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A method is proposed of fabricating tubular constructs from tissue spheroids (conglomerates of cells up to 200 μm in size) in a nutrient fluid using the acoustic radiation force. The source of the acoustic field is a hollow piezoceramic cylinder with a resonant frequency of 800 kHz. Keeping the obtained structure at 37°C for 24 h fuses the spheroids into a solid tubular viable tissue construct.

The ability to measure microtissue contraction in vitro can provide important information when modeling cardiac, cardiovascular, respiratory, digestive, dermal, and skeletal tissues. However, measuring tissue contraction in vitro often requires the use of high number of cells per tissue construct along with time-consuming microscopy and image analysis. Here, we present an inexpensive, versatile, high-throughput platform to measure microtissue contraction in a 96-well plate configuration using one-step batch imaging. More specifically, optical fiber microprobes are embedded in microtissues, and contraction is measured as a function of the deflection of optical signals emitted from the end of the fibers. Signals can be measured from all the filled wells on the plate simultaneously using a digital camera. An algorithm uses pixel-based image analysis and computer vision techniques for the accurate multiwell quantification of positional changes in the optical microprobes caused by the contraction of the microtissues. Microtissue constructs containing 20,000-100,000 human ventricular cardiac fibroblasts (NHCF-V) in 6 mg/mL collagen type I showed contractile displacements ranging from 20-200 μm. This highly sensitive and versatile platform can be used for the high-throughput screening of microtissues in disease modeling, drug screening for therapeutics, physiology research, and safety pharmacology.

Tissue-engineered heart valves can be an alternative to prosthetic valves in heart valve replacement; however, they are not fully efficient in terms of long-lasting functionality as leaflets in engineered valves do not possess the native trilayered leaflet structure. Previously, we developed flat, trilayered, oriented nanofibrous (TN) scaffolds mimicking the trilayered structure and orientation of native heart valve leaflets. In-vivo tissue engineering – a practical regenerative medicine technology – can be used to develop autologous heart valves. Thus, in this study, we used our flat, trilayered, oriented nanofibrous scaffolds to develop trilayered tissue structures with native leaflet orientations through in-vivo tissue engineering in a rat model. After two months of in-vivo tissue engineering, infiltrated cells and their deposited collagen fibrils were found aligned in the circumferential and radial layers, and randomly oriented in the random layer of the scaffolds; i.e., trilayered tissue constructs (TTCs) were developed. The tensile properties of the TTCs were higher than that of the control tissue constructs (without any scaffolds) due to the influence of fibers of the scaffolds in tissue engineering. Different extracellular matrix components – collagen, glycosaminoglycans and elastin that exist in native leaflets were observed in the TTCs. Gene expression of the cells in the TTCs indicated that the tissue constructs were in the growing stage. There was no sign of calcification in the tissue constructs. Thus, the TTCs developed with the flat TN scaffolds signify that autologous leaflet-shaped, trilayered tissue constructs that can function as native leaflets can be developed.

Aim: We aimed to develop a leaflet-shaped trilayered tissue construct mimicking the morphology of native heart valve leaflets. Materials & methods: Electrospinning and in vivo tissue engineering methods were employed. Results: We developed leaflet-shaped microfibrous scaffolds, each with circumferentially, randomly and radially oriented three layers mimicking the trilayered, oriented structure of native leaflets. After 3 months in vivo tissue engineering with the scaffolds, the generated leaflet-shaped tissue constructs had a trilayered structure mimicking the orientations of native heart valve leaflets. Presence of collagen, glycosaminoglycans and elastin seen in native leaflets was observed in the engineered tissue constructs. Conclusion: Trilayered, oriented fibrous scaffolds brought the orientations of the infiltrated cells and their produced extracellular matrix proteins into the constructs.

Tissue engineering strategies provide viable replacement of structurally or functionally damaged native tissues. Development of its key component, the biomaterial matrices is assisted with nano-, micro-, and macrofabrication technologies to provide microenvironment to proliferating cells and regenerating tissues. Electrospinning has been introduced to this field for controlled pore architecture and distribution, with nanofibrous matrices having inducing impact over cell–material interaction and guiding tissue regeneration of various lineages. This chapter is dedicated to the use of electrospinning for tissue engineering applications. The process and control parameters of electrospinning with respect to different tissue engineering applications is also discussed that affect structure and functionality of developed tissue construct.

Cell culture studies are essential tool for improving our understanding in cell and molecular biology, development of tissue construct as well as new drugs and exploring their mechanism of action. Till date the two dimensional cell culture model is dominant method in most of the biological studies. However, considering the complexity of biological system, three dimensional cell culture is considered as promising approach. Specifically in pharamacetical application, three dimensional culture systems is now received remarkable attention for drug discovery and drug trial. Here, we discuss the application of two dimensional and three dimensional cell culture methods in the field of pharmaceutical applications.

We are introducing the FABRICA, a bioprinter-agnostic 3D-printed bioreactor platform designed for 3D-bioprinted tissue construct culture, perfusion, observation, and analysis. The computer-designed FABRICA was 3D-printed with biocompatible material and used for two studies: (1) Flow Profile Study: perfused 5 different media through a synthetic 3D-bioprinted construct and ultrasonically analyzed the flow profile at increasing volumetric flow rates (VFR); (2) Construct Perfusion Study: perfused a 3D-bioprinted tissue construct for a week and compared histologically with a non-perfused control. For the flow profile study, construct VFR increased with increasing pump VFR. Water and other media increased VFR significantly while human and pig blood showed shallow increases. For the construct perfusion study, we confirmed more viable cells in perfused 3D-bioprinted tissue compared to control. The FABRICA can be used to visualize constructs during 3D-bioprinting, incubation, and to control and ultrasonically analyze perfusion, aseptically in real-time, making the FABRICA tunable for different tissues.

This study describes the development of a Time-to-Toxicity approach for liquids (TTL) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1). Briefly, the Time-to-Toxicity of 56 liquids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for three different time periods (5-min, 16-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 50 liquids, was 80.0%. Furthermore, 84.3% Cat. 1 (N = 17), 79.4% Cat. 2 (N = 21) and 72.2% No Cat. (N = 18) were correctly identified with the SkinEthic™ HCE TTL test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) is capable of distinguishing Cat. 2 liquids from Cat. 1 liquids. This is an advantage compared to the SkinEthic™ HCE EITL method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).

This study describes the development of a Time-to-Toxicity approach for solids (TTS) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1). Briefly, the time-to-toxicity of 69 solids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for two different time periods (30-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 48 solids, was 93.7%. Furthermore, 73.6% Cat. 1 (N = 24), 55.6% Cat. 2 (N = 15) and 72.2% No Cat. (N = 30) were correctly identified with the SkinEthic™ HCE TTS test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) can distinguish Cat. 2 solids from Cat. 1 solids. This is an added value compared to the SkinEthic™ HCE EITS method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).

Current neural tissue engineering strategies involve the development and application of neural tissue constructs produced by using an anisotropic polymeric scaffold. This study reports a scaffold-free method of tissue engineering to create a tubular neural tissue construct containing unidirectional neuron bundles. The surface patterning of a thermoresponsive culture substrate and a coculture system of neurons with patterned astrocytes can provide an anisotropic structure and easy handling of the neural tissue construct without the use of a scaffold. Furthermore, using a gelatin gel-coated plunger, the neuron bundles can be laid out in the same direction at regulated intervals within multilayered astrocyte sheets. Since the 3D tissue construct is composed only by neurons and astrocytes, they can communicate physiologically without obstruction of a scaffold. The medical benefits of scaffold-free tissue generation provide new opportunities for the development of human cell-based tissue models required to better understand the mechanisms of neurodegenerative diseases. Therefore, this new tissue engineering approach may be useful to establish a technology for regenerative medicine and drug discovery using the patient's own neurons.

The objective of this study was to create a method for studying cell–matrix interactions in a physiologically relevant 3D protein-based tissue construct that could be scaled up to perform large-scale screens, study cell–matrix interactions on a population basis, or be remodeled by cells to build larger tissues. We have developed an easy-to-use method to miniaturize protein-based tissue constructs that maintains the 3D in vitro environment, while alleviating several obstacles associated with larger avascular tissue constructs. In this study, we demonstrate that (i) cells can interact with the 3D environment both while encapsulated or while interacting only with the surface of the microtissues, (ii) encapsulated cells are highly viable and, for the first time, (iii) microtissues on this size scale (~200 μm) can be used to quantify cell contractility. This versatile platform should facilitate large-scale screens in 3D in vitro culture conditions for drug development and high throughput mechanistic biology.

Bioprinting is a tool increasingly used in tissue engineering laboratories around the world. As an extension to classic tissue engineering, it enables high levels of control over the spatial deposition of cells, materials, and other factors. It is a field with huge promise for the production of implantable tissues and even organs, but the availability of functional bioinks is a barrier to success. Extrusion bioprinting is the most commonly used technique, where high-viscosity solutions of materials and cells are required to ensure good shape fidelity of the printed tissue construct. This is contradictory to hydrogels used in tissue engineering, which are generally of low viscosity prior to cross-linking to ensure cell viability, making them not directly translatable to bioprinting. This review provides an overview of the important rheological parameters for bioinks and methods to assess printability, as well as the effect of bioink rheology on cell viability. Developments over the last five years in bioink formulations and the use of suspended printing to overcome rheological limitations are then discussed.

3D bioprinting techniques have shown great promise in various fields of tissue engineering and regenerative medicine. Yet, creating a tissue construct that faithfully represents the tightly regulated composition, microenvironment, and function of native tissues is still challenging. Among various factors, biomechanics of bioprinting processes play fundamental roles in determining the ultimate outcome of manufactured constructs. This review provides a comprehensive and detailed overview on various biomechanical factors involved in tissue bioprinting, including those involved in pre, during, and post printing procedures. In preprinting processes, factors including viscosity, osmotic pressure, and injectability are reviewed and their influence on cell behavior during the bioink preparation is discussed, providing a basic guidance for the selection and optimization of bioinks. In during bioprinting processes, we review the key characteristics that determine the success of tissue manufacturing, including the rheological properties and surface tension of the bioink, printing flow rate control, process-induced mechanical forces, and the in situ cross-linking mechanisms. Advanced bioprinting techniques, including embedded and multi-material printing, are explored. For post printing steps, general techniques and equipment that are used for characterizing the biomechanical properties of printed tissue constructs are reviewed. Furthermore, the biomechanical interactions between printed constructs and various tissue/cell types are elaborated for both in vitro and in vivo applications. The review is concluded with an outlook regarding the significance of biomechanical processes in tissue bioprinting, presenting future directions to address some of the key challenges faced by the bioprinting community.

There is an increasing interest in organ-level 3D tissue constructs, owing to their mirroring of in vivo-like features. This has resulted in a wide range of preclinical applications to obtain cell- or tissue-specific responses. Additionally, the development and improvement of sophisticated technologies, such as organoid generation, microfluidics, hydrogel engineering, and 3D printing, have enhanced 3D tissue constructs to become more elaborate. In particular, recent studies have focused on including complex compartments, i.e., vascular and innervation structured 3D tissue constructs, which mimic the nature of the human body in that all tissues/organs are interconnected and physiological phenomena are mediated through vascular and neural systems. Here, the strategies are categorized according to the number of dimensions (0D, 1D, 2D, and 3D) of the starting materials for scaling up, and novel approaches to introduce increased complexity in 3D tissue constructs are highlighted. Recent advances in preclinical applications are also investigated to gain insight into the future direction of 3D tissue construct research. Overcoming the challenges in improving organ-level functional 3D tissue constructs both in vitro and in vivo will ultimately become a life-saving tool in the biomedical field.

This textbook has demonstrated that 3D-bioprinted in vitro tissue/organ models using tissue-specific bioinks open up exciting prospects in engineering a more realistic and predictable testing platform. Despite this potential, there are several concerns to be addressed. One of the main concerns is that the productivity and reproducibility of current bioprinted dECM-based tissue models are very limited; only one 3D tissue construct has been built using the 3D bioprinting system. In order to obtain highly accurate and reliable results applicable clinically, high-throughput screening on 3D-bioprinted tissue models should be considered. Because 3D bioprinting tissue models facilitate simultaneous incorporation of a high-contents and high-throughput system, an advanced technique focusing on high-throughput screening would be conducive to accurate prediction.

Regenerative engineering is defined as the integration of tissue engineering with advanced material science, stem cell science, biophysical stimulation, and areas of developmental biology. This article seeks to provide some guidance when considering the characterization of tissue constructs designed for regenerative engineering. Specifically, an overview of mechanical testing parameters and considerations, cellular and cell-material analysis, and potential preclinical models of tissue defect and repair are provided. This article is meant to serve as a starting point for these areas to inform the regenerative engineer about the most salient aspects of tissue construct characterization.

Many living organisms undergo conspicuous or abrupt changes in body structure, which is often accompanied by a behavioral change. Inspired by the natural metamorphosis, robotic systems can be designed as reconfigurable to be multifunctional. Here, a tissue-engineered transformable robot is developed, which can be remotely controlled to assume different mechanical structures for switching locomotive function. The soft robot is actuated by a muscular tail fin that emulates the swimming of whales and works as a cellular engine powered by the synchronized contraction of striated cardiac microtissue constructs. For a transition of locomotive behavior, the robot can be optically triggered to transform from a spread to a retracted form, which effectively changes the bending stiffness of the tail fins, thus minimizing the propulsion output from the "tail fin" and effectively switching off the engine. With the unprecedented controllability and responsiveness, the transformable robot is implemented to work as a cargo carrier for programmed delivery of chemotherapeutic agents to selectively eradicate cancer cells. It is believed that the realization of the transformable concept paves a pathway for potential development of intelligent biohybrid robotic systems.

Vascularization is of great importance in the successful translation of tissue engineered constructs into clinically relevant application. The lack of a general approach to rapidly construct vascular networks in engineered constructs remains a major challenge. Herein, an adhesive hydrogel-based tissue construct, in which cell-affinitive domains and interconnected channels were concurrently constructed, was put forward to enhance vascularization. Hydrogel matrix was modified with Arg-Gly-Asp (RGD) peptide to supply cell adhesion sites. Collagen fibers were added into the hydrogel matrix to produce interconnected vessel-like channels via enzyme mediated degradation. In a bone-like model, the successful outspread morphology and intensive function expression of osteo-like cells and the formation of endothelial cells-lined channels were observed, suggesting it's flexible to functionalize extracellular matrix with vessel-like channels via the introduction of endothelial cells-laden fibers. Our approach furnishes a particular strategy to build vascular architecture and is especially attractive in the bioengineering of rich vascularized tissues.

3D tissue based on human mesenchymal stem cell (hMSC) sheets offers many interesting opportunities for regenerating multiple types of connective tissues. Prevascularizing hMSC sheets with endothelial cells (ECs) will improve 3D tissue performance by supporting cell survival and accelerating integration with host tissue. It is hypothesized that hypoxia cultured hMSC sheets can promote microvessel network formation and preserve stemness of hMSCs. This study investigates the vascularization of hMSC sheets under different oxygen tensions. It is found that the HN condition, in which hMSC sheets formed under physiological hypoxia (2% O2 ) and then cocultured with ECs under normoxia (20% O2 ), enables longer and more branched microvessel network formation. The observation is corroborated by higher levels of angiogenic factors in coculture medium. Additionally, the hypoxic hMSC sheet is more uniform and less defective, which facilitates fabrication of 3D prevascularized tissue construct by layering the prevascularized hMSC sheets and maturing in rotating wall vessel bioreactor. The hMSCs in the 3D construct still maintain multilineage differentiation ability, which indicates the possible application of the 3D construct for various connective tissues regeneration. These results demonstrate that hypoxia created hMSC sheets benefit the microvessel growth and it is feasible to construct 3D prevascularized tissue construct using the prevascularized hMSC sheets.

3D biodegradable and highly regular foamlike cell scaffolds based on biocompatible side-chain liquid crystal elastomers have been prepared. Scaffolds with a primary porosity characterized by spatially interlaced, interconnected microchannels or an additional secondary porosity featuring interconnected microchannel networks define the novel elastomeric scaffolds. The macroscale morphology of the dual porosity 3D scaffold resembles vascular networks observed in tissue. 3D elastomer foams show four times higher cell proliferation capability compared to conventional porous templated films and within the channels guide spontaneous cell alignment enabling the possibility of tissue construct fabrication toward more clinically complex environments.

A grand challenge of tissue engineering is the fabrication of large constructs with a high density of living cells. By adapting the principles of pick-and-place machines used in the high-speed assembly of electronics, we have developed an innovative instrument, the Bio-Pick, Place, and Perfuse (Bio-P3), which picks up large complex multicellular building parts, transports them to a build area, and precisely places the parts at desired locations while perfusing the parts. These assembled parts subsequently fuse to form a larger contiguous tissue construct. Multicellular microtissues were formed by seeding cells into nonadhesive micro-molds, wherein cells self-assembled scaffold-free parts in the shape of spheroids, toroids, and honeycombs. After removal from the molds, the parts were gripped, transported (using an x, y, z controller), and released using the Bio-P3 with little to no effect on cell viability or part structure. As many as 16 toroids were stacked over a 170 μm diameter post where they fused over the course of 48 h to form a single tissue. Larger honeycomb parts were also gripped and stacked onto a build head that, like the gripper head, provided fluid suction to hold and perfuse the parts during assembly. Scaffold-free building parts help to address several of the engineering and biological challenges to large tissue biofabrication, and the Bio-P3 described in this article is a novel instrument for the controlled gripping, placing, stacking, and perfusing of living building parts for solid organ fabrication.

Scaffold-free tissue engineering can be considered as a rapidly developing technique in the field of tissue engineering. In the areas of regenerative medicine and wound healing, there is a demand of techniques where no scaffolds are used for the development of desired tissue. These techniques will overcome the problems of rejection and tissue failure which are common with scaffolds. Main breakthrough of scaffold free tissue engineering was after invention of 3-D printers which made it possible to print complex tissues which were not possible by conventional methods. Mathematical modeling is a prediction technique is used in tissue engineering for simulation of the model to be constructed. Coming to scaffold-free technique, mathematical modeling is necessary for the processing of the model that has to be bio-printed so as to avoid and changes in the final construct. Tissue construct is developed by use of non-destructive imaging techniques i.e. computed tomography (CT) and magnetic resonance imaging (MRI).In this review, we discussed about various mathematical models and the models which are widely used in bioprinting techniques such as Cellular Potts Model (CPM) and Cellular Particle Dynamic (CPD) model. Later, developed of 3-D tissue construct using micro CT scan images was explained. Finally, we discussed about scaffold free techniques such as 3-D bioprinting and cell sheet technology. In this manuscript, we proposed a cell sheet based bioprinting technique where mesenchymal stem cells (MSCs) on the surface of thermoresponsive polymer were subjected to mechanosensing either by introducing acoustic energies or stress created by polymeric strain energy function. Mechanosensing stimulus will trigger the intracellular signal transduction pathway leading to differentiation of the MSCs into desired cells.

Hydrogel plays a vital role in cell-laden three dimensional (3D) bioprinting, whereas those hydrogels mimic the physical and biochemical characteristics of native extracellular matrix (ECM). The complex microenvironment of the ECM does not replicate from the traditional static microenvironment of the hydrogel, but the evolution of the 3D bioprinting facilitates to accommodate the dynamic modulation and spatial heterogeneity of the hydrogel system. Selection of hydrogel for 3D bioprinting depends on the printing techniques including microextrusion, inkjet, laser-assisted printing, and stereolithography. In this review, we specifically cover the 3D printable hydrogels where cells can be encapsulated without significant reduction in the cell viability. The recent research highlights of the most widely used hydrogel materials are elucidated in terms of stability of the hydrogel system, cross-linking method, support cell types and their post-printing cell viability. Also, the techniques used to improve the mechanical and biological properties of the hydrogels, such as adding various organic and inorganic materials and making microchannels, are discussed. Furthermore, the recent advances in vascularized tissue construct and scaffold-free bioprinting as a promising method for vascularization are covered in this review. The recent trends in four-dimensional (4D) bioprinting as a stimuli-responsive formation of new organs, and 3D bioprinting based organ-on-chip systems are also discussed.

Tissue anisotropy directed by cell sheets: Aligned myoblasts can be harvested as an anisotropic cell sheet using a micropatterned thermoresponsive substrate. Neurons and endothelial cells sandwiched between multiple anisotropic cell sheets self-organize oriented cellular networks in the tissue construct. This simple tissue engineering technique is useful for the creation of biomimetic microstructures in complex tissue, required for future advances in regenerative medicine.