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Senescent Cells are Large, which Suggests a Few Simpler Paths to Assays for Senescence Level in Human Subjects

The rise in number of senescent cells with age is one of the root causes of degenerative aging. Somatic cells become senescent when they reach the Hayflick limit on replication, or become damaged, or encounter a toxic environment. They cease replication, and either self-destruct or are destroyed by the immune system. Some small fraction of the countless cells that become senescent each and every day manage to evade destruction, however. They linger, in ever greater numbers with each passing year, and the potent mix of signal molecules they secrete contributes to many forms of tissue dysfunction and organ failure, ranging from fibrosis and loss of regenerative capacity to increased inflammation and loss of tissue elasticity.

Fortunately, we stand just a few years removed from of the clinical availability of therapies that can destroy some fraction of these cells. A few startup companies are working on senolytic treatments ranging from repurposed chemotherapeutic drugs to gene therapies to antibody therapies. Senolytic treatments that work in humans will literally produce rejuvenation to some degree, turning back one of the causes of aging. They don't even have to be expensive in the case of repurposed drug candidates, though these are unlikely to be as effective as the final results of further development efforts. Indeed, the adventurous can order and use some of these drug candidates even now, and experiment upon themselves, with very little outlay of funds. Caution is always recommended, of course.

Perhaps the most important objection to self-experimentation in the matter of the first legitimate rejuvenation therapies is that there is no readily available measure that will determine just how many senescent cells have been destroyed by a treatment. This is a challenge for formal human pilot trials as well. It means that secondary measures or expensive laboratory work are required, and in a basically healthy individual in middle age, it may well be the case that it is hard to separate out the effects of a crude but legitimate rejuvenation therapy from the noise. Aging has numerous distinct contributing causes, forms of cell and tissue damage that mingle to produce the initially slow decline. It is reasonable to expect it to be hard to see the short-term effect of removing 25% of senescent cells in, say, a 50-something individual who has yet to develop a severe manifestation of any of the age-related conditions most strongly linked to cellular senescence. On the other hand, maybe it will produce meaningful impact in cardiovascular measures such as blood pressure and pulse wave velocity. Without referencing a body of data that doesn't yet exist, it is hard to say which of these is the case.

Measures of senescent cell counts in a living individual would be inarguable, however, assuming they could be carried out without generating injury in the process of obtaining that information, as senescent cells are generated temporarily as a part of the response to wounding. Given such a metric, assessed before and after a treatment, one could definitively say whether or not the treatment achieved the intended result - and then all the uncertainty in secondary benefits achieved at any given age and health status will become more a matter of interesting further research than an outright roadblock. Unfortunately, we don't yet have a useful and broadly available tool to achieve this result. Possibilities include, say, some form of blood sample analysis that looks for the distinctive pattern of signal molecules produced by senescent cells. That is a reasonable research and development project for any group capable of proteomics-based analysis of blood, and perhaps there are people out there working away on it, quietly.

The authors of the open access paper below present an intriguing alternative, and one that might be more easily established and deployed. It is based on the observation that senescent cells are large, some twice as large as normal cells, or even larger. Thus these cells could be filtered from blood via the use of suitably sized fluid channel devices and then counted via flow cytometry, a task that is well-established and business as usual in the microfluidic device industry. Is it reasonable to expect that a higher burden of senescence throughout the body would be more or less accurately reflected by a larger number of senescent cells in the bloodstream? Possibly, but again, the work to prove and quantify all of that has yet to be accomplished. Still, the microfluidics approach to a senescence assay seems a very promising direction for further development.

Senescence chips for ultrahigh-throughput isolation and removal of senescent cells

Cellular senescence is a state of permanent cell cycle arrest due to genotoxic stresses and has been shown to be involved in organismal aging and tumorigenesis. Therefore, cellular senescence is an important biomarker for aging as well as genotoxic stresses such as ionizing radiation. However, the small number of senescent cells in biofluids such as whole blood limits their quick and sensitive detection. An effective isolation approach is highly desired for senescent-cell-based point-of-care diagnostics such as radiation biodosimetry. Moreover, recent animal studies have demonstrated the potential of therapeutic targeting of senescent cells for anti-aging and age-related diseases. Because pathways up- or downregulated in senescent cells, such as those involving p16, p21, and p53, also function at various degrees in their healthy counterparts throughout the tissues and organs, conventional methods by targeting these pathways with small molecules and protein drugs could result in side effects in humans. Alternatively, physical means by taking advantage of the cell size increase during cellular senescence provides an attractive novel approach to selectively remove senescent cells from their nonsenescent counterparts and other background cells.

Different microfluidic techniques have been developed for cell separation based on their physical properties. Among those techniques, filtration is the most promising approach to process undiluted whole blood for rare cell separation, and easily scaled up for high throughput. However, several challenges need to be overcome before this technique could be widely used. In dead-end flow filtration which has the flow direction perpendicular to the filter surface, a common issue is the clogging and saturation of the filter, resulting in low separation efficiency, sample purity, and device robustness. In some studies, a periodic reversed flow or fluidic oscillation was adopted to address clogging. To avoid cell damage and clogging issue, cross-flow filtration in microfluidics was developed with a flow direction parallel to the filter surface. Therefore, a shear force was generated to bring the bigger particles to the downstream instead of entering the filtration pores. However, to ensure effective cell separation in a parallel-flow configuration, the cross-flow filtration typically has a much longer channel with a throughput usually lower than 1 ml/hr. Despite the inherent low throughput for microfluidic devices, a higher throughput (e.g., more than 1 ml/min) is highly desired to process a large volume of whole blood samples. High throughput is particularly challenging for a continuous flow because of the difficulties in system integration and fluidic control for multiplexing on a microfluidic chip.

To overcome the clogging and cell damage issue while still achieve a high throughput and recovery rate, we developed a microdevice (senescence chip) for three-dimensional size sieving by taking advantages of both dead-end flow and cross-flow filtrations. A slanted micropillar array was fabricated with an inclination angle relative to the fluidic flow (between 0° to 90°). Therefore, the particles could not only be sieved efficiently but also experience a fluidic shear force to reduce clogging and preserve cell integrity. Moreover, the micropillars worked as cantilevers, which had only one end fixed. Their flexibility allowed small deformation when experiencing a fluidic pressure, creating hundreds of shutters in the vertical direction responsive to the flow rate. These shutters helped to release backpressure, reduce clogging, and dramatically improve separation throughput.

We utilized our senescence chip to isolate and analyze senescent cells in undiluted whole blood and mouse bone marrow. We chose mesenchymal stem cells (MSCs) because we have previously characterized their ionizing radiation-induced senescence progression. In this study, we utilized H2O2- and X-ray-induced senescent human MSCs spiked in whole blood, as a model biological system, to demonstrate the rapid separation and analysis of senescent cells using our senescence chip. The optimized device was then used for an animal study to isolate senescent cells from the bone marrow of mice undergone total body irradiation (TBI) of X-ray. To achieve ultrahigh-throughput removal of senescent cells for blood purification, we enlarged the chip dimensions and stacked multiple chips to build a multiplexed system. We demonstrated that our scaled-up senescent chip could achieve a parallel processing with a throughput up to 300 ml/hr.

Comments

Maybe there are other ways to take out these large senescent cells other than filtering them out of the whole blood, such as developing macrophages that will attack and destroy the senescent cells or injecting them with a killing agent or an irradiated particle.

Posted by: Biotechy at January 19th, 2018 4:58 PM

@Biotechy: This isn't relevant from a removal point of view, I think. The entire utility is detection and assessment in a variety of scenarios.

Posted by: Reason at January 19th, 2018 5:06 PM

Wow, if this works it could be super important. Even if your drug/treatment successfully removes senescent cells, if you cannot directly measure that and instead have to rely on trying to measure the downstream effects of senescent cell removal, trials will be long and expensive (and results will be more easily doubted and disputed).

A quicker and cheaper test of SC removal effectiveness would also be a boon for the "off shore" senescent cell removal industry.

Posted by: Jim at January 20th, 2018 5:42 AM

Take this with a grain of salt. The varicose veins (near my ankles) that I had developed over the past decade, have exhibited about a 50% reduction since I began my various Senolytic therapies 9 months ago. I only just recently noticed this. Of course I do not have before pictures as I was not anticipating any such phenomenon.

Posted by: JohnD at January 20th, 2018 12:18 PM

Hi john, could you elaborate your senolytic protocol?

Posted by: chris at January 21st, 2018 5:08 AM

Also John, can you tell us your age for reference?

Posted by: Robert at January 21st, 2018 8:14 PM

@Robert and Chris. I am 57. I take 1 of 4 potential Senolytics one day every week on a rotating basis. Quercetin, Fistein, Black Ginger, and Long Pepper. The Long Pepper is a source of piperlongumine, but I have no idea of the concentration. I started at low levels and have worked my way up to the following doses, 7,000mg, 1,600mg, 1,800mg, 5,200mg. I am starting to experience some skin rashes on those Q and F doses, and will be cutting back to 4,000mg and 1,000mg respectively.

Posted by: JohnD at January 25th, 2018 2:33 PM

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