Advanced Biosystems

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

Jina Ko

Yongcheng Wang

Jonathan C. T. Carlson

Angela Marquard

Jeremy Gungabeesoon

Alain Charest

David Weitz

Mikael J. Pittet

Ralph Weissleder

Single Extracellular Vesicle Protein Analysis Using Immuno‐Droplet Digital Polymerase Chain Reaction Amplification

使用免疫液滴数字聚合酶链反应扩增的单个细胞外囊泡蛋白质分析

Abstract Schistosomiasis is an acute and chronic disease caused by tropical parasitic worms of the genus Schistosoma , which parasitizes annually over 200 million people worldwide. Screening of antischistosomal compounds is hampered by the low throughput and potential subjectivity of the visual evaluation of the parasite phenotypes, which affects the current drug assays. Here, an impedance‐based platform, capable of assessing the viability of Schistosoma mansoni schistosomula exposed to drugs, is presented. This automated and parallelized platform enables unbiased and continuous measurements of dose–response relationships for more than 48 h. The platform performance is established by exposure of schistosomula to three test compounds, praziquantel, oxethazaine, and mefloquine, which are known to affect the larvae phenotypes. The system is thereafter used to investigate the response of schistosomula to methiothepine, an antipsychotic compound, which causes complex drug‐induced effects. Continuous monitoring of the parasites reveals transient behavioral phenotypes and allows for extracting temporal characteristics of dose–response curves, which are essential for selecting drugs that feature high activity and fast kinetics of action. These measurements demonstrate that impedance‐based detection provides a wealth of information for the in vitro characterization of candidate antischistosomals and, represents a promising tool for the identification of new lead compounds.

Paolo S. Ravaynia

Flavio C. Lombardo

Stefan Biendl

Matthias A. Dupuch

Jennifer Keiser

Andreas Hierlemann

Mario M. Modena

Parallelized Impedance‐Based Platform for Continuous Dose‐Response Characterization of Antischistosomal Drugs

基于阻抗的并行化平台用于抗血吸虫药物连续剂量-反应特性研究

Adipose-derived mesenchymal stromal cells (AdMSC) release numerous soluble factors capable of stimulating angiogenesis. Improved methods for delivering these cells to maximize their potency are now sought that ideally they retain viable cells in the target tissue while promoting the secretion of angiogenic factors. Substrate surface topography is a parameter that can be used to manipulate the behavior of AdMSC but challenges exist with translating this parameter into materials compatible with minimally invasive delivery into tissues for in situ delivery of the angiogenic secretome. The current study investigates three compositions of hierarchically structured, porous biodegradable microcarriers for the culture of AdMSC and the influence of their surface topographies on the angiogenic secretome. All three compositions perform well as cell microcarriers in xeno-free conditions. The attached AdMSC retain their capacity for subsequent trilineage differentiation. The secretome of AdMSC attached to the microcarriers consists of multiple proangiogenic factors, including significantly elevated levels of vascular endothelial growth factor, which stimulates angiogenesis in vitro. The unique properties of hierarchically structured, porous biodegradable microcarriers investigated in this study offer a radically transformative approach for achieving targeted in vivo delivery of AdMSC and enhancing the potency of their proangiogenic activity to induce neovascularization in ischemic tissue.

Chara Simitzi

Eseelle Hendow

Zhuangnan Li

Richard M. Day

Promotion of Proangiogenic Secretome from Mesenchymal Stromal Cells via Hierarchically Structured Biodegradable Microcarriers

通过层级结构可降解微载体促进间充质基质细胞的促血管生成分泌组

Abstract Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi‐layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD.

Anna R. O’Callaghan

Marc A. Dziasko

Radhika Sheth‐Shah

Mark P. Lewis

Julie T. Daniels

Oral Mucosa Tissue Equivalents for the Treatment of Limbal Stem Cell Deficiency

用于治疗角膜缘干细胞缺陷的口腔黏膜组织等效物

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor and despite optimal treatment, long‐term survival remains uncommon. GBM can be roughly divided into three different molecular subtypes, each varying in aggressiveness and treatment resistance. Recent evidence shows plasticity between these subtypes in which the proneural (PN) glioma stem‐like cells (GSCs) undergo transition into the more aggressive mesenchymal (MES) subtype, leading to therapeutic resistance. Extracellular vesicles (EVs) are membranous structures secreted by nearly every cell and are shown to play a key role in GBM progression by acting as multifunctional signaling complexes. Here, it is shown that EVs derived from MES cells educate PN cells to increase stemness, invasiveness, cell proliferation, migration potential, aggressiveness, and therapeutic resistance by inducing mesenchymal transition through nuclear factor‐κB/signal transducer and activator of transcription 3 signaling. The findings could potentially help explore new treatment strategies for GBM and indicate that EVs may also play a role in mesenchymal transition of different tumor types.

Markus W. Schweiger

Mao Li

Alberta Giovanazzi

Renata L. Fleming

Elie I. Tabet

Ichiro Nakano

Thomas Würdinger

Ennio Antonio Chiocca

Tian Tian

Bakhos A. Tannous

Extracellular Vesicles Induce Mesenchymal Transition and Therapeutic Resistance in Glioblastomas through NF‐κB/STAT3 Signaling

胞外囊泡通过NF‐κB/STAT3信号通路诱导胶质母细胞瘤间质转化及治疗抵抗

Cells use vesicles to efficiently compartmentalize bimolecular processes and without them, higher eukaryotic life would be unthinkable. Over 20 years ago, researchers realized that many cells also shed vesicles and that a fraction of these find their way into circulation. Initially believed to be “detritus” and “purification artifacts”, it became clear that these extracellular vesicles (EV) had important biological functions and consequences. Next, different types of vesicles were discovered, and the race to fundamentally understand their biogenesis, composition and biological functions was on. Today EV are used for therapeutic and diagnostic (liquid biopsy) purposes. Their role in diverse immunologic, proliferative and repair, processes continues to be elucidated. Extracellular vesicles are abundant in plasma and other biofluids, stable in circulation, and produced by all cell types. In the normal human organism, the majority of EV are produced by endothelial cells and red blood cells. Tumor cells can produce large quantities of EV (∼4,000x more than normal host cells). These tumor cell derived EV (tEV) partly resemble parental tumor cells in their molecular make-up, reason why they are so attractive for diagnostics. The main challenges with EV diagnostics have largely centered around the following questions: which type of circulating vesicle (exosome, microvesicles, others) is diagnostically most relevant; which molecular biomarker (or combination) is best suited to detect tEV; how does one best separate tumor and host cell derived EV; given the heterogeneity of EV, will future analyses have to rely on single EV diagnostic tools; and what is the best technology to analyze human plasma samples at high throughput and reasonable cost so that patients can benefit from new discoveries? Needless to say, these questions are also relevant for therapeutic applications of EV. This issue of Advanced Biosystems focuses on diagnostic aspects of EV analysis. The compilation of 12 articles have been prepared by current or past members of a large exosome consortium in Boston dedicated to basic and translational biology. Article number 1900305 deals with the pharmacokinetics and biological distribution of EV. Using mathematical modeling, this manuscript lays the groundwork to estimate detectable tumor burden by EV analysis. One take-home message is that tumors as small as 1 mm3 are theoretically detectable in patients when single EV analytical techniques are used for diagnosis. A second take-home message is that single EV analysis will ultimately enable cancer detection multiple years before mass lesions become detectable by imaging. Article 2000069 reviews the different proteomic analysis of EV. One of the current challenges in EV research is the selective isolation of EV subtypes at quantities sufficient for efficient downstream analyses. The review focuses on the diverse techniques and protocols used to isolate and purify EVs with a special emphasis on their adequacy for proteomics applications. Articles 1900310, 2000003, 2000007, and 2000203 describe new EV analytical techniques. Article 1900307 showcases a new way to study EV in vivo by genetically engineering them to display a membrane-bound form of Sortase A, a bacterial transpeptidase that can catalyze the transfer of reporter molecules on the much bigger surface of EV-binding cells. One of the most anticipated clinical role of EV diagnostics is for CNS diseases and glioblastoma where biopsies are not always feasible, carry considerable morbidity and can not be performed serially. Articles 2000035, 2000029, and 1900312, focus on various aspects of EV biology in glioblastomas. Article 2000017 focuses on the recently discovered 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 tumor cells from T cell killing. Article 1900309 is dedicated to neuroinflammation and degenerative CNS diseases. We hope the articles in this issue of Advanced Biosystems will inspire readers to develop new methods, applications, therapeutic uses or simply understand human EV biology using the newly developed tools. Ralph Weissleder is the Thrall Professor of Radiology at Harvard Medical School, Director of the Center for Systems Biology at Massachusetts General Hospital (MGH), and Attending Clinician (Interventional Radiology) at MGH. Dr. Weissleder is also Professor of Systems Biology and a faculty a member of the Department of Systems Biology at HMS and the Dana Farber Harvard Cancer Center. The focus of his research lab is to obtain a deeper understanding of human biology in health and disease, to translate new biological understanding into clinically useful diagnostics, and to identify new therapeutic approaches and drug targets. His research has been translational and several of his developments have been licensed to companies and led to advanced clinical trials.

The Magical World of Circulating Vesicles

循环囊泡的神奇世界

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.

Mesenchymal Transition: Extracellular Vesicles Induce Mesenchymal Transition and Therapeutic Resistance in Glioblastomas through NF‐κB/STAT3 Signaling (Adv. Biosys. 12/2020)

间质转化：外泌体通过NF‐κB/STAT3信号通路诱导胶质母细胞瘤间质转化及治疗抵抗（Adv. Biosys. 12/2020）

In article number 2000003, Hyungsoon Im and co-workers label extracellular vesicles, which are nanoscale phospholipid vesicles secreted by cells, with fluorescently conjugated antibodies. The fluorescence signals are amplified by periodic gold nanohole structures based on surface plasmon resonance. Multi-fluorescence imaging enables multiplexed analyses of proteins with improved sensitivities.

Jouha Min

Taehwang Son

Jae‐Sang Hong

Pike See Cheah

Andreas Wegemann

Koushik Murlidharan

Hakho Lee

Hyungsoon Im

Plasmon‐Enhanced Biosensing: Plasmon‐Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles (Adv. Biosys. 12/2020)

等离子体增强生物传感：用于细胞外囊泡多重分析的等离子体增强生物传感（Adv. Biosys. 12/2020）

In article number 2000127, Clara R. Correia, João F. Mano, and co-workers describe the high-throughput production of alginate-based microgels with defined geometries using an in situ crosslinking procedure. This encapsulation system is surrounded by a permselective polymeric membrane that wraps cells and microparticles in a liquefied environment. The proposed system can potentially be explored as building blocks for bottom-up strategies within the concept of modular tissue engineering.

Sara Nadine

Sónia G. Patrício

Cristina C. Barrias

Insung S. Choi

Michiya Matsusaki

Clara R. Correia

João F. Mano

Liquefied Capsules: Geometrically Controlled Liquefied Capsules for Modular Tissue Engineering Strategies (Adv. Biosys. 11/2020)

液化胶囊：几何控制液化胶囊用于模块化组织工程策略（Adv. Biosys. 11/2020）

Abstract The extent to which biologic payloads can be effectively delivered to cells is a limiting factor in the development of new therapies. Limitations arise from the lack of pharmacokinetic stability of biologics in vivo. Encapsulating biologics in a protective delivery vector has the potential to improve delivery profile and enhance performance. Coacervate microdroplets are developed as cell‐mimetic materials with established potential for the stabilization of biological molecules, such as proteins and nucleic acids. Here, the development of biodegradable coacervate microvectors (comprising synthetically modified amylose polymers) is presented, for the delivery of biologic payloads to cells. Amylose‐based coacervate microdroplets are stable under physiological conditions (e.g., temperature and ionic strength), are noncytotoxic owing to their biopolymeric structure, spontaneously interacted with the cell membrane, and are able to deliver and release proteinaceous payloads beyond the plasma membrane. In particular, myoglobin, an oxygen storage and antioxidant protein, is successfully delivered into human mesenchymal stem cells (hMSCs) within 24 h. Furthermore, coacervate microvectors are implemented for the delivery of human bone morphogenetic protein 2 growth factor, inducing differentiation of hMSCs into osteoprogenitor cells. This study demonstrates the potential of coacervate microdroplets as delivery microvectors for biomedical research and the development of new therapies.

Wenjin Xiao

Monika D. Jakimowicz

Ioannis Zampetakis

Sarah Neely

Fabrizio Scarpa

Sean A. Davis

David S. Williams

Adam W. Perriman

Biopolymeric Coacervate Microvectors for the Delivery of Functional Proteins to Cells

生物高分子凝聚微载体用于向细胞递送功能蛋白

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Journal Impact	4.29
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u9zg3I	生物化学遗传学生物化学医学内科学细胞生物学物理基因心理学材料科学工程类病理计算机科学神经科学免疫学纳米技术内分泌学有机化学
+5d/bs	e+/GRg: Wiley-VCH Verlag，uzYam8: ，dxwaWB: journal，8x2mMQ: TY4X7V
ak9yM6	A7IHrH: 2017，sQJ/D8: 0.00%，sE9og+:0.00%， wzlKC/: 33.18%

Advanced Biosystems

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Single Extracellular Vesicle Protein Analysis Using Immuno‐Droplet Digital Polymerase Chain Reaction Amplification

Parallelized Impedance‐Based Platform for Continuous Dose‐Response Characterization of Antischistosomal Drugs

Promotion of Proangiogenic Secretome from Mesenchymal Stromal Cells via Hierarchically Structured Biodegradable Microcarriers

Oral Mucosa Tissue Equivalents for the Treatment of Limbal Stem Cell Deficiency

Extracellular Vesicles Induce Mesenchymal Transition and Therapeutic Resistance in Glioblastomas through NF‐κB/STAT3 Signaling

The Magical World of Circulating Vesicles

Mesenchymal Transition: Extracellular Vesicles Induce Mesenchymal Transition and Therapeutic Resistance in Glioblastomas through NF‐κB/STAT3 Signaling (Adv. Biosys. 12/2020)

Plasmon‐Enhanced Biosensing: Plasmon‐Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles (Adv. Biosys. 12/2020)

Liquefied Capsules: Geometrically Controlled Liquefied Capsules for Modular Tissue Engineering Strategies (Adv. Biosys. 11/2020)

Biopolymeric Coacervate Microvectors for the Delivery of Functional Proteins to Cells