Agitation as transfection efficiency booster in in vitro optogenetics
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1
George Mason University, Department of Bioengineering, United States
Motivation/ Background
Optical stimulation of genetically modified neuronal cultures has emerged as a powerful tool for in vitro research application as it allows for the activation of localized groups of neurons with more spatial precision than electrical stimulation. While the localized activation of photosensitized neurons has been demonstrated in numerous publications[1]–[3], the robust, uniform, and reliable transduction of genetic material to the cell culture still appears to pose an obstacle, forcing researchers to establish their own protocols prior to beginning of intended experimentation. Therefore, the purpose of this study was to determine viral transfection efficiency utilizing varying viral concentration and in response to the introduction of mechanical load in form of slow frequency agitation.
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Material and Methods
Cell culture, optogenetic transfection and mechanical load
Neural cells were plated on multi-electrode arrays and multi-wells (E17 mouse, cortical tissue, 100,000 neurons/dish). After 14 days of incubation, cultures were transduced with AAV-CAG-ChR2-YFP at differing concentrations from 2,000 MOI up to 100,000 MOI and divided into 2 agitated groups (at different experimental conditions) and 1 (not-agitated) control group.
Fluorescence Imaging and estimation of fluorescence
Transduction efficiency was quantified by analyzing fluorescence response of cells showing yellow fluorescent protein (YFP) under a fluorescence microscope. As it is nearly impossible to count total number of neurons in high density cultures, we deployed a method based on the count of pixels corresponding to the fluorescence signal in each image.
Electrophysiology and optical stimulation
To rule out negative effects of slow frequency agitation on cell network physiology and health, extracellular recordings were taken of agitated cultures and compared with non-agitated cultures.
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Results
Transfection efficiency
Viral concentrations below 6,000 MOI seem only sufficient to trigger ChR2 expression in 2% to 5% of cells, no matter which additional steps to increase expression rate were taken. Highly saturated concentrations of 100,000 MOI result in higher transfection rates. However, for non-agitated samples of this particular concentration the transduction efficiency was consistent among cultures showing an average efficiency varying from 25% to 30%. More interesting are the achieved values of the agitated samples averaging between 50% to 60% for agitations in incubation and 75% to 80% and agitation at room temperature outside of incubator. While the expression efficiency of culture samples transfected with 8,000 MOI and 10,000 MOI and agitated in incubation remained within similar range of maximal 5% to 6%, the culture samples agitated outside of incubation followed the same trend as the highly saturated concentration samples. The 10,000 MOI samples averaged at 15 % efficiency.
Electrophysiological recording
Comparing baseline activity of transfected non-agitated cultures with transfected agitated cultures indicates no negative effects of slow frequency agitation on network health. We were able to successfully evoke network responses to whole field blue light stimulation (1 to 5 ms long pulses at 400-700 mA), and evoked responses stimulating array area covering 4 center electrodes (1 to 3 ms, rectangular pulses at varying patterns and duration). Figure 2 shows an example raster plot with 8 active channels recorded during optical stimulation (300 seconds long plot).
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Discussion
Comparing agitated cultures to non-agitated ones, we found that agitation has the potential to significantly boost ChR2 expression rates. This is true at least for viral concentrations above 6,000 MOI. Viral concentrations below this value seemed only sufficient to trigger ChR2 expression in 2% - 5% of cells, no matter which additional steps to increase expression rate are taken. While highly saturated concentrations of 100,000 MOI generally result in higher transduction rates, the difference in agitated and non-agitated probes is quite significant (as seen in Fig. 3).
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Conclusion
We presented results providing guidance for optogenetic cell culture studies. Depending on experimental design, whole field or highly localized optical stimulation it could be sufficient and economically more effective to choose the non-saturating viral concentration of 8,000 MOI to 10,000 MOI and still reliably achieve rhodopsin expression efficiencies of 15% and more if coupled with low frequency agitation. On the other hand if the experimental design requires the spatially highly localized stimulation in order to selectively probe network responses or connectivity, it is recommended to use a higher concentrated viral particles dosage of 100,000 MOI. In any case we have shown that the introduction of mechanical load in the form of slow agitation is capable of significantly boosting transfection rates without negatively impacting neural network physiology.
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References
[1] A. El Hady, G. Afshar, K. Bröking, O. M. Schlüter, T. Geisel, W. Stühmer, and F. Wolf, “Optogenetic stimulation effectively enhances intrinsically generated network synchrony,” Front. Neural Circuits, vol. 7, 2013.
[2] M. R. Dranias, H. Ju, E. Rajaram, and A. M. J. VanDongen, “Short-Term Memory in Networks of Dissociated Cortical Neurons,” J. Neurosci., vol. 33, no. 5, pp. 1940–1953, Jan. 2013.
[3] E. S. Boyden, F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth, “Millisecond-timescale, genetically targeted optical control of neural activity,” Nat. Neurosci., vol. 8, no. 9, pp. 1263–1268, Sep. 2005.
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Figure Legend
Figure 1: Fluorescence image of a neuronal cell culture after AAV-CAG-ChR2-YFP transduction. After expression of ChR2 and YFP cells and neurites show a clear fluorescence signal and can therefore be easily identified, which allows the quantification of transfection efficiency.
Figure 2: Raster plot of cell network activity in response to optical stimulation. Red squares indicate phases with applied blue light stimulation (1 to 5 ms long pulses at 400-700 mA).
Figure 3: Comparison of transfection efficiencies for viral particle concentrations of 10k MOI and 100k MOI over time at the 3 test conditions agitation outside incubator, incubated agitation and non-agitated control group.
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Acknowledgements
This work was supported by NSF grant CMMI-1300007.
Keywords:
optogenetics,
channelrhodopsin2,
Transfection efficiency,
Primary cortical neurons
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Kaiser
A,
Jose
S and
Peixoto
N
(2016). Agitation as transfection efficiency booster in in vitro optogenetics.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00016
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Alex Kaiser, George Mason University, Department of Bioengineering, Fairfax, United States, akaiser4@gmu.edu