103 results about "Serum free" patented technology

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 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 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 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 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 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 application provides a kind of nasal mucosa in vitro culture method and culture fluid, the composition of the culture fluid includes Wnt / β-catenin signal pathway activator 0.1-20 μg / ml, fibroblast growth factor 10-150 ng / ml, TGF-β inhibitor 0.02-3 μg / ml, ROCK inhibitor 0.1-100 μmol / L, P38 MAPK inhibitor 0.1-1 μmol / L, serum-free additive 1X, N-acetyl cysteine 1-10 mmol / L, nicotinamide 1-20 mmol / L, GlutaMax additive 1X, antibiotic, DMEM / F12 culture medium 1X.The application can carry out in vitro culture to nasal mucosa, can keep at least 2 weeks of activity, and have complete epithelial barrier and immune system, can realize ex vivo nasal mucosa virus infection experiment.

This invention relates to the field of biomedical technology, specifically to a serum-free and protein-free culture medium and its preparation method for enhancing NK cell expansion and cytotoxic activity. The serum-free and protein-free culture medium comprises a basal medium, an AMPK-HIF1α regulator, immunomodulatory factors, mitochondrial function enhancers, antioxidants, trace elements, and a pH buffer. The AMPK-HIF1α regulator is composed of α-ketoglutarate at a final concentration of 1.4-1.6 mmol / L and AICAR at 0.4-0.6 mmol / L. This culture medium activates the AMPK-HIF1α pathway through precise formulation of α-ketoglutarate and AICAR, and optimizes the metabolic-antioxidant network in synergy with nicotinamide ribose and glutathione, thus solving the industry problem of the incompatibility between expansion and function in serum-free NK cell culture.

The invention discloses a method for large-scale cryopreservation of animal cells. The method comprises the following steps: carrying out first-stage amplification on to-be-preserved cells under the support of matrigel; performing second-stage amplification on the to-be-preserved cells subjected to the first-stage amplification under the support of a microcarrier; mixing the to-be-preserved cells with a serum-free cryopreservation solution to prepare a micro-capsule suspension; and putting the microcapsule suspension into a cryopreservation container to carry out cryopreservation. According to the invention, through amplification of two stages, the to-be-preserved cells have reached a high density of 109 orders of magnitude before cryopreservation, a re-amplification link in a cell recovery stage can be greatly shortened, and the to-be-preserved cells are subjected to microencapsulation through the micro-fluidic chip and then cryopreserved, so that physical protection is given to the to-be-preserved cells; the damage of ice crystals in the cryopreservation process is reduced, the survival rate after resuscitation is improved, and the microencapsulated cells can also be directly subcultured, so that the resuscitation operation is simplified; according to the cell cryopreservation device, the cryopreservation container with a large volume is used, so that the cost of cell cryopreservation is reduced.

The invention discloses a stem cell-based microvesicle as well as a preparation method and application thereof, belongs to the technical field of biological tissue engineering, and particularly relates to a preparation method of the stem cell-based microvesicle, which comprises the following steps: culturing stem cells until the cell fusion degree is 80-90%, performing hypoxia treatment, and cleaning for 2-3 times; standing and culturing the cleaned stem cells, then adding a calcium ion carrier, and carrying out ultrasonic treatment; the serum-free basic culture medium containing the calcium ion carrier is immediately absorbed and removed, the cells are cleaned through PBS, culture is conducted for 24 h, and cell supernatant is collected and merged respectively; performing first centrifugation to remove cell debris; performing second centrifugation to remove dead cells and large cell debris; carrying out third centrifugation to remove organelles; fourth centrifugation is carried out, large vesicles are removed, and a final supernatant is obtained; and filtering and centrifuging to obtain a microvesicle precipitate. The preparation method of the microvesicles aims at systematically improving the yield and biological activity of the stem cell microvesicles through low-oxygen pre-stimulation, ultrasonic-calcium ion carrier synergistic induction and differential centrifugation.

The application discloses a composition for inducing T cell reprogramming into NK-like cells and application thereof. The composition comprises a DNA methyltransferase inhibitor, a histone deacetylase inhibitor and a vitamin C phosphate derivative; the molar ratio of the DNA methyltransferase inhibitor and the vitamin C phosphate derivative is 1: (40-2500); and the final concentration of the histone deacetylase inhibitor is 0.05-2 muM. The vitamin C phosphate derivative is used to replace common vitamin C, and the defects of common vitamin C, such as weak reprogramming promotion effect in a platelet lysate system, limited cell proliferation and low receptor expression, are overcome, so that the composition is more suitable for a clinical serum-free large-scale preparation scene.

This invention relates to the fields of stem cell culture and serum-free culture media, specifically to a serum-free culture medium for the proliferation of bovine muscle stem cells and its applications. The serum-free culture medium is based on a serum-free basal culture medium, to which food-derived small molecule compounds are added. Through further combination optimization, four food-derived small molecule compounds with synergistic effects have been obtained. This serum-free culture medium can significantly promote the proliferation of bovine muscle stem cells, effectively maintain cell proliferation activity, and cells passaged using this serum-free culture medium can still maintain good differentiation potential. This provides a stable, sufficient, and safe cell source for the industrial production of cultured meat, which is beneficial for the large-scale production of bovine muscle stem cells.

The application discloses a kind of modular serum-free cell culture medium and its application, belong to biomedical technology field.The culture medium includes: epidermal growth factor, recombinant insulin, cholesterol-phospholipid nanoparticles, beta-mercaptoethanol, pluronic F-68, glucose / glutamine, trace element mixed solution.The modular serum-free cell culture medium provided by the application can not only meet the large-scale culture of stem cells, but also be used for the industrial production of in vitro diagnostic reagent raw materials such as recombinant antigen, viral vector, recombinant protein and monoclonal antibody, with wide cell adaptability, low production cost, significant economy, stable process, and strict requirements of FDA / EMA for biological products.

This invention discloses a strain of Rhodotorula glutinis I7Y2 that promotes cell proliferation in a serum-free environment and its applications. Rhodotorula glutinis ( Rhodotorula mucilaginosa The yeast strain I7Y2 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 13, 2026, with accession number GDMCC No: 67951. This invention has a good effect on promoting Vero cell proliferation, specifically manifested in: (1) the yeast involved in this invention can produce various active substances such as amino acids, polypeptides, sanshool, and inositol to meet the nutritional needs of cell growth; (2) it can effectively improve the viability and density of Vero cells in the basal culture medium; (3) it can effectively improve the proliferation rate and migration rate of Vero cells in the basal culture medium; and (4) it can effectively reduce the apoptosis rate of Vero cells and the expression of apoptosis-related genes in the basal culture medium. Therefore, Rhodotorula glutinis I7Y2 has great application potential in promoting cell proliferation, especially in serum-free culture products.

This invention relates to a method for producing extracellular matrix using connective tissue cell culture and to the uses of the produced extracellular matrix. More specifically, it relates to a method for culturing connective tissue cells using a serum-free and heterogeneous culture medium, which can be effectively used to increase the amount of extracellular matrix produced. Furthermore, the extracellular matrix produced by the production method of this invention is derived from human cells and does not contain any heterogeneous components such as fetal bovine serum, making it safe and useful for future development of cell therapy agents, medical supplies, medical devices for tissue treatment, or cosmetic raw materials.

The invention belongs to the technical field of cell therapy, and relates to a serum-free and heterologous component-free NK cell culture medium as well as a preparation method and application thereof. The culture medium comprises a classical culture medium, a proliferation promoting component, vitamins, trace elements, lipids, transferrin, an energy substance and beta-mercaptoethanol, the proliferation promoting component is prepared from human albumin, resveratrol, insulin and hydroxyproline. On the basis of a serum-free and heterologous component-free NK cell culture medium, IL-12, rapamycin, polyinosinic-polycytidylic acid and human AB plasma are additionally added to obtain an activated culture medium, the activated culture medium and an amplification culture medium are jointly used, the culture effect is better, and amplification components IL-2 and human AB plasma are additionally added to obtain an amplification culture medium. Through precise component design, the NK cell in-vitro proliferation efficiency, motility, purity and killing ability are remarkably improved, and key problems existing in a traditional culture medium are efficiently solved.

This invention provides a method for detecting AAV antibody titers, belonging to the field of biomedical technology, comprising: S1 preparing serum-free AAV vectors with different MOI values; preparing a mixture containing different volumes of test serum samples and AAV vectors with known MOI values ​​for co-incubation; S2: adding plasmids containing the CRISPR-Cas9 gene editing system to electroporation buffer, performing electroporation on a known number of cells, adding the serum-free AAV vectors and mixture from step S1 to the electroporated cells respectively, and detecting the expression efficiency of the reporter gene after electroporation; S3: calculating the AAV antibody titer in the test serum sample based on the detection results. The main advantage of this invention is that it can display AAV antibody titers digitally and quantitatively, facilitating understanding and communication, and obtaining stable and reliable results using different cell lines and different AAV transduction doses. This method is easy to standardize and validate, saving time, effort, and detection costs compared to traditional methods, and directly reflects antibody neutralizing activity.

The invention belongs to the technical field of tissue engineering and aging resistance, and particularly relates to a composition for delaying cell aging and maintaining tissue homeostasis as well as a preparation method and application of the composition. Specifically, research finds that adipose tissues, skin tissues and the like are placed in a serum-free basal culture medium, adherent culture is performed, the obtained supernate is collected, and the obtained serum-free conditioned culture medium preparation can effectively inhibit aging of cells (such as ADSCs or HDFs) and maintain the functional steady state of the cells in in-vitro culture. Meanwhile, the culture medium product provided by the invention is also proved to be capable of inhibiting an IL-6 / STAT3 signal channel, and the channel is revealed to be a core mechanism of the anti-aging effect of the culture medium product, so that mechanism support is provided for the application of the culture medium product to cell aging resistance, and the culture medium product has good practical application value.

Provided is a highly safe articular cartilage regeneration material. This articular cartilage regeneration material contains: a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured with a serum-free medium form a three-dimensional structure; and an extracellular matrix derived from mesenchymal stem cells. The articular cartilage regeneration material is implanted so as to be attached to a site of lost or damaged articular cartilage.

The invention belongs to the technical field of biology, and particularly relates to preparation of DMSO-free cryopreservation liquid and application of the DMSO-free cryopreservation liquid in cell cryopreservation. The invention provides a DMSO (dimethyl sulfoxide)-free serum-free cell cryopreservation solution. Each 100 mL of the cell cryopreservation solution contains 1-100 mg of thermo-sensitive nanogel, 0.1-10 mg of trehalose, 10-100 [mu] L of glycerol and a basic culture medium. The nanogel (preferably pNIPAM) forms a protective layer at low temperature to inhibit ice crystal damage, and is rapidly dissociated during rewarming; and trehalose-glycerol synergistically stabilizes cell membranes. The cryopreservation liquid does not need programmed cooling, does not need centrifugation after resuscitation, is suitable for sensitive cells such as CAR-T cells and stem cells, has the resuscitation survival rate of more than 90%, and completely avoids the toxicity of DMSO and the risk of serum pollution.

The invention relates to a culture medium for serum-free suspension culture of F81 cells and application of the culture medium, the culture medium comprises the following components in concentration: 4000-4500 mg / L of amino acid, 80-120 mg / L of vitamin, 5000-5100 mg / L of inorganic salt, 5000-5500 mg / L of glucose, 1000-1030 mg / L of adherence promoting substance, 20-40 mg / L of growth factor, 2000-2040 mg / L of buffering agent and 2-2.4 mg / L of antioxidant, the pH value of the culture medium is 7.0-7.2, and the pH value of the culture medium is 0.5-1.5. The antioxidant is prepared from partially hydroxylated glutathione and a carboxymethyl chitosan-gallic acid grafted copolymer. The partially hydroxylated glutathione and the carboxymethyl chitosan-gallic acid graft copolymer are compounded to serve as an antioxidant of the culture medium, so that the oxidation resistance of the culture medium is enhanced synergistically, and the cell survival rate is increased.

The invention belongs to the field of biological medicine, and discloses a mesenchymal stem cell-derived exosome and active factor secretion composition and a directional preparation method thereof. According to the method, umbilical cord tissues of healthy mammals are used as raw materials, primary stem cells are separated through enzymolysis, and amplification and passage are conducted to 3-5 generations through an alpha-MEM culture medium; a serum-free induction culture medium containing multiple functional components is adopted for directional induction, a conditioned culture medium is collected, gradient centrifugation and ultrafiltration concentration are performed, and the protein concentration is calibrated to be 2 mg / mL, so that a finished product is obtained. The product can synergistically realize moisturizing, anti-wrinkle and tightening effects. Specifically, the exosome and active factor secreted composition is a composite system which is obtained by separating and concentrating mesenchymal stem cells from a conditioned medium of the mesenchymal stem cells after the mesenchymal stem cells are directionally induced by the method and is mainly composed of the exosome, growth factors self-secreted by the stem cells, cell factors and macromolecular active protein.

The present application relates to a kind of preparation methods of Vero cell influenza virus vaccine, belong to the technical field of virus culture.In the culture of influenza virus, medium is added with red algae protein and soybean protein hydrolysate, the former is precipitated by low-temperature ammonium sulfate and purified by PD-10 desalting column, maintains native conformation, can activate integrin-FAK-Src and IGF-1R signal pathway, promotes cell adhesion and proliferation;The latter contains soybean protein isolate, polysaccharide and RGD motif peptide, can form adhesive film on the substrate and adhere through integrin mediation.Both of them improve cell adhesion, metabolic homeostasis and antioxidant capacity, so that Vero cell can grow in high density under serum-free condition and efficiently replicate influenza virus, virus titer (TCID 50 ) reaches 8.5 log 10 / mL, suitable for industrialized vaccine production.

The invention provides a low-autophagy Vero cell line based on a DDIT3 inhibition gene as well as a construction method and application of the low-autophagy Vero cell line. The cell line stably knocks down DDIT3 expression through lentivirus-mediated shRNA, the autophagy level under the condition of nutrition deficiency is remarkably reduced, the cell proliferation capacity is enhanced, and the yield of influenza A H1N1 viruses is remarkably increased. The construction method comprises the following steps: cloning shRNA (short hairpin ribonucleic acid) to a GV493 lentiviral vector, co-transfecting 293T cell packaging virus with psPAX2 / pMD2. G helper plasmid, infecting Vero cells, and screening through puromycin to obtain a stable cell line. The cell line solves the problem of growth stagnation caused by over-activation of autophagy in serum-free suspension culture of traditional Vero cells, and is suitable for large-scale vaccine production.

The invention belongs to the technical field of cytobiology and immune cell therapy, and particularly relates to a gamma delta T cell serum-free additive culture medium composition with definite components and application and a product thereof. The serum-additive-free culture medium composition comprises a serum-additive-free culture medium A, a serum-free culture addition reagent B, a serum-additive-free culture medium C, a serum-additive-free culture medium D and a serum-additive-free culture medium E, the main components comprise ethanolamine, sodium selenite, recombinant human albumin, IL-15 and IL-2. According to the gamma delta T cell serum-free additive culture medium composition provided by the invention, the components are clear, the serum-free culture additives without animal-derived components can avoid potential safety hazards caused by the animal-derived components, the amplification efficiency is improved, the cell high activity is maintained, the GMP standard is met, and the gamma delta T cell serum-free additive culture medium composition is suitable for large-scale production and clinical transformation.