In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for
Blastocyst ; Embryo Culture Techniques ; Embryonic Development ; Fertilization in Vitro

The genus Paxillus is characterized by the difficulty of species identification, which results in reproducibility problems, as well as the need for large quantities of fungal inoculum. In particular, studies of Paxillus ammoniavirescens have reported divergent results in the in vitro growth while little is known of its capacity to degrade organic matter. For all the above, and assuming that this variability could be due to an inappropriate culture media, the aim of this study was to analyse growth in different culture media (MMN, MS, and 1/2 MS) and in the case of MMN in presence/absence of two types of organic matter (fresh litter and senescence litter) to probe the saprophytic ability of P. ammoniavirescens . We also evaluated the effects of pH changes in the culture media. Growth kinetics was assessed by weekly quantification of the area of growth in solid culture media over 5 wk, calculating the growth curves and inflection points of each culture media. In addition, final biomass after 5 wk in the different culture media was calculated. Results showed that best culture media are MS and 1/2 MS. Moreover, an improvement in growth in culture media containing decomposing fall litter was observed, leading to confirm differences in the culture media of this species with others of the same genus. Further, we established that all growth media suffered a significant acidification after fungal growth.
Aging ; Biomass ; Culture Media* ; Hydrogen-Ion Concentration ; In Vitro Techniques ; Kinetics

In vitro culture of Toxoplasma gondii in HL-60 cells cnd cell-mediated immunity against Toxoplasma in dimethylsulfoxide(DMSO)-induced HL-60 cells, i.e., differentiation into granulocytes, were pursued. HL-60 cells were treated with various concentrations of DMSO, and 1.3%(v/v) for 3 day incubation was chosen as the optimal condition for differentiation into granulocytes. The degree of differentiation was assayed in physiological and functional aspects in addition to morphological point. When treated with 1.3% DMSO for 3 days, HL-60 cells did not synthesize DNA materials beyond background level, and showed active chemotactic response to chemotactic peptide, formyl-methionyl-leucyl-phenylalanine(FMLP). Morphologically promyelocytes of high nuclear/cytoplasmic(N/C) ratio changed to granulocytes of relatively low N/C ratio. The relationships between HL-60 cells or DMSO-induced HL-60 cells and Toxoplasma were examined after stain with Giemsa and fluorescent dye (acridine orange). HS-60 cells did not show any sign of toxoplasmacidal activity but showed intracellular proliferation of Toxoplasma to form rosette for 72 hr co-culture. In contrast, DMSO-induced HL-60 cells phagocytosed Toxoplasma within 1 hr, and performed a process of intracellular digestion of Toxoplasma thereafter. With the above results, it is suggested that phagosome-lysosome fusion is one of the critical events for the parasitism by Toxoplasma or for susceptibility of host cells. The in vitro culture system of this study has offered a defined condition to study the protozoan parasite-host cell interactions.
parasitology-protozoa ; Toxoplasma gondii ; HL-60 cells ; dimethylsulfoxide ; in vitro culture ; dimethylsulfoxide

OBJECTIVEThe purpose of this study was to establish a palatal organ culture method and to investigate the palatogenesis in vitro.

METHODS20 pregnant 14-day mice were killed, embryos were separated ascetically, and palatal shelves were dissected and placed on a modified Trowell's system. All explants were cultured 24 h and 48 h respectively. Finally, all explants were embedded and stained by Hematoxylin and Eosin.

RESULTSAll explants grew healthy. After incubation for 24 h, medial edge epithelium maintained, whereas after 48 h, medial edge epithelium disappeared, bilateral mesenchymal cells contacted, palates fused.

CONCLUSIONThis method provides an effective way for investigating the etiology of cleft palate in vitro.

Animals ; Cleft Palate ; Epithelium ; Female ; In Vitro Techniques ; Mice ; Organ Culture Techniques ; Palate ; cytology ; Pregnancy

OBJECTIVETo investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development.

METHODSSpent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed.

RESULTSIn the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02).

CONCLUSIONELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

Biomarkers ; chemistry ; Chorionic Gonadotropin ; chemistry ; Culture Media ; chemistry ; Embryo Transfer ; Embryonic Development ; Fertilization in Vitro ; Humans

OBJECTIVETo establish an in vitro culture model of single human hair follicle, and observe their morphological and histological changes.

METHODSHuman hair follicles were isolated from the volunteer patients. After dissecting follicles into single, follicles in growth phase were cultured in Williams E without any serum. This experiment included 3 groups: single follicle without sebaceous gland and other surrounding tissue (control group); single follicle with sebaceous gland and without the other surrounding tissue( experiment group A); single follicles with sebaceous gland and the other surrounding tissue (experiment group B). The survival rate, survival time, growth rate, multiplication capacity and apoptosis of cultured follicles and their morphological and histological changes were observed sequentially.

RESULTSThe hair follicles in experiment groups showed a better viability and a higher growth rate than those in control group. And the follicles in group B could keep growing for more than 25 days, which was longer than those in group A. Moreover, the sebaceous gland and the other surrounding tissue in group B showed great induction effect on follicle-cell proliferation and anti-apoptosis.

CONCLUSIONThe in vitro culture model of signal human hair follicles (single follicles including epidermis, sebaceous gland and the other surrounding tissue) had optimized internal environment which is similar to in vivo internal environment.

Culture Techniques ; Hair Follicle ; anatomy & histology ; cytology ; growth & development ; Humans ; In Vitro Techniques ; Sebaceous Glands ; Time Factors

Whether the type of culture media utilized in assisted reproductive technology has impacts on laboratory outcomes and birth weight of newborns in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) was investigated. A total of 673 patients undergoing IVF/ICSI and giving birth to live singletons after fresh embryo transfer on day 3 from Jan. 1, 2010 to Dec. 31, 2012 were included. Three types of culture media were used during this period: Quinn's Advantage (QA), Single Step Medium (SSM), and Continuous Single Culture medium (CSC). Fertilization rate (FR), normal fertilization rate (NFR), cleavage rate (CR), normal cleavage rate (NCR), good-quality embryo rate (GQER) and neonatal birth weight were compared using one-way ANOVA and χ (2) tests. Multiple linear regression analysis was performed to determine the impact of culture media on laboratory outcomes and birth weight. In IVF cycles, GQER was significantly decreased in SSM medium group as compared with QA or CSC media groups (63.6% vs. 69.0% in QA; vs. 71.3% in CSC, P=0.011). In ICSI cycles, FR, NFR and CR were significantly lower in CSC medium group than in other two media groups. No significant difference was observed in neonatal birthweight among the three groups (P=0.759). Multiple linear regression analyses confirmed that the type of culture medium was correlated with FR, NFR, CR and GQER, but not with neonatal birth weight. The type of culture media had potential influences on laboratory outcomes but did not exhibit an impact on the birth weight of singletons in ART.
Birth Weight ; Culture Media ; Female ; Fertilization in Vitro ; Humans ; Infant, Newborn ; Male ; Pregnancy ; Pregnancy Outcome

Immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-PD-L1 Abs, have shown efficacy for the treatment of various cancers. Although research has actively sought to develop new ICIs and immunomodulators, no efficient in vitro assay system is available to evaluate their functional activities. In the present study, we established a two-round MLR with human PBMCs for evaluation of the T cell-activating capacity of anti-PD-1 and other immunomodulators. We initially performed conventional MLR for this purpose. However, anti-PD-1 blocking Abs could not increase the proliferation of allo-reactive T cells in conventional MLR because PD-L1+ and PD-L2+ cells disappeared gradually during MLR. Therefore, we re-applied the same stimulator PBMCs to the allo-stimulated responder cells as a second-round MLR on day 6 when anti-PD-1 or immunomodulators were also added. In this two-round MLR, the proliferation of allo-reactive T cells was enhanced by anti-PD-1 in a dose-dependent manner or by immunomodulators, such as lenalidomide and galunisertib, a TGF-β receptor-1 inhibitor. Proliferation was further increased by the combination of immunomodulators with anti-PD-1. Here, we established a modified two-round MLR method with human PBMCs for evaluation of the functional activities of anti-PD-1 and immunomodulators.
Humans ; Immunologic Factors* ; In Vitro Techniques ; Lymphocyte Culture Test, Mixed* ; Methods ; T-Lymphocytes

vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.]]>
Buffaloes ; Cells, Cultured ; In Vitro Techniques ; Primary Cell Culture ; Stem Cells ; Tretinoin

OBJECTIVE: In vitro culture of preimplantation embryos is improved by grouping embryos together in a drop of media. Individually cultured embryos are deprived of paracrine factors; with this in mind, we investigated whether the addition of a single embryo-secreted factor, interleukin-6 (IL-6), could improve the development of individually cultured embryos. METHODS: Mouse embryos were cultured individually in 2 µL of G1/G2 media in 5% oxygen and supplemented with a range of doses of recombinant mouse or human IL-6. RESULTS: Mouse IL-6 increased hatching at doses of 0.01 and 10 ng/mL compared to the control (93% and 93% vs. 78%, p<0.05) and increased the total number of cells at a dose of 0.1 ng/mL compared to the control (101.95±3.36 vs. 91.31±3.33, p<0.05). In contrast, the highest dose of 100 ng/mL reduced the total number of cells (79.86±3.29, p<0.05). Supplementation with human IL-6 had a different effect, with no change in hatching or total cell numbers, but an increase in the percentage of inner cell mass per embryo at doses of 0.1, 1, and 100 ng/mL compared to the control (22.9%±1.1%, 23.3%±1.1%, and 23.1%±1.1% vs. 19.5%±1.0%, p<0.05). CONCLUSION: These data show that IL-6 improved mouse embryo development when cultured individually in complex media; however, an excess of IL-6 may be detrimental. Additionally, these data indicate that there is some cross-species benefit of human IL-6 for mouse embryos, but possibly through a different mechanism than for mouse IL-6.
Animals ; Blastocyst* ; Cell Count* ; Culture Media ; Cytokines ; Embryology ; Embryonic Development ; Embryonic Structures* ; Female ; Fertilization in Vitro ; Humans ; In Vitro Techniques ; Interleukin-6* ; Mice* ; Oxygen ; Pregnancy