Comparison of methods to generate multicellular spheroids with characteristics compliant with 3D high-content screening experiments

OBJECTIVE: High-content screening (HCS) is a powerful approach to discover new drugs and carry out basic biological research. HCS is widely performed in pharmaceutical and biotechnology industries, but always by using 2D cell cultures. In the literature, there are several papers proving that many cell lines behave differently when cultured as monolayer rather than when cultured in 3D. Accordingly, in vitro-built 3D multicellular spheroids are receiving increased interest with evidence showing that the biological response is closer to that of in vivo cells. Today, more and more biological laboratories are using spheroids to test drugs and radiotherapy treatments. However, a well-established and general purpose validated protocol to generate spheroids with characteristics compliant with 3D HCS experiments does not exist. Here we compared several generation methods with the aim of investigating the possibility to generate in a short time hundreds of homogeneous 3D spheroids, suitable for HCS experiments. MATERIALS AND METHODS: We analysed dimension, shape, and homogeneity of Mesenchymal Stromal Cell (MSC) and A549 lung cancer cell spheroids generated by using: (a) a NASA rotatory bioreactor (Synthecon, Houston, USA); (b) several Perfecta3D (3DBiomatrix, Ann Arbor, USA) and GravityPLUS (InSphero, Schlieren, Switzerland) hanging drop plates; (c) magnetic levitation method with magnetic nanoparticle; (d) pellet culture method. To compute the morphological features we used AnaSP (http://sourceforge.net/p/anasp/) and ReVISP (http://sourceforge.net/p/revisp/). RESULTS: Spheroids generated by using magnetic levitation and hanging drop plates are quite small (diameter < 500 µm) and irregular (sphericity index < 0.90). Instead, spheroids generated with the NASA bioreactor are irregular, but characterized by a large dimension. In addition, after waiting few days, the so-called spheroidization time, they become much more spherical. Spheroids generated with the pellet culture method have a large dimension and a spherical shape. As regards the number of spheroids generated, magnetic levitation and NASA bioreactor provide an easy solution to generate a number of spheroids compliant with HCS experiments in a very short time. The pellet culture method depends on the number of chambers in the centrifuge, and more rounds are typically required to generate a sufficient number of spheroids. A high number of hanging drop plates has to be used to generate a sufficient amount of spheroids. To generate the spheroids the NASA bioreactor required two weeks, magnetic levitation and hanging drop plates one week, pellet culture method one day. CONCLUSION: An absolute best solution does not exist to generate spheroids for HCS experiments. Both NASA bioreactor and pellet culture method seem to be a good choice if a number of large spherical spheroids is required. However, due to the short time needed to generate the spheroids, and the absence of a spheroidization time, the pellet culture method seems to be the best solution at one’s disposal.