Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real-time without cell damage and generally are based on measuring ohmic resistance or measuring impedance across a wide spectrum of frequencies. TEER measurements for various cell types have been reported with commercially available measurement systems and also with custom built microfluidic implementations. Some of the barrier models that have been widely characterized utilizing TEER include the blood-brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary models. Variations in TEER value can arise due to factors such as temperature, medium formulation and passage number of cells. The aim of this paper is to review the different TEER measurement techniques and analyze their strengths and weaknesses, the significance of TEER in drug toxicity studies, examine the various in vitro models and microfluidic organs-on-chips implementations utilizing TEER measurements in some widely studied barrier models (BBB, GI tract and pulmonary), and discuss the various factors that can affect TEER measurements.

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. Highfidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 V Á cm 2 on day 3 on chip and were sustained above 2000 V Á cm 2 up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response.

Human skin constructs (HSCs) have the potential to provide an effective therapy for patients with significant skin injuries and to enable human-relevant drug screening for skin diseases; however, the incorporation of engineered skin appendages, such as hair follicles (HFs), into HSCs remains a major challenge. Here, we demonstrate a biomimetic approach for generation of human HFs within HSCs by recapitulating the physiological 3D organization of cells in the HF microenvironment using 3D-printed molds. Overexpression of Lef-1 in dermal papilla cells (DPC) restores the intact DPC transcriptional signature and significantly enhances the efficiency of HF differentiation in HSCs. Furthermore, vascularization of hair-bearing HSCs prior to engraftment allows for efficient human hair growth in immunodeficient mice. The ability to regenerate an entire HF from cultured human cells will have a transformative impact on the medical management of different types of alopecia, as well as chronic wounds, which represent major unmet medical needs.

Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival, functionality, and viable engraftment. In this study, we used the latest microfabrication techniques and established a novel bioengineering approach to micropattern spatially-controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs). Using 3D printing technology, we were able to control the geometry of the micropatterned vascular networks. We verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function, which allows for the recapitulation of both topical and systemic delivery of drugs. In addition, we examined the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice and demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall, this innovative bioengineering approach could enable in vitro evaluation of topical and systemic drug delivery, as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds.

Advances in bio-mimetic in vitro human skin models increase the efficiency of drug screening studies. In this study, we designed and developed a microfluidic platform that allows for long-term maintenance of full thickness human skin equivalents (HSE) which are comprised of both the epidermal and dermal compartments. The design is based on the physiologically relevant blood residence times in human skin tissue and allows for the establishment of an air-epidermal interface which is crucial for maturation and terminal differentiation of HSEs. The small scale of the design reduces the amount of culture medium and the number of cells required by 36 fold compared to conventional transwell cultures. Our HSE-on-a-chip platform has the capability to recirculate the medium at desired flow rates without the need for pump or external tube connections. We demonstrate that the platform can be used to maintain HSEs for three weeks with proliferating keratinocytes similar to conventional HSE cultures. Immunohistochemistry analyses show that the differentiation and localization of keratinocytes was successfully achieved, establishing all sub-layers of the epidermis after one week. Basal keratinocytes located at the epidermal-dermal interface remain in a proliferative state for three weeks. We use a transdermal transport model to show that the skin barrier function is maintained for three weeks. We also validate the capability of the HSE-on-a-chip platform to be used for drug testing purposes by examining the toxic effects of doxorubucin on skin cells and structure. Overall, the HSE-on-a-chip is a user-friendly and cost-effective in vitro platform for drug testing of candidate molecules for skin disorders.

Advances in maintaining multiple human tissues on microfluidic platforms has led to a growing interest in developing microphysiological systems for drug development studies. Determining the proper design principles and scaling rules for body-on-a-chip systems is critical for their strategic incorporation into physiologically based pharmacokinetic (PBPK)/pharmacodynamic model (PD) -aided drug development. While the need for a functional design considering organ-organ interactions has been considered, robust design criteria and steps to build such systems have not yet been defined mathematically. In this paper, we first discuss strategies for incorporating body-on-a-chip technology into current PBPK modeling-based drug discovery to provide a conceptual model. We propose two types of platforms that can be involved in different stages of PBPK modeling and drug development; these are a μOrgans-on-a-chip and a μHuman-on-a-chip. Then we establish design principles for both types of systems and develop parametric design equations that can be used to determine dimensions and operating conditions. In addition, we discuss the availability of the critical parameters required to satisfy the design criteria, consider possible limitations on estimating such parameter values and propose strategies to address such limitations. This paper is intended to be a useful guide to the researchers focused on designing microphysiological platforms for PBPK/PD based drug discovery.

Hypoxia plays an important role in vascular development through hypoxia-inducible factor-1alpha (HIF-1alpha) accumulation and downstream pathway activation. We sought to explore the in vitro response of cultures of human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), human endothelial progenitor cells (hEPCs), and human umbilical cord vein endothelial cells (HUVECs) to normoxic and hypoxic oxygen tensions. We first measured dissolved oxygen (DO) in the media of adherent cultures in atmospheric (21% O(2)), physiological (5% O(2)), and hypoxic oxygen conditions (1% O(2)). In cultures of both hEPCs and HUVECs, lower oxygen consumption was observed when cultured in 1% O(2). At each oxygen tension, feeder-free cultured hESCs and iPSCs were found to consume comparable amounts of oxygen. Transport analysis revealed that the oxygen uptake rate (OUR) of hESCs and iPSCs decreased distinctly as DO availability decreased, whereas the OUR of all cell types was found to be low when cultured in 1% O(2), demonstrating cell adaptation to lower oxygen tensions by limiting oxygen consumption. Next, we examined HIF-1alpha accumulation and the expression of target genes, including VEGF and angiopoietins (ANGPT; angiogenic response), GLUT-1 (glucose transport), BNIP3, and BNIP3L (autophagy and apoptosis). Accumulations of HIF-1alpha were detected in all four cell lines cultured in 1% O(2). Corresponding upregulation of VEGF, ANGPT2, and GLUT-1 was observed in response to HIF-1alpha accumulation, whereas upregulation of ANGPT1 was detected only in hESCs and iPSCs. Upregulation of BNIP3 and BNIP3L was detected in all cells after 24-h culture in hypoxic conditions, whereas apoptosis was not detectable using flow cytometry analysis, suggesting that BNIP3 and BNIP3L can lead to cell autophagy rather than apoptosis. These results demonstrate adaptation of all cell types to hypoxia but different cellular responses, suggesting that continuous measurements and control over oxygen environments will enable us to guide cellular responses.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in theCOL7A1gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in theCOL7A1gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal–epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient’s quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in theCOL7A1gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.