Abstract
OBJECTIVES: To isolate liver-derived mesenchymal stem cells (L-MSCs) from neonatal rats using tissue explant method combined with enzymatic digestion. METHODS: Liver tissues aseptically harvested from 1-week-old SD rats were rinsed, minced, and digested with D-Hanks solution containing 0.1% type-II collagenase and 0.05% type-IV collagenase for 15-20 min. After filtration through a 100-mesh screen, the cells were cultured for primary L-MSC isolation. The target cells were identified by morphological observation, flow cytometric analysis of surface CD markers, and adipogenic/osteogenic differentiation assays. RESULTS: Six to eight days after primary seeding, short spindle-shaped adherent cells formed a confluent monolayer arranged in a swirling pattern. Flow cytometry of passage-4 cells showed high expressions of CD90 [(99.33±0.06)%], CD73 [(99.60±0.10)%], CD44 [(99.50±0.10)%], and CD29 [(97.60±0.17)%] with an average positive rate of (99.01±0.86)% and low expressions of CD45 [(0.87±0.12)%], CD34 [(0.95±0.22)%], and CD11b/c [(1.71±0.28)%] with an average positive rate of (1.18±0.44)%, which was significantly lower than that of CD90, CD73, CD44, and CD29 (P<0.01), consistent with the immunophenotypic characteristics of MSCs. Both adipogenic and osteogenic induction tests of the cells yielded positive results. CONCLUSIONS: L-MSCs can be successfully isolated from neonatal rat liver using tissue explant method combined with enzymatic digestion.