Vascular Tissue Models Research Articles

A method to generate perfusable physiologic-like vascular channels within a liver-on-chip model.

The human vasculature is essential in organs and tissues for the transport of nutrients, metabolic waste products, and the maintenance of homeostasis. The integration of vessels in in vitro organs-on-chip may, therefore, improve the similarity to the native organ microenvironment, ensuring proper physiological functions and reducing the gap between experimental research and clinical outcomes. This gap is particularly evident in drug testing and the use of vascularized models may provide more realistic insights into human responses to drugs in the pre-clinical phases of the drug development pipeline. In this context, different vascularized liver models have been developed to recapitulate the architecture of the hepatic sinusoid, exploiting either porous membranes or bioprinting techniques. In this work, we developed a method to generate perfusable vascular channels with a circular cross section within organs-on-chip without any interposing material between the parenchyma and the surrounding environment. Through this technique, vascularized liver sinusoid-on-chip systems with and without the inclusion of the space of Disse were designed and developed. The recapitulation of the Disse layer, therefore, a gap between hepatocytes and endothelial cells physiologically present in the native liver milieu, seems to enhance hepatic functionality (e.g., albumin production) compared to when hepatocytes are in close contact with endothelial cells. These findings pave the way to numerous further uses of microfluidic technologies coupled with vascularized tissue models (e.g., immune system perfusion) as well as the integration within multiorgan-on-chip settings.

Coaxial 4D Printing of Vein‐Inspired Thermoresponsive Channel Hydrogel Actuators

AbstractAlthough significant progress has been made in coaxial printing of vascularized tissue models, this technique has not yet been used to fabricate stimulus‐responsive scaffolds capable of shape change over time. Here, a new method of direct ink printing (DIP) is proposed with a coaxial nozzle, coaxial 4D printing, enabling the manufacturing of thermoresponsive constructs embedded with a network of interconnected channels. In this approach, a poly(N‐isopropylacrylamide) (PNIPAAm)‐based thermoink is coaxially extruded into either core/sheath microfibers or microtubes. PNIPAAm renders a hydrogel temperature‐sensitive and endows it with a shape‐morphing property both at the micro‐ and macroscale. Specifically, the lumen diameter of the microtubes can be controlled by temperature by 30%. The macrostructural soft actuators can undergo programmed and reversible temperature‐dependent shape changes due to the structural anisotropy of the hydrogel. The permeability tests demonstrate that the hydrogel can possess enough strength to maintain the hollow channels without breaking. In vitro tests confirm the biocompatibility of the material with EA.hy926 cells, paving the avenue for new perfusable soft robots, or active implants. Finally, microalgae Chlamydomonas reinhardtii is combined with the hydrogels to fabricate materials having functions of both living microorganisms and stimuli‐responsive polymers toward creating engineered living materials (ELMs) with a vein‐like geometry.

Development of in vitro cardiovascular tissue models within capillary circuit microfluidic devices fabricated with 3D stereolithography printing

This study presents the development and morphology analysis of bioinspired 3D cardiovascular tissue models cultured within a dynamic capillary circuit microfluidic device. This study is significant because our in vitro 3D cardiovascular tissue models retained within a capillary circuit microfluidic device provide a less expensive, more controlled, and reproducible platform for more physiologically-relevant evaluation of cellular response to microenvironmental stimuli. The overall aim of our study is to demonstrate our cardiovascular tissue model (CTM) and vascular tissue model (VTM) actively changed their cellular morphology and exhibited structural reorganization in response to biophysical stimuli provided by microposts within the device tissue culture chambers during a 5-day period. The microfluidic device in this study was designed with the Young–Laplace and Navier–Stokes principles of capillary driven fluid flow and fabricated with 3D stereolithography (SLA) printing. The cardiac tissue model and vascular tissue model presented in this study were developed by encapsulating AC16 cardiomyocytes (CTM) and Human umbilical vein endothelial cells (VTM) in a fibrin hydrogel which were subsequently loaded into a capillary circuit microfluidic device. The cardiovascular tissue models were analyzed with fluorescent microscopy for morphological differences, average tube length, and cell orientation. We determined the VTM displayed capillary-like tube formation and the cells within both cardiovascular tissue models continued to elongate around microposts by day-5 which indicates the microfluidic system provided biophysical cues to guide cell structure and direction-specific organization.

3D bioprinting strategy for engineering vascularized tissue models.

Leveraging three-dimensional (3D) bioprinting in the fields of tissue engineering and regenerative medicine has rapidly accelerated progress toward the development of living tissue constructs and biomedical devices. Ongoing vigorous research has pursued the development of 3D in vitro tissue models to replicate the key aspects of human physiology by incorporating relevant cell populations and adequate environmental cues. Given their advantages of being able to intimately mimic the heterogeneity and complexity of their native counterparts, 3D in vitro models hold promise as alternatives to conventional cell cultures or animal models for translational application to model human physiology/pathology and drug screening. Research has highlighted the importance of in vitro models, and a sophisticated biomanufacturing strategy is vitally required. In particular, vascularization is critical for the prolonged survival and functional maturation of the engineered tissues, which has remained one of the major challenges in the establishment of physiologically relevant 3D in vitro models. To this end, 3D bioprinting can efficiently generate solid and reproducible vascularized tissue models with high architectural and compositional similarity to the native tissues, leading to improve the structural maturation and tissue-specific functionality. Multiple bioprinting strategies have been developed to vascularize in vitro tissues by spatially controlled patterning of vascular precursors or generating readily perfusable vascular structures. This review presents an overview of the advanced 3D bioprinting strategies for vascularized tissue model development. We present the key elements for rebuilding functional vasculature in 3D-bioprinted tissue models and discuss the recent achievements in the engineering of 3D vascularized in vitro models using 3D bioprinting. Finally, we delineate the current challenges and future outlooks of 3D bioprinting-based vascularized tissue models.

Small-artery-mimicking multi-layered 3D co-culture in a self-folding porous graphene-based film.

In vitro vessel-mimicking models have been spotlighted as a powerful tool for investigating cellular behaviours in vascular development and diseases. However, it is still challenging to create micro-scale tubular tissues while mimicking the structural features of small arteries. Here, we propose a 3D culture model of small vascular tissue using a self-folding graphene-based porous film. Vascular endothelial cells were encapsulated within the self-folding film to create a cellular construct with a controlled curvature radius ranging from 10 to 100 μm, which is comparable to the size of a human arteriole. Additionally, vascular endothelial cells and smooth muscle cells were separately co-cultured on the inner and outer surfaces of the folded film, respectively. The porous wall worked as a permeable barrier between them, affecting the cell-cell communications like the extracellular layer in the artery wall. Thus, the culture model recapitulates the structural features of a small artery and will help us better understand intercellular communications at the artery wall in physiological and pathological conditions.

Physical Models from Physical Templates Using Biocompatible Liquid Crystal Elastomers as Morphologically Programmable Inks For 3D Printing.

Advanced manufacturing has received considerable attention as a tool for the fabrication of cell scaffolds however, finding ideal biocompatible and biodegradable materials that fit the correct parameters for 3D printing and guide cells to align remain a challenge. Herein, a photocrosslinkable smectic-A (Sm-A) liquid crystal elastomer (LCE) designed for 3D printing is presented, that promotes cell proliferation but most importantly induces cell anisotropy. The LCE-based bio-ink allows the 3D duplication of a highly complex brain structure generated from an animal model. Vascular tissue models are generated from fluorescently stained mouse tissue spatially imaged using confocal microscopy and subsequently processed to create a digital 3D model suitable for printing. The 3D structure is reproduced using a Digital Light Processing (DLP) stereolithography (SLA) desktop 3D printer. Synchrotron Small-Angle X-ray Diffraction (SAXD) data reveal a strong alignment of the LCE layering within the struts of the printed 3D scaffold. The resultant anisotropy of the LCE struts is then shown to direct cell growth. This study offers a simple approach to produce model tissues built within hours that promote cellular alignment.

Angiodynamic and optical coupling analysis of skin tissue model under finite pressure

The pulse amplitude of fingertip volume could be improved by selecting the vascular dense area and applying appropriate pressure above it. In view of this phenomenon, this paper used Comsol Multiphysics 5.6 (Comsol, Sweden), the finite element analysis software of multi-physical field coupling simulation, to establish the vascular tissue model of a single small artery in fingertips for simulation. Three dimensional Navier-Stokes equations were solved by finite element method, the velocity field and pressure distribution of blood were calculated, and the deformation of blood vessels and surrounding tissues was analyzed. Based on Lambert Beer's Law, the influence of the longitudinal compression displacement of the lateral light surface region and the tissue model on the light intensity signal is investigated. The results show that the light intensity signal amplitude could be increased and its peak value could be reduced by selecting the area with dense blood vessels. Applying deep pressure to the tissue increased the amplitude and peak of the signal. It is expected that the simulation results combined with the previous experimental experience could provide a feasible scheme for improving the quality of finger volume pulse signal.

Abstract 4038: Study of therapeutic effects of polyisoprenylated cysteinyl amide inhibitors on lung cancer cells of black patients using 3D-Printed alveolar model

Abstract Lung cancer is one of the top five deadliest cancers, and black Americans have a higher mortality rate when compared to white Americans even though they smoke relatively fewer cigarettes. Lung adenocarcinoma (LUAD) is the most common histological lung cancer subtype in all population groups including blacks. There are no in vitro model systems to study lung cancer in black subjects, and there is only one lung adenocarcinoma cell line from a black patient (NCI-H23). Here we aim to address the lack of in vitro models to study black LUAD. Using three-dimensional (3D) biofabrication technologies, we’ve developed a thick vascularized tissue model to recapitulate physiologically relevant heterogeneous human alveoli. Specifically, the blood-air barrier of the alveolar model is simulated with two perfusable channels embedded in a thick vascularized tissue construct: a helical blood channel and a straight air channel. Spatially, the air channel is in the center and is surrounded by the blood channel. Human endothelial cells are seeded onto the inner surface of the blood channel and NCI-H23 cells (which carry a KRAS mutation) or untransformed alveolar epithelial cells derived from black subjects, are seeded on the inner surface of the air channel. The blood and air channels are perfused using cell medium and oxygenated air, respectively. The model is used for the study of potential LUAD therapeutic agents such as polyisoprenylated cysteinyl amide inhibitors (PCAIs), which target and disrupt the RAS-mediated signaling pathway. KRAS is the most commonly mutated oncogene in LUAD in blacks, and our experiments are aimed at ensuring that PCAIs are effective in black LUAD and may offer better treatment outcomes for ethnically/racially diverse patients in whom the RAS signaling pathway is frequently altered and for whom pharmaceutical therapy is necessitated by the late diagnoses. Citation Format: Nadine Belony, Bing Ren, Phuc Pham, Matthew Gregory, Pablo E. Puente, Nazarius S. Lamango, Ite A. Offringa, Yong Huang. Study of therapeutic effects of polyisoprenylated cysteinyl amide inhibitors on lung cancer cells of black patients using 3D-Printed alveolar model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4038.

Peptide nanogels as a scaffold for fabricating dermal grafts and 3D vascularized skin models

Millions of people worldwide suffer from skin injuries, which create significant problems in their lives and are costly to cure. Tissue engineering is a promising approach that aims to fabricate functional organs using biocompatible scaffolds. We designed ultrashort tetrameric peptides with promising properties required for skin tissue engineering. Our work aimed to test the efficacy of these scaffolds for the fabrication of dermal grafts and 3D vascularized skin tissue models. We found that the direct contact of keratinocytes and fibroblasts enhanced the proliferation of the keratinocytes. Moreover, the expression levels of TGF-β1, b-FGF, IL-6, and IL-1α is correlated with the growth of the fibroblasts and keratinocytes in the co-culture. Furthermore, we successfully produced a 3D vascularized skin co-culture model using these peptide scaffolds. We believe that the described results represent an advancement in the fabrication of skin tissue equivalent, thereby providing the opportunity to rebuild missing, failing, or damaged parts. Graphical abstract

Flexibly Deformable Collagen Hydrogel Tube Reproducing Immunological Tissue Deformation of Blood Vessels as a Pharmacokinetic Testing Model.

Since the biochemical reaction of blood vessels plays an essential role in immune response or various diseases, in vitro vascular models have high demand from medical fields. However, vascular models often tend to be difficult to mimic the biomedical reaction faithfully because of the lack of implementation of the tissue deformation. Here, an inflammatory mediator-induced deformation reaction of a blood vessel on a flexibly deformable collagen hydrogel tube device is reproduced. A self-standing collagen tube enables the tissue to deform flexibly in biochemical reaction and achieves contraction both at tissue and cell level. The contraction of tissue relieves the stress between cells under reaction to maintain cellular junctions even tight junctions are broken. Also, the drug perfusion test with antihistamine chemical is easily performed due to the connector part and properly inhibits the inflammatory reaction. Moreover, the traction force on endothelial cells is analyzed as about 0.9 µN on two types of scaffolds with different stiffness. It is believed that the potential of the flexible tissue model to reproduce biochemical reactions can contribute to the fabrication of vascular tissue models mimicking in vivo in high similarity as a platform for biomedical researches and pharmacokinetic testing.

3D Bioprinting-Based Vascularized Tissue Models Mimicking Tissue-Specific Architecture and Pathophysiology for in vitro Studies.

A wide variety of experimental models including 2D cell cultures, model organisms, and 3D in vitro models have been developed to understand pathophysiological phenomena and assess the safety and efficacy of potential therapeutics. In this sense, 3D in vitro models are an intermediate between 2D cell cultures and animal models, as they adequately reproduce 3D microenvironments and human physiology while also being controllable and reproducible. Particularly, recent advances in 3D in vitro biomimicry models, which can produce complex cell structures, shapes, and arrangements, can more similarly reflect in vivo conditions than 2D cell culture. Based on this, 3D bioprinting technology, which enables to place the desired materials in the desired locations, has been introduced to fabricate tissue models with high structural similarity to the native tissues. Therefore, this review discusses the recent developments in this field and the key features of various types of 3D-bioprinted tissues, particularly those associated with blood vessels or highly vascularized organs, such as the heart, liver, and kidney. Moreover, this review also summarizes the current state of the three categories: (1) chemical substance treatment, (2) 3D bioprinting of lesions, and (3) recapitulation of tumor microenvironments (TME) of 3D bioprinting-based disease models according to their disease modeling approach. Finally, we propose the future directions of 3D bioprinting approaches for the creation of more advanced in vitro biomimetic 3D tissues, as well as the translation of 3D bioprinted tissue models to clinical applications.

3D‐Printed Tissue Models: Engineering a Novel 3D Printed Vascularized Tissue Model for Investigating Breast Cancer Metastasis to Bone (Adv. Healthcare Mater. 15/2020)

3D‐Printed Tissue Models: Engineering a Novel 3D Printed Vascularized Tissue Model for Investigating Breast Cancer Metastasis to Bone (Adv. Healthcare Mater. 15/2020)

Electrochemical measurement of respiratory activity for evaluation of fibroblast spheroids containing endothelial cell networks

Vascularized three-dimensional (3D) cultures such as spheroids overcome the problems of insufficient nutrient and oxygen supply and poor cell viability observed with non-vascularized spheroids. However, the capacity of drug penetration may differ with the presence of vascular structures, and needs to be evaluated. Co-culture of endothelial cells (ECs) with other cell types such as fibroblasts is one of the promising methods to engineer vascularized 3D tissue models. At a first step for fabricating vascularized tissues, it is important to prepare EC networks inside the tissue models. Here, we present the first attempt to apply electrochemical measurements of respiratory activity to the fibroblast spheroids containing EC networks, aiming to develop an evaluation technique for vascularized tissue models. The spheroids were exposed to doxorubicin (DOX), an inhibitor of DNA and RNA synthesis, to investigate their drug sensitivity that may differ with the presence of ECs, though rarely studied with regard to respiratory activity. Respiratory activity was measured using two electrochemical devices, a scanning electrochemical microscope (SECM) and a large scale integration (LSI)-based amperometric device. The fibroblast spheroids containing EC networks were constructed with a simple method involving co-culture of normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs) at different concentrations. Fluorescence images showed that HUVECs formed partially networked structure in the fibroblast spheroids. Respiratory activity of the spheroids as measured by SECM varied according to the proportion of HUVECs. In particular, the respiratory activity decreased more in spheroids with a larger proportion of HUVECs after DOX treatment, indicating that drug sensitivity of the spheroids became higher upon incorporation of EC networks. The results with the LSI device also corresponded to those with SECM. Overall, the electrochemical measurements provide meaningful information regarding the EC networks and play an important role in the analysis of 3D cell structures.

Vascular Microphysiological Systems to Model Diseases.

Human vascular microphysiological systems (MPS) represent promising three-dimensional in vitro models of normal and diseased vascular tissue. These systems build upon advances in tissue engineering, microfluidics, and stem cell differentiation and replicate key functional units of organs and tissues. Vascular models have been developed for the microvasculature as well as medium-size arterioles. Key functions of the vascular system have been reproduced and stem cells offer the potential to model genetic diseases and population variation in genes that may increase individual risk for cardiovascular disease. Such systems can be used to evaluate new therapeutics options.

Ultrastructure of royal palm (Roystonea regia) leaf sheath

Abstract. Adzkia U, Nugroho N, Siregar IZ, Karlinasari L. 2020. Ultrastructure of royal palm (Roystonea regia) leaf sheath. Biodiversitas 21: 967-974. The royal palm (Roystonea regia (Kunth) F.Cook is one of the palm species that are many planted along the streets as part of urban landscapes. As biologically products, in nature they can self-cleaning with drop off their big leaves. The study aims to examine the morphological characteristics of ultrastructure and elemental content, crystallinity, as well as fibers derivation characteristics from the royal palm. The samples were taken out from leaf sheaths that divided into three sections along the width of i.e. outside, middle, and inside; in two conditions of leaf sheaths namely green and brownish-dry. The morphology structure and elemental content were observed by the Scanning Electron Microscopy (SEM) and the Energy Dispersive X-ray analysis (EDX), while the crystallinity was analyzed using X-ray diffraction (XRD), and fiber characteristics were measured to determine cell dimensions as well as to calculate Runkle ratio (RR), muhlsteph ratio (MR), coefficient of rigidity (CR), felting powder (FP), and flexibility ratio (FR). The diameter of palm fiber bundles was decreased from outside towards inside. The model of vascular tissue was rounded which located in the central region of the fiber bundles. The silicon content in the inside section was lower than those in the middle and outside sections. It was in line with the degree of crystallinity in the inside section which that higher than other sections. The highest degree of crystallinity was about 18 %. The fiber length of royal palm leaf sheath was in values around 6000 μm. However, the royal palm leaf sheath had thick fiber walls and thin lumen, as well as other fiber derivation characteristics of muhlsteph ratio, the coefficient of rigidity and the flexibility ratio, were in low-quality values. For that reason, the study provides information that the royal palm sheath was recommended as a composite reinforcing material.

Engineering a Novel 3D Printed Vascularized Tissue Model for Investigating Breast Cancer Metastasis to Bone.

Cancer metastases are a challenge for cancer treatment due to their organ specificity and pathophysiological complexity. Engineering 3D in vitro models capable of replicating native cancer dissemination can significantly improve the understanding of cancer biology and can help to guide the development of more effective treatments. In order to better mimic the behavior of native cancer, a triculture metastatic model is created using a stereolithography printing technique with optimized inks for investigating the invasion of breast cancer (BrCa) cells into vascularized bone tissue. The printed system allows to study transendothelial migration and the colony-forming behavior of metastatic BrCa cells. The key steps of BrCa cell progression including expansion, migration, and colonization are continuously monitored and the interactions between cancer cells, vascular cells, and bone cells are systematically investigated. The study results demonstrate that the 3D printed tissue construct by incorporating multiple cells and various favorable ink matrices provides a suitable model to study the interaction between these cells in a complex vascular microenvironment. As such, the 3D printed tricultured model may serve as a valuable tool for studying metastatic breast cancer progression in bone.

Abstract 54: Characterization of patient-derived organoids cultured on a gas-rich, liquid-liquid interface

Abstract Modeling tissues in vitro has long consisted of two-dimensional (2D) monolayer cultures grown in a dish, flask, or multi-well plate. While these methods remain adequate for some applications, the advent of personalized cancer therapies has necessitated the development of more complex models that more accurately represent the in vivo environment. Attempting to characterize tumor cell biology and response to candidate pharmaceuticals in 2D cultures has led to results that fail to translate to clinical therapies. A key source of this failure is the loss of cell diversity due to selective pressure resulting in cell populations that favor adhering to a rigid substrate. Three-dimensional (3D) cell culture methods have produced models that capture many of the dynamics of the in vivo tissue environment. Patient-derived organoids (PDOs) are quickly becoming the most attractive solution for creating predictive, patient-specific tumor models in vitro. Typically grown as spheroids, these models still present limitations in terms of cell survival and recapitulation of tumor development. Lena Biosciences has previously developed complex, vascularized tissue models using patented 3D scaffolds and a perfusion system. Hepatocytes grown in this system demonstrated increased cell viability and recovery of cytochrome P450 enzyme activity compared to 2D controls. Integral to the perfusion system is a pure, synthetic liquid that is twice as dense as and immiscible with cell culture medium and reagents. It is permissive to atmospheric gases, providing a gas-rich, liquid-liquid interface below 3D cultures maintained in the system. We hypothesized that PDOs could be cultured without a 3D scaffold, directly at the interface between the cell culture medium and this synthetic liquid. A specific preparation of this interface resulted in lung-derived PDOs that spontaneously formed dense, 3D, tissue-like structures that showed increased viability (as measured by a 50% reduction in LDH release) and cellular respiration (three-fold increase as measured by an alamarBlue assay). Ongoing studies are focused on characterizing PDOs from other patients and other tissues as they develop in long-term in vitro studies. Should this PDO model prove to be a faithful representation of in vivo tumor development, this system would be ideal for applications such as testing drug efficacy and studying mechanisms of tumor metastasis. Citation Format: James T. Shoemaker, Katherine R. Richardson, Jamie Arnst, Adam Marcus, Jelena Vukasinovic. Characterization of patient-derived organoids cultured on a gas-rich, liquid-liquid interface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 54.

Generation of Cost-Effective Paper-Based Tissue Models through Matrix-Assisted Sacrificial 3D Printing.

Due to the combined advantages of cellulose and nanoscale (diameter 20-60 nm), bacterial cellulose possesses a series of attractive features including its natural origin, moderate biosynthesis process, good biocompatibility, and cost-effectiveness. Moreover, bacterial cellulose nanofibers can be conveniently processed into three-dimensional (3D) intertwined structures and form stable paper devices after simple drying. These advantages make it suitable as the material for construction of organ-on-a-chip devices using matrix-assisted sacrificial 3D printing. We successfully fabricated various microchannel structures embedded in the bulk bacterial cellulose hydrogels and retained their integrity after the drying process. Interestingly, these paper-based devices containing hollow microchannels could be rehydrated and populated with relevant cells to form vascularized tissue models. As a proof-of-concept demonstration, we seeded human umbilical vein endothelial cells (HUVECs) into the microchannels to obtain the vasculature and inoculated the MCF-7 cells onto the surrounding matrix of the paper device to build a 3D paper-based vascularized breast tumor model. The results showed that the microchannels were perfusable, and both HUVECs and MCF-7 cells exhibited favorable proliferation behaviors. This study may provide a new strategy for constructing simple and low-cost in vitro tissue models, which may find potential applications in drug screening and personalized medicine.

Formation of pressurizable hydrogel-based vascular tissue models by selective gelation in composite PDMS channels.

Vascular tissue models created in vitro are of great utility in the biomedical research field, but versatile, facile strategies are still under development. In this study, we proposed a new approach to prepare vascular tissue models in PDMS-based composite channel structures embedded with barium salt powders. When a cell-containing hydrogel precursor solution was continuously pumped in the channel, the precursor solution in the vicinity of the channel wall was selectively gelled because of the barium ions as the gelation agent supplied to the flow. Based on this concept, we were able to prepare vascular tissue models, with diameters of 1–2 mm and with tunable morphologies, composed of smooth muscle cells in the hydrogel matrix and endothelial cells on the lumen. Perfusion culture was successfully performed under a pressurized condition of ∼120 mmHg. The presented platform is potentially useful for creating vascular tissue models that reproduce the physical and morphological characteristics similar to those of vascular tissues in vivo.

Double-layer perfusable collagen microtube device for heterogeneous cell culture

In vitro perfusable 3D tissue models mimic in vivo tissues and have several benefits in drug testing. However, processes used to fabricate these models often tend to be complicated. Here, we present a double-layer perfusable collagen tube device for multilayered in vitro 3D cell culture. The device is simply made by the repetition of a molding process. The thicknesses of the collagen layers in the tube device can be flexibly designed, and heterogeneous cell types can be co-cultured in/on each collagen layer. Moreover, while our collagen tube is directly attached to silicone tubes, the collagen tube can easily be connected to an external pump system for perfusion culture. We fabricated six different sizes of collagen devices (inner diameter approximately 300–1000 μm) using different molds, and successfully controlled the coefficients of variation to be below 5% for the diameters of each layer for all six device sizes. The device is strong enough to manipulate with tweezers, and can remain stable for more than 3 months in a medium. For the cell culture, we successfully and correctly encapsulated cells in the layer shape at the desired position, and confirmed cell migration. Using the perfusion culture, we demonstrated that the alignments of the HUVEC actin filaments become parallel to the flow direction. We believe that our device could advance the easy fabrication of various tissue models (that is, models mimicking in vivo tissues). In particular, the device could help fabricate vascularized tissue models, and contribute to the development of pharmacokinetic testing platforms and regenerative medicine.