Establishment and Long-Term Expansion of Small Cell Lung Cancer Patient-Derived Tumor Organoids.

Differential chemo-sensitivity of cancer cells, which is attributed to the cellular heterogeneity and phenotypic variation of cancer cells, is considered to be the main reason for tumor recurrence after chemotherapy. Here, we generated small cell lung cancer patient-derived tumor organoids and subjected them to long-term expansion with the addition of WNT3A or R-spondin1. We confirmed that the organoids have similar genetic profiles, molecular characteristics, and morphological architectures to the corresponding patient tumor tissue during and after long-term expansion. Interestingly, the cellular heterogeneity of organoids is reflected in their differential response to cisplatin or etoposide. We propose to utilize the organoids as small cell lung cancer patient avatar models that would be ideal for investigating the mechanisms underlying tumor recurrence after chemotherapy, and would ultimately help to develop personalized medicine.

In vitro lung cancer multicellular tumor spheroid formation using a microfluidic device.

The purpose of this study was to demonstrate self-organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate. The dose response to an MMPs inhibitor was investigated to demonstrate the effects of biochemical substances. The result of long-term stability of MCTSs revealed that continuous perfusion of cell culture medium is one of the major factors for the successful MCTS formation. A continuous flow of cell culture medium in the in vitro TME greatly affected both the proliferation of cancer cells in the micro-wells and the sustainability of the endothelial cell-layer integrity in the lumen of microfluidic channels. Addition of MMP inhibitor to the cell culture medium improved the stability of the collagen matrix by preventing the detachment and shrinkage of the collagen matrix surrounding the MCTSs. In summary, the present constant flow assisted microfluidic system is highly advantageous for long-term observation of the MCTS generation, tumorous tissue formation process and drug responses. MCTS formation in a microfluidic system may serve as a potent tool for studying drug screening, tumorigenesis and metastasis.

Proposal for considerations during human iPSC-derived cardiac organoid generation for cardiotoxicity drug testing.

Human iPSC-derived cardiac organoids (hiPSC-COs) for cardiotoxicity drug testing via the variety of cell lines and unestablished protocols may lead to differences in response results due to a lack of criteria for generation period and size. To ensure reliable drug testing, it is important for researchers to set optimal generation period and size of COs according to the cell line and protocol applied in their studies. Hence, we sought to propose a process to establish minimum criteria for the generation duration and size of hiPSC-COs for cardiotoxic drug testing. We generated hiPSC-COs of different sizes based on our protocol and continuously monitored organoids until they indicated a minimal beating rate change as a control that could lead to more accurate beating rate changes on drug testing. Calcium transients and physiological tests to assess the functionality of hiPSC-COs on selected generation period, which showed regular cardiac beating, and immunostaining assays to compare characteristics were performed. We explained the generation period and size that exhibited and maintained regular beating rate changes on hiPSC-COs, and lead to reliable response results to cardiotoxicity drugs. We anticipate that this study will offer valuable insights into considering the appropriate generation period and size of hiPSC-COs ensuring reliable outcomes in cardiotoxicity drug testing.

Generation of sarconoids from angiosarcoma patients as a systematic-based rational approach to treatment.

Angiosarcoma is a rare subtype of malignant neoplasm originating from vascular or lymphatic endothelial cells; its low incidence has posed significant challenges for comprehensive investigations into its pathogenic mechanisms and the development of innovative treatment modalities through in vitro and in vivo models. Recent endeavors spearheaded by patient-partnered research initiatives have aimed to elucidate the intricacies of angiosarcomas by leveraging biological omics approaches, with the overarching objective of enhancing prognostic indicators and therapeutic options for this uncommon pathology. To bridge the gap between preclinical research and translational applications, we engineered angiosarcoma-derived organoids from surgically resected primary tumors, hereafter referred to as "sarconoids," as a proof-of-concept model. A novel protocol for the establishment of these sarconoids has been developed and validated. To ensure that the sarconoids faithfully recapitulate the heterogeneity and complexities of the patients' original tumors, including transcriptomic signatures, cell-type specificity, and morphological traits, exhaustive histological and transcriptomic analyses were conducted. Subsequently, we expanded the scope of our study to include an evaluation of a sarconoid-based drug screening platform; for this purpose, a drug library (AOD IX), supplied by the National Cancer Institute's Developmental Therapeutics Program, was screened using 96-well plates. Our findings suggest that sarconoids can be reliably generated from angiosarcoma patient-derived tissues and can serve as accurate models for evaluating therapeutic responses, thereby holding far-reaching implications for translational research and clinical applications aimed at advancing our understanding and treatment of angiosarcoma.

Digitally tunable physicochemical coding of material composition and topography in continuous microfibres.

Heterotypic functional materials with compositional and topographical properties that vary spatiotemporally on the micro- or nanoscale are common in nature. However, fabricating such complex materials in the laboratory remains challenging. Here we describe a method to continuously create microfibres with tunable morphological, structural and chemical features using a microfluidic system consisting of a digital, programmable flow control that mimics the silk-spinning process of spiders. With this method we fabricated hydrogel microfibres coded with varying chemical composition and topography along the fibre, including gas micro-bubbles as well as nanoporous spindle-knots and joints that enabled directional water collection. We also explored the potential use of the coded microfibres for tissue engineering applications by creating multifunctional microfibres with a spatially controlled co-culture of encapsulated cells.

Sprouting angiogenesis under a chemical gradient regulated by interactions with an endothelial monolayer in a microfluidic platform.

Microfluidic cell culture assays are versatile tools for studying cell migration, particularly angiogenesis. Such assays can deliver precisely controlled linear gradients of chemical stimuli to cultured cells in a microfluidic channel, offering excellent optical resolution and in situ monitoring of cellular morphogenesis in response to a gradient. Microfluidic cell culture assays provide a chemical gradient subject to molecular diffusion, although cellular metabolism can perturb it. The actual gradient perturbed by cells has not been precisely described in the context of regulated cellular morphogenesis. We modeled the chemical gradient in a microfluidic channel by simulating the analyte(VEGF) distribution during cellular interactions. The results were experimentally verified by monitoring sprouting angiogenic response from a monolayer of human umbilical vein endothelial cells (hUVECs) into a type 1 collagen scaffold. The simulation provided a basis for understanding a real distribution of the analyte interrupted by cells in microfluidic device. The new protocol enables one to quantify the morphogenesis of hUVECs under a flat, less-steep, or steep gradient.

Microfluidic assay of endothelial cell migration in 3D interpenetrating polymer semi-network HA-Collagen hydrogel.

Cell migration through the extracellular matrix (ECM) is one of the key features for physiological and pathological processes such as angiogenesis, cancer metastasis, and wound healing. In particular, the quantitative assay of endothelial cell migration under the well-defined three dimensional (3D) microenvironment is important to analyze the angiogenesis mechanism. In this study, we report a microfluidic assay of endothelial cell sprouting and migration into an interpenetrating polymer semi-network HA-Collagen (SIPNs CH) hydrogel as ECM providing an enhanced in vivo mimicking 3D microenvironment to cells. The microfluidic chip could provide a well-controlled gradient of growth factor to cells, whereas the hydrogel could mimic a well-defined 3D microenvironment in vivo. (In addition/Furthermore, the microfluidic chip gives a well-controlled gradient of growth factor to cells) For this reason, three types of hydrogel, composed of semi-interpenetrating networks of collagen and hyaluronic acid were prepared, and firstly we proved the role of the hydrogel in endothelial cell migration. The diffusion property and swelling ratio of the hydrogel were characterized. It modulated the migration of endothelial cells in quantified manner, also being influenced by additional synthesis of Matrix metalloproteinase(MMP)-sensitive remodeling peptides and Arginine-glycine-lycinee (RGD) cell adhesion peptides. We successfully established a novel cell migration platform by changing major determinants such as ECM material under biochemical synthesis and under growth factor gradients in a microfluidic manner.

A durability study of a paracorporeal pulsatile electro-mechanical pneumatic biventricular assist device.

In 2002, the paracorporeal pulsatile electro-mechanical pneumatic ventricular assist device (VAD) began to be developed by the Korea Artificial Organ Center at Korea University under a Health & Medical Technology Research and Development program which finished in 2008. In vitro durability testing was conducted on the paracorporeal pulsatile pneumatic VAD to determine device durability and to evaluate device failures. The 1- and 2-year reliability of the paracorporeal pulsatile pneumatic VAD was shown to be 91.2% and 54.9%, respectively, with an 80% confidence level. Failure modes were analyzed using fault tree analysis, with customized software continuously acquiring data during the test period. After this period, 21 in vivo animal tests were done, with 14 cases of left atrium to left ventricle (LV) inflow cannulation (36Fr)/outflow grafting to descending aorta, and seven cases of apex cannulation of LV to descending aorta (12 mm). The longest postoperative day (182 days) in Korea was recently recorded in in vivo animal testing (bovine, 90 kg, male, 3.5-4.0 L/min flow rate, and 55 bpm).

Sporadic cell death in macroscale 3D tumor grafts with high drug resistance by activating cell-ECM interactions.

In the tumor microenvironment (TME), the extracellular matrix (ECM) provides a dynamic structure for cell adhesion and cancer cell motility, such as migration and invasion, as well as remodeling. Matrix metalloproteinases (MMPs) promote cancer cell motility, which contributes to inducing drug resistance and thereby acquiring aggressive features. The drug resistance-induced 3Din vitrotumor model can be an effective model for therapeutic strategies for anticancer drugs targeting aggressive cancer cells. Here, we describe highly drug-resistant multicellular tumoroids (MCTs)-ECM tumor grafts under a macroscale dense 3Din vitromodel through a combination of numerous MCTs and a collagen matrix. MCTs-ECM tumor grafts promote the high activity of MMP2 and MMP9 compared to general MCTs and induced cancer cell motility. Then, after the administration of anticancer drugs, the tumor grafts show increased drug resistance, with both the sporadic distribution of necrotic cells and the reduction of apoptotic portions, by activating cancer cell motility. MCTs-ECM tumor graft could be useful as a macroscale tumor graft model for inducing drug resistance by activating cancer cell motility and evaluating the efficacy of anticancer drugs targeting cancer with aggressive features.

Applications of micromixing technology.

This review presents an application of micromixer technologies, which have driven a number of critical research trends over the past few decades, particularly for chemical and biological fields. Micromixer technologies in this review are categorized according to their applications: (1) chemical applications, including chemical synthesis, polymerization, and extraction; (2) biological applications, including DNA analysis, biological screening enzyme assays, protein folding; and (3) detection/analysis of chemical or biochemical content combined with NMR, FTIR, or Raman spectroscopies. In the chemical application, crystallization, extraction, polymerization, and organic synthesis have been reported, not only for laboratory studies, but also for industrial applications. Microscale techniques are used in chemical synthesis to develop microreactors. In clinical medicine and biological studies, microfluidic systems have been widely applied to the identification of biochemical products, diagnosis, drug discovery, and investigation of disease symptoms. The biological and biochemical applications also include enzyme assays, biological screening assays, cell lysis, protein folding, and biological analytical assays. Nondestructive analytical/detection methods have yielded a number of benefits to chemical and biochemical processes. In this chapter, we introduce analytical methods those are frequently integrated into micromixing technologies, such as NMR, FT-IR, and Raman spectroscopies. From the study of micromixers, we discovered that the Re number and mixing time depends on the specific application, and we clustered micromixers in various applications according to the Re number and mixing performance (mixing time). We expect that this clustering will be helpful in designing of micromixers for specific applications.

Flow enhances phenotypic and maturation of adult rat liver organoids.

A biologically relevant in vitro model of hepatic microtissue would be a valuable tool for the preclinical study of pharmacokinetics and metabolism. Although considerable advances have been made in recent years in the establishment of alternative in vitro culture systems that mimic liver tissue, generating an effective liver model remains challenging. Specifically, existing model systems still exhibit limited functions for hepatocellular differentiation potential and cellular complexity. It is essential to improve the in vitro differentiation of liver progenitor cells (LPCs) for disease modeling and preclinical pharmatoxicological research. Here, we describe a rat liver organoid culture system under in vivo-like steady-state flow conditions; this system is capable of controlling the expansion and differentiation of rat liver organoids over 10-15 d. LPCs cultured in medium flow conditions become self-assembled liver organoids that exhibit phenotypic and functional hepato-biliary modeling. In addition, hepatocytes that are differentiated using liver organoids produced albumin and maintained polygonal morphology, which is characteristic of mature hepatocytes.

Gaining New Biological and Therapeutic Applications into the Liver with 3D In Vitro Liver Models.

BACKGROUND: Three-dimensional (3D) cell cultures with architectural and biomechanical properties similar to those of natural tissue have been the focus for generating liver tissue. Microarchitectural organization is believed to be crucial to hepatic function, and 3D cell culture technologies have enabled the construction of tissue-like microenvironments, thereby leading to remarkable progress in vitro models of human tissue and organs. Recently, to recapitulate the 3D architecture of tissues, spheroids and organoids have become widely accepted as new practical tools for 3D organ modeling. Moreover, the combination of bioengineering approach offers the promise to more accurately model the tissue microenvironment of human organs. Indeed, the employment of sophisticated bioengineered liver models show long-term viability and functional enhancements in biochemical parameters and disease-orient outcome. RESULTS: Various 3D in vitro liver models have been proposed as a new generation of liver medicine. Likewise, new biomedical engineering approaches and platforms are available to more accurately replicate the in vivo 3D microarchitectures and functions of living organs. This review aims to highlight the recent 3D in vitro liver model systems, including micropatterning, spheroids, and organoids that are either scaffold-based or scaffold-free systems. Finally, we discuss a number of challenges that will need to be addressed moving forward in the field of liver tissue engineering for biomedical applications. CONCLUSION: The ongoing development of biomedical engineering holds great promise for generating a 3D biomimetic liver model that recapitulates the physiological and pathological properties of the liver and has biomedical applications.

Correction: Multidimensional assembly using layer-by-layer deposition for synchronized cardiac macro tissues.

[This corrects the article DOI: 10.1039/D0RA01577F.].

Evaluation of Bystander Infection of Oncolytic Virus using a Medium Flow Integrated 3D In Vitro Microphysiological System.

Replicable oncolytic viruses (OVs) induce tumor cell lysis and release viral progeny. The released progeny virions and cell debris can spread within surrounding tumor cells or blood vessels. These released molecules may also induce bystander damage in additional tumor cells through spreading within surrounding tumor cells or blood vessels. However, this effect has not been clearly demonstrated due to the difficulty of direct observation. Here, the bystander infection of OVs by vessel delivery and selective infection in 3D multicellular tumoroids (MCTs) in an in vitro microphysiological system (MPS) with integrated medium flow is demonstrated. This study uses replicable vesicular stomatitis virus (VSV)-green fluorescence protein (GFP) to identify the location of infection in 3D MCTs. Using this MPS, the oncoselective infection by VSV-GFP and the spreading by delivery of OVs through flow via block-to-block linkage of the primary infected MPS with uninfected 3D MCTs in an integrated MPS is observed. This MPS enables real-time monitoring and various analysis for the bystander infection of OVs. It is expected that the 3D in vitro MPS can be suitable to investigate the oncoselective spreading and bystander infection of OVs.

Real-time monitoring of oncolytic VSV properties in a novel in vitro microphysiological system containing 3D multicellular tumor spheroids.

As a new class of cancer therapeutic agents, oncolytic viruses (OVs) have gained much attention not only due to their ability to selectively replicate in and lyse tumor cells, but also for their potential to stimulate antitumor immune responses. As a result, there is an increasing need for in vitro modeling systems capable of recapitulating the 3D physiological tumor microenvironment. Here, we investigated the potential of our recently developed microphysiological system (MPS), featuring a vessel-like channel to reflect the in vivo tumor microenvironment and serving as culture spaces for 3D multicellular tumor spheroids (MCTSs). The MCTSs consist of cancer A549 cells, stromal MRC5 cells, endothelial HUVECs, as well as the extracellular matrix. 3D MCTSs residing in the MPS were infected with oncolytic VSV expressing GFP (oVSV-GFP). Post-infection, GFP signal intensity increased only in A549 cells of the MPS. On the other hand, HUVECs were susceptible to virus infection under 2D culture and IFN-ß secretion was quite delayed in HUVECs. These results thus demonstrate that OV antitumoral characteristics can be readily monitored in the MPS and that its behavior therein somewhat differs compared to its activity in 2D system. In conclusion, we present the first application of the MPS, an in vitro model that was developed to better reflect in vivo conditions. Its various advantages suggest the 3D MCTS-integrated MPS can serve as a first line monitoring system to validate oncolytic virus efficacy.

Long-term reversal of diabetes by subcutaneous transplantation of pancreatic islet cells and adipose-derived stem cell sheet using surface-immobilized heparin and engineered collagen scaffold.

OBJECTIVE: Esterified collagen (EC) can be functionalized with heparin to enhance islet graft stability. Growth factors secreted by human adipose-derived stem cells (hADSCs) can bind efficiently to EC-heparin (EC-Hep), which enhances revascularization and cell protection. We investigated the therapeutic potential of a combined heparin-esterified collagen-hADSC (HCA)-islet sheet to enhance islet engraftment. RESEARCH DESIGN AND METHODS: This study was designed to assess the efficiency of using EC-Hep as a scaffold for subcutaneous islet transplantation in diabetic athymic mice. After the hADSC-cocultured islets were seeded in the EC-Hep scaffold, islet function was measured by glucose-stimulated insulin secretion test and growth factors in the culture supernatants were detected by protein array. Islet transplantation was performed in mice, and graft function and survival were monitored by measuring the blood glucose levels. ß-Cell mass and vascular densities were assessed by immunohistochemistry. RESULTS: The EC-Hep composite allowed sustained release of growth factors. Secretion of growth factors and islet functionality in the HCA-islet sheet were significantly increased compared with the control groups of islets alone or combined with native collagen. In vivo, stable long-term glucose control by the graft was achieved after subcutaneous transplantation of HCA-islet sheet due to enhanced capillary network formation around the sheet. CONCLUSIONS: The findings indicate the potential of the HCA-islet sheet to enhance islet revascularization and engraftment in a hADSC dose-dependent manner, following clinical islet transplantation for the treatment of diabetes mellitus.

Optimal pressure regulation of the pneumatic ventricular assist device with bellows-type driver.

The bellows-type pneumatic ventricular assist device (VAD) generates pneumatic pressure with compression of bellows instead of using an air compressor. This VAD driver has a small volume that is suitable for portable devices. However, improper pneumatic pressure setup can not only cause a lack of adequate flow generation, but also cause durability problems. In this study, a pneumatic pressure regulation system for optimal operation of the bellows-type VAD has been developed. The optimal pneumatic pressure conditions according to various afterload conditions aiming for optimal flow rates were investigated, and an afterload estimation algorithm was developed. The developed regulation system, which consists of a pressure sensor and a two-way solenoid valve, estimates the current afterload and regulates the pneumatic pressure to the optimal point for the current afterload condition. Experiments were performed in a mock circulation system. The afterload estimation algorithm showed sufficient performance with the standard deviation of error, 8.8 mm Hg. The flow rate could be stably regulated with a developed system under various afterload conditions. The shortcoming of a bellows-type VAD could be handled with this simple pressure regulation system.

Development of a closed air loop electropneumatic actuator for driving a pneumatic blood pump.

In this study, we developed a small pneumatic actuator that can be used as an extracorporeal biventricular assist device. It incorporated a bellows-transforming mechanism to generate blood-pumping pressure. The cylindrical unit is 88 +/- 0.1 mm high, has a diameter of 150 +/- 0.1 mm, and weighs 2.4 +/- 0.01 kg. In vitro, maximal outflow at the highest pumping rate (PR) exceeded 8 L/min when two 55 mL blood sacs were used under an afterload pressure of 100 mm Hg. At a pumping rate of 100 beats per minute (bpm), maximal hydraulic efficiency was 9.34% when the unit supported a single ventricle and 13.8% when it supported both ventricles. Moreover, pneumatic efficiencies of the actuator were 17.3% and 33.1% for LVAD and BVAD applications, respectively. The energy equivalent pressure was 62.78 approximately 208.10 mm Hg at a PR of 60 approximately 100 bpm, and the maximal value of dP/dt during systole was 1269 mm Hg/s at a PR of 60 bpm and 979 mm Hg/s at a PR of 100 bpm. When the unit was applied to 15 calves, it stably pumped 3 approximately 4 L/min of blood at 60 bpm, and no mechanical malfunction was experienced over 125 days of operation. We conclude that the presently developed pneumatic actuator can be utilized as an extracorporeal biventricular assist device.

A one-stop microfluidic-based lung cancer organoid culture platform for testing drug sensitivity.

Microfluidic devices as translational research tools provide a potential alternative to animal experiments due to their ability to mimic physiological parameters. Several approaches that can be used to predict the efficacy or toxicity of anticancer drugs are available. In general, standard cell culture systems have the advantages of being relatively cost-effective, having high-throughput capability, and providing convenience. However, these models are inadequate to accurately recapitulate the complex organ-level physiological and pharmacological responses. Here, we present a one-stop microfluidic device enabling both 3-dimensional (3D) lung cancer organoid culturing and drug sensitivity tests directly on a microphysiological system (MPS). Our platform reproducibly yields 3D lung cancer organoids in a size-controllable manner and demonstrates for the first time the production of lung cancer organoids from patients with small-cell lung cancer. Lung cancer organoids derived from primary small-cell lung cancer tumors can rapidly proliferate and exhibit disease-specific characteristics in our MPS. Cisplatin and etoposide, the standard regimen for lung cancer, showed increased apoptosis induction in a concentration-dependent manner, but the organoids contained chemo-resistant cells in the core. We envision that this system may provide important information to guide therapeutic approaches at the preclinical level.

A cell-loss-free concave microwell array based size-controlled multi-cellular tumoroid generation for anti-cancer drug screening.

The 3D multi-cellular tumoroid (MCT) model is an in vivo-like, avascular tumor model that has received much attention as a refined screening platform for drug therapies. Several types of research have been efforted to improve the physiological characteristics of the tumor microenvironment (TME) of the in vivo-like MCTs. Size-controlled MCTs have received much attention for obtaining highly reproducible results in drug screening assays and achieving a homogeneous and meaningful level of biological activities. Here, we describe an effective method for fabricating the size-controlled in vivo-like MCTs using a cell-loss-free (CLF) microwell arrays. The CLF microwell arrays was fabricated by using the simple operation of laser carving of a poly (methyl methacrylate) (PMMA) master mold. We also demonstrated the biophysicochemical effect of tumor microenvironment (TME) resident fibroblasts through the expression of TGFß, αSMA, Type I-, IV collagen, angiogenesis related markers on tumorigenesis, and confirmed the drug response of MCTs with anti-cancer agents. This technology for the fabrication of CLF microwell arrays could be used as an effective method to produce an in vitro tumor model for cancer research and drug discovery.