Santiago Mille Fragoso

All Publications

• Microfluidic bioprinting of tough hydrogel-based vascular conduits for functional blood vessels. Science advances Wang, D., Maharjan, S., Kuang, X., Wang, Z., Mille, L. S., Tao, M., Yu, P., Cao, X., Lian, L., Lv, L., He, J. J., Tang, G., Yuk, H., Ozaki, C. K., Zhao, X., Zhang, Y. S. 2022; 8 (43): eabq6900

  Abstract

  Three-dimensional (3D) bioprinting of vascular tissues that are mechanically and functionally comparable to their native counterparts is an unmet challenge. Here, we developed a tough double-network hydrogel (bio)ink for microfluidic (bio)printing of mono- and dual-layered hollow conduits to recreate vein- and artery-like tissues, respectively. The tough hydrogel consisted of energy-dissipative ionically cross-linked alginate and elastic enzyme-cross-linked gelatin. The 3D bioprinted venous and arterial conduits exhibited key functionalities of respective vessels including relevant mechanical properties, perfusability, barrier performance, expressions of specific markers, and susceptibility to severe acute respiratory syndrome coronavirus 2 pseudo-viral infection. Notably, the arterial conduits revealed physiological vasoconstriction and vasodilatation responses. We further explored the feasibility of these conduits for vascular anastomosis. Together, our study presents biofabrication of mechanically and functionally relevant vascular conduits, showcasing their potentials as vascular models for disease studies in vitro and as grafts for vascular surgeries in vivo, possibly serving broad biomedical applications in the future.

  View details for DOI 10.1126/sciadv.abq6900

  View details for PubMedID 36288300

• An expanded whole-cell model of E. coli links cellular physiology with mechanisms of growth rate control. NPJ systems biology and applications Ahn-Horst, T. A., Mille, L. S., Sun, G., Morrison, J. H., Covert, M. W. 2022; 8 (1): 30

  Abstract

  Growth and environmental responses are essential for living organisms to survive and adapt to constantly changing environments. In order to simulate new conditions and capture dynamic responses to environmental shifts in a developing whole-cell model of E. coli, we incorporated additional regulation, including dynamics of the global regulator guanosine tetraphosphate (ppGpp), along with dynamics of amino acid biosynthesis and translation. With the model, we show that under perturbed ppGpp conditions, small molecule feedback inhibition pathways, in addition to regulation of expression, play a role in ppGpp regulation of growth. We also found that simulations with dysregulated amino acid synthesis pathways provide average amino acid concentration predictions that are comparable to experimental results but on the single-cell level, concentrations unexpectedly show regular fluctuations. Additionally, during both an upshift and downshift in nutrient availability, the simulated cell responds similarly with a transient increase in the mRNA:rRNA ratio. This additional simulation functionality should support a variety of new applications and expansions of the E. coli Whole-Cell Modeling Project.

  View details for DOI 10.1038/s41540-022-00242-9

  View details for PubMedID 35986058

• Photoacoustic imaging of 3D-printed vascular networks. Biofabrication Ma, C., Li, W., Li, D., Chen, M., Wang, M., Jiang, L., Mille, L. S., Garciamendez, C. E., Zhao, Z., Zhou, Q., Zhang, Y. S., Yao, J. 1800; 14 (2)

  Abstract

  Thrombosis in the circulation system can lead to major myocardial infarction and cardiovascular deaths. Understanding thrombosis formation is necessary for developing safe and effective treatments. In this work, using digital light processing (DLP)-based 3D printing, we fabricated sophisticatedin vitromodels of blood vessels with internal microchannels that can be used for thrombosis studies. In this regard, photoacoustic microscopy (PAM) offers a unique advantage for label-free visualization of the 3D-printed vessel models, with large penetration depth and functional sensitivity. We compared the imaging performances of two PAM implementations: optical-resolution PAM and acoustic-resolution PAM, and investigated 3D-printed vessel structures with different patterns of microchannels. Our results show that PAM can provide clear microchannel structures at depths up to 3.6 mm. We further quantified the blood oxygenation in the 3D-printed vascular models, showing that thrombi had lower oxygenation than the normal blood. We expect that PAM can find broad applications in 3D printing and bioprinting forin vitrostudies of various vascular and other diseases.

  View details for DOI 10.1088/1758-5090/ac49d5

  View details for PubMedID 35008080

• Biomimetic models of the glomerulus NATURE REVIEWS NEPHROLOGY Valverde, M. G., Mille, L. S., Figler, K. P., Cervantes, E., Li, V. Y., Bonventre, J., Masereeuw, R., Zhang, Y. 2022; 18 (4): 241-257

  Abstract

  The use of biomimetic models of the glomerulus has the potential to improve our understanding of the pathogenesis of kidney diseases and to enable progress in therapeutics. Current in vitro models comprise organ-on-a-chip, scaffold-based and organoid approaches. Glomerulus-on-a-chip designs mimic components of glomerular microfluidic flow but lack the inherent complexity of the glomerular filtration barrier. Scaffold-based 3D culture systems and organoids provide greater microenvironmental complexity but do not replicate fluid flows and dynamic responses to fluidic stimuli. As the available models do not accurately model the structure or filtration function of the glomerulus, their applications are limited. An optimal approach to glomerular modelling is yet to be developed, but the field will probably benefit from advances in biofabrication techniques. In particular, 3D bioprinting technologies could enable the fabrication of constructs that recapitulate the complex structure of the glomerulus and the glomerular filtration barrier. The next generation of in vitro glomerular models must be suitable for high(er)-content or/and high(er)-throughput screening to enable continuous and systematic monitoring. Moreover, coupling of glomerular or kidney models with those of other organs is a promising approach to enable modelling of partial or full-body responses to drugs and prediction of therapeutic outcomes.

  View details for DOI 10.1038/s41581-021-00528-x

  View details for Web of Science ID 000745398900001

  View details for PubMedID 35064233

  View details for PubMedCentralID 6620109

• Digital Light Processing Based Bioprinting with Composable Gradients ADVANCED MATERIALS Wang, M., Li, W., Mille, L. S., Ching, T., Luo, Z., Tang, G., Garciamendez, C., Lesha, A., Hashimoto, M., Zhang, Y. 2022; 34 (1): e2107038

  Abstract

  Recapitulation of complex tissues signifies a remarkable challenge and, to date, only a few approaches have emerged that can efficiently reconstruct necessary gradients in 3D constructs. This is true even though mimicry of these gradients is of great importance to establish the functionality of engineered tissues and devices. Here, a composable-gradient Digital Light Processing (DLP)-based (bio)printing system is developed, utilizing the unprecedented integration of a microfluidic mixer for the generation of either continual or discrete gradients of desired (bio)inks in real time. Notably, the precisely controlled gradients are composable on-the-fly by facilely by adjusting the (bio)ink flow ratios. In addition, this setup is designed in such a way that (bio)ink waste is minimized when exchanging the gradient (bio)inks, further enhancing this time- and (bio)ink-saving strategy. Various planar and 3D structures exhibiting continual gradients of materials, of cell densities, of growth factor concentrations, of hydrogel stiffness, and of porosities in horizontal and/or vertical direction, are exemplified. The composable fabrication of multifunctional gradients strongly supports the potential of the unique bioprinting system in numerous biomedical applications.

  View details for DOI 10.1002/adma.202107038

  View details for Web of Science ID 000710109100001

  View details for PubMedID 34609032

  View details for PubMedCentralID PMC8741743

• A Smartphone-Enabled Portable Digital Light Processing 3D Printer ADVANCED MATERIALS Li, W., Wang, M., Mille, L., Robledo Lara, J., Huerta, V., Uribe Velazquez, T., Cheng, F., Li, H., Gong, J., Ching, T., Murphy, C. A., Lesha, A., Hassan, S., Woodfield, T. F., Lim, K. S., Zhang, Y. 2021; 33 (35): e2102153

  Abstract

  3D printing has emerged as an enabling approach in a variety of different fields. However, the bulk volume of printing systems limits the expansion of their applications. In this study, a portable 3D Digital Light Processing (DLP) printer is built based on a smartphone-powered projector and a custom-written smartphone-operated app. Constructs with detailed surface architectures, porous features, or hollow structures, as well as sophisticated tissue analogs, are successfully printed using this platform, by utilizing commercial resins as well as a range of hydrogel-based inks, including poly(ethylene glycol)-diacrylate, gelatin methacryloyl, or allylated gelatin. Moreover, due to the portability of the unique DLP printer, medical implants can be fabricated for point-of-care usage, and cell-laden tissues can be produced in situ, achieving a new milestone for mobile-health technologies. Additionally, the all-in-one printing system described herein enables the integration of the 3D scanning smartphone app to obtain object-derived 3D digital models for subsequent printing. Along with further developments, this portable, modular, and easy-to-use smartphone-enabled DLP printer is anticipated to secure exciting opportunities for applications in resource-limited and point-of-care settings not only in biomedicine but also for home and educational purposes.

  View details for DOI 10.1002/adma.202102153

  View details for Web of Science ID 000674180900001

  View details for PubMedID 34278618

  View details for PubMedCentralID PMC8416928

• Microfluidic integration of regeneratable electrochemical affinity-based biosensors for continual monitoring of organ-on-a-chip devices NATURE PROTOCOLS Aleman, J., Kilic, T., Mille, L. S., Shin, S., Zhang, Y. 2021; 16 (5): 2564-2593

  Abstract

  Organs-on-chips have emerged as viable platforms for drug screening and personalized medicine. While a wide variety of human organ-on-a-chip models have been developed, rarely have there been reports on the inclusion of sensors, which are critical in continually measuring the microenvironmental parameters and the dynamic responses of the microtissues to pharmaceutical compounds over extended periods of time. In addition, automation capacity is strongly desired for chronological monitoring. To overcome this major hurdle, in this protocol we detail the fabrication of electrochemical affinity-based biosensors and their integration with microfluidic chips to achieve in-line microelectrode functionalization, biomarker detection and sensor regeneration, allowing continual, in situ and noninvasive quantification of soluble biomarkers on organ-on-a-chip platforms. This platform is almost universal and can be applied to in-line detection of a majority of biomarkers, can be connected with existing organ-on-a-chip devices and can be multiplexed for simultaneous measurement of multiple biomarkers. Specifically, this protocol begins with fabrication of the electrochemically competent microelectrodes and the associated microfluidic devices (~3 d). The integration of electrochemical biosensors with the chips and their further combination with the rest of the platform takes ~3 h. The functionalization and regeneration of the microelectrodes are subsequently described, which require ~7 h in total. One cycle of sampling and detection of up to three biomarkers accounts for ~1 h.

  View details for DOI 10.1038/s41596-021-00511-7

  View details for Web of Science ID 000645191100006

  View details for PubMedID 33911259

• Symbiotic Photosynthetic Oxygenation within 3D-Bioprinted Vascularized Tissues MATTER Maharjan, S., Alva, J., Camara, C., Rubio, A. G., Hernandez, D., Delavaux, C., Correa, E., Romo, M. D., Bonilla, D., Santiago, M., Li, W., Cheng, F., Ying, G., Zhang, Y. 2021; 4 (1): 217-240

  Abstract

  In this study, we present the photosynthetic oxygen (O2) supply to mammalian cells within a volumetric extracellular matrix-like construct, whereby a three-dimensional (3D)-bioprinted fugitive pattern encapsulating unicellular green algae, Chlamydomonas reinhardtii (C. reinhardtii), served as a natural photosynthetic O2-generator. The presence of bioprinted C. reinhardtii enhanced the viability and functionality of mammalian cells while reducing the hypoxic conditions within the tissues. We were able to subsequently endothelialize the hollow perfusable microchannels formed after enzymatic removal of the bioprinted C. reinhardtii-laden patterns from the matrices following the initial oxygenation period, to obtain biologically relevant vascularized mammalian tissue constructs. The feasibility of co-culture of C. reinhardtii with human cells, the printability and the enzymatic degradability of the fugitive bioink, as well as the exploration of C. reinhardtii as a natural, eco-friendly, cost-effective, and sustainable source of O2 would likely promote the development of engineered tissues, tissue models, and food for various applications.

  View details for DOI 10.1016/j.matt.2020.10.022

  View details for Web of Science ID 000608248900003

  View details for PubMedID 33718864

  View details for PubMedCentralID PMC7945990

• Recent Advances in Formulating and Processing Biomaterial Inks for Vat Polymerization-Based 3D Printing ADVANCED HEALTHCARE MATERIALS Li, W., Mille, L. S., Robledo, J. A., Uribe, T., Huerta, V., Zhang, Y. 2020; 9 (15): e2000156

  Abstract

  3D printing and bioprinting have become a key component in precision medicine. They have been used toward the fabrication of medical devices with patient-specific shapes, production of engineered tissues for in vivo regeneration, and preparation of in vitro tissue models used for screening therapeutics. In particular, vat polymerization-based 3D (bio)printing as a unique strategy enables more sophisticated architectures to be rapidly built. This progress report aims to emphasize the recent advances made in vat polymerization-based 3D printing and bioprinting, including new biomaterial ink formulations and novel vat polymerization system designs. While some of these approaches have not been utilized toward the combination with biomaterial inks, it is anticipated their rapid translation into biomedical applications.

  View details for DOI 10.1002/adhm.202000156

  View details for Web of Science ID 000539585500001

  View details for PubMedID 32529775

  View details for PubMedCentralID PMC7473482