SD大鼠脂肪源间充质干细胞分离培养特性及影响因素

Isolation and culture characteristics and influential factors of Sprague Dawley rat adipose tissue-derived mesenchymal stem cells

背景:脂肪源间充质干细胞因为容易大量获得、抽取后残留于供区的脂肪组织仍可扩增、供区的损伤小等优点,成为干细胞生物工程研究的理想种子细胞来源。但是,脂肪源间充质干细胞分离培养有较大困难。目的:探索脂肪源间充质干细胞的分离培养方法及其生长特性。方法:分别取4周龄和10月龄不同性别的SD大鼠腹股沟、肩部和腹腔脂肪组织,经细胞培养液洗涤、离心后接种于细胞培养瓶,放置体积分数5%CO2孵箱中培养,分别于24,48,72h后换液。待细胞密度达到约80%时,用0.25%胰酶-EDTA消化,传代培养。分析SD大鼠的年龄、性别、采集脂肪组织的部位、胶原酶Ⅰ的浓度和消化时间,以及原代细胞的首次换液时间等对脂肪源间充质干细胞培养的影响。结果与结论:原代脂肪源间充质干细胞呈梭形或多角形。随着传代和培养时间的延长,脂肪源间充质干细胞由多边形、成巢分布,逐渐连接成片,伸展为梭形或多角形。培养48h后,含脂滴的细胞数量逐渐增多,但仍有90%的脂肪源间充质干细胞始终不表现向脂肪细胞自发分化的趋势。4周龄雌性SD大鼠腹腔脂肪组织在一定条件下传代培养最佳。提示,从SD大鼠腹腔脂肪组织中分离的脂肪源间充质干细胞可在体外稳定传代,SD大鼠的年龄、性别、采集脂肪组织的部位、胶原酶Ⅰ的浓度和消化时间,以及原代细胞的首次换液时间等均影响脂肪源间充质干细胞的分离培养效果。

BACKGROUND：Adipose tissue-derived mesenchymal stem cells （AD-MSCs） could be obtained easily with abundant quantities; the fat cells left in the donation area have the capacity of proliferation and the injury is pretty mild. Therefore,AD-MSCs have been an ideal source of seed cells for tissue engineering. However,the isolation and culture of AD-MSCs still remain difficulties. OBJECTIVE：To explore the method for isolation and culture of AD-MSCs and observe their growing properties. METHODS：The adipose tissues were obtained from the inguinal groove,shoulder and abdominal cavity of Sprague Dawley rats aged 4 weeks and 10 months. Following washes and centrifugation,specimens were incubated in cell culture flask,and placed in 5%CO2 incubator. The liquid was replaced at 24,48 and 72 hours. At a fusion of 80%,specimens were digested with 0.25% trypsogen-EDTA and passaged. Effects of age,gender,and the region of collecting adipose tissue,collagenase Ⅰ concentration and digestion time,and first time of liquid change on the culture of AD-MSCs were analyzed. RESULTS AND CONCLUSION：Primary AD-MSCs presented spindle or multangular shape. While AD-MSCs passaged and cultured longer,the newborn AD-MSCs,with polygon shape in clusters,joined to pieces and stretched into spindle or multangular shape gradually. A few fat cells appeared after 48 hours in culture and the cell number increased when the culture time was prolonged. However,about 90% of the isolated cells still kept the shape of AD-MSCs. The AD-MSCs could be isolated successfully from the fat harvested from the 4-week female Sprague Dawley rat abdominal cavity,and be passaged stably in vitro. The Sprague Dawley rats＇ age,sex,collection site of adipose tissue,the concentration of collagenase Ⅰ and its digestion time,as well as the time of the first change of medium have an impact on the isolation and culture of AD-MSCs.