Facile Tumor Spheroids Formation in Large Quantity with Controllable Size and High Uniformity

Abstract

A facile method for generation of tumor spheroids in large quantity with controllable size and high uniformity is presented. HCT-116 cells are used as a model cell line. Individual tumor cells are sparsely seeded onto petri-dishes. After a few days of growth, separated cellular islets are formed and then detached by dispase while maintaining their sheet shape. These detached cell sheets are transferred to dispase-doped media under orbital shaking conditions. Assisted by the shear flow under shaking and inhibition of cell-to-extracellular matrix junctions by dispase, the cell sheets curl up and eventually tumor spheroids are formed. The average size of the spheroids can be controlled by tuning the cell sheet culturing period and spheroid shaking period. The uniformity can be controlled by a set of sieves which were home-made using stainless steel meshes. Since this method is based on simple petri-dish cell culturing and shaking, it is rather facile for forming tumor spheroids with no theoretical quantity limit. This method has been used to form HeLa, A431 and U87 MG tumor spheroids and application of the formed tumor spheroids in drug screening is also demonstrated. The viability, 3D structure, and necrosis of the spheroids are characterized.

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“…HeLa cells grown in spheroids were more resistant to drug treatment compared to 2D monolayer culture (Figure ): for doxorubicin IC50 were ≈6 and <0.05 × 10 −6 m ; for oxaliplatin ≈2.3 and ≈0.1 × 10 −6 m ; for 5‐fluoruracil 1 × 10 −6 m and ≈0.4 × 10 −6 m for 3D and 2D cell culture, respectively. Increased resistance of tumor spheroids to anticancer compounds compared to monolayer culture of the same type of cells was reported multiple times previously …”

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“…g) 3D OCT image of a spheroid consisting of human colorectal cancer cells to confirm the spherical shape is maintained. Reproduced under the terms and conditions of the CC‐BY‐4.0 license . Copyright 2018, The Authors, published by Springer Nature.…”

Heparin-based hydrogel scaffolding alters the transcriptomic profile and increases the chemoresistance of MDA-MB-231 triple-negative breast cancer cells

“…Recent statistics estimate approximately 90% of chemotherapies and immunotherapies fail when translated to in vivo solid tumors, despite promising in vitro experments. 6 The hanging-drop method, 7 the use of non-adhesive substrates, 8 and orbital shaking 9 have been previously used to form spheroids in aqueous culture. However, none of these methods encourage self-assembly, but are rather focused on external physical stimuli to encourage cell aggregation.…”

“…The hanging-drop method, 7 the use of non-adhesive substrates, 8 and orbital shaking 9 have been previously used to form spheroids in aqueous culture. However, none of these methods encourage self-assembly, but are rather focused on external physical stimuli to encourage cell aggregation.…”