66 results about "Adherent cell" patented technology

The invention relates to the technical field of biology, and particularly discloses a device and a method for separating an adherent cell through a vortex sound field, an ultrasonic module, piezoelectric ceramics and an Archimedes spiral structure concave lens are arranged to generate the vortex sound field to act on a cell culture dish, so that an adjustable shear flow is generated on the cell culture dish, and the adherent cell is separated from the cell culture dish. And locally separating the adherent cells in the cell culture dish. According to the concave lens with the Archimedes spiral structure, the problem of defocusing of a high-order vortex sound field can be solved, and the falling control accuracy of adherent cells is improved. The proposed method does not require any physical contact with adherent cells, and does not require insertion of an instrument into a cell culture dish, thereby reducing the risk of contamination and cell damage. The stable flow generated by absorbing sound energy by the liquid is used for indirectly separating the adherent cells, the method is extremely low in energy density and has good biocompatibility, and the activity of the cells is ensured while the cells are efficiently separated.

The utility model discloses a differential adherent cell separation device which comprises a separation box body and an upper box cover, culture cavities and a one-way conducting assembly are arranged in the separation box body, the one-way conducting assembly is arranged between the culture cavities, the side face of the separation box body extends outwards to form a supporting part, the side edge of the supporting part protrudes upwards to form a baffle ring part, and the baffle ring part is connected with the upper box cover. A limiting slot is formed among the baffle ring part, the supporting part and the separating box body, and the four sides of the upper box cover extend downwards to form side ring parts. According to the utility model, the one-way conduction between the culture cavities is realized through the one-way conduction assembly, the communication state between the culture cavities can be controlled at a proper stage according to the actual condition of cell wall attachment, the cells are ensured to flow according to the expected direction and mode, and the accuracy and purity of cell separation are effectively improved; the upper box cover and the separation box body are tightly and stably connected, the upper box cover is prevented from loosening or displacing in the using process, and the overall sealing performance and stability of the device are guaranteed.

The application relates to a biological cell culture device and a culture method, and relates to the technical field of cell culture. The device comprises a culture box main body with an open upper side. A placing mechanism and a pressing mechanism are arranged in the culture box main body. Before a cylinder is removed, the upper end of the cylinder is first sealed by a sealing plate. Therefore, after the culture dish is removed, external air cannot enter the culture box main body from the corresponding cylinder. When the subsequent culture dish enters, only the gas with the same volume as the cylinder enters the box, and the influence on the gas concentration in the culture box main body is not obvious. The gas concentration environment in the box is protected. Then, the adherent cells are separated from the inner wall to form a suspension liquid through centrifugation. The splashing of the culture liquid is avoided by the sealing plate. Then, the culture liquid is stirred at a low speed by a stirring mechanism. Since the adherent cells are separated from the inner wall in advance and move with the culture liquid, the damage to the cells in the stirring process is reduced, and the uniform distribution of the culture liquid is ensured.

The disclosed invention relates to a method and system 10 for releasing adherent cells 12 in a cell container 14 with a plurality of acoustic transducers 16a, 16b, 16c, 16h. The method comprises: gene

This invention provides a method for processing live single-cell extraction based on deformation trends, comprising the following steps: Step S1: Controlling a microfluidic chip to immerse itself in a culture medium containing adherent cells while maintaining a preset gap, controlling the injection flow rate of the injection channel and the aspiration flow rate of the aspiration channel to form a microjet acting on the cells; Step S2: Controlling an image processing module to receive and process cell morphology images on the culture substrate acquired by a microscopic imaging module, and calculating deformation parameters based on the comparison between the cell area in the current frame image and the initial cell area; Step S3: Comparing the deformation parameters with critical deformation parameters obtained through experimental calibration, and when the deformation parameters are greater than the critical deformation parameters, controlling a fluid drive module to reduce the injection flow rate and / or the aspiration flow rate; or, calculating a comprehensive damage risk index based on the deformation parameters, and when it is greater than 1, reducing the injection flow rate and / or the aspiration flow rate.

A substrate for culturing cells that comprises a modified prolamin having a net positive charge is provided. The modified prolamin may be a prolamin that has been modified through amidation or esterification, such as the addition of a methyl-ester group or an ethanolamine group. The modified prolamin is animal-free may be food-grade and / or may be free of cross-linkers. The modified prolamin may be in a coating that can be applied to a surface for culturing adherent cells, such as a microcarrier (e.g., beads) or well plates. Substrates coated with the modified prolamin are able to support cell expansion and can be used for food-grade applications such as cultured meat.

The disclosure is directed to a method for seed train expansion of adherent cells comprising culturing cells with a serum-supplemented growth medium in a N-2 vessel; removing the cells from the serum-supplemented medium; inoculating the cells from step into a serum-free growth medium in a N-1 vessel; culturing the cells in the N-1 vessel under suspension conditions; and inoculating a growth medium in a bioreactor with the suspension-cultured cells. In some aspects, the adherent cells are not suspension-adapted. In some aspects, the adherent cells are suspension-adapted. In some aspects, the adherent cells produced by the seed train expansion method are used to produce viral vectors. In some aspects, the viral vectors are AAV vectors.

Disclosed is a system for automating passaging of anchorage dependent or adherent mammalian cells, having a fluid distribution cartridge where fluid channels each has an associated pneumatically controlled valve, and the valves can be selectively opened and closed to allow fluid which is pumped into channels in the cartridge to be selectively passed or interrupted. The channels are connected with a series of vessels each held in a housing structure, and a first frame rests on a surface and a second frame is attached with the first frame such that the second frame can tilt on a first axis with respect to the first frame, and the housing is attached to the second frame such that the housing can tilt on a second axis with respect to the second frame, and wherein the second axis is orthogonal to the first axis. A digital system that controls selective opening and closing of the valves by controlling the pressurized air provided to the valves, and also controls the movement of the frames and housing under feedback from the leveling system.

Methods comprise exposing a culture of human pluripotent stem cells adherent on a first substrate comprising a first laminin to a differentiation medium for a first time period of 15 days to 50 days to obtain a first population of adherent cells comprising RPE cells and / or progenitors thereof; at the end of the first time period, dissociating the first population from the first substrate; replating the dissociated first population of cells on a second substrate comprising a second laminin; and culturing the replated first population of cells on the second substrate for a second time period to obtain an expanded and matured second population of cells comprising the RPE cells. The first and second laminins are independently selected from LN-521, LN-511, LN-111 and LN-121, and are an intact protein or protein fragment. Also included are RPE cells and materials and compositions utilizing such RPE cells for various treatments.

The present application relates to the technical field of biology, and particularly discloses a method for culturing and obtaining foregut endoderm cell spheroids and application thereof. The method for culturing and obtaining foregut endoderm cell spheroids of the present application obtains foregut endoderm cell spheroids by inducing differentiation of stem cells on a culture surface with a PDA-matrigel coating. The culture method of the present application can ensure the differentiation efficiency of DE and AFE adherent cells and spheroids, promote the generation of AFE cell spheroids and the separation from the adherent cell layer, reduce the edge rolling of AFE adherent cells, prolong the production time of AFE cell spheroids, significantly improve the yield of AFE cell spheroids, and provide a basis for subsequent differentiation and culture of lung organoids.

This invention relates to the field of cell engineering technology, and more particularly to a method for preparing immunomodulatory stem cells for treating graft-versus-host disease (GVHD). The invention provides a method for preparing immunomodulatory stem cells for treating GVHD, comprising the following steps: (1) washing human fat aspirate and digesting it with collagenase to obtain monocytes; (2) inoculating the monocytes obtained in step (1) into differentiation medium and culturing for 2–3 days, then continuing to culture the adherent cells for 18–22 days to obtain mesenchymal stem cells; (3) inoculating the mesenchymal stem cells obtained in step (2) into induction medium for induction culture, and after screening, obtaining immunomodulatory stem cells. The preparation method provided by this invention solves the technical problems of unstable sources, functional decline, and poor therapeutic effects of immunomodulatory stem cells in the prior art, providing a new, efficient, safe, and standardized solution for the treatment of GVHD.

Provided herein are altered cell lines comprising a suspension cell line adapted from an adherent cell line having a different expression profile from a corresponding non-altered adherent cell line, methods for generating altered cell lines, and methods of characterizing altered expression profiles for a gene, a transcript, or a protein in an altered cell line.

The invention belongs to the field of cell culture automation and intelligent regulation and control, and discloses an optogenetics-based adherent cell culture optimization method and system. The method comprises the following steps: inputting an adherent cell type and a culture target, and calling an adherent cell-optical genetic parameter joint database; performing targeted pretreatment on the target adherent cells; processing through a multi-parameter optimization algorithm hardware platform to generate an executable customized photostimulation scheme; performing accurate light stimulation on the positive adherent cells; comparing the obtained detection data with normal adherent cell parameters in a joint database, and evaluating the influence of a photostimulation scheme on the cells; updating the parameters of the multi-parameter optimization algorithm and the prediction model; and inputting the adherent cell type and the culture target to the updated prediction model, and outputting an optimal light stimulation scheme. The core problems of low photostimulation accuracy and poor targeting in the prior art are solved, and high-activity and high-consistency light-sensitive adherent cells are stably produced.

The application discloses a kind of based on deep learning's tumor cell image extraction method and system, by obtaining tumor cell image data in microscope, data preprocessing is carried out to the tumor cell image data;Using Retinex theory is combined with guided filter to eliminate the uneven illumination in the initial tumor cell image data slice, enhance the contrast of cell nucleus;Based on DCPM hollow convolution pyramid module captures multiscale cell morphological characteristics and utilizes SE compression-excitation mechanism light weight module to strengthen intercellular topological relationship;Through the dynamic weighting fusion of double-branch output of gate attention mechanism, the cytoplasmic center distance constraint of the feature tumor cell image is calculated based on the connected component analysis algorithm of adherent cell separation algorithm, can accurately separate tumor adherent cell.Combined with minimum cut algorithm and area, shape constraint condition, avoid over-segmentation and under-segmentation problem.

The invention discloses an upstream process for producing a virus. The process includes cell expansion of adherent cell cultures grown on cell culture containers and on microcarriers in bioreactors. In particular, a scalable process for the upstream manufacture of a virus is described, which includes optimization of the amount of cell detachment agent used per surface area of a microcarrier to enhance cell growth in production bioreactors.

The current invention pertains to method for producing a microcarrier, and a microcarrier optionally obtainable by the method. The microcarrier comprises an ionically crosslinkable polysaccharide crosslinked with a collagen derivative, preferably characterized by covalent binding between the polysaccharide and the collagen derivative. The microcarrier is particularly suitable for large scale expansion of adherent cells in applications biologics manufacturing, regenerative medicine, cell therapy or cultivated meat or fish production.

The invention discloses a culture method of cornu cervi pantotrichum stem cells, and belongs to the technical field of tissue engineering. The culture method comprises the following steps: taking antler stem periosteum tissue, refrigerating and transporting the antler stem periosteum tissue through a special transportation preserving fluid, cutting, cleaning and centrifuging to obtain tissue precipitate; resuspending the precipitate by using an optimized serum-free culture medium, inoculating to a culture dish pre-coated with gelatin according to a specific density, standing and culturing, and removing non-adherent cells; after trypsinization and centrifugation, cells are resuspended by using a sorting buffer solution, and a cell population of a specific surface marker is screened by using a flow cytometry sorter, so that the antler stem cells are obtained; and finally, carrying out passage amplification under a low-oxygen condition to obtain the pilose antler stem cells after passage amplification. By optimizing a transportation and preservation system, a culture environment and a sorting process, the purity, the activity and the proliferation and differentiation capacity of the pilose antler stem cells are effectively improved, the process standardization degree is high, and reliable technical support is provided for application of the pilose antler stem cells in the fields of tissue engineering, regenerative medicine and the like.

A cell culture matrix for culturing cells in a fixed bed reactor is provided. The cell culture matrix includes a first substrate material with an ordered and regular array of openings passing through the layer, the openings being separated by the substrate material having a physical structure that is substantially regular and uniform and that is configured for growing cell thereon. The physical structure and array of openings are configured such that, when the first substrate material is rolled into a rolled substrate matrix comprising rolled layers of the first substrate material, a variation in packing density of the rolled layers is less than about 20×, less than about 10×, less than about 5×, or less than about 2× throughout the rolled substrate matrix.

The application provides a novel adherent cell culture device, which comprises a PCB circuit board, an image acquisition device arranged on the upper surface of the PCB circuit board, the image acquisition device being connected with a controller, a cell culture container for providing a container for adherent cells, the cell culture container being arranged above the image acquisition device and the bottom of the sidewall of the cell culture container being abutted against the upper surface of the image acquisition device, the bottom of the cell culture container being provided with an opening for exposing the image acquisition device, the image acquisition device acquiring images of the adherent cells through the opening and outputting the acquired images to the controller, and the controller processing the images to obtain growth data of the adherent cells and transmitting the growth data to an externally connected display terminal. The adherent cells in the cell culture container can be monitored in real time, the monitoring precision of the adherent cells is improved, the real-time monitoring space is saved, and the monitoring cost is reduced.

The invention discloses a lymphatic endothelial cell cyst fluid extraction method for lymphatic deformity, and belongs to the technical field of biological cell extraction. Comprising the following steps: S1, sample collection: collecting 5-10 mL of cystic fluid of a patient with macrocystic lymphangial malformation, and placing the collected cystic fluid in a sterile container; s2, primary centrifugation: centrifuging at a low speed of 1000rpm for 10 minutes, and retaining cell precipitates; s3, red blood cell lysis: adding a red blood cell lysis solution, placing at 4 DEG C for 3-5 minutes, centrifuging, and washing and purifying with PBS (Phosphate Buffer Solution); s4, cell culture: resuspending the cells in a culture medium containing an endothelial growth factor replenishing liquid, culturing at 37 DEG C in 5% CO2, and changing the liquid for 24 hours to remove non-adherent cells; and S5, cell identification: carrying out immunofluorescence staining identification 48-72 hours after cell adherence. The method is simple, convenient and efficient to operate, does not need complex digestion, can enrich high-purity and high-activity lymphatic endothelial cells, provides a stable cell model for related pathogenesis research and drug screening, and has important scientific research and clinical value.

FIELD: biotechnology.SUBSTANCE: method for producing a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by propagating cells in a hybrid seed bioreactor system is described, comprising: (a) culturing adherent cells under adherent conditions with a first growth medium containing serum in container N-2; (b) removing adherent cells from the first medium; (c) inoculating adherent cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than in the first medium in container N-1; (d) culturing adherent cells in container N-1 under suspension conditions; (e) inoculating adherent cells from step (d) into a third medium in a bioreactor for growing adherent cultures; and (f) transfecting the adherent cells with a transgenic plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenoviral helper plasmid, to produce rAAV containing the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO: 7.EFFECT: expanding the scope of treatments for muscular dystrophy.18 cl, 8 tbl, 11 ex

The inventive subject matter provides a diagnostic and clinical application scale cell engineering platform for vector-free or viral delivery of payload / cargo compounds and compositions into non-adherent cells. The platform enables rapid delivery of large numbers of cells into a closed system. Related instruments, systems, techniques, articles, and compositions are also described herein.

The culture method comprises the following steps: carrying out precipitation culture on obtained cells for 6 hours, separating adherent cells, continuously culturing, and collecting non-adherent cells in a culture medium to obtain a component containing immune cells; the adipose tissue-derived stem cells are obtained; mixing the components containing the immune cells with the adipose-derived mesenchymal stem cells to obtain a total cell population, and performing three-dimensional culture by using an adipose organoid growth medium to obtain organoid; and inducing to form fat organs. By means of the culture method, the in-vitro model capable of truly reflecting the in-vivo fat development mode can be established, the formed fat-like organ contains the fat precursor stem cells and also contains rich immune cell populations, the cell interaction in real fat tissue is simulated, and the fat-like organ can be used for drug screening or metabolic disease model construction.

The invention relates to the field of bioengineering, in particular to a preparation method of B lymphocytes. The invention provides a preparation method of B lymphocytes. The preparation method comprises the following steps: S1, obtaining a single-cell suspension; s2, performing density gradient centrifugation on the single-cell suspension to obtain interface layer cells rich in mononuclear cells; s3, carrying out T cell lysis on the interface layer cells by adopting a specific antibody and a specific complement of an anti-T cell surface antigen, centrifuging, and collecting a precipitate; and S4, performing adherent purification on the cells obtained by collecting the precipitates in the step S3, and removing non-adherent cells to obtain the B lymphocytes. According to the extraction and purification method disclosed by the invention, researchers can efficiently enrich and obtain high-purity B lymphocyte populations from complex tissues under conventional laboratory conditions without purchasing professional sorting equipment, and the purity can meet strict requirements of various subsequent cell experiments and immune experiments.

Provided is a simpler, more efficient method for producing Leydig-like cells derived from human pluripotent stem cells, said method capable of reducing the risk of biological contamination and the like and involving a cell culturing method with which mass culturing is possible. Provided is a method in which a process for producing Leydig-like cells derived from human pluripotent stem cells includes a culturing process in which adherent culturing is performed after suspension culturing inside a closed-system cell culture vessel having, on the interior thereof, a culture surface on which suspension cells can be subjected to suspension culturing, and a culture surface to which adherent cells can be adhered.

A method of culturing cells for vaccine production comprises culturing cells in the presence of microcarriers with cell culture media and infecting the cells with a virus. The microcarrier comprises a bead and a coating. The coating comprises a thermo-responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C. The cells adhere to the coating of the microcarrier at a temperature above the LCST. The microcarrier comprises a polymeric bead, and a hydrophobic polymer. The hydrophobic polymer is a block copolymer which is connected to the bead by a covalent bond or by physical adsorption. The block copolymer comprises at least one hydrophobic block and at least one thermo-responsive block.

The invention discloses an HEK293 cell capable of stably expressing pigeon adenovirus I type Fiber2 protein as well as a construction method and application of the HEK293 cell. A pigeon adenovirus I type Fiber2 protein recombinant expression vector is constructed and introduced into an HEK293 adherent cell to obtain a cell QYH-PAF2-A1 capable of expressing the pigeon adenovirus I type Fiber2 protein, and the cell QYH-PAF2-A1 is domesticated into a suspension cell QYH-PAF2-A1. The cell suspension growth state is good, the passage is stable, and the Fiber2 protein can still be stably expressed after 10 generations of continuous culture; then, the Fiber2 protein expressed by the cell is prepared into a subunit vaccine, the protection rate of the subunit vaccine reaches 80% through an immune challenge test, and pigeon adenovirus I type infection can be effectively prevented and controlled. The suspension cell constructed by the invention can be applied to research and development of pigeon adenovirus I type subunit vaccines, preparation of diagnostic reagents and basic research of virus invasion mechanisms, and has a wide application prospect.

A cell culture bioreactor system is provided that includes a medium conditioning vessel (MCV) having a conditioning inlet into the MCV and a conditioning outlet out of the MCV; and a bioreactor container for accommodating a cell substrate, the cell substrate having a surface for attaching cells. The bioreactor vessel includes a bioreactor inlet and a bioreactor outlet. The system further includes a perfusion flow circuit fluidly connecting the conditioning outlet to the bioreactor inlet and fluidly connecting the bioreactor outlet to the conditioning inlet; and a fluid bypass configured to fluidly connect the conditioning outlet to the conditioning inlet without flowing fluid through the bioreactor vessel. The system is operable in both a suspension culture mode in which the cell culture medium flows through the bypass and bypasses the bioreactor vessel and an adherent culture mode in which the cell culture medium flows through the bioreactor vessel.

A method of culturing cells for vaccine production comprises culturing cells in the presence of microcarriers with cell culture media and infecting the cells with a virus. The microcarrier comprises a bead and a coating. The coating comprises a thermo-responsive polymer having a lower critical solution temperature (LCST) of between about 20 °C and about 34 °C. The cells adhere to the coating of the microcarrier at a temperature above the LCST. The microcarrier comprises a polymeric bead, and a hydrophobic polymer. The hydrophobic polymer is a block copolymer which is connected to the bead by a covalent bond or by physical adsorption. The block copolymer comprises at least one hydrophobic block and at least one thermo-responsive block.

The invention discloses a dispersion liquid for serum-free suspension domestication of CRFK cells and a use method of the dispersion liquid. The dispersion liquid is composed of a liquid A and a liquid B, the liquid A is an enzyme active component (containing 0.1%-0.2% of trypsin), and the liquid B is a protective component (containing 1%-3% of a serum substitute and 0.01%-0.05% of Prusich F-68). The core of the invention is a dynamic proportion adjustment application method, wherein the ratio of the solution A to the solution B is 2.5: 1 in the initial stage of domestication (1-3 generations); the proportion in the middle stage of domestication (the fourth generation to the fifth generation) is reduced to 1.5: 1; at the later domestication stage (after the sixth generation), the proportion is reduced to 1: 1, and meanwhile, serum-free TransVA-03 is used as a culture medium for culture under the shaking table conditions of 36.5 DEG C, 7% CO2 and 110r / min. The method solves the problem of agglomeration in the serum-free suspension domestication process of the CRFK cells, the viability of the domesticated cells is greater than 95%, the agglomeration rate is less than 10%, and the method is suitable for serum-free suspension domestication of various adherent cells and has important industrial application value.