Effect of Cell Seeding Density and Inflammatory Cytokines on Adipose Tissue-Derived Stem Cells: an in Vitro Study

Adipose tissue-derived stem cells (ASCs) are known to be able to promote repair of injured tissue via paracrine factors. However, the effect of cell density and inflammatory cytokines on the paracrine ability of ASCs remains largely unknown. To investigate these effects, ASCs were cultured in 8000 cells/cm2, 20,000 cells/cm2, 50,000 cells/cm2, and 400,000 cells/cm2 with and without 10 or 20 ng/ml tumor necrosis factor alpha (TNFα) and 25 or 50 ng/ml interferon gamma (IFNγ). ASC-sheets formed at 400,000 cells/cm2 after 48 h of culture. With increasing concentrations of TNFα and IFNγ, ASC-sheets with 400,000 cells/cm2 had increased production of angiogenic factors Vascular Endothelial Growth Factor and Fibroblast Growth Factor and decreased expression of pro-inflammatory genes TNFA and Prostaglandin Synthase 2 (PTGS2) compared to lower density ASCs. Moreover, the conditioned medium of ASC-sheets with 400,000 cells/cm2 stimulated with the low concentration of TNFα and IFN

Effects of adipose stem cell sheets on colon anastomotic leakage in an experimental model: Proof of principle

The most dreaded complication of colorectal surgery is anastomotic leakage. Adipose tissue-derived stem cell sheets (ASC sheets) prepared from temperature-responsive culture surfaces can be easily transplanted onto tissues. These sheets are proposed to improve cell transplant efficiency and enhance wound healing. The aim of this study was to investigate whether application of ASC sheets could prevent leakage of sutured colorectal anastomoses. Insufficient suturing of colorectal anastomoses was performed in Wistar rats to create a colorectal anastomotic leakage model. Rats were randomized to ASC sheet application or control group. Leakage, abscess formation, adhesion formation, anastomotic bursting pressure (ABP), and histology were evaluated on postoperative day 3 or 7. ASC shee

Adipose tissue-derived stem cell sheets for tissue repair

Impaired tissue healing has become a major challenge to healthcare systems worldwide. Although research advances in the field have led to solutions that have been effective in improving patient care, the economic burden and an aging population call for allocation of a higher level of attention and resources. Using stem cells in cell-based therapies could provide an alternative treatment option. Mesenchymal stem cells such as adipose tissue-derived stem cells (ASC) can enhance tissue healing by their paracrine potential and ability to modulate inflammatory responses. Traditionally, ASC were delivered to injured tissues via injection or scaffold-based constructs. This thesis explored a relatively new method for ASC application to injured tissues: adipose tissue-derived stem cells (ASC) cultured in a cell sheet (ASC sheet). The rationale of this method is that applying the ASCs as a sheet improves their retention and engraftment after transplantation. In addition, it might avoid a foreign body response. However, detailed information on ASC sheet therapeutic potential in the healing of various tissues is limited. This body of work attempts to provide insight in the mechanisms by which ASC sheet can contribute to tissue repair. In addition, this thesis describes the methodology and effects of transplantation of ASC sheet in an in vivo intestinal disease model for the first time

Human mesenchymal stromal cell sheets induce macrophages predominantly to an anti-inflammatory phenotype

Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that mesenchymal stromal cells can modulate macrophage-induced inflammation and therefore are promising candidates for cell-based therapies aimed at promoting tissue repair. Recently, cell sheets were introduced as a new method of delivering stromal cells to the repair-site. The goal of the current study was to compare the effect of different types of stromal cell sheets on the inflammatory state of macrophages in vitro. We compared the effects of adipose tissue-derived stromal cell (ASC) sheets, bone marrow derived stromal cell (BMSC) sheets and fibroblast sheets on macrophage functional phenotype using flow cytometric analysis, gene expression as well as cell sheet protein secretion. This was evaluated with and without inflammatory stimulation. Viability and senescence for the different types of sheet were also evaluated. Macrophages cultured in ASC sheet conditioned medium (CM) displayed a higher fluorescence intensity of the anti-inflammatory CD206 surface marker than when cultured in BMSC sheet CM, and expressed more CCL18 and IL1RA than when cultured in fibroblast sheet CM. Moreover, ASC sheets had higher cell viability and less senescent cells than BMSC sheets and fibroblast sheets. Taken together, ASC and BMSC can stimulate the anti-inflammatory macrophage (M2) phenotype to a better extent than fibroblasts. It is suggested, that ASC sheets might outperform BMSC sheets in an inflammatory situation since ASC sheet CM induced-macrophages have more M2 characteristics and ASC in the sheet were more viable

Human mesenchymal stromal cell sheets induce macrophages predominantly to an anti-inflammatory phenotype

Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that mesenchymal stromal cells can modulate macrophage-induced inflammation and therefore are promising candidates for cell-based therapies aimed at promoting tissue repair. Recently, cell sheets were introduced as a new method of delivering stromal cells to the repair-site. The goal of the current study was to compare the effect of different types of stromal cell sheets on the inflammatory state of macrophages in vitro. We compared the effects of adipose tissue-derived stromal cell (ASC) sheets, bone marrow derived stromal cell (BMSC) sheets and fibroblast sheets on macrophage functional phenotype using flow cytometric analysis, gene expression as well as cell sheet protein secretion. This was evaluated with and without inflammatory stimulation. Viability and senescence for the different types of sheet were also evaluated. Macrophages cultured in ASC sheet conditioned medium (CM) displayed a higher fluorescence intensity of the anti-inflammatory CD206 surface marker than when cultured in BMSC sheet CM, and expressed more CCL18 and IL1RA than when cultured in fibroblast sheet CM. Moreover, ASC sheets had higher cell viability and less senescent cells than BMSC sheets and fibroblast sheets. Taken together, ASC and BMSC can stimulate the anti-inflammatory macrophage (M2) phenotype to a better extent than fibroblasts. It is suggested, that ASC sheets might outperform BMSC sheets in an inflammatory situation since ASC sheet CM induced-macrophages have more M2 characteristics and ASC in the sheet were more viable

Adipose tissue-derived stem cell sheet application for tissue healing in vivo; a systematic review

Adipose tissue-derived stem cells (ASCs) are known to be tissue-healing promoters due to their cellular plasticity and secretion of paracrine factors. Cultured ASC sheets provide a novel method of ASCs application and can retain ASCs at the targeted tissue. The purpose of this systematic review is to evaluate preclinical studies using adipose tissue-derived cell (ASC) sheet transplantation therapy for promoting tissue healing. Firstly, we searched databases to identify studies of ASC sheet therapy in different experimental animal models, and then determined the quality score of studies using SYRCLE's risk bias tool. A total of 18 included studies examined the role of ASC sheets on tissue healing and function in models for myocardial infarction, dilated cardiomyopathy, full-thickness skin wounds, hind limb ischemia, esophageal strictures and oral ulcers. ASC sheet application after myocardial infarction improved survival rate, cardiac function and capillary density and reduced the extent of fibrosis. Application of ASC sheets to a full thickness skin wound decreased the wound size and stimulated wound maturation. In the hind limb ischemia model, ASC sheet application improved limb perfusion, and capillary density and decreased the amount of ischemic tissue and inflammation. ASC sheet application to mucosal wounds of the gastrointestinal system accelerated wound healing and decreased the degree of stricture and fibrosis. Taken together, transplanted ASC sheets had a positive effect on tissue healing and reconstruction in these pre-clinical studies. The reported favorable effects of ASC sheet therapy in various tissue healing applications may be implemented in future translational studies. It is suggested that future preclinical animal model studies of ASC sheet therapy should concern standardization of culture techniques and investigate the mechanisms of action. In addition, clearly indicated experimental set-ups according to the SYRCLE's guidelines should improve study quality and validity

Adipose tissue-derived stem cell sheet application for tissue healing in vivo; a systematic review

Adipose tissue-derived stem cells (ASCs) are known to be tissue-healing promoters due to their cellular plasticity and secretion of paracrine factors. Cultured ASC sheets provide a novel method of ASCs application and can retain ASCs at the targeted tissue. The purpose of this systematic review is to evaluate preclinical studies using adipose tissue-derived cell (ASC) sheet transplantation therapy for promoting tissue healing. Firstly, we searched databases to identify studies of ASC sheet therapy in different experimental animal models, and then determined the quality score of studies using SYRCLE's risk bias tool. A total of 18 included studies examined the role of ASC sheets on tissue healing and function in models for myocardial infarction, dilated cardiomyopathy, full-thickness skin wounds, hind limb ischemia, esophageal strictures and oral ulcers. ASC sheet application after myocardial infarction improved survival rate, cardiac function and capillary density and reduced the extent of fibrosis. Application of ASC sheets to a full thickness skin wound decreased the wound size and stimulated wound maturation. In the hind limb ischemia model, ASC sheet application improved limb perfusion, and capillary density and decreased the amount of ischemic tissue and inflammation. ASC sheet application to mucosal wounds of the gastrointestinal system accelerated wound healing and decreased the degree of stricture and fibrosis. Taken together, transplanted ASC sheets had a positive effect on tissue healing and reconstruction in these pre-clinical studies. The reported favorable effects of ASC sheet therapy in various tissue healing applications may be implemented in future translational studies. It is suggested that future preclinical animal model studies of ASC sheet therapy should concern standardization of culture techniques and investigate the mechanisms of action. In addition, clearly indicated experimental set-ups according to the SYRCLE's guidelines should improve study quality and validity