Baumgart J, Bintig W, Ngezahayo A, Lubatschowski H, Heisterkamp A
Laser Zentrum Hannover eV, Hollerithallee 8, Hannover, Germany.
Opt Express. 2010 Feb 1;18(3):2219-29. doi: 10.1364/OE.18.002219.
Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.
基于飞秒激光的光穿孔技术是一种将基因导入干细胞或原代细胞等敏感细胞的温和方法。其高选择性和对细胞的低损伤导致了高转染效率。然而，由于细胞内外介质的交换以及激光照射导致生物分子结构的解体，存在一些会给细胞带来应激的副作用。此外，光转染的机制仍不清楚。在本文中，我们展示了我们对细胞手术过程中，特别是激光诱导膜穿孔过程中钙（Ca(2+)）稳态的研究。我们表明，对细胞的操作可导致胞质Ca(2+)浓度升高。如果在没有细胞外钙的情况下对细胞进行操作，则不会观察到这种升高，这表明Ca(2+)摄取的重要性。我们发现，细胞外Ca(2+)的摄取强烈依赖于激光脉冲的重复率和照射时间。暴露于kHz脉冲数秒甚至会诱导Ca(2+)诱导的Ca(2+)释放。根据穿孔的位置，可能在细胞内钙库附近，可诱导瞬时细胞内Ca(2+)释放。由于Ca(2+)可能参与细胞手术的负面副作用，我们建议在名义上无Ca(2+)的外部溶液中应用光穿孔技术。
