Goodbye flat biology - time for the 3rd and the 4th dimensions.

It is nowmore than a decade since Allison Abbot of Nature wrote an editorial using the first part of the above title (Abbott, 2003a) and, inside the same issue, under the title of ‘Nature’s Third Dimension’(Abbott, 2003b), described the efforts of a number of laboratories that had come to terms with using three-dimensional (3D) strategies, and described in more detail our laboratory’s development of 3D assays, which involved growing cells inside laminin-rich gels. These assays could be used for both functional studies of tissue-specific gene expression in rodents (Barcellos-Hoff et al., 1989) and, in collaboration with Ole Petersen, to develop a simple and robust assay to distinguish normal and/or non malignant breast cells from those that were premalignant and malignant (Petersen et al., 1992). The assays we developed are already more than 25 years old, and the procedures were described further in Nature Methods in 2007 (Lee et. Al., 2007). Thesewere the beginnings of the 3D cell biology field, although they followed a respectable body of literature that had been published years earlier describing many attempts to cultivate cells to restore function in culture using everything from weighing papers floated on top of the media to floating collagen gels from rat tails (for a timeline of some such attempts, please see Simian and Bissell, 2017). The defining difference between the use of laminin-rich gels and the previous attempts to culture cells was that, with time, the lamininrich cultures developed the foot prints of both functional and morphological characteristics of the tissue of origin. In the case of the mammary gland, such cultures could make copious amounts of milk and also form acini that were similar to the unit structure of mammary glands of pregnant mice. Appreciable numbers of laboratories worldwide have begun to switch from flat substrata to laminin-rich cultures to test drug behavior with promising data. Many companies not only provide different kinds of scaffolds but are rushing also to provide different high-throughput techniques and pre-fabricated substrata, and different disciplines have joined the game of who can provide better ‘designer microenvironments’(Stoker et al., 1990)! I must admit, however, that in my many invited travels across the USA and other countries to lecture on our work, I am often astonished at how many people in my audience have either never heard of 3D cultures or have not given it a serious thought, and thus are startled to see how profoundly results differ with changes in context. There are many differences in the integration of signaling pathways and in the conclusions reached between 2D and 3D assays, with the latter providing results that are much closer to the in vivo situation. In fact, quite a few scientists still don’t see any need to change the ease of culturing on tissue culture plastic, and they don’t want to deal with what they perceive to be the complexities and expense of using 3D cultures, and so continue to look under the light. But I think that the time is now to make the switch to 3D if functional integrity is the end point. I attribute this lack of knowledge and interest to the real gaps in our teachings and our textbooks in the classrooms. Of course we all know some organisms make excellent 3D models. We would not be anywhere close to where we are in our understanding of biology without bacteria, worms, Drosophila, zebrafish and rodents, and many of us use one or more of these organisms as well as 3D cultures. The animal models have provided rich genetic and functional data, and have contributed immensely to our understanding of biological systems through high-throughput mutational analysis, physiological studies and useful screens. Yet the 3D culture systems are unique in their ability to allow us to study human cells and tissues, and also to unravel regulatory circuits and pathways that differ between other species and humans (e.g. for example, see Bissell et al., 1987). An offshoot of thinking in three-dimensions is the past few years’ excitement and the literature of what is being referred to as ‘organoids’. Organoids are great 3D models, but treating them as if it is a new field is like calling ‘a rose by any other name’! What others and we have developed and reported in the literature in the past three decades is nothing if not organoids. Anything that can be cultivated on a substratum that is not tissue culture plastic or any other flat surface, and that would allow imitation of form and, hopefully, function in an organand tissue-specific manner fits under the umbrella of 3D culture and/or organoids. Thus, it is with pleasure that I accepted the invitation to write a short foreword to this impressive volume, conceived by the Journal of Cell Science editorial team and expertly edited by Andrew Ewald. He has also penned the introduction to the volume, in which he pays specific attention to the contributed articles. The past five years have produced a bonanza of reviews and articles from a number of other laboratories, including ours, and in other journals in addition to this volume (Shamir and Ewald, 2014; Lancaster and Knoblich, 2014; Clevers, 2016; Fatehullah et al., 2016). So my intent here is to simply acknowledge the past and raise a few questions and concepts for the future. I see little gain in repeating and re-embellishing 3D and organoid culture history, and so look forward to where we need to go.