基于微流控的真菌单细胞捕获和培养

Single cell capture and culture of fungi based on microfluidic

【背景】真菌单细胞培养在研究细胞异质性及细胞生长特性等方面十分重要,因此需要建立简单便捷的方法对真菌单细胞进行培养与观察。【目的】基于微流控建立一种真菌单细胞的捕获及培养方法,同时直观地对单细胞进行定位和实时观察。【方法】利用L-edit设计芯片图案并利用等离子键合的方法制备微流控芯片;通过注射泵将红酵母菌溶液及里氏木霉孢子溶液进样以实现单细胞捕获;采用台盼蓝染色法测定酵母细胞的存活率;利用显微镜对酵母单细胞及木霉孢子的萌发、生长、繁殖过程进行观察。【结果】所制备的芯片形状完好,可实现酵母或孢子的单细胞捕获;酵母的捕获率为25.00%±1.38%;分别于0、2、4、6h对酵母进行观察,可看到酵母出芽过程;培养至48h,芯片上酵母细胞的存活率与游离培养条件下的存活率无显著性差异;分别于0、3、6、9 h对单个孢子进行观察,可以看到孢子萌发以及菌丝生长情况,且直至120h菌丝仍在生长。【结论】设计并制备了一种用于真菌单细胞捕获及定位培养的微流控芯片,这是此种芯片在真菌单细胞培养中的首次应用。细胞可在此微流控芯片上正常生长至少2 d,并可实现5 d及更长时间的培养,此方法可对真菌单细胞进行直观、定位的实时观察,有望用于多种微生物单细胞的生理、遗传性状研究,以及原生质体融合育种研究。

[Background] Single cell culture of fungi is very important to study cell heterogeneity and cell growth characteristics. Therefore, it is necessary to establish a simple and convenient method for culturing and observing fungal single cells. [Objective] To establish a method for capturing and culturing the single cell of fungi based on microfluidic. At the same time, localization and real-time observation of single cell will be done. [Methods] L-edit was used to design the pattern and the plasma bonding was used to produce the microfluidic chip. Rhodotorula glutinis solution and Trichoderma reesei spore solution were injected by syringe pump to capture single cell. Trypan blue was used to determine the survival rate of yeast cells. Germination, growth and reproduction of yeast single cells and spore were observed under microscope. [Results] The microfluidic chip was intact and can be used in single cell capture of yeast or spore. The capture rate of the yeast was 25.00%±1.38%. The budding process of yeast cells was observed at 0, 2, 4 and 6 h. There was no significant difference between the survival rate of yeast cultured in chip and in shaking culture until 48 h. The process of spore germination and mycelium growth was observed at0, 3, 6 and 9 h. Moreover, the mycelium kept growing until 120 h. [Conclusion] A microfluidic chip was designed and produced to capture, culture and localization the single cell of Fungi. This is the first application of this kind of chip in the fungal single cell culture. The cell can grow normally in this chip for at least 2 days, and 5 days even longer cell culture could be achieved by this way. The microfluidic chip can achieve intuitionistic localization and real-time observation of fungal single cell. It has the potential to study physiological and genetic characters of a variety of microorganism.