114 results about "Spermatogonial stem cells" patented technology

A method of in vitro maturation of adult human germ line cells in an artificial biological environment, which entails:a) isolating human spermatogonial stem cells (SSCs), and optionally purifying the same; andb) co-culturing the isolated and optionally purified SSCs with a suitably adjusted Sertoli cell environment to obtain haploid germ cells.

The invention relates to organisms and compositions comprising one or more stem cells or one or more embryos, wherein the one or more stem cells or one or more embryos comprise one or more of the following mutations: (i) a deletion mutation; (ii) a knockout mutation; and / or (iii) an addition of a heterologous nucleic acid sequence; wherein the one or more mutations of (i), (ii), and / or (iii) are site-specific mutations caused by a Xanthomonas TAL nuclease (XTN). The invention also relates to method of mutating an embryo, iPS cell, stem cell, or more particularly a spermatogonial stem cell by exposing the nucleic acid sequence contained within such embryos or cell with a Xanthomonas TAL nuclease.

The present invention relates to methods of identifying and enriching rat spermatogonioal stem cells, and compositions thereof. Further, the invention relates to methods and compositions for the isolation, maintenance and proliferation of rat spermatogonial stem cells, as well as methods and compositions for the identification and use of factors influencing rat spermatogonial stem cell maintenance and proliferation.

The invention discloses a method for separating and culturing mouse primitive spermatogonia. In the method, the mouse primitive spermatogonia are separated by utilizing the characteristics of neutral protease, such as mild digestibility performance, high efficiency and small stimulation to cells; the step of enrichment and subculture of the mouse primitive spermatogonia are organically combined; and adherent mouse primitive spermatogonium colony is digested by utilizing the neutral protease, so a better enrichment effect is achieved. The method for separating and culturing the mouse primitive spermatogonia is characterized by simple separating and culturing operation, short period, good effect and low cost.

The present invention relates to methods of identifying and enriching spermatogonioal stem cells, and compositions thereof. Further, the invention relates to methods and compositions for the isolation, maintenance and proliferation of spermatogonial stem cells, as well as methods and compositions for the identification and use of factors influencing spermatogonial stem cell maintenance and proliferation.

A cell composition composed of spermatogonial stem cells, Sertoli cells, Leydig cells and optionally peritubular cells, is provided, as is a culture composition, artificial testicular construct, hydrogel composition, and device containing the same. A method for using the device as a physiologically relevant in vitro model of human testicular function to screen compounds for pharmacological or toxicological activity is also provided.

The invention relates to a method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells. The method includes the steps that various solutions are prepared, 2-3 g of tissue blocks of the seminiferous tubule are taken out from the testis of a sheep of 3-4 mouths of age, single-cell suspension is prepared through enzymolysis and digestion of two steps, purified spermatogonia stem cells are separated according to the difference adherence method, the long-term passage cultivation of the sheep spermatogonia stem cells is achieved through an SSCM culture solution, and the sheep spermatogonia stem cells are cryopreserved and recovered after the long-term passage cultivation. By the technical scheme, the sheep spermatogonia stem cells with high purity can be obtained without using specific sheep spermatogonia stem cell antibodies, a long-term passage cultivation system of the sheep spermatogonia stem cells is built, and the method for preserving the sheep spermatogonia stem cells for a long term through a liquid nitrogen freezing method is successfully provided. The sheep spermatogonia stem cells achieving separation, purification and passage according to the method are cultivated at least for more than 40 generations, and the longest cultivation period reaches four years if the cryopreservation time is included.

The invention relates to an erythroculter ilishaeformis spermatogonia stem cell separation and culture method. The method comprises the following steps: (1) collecting spermatic tissues: collecting spermary of erythroculter ilishaeformis with an age of seven months in a sterile environment, then rinsing spermary in PBS containing double-antibody, and finally grinding the spermary to disperse the spermary tissues; (2) digesting and separating spermatogonia stem cell: adding IV type collagenase (0.1%) into the dispersed spermary tissues, subjecting the digestive fluid to centrifugal separation, collecting the precipitate, digesting the precipitate by trypsin (0.25%), stopping the digestion by a culture medium containing FBS (10%), and collecting the cells; (3) carrying out primary culture of spermatogonia stem cell: using a DMEM / F12 complete medium containing cell factors to re-suspend the cells obtained in the step (2), adjusting the cell concentration, then paving the suspension liquid on a 24-hole cell culture plate coated by gelatin, and culturing the cells at a temperature of 26 DEG C. Trough the provided method, the in-vitro growth of primary cells of erythroculter ilishaeformis spermatogonia stem cell becomes easier, and an effective cell platform is provided for the research on reproduction and growth of erythroculter ilishaeformis.

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 relates to a long-term culture method for spermatogonial stem cells in vitro without a feeder layer. According to the method, the spermatogonial stem cells of a pig can be cultured in vitro to three months by screening different extracellular matrices and applying chemical small molecule substances to long-term culture of the spermatogonial stem cells of the pig. A 2D natural artificial synthetic polypeptide matrix coating is utilized as an extracellular matrix, a typical grape beaded spermatogonial stem cell cluster is observed during the culture process, different chemical small molecule combinations are screened, the spermatogonial stem cells of the pig are cultured in vitro for three months by adding Chir99021, Repsox and a CD lipid concentrate with good spermatogonial stem cell characteristics maintained.

A transgenic rat expresses a gene of interest, such as EGFP, exclusively in the germ cells of both males and females. From such a transgenic rat one can isolate a line of spermatogonial stem cells, which can renew and proliferate in culture.

The invention relates to a method for separating spermatogonial stem cells of seawater fishes, in particular to a method for separating spermatogonial stem cells of turbot. The method comprises the steps as follows: pretreated complete gonads of the turbot which is 1-1.5 years old are washed with an L-15 medium containing double antibodies (penicillin / streptomycin) and digested by enzymes, digested digestive fluid is subjected to density gradient centrifugation, and accordingly, the spermatogonial stem cells with high purity and high density are obtained. According to the method for separatingthe spermatogonial stem cells of the seawater fishes, the spermatogonial stem cells separated with the method have complete morphological structure, high purity and large density, and can be used forpreservation of the spermatogonial stem cells and transplantation research of the stem cells. According to the method, a new method and a new idea are provided for preservation of marine fish germplasm resources and cultivation of new varieties.

The invention belongs to the field of biological engineering and provides a method for separating human spermatogonial stem cells. The method comprises the following steps of: mechanically separating a testicular tissue; processing the testicular tissue into a semi-liquid state; adding digestive enzyme solution I into the liquid testicular tissue in the semi-liquid state; digesting the testiculartissue into a single convoluted tubule; adding digestive enzyme solution II in the convoluted tubule; digesting the convoluted tubule into a single cell; separating reproductive cells from supportingcells in the single cell; separating G protein-coupled receptor 125 (GPR125) positive spermatogonial stem cells from glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 1 (GFRA1)positive spermatogonial stem cells by adopting an immunomagnetic bead cell sorting method; and identifying the separated GPR125 positive spermatogonial stem cells and the GFRA1 positive spermatogonial stem cells by adopting an immune cell chemical method. By the method, a large number of the high-purity human spermatogonial stem cells are obtained, and sufficient cell sources are provided for theresearch of the human spermatogonial stem cells and the application of the human spermatogonial stem cells in regenerative medicine and reproductive medicine.

The present invention provides a method of growing spermatogonial stem cells of mammals and the like in vitro, which is characterized in that glial cell-derived neurotrophic factor (GDNF) or an equivalent thereto, and leukemia inhibitory factor (LIF) are contained in a medium (culture broth) for culturing spermatogonial stem cells. According to the method of the present invention, spermatogonial stem cells can be grown in vitro to the extent that enables use thereof for developmental engineering.

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 discloses a method for efficiently separating mouse spermatogonial stem cells. The method includes the following steps that firstly, mouse testis are collected, and a testicular cell suspension is prepared through a two-step enzyme digestion method; secondly, the testicular cell suspension is inoculated in a porous plate according to a certain density, and a spermatogonial stem cell culture solution is added for in-vitro culture; thirdly, the culture solution is removed, testis cells of different times are cultured through tryptic digestion, and spermatogonial stem cells are sorted from the testis cells through the magnetic activated cell sorting; fourthly, the sorted spermatogonial stem cells are inoculated to an STO trophoblast, and a spermatogonial stem cell culture solution is added for primary culture and subculture of the spermatogonial stem cells. A mouse spermatogonial stem cell system is built through the method including the steps of separation, culture, re-separation and re-culture. The method can achieve the aim of efficiently enriching the spermatogonial stem cells in vitro, and is economical and easy to implement, so that a direction is provided for protection of rare animal varieties and treatment of male infertility.

The invention discloses a method for directionally inducing and differentiating pig pluripotent stem cells into male germ cells and a special culture medium. The invention provides the special culture medium. The special culture medium comprises an EpiLC inducing medium, a PGCLC (primordial germ cell-like cell) inducing medium and an SSCLC (spermatogonial stem cell-like cell) inducing medium. The invention provides the method for directionally inducing and differentiating the pig pluripotent stem cells into PGCLC in vitro; experiments prove that the PGCLC can be further induced and differentiated to form SSCLC; in vivo, induced cells are injected into seminiferous tubules of mice of which endogenous sperms are removed, and the PGCLC and the SSCLC can survive and are further developed. Therefore, the invention provides a feasible and useful scheme for directional differentiation from the pig pluripotent stem cells to the germ cells, and lays a foundation for exploring a development mechanism and the like of the germ cells.

The invention discloses a livestock spermatogonial stem cell medium (SSCM). The preparation method of the livestock SSCM comprises a step of uniformly mixing 1ml of SSCB, 1mul of each of three cytokine stock solutions, 1mul of a penicillin stock solution, 1mul of a streptomycin stock solution and 1mul of a Fungizone stock solution before use. The livestock SSCM is suitable for the long-term culture of livestock SSCs. The invention simultaneously discloses a livestock SSC long-term culture method, a livestock SSC immunofluorescence dyeing identification method, a livestock SSC cryopreservation method and the like.

The invention relates to a spermatogonial stem cell culture system without adding recombinant growth factors and application thereof. The spermatogonial stem cell culture system comprises a well-defined spermatogonial stem cell culture medium and a trophoblast cell containing glial-derived neurotrophic factors (GDNF). By utilizing a method provided by the invention, a spermatogonial stem cell can be cultured in vivo for a long term; a transplantation experiment verifies that the characteristics of the stem cell are no different from those of a spermatogonial stem cell cultured by a traditional culture method. Compared with the traditional culture method, the method provided by the invention has the benefits that any expensive recombinant growth factor and fetal bovine serum are not required to be added, and the culture medium is very clear in component and good in repeatability. The spermatogonial stem cell cultured by applying the method provided by the invention has a complete regeneration potential and an ability of infinite proliferation. The method provided by the invention is simple and convenient to culture, economic and efficient.

The invention relates to a three-dimensional inducing method for inducing a spermatogonia stem cell to differentiate into a functional sperm cell in vitro, and belongs to the field of bioengineering. On the basis of acquiring the spermatogonia stem cell, a three-dimensional culture system is established through Matrigel, a key factor for inducing differentiation is added, the spermatogonia stem cell is induced to differentiate in vitro to obtain the sperm cell having a fertilization ability, the sperm cell obtained through induction is identified through an immunocytochemical method, the chromosome number of the sperm cell obtained through induction is detected through fluorescence in situ hybridization (FISH), and the fertilization ability and the viability of the sperm cell obtained through induction are detected through round spermatid injection (ROSI). The application provides an excellent model for the molecular mechanism of the human sperm, and in addition, a functional gamete is provided for an azoospermia patient, so that a male infertility patient can have a child of his own.

The present invention relates to the production and culture of undifferentiated spermatogonial stem cells that can be maintained long term and are feeder free. The resultant feeder-free populations can be used in any of a number of protocols including the generation of progeny bulls. The present invention includes novel methods required for the successful enrichment of bovine spermatogonial stem cells, novel cell lines and other components used for the same, as well as the resultant stem cell compositions.

The invention relates to an in vitro culture solution and cryopreservation solution of fish spermatogonial stem cells (SSCs), and an in vitro culture and cryopreservation method, and solves the existing problems of relatively short in vitro culture time of SSCs and complex components of SSCs medium. The in vitro culture solution is prepared by taking an L-15 medium as the basis and adding HEPES, penicillin, streptomycin, NaHCO3, L-glutamic acid, fetal bovine serum, catfish serum and bFGF. The in vitro culture method includes: 1. Ietalurus Punetaus testis cell separation and SSCs enrichment; and 2. SSCs culture. The in vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells provided by the invention have the characteristics of simple formula components, easy acquisition and preparation, and low price, and can realize long-term in vitro culture and cryopreservation of Ietalurus Punetaus spermatogonial stem cells.

The invention relates to a method for separating pig spermatogonia stem cells. The method comprises steps of preparing solutions and separating spermatogonia stem cells, wherein in the step of preparing solutions, the solutions to be prepared comprises nutrient solution, digestive enzyme solution, buffer solution, collagen solution, gelatin solution and laminin solution, the step of separating spermatogonia stem cells comprises substeps of cutting pig testicular tissue into pieces and adding enzyme for digestion, filtering by a cell strainer, carrying out enrichment culture and difference adherent culture of the spermatogonia stem cells, finally collecting the spermatogonia stem cells, wherein the difference adherent culture comprises collagen coating adherent culture and laminin coating adherent culture. The method is characterized in that the nutrient solution comprises DMEM / F12, FBS (fetal bovine serum), sodium pyruvate, penicillin and streptomycin; according to the collagen coating adherent culture, after being cultured for required time, the cell is blown and beaten and then continuously cultured. In the collagen coating adherent culture, the spermatogonia stem cell is slightly blown and beaten and is not attached to the wall of the collagen, so that loss of the spermatogonia stem cell is reduced, and the yield of the spermatogonia stem cell is improved.

A spermatogonial stem cell line that is derived from testes rats characterized by a desirable genetic background can serve as a source for cells to transplant into male-sterile recipient animals that are immuno-compatible with the spermatogonial line. Rat cells thus transplanted readily develop into fertilization-competent, haploid male gametes, with little or no endogenous sperm competition generated by the testes of the male-sterile recipient. This approach, constituting the first vector system for the use of rat spermatogonial lines from in vitro culture in generating mutant rats on a desired genetic background, effects maximal germline transmission of donor haplotypes from the transplanted spermatogonial cells.

The present invention relates to a method for long-term culturing of avian spermatogonial stem cells, which comprises the steps of: (a) preparing an avian testis; (b) isolating a population of testicular cells from said avian testis; and (c) culturing said avian spermatogonial stem cells in said population of testicular cells on a feeder cell layer in a medium containing a cell growth factor, a population of avian spermatogonial stem cells and a method for producing transgenic ayes.

The invention belongs to the technical field of biology and relates to a method of introducing random insertion mutation to genome of in-vivo spermatogonial stem cells. In the introduction of insertion mutation, provided is a non-integrated lentivirus composite carrier carrying a piggyBac transposon system and its preparation method. The carrier and the method are applicable to efficiently preparing non-human gene mutation animals. A gene mutation animal prepared by the method is applicable to animal phenotypic analysis, gene function research, Human genetic disease simulation, novel drug screening and novel drug target finding.

The invention discloses a method for separating and culturing pig spermatogonial stem cells by a mechanical process. The method of the invention is characterized in that the characteristics of animal testicular tissue are combined, and the testicular tissue can be separated by the mechanical process and then cultured, so that the method for separating and purifying spermatogonial stem cells can be enriched. According to the invention, the pig spermatogonial stem cells can be obtained without enzyme digestion, the method of the invention has the advantages of fast cells clone and good state ofcells, and solves the problems of difficult separation, small quantity and high requirement of culture condition of spermatogonial stem cells. Compared with the separating technology of the prior art, the method of the invention has simple operation, and is capable of reducing the difficulty to obtain cells, substantially reducing the experimental cost and raising the success rate of the experiments.

The present invention provides a method of producing a non-human vertebrate that harbours germ cells having a desired gene transferred thereto, comprising injecting the desired gene to the testis of a non-human vertebrate wherein no tight junction exists between Sertoli cells to transfer the desired gene to germ cells, so as to obtain a non-human vertebrate that harbours the germ cells having the desired gene transferred thereto. Using the method of the present invention, even in animal species and lines for which in vitro transduction has been difficult to date, it is possible to obtain an individual harbouring germ cells, particularly spermatogonial stem cells, having a desired gene transferred thereto, at extremely high efficiency. Also, the fertility of the male to receive an injection of the gene is retained, compared to in vitro transduction of germ cells, because gene transfer is achieved without reducing the number of spermatogonial stem cells in the testis and transgenic sperms and transgenic animals can easily be prepared.

The present invention provides a method of producing offspring originating in a spermatogonial stem cell of a donor animal which comprises generating spermatozoa in the reproductive organ of a male recipient animal with the use of frozen spermatogonial stem cells originating in the donor animal to give a male individual for reproduction, and then producing animal individual originating in the spermatogonial stem cell of the donor using the said individual for reproduction.