Adipose Derived-Mesenchymal Stem Cells Viability and Differentiating Features for Orthopaedic Reparative Applications: Banking of Adipose Tissue

Abstract

Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at -80°C and -196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at -80°C and -196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.

Figure 1

Local anesthetic did not interfere with adipose tissue morphology. H&E staining showed a similar morphology of adipose tissue harvested with or without local anesthetic. No evidence of adipose tissue degeneration or necrosis was evident (a, b). IHC analysis showed comparable staining for CD68 and CD31, in both samples harvested with or without anesthetic (c–f).

Figure 2

Local anesthetic reduced ATD-MSCs growth. (a) Box and whisker plot showed a significant difference in the number of cells isolated from adipose tissues harvested with or without anesthetic. (b) The regression analysis showed that, after 7 days, the number of cells grown in culture was reduced when the anesthetic was added to the solution of infiltration, particularly in relation to the increasing of the patients' age, p < 0.001.

Figure 3

DMSO cryopreserved adipose tissue better than trehalose. ATD-MSCs isolated from DMSO cryopreserved lipoaspirates showed a better morphology compared with trehalose ((a) and (b)). The IHC staining for CD31 (line (c)) and CD105 (line (d)) showed that the different cellular structures were better preserved in samples cryopreserved with DMSO than with trehalose.

Figure 4

Adipose tissue is better preserved at −80°C than −20°C. The samples cryopreserved at −20°C (b) showed a damaged structure compared to fresh (a) and −80°C (c).

Figure 5

Cryopreservation at −80°C and −196°C guaranteed viability of adipose tissue and ATD-MSCs. The percentages of viability of adipose tissue normalized on fresh sample are reported (a). 7-AAD staining showed a significant increase of dead cells in cryopreserved compared to fresh samples, p < 0.05, but there was not any difference between the two temperatures of cryopreservation (b).

Figure 6

Cryopreservation at −80°C and −196°C maintained morphology of adipose tissue and the content of ATD-MSCs. The morphology of cryopreserved adipose tissue at −80°C and −196°C was maintained, without evidence of tissue degeneration or necrosis, comparable to the fresh sample (a–c). The percentage of CD105, CD44, and CD73+ ATD-MSCs and the CD271+ subpopulation was comparable to fresh samples and also between the two temperatures (d, e).

Figure 7

ATD-MSCs differentiating abilities in vitro were preserved at both temperatures of cryopreservation. After cell culture, ATD-MSCs assumed the typical fibroblast-like morphology (a) and expressed CD105, CD44, CD73, and CD271 (b–e). The immunophenotype of MSCs expressing CD105/CD44/CD73 and of the subpopulation CD105/CD44/CD73/CD271+ cells was comparable with both the temperatures of cryopreservation (f, g). Chondrocytes (h, i) and osteoblasts (j, k) formed from both adipose tissues cryopreserved at −80°C and –196°C. von Kossa staining showed that osteoblasts had comparable ability to mineralize at both temperatures (l, m).