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@#Abstract: Objective To screen out a more universally applicable culture medium for the isolation and culturing of pathogenic fungi through comparing the performance of various universal fungal culture media, to optimize the fungal culturomics technique, and to better apply it to the culturomics research of pathogenic fungi. Methods Multiple common fungal culture media Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), modified Dixon (mDixon), modified LeemingNotman agar (MLNA), etc., and a new pan-fungal medium (PF) were used to culture 40 strains of common pathogenic fungi to determine the growth states of strains under different conditions. Based on that, PF, SDA, PDA, mDixon and MLNA, a total of 5 culture media, were used to isolate and culture a simulated sample (suspension of Candida albicans and Aspergillus fumigatus), 10 human samples (4 fecal samples and 6 vaginal secretion samples) and 3 environmental samples. Results The positive growth rates of 40 strains of pathogenic fungi in the 7 media were as follows: PDA 95.0% (38/40), SDA 95.0% (38/40), BHI 95.0% (38/40), YPD 90.0% (36/40), mDixon 95.0% (38/40), MLNA 87.5% (35/40), PF 100.0% (40/40). For the simulated samples, PF could effectively promote the self-limited growth of filamentous fungi, performing better in isolation and culture. For the human samples and environmental samples, PF showed the same versatility as SDA and PDA. Conclusions In the isolation and culturing of pathogenic fungi, PF medium can effectively isolate and culture most fungal species. Meanwhile, PF can make the fast-growing fungi show self-limited growth and clear edges, and not easy to cross-contamination, which indicates it is conducive to the isolation and identification of single colonies. PF medium outperforms other common media in isolating strains from unknown samples in culturomics, which illustrates PF medium can be effectively used for the study of fungal culturomics.
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Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kit
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Objective@#To establish the isolation and culture methods of skeletal muscle stem cells, derived from human orbicularis oculi muscle (OOMSCs), and to identify their multi-directional differentiation potential in vitro.@*Methods@#The cellswere isolated from pretarsal orbicularis oculi muscle (OOM), obtained in routine blepharoplasty surgery.The tissue was digested using collagenase type I combined with re-plating methods. Specific cell surface antigen markers were detected using flow cytometry analysis. OOMSCs were cultured under different inductive conditions, to identify their pluripotent differentiation ability.@*Results@#OOMSCs exhibited similar fibroblast-like morphology as mesenchymal stem cells with high expression of skeletal muscle-derived stem cell surface markers. OOMSCs were able to differentiate into adipocytes, osteoblasts and chondrocytes in vitro, in the presence of lineage-specific inductive media. Moreover, after myogenic induction, the differentiated cells were fused into multinucleated myotube-like structure, and positive for myogenic-related marks, Pax3, Pax7, Myf5 and MyoD.@*Conclusions@#Muscle-derived stem cells can be isolated from human OOM with myogenic differentiation properties, showing further applications potential intissue regeneration and medical therapies of muscle diseases.
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Objective · To investigate the effect of poly (L-lactic acid caprolactone) (PLCL)/gelatin electrospinning on the angiogenesis differentiation of endothelial progenitor cells (EPCs).Methods· Rat bone marrow-derived EPCs were isolated and cultured,then identification was performed.After preparation of PLCL/gelatin blend electrospun scaffold,scanning electron microscopy and water contact angle test were carried out.EPCs were grown on PLCL/gelatin electrospinning and CCK8 was used to detect cell proliferation.The expression of vascular endothelial growth factor (Vegf) and kinases insert region receptor (Kdr) was observed by RT-PCR and the expression of VEGF protein was observed by Western blotting.Results· The density gradient centrifugation combined with differential adherence method could effectively isolate EPCs.PLCL/gelatin electrospun nanofibers were porous,and the hydrophilic properties were favorable for cell adhesion,and EPCs grew well on the scaffold.The expression of Vegfand Kdr gene in PLCL/gelatin group was higher than that in control group (P=0.000),and the expression of VEGF protein was also increased (P=0.000).Conclusion · PLCL/gelatin is an ideal scaffold for tissue engineering,and it can promote the angiogenesis differentiation of EPCs.
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Objective: To investigate the effect of poly (L-lactic acid caprolactone) (PLCL) /gelatin electrospinning on the angiogenesis differentiation of endothelial progenitor cells (EPCs). Methods: Rat bone marrow-derived EPCs were isolated and cultured, then identification was performed. After preparation of PLCL/gelatin blend electrospun scaffold, scanning electron microscopy and water contact angle test were carried out. EPCs were grown on PLCL/gelatin electrospinning and CCK8 was used to detect cell proliferation. The expression of vascular endothelial growth factor (Vegf ) and kinases insert region receptor (Kdr) was observed by RT-PCR and the expression of VEGF protein was observed by Western blotting. Results: The density gradient centrifugation combined with differential adherence method could effectively isolate EPCs. PLCL/gelatin electrospun nanofibers were porous, and the hydrophilic properties were favorable for cell adhesion, and EPCs grew well on the scaffold. The expression of Vegf and Kdr gene in PLCL/gelatin group was higher than that in control group (P=0.000), and the expression of VEGF protein was also increased (P=0.000). Conclusion: PLCL/gelatin is an ideal scaffold for tissue engineering, and it can promote the angiogenesis differentiation of EPCs.
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Objective In order to study the biological characteristics of macrophages and provide the materials to study the survival mechanism of intracellular parasites, we conducted this study to establish a high-purity alveolar macrophage isolation and culture method.Methods Goat lungs were lavaged with normal saline in sterile environment several times, and cells were collected and then goat alveolar macrophages were purified by density gradient centrifugation using peripheral blood mononuclear cells (PBMC) solution.The isolated goat alveolar macrophages were cultured in cell culture medium containing 10% fetal bovine serum and cell morphology was observed under an inverted microscope every day,and the phagocytic activity of the cells was detected by chicken red blood cell phagocytosis test.Flow cytometry was used to detect CD14, a characteristic monocyte-macrophage surface marker.Results The adherent cells were characterized by typical macrophage morphology, pseudopodia and protrusions, showing round and irregular shape, rich cytoplasm, and large cell body.Of the cultured macrophages, 54.5% could phagocytize chicken erythrocytes and showed good phagocytic activity.After one month of in vitro culture, 93.7% of the cells were able to express CD14 antigen, which had a macrophage-specific immunophenotype.Conclusions The alveolar macrophages obtained in this study have high purity and good bioactivity, thus provide a cell model for studying the immune mechanism of intracellular parasites.
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BACKGROUND: There are different methods to isolate and culture human nucleus pulposus cells, and the differences in digestive enzymes components and digestion time quite are significant. So how to rapidly and efficiently harvest human nucleus pulposus cells has become a research hotspot. OBJECTIVE: To optimize the digestive enzymes components and digestion methods for the preparation of human nucleus pulposus cells. METHODS: Nucleus pulposus tissue specimens were selected from three adult discs in the Department of Orthopedics, China-Japan Union Hospital of Jilin University. The acute traumatic disc tissues that outstanding to the spinal canal were taken under aseptic conditions, and then the peripheral white annulus and jel y-like nucleus pulposus in the center could be seen. According to different mixed enzyme concentration ratio, the samples were divided into two groups. The enzyme Ⅰ group was treated with 0.2% Ⅱ col agenase; and the mixed enzymeⅡ group was digested with 0.25% trypsin for 30 minutes, and then treated with 0.2% Ⅱ col agenase. According to digestion time, each group was divided into three subgroups: 2 hours group, 4 hours group, and overnight group. Final y, suspended cel volume was decided as 2 mL to count cells. Dulbecco’s modified Eagle’s medium containing fetal bovine serum was used for cel culture in vitro. Trypan blue staining was performed to count total cel number and ratio of living cells. Methylthiazolyldiphenyl-tetrazolium bromide assay was used to detect the growth curve of nucleus pulposus cells. RESULTS AND CONCLUSION: Based on the two digestion enzyme concentration, the number of digested cells in the enzyme Ⅰ group was larger than that in the enzyme Ⅱ group after digested for 2 and 4 hours, but the difference was not significant (P > 0.05). Overnight, cellsurvival rate was decreased in the enzyme Ⅰ group after digested for 2 and 4 hours when compared with the enzyme Ⅱ group, and the difference was significant (P < 0.05). After digested for 4 hours, tissue blocks disappeared, and the number of cells reached maximum. The results indicate that enzyme Ⅰgroup composite with Ⅱ col agenase is benefit for the separation of nucleus pulposus cells, and the digestion time is appropriate to 4 hours. This condition has the advantages of simple operation, high efficiency and low cost, and it considered that digestion of nucleus pulposus tissues with 0.2% Ⅱ col agenase for 4 hours is the best condition to obtain nucleus pulposus cells.
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10.3969/j.issn.2095-4344.2013.23.021
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Tricholoma giganteum is a famous species product of Jishou in the wes t of Hunan.It have not only special biological characteristics such as formatio n and ecological enviromment,but also delicious,fragrant with abundant nutrien t.It's mycelium can be isolated and growing well in medium,but its fruiting bo dy is difficult to be formed.