Posology and Serum-/Xeno-Free Engineered Adipose Stromal Cells Cell Sheets

Abstract

Well-characterized adipose stem cells and chemically defined culture media are important factors that control the production of the cell sheet, used in translational medicine. In this study, we have developed and engineered multilayer adipose stem cell cell sheets (ASCCSs) using chemically defined/serum-free culture media: undifferentiated or differentiated into osteoblasts and chondrocytes. In addition, using the cell sheet transmittance, we estimated the number of cells per cell sheet. Undifferentiated ASCCSs were engineered in 10 days, using serum-free/xeno-free culture media. They were CD29⁺, CD73⁺, CD90⁺, CD105+, HLA-A+, and HLA-DR-. ASCCSs differentiated into chondrocytes and osteoblasts were also engineered using chemically defined and animal-free culture media, in only 14 days. The addition of an ROCK inhibitor improved the chondrocyte cell sheet engineering. The decrease in the cell sheet transmittance rate was higher for the osteoblast cell sheets due to the intracellular Ca²⁺ accumulation. The estimation of cell number per cell sheet was carried out with the transmittance, which will provide important information for cell sheet posology. In conclusion, three types of ASCCSs were engineered using serum-free, xeno-free culture media, expressing their specific markers. Their transmittance measurement allowed estimating the number of cells per cell sheet, with a non-invasive methodology.

Keywords: cell sheets; mesenchymal stem cells; multilayer; posology; serum-/xeno free; transmittance.

FIGURE 1

(A) Pictures of the cell sheets, during the differentiation time (the scale bars are 200 µm). (B) Pictures of the harvested cell sheets, placed in 35-mm cell culture dishes.

FIGURE 2

Dye of the undifferentiated ASCCSs, chondrocyte cell sheets, and osteoblast cell sheets with Alcian blue and Alizarin red. Scale bars are 150 µm.

FIGURE 3

Immunostaining shows the positive expression of HLA-A, CD29, CD73, CD105, and Oct3/4. The proteins HLA-DR and CD19 were not detected. The scale bars are 150 µm.

FIGURE 4

(A) Immunostaining of chondrocyte cell sheets, with HLA-A⁺, SPARC⁺, collagen II⁺, aggrecan⁺, and HLA-DR^-. SPARC, collagen II, and aggrecan being specific markers of cartilage. (B) Immunostaining on osteoblast cell sheets with osteocalcin, a specific marker of osteoblast. Scale bars are 150 µm.

FIGURE 5

Measurement of undifferentiated, osteoblast, and chondrocyte cell sheets. ASCCSs cultured with skeletal muscle differentiation culture media were used as a control to show the unchanged transmittance over time. Number of days in culture is the number of days after the cells reached confluence (day 4) (number of cell sheets measured per point n = 5, except for days 0, n = 10).

FIGURE 6

(A) Standard curve of total DNA content per number of single adipose stromal cells. The equation y = 17.236x was used to estimate the number of cells from each cell sheet. (B) Graph showing the correlation between the number of cells per type of cell sheet and the transmittance.