82results about How to "Maintain biological properties" patented technology

The invention relates to the technical field of cell preservation, in particular to a storage method of immune cells and a cell freezing medium. The method includes: separating and collecting peripheral blood mononuclear cells, and adding the cell freezing medium to store the cells in a frozen manner, wherein the cell freezing medium comprises, by weight percentage, 0-62% of serum-free liquid culture medium, 0.5-1.5% of penicillin-streptomycin solution, 5-10% of dimethyl sulfoxide and 30-92% of autologous plasma. The storage method of the immune cells has the advantages that the method is simple and effective, the infectious disease risks of exogenous serum are avoided, the survival rate of the revived freezing-stored cells after induction culture is above 90%, the various biological indexes of the cells satisfy the requirements, and a good storage effect is achieved; the method can monitor and trace cell bank samples in real time, accurately record information such as cell storage positions, temperature and cell sources, each cell has a unique identifier, and the method is convenient to use and safe and reliable.

The invention relates to the technical field of cell culture, and particularly relates to a culture method of mesenchymal stem cells. In the culture method, a serum-free culture medium optimized by the invention is used for culturing the mesenchymal stem cells. In addition, the invention further provides a better culture method for 3D culture. The cells obtained by the culture method disclosed bythe invention are strong in proliferation capacity, intact in shape and strong in secretion capacity, and are in accord with the international quality control standards of the mesenchymal stem cells.Meanwhile, according to the culture method disclosed by the invention, a large number of mesenchymal stem cells (particularly human umbilical cord mesenchymal stem cells) can be obtained by amplifyinga small number of mesenchymal stem cells under the conditions of small space occupation, less culture medium consumption, simpler and more convenient operation and greatly reduced workload; and a solid technical scheme and a theoretical basis are provided for obtaining a large number of high-quality mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) and secretions thereof in the clinical field.

The present invention describes a bioconjugation strategy and compounds that are useful therein in which a fluorescent signal is produced when two molecular or supramolecular entities are linked by chemoselective combination of one linker having an azido or halide substituent group with another linker having a cyano or an alkyne substituent group. A kit is also provided.

The invention relates to a serum-free cryopreservation medium and method for peripheral blood monouclear cells. The serum-free cryopreservation medium comprises recombinant human interleukin-2, humanserum albumin, polyethylene glycol, trehalose and a basic culture medium or sodium chloride for injection. The serum-free cryopreservation medium has the advantages that the recombinant human interleukin-2 (IL-2) is added into the cryopreservation medium, so that the activity stability of immune cells cultured by thawed cells can be increased greatly, the high activity of original cells can be kept, the immune cells can well keep the physiological function and biological feature of the thawed cells, and the problem of direct large-scale amplification of the thawed cells can be solved effectively; the polyethylene glycol and the trehalose are added into the cryopreservation medium, the cryopreservation medium can replace an existing dimethyl sulfoxide-based (DMSO-based) cryopreservation medium, the toxicity of the cryopreservation medium to the cells is lowered effectively, cell stability is maintained, the direct application safety of the thawed cells is increased, and the thawed cellscan be directly used.

The invention discloses an immune cell cryopreservation liquid, and a preparation method, a cryopreservation method and an application thereof; the immune cell cryopreservation liquid includes the components by weight: 5%-15% of DMSO, 1%-5% of propylene glycol, 2%-8% of trehalose, 3%-8% of methyl cellulose, 0.3%-1.2% of glutathione, 0.06%-0.14% of sodium pyruvate, and 60%-80% of human donor plasma; the cryopreservation effect is quite good, the immune cells can be effectively protected from freezing injury, physiological functions and biological characteristics of the immune cells after resuscitation are maintained, cell biological characteristics are not changed, and the immune cell cryopreservation liquid can be used in clinical treatment; the immune cell cryopreservation liquid is suitable for long-term frozen preservation of human mononuclear cells and other kinds of immune cells.

The invention relates to an anti-tumor drug screening kit based on a three-dimensional micro-tissue level and an application method thereof. The anti-tumor drug screening kit comprises a three-dimensional cell culture material, a tissue cell dispersion reagent, a proteinase inhibitor, a basic cell culture medium, blood serum, antibiotics, a balance salt solution, a cell activity detection reagentand a cell filter screen. The application method comprises the following steps: dispersing tumor tissues of a patient to obtain tissue blocks, tissue fragments, cell aggregates and/or dispersed singlecells; then culturing the dispersed tumor tissues of the patient and a cell sample through a three-dimensional culture method and enabling the cells to grow in a three-dimensional space to form a spherical structure or an irregular three-dimensional structure with a stereoscopic shape; finally, carrying out a drug sensitivity test on tumor micro-tissues obtained by three-dimensional culture. Theanti-tumor drug screening kit based on the three-dimensional micro-tissue level is simple to operate and is suitable for the drug sensitivity tests of various solid tumors.

The invention relates to a multi-enzyme system used for separating patient tumor tissue-originated primary cells. The system comprises components of, by weight: 0.02 to 0.2% of collagenase I-IV, 0.001 to 0.01% of hyaluronidase, 0.01 to 0.1% of trypsin, 0.02 to 0.2% of proteinase neutral, 0.001 to 0.01% of deoxyribonuclease I, and 5 to 50U/ml of elastase. The invention also relates to an application method of the system, an application of the system, and a kit formed by the system. The multi-enzyme system is advantaged in reasonable design. With the system, patient tumor tissue-originated primary cells can be separated with high specificity and high sensitivity. The separated primary cells are characterized by high survival rate, high purity, easy cryopreservation and easy resuscitation. The system and the kit can be used in medicine experimental evaluation, medicine toxicity testing, and medicine therapeutic performance determination. The system, the method and the kit are suitable to be widely popularized.

The invention relates to a mesenchymal stem cells serum-free culture medium. The serum-free culture medium comprises a basal culture medium and an additive component, wherein the additive component comprises the following components: rhEGF, rhVEGF, rhPDGF-BB, rhFGF-b, rhTGF-beta1, human serum albumin, recombinant deferritin, a recombinant human insulin growth factor and hydrocortisone. The invention also provides an application of the mesenchymal stem cell serum-free medium. The composition is clear and stable; the instability of serum culture is eliminated, and meanwhile, all factors have thesynergistic effect, the umbilical cord mesenchymal stem cells can be efficiently amplified, the cell cycle is shortened, the proliferation is rapid, the yield of three generations of cultured cells reaches 1.5 times or more of that of serum culture, the cell consistency is good, and the biological characteristics and differentiation potential of the umbilical cord mesenchymal stem cells can be efficiently maintained due to the synergistic effect of all factors.

The invention belongs to the field of tissue engineering, and particularly relates to a carrier-free cell sheet and a preparation method and application thereof. The preparation method of the carrier-free cell sheet comprises the steps of transfecting the suicide gene into a supporting cell; planting the supporting cell with the transfected suicide gene at the bottom of a culture container; planting target cells on the supporting cell; after realizing good fusion between the target cells and establishing intercellular connection, starting the suicide gene in the supporting cell for apoptosis to obtain a carrier-free cell sheet containing pure target cells. By adopting the method provided by the invention, a carrier-free cell sheet with good biological characteristics can be obtained, which is a new breakthrough in establishing a carrier-free cell sheet in the field of tissue engineering; meanwhile, the product also provides a feasible scheme for clinical disease treatment. The principle is scientific and reliable, and the process is simple and flexible.

Compositions of exosomes are provided that include a plurality of exosomes and a biocompatible cryoprotectant, such that the exosomes are suspended in the biocompatible cryoprotectant as a colloidal suspension of exosomes. Preferably, the cryoprotectant is a carboxylated E-poly-1-lysine (COOH-PLL) cryoprotectant, but other moieties might be extended to the claim of hybridization polymers to incorporate preferred embodiments for lineage and tissue specific intentions. The colloidal suspension of exosomes can be frozen at −65 degrees C. or colder and thereafter stored as a frozen composition of exosomes or can be freeze-dried and thereafter stored at ambient conditions in a vacuum sealed container. Also provided are kits comprising the composition of exosomes and methods of making the compositions of exosomes.

The invention relates to a pelodiscus sinensis heart cell continuous cell line which is preserved in the common microorganism center of China Committee for Culture Collection of Microorganisms and has a preservation number of CGMCC No.10597, and further relates to an establishing method and ultra-low-temperature cryopreservation method of the pelodiscus sinensis heart cell continuous cell line. The establishing method includes the following steps of firstly, conducting primary culture, wherein a pelodiscus sinensis heart is cut into tissue small pieces after being preprocessed, trypsin is inoculated into a culture bottle after being digested at the room temperature, and subculture is not started until cells are fully laid on 90% or more of the bottle bottom; secondly, conducting subculture, wherein air is blown on the bottle bottom through a trypsin digesting method to make adherent cells fall down so that subculture can be conducted. By means of the established pelodiscus sinensis heart cell continuous cell line, the research on viral diseases of pelodiscus sinensis and other turtle animals and the research on related therapy drugs for instance the analysis of chromosome and the research of iridescent virus can be conducted on the level of molecular cells, and the requirements for pelodiscus sinensis germplasm resource conservation and theoretical research and application can be met.

The invention relates to a culture and cryopreservation method of amniotic mesenchymal stem cells. The culture method comprises amniotic tissue separation, amniotic mesenchymal stem cell separation, P0 generation amniotic mesenchymal stem cell culture and amplification culture. According to the culture method of the amniotic mesenchymal stem cells, at a separating stage of the amniotic mesenchymalstem cells, a special mixed enzyme digestive juice system (the final concentrations of components in the digestive juice are as follows: 1.5-2U/mL of neutral protease, 0.5mg/mL of deoxyribonuclease Iand 1mg/mL of collagenase IV) is adopted for digestion, so that the amniotic mesenchymal stem cells can be more effectively separated from amniotic tissues, and then the yield of P0 generation cellsis obviously improved. Compared with conventional two-step digestion, the culture method provided by the invention has the advantages that a one-step digestion method is adopted, operation is more convenient, and the cultured amniotic mesenchymal stem cells have good activity, high yield, high purity and good repeatability.

The invention belongs to the technical field of biology, and particularly discloses a mesenchymal stem cell culture medium. The mesenchymal stem cell culture medium comprises a basal culture medium and additives, wherein the additives comprise non-essential amino acids, a pH regulator, antibiotics, organic substance components for promoting cell membrane synthesis, cell matrix components, cell growth factors, vitamins, trace elements and an antioxidant. The mesenchymal stem cell culture medium can provide nutrient substances required by cell growth for stem cell growth, maintain the cell growth environment and the cell activity, promote stem cell division and proliferation, increase the stem cell proliferation speed and shorten the stem cell culture time; meanwhile, stem cell differentiation can be inhibited, but the differentiation potential of stem cells is not influenced; after the culture medium is used for carrying out passage on the stem cells for multiple times, the stem cells can still keep the original biological characteristics and multidirectional differentiation potential.

The invention discloses a mouse fat stem cell culture solution. The culture solution consists of the following components: 20 percent of fetal calf serum, 100 IU/ml of penicillin, 100 mug/ml of streptomycin, 2 mmol/L of L-glutamine, 10 ng/ml of basic fibroblast growth factor, 50 mug/ml of vitamin C, and the balance of Dulbecco's modified eagle medium F12 (DMEM/F12). The culture solution has simple component and low cost, can effectively maintain in vitro long-term propagation of mouse fat stem cells, and can well maintain the biological properties of the fat stem cells.

The invention relates to a method for proliferating avian influenza virus by adopting an MDCK cell line. The method comprises the following steps: domesticating adherent MDCK cells into a serum-free full-suspension culture type MDCK cell line which is stably proliferated, inoculating a bioreactor with the MDCK cell line, completing primary cell culture, preparing avian influenza H5N1 subtype virusseed viruses for suspension MDCK cell culture, inoculating suspension MDCK cells with the avian influenza H5N1 subtype virus seed viruses, performing culturing, and separating and purifying virus particles. The cells obtained by domestication are stable in passage, good in cell morphology, high in quality, capable of keeping sensitivity and biological characteristics to viruses, high in virus multiplication capacity and capable of effectively multiplying avian influenza virus strains.

The invention discloses a culture medium for obtaining mesenchymal stem cells and exosomes thereof and a preparation method of the culture medium. DMEM/F12 is used as a basic culture medium, and the following components are added: 10-22 g/mL of a serum substitute, 20-55 ng/mL of a growth promoting factor, 5-10 ng/mL of a muscle cell repair factor HCGF, 5-16 mM of an anti-aging factor, a buffer agent, 0.5-3 mg/L of an antioxidant, 1-20 mg/L of vitamins, antibiotics, 5-65 mg/L of trace elements and 1-16 [mu] g/mL of a protective agent. The mesenchymal stem cell culture medium is definite in component, free of serum components, controllable in quality and capable of being produced on a large scale, stem cells can normally grow when the mesenchymal stem cell culture medium is used for in-vitro culture of the stem cells, moreover, the mesenchymal stem cell culture medium is beneficial to division and proliferation of the stem cells, the stem cell amplification rate is greatly increased, and the mesenchymal stem cell culture medium is suitable for large-scale production. Compared with the prior art, the stem cell culture medium has the advantages that stem cell culture time is shortened, and stem cells still can keep original biological characteristics and multidirectional differentiation potential after being subjected to multiple passage times by the aid of the stem cell culture medium.

The invention discloses a rapid propagation method for stem segments of Dendrobium huoshanense, belonging to the technical field of plant cultivation. The method comprises the following steps: explant preparation, induced culture, enrichment culture, and strong seedling rooting culture. According to the invention, materials are easily available and are not limited by seasons; a stem segment culture manner of bud generation from buds has simple generating process and can well maintain biological characters of female parentd; thus, medicinal values of Dendrobium huoshanense are guaranteed. The propagation method is simple and practicable and solves the technical problems of insufficient explant sterilization in asexual propagation of Dendrobium huoshanense, slow protocorm differentiation, long propagation cycle, high production cost, etc. The rapid propagation method provided by the invention can be applied in actual production, is capable of providing guarantee for planting development of Dendrobium huoshanense, and has good economic benefits and social benefits.

The invention relates to the field of cell culture, in particular to a domestication method of a full suspension culture-type MDCK cell line. According to the domestication method, adherent MDCK cellsare domesticated into a low-serum full suspension culture-type MDCK cell line and a serum-free full suspension culture-type MDCK cell line, so that traditional production of an avian influenza virusthrough chicken embryos can be replaced, the subsequent separation and purification efficiency is improved, further upgrade of a process is promoted, a culture process is also simplified while the cost is reduced, and the domestication method is easier to industrialize.