithful CLL model, recapitulating the most common genomic aberration of human disease, del(13q). Since then, a series of models generated by our group and others have allowed to uncover key functional changes underlying disease onset and transformation, including in the context of combined mutation in the splicing factor Sf3b1 and the DNA damage response gene Atm, sole mutation in the B cell developmental factor Ikzf3, the nuclear export receptor Xpo1, the ribosomal protein Rps15, and the DNA methyltransferase Dnmt3a (the latter not a CLL lesion per se, but recapitulating disordered methylation patterns typical of CLL). The CRISPR-Cas9 gene editing technology has further enabled B-cell restricted introduction of multiple combinatorial loss-of-function lesions, allowing the generation of faithful models not only of CLL but, importantly, of transformation into RS. In this presentation, I will describe the application of mouse models to studies of CLL and RS pathogenesis and treatment, with a focus on key aspects uncovered through the characterization of genetically- inspired mouse lines of recurrent disease drivers. I will provide a perspective on how these novel models combined with new technology allowed the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible, representing valuable platforms for functional genomic analyses. Citation Format: Elisa ten Hacken. Understanding CLL and Richter’s syndrome biology through mouse models of human genetics [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr IA26.

Periodic switching of acoustic radiation force with beat created by multitone field

Acoustic radiation force plays a key role in microfluidic systems for particle and cell manipulation. In this study, we investigate the acoustic radiation force resulting from synthesized ultrasounds that are emitted from multiple sound sources with slightly different oscillation frequencies. Due to the synthesized field, the acoustic radiation force is expressed as the sum of a dc component and harmonics of fundamental frequencies of a few hertz. This induces the beat of the acoustic radiation force. We demonstrate that the synthesized field provides the periodic on/off switching of the acoustic radiation force associated with the one denominational planar standing wave in a straight microfluidic channel. Consequently, our system can temporally manipulate acoustic radiation force without active controls.

Cell bioinformatics and technology

<p indent="0mm">Cell is the basic unit of life. The characteristics and activity process of cells are directly related to the physiology and pathology of life. In recent years, the research on (single) cells has made rapid progress. Understanding cell fate, manipulation and biomedical applications must firstly be supported by cell information acquisition and analysis. Due to the insurmountable limitations in technical principles and methods, relevant research needs to be broken through. This article introduces origin and classification of cell bioinformatics, summarizes the main progress in cell bioinformatics acquisition and analysis, and discusses the future development trend and challenges.

Two forty-years for researches on sexual plant reproduction in Wuhan University

<p indent="0mm">Investigations on sexual plant reproduction in Wuhan University was promoted by Prof. Hong-Yuan Yang and Prof. Chang Zhou. After forty-years’ effort remarkable progress has been made in experimental sexual reproductive biology, especially in the field of unfertilized ovule culture and <italic>in vitro</italic> sexual cell manipulation. Their contributions in this field have been summarized in a Prof. Yang-edited book named “forty-year researches on experimental sexual plant reproduction”, which is a remarkable indicator of the transition from traditional descriptive sexual plant reproduction to experimental sexual plant reproduction in China. As a student of Prof. Yang, Prof. Meng-xiang Sun has been also working in this field for forty years and experienced a quick transition from sexual plant reproductive biology to developmental biology of sexual plant reproduction, in which regulatory mechanism of sexual reproduction process is deeply investigated under the guidance of basic theory of developmental biology and based on the combination of molecular and cell biological techniques. After years’ effort they finally catch up with the moving frontier of the field and add new pages in the book of sexual plant reproduction research in Wuhan University. This paper summarizes what they experienced, what they thought and what they did in the two overlapped forty-years respectively, to present some aspects of the researches in Wuhan University.

Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport.

The oncostatic effects of melatonin correlate with increased reactive oxygen species (ROS) levels, but how melatonin induces this ROS generation is unknown. In the present study, we aimed to elucidate the two seemingly opposing actions of melatonin regarding its relationship with free radicals. We analyzed the effects of melatonin on head and neck squamous cell carcinoma cell lines (Cal‐27 and SCC‐9), which were treated with 0.5 or 1 mM melatonin. We further examined the potential effects of melatonin to induce ROS and apoptosis in Cal‐27 xenograft mice. Here we report that melatonin mediates apoptosis in head and neck cancer by driving mitochondrial reverse electron transport (RET) to induce ROS production. Melatonin‐induced changes in tumoral metabolism led to increased mitochondrial activity, which, in turn, induced ROS‐dependent mitochondrial uncoupling. Interestingly, mitochondrial complex inhibitors, including rotenone, abolished the ROS elevation indicating that melatonin increased ROS generation via RET. Melatonin also increased membrane potential and CoQ10H2/CoQ10 ratio to elevate mitochondrial ROS production, which are essential conditions for RET. We found that genetic manipulation of cancer cells with alternative oxidase, which transfers electrons from QH2 to oxygen, inhibited melatonin‐induced ROS generation, and apoptosis. RET restored the melatonin‐induced oncostatic effect, highlighting the importance of RET as the site of ROS production. These results illustrate that RET and ROS production are crucial factors in melatonin's effects in cancer cells and establish the dual effect of melatonin in protecting normal cells and inducing apoptosis in cancer cells.

Ethical Challenges and Controversies in the Practice and Advancement of Gene Therapy

One of the most important technologies in modern medicine is gene therapy, which allows therapeutic genes to be introduced into cells of the body. The approach involves genetics and recombinant DNA techniques that allow manipulating vectors for delivery of exogenous material to target cells. The efficacy and safety of the delivery system are a key step towards the success of gene therapy. Somatic cell gene therapy is the easiest in terms of technology and the least problematic in terms of ethics. Although genetic manipulation of germline cells at the gene level has the potential to permanently eradicate certain hereditary disorders, major ethical issues such as eugenics, enhancement, mosaicism, and the transmission of undesirable traits or side effects to patients’ descendants currently stymie its development, leaving only somatic gene therapy in the works. However, moral, social, and ethical arguments do not imply that germline gene therapy should be banned forever. This review discusses in detail the current challenges surrounding the practice of gene therapy, focusing on the moral arguments and scientific claims that affect the advancement of the technology. The review also suggests precautionary principles as a means to navigate ethical uncertainties.

Composite ultrasound transducer for multi-size of tweezer manipulation

The application of acoustic tweezers in many fields has driven the innovation of device optimization, such as cell characterization and manipulation, drug extraction and movement. The current application of acoustic tweezers faces certain challenges because it does not enable the capture of multi-size particles. In order to improve the adaptability of acoustic tweezers and overcome the forbidden band of radiation force trapping, this paper presents the optimizer design, modeling and implementation of an acoustic tweezer with high sensitivity and broad bandwidth. The acoustic focusing beam and length between the numerical results and experimental results show fair agreements. Particularly, the acoustic radiation force on the elastic sphere with a diameter from 100 to 500 μm is calculated at 3.5 MHz, 5.5 MHz, 7.5 MHz, and 9.5 MHz. Experimental particle trapping demonstrates the effective trapped size range from 100 to 400 μm, which verifies the calculation of acoustic radiation force. The results show this transducer achieves a good focusing effect in a wide frequency band which realizes multi-dimensional control of particles in water at different working frequencies. In the future, this research for novel acoustic tweezers has great potential and value for industrial, chemical, medical and biological applications.

Microfluidic device for the Separation of non-metastatic (MCF-7) and non-tumor (MCF-10A) breast cancer cells using AC Dielectrophoresis.

Dielectrophoretic devices are capable of the detection and manipulation of cancer cells in a label-free, cost-effective, robust, and accurate manner using the principle of the polarization of the cancer cells in the sample volume by applying an external electric field. This article demonstrates how a microfluidic platform can be utilized for high-throughput continuous sorting of non-metastatic breast cancer cells (MCF-7) and non-tumor breast epithelial cells (MCF-10A) using hydrodynamic dielectrophoresis (HDEP) from the cell mixture. By generating an electric field between two electrodes placed side-by-side with a micron-sized gap between them in an HDEP microfluidic chip, non-tumor breast epithelial cells (MCF-10A) can be pushed away, exhibiting negative DEP inside the main channel, while the non-metastatic breast cancer cells follow their course unaffected when suspended in cell medium due to having conductivity higher than the membrane conductivity. To demonstrate this concept, simulations were performed for different values of medium conductivity, and the sorting of cells was studied. A parametric study was carried out, and a suitable cell mixture conductivity was found to be 0.4 S/m. By keeping the medium conductivity fixed, an adequate AC frequency of 0.8 MHz was established, giving maximum sorting efficiency, by varying the electric field frequency. Using the demonstrated method, after choosing the appropriate cell mixture suspension medium conductivity and frequency of the applied AC, maximum sorting efficiency can be achieved.

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications.

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

Recursive Editing improves homology-directed repair through retargeting of undesired outcomes

CRISPR-Cas induced homology-directed repair (HDR) enables the installation of a broad range of precise genomic modifications from an exogenous donor template. However, applications of HDR in human cells are often hampered by poor efficiency, stemming from a preference for error-prone end joining pathways that yield short insertions and deletions. Here, we describe Recursive Editing, an HDR improvement strategy that selectively retargets undesired indel outcomes to create additional opportunities to produce the desired HDR allele. We introduce a software tool, named REtarget, that enables the rational design of Recursive Editing experiments. Using REtarget-designed guide RNAs in single editing reactions, Recursive Editing can simultaneously boost HDR efficiencies and reduce undesired indels. We also harness REtarget to generate databases for particularly effective Recursive Editing sites across the genome, to endogenously tag proteins, and to target pathogenic mutations. Recursive Editing constitutes an easy-to-use approach without potentially deleterious cell manipulations and little added experimental burden.

Manipulation of single cells via a Stereo Acoustic Streaming Tunnel (SteAST)

At the single-cell level, cellular parameters, gene expression and cellular function are assayed on an individual but not population-average basis. Essential to observing and analyzing the heterogeneity and behavior of these cells/clusters is the ability to prepare and manipulate individuals. Here, we demonstrate a versatile microsystem, a stereo acoustic streaming tunnel, which is triggered by ultrahigh-frequency bulk acoustic waves and highly confined by a microchannel. We thoroughly analyze the generation and features of stereo acoustic streaming to develop a virtual tunnel for observation, pretreatment and analysis of cells for different single-cell applications. 3D reconstruction, dissociation of clusters, selective trapping/release, in situ analysis and pairing of single cells with barcode gel beads were demonstrated. To further verify the reliability and robustness of this technology in complex biosamples, the separation of circulating tumor cells from undiluted blood based on properties of both physics and immunity was achieved. With the rich selection of handling modes, the platform has the potential to be a full-process microsystem, from pretreatment to analysis, and used in numerous fields, such as in vitro diagnosis, high-throughput single-cell sequencing and drug development.