A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Summary

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Abstract

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels¹or in biomaterial scaffolds². Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

Protocol

1. Preparation of a Single Cell Suspension

1. Adherent cell cultures should be grown to 90% confluence, whereupon monolayers should be rinsed twice with PBS. After draining well, add 2 mls (for 100 mm plates) of 0.05% trypsin-1 mM EDTA, and incubate at 37°C until cells detach. Stop trypsinization by adding 2 mls of complete medium and gently use a 5 ml pipette to triturate the mixture until cells are in suspension. Transfer cells to a 15 ml conical tube.
2. Add 40 μl of a 10 mg/ml DNAse stock and incubate for 5 minutes at RT. Vortex briefly and centrifuge at 200 XG for 5 minutes.
3. Discard supernatant and wash pellet with 1 ml complete tissue ....

Discussion

Studies have shown that culturing cells in a three-dimensional context (3D) produces distinct cellular morphology and signaling when compared to a rigid two-dimensional (2D) culture system⁹. For example, fibroblast-populated collagen gels demonstrate that fibroblast morphology in 3D is quite distinct from that observed in 2D ^10,11. Similarly, 3D culture can induce tissue-specific differentiation of mammary epithelial cells. 3D culture systems have been used to distinguish between normal and malignan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
automatic cell counter	Bio-Rad	TC10
shaking water bath with CO2 gable cover	Labline Instruments	3540