present 3D neurospheroids with self-standing silicon microdevices internalized at different levels. Results are obtained by uncovering the control capability of silicon surface engineering to drive microdevices at predefined sites during cellular aggregation and 3D growth. This work paves the way to bionic brain-tissue models with inbuilt untethered bioelectronic sensors and actuators. 3D Neural SpheroiDS
A plethora of bioinspired cell‐laden hydrogels are being explored as building blocks that once assembled are able to create complex and highly hierarchical structures recapitulating the heterogeneity of living tissues. Yet, the resulting 3D bioengineered systems still present key limitations, mainly related with limited diffusion of essential molecules for cell survival, which dictates the failure of most strategies upon implantation. To maximize the hierarchical complexity of bioengineered systems, while simultaneously fully addressing the exchange efficiency of biomolecules, the high‐throughput fabrication of liquefied capsules is proposed using superhydrophobic–superhydrophilic microarrays as platforms to produce the initial structures with high fidelity of geometry and size. The liquefied capsules are composed by i) a permselective multilayered membrane; ii) surface‐functionalized poly(ε‐caprolactone) microparticles loaded into the liquefied core acting as cell adhesion sites; and iii) cells. It is demonstrated that besides the typical spherical liquefied capsules, it is also possible to obtain multi‐shaped blocks with high geometrical precision and efficiency. Importantly, the internal gelation approach used to produce such blocks does not jeopardize cell viability, evidencing the mild conditions of the proposed cell encapsulation technique. The proposed system is intended to be used as hybrid devices implantable using minimally invasive procedures for multiple tissue engineering applications.
In article number 1900300, Naside Gözde Durmus, Sean M. Wu, Utkan Demirci, and co-workers show that magnetic levitation defines the fine balance of living cells where fatty and healthy populations of iPS-derived cardiomyocytes levitate to a precise height under the magnetic field inside a microfluidic setup engineered as meticulously as the geometry of the pyramids.
In article number 2000056, Kristopher A. Kilian and co-workers introduce a templating approach to build 3D geometrically structured tumor-mimics in virtually any hydrogel material, with scope for controlling tumor geometry and matrix properties in orthotopic xenografts.
Cancers are a complex conglomerate of heterogeneous cell populations with varying genotypes and phenotypes. The intercellular heterogeneity within the same tumor and intratumor heterogeneity within various tumors are the leading causes of resistance to cancer therapies and varied outcomes in different patients. Therefore, performing single-cell analysis is essential to identify and classify cancer cell types and study cellular heterogeneity. Here, the development of a machine learning-assisted nanoparticle-printed biochip for single-cell analysis is reported. The biochip is integrated by combining powerful machine learning techniques with easily accessible inkjet printing and microfluidics technology. The biochip is easily prototype-able, miniaturized, and cost-effective, potentially capable of differentiating a variety of cell types in a label-free manner. n-feature classifiers are established and their performance metrics are evaluated. The biochip's utility to discriminate noncancerous cells from cancerous cells at the single-cell level is demonstrated. The biochip's utility in classifying cancer sub-type cells is also demonstrated. It is envisioned that such a chip has potential applications in single-cell studies, tumor heterogeneity studies, and perhaps in point-of-care cancer diagnostics-especially in developing countries where the cost, limited infrastructures, and limited access to medical technologies are of the utmost importance.
Framework nucleic acid (FNA) is an emerging drug carrier platform for systemic and transdermal delivery. In article number 1900169, Chenjie Xu and co-workers describe an optically clear keratinocyte/fibroblast co-culture system to elucidate FNA-skin cell interactions. This system enables facile, real-time evaluation of size-dependent FNA interactions and cellular internalization, as well as direct toxicity assessment on both cell types.
Long before the age of microelectronics, a group of filamentous microorganisms named cable bacteria developed an electrical network within their own cell structure. In article number 2000006, Jean V. Manca and co-workers present the local electrical pathways for cable bacteria using conductive atomic force microscopy, showing a fail-safe electrical structure conducted by parallel and electrically interconnected biological fibers.
Intratumoral heterogeneity plays a major role in rendering conventional and targeted therapies for glioblastoma ineffective. In article number 1900312, Bakhos A. Tannous and co-workers demonstrate that extracellular vesicles can induce mesenchymal transition and treatment resistance in recipient proneural glioblastoma stem cells. The findings may point to novel treatment strategies for glioblastoma and indicate the involvement of extracellular vesicles in mesenchymal transition of other tumor types.
Extracellular vesicles (EVs) are now well established as important mediators of intercellular communication. EVs constitute a diverse group of secreted vesicles which function by the delivery of protein and nucleic acid cargoes from donor to recipient cells. In cancer, tumor cell-derived EVs are shown to promote disease progression by facilitating local reprogramming of the tumor microenvironment. EVs also have more distant systemic effects via transport in biofluids, and therefore have great potential as biomarkers for disease detection and monitoring. Recently, the discovery that EVs derived from glioblastoma cells can mediate immunosuppression by activation of immune checkpoint signaling and T cell dysfunction was reported. Mechanistically we showed that this occurs via direct binding of PD-L1 secreted in EVs, to its receptor PD1 expressed on the surface of activated T cells. This previously unidentified mechanism of tumor immunosuppression has been confirmed in subsequent independent studies, which have demonstrated the biologic importance of this mechanism across multiple tumor types. These studies have established a new and significant paradigm in which PD-L1 containing tumor cell-derived EVs cause immune suppression by the direct engagement of PD1 on T cells, decreasing their activation and providing a further barrier to protect tumors from T cell killing.
Islet transplantation has been demonstrated to be a promising therapy for type 1 diabetes mellitus. Although it is a minimally invasive operating procedure and provides easy access for graft monitoring, subcutaneous transplantation of the islet only has limited therapeutic outcomes, owing to the poor capacity of skin tissue to foster revascularization in a short period. Herein, 3D cell spheroids of clinically accessible umbilical cord blood mesenchymal stem cells and human umbilical vein endothelial cells are formed and employed for codelivery with β cells subcutaneously. The 3D stem cell spheroids, which can secrete multiple proangiogenic and prosurvival growth factors, induce robust angiogenesis and prevent β cell graft death, as indicated by the results of in vivo bioluminescent tracking and histological analysis. These experimental data highlight the efficacy of the 3D stem cell spheroids that are fabricated using translationally applicable cell types in promoting the survival and function of subcutaneously transplanted β cells.
A number of natural polymer biomaterial-based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross-linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross-linking agents. In this study, collagen-based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross-linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross-linking is developed, by incorporating genipin into solution prior to freeze-drying the NGCs. This leads to successful cross-linking as demonstrated by higher cross-linking degree and similar tensile strength of genipin cross-linked conduits compared to formaldehyde cross-linked conduits. Genipin cross-linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross-linked conduits, supports the viability of Schwann cells. Moreover, genipin cross-linked conduits direct macrophages away from a pro-inflammatory and toward a pro-repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti-inflammatory alternative to formaldehyde for cross-linking clinical grade NGCs.
Living organisms are programmed to perform multiple functions by sensing intra- and extra-cellular environments and by controlling gene expressions. Synthetic biologists aim to program cells by mimicking, designing, and constructing genetic circuits. Synthetic mRNA-based genetic switches and circuits have attracted attention for future therapeutic applications because of their safety and functional diversity. Here, the mRNA-based switches and circuits that detect specific microRNAs or proteins expressed in a target cell to control transgene expression and cell fate are reviewed. Future perspectives of artificial RNA systems for cell engineering will also be addressed.
Nature uses vascular systems to permit large-area control over the functionality of surfaces that lie above them. In this work, the application of this concept to the control of a hybrid living-nonliving system is demonstrated. Defined arrangements of vascular channels are created in agar using a fugitive ink printing method. The antibiotic gentamicin is then introduced into the vascular network where it diffuses to the surface and interacts with a model system of Escherichia coli cells. The cells either live or die depending on their distance from the underlying channels, permitting spatial control over the biological system. Using single-channel systems to define critical parameters, a theoretical model is developed to define the final surface pattern based solely on the arrangement of the underlying vascular channels. The model is then successfully used to create more complex arrangements of cells at the surface. Finally, by introducing different types of active compounds into separate vascular channels, a mixture of bacterial species is separated and localized at defined points. This work demonstrates the ability of bioinspired embedded vascular systems to predictably control a biological system at a surface, laying the groundwork for future spatially and temporally controlled biointerfaces in both industry and medicine.
As we enter 2020, it is once again time to look back at the previous year, and it is time to see what the new year might have in store. In 2019, submissions to Advanced Biosystems grew further. However, we not only received more submissions, but we also increased the number of published articles from 109 in 2018 to 119 last year. Some of these articles were published as part of the two special issues we assembled. The June 2019 issue was dedicated to “Synthetic Cells” and was guest-edited by Katharina Landfester and Kai Sundmacher. In November we published a special issue on “Single Cell Technology” guest-edited by Angela Wu and Jianbin Wang. Both special issues were well received. This is, for example, reflected by the fact that only half a year after the publication of the “Synthetic Cells” special issue, eight of the twelve articles have already received at least one citation. The article “Microfluidic Handling and Analysis of Giant Vesicles for Use as Artificial Cells: A Review” by Tom Robinson published in that issue has even been cited six times to date. In addition to the special issues, we have also created an online collection with our best articles published to date. These articles in our Editors' Choice collection showcase the diversity of the topics we cover in Advanced Biosystems, from metabolic engineering to 3D culture techniques and medical applications to name just a few. The collection can be accessed here: https://onlinelibrary.wiley.com/doi/toc/10.1002/(ISSN)2366-7478.editors-choice and articles are free to read for a limited time. We hope you enjoy reading these selected articles, which will be updated on a regular basis. Advanced Biosystems continues to be ever more widely recognized. Compared to 2018, our downloads have approximately doubled. On the one hand, this is certainly owing to the fact that the number of articles available for downloading is growing with every issue we publish. On the other hand, the indexing of Advanced Biosystems in Scopus and Clarivate Analytics' Emerging Sources Citation Index has also ensured that the content is more easily discoverable. The year 2019 also brought a change in the leadership of Advanced Biosystems. In May, I took over the role of Editor-in-Chief from Lorna Stimson. Having worked as an editor for more than nine years—among others on the journal BioEssays for which I continue to be Deputy Editor—and having been an editor for Advanced Biosystems ever since its launch in late 2016, I am very excited about this new role. I am looking forward to further developing the journal, and I would also like to take this opportunity to thank Lorna for her vital work on getting Advanced Biosystems “off the ground”. Thanks are also due to our authors, reviewers, and readers as well as to the members of our editorial advisory board without whom Advanced Biosystems would not be in such a good position. Your continued support of the journal is much appreciated. Looking ahead at 2020, we are very grateful that our executive advisory board member Ralph Weissleder has agreed to guest-edit a special issue on the topic of “Extracellular Vesicles”. In addition, we are planning to publish a focus issue dedicated to “Cancer Immunity”, guest-edited by Fangfang Zhou. On a more technical side, we are happy to announce that beginning in January 2020, we will enable ORCID reviewer recognition. This means that people who are reviewing articles for Advanced Biosystems will be able to connect the reviews they have undertaken for the journal to their ORCID profile. In order to not give away the identity of the reviewers, ORCID will not include any details on individual manuscripts. In addition, posting of recognition to ORCID will be delayed in order to mask the exact date on which the review was completed. In the first issue of 2020 we once again bring you a wide variety of different topics. In article 1900188, Wilhelm Huck and colleagues review how droplet microfluidics has revolutionized single cell analyses such as single-cell (epi)genomics, transcriptomics, as well as proteomics and metabolomics. Torben van der Boon et al. introduce a new 96-well plate with a topography gradient allowing for high-throughput screening of cell-surface interactions. Importantly, these cell culture dishes are compatible with general laboratory and imaging equipment (see article 1900218). Chenxiang Lin and colleagues provide an overview on how lipid membranes might be engineered with programmable DNA nanostructures. Such lipid-interacting DNA devices open new opportunities for synthetic biology as well as for therapeutic applications (see article 1900215). Christian Wiraja et al. have developed a keratinocyte/fibroblast co-culture system. Please take a look at article 1900169 to see how they have used this system for the real-time visualization of framework nucleic acid-cell interactions. Tilo Pompe et al. established a direct coculture of primary human cells in a 3D Coll I matrix as a tool to mimic processes typically occurring during the late stages of wound healing. They demonstrated that the secretion of IL-10 by macrophages drives myofibroblast dedifferentiation in a paracrine manner (see article 1900220). In article 1900225, Blake N. Johnson and colleagues presented a 3D printed migration assay for the analysis of chemotactic responses in the presence of spatially distributed sources of chemoattractants. The device enabled the study of the effect of spatially opposing gradients of bradykinin on the migration response of glioblastoma cells toward epidermal growth factor sources. Koji Nagahama et al. generated an artificial nuclear nanotransporter. Their NucPorter demonstrates rapid and highly efficient nuclear transport of both enzymes and synthetic anticancer drugs and therefore provides a promising route for the generation of novel delivery systems to the nucleus (see article 1900189). Francesco Decataldo et al. used organic electrochemical transistors (OECTs) for the real-time monitoring of the toxic effects induced by nanoparticles. In article 1900204, you will find more information on how this technology provides further insights for our understanding of the interaction between nanomaterials and cells. By using a fugitive ink printing method, Caitlin Howell and colleagues created a defined arrangement of vascular channels in agar. By filling these channels with antibiotics, they were able to spatially and temporally control the growth of bacteria on the agar surface (see article 1900216). In article 1900224, Claire Repellin et al. engineered an ovarian cancer cell line and used this cell line to assess the cytolytic function and specificity of primary T cells engineered with chimeric antigen receptors. The authors point out that the strategy described in their article can be more widely used as a quality control tool to validate biologics for their target-specific functions. We hope you enjoy reading these articles in the first issue of Advanced Biosystems' volume 4, and we encourage you to come back in the coming months to discover more exciting content as we progress through the year. Kerstin Brachhold Editor-in-Chief
Adipose tissue (AT) has a dynamic extracellular matrix (ECM) surrounding adipocytes that allows for remodeling during metabolic fluctuations. During the progression of obesity, AT has increased ECM deposition, stiffening, and remodeling, resulting in a pro-fibrotic dysfunctional state. Here, the incorporation of ethylene glycol-bis-succinic acid N-hydroxysuccinimide ester (PEGDS) allows for control over 3D collagen hydrogel stiffness and architecture to investigate its influence on adipocyte metabolic and fibrotic function. Upon stiffening and altering ECM architecture, adipocytes did not alter their expression of key adipokines, leptin, and adiponectin. However, they do increase actin cytoskeletal fiber formation, pro-fibrotic gene expression, ECM deposition, and remodeling within a stiffer, 3D collagen hydrogel. For example, COL6A3 gene expression is upregulated approximately twofold, resulting in increased deposition of pericellular collagen VI alpha 3 surrounding adipocytes. Furthermore, inhibition of actin contractility results in a reversal of pro-fibrotic gene expression and ECM deposition, indicating that adipocytes are mediating mechanical cues through actin cytoskeletal networks. This study demonstrates that ECM stiffness and architecture plays a critical regulatory role in adipocyte fibrotic function and contributes to the overall pro-fibrotic dysfunctional state of AT during the progression of obesity and AT fibrosis.
Achieving vascularization of engineered tissues or structures is a major challenge in the field of tissue engineering. Hitherto, studies on vascularization have demonstrated limited control of vascular network geometry, such as vasculature direction and network density. An open vascular lumen is crucial to ensure that cells survive and that metabolic activity is fully functional in large-sized tissues. Herein, a method based on high water-dispersible collagen microfibers (CMF) to fabricate capillary orientation-controllable 3D tissue with an open vascular lumen using a dispensing machine is reported. A twenty micrometers-long CMF (CMF-20) with high dispersion property are shown to be more effective for dispensing a homogenous tissue and inducing formation of an interconnected capillary network than two hundred micrometers-long CMF (CMF-200). One of the advantages is the prevention of shrinkage on the z-axis of hydrogel-based tissue which acts as a microscaffold. The gaps between the fibers can support endothelial cell migration and maturation, thus forming a larger vascular lumen compared to CMF-free controls. Besides, shear forces produced by the dispensing process cause the collagen microfibers to align, and these microfibers guide cell alignment by integrin-induced adhesion. The findings based on CMF to allow blood capillary alignment and vascular lumen stabilization will be an important technology in tissue engineering.
Epithelial to mesenchymal transition (EMT) is integral for cells to acquire metastatic properties, and ample evidence links it to bioorganic framework of the tumor microenvironment (TME). Hydroxymethyl‐functionalized 3,4‐ethylenedioxythiophene polymer (PEDOT‐OH) enables construction of diverse nanotopography size and morphologies and is therefore exploited to engineer organic artificial microenvironments bearing nanodots from 300 to 1000 nm in diameter to understand spatiotemporal EMT regulation by biophysical components of the TME. MCF‐7 breast cancer cells are cultured on these artificial microenvironments, and temporal regulation of cellular morphology and EMT markers is investigated. The results show that upon physical stimulation, cells on 300 nm artificial microenvironments advance to EMT and display a decreased extracellular matrix (ECM) protein secretion. In contrast, cells on 500 nm artificial microenvironments are trapped in EMT‐imbalance. Interestingly, cells on 1000 nm artificial microenvironments resemble those on control surfaces. Upon further investigation, it is found that EMT induction is triggered via transforming growth factor β (TGF‐β) and ECM cleaving protein, matrix metalloproteinease‐9. Immunostaining EMT proteins highlighted that EMT induction is achieved through attenuation of cell–cell and cell–microenvironment adhesions. The physical stimulation‐induced TGF‐β perturbation can have a profound impact on the understanding of tumor‐promoting signaling cascades originated by cellular microenvironment.
