184 results about "Single cell suspension" patented technology

The invention discloses a temperature-controllable preparation method for tissue temperature dissociation into single cells and application, the temperature-controllable preparation method for tissue temperature dissociation into single cells comprises the following steps: S1, providing a tissue sample, and pre-treating the tissue sample to obtain a sample block; s2, mixing the sample blocks with a compound digestive enzyme, and filtering to obtain a single-cell suspension after digestion treatment is completed; wherein the compound digestive enzyme is prepared from collagenase, hyaluronidase and papain; and selectively, S3, carrying out red removing treatment. The invention not only provides an improved preparation method of the single-cell suspension, but also has higher efficiency on the premise of ensuring the yield and activity of single cells, and provides a new strategy and tool for cancer research and treatment by particularly enriching CSCs.

The invention provides a single-cell suspension and a preparation method thereof. Specifically, the preparation method comprises the following steps: 1) mixing a condensed-state cell sample to be dispersed with a pretreatment solution, and carrying out ultrasonic treatment; 2) mixing the sample obtained in the step 1) with the enzymatic hydrolysate, and carrying out an enzymatic hydrolysis reaction; 3) adding a nanometer material with the surface modified with a target cell specific marker, controlling the distribution of the nanometer material through an external magnetic field, and enriching target single cells; 4) separating the sample obtained in the step 3) to obtain a target single-cell suspension; wherein the conditions of the ultrasonic treatment and the enzymolysis reaction are controlled by an intelligent control system. According to the technical scheme, ultrasonic treatment and enzymolysis reaction are combined, an intelligent control system is used for controlling ultrasonic conditions and enzymolysis conditions respectively, and a nano material labeling technology is combined, so that the finally obtained cell suspension is high in cell viability and high in purity.

The embodiment of the invention relates to the technical field of drug detection, in particular to an anti-CD38 antibody drug receptor occupation detection method and system, a peripheral blood sample is used for preparing a single-cell suspension, the single-cell suspension is divided into a TEST tube and an FMO tube, a cell surface antibody and an RO detection antibody are added into the TEST tube, only the cell surface antibody is added into the FMO tube, and only the RO detection antibody is added into the FMO tube. The TEST tube and the FMO tube are subjected to sample detection analysis, so that the average fluorescence intensity of RO detection antibodies of the TEST tube and the FMO tube is obtained. And performing RO detection analysis by adopting a dissociation method, taking the tubes which are not combined with the medicine as a total receptor group, taking the tubes which are combined with the medicine as a dissociation receptor group, and calculating an RO detection result according to the average fluorescence intensity of the TEST tube and the FMO tube.

The invention discloses a preparation method of a seawater shellfish single-cell suspension suitable for dynamic detection. The preparation method comprises the following steps: extracting gill filament tissues from living shellfish; washing gill filaments, and absorbing moisture; cutting the gill filament tissue into pieces; carrying out enzymolysis digestion on the gill filament tissue fragments by using an enzyme mixed solution consisting of collagenase II, hyaluronidase and deoxyribonuclease I; centrifuging and resuspending the cells; and filtering to obtain a single-cell suspension. Aiming at the condition that the shellfish tissue structure is relatively loose, cleaning liquid, enzyme liquid components, enzymolysis time, centrifugal speed and the like are optimized, and the high-quality single-cell suspension is obtained. According to the preparation method of the single-cell suspension, non-lethal sampling of seawater shellfish is conducted, the requirement for dynamic detection of shellfish traits in the industry can be met, enzymolysis is more sufficient, experiment time is shorter, operation steps are simple and convenient, obtained cells are more complete in morphology, the caking rate is extremely low, and the living cell rate is higher; and the requirements of construction of various single cell sequencing platform libraries can be fully met.

The invention provides a new bunyavirus neutralizing antibody screening method based on single cell BCR sequencing. The new bunyavirus neutralizing antibody screening method comprises the following steps: S1, collecting peripheral blood B cells of acute stage and recovery stage patients and healthy volunteers of new bunyavirus infected persons; s2, carrying out CD19 < + > CD38 < + > CD138 < + > magnetic bead separation to obtain a single-cell suspension; s3, constructing a single cell transcriptome and a BCR library, separating single cells by using a microfluidic system, and performing high-throughput sequencing; s4, analyzing a B cell subset, a BCR cloning amplification mode and a VJ gene pair enrichment characteristic through bioinformatics analysis; and S5, screening a specific BCR clone type related to the severity of the disease as a neutralizing antibody candidate target. The method provided by the invention provides powerful support for immune monitoring and vaccine design of new bunyavirus infection, and is expected to provide new strategies and methods for clinical treatment and prevention.

The present invention relates to a cartilage tissue single cell extraction kit and an extraction method thereof, the kit comprises: (1) a first enzyme for enzymatic hydrolysis, the first enzyme for enzymatic hydrolysis comprising one or more of trypsin, chondroitinase ABC and fucosidase; (2) a second enzyme for enzymolysis, wherein the second enzyme for enzymolysis comprises collagenase II, neutral protease II, hyaluronidase and Dnase I; and (3) a basic solution, wherein the basic solution comprises a basic culture medium and / or a buffer solution. The invention also provides a method for extracting single cells from cartilage tissues by using the kit. The cartilage tissue single-cell suspension prepared by the method has the advantages of high cell yield, high activity and the like, and has a wide application prospect.

The invention provides a method for culturing primary spermatogonial stem cells of Tibetan sheep, and belongs to the technical field of cell separation and culture. The culture method provided by the invention comprises the following steps: disinfecting and cleaning testis tissues of Tibetan sheep lambs; shearing tissue blocks, and digesting and cleaning to obtain a single-cell suspension of the seminiferous tubule; purifying the single-cell suspension, and culturing without a feeding layer or with the feeding layer; the culture without the feeding layer refers to culture in a laminin-coated culture container by using a feeding-layer-free MEM alpha culture medium or a StemProTM-34SFM complete culture medium; culture with a feeding layer refers to culture in a mouse fibroblast line feeding layer or a Tibetan sheep primary testicular sertoli feeding layer, and a feeding layer MEMS alpha culture medium is added. According to the culture method provided by the invention, the Tibetan sheep spermatogonial stem cells with higher purity can be obtained through separation, the subsequent proliferation of the spermatogonial stem cells can be remarkably promoted, and the apoptosis of the spermatogonial stem cells can be inhibited.

The invention belongs to the technical field of biology, and particularly relates to a preparation method of macaca fascicularis liver tissue single-cell suspension. The method comprises the following steps: cleaning macaca fascicularis liver tissues with a pre-cooling buffer solution, and processing into tissue fragments; mixing the tissue fragments with a digestion solution, wherein the digestion solution comprises collagenase I with the final concentration of 1 + / -0.2 mg / ml, collagenase II with the final concentration of 1 + / -0.2 mg / ml, bovine serum albumin with the final concentration of 0.04 + / -0.01% (w / v), DNase I with the final concentration of 10 + / -2 U / ml and the balance of a buffer solution; oscillating and incubating for 30 + / -10min under the conditions of 37 + / -2 DEG C and 150 + / -50rpm, and performing tissue digestion; centrifuging the digested suspension, removing the supernatant, resuspending with a buffer solution, and filtering with a cell sieve to obtain a single-cell primary suspension; and carrying out red blood cell lysis, washing and resuspension on the single-cell primary suspension to prepare the cynomolgus monkey liver tissue single-cell suspension. The method significantly improves the tissue dissociation efficiency, reduces cell adhesion and fragments, effectively protects cell surface antigen epitopes, and has the advantages of simple operation, economy and high efficiency.

The invention belongs to the technical field of immunology, and particularly discloses an in-vitro preparation method and application of an induced follicular regulatory T cell iTfr.The in-vitro preparation method comprises the steps that mouse spleen single-cell suspension or healthy human peripheral blood is obtained, peripheral blood mononuclear cells are obtained, then initial CD4 + T cells are obtained through immunomagnetic bead sorting, and iTfr cells are obtained; then carrying out induced differentiation by using an antibody and a specific cytokine, and carrying out continuous stimulation and culture by adopting two different cytokine polarization conditions, so as to obtain the iTfr cell. The invention provides a method for preparing the iTfr cells in vitro, which is expected to obtain an enough number of induced follicular regulatory T cells, and provides an important tool and a treatment means for in vitro research of immune tolerance related diseases such as autoimmune diseases and organ transplantation.

The invention discloses a reagent for reducing the agglomeration rate of suspended HEK293F cells, a culture method and a culture medium. The reagent comprises the following components: 0.01-0.1% (v / v) of a nonionic surfactant, 0.05-0.2% (v / v) of polysaccharide or a derivative thereof, 1-5mM of L-glutamine or a derivative thereof, 0.001-0.01 mM of FeCl3, 14-16mM of HEPES and 50-100mu M of an RGD peptide inhibitor. According to the anti-caking agent, the nonionic surfactant, the polysaccharide or the polysaccharide derivative, the L-glutamine or the L-glutamine derivative, the FeCl3, the HEPES and the RGD peptide inhibitor are optimally proportioned, so that the HEK293F cell caking rate is remarkably reduced to 30% or below, after the anti-caking agent is added, cells are in a uniform single-cell suspension state, macroscopic cell agglomerates do not exist, and the anti-caking effect is good. The uptake efficiency of cells on nutrient substances is obviously improved. Compared with a commercially available PEG (Polyethylene Glycol)-containing anti-caking agent, the anti-caking agent disclosed by the invention has the advantages that the anti-caking rate is lower than 35% (the anti-caking rate is only 30% vs after being cultured for 48 hours, and the anti-caking rate is 40%) while the cell viability is maintained to be 95% or above, and no cytotoxic accumulation exists after long-term passage (more than or equal to 20 generations).

The invention provides a preparation method of a single-cell sequencing suspension fixedly preserved in situ. The invention provides digestive juice for preparing a single-cell suspension. The digestive juice is a solution composed of collagenase II and neutral protease. The invention also provides a kit containing the digestive juice. The invention provides application of the digestive juice to preparation of a kit and application of the kit to in-situ fixed preservation of cells and preparation of a single-cell suspension. The invention also provides a preparation method of the in-situ immobilized preserved cell and the single-cell suspension, and the single-cell suspension prepared by the preparation method.

The invention discloses a method for simultaneously extracting multiple types of cells from the same liver tissue, which is characterized by comprising the following steps: preparing a single-cell suspension; extracting parenchymal hepatic cells; extracting bile duct epithelial cells; extracting hepatic stellate cells; wherein the extraction step of the parenchymal hepatic cells, the bile duct epithelial cells and the hepatic stellate cells is carried out step by step according to the sequence. According to the technical scheme, efficient extraction of liver tissue cells and culture of functional organoid are achieved, various defects in the prior art are overcome, meanwhile, the technical scheme has a wide application range and is suitable for various liver tissues, and important technical support is provided for research and treatment of liver diseases.

The invention discloses a construction and evaluation method of a liver injury organoid model, which comprises the following steps: (1) selecting mouse liver cells, perfusing a mouse with pancreatin through a sterile operation technology, removing blood in the liver, collecting liver tissues, and carrying out fine enzymolysis digestion treatment to obtain a single-cell suspension; (2) inoculating the single-cell suspension into a three-dimensional culture system, and carrying out preliminary stimulation by adopting a culture medium combined with growth factors and inhibition small molecules, so as to promote the growth, proliferation and differentiation of cells in a three-dimensional space; according to the cell growth condition, subculture operation is carried out in good time, so that continuous culture and amplification of liver organs are ensured; and (3) exposing the maturely cultured liver organoid in a stimulating factor for simulating a liver injury pathological environment, and constructing different types of liver injury organoid models. According to the liver injury organ-like model construction method, model construction can be completed in a short time, the research period is greatly shortened, the efficiency is high, and the cost is low.

The invention discloses a single cell ion dynamic change detection method (scIon-seq) based on a nucleic acid aptamer, which comprises the following steps: anchoring a hydrophobic lipid modified capture probe on the surface of a cell membrane, and when metal ions are released out of the cell from the cell, detecting the dynamic change of the single cell ions. The detection probe with the sequencing joint and the capture probe on the membrane form a secondary structure through inter-chain folding in the presence of the metal ions; and then in a single cell suspension system, carrying out high-throughput sequencing on the detection probe with the sequencing joint in the secondary structure to obtain the dynamic change information of the metal ions on the single cell level. According to the invention, a research means is provided for researching the physiological function of the metal ions at the single cell level on the technical aspect by utilizing the characteristics that the nucleic acid aptamers can recognize the metal ions in a high-specificity manner and can be compatible with high-throughput sequencing; the development of multiple omics such as metabonomics, proteomics and transcriptomics on the single cell level of the nucleic acid aptamer can be promoted.

The invention relates to a human thymus primary cell separation kit and a separation method, the kit comprises an extraction reagent for preparing a thymus tissue single-cell suspension and a separation reagent for purification, and by combining the reagents in the kit and matching with corresponding operation steps, the human thymus primary cell can be separated. And one or a combination of more of thymus epithelial cells, thymus dendritic cells, thymus T cells and thymus B cells can be accurately extracted from human thymus tissues. The method has the beneficial effects that the target primary cells in the thymus tissue sample can be separated, the specificity is high, different primary cell components can be separated through one-time operation by combining different reagents, and various target primary cells with relatively high purity are obtained; and the success rate of separating the primary cell component in the thymus tissue sample by using the method is as high as 90%, and the sample loss is reduced.

The invention discloses a brain glioma animal model construction method based on an AAV mediated immune microenvironment. The method comprises the following steps: selecting an immunodeficient mouse at the right age; human CD34 + hematopoietic stem cells are injected for immune system reconstruction; human CD45 + immune cells in the peripheral blood PBMC are detected; the method comprises the following steps: selecting tumor tissues with high-expression brain glioma marker genes, carrying out rapid qPCR (quantitative polymerase chain reaction) or targeted RNA-seq immunohistochemical multiple verification, and screening consistent tumor tissues; the method comprises the following steps: physically dicing tumor tissues, treating the tumor tissues with pretreatment liquid, digesting the tumor tissues into single-cell suspension, and inoculating the single-cell suspension into a mouse cranium; an AAV vector carrying a specific immune regulation gene is injected into the tail vein of the mouse, and the brain glioma immune microenvironment is regulated; when it is observed that the tumor tissue grows stably and has immune cell infiltration characteristics, construction of the brain glioma animal model is completed. Compared with an existing model, the brain glioma model which is more stable, more scientific and closer to the human immune background is provided, and the brain glioma model is suitable for related immunotherapy drug screening and mechanism research.

The present disclosure relates to a method of generating pancreatic lineage cells, e.g. islet-like cell aggregates comprising pancreatic beta cells, comprising the steps of providing a single cell suspension of a population of endocrine progenitor cells (EP), allowing the EP cells in the single cell suspension to form 3D structures, and culturing the cells under conditions allowing differentiation into pancreatic mono-hormonal beta cells. The present disclosure also relates to islet-like cell aggregates obtainable by said method and their medical use.

This invention relates to a method for detecting colorectal cancer tumor cell-associated microorganisms, specifically a method for eliminating intestinal fecal microbial contamination and detecting colorectal cancer tumor-associated microorganisms, belonging to the field of biotechnology. This invention initially eliminates fecal microbial contamination from the sample structure by coarsely shearing and enzymatically digesting the intestinal folds and villi; then, it uses a 40μm cell sieve to filter out incompletely digested tissue fragments, further eliminating intestinal fecal bacteria contamination, and collecting a single-cell suspension; subsequently, the collected single-cell suspension is repeatedly washed with 300g centrifugation to remove as many intercellular or loosely bound "transient bacteria" as possible, retaining intracellular and firmly colonized microorganisms. Ultimately, this eliminates the severe contamination from the large and complex fecal microorganisms, allowing the detection of tumor cell-associated microorganisms, which are already present in low biomass. This method is suitable for detecting microorganisms in gastrointestinal tissues that are in direct contact with the environment.

The invention belongs to the cross technical field of veterinary medicine and biomedicine, and relates to a construction and evaluation method of a dog spontaneous breast cancer organoid model, which specifically comprises the following steps: (1) obtaining dog spontaneous breast cancer tissues through operation or biopsy, separating tumor tissues under sterile conditions, and carrying out fine enzymolysis digestion treatment to obtain dog spontaneous breast cancer tissues; obtaining a single-cell suspension; and (2) inoculating the single-cell suspension into a three-dimensional culture system, carrying out three-dimensional culture by adopting a culture medium containing EGF, FGF and B27 in combination with a growth factor, promoting the growth and amplification of the breast cancer organs, and carrying out timely passage according to the growth condition so as to maintain the activity of the model. The model constructed by the invention completely retains genetic heterogeneity and pathological microenvironment of primary tumors, does not need gene editing, is simple and convenient to operate and low in cost, is particularly suitable for individual drug sensitivity detection of pet dogs, and provides an efficient and high-simulation experimental platform for dog breast cancer mechanism research and clinical precise medication.

The invention discloses a non-tumorigenic MDCK cell strain, a screening method and application. The non-tumorigenic MDCK cell strain is MDCK-CA027 (the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2023329). The tumor formation rate of the cell strain is 0% when the cell strain is observed for at least 16 weeks in a nude mouse in-vivo tumor formation test, and the cell strain has good sensitivity to various influenza viruses (such as H1N1, H3N2, BY and BV). The invention also provides a method for screening the cell strain. The method comprises the following steps: preparing a single-cell suspension, carrying out limited dilution to obtain monoclone, carrying out enlarged culture, carrying out virus sensitivity detection, carrying out in-vitro tumorigenicity related detection (clone formation test / soft agar test), carrying out nude mouse in-vivo tumorigenicity test and the like. The non-tumorigenic MDCK cell strain is high in safety, can efficiently support influenza virus replication, can be safely and effectively applied to influenza vaccine production and influenza virus amplification, overcomes the tumorigenic risk of traditional MDCK cells, and has important scientific significance and application prospects.

This invention provides a method and apparatus for splitting single-cell mixed sample sequencing data, relating to the field of biotechnology. The splitting method includes: capturing and sequencing single-cell suspensions using a single-cell platform; then performing reference genome alignment, cell identification, and gene expression level quantification on the sequencing data using Cellranger; splitting the cell data identified in step a into two groups of cell data for different sexes based on SNP locus information from the 1000 Genomes Project; and distinguishing the two groups of cell data from male or female samples based on the proportion of sex-specific genes expressed in the two groups of cell data. This splitting method eliminates the need for additional experimental operations such as protein labeling and genome sequencing, and can provide accurate and reliable data splitting even when individual SNP information is unavailable.

The application provides a preparation method of organoids based on a single cell chip, which comprises microfluidic chip preparation, a two-water-phase solution preparation, a single cell suspension preparation, microfluidic manipulation, single cell-loaded microgel generation and organoid construction and the like. The application takes a microfluidic chip integrated with a normally closed pneumatic pump valve as a technical platform, takes a single cell suspension pre-incubated with a crosslinking agent as a chemical reaction core, takes a water-in-water droplet as a forming template, and realizes efficient in-situ loading of single dispersed seed cells in a micro hydrogel carrier through a one-step method, and simply removes empty droplets without cells in a subsequent transfer step. The single cells in the microgel can be cultured, expanded and self-organized to form required organoids. The application improves the consistency of the starting point of the organoids, has the high-throughput characteristics of the droplet microfluidic technology, realizes efficient and highly reproducible in-vitro construction of the organoids, and can play a great role in the fields of basic research and transformation application related to the organoids.

The invention belongs to the field of immunotoxicology, and particularly relates to a method for immunotyping bone marrow cells by flow cytometry. The flow cytometry immune typing method for bone marrow cells comprises the following steps: collecting macaca fascicularis bone marrow fluid, and preparing the macaca fascicularis bone marrow fluid into single-cell suspension; dyeing the obtained cells by using an antibody combination containing a plurality of fluorescent labels; then, a flow cytometer is used for collecting cell fluorescence signals, a standardized gate setting strategy is executed, and based on collected data, the percentage of each cell subset in CD45 + living white blood cells is calculated to complete immune typing. According to the flow cytometry immune typing method for the bone marrow cells, the problems of low efficiency, low repeatability, low sensitivity and poor specificity of related technologies are solved, and more accurate and reliable data support is provided for early discovery, risk assessment and mechanism research of drug immunotoxicity.

The application discloses a kind of mesenchymal stem cell ball fractional dissociation digestive juice, its preparation method and application, it is related to cell preparation and three-dimensional cell culture technical field, specifically at to provide C liquid, D liquid and E liquid three kinds of gradient concentration working solution.When dissociating, the mesenchymal stem cell ball to be dissociated is determined its average particle size;According to the average particle size, select C liquid, D liquid or E liquid as digestive juice as described above;The selected digestive juice is mixed with the mesenchymal stem cell ball, and digestion reaction is carried out under oscillation condition, and single cell suspension is obtained.The application establishes the hierarchical matching rule of mesenchymal stem cell ball particle size and specific digestive juice concentration, and optimizes oscillation digestion condition, so as to maintain high activity rate and complete phenotype characteristics while efficiently dissociating, realizes the standardization of process and the controllable of dissociation quality, provides stable and reliable solution for the large-scale preparation and quality control of mesenchymal stem cell ball.

The invention discloses a cell crude sample sex determination primer suitable for chickens, ducks or pigeons and a PCR detection method, and belongs to the technical field of biological detection. A pair of gender-specific universal primers for various poultry such as chicken, duck and pigeon is designed for the first time, a cell crude sample is adopted as a PCR template, stable amplification can be realized without DNA extraction, the operation process is greatly simplified, the detection time is shortened, and the detection cost is reduced. Meanwhile, TrypLE enzyme digestion is very mild to cells, and the method is also suitable for preparation of single-cell suspensions related to single-cell omics. The method is suitable for the fields of early sex identification of poultry, breeding poultry breeding, molecular breeding and the like, and has wide application prospects.

The invention discloses a method for high-throughput screening of high-activity lactic acid bacteria based on multi-index fusion and application, and belongs to the field of lactic acid bacteria screening, the method comprises the steps of bacterial cell enrichment, single-cell suspension preparation, physiological status and activity evaluation of cells, flow cytometry analysis, cell sorting and strain identification; in the step of enrichment of the bacterial cells, a sample is activated and enriched by using an improved liquid culture medium and an improved solid culture medium, and activated and enriched bacterial suspension is obtained. The invention provides the method for detecting the activity of the fermentation product and the lactic acid bacteria stored for a long time, the method is convenient and rapid, the screening time is greatly shortened, and the screening efficiency is improved.

The invention relates to the field of tissue dissociation and cell separation, and discloses a method for rapidly digesting mouse skin tissue into single cells. According to the method, the skin tissue on the back of an adult mouse is taken as an example, different enzyme digestion modes and mechanical treatment modes are combined and used, and the optimal dissociation condition is screened out, so that the skin tissue can be dissociated into the single-cell suspension within a short time. According to the technical scheme, the efficient dissociation of the skin tissue is realized through the optimal combination of the enzyme type, the concentration and the mechanical treatment mode, and the required treatment time is greatly shortened; the cell purity and the cell viability are effectively improved, and an ideal cell sample is provided for subsequent single cell omics research; and reliable technical support is provided for subsequent flow cytometry analysis, cell sorting, high-throughput sequencing and other researches.

The utility model discloses a single-person continuously operated pre-flow sample loading cell filtering device which comprises an upper platform and a lower platform, the upper platform and the lower platform are fixed through a plurality of supporting columns, a filtering mechanism is arranged on the upper surface of the upper platform and used for fixing and replacing a filter screen, and a test tube rack is movably arranged on the lower platform and used for fixing and replacing the filter screen. A plurality of flow type test tubes are fixedly arranged in the test tube rack. The single-cell suspension filtering device is simple in structure, easy to assemble and convenient in filtering operation, and solves the problems of inconvenience in operation, time and labor consumption and easiness in polluting an experimental sample in the process of filtering a cell sample into a single-cell suspension.

The present invention relates to the field of biomedical technologies and provides a method for efficiently capturing neutrophils from peripheral blood for single-cell sequencing by lysing red blood cells. The method includes the following steps: collecting human peripheral blood into an anticoagulant tube containing ethylenediaminetetraacetic acid; taking the ethylenediaminetetraacetic acid anticoagulated whole blood, adding a red blood cell lysate, lysing at room temperature, waiting until the turbid mixed blood solution becomes clear and transparent, waiting for 1 minute, then adding a FACS cell resuspension solution to terminate red blood cell lysis, and centrifuging; adding the FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding the FACS cell resuspension solution to slowly resuspend the cells, and centrifuging; adding a phosphate buffer solution to slowly resuspend the cells, and centrifuging. By selecting a suitable anticoagulant, precisely controlling the red blood cell lysis conditions, and performing meticulous cell resuspension and centrifugation operations, the present invention can ensure high neutrophil activity and a low cell debris rate, provide a suitable single-cell suspension for single-cell RNA sequencing, and help analyze the characteristics and functions of various neutrophil subsets.

According to the application of the cell sheet fragments in preparation of the medicine for treating lower limb ischemia, specifically, the cell sheet fragments formed after injection administration of the cell sheet can prevent limb necrosis caused by lower limb ischemia, have a good limb protection effect, can also effectively prevent muscular atrophy of an affected limb, and can be used for preparing the medicine for treating lower limb ischemia. The limb protection effect and the effect of preventing muscle atrophy of an affected limb are equivalent to those of a cell patch, and are remarkably superior to those of a single-cell suspension.