Ex vivo organ culture of adipose tissue for in situ mobilization of adipose-derived stem cells and defining the stem cell niche.

In spite of the advances in the knowledge of adipose-derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix-supported three-dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and alpha-smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31(-)/CD34(-)/CD146(+)/SMA(+) interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix-supported 3D environment can recapitulate the ASC niche in vitro.

Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing.

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm' or TegadermT' alone. A significant increase in re-epithelialization was observed on day 5 (p < 0.001) and day 7 (p < 0.05) for wounds treated with a GAG film plus Tegaderm versus those treated with Tegaderm alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components. presentation of growth and differentiation factors, and cell migration can readily occur.

Fibrin matrix-supported three-dimensional organ culture of adipose tissue for selective outgrowth, expansion, and isolation of adipose-derived stem cells.

Conventional systems for isolating adipose-derived stem cells (ASC) require enzymatic digestion of adipose tissue (AT), followed by monolayer culture to the enrich the stem cell population. However, these systems are hindered by low cell yields and a lack of reproducibility. The present study was aimed at developing a unique strategy for isolating ASC based on fibrin matrix-supported three-dimensional (3-D) organ culture of native AT. Furthermore, we tried to optimize the fibrin composition by adjusting the fibrinogen and thrombin concentrations to allow rapid outgrowth and proliferation of ASC in the 3-D fibrin matrix. Human cutaneous AT fragments were encapsulated within the fibrin matrix to construct a 3-D environment and cultured under dynamic conditions. During in vitro culture the fibrin matrix provided physical support for the AT and also allowed selective outgrowth of ASC from embedded AT fragments. In situ expanded outgrown cells were recovered from the fibrin matrix by selective fibrinolysis and propagated under monolayer culture conditions. The cultured cells fulfilled the following criteria for ASC: adhesion to culture plastic, multipotent differentiation, correct immunophenotypic profile. Fibrin matrix-supported 3-D organ culture produced ASC that with high competency in terms of growth and differentiation capabilities, and resulted in a larger and more consistent cell yield than obtained with conventional culture systems. The fibrinogen and thrombin concentrations inversely affected spreading, migration, and ASC outgrowth from native AT. Our results indicate that this 3-D organ culture system for AT can be used as an efficient and reproducible method for ASC isolation.

Release of basic fibroblast growth factor from a crosslinked glycosaminoglycan hydrogel promotes wound healing.

We describe synthetic extracellular matrix (sECM) hydrogel films composed of co-crosslinked thiolated derivatives of chondroitin 6-sulfate (CS) and heparin (HP) for controlled-release delivery of basic fibroblast growth factor (bFGF) to full-thickness wounds in genetically diabetic (db/db) mice. In this model for chronic wound repair, full-thickness wounds were treated with CS, CS-bFGF, or CS-HP-bFGF films. At 2 and 4 weeks postinjury, wound closure and formation of the new epidermis and dermis were determined. Both CS and CS-HP hydrogel films accelerated wound repair, even without bFGF. Addition of bFGF to CS films showed partial dose-dependent acceleration of wound repair. Importantly, addition of bFGF to co-crosslinked CS-HP sECM films showed a dramatic bFGF dose-dependent acceleration of wound healing, as well as improved dermis formation and vascularization. Compared with 27% wound closure in 2 weeks in the controls, 89% wound closure was observed for mice treated with the CS-HP-bFGF films. The synthetic CS-HP sECM films mimic the chemistry and biology of heparan sulfate proteoglycans, and may have clinical potential for topical delivery of growth factors to patients with compromised wound healing.