147 results about "Perfusion Culture" patented technology

The invention discloses automatic perfusion culture equipment for NK (Natural Killer) cell culture, which belongs to the technical field of cell culture equipment and comprises a liquid storage cylinder, a waste liquid cylinder, a mounting rack, a culture box, a first conveying mechanism, a second conveying mechanism, a driving mechanism and an anti-skid fixing mechanism. According to the automatic perfusion culture equipment for NK cell culture, through the arrangement of the first conveying mechanism, the second conveying mechanism and the driving mechanism, a nutrient solution in the liquid storage cylinder can be automatically conveyed into the culture box, a waste nutrient solution in the culture box can be automatically conveyed into the waste liquid cylinder, automatic perfusion culture of NK cells is achieved, and through flowing of the nutrient solution, the culture efficiency is improved. The mass transfer between nutrient substances and metabolic waste products and cells can be realized, so that the concentration accumulation of harmful metabolic waste is reduced, and the culture of NK cells is facilitated.

The invention belongs to the technical field of biomedical engineering and microfluidics, and particularly discloses a vascularized organ chip and method for large-scale culture, the vascularized organ chip comprises an upper runner layer and a lower culture layer, and the runner layer and the culture layer are fixedly sealed; a liquid storage pipe matched with the flow channel layer is adhered to the upper surface of the flow channel layer; according to the vascularized organ chip, the flow channel layer is used for pouring a culture solution to provide fluid stimulation and nutrients for cells; the three parallel-connected large-scale culture chambers of the culture layer are used for culturing cells, so that blood circulation of a microvascular system in a human body can be simulated, and a microenvironment in tissues can be reproduced more truly; meanwhile, the requirement for culturing a large number of tissue cells through the chip can be met, and the cell yield and the experiment scale can be effectively increased.

The invention belongs to the technical field of micro-fluidic chips, and discloses a hepatic lobule chip which comprises a single cell capture array chip, a micropore array chip and an antibody slide, a capture array on the single cell capture array chip and a micropore array on the micropore array chip are in a polygon shape, perfusion culture solution channels are arranged in the diagonal directions of all vertexes of the polygon, and the capture array and the micropore array are in one-to-one correspondence after being assembled. The liver lobule chip overcomes the dependence of research on spatial heterogeneity of liver cells on precise liver anatomical slices, avoids the defects that the liver slices are difficult to obtain and store, can directly reveal spatial distribution characteristics of secretion activity of the liver cells in the liver lobule, and becomes an ideal tool for spatial heterogeneity secretion of the liver cells. The invention further discloses application of the hepatic lobule chip in hepatic lobule spatial heterogeneity single cell secretion analysis, operation is easy, large experimental instruments are not needed, cost is low, and the hepatic lobule chip is easy to master.

Disclosed herein are compositions and methods related to differentiation of stem cells into pancreatic islet cells. In some embodiments, the methods provided herein relate to generation of pancreatic β cell, α cell, δ cells, and EC cells in vitro. In some embodiments, the disclosure provides pharmaceutical compositions including the cells generated according to the methods disclosed herein, as well as methods of treatment making use thereof.

The invention relates to a method for producing an antibody by improving cell inoculation density through seed perfusion culture and application. In N-1 seed cell culture, through a perfusion culture process mode, especially according to the cell density, the perfusion rate is adjusted to timely increase nutrient substances in a cell culture system and discharge metabolic wastes in cell culture to increase the density of living cells, so that the initial density of N-generation cells is greatly improved. The method is convenient to amplify and simple to operate, the antibody yield can be increased in the same culture period, or a target product can be obtained in a shorter culture period, and the production cost is greatly reduced.

The present invention provides a continuous upstream manufacturing process for the production of bispecific antibody products, which comprise at least two binding domains. The process comprises at least the steps of (i) providing in a perfusion bioreactor at least one mammalian cell culture, which is capable of expressing the bispecific antibody product, (ii) growing the mammalian cell culture at a first perfusion rate until a set point viable cell density is reached, and (iii) maintaining perfusion culture at a second perfusion rate, wherein the bispecific antibody product concentration in the bioreactor is kept below a threshold value. The bispecific antibody product is then subject to subsequent downstream processing. Moreover, the invention provides a bispecific antibody product produced by the continuous upstream manufacturing process.

The invention provides a perfusion culture preparation method and application of a recombinant human anti-rabies virus monoclonal antibody R71, and relates to the technical field of biological medicines. According to the method, key process parameters in the culture process of the engineering cell strain expressing the R71 antibody are optimized, the key processes comprise a culture medium, a rotating speed, pH, an ATF P-flow set value and a dissolved oxygen (DO) range, and experimental results show that the receiving amount of R71 in each batch of the optimized perfusion culture method can reach more than 200g and is 5-8 times that of fed-batch culture under the same culture scale.

The utility model discloses a micro-fluidic chip for in-vitro 3D hepatic organ co-culture, which belongs to the technical field of biomedical engineering and comprises a perfusion layer and a culture layer, and the perfusion layer is provided with a first culture solution inlet and a first culture solution outlet; the culture layer is provided with a circulating microfluidic perfusion culture system; the chip comprises an upper layer and a lower layer formed by bonding a perfusion layer and a culture layer; the circulating micro-fluidic perfusion culture system comprises two groups of micro-fluidic multi-chamber co-culture chips which are connected in series; the series micro-fluidic multi-chamber co-culture chip comprises a second culture solution inlet, a micro-fluid channel, a compound cell co-culture area and a second culture solution outlet, and the second culture solution inlet, the micro-fluid channel, the compound cell co-culture area and the second culture solution outlet are communicated with one another. The problem that in the prior art, while complex physiological environment simulation is maintained, the reliability and practicability of the model are improved is solved. The method has the advantages of high efficiency and accuracy.

An object of the present invention is to provide a method in which the amount of mannose 5 in a product can be reduced in the production method for the product by cell culture, and a product that is produced by the method. According to the present invention, there is provided a production method for a product, including culturing cells in a culture container, in which the culturing is a perfusion culture method, Expressions (1) to (5) are satisfied in a production period of a product after reaching a target viable cell density, a variation of VCD during the production period is ±20% or less with respect to an average viable cell density during the production period, and a variation of VCV during the production period is ±20% or less with respect to an average value of the VCV during the production period. 0≤Gln−C≤2.5 0.5≤Gluc−C≤8.0 1.0≤NH4−C≤8.0 50≤VCD≤300 0.6×105≤VCV≤4.0×105 The meanings of the symbols in the expressions are as defined in the present specification.

Provided are methods for the creation of ultra-high density cryopreserved cell banks. In certain embodiments, these methods employ altered perfusion culture techniques that allow for production of ultra-high density cell cultures that can be cryopreserved at unexpectedly high cell densities without the need for any cell concentration steps, while retaining excellent cell viability and quality.

The invention relates to the technical field of bone defect repair, in particular to a composite cylindrical bionic bone scaffold and a preparation method thereof.The preparation method comprises the following steps of regenerated material preparation, ZP scaffold preparation, composite hydrogel preparation, PH scaffold preparation and scaffold assembling, specifically, the ZP scaffold and the PH scaffold are subjected to ultraviolet disinfection treatment, then a phosphate buffer solution is used for flushing treatment, and the composite cylindrical bionic bone scaffold is obtained; the preparation method comprises the following steps: implanting endothelial cells into a PH scaffold according to 1 * 10 < 5 > mL, carrying out perfusion culture for 6-8 days, and combining a ZP scaffold with the PH scaffold in which the endothelial cells are implanted to form the macro-microstructure composite cylindrical bionic bone scaffold. According to the composite cylindrical bionic bone scaffold and the preparation method thereof, the ZP scaffold and the PH scaffold are combined to form the vascularized composite cylindrical bionic bone scaffold containing internal and external bionics, so that the composite cylindrical bionic bone scaffold has satisfactory biocompatibility, and the angiogenesis and osteogenesis processes are obviously promoted.

A multi-cell barrier tissue model of a bionic sequential development mechanism has a multi-layer multi-cell structure simulating a physiological barrier, related cells of different barriers are distributed in different layers according to the physiological structure, and the development time and sequence of the cells of different layers are regulated and controlled according to natural barrier multi-cell development physiological characteristics; the multi-cell three-dimensional structure of in-vivo barrier tissue can be simulated, regulation and control of cell development behaviors are achieved by regulating and controlling the matrix environment, and meanwhile, the model has enough overall mechanical support through the soft / hard chimeric structural design so as to adapt to perfusion culture and operation requirements; the model can realize functional bionics of natural barrier tissues at the same time in the spatial structure dimension and the time development dimension so as to realize application in the aspects of disease modeling, drug screening and the like.

Provided are a perfusion culture method and the like that are able to improve the productivity in the production of recombinant protein. Specifically, a method for improving recombinant protein production in perfusion culture of an animal cell having a capacity to produce a recombinant protein, the method comprising adding cystine and / or ammonium iron citrate to a cell culture medium, and the like, are provided.

The present invention provides a novel method for producing natural killer (NK) cells, which enables production of a large amount of high-quality NK cells easily, inexpensively, and safely in a short period of time from pluripotent stem cells. Specifically, a method for producing NK cells is provided, including(1) a step of forming pluripotent stem cell spheres having an average particle size of not less than 200 μm in a first medium;(2) a step of inducing the pluripotent stem cell spheres formed in step (1) into a cell population including hematopoietic progenitor cells by three-dimensional culture using a second medium; and(3) a step of inducing the cell population including hematopoietic progenitor cells obtained in step (2) into a cell population including NK cells by three-dimensional culture using a third medium,wherein the steps (1) to (3) are performed by a perfusion culture method.

The invention belongs to the field of perfusion culture chips, and provides a perfusion culture microfluidic device and system. A perfusion culture microfluidic device comprises a main body, a culture support and a sealing structure, the main body is divided into an upper layer structure, a middle layer structure and a bottom layer structure, the upper layer is provided with a liquid inlet and a liquid outlet which are respectively connected with a channel between the upper layer and the middle layer, and a channel between the middle layer and the bottom layer; the channel penetrates through the culture bracket in the middle layer, and the culture bracket is provided with an upper circular ring and a lower circular ring which are separated by a porous membrane; the sealing structures are respectively arranged on the upper layer and the middle layer of the main body and on the bottom layer of the main body, and seal the upper-layer circular ring, the porous membrane and the lower-layer circular ring of the culture bracket into two culture chambers. According to the perfusion culture micro-fluidic device and system and the double-channel perfusion micro-fluidic device based on the porous membrane culture support, the growth requirements of various tissue or organ simulants can be met, the sealing structure enables the device to be disassembled and assembled without the help of auxiliary equipment, inoculation and characterization of cells are facilitated, and the operation is simple and convenient. The whole device can be repeatedly used.

The utility model discloses an organoid automatic perfusion culture device, and the device comprises: upper cover structure, upper cover structure is equipped with liquid inlet and a plurality of shunt passageways for guiding perfusion liquid to a plurality of culture cavities below respectively, modular culture unit, modular culture unit includes a plurality of culture cavities, and the vertical fluid passage for liquid through is arranged below every culture cavity and extends along the vertical direction, modular culture unit has stackable configuration, and the four corners of every modular culture unit are provided with limit cooperation structure and are used for aligning and positioning with adjacent unit, the upper surface or lower surface of modular culture unit is equipped with annular sealing groove, and the O type sealing ring is embedded in sealing groove and is used for forming sealed contact under the stacked state, base structure, and the vertical fluid passage export of the lowermost layer modular culture unit is communicated with base structure, and the inside is provided with the confluence cavity and liquid outlet.

The invention discloses a dynamic perfusion blood tube chip based on micro-fluidic core-shell fibers and a preparation method thereof, and belongs to the technical field of biomedical materials.The preparation method comprises the steps that 1, hydrogel fibers with hollow inner cavities are prepared through the micro-fluidic technology; step 2, planting human coronary artery smooth muscle cells on the outer surface of the hydrogel fiber, and planting human umbilical vein endothelial cells in the cavity of the hydrogel fiber to realize three-dimensional co-culture of the two cells; and step 3, soaking the hydrogel fiber planted with the cells in a human coronary artery smooth muscle cell complete medium, connecting the hydrogel fiber with the human umbilical vein endothelial cell complete medium, and continuously pouring the human umbilical vein endothelial cell complete medium into an inner cavity of the hydrogel fiber planted with the cells to obtain the dynamic perfusion blood tube chip. The chip provided by the invention not only can realize three-dimensional co-culture of cells, but also realizes dynamic perfusion culture, and better simulates the structure and function of human blood vessels.

The present invention relates to a serum-free cell culture perfusion medium comprising medium components that make up at least three separate aqueous concentrated feeds and a diluent wherein the resulting serum-free cell culture perfusion medium, upon mixing, adjusts the pH to a neutral pH. The invention also provides a method for preparing the serum-free cell culture perfusion medium. The invention further relates to methods of culturing mammalian cells or producing proteins of interest in perfusion cultures using the serum-free cell culture perfusion media, which achieve high productivity at low cell specific perfusion rates. The invention further relates to the use of novel and improved serum-free cell culture perfusion media to control the osmotic pressure of a perfusion cell culture, where an increase in total productivity and / or cell specific productivity is caused by inhibiting cell growth, increasing osmotic pressure during cell culture, for example during the production phase of perfusion cell culture. Inhibiting cell growth particularly reduces or eliminates the need for wasted cell expulsion.

The utility model belongs to the technical field of perfusion culture, and particularly relates to a liquid level control device for perfusion culture, which comprises a bottom plate, a floating plate and a laser range finder, supporting legs are fixedly installed at the bottom of the bottom plate, a fixing support is fixedly installed at the top of the bottom plate, a tank body is fixedly installed on the inner wall of the fixing support, a liquid inlet assembly is arranged at the top of the tank body, a liquid outlet assembly is arranged at the bottom of the bottom plate, and a supporting assembly is arranged on the inner wall of the tank body. A measuring assembly is arranged on the inner wall of the tank body; through the arrangement of a liquid inlet pump, a first liquid conveying pipe, a liquid outlet pump and a second liquid conveying pipe, the liquid inlet pump and the first liquid conveying pipe are matched for use, the liquid inlet amount can be accurately controlled, the height of liquid in the tank body can be effectively controlled, and the liquid outlet pump and the second liquid conveying pipe are matched for use, so that the water outlet amount of the liquid in the tank body can be accurately controlled; and the water level can be accurately controlled by matching with the liquid inlet pump.

The invention provides a cell perfusion culture method and device, and the method comprises the steps: configuring a rotatable container capable of accommodating liquid, the container being provided with a first opening located in a bottom central region and a second opening located in a top central region; a cell-containing culture solution in a cell culture tank is injected into the container through the first opening for feeding; the container is controlled to rotate, and the cells are pushed to the position close to the container wall of the container through centrifugal force; liquid is continuously discharged from the second opening, so that continuous concentration of the cells is realized; the cell concentration is detected in real time, and when the cell concentration reaches a preset value, feeding, rotating and discharging operation is stopped; the conveying direction of the first opening is switched, the concentrated cells are conveyed back to the cell culture tank, perfusion culture is achieved, efficient, low-shear, low-cost and sustainable cell concentration and separation are achieved, perfusion stability and productivity are improved, and the cell concentration and separation device is suitable for large-scale cell production and industrial biopharmaceutical processes.

The present application provides a method for maintaining a steady state of a cell perfusion culture system, and relates to the field of cell culture technology. The method includes: placing cells in a bioreactor and performing a first culture at a temperature of T0; when the density of live cells in the bioreactor reaches the first target value, the temperature of T0 is reduced to the temperature of T1, and a perfusion medium is used for the second culture, and after maintaining for 4-5 days, the temperature of T1 is increased to the temperature of T2, and the third culture is continued, and the second and third cultures are not discharged; or, when the density of live cells in the bioreactor reaches the second target value, the temperature of T0 is reduced to the temperature of T2, and a perfusion medium is used for the fourth culture, and discharge is required in the fourth culture; wherein, T0>T2>T1. The present application adopts the means of two-step cooling, and according to the law of cell first-order kinetic multiplication, a fixed discharge rate is used for discharge, so as to maintain the steady-state growth of the cells.

The invention mainly provides a method for producing oncolytic viruses by using a bioreactor. Through perfusion culture, the Vero cell quantity during virus infection is optimized, so that the purposes of increasing the virus yield, reducing the batch difference and finally producing the oncolytic virus can be achieved.

The invention discloses a patient-specific bile duct cancer chip and a method thereof in dynamic drug resistance evolution analysis, and relates to the technical field of biomedicine, the patient-specific bile duct cancer chip comprises a central tumor microenvironment chamber, the central tumor microenvironment chamber is internally provided with a plurality of independent three-dimensional culture chambers, and patient-derived cells are embedded and loaded in a matrix; the perfusion channel network is connected with the plurality of three-dimensional culture chambers to form a flow channel and is used for continuously perfusing a culture medium, a medicine or a stromal cell suspension and simulating blood flow and interstitial fluid flow, and the microenvironment regulation and control unit is integrated with the perfusion channel network to control the flow channel. The chip provided by the invention integrates tumor cells, cancer-related fibroblasts and endothelial cells, so that the tumor cells, the cancer-related fibroblasts and the endothelial cells grow in a 3D matrix to form organoids with spatial structures and interstitial infiltration, and the microfluidic perfusion simulates blood / interstitial fluid flow, so that the drug test environment is closer to the actual situation of a human body, and the obtained data has higher physiological prediction value.

The present invention relates to hollow fiber tangential flow depth filtration media adapted for perfusion bioprocessing, and related TFDF filtration units, bioprocessing systems, and methods, including methods for perfusion cell culture. The hollow fiber elements of the present invention combine a relatively thin porous wall with a relatively low porosity to provide filtration media resistant to fouling during perfusion culture.

The invention discloses a culture chip, a culture module and cell perfusion culture equipment, and belongs to the field of cell culture, a first connector of a main body is used for perfusion culture, the first connector is communicated with a culture area, a grid assembly forms a plurality of culture spaces, and the top of the grid assembly is of an open structure so that cells can enter; each culture space is provided with a plurality of gaps, the size of the gaps is smaller than that of cells to be cultured in the culture spaces, so that the cells to be cultured are restrained in the culture spaces, the gaps enable every two adjacent culture spaces to be communicated and enable the culture spaces to be communicated with the culture areas, and the sealing piece is installed in the chip seat and located above the grid assembly. The sealing piece prevents to-be-cultured cells from flowing out of the top of the culture space, a gap is formed between the sealing piece and the top of the grid assembly and used for breaking liquid tension during liquid change, and during cell culture, perfusion is performed through the first connector and the grid assembly, shearing force is generated, and the internal environment of the human body is simulated; the culture solution can be replaced in the solution replacement area.

A production method for a bispecific antibody, comprising constructing cells for producing a bispecific antibody, screening the cells, culturing the cells obtained in step one to obtain a culture solution, and performing separation and purification to obtain the bispecific antibody. The cells comprise CHO cells. The culture mode comprises fed-batch culture or perfusion culture. Various influence factors in fermentation culture and separation and purification are comprehensively analyzed, and by effective control, all factors can effectively cooperate, so that a high-purity bispecific antibody is efficiently produced, the daily output can reach 3 g / L or above, and the purity of the purified bispecific antibody can reach 90% or above. Moreover, the production process is amplified and verified, the process is stable and reliable, the cost is low, a great breakthrough is achieved compared with an existing production method. The present application has great significance for wide clinical application of bispecific antibodies.

An in-vivo rapid recellularization tissue engineered blood vessel and a preparation method thereof are provided. The preparation method includes: dissolving polyglycolic acid and polyethylene glycol in an organic solvent to prepare a spinning solution, and performing electrostatic spinning according to a certain proportion to prepare a tubular polymer composite support of a special tube wall structure, the fiber on the outer side of the tube wall support containing a higher proportion of polyethylene glycol; planting seed cells on the tubular polymer support of the special tube wall structure, and under certain culture conditions, simulating body arteriopalmus flow conditions and performing perfusion culture for 3 weeks to 3 months; and removing cell components from the tube wall of the obtained engineered blood vessel by using a decellularization method to give a tissue engineered blood vessel composed of an extracellular matrix of a bionic structure.

The present invention provides a microfluidic chip and its packaging structure. The microfluidic chip has multiple functional units, and the functional units have cell sorting structures and cell culture chambers. The cell sorting structure sorts single cells from a cell suspension, and the single cells enter the cell culture chamber and are cultured into a cell group. The cell group is screened in the cell culture chamber and then exported. The microfluidic chip provided by the present invention integrates single cell sorting, culture, cell group screening and exporting into one, and the operation is simple and efficient; and the cell sorting structure in the microfluidic chip can improve the capture rate of single cells, avoiding the problem of multi-cell acquisition; in addition, combined with thermal bubble printing technology, the liquid flow of each functional unit can be controlled separately, achieving higher throughput, making the whole process more controllable, convenient and efficient. The packaging structure provided by the present invention is used to package the above-mentioned microfluidic chip, which can realize free switching of samples, convenient perfusion culture and reagent introduction analysis, and is simple and efficient to operate.

The invention discloses a method for preparing a vascular organ chip from human umbilical artery stem cells, and belongs to the field of biomedical engineering and tissue engineering. The method comprises the following steps: separating and culturing human umbilical artery stem cells to the third generation; preparing a PDMS micro-fluidic chip containing a liquid inlet channel, a liquid outlet channel, a growth channel and a central culture chamber; a blood vessel precursor aggregate is obtained through cell digestion, aggregate construction, mesoderm induction and blood vessel pedigree induction, the blood vessel precursor aggregate is inoculated into a Coll-1-Matrigel gel modified chip through a two-step method, and dynamic perfusion culture is conducted in combination with a culture medium containing VEGF-A and FGF-2. And finally, the structure and the function of the vascular organ are verified through immunofluorescent staining and fluorescent microbead perfusion. The problems that in the prior art, cell sources are limited, culture conditions are single and the like are solved, the high angiogenesis potential of the human umbilical artery stem cells and a dynamic culture system are utilized to construct a high-bionic-degree vascular network, and the method can be applied to drug screening, vascular pathological model construction and regenerative medicine research.

The invention discloses a micro-fluidic blood vessel chip for diagnosing incomplete Kawasaki disease as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials.The preparation method comprises the following steps: step 1, preparing a micro-fluidic chip containing a collagen channel; step 2, planting the HCASMCs in the collagen channel, planting the HUVECs after the HCASMCs converge into a single layer to cover the collagen channel, enabling the HUVECs to converge into a single layer to cover the surface of the HCASMCs, and then dynamically pouring a culture medium to obtain the micro-fluidic blood vessel chip. According to the invention, the tissue structure of the blood vessel can be effectively simulated, and the blood flow of the blood vessel can be simulated through continuous dynamic perfusion culture of cells. In practical application, serum to be diagnosed is injected into the microfluidic blood vessel chip, and the physical change of a collagen channel and the change of a cell inflammatory reaction antibody are observed, so that the incomplete Kawasaki disease can be diagnosed. The invention provides a brand new thought and technical platform for diagnosis and treatment of Kawasaki disease, and screening of incomplete Kawasaki disease can be carried out in a short time.