Publications by authors named "Shaochen Chen"

109 Publications

Bioprinting of dual ECM scaffolds encapsulating limbal stem/progenitor cells in active and quiescent statuses.

Biofabrication 2021 08 13;13(4). Epub 2021 Aug 13.

Department of NanoEngineering, University of California San Diego, La Jolla, CA 92093, United States of America.

Limbal stem cell deficiency and corneal disorders are among the top global threats for human vision. Emerging therapies that integrate stem cell transplantation with engineered hydrogel scaffolds for biological and mechanical support are becoming a rising trend in the field. However, methods for high-throughput fabrication of hydrogel scaffolds, as well as knowledge of the interaction between limbal stem/progenitor cells (LSCs) and the surrounding extracellular matrix (ECM) are still much needed. Here, we employed digital light processing (DLP)-based bioprinting to fabricate hydrogel scaffolds encapsulating primary LSCs and studied the ECM-dependent LSC phenotypes. The DLP-based bioprinting with gelatin methacrylate (GelMA) or hyaluronic acid glycidyl methacrylate (HAGM) generated microscale hydrogel scaffolds that could support the viability of the encapsulated primary rabbit LSCs (rbLSCs) in culture. Immunocytochemistry and transcriptional analysis showed that the encapsulated rbLSCs remained active in GelMA-based scaffolds while exhibited quiescence in the HAGM-based scaffolds. The primary human LSCs encapsulated within bioprinted scaffolds showed consistent ECM-dependent active/quiescent statuses. Based on these results, we have developed a novel bioprinted dual ECM 'Yin-Yang' model encapsulating LSCs to support both active and quiescent statues. Our findings provide valuable insights towards stem cell therapies and regenerative medicine for corneal reconstruction.
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http://dx.doi.org/10.1088/1758-5090/ac1992DOI Listing
August 2021

Rapid 3D Bioprinting of Glioblastoma Model Mimicking Native Biophysical Heterogeneity.

Small 2021 04 27;17(15):e2006050. Epub 2021 Jan 27.

Department of NanoEngineering, University of California San Diego, La Jolla, CA, 92093, USA.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor characterized by high cellular and molecular heterogeneity, hypervascularization, and innate drug resistance. Cellular components and extracellular matrix (ECM) are the two primary sources of heterogeneity in GBM. Here, biomimetic tri-regional GBM models with tumor regions, acellular ECM regions, and an endothelial region with regional stiffnesses patterned corresponding to the GBM stroma, pathological or normal brain parenchyma, and brain capillaries, are developed. Patient-derived GBM cells, human endothelial cells, and hyaluronic acid derivatives are used to generate a species-matched and biochemically relevant microenvironment. This in vitro study demonstrates that biophysical cues are involved in various tumor cell behaviors and angiogenic potentials and promote different molecular subtypes of GBM. The stiff models are enriched in the mesenchymal subtype, exhibit diffuse invasion of tumor cells, and induce protruding angiogenesis and higher drug resistance to temozolomide. Meanwhile, the soft models demonstrate enrichment in the classical subtype and support expansive cell growth. The three-dimensional bioprinting technology utilized in this study enables rapid, flexible, and reproducible patient-specific GBM modeling with biophysical heterogeneity that can be employed by future studies as a tunable system to interrogate GBM disease mechanisms and screen drug compounds.
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http://dx.doi.org/10.1002/smll.202006050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049977PMC
April 2021

Biomaterials and 3D Bioprinting Strategies to Model Glioblastoma and the Blood-Brain Barrier.

Adv Mater 2021 Feb 16;33(5):e2004776. Epub 2020 Dec 16.

Department of NanoEngineering, University of California, San Diego, La Jolla, CA, 92093, USA.

Glioblastoma (GBM) is the most prevalent and lethal adult primary central nervous system cancer. An immunosuppresive and highly heterogeneous tumor microenvironment, restricted delivery of chemotherapy or immunotherapy through the blood-brain barrier (BBB), together with the brain's unique biochemical and anatomical features result in its universal recurrence and poor prognosis. As conventional models fail to predict therapeutic efficacy in GBM, in vitro 3D models of GBM and BBB leveraging patient- or healthy-individual-derived cells and biomaterials through 3D bioprinting technologies potentially mimic essential physiological and pathological features of GBM and BBB. 3D-bioprinted constructs enable investigation of cellular and cell-extracellular matrix interactions in a species-matched, high-throughput, and reproducible manner, serving as screening or drug delivery platforms. Here, an overview of current 3D-bioprinted GBM and BBB models is provided, elaborating on the microenvironmental compositions of GBM and BBB, relevant biomaterials to mimic the native tissues, and bioprinting strategies to implement the model fabrication. Collectively, 3D-bioprinted GBM and BBB models are promising systems and biomimetic alternatives to traditional models for more reliable mechanistic studies and preclinical drug screenings that may eventually accelerate the drug development process for GBM.
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http://dx.doi.org/10.1002/adma.202004776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854518PMC
February 2021

Rapid bioprinting of conjunctival stem cell micro-constructs for subconjunctival ocular injection.

Biomaterials 2021 01 23;267:120462. Epub 2020 Oct 23.

Department of NanoEngineering, University of California San Diego, La Jolla, CA, 92093, USA; Materials Science and Engineering Program, University of California San Diego, La Jolla, CA, 92093, USA; Department of Bioengineering, University of California San Diego, La Jolla, CA, 92093, USA. Electronic address:

Ocular surface diseases including conjunctival disorders are multifactorial progressive conditions that can severely affect vision and quality of life. In recent years, stem cell therapies based on conjunctival stem cells (CjSCs) have become a potential solution for treating ocular surface diseases. However, neither an efficient culture of CjSCs nor the development of a minimally invasive ocular surface CjSC transplantation therapy has been reported. Here, we developed a robust in vitro expansion method for primary rabbit-derived CjSCs and applied digital light processing (DLP)-based bioprinting to produce CjSC-loaded hydrogel micro-constructs for injectable delivery. Expansion medium containing small molecule cocktail generated fast dividing and highly homogenous CjSCs for more than 10 passages in feeder-free culture. Bioprinted hydrogel micro-constructs with tunable mechanical properties enabled the 3D culture of CjSCs while supporting viability, stem cell phenotype, and differentiation potency into conjunctival goblet cells. These hydrogel micro-constructs were well-suited for scalable dynamic suspension culture of CjSCs and were successfully delivered to the bulbar conjunctival epithelium via minimally invasive subconjunctival injection. This work integrates novel cell culture strategies with bioprinting to develop a clinically relevant injectable-delivery approach for CjSCs towards the stem cell therapies for the treatment of ocular surface diseases.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719077PMC
January 2021

3D Printable Non-Isocyanate Polyurethanes with Tunable Material Properties.

Polym Chem 2019 Sep 19;10(34):4665-4674. Epub 2019 Jul 19.

Department of NanoEngineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093.

Green chemistry-based non-isocyanate polyurethanes (NIPU) are synthesized and 3D-printed via rapid, projection photopolymerization into compliant mechanisms of 3D structure with spatially-localized material properties. Trimethylolpropane allyl ether-cyclic carbonate is used to couple the unique properties of two types of reaction chemistry: (1) primary diamine-cyclic carbonate ring-opening conjugation for supplanting conventional isocyanate-polyol reactions in creating urethane groups, with the additional advantage of enabling modular segment interchangeability within the diurethane prepolymers; and (2) thiol-ene (click) conjugation for non-telechelic, low monodispersity, quasi-crystalline-capable, and alternating step-growth co-photopolymerization. Fourier Transform Infrared Spectroscopy is used to monitor the functional group transformation in reactions, and to confirm these process-associated molecular products. The extent of how these processes utilize molecular tunability to affect material properties were investigated through measurement-based comparison of the various polymer compositions: frequency-related dynamic mechanical analysis, tension-related elastic-deformation mechanical analysis, and material swelling analysis. Stained murine myoblasts cultured on NIPU slabs were evaluated via fluorescent microscopy for "green-chemistry" affects on cytocompatibility and cell adhesion to assess potential biofouling resistance. 3D multi-material structures with micro-features were printed, thus demonstrating the capability to spatially pattern different NIPU materials in a controlled manner and build compliant mechanisms.
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http://dx.doi.org/10.1039/c9py00999jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577587PMC
September 2019

3D printing of a biocompatible double network elastomer with digital control of mechanical properties.

Adv Funct Mater 2020 Apr 19;30(14). Epub 2020 Feb 19.

Department of NanoEngineering, Chemical Engineering Program, Materials Science and Engineering Program, Department of Bioengineering, University of California San Diego, La Jolla, CA 92093.

The majority of 3D-printed biodegradable biomaterials are brittle, limiting their potential application to compliant tissues. Poly (glycerol sebacate) acrylate (PGSA) is a synthetic biodegradable and biocompatible elastomer, compatible with light-based 3D printing. In this work we employed digital-light-processing (DLP)-based 3D printing to create a complex PGSA network structure. Nature-inspired double network (DN) structures with two geometrically interconnected segments with different mechanical properties were printed from the same material in a single shot. Such capability has not been demonstrated by any other fabrication technique. The biocompatibility of PGSA after 3D printing was confirmed via cell-viability analysis. We used a finite element analysis (FEA) model to predict the failure of the DN structure under uniaxial tension. FEA confirmed the soft segments act as sacrificial elements while the hard segments retain structural integrity. The simulation demonstrated that the DN design absorbs 100% more energy before rupture than the network structure made by single exposure condition (SN), doubling the toughness of the overall structure. Using the FEA-informed design, a new DN structure was printed and the FEA predicted tensile test results agreed with tensile testing of the printed structure. This work demonstrated how geometrically-optimized material design can be easily and rapidly achieved by using DLP-based 3D printing, where well-defined patterns of different stiffnesses can be simultaneously formed using the same elastic biomaterial, and overall mechanical properties can be specifically optimized for different biomedical applications.
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http://dx.doi.org/10.1002/adfm.201910391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566974PMC
April 2020

Bioprinting of Complex Vascularized Tissues.

Methods Mol Biol 2021 ;2147:163-173

Department of NanoEngineering, University of California, San Diego (UCSD), La Jolla, CA, USA.

Functional vasculature is crucial for the maintenance of living tissues via the transport of oxygen, nutrients, and metabolic waste products. As a result, insufficient vascularization in thick engineered tissues will lead to cell death and necrosis due to mass transport and diffusional constraints. To circumvent these limitations, we describe the development of a microscale continuous optical bioprinting (μCOB) platform for 3D printing complex vascularized tissues with superior resolution and speed. By using the μCOB system, endothelial cells and other supportive cells can be printed directly into hydrogels with precisely controlled distribution and subsequent formation of lumen-like structures in vitro.
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http://dx.doi.org/10.1007/978-1-0716-0611-7_14DOI Listing
March 2021

High-fidelity 3D Printing using Flashing Photopolymerization.

Addit Manuf 2019 Dec 19;30. Epub 2019 Aug 19.

Department of NanoEngineering, University of California San Diego, 9500 Gilman Drive, La Jolla, California, 92093, USA.

Photopolymerization-based 3D printing has emerged as a promising technique to fabricate 3D structures. However, during the printing process, polymerized materials such as hydrogels often become highly light-scattering, thus perturbing incident light distribution and thereby deteriorating the final print resolution. To overcome this scattering-induced resolution deterioration, we developed a novel method termed flashing photopolymerization (FPP). Our FPP approach is informed by the fundamental kinetics of photopolymerization reactions, where light exposure is delivered in millisecond-scale 'flashes', as opposed to continuous light exposure. During the period of flash exposure, the prepolymer material negligibly scatters light. The material then polymerizes and opacifies in absence of light, therefore the exposure pattern is not perturbed by scattering. Compared to the conventional use of a continuous wave (CW) light source, the FPP fabrication resolution is improved. FPP also shows little dependency on the exposure, thus minimizing trial-and-error type optimization. Using FPP, we demonstrate its use in generating high-fidelity 3D printed constructs.
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http://dx.doi.org/10.1016/j.addma.2019.100834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442265PMC
December 2019

A sequential 3D bioprinting and orthogonal bioconjugation approach for precision tissue engineering.

Biomaterials 2020 11 9;258:120294. Epub 2020 Aug 9.

Department of NanoEngineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA; Materials Science and Engineering Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA; Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA; Chemical Engineering Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. Electronic address:

Recent advances in 3D bioprinting have transformed the tissue engineering landscape by enabling the controlled placement of cells, biomaterials, and bioactive agents for the biofabrication of living tissues and organs. However, the application of 3D bioprinting is limited by the availability of cytocompatible and printable biomaterials that recapitulate properties of native tissues. Here, we developed an integrated 3D projection bioprinting and orthogonal photoconjugation platform for precision tissue engineering of tailored microenvironments. By using a photoreactive thiol-ene gelatin bioink, soft hydrogels can be bioprinted into complex geometries and photopatterned with bioactive moieties in a rapid and scalable manner via digital light projection (DLP) technology. This enables localized modulation of biophysical properties such as stiffness and microarchitecture as well as precise control over spatial distribution and concentration of immobilized functional groups. As such, well-defined properties can be directly incorporated using a single platform to produce desired tissue-specific functions within bioprinted constructs. We demonstrated high viability of encapsulated endothelial cells and human cardiomyocytes using our dual process and fabricated tissue constructs functionalized with VEGF peptide mimics to induce guided endothelial cell growth for programmable vascularization. This work represents a pivotal step in engineering multifunctional constructs with unprecedented control, precision, and versatility for the rational design of biomimetic tissues.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489302PMC
November 2020

Medical imaging of tissue engineering and regenerative medicine constructs.

Biomater Sci 2021 Jan 10;9(2):301-314. Epub 2020 Aug 10.

Departments of NanoEngineering, University of California, San Diego, USA.

Advancement of tissue engineering and regenerative medicine (TERM) strategies to replicate tissue structure and function has led to the need for noninvasive assessment of key outcome measures of a construct's state, biocompatibility, and function. Histology based approaches are traditionally used in pre-clinical animal experiments, but are not always feasible or practical if a TERM construct is going to be tested for human use. In order to transition these therapies from benchtop to bedside, rigorously validated imaging techniques must be utilized that are sensitive to key outcome measures that fulfill the FDA standards for TERM construct evaluation. This review discusses key outcome measures for TERM constructs and various clinical- and research-based imaging techniques that can be used to assess them. Potential applications and limitations of these techniques are discussed, as well as resources for the processing, analysis, and interpretation of biomedical images.
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http://dx.doi.org/10.1039/d0bm00705fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262082PMC
January 2021

Direct 3D bioprinting of cardiac micro-tissues mimicking native myocardium.

Biomaterials 2020 10 22;256:120204. Epub 2020 Jun 22.

Materials Science and Engineering Program, University of California San Diego, USA; Department of NanoEngineering, University of California San Diego, USA; Department of Bioengineering, University of California San Diego, USA. Electronic address:

The heart possesses a complex three-dimensional (3D) laminar myofiber organization; however, because engineering physiologically relevant 3D tissues remains a technical challenge, the effects of cardiomyocyte alignment on excitation-contraction coupling, shortening and force development have not been systematically studied. Cellular shape and orientations in 3D can be controlled by engineering scaffold microstructures and encapsulating cells near these geometric cues. Here, we show that a novel method of cell encapsulation in 3D methacrylated gelatin (GelMA) scaffolds patterned via Microscale Continuous Optical Printing (μCOP) can rapidly micropattern neonatal mouse ventricular cardiomyocytes (NMVCMs) in photocrosslinkable hydrogels. Encapsulated cardiomyocytes preferentially align with the engineered microarchitecture and can display morphology and myofibril alignment phenotypic of myocardium in vivo. Utilizing the μCOP system, an asymmetric, multi-material, cantilever-based scaffold was directly printed, so that the force produced by the microtissue was transmitted onto a single deformable pillar. Aligned 3D encapsulated NMVCM scaffolds produced nearly 2 times the force compared to aligned 2D seeded samples. To further highlight the flexibility of μCOP, NMVCMs were encapsulated in several patterns to compare the effects of varying degrees of alignment on tissue displacement and synchronicity. Well aligned myofiber cultured patterns generated 4-10 times the contractile force of less anisotropically patterned constructs. Finally, normalized fluo-4 fluorescence of NMVCM-encapsulated structures showed characteristic calcium transient waveforms that increased in magnitude and rate of decline during treatment with 100 nM isoproterenol. This novel instrumented 3D cardiac microtissue serves as a physiologically relevant in vitro model system with great potential for use in cardiac disease modeling and drug screening.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423764PMC
October 2020

Noninvasive in vivo 3D bioprinting.

Sci Adv 2020 Jun 5;6(23):eaba7406. Epub 2020 Jun 5.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, P. R. China.

Three-dimensional (3D) printing technology has great potential in advancing clinical medicine. Currently, the in vivo application strategies for 3D-printed macroscale products are limited to surgical implantation or in situ 3D printing at the exposed trauma, both requiring exposure of the application site. Here, we show a digital near-infrared (NIR) photopolymerization (DNP)-based 3D printing technology that enables the noninvasive in vivo 3D bioprinting of tissue constructs. In this technology, the NIR is modulated into customized pattern by a digital micromirror device, and dynamically projected for spatially inducing the polymerization of monomer solutions. By ex vivo irradiation with the patterned NIR, the subcutaneously injected bioink can be noninvasively printed into customized tissue constructs in situ. Without surgery implantation, a personalized ear-like tissue constructs with chondrification and a muscle tissue repairable cell-laden conformal scaffold were obtained in vivo. This work provides a proof of concept of noninvasive in vivo 3D bioprinting.
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http://dx.doi.org/10.1126/sciadv.aba7406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269646PMC
June 2020

Three-dimensional bioprinted glioblastoma microenvironments model cellular dependencies and immune interactions.

Cell Res 2020 10 4;30(10):833-853. Epub 2020 Jun 4.

Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, La Jolla, CA, 92037, USA.

Brain tumors are dynamic complex ecosystems with multiple cell types. To model the brain tumor microenvironment in a reproducible and scalable system, we developed a rapid three-dimensional (3D) bioprinting method to construct clinically relevant biomimetic tissue models. In recurrent glioblastoma, macrophages/microglia prominently contribute to the tumor mass. To parse the function of macrophages in 3D, we compared the growth of glioblastoma stem cells (GSCs) alone or with astrocytes and neural precursor cells in a hyaluronic acid-rich hydrogel, with or without macrophage. Bioprinted constructs integrating macrophage recapitulate patient-derived transcriptional profiles predictive of patient survival, maintenance of stemness, invasion, and drug resistance. Whole-genome CRISPR screening with bioprinted complex systems identified unique molecular dependencies in GSCs, relative to sphere culture. Multicellular bioprinted models serve as a scalable and physiologic platform to interrogate drug sensitivity, cellular crosstalk, invasion, context-specific functional dependencies, as well as immunologic interactions in a species-matched neural environment.
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http://dx.doi.org/10.1038/s41422-020-0338-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608409PMC
October 2020

Photopolymerizable Biomaterials and Light-Based 3D Printing Strategies for Biomedical Applications.

Chem Rev 2020 10 23;120(19):10695-10743. Epub 2020 Apr 23.

Department of NanoEngineering, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States.

Since the advent of additive manufacturing, known commonly as 3D printing, this technology has revolutionized the biofabrication landscape and driven numerous pivotal advancements in tissue engineering and regenerative medicine. Many 3D printing methods were developed in short course after Charles Hull first introduced the power of stereolithography to the world. However, materials development was not met with the same enthusiasm and remained the bottleneck in the field for some time. Only in the past decade has there been deliberate development to expand the materials toolbox for 3D printing applications to meet the true potential of 3D printing technologies. Herein, we review the development of biomaterials suited for light-based 3D printing modalities with an emphasis on bioprinting applications. We discuss the chemical mechanisms that govern photopolymerization and highlight the application of natural, synthetic, and composite biomaterials as 3D printed hydrogels. Because the quality of a 3D printed construct is highly dependent on both the material properties and processing technique, we included a final section on the theoretical and practical aspects behind light-based 3D printing as well as ways to employ that knowledge to troubleshoot and standardize the optimization of printing parameters.
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http://dx.doi.org/10.1021/acs.chemrev.9b00810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572843PMC
October 2020

High throughput direct 3D bioprinting in multiwell plates.

Biofabrication 2020 Apr 16. Epub 2020 Apr 16.

Nanoengineering, University of California San Diego, La Jolla, California, UNITED STATES.

Advances in three dimensional (3D) bioprinting have enabled the fabrication of sophisticated 3D tissue scaffolds for biological and medical applications, where high speed, high throughput production in well plates is a critical need. Here, we present an integrated 3D bioprinting platform based on microscale continuous optical printing, capable of high throughput in situ rapid fabrication of complex 3D biomedical samples in multiwell plate formats for subsequent culture and analysis. Our high throughput 3D bioprinter (HT-3DP) was used to showcase constructs of varying spatial geometries of biomimetic significance, tunable mechanical properties, as well as reproducibility. Live hepatocellular carcinoma 3D tissue scaffolds were fabricated in situ in multiwell plates, after which a functional drug response assay against the chemotherapy drug doxorubicin was performed. Dual cell-type populations involving both live hepatocellular carcinoma as well as human umbilical vein endothelial cells were also printed to demonstrate dual-tissue fabrication capability. This work demonstrates a significant advancement in that the production rate of 3D bioprinted tissue scaffolds with controllable spatial architectures and mechanical properties can now be done on a high throughput scale, enabling rapid generation of in vitro 3D tissue models within conventional multiwell cell culture plates for high throughput preclinical drug screening and disease modeling.
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http://dx.doi.org/10.1088/1758-5090/ab89caDOI Listing
April 2020

Bionic 3D printed corals.

Nat Commun 2020 04 9;11(1):1748. Epub 2020 Apr 9.

Bioinspired Photonics Group, Department of Chemistry, University of Cambridge, Cambridge, UK.

Corals have evolved as optimized photon augmentation systems, leading to space-efficient microalgal growth and outstanding photosynthetic quantum efficiencies. Light attenuation due to algal self-shading is a key limiting factor for the upscaling of microalgal cultivation. Coral-inspired light management systems could overcome this limitation and facilitate scalable bioenergy and bioproduct generation. Here, we develop 3D printed bionic corals capable of growing microalgae with high spatial cell densities of up to 10 cells mL. The hybrid photosynthetic biomaterials are produced with a 3D bioprinting platform which mimics morphological features of living coral tissue and the underlying skeleton with micron resolution, including their optical and mechanical properties. The programmable synthetic microenvironment thus allows for replicating both structural and functional traits of the coral-algal symbiosis. Our work defines a class of bionic materials that is capable of interacting with living organisms and can be exploited for applied coral reef research and photobioreactor design.
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http://dx.doi.org/10.1038/s41467-020-15486-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145811PMC
April 2020

Oncogenic human herpesvirus hijacks proline metabolism for tumorigenesis.

Proc Natl Acad Sci U S A 2020 04 25;117(14):8083-8093. Epub 2020 Mar 25.

Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033;

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis gene and proline degradation gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.
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http://dx.doi.org/10.1073/pnas.1918607117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149499PMC
April 2020

Modulating physical, chemical, and biological properties in 3D printing for tissue engineering applications.

Appl Phys Rev 2018 Dec;5(4)

Department of NanoEngineering, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

Over the years, 3D printing technologies have transformed the field of tissue engineering and regenerative medicine by providing a tool that enables unprecedented flexibility, speed, control, and precision over conventional manufacturing methods. As a result, there has been a growing body of research focused on the development of complex biomimetic tissues and organs produced via 3D printing to serve in various applications ranging from models for drug development to translational research and biological studies. With the eventual goal to produce functional tissues, an important feature in 3D printing is the ability to tune and modulate the microenvironment to better mimic conditions to improve tissue maturation and performance. This paper reviews various strategies and techniques employed in 3D printing from the perspective of achieving control over physical, chemical, and biological properties to provide a conducive microenvironment for the development of physiologically relevant tissues. We will also highlight the current limitations associated with attaining each of these properties in addition to introducing challenges that need to be addressed for advancing future 3D printing approaches.
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http://dx.doi.org/10.1063/1.5050245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959479PMC
December 2018

3D bioprinting of hydrogels for retina cell culturing.

Bioprinting 2018 Dec 5;11. Epub 2018 Sep 5.

Materials Science and Engineering Program, University of California San Diego, La Jolla, CA 92093, USA.

Recapitulating native retina environment is crucial in isolation and culturing of retina photoreceptors (PRs). To date, maturation of PRs remains incomprehensible . Here we present a strategy of integrating the physical and chemical signals through 3D-bioprinting of hyaluronic acid (HA) hydrogels and co-differentiation of retinal progenitor cells (RPCs) into PRs with the support of retinal-pigment epithelium (RPEs). To mimic the native environment during retinal development, we chemically altered the functionalization of HA hydrogels to match the compressive modulus of HA hydrogels with native retina. RPEs were incorporated in the culturing system to support the differentiation due to their regeneration capabilities. We found that HA with a specific functionalization can yield hydrogels with compressive modulus similar to native retina. This hydrogel is also suitable for 3D bioprinting of retina structure. The results from cell study indicated that derivation of PRs from RPCs was improved in the presence of RPEs.
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http://dx.doi.org/10.1016/j.bprint.2018.e00029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941869PMC
December 2018

Controlled Growth Factor Release in 3D-Printed Hydrogels.

Adv Healthc Mater 2020 08 7;9(15):e1900977. Epub 2019 Nov 7.

Materials Science and Engineering Program, University of California San Diego, La Jolla, CA, 92093, USA.

Growth factors (GFs) are critical components in governing cell fate during tissue regeneration. Their controlled delivery is challenging due to rapid turnover rates in vivo. Functionalized hydrogels, such as heparin-based hydrogels, have demonstrated great potential in regulating GF release. While the retention effects of various concentrations and molecular weights of heparin have been investigated, the role of geometry is unknown. In this work, 3D printing is used to fabricate GF-embedded heparin-based hydrogels with arbitrarily complex geometry (i.e., teabag, flower shapes). Simplified cylindrical core-shell structures with varied shell thickness are printed, and the rates of GF release are measured over the course of 28 days. Increasing the shell layers' thickness decreases the rate of GF release. Additionally, a mathematical model is developed, which is found capable of accurately predicting GF release kinetics in hydrogels with shell layers greater than 0.5 mm thick (R > 0.96). Finally, the sequential release is demonstrated by printing two GFs in alternating radial layers. By switching the spatial order, the delivery sequence of the GFs can be modulated. This study demonstrates how 3D printing can be utilized to fabricate user-defined structures with unique geometry in order to control the rate of GF release in hydrogels.
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http://dx.doi.org/10.1002/adhm.201900977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202999PMC
August 2020

Rapid 3D bioprinting of cardiac tissue models using human embryonic stem cell-derived cardiomyocytes.

Bioprinting 2019 Mar 10;13. Epub 2019 Jan 10.

Materials Science and Enginering Program, University of California, San Diego, La Jolla, CA 92093, USA.

There is a great need for physiologically relevant 3D human cardiac scaffolds for both short-term, the development of drug testing platforms to screen new drugs across different genetic backgrounds, and longer term, the replacement of damaged or non-functional cardiac tissue after injury or infarction. In this study, we have designed and printed a variety of scaffolds for diagnostics using light based Micro-Continuous Optical Printing (μCOP). Human embryonic stem cell-derived cardiomyocyte (hESC-CMs) were directly printed into gelatin hydrogel on glass to determine their viability and ability to align. The incorporation of Green Fluorescent Protein/Calmodulin/M13 Peptide (GCaMP3)-hESC-CMs allowed the ability to continuously monitor calcium transients over time. Normalized fluorescence of GCaMP3-hESCCMs increased by 18 ± 6% and 40 ± 5% when treated with 500 nM and 1 μM of isoproterenol, respectively. Finally, GCaMP3-hESC-CMs were printed across a customizable 3D printed cantilever-based force system. Along with force tracking by visualizing the displacement of the cantilever, calcium transients could be observed in a non-destructive manner, allowing the samples to be examined over several days. Our μCOP-printed cardiac models presented here can be used as a powerful tool for drug screening and to analyze cardiac tissue maturation.
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http://dx.doi.org/10.1016/j.bprint.2019.e00040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768568PMC
March 2019

Canonical Wnt5b Signaling Directs Outlying Nkx2.5+ Mesoderm into Pacemaker Cardiomyocytes.

Dev Cell 2019 09 8;50(6):729-743.e5. Epub 2019 Aug 8.

Department of Medicine, Division of Cardiology, University of California, San Diego, La Jolla, CA 92093, USA; Institute for Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.
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http://dx.doi.org/10.1016/j.devcel.2019.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759400PMC
September 2019

Rapid continuous 3D printing of customizable peripheral nerve guidance conduits.

Mater Today (Kidlington) 2018 Nov 27;21(9):951-959. Epub 2018 Apr 27.

Department of NanoEngineering, University of California San Diego, La Jolla, CA 92093, United States.

Engineered nerve guidance conduits (NGCs) have been demonstrated for repairing peripheral nerve injuries. However, there remains a need for an advanced biofabrication system to build NGCs with complex architectures, tunable material properties, and customizable geometrical control. Here, a rapid continuous 3D-printing platform was developed to print customizable NGCs with unprecedented resolution, speed, flexibility, and scalability. A variety of NGC designs varying in complexity and size were created including a life-size biomimetic branched human facial NGC. implantation of NGCs with microchannels into complete sciatic nerve transections of mouse models demonstrated the effective directional guidance of regenerating sciatic nerves via branching into the microchannels and extending toward the distal end of the injury site. Histological staining and immunostaining further confirmed the progressive directional nerve regeneration and branching behavior across the entire NGC length. Observational and functional tests, including the von Frey threshold test and thermal test, showed promising recovery of motor function and sensation in the ipsilateral limbs grafted with the 3D-printed NGCs.
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http://dx.doi.org/10.1016/j.mattod.2018.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538503PMC
November 2018

Projection Printing of Ultrathin Structures with Nanoscale Thickness Control.

ACS Appl Mater Interfaces 2019 May 18;11(17):16059-16064. Epub 2019 Apr 18.

Department of NanoEngineering , University of California San Diego , La Jolla , California 92093 , United States.

Spatial control of photon energy has been a central part of many light-based manufacturing processes. We report a direct projection printing method for ultrathin structures with nanoscale thickness control by using a patterned evanescent field. The evanescent field is induced by total internal reflection at the interface between the substrate and a prepolymer solution, and it is patterned by a phase-only spatial light modulator. The ultrathin structure is printed on a high-refractive-index glass substrate through photopolymerization. An iterative algorithm is used to calculate the phase pattern for generating arbitrary holography images and making the image plane to coincide with the interface. The thickness of the pattern is limited by the penetration depth of the evanescent field. Experiment results demonstrated that polymer structures as thin as 200 nm can be patterned without significant process optimization. Such fine control in thickness could transform many techniques such as light-based 3D printing and laser direct-write manufacturing.
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http://dx.doi.org/10.1021/acsami.9b02728DOI Listing
May 2019

Rapid 3D printing of functional nanoparticle-enhanced conduits for effective nerve repair.

Acta Biomater 2019 05 28;90:49-59. Epub 2019 Mar 28.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610065, China.

Nerve conduits provide an advanced tool for repairing the injured peripheral nerve that often causes disability and mortality. Currently, the efficiency of conduits in repairing peripheral nerve is unsatisfying. Here, we show a functional nanoparticle-enhanced nerve conduit for promoting the regeneration of peripheral nerves. This conduit, which consists of gelatin-methacryloyl (GelMA) hydrogels with drug loaded poly(ethylene glycol)- poly(3-caprolactone) (MPEG-PCL) nanoparticles dispersed in the hydrogel matrix, is rapidly fabricated by a continuous three-dimensional (3D) printing process. While the 3D-printed hydrogel conduit with customized size, shape and structure provides a physical microenvironment for axonal elongation, the nanoparticles sustained release the drug to facilitate the nerve regeneration. The drug, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino) benzenesulfonamide, is a Hippo pathway inhibitor with multiple functions including improving the proliferation and migration of Schwann cells and up-regulating neurotrophic factors genes. The descried functional nerve conduit efficiently induced the recovery of sciatic injuries in morphology, histopathology and functions in vivo, showing the potential clinical application in peripheral nerve repair. STATEMENTS OF SIGNIFICANCE: Functional nerve conduit provides a promising strategy alternative to autografts. In this work, we rapidly customized a nanoparticle-enhanced conduit by the continuous bioprinting process. This nanoparticle in the conduit can release a Hippo pathway inhibitor to facilitate the nerve regeneration and function restoration. The efficacy of the conduits is comparable to that of autograft, suggesting the potential clinical applications.
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http://dx.doi.org/10.1016/j.actbio.2019.03.047DOI Listing
May 2019

Biomimetic 3D-printed scaffolds for spinal cord injury repair.

Nat Med 2019 02 14;25(2):263-269. Epub 2019 Jan 14.

Department of Neuroscience, University of California San Diego, La Jolla, CA, USA.

Current methods for bioprinting functional tissue lack appropriate biofabrication techniques to build complex 3D microarchitectures essential for guiding cell growth and promoting tissue maturation. 3D printing of central nervous system (CNS) structures has not been accomplished, possibly owing to the complexity of CNS architecture. Here, we report the use of a microscale continuous projection printing method (μCPP) to create a complex CNS structure for regenerative medicine applications in the spinal cord. μCPP can print 3D biomimetic hydrogel scaffolds tailored to the dimensions of the rodent spinal cord in 1.6 s and is scalable to human spinal cord sizes and lesion geometries. We tested the ability of µCPP 3D-printed scaffolds loaded with neural progenitor cells (NPCs) to support axon regeneration and form new 'neural relays' across sites of complete spinal cord injury in vivo in rodents. We find that injured host axons regenerate into 3D biomimetic scaffolds and synapse onto NPCs implanted into the device and that implanted NPCs in turn extend axons out of the scaffold and into the host spinal cord below the injury to restore synaptic transmission and significantly improve functional outcomes. Thus, 3D biomimetic scaffolds offer a means of enhancing CNS regeneration through precision medicine.
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http://dx.doi.org/10.1038/s41591-018-0296-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559945PMC
February 2019

Sustainable Synthesis and Characterization of Bisphenol A-Free Polycarbonate from Six-Membered Dicyclic Carbonate.

Polym Chem 2018 Jul 18;9(27):3798-3807. Epub 2018 Jun 18.

Biotechnology, Department of Chemistry, Center for Chemistry and Chemical Engineering, Lund University, SE-22100 Lund, Sweden.

Bisphenol A, (2,2-bis(4-hydroxyphenyl)propane, BPA)-free polycarbonate (PC) from six-membered di-cyclic carbonate, di-trimethylolpropane di-cyclic carbonate (DTMPC) was developed as a new type of PC by ring opening homo-polymerization. The polymerization was controlled by using metal-free organic-based catalyst systems. The results indicated that the conversion rate depends on the basicity of the catalyst in the order of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 4-dimethylaminopyridine (DMAP), triethylamine (TEA) from high to low. Over 99% conversion of DTMPC was obtained at 130°C within 15 min by TBD, DBU and DMAP. The resulting PC as a homo-polymer showed high optical transparency and hardness, low swelling property in organic solvents, and thermally stable at temperatures as high as 200 °C. High cell viability as the cyto-compatibility of C3H 10T1/2 cells seeded directly on the surface of PC films was obtained. This implied that PC is a viable material for biomedical and consumer products applications where safety is an important consideration.
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http://dx.doi.org/10.1039/C8PY00676HDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300155PMC
July 2018

3D printed micro-scale force gauge arrays to improve human cardiac tissue maturation and enable high throughput drug testing.

Acta Biomater 2019 09 19;95:319-327. Epub 2018 Dec 19.

Department of Bioengineering, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; Department of Materials Science and Engineering Program, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; Department of NanoEngineering, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; Chemical Engineering Program, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. Electronic address:

Human induced pluripotent stem cell - derived cardiomyocytes (iPSC-CMs) are regarded as a promising cell source for establishing in-vitro personalized cardiac tissue models and developing therapeutics. However, analyzing cardiac force and drug response using mature human iPSC-CMs in a high-throughput format still remains a great challenge. Here we describe a rapid light-based 3D printing system for fabricating micro-scale force gauge arrays suitable for 24-well and 96-well plates that enable scalable tissue formation and measurement of cardiac force generation in human iPSC-CMs. We demonstrate consistent tissue band formation around the force gauge pillars with aligned sarcomeres. Among the different maturation treatment protocols we explored, 3D aligned cultures on force gauge arrays with in-culture pacing produced the highest expression of mature cardiac marker genes. We further demonstrated the utility of these micro-tissues to develop significantly increased contractile forces in response to treatment with isoproterenol, levosimendan, and omecamtiv mecarbil. Overall, this new 3D printing system allows for high flexibility in force gauge design and can be optimized to achieve miniaturization and promote cardiac tissue maturation with great potential for high-throughput in-vitro drug screening applications. STATEMENT OF SIGNIFICANCE: The application of iPSC-derived cardiac tissues in translatable drug screening is currently limited by the challenges in forming mature cardiac tissue and analyzing cardiac forces in a high-throughput format. We demonstrate the use of a rapid light-based 3D printing system to build a micro-scale force gauge array that enables scalable cardiac tissue formation from iPSC-CMs and measurement of contractile force development. With the capability to provide great flexibility over force gauge design as well as optimization to achieve miniaturization, our 3D printing system serves as a promising tool to build cardiac tissues for high-throughput in-vitro drug screening applications.
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http://dx.doi.org/10.1016/j.actbio.2018.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584548PMC
September 2019

Scanningless and continuous 3D bioprinting of human tissues with decellularized extracellular matrix.

Biomaterials 2019 02 10;194:1-13. Epub 2018 Dec 10.

Department of NanoEngineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA; Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA; Materials Science and Engineering Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA; Chemical Engineering Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. Electronic address:

Decellularized extracellular matrices (dECMs) have demonstrated excellent utility as bioscaffolds in recapitulating the complex biochemical microenvironment, however, their use as bioinks in 3D bioprinting to generate functional biomimetic tissues has been limited by their printability and lack of tunable physical properties. Here, we describe a method to produce photocrosslinkable tissue-specific dECM bioinks for fabricating patient-specific tissues with high control over complex microarchitecture and mechanical properties using a digital light processing (DLP)-based scanningless and continuous 3D bioprinter. We demonstrated that tissue-matched dECM bioinks provided a conducive environment for maintaining high viability and maturation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and hepatocytes. Microscale patterning also guided spontaneous cellular reorganization into predesigned striated heart and lobular liver structures through biophysical cues. Our methodology enables a light-based approach to rapidly bioprint dECM bioinks with accurate tissue-scale design to engineer physiologically-relevant functional human tissues for applications in biology, regenerative medicine, and diagnostics.
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http://dx.doi.org/10.1016/j.biomaterials.2018.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339581PMC
February 2019
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