A morphological study of adipose-derived stem cell sheets created with temperature-responsive culture dishes using scanning electron microscopy.

Adipose-derived stem cell (ADSC) sheets have potential to be effective in various therapies. In this study, we first demonstrated that a cell sheet composed of human ADSCs could be created using a new temperature-responsive culture dish from the DIC Corporation. The dish can cause detachment of adherent cells due to temperature changes, but a few morphological analyses have evaluated the presence or absence of damage on the detached surface of cell sheet. To characterize our ADSC sheet, we tried to observe the surface of ADSC sheets with scanning electron microscope (SEM) using the ionic liquid, which enables the rapid preparation of samples. No damage was found on the surface of the ADSC sheets on the side that had been in contact with the surface of the culture dishes. In addition, when the transcriptomes of the harvested cell sheets were compared with those of monolayer cultures, no up-regulation of cell death related genes were detected. These results propose that the detachment from temperature-responsive culture dish causes no serious damage on the prepared ADSC sheet. It is also suggested that the SEM with ionic liquids is a useful and rapid method for the analysis of ADSC sheets for therapy.

Tissue-Engineered Hepatocyte Sheets Supplemented with Adipose-Derived Stem Cells.

The ability to engineer biologically viable hepatocytes and tissue matrices with long-term functional maintenance has attracted considerable interest in the fields of hepatocyte transplantation and liver tissue engineering. Here, newly developed hepatocyte sheets supplemented with adipose-derived stem cells (ADSCs) were evaluated to assess the effects of ADSCs on hepatocyte function and engraftment into the subcutaneous space. Eight-week-old male C57BL/6J mice were used as donors, and 6-week-old male C.B-17/Icr-scid/scid mice were used as recipients. Hepatocyte-ADSC composite sheets were developed using temperature-responsive culture dishes. Hepatocyte viability in the hepatocyte-ADSC composite sheets was evaluated in an in vitro assay, and the outcome of subcutaneous transplantation of the sheet was evaluated. Hepatocyte viability was sustained in the hepatocyte-ADSC composite sheets in vitro. Albumin secretion was significantly higher (p = 0.015) in the hepatocytes of the hepatocyte-ADSC composite sheets (70.5 µg/mL) than in hepatocyte-only sheets (24.0 µg/mL). Cytokine assays showed that hepatocyte growth factor and interleukin-6 were contributed by ADSCs and not hepatocytes, which were not capable of constitutively secreting them. Immunohistochemically, phosphorylated STAT3 and c-MET expression in hepatocytes in the hepatocyte-ADSC composite sheets was significantly higher than that in the hepatocyte-only sheets. Engraftment of the transplanted hepatocyte-ADSC composite sheets was significantly enhanced without pretreatment of the subcutaneous tissue to induce a vascular network. In the hepatocyte-ADSC composite sheets, the viability of the hepatocytes was significantly maintained as the co-cultured ADSCs provided cytokines, enhancing pivotal cell signaling necessary for hepatocyte activity. Impact statement Hepatocyte transplantation is a safe, less invasive bridge treatment for liver transplantation, but its effectiveness is low and transitory. Herein, we introduce newly developed hepatocyte-adipose-derived stem cell composite sheets with improved strength, easier transplantation, and increased hepatocyte viability in the subcutaneous transplantation compared with hepatocyte-only sheets.

Transplantation of hybrid adipose-derived stem cell sheet with autologous peritoneum: An in vivo feasibility study.

Introduction: In regenerative medicine, cell sheet engineering has various advantages, including the retention of cells at the transplantation site for a longer period and the local delivery of growth factors and cytokines. Adipose-derived stem cell (ASC) is widely used owing to their various functions such as wound healing, immunomodulation, and nerve regeneration, in addition to their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. ASC sheet generated using cell sheet engineering is considered effective in preventing anastomotic leakage, a serious postoperative complication in gastrointestinal surgery. However, the ASC sheet is too soft, thin, and brittle to handle with laparoscopic forceps during the operation. Therefore, we considered using the peritoneum, which is stiff and easy to collect while operating, as an alternative support. In this study, we explored the feasibility of using the peritoneum as a support for the precise transplantation of ASC sheets during surgery. Methods: ASCs were isolated from the subcutaneous fat of the inguinal region of Sprague-Dawley (SD) transgenic rats expressing green fluorescent protein. ASCs were cultured until passage 3, seeded in temperature-responsive culture dishes, and the resulting ASC sheet was harvested at more than 80% confluency. Non-transgenic SD rats were used for transplant experiments. The wall peritoneum was harvested from SD rats following laparotomy, and hybrid adipose-derived stem cell (HASC) sheet was prepared by laminating the peritoneum with ASC sheet. The cell sheets were transplanted on the backs of SD rats following the incision. On post-transplantation days 3 and 7, the specimens were extracted. ASC and HASC sheets were then compared macroscopically and histopathologically. Results: HASC sheet transplantation was macroscopically and histopathologically more effective than ASC sheet transplantation. The peritoneum provided sufficient stiffness as a support for precise transplantation. Conclusion: The newly developed HASC sheet, which combine the advantages of ASC sheet with those of the peritoneum, could be more useful for clinical application than the ASC sheet alone.

Characterization of layered chondrocyte sheets created in a co-culture system with synoviocytes in a hypoxic environment.

Endeavouring to repair and regenerate articular cartilage using cell sheets, we have previously established a co-culture system of chondrocytes and synoviocytes, and have reported the successful and rapid production of chondrocyte sheets. In the present study, to examine the effects of oxygen concentration on the chondrocyte sheets, we co-cultured human articular chondrocytes and human synoviocytes in 2%, 5% and 21% oxygen, and measured chondrocyte metabolic activity and proliferation activities under each condition for 14 days in culture. Layered chondrocyte sheets were also created under each condition and the proteoglycan (PG) level was compared with the gene expression of type I collagen (COL1), COL2, COL27, tissue metallopeptidase inhibitor 1 (TIMP1), fibronectin-1 (FN1), SRY-related HMG Box 9 (SOX9), aggrecan-1 (ACAN), integrin-α10 (ITGα10), matrix metalloproteinase 3 (MMP3), MMP13 and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5). Compared with 5% and 21% oxygen, the 2% condition caused significantly greater cell metabolic activity and proliferation (p < 0.05). The 2% condition produced a 10% greater PG level compared with 21% oxygen (p < 0.05). All conditions increased the expression of chondrocyte-specific genes, such as COL2, and were associated with low expression levels of catabolic factors, such as MMP3 and MMP13. These observations indicated that the specificity of the chondrocyte sheets was maintained under all conditions. The culture times did not differ between the 5% and 21% conditions. Compared with 21% oxygen, layered chondrocyte sheets rich in extracellular matrix were created 2.85 days earlier in 2% oxygen, which is similar to the level found in deep cartilage. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of chondrocyte sheets prepared using a co-culture method with temperature-responsive culture inserts.

Conventional culture methods using temperature-responsive culture dishes require 4-5 weeks to prepare layered chondrocyte sheets that can be used in articular cartilage repair and regeneration. This study investigated whether the use of synovial tissue obtained from the same joint as the chondrocyte nutritive supply source could more quickly facilitate the preparation of chondrocyte sheets. After culturing derived synoviocytes and chondrocytes together (i.e. combined culture or co-culture) on temperature-responsive inserts, chondrocyte growth was assessed and a molecular analysis of the chondrocyte sheets was performed. Transplantable tissue could be obtained more quickly using this method (average 10.5 days). Real-time polymerase chain reaction and immunostaining of the three-layer chondrocyte sheets confirmed the significant expression of genes critical to cartilage maintenance, including type II collagen (COL2), aggrecan-1 and tissue metallopeptidase inhibitor 1. However, the expression of COL1, matrix metalloproteinase 3 (MMP3), MMP13 and A-disintegrin and metalloproteinase with thrombospondin motifs 5 was suppressed. The adhesive factor fibronectin-1 (FN1) was observed in all sheet layers, whereas in sheets generated using conventional preparation methods positive FN1 immunostaining was observed only on the surface of the sheets. The results indicate that synoviocyte co-cultures provide an optimal environment for the preparation of chondrocyte sheets for tissue transplantation and are particularly beneficial for shortening the required culture period. Copyright © 2013 John Wiley & Sons, Ltd.