hBMSCs的生物打印初步研究

PRELIMINARY STUDY ON BIOLOGICAL PRINTING OF hBMSCs

目的初步确立hBMSCs的二维生物打印方法,实现对细胞喷射过程的控制并保持打印后细胞活力。方法取健康志愿者骨髓5mL,常规培养hBMSCs至第2代,调整为1×106个/mL单细胞悬液。实验分3组:打印组1细胞先行碘化丙啶(propidium iodide,PI)荧光标记,快速成型组织打印机进行二维细胞打印,x轴间隔300μm,y轴间隔1500μm,激光共聚焦显微镜观察。打印组2细胞未行PI标记,经生物打印后培养2h,Live/Dead viability Kit测定细胞活力,激光共聚焦显微镜观察细胞荧光染色情况;取1份未经Live/Dead viability Kit测试的打印组2细胞,培养7d后倒置显微镜观察细胞形态,贴壁后常规培养,动态观察细胞生长状态。对照组除细胞悬液不行打印,其余操作同打印组2。结果打印组1细胞激光共聚焦显微镜观察,"细胞墨滴"在二维组织中规则且均匀分布,满足二维设计细胞打印要求;每个"细胞墨滴"包含细胞15～35个。打印组2细胞经活力测试,细胞孵育30min后激光共聚焦显微镜观察显示细胞荧光染色情况。对照组细胞活力与打印组2无明显区别。打印后细胞常规培养7d,细胞可正常贴壁生长,形态、生长状态良好。结论通过生物打印技术可实现hBMSCs在二维平面上的定向、定量规则分布,并为进一步的细胞三维打印乃至器官打印体系奠定基础。

Objective To establ ish a two-dimensional biological printing technique of hBMSCs so as to control the cell transfer process and keep cell viabil ity after printing. Methods Bone marrow （5 mL） was obtained from healthy volunteer. The hBMSCs were regularly subcultured to harvest cells at passage 2, which were adjusted to the single cell suspension at a density of 1 × 106/mL. The experiment was divided into 3 groups： printing group 1 in which cells underwent propidium iodide （PI） fluorescent label ing, then were transferred by rapid prototype biological printer （interval in x-axis 300 μm, interval in y-axis 1 500 μm）, and laser scanning confocal microscope was appl ied to observe cell fluorescence; printing group 2 in which cells received no PI label ing and were cultured for 2 hours after transfer, Live/Dead viabil ity Kit was adopted to detect cell viabil ity and laser scanning confocal microscope was appl ied to observe cell fluorescence; half of the cells in printing group receiving no Live/Dead viabil ity Kit detection were cultured for 7 days, then inverted microscope was used to observe cell morphology, routine culture was conducted after the adherence of cells, the growth condition of cells was observed dynamically; control group in which steps were the same as the printing group 2 except that cell suspension received no printing. Results Laser scanning confocal microscope observation on the cells in printing group 1 revealed the ＂cell ink droplets＂ were distributed regularly and evenly in the two-dimensional layer and each contained 15-35 cells, meeting the requirement of designing two-dimensional cell printing. The cells in printing group 2 went through cell viabil ity test, laser scanning confocal microscope observation showed the fluorescence of cells 30 minutes after cell incubation. There was no significant difference between the control group and the printing groups in terms of cell viabil ity. The printed cells presented normal adherence, good morphology and good growth state 7 days after routine culture. Conclusion Biological printing technique can real ize the oriented, quantificational and regular distribution of hBMSCs in the two-dimensional plane and lays the foundation for the construction of three-dimensional cell printing or even organ printing system.