Cell Printing Research Articles

Visible light-crosslinkable tyramine-conjugated alginate-based microgel bioink for multiple cell-laden 3D artificial organ

While the natural carbohydrate alginate has enabled effective three-dimensional (3D) extrusion bioprinting, it still suffers from some issues such as low printability and resolution and limited cellular function due to ionic crosslinking dependency. Here, we prepared a harmless visible light-based photocrosslinkable alginate by chemically bonding tyrosine-like residues onto alginate chains to propose a new microgel manufacturing system for the development of 3D-printed bioinks. The photocrosslinkable tyramine-conjugated alginate microgel achieved both higher cell viability and printing resolution compared to the bulk gel form. This alginate-based jammed granular microgel bioink showed excellent 3D bioprinting ability with maintained structural stability. As a biocompatible material, the developed multiple cell-loaded photocrosslinkable alginate-based microgel bioink provided excellent proliferation and migration abilities of laden living cells, providing an effective strategy to construct implantable functional artificial organ structures for 3D bioprinting-based tissue engineering.

Enhancing Mechanical Properties of 3D Printing Metallic Lattice Structure Inspired by Bambusa Emeiensis.

Metallic additive manufacturing process parameters, such as inclination angle and minimum radius, impose constraints on the printable lattice cell configurations in complex components. As a result, their mechanical properties are usually lower than their design values. Meanwhile, due to unavoidable process constraints (e.g., additional support structure), engineering structures filled with various lattice cells usually fail to be printed or cannot achieve the designed mechanical performances. Optimizing the cell configuration and printing process are effective ways to solve these problems, but this is becoming more and more difficult and costly with the increasing demand for properties. Therefore, it is very important to redesign the existing printable lattice structures to improve their mechanical properties. In this paper, inspired by the macro- and meso-structures of bamboo, a bionic lattice structure was partitioned, and the cell rod had a radius gradient, similar to the macro-scale bamboo joint and meso-scale bamboo tube, respectively. Experimental and simulated results showed that this design can significantly enhance the mechanical properties without adding mass and changing the printable cell configuration. Finally, the compression and shear properties of the Bambusa-lattice structure were analyzed. Compared with the original scheme, the bamboo lattice structure design can improve the strength by 1.51 times (β=1.5). This proposed strategy offers an effective pathway to manipulate the mechanical properties of lattice structures simultaneously, which is useful for practical applications.

Pathophysiological Reconstruction of a Tissue‐Specific Multiple‐Organ On‐A‐Chip for Type 2 Diabetes Emulation using 3D Cell Printing

AbstractType 2 diabetes mellitus (T2D), a chronic complex disease with high prevalence, causes severe damage to various organs. T2D has many etiologies that have not been fully elucidated. Although various models have been created to understand T2D, reconstructing the tissue‐specific microenvironment and complications of T2D remain difficult. Therefore, a dynamic multiple‐organ on‐a‐chip that mimics T2D in a hyperglycemic environment and visceral adipose tissue (vAT) using 3D cell printing with decellularized extracellular matrix (dECM) bioinks is developed. This chip composed of separate compartments for the pancreas, adipose tissue, and liver, closely related to T2D, and a retinal compartment is added to confirm T2D complications. Furthermore, the pathological features of T2D are clearly revealed in the chip with vAT‐derived dECM bioink in a hyperglycemic environment. These pathological features caused cellular dysfunctions of the retinal compartment. Moreover, treated T2D medications on the chip showed clinically identical efficacy. The chip is suitable for recapitulating the key features of T2D and is a promising platform for drug testing and T2D complication research.

Design of a novel bioink suitable for the 3D printing of lymphoid cells

Introduction: For decades, in vitro 2D cell culture techniques have been employed in research, but they fail to recapitulate the complexity of natural tissues. 3D bioprinting could potentially overcome this drawback due to the possibility to control the spatial disposition of living cells and the geometry of the 3D scaffold.Materials and methods: This study reports the design and characterization of a novel bioink for extrusion bioprinting, analyzing different blend formulations composed of alginate, gelatin, and methylcellulose, suitable as cell-laden bioink for lymphoid cells, in particular those isolated from patients with Chronic Lymphocytic Leukemia (CLL). The rheological properties as a function of temperature and the printability of the formulations were investigated to define the optimal printing parameters. In vitro stability of the printed scaffolds was investigated under culture conditions and compression tests were performed on printed and bioprinted scaffolds to compare their mechanical properties with those of fresh lymphoid tissue. Finally, MEC1, a CLL cell line, was bioprinted to investigate cell viability, cell density, and cell capability to be released from the scaffold over time.Results and discussion: Results showed that, for the selected blends, good shape fidelity and printing accuracy were achieved with a limitation on the number of printed layers. Scaffolds withstood culture conditions showing stability for up to 3 weeks and their mechanical properties were similar to those of lymphoid tissues already reported in the literature. High cell viability after 21 days was observed for both MEC1 and primary peripheral mononuclear cells, confirming the possibility to use the selected formulation to successfully bioprint lymphoid cells by possibly mimicking their native lymphoid microenvironment.

Nanocomposite Bioprinting for Tissue Engineering Applications.

Bioprinting aims to provide new avenues for regenerating damaged human tissues through the controlled printing of live cells and biocompatible materials that can function therapeutically. Polymeric hydrogels are commonly investigated ink materials for 3D and 4D bioprinting applications, as they can contain intrinsic properties relative to those of the native tissue extracellular matrix and can be printed to produce scaffolds of hierarchical organization. The incorporation of nanoscale material additives, such as nanoparticles, to the bulk of inks, has allowed for significant tunability of the mechanical, biological, structural, and physicochemical material properties during and after printing. The modulatory and biological effects of nanoparticles as bioink additives can derive from their shape, size, surface chemistry, concentration, and/or material source, making many configurations of nanoparticle additives of high interest to be thoroughly investigated for the improved design of bioactive tissue engineering constructs. This paper aims to review the incorporation of nanoparticles, as well as other nanoscale additive materials, to printable bioinks for tissue engineering applications, specifically bone, cartilage, dental, and cardiovascular tissues. An overview of the various bioinks and their classifications will be discussed with emphasis on cellular and mechanical material interactions, as well the various bioink formulation methodologies for 3D and 4D bioprinting techniques. The current advances and limitations within the field will be highlighted.

Coaxial cell printing of a human glomerular model: an in vitro glomerular filtration barrier and its pathophysiology

Much effort has been expended in emulating the kidney’s glomerular unit because of its limitless potential in the field of drug screening and nephrotoxicity testing in clinics. Herein, we fabricate a functional bilayer glomerular microvessel-on-a-chip that recapitulates the specific arrangement of the glomerular endothelial cell, podocyte layers, and the intervening glomerular basement membrane (GBM) in a single step. Our perfusable chip allows for the co-culture of monolayer glomerular endothelium and podocyte epithelium, which display mature functional markers of glomerular cells, and their proper interactions produce GBM proteins, which are the major components of the GBM in vivo. Furthermore, we test the selective permeability capacity, a representative hallmark function of the glomerular filtration barrier. Lastly, we evaluate the response of our glomerular model to Adriamycin- and hyperglycemia-induced injury to evaluate its applicability for drug screening and glomerular disease modeling.

Towards a Novel Cost-Effective and Versatile Bioink for 3D-Bioprinting in Tissue Engineering

3D-bioprinting for tissue regeneration relies on, among other things, hydrogels with favorable rheological properties. These include shear thinning for cell-friendly extrusion, post-printing structural stability as well as physiologically relevant elastic moduli needed for optimal cell attachment, proliferation, differentiation and tissue maturation. This work introduces a cost-efficient gelatin-methylcellulose based hydrogel whose rheological properties can be independently optimized for optimal printability and tissue engineering. Hydrogel viscosities were designed to present three different temperature regimes: low viscosity for eased cell suspension and printing with minimal shear stress, form fidelity directly after printing and long term structural stability during incubation. Enzymatically crosslinked hydrogel scaffolds with stiffnesses ranging from 5 to 50 kPa were produced, enabling the hydrogel to biomimic cell environments for different types of tissues. The bioink showed high intrinsic cytocompatibility and tissues fabricated by embedding and bioprinting NIH 3T3 fibroblasts showed satisfactory viability. This novel hydrogel uses robust and inexpensive technology, which can be adjusted for implementation in tissue regeneration, e.g., in myocardial or neural tissue engineering.

Enhanced Glutaminolysis Drives Hypoxia-Induced Chemoresistance in Pancreatic Cancer.

Increased glutaminolysis induces hypoxia via oxidative phosphorylation-mediated oxygen consumption and drives chemoresistance in pancreatic cancer, revealing a potential therapeutic strategy of combining glutaminolysis inhibition and chemotherapy to overcome resistance.

Continuous trapping, elasticity measuring and deterministic printing of single cells using arrayed microfluidic traps.

Analysis of single cells after elasticity measurement may construct a linkage between biophysics and other cellular properties, e.g., cell signaling and genetics. This paper reports a microfluidic technology integrating trapping, elasticity measurement, and printing of single cells based on the precise regulation of pressure across an array of U-shaped traps. Both numerical and theoretical analyses revealed that the positive and negative pressure drop across each trap correspondingly contributed to the capture and release of single cells. Afterward, microbeads were employed to demonstrate the capabilities in rapid capturing of single beads. As the printing pressure increased from 0.64 to 3.03 kPa, all beads were released from traps one by one and dispensed into individual wells with an efficiency of 96%. Cell experiments demonstrated that all traps captured K562 cells within 15.25 ± 7.63 seconds. The single-cell trapping efficiency (75.86-95.31%) was proportional to the sample flow rate. Based on the protrusion of each trapped cell and the relevant pressure drop, the stiffness of passages 8 and 46 K562 cells was respectively determined as 171.15 ± 73.35 Pa and 13 959 ± 6328 Pa. The former was consistent with previous studies and the latter was extremely elevated, owing to the cell property variation during a long culture period. Finally, the single cells with known elasticity were deterministically printed into well plates with an efficiency of 92.62%. This technology is a powerful tool for both continuous single cell dispensing and innovatively enabling the relation of cell mechanics to biophysical properties using traditional equipment.

Towards skin-on-a-chip for screening the dermal absorption of cosmetics.

Over the past few decades, there have been increasing global efforts to limit or ban the use of animals for testing cosmetic products. This ambition has been at the heart of international endeavours to develop new in vitro and animal-free approaches for assessing the safety of cosmetics. While several of these new approach methodologies (NAMs) have been approved for assessing different toxicological endpoints in the UK and across the EU, there remains an absence of animal-free methods for screening for dermal absorption; a measure that assesses the degree to which chemical substances can become systemically available through contact with human skin. Here, we identify some of the major technical barriers that have impacted regulatory recognition of an in vitro skin model for this purpose and propose how these could be overcome on-chip using artificial cells engineered from the bottom-up. As part of our future perspective, we suggest how this could be realised using a digital biomanufacturing pipeline that connects the design, microfluidic generation and 3D printing of artificial cells into user-crafted synthetic tissues. We highlight milestone achievements towards this goal, identify future challenges, and suggest how the ability to engineer animal-free skin models could have significant long-term consequences for dermal absorption screening, as well as for other applications.

High-speed printing of a bulk-heterojunction architecture in organic solar cells films

A polymer self-doping strategy can improve the high-speed processability of the active layer without compromising performance to reduce minimum sustainable price, which guide the lab-to-fab of the high coating speed printing of organic solar cells.

Multimodal 3D printing of biodegradable shape memory elastomer resins for patient specific soft tissue repair

Acrylated poly(glycerol-dodecanedioate) (APGD), a biodegradable shape memory polymer, is a photocurable resin developed for 3D printing of biomaterial implants that is especially suited for minimally invasive procedures. 3D printing has numerous advantages over traditional manufacturing modalities including production of complex porous geometries, improved feature resolution, and lower production costs on a per part basis for low volume clinical production. Material extrusion (ME) and vat photopolymerization (VP) printing are two prevalent 3D printing modalities used in tissue engineering. Compared to VP printing, ME printing offers a higher degree of customization by allowing co- or multi-material prints, along with biologic cell printing. VP printing offers higher throughput and capability for printing more complex geometries, versus ME printing, with shorter manufacturing times, making it a more commercially viable choice for clinically translatable materials. Motivated by each printing modalities’ unique advantages, APGD resins for ME and VP printing were developed and assessed for crosslinker and photoinitiator (PI) concentration effect on resin time to gel point, storage modulus and viscosity. Increasing crosslinker concentration decreased the gel time and increased the storage modulus of cured resin. Significant differences in printed sample geometry along with material and mechanical properties resulted from using the two printing modalities. ME samples had larger void volume (6.1 ± 1.2% vs. 0.2 ± 0.4%), lower gel content (55.3 ± 2.6% vs. 68.7 ± 1.9%), and less shape fixity (77.4 ± 4.7 vs. 97.3 ± 3.7%) compared to VP samples. VP printing generated complicated 3D anatomic shapes of higher quality than ME printing. In conclusion, this manuscript provides the first report of APGD 3D printing that can be applied to shape memory, resorbable medical devices.

Integrated photoelasticity in a soft material: phase retardation, azimuthal angle, and stress-optic coefficient

Integrated photoelasticity is investigated for a soft material subjected to a three-dimensional stress state with large deformation. Our measurement target is designed based on the axisymmetric stress field (Hertzian contact problem). In the experiment, a solid sphere is pressed against a gelatin gel (Young’s modulus is about 4.2 kPa) with varying applied forces from zero to the maximum force that deforms the gel up to approximately 4.5 mm. For soft materials, two-dimensional photoelasticity, as conventionally practiced, is difficult to be used. This is because two-dimensional slices of highly deformable materials, such as the gelatin used in this study, do not allow stable experiments. This requires a three-dimensional body with sufficient thickness for stability, where integrated photoelasticity is demanded. The stressed gel’s photoelastic parameters (phase retardation and azimuthal angle) are measured using a polarization camera. The measured phase retardation and azimuthal angle are compared with the analytical prediction based on Hertzian contact problem. Remarkably, experimental and analytical results of the photoelastic parameters show a reasonable agreement not only in the phase retardation but also in the azimuthal angle that is related to the direction of secondary principal stresses. Never before validated in previous studies, which is crucial for reconstructing three-dimensional stress fields in soft materials. In addition, the stress-optic coefficient of the gelatin gel used is measured to be 3.12×10−8 1/Pa. Such findings proved that integrated photoelasticity benefits measure the three-dimensional stress field in soft materials, which is essential in biomedical engineering and cell printing applications.

Thermal Inkjet Printing: Prospects and Applications in the Development of Medicine

Over the last 10 years, inkjet printing technologies have advanced significantly and found several applications in the pharmaceutical and biomedical sector. Thermal inkjet printing is one of the most widely used techniques due to its versatility in the development of bioinks for cell printing or biosensors and the potential to fabricate personalized medications of various forms such as films and tablets. In this review, we provide a comprehensive discussion of the principles of inkjet printing technologies highlighting their advantages and limitations. Furthermore, the review covers a wide range of case studies and applications for precision medicine.

κ-Carrageenan and PVA blends as bioinks to 3D print scaffolds for cartilage reconstruction

3D printing of polymeric scaffolds and autologous stem cells is a promising tool for damaged facial cartilage reconstruction surgeries. To this end, suitable bioinks are needed to generate scaffolds with the required morphological and functional features. We formulated hydrogel bioinks using k-Carrageen (kC) and poly(vinyl alcohol) (PVA) in three different weight ratios. The kC gives the systems the ability to undergo rapid sol-to-gel transitions upon cooling from 60 °C and above to body temperature, while the PVA is used as rheology modifier and porogen. The latter is crosslinked after molding or printing by freeze-thaw cycling for 1 day (FT1) or 5 days (FT5). To select the most suitable formulation for 3D printing, the sol-to-gel transition and the physico-chemical, mechanical and morphological properties of obtained hydrogels were studied. Moreover, the absence of cytotoxic effects of the material on SASCs was assessed in both stemness-preserving or chondro-inductive media. Printing trials were performed to identify optimal process parameters and co-printing and post-printing seeding approaches of SASCs were evaluated. Cells were found to be viable after co-printing and also after the FT1 treatment. Viable adherent cells were also found in the FT5 system, where cells were plated after freezing and thawing treatment.

Biocompatibility of 3D-Printed PLA, PEEK and PETG: Adhesion of Bone Marrow and Peritoneal Lavage Cells.

Samples in the form of cylindrical plates, additively manufactured using the fused deposition modelling (or filament freeform fabrication, FDM/FFF) technology from polylactide (PLA), polyethylene terephthalate glycol (PETG) and polyetheretherketone (PEEK), were studied in series of in-vitro experiments on the adhesion of rat bone-marrow cells and rat peritoneal cells. Methods of estimation of the absolute number of cells and polymer samples’ mass change were used for the evaluation of cells adhesion, followed by the evaluation of cell-culture supernatants. The results of experiments for both types of cells demonstrated a statistically significant change in the absolute number of cells (variation from 44 to 119%) and the weight of the polymer samples (variation from 0.61 to 2.18%), depending on roughness of sample surface, controlled by a nozzle diameter of a 3D printer as well as printing layer height. It was found that more cells adhere to PLA samples with a larger nozzle diameter and layer height. For PETG samples, the results did not show a clear relationship between cell adhesion and printing parameters. For PEEK samples, on the contrary, adhesion to samples printed with a lower nozzle diameter (higher resolution) is better than to samples printed with a larger nozzle diameter (lower resolution). The difference in results for various polymers can be explained by their chemical structure.

Embedded 3D Printing in Self‐Healing Annealable Composites for Precise Patterning of Functionally Mature Human Neural Constructs (Adv. Sci. 25/2022)

Embedded 3D Printing In article number 2201392, Janko Kajtez, Jenny Emnéus, and co-workers present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside self-healing annealable particle-extracellular matrix (SHAPE) composites. The approach allows direct freeform patterning and functional maturation of human neural constructs. The image depicts deposition of cells inside SHAPE support to build human brain tissue.

Hybrid cell constructs consisting of bioprinted cell-spheroids.

Bioprinted cell constructs have been investigated for regeneration of various tissues. However, poor cell-cell interactions have limited their utility. Although cell-spheroids offer an alternative for efficient cell-cell interactions, they complicate bioprinting. Here, we introduce a new cell-printing process, fabricating cell-spheroids and cell-loaded constructs together without preparation of cell-spheroids in advance. Cells in mineral oil droplets self-assembled to form cell-spheroids due to the oil-aqueous interaction, exhibiting similar biological functions to the conventionally prepared cell-spheroids. By controlling printing parameters, spheroid diameter and location could be manipulated. To demonstrate the feasibility of this process, we fabricated hybrid cell constructs, consisting of endothelial cell-spheroids and stem cells loaded decellularized extracellular matrix/β-tricalcium phosphate struts for regenerating vascularized bone. The hybrid cell constructs exhibited strong angiogenic/osteogenic activities as a result of increased secretion of signaling molecules and synergistic crosstalk between the cells.

Controlling Phase Transition toward Future Low-Cost and Eco-friendly Printing of Perovskite Solar Cells.

Perovskite solar cells (PSCs) have grown increasingly popular over the past few years and are considered to be game-changers in the energy conversion market. It has became vital to transfer the deep understanding of the perovskite film formation process during lab-scale fabrication to large-scale production. Complex phase transition during film formation has been revealed by in situ strategies. However, there is still debate which phase transition is the right route for a future scalable approach. Herein, we briefly summarize perovskite crystallization during scalable printing processes. The critical information about the intermediates involved in phase transition from precursors to perovskite crystals are discussed because it deeply impacts the morphology of printed films. Finally, important strategies to control phase transition and challenges toward future low-temperature and eco-friendly printing of perovskite solar cells are proposed. The information provided by this Perspective will assist the screening and development of the perovskite phase transition for future cost-efficient printed perovskite panels.

Impact dynamics of air-in-liquid compound droplets

Compound droplets are utilized in applications ranging from preparation of emulsion to biological cell printing and additive manufacturing. Here, we report on the impact dynamics of an air-in-liquid hollow compound droplet on a solid substrate. Contrary to the impact of pure droplets and compound droplets with liquids of similar densities, a compound droplet with an encapsulated air bubble demonstrates the formation of a counterjet in addition to the lamella. We experimentally investigate the influence of the size of the air bubble, liquid viscosity, and height of impact on the evolution of counterjet and the spreading characteristics of the lamella. For a given hollow droplet, the volume of the counterjet is observed to depend on the volume of air and liquid in the droplet and is independent of the viscosity of the liquid and impact velocity of the droplet. We observe that the spread characteristics, counterintuitively, do not vary significantly compared to that of a pure droplet having an identical liquid volume as the hollow droplet. We propose a model to predict the maximum spread during the impact of a hollow droplet based on the energy interaction between the spreading liquid and the liquid in the counterjet during the impact process.