286 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 discloses a serum-free culture system and culture method of NK cells. The invention firstly discloses a serum-free culture system of the NK cells, wherein the serum-free culture system comprises a coating solution, an amplification culture medium, an activation culture medium and autologous plasma; the invention further discloses a culture method for NK cells by using the serum-free culture system. The method for culturing the NK cells by using the serum-free culture system disclosed by the invention reduces the culture cost of the NK cells, can simply, quickly, efficiently and safely obtain a large number of NK cells with high activity, high purity and strong killing ability in vitro, provides a better cell source for clinical application of the NK cells, and promotes further development of an NK cell treatment technology.

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 relates to a method for efficiently inducing human hematopoietic stem progenitor cell erythroid to be directionally differentiated into mature erythrocytes in a serum-free and feeder-layer-free manner, which adopts a serum-free and feeder-layer-cell-free induction culture system, and uses an mTOR pathway inhibitor rapamycin to regulate the activity of an mTOR pathway in the erythroid differentiation process. The use dosage and the treatment mode of the mTOR pathway inhibitor rapamycin are further optimized, and the erythrocyte denucleation efficiency of an in-vitro induced differentiation system is remarkably improved to 70% under the condition that erythrocyte proliferation and differentiation are not affected or the cell state is not reduced. According to the method, heterologous pollution caused by animal serum or heterologous cells is effectively avoided, the clinical safety of in-vitro preparation of transfusion-level red blood cells in the future is ensured, the erythroid directional induced differentiation final maturation efficiency of the human hematopoietic stem progenitor cells is improved, meanwhile, the reagent cost is reduced, and large-scale preparation and clinical transformation are facilitated.

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 relates to the technical field of biology, and aims to provide a zinc ion-containing influenza A virus culture medium and a preparation method thereof. The culture medium contains a serum-free cell culture medium, water, a PBS (Phosphate Buffer Solution), bovine serum albumin with the final concentration of 20g / L, TPCK-Trypsin with the final concentration of 2mu g / mL and zinc ions with the final concentration of 0.009-0.015 mmol / L. According to the invention, zinc ions which are originally considered to generate a virus inhibition effect under a relatively low concentration condition can be used for promoting virus replication after the use concentration range is adjusted; therefore, a brand-new culture medium which can be applied to research on common cell models and virus in-vitro culture models of the influenza A virus is provided, and a new solution is provided for optimizing a vaccine production process. The culture medium is simple in formula and convenient to prepare; all the raw material components are common consumables in laboratories, and the required liquid components can be prefabricated in advance and refrigerated for later use; and large-scale vaccine production can be supported.

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.

The invention relates to the technical field of culture media, in particular to a serum-free complete culture medium for human umbilical cord mesenchymal stem cells and a preparation method thereof.The serum-free complete culture medium takes DMEM / F12 as a basic culture medium and is matched with UltraGRO-Advanced, a penicillin-streptomycin double-antibody solution, a recombinant human epidermal growth factor and a basic fibroblast growth factor, and the serum-free complete culture medium for the human umbilical cord mesenchymal stem cells is obtained. The serum-free complete culture medium can replace a serum culture medium in a conventional culture system of mesenchymal stem cells, the characteristics of normal adherence, good growth state and stem cells of the cells are kept, the serum-free complete culture medium reduces the risks of heterologous contamination and immunological rejection to the maximum extent, the added components capable of promoting cell growth and the content can be determined and controlled, and the culture medium is suitable for large-scale popularization and application. The use is safer.

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 discloses a preparation method and application of mesenchymal stem cells based on neural crest organics.The preparation method comprises the following steps that 1, human induced pluripotent stem cells are added into digestive juice, and after digestion, a culture medium is added for dilution; 2, collecting cells, centrifugally separating the cells, removing supernatant, suspending the cells in a culture medium, and adjusting the size of a cell block to 50-80 microns; 3, culturing the cells, and then carrying out induced differentiation on neural crest organs; 4, digesting the neural crest organs into single cells, transferring the single cells into a serum-free mesenchymal stem cell culture medium for adherent culture, and then performing amplification to obtain the mesenchymal stem cells. According to the invention, the induced pluripotent stem cells ihPSCs3D are induced and differentiated to obtain neural crest organs, the neural crest-derived mesenchymal stem cells are obtained, and the obtained NCC-MSCs have improvement and repair effects on pancreatitis.

The invention discloses a construction method of a tumor-associated meningeal metastasis model, and particularly relates to the technical field of biology. The method comprises the following steps: treating tumor cells according to a cell passage mode and centrifugal parameters, adding a serum-free culture medium, centrifuging again, adjusting the cell density by using a serum-free fresh culture medium, and sub-packaging to obtain a tumor cell suspension; the tumor cell suspension is injected into the L4-L5 intervertebral space of the mouse. After injection, the mouse resuscitates and normally diets; the growth of the tumor is monitored through a small animal living body imaging technology, and the dissected organ is subjected to in-vitro imaging to judge the invasion range of the tumor. The mouse tumor meningeal metastasis model is constructed in a simple, repeatable and efficient manner.

The invention belongs to the technical field of cell culture, and particularly relates to a serum-free mesenchymal stem cell culture medium and a preparation method thereof. The serum-free mesenchymal stem cell culture medium provided by the invention is prepared by taking a DMEM (Dulbecco Modified Eagle Medium) / F12 basal culture medium as a basis and adding supplementary factors, namely yeast hydrolysate, insulin, albumin, dexamethasone, taurine, salidroside, soy isoflavone, baicalein, human platelet lysate, momordica saponin and hyperoside. According to the serum-free mesenchymal stem cell culture medium provided by the invention, the normal form of cells can be maintained without supplementing serum, rapid proliferation is realized, and the biological characteristics of the mesenchymal stem cells are maintained.

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 present invention discloses the use of umbilical cord extract in improving the effect of anti-hair loss, increasing hair density and hair diameter of Platycladus orientalis leaves. The preparation of the umbilical cord extract is as follows: the animal umbilical cord is washed and cut into pieces, digested with trypsin, added with serum-free mesenchymal stem cell culture medium, blown and dispersed tissue, filtered, the filtered tissue fluid is incubated for 10~12 h after low-energy red light irradiation, an equal volume of serum-free mesenchymal stem cell culture medium is added, and the incubation is continued for 48~72 h, an equal volume of serum-free mesenchymal stem cell culture medium is added, and mir‑135b is added at the same time, and the tissue fluid is processed for 48~72 h, and the tissue fluid is taken out, and freeze-thawed, ultrasonically treated, filtered, and the filtrate is diluted with sterile water to obtain. The umbilical cord extract of the present invention can significantly improve the effect of anti-hair loss, increasing hair density and hair diameter of Platycladus orientalis leaves, and it is mixed with Platycladus orientalis leaves extract, and can be used to prepare cosmetics and medicines with the effect of anti-hair loss, increasing hair density and hair diameter.

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.