Abstract: BACKGROUND: Periprostatic adipose tissue is the white visceral adipose tissue that closest to the prostate, which is part of the prostate cancer microenvironment and plays a key role in the occurrence and progression of prostate cancer.
OBJECTIVE: To investigate the ability of adipose-derived stem cells derived from periprostatic adipose tissue to form three-dimensional cell sheets.
METHODS: Periprostatic adipose tissue was harvested from patients undergoing radical prostatectomy. Adipose-derived stem cell suspensions were prepared using a combination of enzymatic digestion and mechanical dissection. Adipose-derived stem cell proliferation was assessed using a CCK-8 assay. Expression of stem cell-associated antigens CD34/CD44/CD45/CD90/CD105 was determined by flow cytometry. Multidirectional differentiation potential of the stem cells was assessed using osteogenic/adipogenic/chondrogenic differentiation assays. Adipose-derived stem cells were cultured for three weeks in low-glucose DMEM supplemented with 100 μg/mL vitamin C and 10% fetal bovine serum to form cell sheets. Histological analysis and scanning electron microscopy were performed. 
RESULTS AND CONCLUSION: (1) The cell morphology of periprostatic adipose-derived stem cells was long spindles or long spindles, grew in the same direction and relatively consistent. After 9-10 days of primary culture, 95% of the cells were fused, and the cell activity was good. No obvious signs of cell senescence were seen even when the cells were passed through passage 15. (2) The expression rates of CD44, CD90, and CD105 were 98.24%, 84.99% and 89.14%, respectively. The expression rates of CD34 and CD45 were 0.64% and 1.02%. After 3 weeks of osteogenic/lipogenic/chondrogenic induction, periprostatic adipose tissue-adipose-derived stem cells could be induced into osteoblast, adipoblast, and chondroblast in multiple directions. (3) Continuous culture of periprostatic adipose tissue-adipose-derived stem cells for 3 weeks could form three-dimensional cell sheet tissue. The surface of the sheet was smooth with uniform texture, and it was rich in extracellular matrix, such as fibronectin and type 1 collagen. The results of scanning electron microscopy observation showed that the surface of the cell sheet was smooth and the long spindle-shaped cells were arranged in a consistent direction, with a large amount of extracellular matrix deposited between cells. In this study, adipose-derived stem cells were successfully isolated from prostate cancer patient derived periprostatic adipose tissue. Vitamin C was used to stimulate extracellular matrix secretion, and three-dimensional cell membranes were successfully constructed after three weeks of continuous culture.

Key words: prostate cancer, periprostatic adipose tissue, adipose-derived stem cells, extracellular matrix, cell sheet