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Liver is dynamic, heterogeneous, and each cell type acts in concert to regulate its function. Currently, heterogeneous liver tissues are typically built from single cells using bioprinting, making crosstalk between cells difficult. Therefore, in vitro morphogenesis is limited, and self-assembled biliary and blood vessels system are absent from manufactured liver tissues. The combination of bioprinting and organoid technique offers spatial and cellular control over three-dimensional (3D) organ tissue manufacturing, allowing to build liver tissues with self-assembled structure in vitro. We developed a high-throughput PDMS microwell platform (PMP) generating uniform and functional hepatic organoid building blocks (HOBBs) which displayed cellular crosstalk and self-assembled structure. For bioprinting process, we developed three-level temperature control system and new quadratic material, i.e., alginate-gelatin-collagen-laminin (AGCL) biomaterial, realizing reproducible construction of liver tissues with requisite cellular density. Under long-term differentiation, bioprinted liver tissues exhibited enhanced hepatobiliary function, intrahepatic bile duct networks and angiogenic potential. Heterogeneous tissues with coexistence of cholangiocytes, endothelial cells, and hepatocytes was constructed. The heterogeneous liver tissues with angiogenesis and bile duct component (HABs) provides a novel tool for morphogenesis study, liver regeneration, drug testing, and disease research.

Extrusion-based bioprinting is a widely used approach to construct artificial organs or tissues in the medical fields due to its easy operation and good ability to combine multi-material. Nevertheless, the current technology is limited to some printing errors when combining multi-material printing, including mismatch between printing filaments of different materials and error deposited materials (e.g., under-extrusion and over-extrusion). These errors will affect the function of the printed structure (e.g., mechanical and biological properties), and the traditional manual correction methods are inefficient in time and material, so an automatic procedure is needed to improve multi-material printing accuracy and efficiency. However, to the best of our knowledge, very few automated procedure can achieve the registration between printing filaments of different materials. Herein, we utilized optical coherence tomography (OCT) to monitor printing process and presented a multi-material static model and a time-related control model in extrusion-based multi-material bioprinting. Specifically, the multi-material static model revealed the relationship between printed filament metrics (filament size and layer thickness) and printing parameters (printing speeds or pressures) with different materials, which enables the registration of printing filaments by rapid selection of printing parameters for the materials, while time-related control model could correct control parameters of nozzles to reduce the material deposition error at connection point between nozzles in a short time. According to the experimental results of single-layer scaffold and multi-layer scaffold, material deposition error is eliminated, and the same layer thickness between different materials of the same layer is achieved, which proves the accuracy and practicability of these models. The proposed models could achieve improved precision of printed structure and printing efficiency.

Biphasic scaffolds with biomimetic structure are essential to osteochondral tissue engineering. However, insufficient mechanical properties limit their applications. Here, a novel biphasic scaffold was designed and fabricated using a multi-nozzle three-dimensional (3D) printer with hydroxyapatite (HAp) and polycaprolactone (PCL). HAp was first printed as the subchondral layer and strengthened by sintering, followed by printing PCL as the cartilage layer on top of the HAp layer. High interface bonding strength between the two layers was obtained by controlling the temperature of the printing platform. Compression tests showed that the biphasic scaffold had properties between those of pure HAp and PCL scaffolds, and its compressive modulus was about 18 MPa at the strain of 0–5% and 143 MPa at the strain of 5–10%, which was separately at the range of native cartilage and subchondral bone. Finite element simulation was adopted to investigate the variation of inner structure of the biphasic scaffold under compression, demonstrating a biomimetic strengthened structure with a deformable cartilage layer. Therefore, the biphasic HAp/PCL scaffold takes advantage of both the rigidity of HAp and the elasticity of PCL, thus has biomimetic mechanical properties for its further applications.

Extrusion-based three-dimensional (3D) bioprinting is one of the most common methods used for tissue fabrication and is the most widely used additive manufacturing technique in all industries. In extrusion-based bioprinting, printing defects related to material deposition errors lead to a significant deviation from shape to function between the printed construct and design model. Using 3D extrusion-based bioprinter-associated optical coherence tomography (3D P-OCT), an in situ defect detection and feedback system was presented based on the accurate defect analysis and location, and a pre-built feedback mechanism. Using 3D P-OCT, multi-parameter quantification of the material deposition was carried out in real time, including the filament size, layer thickness, and layer fidelity. The material deposition errors under different paths were quantified and located specifically, including the start-stop points, straight-line path, and turnarounds. The pre-built feedback mechanism involving the control inputs, such as printing path, pressure, and velocity, provided the basis for in situ defect detection and real-time feedback control. In particular, the second printing repair can be performed after the broken filament defect is detected and located. After printing, fidelity can be quantitatively analyzed based on the point cloud registration between the 3D P-OCT result and the design model. In conclusion, 3D P-OCT enables in situ defect detection and feedback control, broken filament repair, and 3D fidelity analysis to achieve high-fidelity printing from shape to function.

Patient-derived organoids (PDOs) serve as excellent tools for personalized drug screening to predict clinical outcomes of cancer treatment. However, current methods for efficient quantification of drug response are limited. Herein, we develop a method for label-free, continuous tracking imaging and quantitative analysis of drug efficacy using PDOs. A self-developed optical coherence tomography (OCT) system was used to monitor the morphological changes of PDOs within 6 days of drug administration. OCT image acquisition was performed every 24 h. An analytical method for organoid segmentation and morphological quantification was developed based on a deep learning network (EGO-Net) to simultaneously analyze multiple morphological organoid parameters under the drug's effect. Adenosine triphosphate (ATP) testing was conducted on the last day of drug treatment. Finally, a corresponding aggregated morphological indicator (AMI) was established using principal component analysis (PCA) based on the correlation analysis between OCT morphological quantification and ATP testing. Determining the AMI of organoids allowed quantitative evaluation of the PDOs responses to gradient concentrations and combinations of drugs. Results showed that there was a strong correlation (correlation coefficient >90%) between the results using the AMI of organoids and those from ATP testing, which is the standard test used for bioactivity measurement. Compared with single-time-point morphological parameters, the introduction of time-dependent morphological parameters can reflect drug efficacy with improved accuracy. Additionally, the AMI of organoids was found to improve the efficiency of 5-fluorouracil(5FU) against tumor cells by allowing the determination of the optimum concentration, and the discrepancies in response among different PDOs using the same drug combinations could also be measured. Collectively, the AMI established by OCT system combined with PCA could quantify the multidimensional morphological changes of organoids under the drug's effect, providing a simple and efficient tool for drug screening in PDOs.

Full-field optical coherence tomography (FF-OCT) enables en face views of scattering samples at a given depth with subcellular resolution, similar to biopsy without the need of sample slicing or other complex preparation. This noninvasive, high-resolution, three-dimensional (3D) imaging method has the potential to become a powerful tool in biomedical research, clinical applications, and other microscopic detection.Our review provides an overview of the disruptive innovations and key technologies to further improve FF-OCT performance, promoting FF-OCT technology in biomedical and other application scenarios.A comprehensive review of state-of-the-art accomplishments in OCT has been performed. Methods to improve performance of FF-OCT systems are reviewed, including advanced phase-shift approaches for imaging speed improvement, methods of denoising, artifact reduction, and aberration correction for imaging quality optimization, innovations for imaging flux expansion (field-of-view enlargement and imaging-depth-limit extension), new implementations for multimodality systems, and deep learning enhanced FF-OCT for information mining, etc. Finally, we summarize the application status and prospects of FF-OCT in the fields of biomedicine, materials science, security, and identification.The most worth-expecting FF-OCT innovations include combining the technique of spatial modulation of optical field and computational optical imaging technology to obtain greater penetration depth, as well as exploiting endogenous contrast for functional imaging, e.g., dynamic FF-OCT, which enables noninvasive visualization of tissue dynamic properties or intracellular motility. Different dynamic imaging algorithms are compared using the same OCT data of the colorectal cancer organoid, which helps to understand the disadvantages and advantages of each. In addition, deep learning enhanced FF-OCT provides more valuable characteristic information, which is of great significance for auxiliary diagnosis and organoid detection.FF-OCT has not been completely exploited and has substantial growth potential. By elaborating the key technologies, performance optimization methods, and application status of FF-OCT, we expect to accelerate the development of FF-OCT in both academic and industry fields. This renewed perspective on FF-OCT may also serve as a road map for future development of invasive 3D super-resolution imaging techniques to solve the problems of microscopic visualization detection.

Significance: Artificial skin (AS) is widely used in dermatology, pharmacology, and toxicology, and has great potential in transplant medicine, burn wound care, and chronic wound treatment. There is a great demand for high-quality AS product and a non-invasive detection method is highly desirable. Aim: To quantify the constructure parameters (i.e., thickness and surface roughness) of AS samples in the culture cycle and explore the growth regularities using optical coherent tomography (OCT). Approach: An adaptive interface detection algorithm is developed to recognize surface points in each A-scan, offering a rapid method to calculate parameters without constructing OCT B-scan pictures and further achieving realizing real-time quantification of AS thickness and surface roughness. Experiments on standard roughness plates and H&E-staining microscopy were performed as a verification. Results: As applied on the whole cycle of AS culture, our method’s results show that during the air–liquid culture, the surface roughness of the skin first decreases and then exhibits an increase, which implies coincidence with the degree of keratinization under a microscope. And normal and typical abnormal samples can be differentiated by thickness and roughness parameters during the culture cycle. Conclusions: The adaptive interface detection algorithm is suitable for high-sensitivity, fast detection, and quantification of the interface with layered characteristic tissues, and can be used for non-destructive detection of the growth regularity of AS sample thickness and roughness during the culture cycle.

Vascularization is essential for the regeneration of three-dimensional (3D) bioprinting organs. As a general method to produce microfluidic channels in 3D printing constructs, coaxial extrusion has attracted great attention. However, the biocompatible bioinks are very limited for coaxial extrusion to fabricate microchannels with regular structure and enough mechanical properties. Herein, a hybrid bioink composed of alginate (Alg) and silk fibroin (SF) was proposed for 3D bioprinting of microchannel networks based on coaxial extrusion. The rheological properties of the bioink demonstrated that the hybrid Alg/SF bioink exhibited improved viscosity and shear thinning behavior compared with either pure Alg or SF bioink and had similar storage and loss modulus in a wide range of shear frequency, indicating a sound printability. Using a coaxial extrusion system with calcium ions and Pluronic F127 flowing through the core nozzle as cross-linkers, the Alg/SF bioink could be extruded and deposited to form a 3D scaffold with interconnected microchannels. The regular structure and smooth pore wall of microchannels inside the scaffold were demonstrated by optical coherence tomography. Micropores left by the rinse of F127 were observed by scanning electron microscope, constituting a hierarchical structure together with the microchannels and printed macropores. Fourier transform infrared spectroscopy analysis proved the complete rinse of F127 and the formation of β-sheet SF structure. Thus, Alg/SF could form a double cross-linked network, which was much stronger than the pure Alg network. Moreover, cells in the Alg/SF scaffold showed higher viability and proliferation rate than in the Alg scaffold. Therefore, Alg/SF is a promising bioink for coaxial extrusion-based 3D bioprinting, with the printed microchannel network beneficial for complex tissue and organ regeneration.

In three-dimensional (3D) bioprinting, errors during the printing process leads to lower structural fidelity, and the desired structural and functional performances cannot be fully satisfied. Process monitoring with high-speed imaging techniques is helpful for identifying material deposition errors. 3D reconstruction of printed objects enables comprehensive evaluation of structural characteristics, with wide-field full-depth imaging being necessary for large objects. In this study, we demonstrate 3D extrusion-based bioprinter-associated optical coherence tomography (3D P-OCT) to achieve high-speed, wide-field and full-depth imaging, and provide in situ process monitoring and comprehensive evaluation for 3D bioprinting. In 3D P-OCT, a wide field was achieved with a lateral image mosaic using the simplified iterative closest point algorithm, and the extension of imaging depth was achieved with a longitudinal image mosaic using image fusion based on maximum intensity values. During the printing process, 3D P-OCT enables real-time multi-parameter quantification for intelligent feedback. In conclusion, with in situ process monitoring and evaluation, 3D P-OCT contributes to ensure better structural and functional performances of 3D functional tissue constructs.

Three-dimensional bio-printing technology is the most advanced new technology in the manufacture of complex multi-layered structures of the skin tissue. This computer-controlled technology is used to precisely place a variety of biological materials and cells at the designated position so that they can be assembled to form three-dimensional tissue layers. Based on the analysis of the structure and composition of human skin, this paper summarizes the basic processes and key elements of three-dimensional bio-printed skin structure modeling, biomaterials, and seed cells. Three types of printers, laser-assisted, inkjet, and extrusion printers, are described and their advantages and disadvantages analyzed. The future development of 3D bio-printing is discussed, and its value in clinical treatment, drug screening, and basic research is summarized.

Ming Yan,1,* Yun Zhang,2,* Haiyan Qin,3 Kezhou Liu,1 Miao Guo,1 Yakun Ge,1 Mingen Xu,1 Yonghong Sun,4 Xiaoxiang Zheng4 1Department of Biomedical Engineering, College of Life Information Science and Instrument Engineering, Hangzhou Dianzi University, Hangzhou, 2Basic Medical Sciences, College of Medicine, Shaoxing University, Shaoxing, 3Department of Chemistry, Zhejiang University, 4Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Department of Biomedical Engineering, Zhejiang University, Hangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Cadmium telluride quantum dots (CdTe QDs) have been proposed to induce oxidative stress, which plays a crucial role in CdTe QDs-mediated mitochondrial-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). However, the direct interactions of CdTe QDs with HUVECs and their potential impairment of other organelles like endoplasmic reticulum (ER) in HUVECs are poorly understood. In this study, we reported that the negatively charged CdTe QDs (–21.63±0.91mV), with good dispersity and fluorescence stability, were rapidly internalized via endocytosis by HUVECs, as the notable internalization could be inhibited up to 95.52% by energy depletion (NaN3/deoxyglucose or low temperature). The endocytosis inhibitors (methyl-β-cyclodextrin, genistein, sucrose, chlorpromazine, and colchicine) dramatically decreased the uptake of CdTe QDs by HUVECs, suggesting that both caveolae/raft- and clathrin-mediated endocytosis were involved in the endothelial uptake of CdTe QDs. Using immunocytochemistry, a striking overlap of the internalized CdTe QDs and ER marker was observed, which indicates that QDs may be transported to ER. The CdTe QDs also caused remarkable ER stress responses in HUVECs, confirmed by significant dilatation of ER cisternae, upregulation of ER stress markers GRP78/GRP94, and activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4). CdTe QDs further promoted an increased C/EBP homologous protein expression, phosphorylation of c-JUN NH2-terminal kinase, and cleavage of ER-resident caspase-4, while the specific inhibitor (SP600125, Z-LEVD-fmk, or salubrinal) significantly attenuated QDs-triggered apoptosis, indicating that all three ER stress-mediated apoptosis pathways were activated and the direct participation of ER in the CdTe QDs-caused apoptotic cell death in HUVECs. Our findings provide important new insights into QDs toxicity and reveal potential cardiovascular risks for the future applications of QDs. Keywords: quantum dots, human umbilical vein endothelial cells, endocytosis, ER stress, apoptosis

Cell therapy, the treatment of diseases using living cells, offers a promising clinical approach to treating refractory diseases. The global market for cell therapy is growing rapidly, and there is an increasing demand for automated methods that can produce large quantities of high quality therapeutic cells. Biomaterials can be used during cell production to establish a biomimetic microenvironment that promotes cell adhesion and proliferation while maintaining target cell genotype and phenotype. Here we review recent progress and emerging techniques in biomaterial-assisted cell production. The increasing use of auxiliary biomaterials and automated production methods provides an opportunity to improve quality control and increase production efficiency using standardized GMP-compliant procedures.

One of the major obstacles in drug discovery is the lack of in vitro three-dimensional (3D) models that can capture more complex features of a disease.Here we established a in vitro physiological model of the metabolic syndrome (MS) using cell-assembly technique (CAT), which can assemble cells into designated places to form complex 3D structures. Adipose-derived stromal (ADS) cells were assembled with gelatin/alginate/fibrinogen. Fibrin was employed as an effective material to regulate ADS cell differentiation and self-organization along with other methods. ADS cells differentiated into adipocytes and endothelial cells, meanwhile, the cells were induced to self-organize into an analogous tissue structure. Pancreatic islets were then deposited at designated locations and constituted the adipoinsular axis with adipocytes. Analysis of the factors involved in energy metabolism showed that this system could capture more pathological features of MS. Drugs known to have effects on MS showed accordant effects in this system, indicating that the model has potential in MS drug discovery. Overall, this study demonstrated that cell differentiation and self-organization can be regulated by techniques combined with CAT. The model presented could result in a better understanding of the pathogenesis of MS and the development of new technologies for drug discovery.

One of the major obstacles in developing multifunctional drugs for the metabolic syndrome (MS) is lack of in vitro models that capture more complex features of the disease. Here we give the first report that establishes an energy metabolic system model using cell-assembly technique which can assemble cells into designated places to form complex three-dimensional structures. Adipose-derived stromal cells were assembled and induced differentiation into adipocytes and endothelial cells; pancreatic islets were then deposited at designated locations and constituted adipoinsular axis with adipocytes. Analysis of the factors involved in energy metabolism showed our system could capture more physiological and pathophysiological features of the in vivo energy metabolism. Drugs known to have effects on MS showed accordant effects in the systems. Construction and study of such multicellular systems could help us better understand pathogenesis of MS, develop new technologies for drug discovery, and foster applications in tissue engineering and metabolomics profiling.