232 results about "Serum free" patented technology

The present invention provides a serum-free culture medium for in vitro suspension culture of porcine muscle stem cells and its application, which can help porcine muscle stem cells expand and be suspended under serum-free and carrier-free conditions. Compared with traditional porcine muscle stem cell culture protocols and culture media, this serum-free suspension culture medium enables porcine muscle stem cells to be cultured continuously at high densities in both batch and fed-batch culture processes under serum-free and carrier-free suspension culture conditions, while maintaining high cell viability and achieving a peak cell density of 1×10 7 The beneficial effect of the present invention is to effectively solve the problem of serum-free and carrier-free efficient suspension expansion of cell cultured meat seed cells, and provide a technical and raw material basis for the industrial production of cell cultured meat.

The invention provides a serum-free culture medium for in-vitro suspension culture of porcine muscle stem cells and application of the serum-free culture medium. The serum-free culture medium can help the porcine muscle stem cells to be subjected to suspension enlarged culture under serum-free and carrier-free conditions. Compared with a traditional porcine muscle stem cell culture scheme and culture medium, the serum-free suspension culture medium has the advantages that high-density continuous culture of porcine muscle stem cells can be realized in a batch culture process and a fed-batch culture process under the serum-free carrier-free suspension culture condition, high cell viability is maintained, and the survival rate of the porcine muscle stem cells is increased. The peak density of the cells reaches 1 * 10 < 7 > cells / mL or above, and the cell viability is maintained to be 90% or above. The method has the beneficial effects that the problem of serum-free carrier-free efficient suspension amplification of the seed cells of the cell culture meat is effectively solved, and a technical and raw material basis is provided for industrial production of the cell culture meat.

The invention discloses a cord blood-derived regulatory T cell amplification culture medium and a use method thereof, and relates to the field of animal cell culture and immune cell therapy, the culture medium is a serum-free culture medium, the culture medium is composed of a basic culture medium, a serum substitute combination, a combination of four cell factors, an immunosuppressor, an apparent stabilizer and an antioxidant, and all the components play a role synergistically; according to the use method, cell amplification is realized through a collaborative process of staged environmental regulation and targeted activation. According to the method, the problems of exogenous pollution and batch difference caused by dependence of a serum-containing system on cord blood-derived regulatory T cell amplification in the prior art are solved, the defect that a serum-free scheme is difficult to consider the amplification efficiency and the function stability at the same time is overcome, the cell amplification quality and the clinical application safety are guaranteed, and the method is suitable for large-scale clinical transformation requirements.

The specification describes an improved serum-free animal cell culture medium, which can used for the production of a protein of interest. Ornithine, or a combination of ornithine and putrescine can be added to serum-free media or chemically defined media to improve viable cell density, to reduce cell doubling time, and to increase the production of a protein of interest.

The invention discloses a serum-free full-suspension MDCK (Madin-Darby Canine Kidney) cell and application thereof in preparation of influenza vaccines, and belongs to the field of virology and biological medicines. The serum-free full-suspension MDCK cell is preserved in the China Center for Type Culture Collection, is classified and named as a canine kidney cell MDCK-JHS, and has a preservation number of CCTCC NO: C2024373. The invention further provides a method for culturing the influenza virus by using the cell line, high-titer amplification of the influenza virus can be realized, and the method is suitable for industrial production.

The invention discloses a recombinant subunit vaccine for resisting trichomonas pigeonae infection and a preparation method of the recombinant subunit vaccine. The vaccine comprises two recombinant proteins with sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively and a pharmaceutically acceptable carrier. According to the invention, Sf9 cells are used for respectively expressing trichomonas pigeonae adhesion protein 33 and adhesion protein 65, the immunogenicity of the obtained recombinant protein is similar to that of natural protein, the expression level is high, the immunogenicity is strong, a very small amount of recombinant protein can provide a better immune protection effect, and the recombinant protein has no pathogenicity to pigeons, and can be used for preparing the recombinant protein for preventing and treating trichomonas pigeonae. Meanwhile, large-scale serum-free suspension culture can be carried out through a bioreactor, so that the vaccine production cost is greatly reduced.

The invention provides a serum free light chain detection reagent and method, and the reagent comprises a first reagent which comprises a reaction buffer solution and an anti-human IgG-Fab polyclonal antibody; the second reagent comprises an anti-human FLC polyclonal antibody marked by nano microspheres; wherein the anti-human IgG-Fab polyclonal antibody is used for blocking a cross reaction between a binding light chain in immune globulin in a sample and the anti-human FLC polyclonal antibody. The anti-human IgG-Fab polyclonal antibody is added into the reaction buffer solution, so that the problem of cross reaction between the anti-FLC polyclonal antibody and a light chain combined on immune globulin can be effectively solved, and meanwhile, the long-term stability of the reagent is ensured.

The invention relates to the technical field of biomedicine, in particular to an umbilical cord mesenchymal stem cell exosome and a preparation method and application thereof.The umbilical cord mesenchymal stem cell exosome is derived from umbilical cord mesenchymal stem cells expressing S100g-MTOR binding domain fusion protein and can inhibit excessive autophagy through an S100g / MTOR signal channel, and therefore intestinal mucosal barrier damage caused by antibiotics is relieved. The preparation method comprises the following steps: constructing an expression vector, transfecting and screening a stable expression cell strain, performing serum-free culture, collecting a culture supernatant, purifying the exosome and the like. The exosome disclosed by the invention can be used for preparing a medicine for treating antibiotic-related intestinal mucosal barrier injury, has the advantages of strong targeting property, definite action mechanism, remarkable curative effect and the like, and provides a new choice for preventing and treating antibiotic-related intestinal adverse reactions.

The invention discloses a method for establishing a cell line by serum-free culture of PGCs (Primordial Germ Cells) of breeding poultry, the cell line is prepared by blending calcium-free DMEM (Dulbecco Modified Eagle Medium), water and Knockout-DMEM, the cell line is more suitable for osmotic pressure and calcium ion concentration required by proliferation of the PGCs of the breeding poultry, various components of serum-free culture are perfected by using fresh egg white, and the cell line is more suitable for proliferation of the PGCs of the breeding poultry. The combination of growth factors required by primordial germ cells is optimized by adding the FGF-1, so that 100% of cell line establishment efficiency and rapid growth of cells from reproduction crescent, blood and gonad tissues of chicken embryos are realized, and a technical basis is provided for poultry frozen breed conservation, stem cell propagation and gene editing operation.

The invention relates to a culture medium for high-density serum-free suspension culture of BHK-21 cells and application of the culture medium. The culture medium contains inorganic salt, amino acid, vitamins, energy substances, a buffer substance, an antioxidant, sterol, polyamine, an additive and an indicator, and the antioxidant comprises dihydrolipoic acid and S-acetyl-L-glutathione. According to the invention, the culture medium for serum-free suspension culture of the BHK-21 cells, which is clear in components and remarkable in culture effect, can be provided. The added dihydrolipoic acid and S-acetyl-L-glutathione are combined as antioxidants of the culture medium, so that the survival rate of BHK-21 cells cultured in the culture medium is improved while the oxidation resistance of the serum-free suspension culture medium is improved.

The present invention provides methods of producing in vitro derived neutrophils or macrophages in xenogen- and serum-free conditions from pluripotent stem cells and in vitro derived populations of neutrophils and macrophages. Methods of treatment using in vitro derived neutrophils or macrophages are also contemplated.

The invention discloses a cat kidney suspension culture cell line suitable for serum-free suspension culture and application of the cat kidney suspension culture cell line. The name of the cell line is F81-B4, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.46350. After 50 generations of continuous subculture, the cells still can maintain the original proliferation level and cellular morphology, the cell number can reach 9.0 * 10 < 6 > / mL after the cells are subcultured at the density of 1.0 * 10 < 6 > / mL for three days, the multiplication time is 22-26 hours, and the cell proliferation speed is equivalent to that in a shake flask when the cells are amplified to a 10L bioreactor for culture. The cell line is highly susceptible to feline parvovirus, feline calicivirus, feline herpes virus, canine parvovirus and canine distemper virus, the virus content of a virus solution harvested 72 h after FPV virus inoculation can reach 107.5 TCID50 / mL, the virus content of a virus solution harvested 24 h after FCV virus inoculation can reach 1010.8 TCID50 / mL, the virus content of a virus solution harvested 48 h after FHV-1 virus inoculation can reach 107.5 TCID50 / mL, compared with an existing F81 adherent cell production process, the production process has the advantages that the production efficiency is high, and the production cost is low. The method has the characteristics of rapid lesion, short virus collection time and high titer. The cell matrix is an ideal cell matrix which can be used for culturing the three viruses.

Described herein are serum-free and feeder-free methods and compositions for the tunable differentiation and production of CD4+ (single positive) T cells and CD8+ (single positive) T cells, by adjusting T cell receptor (TCR) stimulation and notch activation (or notch signaling activation, stimulation or inducement). In some other aspects the invention provides a method and in vitro niche and cell culture media that results in enhancing CD4+ (SP) T cell generation, including naive mature CD4+ single positive T cells from double positive (DP) CD4+8+cells or CD8+(SP) T cells by TCR stimulation while having low or no notch signaling activation, stimulation or inducement. In yet other aspects, the invention provides a method, niche and culture media for producing cells derived from CD4+ (SP) T cells, including Treg cells.

The invention provides a serum-free stem cell culture medium and a preparation method thereof, and the culture medium realizes long-term stable culture of stem cells under a completely serum-free condition by optimizing basic culture medium components and adding a specific growth factor combination, an extracellular matrix simulant and a metabolism regulator. The culture medium comprises a basic culture medium, a recombinant human protein substitute, a cell adhesion promoting factor, a growth factor combination, an antioxidant and a metabolism regulator. Compared with the prior art, the invention has the following advantages: 1) animal-derived components are completely avoided, and the immunogenicity and the difference between batches are reduced; 2) maintaining the dryness of the stem cells through a specific growth factor combination; 3) adding a metabolism regulator to optimize cell energy metabolism; 4) the cost is obviously lower than that of commercially available like products; and 5) supporting long-term culture of stem cells without spontaneous differentiation. The invention also provides a preparation method of the culture medium and a method for culturing stem cells by using the culture medium.

The invention relates to a preparation method of a Vero cell influenza virus vaccine, and belongs to the technical field of virus culture. In the culture of the influenza virus, red algae protein and soybean protein hydrolysate are added into a culture medium at the same time, the red algae protein and soybean protein hydrolysate are subjected to low-temperature ammonium sulfate precipitation and PD-10 desalination column purification, natural conformation is kept, integrin-FAK-Src and IGF-1R signal channels can be activated, and cell adhesion and proliferation are promoted; the latter contains soybean protein isolate, polysaccharide and RGD motif peptide, and can form an adhesion film on a substrate and mediate adhesion through integrin. According to the invention, the Vero cells and the recombinant plasmid are combined to improve cell adherence, metabolic homeostasis and oxidation resistance, so that the Vero cells grow at high density and efficiently replicate influenza viruses under a serum-free condition, the virus titer (TCID50) reaches 8.5 log10 / mL, and the Vero cells and the recombinant plasmid are suitable for industrial vaccine production.

The invention discloses an NKT cell in-vitro amplification method based on a combined stimulant, and belongs to the technical field of biology. The method comprises the following steps: firstly separating mononuclear cells in autologous peripheral blood, and then performing induced activation and amplification on the NKT cells in a serum-free culture system by adding a group A streptococcus preparation, a zoledronic acid preparation and a cytokine IL-2 as a combined stimulant. The method is easy to operate, efficient amplification of the NKT cells can be achieved only through three key reagents, the obtained NKT cells have the advantages of being large in number, high in purity, high in cytotoxicity and the like, and the requirements of clinical treatment can be met. Compared with a traditional method, the culture cost is remarkably reduced, the operation process is simplified, large-scale production, popularization and application are easy, and an efficient and economical technical scheme is provided for NKT cellular immunotherapy.

The invention relates to the technical field of stem cells, in particular to a serum-free induction medium with definite components for promoting and inducing mesenchymal stem cells to be differentiated into dermal papilla cells and an induction method. The small molecule compound composition is prepared from all-transretinoic acid, calomel, CHIR-99021 and minoxidil according to the molar ratio of (0.2 to 5): (2 to 50): (0.2 to 10): (0.2 to 10). According to the scheme, a plurality of small molecule compounds are adopted and have a synergistic effect, so that differentiation and function improvement of dermal papilla cells are promoted, and transformation of mesenchymal stem cells to the dermal papilla cells is promoted. The former three promote dermal papilla cell differentiation, and the latter promotes dermal papilla cell proliferation and inhibits apoptosis. According to the technical scheme, the technical problems that cell factors need to be added in the process of forming dermal papilla cells from stem cells through induced differentiation, so that the process cost is high, and the clinical safety is not ideal can be solved, the induction process is accelerated, and the method has ideal popularization and application prospects.

The main objective of this invention is to provide a novel manufacturing method capable of efficiently and directly converting or inducing somatic cells into insulin-producing cells. As an example, a method for manufacturing insulin-producing cells is provided, which involves directly differentiating and inducing insulin production from somatic cells. This method includes a step of culturing somatic cells in a serum-free differentiation-induction medium; or a step of culturing somatic cells in a differentiation-induction medium containing 5 μg / mL or more of insulin. According to this invention, insulin-producing cells with high insulin secretion capacity can be efficiently and directly manufactured from somatic cells. The insulin-producing cells obtained by this invention are useful in regenerative medicine and the like.

The invention relates to a mesenchymal stem cell serum-free culture medium with limited chemical components as well as application and a method thereof, the serum-free culture medium is completely serum-free and has definite components: all additives are known chemical substances and meet the production requirements of cGMP (Complementary Good Manufacturing Practice); telomere shortening can be obviously delayed; replicative senescence is slowed down by activating TERT expression; the multiplication capacity can also be enhanced; the P10 generation cells still have the active division capacity; multidirectional differentiation is supported; the osteogenic differentiation efficiency is superior to that of a traditional serum-containing system; and the method is suitable for industrial production and clinical transformation.

The invention relates to the technical field of cell culture, and provides an NK cell in-vitro amplification culture method which comprises the following steps: S1, separating to obtain a mononuclear cell of peripheral blood, resuspending the mononuclear cell by using a serum-free NK cell basic culture medium, and then inoculating the mononuclear cell to an NK cell activation culture medium for incubation; s2, culturing to the third day, supplementing the NK cell to activate the culture medium for amplification, then supplementing the NK cell to activate the culture medium once every other day, and culturing to the 19th day to obtain high-purity NK cells; the NK cell activation culture medium is a serum-free NK cell basal culture medium which contains IL-2 (Interleukin-2), IL-12 (Interleukin-12), IL-18 (Interleukin-18), IL-21 (Interleukin-21), a CD16 antibody, a CD56 antibody, 4-1BBL (4-1BBL), nicotinamide, sodium diethyldithiocarbamate, polyinosinic acid and plasma. The method is simple and convenient to operate and short in culture period, the amplification efficiency reaches up to 1050 times within 19 days, and the NK cells are high in purity and accord with clinical treatment standards.

The invention relates to a combined additive, a CHO cell culture medium, a preparation method and a culture method. The combined additive comprises cyclohexyl alanine and 3-(2-naphthyl)-L-alanine. The CHO cell culture medium comprises amino acids, vitamins, trace elements, inorganic salts, lipids, carbohydrates and a combined additive, the preparation method is used for preparing the CHO cell culture medium. The culture method uses a CHO cell culture medium to culture CHO cells. The combined additive is obtained through structural domain virtual docking and hydrophobicity regulation and control screening, G1 / S conversion can be mildly delayed, cell apoptosis can be inhibited, and an expression window can be prolonged. The culture medium contains amino acids, vitamins, trace elements and the like, and preferably selects a functional additive to replace serum and foreign protein functions. According to the application, high proliferation of CHO cells and efficient target protein expression can be realized under the conditions of no serum and no foreign protein, meanwhile, the accumulation of lactic acid and ammonia is reduced, the cell caking phenomenon is reduced, and the application has remarkable technical advantages.

The invention relates to the technical field of cell biology, and particularly discloses an isolated culture method of primary meibomian gland epithelial cells, the obtained primary meibomian gland epithelial cells and application of the primary meibomian gland epithelial cells. The method comprises the following steps: efficiently and mildly digesting meibomian gland tissues by adopting a compound digestive juice consisting of I-type collagenase, dispersing enzyme and trypsin in a specific ratio; stopping digestion by using a serum-containing neutralizing solution and collecting cells; the method comprises the following steps: promoting cell attachment by using a serum-containing initial culture medium, and carrying out selective amplification by using a serum-free proliferation culture medium to finally obtain high-purity and high-activity primary meibomian gland epithelial cells. The method solves the problems of long digestion time, low cell yield, poor purity and the like in the traditional method. The obtained cell can be used for constructing a meibomian gland dysfunction model, screening drugs and researching lipid metabolism, and an important technical platform and a cell model are provided for research and treatment of related eye diseases.

The present invention relates to the field of T cells and provides methods of producing autologous T cells and compositions thereof for the treatment of B-cell malignancies and other cancers. A method of making T cells expressing a cell surface receptor that recognizes a specific antigen moiety on the surface of a target cell, the method comprising enriching a population of lymphocytes; stimulating the population of lymphocytes with one or more T cell stimulators to produce a population of activated T cells, the stimulation being performed in a closed system using a serum-free culture medium; transduction of the activated population of T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor, producing a transduction population of T cells using single cycle transduction, the transduction being performed in a closed system using a serum-free culture medium; the transduced population of T cells is expanded for a predetermined time resulting in an engineered population of T cells, the expansion being performed in a closed system using a serum-free culture medium. The methods and processes described herein can be completed in significantly shorter time.

The invention discloses a dipeptide modified polymer and application thereof in intracellular delivery of protein. The screened polymer and protein form a compound and are subjected to phase transformation, solute in a liquid phase is transformed into solid-phase particles insoluble in water, early release of the target protein is reduced, and delivery of the protein in the presence of serum is achieved. The protein intracellular delivery method provided by the invention has relatively high intracellular delivery efficiency in the presence of serum-free protein and serum, and the delivery efficiency is higher than that of a commercial protein transfection reagent PULSin; after beta-Gal and other enzyme proteins are delivered into cells, the activity can be recovered, a catalytic substrate is converted into a monitorable product, and the amount of the delivered and active recovered protein is less affected by serum protein in incubation conditions; in addition, the material has low cytotoxicity, the cell activity is higher than 90% under the experimental condition of protein delivery, and the material has good biocompatibility.

The invention relates to the technical field of cell culture, in particular to an NK cell in-vitro amplification method and application thereof. The invention provides a method for amplifying NK cells, which comprises the following steps: mixing the NK cells with aAPC, activating the NK cells, and culturing the activated NK cells in a culture medium to realize multiplication culture of the NK cells, the aAPC expresses human-derived CD64 protein, and the activating treatment comprises incubation treatment with the NK cells and the aAPC by using an anti-CD56 antibody. The method is simple, efficient and low in risk, a serum-free and animal-source-component-free culture system is adopted in the whole process, the amplification multiple of the NK cells can reach hundreds of thousands of times or above after the NK cells are cultured for 28 days, the purity of the NK cells can reach 95% or above, the requirements of clinical immune cell therapy are met, and the method has high clinical transformation potential.

The present invention belongs to the field of biomedicine, and particularly relates to a fibroblast cell line of mallard duck embryos, a construction method thereof, and an application thereof. Through a large amount of creative work by the inventors, the present invention for the first time isolated and obtained a fibroblast cell line from mallard duck embryos, and successfully domesticated it in serum-free suspension. The name of the cell line is DEF-Sfs, fibroblast suspension cells of mallard duck embryos, which was deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 28, 2024, with the deposit number of CCTCC NO: C2024191. This cell line can be used for culturing, isolating, and detecting avian viruses, preparing avian vaccines, and screening drugs for preventing or treating diseases caused by avian virus infections.

The invention belongs to the technical field of biological medicine, and particularly relates to a frozen umbilical cord blood NK cell amplification method which comprises the following steps: S1, umbilical cord blood NK cell separation; s2, performing serum-free culture; s3, performing dynamic amplification; and S4, performing cryopreservation and resuscitation verification. According to the invention, after unfreezing, a culture medium containing a TGF-beta inhibitor (Galunissertib) is firstly used for pretreatment for 24 hours, so that an inhibitory signal is blocked, and the survival rate is increased. The dynamic amplification adopts three-stage metabolism regulation: stage 1: in a low-oxygen environment, using a culture medium containing SCF + FLT3L + IL-7 to promote activation of NK cells in a stationary period; stage 2: under a normal oxygen condition, switching to IL-12 + IL-18 + IL-21, and adding an antioxidant NAC to reduce ROS accumulation; and stage 3: pulse-type stimulation: adding CD16 cross-linked magnetic beads every 48 hours to enhance cytotoxicity.

The invention discloses a PDCoV-S1 recombinant protein antigen as well as a preparation method and a vaccine thereof, and belongs to the technical field of recombinant protein vaccines. The amino acid sequence of the PDCoV-S1 recombinant protein antigen disclosed by the invention is as shown in SEQ ID NO: 1; the nucleotide sequence of the PDCoV-S1 recombinant protein antigen is as shown in SEQ ID NO: 2. The vaccine provided by the invention only contains recombinant PDCoV-S1 protein and does not contain genetic materials of viruses, so that all risks of virus replication, virulence reversion or gene recombination are fundamentally avoided. Live viruses do not need to be operated in the production process, so that the biological safety risk and the requirement on the production environment are greatly reduced. The constructed stable CHO engineering cell strain is combined with a serum-free suspension culture technology, so that high-density, large-scale and standardized production of the PDCoV-S1 protein can be realized in a bioreactor. The production process is stable and reliable, the batch-to-batch consistency is good, and the uncertainty and volatility caused by the virus culture link of the traditional vaccine are overcome.

The invention discloses a method for obtaining Vgamma9Vdelta2T cells with strong killing ability through serum-free specific amplification, a serum-free culture system is used in the method, the scheme is simple, the operation is convenient and fast, the amplification efficiency and the killing effect on solid tumor cells can be greatly improved, and in addition, the method is suitable for large-scale production. The Vgamma9Vdelta2T cell prepared by adopting the method provided by the invention is high in positive rate and strong in killing ability for solid tumors, and has a good application prospect and important transformation significance in the technical field of tumor immunotherapy.

To provide a method for manufacturing a hepatoblast, a hepatoblast, and a method for manufacturing a hepatocyte.SOLUTION: A method for manufacturing a hepatoblast includes a step of culturing an iPS cell-derived entodermal cell serum-free medium containing FGF2, HGF, oncostatin M and dexamethasone and differentiating the cell into a hepatoblast. As one aspect, the medium further contains nicotinamide, or the serum-free medium is serum-free differentiation medium, or an iPS cell is derived from a human, or a culture period of the iPS cell-derived entodermal cell is 5 days or more and less than 10 days.SELECTED DRAWING: Figure 2-1