1. Introduction

The aging process is defined as the “accumulation of consequences of life, such as molecular and cellular damage, that leads to functional decline, chronic diseases, and ultimately mortality” [1]. Longevity refers to the length of time an organism lives and is characterized by progressive decline in tissue and organ function and increased risk of mortality. Longevity is also often associated with a healthy and fulfilling life using the term extended healthspan instead of lifespan [2,3]. It involves a number of factors, including genetics, lifestyle choices, environmental influences and access to healthcare. In addition, advances in medical science have led to preventive treatments that improve health in older age. Promoting healthy ageing not only improves the quality of life of individuals, but also reduces the burden on healthcare systems [4]. Increased age is the largest risk factor for chronic diseases such as cancer, neurodegeneration and cardiovascular disease [5]. Moreover, reactive oxygen species have also been proposed as key biomarkers of the aging process [6-8].

Cellular health as one prerequiste for longevity is controlled by a number of regulatory pathways which coordinate the various processes of cell aging. Because cell health and vitality is regulated at many molecular levels, the regulatory processes are interconnected. Each of these processes is linked to the regulation of aging at the cellular level, which ultimately affects the control of aging in the whole organism [9-11]. Although cellular models of human disease provide valuable mechanistic information, they are limited as they may not represent the in vivo biology of the human body [12]. Nevertheless, cellular models allow to examine several cellular and molecular aspects of aging more detailed [13].

L.I.F.E. technology was created to promote the replicative lifespan, measured by the number of mitotic divisions until replicative senescence occurs and was studied here with primary cultured eucaryotic cells of the respiratory tract possessing a limited lifespan.

2. Materials and methods

2.1. L.I.F.E. (Light Inner for Ever) technology

The technology was created by Nexus Research Solutions, Dubai, United Arab Emirates, and is based on a biomimetic innovation that utilizes specific bioactive agents, which were rigorously selected and optimized. Inspired by adaptive mechanisms observed within complex biological structures, this innovation replicates their intrinsic abilities to modify their structural organization or growth dynamics under controlled environments. The bioactive agents, such as organic polymers or functional macromolecules are extracted, refined and subjected to advanced physico-chemical transformation processes to maximize their efficiency. The material of L.I.F.E. technology was applied to the cells in form of a small circular object which was provided by Nexus Research Solutions for the duration of the experiments.

2.2. Primary cell cultures

Respiratory epithelial cells were isolated from the tissue of the trachea and biforkatio of slaughter cattle as described by Cozens, et al. [14]. Cells were cultivated for four passages in Dulbecco's modified Eagle's medium (4.5 g/L glucose) and Ham’s F12 medium (1:1) supplemented with 10% growth mixture and 0.5% gentamycin in a gassed incubator at 37°C in an atmosphere of 5% CO₂ and 95% air at more than 90% humidity. Cells were routinely subcultured once a week by trypsin/EDTA treatment. From our own observations these primary epithelial cells of the respiratory tract have a limited replicative lifespan below 10 passages before senescence occurs and the cells remain in post-mitotic stage. This is valid for cells which were not cultivated at air liquid interface conditions. Therefore, cells were taken for the experiments in their fourth passage (see below).

 2.3. Experimental design

Respiratory epithelial cells of fourth passage were seeded at extremely low density (20,000 cells/75 cm² flasks in 20 ml culture medium). This was the first passage (P1) of the subsequent longevity experiments with and without L.I.F.E. technology. The circular object with L.I.F.E. technology was always placed directly beneath the cell culture flasks. To avoid any unwanted interferences between the cell cultures, cells were cultivated with and without L.I.F.E. technology in two separated mini-incubators at 37°C with a distance of at least 5 meters with two inner house walls between them.

At definite time points (see results section) cells were subcultured by trypsin/EDTA treeatment and seeded at the same densities in new cell culture flasks with fresh culture medium. This procedure was done four times (P1 ð P4) until the cells became senescent and remained in a post-mitotic state without further cell divisions. At each time point the cell densities in the flasks were examined by using a specialized software (confluence assay v2.1.0 from IKOSA AI software; KML Vision, Graz, Austria. The total incubation time of the cell cultures (P1 ð P4) with and without L.I.F.E. technology was 28 days representing about 25-30 mitotic cell divisions. Although the mitotic activity of the cells in an organism is related to organ-specifity, this in vitro period is equivalent to at least several months in vivo.

4. Statistical Analysis

Statistical analysis was done using the parameter-free two-tailed Wilcoxon-Mann-Whitney rank-sum test.

5. Results and Discussion

During the experimental cultivation period of the cells P1 ð P4, their mitotic activity decreased with the numbers of subcultures or in vitro-age, respectively (Figure 1A). However, this natural senescence of primary cells was much lower in all passages for cell cultures which were exposed to L.I.F.E technology compared to untreated cell cultures at all measurement time points. As depicted in Figure 1B, the improvement by L.I.F.E. technology became even more pronounced with increasing in vitro-age or cultivation time, respectively. This means, that after only 6 days of cultivation, the increase was quite moderate with about 20%, but was very prominent with more than 150% after 28 days. However, the differences between both cell cultures with and without L.I.F.E. technology were always statistically significant (p ≤ 0.01). Figures 2 and 3 show the micrographs of the time sequence of the cell cultures with and without L.I.F.E. technology in direct relation.

The main finding of this study demonstrates that untreated cells became earlier replicative senescent with a much lower replicative lifespan compared to cells exposed to L.I.F.E. technology. To achieve a prolonged lifespan and healthspan in humans, one must understand the complexity of cellular programs responsible for aging. Aging is not only a passive process; it involves biological mechanisms that govern cellular function and integrity. Key processes such as telomere shortening, oxidative stress and mitochondrial dysfunction contribute to the gradual deterioration of cellular health [11,15-17]. Dysregulation of these programs can lead to cellular senescence, a state in which cells lose their ability to divide and function optimally. Investigating the molecular pathways and signals that drive these changes is essential for developing interventions that may decrease the effects of aging and enhance healthspan.

In summary, longevity is a multifaceted concept influenced by a combination of genetic, environmental, lifestyle and healthcare factors. Especially the key processes on cellular and molecular level seem to be responsible for a longer and healthier life and remains a priority for encouraging individuals and communities to adopt practices that support well-being and longevity. However, as shown in this study by the limited lifespan of primary respiratory cells, there might be additional options by the use of L.I.F.E. technology to promote a prolonged ageing which associated with improved individual systemic health.

 6. References

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