3D生物打印兔肺成纤维细胞水凝胶结构体片段

3D bioprinting hydrogel structure lamella of rabbit's lung fibroblasts

目的通过三维(3D)生物打印兔肺成纤维细胞-海藻酸钠-明胶共混物,探讨3D生物打印技术构建肺成纤维细胞水凝胶结构体片段的可行性。方法用轻度Ⅳ型胶原酶消化结合组织块培养法分离纯化新生兔肺成纤维细胞,并对分离纯化的肺成纤维细胞进行原代和传代培养;制备海藻酸钠-明胶水溶胶,与兔肺成纤维细胞共混;用圆柱状微丝逐层交错堆积技术,进行兔肺成纤维细胞-海藻酸钠-明胶共混物的3D生物打印,获得三维结构体。用钙黄绿素-AM和碘化丙啶溶液对该结构体进行活/死细胞双荧光染色,评价打印后及培养后兔肺成纤维细胞的活性,计算细胞存活率。结果通过按照设计的参数,打印出含有兔肺成纤维细胞的网格状水凝胶结构体。该结构体的大小约为9 mm×9 mm×1.2 mm,微丝间的孔隙相互通连,孔隙大小约600μm×600μm。打印后的兔肺成纤维细胞在三维结构体中仍可增殖,活/死细胞荧光染色结果表明结构体中兔肺成纤维细胞的存活率约为84%±3%,兔肺成纤维细胞在结构体中分布均匀。结论本实验实现了兔肺成纤维细胞共混物结构体的3D生物打印,为构建类肺组织工程结构体片段提供新思路。

Objective To construct three dimensional（ 3 D） structure lamella containing rabbit＇s lung fibroblasts/alginate/gelatin blend by 3 D bioprinting technique. Methods Rabbit lung fibroblasts were isdated from lung tissue pieces of newborn rabbit using collagenase digestion combined with tissue pieces culture method. Alginate and gelatin were mixed to form hydrosol,and rabbit lung fibroblasts were added into the hydroso1. A 3 D bioprinting system was used to construct 3 D printed lung-like structures using rabbit lung fibroblasts-alginate-gelatin blend. The likelung hydrogel structures were cultured,and the cells＇ survival rate was detected by live/dead cells double fluorescence staining. Results The 3 D structures with the grid hydrogel composed of multilayer staggered cylindrical microfilament were obtained. The size of the structure was about 9 mm × 9 mm × 1. 2 mm,and pore size was about600 μm× 600 μm. The results of live/dead cells by fluorescence staining showed that the lung fibroblasts survival rate was 84% ± 3% after printing. Conclusions 3 D bioprinting of lung fibroblasts is feasible,which provides amovo approach to construct lung-like tissue engineering.