• Density gradient centrifugation
• Fertility preservation
• Flow cytometry
• Immunomagnetic bead separation
• Spermatogonial stem cell
• Tumor

• Animals
• Cells, Cultured
• Female
• Humans
• Male
• Mice
• Mice, Nude
• Spermatogenesis/genetics
• Spermatogonia/metabolism
• Stem Cell Transplantation/methods
• Xenograft Model Antitumor Assays

• Ningxia Medical University Scientific Research Project
• National Natural Science Foundation of China

Laryngopharyngeal reflux (LPR) induces a differential damage effect on several anatomic sites within the larynx and hypopharynx; therefore, an in vitro model is needed for each anatomic site. This study aimed to establish a primary culture method for human laryngeal and hypopharyngeal epithelial cells derived from multiple anatomic sites. Surgical mucosa specimens were treated with a two-step enzymatic strategy to establish a primary culture. Of the 46 samples, primary cultivation was achieved successfully with 36 samples, and the positive ratio was 78.3%. In addition, flow cytometry revealed that these primary cells were epithelial cells with a purity of 94.9%. The proliferative ability was confirmed by positive staining for Ki-67. Laryngeal and hypopharyngeal epithelial cells from multiple sites exhibited similar epithelial morphology and positive cytokeratin expression. These cells can be cultured to passage 4. In summary, we successfully established the in vitro epithelial model of larynx and hypopharynx subsites, which may potentially be used as a platform for reflux research, especially for site-specific damage effect.

• epithelial cell
• hypopharynx
• laryngopharyngeal reflux
• larynx
• site-specific heterogeneity

• in vitro differentiation
• intermediate mesoderm
• mouse embryonic stem cell
• sertoli cell

To evaluate the efficiency of two vitrification protocols for rat immature testicular tissue and heterotopic transplantation.

Twenty-four pre-pubertal Wistar rats were divided into three groups (n=8). After orchiectomy, testicular fragments (3mm) from Groups 1 and 2 were vitrified with different cryoprotectant concentration solutions, using sterile inoculation loops as support. After warming up, the fragments were submitted to cell viability assessment by Trypan blue and histological evaluation. Vitrified (Groups 1 and 2) and fresh (Group 3) fragments were grafted to the animals periauricular region. After 8 weeks of grafting, the implant site was histologically analyzed.

The viability recovery rate from Group 1 (72.09%) was higher (p=0.02) than that from Group 2 (59.19%). Histological analysis showed similar tubular integrity between fresh fragments from Groups 1 and 3. Group 2 samples presented lower tubular integrity. We ran histological analyses in the grafts from the Groups. In all groups, it was possible to see the implant site, however, no fragment of testicular tissue or signs of inflammation were histologically found in most samples from Groups 1 and 3. In one sample from Group 2, we found degenerated seminiferous tubules with necrosis and signs of an inflammatory process. In another sample from Group 2, we found seminiferous tubules in the implant site.

The vitrification of pre-pubertal testicular tissue of rats showed little damage to cell viability through histological analysis when we used cryoprotectants in a lower concentration. Heterotopic transplantation could not preserve the structural organization of the testicular tissue.

• cryopreservation
• fertility preservation
• prepubertal
• transplantation

Spermatogonial stem cells, which exist in the testicles since birth, are progenitors cells of male gametes. These cells are critical for the process of spermatogenesis, and not able to produce mature sperm cells before puberty due to their dependency of hormonal stimuli. This characteristic of the reproductive system limits the preservation of fertility only to males who are able to produce an ejaculate. This fact puts some light on the increase in survival rates of childhood cancer over the past decades because of improvements in the diagnosis and effective treatment in pediatric cancer patients. Therefore, we highlight one of the most important challenges concerning male fertility preservation that is the toxic effect of cancer therapy on reproductive function, especially the spermatogenesis. Currently, the experimental alternative for fertility preservation of prepubertal boys is the testicular tissue cryopreservationfor, for future isolation and spermatogonial stem cells transplantation, in order to restore the spermatogenesis. We present a brief review on isolation, characterization and culture conditions for the in vitro proliferation of spermatogonial stem cells, as well as the future perspectives as an alternative for fertility preservation in prepubertal boys. The possibility of restoring male fertility constitutes a research tool with an huge potential in basic and applied science. The development of these techniques may be a hope for the future of fertility preservation in cases that no other options exist, e.g, pediatric cancer patients.

• avian
• genome editing
• germline competent cell
• germline modification

• Animals
• Animals, Genetically Modified
• Cells, Cultured
• Chick Embryo
• Chickens/genetics
• Genetic Engineering/methods
• Genome
• Germ Cells/physiology
• Humans

The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0.05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ± 0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51% for GFRα-1, P<0.05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.

Spermatogonial stem cells (SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.

• Cell culture
• Spermatogonial stem cells
• Spermatogenesis
• Pluripotency
• Transplantation
• Fertility