Engineering organoid microfluidic system for biomedical and health engineering: A review

In recent years, organoid technology, i.e., in vitro three-dimensional (3D) tissue culture, has attracted increasing attention in biomedical engineering. Organoids are cell complexes induced by differentiation of stem cells or organ-progenitor cells in vitro using 3D culture technology. They can replicate the key structural and functional characteristics of the target organs in vivo. With the opening up of this new field of health engineering, there is a need for engineering-system approaches to the production, control, and quantitative analysis of organoids and their microenvironment. Traditional organoid technology has limitations, including lack of physical and chemical microenvironment control, high heterogeneity, complex manual operation, imperfect nutritional supply system, and lack of feasible online analytical technology for the organoids. The introduction of microfluidic chip technology into organoids has overcome many of these limitations and greatly expanded the scope of applications. Engineering organoid microfluidic system has become an interdisciplinary field in biomedical and health engineering. In this review, we summarize the development and culture system of organoids, discuss how microfluidic technology has been used to solve the main technical challenges in organoid research and development, and point out new opportunities and prospects for applications of organoid microfluidic system in drug development and screening, food safety, precision medicine, and other biomedical and health engineering fields.     

Modeling Intestinal Stem Cell Function with Organoids

Intestinal epithelial cells (IECs) are crucial for the digestive process and nutrient absorption. The intestinal epithelium is composed of the different cell types of the small intestine (mainly, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and tuft cells). The small intestine is characterized by the presence of crypt-villus units that are in a state of homeostatic cell turnover. Organoid technology enables an efficient expansion of intestinal epithelial tissue in vitro. Thus, organoids hold great promise for use in medical research and in the development of new treatments. At present, the cholinergic system involved in IECs and intestinal stem cells (ISCs) are attracting a great deal of attention. Thus, understanding the biological processes triggered by epithelial cholinergic activation by acetylcholine (ACh), which is produced and released from neuronal and/or non-neuronal tissue, is of key importance. Cholinergic signaling via ACh receptors plays a pivotal role in IEC growth and differentiation. Here, we discuss current views on neuronal innervation and non-neuronal control of the small intestinal crypts and their impact on ISC proliferation, differentiation, and maintenance. Since technology using intestinal organoid culture systems is advancing, we also outline an organoid-based organ replacement approach for intestinal diseases.     

Wnt Modulation Enhances Otic Differentiation by Facilitating the Enucleation Process but Develops Unnecessary Cardiac Structures

Otic organoids have the potential to resolve current challenges in hearing loss research. The reproduction of the delicate and complex structure of the mammalian cochlea using organoids requires high efficiency and specificity. Recent attempts to strengthen otic organoids have focused on the effects of the Wnt signaling pathway on stem cell differentiation. One important aspect of this is the evaluation of undesirable effects of differentiation after Wnt activation. In the present study, we differentiated mouse embryonic stem cell embryoid bodies (EB) into otic organoids and observed two morphologies with different cell fates. EBs that underwent a core ejection process, or ‘enucleation,’ were similar to previously reported inner ear organoids. Meanwhile, EBs that retained their core demonstrated features characteristic of neural organoids. The application of a Wnt agonist during the maturation phase increased enucleation, as well as otic organoid formation, in turn leading to sensory hair cell-like cell generation. However, with a longer incubation period, Wnt activation also led to EBs with ‘beating’ organoids that exhibited spontaneous movement. This observation emphasizes the necessity of optimizing Wnt enhancement for the differentiation of specific cells, such as those found in the inner ear.     

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.     

Retinal Organoid Technology: Where Are We Now?

It is difficult to regenerate mammalian retinal cells once the adult retina is damaged, and current clinical approaches to retinal damages are very limited. The introduction of the retinal organoid technique empowers researchers to study the molecular mechanisms controlling retinal development, explore the pathogenesis of retinal diseases, develop novel treatment options, and pursue cell/tissue transplantation under a certain genetic background. Here, we revisit the historical background of retinal organoid technology, categorize current methods of organoid induction, and outline the obstacles and potential solutions to next-generation retinal organoids. Meanwhile, we recapitulate recent research progress in cell/tissue transplantation to treat retinal diseases, and discuss the pros and cons of transplanting single-cell suspension versus retinal organoid sheet for cell therapies.     

Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Among many different roles, MeCP2 has a high phenotypic impact during the different stages of brain development. Thus, it is essential to intensively investigate the function of MeCP2, and its regulated targets, to better understand the mechanisms of the disease and inspire the development of possible therapeutic strategies. Several animal models have greatly contributed to these studies, but more recently human pluripotent stem cells (hPSCs) have been providing a promising alternative for the study of RTT. The rapid evolution in the field of hPSC culture allowed first the development of 2D-based neuronal differentiation protocols, and more recently the generation of 3D human brain organoid models, a more complex approach that better recapitulates human neurodevelopment in vitro. Modeling RTT using these culture platforms, either with patient-specific human induced pluripotent stem cells (hiPSCs) or genetically-modified hPSCs, has certainly contributed to a better understanding of the onset of RTT and the disease phenotype, ultimately allowing the development of high throughput drugs screening tests for potential clinical translation. In this review, we first provide a brief summary of the main neurological features of RTT and the impact of MeCP2 mutations in the neuropathophysiology of this disease. Then, we provide a thorough revision of the more recent advances and future prospects of RTT modeling with human neural cells derived from hPSCs, obtained using both 2D and organoids culture systems, and its contribution for the current and future clinical trials for RTT.     

The Rise of Retinal Organoids for Vision Research

Retinal degenerative diseases lead to irreversible blindness. Decades of research into the cellular and molecular mechanisms of retinal diseases, using either animal models or human cell-derived 2D systems, facilitated the development of several therapeutic interventions. Recently, human stem cell-derived 3D retinal organoids have been developed. These self-organizing 3D organ systems have shown to recapitulate the in vivo human retinogenesis resulting in morphological and functionally similar retinal cell types in vitro. In less than a decade, retinal organoids have assisted in modeling several retinal diseases that were rather difficult to mimic in rodent models. Retinal organoids are also considered as a photoreceptor source for cell transplantation therapies to counteract blindness. Here, we highlight the development and field’s improvements of retinal organoids and discuss their application aspects as human disease models, pharmaceutical testbeds, and cell sources for transplantations.     

High Glucose Exposure Impairs L-Cell Differentiation in Intestinal Organoids: Molecular Mechanisms and Clinical Implications

Intestinal organoids are used to analyze the differentiation of enteroendocrine cells (EECs) and to manipulate their density for treating type 2 diabetes. EEC differentiation is a continuous process tightly regulated in the gut by a complex regulatory network. However, the effect of chronic hyperglycemia, in the modulation of regulatory networks controlling identity and differentiation of EECs, has not been analyzed. This study aimed to investigate the effect of glucotoxicity on EEC differentiation in small intestinal organoid platforms. Mouse intestinal organoids were cultured in the presence/absence of high glucose concentrations (35 mM) for 48 h to mimic glucotoxicity. Chronic hyperglycemia impaired the expression of markers related to the differentiation of EEC progenitors (Ngn3) and L-cells (NeuroD1), and it also reduced the expression of Gcg and GLP-1 positive cell number. In addition, the expression of intestinal stem cell markers was reduced in organoids exposed to high glucose concentrations. Our data indicate that glucotoxicity impairs L-cell differentiation, which could be associated with decreased intestinal stem cell proliferative capacity. This study provides the identification of new targets involved in new molecular signaling mechanisms impaired by glucotoxicity that could be a useful tool for the treatment of type 2 diabetes.     

Functionalized nanostructures with application in regenerative medicine

In the last decade, both regenerative medicine and nanotechnology have been broadly developed leading important advances in biomedical research as well as in clinical practice. The manipulation on the molecular level and the use of several functionalized nanoscaled materials has application in various fields of regenerative medicine including tissue engineering, cell therapy, diagnosis and drug and gene delivery. The themes covered in this review include nanoparticle systems for tracking transplanted stem cells, self-assembling peptides, nanoparticles for gene delivery into stem cells and biomimetic scaffolds useful for 2D and 3D tissue cell cultures, transplantation and clinical application.     

Epigenetic Regulation of Cardiomyocyte Differentiation from Embryonic and Induced Pluripotent Stem Cells

With the intent to achieve the best modalities for myocardial cell therapy, different cell types are being evaluated as potent sources for differentiation into cardiomyocytes. Embryonic stem cells and induced pluripotent stem cells have great potential for future progress in the treatment of myocardial diseases. We reviewed aspects of epigenetic mechanisms that play a role in the differentiation of these cells into cardiomyocytes. Cardiomyocytes proliferate during fetal life, and after birth, they undergo permanent terminal differentiation. Upregulation of cardiac-specific genes in adults induces hypertrophy due to terminal differentiation. The repression or expression of these genes is controlled by chromatin structural and epigenetic changes. However, few studies have reviewed and analyzed the epigenetic aspects of the differentiation of embryonic stem cells and induced pluripotent stem cells into cardiac lineage cells. In this review, we focus on the current knowledge of epigenetic regulation of cardiomyocyte proliferation and differentiation from embryonic and induced pluripotent stem cells through histone modification and microRNAs, the maintenance of pluripotency, and its alteration during cardiac lineage differentiation.     

Polymeric design of cell culture materials that guide the differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the potential to differentiate into many cell types that originate from the three germ layers, such as dopamine-secreting cells and insulin-secreting cells for the treatment of Alzheimer's disease and diabetes, respectively. However, it is challenging to guide hPSC differentiation into desired cell lineages due to their varying differentiation ability. A reasonable strategy is to mimic the stem cell microenvironment for the differentiation of hPSCs into specific cell lineages using optimal polymeric biomaterials for hPSC culture. This review summarizes various methods for differentiating hPSCs cultured on polymeric biomaterials and discusses the optimal methods and cell culture polymeric biomaterials for hPSC differentiation into specific cell lineages. The recent trend in protocols avoids embryoid body (EB, aggregated cells) formation because EBs contain different types of cells. The combination of appropriate differentiation protocols and cell culture polymeric biomaterials for the differentiation of hPSCs into specific cell lineages will produce a large quantity of highly pure GMP-grade differentiated cells for use in translational medicine.     

Metformin and Vitamin D Modulate Inflammation and Autophagy during Adipose-Derived Stem Cell Differentiation

Adipose-derived stem cells (ADSCs) came out from the regenerative medicine landscape for their ability to differentiate into several phenotypes, contributing to tissue regeneration both in vitro and in vivo. Dysregulation in stem cell recruitment and differentiation during adipogenesis is linked to a chronic low-grade inflammation and macrophage infiltration inside the adipose tissue, insulin resistance, cardiovascular disease and obesity. In the present paper we aimed to evaluate the role of metformin and vitamin D, alone or in combination, in modulating inflammation and autophagy in ADSCs during adipogenic commitment. ADSCs were cultured for 21 days in the presence of a specific adipogenic differentiation medium, together with metformin, or vitamin D, or both. We then analyzed the expression of FoxO1 and Heat Shock Proteins (HSP) and the secretion of proinflammatory cytokines IL-6 and TNF-α by ELISA. Autophagy was also assessed by specific Western blot analysis of ATG12, LC3B I, and LC3B II expression. Our results showed the ability of the conditioned media to modulate adipogenic differentiation, finely tuning the inflammatory response and autophagy. We observed a modulation in HSP mRNA levels, and a significant downregulation in cytokine secretion. Taken together, our findings suggest the possible application of these molecules in clinical practice to counteract uncontrolled lipogenesis and prevent obesity and obesity-related metabolic disorders.     

Modified Industrial Three-Dimensional Polylactic Acid Scaffold Cell Chip Promotes the Proliferation and Differentiation of Human Neural Stem Cells

In this study, we fabricated a three-dimensional (3D) scaffold using industrial polylactic acid (PLA), which promoted the proliferation and differentiation of human neural stem cells. An industrial PLA 3D scaffold (IPTS) cell chip with a square-shaped pattern was fabricated via computer-aided design and printed using a fused deposition modeling technique. To improve cell adhesion and cell differentiation, we coated the IPTS cell chip with gold nanoparticles (Au-NPs), nerve growth factor (NGF) protein, an NGF peptide fragment, and sonic hedgehog (SHH) protein. The proliferation of F3.Olig2 neural stem cells was increased in the IPTS cell chips coated with Au-NPs and NGF peptide fragments when compared with that of the cells cultured on non-coated IPTS cell chips. Cells cultured on the IPTS-SHH cell chip also showed high expression of motor neuron cell-specific markers, such as HB9 and TUJ-1. Therefore, we suggest that the newly engineered industrial PLA scaffold is an innovative tool for cell proliferation and motor neuron differentiation.     

人脂肪干细胞复合胶原/壳聚糖支架在生物反应器中扩增的研究

研究表明脂肪组织是多潜能干细胞的又一新来源,脂肪组织来源的干细胞可被用于细胞治疗及组织工程。然而,传统的培养方法很难满足临床需求。为获得大量的脂肪干细胞以满足临床需求,现将脂肪干细胞接种到胶原/壳聚糖支架上,比较细胞在静态环境和在转瓶中动态扩增的情况。通过CCK-8检测细胞增殖情况;并分析葡萄糖和乳酸的代谢情况;14d后扫描电镜观察细胞在支架内的生长情况;流式细胞仪检测干细胞相关表面标记表达;RT-PCR检测干细胞相关转录因子的表达;并检测扩增后的干细胞的多向分化潜能。结果表明:在动态微环境中扩增14d后,与静态条件下相比,支架内的细胞具有更强的增殖活性和更好的多向分化潜能。所扩增的细胞能够保持原有干细胞的特性。结论:所设计的支架-转瓶培养系统是一个简便有效的扩增脂肪干细胞的方法。     

The Role of BMP Signaling in Female Reproductive System Development and Function

Bone morphogenetic proteins (BMPs) are a group of multifunctional growth factors that belong to the transforming growth factor-β (TGF-β) superfamily of proteins. Originally identified by their ability to induce bone formation, they are now known as essential signaling molecules that regulate the development and function of the female reproductive system (FRS). Several BMPs play key roles in aspects of reproductive system development. BMPs have also been described to be involved in the differentiation of human pluripotent stem cells (hPSCs) into reproductive system tissues or organoids. The role of BMPs in the reproductive system is still poorly understood and the use of FRS tissue or organoids generated from hPSCs would provide a powerful tool for the study of FRS development and the generation of new therapeutic perspectives for the treatment of FRS diseases. Therefore, the aim of this review is to summarize the current knowledge about BMP signaling in FRS development and function.     

Gelatin electrospun nanofibrous matrices for cardiac tissue engineering applications

The generation of in vitro tissue constructs using biomaterials and cardiac cells is a promising strategy for screening novel therapeutics and their effects on cardiac regeneration. Current cardiac mimetic tissue constructs are unable to stably maintain functional characteristics of cardiomyocytes for long-term cultures. The objective of our study was to fabricate and characterize nanofibrous matrices of gelatin for prolonged cultures of primary cardiomyocytes which previously has been used as copolymer or hydrogels. Gelatin nanofibrous matrices were successfully electrospun using a benign binary solvent, cross-linked without swelling and fusing and evaluated by scanning electron microscopy (SEM) and uniaxial tensile measurement. Scaffolds exhibited modulus 19.6 ± 3.6 kPa similar to native human myocardium tissue with fiber diameters of 200–600 nm and average porosity percentage of 49.9 ± 5.6. Myoblasts showed good cell adhesion and proliferation. Neonatal rat cardiomyocytes cultured on gelatin nanofibrous matrices showing synchronized contracting cardiomyocytes (beating) for 27 days were studied by video microscopy. Confocal microscopic analysis of immunofluorescence stained sections indicated the presence of cardiac specific Troponin T in long-term cultures. Semiquantitative RT-PCR analysis of 3D versus 2D cultures revealed enhanced expression of contractile protein desmin. Our studies show that the biophysical and mechanical properties of electrospun gelatin nanofibers are ideal for in vitro engineered cardiac constructs (ECC), to explore cardiac function in drug testing and tissue replacement. Together with stem cell techniques, they may be an ideal platform for prolongedin vitro studies in alternatives to animal usage for the pharmaceutical industry.     

Assessment of Normal Tissue Radiosensitivity by Evaluating DNA Damage and Repair Kinetics in Human Brain Organoids

DNA-double strand break (DSB), detected by immunostaining of key proteins orchestrating repair, like γH2AX and 53BP1, is well established as a surrogate for tissue radiosensitivity. We hypothesized that the generation of normal brain 3D organoids (“mini-brains”) from human induced pluripotent stem cells (hiPSC) combined with detection of DNA damage repair (DDR) may hold the promise towards developing personalized models for the determination of normal tissue radiosensitivity. In this study, cerebral organoids, an in vitro model that stands in its complexity between 2D cellular system and an organ, have been used. To quantify radiation-induced response, immunofluorescent staining with γH2AX and 53BP1 were applied at early (30 min, initial damage), and late time points (18 and 72 h, residual damage), following clinical standard 2 Gy irradiation. Based on our findings, assessment of DDR kinetics as a surrogate for radiosensitivity in hiPSC derived cerebral organoids is feasible. Further development of mini-brains recapitulating mature adult neuronal tissue and implementation of additional signaling and toxicity surrogates may pave the way towards development of next-generation personalized assessment of radiosensitivity in healthy neuronal tissue.