| It is the precondition to harvest seed cells maintaining stable phenotype, highproliferous activity and with broad extensive source when engineered cartilages wereconstructed Ex Vivo to repaire joint cartilage defects and the other cartilage defects.Heterocartilage chondrocytes have many advantages such as extensive source,materials got easily, without extra-suffering compared with autochondrocytes,periosteum cells, marrow menchymal stem cells, embryonic stem cells. In this studywe adopted Guanzhong Black Porcine as experimental animals in study methods ofisolation, culture in vitro and identification of auricular chondrocytes, and we slightlyattempt the method of reconstruction of engineering cartilage in vitro withchondrocytes isolated from auricular chondrocytes In this study chondrocytes were isolated with 0.25% trypsine and 0.2%collagenase type Ⅱ . While chondrocytes were cultured in vitro, morphologicalchanges of chondrocytes were observed through microscope; chondrocytes clone rateswere counted; growth curve was depicted and its secretion of GAG was detected afterstained with acian blue and collagen type Ⅱ was detected after immunostaining withanti-collagen type Ⅱ antibodies. The results showed that Guanzhong Black Porcineauricular chondrocytes could be isolated successfully with trypsine and collagenasetype Ⅱ. When cultured in vitro, chondrocytes presented different shapes includinground, short shuttle-like, multi-angle form. As passed more and more, morphology ofchondrocytes changed into fibre-like, and hypertrophic chondrocytes appearedoccasionally；glycosaminoglycans(GAG) and collagen type Ⅱ secreted less. fibre-like chondrocytes existed more and more after being passed 3 times in vitro, whilemost chondrocytes could maintain short shuttle-like after being passed 5～6 times invitro when cultured with some digested remainder, some secretion vesicles couldobserved in cell plasma. This paper also studied chondrocytes proliferous ability and morphologicalchanges when cultured in media F12 with 10％FBS,5％BFF,10％BFF,20％BFF,5ng/mlEGF,20ng/ml or 40ng/ml. Results proved that BFF could promotechondrocytes proliferation, group II had the best proliferous effect among the 3groups; the difference was prominent (P<0.05). Madium F12 with EGF could alsopromote chondrocytes multiplication; group II exhibits strongest promotingproliferation activity; the difference was extremely prominent (P<0.01). And thedifference was also extremely prominent between the results of BFF group II andEGF group II (P<0.01). And we also attempt to reconstruct engineering cartilage in centrifuge tubewithout scaffold. Four cartilage-like tissues were obtained after culturing for 14 days.Three of them were smashed when fetched with nippers. The residual cartilage-liketissue appeared milk-white, and lustrous slightly, with its diameter at about 3 mm. According to the results, we can draw conclusions as follows: 1. With 0.25% trypsine and 0.2% collagenase type Ⅱ,auricular chondrocytes can be isolated successfully from Guanzhong Black pigs'ears. 2. Co-culture with some digested remainders chondrocytes can maintain normal proliferous ability and stable phenotype similar to physiological condition. 3. BFF could promote chondrocytes proliferation, and keep the normal phenotype. Among the three groups adding BFF at 10% concentration expressed the best proliferous effect. 4. EGF could promote chondrocytes proliferation, adding at 20ng/ml concentration expressed the best proliferous effect, but it couldn't inhibit chondrocytes changed in fibre-like cells. 5. Cartilage-like tissues could be obtained when reconstruction in centrifuge tube without scaffold.
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