Eun‐Jin Lee

The interaction of chemicals, nanoparticles, and circulating cells with the endothelium depends on the magnitude of the mechanical shear produced by the flow of blood. When simulating those interactions with microphysiological systems (MPSs), it is critical to reproduce those shear conditions faithfully. For example, unidirectional flow of a specific magnitude keeps the endothelium healthy with normal barrier tissue function, while bidirectional flow mimics disease conditions with compromised barrier function. Additionally, in MPS, recirculating fluid may be necessary to retain tissue-derived factors and metabolites. However, existing MPS designs struggle to achieve medium recirculation of small volumes of liquid with precise flow control. Here, we present an MPS design that is highly versatile and overcomes this limitation. We demonstrate how the device can produce a wide range of fluidic flow rates that can accommodate both low shear conditions suitable for tissues that typically are only exposed to interstitial flow and high shear conditions suitable for barrier tissues that experience blood flow. We demonstrate the device’s functionality by culturing human umbilical vein endothelial cells (HUVEC) and confirming their flow-aligned morphology through immunostaining of the adherens junction protein (VE-cadherin) and actin filaments. Furthermore, we present a mathematical model that can be used to calculate operating parameters for culturing any tissue under optimum conditions. We also discuss how the device can be adjusted to recirculate liquid volumes ranging from 100 µL to 5 mL. This versatile system holds promise for commercial applications, including the investigation of expensive compounds that are limited to very small volume samples such as rare cells (e.g., circulating tumor cells) or engineered therapeutic cells with barrier tissues. By offering precise control over a wide range of flow conditions with medium recirculation of small liquid volumes, our device addresses a critical gap in current MPS technology.

Glucocorticoids (GCs) are standard‐of‐care treatments for inflammatory and immune disorders, and their long‐term use increases the risk of osteoporosis. Although GCs decrease bone functionality, their role in bone microvasculature is incompletely understood. Herein, the study investigates the mechanisms of bone microvascular barrier function via osteoblast‐endothelial interactions in response to GCs. The animal data shows that prednisolone (Psl) downregulated the osteoblast function and microvessel number and size. To investigate the role of GCs in bone endothelial barrier function further, a bicellular microfluidic in vitro system is developed and utilized, which consists of three‐dimensional (3D) perfusable microvascular structures embedded in collagen I/osteoblast matrix. Interestingly, it is demonstrated that GCs significantly inhibit osteogenesis and microvascular barrier function by interfering with endothelial‐osteoblast interactions. This effect is triggered by MAPK‐induced phosphorylation of connexin43 (Cx43) at Ser282. Collectively, this study sheds light on microvascular function in bone disorders, as osteoporosis, and permits to capture dynamic changes in endothelial‐bone interactions under GCs by dissecting the MAPK/Cx43 mechanism and proposing this as a potential target for bone diseases.

Background Interleukin-2 (IL-2) is a pleiotropic cytokine that plays pivotal roles in the immune response. It induces the proliferation of T cells and NK cells and demonstrates therapeutic anti-tumor activity. CD80 expressed on antigen-presenting cells provides co-stimulatory signals for T cell activation and immune responses via CD28 binding. Conversely, CD80 acts as a checkpoint molecule when bound to CTLA-4. Despite the active and progressive efforts put into the development of immune checkpoint inhibitors for cancer therapy, notable unmet medical needs continue to persist. To address these challenges and provide a safe and effective immune-boosting agent, we generated a novel CD80-IgG4-IL2v3 bispecific fusion protein which allows tumor-targeted delivery of IL-2 and robust proliferation of anti-tumor effector cells. Methods The evaluation of anti-tumor efficacy and tumor-infiltrating lymphocytes (TIL) was conducted using the EMT6 syngeneic tumor model. Analysis of peripheral lymphocyte alterations was carried out in nonhuman primates (NHP). Intra-tissue concentration analysis of GI-102 was performed utilizing the LL/2 syngeneic tumor model. Results The N-terminal CD80 domain of GI-102 is designed to target and block CTLA-4, while the C-terminal IL-2v3 domain has no binding affinity to IL-2Rα, while maintaining the affinity towards IL-2Rβγ. In the EMT6 syngeneic mouse model, GI-102-treated group showed significantly reduced tumor volume and percent of tumor growth rate compared with other groups. The analysis of the TIL revealed that GI-102 induced a robust expansion of both CD8⁺ T and NK cells in tumor microenvironment (TME), relative to vehicle control and Proleukin^®. However, in comparison to Proleukin^®, injection of GI-102 did not lead to an expansion of Tregs population in TME compared. A similar pattern was observed in peripheral lymphocytes of NHP that were administered GI-102 and Proleukin^®. Human PBMC treated with GI-102 were analyzed for cytokine release, and no increase in inflammatory cytokines associated with cytokine release syndrome was observed with GI-102. Following the injecting GI-102 into mice bearing LL/2 tumors, elevated levels of GI-102 were detected from serum, tumor and lymphoid organs, suggesting the CD80-guided distribution of the molecule towards area reach in lymphocytes. Conclusions GI-102 is a novel anti-cancer agent that activates immune cells and modulates tumor-infiltrating lymphocytes. This potent IL-2-based bispecific fusion protein has great potential to deliver effective therapeutic outcomes for a various types of cancer. It exhibits low toxicity and does not activate Treg, further enhancing its therapeutic value as the next-generation IL-2.

Fluidic microphysiological systems (MPS) are microfluidic cell culture devices that are designed to mimic the biochemical and biophysical in vivo microenvironments of human tissues better than conventional petri dishes or well‐plates. MPS‐grown tissue cultures can be used for probing new drugs for their potential primary and secondary toxicities as well as their efficacy. The systems can also be used for assessing the effects of environmental nanoparticles and nanotheranostics, including their rate of uptake, biodistribution, elimination, and toxicity. Pumpless MPS are a group of MPS that often utilize gravity to recirculate cell culture medium through their microfluidic networks, providing some advantages, but also presenting some challenges. They can be operated with near‐physiological amounts of blood surrogate (i.e., cell culture medium) that can recirculate in bidirectional or unidirectional flow patterns depending on the device configuration. Here we discuss recent advances in the design and use of both pumped and pumpless MPS with a focus on where pumpless devices can contribute to realizing the potential future role of MPS in evaluating nanomaterials.This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials

Epithelial cell function is modulated by mechanical forces imparted by the extracellular environment. The transmission of forces onto the cytoskeleton by modalities such as mechanical stress and matrix stiffness is necessary to address by the development of new experimental models that permit finely tuned cell mechanical challenges. Herein, we developed an epithelial tissue culture model, named the 3D Oral Epi-mucosa platform, to investigate the role mechanical cues in the epithelial barrier. In this platform, low-level mechanical stress (0.1 kPa) is applied to oral keratinocytes, which lie on 3D fibrous collagen (Col) gels whose stiffness is modulated by different concentrations or the addition of other factors such as fibronectin (FN). Our results show that cells lying on intermediate Col (3 mg/mL; stiffness = 30 Pa) demonstrated lower epithelial leakiness compared with soft Col (1.5 mg/mL; stiffness = 10 Pa) and stiff Col (6 mg/mL; stiffness = 120 Pa) gels, indicating that stiffness modulates barrier function. In addition, the presence of FN reversed the barrier integrity by inhibiting the interepithelial interaction via E-cadherin and Zonula occludens-1. Overall, the 3D Oral Epi-mucosa platform, as a new in vitro system, will be utilized to identify new mechanisms and develop future targets involved in mucosal diseases.

Orthopedic and craniofacial surgical procedures require the reconstruction of bone defects caused by trauma, diseases, and tumor resection. Successful bone restoration entails the development and use of bone grafts with structural, functional, and biological features similar to native tissues. Herein, we developed three-dimensional (3D) printed fine-tuned hydroxyapatite (HA) biomimetic bone structures, which can be applied as grafts, by using calcium phosphate cement (CPC) bioink, which is composed of tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), and a liquid [Polyvinyl butyral (PVB) dissolved in ethanol (EtOH)]. The ink was ejected through a high-resolution syringe nozzle (210 µm) at room temperature into three different concentrations (0.01, 0.1, and 0.5) mol/L of the aqueous sodium phosphate dibasic (Na₂HPO₄) bath that serves as a hardening accelerator for HA formation. Raman spectrometer, X-ray diffraction (XRD), and scanning electron microscopy (SEM) demonstrated the real-time HA formation in (0.01, 0.1, and 0.5) mol/L Na₂HPO₄ baths. Under those conditions_, HA was formed at different amounts, which tuned the scaffolds’ mechanical properties, porosity, and osteoclast activity. Overall, this method may pave the way to engineer 3D bone scaffolds with controlled HA composition and pre-defined properties, which will enhance graft-host integration in various anatomic locations.

Eun Jin Lee, Miyoung Kim, Eunyup Lee, Kibum Jeon, Jiwon Lee, Han-Sung Kim, Hee Jung Kang, and Young Kyung Lee. J Lab Med Qual Assur 2018;40:27-37. https://doi.org/10.15263/jlmqa.2018.40.1.27

Gamma-aminobutyric acid (GABA) is an important retinal neurotransmitter. We studied the expression of GABA, glutamate decarboxylase 65 (GAD65) and GAD67 by immunocytochemistry and Western blot, in the retinas of control and dark-reared C57BL/6J black mice. This study asked three questions. First, is visual input necessary for the normal expression of GABA, GAD65 and GAD67? Second, can the retina recover from the effects of dark-rearing if returned to a normal light-dark cycle? Third, does BDNF prevent the influence of dark-rearing on the expression of GABA and GAD? At postnatal day 10 (P10), before eye opening, GABA immunoreactivity was present in the ganglion cell layer (GCL), in the innermost rows of the inner nuclear layer (INL) and throughout the inner plexiform layer (IPL) of control and dark-reared retinas. In P30 control retinas, GABA immunoreactivity showed similar patterns to those at P10. However, in P30 dark-reared retinas, the density of GABA-immunoreactive cells was lower in both the INL and GCL than in control retinas. In addition, visual deprivation retarded GABA immunoreactivity in the IPL. Western blot analysis showed corresponding differences in the levels of GAD65 but not of GAD67 expression between control and dark-rearing conditions. In our study, dark-rearing effects were reversed when the mice were put in normal cyclic light-dark conditions for 2 weeks. Moreover, dark-reared retinas treated with BDNF showed normal expression of both GABA and GAD65. Our data indicate that normal expression of GABA and GAD65 is dependent on visual input. Furthermore, the data suggest that BDNF controls this dependence.

We serendipitously found a distal Disabled‐1 (Dab1)‐immunoreactive cell in retina of the C57BL/6J black mouse. The somata of these cells are located in the outermost part of the inner nuclear layer (INL). Their processes extend toward the outer plexiform layer (OPL), receiving synaptic inputs from horizontal and interplexiform cells. In the current study, we name this cell the “distal Dab1‐immunoreactive cell.” Double‐labeling experiments demonstrate that the distal Dab1‐immunoreactive cell is not a horizontal cell. Rather, the distal Dab1 cell appears to be a misplaced AII cell, by being glycine transporter‐1‐immunoreactive and by resembling the latter cell in an electron microscopic analysis. A distal Dab1 cell had been reported in the FVB/N mouse retina, a model of retinitis pigmentosa (Park et al. [2004] Cell Tissue Res 315:407–412). However, here, we found this distal Dab1‐immunoreactive cell in the adult and normal developing mouse retinas. Hence, we show that such cells do not require the loss of photoreceptors as suggested previously (Park et al. [2004] Cell Tissue Res 315:407–412). Instead, two other pieces of data suggest an alternative explanation sources for distal Dab1 cells. First, we find a correlation between the number of these cells in the left and right eyes Second, developmental analysis shows that the distal Dab1‐immunoreactive cell is first observed shortly after birth. At the same time, AII cells emerge, extending their neurites into the inner retina. These data suggest that distal Dab1‐immunoreactive cells are misplaced AII amacrine cells, resulting from genetically modulated anomalies owing to migration errors. J. Comp. Neurol. 494:651–662, 2006.