With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.

A lack of physiological parity between 2D cell culture and in vivo, has paved the way towards more organotypic models. Organoids exist for a number of tissues, including the liver. However, current approaches to generate hepatic organoids suffer drawbacks, including a reliance on extracellular matrices (ECM), the requirement to pattern in 2D culture, costly growth factors and a lack of cellular diversity, structure and organisation. Current hepatic organoid models are generally simplistic, composed of hepatocytes or cholangiocytes, which renders them less physiologically relevant when compared to native tissue. Here we aim to address these drawbacks. To address this, we have developed an approach that does not require 2D patterning, is ECM independent combined with small molecules to mimic embryonic liver development that produces massive quantities of liver like organoids. Using single cell RNA sequencing and immunofluorescence we demonstrate a liver like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, innervation and a population of resident macrophage, the Kupffer cells. The organoids exhibit key liver functions including drug metabolism, serum protein production, coagulation factor production, bilirubin uptake and urea synthesis. The organoids can be transplanted and maintained in mice producing human albumin long term. The organoids exhibit a complex cellular repertoire reflective of the organ, have de novo vascularization and innervation, enhanced function and maturity. This is a prerequisite for a myriad of applications from cellular therapy, tissue engineering, drug toxicity assessment, disease modeling, to basic developmental biology.

In vitro screening methods for compound efficacy and toxicity to date mostly include cell or tissue exposure to preset constant compound concentrations over a defined testing period. Such concentration profiles, however, do not represent realistic in vivo situations after substance uptake. Absorption, distribution, metabolism and excretion of administered substances in an organism or human body entail gradually changing pharmacokinetic concentration profiles. As concentration profile dynamics can influence drug effects on the target tissues, it is important to be able to reproduce realistic concentration profiles in in vitro systems. We present a novel design that can be integrated in tubing-free, microfluidic culture chips. These chips are actuated by tilting so that gravity-driven flow and perfusion of culture chambers can be established between reservoirs at both ends of a microfluidic channel. The design enables the realization of in vivo -like substance exposure scenarios. Compound gradients are generated through an asymmetric Y-junction of channels with different hydrodynamic resistances. Six microtissues (MTs) can be cultured and exposed in compartments along the channel. Changes of the chip design or operation parameters enable to alter the dosing profile over a large range. Modulation of, e.g., the tilting angle, changes the slope of the dosing curves, so that concentration curves can be attained that resemble the pharmacokinetic characteristics of common substances in a human body. Human colorectal cancer (HCT 116) MTs were exposed to both, gradually decreasing and constant concentrations of Staurosporine. Measurements of apoptosis induction and viability after 5 h and 24 h showed different short- and long-term responses of the MTs to dynamic and linear dosing regimes

Pharmacokinetic DDIs arise upon modulation of the absorption, distribution, and metabolism or excretion (ADME) properties of one drug by co-administration of another drug and can lead to massive changes of drug plasma concentrations with potentially life-threatening consequences. [6] Most commonly, a perpetrator drug modulates the metabolism of a victim drug by chemical inhibition or transcriptional induction of drug-metabolizing enzymes. In both cases, such an interaction leads to an altered metabolic fate of the victim drug. [7] The clinical implications of such interactions can result in a lack of efficacy, especially in the context of so-called prodrugs. Prodrugs are drug variants with enhanced solubility or lifetime, which rely on in vivo bioconversion to exert their pharmacological activity. [8] Positive treatment outcomes in prodrug therapies strongly depend on the ability of the patient to endogenously bioactivate the prodrug compound to its pharmacologically active metabolite(s). [9] Drug metabolism predominantly occurs in the liver and is mainly catalyzed by enzymes of the cytochrome P450 (CYP) family. These enzymes oxidize, reduce, and hydrolyze a wide range of drugs or chemical entities. Therefore, it does not come as a surprise that inhibition or induction of CYP enzymes is largely involved in clinical DDIs and has led to severe ADRs and the withdrawal of multiple drugs from the market. [10] In many cases, DDIs manifest themselves in secondary organs, to which liver-generated metabolites are transported through systemic circulation. Such tissues include kidney, heart, brain, gut, and lungs, and drug target tissues, such as tumors. [11] Oncology patients are considered especially prone to toxic DDI events owing to i) the wide use of anticancer prodrugs, [12,13] ii) the inherent toxicity of anticancer agents and their very narrow therapeutic index, iii) the likelihood of cancer patients to receive many different medications for the management of other illnesses, [14] and iv) the increasing use of combination therapies with more than one anticancer medication. [15] Hence, clinicians are confronted with the growing risk of prescribing drug combinations that can inadvertently lead to severe clinical implications. [16] Additionally, genetic polymorphisms in the liver can lead to individual patterns of CYP-enzyme-mediated metabolism for different patients. [17] Management of DDIs is, therefore, crucial in oncology therapy, where the altered Drug-drug interactions (DDIs) occur when the pharmacological activity of one drug is altered by a second drug. As multimorbidity and polypharmacotherapy are becoming more common due to the increasing age of the population, the risk of DDIs is massively increasing. Therefore, in vitro testing methods are needed to capture such multiorgan events. Here, a scalable, gravity-driven microfluidic system featuring 3D microtissues (MTs) that represent different organs for the prediction of drug-drug interactions is used. Human liver microtissues (hLiMTs) are combined with tumor m...