Published: August 4th, 2017
Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.
Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.
The population of diabetic patients is increasing worldwide and reached 400 million in 20151; an estimated 15 - 25% of patients with diabetes are at risk from the progression of a lower-extremity diabetic ulcer2. Lower-extremity diabetic ulcers are intractable and might require a prolonged therapeutic period with rehabilitation training after complete recovery. A long therapy period often results in a significant reduction in patient quality of life. Thus, new therapies that decrease or prevent aggravation must be developed for the treatment of diabetic wounds. To evaluate diabetic wound healing, we optimized a diabetic ....
All experimental protocols presented below were approved by the Animal Welfare Committee of Tokyo Women's Medical University School of Medicine and abided by all requirements of the Guidelines for Proper Conduct of Animal Experiments.
1. Preparation of Animals, Instruments, Culture Media, and Dishes
This protocol attempted to establish a new cell-based therapy for intractable diabetic wounds. Briefly (as illustrated in Figure 1), allogeneic rASC sheets were created from normal rats using cell-sheet engineering and were then transplanted using a bilayer of artificial skin onto a full-thickness skin defect on a diabetic rat. Light microscope images of a good example of an rASC sheet (Figure 2A) and a bad example of an rASC sheet (Figure 2B
The most critical steps for successfully culturing an rASC sheet are as follows: 1) The temperature must be maintained at approximately 37 °C during culturing on the temperature-responsive culture dishes. During the creation of an rASC sheet, every procedure was performed on a 37 °C thermo-plate, and every reagent was warmed to 37 °C to prevent the cells from spontaneously detaching from the dish31. 2) The recipient ZDF rats must be monitored to prevent the removal of the non-adhesi.......
The authors thank Dr. Yukiko Koga of the Department of Plastic and Reconstructive Surgery, Juntendo University School of Medicine, for providing practical advice. We also thank Mr. Hidekazu Murata of the Diabetic Center of Tokyo Women’s Medical University School of Medicine for excellent technical support. This study was supported by the Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program of the Project for Developing Innovation Systems “Cell Sheet Tissue Engineering Center (CSTEC)” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.....
|Fetal bovine serum (FBS)
|Japan Bioserum Co Ltd.
|10 103 578 001
|60-cm2 Primaria tissue culture dish
|Franklin Lakes, NJ
|Dulbecco's Phosphate Buffer Saline (PBS)
|0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)
|L-ascorbic acid phosphate magnesium salt n-hydrate
|35-mm temperature-responsive culture dish (UpcellTM)
|Microwarm plate (MP-1000)
|Kitazato Science Co., Ltd.
|Rodent mechanical ventilator
|Wood Dale, IL
|Artificial skin (Pelnac®)
|Smith & Nephew
|Non-adhesive dressing (Hydrosite plus®)
|Smith & Nephew
|Known as Allevyn non-adhessing® in the United State
|5-0 nylon suture
|20 CELLSTAR TUBES
|15mL Centrifuge Tube
|14 GOLDMAN-FOX PERIOSTEAL
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