Multicellular tumor spheroids and high-throughput preparation method thereof

Abstract

The invention discloses a high-throughput preparation method of multicellular tumor spheroids. The preparation method comprises the following steps: S1, quickly mixing a cell suspension of tumor cellswith a basement membrane matrix, and transferring the obtained cell suspension into a low-attachment well plate; and S2, performing centrifuging, culturing the cell suspension in an incubator after centrifugation, and maintaining the cell suspension during culture to form the multicellular tumor spheroids combined with the basement membrane matrix. The added substance used in the technical schemeis the basement membrane matrix Matrigel which can promote the formation and growth of the multicellular tumor spheroids, thereby promoting the three-dimensional structure of the formed multicellulartumor spheroids; meanwhile, the low-attachment well plate is used, so the tumor cells spontaneously aggregate into spheroids; and combined with culture after centrifugation of the well plate and thedesign of the step of maintaining the cell suspension during culture, cell aggregation is artificially promoted by centrifugation, so the subsequently formed cell spheroids are more compact and have amore three-dimensional structure. Therefore, the prepared tumor spheroids are in a state of uniform diameter and concentrated particle size distribution.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a multicellular tumor sphere and a high-throughput preparation method thereof. Background technique [0002] In the traditional two-dimensional culture, the cells grow in a plane, lack the interaction of various cytokines, and cannot construct a three-dimensional microenvironment, which is far from the state of cells in vivo. Multicellular tumorspheres are compact cell aggregates, similar to solid tumors in vivo, avascular tumor nodules, or tumor tissue structures near capillaries, and can simulate specific cell populations of tumor tissue in time and space. Multicellular tumorspheres are ideal in vitro models for studying tumor formation mechanisms and tumor drug resistance studies. [0003] Currently, several commonly used preparation methods of multicellular tumor spheres are: hanging drop method, liquid covering method, collagen and hydrogel scaffold method, ...

AI Technical Summary

Problems solved by technology

However, its disadvantages are: due to the small volume of culture medium (5-20 μL) in the hanging drop method, the existence of evaporation makes it difficult to maintain the microenvironment of cell culture for a long time, and there are certain difficulties in the subsequent isolation and purification of tumor spheroids. Difference

However, the disadvantage of the liquid covering method is that the operation is relatively simple, but there are many influencing factors, and it is not easy to control the growth state of the cell spheroid, which may lead to non-repeatable

However, the disadvantage of the scaffold method is that the stability of the collagen and hydrogel scaffold methods is poor, and the scaffold may change the microenvironment of tumor cells and affect subsequent analysis.

However, the disadvantages of the rotary culture method are: it is not suitable for cells sensitive to shear force or with low adhesion, continuous stirring is not conducive to visually observing the aggregation of cells, and the equipment structure is relatively complicated, and it is cumbersome to implement, which limits its wide application. (Ruei-Zhen Lin, Hwan-You Chang. Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnology Journal, 2008, 3, 1172–1184.)

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