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. 2018 Jul 5;19(7):1963.
doi: 10.3390/ijms19071963.

Isolation of Adipose-Derived Stem/Stromal Cells From Cryopreserved Fat Tissue and Transplantation Into Rats With Spinal Cord Injury

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Free PMC article

Isolation of Adipose-Derived Stem/Stromal Cells From Cryopreserved Fat Tissue and Transplantation Into Rats With Spinal Cord Injury

Yuki Ohta et al. Int J Mol Sci. .
Free PMC article

Abstract

Adipose tissue contains multipotent cells known as adipose-derived stem/stromal cells (ASCs), which have therapeutic potential for various diseases. Although the demand for adipose tissue for research use remains high, no adipose tissue bank exists. In this study, we attempted to isolate ASCs from cryopreserved adipose tissue with the aim of developing a banking system. ASCs were isolated from fresh and cryopreserved adipose tissue of rats and compared for proliferation (doubling time), differentiation capability (adipocytes), and cytokine (hepatocyte growth factor and vascular endothelial growth factor) secretion. Finally, ASCs (2.5 × 10⁶) were intravenously infused into rats with spinal cord injury, after which hindlimb motor function was evaluated. Isolation and culture of ASCs from cryopreserved adipose tissue were possible, and their characteristics were not significantly different from those of fresh tissue. Transplantation of ASCs derived from cryopreserved tissue significantly promoted restoration of hindlimb movement function in injured model rats. These results indicate that cryopreservation of adipose tissue may be an option for clinical application.

Keywords: adipose tissue; adipose-derived stem/stromal cells; cryopreservation; spinal cord injury; tissue bank; transplantation.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Doubling time of adipose-derived stem/stromal cells (ASCs). ASCs derived from fresh or cryopreserved adipose tissue were cultured for up to seven passages. Doubling time (DT) was determined at the indicated number of passages (as described in the Materials and Methods section). Data are expressed as the mean ± standard error (SE) (n = 4). * p < 0.05 vs. Fresh.
Figure 2
Figure 2
Adipogenic differentiation in ASCs. Fresh or cryopreserved adipose-derived stem/stromal cells (Fresh- or Cryo-ASCs) were differentiated into adipocytes (as described in the Materials and Methods section). Adipogenesis was revealed by Oil red O staining for lipid droplets. (−): confluent state without adipogenic induction; (+): 28 days after adipogenic induction.
Figure 3
Figure 3
Osteogenic differentiation in ASCs. Fresh or Cryo-ASCs were differentiated into osteoblasts (as described in the Materials and Methods section). Osteogenesis was revealed by alkaline phosphatase staining. (−): confluent state without osteogenic induction; (+): 21 days after osteogenic induction. Scale bars = 500 µm.
Figure 4
Figure 4
Cytokine secretion from ASCs. P2-ASCs were cultured for four days. Supernatants were harvested for cytokine assay. Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in the culture medium were measured by enzyme-linked immunosorbent assay. Data are expressed as the mean ± SE (n = 4).
Figure 5
Figure 5
Functional recovery following ASC transplantation. On day eight after injury (arrow), 12-week-Cryo-ASCs (2.5 × 106 cells) were infused into spinal cord injury (SCI) rats via the tail vein (n = 8). Basso–Beattie–Bresnahan (BBB) scores were determined before and after transplantation. Black and white circles are ASC and saline group, respectively. The median score is plotted; bars show the inter quartile range (IQR). * p < 0.05 vs. saline controls (n = 10).
Figure 6
Figure 6
Long-term cryopreserved adipose-tissue-derived cells. ASCs were isolated from 24-week-cryopreserved tissues and examined for proliferation and adipogenic differentiation: (A) DT; (B) Oil red O staining. Data are expressed as the mean ± SE (n = 4).

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