A SYNTHETIC HUMAN BRAIN ECM HYDROGEL FOR TIGHT CONTROL OF ASTROCYTE ACTIVATION

Bioengineers have aimed to design instructive extracellular matrix (ECM) models that can tailor the protein composition and biomechanics of the brain in vitro in order to study how astrocytes remodel the brain during trauma and inflammation. However, these parameters cannot be independently controlled in protein-based models, and although tunable in synthetic systems, current astrocyte cultures fail to retain their characteristic stellate morphology without becoming activated. To this date, there is no biomaterial model that can retain astrocyte quiescence in vitro. This dissertation sought to develop such an in vitro model that would enable the study of specific ECM factors that control astrocyte activation while retaining quiescent astrocytes in vitro. Here we introduce a synthetic hydrogel, that for the first time shows maintenance of astrocyte quiescence and control over activation on demand. We first characterized the human brain ECM via proteomics, and the brain biomechanics via needle-induced cavitation rheology and volume-controlled cavity expansion and incorporated the top ECM components responsible for integrin-mediated and MMP-mediated degradation alongside matched mechanical properties into a fully synthetic hydrogel. Using this hydrogel, composed of just PEG and peptides, we demonstrate control over astrocyte activation via tuning of the integrin-binding and MMP-degradable profile or via cytokine molecules, in contrast to other protein-based models like collagen where astrocytes remain in a reactive state. Finally, to aid with the implementation of biomaterials as in vitro platforms to predict in vivo physiology, the correlation between current 2D, 3D and in vivo studies of glioblastoma motility was explored, and how an effect size can help standardize comparison across labs and culture dimensions. An additional study highlighted the importance of adopting growth rate in drug metric responses and how these can be implemented in current biomaterial platforms. Overall, this work can help integrate biomaterials as models to predict in vivo physiology. This brain hydrogel system can be used as a new platform to model the physiological state of quiescent astrocytes and their reactivity upon injury, for the first time, in vitro

A Student-Created, Open Access, Living Textbook

Textbooks are expensive, updated infrequently, and rarely used effectively by students. We discuss here a way for students to create the textbook for the course, helping them feel ownership over the course material. This Wiki-based, student-created textbook is online free for use, widely accessible by all, and editable during the course of and as topics evolve. This type of textbook format is particularly well suited to upper-level electives on topics that are rapidly emerging. We have nucleated a student created textbook here, fully online and open access, for two upper elective courses in chemical engineering. Wikis offer an easy-to-learn platform that does not require previous training in coding, and we have found it to be an excellent way to increase student learning while encouraging student buy-in and ownership of their course textbook

Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel

Bioengineers have designed numerous instructive brain extracellular matrix (ECM) environments with tailored and tunable protein composition and biomechanical properties in vitro to study astrocyte reactivity during trauma and inflammation. However, a major limitation of both protein-based and model microenvironments is that astrocytes within fail to retain their characteristic stellate morphology and quiescent state without becoming activated under “normal” culture conditions. Here we introduce a synthetic hydrogel, that for the first time demonstrates maintenance of astrocyte quiescence and activation on demand. With this synthetic brain hydrogel, we show the brain-specific integrin-binding and matrix metalloprotease (MMP)-degradable domains of proteins control astrocyte star-shaped morphologies, and we can achieve an ECM condition that maintains astrocyte quiescence with minimal activation. In addition, we can induce activation in a dose-dependent manner via both defined cytokine cocktails and low molecular weight hyaluronic acid. We envision this synthetic brain hydrogel as a new tool to study the physiological role of astrocytes in health and disease

2D or 3D? How cell motility measurements are conserved across dimensions in vitro and translate in vivo

Cell motility is a critical aspect of several processes such as wound healing and immunity; however, it is dysregulated in cancer. Current limitations of imaging tools make it difficult to study cell migration in vivo. To overcome this, and to identify drivers from the microenvironment that regulate cell migration, bioengineers have developed 2D and 3D tissue model systems in which to study cell motility in vitro, with the aim of mimicking elements of the environments in which cells move in vivo. However, there has been no systematic study to explicitly relate and compare cell motility measurements between these geometries or systems. Here, we provide such analysis on our own data, as well as across data in existing literature to understand whether, and which, metrics are conserved across systems. To our surprise, only one metric of cell movement on 2D surfaces significantly and positively correlates with cell migration in 3D environments (percent migrating cells, and cell invasion in 3D has a weak, negative correlation with glioblastoma invasion in vivo. Finally, to compare across complex model systems, in vivo data, and data from different labs, we suggest that groups report an effect size, a statistical tool that is most translatable across experiments and labs, when conducting experiments that affect cellular motility

Control of thiol-maleimide reaction kinetics in PEG hydrogel networks

Michael-type addition reactions are widely used to polymerize biocompatible hydrogels. The thiol-maleimide modality achieves the highest macromer coupling efficiency of the reported Michael-type pairs, but the resulting hydrogel networks are heterogeneous, because polymerization is faster than the individual components can be manually mixed. The reactivity of the thiol dictates the overall reaction speed, which can be slowed in organic solvents and acidic buffers. Since these modifications also reduce the biocompatibility of resulting hydrogels, we investigated a series of biocompatible buff­ers and crosslinkers to decelerate gelation while maintaining high cell viability. We found that lowering the polymer weight percentage (wt%), buffer concentration, and pH slowed gelation kinetics, but crosslinking with an electronegative peptide was optimal for both kinetics and cell viability. Including a high glucose medium supplement in the polymer solvent buffer improved the viability of the cells being encapsulated without impacting gelation time. Slowing the speed of polymerization resulted in more uniform hydrogels, both in terms of visual inspection and the diffusion of small molecules through the network. However, reactions that were too slow resulted in non-uniform particle dispersion due to settling, thus there is a trade-off in hydrogel network uniformity versus cell distribution in the hydrogels when using these networks in cell applications

Applicability of Drug Response Metrics for Cancer Studies using Biomaterials

Bioengineers have built models of the tumour microenvironment (TME) in which to study cell–cell interactions, mechanisms of cancer growth and metastasis, and to test new therapies. These models allow researchers to culture cells in conditions that include features of the in vivo TME implicated in regulating cancer progression, such as extracellular matrix (ECM) stiffness, integrin binding to the ECM, immune and stromal cells, growth factor and cytokine depots, and a three-dimensional geometry more representative of the in vivo TME than tissue culture polystyrene (TCPS). These biomaterials could be particularly useful for drug screening applications to make better predictions of efficacy, offering better translation to preclinical models and clinical trials. However, it can be challenging to compare drug response reports across different biomaterial platforms in the current literature. This is, in part, a result of inconsistent reporting and improper use of drug response metrics, and vast differences in cell growth rates across a large variety of biomaterial designs. This study attempts to clarify the definitions of drug response measurements used in the field, and presents examples in which these measurements can and cannot be applied. We suggest as best practice to measure the growth rate of cells in the absence of drug, and follow our ‘decision tree’ when reporting drug response metrics

The Predictive Link between Matrix and Metastasis

Cancer spread (metastasis) is responsible for 90% of cancer-related fatalities. Informing patient treatment to prevent metastasis, or kill all cancer cells in a patient\u27s body before it becomes metastatic is extremely powerful. However, aggressive treatment for all non-metastatic patients is detrimental, both for quality of life concerns, and the risk of kidney or liver-related toxicity. Knowing when and where a patient has metastatic risk could revolutionize patient treatment and care. In this review, we attempt to summarize the key work of engineers and quantitative biologists in developing strategies and model systems to predict metastasis, with a particular focus on cell interactions with the extracellular matrix (ECM), as a tool to predict metastatic risk and tropism

Tumor cell–organized fibronectin maintenance of a dormant breast cancer population

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, nonproliferative state before reactivation and outgrowth. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system with carefully controlled ECM substrates to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle–arrested, and actively proliferating cells. Cell populations capable of entering dormancy formed an organized, fibrillar fibronectin matrix via αvβ3 and α5β1 integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation, and cancer cell outgrowth after dormancy required MMP-2–mediated fibronectin degradation. We propose this approach as a useful, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation

Tumor cell-organized fibronectin is required to maintain a dormant breast cancer population [preprint]

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, non-proliferative state before reactivation and outgrowth. For a patient, these post-remission tumors are often drug resistant and highly aggressive, resulting in poor prognosis. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system that combines carefully controlled ECM substrates with nutrient deprivation to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle arrested, and actively proliferating cells. Cell populations that endured extended periods of serum-deprivation-induced dormancy formed an organized, fibrillar fibronectin matrix via αvβ3 and α5β1 integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation. We surmised that the fibronectin matrix was primarily a mediator of cell survival, not proliferation, during the serum-deprivation stress, bacause cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. Given the difficulty of animal models in observing entrance and exit from dormancy in real-time, we propose this approach as a new, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation

Applicability of Drug Response Metrics for Cancer Studies Using Biomaterials

Bioengineers have built models of the tumour microenvironment (TME) in which to study cell–cell interactions, mechanisms of cancer growth and metastasis, and to test new therapies. These models allow researchers to culture cells in conditions that include features of the in vivo TME implicated in regulating cancer progression, such as extracellular matrix (ECM) stiffness, integrin binding to the ECM, immune and stromal cells, growth factor and cytokine depots, and a three-dimensional geometry more representative of the in vivo TME than tissue culture polystyrene (TCPS). These biomaterials could be particularly useful for drug screening applications to make better predictions of efficacy, offering better translation to preclinical models and clinical trials. However, it can be challenging to compare drug response reports across different biomaterial platforms in the current literature. This is, in part, a result of inconsistent reporting and improper use of drug response metrics, and vast differences in cell growth rates across a large variety of biomaterial designs. This study attempts to clarify the definitions of drug response measurements used in the field, and presents examples in which these measurements can and cannot be applied. We suggest as best practice to measure the growth rate of cells in the absence of drug, and follow our ‘decision tree’ when reporting drug response metrics