A multi-organ-chip co-culture of liver and testis equivalents: a first step toward a systemic male reprotoxicity model

Abstract

STUDY QUESTION Is it possible to co-culture and functionally link human liver and testis equivalents in the combined medium circuit of a multi-organ chip? SUMMARY ANSWER Multi-organ-chip co-cultures of human liver and testis equivalents were maintained at a steady-state for at least 1 week and the co-cultures reproduced specific natural and drug-induced liver–testis systemic interactions. WHAT IS KNOWN ALREADY Current benchtop reprotoxicity models typically do not include hepatic metabolism and interactions of the liver–testis axis. However, these are important to study the biotransformation of substances. STUDY DESIGN, SIZE, DURATION Testicular organoids derived from primary adult testicular cells and liver spheroids consisting of cultured HepaRG cells and hepatic stellate cells were loaded into separate culture compartments of each multi-organ-chip circuit for co-culture in liver spheroid-specific medium, testicular organoid-specific medium or a combined medium over a week. Additional multi-organ-chips (single) and well plates (static) were loaded only with testicular organoids or liver spheroids for comparison. Subsequently, the selected type of medium was supplemented with cyclophosphamide, an alkylating anti-neoplastic prodrug that has demonstrated germ cell toxicity after its bioactivation in the liver, and added to chip-based co-cultures to replicate a human liver–testis systemic interaction in vitro. Single chip-based testicular organoids were used as a control. Experiments were performed with three biological replicates unless otherwise stated. PARTICIPANTS/MATERIALS, SETTING, METHODS The metabolic activity was determined as glucose consumption and lactate production. The cell viability was measured as lactate dehydrogenase activity in the medium. Additionally, immunohistochemical and real-time quantitative PCR end-point analyses were performed for apoptosis, proliferation and cell-specific phenotypical and functional markers. The functionality of Sertoli and Leydig cells in testicular spheroids was specifically evaluated by measuring daily inhibin B and testosterone release, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Co-culture in multi-organ chips with liver spheroid-specific medium better supported the metabolic activity of the cultured tissues compared to other media tested. The liver spheroids did not show significantly different behaviour during co-culture compared to that in single culture on multi-organ-chips. The testicular organoids also developed accordingly and produced higher inhibin B but lower testosterone levels than the static culture in plates with testicular organoid-specific medium. By comparison, testosterone secretion by testicular organoids cultured individually on multi-organ-chips reached a similar level as the static culture at Day 7. This suggests that the liver spheroids have metabolised the steroids in the co-cultures, a naturally occurring phenomenon. The addition of cyclophosphamide led to upregulation of specific cytochromes in liver spheroids and loss of germ cells in testicular organoids in the multi-organ-chip co-cultures but not in single-testis culture. LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The number of biological replicates included in this study was relatively small due to the limited availability of individual donor testes and the labour-intensive nature of multi-organ-chip co-cultures. Moreover, testicular organoids and liver spheroids are miniaturised organ equivalents that capture key features, but are still simplified versions of the native tissues. Also, it should be noted that only the prodrug cyclophosphamide was administered. The final concentration of the active metabolite was not measured. WIDER IMPLICATIONS OF THE FINDINGS This co-culture model responds to the request of setting up a specific tool that enables the testing of candidate reprotoxic substances with the possibility of human biotransformation. It further allows the inclusion of other human tissue equivalents for chemical risk assessment on the systemic level. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by research grants from the Scientific Research Foundation Flanders (FWO), Universitair Ziekenhuis Brussel (scientific fund Willy Gepts) and the Vrije Universiteit Brussel. Y.B. is a postdoctoral fellow of the FWO. U.M. is founder, shareholder and CEO of TissUse GmbH, Berlin, Germany, a company commercializing the Multi-Organ-Chip platform systems used in the study. The other authors have no conflict of interest to declare.

Predicting Metabolism‐Related Drug–Drug Interactions Using a Microphysiological Multitissue System

“…[24][25][26][27] Probing off-target toxicity of compounds in multiorgan systems has proven to be particularly useful for prodrugs that are inactive without hepatic metabolism. [26][27][28][29][30][31] Multiorgan systems represent a potential solution to capture the full extent of complex DDI eventsranging from metabolizing organs to drug target organs-and to predict the safety of critical drug combinations. [32] Here, we present the use of a previously described microfluidic chip featuring multiple tissue compartments to detect and predict metabolism-related DDIs.…”

“…[ 24–27 ] Probing off‐target toxicity of compounds in multiorgan systems has proven to be particularly useful for prodrugs that are inactive without hepatic metabolism. [ 26–31 ] Multiorgan systems represent a potential solution to capture the full extent of complex DDI events—ranging from metabolizing organs to drug target organs—and to predict the safety of critical drug combinations. [ 32 ]…”

Mechanical Stimulation: A Crucial Element of Organ-on-Chip Models

“…Fluid Flow: 10 nL/min-10 µL/min Fluid shear: 0.001-2 Pa Blood-brain barrier (Prabhakarpandian et al, 2013;Deosarkar et al, 2015;Tang et al, 2018;Brown et al, 2019;Da Silva-Candal et al, 2019) Blood vessel (Silvani et al, 2019) Blood vessel: Microvascular network (Rosano et al, 2009;Prabhakarpandian et al, 2011;Lamberti et al, 2013) Cancer models (Tang et al, 2017;Terrell-Hall et al, 2017;Vu et al, 2019) Lung (Kolhar et al, 2013;Liu et al, 2019;Soroush et al, 2020) TissUse https://www.tissuse.com/en/ Fluid shear: 0.02-2 Pa Multi-tissue models: Intestine-Liver-Brain-Kidney (Ramme et al, 2019) Intestine-Liver-Skin-Kidney (Maschmeyer et al, 2015b) Liver-Brain (Materne et al, 2015) Liver-Intestine (Maschmeyer et al, 2015a) Liver-Kidney (Lin et al, 2020) Liver-Lung (Schimek et al, 2020) Liver-Pancreatic islets (Bauer et al, 2017) (Continued) Liver-Skin (Wagner et al, 2013) Liver-Skin-Vasculature (Maschmeyer et al, 2015a) Liver-Testis (Baert et al, 2020) Skin-Lung cancer (Hubner et al, 2018) Single tissue models: Blood vessels (Schimek et al, 2013;Maschmeyer et al, 2015b) Blood vessels: Micro capillaries (Hasenberg et al, 2015) Bone marrow (Sieber et al, 2018) Brain (Materne et al, 2015) Hair follicle biopsies (Atac et al, 2013) Intestine (Maschmeyer et al, 2015a) Kidney (Ramme et al, 2019) Liver (Maschmeyer et al, 2015a;Materne et al, 2015;…”

Advanced 3D Cell Culture Techniques in Micro-Bioreactors, Part II: Systems and Applications

“…The idea of OoC was born about 10 years ago and is constantly evolving [227]. One trend here is to move towards body-or human-on-a-chip models by using multi-organ chips [217,228,229]. The topic of personalized medicine as well as disease models on chips, so-called micro-pathophysiological systems (MPPS), also has an important influence on the development of OoC [148,[230][231][232].…”