Fabrication of protease XIV-loaded microspheres for cell spreading in silk fibroin hydrogels.

Due to their excellent mechanical strength and biocompatibility, silk fibroin(SF) hydrogels can serve as ideal scaffolds. However, their slow rate of natural degradation limits the space available for cell proliferation, which hinders their application. In this study, litchi-like calcium carbonate@hydroxyapatite (CaCO3@HA) porous microspheres loaded with proteases from Streptomyces griseus (XIV) were used as drug carriers to regulate the biodegradation rate of SF hydrogels. The results showed that litchi-like CaCO3@HA microspheres with different phase compositions could be prepared by changing the hydrothermal reaction time. The CaCO3@HA microspheres controlled the release of Ca ions, which was beneficial for the osteogenic differentiation of mesenchymal stem cells (MSCs). The adsorption and release of protease XIV from the CaCO3@HA microcarriers indicated that the loading and release amount can be controlled with the initial drug concentration. The weight loss test and SEM observation showed that the degradation of the fibroin hydrogel could be controlled by altering the amount of protease XIV-loaded CaCO3@HA microspheres. A three-dimensional (3D) cell encapsulation experiment proved that incorporation of the SF hydrogel with protease XIV-loaded microspheres promoted cell dispersal and spreading, suggesting that the controlled release of protease XIV can regulate hydrogel degradation. SF hydrogels incorporated with protease XIV-loaded microspheres are suitable for cell growth and proliferation and are expected to serve as excellent bone tissue engineering scaffolds.

Microfluidic device for the high-throughput and selective encapsulation of single target cells.

Droplet-based microfluidic technologies for encapsulating single cells have rapidly evolved into powerful tools for single-cell analysis. In conventional passive single-cell encapsulation techniques, because cells arrive randomly at the droplet generation section, to encapsulate only a single cell with high precision, the average number of cells per droplet has to be decreased by reducing the average frequency at which cells arrive relative to the droplet generation rate. Therefore, the encapsulation efficiency for a given droplet generation rate is very low. Additionally, cell sorting operations are required prior to the encapsulation of target cells for specific cell type analysis. To address these challenges, we developed a cell encapsulation technology with a cell sorting function using a microfluidic chip. The microfluidic chip is equipped with an optical detection section to detect the optical information of cells and a sorting section to encapsulate cells into droplets by controlling a piezo element, enabling active encapsulation of only the single target cells. For a particle population including both targeted and non-targeted particles arriving at an average frequency of up to 6000 particles per s, with an average number of particles per droplet of 0.45, our device maintained a high purity above 97.9% for the single-target-particle droplets and achieved an outstanding throughput, encapsulating up to 2900 single target particles per s. The proposed encapsulation technology surpasses the encapsulation efficiency of conventional techniques, provides high efficiency and flexibility for single-cell research, and shows excellent potential for various applications in single-cell analysis.

3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber.

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.