54 results about "Spermatogonium" patented technology

The invention discloses a method for in-vitro separation and culture of goat male germ stem cells. Spermatogonia are obtained from the testicular convolutedtubules of a male goat. A method for separating and purifying the goat male germ stem cells adopts 0.2% of gelatin and a Matrigel differential adherence combined cloning method to differentiate and separate un-adhered goat male germ stem cellsfrom other cells for adherence culture; and the culture of the separated goat male germ stem cells is MEF feeder-layer culture of feeder-layer-free culture. The separated goat male germ stem cells have ES cell characteristics and the potential of differentiation into nerve cells, cardiac myocytes and sperm sample cells.

The present invention provides a composition and method for making a modified germ cell (MGC) comprising a primordial sex cell, (PSC) or nucleus thereof, combined with an enucleated ovum. The PSC is a spermatogonia or an oogonia from a donor animal or mammal. Similarly, the enucleated ovum is from the same species donor animal or mammal as the donor of the PSC. The present invention also provides a method of maturing the MGC in vitro using a specialized cell culture apparatus or bioreactor chamber. The present invention also provides for a method of screening the MGCs for receptor sites, to determine if they are sufficiently matured to be translocated into a host animal or mammal in vivo and/or tissue in vitro. The present invention also provides for a specialized cell culture chamber or bioreactor chamber to grow, expand, maintain, sustain and mature the MGCs, or other types of cells.

The invention discloses to the method used to culture bird stem spermatogonium in vitro. It includes the following steps: inoculating the bird stem spermatogonium to CEF feeder layer; adding 10% calf blood serum, 2% chicken blood serum, 2mmol/L L-glutamine, 1mmol/L sodium pyruvate, 5.5*10-15ng/ml beta-mercaptoethanol, 10 mul/ml non-essential amino acid, 15-20ng/ml hSCF, 10U/ml mLIF, 10-15ng/ml bFGF, 0.04ng/ml hIL-11, 10ng/ml IGF, 100U/ml gentamicin sulphate culture solution into DMEM high glucose culture medium. This culturing method can process vitro operation for chicken stem spermatogonium, and lay study foundation for chicken embryo stem spermatogonium further series building.

The invention provides skin spermatogonium protection liquid and a preparation method thereof. The protection liquid is mainly formed by dissolving aminoglycoside antibiotics in DMEM (Dulbecco's modified eagle medium) culture medium water solution, 80-120U of the aminoglycoside antibiotics are dissolved in every mL of the DMEM culture medium water solution, and the concentration of the DMEM culture medium water solution is 13-25mg/mL. The skin spermatogonial stem cell protection liquid sufficiently simulates a human internal environment and is more suitable for survival of skin stem cells and spermatogonial stem cells and stable in property, the survival rate of the skin stem cells and the spermatogonial stem cells can be remarkably improved, and cultivation of the skin stem cells and the spermatogonial stem cells can be further enlarged while the skin stem cells and the spermatogonial stem cells are preserved.

The invention provides a testicle cryopreservation agent for marine fishes and a preparation method of spermatogonium. The preparation method of spermatogonium comprises the following main steps: preparing a cryopreservation agent formula suitable for sciaenops ocellatus; establishing a testicle cryopreservation and unfreezing method; preparing the transplantable spermatogonium. The invention establishes a cryopreservation method for the spermatogonium of the sciaenops ocellatus and technical guarantees are provided for a reproductive cell transplanting technology. According to the testicle cryopreservation agent for the marine fishes and the preparation method of the spermatogonium, a donor source for transplanting reproductive cells is expanded; a testicle is cryopreserved for a long period and can be used according to requirements; guarantees are provided for the donor source for transplanting the reproductive cells and an application range of transplanting of the reproductive cells of fishes is greatly widened.

The invention discloses a preparation of pairing monoclonal antibody aiming to dairy cow Y sperm specificity and application thereof. Dairy cow spermatogonium is adopted to directly mithridatize a Balb/c mouse; after dairy cow spermatogonium is blended with Balb/c mice, tumor strain is built to screen; tumor cells are injected into the abdominal cavity of a mouse to produce hydroperitoneum; the hydroperitoneum of the mouse is extracted to be purified and screened to obtain pairing monoclonal antibody aiming to dairy cow Y sperm specificity; the pairing monoclonal antibody determines clusters respectively according to different antigens of H-Y antigen. The obtained pairing monoclonal antibody has high titer and strong specificity; testing proves that the pairing monoclonal antibody has no binding force to X sperm; based on principles of immunology, H-Y antibodies can be combined with Y sperm specificity to form an antigen-antibody compound; X sperm and Y sperm of the dairy cow can be separated by direct input method, IMB technology, centrifugal process, immune affinity and column chromatography so as to control the sex of dairy cows. The invention has the advantages of high efficiency, safety and low cost, is suitable for breeding stirps female on large scale and has higher economic benefits.

The invention discloses novel application of 12-HEPE or pharmaceutically acceptable fatty acid thereof, and comprises the application of the 12-HEPE or the pharmaceutically acceptable fatty acid thereof in preparation of a medicine for treating NOA. The 12-HEPE or the pharmaceutically acceptable fatty acid thereof can play a therapeutic role by promoting proliferation and differentiation of the endogenous spermatogonium of a mouse damaged by busulfan, and experiments prove that recovery of the thickness and the integrity of the spermatogenic epithelium of the testis and the recovery of the sperm concentration of the tail of epididymiscan be observed through intragastric administration of the 12-HEPE into the NOA mouse induced by the busulfan, meanwhile, proliferation and differentiation of spermatogonium are promoted, expression of the paracrine factors of cells is supported, so that the 12-HEPE can be used as a new target for alleviating the spermatogenesis dysfunction of the testis of the NOA mouse, and a new method is provided for treating the non-obstructive azoospermia.

The invention discloses the method used to directionally induce chicken embryo stem spermatogonium to differentiate into bone cell. Its features are that inoculating the chicken stem spermatogonium to culture bottle without feeder layer; using conditioned medium to induce culturing which is that the DMEM high glucose contains 10% calf blood serum, 2% chicken blood serum, 2mmol/L L-glutamine, 1mmol/L sodium pyruvate, 5.5*10-15ng/ml beta-mercaptoethanol, 10 mul/ml non-essential amino acid, 10-7mol/L dexamethasone, 0.01mol/L beta-phospho glycerol, 0.05g/L vitamin C. The invention has the advantages of advanced technology, simple operation, strong repeatability, providing possible for realizing tissue organ rebirthing and replacing. Thereby hopefully to find out the approach for build cell platform in large scale production, further a new study prospect can be exploited in developmental biology, medicine, genetics fields etc.

The invention provides a dissociation and separation method for shellfish spermary spermatogenic cells, and belongs to the technical field of the separation of ocean shellfish cells. The dissociationand separation method comprises the following steps of: the spermary of a shellfish during a proliferating phase is broken and is digested by a collagenase IV solution of which the mass concentrationis 0.1% and a trypsin solution of which the mass concentration is 0.25% in sequence, and spermary dissociation is carried out to obtain a single-cell suspension; and Percoll solutions of which the volume concentration is independently 10%, 22% and 35% is prepared, and the Percoll solutions are loaded in the same centrifuge tube in a descending order, the single-cell suspension of the shellfish spermatogenic cells is added into the top layer of the centrifuge tube, and centrifugation is carried out to independently collect three layers of cells. By use of a two-step enzymolysis method, spermatogenic cells in spermary tissues can be successfully dissociated into the single-cell suspension, a cell survival rate can be as high as 96.62%, and the cells can be normally subjected to in vitro normal culture. Meanwhile, high-purity spermatogonia and spermatocytes can be separated, an important material is provided for researching a shellfish gamete generating and breeding mechanism, and the dissociation and separation method has a high guidance meaning and a good application value.

Provided are methods of in vitro maturation of human spermatogonium, comprising culturing the spermatogonium in a three-dimensional methylcellose culture system (MCS) under conditions capable of differentiating said human spermatogonium into an elongated spermatid, thereby in vitro maturing the human spermatogonium. Also provided is an in vitro matured sperm obtainable according to the method of the invention and methods of treating subjects in need of mature sperm cells.

The invention relates to a spermatogonial cell culture medium and a culture method of hexagrammos otakii, and belongs to the field of cell biology, the spermatogonial cell culture medium comprises the following components: a mixed culture medium, MEM non-essential amino acid with the volume ratio of 1%, double antibodies with the volume ratio of 1%, fish serum with the volume ratio of 1%, growth factors, nutrient substances and fetal calf serum with the volume ratio of 5%. The mixed culture medium is formed by mixing L-15 and DMEM/F12 according to the mass ratio of 1: 1. The culture medium and the culture method are used for culturing the Hexagrammos otakii spermatogonium which is separated and purified in vitro, the spermatogonium which is relatively high in purity and has dryness and proliferative activity can be obtained, and a basic experimental material is provided for research on a regulation mechanism of marine fish spermatogonium transplantation and spermatogonium proliferation and differentiation.

The present invention provides a hybrid stem comprising an enucleated adult stem cell having a nucleus derived from a primordial sex cell or an embryonic stem cell. The primordial sex cell may be a spermatogonia or an oogonia from a donor animal or mammal. The enucleated adult stem cell may be fused to a primordial sex cell or an embryonic stem cell by many methods including but not limited to electrofusion, virus-based fusion methodology, chemical fusion or mechanical-based fusion.

The method of obtaining transgenic chicken relates to gene engineering technology. Exogenous gene pEGFP-N1 plasmid is used to transfect stem spermatogonium, which is then differentiated inside body to obtain sperms carrying exogenous gene. Utilizing these sperms carrying exogenous gene can obtain great amount of transgenic descendents for improving chickení»s production performance, preserving chickení»s germ plasm resource, breeding disease variety, establishing disease mold, genetic treatment and development control, making bioreactor, and producing various kinds of bioactive medical protein hard to obtain in other methods.

The invention relates to a testicular tissue cryopreservation method based on single seminiferous tubule perfusion, which comprises the following steps: making testicular tissue into the single seminiferous tubule, soaking a single seminiferous tubule in a cryopreservation protective agent 1 for 10 minutes, and soaking and perfusing the seminiferous tubule by using a cryopreservation protective agent 2, so as to maximally reduce the permeation damage of the protective agent while achieve vitrification cooling, quickly transferring the poured spermatogenic tubule to a Cryo-genic single sperm cryopreservation slice, after sleeving of the Cryo-genic tubule, directly conducting immersing in liquid nitrogen to quickly achieve cooling, and quickly conducting soaking in a recovery solution aftercooling. Compared with a traditional testicular cell suspension cryopreservation method, the method disclosed by the invention has the advantages that (1) the complete structure of the seminiferous tubule is reserved; (2) the oxidative stress level of testicular cells is effectively reduced; (3) the death of a large number of spermatogonial cells and supportive cells after cryopreservation is effectively reduced; and (4) the recovery rate and activity of sperms in the spermatogenic tubules are high.

The invention discloses a method for producing functional sperms by 3D in-vitro culture of bostrichthys sinensis spermatogonium, which comprises the following steps: (1) disinfecting, cleaning and cutting separated bostrichthys sinensis testis, adding a testis digestive juice for digestion, and filtering by a filter screen to remove undigested cell debris or cell clusters to prepare a testis single-cell suspension; adding the testis single-cell suspension into a percoll gradient solution for centrifugation, and separating to obtain spermatogonium; the percoll gradient solution is composed of a percoll solution with two concentrations of 22%-30% and 35%-45%; (2) transferring the spermatogonium into a 3D culture dish, and culturing the spermatogonium with a sperm induction culture medium; the sperm induction culture medium comprises a basic sperm culture medium and sex hormone, and further comprises melatonin. According to the method, the separation, culture and induction conditions of the spermatogonium are optimized and improved, and the efficiency of generating functional sperms by culturing the spermatogonium in vitro is improved.

The invention relates to application of dicabaine in preparation of a medicine for treating spermatogenic dysfunction, and belongs to the technical field of medicines. The invention provides an application of dibucaine in preparation of a medicine for preventing and/or treating spermatogenic dysfunction caused by radiation, and research finds that dibucaine can significantly increase the formation number of long sperms in a spermatogenic tubule cavity of an irradiated mouse, accelerate differentiation from spermatogonium cells to spermatids of the irradiated mouse, improve the spermatogenic dysfunction of the irradiated mouse, and improve the spermatogenic dysfunction of the irradiated mouse. The compound has a great application prospect in prevention and/or treatment of spermatogenic dysfunction caused by radiation.