Method for conditioning cells and inducing a regenerative phenotype

Abstract

This development represents a method for conditioning cells grown under in vitro conditions in the presence of biochemical and / or metabolic stimuli, specifically cellular reprogramming factors, emergency stimuli, and metabolic activity stimulants. This method is intended for the reversal of the aging of treated cells, restoring their regenerative and proliferative capacities, and generating conditioned media with properties that restore cell youthfulness and proliferation.

Description

[0001] 8882862

[0002] - 1 -

[0003] METHOD FOR CONDITIONING CELLS AND INDUCING A REGENERATIVE PHENOTYPE

[0004] Cross-reference to related applications

[0005] This patent application claims the benefit of priority application SENADI-2024-91641 filed 11 December 2024 which is herein incorporated in its entirety.

[0006] Technical field

[0007] This development is related to the field of cell biology, particularly to methods for conditioning and reprogramming cells, allowing for the restoration, stimulation, amplification, and direction of their regenerative capacity.

[0008] Traditionally, cellular aging has been considered a natural and irreversible phenomenon resulting from the passage of time. However, modern science is working to change this concept. Aging is more accurately defined as a biological state characterized by a decline in physical capacities, influenced by factors such as chronic inflammation, altered cellular communication, mitochondrial dysfunction, and cellular senescence. The latter, senescence, is a distinctive feature of aging that represents a challenge for public health due to its involvement in various pathologies (Lopez-Otin, C. et al. 2013. The hallmarks of aging. Cell; 153(6): 1194-1217).

[0009] In aging research, cellular senescence is relevant as it causes the irreversible cessation of cell growth and regeneration of damaged tissue. Understanding the mechanisms for slowing and even reversing the state of senescence is key to recovering the biological capacities of a cell and an individual, regardless of the time elapsed.

[0010] In general, the methodologies described in the state of the art to reverse or mitigate cellular aging are conditioned by the system in which the cells are found, whether in vitro or in vivo.

[0011] In an in vitro system, cell conditioning or reprogramming can be carried out using inducers of a physical nature (e.g., pH, electricity, oxygen concentration), biological nature (metabolites, cytokines, growth and transcription factors), and / or pharmacological nature (e.g., rapamycin, metformin, acarbose, nicotinamide mononucleotide (NMN), lithium, and nonsteroidal anti-inflammatory drugs (NSAIDs), curcumin). These factors promote the activation of different cell signaling pathways related to proliferation, apoptosis, and resistance to oxidative stress in treated cells (Moeinabadi-Bidgoli, K. et al. 2021. Translational insights into stem cell preconditioning: From molecular mechanisms to preclinical applications. Biomedicine & Pharmacotherapy; 142: 112026). 8882862

[0012] - 2 - ln an in vivo system, different methodologies to prolong an optimal state of health over time include caloric restriction, the use of active agents, hormones, and others with a therapeutic focus.

[0013] A relevant example of in vitro conditioning is found in patent US10047345, which discloses a method for culturing mesenchymal stem cells in a medium comprising nicotinamide and fibroblast growth factor 4 (FGF4). This method allows for the expansion of mesenchymal stem cells by culturing a population of said cells for a time sufficient for their expansion, alternating the medium with and without nicotinamide and FGF4. Mesenchymal stem cells derived from this method can be transplanted directly into a subject in need of them and used for tissue regeneration or the treatment of various diseases, including autoimmune and proliferative diseases.

[0014] Another example is patent application US20160115455, which refers to a method for reprogramming cells based on the expression of exogenous polycistronic nucleic acids encoding cellular reprogramming factors and immortalization factors. The resulting cells can proliferate through 40 or more duplication cycles and can be used to identify agents related to nuclear reprogramming, differentiation, proliferation, and cell viability. Furthermore, they can be used to treat patients to restore, enhance, and / or provide functions of interest.

[0015] In another state-of-the-art example, Shinya Yamanaka's team described a method for the successful reprogramming of differentiated human somatic cells to a pluripotent state (iPS), which allows the creation of patient-specific stem cells and the modeling of different diseases and injuries. The generation of iPS cells from adult human dermal fibroblasts is demonstrated using four factors: Oct3 / 4, Sox2, Klf4, and c-Myc. The obtained human iPS cells showed phenotypic similarities with embryonic stem cells and telomerase activity (Takahashi, K. et al. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell; 131 (5): 861-872). Furthermore, these cells were able to differentiate into cell types of the three germ layers in vitro and into teratomas. These findings demonstrate that it is possible to generate iPS cells through a reprogramming method from transcription factors, using adult human fibroblasts, and provide a valuable tool for regenerative medicine and the study of diseases.

[0016] Despite the progress made, the development of effective therapies to treat or delay the effects of senescence remains limited due to their high costs, low levels of effectiveness, and complex production (Amaya-Montoya, M. et al. 2020. Cellular senescence as a therapeutic target for age-related diseases: a review. Advances in Therapy; 37: 1407- 1424). In the study of aging and senescence, in vitro models, such as cell cultures, have allowed a detailed and controlled investigation of these processes (Campisi, J. & d'Adda di Fagagna, F. 2007. Cellular senescence: when bad things happen to good cells. Nature Reviews Molecular Cell Biology; 8: 729-740). These models facilitate the creation of a controlled environment and the use of precise factors in order to reverse cellular aging or senescence, restoring a youthful or healthy phenotype in cells. It is crucial to seek safe and more affordable methodologies to accelerate the discovery and development of effective anti-aging therapies, including the generation of cells with a rejuvenated 8882862

[0017] - 3 - phenotype, their conditioning, stimulation, amplification, and targeting of their regenerative capacity.

[0018] After the priority date of the present application a poster briefly reporting some of the results described herein was made available by the inventors in connection with the 31st Annual International Society for Cell & Gene Therapy (ISCT) Meeting held from 7 May 2025 with an online abstract published 30 April 2025.

[0019] Brief description

[0020] This development corresponds to a method for conditioning cells in culture in the presence of cellular reprogramming factors, emergency stimuli, and metabolic activity stimulants. The method allows for the generation of cells with improved regenerative capacity against damage and senescence. In turn, the method allows for the generation of a conditioned medium through the production of regenerative substances, which can regenerate or enhance metabolic capacities and have an anti-senescence effect. Cells conditioned using this method can be used directly in the body for the treatment of various diseases or damage, as well as for the production of enriched media that promote tissue repair or rejuvenation.

[0021] Brief description of the fiaures

[0022] FIG. 1 Schematic of the “Growth Hormone, Epinephrine, Mitochondria” (GHEM) method for conditioning cells and inducing a regenerative phenotype.

[0023] FIG. 2 Analysis of senescence levels in fibroblasts after conditioning using the GHEM method, assessed by flow cytometry.

[0024] FIG. 3 Evaluation of morphological changes in senescent fibroblasts exposed to two GHEM stimuli.

[0025] FIG. 4 Determination of P53 (A) and P21 (B) expression levels in fibroblasts with different passage numbers after 72 hours of treatment with GHEM medium by qPCR.

[0026] FIG. 5 Quantification of cell-free DNA in mesenchymal stem cell cultures before (A) and after (B) cell lysis.

[0027] FIG. 6 Microscopy of MSCs after exposure to GHEM for 24, 48, 72, and 96 hours.

[0028] FIG. 7A Microscopy of fibroblasts (20 passages) after exposure to GHEM for 48, 72, and 96 hours.

[0029] FIG. 7B Microscopy of fibroblasts (36 passages) after exposure to GHEM for 48, 72, and 96 hours. 8882862

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[0031] FIG. 8 Proliferation of fibroblasts treated with GHEM (single conditioning), showing the number of cells after 48 hours of treatment.

[0032] Detailed description

[0033] For the purpose of interpreting the terms used throughout this document, their usual meaning in the technical field must be taken into account, unless a specific definition is incorporated or the context clearly indicates otherwise. Additionally, terms used in the singular form will also include the plural form. Unless otherwise indicated, all concentrations and percentages in this Descriptive Chapter correspond to weight / volume (w / v).

[0034] Method for conditioning cells or producing conditioned media

[0035] The present development is directed to a method for conditioning cells or producing conditioned media comprising the following steps:

[0036] (a) depositing cells in a culture medium;

[0037] (b) adding a cell reprogramming factor to the culture medium of (a) and incubating;

[0038] (c) introducing an emergency stimulus to the culture medium obtained in (b) and incubating; and

[0039] (d) adding a metabolic activity enhancer to the culture medium obtained in (c) and incubating; wherein the metabolic activity enhancer is selected from mitochondria, macro- and micronutrient cocktails, or a combination thereof.

[0040] As explained herein, the methods of the invention generate conditioned cells and produce enriched regenerative medium which can be used together or separately in therapeutic or cosmetic methods, for example for tissue repair or rejuvenation.

[0041] In one preferred embodiment the steps (b)-(d) are carried out sequentially in the order stated.

[0042] In another embodiment the steps (b)-(d) are carried out sequentially in any order.

[0043] In another embodiment one or more, or all, steps (b)-(d) are carried out simultaneously.

[0044] In some embodiments, the cells conditioned using the method described herein correspond to animal cells, wherein said cells are selected from stem cells and support cells. 8882862

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[0046] Stem cells are undifferentiated cells vital to the maintenance of tissues in an organism, largely attributed to their ability to secrete factors that help preserve cell function in their environment. Interest in the regenerative properties of this type of cell, and their consequent potential for disease prevention and treatment, has been increasing in recent years, giving rise to a variety of applications in the pharmaceutical and biotechnology fields. However, one of the most important limitations in the physiology and application of stem cells is their aging, accentuated by the damage caused by the stress they may be subjected to in a particular environment.

[0047] In particular embodiments, the stem cells are selected, but not limited to, totipotent stem cells, pluripotent stem cells, multipotent stem cells, unipotent stem cells, induced reprogrammed stem cells, or a combination thereof.

[0048] For the purposes of this disclosure, "support cells" are understood to be those that perform crucial functions in various biological systems such as somatic cells, supporting stromal cells, immune cells, endothelial cells, fibroblasts, muscle cells, epithelial cells etc.

[0049] In particular embodiments, said support cells are selected, but are not limited to, astrocytes, oligodendrocytes, Schwann cells, microglia, ependymal cells and satellite cells, fibroblasts, adipocytes, reticular cells, mesenchymal cells, chondrocytes, osteoblasts, osteocytes, osteoclasts, dendritic cells, macrophages, plasma cells, Langerhans cells, Merkel cells, pericytes, Sertoli cells, Leydig cells, granulosa cells, theca cells, and Ito cells. In more specific embodiments, support cells correspond to fibroblasts.

[0050] In the first stage of the process described herein, the cells to be conditioned are cultured in a suitable culture medium under favorable temperature and agitation conditions for their growth. Any culture medium currently known in the art that allows stem cell growth can be used in the method described herein. In specific embodiments, the culture medium used in step (a) is Dulbecco's Modified Eagle's Medium (DMEM). In more specific embodiments, the DMEM medium is supplemented with fetal bovine serum and antibiotics.

[0051] In some embodiments, the culture in step (a) lasts between 1 hour and 1 month. In specific embodiments, step (a) lasts between 1 hour and 1 week. In more specific embodiments, step (a) lasts between 12 hours and 48 hours. In even more specific embodiments, the culture in step (a) lasts between 12 and 44 hours; 16 and 48 hours; 16 and 44 hours; 20 and 44 hours; 20 and 40 hours; 24 and 40 hours; or 24 and 36 hours.

[0052] In some embodiments, the cells obtained in step (a) are transferred to a minimal culture medium, where they are subjected to stress due to a decrease in the amount of available nutrients. In specific embodiments, this nutrient reduction corresponds to a reduction in the amount of fetal bovine serum (FBS) from 10% to 1%. In turn, other nutrients, such as inorganic and organic compounds, can be reduced to induce metabolic stress in the cells to be conditioned with GHEM. In more specific embodiments, nutrients include carbohydrates, proteins, lipids, vitamins (such as vitamins A, B, C, D, E, and K), minerals (such as calcium, phosphorus, potassium, sodium, magnesium, iron, zinc, copper, 8882862

[0053] - 6 - manganese, iodine, fluoride, and selenium), essential fatty acids, essential amino acids, nucleotides, antioxidants, electrolytes, trace elements, dietary fiber, and other bioactive compounds that contribute to the growth, maintenance, and proper functioning of cells.

[0054] In particular developmental modalities, the final concentration of nutrients in the culture medium can be adjusted to optimize the metabolic stress induced in the cells. In more particular modalities, the final concentration of macronutrients (such as carbohydrates, proteins, and lipids) and micronutrients (such as vitamins and minerals) can be adjusted within specific ranges. For macronutrients, the concentrations can vary between 10 ng / mL and 100,000 ng / mL, 100,000 ng / mL and 200,000 ng / mL, 200,000 ng / mL and 300,000 ng / mL, 300,000 ng / mL and 400,000 ng / mL, 400,000 ng / mL and 500,000 ng / mL. For micronutrients, concentrations can range from 10 ng / mL to 10,000 ng / mL, 10,000 ng / mL to 20,000 ng / mL, 20,000 ng / mL to 30,000 ng / mL, 30,000 ng / mL to 40,000 ng / mL, and 40,000 ng / mL to 50,000 ng / mL, depending on the specific needs of the experiment and the characteristics of the cells being studied.

[0055] In the context of the present invention, a "cellular reprogramming factor" is used to a denote an agent capable of shifting a cell from a resting or low-activity state into an altered metabolic, proliferative, functional, or rejuvenated state. More specifically an agent that shift cells from a quiescent or aged state toward enhanced metabolic activity, growth, rejuvenation, or functionality.

[0056] The term “cellular reprogramming factor" therefore includes, but is not limited to, growthstimulating hormones (such as HGH), rejuvenation factors, classical transcription-based reprogramming factors, and secreted metabolic regulators.”

[0057] In one embodiment it may correspond to a compound capable of converting a differentiated cell into a less differentiated cell.

[0058] In particular embodiments, the cellular reprogramming factor is selected from inorganic compounds and organic compounds. In more particular embodiments, said inorganic compounds are selected from metals such as calcium, magnesium, iron, zinc, copper, and manganese; signaling gases such as nitric oxide, carbon monoxide, and hydrogen sulfide, among others. In other embodiments, the reprogramming factors correspond to organic compounds selected from peptides, nucleotides, sugars, lipids, or a combination thereof.

[0059] In even more specific embodiments, the cellular reprogramming factor is selected from oligopeptides, polypeptides, carbohydrates, glycoproteins, glycolipids, proteoglycans, mononucleotides or derivatives thereof, polynucleotides, oligosaccharides, saturated and unsaturated fatty acids, eicosinoids, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), endocannabinoids, oleoylethanolamide (OEA), palmitoylethanolamide (PEA), sphingolipids, acylglycerides, cerides, phospholipids, arachidonic acid, hydroxyeicosatetraenoic acid, leukotriene acid, lipids, steroid hormones, glucocorticoids, mineralocorticoids, androgens, estrogens, progesterone, thyroid hormones, vitamin D, 8882862

[0060] - 7 - retinoic acid, lipoproteins, ribozymes; transcription factors, epigenetic modifiers, among others.

[0061] In one embodiment, the cellular reprogramming factor is growth hormone such as human growth hormone (HGH).

[0062] HGH functions not merely as a trophic or growth-promoting molecule, but as a master metabolic and transcriptional regulator capable of inducing cellular reprogramming. HGH activates the GH receptor / JAK2 / STAT axis, PI3K-AKT-mTOR signaling, and MAPK pathways, resulting in coordinated modulation of gene expression, translation, mitochondrial metabolism, protein turnover, and cellular fate programs. Through its canonical endocrine action and autocrine / paracrine signaling, HGH can reconfigure metabolic states by enhancing oxidative phosphorylation, glycolytic flux, amino acid uptake, and nitrogen retention; Increases protein synthesis and ribosomal biogenesis, thereby altering the proteome and structural identity of a cell; Enhances survival, anti- apoptotic responses, and stress tolerance via STAT5 / AKT-dependent transcription of survival genes; Promotes proliferation and lineage progression while modulating checkpoints and senescence programs; Influences epigenetic remodeling, including chromatin accessibility, histone acetylation states, and transcriptional priming through STAT-mediated recruitment of chromatin-regulating complexes; Modulates sternness and differentiation trajectories through downstream insulin-like growth factor (IGF-1) signaling, Wnt / p-catenin cooperation, and crosstalk with Notch, Hedgehog, and retinoid receptor pathways; and Acts as a system-level program switch, shifting cells from quiescent or catabolic states toward growth, repair, biosynthetic activity, and altered functional identity.

[0063] Accordingly, HGH can be understood as a cellular reprogramming effector capable of respecifying metabolic identity, modifying transcriptomic profiles, influencing epigenetic landscapes, and biasing phenotypic outcomes, including differentiation, activation, or regenerative competence.

[0064] In certain embodiments, HGH acts alone or synergistically with additional reprogramming factors — including steroids, cytokines, transcription factors, epigenetic modifiers, or metabolic ligands — to generate context-specific shifts in cell functionality, phenotype, and regenerative behavior.

[0065] In particular development modalities, the final concentration of the cellular reprogramming factor added to the culture medium in step (b) is 0.02 ng / mL to 900 ng / mL. In more particular embodiments, the final concentration of the reprogramming factor is between 0.02 ng / mL and 10 ng / mL, 10 ng / mL and 50 ng / mL, 50 ng / mL and 100 ng / mL, 100 ng / mL and 200 ng / mL, 200 ng / mL and 300 ng / mL, 300 ng / mL and 400 ng / mL, 400 ng / mL and 500 ng / mL, 500 ng / mL and 600 ng / mL, 600 ng / mL and 700 ng / mL, 700 ng / mL and 800 ng / mL, 800 ng / mL and 900 ng / mL.

[0066] In the present disclosure, the term "emergency stimulus" refers to a molecule that triggers a biochemical response in the cell associated with its maintenance and / or repair. In some 8882862

[0067] - 8 - embodiments, the emergency stimulus used in step (c) of the disclosed method is selected from peptide hormones, amino acid-derived hormones, lipid-derived hormones (DAMPs), proinflammatory factors, or a combination thereof.

[0068] In a more particular embodiment, the emergency stimulus is an amino acid-derived hormone, such as epinephrine, or an analog thereof.

[0069] Epinephrine represents a broader class of biogenic amines and catecholaminergic mediators derived from amino acid precursors (for example, tyrosine, phenylalanine, or tryptophan) that act as rapid-response signaling molecules capable of triggering acute metabolic reprogramming, stress adaptation, and survival states. Accordingly, the emergency stimulus may encompass monoamine hormones, neurotransmitters, and paracrine or autocrine regulators, including catecholamines such as epinephrine, norepinephrine, and dopamine; indoleamines such as serotonin or melatonin; histamine- type amines; trace amine modulators; decarboxylated amino-acid derivatives; as well as synthetic, chemically modified, or receptor-selective analogues thereof. Example analogs include ephedrine / pseudoephedrine.

[0070] These molecules function as fast-acting cellular modifiers that alter second-messenger fluxes (including cAMP, calcium, and IP3), modulate mitochondrial performance, reconfigure transcriptional programming, and redirect survival and metabolic pathways. Thus, in some embodiments, the emergency stimulus comprises monoamines, sympathoadrenal hormones, or other amino acid-derived mediators capable of inducing acute cellular reprogramming, shifting cells toward fight-or-flight metabolic biasing, increasing glycolytic flux and ATP mobilization, suppressing anabolic processes with redirection of biosynthetic priorities, modulating apoptosis and stress responses, and activating transcriptional programs associated with adaptation, repair, and survival. In certain embodiments, the emergency stimulus may further include modified, stabilized, synthetic, receptor-selective, or bioengineered analogues of amino acid-derived hormones engineered to optimize potency, stability, pharmacokinetics, or specific reprogramming outcomes.

[0071] In particular embodiments, the final concentration of the emergency stimulus in the culture medium in step (c) is 10 mM to 1000 mM. In more particular embodiments, the final concentration of the emergency stimulus is between 10 mM and 100 mM, 100 mM and 200 mM, 200 mM and 300 mM, 300 mM and 400 mM, 400 mM and 500 mM, 500 mM and 600 mM, 600 mM and 700 mM, 700 mM and 800 mM, 800 mM and 900 mM, 900 mM and 1000 mM.

[0072] In the context of the present invention, a “metabolic activity enhancer” refers to any agent that increases the rate of chemical reactions and biological processes within cells, thereby improving energy production and nutrient utilization. This may include, but is not limited to, hormones, growth factors, vitamins, minerals, and bioactive compounds that facilitate cellular metabolism, promoting cell proliferation, differentiation, and tissue repair. 8882862

[0073] - 9 - ln particular developmental embodiments, the metabolic activity enhancer is selected from mitochondria, macro- and micronutrient cocktails, or a combination thereof. In other embodiments, the metabolic activity enhancer corresponds to mitochondria. In other embodiments of the disclosure, mitochondria as metabolic enhancers are those that are structurally intact, which can be taken up by cells through active processes, primarily, which have the capacity to induce positive changes in a given biological function.

[0074] These positive changes include rejuvenation of cellular function, anti-stress, anti-aging, and anti-apoptotic effects, counteracting the main effects of aging such as genomic instability, telomere shortening, epigenetic alterations, loss of proteostasis, dysregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and altered intercellular communication.

[0075] These mitochondria can shed their mitochondrial DNA, integrate into the endogenous mitochondrial network, or, if not integrated, generate changes in the cell that enhance its activity to produce more mitochondria, grow, proliferate, and produce active agents that can change cell fate or even regenerate the rejuvenating capabilities of the recipient cell.

[0076] By stimulating the production of regenerative agents, the cells can be used for therapeutic purposes.

[0077] In particular embodiments, the final concentration of the metabolic activity promoter added to the culture medium in step (c) is 0.1 ng / pL to 1 pg / pL. In more particular embodiments, the final concentration of the metabolic activity promoter is between 0.1 ng / pL and 10 ng / pL, 10 ng / pL and 50 ng / pL, 50 ng / pL and 100 ng / pL, 100 ng / pL and 200 ng / pL, 200 ng / pL and 500 ng / pL, 500 ng / pL and 1000 ng / pL.

[0078] In some embodiments, the incubation in step (d) lasts between 1 hour and 1 week. In more specific embodiments, the incubation in step (d) lasts between 12 and 48 hours. In even more specific embodiments, the incubation in step (d) lasts between 12 and 44 hours; 16 and 48 hours; 16 and 44 hours; 20 and 44 hours; 20 and 40 hours; 24 and 40 hours; or 24 and 36 hours.

[0079] In some embodiments, a single incubation is performed after adding the cellular reprogramming factor, the emergency stimulus, and the metabolic activity stimulant. In specific embodiments, this incubation step lasts from 24 to 72 hours. In even more specific embodiments, this incubation step lasts 48 hours.

[0080] Other aspects of the invention

[0081] The conditioned cells produced by the methods of the invention form one aspect of the invention, as does the medium in which they are conditions (“conditioned medium”) which may include the cells or be cell-free. All of these things have utility in regenerative medicine, anti-aging applications, and skin-repair strategies, in both therapeutic and cosmetic settings. 8882862

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[0083] Conditioned-cells obtained by the methods of the invention can be metabolically activated or rejuvenated cells, including but not limited to, hypoxia-conditioned cells.

[0084] Conditioned medium produced by such cells may be enriched in exosomes, extracellular vesicles, cytokines, peptides, and bioactive molecules capable of exerting therapeutic or cosmetic benefits.

[0085] Use of these things in therapeutic or cosmetic methods, or in the preparation of therapeutic or cosmetic products forms a further aspect of the invention, as do the cells or medium for use in such methods.

[0086] For example, conditioned cells can then be used directly in regenerative or dermatological or epidermal procedures “ex vivo”.

[0087] The conditioned medium can, if desired, be used independently of the cells. When the conditioned cells are exposed to the methods of the invention they release a wide variety of regenerative compounds into their surrounding medium, including exosomes, extracellular vesicles, cytokines, peptides, nucleic acids, and other bioactive molecules. The resulting medium is enriched in factors that promote regeneration, rejuvenation, and cellular repair. This conditioned medium can be used as a standalone therapeutic or cosmetic product when applied to the human or animal body “in vivo”, particularly in topical formulations intended for skin application.

[0088] Topical usage is particularly preferred. UV radiation is well known to induce skin aging, impair dermal and epidermal cell function, and reduce the regenerative capacity of skin tissue. The present invention therefore offers a way to improve the quality of the factors secreted by cells, making the conditioned medium suitable for repairing UV-induced damage and enhancing skin appearance or function.

[0089] A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0090] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0091] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates 8882862

[0092] - 11 - otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0093] Ranges are often expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0094] Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

[0095] The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

[0096] The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by crossreference.

[0097] The technological development is presented in detail through the following examples, which are provided for illustrative purposes only and are not intended to limit its scope.

[0098] Example 1. Cell Conditioning and Induction of Regenerative Phenotype

[0099] Assays of the method described here were performed, specifically following the "GHEM" methodology outlined in FIG. 1.

[0100] Mesenchymal Stem / Stromal Cells (MSCs) are multipotent cells widely used in regenerative medicine as a standard biologic model. In this Example MSCs are used to demonstrate that the described factors can induce measurable functional and metabolic changes in a paradigmatic responsive cell type.

[0101] Specifically, 50,000 mesenchymal stem cells (MSCs) were added to each well of a P6 plate with DMEM medium (4.5 g / L glucose, 1% glutamine, 1% penicillin / streptomycin) supplemented with 10% FBS under standard culture conditions on the first day. On the second day, the DMEM medium was changed to a fresh complete medium containing 1% FBS with similar characteristics and 200 pg / mL of human growth hormone (hGH). 10 pM epinephrine was added to induce cellular stress, and 1 ,000 ng of mitochondria diluted in 1 ,000 pL were added to the medium, obtained as described using the Thermo Scientific™ Mitochondrial Isolation Kit 89801. The cells were incubated at 37°C in a 5% CO2atmosphere to maintain the medium pH between 7.2 and 7.4, with an adequate level of humidity to prevent medium evaporation. Finally, on the fourth day, the treated cells and conditioned medium were harvested and separated using methods known in the art. 8882862

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[0103] Example 2. Senescence Assessment in Fibroblasts by Flow Cytometry

[0104] The level of senescence in fibroblasts was assessed through 28 passages by flow cytometry using the cellular senescence marker CellSenescence™, which allows the identification and quantification of senescent cells and the intensity of this phenotype in a fluorimetric reaction. Fibroblasts were exposed to GHEM during initial conditioning for 48 hours, and then the medium was renewed with GHEM for an additional 48 hours until the day of the assay. Conditions were used with 100% GHEM medium and a mixture of 50% GHEM with 50% fresh medium, assessed using the CellSenescence™ marker with flow cytometry.

[0105] On the first day, 250,000 fibroblasts were seeded per well in a P6 plate in DMEM medium supplemented with 10% FBS. On the second day, cells were exposed to 50 mM hydrogen peroxide for 30 minutes and then to 3 minutes of UV radiation. After this senescence stimulus, the medium was changed to a mixture of 50% GHEM and 50% DMEM with 1% FBS and 100% GHEM.

[0106] On the third day, cell viability was checked. On the fourth day, the medium was renewed again with the same mixing conditions. On the fifth day, cell viability was checked again.

[0107] On the sixth day, the medium was removed from each well, washed with 1 mL of PBS, and trypsinized with 500 pL of trypsin, incubating for 5 minutes at 37°C with 5% CO2. The trypsin was then inactivated with 2 mL of inactivation medium, and the medium with the cells was transferred to 2 mL tubes, centrifuging them at 1500 g for 5 minutes at 4°C. The supernatant was removed, and PBS was added to resuspend the cells. 3.7% PFA was added to each sample with PBS, resuspending the cells, and incubating for 10 minutes at room temperature, protected from light. The cells were centrifuged at 1500 g for 5 minutes at 4°C. The supernatant was removed, washed with 1% bovine serum albumin (BSA) in PBS, centrifuged again at 1500 g for 5 minutes at 4°C, and stained with the CellSenescence™ marker according to the manufacturer's instructions. The cells were incubated for 2 hours at 37°C in a heat block outside the incubator and protected from light. The mixture was then centrifuged at 1500 g for 5 minutes at 4°C. The resulting supernatant was removed, and the cells were resuspended in 200 pl of 1% PBS in BSA, after which they were transferred to cytometry tubes for further analysis.

[0108] As can be seen in FIG. 2, in both 100% GHEM and 50% GHEM and 50% fresh medium treatments, fibroblasts showed a highly significant reduction in the intensity of the marker associated with cellular senescence after exposure to GHEM medium. A nonparametric Mann-Whitney analysis was also performed, showing highly significant results (P < 0.01) in the reduction of senescence in GHEM-treated fibroblasts.

[0109] These results indicate that GHEM medium has a regenerative effect on the aging observed in this assay and may improve fibroblast viability and functionality. 8882862

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[0111] Example 3. Evaluation of cell status by microscopy after senescence induction and culture in the presence of GHEM

[0112] The level of senescence in fibroblasts was assessed over 28 passages using conventional (phase-contrast) microscopy, which allows for the identification of senescent cells and the intensity of this phenotype in relation to their size, granularity, and shape. Fibroblasts were exposed to GHEM during initial conditioning for 48 hours, and then the medium was renewed with GHEM for an additional 48 hours until the day of the assay. Conditions were used with 100% GHEM medium and a mixture of 50% GHEM with 50% fresh medium. One mixture was 100% GHEM and another with a mixture of 50% GHEM and 50% DMEM supplemented with 10% FBS.

[0113] On the first day, 250,000 fibroblasts were seeded per well in a P6 plate in DMEM medium supplemented with 10% FBS. On the second day, cells were exposed to 50 mM hydrogen peroxide for 30 minutes and then to 3 minutes of UV radiation. After this senescence stimulus, the medium was changed to a mixture of 50% GHEM and 50% DMEM with 1% FBS and 100% GHEM.

[0114] On the third day, cell viability was checked. On the fourth day, the medium was renewed again with the same mixing conditions. On the fifth day, cell viability was checked again.

[0115] On the sixth day, the medium was removed from each well, washed with 1 mL of PBS, and samples were taken for subsequent analysis of size, granularity, and shape. It can be estimated that under normal conditions (fibroblasts without double shock), fibroblasts present an elongated, wave-like shape, in a normal distribution. It can then be observed that cells exposed to H2O2 (ROS) and ultraviolet (UV) radiation for 3 minutes changed their shape, decreasing their size due to the stress induced by the factors that cause senescence, as demonstrated in FIG. 2. After culture with 50% GHEM and 100% GHEM, a complete recovery of the normal phenotype of cells exposed to senescence inducers is observed. These images demonstrate the usefulness of the conditioned medium using the GHEM methodology (FIG. 3).

[0116] Example 4. Evaluation of P21 and P53 levels in fibroblasts exposed to GHEM medium

[0117] Senescence is a complex phenotype where multiple factors, such as those related to the beta-galactosidase activity of these cells and the regulation of genes associated with this phenotype, are key to defining it and determining the effect of factors that could lessen or reverse its occurrence. In relation to the previous essay, which determined the decrease in the senescence marker in cells exposed to GHEM, it is imperative to understand the expression of the genes associated with this phenotype. That is, the expression of two genes, P21 and P53, overexpressed in senescent cells is analyzed. These genes are critical in regulating the cell cycle and stress response. P53 acts as a guardian of the genome, inducing the expression of P21 in response to DNA damage, leading to cell cycle arrest and, in extreme cases, to cellular senescence or apoptosis. P21 is an inhibitor of cyclin-dependent kinases (CDKs), and its overexpression is associated with 8882862

[0118] - 14 - the induction of cellular senescence, a state of decreased growth that contributes to cellular aging.

[0119] To assess the senescence of cells treated with GHEM medium, gene expression levels of P21 and P53 were measured in fibroblasts with different passage numbers (26 and 32) after 72 hours of exposure to GHEM medium. PUM1 was used as a reference gene.

[0120] In cells with lower passage numbers (26), higher levels of P21 and P53 were observed, respectively. In those with higher passage numbers (32), only P53 was monitored.

[0121] As demonstrated by the results presented in FIG. 4, after exposure to GHEM medium for 72 hours, reduced levels of P21 and P53 were observed. This finding suggests that GHEM medium reduces cellular stress and the induction of senescence, thereby improving fibroblast viability and function regardless of their passage number. This indicates that GHEM medium has a rejuvenating effect on cells, mitigating the molecular signals associated with cellular aging and P21 -induced senescence.

[0122] Example 5. Induction of DNA-laden membranous structures in GHEM-conditioned mesenchymal stem cells

[0123] In this study, the induction of vesicle-associated membranous structures loaded with genetic material (DNA) from MSCs using GHEM medium was evaluated using the method described below.

[0124] A total of 250,000 MSCs were used, which were exposed for 48 hours to treatment with 10 pM epinephrine, 50 ng / mL of growth hormone, and 1000 ng / ml of mitochondria isolated from MSCs. The conditioned medium was taken from these cells and mixed with reagents that allow DNA to be detected and estimated by spectrophotometry and fluorometry.

[0125] The results showed significant differences in the amount of extracellular genetic material between cells treated with GHEM and controls (FIG. 5). Analysis of the conditioned medium using the GHEM method revealed the absence of free genetic material, as determined by a DNA quantification method. Upon treatment of the GHEM medium with lysis buffer, DNA release was detected, suggesting the significant presence of vesicles loaded with genetic material. The initial absence of free DNA, followed by its post-lysis release, indicates that these vesicles contained such genetic material. This finding suggests that GHEM medium can induce the secretion of potentially DNA-laden structures, which could have important implications for the regenerative activity of cells exposed to GHEM.

[0126] Example 6. Microscopic examination of fibroblasts and MSCs exposed to GHEM medium 8882862

[0127] - 15 -

[0128] The morphology of MSCs at passage 26 and fibroblasts at passages 20 and 36 was evaluated after exposure to the GHEM generation steps for 24, 48, 72, and 96 hours. The previously described cell conditioning steps were followed.

[0129] The images obtained show that there was no induction of stress or cell death due to the use of GHEM. Furthermore, a favorable morphological change was observed, indicating a recovery of a profile not subject to stress or cellular senescence, especially in passage 36 MSCs. In phase contrast photographs, a decrease in growth is evident, particularly between 48 and 72 hours (FIG. 6 & 7A-B).

[0130] Example 7. Evaluation of the Effect of GHEM on Fibroblast Proliferation

[0131] Many differentiated or partially differentiated cells can undergo significant functional or metabolic changes in response to appropriate stimuli. For instance somatic cells can be metabolically reprogrammed toward a more active or regenerative state. Support cells (fibroblasts, endothelial cells, immune cells) are known to undergo functional “reprogramming” when exposed to cytokines, hormones, or metabolic regulators.

[0132] In this example, the effect of GHEM on the growth and morphology of passage 18 to 28 fibroblasts was evaluated. 50,000 cells were seeded in p12 plates and, after 48 hours of culture, treated with GHEM medium for 24 hours. At the end of the process, a parametric t-test analysis of the results revealed highly significant differences (P < 0.0001) in the induction of proliferation of fibroblasts treated with GHEM (FIG. 8).

Claims

8882862- 16 -CLAIMS1 . A method for conditioning cells comprising the following steps:(a) depositing cells in a culture medium;(b) adding a cell reprogramming factor to the culture medium in (a) and incubating;(c) introducing an emergency stimulus to the culture medium obtained in (b) and incubating; and(d) adding a metabolic activity stimulant to the culture medium obtained in (c) and incubating; wherein the metabolic activity stimulant is selected from mitochondria, macro- and micronutrient cocktails, or a combination thereof;2. The method according to Claim 1 , wherein the culture medium used in step (a) is or has been adjusted to decrease the nutrient concentration.

3. The method according to Claim 1 or Claim 2, wherein a single incubation is performed for 24 to 72 hours after adding the cellular reprogramming factor, the emergency stimulus, and the metabolic activity stimulant.

4. The method according to any one of Claims 1 to 3, wherein the cells in step (a) are stem cells or support cells.

5. The method according to Claim 4, wherein the stem cells are selected from totipotent stem cells, pluripotent stem cells, multipotent stem cells, unipotent stem cells, induced reprogrammed stem cells, or a combination thereof.***6. The method according to any one of Claims 1 to 5, wherein the cellular reprogramming factor is selected from proteins, nucleic acids, lipids, or a combination thereof.

7. The method according to Claim 6, wherein the cellular reprogramming factor is a growth hormone, which is optionally HGH.

8. The method according to any one of Claims 1 to 7, wherein the final concentration of the cellular reprogramming factor is 0.02 ng / mL to 900 ng / mL.***9. The method according to any one of Claims 1 to 8, wherein the stimulant is selected from peptide hormones, amino acid-derived hormones, lipid-derived hormones, Damage- Associated Molecular Patterns (DAMPs), proinflammatory factors, or a combination thereof.

10. The method according to Claim 9 wherein the stimulant is epinephrine or an analog thereof.8882862- 17 -11. The method according to any one of Claims 1 to 10, wherein the final concentration of the stimulant is 10 mM to 1000 mM.

12. The method according to any one of Claims 1 to 11 , wherein the metabolic activity stimulant is mitochondria.

13. The method according to any one of Claims 1 to 12, wherein the final concentration of the metabolic activity stimulant is 0.1 ng / pL to 1 ng / pL.14 The method according to any one of Claims 1 to 13 wherein steps (b)-(d) are carried out sequentially in the order stated.15 The method according to any one of Claims 1 to 14 wherein the method is for restoring the regenerative and / or proliferative and / or regenerative capacity of the cells.16 The method according to any one of Claims 1 to 15 further comprising separating the conditioned cell from the culture medium after steps (a)-(d).***17 A conditioned cell obtained or obtainable by a method as claimed in any one of Claims 1 to 16.18 A conditioned culture medium, which is optionally cell-free, obtained or obtainable by performing a method as claimed in any one of Claims 1 to 16, wherein said conditioned medium includes one or more additional cellular-derived extracellular vesicles, cytokines, peptides, or other bioactive molecules compared to the culture medium used in step (a).19 Use of a conditioned cell or conditioned medium of Claim 17 or Claim 18 in a method of treatment of a human or animal subject.20 A therapeutic or cosmetic method of a subject in need of the same, the method comprising administering a conditioned cell or conditioned medium of Claim 17 or Claim 18 to the subject21 The therapeutic or cosmetic method according to claim 20, the method comprising applying the medium topically to the skin of the subject8882862- 18 -22 The therapeutic or cosmetic method according to claim 21 for repairing UV- induced damage and / or enhancing skin appearance or function.

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