Increasing the Survival Rate of Manipulated Cells

Gentle Single Cell Manipulation

  • © dra_schwartz© dra_schwartz
  • © dra_schwartz
  • Fig. 1: Fluorescence/whitelight overlay time-lapse images. Observation of single cells injected with dextran-Alexa 647 by a 100 nm pipette during proliferation. “p” marks parental cells, the arrows indicate a proliferation event and “d” is for the corresponding daughter cells. Images were acquired every 10 min at an integration time of 200 ms and 10ms for the red (632 nm) and whitelight excitation respectively. Scale bar, 20 µm.
  • Fig. 2: Statistics of cell status 24 hours after the injection of Dextran Alexa Fluor (DAF) with a 100 nm and 500 nm pipette into the nucleus and cytoplasm. (a) shows the viability after the different experiments after 24 h. For cytoplasmic injection it is 92% (N=68) for 100 nm and 40% (N=50) for 500 nm. Injection into the nucleus leads to a viability of 85% (N=71) and 36% (N=50) respectively. (b)Comparison of the surviving cells only, with regard to the proliferation percentage. 81% (N=116) of the nanoinjected cells divided, whereas only 47% (N=36) of the cells treated with the 500nm pipette showed proliferation.
  • Fig. 3: Statistics of cell viability 24 hours after injection with two different voltages and timings. The ‘injection’ was carried out with nanopipettes (100 nm), loaded only with PBS. Approached and inserted into the cytoplasm of single living cells, the pipette was left inside each cell either for 1 min or 5 min. The applied voltage was set to either 1 V or 0.5 V. Afterwards the ‘injected’ cells were observed for 24 h. The result shows that for 1 min ’injection’ time the voltage has no impact on viability with 94% (N=35) for 1V and 97% (N= 32) for 0.5 V. After 5min however, the difference clearly shows that only 49% (N=26) of cells treated with 1 V survived the ‘injection’ whereas the viability at 0.5 V is at 85% (N= 17).
The investigation of living cells is of high relevance in the biosciences. To observe intracellular structures of living cells, specialized tools in combination with fluorescent probes can be used, which grant access to their complex machinery. A possible strategy to investigate these intracellular structures of single living cells is to combine specialized tools with functionalized fluorescent probes. These tools overcome the plasma membrane of a living cell by penetration with pipettes or cantilevers to deliver the fluorescent probes. But these tools affect the survival of a cell significantly. 
 
Introduction
 
Single cell manipulation is of high interest in the biosciences, either by generating external stimuli, by injection of fluorescent probes or other molecules of interest or extraction of an intracellular sample. Single cell manipulation allows to follow the reaction of a specific cell, determine the formation of intracellular structures, visualize intracellular processes or enable the direct analysis of intracellular specimen. It also allows visualizing intercellular communication by e.g. exchange of molecules within an ensemble of cells. So far, only a few available tools for cellular manipulation enable genuine single cell resolution, e.g. microinjection, fluidFM [1], the attosyringe [2] and nanoinjection [3]. Using these tools, various tasks can be fulfilled at the single cell level. But all these tools have one thing in common: They are penetrating tools, which physically penetrate the outer cellular plasma membrane by either a hollow glass pipette or a cantilever. This penetration process influences the survival probability of each manipulated cell, as the plasma membrane is disrupted at a defined area and subsequently has to be repaired by the cell. Thus, there is a need to develop precise tools and carry out single cell manipulation as gentle as possible, and - in the optimal case - with no (measurable) effect on the cell or cellular behavior.
 
Precise Manipulation of Single Cells 
 
Recently, the nanoinjection tool for delivery of fluorescent molecules was developed.

It employs a small hollow glass capillary with a diameter of approx. 100 nm at the tip of the so-called nanopipette. In combination with a precise axial feedback system generated by a low ionic current flowing between two electrodes inside the nanopipette and the bath solution, precise positioning of the nanopipette tip inside the cell is possible. This feedback system enables the tool to deliver molecules precisely to subcellular compartments, e.g. the nucleus of a living cell. Ejecting molecules out of the nanopipette by (di-)electrophoretic forces, this tool is also capable of monitoring the labeling process of a single cell in real-time by a fluorescence microscope. This allows the user to ensure a proper and sufficient labeling of the target cell. Nanoinjection also allows the injection of different fluorescent probes within a single injection step to visualize various intracellular structures successively or in parallel. As nanoinjection has several advantages for the precise manipulation of single cells, a long-term observation to determine the effect of the penetrating nanopipette and subsequent injection step on living cells was still missing.

 
Cell Survival after Nanoinjection
 
Now, a detailed study on the survival of living cells which were manipulated by nanoinjection was carried out, comprising over 300 single injected cells. The cells were injected by nanopipettes with a typical diameter of approx. 100 nm. For comparison, additional injection was carried out with larger microinjection pipettes (typically 500 nm). Both pipette types were used to inject a specific amount of fluorescently labeled dextran, which is known to be live cell compatible and even remains inside the cells upon proliferation, allowing to track and investigate the behavior of single injected cells over at least 24 h (fig. 1). The injection was carried out on a standard wide-field fluorescence microscope with human osteosarcoma (U2OS) cells. The cells were given at least 24 h to settle down prior to the measurements. For each injection experiment, the cells were placed on a temperature controlled microscope stage, heated to 37°C. Beforehand, the culture medium (DMEM) was replaced with pre-warmed PBS. A typical injection of the dextran-Alexa 647 conjugate to a single cell was carried out typically in 5-10 s, at very low laser illumination intensity, which was used to confirm the successful delivery of the dextran. The injection experiments were performed for a maximum time of one hour per culture dish. After the experiment, the PBS was replaced with pre-warmed culture medium and the cells were put back in the incubator. After 24 h, the cells were investigated to determine the survival rate and the proliferation rate of injected cells. It turns out, that a decreased tip diameter of 100 nm of the injection pipette has a tremendous effect on the survival rate of injected cells. Cells which were injected by 500 nm pipettes have a survival probability of 40%, whereas the survival probability of cells injected by smaller nanopipettes with a tip diameter of 100 nm increases to 92%. Additionally, the proliferation behavior of survived cells was investigated, representing another crucial indicator for the health state of a cell. A proliferation rate of 81% was observed within 24 h among the survived cells injected by a 100 nm nanopipette and in contrast a rate of 47% for cells injected by a 500 nm pipette (fig. 2). As not only the pure penetration of the nano- and micropipette could influence the survival of injected cells, also the strength of the electric field generated during the ejection of molecules was investigated with regards to the survival rate of injected cells. Applying a voltage of 0.5 V or 1 V for 1 min to a cell during injection results in no measurable effects on the survival rate. At 0.5 V for 5 min, the survival rate decreases to 85%. Applying 1 V for 5 min lead to a decrease in the survival rate to 49 % (fig. 3). As the typical injection for dextran was carried out in typically 5-10 s, the influence of the electric field can be neglected for the previously described survival experiments.
 
As many factors influences the survival and behavior of single living cells during the observation with fluorescence microscopes (e.g. the labeling itself [4] and the laser illumination power [5]) it is crucial to minimize the overall stress of investigated cells. This will pave the way for reliable results, originating from living cells. As a precise manipulation of single cells combined with a very high survival rate of injected cells can be achieved, nanoinjection offers excellent conditions for single cell manipulation. Furthermore, as the method is single cell sensitive and the influence of the electric field is restricted to a confined area, other unaffected cells within the ensemble can serve as a control group for single cell experiments, increasing the reliability of findings in relation to the control group and therefore making further experiments unnecessary. This will in conclusion lead to new experimental layouts in the field of biosciences.
 
Authors
Matthias Simonis1 and Simon Hennig1
 
Affiliation
1 Bielefeld University, Faculty of Physics, Biomolecular Photonics, Bielefeld, Germany 
 
Contact
Dr. Simon Hennig
Bielefeld University
Biomolecular Photonics
Bielefeld, Germany
shennig@physik.uni-bielefeld.de
 

References
[1] Guillaume-Gentil, O. et al.; Tunable Single-Cell Extraction for Molecular Analyses; Cell 2016, 166, 2, 506 – 516; DOI: 10.1016/j.cell.2016.06.025
[2] Laforge, F.O. et al.; Electrochemical attosyringe; PNAS 2007, 104 (29), 11895-11900; DOI: 10.1073/pnas.0705102104
[3] Hennig, S. et al.; Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes; Nano Lett., 2015, 15 (2), pp 1374–1381, DOI: 10.1021/nl504660t
[4] Alford, R. et al.; Toxicity of Organic Fluorophores Used in Molecular Imaging: Literature Review; Mol Imaging. 2009 Dec;8(6):341-54; DOI: 10.2310/7290.2009.00031
[5] Wäldchen, S. et al.; Light-induced cell damage in live-cell super-resolution microscopy; Sci. Rep. 2015, 5, 15348;  DOI:10.1038/srep15348

 

 

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