Abstract：For mammals, ear is an important auditory organ for the recognition of sound waves and the identification of orientation. Considering that ear size of pig (Sus scrofa) has a very large variation after long-term domestication and artificial breeding, it is an ideal model animal for human auricular disease. So far, the study of pig ear size has mainly focused on the genetic mechanism, yet the research on the pig auricle cells is limited. In this study, using enzymatic method, cells in cartilage of pig ear was isolated and cultured in vitro. The cells in cartilage of pig ear were continuously subcultured and detected their size and distribution during the 3~10 passage by flow cytometry, respectively. The cells in cartilage of pig ear were mainly devided into fibrillar type (P1) and oval type (P2). P1 was of bigger size account for the vast majority of the cells (69.4%), and P2 was the minority (11.0%). Then the combination capacity of the 2 kinds of cells was detected using the positive markers of cartilage stem cells CD44 and CD90. The results showed that cells P1 could hardly combine with CD44, cells P2 had a strong combination capacity with CD44. However, both P1 and P2 could bind with CD90, which the binding capacity of P2 was slightly stronger than P1. According to the analysis results, pig auricle cells were comprised by chondrocytes (P1) and cartilage progenitor cells (P2). Subsequently, in order to study the quantity and physiological state changes of P1 and P2 cells during passages, the cells were incubated with FITC-Annexin V/PI to analyze apoptosis using flow cytometry. The result showed that apoptosis was a common phenomenon in the pig ear cartilage cells cutured in vitro from the 3~6 passages with a rising trend of apoptosis, and after the 6 generations, the situation of apoptosis was basically constant. Further apoptosis analysis result showed that P1 cells were only a small amount of apoptosis or necrosis, with cell viability of (96.61±1.32)%, indicating that P1 cell could maintain a stable state cultured in vitro. However, living cells accouts ＜16.5% in P2 cells from the 3~10 generation, and more than 83% of P2 cells were in the late stage of apoptosis or necrosis. This suggested that the P2 cells in the adult porcine ear were prone to apoptosis under in vitro culture conditions. As the number of passages increased and the time of culture prolonged, the proportion of P2 cells in primary cells decreased significantly. These limitations clearly illustrated the need to develop cell culture conditions which enabled prolonged expansion of cartilage progenitor cell. Meanwhile, the results also are valuable to the research on pig auricle as a model for human elastic cartilage regeneration medicine in the future.
[1]Alasti, F.Van CampGenetics of microtia and associated syndromes[J].J Med Genet, 2009, 46(6):361-369[2]Corsi, A, Riminucci, M.Fisher,LW. et al..Achondrogenesis type IB: agenesis of cartilage interterritorial matrix as the link between gene defect and pathological skeletal phenotype[J].Arch Pathol Lab Med, 2001, 125(10):1375-1378[3]Dickhut, A, Pelttari, K.Janicki,Pet al..Calcification or dedifferentiation: requirement to lock mesenchymal stem cells in a desired differentiation stage[J].J Cell Physiol, 2009, 219(1):219-226[4]Gerter, R, Kruegel, J.Miosge,N.New insights into cartilage repair — The role of migratory progenitor cells in osteoarthritis[J].Matrix Biology, 2012, 31(3):206-213[5]Hsu, PD.Lander,ES. & Zhang,F..Development and applications of CRISPR-Cas9 for genome engineering[J].Cell, 2014, 157(6):1262-1278[6]Ka, C, W.O. B& I.,V. D. M. P..The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels[J].Journal of Orthopaedic Research, 2004, 22(2):446-455[7]Kobayashi, S, Takebe, T.Inui,Met al..Reconstruction of human elastic cartilage by a CD44+ CD90+ stem cell in the ear perichondrium[J].Proc Natl Acad Sci U S A, 2011, 108(35):14479-14484[8]Ren, J, Duan, Y.Qiao,Ret al..A missense mutation in PPARD causes a major QTL effect on ear size in pigs[J].PLoS Genet, 2011, 7(5):e1002043-e1002053[9]Yang, L, Guell, M.Niu,Det al..Genome-wide inactivation of porcine endogenous retroviruses (PERVs)[J].Science, 2015, 350(6264):1101-1104
