NEPA21_v_CUY21EDIT_CUY21SC_Amaxa_BTXECM830

The following is useful information about the NEPA21 system viz-a-viz our old CUY21SC and CUY21EDIT systems and the Amaxa and BTX ECM830 systems.

The attached ‘Output Comparison NEPA21 v BTX’ will give you a clear understanding of how well the NEPA21 outperforms the BTX.

– You will clearly see the following major differences depicted on the graphical illustration of the delivered electrical energy. These differences each individually have a marked impact on the quality and results of your experiments. When taken together, the impact is even more stark

When you compare graphical illustrations for the BTX and the NEPA21, the most obvious point of difference is the confirmation of the delivered Voltage
- Clearly the BTX does not deliver the set voltage parameter
- The NEPA21 delivers precisely the exact set voltage parameter
If you observe the quality of the Squarewave
- It is clear that the NEPA21 delivers a Perfect Squarewave THROUGHOUT the delivered pulse
- It is clear that the quality of the delivered BTX squareware is impaired THROUGHOUT its delivered pulse
When you compare the measured Pulse Intervals
- It is clear that the NEPA21 delivers the exact set pulse interval
- It is clear that the BTCX device does not deliver the exact set pulse interval

For your information, this comparison information was created with our NEPA Monitoring System.

Many of our clients also purchase this system when they purchase the NEPA21 system. Effectively, with the NEPA Monitoring System, researchers can for the first-time see in Real-Time the delivery of the electrical pulse to the target. This is important particularly when one is trouble-shooting in order to ascertain why a particular experiment did not give the expected results.

It is also even more significant in that it can potentially save the researcher Time and Money. In real-time, you can now measure and see if the set electrical pulse (AND all the other factors critical to successful electroporation) was delivered (by simply viewing the graphical interface on the NEPA Monitoring System). For example, if you observe from the Monitoring System are that the electrical field did not appear to be delivered for a particular sample/s (or just did not look right), you can straight away re-do that sample/s. Researchers who do not have the NEPA21 Monitoring System have to wait (sometimes days) until after they complete the full after-experiment processing of their work (staining/imaging, data-crunching etc.) before they can tell if the experiment was successful or not. If unsuccessful, they then have to find time to re-perform the full experiment which means having to re-peat their earlier efforts and prepare all the consumables, booking lab-space and post-experiment equipment. (Also, below, there are further links that directly compare the CUY21 system with the BTX.)

Also Note the following links to:

– a comparison of the NEPA21 with the Amaxa-Nucelofector device (for a series of difference cell-line examples): ‘NEPA21 v Amaxa-Nucleofector etc’

– a comparison of the NEPA21 with the Bio-Rad device for the delivery of vectors into ES Cells: ‘Dr Tokunaga Comparison with Bio-Rad’

You will observe better Viability and Transfection Efficiency (at quite a lower Voltage setting)

Since launch last year, The NEPA21 system has taken over 70% of the Japanese electroporation transfection market (principally from the Anaxa-Lonza market). The NEPA21 system is much cheaper to purchase and the cost per electroporation is less than 2 Euro (if you only use the cuvette once). Amaxa-Lonza cost over 19 Euro per electroporation. That said, the prime reason for the NEPA21’s success is not just price. A device that performs both In Vitro and In Vivo applications is also an attractive proposition for a busy and diverse laboratory. But what differentiates the system is that it ameliorates the delivery of the output pulse into two separate pulses (a high-voltage short duration pulse followed by a low-voltage longer duration pulse). Competing devices merely store charge on a capacitor and brutally ‘dump’ that charge in one pulse. This 2-Pulse capability of the NEPA21 accounts for the increased Viability statistics we are getting (please see the list of Cell Lines and result data in my very first email). Enhanced Transfection Efficiency is achieved by applying the Polarity Change function (with voltage decay – another important differentiating factor in what we offer).

To give you an idea of the support we can offer your cleint’s laboratory post sale and delivery and to get a feel for the depth of our expertise please note the following In Vivo Protocols and Electrode Recommendations link (password: OCT2010*UjU). No other company in the world can compete with our range of in vivo electrodes. We have over 250 different electrode configurations to chose from. This significantly increase the in vivo application range of the NEPA21. No other device on the market can come close to this range of application-focused support.

With being able to split the NEPA21 output pulse into two separate pulses (followed by polarity exchange and optional voltage decay functionality) the researcher now has an ability to experiment with pulse-configuration options not previously available.

For your further information, our experience in this field demonstrates that lower-voltage parameters produce the best results, hence our focus on voltage control in our devices.

Another very important point to get across is the distinctiveness of the NEPA21 device compares to competing devices. The NEPA21 device is what we call a current-controlled device. Please note below for a fuller explanation of this. In summary it means that our device (unlike other on the market) respects the formula [V = IR] by testing for and accounting for the sample’s resistance prior to delivery of the electric pulse.

For your information, I forward the following background on the NEPA21 system. This information compares the NEPA21 with competing devices and describes why/how it is superior.

– Down Load NEPA21 Presentation

– Down Load Comparison of NEPA21 with Nucleofector Neon

NEPA21 v CUY21EDIT and CUY21SC

The NEPA21 system is our latest and most sophisticated electroporation device to-date. It is the successor to our hugely successful CUY21EDIT and CUY21SC electroporator range. The NEPA21 is based on the same platform and core technology that made the EDIT and SC devices leaders in their class.

The NEPA21:

– since launch last year in Japan, has taken over 70% of the Japanese electroporation transfection market (principally the Amaxa-Lonza market).

– Unique to the NEPA21, its electroporation protocol is a 3-Step process.

Step 1: is a high-voltage short duration pulse
Step 2: is low-voltage longer duration pulse
Step 3: reverses the polarity of the pulse and drives the pay-load (e.g., DNA) in the opposite direction

– Competing devices merely store charge on a capacitor and brutally ‘dump’ that charge in one pulse.

Effectively and uniquely, the first two steps of the NEPA21 electroporation process ameliorate the destructive impact of the delivery of an electrical pulse.
Together, these first two steps account for the increased Viability statistics achievable with the NEPA21.
Enhanced Transfection Efficiency is achieved by applying the integrated and unique Polarity Exchange function, Step 3. (Link to photo results).
Voltage Decay can also be programmed into the electroporation protocol – another important differentiating feature of the NEPA21 compared with other devices. We have found this feature to contribute to both the enhanced Viability and Transfection Efficiency obtainable with the NEPA21.

– covers the full In Vitro application range

for both Suspended Cells and Adherent Cells
compared with, for instance, the Amaxa/Lonza devices, its cost per electroporation is massively less. Less than Euro 2.00 per electroporation (if you only use the cuvette once) as against approximately Euro 19.00 per electroporation for Amax-Lonza devices
there are no hidden extra costs. The system does not require expensive buffers

– covers the full In Vivo application range

previously if you wished to cover the full in vivo application range with our systems you had to purchase both the CUY21EDIT and CUY21SC devices. Together they cost over Euro 30,000. But now with the NEPA21 you get full in vivo application range in the one device for almost half the previous cost. It combines into one device BOTH the wider voltage range of the EDIT and the enhanced sensitivity at lower voltages of the SC.

– can still be set-up to output the same single output-pulse wave-form found in the EDIT and SC devices.

– Unlike competing devices, the NEPA21 is current controlled device.

Bearing in mind the formula [V = IR]: What this means is that the target sample is first tested for R, and then the delivered current is precisely controlled to obtain the appropriate V. The net effect of this is that the electric fields delivered into the target are much more highly controlled which, in turn, makes for more precise electroporation from one experiment to the next and enhanced Viability and Transfection Efficiencies.
Many of the competing devices on the market are what we term voltage controlled devices. What this means is that researchers (when they are performing their experiments) are encouraged to almost exclusively focus on voltage settings. Such a ‘voltage only’ approach ignores the biological fact that current varies greatly with the impedance of the target. The net effect is that many competing devices simply store charge on a capacitor and then release the energy into the target without regard for or measuring the resistance (impedance).

– the NEPA21 delivers a precise Square Wave throughout pulse delivery.

For many competing devices, the quality of the square wave decays during the delivery of thepulse. This is not the case for the NEPA21.

It is very easy to use the NEPA21 device like a CUY21 EDIT or a CUY21 SC device. To use the NEPA21 system like an EDIT or SC system, kindly note the following simple methodology.

The Single-Step Simple Pulse Regime

In addition to the 2-Step Poring and Transfer Pulse Regime described above, the NEPA21 device also allows the researcher to configure and deliver a Single-Step Simple Pulse Regime. The Single-Step Pulse Regime function of the NEPA21 device delivers the exact same conventional pulse regimes of the legacy CUY21EDIT and CUY21SC electroporation systems. In effect, the NEPA21 device is three devices in one comprising the new NEPA21 platform and the legacy CUY21EDIT and CUY21SC devices. Indeed, excellent transfection efficiency and viability is achievable using the Single-Step simple square pulse regime. But the new NEPA21 device provides the user with the ability to deliver optimised continuous combination pulse streams in an uninterrupted manner and to exercise a more refined control over the output pulse.

On delivery of the Single-Step Pulse Regime, the NEPA21 device offers the researcher the further option of reversing the electrode polarities. This has the effect of making the DNA move in the opposite direction and this can increase transfection efficiency. We believe this would be particularly important for applications where cells are cultured in 3D gels/matrices.

To configure the NEPA21 device to deliver the Single-Step Pulse Regimes of the CUY21EDIT or CUY21SC devices: set the pulse number for the Poring Pulse to zero, the voltage decay percentage for the Transfer Pulse to zero and the Polarity Switching option to off and then set the other Transfer Pulse parameters according to the protocol you wish to replicate. For protocols where the required voltage is greater than 100V, set the pulse number for the Transfer Pulse to zero and the voltage decay percentage for the Poring Pulse to zero and then set the other Poring Pulse parameters according to the protocol you wish to replicate.

Pulse parameters can be modified easily on NEPA21. Preliminary experiments may be needed to optimize the parameters in order to achieve both high transfection efficiency and high cell viability. To better understand the pulse parameters, please refer to the measured output values of voltage, current and energy.

Below and attached is some further information demonstrating the difference between the quality of the output square-wave form of the NEPA21 v the BTX, which, in turn, explains the better reproducibility and results of the NEPA21 and CUY21 systems.

NEPA21 v BTX

Please note that there is an enormous difference between the NEPA21 system and the BTX system BOTH in terms of application range and quality of delivered output and as such experimental result and reproducibility of experimental results.

We are very familiar with the BTX ECM830. For your information, the majority of our clients are former BTX users. Their main dissatisfaction with the BTX system is the variability between what the system says it outputs and what is actually measured as its output. They are not the same. And especially at the lower voltage levels (at which most electroporation is performed now) and for sensitive targets, this variability can impact adversely on experiment results. For a summary of the comparison of the NEPA21 v the BTX device please see the attached ‘NEPA21 Comparison with BTX.pptx’. As you will see, the NEPA21 also incorporates a Voltage Decay function that is not present in the BTX.

To compare the NEPA21/CUY21 system with the BTX ECM830 is to compare ‘oranges with carrots’. The results in the following link clearly demonstrate the superiority of the NEPA21/CUY21 system and the accuracy of the delivered electroporation parameters compared to those of the BTX ECM830 device: Comparison of the NEPA21/CUY21SC and BTX ECM830 Square Waves at various voltages. (The page password is: PDUo7Y6).

On the first page of the above link we list all the advantages of the NEPA21/CUY21 system v the BTX ECM830. There are also further links to where we have compared the oscilloscope measured output of the NEPA21/CUY21 system with that of the BTX system at various voltages. In summary those results are:

Set Voltage on Device	Measured Voltage for BTX System	Measured Voltage for NEPA21/CUY21 System
15V	23.4V	15.2V
20V	28.6V	20.0V
25V	32.8V	25.0V
30V	38.0V	30.4V
40V	48.8V	40.4V
50V	58.4V	50.4V
100V	108V	101V

What distinguishes the NEPA21/CUY21 Electroporation System is that it is optimised for all the key critical success factors required to achieve successful, consistent and reproducible electroporation results for each and every electroporation event.

Current Controlled Device:

The NEPA21/CUY21 Electroporation System (unlike its main competitors) is a current controlled device. Bearing in mind the formula [V = IR]: What this means is that the target sample is first tested for R, and then the delivered current is precisely controlled to obtain the appropriate V. The net effect of this is that the electric fields delivered into the target are much more highly controlled which, in turn, makes for more precise electroporation from one experiment to the next.

Many of the competing devices on the market (like the BTX system) are what we term voltage controlled devices. What this means is that researchers (when they are performing their experiments) are encouraged to almost exclusively focus on voltage settings. Such a ‘voltage only’ approach ignores the biological fact that current varies greatly with the impedance of the target. The net effect is that many competing devices simply store charge on a capacitor and then release the energy into the target without regard for or measuring the resistance (impedance).

Moreover, and in addition to its superior current control and delivery, the NEPA21/CUY21 series delivers a precision Square Wave. For many competing devices, the quality of the square wave decays during the pulse. This is not the case for the NEPA21/CUY21 series.

NEPA21 v AMAZA/LONZA DEVICE:

I do not believe I sent you this information previously. It illustrates what new avenues clients are exploring with the NEPA21 device.

The client in question tested the NEPA21 for rat primary cerebellar granule neurons.

He has been using the Nucleofector (amaxa: Lonza) for 5 years. He is a heavy user of the Nucleofector and has spent more than JPY 20,000,000 (Euro 171,000) on Nucleofector Kits to-date.

In his feedback, he has said that he is very surprised and pleased with the NEPA21 results. He said that the NEPA21 results are “incomparably” better than those he gets with the Nucleofector.

He has sent us some cell image results. Please find attached (Nepa v Amaxa) file. You will see the direct comparison with the Amaxa:Lonza device.

I also translate his comments into English as follows:

Cuvette EP:

“#3 or #4 seems to be the best.

On day 1, the other numbers look good as well.

But, on day 4, most neurons of the other numbers were dead. (On the other hand, it looks like glial cells were growing.)

The neurons seem to be weak at higher voltage.

With all things considered, #3 or #4 seems to be the best.

Obviously the results were incomparably better than Amaxa.”

Adherent-Cell EP:

“#6 had the best transfection efficiency on day 1, but most of the cells of #6 were dead on day 4.

On the other hand, the samples with lower-voltage pulses seem to survive longer.

Therefore, #2 or “3 seems to be the best.”

For your information:

– A consistent thread in the feedback we are getting from Nucleofector (Amaxa) users is a dissatisfaction with the poor level of cell viability (post ‘nucleofection’)

– For example, neuron researchers say that axons of neurons do not grow after ‘nucleofection’ or that neurons die a few days after ‘nucleofection’.

As you will see from the comparison data (NEPA21 v Amaxa), viz-a-viz primary cerebellar granule neurons. The customer who gave us that data had the same complaint about poor cell viability. But he now believes that he can keep neurons alive much longer if he switches to the NEPA21 system. In addition, we believe that our adherent-cell EP system might offer another viable solution to the poor cell viability problems relating to the use of the Nucleofector technology. We believe that this can be big advantages for NEPA21.