103 results about "Perfusion Culture" patented technology

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 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.

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.

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 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 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 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.

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 present application provides a cell culture device and a bioreactor. The culture device comprises: a mixer for mixing substances required for cell culture, the mixer comprising a mixing container and a mixing drive member, the mixing container providing a sealed mixing space and being in communication with the outside via a first interface and a second interface, and the mixing drive member being capable of driving substances to be mixed to move and mix; and a bioreactor comprising a reaction container and a plurality of culture dishes, the reaction container providing a closed space, and the plurality of culture dishes being disposed within the closed space. The closed space is in communication with the outside via a third interface and a fourth interface, the third interface may communicate with the second interface of the mixer, and the fourth interface may communicate with the first interface of the mixer, thereby providing perfusion culture within the bioreactor.

The utility model relates to the technical field of organoid perfusion culture, and provides an organoid perfusion culture chip which comprises a box cover, a bottom box and a partition plate, the partition plate is fixedly connected to the interior of the bottom box, and a plurality of partition rings are fixedly connected to the inner wall of the partition plate; a plurality of mounting through holes are formed in the box cover, first culture barrels are inserted into the mounting through holes, and second culture barrels are inserted into the inner walls of the mounting through holes; sealing rings are fixedly connected to the outer walls of the first culture barrel and the second culture barrel. The bottom box is divided into an upper space and a lower space through the partition plate, the microporous membrane of the first culture barrel can be located in the space below the partition plate through the partition ring, the second culture barrel can be located in the space above the partition plate, and deviation of a final result caused by mutual interference when liquid is input into the culture box is avoided through the sealing ring.

The disclosure provides methods of purifying a target protein, e.g., an antibody, from a host cell such as a mammalian cell using purification protocols incorporating harvest recovery operations comprising a continuous solids discharge disc stack centrifugation step followed by flocculation and depth filtration steps. The protein harvest methods of the disclosure recover high yields of purified target protein from perfusion cultures having a packed cell volume of greater than or equal to 16%, using an unconventional yet effective process.

The present disclosure provides a bioengineered kidney construction method based on organoids and acellular scaffolds, which comprises first injecting pro-vascular stem cells into a kidney acellular scaffold material, and then culturing for several days through fluid circulation perfusion, and then injecting kidney organoids (KIO) into the scaffold material for perfusion culture after the kidney acellular scaffold material is recellularized. The bioengineered kidney obtained by the method of the present disclosure can be used as a graft for replacing kidney function. The present disclosure also provides a kidney organoid culture and expansion method, and the organoids obtained by the method contain not only kidney parenchymal cells such as glomerular cells and renal tubular cells, but also blood vessels and immune cells, which provides a good source of kidney parenchymal cells for the preparation of tissue-engineered kidneys.

The present application belongs to the technical field of biopharmaceutical equipment, and particularly relates to a flow control device for cell perfusion culture, a perfusion culture device and a use method thereof. In the flow control device for cell perfusion culture, the combination of a control system and an automatic pressure regulating valve is used to realize self-adaptive regulation and control of air pressure, and is combined with the way of limiting flow through a plurality of capillary bodies with different lengths and / or inner diameters, so that continuous and accurate regulation of gas flow is realized at low cost, thereby meeting the dynamic flow requirements in different stages of the cell culture process.

The present invention provides a multi-material bionic liver tissue organ chip capable of perfusion culture and a preparation method thereof. The multi-material bionic liver tissue organ chip comprises an upper vascular network module, a bionic liver tissue module, and a bottom vascular network trough module, which are sequentially connected. The upper vascular network module is provided with a perfusion inlet and a first bionic vascular network, and the bottom vascular network trough module is provided with a second bionic vascular network and a perfusion outlet. The bionic liver tissue module comprises an outer shell, within which are disposed N hepatic lobules, where N is greater than or equal to 3. Each hepatic lobule is provided with hepatic lobular connective tissue on the outer side, and a central hepatic lobule vein is provided running through the middle of each hepatic lobule from top to bottom. The first and second bionic vascular networks are provided with bionic central veins and bionic vascular connective tissue corresponding to the central hepatic lobule vein and hepatic lobular connective tissue of each hepatic lobule. The technical solution of the present invention has excellent biocompatibility and meets the requirements of perfusion culture.

The invention discloses a micro-fluidic chip for droplet generation, solidification and deoiling integration and a method thereof. The micro-fluidic chip comprises a spiral micro-channel, a chip sample inlet, a capturing structure and a chip sample outlet, wherein the spiral micro-channel is internally provided with a capturing structure which is used for liquid drop generation, solidification and deoiling and is adjustable in size. When in use, the channel is infiltrated by an oil phase, then hydrogel wrapping cells is introduced into the spiral channel, and finally the oil phase is introduced, so that liquid drops are formed at the capturing structure and are captured; then, the liquid drops are solidified in situ in the channel, a degreaser is introduced to remove the surrounding oil phase, and finally, the liquid drops are cleaned through a culture medium and subjected to perfusion culture or transfer. The flow speed can be accurately controlled without using an injection pump in a matched manner, the liquid drops can be generated by manual operation only through the pipette, the cost is low, and the operation is simple. The micro-fluidic chip lays a good technical foundation for subsequent development of a high-throughput and automatic organoid culture or drug screening platform.

The invention relates to a method of culturing mammalian cells expressing a heterologous protein in a perfusion cell culture comprising increasing the potassium concentration and decreasing the molar ratio of sodium to potassium to reduce wasteful cell bleed and to increase protein production. The invention further relates to a serum-free perfusion medium comprising a high potassium ion concentration and a low molar ratio of sodium to potassium and to the use of this medium for use in culturing cells in a perfusion culture during production phase or for reducing the cell bleed volume during production phase.

The invention discloses a tangential flow filtering device with a cleaning device and a perfusion culture system and method.The tangential flow filtering device comprises a hollow fiber column assembly, a pressure detection meter, a first diaphragm pump and a second diaphragm pump, the hollow fiber column assembly comprises a hollow fiber column and a sleeve, and the sleeve is arranged outside the hollow fiber column in a sleeving mode; a waste liquid cavity is formed between the inner wall of the sleeve and the hollow fiber column; a first butt-joint port and a second butt-joint port are formed in the upper end and the lower end of the hollow fiber column assembly respectively, and a first liquid outlet and a second liquid outlet are formed in the upper end side wall and the lower end side wall of the hollow fiber column assembly respectively; the first diaphragm pump is in butt joint with the second butt joint opening, the second diaphragm pump is in butt joint with the second liquid outlet, and the pressure detection meter is connected with the waste liquid cavity through the second liquid outlet. The blockage condition of the hollow fiber column is judged by monitoring the pressure change of the liquid outlet through the pressure detection meter, and liquid is driven by the second diaphragm pump to perform back flushing on the hollow fiber column, so that the smoothness of the hollow fiber column is ensured.

This invention discloses a microfluidic chip and its fabrication method for constructing modular cell cultures, relating to the field of cell culture technology. The chip includes an upper plate and a lower plate. The upper plate includes a first flow channel and a second flow channel, while the lower plate includes multiple spaced-apart culture chambers, each with a different cross-sectional shape. This allows each chamber to be independently formed into cultures of different shapes. The cultures can be stacked and combined after removal to construct a multi-cell co-culture model with functional partitions, significantly improving structural complexity and biomimicry. The upper plate slides to switch the connection between the first and second flow channels and the culture chambers, enabling inoculation and culture modes. During inoculation, cultured cells are injected into the culture chamber through the first flow channel; during culture, the flow channel is switched to perfuse the culture chamber with culture medium, avoiding damage to the culture during residual gel removal. This simple operation improves the success rate and enhances the reliability of experimental results.

The invention relates to a detachable multi-organ chip co-culture device, and relates to the technical field of biological tissue engineering. Comprising an upper-layer fluid channel layer chip, a lower-layer micro-pit cell culture layer chip and an osmotic pump, the upper-layer fluid channel layer chip and the lower-layer micro-pit cell culture layer chip are respectively divided into five independent functional units, and the structures of the independent functional units are mutually independent; each independent functional unit of the upper fluid channel layer chip comprises a fluid channel structure; each independent functional unit of the lower-layer micro-pit cell culture layer chip comprises a micro-pit array structure and a micro-cavity, and the micro-pit array structure is arranged at the bottom of the micro-cavity; the upper-layer fluid channel layer chip and the lower-layer micro-pit cell culture layer chip are detachably combined and connected through a tenon-and-mortise structure; the osmotic pump is connected to the outlet end of the culture device and is used for constructing a dynamic perfusion culture environment. According to the invention, high-throughput culture of multiple organs can be realized, and the cell viability and proliferation state are good under a dynamic perfusion condition.

A method for producing spheroids and cellular by-products of spheroids, such as extracellular vesicles, is provided. The method includes providing a bioreactor having a cavity for culturing cells, inserting the cells into the cavity to form spheroids, and perfusing cell culture medium through the cavity to culture the spheroids. The porous scaffold may be dissolvable or indissolvable, and cells are seeded into the pores of the porous scaffold, where they aggregate to form spheroids. The bioreactor is continuously perfused for large-scale spheroid production, allowing for the recovery of spheroids and / or extracellular vesicles or other elements from the cell secretome.

In certain embodiments, this disclosure provides novel methods of increasing the viable cell density of an N−1 large-scale bioreactor cell culture, comprising culturing a host cell expressing a recombinant poly peptide of interest in a non-perfusion-based culture system, and wherein the viable cell density is increased to at least 5×106 cells / mL. In certain embodiments, the disclosure provides novel methods for large-scale production of a recombinant polypeptide of interest, comprising: (1) culturing a host cell expressing a recombinant polypeptide of interest in an N−1 stage in a non-perfusion-based culture system, wherein the viable cell density is increased to at least 5×106 cells / mL; and (2) culturing N fed-batch production cells in an enriched media with high-seed density at least 1.5×106 cells / mL, wherein the N fed-batch production cells are inoculated from the N-1 stage in a non-perfusion-based culture system.

The invention provides a cell perfusion culture method for efficiently and correctly expressing the antibody with the asymmetric structure, a rocking tube model suitable for a perfusion culture process, perfusion culture medium development and the like is established, the asymmetric antibody with a correct structure can be obtained, the daily output can reach 2g / L, the asymmetric antibody with higher antibody purity can be obtained after purification, and the method is suitable for large-scale industrial production. Compared with a traditional fed batch (Fed-Batch) culture mode, the method has a great breakthrough, and has great significance in production of asymmetric antibody products with low expression and poor stability.

The application provides a microfluidic perfusion culture integrated chip cartridge for space environment, which comprises a cartridge frame structure, a microfluidic perfusion culture module and a gas-tight electric connector, the cartridge frame structure is formed into a full-sealing structure by a middle frame plate, an upper cover plate and a lower cover plate, the microfluidic perfusion culture module is enclosed in the cartridge frame structure, and the cartridge frame structure is connected with an external control device through the gas-tight electric connector. The microfluidic perfusion culture integrated chip cartridge is full-sealed, the microorganism in the cartridge is isolated from the outside operator, and is suitable for use in a space environment.