peer-reviewed articles
Swathi Swaminathan et al 2019 Biofabrication
Abstract
3D human cancer models provide a better platform for drug efficacy studies than conventional 2D culture, since they recapitulate important aspects of the in vivo microenvironment. While biofabrication has advanced model creation, bioprinting generally involves extruding individual cells in a bioink and then waiting for these cells to self-assemble into a hierarchical 3D tissue. This selfassembly is time consuming and requires complex cellular interactions with other cell types, extracellular matrix components, and growth factors. We therefore investigated if we could directly bioprint pre-formed 3D spheroids in alginate-based bioinks to create a model tissue that could be used almost immediately. Human breast epithelial cell lines were bioprinted as individual cells or as preformed spheroids, either in monoculture or co-culture with vascular endothelial cells. While individual breast cells only spontaneously formed spheroids in Matrigel-based bioink, pre-formed breast spheroids maintained their viability, architecture, and function after bioprinting. Bioprinted breast spheroids were more resistant to paclitaxel than individually printed breast cells; however, this effect was abrogated by endothelial cell co-culture. This study shows that 3D cellular structure bioprinting has potential to create tissue models that quickly replicate the tumor microenvironment.(click here to read more)
Boqing Zhang et al 2018 Elsevier Composites Part B
Abstract
Hierarchical structures with tailored macro/micro-porosity architecture play an important role in bone tissue regeneration. In 3D printing process, the printing ink formulation will influence on the ceramic macro and micro porous architectures. In this paper, HA powders of nano-sized grains (NP) with diameters of 30–50 nm, air jet milling powders (AP) with diameters of 10–30 μm, and spherical powder (SP) with diameters of 10–50 μm, were used as the initial printing materials in the printing ink formulations. The viscosity and the rheological behavior of printing inks were studied. The microstructure and morphology of the printed scaffold were observed and the mechanical properties of different types of scaffolds were tested. The results showed that the initial printing materials would influence on printing performance, both of the AP and SP inks may print porous scaffolds successfully. However, NP printing inks exhibited dramatic shrinkage and it is not suitable for 3D printing of bioceramics. The printing ink formulation also have effects on the ceramic macro and micro porous architectures and mechanical properties. The maximum compressive strength of SPS were 5.5 MPa, 3.2 MPa and 0.9 MPa with porosities of 60%, 70% and 80%, respectively. As the macroporosity decreases, the mechanical properties of the material would drop dramatically. With the same porosities, the compressive strength of APS were slightly higher than that of the SPS specimens.(click here to read more)
Jessica Snyder, et. al., © 2016 IOP Publishing Ltd
Abstract
Bottom-up tissue engineering requires methodological progress of biofabrication to capture key design facets of anatomical arrangements across micro, meso and macro-scales. The diffusive mass transfer properties necessary to elicit stability and functionality require hetero-typic contact, cell-to-cell signaling and uniform nutrient diffusion. Bioprinting techniques successfully build mathematically defined porous architecture to diminish resistance to mass transfer. Current limitations of bioprinted cell assemblies include poor micro-scale formability of cell-laden soft gels and asymmetrical macro-scale diffusion through 3D volumes. The objective of this work is to engineer a synchronized multi-material bioprinter (SMMB) system which improves the resolution and expands the capability of existing bioprinting systems by packaging multiple cell types in heterotypic arrays prior to deposition. This unit cell approach to arranging multiple cell-laden solutions is integrated with a motion system to print heterogeneous filaments as tissue engineered scaffolds and nanoliter droplets. The set of SMMB process parameters control the geometric arrangement of the combined flow’s internal features and constituent material’s volume fractions. SMMB printed hepatocyte-endothelial laden 200 nl droplets are cultured in a rotary cell culture system (RCCS) to study the effect of microgravity on an in vitro model of the human hepatic lobule. RCCS conditioning for 48 h increased hepatocyte cytoplasm diameter 2 μm, increased metabolic rate, and decreased drug half-life. SMMB hetero-cellular models present a 10-fold increase in metabolic rate, compared to SMMB mono-culture models. Improved bioprinting resolution due to process control of cell-laden matrix packaging as well as nanoliter droplet printing capability identify SMMB as a viable technique to improve in vitro model efficacy. (click here to read more)
Jessica Snyder, et. al., © 2016 IOP Publishing Ltd
Abstract
This topical review with original analysis and empirical results compares cell sensitivity to physical stress during printing. The objective is to frame a reproducible causation between printing environment and printed cell morphology, viability and phenotype stability. Content includes: (1) a topical review classifies the overlap between physical stress vectors during printing and mesenchymal stem cell sensitivities. (2) Original flow analysis frames the feasible range of stress duration and intensity during manufacturing. (3) Preliminary empirical results define cell properties as a function of minimum, mean and maximum stress conditions. The review and analytical characterization serve as an essential precursor to interpret surprising empirical results. Results identify key cell properties are stress-dependent and controllable based on printing process parameter selection. Printing’s minimum stress condition preserves cell viability. The maximum stress increases heterogeneity of cell response, induces inelastic ultra-structural distortion of the cell membrane and chromatin, and increases necrotic subpopulations post-printing. The review, analysis and preliminary results support the feasibility of modulating cell properties during fabrication by prescriptively tuning the stress environment. The process control over cell morphology, health and the rate of differentiation is both a direct result of strain during printing and an in-direct result of increased distress signaling from necrotic sub-populations. (click here to read more)
Yu Zhao, et. al., © 2015 IOP Publishing Ltd
Abstract
Three-dimensional (3D) cell printing technology has provided a versatile methodology to fabricate cell-laden tissue-like constructs and in vitro tissue/pathological models for tissue engineering, drug testing and screening applications. However, it still remains a challenge to print bioinks with high viscoelasticity to achieve long-term stable structure and maintain high cell survival rate after printing at the same time. In this study, we systematically investigated the influence of 3D cell printing parameters, i.e. composition and concentration of bioink, holding temperature and holding time, on the printability and cell survival rate in microextrusion-based 3D cell printing technology. Rheological measurements were utilized to characterize the viscoelasticity of gelatin-based bioinks. Results demonstrated that the bioink viscoelasticity was increased when increasing the bioink concentration, increasing holding time and decreasing holding temperature below gelation temperature. The decline of cell survival rate after 3D cell printing process was observed when increasing the viscoelasticity of the gelatin-based bioinks. However, different process parameter combinations would result in the similar rheological characteristics and thus showed similar cell survival rate after 3D bioprinting process. On the other hand, bioink viscoelasticity should also reach a certain point to ensure good printability and shape fidelity. At last, we proposed a protocol for 3D bioprinting of temperature-sensitive gelatin-based hydrogel bioinks with both high cell survival rate and good printability. This research would be useful for biofabrication researchers to adjust the 3D bioprinting process parameters quickly and as a referable template for designing new bioinks. (click here to read more)
Liliang Ouyang, et. al., © 2015 IOP Publishing Ltd
Abstract
With the ability to manipulate cells temporarily and spatially into three-dimensional (3D) tissue-like construct, 3D bioprinting technology was used in many studies to facilitate the recreation of complex cell niche and/or to better understand the regulation of stem cell proliferation and differentiation by cellular microenvironment factors. Embryonic stem cells (ESCs) have the capacity to differentiate into any specialized cell type of the animal body, generally via the formation of embryoid body (EB), which mimics the early stages of embryogenesis. In this study, extrusion-based 3D bioprinting technology was utilized for biofabricating ESCs into 3D cell-laden construct. The influence of 3D printing parameters on ESC viability, proliferation, maintenance of pluripotency and the rule of EB formation was systematically studied in this work. Results demonstrated that ESCs were successfully printed with hydrogel into 3D macroporous construct. Upon process optimization, about 90{7a55f8d36b46e0305fc07d25bcd2dd9e62a9a3eed2b319ac0dd6db4860522d42} ESCs remained alive after the process of bioprinting and cell-laden construct formation. ESCs continued proliferating into spheroid EBs in the hydrogel construct, while retaining the protein expression and gene expression of pluripotent markers, like octamer binding transcription factor 4, stage specific embryonic antigen 1 and Nanog. In this novel technology, EBs were formed through cell proliferation instead of aggregation, and the quantity of EBs was tuned by the initial cell density in the 3D bioprinting process. This study introduces the 3D bioprinting of ESCs into a 3D cell-laden hydrogel construct for the first time and showed the production of uniform, pluripotent, high-throughput and size-controllable EBs, which indicated strong potential in ESC large scale expansion, stem cell regulation and fabrication of tissue-like structure and drug screening studies. (click here to read more)
Jessica Snyder, et. al., © 2015 ASME
Abstract
A PED (precision extrusion deposition)/replica molding process enables scaffold guided tissue engineering of a heterocellular microfluidic device. We investigate two types of cell-laden devices: the first with a 3D microfluidic manifold fully embedded in a PDMS (polydimethylsiloxane) substrate and the second a channel network on the surface of the PDMS substrate for cell printing directly into device channels. Fully embedded networks are leak-resistant with simplified construction methods. Channels exposed to the surface are used as mold to hold bioprinted cell-laden matrix for controlled cell placement throughout the network from inlet to outlet. The result is a 3D cell-laden microfluidic device with improved leak-resistance (up to 2.0 mL/min), pervasive diffusion and control of internal architecture. (click here to read more)
Chengyang Wang, et. al., © 2015 IOP Publishing Ltd
Abstract
Surface properties of biopolymers are crucial for providing topographical and chemical cues to affect cellular behaviors, such as attachment, spreading, viability, proliferation, and differentiation. As an effective surface modification technique, plasma treatment is often applied to enhance surface wettability, adhesion, and biocompatibility of polymers. In this study, an atmospheric-pressure microplasma jet based on dielectric barrier discharge was installed on an automated arm which allows movement in the x–y–z directions at various trajectory presets. Polycaprolactone (PCL) samples were functionalized with helium-oxygen plasma generated by this system and characterized via water contact angle, x-ray photoelectron spectroscopy, and scanning electron microscopy. Mouse osteoblast cells (7F2) were cultured on both treated and native PCL samples and examined by MarkerGene™ Live: Dead/Cytotoxicity and alamarBlue® assaying techniques. The surface and biological characterization results indicate that microplasma treatment improved surface hydrophilicity, as well as cell viability and proliferation. The localized microplasma treatment can lead to the application of bioactive scaffolds with selective surface functionalization. (click here to read more)
Long Zhao, et. al., © 2015 ASME
Abstract
Drop-on-demand (DOD) microdroplet formation and deposition play an important role in additive manufacturing, particularly in printing of three-dimensional (3D) in vitro biological models for pharmacological and pathological studies, for tissue engineering and regenerative medicine applications, and for building of cell-integrated microfluidic devices. In development of a DOD based microdroplet deposition process for 3D cell printing, the droplet formation, controlled on-demand deposition and at the single-cell level, and most importantly, maintaining the viability and functionality of the cells during and after the printing are all remaining to be challenged. This report presents our recent study on developing a novel DOD based microdroplet deposition process for 3D printing by utilization of an alternating viscous and inertial force jetting (AVIFJ) mechanism. The results include an analysis of droplet formation mechanism, the system configuration, and experimental study of the effects of process parameters on microdroplet formation. Sodium alginate solutions are used for microdroplet formation and deposition. Key process parameters include actuation signal waveforms, nozzle dimensional features, and solution viscosity. Sizes of formed microdroplets are examined by measuring the droplet diameter and velocity. Results show that by utilizing a nozzle at a 45 μm diameter, the size of the formed microdroplets is in the range of 52–72 μm in diameter and 0.4–2.0 m/s in jetting speed, respectively. Reproducibility of the system is also examined and the results show that the deviation of the formed microdroplet diameter and the droplet deposition accuracy is within 6{7a55f8d36b46e0305fc07d25bcd2dd9e62a9a3eed2b319ac0dd6db4860522d42} and 6.2 μm range, respectively. Experimental results demonstrate a high controllability and precision for the developed DOD microdroplet deposition system with a potential for precise cell printing. (click here to read more)
Liliang Ouyang, et. al., © 2015 IOP Publishing Ltd
Abstract
3D printing has evolved into a versatile technology for fabricating tissue-engineered constructs with spatially controlled cells and biomaterial distribution to allow biomimicking of in vivo tissues. In this paper, we reported a novel study of 3D printing of cell lines derived from human embryonic kidney tissue into a macroporous tissue-like construct. Nozzle temperature, chamber temperature and the composition of the matrix material were studied to achieve high cell viability (>90{7a55f8d36b46e0305fc07d25bcd2dd9e62a9a3eed2b319ac0dd6db4860522d42}) after 3D printing and construct formation. Long-term construct stability with a clear grid structure up to 30 days was observed. Cells continued to grow as cellular spheroids with strong cell–cell interactions. Two transfected cell lines of HEK 293FT were also 3D printed and showed normal biological functions, i.e. protein synthesis and gene activation in responding to small molecule stimulus. With further refinement, this 3D cell printing technology may lead to a practical fabrication of functional embryonic tissues in vitro. (click here to read more)
Qudus Hamid, et. al., © 2015 IOP Publishing Ltd
Abstract
The utilization of the microfabrication technique to fabricate advanced computing chips has exponentially increased in the last few decades. Needless to say, this fabrication technique offers some unique advantages to develop micro-systems. Though many conventional microfabrication techniques today uses very harsh chemicals, the authors believe that the manipulation of system components and fabrication methods may aid in the utilization of the microfabrication techniques used in fabricating computer chips to develop advanced biological microfluidic systems. Presented in this paper is a fabrication approach in which popular fabrication methods and techniques are coupled together to develop an integrated system that aids in the fabrication of cell-laden microfluidic systems. This system aims to reduce the uses of harsh chemicals and decreases the lengthy fabrication time. Additionally, this integrated system will enable the printing of cells as the microfluidic chip is being fabricated. To demonstrate the unique capabilities of the integrated system, an advanced microfluidic chip is being fabricated and investigated. The advanced chip will feature the investigation of cancer cells in a co-cultured microfluidic environment. The investigations presented demonstrate co-cultures in a microfluidic chip, advanced cell printing with localized surface enhancement, cell integration, and full additive fabrication of a microfluidic chip. (click here to read more)
ISSUED PATENTS
Method and apparatus for computer-aided tissue engineering for modeling, design and freeform fabrication of tissue scaffolds, constructs, and devices
Description
A process and apparatus are provided for manufacturing complex parts and devices which utilize a CAD environment to design a part or device to be created (FIG. 1); Boolean, Scaling, Smoothing, mirroring, or other operations to modify the CAD design; a software interface to convert the CAD designed part (Data Process System) or device into a hetero geneous material and multi-part assembly model (Design Input Model) which can be used for multi-nozzle printing: and a multi-nozzle system to print the designed part or device using different, specialized nozzles (Tissue Substitutes).
Method for Making Artificial Scaffolds Having Porous Three-Dimensional Body Comprising Cells
Description
In on aspect, the invention includes a microcarrier bead having a porous three-dimensional core having (a) a polymeric porous three-dimensional body having porosity of about 15 to about 90{7a55f8d36b46e0305fc07d25bcd2dd9e62a9a3eed2b319ac0dd6db4860522d42} such that at least 99{7a55f8d36b46e0305fc07d25bcd2dd9e62a9a3eed2b319ac0dd6db4860522d42} of pores are interconnected and have diameters of at most 200 microns, (b) an outer protective layer and optionally (c) a filler. In another aspect, the invention includes a method of making an artificial scaffold wherein a scaffolding material is extruded into a coolant and thereby creating a porous material having a porosity of between 15-90{7a55f8d36b46e0305fc07d25bcd2dd9e62a9a3eed2b319ac0dd6db4860522d42} such that at least 99{7a55f8d36b46e0305fc07d25bcd2dd9e62a9a3eed2b319ac0dd6db4860522d42} of pores are interconnected and have diameters of at most 200 microns
INTEGRATABLE ASSISTED COOLING SYSTEM FOR PRECISION EXTRUSION DEPOSITION IN THE FABRICATION OF 3D SCAFFOLDS
Description
The present invention relates to an integrated Assisting Cooling (AC) device, system and method for use with PED devices, allowing use of biopolymers having higher melting points in the fabrication of 3D scaffolds. The AC device cools the filament as it is extruding from the nozzle via low flow convective cooling. The AC device allows for cooling in the +/− direction of motion on an XY plane. The AC device elevates with the material delivery chamber. The AC device allows for scaffold fabrication at applied temperatures as high as about 250° C.
Layered manufacturing utilizing foam as a support and multi-functional material for the creation of parts and for tissue engineering
Description
A solid freeform fabrication method of creating a three-dimensional article built at least in part from scaffolding layers, the method includes providing a scaffolding material, providing a supporting material in a shape of a foamy layer, and contacting the scaffolding material with the foamy layer to form at least one scaffolding layer and thereby creating the three dimensional article.
Micro-Organ Device
Description
A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.
PENDING PATENTS
Method for creating an internal transport system within tissue scaffolds using computer-aided tissue engineering
Methods of generating ultraviolet radiation, plasma-and ultraviolet generating nozzles, printing systems, method of generating a substrate, and substrates fabricated according to the same
