Abstract 483: Novel Evaluation Method of Spheroid

Abstract

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.