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The conceptualization of body-on-a-chip in 2004 resulted in a new approach for studying human physiology in three-dimensional culture. Despite pioneering works and the progress made in replicating human physiology on-a-chip, the stability, reliability, and preservation of cell-culture-treated microfluidic chips remain a challenge. The development of a reliable surface treatment technique to more efficiently and reproducibly modify microfluidic channels would significantly simplify the process of creating and implementing organ-on-a-chip (OOC) systems. In this work, a new flow-based coating technique using bioinspired polymers was implemented to create reliable, reproducible, ready-to-use microfluidic cell culture chips for OOC studies. Single-channel polydimethylsiloxane microfluidic chips were coated with the bioinspired catecholamine polymers, polydopamine (PDA) and polynorepinephrine (PNE), using a flow-based coating technique. The functionality of the resulting microfluidic chips was evaluated by extensive surface characterizations, at 130 °C, in the presence of various cleaning and culture media in static and flow conditions regularly used in OOCs and tested for shelf life by storing the coated microfluidic chips for 4 months at room temperature. Microfluidic chips coated with polycatecholamine were then seeded with the mouse cancer cell line Cath.a.differentiated (CAD) and with the normal human cerebral microvascular endothelial cell line human cerebral microvascular endothelial cells (hCMEC)/D3. Cell viability, cell phenotype, and cell functionality were assessed to evaluate the performance of both the coatings and the surface treatment technique. Both PDA- and PNE-coated microfluidic chips maintained high viability, phenotype, and functionality of CAD cells and hCMEC/D3 cells. In addition, CAD cells retained high viability when they were cultured in both the polymer-coated chips, which were stored at room temperature for up to 120 days. These results suggest that flow-based techniques to coat surfaces with polycatecholamines can be used to generate ready-to-use microfluidic OOC chips that offer long-term stability and reliability for the culture of cell types with application in pathophysiological studies and drug screening.

High concentrations of cryoprotective agents (CPAs) are required to achieve successful vitrification of articular cartilage; however, CPA cytotoxicity causes chondrocyte death. To reduce CPA toxicity, supplementation with research-grade additives, in particular chondroitin sulfate (CS) and ascorbic acid (AA), have previously been shown to improve chondrocyte recovery and metabolic function after exposure to CPAs at hypothermic conditions. However, it is necessary to evaluate the pharmaceutical equivalent clinical grade of these additives to facilitate the supplementation of additives into future vitrification protocols, which will be designed for vitrifying human articular cartilage in tissue banks. We sought to investigate the effectiveness of clinical-grade CS, AA, and N-acetylcysteine (NAC) in mitigating toxicity to chondrocytes during CPA exposure and removal, and determine whether a combination of two additives would further improve chondrocyte viability. We hypothesized that clinical-grade additives would exert chondroprotective effects comparable to those of research-grade additives, and that this protective effect would be enhanced if two additives were combined when compared with a single additive. The results indicated that both clinical-grade and research-grade additives significantly improved cell viability (p 0.10). We demonstrated that supplementation with clinical-grade CS or AA significantly improved chondrocyte viability in porcine cartilage subjected to high CPA concentrations, whereas supplementation with clinical-grade NAC did not benefit chondrocyte viability. Supplementation with clinical-grade additives in CPA solutions can mitigate CPA toxicity, which will be important in translating previously developed effective protocols for the vitrification of articular cartilage to human tissue banks.

Physiologically relevant intracranial aneurysm (IA) models are crucially required to facilitate testing treatment options for IA. Herein, we report the development of a new in vitro tissue-engineered platform, which recapitulates the microenvironment, structure, and cellular complexity of native human IA. A new modified liquid-assisted injection molding technique was developed to fabricate a three-dimensional hollow IA model with clinically relevant IA dimensions within a mechanically tuned Gelatin Methacryloyl (GelMA) hydrogel. An endothelium lining was created inside the IA model by culturing human umbilical vein endothelial cells over pre-cultured human brain vascular smooth muscle cells. These cellularized IA models were subjected to medium perfusion at flow rates between 6.3 and 15.75 mL/min for inducing biomimetic vessel wall shear stress (10–25 dyn/cm2) to the cells for ten days. Both cell types maintained their secretome profiles and showed more than 96% viability, demonstrating the biocompatibility of the hydrogel during perfusion cell culture at such flow rates. Based on the characterized viscoelastic properties of the GelMA hydrogel and with the aid of a fluid-structure interaction model, the capability of the IA model in predicting the response of the IA to different fluid flow profiles was mathematically shown. With physiologically relevant behavior, our developed in vitro human IA model could allow researchers to better understand the pathophysiology and treatment of IA. Statement of significance A three-dimensional intracranial aneurysm (IA) tissue model recapitulating the microenvironment, structure, and cellular complexity of native human IA was developed. • An endothelium lining was created inside the IA model over pre-cultured human brain vascular smooth muscle cells over at least 10-day successful culture. • The cells maintained their secretome profiles, demonstrating the biocompatibility of hydrogel during a long-term perfusion cell culture. • The IA model showed its capability in predicting the response of IA to different fluid flow profiles. • The cells in the vessel region behaved differently from cells in the aneurysm region due to alteration in hemodynamic shear stress. • The IA model could allow researchers to better understand the pathophysiology and treatment options of IA.

Vitrification of mammalian tissues is important in the areas of human assisted reproduction, animal reproduction, and regenerative medicine. Non-permeating cryoprotectants (CPAs), particularly sucrose, are increasingly used in conjunction with permeating CPAs for vitrification of mammalian tissues. Combining non-permeating and permeating CPAs was found to further improve post-thaw viability and functionalities of vitrified mammalian tissues, showing the potential applications of such tissues in various clinical and veterinary settings. With the rising demand for the use of non-permeating CPAs in vitrification of mammalian tissues, there is a strong need for a timely and comprehensive review on the supplemental effects of non-permeating CPAs toward vitrification outcomes of mammalian tissues. In this review, we first discuss the roles of non-permeating CPAs including sugars and high molecular weight polymers in vitrification. We then summarize the supplemental effects of non-permeating CPAs on viability and functionalities of mammalian embryos, and ovarian, testicular, articular cartilage, tracheal, and kidney tissues following vitrification. Lastly, challenges associated with the use of non-permeating CPAs in vitrification of mammalian tissues are briefly discussed.

Large bone defects are a major health concern worldwide. The conventional bone repair techniques (e.g., bone-grafting and Masquelet techniques) have numerous drawbacks, which negatively impact their therapeutic outcomes. Therefore, there is a demand to develop an alternative bone repair approach that can address the existing drawbacks. Bone tissue engineering involving the utilization of human mesenchymal stem cells (hMSCs) has recently emerged as a key strategy for the regeneration of damaged bone tissues. However, the use of tissue-engineered bone graft for the clinical treatment of bone defects remains challenging. While the role of mechanical loading in creating a bone graft has been well explored, the effects of mechanical loading factors (e.g., loading types and regime) on clinical outcomes are poorly understood. This review summarizes the effects of mechanical loading on hMSCs for bone tissue engineering applications. First, we discuss the key assays for assessing the quality of tissue-engineered bone grafts, including specific staining, as well as gene and protein expression of osteogenic markers. Recent studies of the impact of mechanical loading on hMSCs, including compression, perfusion, vibration and stretching, along with the potential mechanotransduction signalling pathways, are subsequently reviewed. Lastly, we discuss the challenges and prospects of bone tissue engineering applications.

Heterogeneity in cell populations poses a significant challenge for understanding complex cell biological processes. The analysis of cells at the single-cell level, especially single-cell RNA sequencing (scRNA-seq), has made it possible to comprehensively dissect cellular heterogeneity and access unobtainable biological information from bulk analysis. Recent efforts have combined scRNA-seq profiles with genomic or proteomic data, and show added value in describing complex cellular heterogeneity than transcriptome measurements alone. With the rising demand for scRNA-seq for biomedical and clinical applications, there is a strong need for a timely and comprehensive review on the scRNA-seq technologies and their potential biomedical applications. In this review, we first discuss the latest state of development by detailing each scRNA-seq technology, including both conventional and microfluidic technologies. We then summarize their advantages and limitations along with their biomedical applications. The efforts of integrating the transcriptome profile with highly multiplexed proteomic and genomic data are thoroughly reviewed with results showing the integrated data being more informative than transcriptome data alone. Lastly, the latest progress toward commercialization, the remaining challenges, and future perspectives on the development of scRNA-seq technologies are briefly discussed.

An outstanding amount of resources has been used in research on manipulation of human stem cells, especially mesenchymal stem cells (MSCs), for various clinical applications. However, human MSCs have not been fully utilized in clinical applications due to restrictions with regard to their certain biosafety and bioefficacy concerns, for example, genetic abnormality, tumor formation, induction of host immune response and failure of homing and engraftment. This review summarizes the biosafety and bioefficacy assessment of human MSCs in terms of genetic stability, tumorigenicity, immunogenicity, homing and engraftment. The strategies used to reduce the biosafety concerns and improve the bioefficacy of human MSCs are highlighted. In addition, the approaches that can be implemented to improve their biosafety and bioefficacy assessment are briefly discussed.

Cryopreservation is the only existing method of storage of human adipose-derived stem cells (ASCs) for clinical use. However, cryopreservation has been shown to be detrimental to ASCs, particularly in term of cell viability. To restore the viability of cryopreserved ASCs, it is proposed to culture the cells in a hypoxic condition. To this end, we aim to investigate the effect of hypoxia on the cryopreserved human ASCs in terms of not only cell viability, but also their growth and stemness properties, which have not been explored yet. In this study, human ASCs were cultured under four different conditions: fresh (non-cryopreserved) cells cultured in 1) normoxia (21% O2) and 2) hypoxia (2% O2) and cryopreserved cells cultured in 3) normoxia and 4) hypoxia. ASCs at passage 3 were subjected to assessment of viability, proliferation, differentiation, and expression of stemness markers and hypoxia-inducible factor-1 alpha (HIF-1α). We found that hypoxia enhances the viability and the proliferation rate of cryopreserved ASCs. Further, hypoxia upregulates HIF-1α in cryopreserved ASCs, which in turn activates chondrogenic genes to promote chondrogenic differentiation. In conclusion, hypoxic-preconditioned cryopreserved ASCs could be an ideal cell source for cartilage repair and regeneration.

Human adipose-derived mesenchymal stem cells (hASCs) are an ideal cell source for regenerative medicine due to their capabilities of multipotency and the readily accessibility of adipose tissue. They have been found residing in a relatively low oxygen tension microenvironment in the body, but the physiological condition has been overlooked in most studies. In light of the escalating need for culturing hASCs under their physiological condition, this review summarizes the most recent advances in the hypoxia effect on hASCs. We first highlight the advantages of using hASCs in regenerative medicine and discuss the influence of hypoxia on the phenotype and functionality of hASCs in terms of viability, stemness, proliferation, differentiation, soluble factor secretion, and biosafety. We provide a glimpse of the possible cellular mechanism that involved under hypoxia and discuss the potential clinical applications. We then highlight the existing challenges and discuss the future perspective on the use of hypoxic-treated hASCs.

Background: Molecularly Imprinted Polymers (MIPs), a type of biomimetic materials have attracted considerable interest owing to their cost-effectiveness, good physiochemical stability, favorable specificity and selectivity for target analytes, and long shelf life. These materials are able to mimic natural recognition entities, including biological receptors and antibodies, providing a versatile platform to achieve the desirable functionality for various biomedical applications. Objective: In this review article, we introduce the most recent development of MIPs to date. We first highlight the advantages of using MIPs for a broad range of biomedical applications. We then review their various methods of synthesis along with their latest progress in biomedical applications, including biosensing, drug delivery, cell imaging and drug discovery. Lastly, the existing challenges and future perspectives of MIPs for biomedical applications are briefly discussed. Conclusion: We envision that MIPs may be used as potential materials for diverse biomedical applications in the near future.

Black phosphorus (BP), also known as phosphorene, has attracted recent scientific attention since its first successful exfoliation in 2014 owing to its unique structure and properties. In particular, its exceptional attributes, such as the excellent optical and mechanical properties, electrical conductivity and electron-transfer capacity, contribute to its increasing demand as an alternative to graphene-based materials in biomedical applications. Although the outlook of this material seems promising, its practical applications are still highly challenging. In this review article, we discuss the unique properties of BP, which make it a potential platform for biomedical applications compared to other 2D materials, including graphene, molybdenum disulphide (MoS2),tungsten diselenide (WSe2) and hexagonal boron nitride (h-BN). We then introduce various synthesis methods of BP and review its latest progress in biomedical applications, such as biosensing, drug delivery, photo acoustic imaging and cancer therapies (i.e., photothermal and photodynamic therapies). Lastly, the existing challenges and future perspective of BP in biomedical applications are briefly discussed. usc Refereed/Peer-reviewed

Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture, and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair.

Food safety issues have recently attracted public concern. The deleterious effects of compromised food safety on health have rendered food safety analysis an approach of paramount importance. While conventional techniques such as high-performance liquid chromatography and mass spectrometry have traditionally been utilized for the detection of food contaminants, they are relatively expensive, time-consuming and labor intensive, impeding their use for point-of-care (POC) applications. In addition, accessibility of these tests is limited in developing countries where food-related illnesses are prevalent. There is, therefore, an urgent need to develop simple and robust diagnostic POC devices. POC devices, including paper- and chip-based devices, are typically rapid, cost-effective and user-friendly, offering a tremendous potential for rapid food safety analysis at POC settings. Herein, we discuss the most recent advances in the development of emerging POC devices for food safety analysis. We first provide an overview of common food safety issues and the existing techniques for detecting food contaminants such as foodborne pathogens, chemicals, allergens, and toxins. The importance of rapid food safety analysis along with the beneficial use of miniaturized POC devices are subsequently reviewed. Finally, the existing challenges and future perspectives of developing the miniaturized POC devices for food safety monitoring are briefly discussed.

Human mesenchymal stem cells (hMSCs), a type of adult stem cells that hold great potential in clinical applications (e.g., regenerative medicine and cell-based therapy) due to their ability to differentiate into multiple types of specialized cells and secrete soluble factors which can initiate tissue repair and regulate immune response. hMSCs need to be expanded in vitro or cryopreserved to obtain sufficient cell numbers required for clinical applications. However, long-term in vitro culture-expanded hMSCs may raise some biosafety concerns (e.g., chromosomal abnormality and malignant transformation) and compromised functional properties, limiting their use in clinical applications. To avoid those adverse effects, it is essential to cryopreserve hMSCs at early passage and pool them for off-the-shelf use in clinical applications. However, the existing cryopreservation methods for hMSCs have some notable limitations. To address these limitations, several approaches have to be taken in order to produce healthy and efficacious cryopreserved hMSCs for clinical trials, which remains challenging to date. Therefore, a noteworthy amount of resources has been utilized in research in optimization of the cryopreservation methods, development of freezing devices, and formulation of cryopreservation media to ensure that hMSCs maintain their therapeutic characteristics without raising biosafety concerns following cryopreservation. Biobanking of hMSCs would be a crucial strategy to facilitate clinical applications in the future.

Human mesenchymal stem cells (hMSCs) hold great promise in cardiac fibrosis therapy, due to their potential ability of inhibiting cardiac myofibroblast differentiation (a hallmark of cardiac fibrosis). However, the mechanism involved in their effects remains elusive. To explore this, it is necessary to develop an in vitro cardiac fibrosis model that incorporates pore size and native tissue-mimicking matrix stiffness, which may regulate cardiac myofibroblast differentiation. In the present study, collagen coated polyacrylamide hydrogel substrates were fabricated, in which the pore size was adjusted without altering the matrix stiffness. Stiffness is shown to regulate cardiac myofibroblast differentiation independently of pore size. Substrate at a stiffness of 30 kPa, which mimics the stiffness of native fibrotic cardiac tissue, was found to induce cardiac myofibroblast differentiation to create in vitro cardiac fibrosis model. Conditioned medium of hMSCs was applied to the model to determine its role and inhibitory mechanism on cardiac myofibroblast differentiation. It was found that hMSCs secrete hepatocyte growth factor (HGF) to inhibit cardiac myofibroblast differentiation via downregulation of angiotensin II type 1 receptor (AT1R) and upregulation of Smad7. These findings would aid in establishment of the therapeutic use of hMSCs in cardiac fibrosis therapy in future.

Cryopreservation is the most promising way for long-term storage of biological samples e.g., single cells and cellular structures. Among various cryopreservation methods, vitrification is advantageous by employing high cooling rate to avoid the formation of harmful ice crystals in cells. Most existing vitrification methods adopt direct contact of cells with liquid nitrogen to obtain high cooling rates, which however causes the potential contamination and difficult cell collection. To address these limitations, we developed a non-contact vitrification device based on an ultra-thin freezing film to achieve high cooling/warming rate and avoid direct contact between cells and liquid nitrogen. A high-throughput cell printer was employed to rapidly generate uniform cell-laden microdroplets into the device, where the microdroplets were hung on one side of the film and then vitrified by pouring the liquid nitrogen onto the other side via boiling heat transfer. Through theoretical and experimental studies on vitrification processes, we demonstrated that our device offers a high cooling/warming rate for vitrification of the NIH 3T3 cells and human adipose-derived stem cells (hASCs) with maintained cell viability and differentiation potential. This non-contact vitrification device provides a novel and effective way to cryopreserve cells at high throughput and avoid the contamination and collection problems.

Cryopreservation represents an efficient way to preserve human mesenchymal stem cells (hMSCs) at early culture/passage, and allows pooling of cells to achieve sufficient cells required for off-the-shelf use in clinical applications, e.g. cell-based therapies and regenerative medicine. To fully apply cryopreserved hMSCs in a clinical setting, it is necessary to evaluate their biosafety, e.g. chromosomal abnormality and tumourigenic potential. To date, many studies have demonstrated that cryopreserved hMSCs display no chromosomal abnormalities. However, the tumourigenic potential of cryopreserved hMSCs has not yet been evaluated. In the present study, we cryopreserved human adipose-derived mesenchymal stem cells (hASCs) for 3 months, using a slow freezing method with various cryoprotective agents (CPAs), followed by assessment of the tumourigenic potential of the cryopreserved hASCs after thawing and subculture. We found that long-term cryopreserved hASCs maintained normal levels of the tumour suppressor markers p53, p21, p16 and pRb, hTERT, telomerase activity and telomere length. Further, we did not observe significant DNA damage or signs of p53 mutation in cryopreserved hASCs. Our findings suggest that long-term cryopreserved hASCs are at low risk of tumourigenesis. These findings aid in establishing the biosafety profile of cryopreserved hASCs, and thus establishing low hazardous risk perception with the use of long-term cryopreserved hASCs for future clinical applications. Copyright © 2016 John WileySons, Ltd.

Cardiac myofibroblast differentiation, as one of the most important cellular responses to heart injury, plays a critical role in cardiac remodeling and failure. While biochemical cues for this have been extensively investigated, the role of mechanical cues, e.g., extracellular matrix stiffness and mechanical strain, has also been found to mediate cardiac myofibroblast differentiation. Cardiac fibroblasts in vivo are typically subjected to a specific spatiotemporally changed mechanical microenvironment. When exposed to abnormal mechanical conditions (e.g., increased extracellular matrix stiffness or strain), cardiac fibroblasts can undergo myofibroblast differentiation. To date, the impact of mechanical cues on cardiac myofibroblast differentiation has been studied both in vitro and in vivo. Most of the related in vitro research into this has been mainly undertaken in two-dimensional cell culture systems, although a few three-dimensional studies that exist revealed an important role of dimensionality. However, despite remarkable advances, the comprehensive mechanisms for mechanoregulation of cardiac myofibroblast differentiation remain elusive. In this review, we introduce important parameters for evaluating cardiac myofibroblast differentiation and then discuss the development of both in vitro (two and three dimensional) and in vivo studies on mechanoregulation of cardiac myofibroblast differentiation. An understanding of the development of cardiac myofibroblast differentiation in response to changing mechanical microenvironment will underlie potential targets for future therapy of cardiac fibrosis and failure.

Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.

Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2) or in situ normoxia (2% O2). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.