3D bioprinting of dECM-incorporated hepatocyte spheroid for simultaneous promotion of cell-cell and -ECM interactions

The cell spheroid technology, which greatly enhances cell-cell interactions, has gained significant attention in the development of in vitro liver models. However, existing cell spheroid technologies still have limitations in improving hepatocyte-extracellular matrix (ECM) interaction, which have a significant impact on hepatic function. In this study, we have developed a novel bioprinting technology for decellularized ECM (dECM)-incorporated hepatocyte spheroids that could enhance both cell-cell and -ECM interactions simultaneously. To provide a biomimetic environment, a porcine liver dECM-based cell bio-ink was developed, and a spheroid printing process using this bio-ink was established. As a result, we precisely printed the dECM-incorporated hepatocyte spheroids with a diameter of approximately 160–220 μm using primary mouse hepatocyte (PMHs). The dECM materials were uniformly distributed within the bio-printed spheroids, and even after more than 2 weeks of culture, the spheroids maintained their spherical shape and high viability. The incorporation of dECM also significantly improved the hepatic function of hepatocyte spheroids. Compared to hepatocyte-only spheroids, dECM-incorporated hepatocyte spheroids showed approximately 4.3- and 2.5-fold increased levels of albumin and urea secretion, respectively, and a 2.0-fold increase in CYP enzyme activity. These characteristics were also reflected in the hepatic gene expression levels of ALB, HNF4A, CPS1, and others. Furthermore, the dECM-incorporated hepatocyte spheroids exhibited up to a 1.8-fold enhanced drug responsiveness to representative hepatotoxic drugs such as acetaminophen, celecoxib, and amiodarone. Based on these results, it can be concluded that the dECM-incorporated spheroid printing technology has great potential for the development of highly functional in vitro liver tissue models for drug toxicity assessment

Utilizations of 3D bioprinting technology

Department of Biomedical Engineeringclos

Bioprinting of Prevascularized Beta-cell Sheet to Improve Islet Transplantation

Type I diabetes is an incurable autoimmune disease, which is failure in producing sufficient insulin because of loss of b cells in pancreas. Islet transplantation has been considered as a viable option for healing the disease. However, absence of vascular network and low mechanical stability of islet construct are still big obstacles in its clinic applications. Therefore, the way to fabricate prevascularized b-cell construct having reliable stability was studied to overcome the difficulties in this research. Mouse insulinoma cell line 6 (Min6), human umbilical vein endothelial cells (HUVEC) and polycaprolactone (PCL) were applied to fabricate a prevascularized b-cell laden sheet by co-bioprinting of the multiple materials. Min6 and HUVEC laden hydrogel were printed individually within PCL lattice structure having 300mm channel. Time-series micrographs of vessel formation within the sheet was observed. And viability, proliferation and functionality test of b cells were also conducted to show its usefulness. These results successfully demonstrated that the bioprinted b cell sheet having prevascular structures has great potential to treat type I diabetes in clinics

Visual Estimation of Bacterial Growth Level in Microfluidic Culture Systems

Microfluidic devices are an emerging platform for a variety of experiments involving bacterial cell culture, and has advantages including cost and convenience. One inevitable step during bacterial cell culture is the measurement of cell concentration in the channel. The optical density measurement technique is generally used for bacterial growth estimation, but it is not applicable to microfluidic devices due to the small sample volumes in microfluidics. Alternately, cell counting or colony-forming unit methods may be applied, but these do not work in situ; nor do these methods show measurement results immediately. To this end, we present a new vision-based method to estimate the growth level of the bacteria in microfluidic channels. We use Fast Fourier transform (FFT) to detect the frequency level change of the microscopic image, focusing on the fact that the microscopic image becomes rough as the number of cells in the field of view increases, adding high frequencies to the spectrum of the image. Two types of microfluidic devices are used to culture bacteria in liquid and agar gel medium, and time-lapsed images are captured. The images obtained are analyzed using FFT, resulting in an increase in high-frequency noise proportional to the time passed. Furthermore, we apply the developed method in the microfluidic antibiotics susceptibility test by recognizing the regional concentration change of the bacteria that are cultured in the antibiotics gradient. Finally, a deep learning-based data regression is performed on the data obtained by the proposed vision-based method for robust reporting of data

Vasculature-On-A-Chip for In Vitro Disease Models

Vascularization, the formation of new blood vessels, is an essential biological process. As the vasculature is involved in various fundamental physiological phenomena and closely related to several human diseases, it is imperative that substantial research is conducted on characterizing the vasculature and its related diseases. A significant evolution has been made to describe the vascularization process so that in vitro recapitulation of vascularization is possible. The current microfluidic systems allow elaborative research on the effects of various cues for vascularization, and furthermore, in vitro technologies have a great potential for being applied to the vascular disease models for studying pathological events and developing drug screening platforms. Here, we review methods of fabrication for microfluidic assays and inducing factors for vascularization. We also discuss applications using engineered vasculature such as in vitro vascular disease models, vasculature in organ-on-chips and drug screening platforms

MineLoC: A Rapid Production of Lab-on-a-Chip Biosensors Using 3D Printer and the Sandbox Game, Minecraft

Here, MineLoC is described as a pipeline developed to generate 3D printable models of master templates for Lab-on-a-Chip (LoC) by using a popular multi-player sandbox game “Minecraft”. The user can draw a simple diagram describing the channels and chambers of the Lab-on-a-Chip devices with pre-registered color codes which indicate the height of the generated structure. MineLoC converts the diagram into large chunks of blocks (equal sized cube units composing every object in the game) in the game world. The user and co-workers can simultaneously access the game and edit, modify, or review, which is a feature not generally supported by conventional design software. Once the review is complete, the resultant structure can be exported into a stereolithography (STL) file which can be used in additive manufacturing. Then, the Lab-on-a-Chip device can be fabricated by the standard protocol to produce a Lab-on-a-Chip. The simple polydimethylsiloxane (PDMS) device for the bacterial growth measurement used in the previous research was copied by the proposed method. The error calculation by a 3D model comparison showed an accuracy of 86%. It is anticipated that this work will facilitate more use of 3D printer-based Lab-on-a-Chip fabrication, which greatly lowers the entry barrier in the field of Lab-on-a-Chip research

Lipopolysaccharide-Induced Vascular Inflammation Model on Microfluidic Chip

Inflammation is the initiation of defense of our body against harmful stimuli. Lipopolysaccharide (LPS), originating from outer membrane of Gram-negative bacteria, causes inflammation in the animal’s body and can develop several diseases. In order to study the inflammatory response to LPS of blood vessels in vitro, 2D models have been mainly used previously. In this study, a microfluidic device was used to investigate independent inflammatory response of endothelial cells by LPS and interaction of inflamed blood vessel with monocytic THP-1 cells. Firstly, the diffusion of LPS across the collagen gel into blood vessel was simulated using COMSOL. Then, inflammatory response to LPS in engineered blood vessel was confirmed by the expression of Intercellular Adhesion Molecule 1 (ICAM-1) and VE-cadherin of blood vessel, and THP-1 cell adhesion and migration assay. Upregulation of ICAM-1 and downregulation of VE-cadherin in an LPS-treated condition was observed compared to normal condition. In the THP-1 cell adhesion and migration assay, the number of adhered and trans-endothelial migrated THP-1 cells were not different between conditions. However, migration distance of THP-1 was longer in the LPS treatment condition. In conclusion, we recapitulated the inflammatory response of blood vessels and the interaction of THP-1 cells with blood vessels due to the diffusion of LPS