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Cell death quantitation and kinetics, toxicity tests and cell-free DNA quantitation, in culture media as well as in blood plasma: a fast, sensitive, non-destructive and reproducible technology, using a simple reagent without purification or amplification, easy to robotize


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SAFAS S.A. - Société Anonyme de Fabrication d'Appareillages Scientifiques
10, quai Antoine 1er
98000 MONACO Principality
MONACO

phone: +377 99 99 52 52
fax: +377 99 99 52 50
e-mail: safas@safas.com
website: www.safas.com


Accurate Cell Death Quantitation is a major challenge for many biological processes : how is it currently usually achieved in cell cultures?

Currently, the most widespread method for cultures uses a reagent which colours living cells. The photometric measurement is not very accurate, as it is affected by the geometry of the cells sample, and subject to interferences. Moreover, estimating cell death from a viability information does not enable to discriminate the antagonist effects of proliferation and death. Last but not least, you must kill your cells to know how many were alive… not very nice when you want to use your cells for other purposes! Duplicates or triplicates must be prepared and sacrificed, which introduces complementary costs, and an additional bias with the main culture.

What are the principle and main advantages of your technology to monitor cell death in cultures?

Each cell releases its DNA inside the culture medium when it dies. Our technology enables to quantify cell-free DNA inside the medium and to know exactly how many cells died, wherever inside the culture and independently of usual interferences. You don’t need any more to sample the cells, as you just analyse the free DNA inside the media or supernatant. Not only you don’t kill or modify any cell, but also the volume required is so small (0.2µl is enough) that you don’t alter your culture. An immediate advantage is that you don’t need any more to prepare replicates: with our indirect technology, your main culture is monitored with accuracy, and remains 100% available for other experiments at the end.

In the human or animal body, the measurement of cell free DNA is well-known since decades, as a very interesting way to follow up all the pathologies associated to cell death or proliferation, as well as their treatments. How is it currently achieved, and what is the advantage of your technology?

Effectively, hundreds of publications prove that cell-free DNA quantitation is an excellent way to monitor a lot of diseases involving cell death, like infarcts, cancer, infections, inflammations, etc. It’s also very useful to evaluate the efficiency of targeted therapies. The number of publications is growing exponentially, but corresponding quantifications were usually achieved by QPCR, and anyway after extraction, purification or amplification, which is too long and expensive to be used in routine analysis.

Do you need to extract, purify or amplify?

No, and this is a strong advantage of our technology: it is not sensitive to the usual interferences and does not require any more to achieve extraction purification or amplification, which avoids corresponding costs and time lost. The measurement is achieved directly in the culture media or the blood plasma; it is perfectly correlated to QPCR analysis, and shows a better sensitivity and reliability. It opens the way to routine and series analysis for this important new parameter, as well as to automation, robotization and HTS.

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What is the total time necessary for the analysis, and what are its linearity and sensitivity levels?

A few minutes are enough to prepare and measure a few dozens of samples; the analysis stage requires only 20 seconds per sample. The sensitivity obtained is very high: the resolution is about 30 cell deaths per millilitre of culture medium, and when monitoring cfDNA inside human or animal body, the sensitivity is around 0.1ng/ml in cuvettes and 4ng/ml in microplates. The analysis is linear, as proved by the curve attached: a correlation around 0.999 can be easily obtained, and reproducibility is good, a CV% of 2 to 3% is obtained on standard levels of cfDNA in blood.

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Considering that preparatory steps are eliminated and that the analysis is fast, can you achieve kinetics of cell death? What about toxicity tests?

Yes, indeed: kinetic curves of cell death can be drawn, so as cell cultures can be optimized in real time, providing useful data for Quality Management. For toxicity tests, our technology enables to obtain a lot of additional information about the molecules tested, which is particularly useful as animal experimentation is being banned, raising new challenges for toxicity tests.

First of all, as it can be seen in the figure here attached, cell death intensity can already be evaluated after a few minutes thanks to the slope of the curve, instead of waiting several hours to get a single value not providing any information about the evolution.

On the figure attached, we can see that the yellow and blue curves may provide the same value if a single measurement would be achieved at the end, but that in reality the intensity and duration were very different: now these two important features will be easily detected and quantified thanks to our kinetic curves.

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Also, some toxic effects can be delayed, for example due to indirect effects of messengers or metabolites, generating a latency period which we can easily quantify. A bimodal pattern can occur as well, and can be detected and specified thanks to our kinetic curve.

Instead of a single and approximate value of toxicity, it’s now a real fingerprint of toxicity which our technology makes available for the scientists, associating several parameters to a single test.

What are the main kinds of samples which can be efficiently analysed by your technology, and in which fields is this quantification the most promising?

Blood plasma, urine, sperm are some examples of liquids where our technology will provide excellent quantification, and get rid of the many interferences which exist in such complex biological samples. Cell cultures and toxicity tests are also examples of applications for which precious information can be provided, for optimization or for characterization, as well as for Quality Management purposes.

This innovation is very interesting for quality control as well as for research, for all the scientists concerned by cell death or DNA measurement: Pharmaceutical and Cosmetic Groups, Biotechnology, Medical Research Laboratories, and obviously clinical biology, cardiology, oncology, infertility, rheumatology, etc… At the moment the reagent is Research Use Only, and must be used accordingly.

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Can you tell us more about this technology ? Can it be installed on SAFAS instruments already in service?

This innovative technology is based on the outstanding technologies and performances of a SAFAS Xenius multidetection microplate reader, fitted with an optional analysis and data-processing software, and using a specific reagent. The SAFAS Xenius is designed for 24/7 use reliability; it is scalable and can be fitted with many options, including high accuracy, auto-priming and auto-washing injectors with 0.1µl accuracy and 6-way stainless steel / ceramic automatic valves, which can help to partially automate the manipulations with outstanding reliability.

For higher throughputs, for example for HTS, the SAFAS Xenius can be coupled to a robot with a direct injection exclusive concept, which enables the robot to pipet directly inside the microplate already inserted in the reader tray holder; this concept also preserves sterility, temperature, moisture and atmosphere around the plate, spares an additional arm, and enables to start measuring immediately after the pipetting. The control is ensured via Ethernet TCP/IP thanks to a modern Web Service, which avoids development of drivers and eliminates the problems of software compatibility when upgrading the software. Moreover the robot and the reader keep 100% of the features of their respective software.

In conformity with the exclusive SUSTAINABLE SPECTROSCOPY concept of SAFAS, our new disruptive technology for cfDNA quantitation can be added on any existing SAFAS flx or SAFAS Xenius already in service, whatever could be its age. It avoids to our customers to have to invest in new models when they want to profit by our innovations: since 1952 this is a strong commitment of our company against programmed obsolescence and for sustainable development.

What are your assets for laboratories interested in cfDNA quantitation and involved in a demanding Quality Management ?

First of all, SAFAS Xenius is already in service in various Pharmaceutical groups, as well as Vaccine Manufacturers, for Military use and as a rule in various laboratories having a demanding Quality Management. We are used to achieve IQ/OQ and corresponding validations, and have several strong additional assets. To the best of our knowledge, the SAFAS Xenius is the only microplate reader in the world which includes built-in standards for daily perfect autocalibration, including for fluorescence and anisotropy. It is also the only one which can be validated in all technologies on certified and connected standards, including in Fluorescence and Bioluminescence! It’s also the only one which can provide absolute fluorescence spectra, thanks to sophisticated instrumental correction. These technologies are important for Quality Management, as well as some features like the accuracy of 0.2nm at any wavelength, which provides perfect compliance to Pharmacopoeia requirements with a good safety margin.

If electronic signature, audit trail and safety of records are required according to FDA 21CFR part11, an optional sophisticated software is available, providing compatibility at 100%. And obviously, additionally to the Cell Death Quantitation / cell-free DNA technology, the Xenius remains available for all the other techniques, in cuvettes as well as in microplates, in Absorbance, Fluorescence, Anisotropy and Bioluminescence, with the best performance for each technique, which enables to use it for a lot of applications, in research as well as for control, and to get a prompt return on investment.

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© SAFAS S.A. - Société Anonyme de Fabrication d'Appareillages Scientifiques
Letzte Änderungen: 29.05.2019


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