In vitro neuromuscular junction functionality

Abolition of contractile activity in in vitro neuromuscular junctions within multi-nodal microfluidic chips using alpha-Bungarotoxin. Chips seeded as described above were allowed to mature for 21 days after seeding. Chips were selected for high contractile activity of myotubes that had visual contact with axons (more than 1 contraction per minute over 5 minutes with at least 1 contraction in each minute, the day prior to the experiment). Video of pre-experiment activity was recorded for 1 min followed by counting of the total number of contractions in 10 min. The cells were allowed to recover for 1 h at 37°C, 5% CO2 before addition of 1:100 of α-BTX at a final concentration of 1.25μM or of 0.75μl sterile PBS and incubation of 10 min at 37°C, 5% CO2. The α-BTX was only added to the central well containing the myotubes, which was fluidically isolated by hydrostatic pressure throughout the incubation. Another 1 min video of activity after intervention was recorded before counting the total number of contractions in 10 min. Cells were fixed immediately after the final count. For quantitative analysis the experiment was repeated with 10 min video being recorded as baseline followed by 1 h recovery at 37°C, 5% CO2 and treatment. Another 10 min video was recorded after treatment, and blinded quantification was carried out for both.

采用α-银环蛇毒素（alpha-Bungarotoxin）消除多节点微流控芯片（multi-nodal microfluidic chips）内体外神经肌肉接头（neuromuscular junctions）的收缩活性。按前述方案接种的芯片于接种完成后成熟培养21天。实验前一日，选取肌管（myotubes）与轴突（axons）形成可视性接触且收缩活性较高的芯片：在5分钟观测期内，每分钟至少出现1次收缩，且平均每分钟收缩次数大于1次。记录实验前1分钟的活动视频，随后统计10分钟内的总收缩次数。将细胞置于37℃、5%CO₂培养条件下恢复培养1小时，随后分别加入按1:100比例稀释、终浓度为1.25μM的α-银环蛇毒素，或0.75μl无菌磷酸盐缓冲液（PBS），并于37℃、5%CO₂条件下孵育10分钟。α-银环蛇毒素仅添加至承载肌管的中央孔道，孵育全程通过静水压（hydrostatic pressure）实现该孔道的流体隔离。干预完成后再次录制1分钟的活动视频，随后统计10分钟内的总收缩次数。完成最终计数后立即对细胞进行固定。为开展定量分析，本实验重复设置如下流程：以10分钟视频作为基线数据，随后于37℃、5%CO₂条件下恢复培养1小时并施加对应处理；处理结束后再次录制10分钟视频，随后对两组样本开展盲法定量分析。