1. Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II  and Stress  Fiber Formation in  Human Chondrocyte Sheets 
Pojchong Chotiyarnwong1, Sopita Wongin2, Wanwipa Siriwatwechakul3, Kwanchanok Viravaidya‐Pasuwat2,4 and Saranatra Waikakul1
1Department of Orthopaedic Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University,  Bangkok 10700, Thailand.
2Biological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology  Thonburi, Bangkok 10140, Thailand 
3School  of  Bio‐Chemical  Engineering  and  Technology,  Sirindhorn  International  Institute  of  Technology, Thammasat University, Pathum Thani 12121, Thailand 
4Department  of  Chemical  Engineering,  Faculty  of  Engineering,  King  Mongkut’s  University  of  Technology Thonburi, Bangkok 10140, Thailand 
Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress  Fiber Formation in Human Chondrocyte Sheets 
Sopita Wongin1, Saranatra Waikakul2, Pojchong Chotiyarnwong2, Wanwipa Siriwatwechakul3 and  Kwanchanok Viravaidya‐Pasuwat1,4* 
1Biological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology  Thonburi, Bangkok 10140, Thailand 
2Department of Orthopaedic Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University,  Bangkok 10700, Thailand. 
3School  of  Bio‐Chemical  Engineering  and  Technology,  Sirindhorn  International  Institute  of  Technology, Thammasat University, Pathum Thani 12121, Thailand 
4Department  of  Chemical  Engineering,  Faculty  of  Engineering,  King  Mongkut’s  University  of  Technology Thonburi, Bangkok 10140, Thailand 

2. Abstract
Cell sheet technology is applied to human articular chondrocytes to construct a tissue‐like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient‐derived chondrocytes using a temperature‐ responsive polymer, and a gelatin manipulator as a transfer carrier. The properties of the cell sheets including sizes, expression levels of collagen type II and I, and the localization of the stress fibers were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue‐like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

3. Objective Methods Future study
To construct a multi-layered chondrocyte cell sheet for the treatment of articular cartilage defects
Cartilage harvest
Methods
SIRB (COA no. Si387/2015)
Future study In

4. Cell Sheet Using Temperature-Responsive Polymer
Methods
Cell Sheet Using Temperature-Responsive Polymer
Gelatin manipulator

5. Results
Quantitative RT‐PCR analysis of (A) collagen type II and (B) collagen type I expressions
in human chondrocyte cells sheet harvested

6. Sizes of the chondrocyte cell sheets harvesting using and without using a gelatin manipulator
Results
1‐layered
cell sheets
3‐layered
cell sheets

7. Results

8. Conclusion
Chondrocytes obtained from patient‐derived cartilage were successfully used to construct multi‐layered cell sheets that were manipulated with and without gelatin discs to maintain size and shape once detached from culture dishes.
Changes of cell shape in the chondrocyte cell sheets affected the production of chondrogenic markers.
Without using a gelatin manipulator, a shrinkage of chondrocyte cell sheet occurred. However, most of these cells exhibited significantly up‐regulation of collagen type II.
The evidence that such a cell sheet manipulated without a gelatin manipulator, exhibiting a tissue‐like structure, may offer high clinical potential in autologous chondrocyte implantation to treat cartilage defects.