多种物理方法分离脂肪源基质血管组分细胞的比较

Separation of adipose-derived stromal vascular fraction cells by a variety of physical methods: a comparative study

背景:脂肪源基质血管组分和脂肪源性干细胞在组织工程中的应用受到越来越多科研工作者的关注。当前,分离脂肪源基质血管组分的方法主要有酶解法和推注法,但这两种方法都存在着不容忽视的缺点。目的:寻找一种更加高活性、安全、简便的制备脂肪源基质血管组分的方法。方法:以无任何处理的脂肪组织为阴性对照,酶解法为阳性对照,通过细胞量、存活率、细胞碎片、细胞活性、增殖率等指标来比较酶解法、普通推注法、改良推注法、玻璃珠破碎法(简称玻璃珠法)及内置式超声波破碎法(简称内置超声波法)的差异。酶解法及普通推注法为目前分离脂肪源基质血管组分细胞普遍使用的方法;改良推注法是在普通推注法的基础上进行改良后得到的方法;玻璃珠法是利用玻璃珠震荡产生的剪切力,在脂肪颗粒中加入玻璃珠后在2500 r/min的条件下震荡9 min以制备基质血管组分细胞;内置超声波法则是利用空化效应,在25 W的功率下对脂肪组织处理36 s以获得基质血管组分细胞。结果与结论:①5种方法获得的基质血管组分细胞的大小差异无显著性意义(P>0.05);②阴性对照组细胞活性最低,酶解法细胞活性最高,酶解法、玻璃珠法及内置超声波法的细胞活性高于改良推注法和普通推注法(P<0.05);③酶解法、玻璃珠法及内置超声波法的细胞存活率、细胞增殖率高于改良推注法和普通推注法(P<0.05);④酶解法、玻璃珠法及内置超声波法细胞碎片比例、细胞凋亡率要远远低于普通推注法和改良推注法(P<0.05);⑤结果表明,玻璃珠法和内置超声波法富集基质血管组分细胞的效果优良,但玻璃珠法加入了外源物质进行处理,增加了污染的风险,而内置超声波法尽管将超声探头插入脂肪组织中,但只要将超声探头彻底灭菌,即可将污染风险降到最小。总的来说,内置超声波法和玻璃珠法优于普通推注法及改良推注法。

BACKGROUND:Increasing attention has been paid to vascular components of the adipose-derived matrix and adipose-derived stem cells in tissue engineering.Existing methods for separating the vascular components of the adipose-derived matrix mainly include enzymatic and bolus injection,both of which have fatal disadvantages.OBJECTIVE:To search for a method for preparing adipose-derived stromal vascular fractions with high efficiency,safety,and simplicity.METHODS:The group without any treatment was used as the negative control,and the enzymatic hydrolysis method served as the positive control.The enzymatic hydrolysis method,traditional bolus method,modified bolus method,glass beads method and built-in ultrasonic waves method were compared through cell volume,survival rate,cell fragments,cell viability,increment rate and detection of microbial infection.The enzymatic hydrolysis method and the common bolus injection method were commonly used in the separation of vascular component cells of the fat source matrix;the improved bolus method was a method obtained by improving on the basis of the ordinary bolus method;the glass bead method was to use the glass bead to oscillate.The shear force generated was obtained by adding glass beads to the fat granules and shaking at 2500 r/min for 9 minutes to prepare stromal vascular fraction cells.Using the built-in ultrasonic method,adipose tissue was treated at 25 W for 36 seconds to obtain stromal vascular fraction cells through a cavitation effect.RESULTS AND CONCLUSION:(1)The size of stromal vascular fraction cells isolated by five methods showed no significant difference(P>0.05).(2)The cell viability was lowest in the negative control group,and highest in the enzymatic hydrolysis group.The cell viability in the enzymatic hydrolysis,glass bead,and built-in ultrasonic wave groups was significantly higher than that in the modified and traditional bolus groups(P<0.05).(3)The cell survival rate and cell proliferation rate in the enzymatic hydrolysis,glass bead,and built-in ultrasonic wave groups were significantly higher than those in the modified and traditional bolus groups(P<0.05).(4)The cell fragmentation rate and cell apoptosis rate in the enzymatic hydrolysis,glass bead,and built-in ultrasonic wave groups were significantly lower than those in the modified and traditional bolus groups(P<0.05).(5)These results indicate that the built-in ultrasonic method and the glass bead method are better in enriching vascular components of the adipose-derived matrix.But glass bead method adds exogenous products,so it increases the risk of pollution.Built-in ultrasonic method inserts the ultrasound probe into the adipose tissue,but as long as the ultrasound probe is thoroughly sterilized,the risk of contamination is minimized.In general,the built-in ultrasonic method and the glass bead method are superior to modified and traditional bolus methods.