Recent Advances in 3D Cultures.

Methods and protocols for creating complex 3D cell culture systems have been rapidly advancing in the past decade from the perspective of biomaterials [...].

Early Impairment of Paracrine and Phenotypic Features in Resident Cardiac Mesenchymal Stromal Cells after Thoracic Radiotherapy.

Radiotherapy-induced cardiac toxicity and consequent diseases still represent potential severe late complications for many cancer survivors who undergo therapeutic thoracic irradiation. We aimed to assess the phenotypic and paracrine features of resident cardiac mesenchymal stromal cells (CMSCs) at early follow-up after the end of thoracic irradiation of the heart as an early sign and/or mechanism of cardiac toxicity anticipating late organ dysfunction. Resident CMSCs were isolated from a rat model of fractionated thoracic irradiation with accurate and clinically relevant heart dosimetry that developed delayed dose-dependent cardiac dysfunction after 1 year. Cells were isolated 6 and 12 weeks after the end of radiotherapy and fully characterized at the transcriptional, paracrine, and functional levels. CMSCs displayed several altered features in a dose- and time-dependent trend, with the most impaired characteristics observed in those exposed in situ to the highest radiation dose with time. In particular, altered features included impaired cell migration and 3D growth and a and significant association of transcriptomic data with GO terms related to altered cytokine and growth factor signaling. Indeed, the altered paracrine profile of CMSCs derived from the group at the highest dose at the 12-week follow-up gave significantly reduced angiogenic support to endothelial cells and polarized macrophages toward a pro-inflammatory profile. Data collected in a clinically relevant rat model of heart irradiation simulating thoracic radiotherapy suggest that early paracrine and transcriptional alterations of the cardiac stroma may represent a dose- and time-dependent biological substrate for the delayed cardiac dysfunction phenotype observed in vivo.

3D Cultures for Modelling the Microenvironment: Current Research Trends and Applications.

The importance of 3D culture systems for drug screening or physio-pathological models has exponentially increased in recent years [...].

Multicellular 3D Models for the Study of Cardiac Fibrosis.

Ex vivo modelling systems for cardiovascular research are becoming increasingly important in reducing lab animal use and boosting personalized medicine approaches. Integrating multiple cell types in complex setups adds a higher level of significance to the models, simulating the intricate intercellular communication of the microenvironment in vivo. Cardiac fibrosis represents a key pathogenetic step in multiple cardiovascular diseases, such as ischemic and diabetic cardiomyopathies. Indeed, allowing inter-cellular interactions between cardiac stromal cells, endothelial cells, cardiomyocytes, and/or immune cells in dedicated systems could make ex vivo models of cardiac fibrosis even more relevant. Moreover, culture systems with 3D architectures further enrich the physiological significance of such in vitro models. In this review, we provide a summary of the multicellular 3D models for the study of cardiac fibrosis described in the literature, such as spontaneous microtissues, bioprinted constructs, engineered tissues, and organs-on-chip, discussing their advantages and limitations. Important discoveries on the physiopathology of cardiac fibrosis, as well as the screening of novel potential therapeutic molecules, have been reported thanks to these systems. Future developments will certainly increase their translational impact for understanding and modulating mechanisms of cardiac fibrosis even further.

Serum and supplement optimization for EU GMP-compliance in cardiospheres cell culture.

Cardiac progenitor cells (CPCs) isolated as cardiospheres (CSs) and CS-derived cells (CDCs) are a promising tool for cardiac cell therapy in heart failure patients, having CDCs already been used in a phase I/II clinical trial. Culture standardization according to Good Manufacturing Practices (GMPs) is a mandatory step for clinical translation. One of the main issues raised is the use of xenogenic additives (e.g. FBS, foetal bovine serum) in cell culture media, which carries the risk of contamination with infectious viral/prion agents, and the possible induction of immunizing effects in the final recipient. In this study, B27 supplement and sera requirements to comply with European GMPs were investigated in CSs and CDCs cultures, in terms of process yield/efficiency and final cell product gene expression levels, as well as phenotype. B27- free CS cultures produced a significantly reduced yield and a 10-fold drop in c-kit expression levels versus B27+ media. Moreover, autologous human serum (aHS) and two different commercially available GMP AB HSs were compared with standard research-grade FBS. CPCs from all HSs explants had reduced growth rate, assumed a senescent-like morphology with time in culture, and/or displayed a significant shift towards the endothelial phenotype. Among three different GMP gamma-irradiated FBSs (giFBSs) tested, two provided unsatisfactory cell yields, while one performed optimally, in terms of CPCs yield/phenotype. In conclusion, the use of HSs for the isolation and expansion of CSs/CDCs has to be excluded because of altered proliferation and/or commitment, while media supplemented with B27 and the selected giFBS allows successful EU GMP-complying CPCs culture.

A vascularized 3D model of the human pancreatic islet forex vivostudy of immune cell-islet interaction.

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively byßcells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of isletßcells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlyingßcell damage in diabetes rely onin vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing-key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel,ex vivoplatform for studying human islet biology in both health and disease.

Biocompatibility and Connectivity of Semiconductor Nanostructures for Cardiac Tissue Engineering Applications.

Nano- or microdevices, enabling simultaneous, long-term, multisite, cellular recording and stimulation from many excitable cells, are expected to make a strategic turn in basic and applied cardiology (particularly tissue engineering) and neuroscience. We propose an innovative approach aiming to elicit bioelectrical information from the cell membrane using an integrated circuit (IC) bearing a coating of nanowires on the chip surface. Nanowires grow directly on the backend of the ICs, thus allowing on-site amplification of bioelectric signals with uniform and controlled morphology and growth of the NWs on templates. To implement this technology, we evaluated the biocompatibility of silicon and zinc oxide nanowires (NWs), used as a seeding substrate for cells in culture, on two different primary cell lines. Human cardiac stromal cells were used to evaluate the effects of ZnO NWs of different lengths on cell behavior, morphology and growth, while BV-2 microglial-like cells and GH4-C1 neuroendocrine-like cell lines were used to evaluate cell membrane-NW interaction and contact when cultured on Si NWs. As the optimization of the contact between integrated microelectronics circuits and cellular membranes represents a long-standing issue, our technological approach may lay the basis for a new era of devices exploiting the microelectronics' sensitivity and "smartness" to both improve investigation of biological systems and to develop suitable NW-based systems available for tissue engineering and regenerative medicine.

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart.

Experimental data suggest that cell-based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit(+) CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild-type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate 'cardiospheres', a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit(+) CSCs.

Injectable Myocardial Matrix Hydrogel Mitigates Negative Left Ventricular Remodeling in a Chronic Myocardial Infarction Model.

A first-in-man clinical study on a myocardial-derived decellularized extracellular matrix hydrogel suggested the potential for efficacy in chronic myocardial infarction (MI) patients. However, little is understood about the mechanism of action in chronic MI. In this study, the authors investigated the efficacy and mechanism by which the myocardial matrix hydrogel can mitigate negative left ventricular (LV) remodeling in a rat chronic MI model. Assessment of cardiac function via magnetic resonance imaging demonstrated preservation of LV volumes and apical wall thickening. Differential gene expression analyses showed the matrix is able to prevent further negative LV remodeling in the chronic MI model through modulation of the immune response, down-regulation of pathways involved in heart failure progression and fibrosis, and up-regulation of genes important for cardiac muscle contraction.

Cardiospheres and tissue engineering for myocardial regeneration: potential for clinical application.

Tissue engineering is an increasingly expanding area of research in the cardiovascular field that involves engineering, chemistry, biology and medicine. Cardiac tissue engineering (CTE) aims to regenerate myocardial damage by combining cells, matrix, biological active molecules and physiological stimuli. The rationale behind CTE applications is that in order to regenerate the ventricular wall after a myocardial infarction it is necessary to combine procedures that regenerate both cardiomyocytes and the extracellular matrix. The application of (stem) cells together with a matrix could represent an environment protected from the inflammatory and pro-apoptotic signals, a stemness/survival reservoir slowly releasing cells and factors promoting tissue regeneration and angiogenesis. This review will focus on the applications and advantages that CTE application could offer compared to conventional cell therapy.

Building an Artificial Cardiac Microenvironment: A Focus on the Extracellular Matrix.

The increased knowledge in cell signals and stem cell differentiation, together with the development of new technologies, such as 3D bioprinting, has made the generation of artificial tissues more feasible for in vitro studies and in vivo applications. In the human body, cell fate, function, and survival are determined by the microenvironment, a rich and complex network composed of extracellular matrix (ECM), different cell types, and soluble factors. They all interconnect and communicate, receiving and sending signals, modulating and responding to cues. In the cardiovascular field, the culture of stem cells in vitro and their differentiation into cardiac phenotypes is well established, although differentiated cardiomyocytes often lack the functional maturation and structural organization typical of the adult myocardium. The recreation of an artificial microenvironment as similar as possible to the native tissue, though, has been shown to partly overcome these limitations, and can be obtained through the proper combination of ECM molecules, different cell types, bioavailability of growth factors (GFs), as well as appropriate mechanical and geometrical stimuli. This review will focus on the role of the ECM in the regulation of cardiac differentiation, will provide new insights on the role of supporting cells in the generation of 3D artificial tissues, and will also present a selection of the latest approaches to recreate a cardiac microenvironment in vitro through 3D bioprinting approaches.

When Stiffness Matters: Mechanosensing in Heart Development and Disease.

During embryonic morphogenesis, the heart undergoes a complex series of cellular phenotypic maturations (e.g., transition of myocytes from proliferative to quiescent or maturation of the contractile apparatus), and this involves stiffening of the extracellular matrix (ECM) acting in concert with morphogenetic signals. The maladaptive remodeling of the myocardium, one of the processes involved in determination of heart failure, also involves mechanical cues, with a progressive stiffening of the tissue that produces cellular mechanical damage, inflammation, and ultimately myocardial fibrosis. The assessment of the biomechanical dependence of the molecular machinery (in myocardial and non-myocardial cells) is therefore essential to contextualize the maturation of the cardiac tissue at early stages and understand its pathologic evolution in aging. Because systems to perform multiscale modeling of cellular and tissue mechanics have been developed, it appears particularly novel to design integrated mechano-molecular models of heart development and disease to be tested in ex vivo reconstituted cells/tissue-mimicking conditions. In the present contribution, we will discuss the latest implication of mechanosensing in heart development and pathology, describe the most recent models of cell/tissue mechanics, and delineate novel strategies to target the consequences of heart failure with personalized approaches based on tissue engineering and induced pluripotent stem cell (iPSC) technologies.

Enzyme-responsive progelator cyclic peptides for minimally invasive delivery to the heart post-myocardial infarction.

Injectable biopolymer hydrogels have gained attention for use as scaffolds to promote cardiac function and prevent negative left ventricular (LV) remodeling post-myocardial infarction (MI). However, most hydrogels tested in preclinical studies are not candidates for minimally invasive catheter delivery due to excess material viscosity, rapid gelation times, and/or concerns regarding hemocompatibility and potential for embolism. We describe a platform technology for progelator materials formulated as sterically constrained cyclic peptides which flow freely for low resistance injection, and rapidly assemble into hydrogels when linearized by disease-associated enzymes. Their utility in vivo is demonstrated by their ability to flow through a syringe and gel at the site of MI in rat models. Additionally, synthetic functionalization enables these materials to flow through a cardiac injection catheter without clogging, without compromising hemocompatibility or cytotoxicity. These studies set the stage for the development of structurally dynamic biomaterials for therapeutic hydrogel delivery to the MI.

Ion cyclotron resonance as a tool in regenerative medicine.

The identification of suitable stem cell cultures and differentiating conditions that are free of xenogenic growth supplements is an important step in finding the clinical applicability of cell therapy in two important fields of human medicine: heart failure and bone remodeling, growth and repair. We recently demonstrated the possibility of obtaining cardiac stem cells (CSCs) from human endomyocardial biopsy specimens. CSCs self-assemble into multi-cellular clusters known as cardiospheres (CSps) that engraft and partially regenerate infarcted myocardium. CSps and cardiosphere-derived-cells (CDCs) were exposed for five days in an incubator regulated for temperature, humidity, and CO(2) inside a solenoid system. This system was placed in a magnetically shielded room. The cells were exposed simultaneously to a static magnetic field (MF) and a parallel low-alternating frequency MF, close to the cyclotron frequency corresponding to the charge/mass ratio of the Ca(++) ion. In this exposure condition, CSps and CDCs modulate their differentiation turning on cardiogenesis and turning off vasculogenesis. Cardiac markers such as troponin I (TnI) and myosin heavy chain (MHC) were up-regulated. Conversely, angiogenic markers such as vascular endothelial growth factor (VEGF) and kinase domain receptor (KDR) were down-regulated as evidenced by immunocytochemistry. Exposure to the 7 Hz calcium ion cyclotron resonance (ICR) frequency can modulate the cardiogenic vs. angiogenic differentiation process of ex vivo expanded CSCs. This may pave the way for novel approaches in tissue engineering and cell therapy. With regard to bone remodeling, it has been suggested that bone marrow-derived mesenchymal stem cells (MSC) may be considered as a potential therapeutic tool. Using the Ca(++)-dependent specific differentiation potential of the ELF-MF 7 Hz ICR, we show here that exposure of human MSC to these same MF conditions enhanced the expression of osteoblast differentiation markers such as alkaline phosphatase, osteocalcin, and osteopontin, as analyzed by real-time quantitative PCR, without affecting cell proliferation. As expected, while the differentiation marker factors were up regulated, the ICR electromagnetic field down regulated osteoprotegerin gene expression, a critical regulator of postnatal skeletal development and homeostasis in humans as well as mice.

A Bioprinted Cardiac Patch Composed of Cardiac-Specific Extracellular Matrix and Progenitor Cells for Heart Repair.

Congenital heart defects are present in 8 of 1000 newborns and palliative surgical therapy has increased survival. Despite improved outcomes, many children develop reduced cardiac function and heart failure requiring transplantation. Human cardiac progenitor cell (hCPC) therapy has potential to repair the pediatric myocardium through release of reparative factors, but therapy suffers from limited hCPC retention and functionality. Decellularized cardiac extracellular matrix hydrogel (cECM) improves heart function in animals, and human trials are ongoing. In the present study, a 3D-bioprinted patch containing cECM for delivery of pediatric hCPCs is developed. Cardiac patches are printed with bioinks composed of cECM, hCPCs, and gelatin methacrylate (GelMA). GelMA-cECM bioinks print uniformly with a homogeneous distribution of cECM and hCPCs. hCPCs maintain >75% viability and incorporation of cECM within patches results in a 30-fold increase in cardiogenic gene expression of hCPCs compared to hCPCs grown in pure GelMA patches. Conditioned media from GelMA-cECM patches show increased angiogenic potential (>2-fold) over GelMA alone, as seen by improved endothelial cell tube formation. Finally, patches are retained on rat hearts and show vascularization over 14 d in vivo. This work shows the successful bioprinting and implementation of cECM-hCPC patches for potential use in repairing damaged myocardium.

Decellularized Extracellular Matrix Hydrogels as a Delivery Platform for MicroRNA and Extracellular Vesicle Therapeutics.

In the last decade, the use of microRNA (miRNA) and extracellular vesicle (EV) therapies has emerged as an alternative approach to mitigate the negative effects of several disease pathologies ranging from cancer to tissue and organ regeneration; however, delivery approaches towards target tissues have not been optimized. To alleviate these challenges, including rapid diffusion upon injection and susceptibility to degradation, porcine-derived decellularized extracellular matrix (ECM) hydrogels are examined as a potential delivery platform for miRNA and EV therapeutics. The incorporation of EVs and miRNA antagonists, including anti-miR and antago-miR, in ECM hydrogels results in a prolonged release as compared to the biologic agents alone. In addition, individual in vitro assessments confirm the bioactivity of the therapeutics upon release from the ECM hydrogels. This work demonstrates the feasibility of encapsulating miRNA and EV therapeutics in ECM hydrogels to enhance delivery and potentially efficacy in later in vivo applications.

Evaluation of Different Decellularization Protocols on the Generation of Pancreas-Derived Hydrogels.

Different approaches have investigated the effects of different extracellular matrices (ECMs) and three-dimensional (3D) culture on islet function, showing encouraging results. Ideally, the proper scaffold should mimic the biochemical composition of the native tissue as it drives numerous signaling pathways involved in tissue homeostasis and functionality. Tissue-derived decellularized biomaterials can preserve the ECM composition of the native tissue making it an ideal scaffold for 3D tissue engineering applications. However, the decellularization process may affect the retention of specific components, and the choice of a proper detergent is fundamental in preserving the native ECM composition. In this study, we evaluated the effect of different decellularization protocols on the mechanical properties and biochemical composition of pancreatic ECM (pECM) hydrogels. Fresh porcine pancreas tissue was harvested, cut into small pieces, rinsed in water, and treated with two different detergents (sodium dodecyl sulfate [SDS] or Triton X-100) for 1 day followed by 3 days in water. Effective decellularization was confirmed by PicoGreen assay, Hoescht, and H&E staining, showing no differences among groups. Use of a protease inhibitor (PI) was also evaluated. Effective decellularization was confirmed by PicoGreen assay and hematoxylin and eosin (H&E) staining, showing no differences among groups. Triton-treated samples were able to form a firm hydrogel under appropriate conditions, while the use of SDS had detrimental effects on the gelation properties of the hydrogels. ECM biochemical composition was characterized both in the fresh porcine pancreas and all decellularized pECM hydrogels by quantitative mass spectrometry analysis. Fibrillar collagen was the major ECM component in all groups, with all generated hydrogels having a higher amount compared with fresh pancreas. This effect was more pronounced in the SDS-treated hydrogels when compared with the Triton groups, showing very little retention of other ECM molecules. Conversely, basement membrane and matricellular proteins were better retained when the tissue was pretreated with a PI and decellularized in Triton X-100, making the hydrogel more similar to the native tissue. In conclusion, we showed that all the protocols evaluated in the study showed effective tissue decellularization, but only when the tissue was pretreated with a PI and decellularized in Triton detergent, the biochemical composition of the hydrogel was closer to the native tissue ECM. Impact Statement The article compares different methodologies for the generation of a pancreas-derived hydrogel for tissue engineering applications. The biochemical characterization of the newly generated hydrogel shows that the material retains all the extracellular molecules of the native tissue and is capable of sustaining functionality of the encapsulated beta-cells.