Transplantation of Fibroblast Sheets with Blood Mononuclear Cell Culture Exerts Cardioprotective Effects by Enhancing Anti-Inflammation and Vasculogenic Potential in Rat Experimental Autoimmune Myocarditis Model

Fulminant myocarditis causes impaired cardiac function, leading to poor prognosis and heart failure. Cell sheet engineering is an effective therapeutic option for improving cardiac function. Naïve blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. Herein, we investigated whether autologous cell sheet transplant with QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM). Fibroblast sheets (F-sheet), prepared from EAM rats, were co-cultured with or without QQMNCs (QQ+F sheet) on temperature-responsive dishes. QQ+F sheet induced higher expression of anti-inflammatory and vasculogenic genes (Vegf-b, Hgf, Il-10, and Mrc1/Cd206) than the F sheet. EAM rats were transplanted with either QQ+F sheet or F-sheet, and the left ventricular (LV) hemodynamic analysis was performed using cardiac catheterization. Among the three groups (QQ+F sheet, F-sheet, operation control), the QQ+F sheet transplant group showed alleviation of end-diastolic pressure–volume relationship on a volume load to the same level as that in the healthy group. Histological analysis revealed that QQ+F sheet transplantation promoted revascularization and mitigated fibrosis by limiting LV remodeling. Therefore, autologous QQMNC-modified F-sheets may be a beneficial therapeutic option for EAM.

Cartilage Regeneration Characteristics of Human and Goat Auricular Chondrocytes

Although cartilage regeneration technology has achieved clinical breakthroughs, whether auricular chondrocytes (AUCs) represent optimal seed cells to achieve stable cartilage regeneration is not clear. In this study, we systematically explore biological behaviors of human- and goat-derived AUCs during in vitro expansion as well as cartilage regeneration in vitro and in vivo. To eliminate material interference, a cell sheet model was used to evaluate the feasibility of dedifferentiated AUCs to re-differentiate and regenerate cartilage in vitro and in vivo. We found that the dedifferentiated AUCs could re-differentiate and regenerate cartilage sheets under the chondrogenic medium system, and the generated chondrocyte sheets gradually matured with increased in vitro culture time (2, 4, and 8 weeks). After the implantation of cartilage sheets with different in vitro culture times in nude mice, optimal neocartilage was formed in the group with 2 weeks in vitro cultivation. After in vivo implantation, ossification only occurred in the group with goat-regenerated cartilage sheet of 8 weeks in vitro cultivation. These results, which were confirmed in human and goat AUCs, suggest that AUCs are ideal seed cells for the clinical translation of cartilage regeneration under the appropriate culture system and culture condition.

Zonal-Layered Chondrocyte Sheets for Repairment of Full-Thickness Articular Cartilage Defect: A Mini-Pig Model

The cell sheet technique is a promising approach for tissue engineering, and the present study is aimed to determine a better configuration of cell sheets for cartilage repair. For stratified chondrocyte sheets (S-CS), articular chondrocytes isolated from superficial, middle, and deep zones were stacked accordingly. Heterogeneous chondrocyte sheets (H-CS) were obtained by mixing zonal chondrocytes. The expressions of chondrocytes, cytokine markers, and glycosaminoglycan (GAG) production were assessed in an in vitro assay. The curative effect was investigated in an in vivo porcine osteochondral defect model. The S-CS showed a higher cell viability, proliferation rate, expression of chondrogenic markers, secretion of tissue inhibitor of metalloproteinase, and GAG production level than the H-CS group. The expressions of ECM destruction enzyme and proinflammatory cytokines were lower in the S-CS group. In the mini-pigs articular cartilage defect model, the S-CS group had a higher International Cartilage Repair Society (ICRS) macroscopic score and displayed a zonal structure that more closely resembled the native cartilage than those implanted with the H-CS. Our study demonstrated that the application of the S-CS increased the hyaline cartilage formation and improved the surgical outcome of chondrocyte implication, offering a better tissue engineering strategy for treating articular cartilage defects.

DeepProjection: Rapid and structure-specific projections of tissue sheets embedded in 3D microscopy stacks using deep learning

The efficient extraction of local high-resolution content from massive amounts of imaging data remains a serious and unsolved problem in studies of complex biological tissues. Here we present DeepProjection, a trainable projection algorithm based on deep learning. This algorithm rapidly and robustly extracts image content contained in curved manifolds from time-lapse recorded 3D image stacks by binary masking of background content, stack by stack. The masks calculated for a given movie, when predicted, e.g., on fluorescent cell boundaries on one channel, can subsequently be applied to project other fluorescent channels from the same manifold. We apply DeepProjection to follow the dynamic movements of 2D-tissue sheets in embryonic development. We show that we can selectively project the amnioserosa cell sheet during dorsal closure in Drosophila melanogaster embryos and the periderm layer in the elongating zebrafish embryo while masking highly fluorescent out-of-plane artifacts.