Stephens Elizabeth H, Carroll Joshua L, Grande-Allen K Jane
Department of Bioengineering, Rice University, Houston, Texas 77251-1892, USA.
J Heart Valve Dis. 2007 Mar;16(2):175-83.
Substantial heart valve research relies on the isolation of valvular interstitial cells (VICs). While a wide variety of conditions have been reported for VIC isolation, the effectiveness of these methods has rarely been compared. It is also likely that valve donor age will influence these valvular tissue dissociation conditions. The study aim was to increase the efficiency and cost-effectiveness of VIC isolation, while taking into account possible differences due to valve donor age.
Aortic valves were obtained from six-month-old (n = 24) and six-week-old (suckling) pigs (n = 45) within 24 h of death. After removal of endothelial cells, the tissues were minced and subjected to a variety of enzymatic digestions for variable lengths of time.
The optimal concentration of collagenase III was determined as 1 mg/ml for six-week-old pigs, and 2 mg/ml for six-month-old pigs. The optimal duration of digestion was 4 h for both ages. The addition of neutral protease (2 mg/ml) further increased yield, while additional DNAse and hyaluronidase had no effect. Yield was not influenced by the volume of enzyme solution, nor the use of previously frozen enzyme solution.
These findings provide age-specific conditions for improving the yield of VIC isolation, which should be of value in experimental studies of valvular cell biology and tissue engineering investigations.
大量心脏瓣膜研究依赖于瓣膜间质细胞（VICs）的分离。虽然已有多种VICs分离条件的报道，但这些方法的有效性鲜有比较。瓣膜供体年龄也可能影响这些瓣膜组织解离条件。本研究的目的是提高VICs分离的效率和成本效益，同时考虑到瓣膜供体年龄可能存在的差异。
在猪死亡后24小时内，从6月龄（n = 24）和6周龄（哺乳仔猪）（n = 45）的猪获取主动脉瓣。去除内皮细胞后，将组织切碎并进行不同时长的多种酶消化处理。
确定6周龄猪胶原酶III的最佳浓度为1 mg/ml，6月龄猪为2 mg/ml。两个年龄段的最佳消化时长均为4小时。添加中性蛋白酶（2 mg/ml）可进一步提高产量，而额外添加DNA酶和透明质酸酶则无效果。产量不受酶溶液体积的影响，也不受使用先前冷冻的酶溶液的影响。
这些发现提供了针对不同年龄的条件，以提高VICs分离的产量，这在瓣膜细胞生物学实验研究和组织工程研究中应具有价值。
