A Comprehensive Review of Stem Cell Conditioned Media Role for Anti-Aging on Skin

Introduction

Anti-aging treatment is an area of study with many unknowns, particularly when it comes to the use of Conditioned Medium (CM). Prior research has shown that MSCs derived from CM possess anti-aging properties. Superior secretory capabilities can be derived from fetal MSCs sourced from human umbilical cord blood (CB).¹ Stem cells secrete a diverse array of growth factors and compounds that induce tissue renewal, all of which are found within CM.² Cellular medicines (CMs) are anticipated to encounter less stringent regulatory constraints when contrasted with stem cell therapy products in their cellular preparation format because they are rich in cytokines and growth factors that meet regulatory requirements.³ CM contains a variety of cytokines and growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-β), which are important in cell growth and maintenance of skin tissue.^4,5 Additionally, CM is rich in vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), macrophage stimulating protein (MSP), keratinocyte growth factor (KGF), and insulin-like growth factor 1 (IGF-1).⁶ As long as the donor of umbilical cord tissue is properly screened, tested, and approved, CM is devoid of cells, thereby eliminating the potential for Graft Versus Host Disease (GvHD) and mitigating adverse consequences.³ Currently, there are no specific regulations governing the use of stem cells in cosmetics, so testing requires approval from a regulatory body before initiating clinical trials.⁷

Skin aging is a multifaceted and progressive biological process associated with both photoaging, induced by exposure to sunlight, and chronological aging, occurring naturally over time.⁸ Photoaging and chronological aging may share similar mechanisms, as evidenced by reduced responses to growth factors associated with chronological aging, The deterioration of the extracellular matrix, reduced production of collagen molecules, and decreased procollagen functionality are evident in photodamaged skin.⁹ Numerous internal and external elements play a role in the phenomenon of skin aging by weakening the skin’s structural integrity and impairing regular physiological processes. UV light, commonly known as a contributor to photoaging, is the most significant external factor among these components.¹⁰ Signs of aging skin include wrinkles, dryness, thinning, sagging, uneven pigmentation, and loss of elasticity.^11,12 Additionally, aging can manifest as drooping skin, age spots or blemishes, changes in pigmentation, loss of skin tone, and dehydration.¹³ The manifestation of skin aging through clinical symptoms like wrinkles, sagging, and irregular texture predominantly stems from alterations in the extracellular matrix (ECM), particularly affecting collagen and elastin components.^14–18 This results in the buildup of impaired large molecules like lipids, proteins, and DNA. Reactive molecules like free radicals, produced during regular cellular metabolism, chemically alter these macromolecules. As people age, the generation of these molecules increases significantly.¹⁹

Ultraviolet (UV) radiation plays a pivotal role in the process of skin aging. The effects of both ultraviolet B (UVB) and ultraviolet A (UVA) rays on the skin cells manifest through the induction of considerable oxidative stress. This stress arises from the interaction between UV rays and specific substances within the cells known as chromophores and photosensitizers. This interaction leads to detrimental effects such as genetic damage, cellular aging, and damage to connective tissues.²⁰ The extent of harm induced by ultraviolet (UV) radiation differs based on the specific wavelength of the light.^21,22 UVB radiation, specifically, has the potential to induce genetic alterations within cellular DNA, resulting in manifestations such as skin erythema and the heightened risk of developing skin malignancies, resulting in approximately 800–1000 times more skin damage than an equivalent dose of UVA radiation.²³ Extended contact with UV radiation leads to solar elastosis, a condition marked by the degradation of the extracellular matrix (ECM) within the human skin, consequently precipitating the formation of wrinkles. Additionally, UV light stimulates the production of reactive oxygen species (ROS) in the skin.²⁴ It is believed that UVB-irradiated fibroblasts contain ROS that activate cytoplasmic signaling pathways associated with both cell growth and the aging process.^25,26 As tissues age, there is typically a rise in the generation of reactive oxygen species (ROS), accompanied by heightened concentrations of 8-Hydroxyguanine (8-oxo-dG) within mitochondrial DNA (mtDNA). These observations imply that the progressive buildup of oxidative damage to DNA plays a substantial role in the aging phenomenon.²⁷ Fibroblasts exposed to UVB radiation exhibit a reduction in the synthesis of type I collagen along with the upregulation of MMP-1 expression.^28–33

Anti-aging medicine, a rapidly evolving field, focuses on utilizing cutting-edge scientific and medical advancements to prevent, detect, treat, and cure age-related dysfunctions. Its primary objective, however, goes beyond merely extending lifespan. Rather, anti-aging medicine aims to sustain a healthy existence for an extended duration. This involves safeguarding against age-related ailments, including atherosclerosis, neurodegenerative disorders, cancer, diabetes, and molecular-level skin wrinkles, by employing antioxidants.^34–36 Several compounds have been identified as having geroprotective properties. These include anesthetics like procaine, plant polyphenols such as antioxidants and resveratrol, rapamycin, and vitamins E, C, and A.³⁷ Other compounds with geroprotective effects include coenzyme Q, lipoic acid, carotenoids, selenium, hormones like growth hormone, thyroid hormones, and adrenaline, as well as sex hormones. Additionally, melatonin, bioregulatory peptides like thymalin and epithalamin, biguanides such as phenformin and metformin, and adaptogens like ginseng have been studied for their anti-aging properties. These geroprotective compounds, which are being researched globally, disrupt the oxidative balance and include both natural components like resveratrol, rapamycin, and procaine, as well as synthetic molecules.³⁵ Substances possessing innate anti-aging attributes, including polysaccharides, hydroxy acids, polyphenols, vitamins, and various other compounds, significantly contribute to skincare.³⁸ Hyaluronic acid, collagen, and elastin play vital roles in maintaining the integrity and appearance of the skin. These three elements act as the building blocks, imparting strength, flexibility, and structure to the skin’s composition. With collagen’s robustness, elastin’s elasticity, and hyaluronic acid’s moisture-retaining properties, the skin achieves a harmonious combination of resilience, suppleness, and smoothness. These structural components work in unison to create a solid foundation for healthy and youthful-looking skin.³⁹

In the development of anti-aging science, many therapies are being explored to achieve the best anti-aging results. Among these, cell therapy stands out, encompassing both unicellular and multicellular therapies, including stem cell-based and non-stem cell approaches. These therapies differ mainly in their characteristics, sources of isolation, and areas of application.⁴⁰ Stem cell research is a rapidly advancing field that holds promise in the realm of regenerative medicine. It offers hope for treating numerous severe illnesses by addressing unmet medical needs.⁴¹ Stem cells possess an inexhaustible potential for proliferation and possess an extraordinary capacity to undergo differentiation into diverse cellular lineages, rendering them a captivating focus of research within the realm of anti-aging investigations. These remarkable cells possess the capacity to mitigate oxidative stress, prevent programmed cell death, and counteract aging effects. They also have the ability to promote the generation of the extracellular matrix (ECM) to rejuvenate the skin, regulate inflammatory reactions, and offer advantages such as reducing wrinkles and enhancing brightness.^42,43 Mesenchymal stem cell (MSC)-derived treatments represent a promising avenue in addressing skin disorders, as they provide replenishing cells that aid in regulating epidermal equilibrium and rejuvenating compromised tissues.^44–46 Human mesenchymal stem cells (hMSCs) possess significant advantages owing to their remarkable capacity for differentiation into diverse cellular lineages. These cells can be readily obtained, cultured, and efficiently expanded within laboratory environments. Furthermore, they possess the versatility to be utilized in both allogeneic therapies and exhibit robust paracrine functions. Additionally, researchers have utilized the capabilities of conditioned medium (CM) obtained from these stem cell cultures for diverse pragmatic purposes.

Material and Methods

The literature search was conducted using the Scopus database to capture all high-quality research articles. The search for articles was not restricted by publication date to ensure the comprehensiveness of the review. Relevant research articles were identified using the following keywords: ((“conditioned medium“ OR “secretome“) AND ‘stem cell’ AND (”anti-aging” OR ”skin rejuvenation” OR ‘skin aging’ OR ‘cellular senescence’)). Following identification, the articles underwent manual screening in accordance with predefined inclusion and exclusion criteria (outlined in Table 1). The initial search yielded 68 articles, which were subsequently subjected to screening. Following a thorough evaluation, 23 articles were deemed to meet the inclusion criteria, whereas 45 articles failed to meet the criteria established for this research endeavor.

Table 1 Inclusion and Exclusion Criteria

Results

Human Skin Aging Process and Factors

Skin aging, characterized primarily by a decrease in elasticity and the development of wrinkles, has puzzled humanity for centuries, driving us to tirelessly combat its effects, yet it remains an enigma. The skin experiences extrinsic aging, influenced by external factors, in addition to intrinsic aging, resulting in alterations in both the structure and function of the skin tissue.^47,48 Other research indicates that aging causes changes in inflammatory responses as well as scar remodeling, maturation, proliferation, and cell migration, all of which play a crucial role in effective skin healing.⁴⁹ The aging process negatively impacts the mechanisms that facilitate wound healing. This is mainly due to a reduced rate of cell migration and proliferation, as well as a deficiency in the production of the components comprising the extracellular matrix. Furthermore, there is a shortage of growth factors that promote cell division. In older animals, wounds exhibit diminished cell proliferation and noticeable alterations in matrix deposition across all biological components, including keratinocytes, fibroblasts, and endothelial cells.⁵⁰ An overview of the various characteristics of aging is provided in Figure 1.

Figure 1 Aging Characteristics. The hallmark indicators of aging include the deterioration of the extracellular matrix (ECM), heightened generation of reactive oxygen species (ROS) within the skin, and raised concentrations of 8-hydroxyguanine (8-oxo-dG) in mitochondrial DNA (mtDNA). These changes manifest in various ways, including wrinkles, dryness, loss of elasticity, thinning, sagging, uneven pigmentation, the appearance of age spots or blemishes, sagging skin, changes in pigmentation, dehydration, and loss of skin tone. Additionally, there is an accumulation of damaged lipids, proteins, and DNA. Reactive molecules like free radicals chemically modify these macromolecules, contributing to the aging process.

Both the processes of intrinsic and extrinsic aging are subject to the influence of diverse mechanisms and conditions. Emerging studies suggest that oxidative harm plays a pivotal role in the initiation of numerous age-related ailments, including but not limited to arthritis, Alzheimer’s disease, and allergic conditions.⁵¹ As individuals grow older, the incidence of these conditions tends to rise steeply. Increased concentrations of reactive oxygen species (ROS) within cellular structures are strongly linked to the aging of cells, with hydrogen peroxide (H2O2) potentially playing a pivotal role in initiating this aging phenomenon. Cells undergoing aging can be identified by enhanced Senescence-Associated β-Galactosidase (SA-β-gal) activity. Senescence can be induced by markers of cellular aging, including p53/p21Waf1/Cip1, INk4a, and Id-1 (inhibitor of differentiation or DNA binding-1). Senescence can inhibit DNA replication and result in cell cycle arrest.^52,53

Anti-Aging Treatment and Care Using Conditioned Medium

CM-MSCs have various developmental aspects that act as chemoattractants, recruiting endothelial and macrophage cells for wound healing through cytokines.⁵⁴ Chondrocyte-derived factors (CM) have been observed to stimulate the proliferation, migration, and synthesis of Extracellular Matrix (ECM) in fibroblasts exposed to exogenous agents. This implies the potential utility of CM in facilitating skin regeneration and wound healing processes.⁵⁵ Therapies combining CM with appropriate cell populations could serve as alternatives or potentially enhance existing procedures. Immunocompatibility is one benefit that could make CMs and their component medicines preferable to cell-based therapies. The absence of the need for donor and recipient selection in therapy due to cell exclusion is another notable benefit.^15,56

Within the cell culture microenvironment, factors such as cell contact inhibition, physical and chemical characteristics, capacity for cell growth and differentiation, and cell aggregation ability all affect its structure.⁵⁷ Like any method, there are uncertainties regarding the application of CM in regenerative medicine, similar to any new technique. A significant concern arises from the absence of universally accepted protocols and criteria governing the biological processing and quality assurance of treatments derived from cell media (CM). This absence results in a diverse array of compositions and deviations, influenced by factors such as the methodology of cultivation and duration.⁵⁸

Furthermore, cells prepared for therapy may undergo cell death in culture or during transport to the injury site, a challenge that CM avoids due to its lack of living material, thereby avoiding such inconveniences.⁵⁹ CM is easy to produce, package, freeze, and transport.¹⁵ The production phase of cell-based therapies requires stringent laboratory sterility measures, while CM application can take place under non-sterile conditions.^15,56

Using secretome MSCs in biomedical contexts is perceived to present fewer risks compared to the utilization of medicinal formulations containing viable cells.^15,60 Aged mouse MSCs display decreased antioxidant capacity.^61,62 It is worth noting that there may be a risk of hypersensitivity reactions associated with the base medium used for cell culture to produce conditioned medium. Due to limited clinical trials in this field, the long-term effects of CM remain poorly understood.⁵⁹

Therefore, regenerative therapies requiring only outpatient conditions are well-suited to CM. Therapies involving CM can also save time and money because media can be obtained from cells introduced once without waiting for them to mature.⁶³ As several researchers indicate, the secretome can sustain its curative capabilities over time.^15,64,65 Through the examination of individual patient reactions to the administration of contrast media via in vitro studies, it becomes possible to gauge to some extent the semi-retention duration of medium constituents within the body. Incorporating patient-specific variables into the medium’s structure during the manufacturing process can also significantly increase the effectiveness of CM-mediated implementation, enabling the advancement of secure and efficient tailored treatments for diverse ailments.⁵⁶

Conditioned Medium as Stem Cell Carrier

Numerous studies have showcased the effectiveness of stem cells conditioned in a medium for combating aging, with a predominant emphasis on non-dermal stem cells, notably those sourced from adipose tissue or umbilical cord blood.^66–68 Umbilical Cord-Conditioned Medium (UC-CM) has been shown in numerous clinical and preclinical studies to support tissue homeostasis by promoting skin regeneration and protecting against harmful skin conditions.^69–72 It is well-known that conditioned media (CM), with an assortment of cytokines and growth factors stimulate the restoration of damaged tissues, facilitating their regeneration. Therefore, stem cell conditioned media represent a cutting-edge technology that can be applied to treat dark spots around the eyes, hair, and skin care.^15,41 Below are examples of various conditioned media formulations, each containing different types of stem cells for anti-aging purposes (Table 2).

Table 2 Conditioned Medium Carrier of Stem Cell as Anti-Aging

Various Anti-Aging Tests

In anti-aging testing, various methods are employed to analyze the effectiveness of conditioned medium containing stem cells. UVB rays can damage DNA, age cells, and potentially trigger skin cell cancer.⁹¹ UVB-induced apoptosis is thought to represent a protective mechanism aimed at maintaining skin integrity while eradicating aberrant precancerous cells.⁹² The G1 phase of the cell cycle is arrested, and the cell’s capacity to divide is reduced due to cellular degeneration.⁹³ Intense light exposure can lead to increased cell apoptosis and inflammation. Cell migration was assessed via a wound healing assay. HaCaT cells were cultured on six-well plates. When they reached full coverage, a deliberate wound was created in the center of each well. The migration rate of HaCaT cells was photographed and quantified using inverted microscopy.⁸⁸

A summary of in vitro tests involving conditioned medium for anti-aging purposes is provided in Table 3. Clinical trials have also been conducted by Sohn et al in 2018,¹⁴ where twenty-five female participants with mild to moderate signs of aging, aged 29 to 69, were enrolled. Over the course of four weeks, participants applied a cosmetic solution infused with 5% EPC-CM to their cheeks twice daily. Initial assessments were conducted to evaluate Fitzpatrick skin attributes and dimensions of crow’s feet wrinkles,⁹⁴ doctor’s global rating scale,⁹⁵ Before the application, measurements of the wrinkle index using the ANTERA 3D system from Miravex in Dublin, Ireland, were conducted. This index encompasses various aspects such as skin texture, skin surface, and the depth of wrinkles (specifically, crow’s feet). The evaluation of these components was performed by two dermatologists. The wrinkle index is calculated by determining the spot with the least facial muscle activity. The deepest point in a given area is used to determine wrinkle depth, while the skin’s surface represents the volume of the entire affected area in a given region. The Ra value of the ANTERA 3D program index itself reflects skin texture, indicating the level of surface roughness. Various tests of conditioned medium as anti-aging agents in vivo and clinically are summarized in Figures 2 and 3. Figure 3 represents the rapid advancement of clinical testing for conditioned media as an anti-aging agent. This progress can provide more safety data on the use of conditioned media, which would be useful for regulatory bodies in each region. However, there are still numerous obstacles in testing that have prevented several countries from issuing specific regulations for the use of conditioned media (CM), particularly in clinical trials. Currently, there are no regulations governing the use of stem cells in cosmetics, so testing requires approval from a regulatory body before starting clinical trials.⁷ The use of CM for regenerative medicine remains a new field with many uncertainties. There is a lack of recommendations and general standards for bioprocesses and quality control of CM-based therapies, leading to significant variations in content that depend on the method and duration of culture.⁹⁶ Furthermore, hypersensitivity reactions may occur due to the components in the basic media used to produce CM. Given the limited number of clinical trials in this field, the long-term effects of its use are not yet fully understood.⁹⁷ The use of stem cells has certain limitations, including maintaining biological activity, quantifying biologically active substances, and managing logistics and distribution.⁹⁸ Storage temperatures must always be low, and storage times must be relatively short.⁴⁶ In some studies, small sample sizes and a lack of pathological assessments are the main limitations.⁷⁵ A challenge that cannot be ignored is the difficulty of maintaining conditioned medium during storage until its application.

Table 3 Various Conditioned Medium Tests as Anti-Aging in vitro

Figure 2 Assessment of Conditioned Medium for Anti-Aging Effects In Vivo. In an experiment comprising 28 male Wistar rats aged between 10 and 12 weeks, the rodents underwent a one-week acclimatization period before being randomly assigned to four distinct groups. For four weeks, ASC-CM was administered twice daily, resulting in a total radiation dose of 4.2 J/cm2 over six weeks. Data analysis was conducted using SPSS. According to the assessment of Transepidermal Water Loss (TEWL) and skin thickness, ASC-CM shows potential as a viable remedy for skin photoaging. Nevertheless, additional investigation is warranted to ascertain the enduring impacts of ASC-CM. In aged mice, the immune response exhibited greater efficacy when conditioned with human cord blood mesenchymal stem cells (CBMSC-CM). This led to a decreased cell count associated with aging in kidney tissue. CBMSC-CM, with its significantly reduced hydrogen peroxide levels, stimulates T-cell responses and reduces oxidative stress, thereby inhibiting cellular senescence.

Figure 3 Test Results of Conditioned Medium as Clinical Anti-Aging. Previous research indicates that the integration of human umbilical cord mesenchymal stem cell-conditioned medium (hUC-MSCs-CM) with Microneedling (MN) resulted in noteworthy enhancements in brightness and pore size, as evidenced by comprehensive assessments conducted before and after treatment. These improvements were found to be significantly superior to those observed with MN as a standalone therapy. Another investigation emphasizes the practical results, wherein a cosmetic formulation comprising 5% EPC-CMs was administered to the facial skin of 25 female participants twice a day over a duration of four weeks. In this clinical investigation, EPC-CMs demonstrated improvements in the signs of skin aging, potentially through the activation of the cellular defense system, as substantiated by in vitro findings.

Mechanism of Stem Cell Condition Medium as Anti-Aging

Zuk et al published the first analysis of adipose-derived stem cells (ASCs) in 2001.⁹⁹ Numerous research investigations have demonstrated the beneficial impacts of these cells, such as anti-apoptotic, immunomodulatory, and wound repair impacts, either through paracrine effects or differentiation.^99,100 Recent research has indicated that ASCs have the potential to improve the emergence of wrinkles resulting from photoaging is accompanied by a simultaneous enhancement in collagen synthesis within fibroblasts cultured in vitro.^29,101

In a separate study conducted by Wang et al in 2015,⁸⁵ HDFs at different points in the aging process underwent UVB exposure followed by cultivation in conditioned medium derived from adipose-derived stem cells (ADSC-CM), which contains the whole crude secretions and offers more convenient storage and safer application compared to ADSCs. Various biochemical markers were assessed to comprehensively observe the aging characteristics of HDFs and evaluate the anti-aging effects of ADSC secretions.

Moreover, it was observed that mesenchymal stem cells obtained from umbilical cord blood (UCB-MSCs) exhibit elevated expressions of wound healing mediators in contrast to various other MSCs.¹⁰² UCB-MSCs promote collagen synthesis, proliferation, and fibroblast migration.¹⁰³ Additionally, conditioned media from UCB-MSCs have demonstrated the ability to improve wound closure and re-epithelialization in animals when administered via subcutaneous injection.¹⁰⁴ The visual representation of the mechanism of conditioned media in helping to prevent skin aging is shown in Figure 4.

Figure 4 Mechanism of Stem Cell Condition Medium as Anti-Aging.

Conclusion and Future Perspective

Skin aging is a problem that arises from natural (intrinsic) and external (extrinsic) factors. The challenge of reducing these skin aging factors has attracted significant attention from researchers today. Conditioned media from stem cells have tremendous potential in reducing skin aging factors. In addition, conditioned media stimulate skin regeneration and recovery while showing low cytotoxicity. The description above provides examples of various types of conditioned media from different stem cell sources, as well as research results demonstrating their anti-aging effects on the skin. Many tests have been conducted to prove the benefits of conditioned media for anti-aging, including in vitro, in vivo, and clinical studies. In the future, numerous areas require further exploration to advance the use of conditioned media, particularly for anti-aging applications. Current research still has gaps that need to be addressed, such as the potential use of methotrexate,¹⁰⁵ mechanistic studies,¹⁰⁶ ECM development and Its influence on scar formation after glaucoma filtration surgery through the p53/Sp1/miR-29b pathway,¹⁰⁷ the impact of endogenous klotho deficiency on the potential for stem cell differentiation during blood vessel calcification,¹⁰⁸ and the interaction between macrophages and MSCs to generate ideas for developing macrophage activators to promote tendon-bone healing.¹⁰⁹ Advances in the exploration and utilization of conditioned media are expected to offer solutions to skin aging problems.