A method for rejuvenating senescent cells based on osmotic pressure.

Abstract

This invention discloses a method for rejuvenating senescent cells based on osmotic pressure induction. Senescent cells typically exhibit cell volume expansion. This method constructs a hypertonic environment by introducing a biocompatible polymeric solute (such as 2% or 0.5% PEG 300), applying controlled hypertonic pressure to senescent cells to induce cytoplasmic dehydration and counteract volume expansion. Experiments have demonstrated that long-term hypertonic compression intervention (such as 7 days of 0.5% PEG treatment) can effectively reset the mechanical state of senescent cells. After removing the pressure and restoring isotonic culture, some senescent cells can regain their proliferative capacity, as evidenced by negative SA-β-gal staining, strong positivity for Ki67 and EdU proliferation markers, and long-term stable passage. This invention provides a non-genetic, physical means to achieve cell rejuvenation, offering a new pathway for the intervention of age-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for using hyperosmotic pressure generated by regulating the osmotic pressure of the extracellular environment to drive senescent cells out of a state of proliferative arrest and achieve rejuvenation. Background Technology

[0002] Cellular senescence is characterized by cell cycle arrest. Traditional interventions primarily focus on inhibiting biochemical pathways or clearing senescent cells with drugs, but existing technologies still have significant limitations. First, although cell volume increase is generally accompanied by senescence, the physical driving mechanisms behind this morphological feature have long been overlooked, making it difficult for current technologies to quantitatively characterize and intervene in the physical dilution effect of the intracellular microenvironment caused by volume expansion. Furthermore, due to the traditional bias that senescence arrest is irreversible, there is currently a lack of effective means to induce senescent cells to re-enter the cell cycle, while existing gene editing or small molecule drug interventions often come with unavoidable off-target effects and cytotoxicity risks. Summary of the Invention

[0003] To overcome the shortcomings of existing technologies, this invention provides a method for reversing cellular senescence phenotypes and achieving cellular rejuvenation by regulating cellular physical characteristics through physical means. The core of this invention lies in its ability to achieve senescence reversal without relying on exogenous genes or chemically toxic drugs, but solely by adjusting the extracellular fluid osmotic pressure to implement controlled physical compression and subsequent release of senescent cells, thereby activating the autophagy mechanism. The specific technical solution is as follows: 1. Overall method: Senescent cells were placed in a hypertonic medium containing a biocompatible hypertonic drug for continuous physical compression culture to induce cell cytoplasmic dehydration, counteract cell volume expansion caused by senescence, and reset the cell mechanical state; subsequently, the hypertonic medium was removed and replaced with a conventional isotonic medium for recovery culture, so that some senescent cells regained their proliferative capacity and were removed from the senescent state.

[0004] 2. Concentration and osmotic pressure range of hypertonic drugs: The hypertonic culture medium is prepared by adding hypertonic drugs to a conventional basal culture medium. Taking polyethylene glycol 300 (PEG 300) as an example, its added mass-volume ratio concentration (w / v) ranges from 0.5% to 5%, with an optimal concentration range of 0.5% to 2%.

[0005] Osmotic pressure conversion range: The osmotic pressure of standard cell culture medium is typically 300 mOsm / L. Based on the molecular weight conversion of PEG 300, adding 0.5% to 2% PEG 300 is equivalent to increasing the osmotic pressure of the basal medium by approximately 17 to 70 mOsm / L. Therefore, the hypertonic culture medium described in this invention, with its overall osmotic pressure controlled between 317 mOsm / L and 370 mOsm / L, is the optimal range for promoting cell rejuvenation.

[0006] 3. The types of hypertonic drugs are clearly defined: the biocompatible hypertonic drugs mentioned are not limited to polyethylene glycol (PEG), but also include, but are not limited to, polyethylene glycols of different molecular weights (such as PEG 200, 400, PEG 1000, etc.), dextran, sucrose, mannitol, and sorbitol, or combinations thereof. Any biomolecule or sugar that can increase extracellular fluid osmotic pressure without producing direct cytochemical toxicity is within the scope of protection of this invention.

[0007] 4. Define the duration of physical compression: The duration of physical compression culture in hypertonic medium is 3 to 7 days (optimally 7 days), during which the hypertonic medium with the same osmotic pressure is replaced every 48 hours to maintain constant physical compression force; the recovery culture time after replacement with isotonic medium is at least 1 to 7 days to observe and maintain cell proliferation.

[0008] 5. Define the observation details and evaluation indicators of the rejuvenation (rejuvenation) state: The characteristics and observation methods for the rejuvenation of senescent cells include: (1) Measurement of morphological and physical characteristics: Microscopic imaging showed that the cell volume and spreading area were significantly reduced, and the diffusion rate of intracellular macromolecules was restored to the level of young cells; (2) Measurement of aging markers: The staining of aging-related β-galactosidase (SA-β-gal) changed from positive (blue) to negative; (3) Measurement of proliferation markers: When cells re-enter mitosis, the DNA synthesis marker EdU staining shows strong positive, the cell proliferation marker protein Ki67 immunofluorescence staining shows strong positive, and stable passage proliferation can be achieved for more than 30 days thereafter.

[0009] Beneficial effects The present invention has significant advantages in the fields of biotechnology and mechanical intervention: First, by adjusting the physical scale (volume), it was demonstrated for the first time that the aging-induced physical dilution effect could be effectively counteracted, and the intracellular physical state (such as macromolecular crowding and diffusion dynamics) could be successfully reset.

[0010] Secondly, it showed extremely high efficiency and stability in terms of intervention effect. After rejuvenation, the cells were completely negative for SA-β-gal staining, and the proliferation markers (Ki67 and EdU) were strongly positive, and they could maintain stable passage for more than a month.

[0011] Furthermore, this method has strong universality and has been consistently validated in various cancer cell lines (A549, H1975) and non-cancer primary cells (senescent mouse fibroblasts) in a variety of backgrounds.

[0012] Finally, this approach has the safety advantage of being a non-genetic procedure, using only biocompatible hypertonic reagents for physical osmotic pressure regulation, without involving the introduction of exogenous genes or harmful chemical toxins, which greatly improves the safety of the technology implementation and its future application prospects in the field of anti-aging medicine. Attached Figure Description

[0013] Figure 1 This is an introduction to the entire experimental procedure. Normal cells were induced to undergo DNA damage by adding the drug Dox, resulting in cellular senescence. The successful construction of the senescent cell model was marked as day 0. The cells were subjected to hyperosmolar compression with 0.5% PEG300 for 7 days, and then cultured under normal isotonic conditions for another 7 days. It was observed that the senescent cells exhibited a youthful phenotype. Figure 2 SA-β-gal staining was used to identify normal and senescent cells.

[0014] Figure 3 The ratio of SA-β-gal positivity in normal cells and senescent cells.

[0015] Figure 4 SA-β-gal staining and statistical analysis were performed on senescent cells after physical compression with 0.5% PEG for 7 days and isotonic recovery for 7 days.

[0016] Figure 5 Live cell imaging within 24 hours of restoring normal culture medium.

[0017] Figure 6 EdU staining images of control, senescent, physically compressed, and isotonic recovery cells.

[0018] Figure 7 The percentage of EdU-positive cells in control, senescent, physically compressed, and isotonic recovery cells.

[0019] Figure 8 Images of Ki67 staining in control, senescent, physically compressed, and isotonic restored cells.

[0020] Figure 9 The percentage of Ki67-positive cells in control, senescent, physically compressed, and isotonic restored cells.

[0021] Figure 10 SA-β-gal staining results for senescent H1975 cells after physical compression with 0.5% PEG for 7 days and isotonic recovery for 7 days.

[0022] Figure 11 The results of SA-β-gal staining of mouse fibroblasts after physical compression with 0.5% PEG for 7 days.

[0023] Figure 12 The percentage of mouse fibroblasts that were not stained with SA-β-gal after 7 days of physical compression with 0.5% PEG.

[0024] Figure 13 The results of p53 staining of mouse fibroblasts after physical compression with 0.5% PEG for 7 days.

[0025] Figure 14 p53 staining results of mouse fibroblasts that were physically compressed with 0.5% PEG for 7 days, compared with those of the control group. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are merely illustrative and are not intended to limit the scope of the invention.

[0027] Note: The senescent cell population used for testing in this embodiment of the invention can be obtained by conventional low-dose doxorubicin (Dox)-induced DNA damage method or natural passage replication senescence method. 0.5% PEG300 was added for hypertonic compression, which restored the abnormally swollen volume of the senescent cells. The cells were then cultured in normal isotonic medium for another seven days. The hypertonic compression operation induced a youthful phenotype in the senescent cells. Figure 1 ).

[0028] Example 1: Induction of A549 Senescent Cell Rejuvenation Using Hyperosmolar Compression (1) A human lung cancer A549 cell senescence model was successfully constructed using 400 nM doxorubicin (Dox). Senescent cells showed increased volume, increased thickness, and strong positive SA-β-gal staining. Figure 2 ), positive rate >90% ( Figure 3 Human lung cancer A549 cells that were confirmed to be in a senescent state were replaced with hypertonic complete culture medium supplemented with 0.5% (w / v) PEG 300 (osmotic pressure increased by approximately 17 mOsm / L compared to the basal medium).

[0029] (2) The cells were cultured in this hypertonic environment for 7 days. Fresh 0.5% PEG 300 hypertonic medium was replaced every 48 hours. During this intervention phase, cytoplasmic dehydration effectively counteracted the volume expansion caused by aging, and under a microscope, the cell morphology changed from flat and enlarged to small and slender. Figure 4 ).

[0030] (3) After 7 days, remove the hypertonic culture medium, wash gently with phosphate buffered saline (PBS), and add conventional isotonic complete culture medium for recovery culture.

[0031] (4) Observation results: Within 1 to 3 days of restoring isotonic culture, "rejuvenated cells" emerged in the senescent population, regaining their proliferative capacity. Live-cell dynamic imaging accurately captured the rejuvenated cells entering mitosis approximately 12 hours after depressurization. Figure 5 Using the kit, the SA-β-gal staining of these rejuvenated cells turned negative; the nuclear EdU signal was strongly positive. Figure 6 , Figure 7 ), and the proliferation marker Ki67 showed strong positive expression ( Figure 8 , Figure 9 This demonstrates that long-term hyperosmolar compression intervention successfully drove A549 senescent cells to break through proliferation arrest and return to the cell cycle.

[0032] Example 2: Induction of H1975 Senescent Cell Rejuvenation Using Hyperosmolar Compression (1) Select human non-small cell lung cancer H1975 cell lines with different gene backgrounds and obtain their senescent cell models.

[0033] (2) Add hypertonic medium containing 1.0% (w / v) PEG 300 to H1975 senescent cells (increasing osmotic pressure by about 34 mOsm / L), and continue physical compression treatment for 7 days, changing the medium every two days.

[0034] (3) After the treatment is completed, the culture medium is replaced with conventional isotonic culture medium.

[0035] (4) Observation results: After 7 days of recovery culture, not only was the cell volume observed to recover to the level of the young cell control group, but also a large number of cells formed confluent monolayer cell clones. Staining of these clones showed that SA-β-gal was negative, and the intracellular macromolecular diffusion kinetics were consistent with those of normal H1975 cells. Figure 10 This embodiment strongly demonstrates the broad biological applicability of the hyperosmolarity-induced rejuvenation method of the present invention in different cell lines.

[0036] Example 3: Rejuvenation of Senescent Mouse Fibroblasts Induced by Hyperosmolar Compression To verify the effectiveness of the method of the present invention on non-cancerous, naturally aging primary mammalian cells, the following experiments were conducted: (1) Mouse fibroblasts that have undergone multiple passages and naturally entered a senescent state were obtained, and their SA-β-gal positivity rate was found to be high. Figure 11 and Figure 12 ), and the aging-related protein p53 is highly expressed ( Figure 13 and Figure 14 ).

[0037] (2) Add hypertonic medium containing 0.5% (w / v) PEG 300 to senescent mouse fibroblasts and perform osmotic pressure physical compression intervention for 7 days.

[0038] (3) Remove the high-permeability environment after 7 days. (4) Observation results: Molecular level detection showed that the proportion of SA-β-gal staining in the 7-day osmotic pressure physical compression intervention group was lower than that in the untreated group ( Figure 11 and Figure 12 ), and p53 expression was downregulated ( Figure 13 and Figure 14 This embodiment further confirms that the rejuvenation strategy based on high osmotic pressure physical compression is not only applicable to cancer cell lines, but also to normal somatic cells of mammals that are physiologically aging.

Claims

1. A method for rejuvenating senescent cells based on physical compression, comprising placing senescent cells in a hypertonic culture medium containing a biocompatible hypertonic drug for continuous physical compression culture, inducing cytoplasmic dehydration, counteracting cell volume expansion caused by senescence, and resetting the cell's mechanical state; observing details and evaluating indicators, removing the hypertonic culture medium, replacing it with a conventional isotonic culture medium for recovery culture, enabling some senescent cells to regain proliferative capacity and escape the senescent state, characterized by: The hypertonic culture medium is prepared by adding hypertonic drugs to a conventional basal culture medium. The hypertonic drugs include biological macromolecules or sugars that increase the osmotic pressure of extracellular fluid without producing direct cellular chemical toxicity. The physical compression culture in the hypertonic medium needs to be carried out continuously and the hypertonic medium with the same osmotic pressure needs to be replaced at regular intervals to maintain a constant physical compression force. The recovery culture time after replacing with isotonic medium needs to be maintained for a certain period of time in order to observe and maintain cell proliferation; the overall osmotic pressure of hypertonic medium should be controlled within a certain range.

2. The method of claim 1, wherein, The hypertonic drug is a biological macromolecule or sugar that increases the osmotic pressure of extracellular fluid without producing direct cytochemical toxicity, including one or more or combinations of polyethylene glycol PEG 200, PEG400, PEG 1000, dextran, sucrose, mannitol, and sorbitol of different molecular weights.

3. The method of claim 1, wherein, The overall osmotic pressure of the hypertonic culture medium is controlled between 317 mOsm / L and 370 mOsm / L.

4. The method of claim 1, wherein, The hypertonic drug is polyethylene glycol PEG300, and its added mass-volume ratio concentration (w / v) ranges from 0.5% to 5%, with an optimal concentration range of 0.5% to 2%.

5. The method of claim 1, wherein, The duration of physical compression culture in the hypertonic medium is 3-7 days, and the interval between replacing the hypertonic medium with the same osmotic pressure is 48 hours.

6. The method of claim 1, wherein, The observation details and evaluation indicators include the following: (1) Measurement of morphological and physical characteristics: Microscopic imaging showed that the cell volume and spreading area were significantly reduced, and the diffusion rate of intracellular macromolecules was restored to the level of young cells; (2) Measurement of aging markers: The staining of aging-related β-galactosidase SA-β-gal changed from positive to negative; (3) Measurement of proliferation markers: When cells re-enter mitosis, the DNA synthesis marker EdU staining shows strong positive, the cell proliferation marker protein Ki67 immunofluorescence staining shows strong positive, and stable passage proliferation can be achieved for more than 30 days thereafter.

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