Effects of various carbohydrates on the in vitro pollen germination of Vinca rosea and Cucumis melo var. utilissimus

Pollen germination is crucial for the process of plant development which strongly depends on the presence of carbohydrates as a primary source of energy. In this study, we analyzed the differential effects of four sugars with varying concentrations on the pollen germination of Vinca rosea Linn. and Cucumis melo var. utilissimus Duth. & Fuller. using Brewbaker and Kwack’s medium as germination medium and hanging drop method after an incubation period of 1h. Sucrose and glucose supported the pollen germination but galactose and fructose did not support and even considerably inhibited the pollen germination of Vinca rosea. Whereas in pollen germination of Cucumis melo var. utilissimus, all the four sugars supported the pollen germination. The study suggests that 15% sucrose, for Vinca rosea, and 12% galactose, for Cucumis melo var. utilissimus, supports in achieving the highest pollen germination percentage when added to the pollen germination medium.

Abstract 483: Novel Evaluation Method of Spheroid

Introduction: The 3D culture methods are growing in importance in various fields, such as tissue engineering, cancer research and drug estimation. There are many methods to create 3D cell aggregation, as known as spheroids, e.g. hanging drop method, low attach U-shape plate, microwell low attach plate, spinning flask and magnetic levitation. In either field, researchers require not only high-scale but also high-quality spheroids creation. However, the method for evaluation of spheroids has not yet established. We established an accurate method of quantification of spheroids by measuring cell density and area of them with NIH Image J. Method: Spheroids were created with a hanging drop method (HD) and 96 well low attach U-shape plate (LAP) by using H9C2 cells, NIH 3T3 cells, human cardiac fibroblast and human dermal fibroblast cells using 22000 cells, 33000 cells and 44000 cells per spheroids. Spheroids were created five times using one of these systems.These spheroids were taken pictures in 10 times magnification with a microscope under constant condition using one tenth of spheroids solution created with HD and LAP and then quantified mean gray value and area using NIH image J. We saw relationship between its cell diameter and these data and also measured the cost and time for creating one spheroid and compared them among these methods. We also compared these data with histological analysis. Result: Spheroids created with HD had no relation between cell number and spheroid quality, whereas those with LAP demonstrated that higher cell numbers created larger and higher density of spheroid consistently. The number of spheroids created with the hanging drop method was higher but more inconstant and the success rate was low. However, Spheroids creation with this method took much less cost and time compared with LAP. We could see a positive correlation between area and cell size and negative correlation between gray value and its cell diameter when we create spheroids with HD with the same kinds of cells, i.e. NIH 3T3 cells, human cardiac fibroblast, and human dermal fibroblast cells. Conclusion: Although HD has the potentiality to create a large number of spheroids at once and this method was cost and time effective, the quality of spheroids was not consistent compared to LAP. The cell diameter influenced the quality of spheroids when we created them with HD.

Intraportally delivered stem cell spheroids localize in the liver and protect hepatocytes against GalN/LPS-induced fulminant hepatic toxicity

Background Systemic inflammatory response syndrome (SIRS) is common in severe fulminant hepatic failure (FHF) and has a high mortality rate (20–50%) due to irreversible cerebral edema or sepsis. Stem cell-based treatment has emerged as a promising alternative therapeutic strategy to prolong the survival of patients suffering from FHF via the inhibition of SIRS due to their immunomodulatory effects. Methods 3D spheroids of adipose-derived mesenchymal stem cells (3D-ADSC) were prepared by the hanging drop method. The efficacy of the 3D-ADSC to rescue FHF was evaluated in a d-galactosamine/lipopolysaccharide (GalN/LPS)-induced mouse model of FHF via intraportal transplantation of the spheroids. Results Intraportally delivered 3D-ADSC better engrafted and localized into the damaged livers compared to 2D-cultured adipose-derived mesenchymal stem cells (2D-ADSC). Transplantation of 3D-ADSC rescued 50% of mice from FHF-induced lethality, whereas only 20% of mice survived when 2D-ADSC were transplanted. The improved transplantation outcomes correlated with the enhanced immunomodulatory effect of 3D-ADSC in the liver microenvironment. Conclusion The study shows that the transplantation of optimized 3D-ADSC can efficiently ameliorate GalN/LPS-induced FHF due to improved viability, resistance to exogenous ROS, and enhanced immunomodulatory effects of 3D-ADSC.

Design, 3D printing and validation of a novel low-cost high-capacity sitting-drop bridge for protein crystallization

Sitting-drop protein crystallization is not used as commonly as the hanging-drop method for crystal optimization owing to the limitations of commercially available sitting-drop bridges, particularly when they are used in conjunction with 24-well crystallization plates. The commercially available sitting-drop bridge, containing space for only a single drop, restricts their wider use. Proteins that preferentially crystallize under sitting-drop conditions therefore require more work, time and resources for their optimization. In response to these limitations, and using 3D printing, a new sitting-drop bridge has been designed and developed, where five crystallization drops can be placed simultaneously in each well of a 24-well crystallization plate. This significantly simplifies the process and increases the potential of sitting drops in crystal optimization, reducing costs and hence overcoming the limitations of current approaches.