ARTICLE Received 3 Aug 2016 | Accepted 13 Jan 2017 | Published 28 Mar 2017 DOI: 10.1038/ncomms14584 OPEN A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle Shuo Xiao1,*, Jonathan R. Coppeta2,*, Hunter B. Rogers1,*, Brett C. Isenberg2, Jie Zhu1,*, Susan A. Olalekan1,*, Kelly E. McKinnon1,*, Danijela Dokic1, Alexandra S. Rashedi1, Daniel J. Haisenleder3, Saurabh S. Malpani1, Chanel A. Arnold-Murray1, Kuanwei Chen1, Mingyang Jiang1, Lu Bai1, Catherine T. Nguyen1, Jiyang Zhang1, Monica M. Laronda1, Thomas J. Hope1, Kruti P. Maniar4, Mary Ellen Pavone1, Michael J. Avram5, Elizabeth C. Sefton1, Spiro Getsios6, Joanna E. Burdette7, J. Julie Kim1, Jeffrey T. Borenstein2 & Teresa K. Woodruff1 The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ–organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies. 1 Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. 2 The Charles Stark Draper Laboratory, Cambridge, Massachusetts 02139, USA. 3 Ligand Assay and Analysis Core, Center for Research in Reproduction, University of Virginia, Charlottesville, Virginia 22908, USA. 4 Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. 5 Department of Anesthesiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. 6 Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. 7 Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60607, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to T.K.W. (email: [email protected]). NATURE COMMUNICATIONS | 8:14584 | DOI: 10.1038/ncomms14584 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14584 he female reproductive tract is required for the production Results of ova, secretion of sex hormones and the maintenance Microfluidic technology enabled dynamic tissue integration. Tof pregnancy throughout the gestation of healthy offspring. The first step of our work was to develop platforms that could Entry and advancement through puberty, normal menstrual sustain tissue-level function for the length of the human menstrual cycles with a potential intervening pregnancy, and endocrine cycle (that is, 28 days). The Solo-MFP and Duet-MFP systems are support of peripheral tissues, such as the bone, brain and heart, based on pneumatic actuation technology, by which the individual are all essential roles that the reproductive tract tissues and their systems are supplied with positive and negative air pressures via a hormones play. The main organs of the female reproductive tract system dock that is connected to a five-channel pressure controller are the ovaries, fallopian tubes, uterus and cervix. Each organ manifold (Fig. 1a,b, refer to Supplementary Table 1 for nomen- has major responsibilities that are either autonomous (for clature). The pressure of individual channels is switched between a example, maturation of oocytes and gestation of the fetus) or vacuum or pressure source using an electromagnetic three-way interdependent such as providing hormonal support for the tracts valve controlled via a personalized pump programme created in through which gametes (eggs and sperm) travel and a location for LabVIEW using a computer interface. The Solo-MFP and Duet- the developing embryo to implant. Moreover, each organ is MFP systems use a universal pneumatic plate that distributes composed of cells from multiple lineages (for example, myome- positive and negative air pressures to specific valve or pump trium and endometrium in the uterus) that provide local function membranes clamped between the pneumatic and fluidic plates. and feedback control. Because of the intimate relationship Sequential application of pressure and vacuum to valve and pump between the cells of each organ and between organs, toxicology membranes creates a peristalsis-like stroke that enables fluid to studies in the female reproductive tract have been difficult to move through individual microfluidic paths within or between design. modules. Four valves arranged in North, South, East and West Methods used to grow mammalian cells outside the body positions that are oriented about a central pump chamber create a have not fundamentally changed in the last 50 years. Preclinical four-port pump structure, enabling multiple bi-directional flows in studies often begin with individual cells, separated from each fluidic circuit. The fluidic plates are specifically designed for cellular and physical contacts that are important for biological either the Solo-MFP or Duet-MFP module configuration. Solo- function1. These dispersed cells must be propagated MFP systems consist of four replicates of a fluidic circuit with two through weekly reduction divisions and maintained on flat connected modules: a coupled donor/acceptor module and a plastic; however, these cells are missing the cell physicochemical module for tissues (Fig. 1a). Duet-MFP systems consist of a single microenvironment, three-dimensional (3D) tissue-specific set of four modules: a donor module, two modules for tissues, and architecture, and blood flow perfusion found in natural a separate acceptor module (Fig. 1b). Pneumatic actuation tissues. Furthermore, typical media composition is based on manipulates the membranes, generating pressure-driven flow in basal nutrients, bovine serum and a few specialized factors that the fluidic paths, in order to transport fresh media from the donor are placed in a static setting with random mixing. As a to tissues, remove older media and secreted factors to the acceptor, consequence, cell–cell and tissue-level cytokine and endocrine or (in the case of Duet-MFP systems) move fluid from the signals are not integrated into signalling pathways. In parallel upstream tissue to the downstream tissue to enable in vitro com- with these developments, the pharmaceutical industry is munication between cultured tissues. challenged by the fact that fewer drugs are emerging to address To integrate five tissues in a single system, a more practical and many unmet needs, including cardiovascular disease, cancer, scalable approach was used for microfluidic control of tissue immune diseases, and new contraceptives2–4. Despite large interaction (Fig. 1c). This was accomplished by embedding investments in research funding, only B8% of drugs for which electromagnetically actuated micropumps within the platform, Investigational New Drug applications have been filed will be termed the Quintet-MFP, obviating the need to supply the platform approved by the FDA5. Innovative methods to culture cells with independent air lines for each pneumatic actuator (Fig. 1d). in vitro to test new compounds are therefore necessary to This design approach allowed each of the 60 actuators of the reinvigorate the drug pipeline. Quintet-MFP to be individually controlled, thus enabling precise Recently, organ-on-a-chip and human-on-a-chip microfluidic flow control over a wide dynamic range. Modules included flow technologies have garnered significant interest and offer promis- ports to permit recirculation within each module, ensuring that the ing approaches to test the efficacy and toxicity of new drugs system was well mixed and enabling homogenous exposure of in vitro16–18. Microfluidics represents an engineered cultured tissues to factors within the media. In addition to manipulation of fluid flow in a set of micrometre-sized recirculation within individual modules, the fluidic path design channels and provides precise control of microlitre volume of allowed for whole-system recirculation (Fig. 1c). The combination fluids. In the current study, Solo-MFP and Duet-MFP systems of whole-system and intermodule recirculation enabled a well- based on pneumatic actuation technology and a Quintet-MFP mixed system within and across all modules of the Quintet-MFP. system based on embedded electromagnetic actuation technology Each fluidic path between modules was controlled by two sets of were designed for single and multiple tissue cultures, respectively. pump actuators. This redundancy acted as a fail-safe in the event of Mouse ovarian tissue was cultured in the Solo-MFP and Duet- actuator failure. A total of twelve modules, including one donor, MFP systems for 28 days, which resulted in follicle production one acceptor, five tissue-specific modules, and five blank modules of 28-day menstrual cycle hormone profiles. To test the ovarian (for increased