Enhancing developmental rate and quality of mouse single blastomeres into blastocysts using a microplatform.

The present work reports the beneficial effects of using a microplatform on the development of mouse single blastomeres (SBs) to the blastocyst stage. Development of blastocysts from SBs separated from two- and four-cell stage embryos (two- and four-cell SBs) can provide a valuable supply both for couples who use fertility-assisted techniques and farm animals. As a step forward, we introduce three chips that provide the possibility of culturing SBs separately, in groups, and in the vicinity of the intact embryo (co-culture), while each well of the chips is assigned to an isolated SB. Two- and four-cell SBs co-cultured with intact embryos showed 97.1% and 76.6% developmental rates and up to 34.1% and 49.1% growth relative to the microdroplet method (control). We examined the quality of developed blastocysts by assessing the total cell number, the number of inner cell mass (ICM) according to the octamer-binding transcription factor 4 marker (OCT4), and trophectoderm (TE). Co-culture of SBs with an intact embryo in a chip with nanoscale culture medium volume also increased the cell population of the developed embryo. The ICM:TE ratio, which is the most important blastocyst quality parameter, also indicated that developed two-cell SBs have a higher degree of similarity to intact embryos despite fewer numbers of total cells.

An integrated microfluidic device for stem cell differentiation based on cell-imprinted substrate designed for cartilage regeneration in a rabbit model.

Separating cells from the body and cultivating them in vitro will alter the function of cells. Therefore, for optimal cell culture in the laboratory, conditions similar to those of their natural growth should be provided. In previous studies, it has been shown that the use of cellular shape at the culture surface can regulate cellular function. In this work, the efficiency of the imprinting method increased by using microfluidic chip design and fabrication. In this method, first, a cell-imprinted substrate of chondrocytes was made using a microfluidic chip. Afterwards, stem cells were cultured on a cell-imprinted substrate using a second microfluidic chip aligned with the substrate. Therefore, stem cells were precisely placed on the chondrocyte patterns on the substrate and their fibroblast-like morphology was changed to chondrocyte's spherical morphology after 14-days culture in the chip without using any chemical growth factor. After chondrogenic differentiation and in vitro assessments (real-time PCR and immunocytotoxicity), differentiated stem cells were transferred on a collagen-hyaluronic acid scaffold and transplanted in articular cartilage defect of the rabbit. After 6 months, the post-transplantation analysis showed that the articular cartilage defect had been successfully regenerated in differentiated stem cell groups in comparison with the controls. In conclusion, this study showed the potency of the imprinting method for inducing chondrogenicity in stem cells, which can be used in clinical trials due to the safety of the procedure.

Oxygen-rich Environment Ameliorates Cell Therapy Outcomes of Cardiac Progenitor Cells for Myocardial Infarction.

To some extent, cell therapy for myocardial infarction (MI) has supported the idea of cardiac repair; however, further optimizations are inevitable. Combined approaches that comprise suitable cell sources and supporting molecules considerably improved its effect. Here, we devised a strategy of simultaneous transplantation of human cardiac progenitor cells (CPCs) and an optimized oxygen generating microparticles (MPs) embedded in fibrin hydrogel, which was injected into a left anterior descending artery (LAD) ligating-based rat model of acute myocardial infarction (AMI). Functional parameters of the heart, particularly left ventricular systolic function, markedly improved and reached pre-AMI levels. This functional restoration was well correlated with substantially lower fibrotic tissue formation and greater vascular density in the infarct area. Our novel approach promoted CPCs retention and differentiation into cardiovascular lineages. We propose this novel co-transplantation strategy for more efficient cell therapy of AMI which may function by providing an oxygen-rich microenvironment, and thus regulate cell survival and differentiation.

Generation of Scalable Hepatic Micro-Tissues as a Platform for Toxicological Studies.

BACKGROUND: Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects of therapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to human hepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and the presence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this study was to generate the scalable and functional hepatic micro-tissues (HMTs). METHODS: The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cells and human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, using an air-driven droplet generator. RESULTS: The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoring glycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretion levels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involved in hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional (2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity to hepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450 enzymes confirmed that the HMTs can be used for in vitro drug screening. CONCLUSION: Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7, with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.

Induced cell migration based on a bioactive hydrogel sheet combined with a perfused microfluidic system.

Endothelial cell migration is a crucial step in the process of new blood vessel formation-a necessary process to maintain cell viability inside thick tissue constructs. Here, we report a new method for maintaining cell viability and inducing cell migration using a perfused microfluidic platform based on collagen gel and a gradient hydrogel sheet. Due to the helpful role of the extracellular matrix components in cell viability, we developed a hydrogel sheet from decellularized tissue (DT) of the bovine heart and chitosan (CS). The results showed that hydrogel sheets with an optimum weight ratio of CS/DT = 2 possess a porosity of around 75%, a mechanical strength of 23 kPa, and display cell viability up to 78%. Then, we immobilized a radial gradient of vascular endothelial growth factor (VEGF) on the hydrogel sheet to promote human umbilical vein endothelial cell migration. Finally, we incorporated the whole system as an entirety on the top of the microfluidic platform and studied cell migration through the hydrogel sheet in the presence of soluble and immobilized VEGF. The results demonstrated that immobilized VEGF stimulated cell migration in the hydrogel sheet at all depths compared with soluble VEGF. The results also showed that applying a VEGF gradient in both soluble and immobilized states had a significant effect on cell migration at limited depths (<100 µm). The main finding of this study is a significant improvement in cell migration using an in vivo imitating, cost-efficient and highly reproducible platform, which may open up a new perspective for tissue engineering applications.

Vascular endothelial growth factor sustained delivery augmented cell therapy outcomes of cardiac progenitor cells for myocardial infarction.

Cell therapy has become a novel promising approach for improvement of cardiac functional capacity in the instances of ventricular remodeling and fibrosis caused by episodes of coronary artery occlusion and hypoxia. The challenge toward enhancing cell engraftment as well as formation of functional tissue, however, necessitated combinatorial approaches. Here, we complemented human embryonic stem cell-derived cardiac progenitor cell (hESC-CPC) therapy by heparin-conjugated, vascular endothelial growth factor (VEGF)-loaded fibrin hydrogel as VEGF delivery system. Transplantation of these cardiac committed cells along with sustained VEGF release could surpass the cardiac repair effects of each constituent alone in a rat model of acute myocardial infarction. The histological sections of rat hearts revealed improved vascularization as well as inclusion of hESC-CPC-derived cardiomyocytes, endothelial, and smooth muscle cells in host myocardium. Thus, co-transplantation of hESC-CPC and proangiogenic factor by a suitable delivery rate may resolve the shortcomings of conventional cell therapy.

A microfabricated platform for the study of chondrogenesis under different compressive loads.

Microfluidic devices are beneficial in miniaturizing and multiplexing various cellular assays in a single platform. Chondrogenesis is known to pertain to chemical, topographical, and mechanical cues in the microenvironment. Mechanical cues themselves have numerous parameters such as strain magnitude, frequency, and stimulation time. Effects of different strain magnitudes on the chondrogenic differentiation of adult stem cells have not been explored thoroughly. Here, a new multilayer microdevice is presented for the unidirectional compressive stimulation of cells in a three-dimensional cell culture. Numerical simulations were performed to evaluate and optimize the design. Results showed a favorable highly uniform axial strain distribution and negligible radial and circumferential strain for the optimized design. Moreover, an experimental study was performed on rabbit adipose-derived stem cells encapsulated in-situ in alginate hydrogel. Strain levels of 20%, 15%, 10%, 5%, and 0% were studied simultaneously on a microfluidic platform. Dynamic mechanical compression positively influenced cellular viability and upregulated collagen II, Sox-9, and aggrecan expression in the absence of exogenous growth factors. The expression of collagen type II as specific marker for articular chondrocytes was further confirmed by immunofluorescence staining of collagen type II. Taking together, 10% strain can be considered as optimal stimulation factor for chondrogenic differentiation of adipose derived stem cells.

Strategies for directing cells into building functional hearts and parts.

The increasing population of patients with heart disease and the limited availability of organs for transplantation have encouraged multiple strategies to fabricate healthy implantable cardiac tissues. One of the main challenges in cardiac tissue engineering is to direct cell behaviors to form functional three-dimensional (3D) biomimetic constructs. This article provides a brief review on various cell sources used in cardiac tissue engineering and highlights the effect of scaffold-based signals such as topographical and biochemical cues and stiffness. Then, conventional and novel micro-engineered bioreactors for the development of functional cardiac tissues will be explained. Bioreactor-based signals including mechanical and electrical cues to control cardiac cell behavior will also be elaborated in detail. Finally, the application of computational fluid dynamics to design suitable bioreactors will be discussed. This review presents the current state-of-the-art, emerging directions and future trends that critically appraise the concepts involved in various approaches to direct cells for building functional hearts and heart parts.