Microbes and Infection 16 (2014) 707e710 www.elsevier.com/locate/micinf


Flatland goes 3D* Upon the acquisition of a novel fish for decorative purposes, the considerate aquarist usually takes care of placing the animal into the ideal conditions, meant to faithfully mimic the natural environment of the water folks and composed of diverse greenery, various cobblestones, colourful miniature shipwrecks and Elvis Presley figurines. Compared to the loving fish owner, the average cellular biologist cares much less about the culture shock of his study objects, kept in monotone monolayers on flat and hard plastic or glass surfaces, with a gratis plush toy from some antibody provider on a shelf as maximal decoration. Scientists from the DSTL Porton Down e a “dark” place obviously fuelling the minds of internet conspiracy enthusiasts thanks to it being a secret scientific and military research institute [2] e correctly address the issue in the present research article [1], the problem being that 2D monolayer culture models are way too often far from being appropriate for the scientific question asked. So what is wrong with flat biology, a process developed in 1907 and one of the major technical advances in life sciences, the fantastic counterpart to living organisms which tend to be complicated, expensive, ethically contestable and to bite occasionally [3,4]? Technically speaking, flat biology is fine as long as the cell is considered as a liquid bag filled with molecules, and as an isolated unit. Given this axiom, a monolayer equals no more than a few million statistical replicas of a single cell. This kind of system is indeed suitable when it comes to the dissection of events at the molecular level, events uniquely due to basic thermodynamics e diffusion, differential degrees of affinity and sterical hindrance, aided by the compartmentalization of the eucaryote cell. Needless to say, the possibility to decipher if molecule A binds to molecule B is far from being negligible, as the quasi-totality of our knowledge of gene expression and signalling pathways derives from Flatland [5,6]. The single unit of an organ, however, is not the isolated cell, but a unit of different cells, a building block of tissue


Article highlight based on “The use of a three-dimensional cell culture model to investigate hostepathogen interactions of Francisella tularensis in human lung epithelial cells” by Jonathan David et al. [1].

repetitively combined into a larger structure, like sinusoids making up lobules, making up a liver [4,5]. François Jacob very correctly pointed out that the laws of each additional layer of complexity are more than the sum of the laws of the previous layer [7], thus preserving the nature of the elementary tissue unit is primordial in most cases. Concretely, this nature splits up into two main compounds, form and composition. The in vitro study of infection, without surprise, is a perfect example. Infection, first of all, is not an individual but a family business [3]. An army ready to meet an invader is not composed uniquely of one single type of soldiers but of cavalry, infantry, generals and drummers, according to the flea market's lead soldiers. Nor is a tissue composed of one single cell type, least of all the immune system. Furthermore, the placement of the army's components is of major strategical importance, still according to the lead soldiers. So is the architecture of the tissue. Tissue heterogeneity is theoretically solved by the coculture of various cell types, however, most attempts to recreate a more faithful in vitro imitation of an in vivo tissue have focused first onto the recapitulation of the natural tissue architecture. Nota bene, 3D culture does not forcefully mean the stacking of various cell layers. An epithelium is considered a 3D culture provided that cells present an apical-basal polarity and the related functions, such as secretion and a correct free/contact- surface ratio. Nowadays, it is broadly accepted that position, contact with neighbouring cells and the extracellular matrix (ECM) and the resulting polarity, mechanical constraints, shear forces and gradients of signalling molecules are fundamental for a cell's function and decisions from survival, proliferation to secretion of specific compounds [5]. And this is exactly where the major deficiencies of the 2D cultures reside. Taking the example of infection studies, the relevance of shape and arrangement is of an overwhelming evidence. Cells in monolayers, lost on a (too) huge and stiff surface and deprived of an ECM, inevitably spread out, loose their polarity and flatten until only 5% of the cell surface contacts its compatriots, while the average epithelial cell shares at least 70% of its surface with the ECM and neighbours and moreover tightly binds to the latter through various junctions, establishing additional barriers [6,8]. As a consequence, no

http://dx.doi.org/10.1016/j.micinf.2014.07.008 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.


Highlight / Microbes and Infection 16 (2014) 707e710

major mathematical abilities are required to foresee that monolayer cultures, offering a huge entry surface to pathogens and being unable to produce antimicrobial substances like mucus, due to the lack of polarity, are way more vulnerable to bacterial entry than their epithelial counterparts [3]. The use of those deliberately handicapped specimens, unable to mount a correct defence mechanism, burdened beyond in vivo occurring difficulties, is highly questionable. Similar basic, geometrical arguments apply to other major branches of research. Tumour cells are well known to form solid masses and for their ability to squeeze through various tissues during metastasis [8]. Nevertheless, 2D cell culturebased approaches represent still the first step of anti- cancer drug screening, in spite of the striking differences of results obtained on the same cell line grown either as a monolayer or as spheroids. While the 2D versions of numerous cancer cell lines display a high sensitivity to chemical drugs or radiation at low doses, they turn out to be much more resistant in form of spheroids, similar to the in vivo scenario, where the limited diffusion of a drug into the tumour has to be taken into account. Contrariwise, hypoxia-activated cytotoxins are more efficient on cancer cell spheroids, whose hypoxic core probably mimics quite accurately the conditions found in the innards of solid tumours. Considering the enormous losses of time and money during dug development e only 10% of compounds manage to complete the entire road from bench to bedside at a coast of 1 billion US dollars per new approved drug e improving the initial steps of compound library screening seems fundamental [4]. But the realization of the limitations of 2D cultures is endemic. Even one of the latest issues en vogue, long noncoding RNAs, are no exception to the rule, as it became clear how big the gap between knock-out phenotypes in vitro and in animal models are [9] e while the KO of famous Hotair in mouse fibroblasts jumbles the methylation and expression of an entire Hox gene cluster, the effects on mouse physiognomy are deceivingly minor. In order to bridge the gap between Petri dish and body, during the last decades, a panoply of techniques and materials have been developed in order to allow both cell lines and primary cells of virtually any organ to feel at home in vitro, and a flock of articles published taking an inventory of them (a small compendium being referenced here). They form an impressive continuum of complexity ranging from floating, moderately organized cell balls, suitable for drug screening on cancer cells, over various epithelia sticking on ECM-coated spheres to “organs on a chip”, elaborated organoids like the optic cup [5,10], sometimes even with integrated vascularization [3]. The most amazing recent advances, which benefitted from intense media coverage, stem without doubt from the field of regenerative medicine, in form of the generation of reasonably functional organs in a test tube: all cellular components of an organ were digested, leaving only the ECM and thus the memory of the organ's shape. The latter was repopulated with stem cells whose differentiation was subsequently induced, giving rise for example to a urineproducing rat kidney [11].

The essence of those novel techniques is an impressive use of the prefix micro- and the definitive move-in of physicists and engineers into the biologists' headquarters, leading to the creation of both jobs and terms such as bioengineering [5,8]. The very rotating-wall vessel bioreactor was originally developed by the NASA in 1992 in order to mimic microgravity while exerting minimal shear force on the cultures [4]. Using fancy terms like soft lithography and laser activation, their principal purpose is to design tools and furniture at cell size [10,12], from artificial stem cell niches with a precise control of the nature and localization of adhesion molecules to microfluidic devices, meant to regulate mechanical and chemical signalling in time and space. The step by step conquest of the cellular microenvironment opens an entirely new playground, where even in 2D much more exciting activities than dull monolayer culture exist, as proven by cells capable of solving mazes and the first World Cell Race in 2011, when over 50 cell lines from laboratories around the world competed on fibronectin coated lines [12]. Considering the evidence of how aberrant monolayer culture often is and the commercial availability of appropriate tools [4], it is surprising how little use is yet made of those techniques. For sure, there is still some way to go until using 3D cultures becomes an automatism. On the one hand, protocols and compounds, like synthetic ECM, have to be standardized and each required degree of complexity of a model assigned to one biological question [10]. One the other hand, this being a major issue by the way, the analysis tools have to catch up with and to adapt to the novel models, especially in the era of highthroughput analyses, aiming to assess every system as a whole and generating monstrous quantities of data [12]. However, confocal microscopy is not adapted to tissue thickness exceeding 320 mm and microarrays to highly heterogeneous samples [5] e the opportunity for less famous but more appropriate techniques such as optical projection tomography and light sheet microscopy to enter the stage [6]… “Either this is madness or it is Hell.” “It is neither,” calmly replied the voice of the Sphere, “it is knowledge; it is three dimensions: open your eye once again and try to look steadily.” [13]. 1. Biosketch e Jonathan David Dr. Jonathan David has a BSc (Hons) in Biochemistry and Genetics and a PhD in Life and Biomolecular Sciences. He started his career in 2003 working for the UK Health Protection Agency working on the Bacillus anthracis vaccine program. Since 2005, he has worked for DSTL and has 10 years experience working with numerous dangerous pathogens (ACDP3), delivering medical countermeasures for UK government. He has been a Senior Scientist at DSTL since 2010. Dr. David's research interests include how the lung epithelium responds to infection and the use of high-throughput technologies (microarray, RNASeq) to investigate the host response to infections in order to identify and test novel therapeutic targets.

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4. What is the take-home message of the article? Really there are two take home messages; 1) always use an appropriate in vitro model to answer your specific scientific question (this is something that is often overlooked in favour of ease and decreased cost) and 2) high-throughput technologies are a fascinating research tool for studying the host response to infection but will not solve all questions. I believe that these technologies can be “over-sold” and care is needed for the complex down-stream analysis and subsequent follow-on work. 5. Do you have a personal motto, quote or leading sentence?

2. Interview with Jonathan David

“If you trust in yourself, believe in your dream and follow your star….you’ll still get beaten by people who spent their time working hard and learning things and weren't so lazy”. Terry Pratchett, 2003.

1. What triggered your interest in applying a 3D cell culture system to Francisella tularensis infection?

6. What advice would you give to the young next-generation scientists?

Many bacterial infections are well studied in immune cell types but the emerging discovery that the lung epithelium plays a complex role in innate immunity is one that I find particularly interesting and inspiring. A passion of mine since graduating has been the use of appropriate in vitro models to answer specific scientific questions. In fact one of my first actions during my doctoral studies was to write a review of various in vitro lung models and their uses. Most of my work is driven towards the end goal of creating therapies for human infection and any model I use in my work must be as closely relevant to the human lung as possible, hence applying 3D cell culture systems as a model to study the infection. I am still fascinated by in vivo-like cell models and my current research is exploring the use of even more in vivo-like models to study infection.

Ask lots and lots of questions. There is no such thing as a silly question and it is only by questioning things that we can make discoveries. 7. What is your favourite hang-out method after a tough day at the lab? Walking my dog, a working Cocker Spaniel called Merry. 8. If you could travel forth in time e what eventual invention would you like to check out? Any invention that would allow me to have more hours in the day would be a good start!

2. What was your first reaction when you faced the results? Did you expect them? The confirmation of the in vivo-like phenotype of the model for use during infection assays was the first big relief as engineering the system to do infections on the beads at high containment would have been very costly and perhaps beyond our reach. The observation of the increase in resistance in infection only served to increase the excitement of carrying out the microarray work and the down-stream biological analysis. It is the application of high-throughput technologies (microarray, RNASeq etc.) and the complex analysis that is the area of science I enjoy the most. 3. How will the project go on? As outlined in the paper, several hypotheses were generated which may yield answers in our quest to find therapies against Francisella. These hypotheses are currently being explored and we have identified some promising targets and host modulators which are biasing the host towards protection against infection.

Background - Rotating-wall vessel (RWV) bioreactors were designed in 1992 by the NASA in order to mimic microgravity. - The system consists in a culture chamber screwed onto a rotator that slowly turns around a horizontal axis, keeping the cells in a permanent gentle fall with low fluid shear, comparable to the conditions between the vili of epithelial body cells. - Cell lines or primary cells either form spontaneous aggregates or attach onto porous extracellular matrix (ECM)-coated beads and differentiate into 3D tissuelike structures. - The system has been successfully used to mimic human intestine, bladder, lung, liver, placenta, neuronal tissue, tonsil and vaginal epithelium.


Highlight / Microbes and Infection 16 (2014) 707e710


In A Nutshell - A549 cells grown in RWV bioreactors display more an in vivo-like phenotype than monolayer cultures by maintaining cell polarity, stratification, mucus production and regulation of the extracellular matrix. - The transcriptomes of A549 cells grown either as a monolayer or in RWV-cultures differ significantly. - Intracellular infection by Francisella tularensis is higher in A549 cells cultured in monolayer than in RWV bioreactors. - The comparison of the transcriptomes of A549 monolayer and RWV-culture cells upon infection by Francisella tularensis reveals pathways implicated in the higher resistance to the pathogen of RWVcultured cells, such as the CDC42 pathway. - Inhibition of CDC42 decreases Francisella tularensis infection in monolayer cultures of A549 cells to the level of RWV-cultures.

[1] David J, Sayer NM, Sarkar-Tyson M. The use of a three-dimensional cell culture model to investigate host-pathogen interactions of Francisella tularensis in human lung epithelial cells. Microbes Infect 2014;16:735e45. [2] http://www.dark-places.org.uk/site/dstl-porton-down. [3] Barrila J, Radtke AL, Crabbe A, Sarker SF, Herbst-Kralovetz MH, Ott CM, et al. Organotypic 3D cell culture models: using the rotating wall vessel to study host- pathogen interactions. Nat Rev Microbiol 2010;8:791e801. [4] Breslin S, O'Driscoll L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today 2013;18:240e9. [5] Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol 2013;32:108e15. [6] Page H, Flood P, Reynaud EG. Three-dimensional tissue cultures: current trends and beyond. Cell Tissue Res 2013;352:123e31. [7] Jacob F. Evolution and tinkering. Science 1977;196:1161e6. [8] Baker BM, Chen CS. Deconstructing the third dimension e how 3D culture microenvironments alter cellular cues. J Cell Sci 2012;125:3015e24. [9] Kohtz JD. Long non-coding RNAs learn the importance of being in vivo. Front Genet 2014;5:45. [10] Gjorevski N, Ranga A, Lutolf MP. Bioengineering approaches to guide stem cell-based organogenesis. Development 2014;141:1794e804. [11] Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, Ott HC. Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat Med 2013;19:646e51. [12] Lautenschl€ager F, Piel M. Microfabricated devices for cell biology: all for one and one for all. Curr Opin Cell Biol 2013;25:114e24. [13] E.A. Abbott. Flatland: a romance of many dimensions.

Sophia H€afner Universite Paris Diderot, Sorbonne Paris Cite, UMR 7216 CNRS, Epigenetics and Cell Fate, 75013 Paris, France E-mail address: [email protected] 20 July 2014

Flatland goes 3D.

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