To compare three different methods for the isolation of cartilage-derived stem/progenitor cells (CSPCs) in rabbits auricula.

Auricular cartilage was harvest from 4-month-old healthy rabbits during January to December 2019, and chondrocytes were obtained by enzymatic digestion, then randomly divided into 4 groups: CSPCs isolated by fibronectin adhesion and low-density culture method (FN+ LD group), CSPCs isolated by fibronectin adhesion (FN group), CSPCs isolated by low-density culture method (LD group), chondrocytes (CC group). The morphology, mesenchymal stem cells (MSC)-specific surface markers CD44 and CD90, colony formation ability and multiple-lineage differentiation potential were evaluated and compared. Independent sample t test was used for comparison between two groups and variance analysis was used for comparison between groups.

CSPCs isolated by FN+ LD, FN and LD methods showed high CD44 and CD90 expression. FN+ LD and FN groups expressed higher CD44 than LD group [(98.13±0.25)% vs. (95.40±0.43)%, t=3.521, P<0.01 and (97.77±1.33)% vs. (95.40±0.43)%,t=4.148, P<0.05]. FN group showed higher CD90 expression than LD group [(95.47±0.66)% vs. (92.07±1.20)%,t=9.087, P<0.01]. Compared with CC group, the colony formation ability in FN+ LD, FN and LD groups was significantly increased [(15.80±2.68)% vs. (4.80±0.75)%,t=8.751, P<0.01; (20.80±1.64)% vs. (4.80±0.75)%,t=19.400, P<0.01; (8.40±2.61)% vs. (4.80±0.75)%,t=2.939, P<0.05]. The colony formation ability in FN+ LD group turned to be higher than LD group [(15.80±2.68)% vs. (8.40±2.61)%,t=4.422, P<0.01] and lower than FN group [(15.80±2.68)% vs. (20.80±1.64)%,t=3.553, P<0.01]. Compared with CC group, multiple-lineage differentiation potential in FN+ LD, FN and LD groups was significantly increased (66.84±1.72 vs. 59.42±2.20,t=5.939, P<0.01; 69.46±1.95 vs. 59.42±2.20,t=7.630, P<0.01; 63.34±1.44 vs. 59.42±2.20,t=3.330, P<0.01 and 26.34±0.57 vs. 13.40±0.84,t=26.080, P<0.01; 26.41±1.12 vs. 13.40±0.84,t=20.820, P<0.01; 26.58±0.76 vs. 13.40±0.84,t=26.080, P<0.01). The osteogenic differentiation capability in FN+ LD group turned to be higher than in LD group (66.84±1.72 vs. 63.34±1.44,t=3.493, P<0.01) and lower than in FN group (66.84±1.72 vs. 69.46±1.95,t=2.257, P<0.05). The adipogenic differentiation capability in three CSPCs groups had no significant changes (26.34±0.57 vs. 26.41±1.12 vs. 26.58±0.76,F=0.107, P>0.05).

Fibronectin adhesion with low-density culture method, fibronectin adhesion method alone and low-density culture method alone could isolate CSPCs, and the difference of cell surface marker, self-renewal and differentiation ability was influenced by the isolation method.

Cartilage-derived stem/progenitor cells; Differential adhesion; Low-density culture; Self-renewal; Multiple-lineage differentiation