Novel Combination Of Antioxidants To Restore Healthy Intercellular Communication In An In vitro Model Using Conditioned Medium From Senescent Cells

Summary

This method studies how senescent skin fibroblasts affect healthy ones. Senescence is induced via UVB exposure, and the conditioned medium is applied to normal fibroblasts. It helps evaluate bioactive compounds targeting altered cell-to-cell communication in skin aging.

Abstract

Skin aging is a multifactorial process driven by alterations at the molecular and cellular level, also known as hallmarks of aging. One of these alterations consists of altered intercellular communication, which contributes to skin tissue dysfunction and structural damage. Here, an in vitro model to mimic these skin aging features by incubating healthy fibroblasts with conditioned medium from senescent fibroblasts is presented. First, UVB-irradiated fibroblasts can be analyzed for changes in gene expression associated with photoaging. Second, conditioned medium from UVB-induced senescent fibroblasts can be harvested and transferred to healthy fibroblasts, leading to downregulation of extracellular matrix proteins and upregulation of proinflammatory cytokine gene expression. These changes are counteracted by using a novel combination of antioxidants (vitamin C + idebenone). These results demonstrate that the in vitro model can be used to assess the efficacy of novel compounds on skin dermal cell aging features, and thus to select compounds that may modulate skin regenerative and rejuvenation cellular processes.

Introduction

Skin aging is the natural process of decreased skin functions and properties, including loss of firmness and elasticity and increased sagging. This is accelerated by external factors, such as ultraviolet (UV) exposure and pollution, which damage cells and cause changes at the molecular level¹. These changes have been well characterized by recent research and identified as hallmarks of aging, including genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, defective macroautophagy, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis². These features explain how subcellular damage translates into tissue dysfunction, thereby favoring the aging process. Interestingly, these alterations do not act independently but rather form an interdependent network signature. Primary damage, such as genomic damage or epigenetic alterations, accumulates and eventually causes cellular senescence. In the case of the skin, this is mainly caused by UV damage, with photoaging being the main contributor to skin aging traits³. Senescent cells show increased production of proinflammatory cytokines, matrix metalloproteinases, and growth factors, which is also known as senescence-associated secretory phenotype (SASP) and includes IL6, IL8, MMP1, MMP3, TNFA, or IL1A, among others^4,5,6. These secreted factors not only affect the structural properties of the skin, such as the dermal extracellular matrix, but also disrupt the function of surrounding cells. This includes the release of cytokines that stimulate melanocyte activation to provoke age-associated skin hyperpigmentation (such as Stem Cell Factor or IL18) or other damaging proteins that alter the function of surrounding healthy fibroblasts, compromising their function and contributing to the enhanced amplification of the senescent phenotype^7,8,9,10,11. As a result, chronic inflammation and altered intercellular communication are key promoters of the damaging state that translates these molecular alterations to visual signs of skin aging, such as wrinkles, sagging, age spots, or loss of firmness.

The specific protein contributors to the detrimental effects of SASP can be studied through well-known techniques such as enzyme-linked immunosorbent assay (ELISA), Luminex, quantitative real-time polymerase chain reaction (qRT-PCR), or Western Blot, among others^5,12. However, this approach is useful for identifying specific proteins from an individual perspective and characterizing a specific mechanism of action, but it does not consider the effects of the full SASP on neighboring cells. Here, an in vitro protocol is proposed to study the effect of the SASP produced by UVB-induced senescent dermal fibroblasts on healthy neighboring fibroblasts, mimicking the altered intercellular communication hallmark of aging. As stated before, UV is the main driver of cell senescence in the skin. Hence, UV damage is selected as a source of damage over replicative or oncogene-induced senescence to build the current model³. For this, the medium containing the factors secreted by senescent fibroblasts (conditioned medium) is used, and markers of cell function are quantified in healthy fibroblasts incubated with this medium. Under this protocol, once the conditioned medium has been transferred from senescent to healthy cells, UVB-induced senescent fibroblasts are harvested to quantify molecular features, such as genes involved in proinflammatory and paracrine signaling. Secondly, healthy fibroblasts treated with conditioned medium from senescent fibroblasts are harvested to characterize the SASP's effect on neighboring cells, quantifying genes involved in extracellular matrix, growth factors, and fibroblast activation. Thus, the effect of compounds of interest (COI) is analyzed both initially in senescent fibroblasts and subsequently in healthy fibroblasts treated with the conditioned medium. Regarding these compounds, previous research has shown that antioxidants are effective in protecting against and correcting signs of photoaging-induced skin aging. Consequently, the combination of two antioxidants (vitamin C and idebenone) is used to revert some of the alterations induced by UVB, both in senescent cells and healthy cells incubated with conditioned medium from senescent cells^13,14,15

Overall, this protocol is designed to study the effects of SASP on skin cells and to identify novel molecules/interventions that restore altered intercellular communication, thereby being appropriate for regenerative and bio-stimulating products in aesthetic medicine and regenerative dermatology.

Protocol

Human dermal fibroblasts used in this study were obtained from a commercial cell line provider. As no primary human samples, patient data, or in vivo experiments were involved, Institutional Review Board (IRB) approval and informed consent were not required for this study.

1. Senescence induction in dermal fibroblasts using UVB

1. Plate human dermal fibroblasts at a density of 6 × 10⁴ cells per well in a 6-well plate using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS).
2. After 24 h, remove the cell culture medium and replace it with 1 mL of phosphate buffered saline (PBS) per well.
3. Irradiate the cells with UVB light at 25 mJ/cm².
   NOTE: This energy has been optimized for the UVB irradiation device used in this protocol (see Table of Materials) to induce sufficient damage to trigger senescence while avoiding excessive damage that may lead to apoptosis. When using other devices, adjust the irradiation energy accordingly.
   CAUTION: UVB radiation is hazardous. Use appropriate protective equipment and shielding to avoid exposure.
4. Remove PBS and add 2 mL of fresh cell culture medium per well.
5. After 72 h, remove the cell culture medium, add 1 mL of PBS per well, and repeat UVB irradiation as described in step 1.3.
6. After 72 h, remove the cell culture medium and add 0.5 mL of 0.05% trypsin-EDTA per well. Incubate for 1–2 min at 37 °C, then harvest the cells using 1.5 mL of fresh cell culture medium.
7. Plate the cells at a density of 8 × 10⁴ cells per well in a 6-well plate using cell culture medium (DMEM supplemented with 10% FBS). Cellular senescence is induced after this step.

2. Treatment of senescent cells with compounds of interest

1. After 24 h, remove the cell culture medium and add 2 mL of cell culture medium containing compounds of interest.
2. After an additional 24 h, plate a separate batch of human dermal fibroblasts (as described in step 1.1) in 6-well plates. These cells will serve as healthy dermal fibroblasts for the transfer of conditioned medium.

3. Conditioned medium transfer from senescent cells to healthy cells

1. At 48 h after compound treatment of senescent cells and 24 h after plating healthy fibroblasts, collect 2 mL of conditioned medium per well from senescent cells and transfer it to the healthy fibroblasts.
   NOTE: Remove the culture medium from healthy fibroblasts immediately before adding the conditioned medium to ensure efficient transfer.
2. Add 1 mL of PBS to the senescent cells and harvest them using trypsin as described in step 1.6. Use these cells for analysis of senescence or inflammation markers by using an ELISA kit or qPCR.
   NOTE: Cell pellets may be stored at −80 °C if downstream assays are not performed immediately.
3. Incubate healthy fibroblasts receiving conditioned medium at 37 °C with 5% CO₂ for 24 h.

4. Healthy cells harvest for fibroblast activation analysis by gene expression analysis

1. After 24 h incubation with conditioned medium, harvest healthy fibroblasts using trypsin as described in step 1.6. Analyze extracellular matrix gene expression by qPCR.
2. Perform qPCR analysis.
   1. Extract RNA from cell pellets according to the manufacturer’s instructions (RNA extraction kit).
   2. Perform reverse transcription of 200 ng RNA according to the manufacturer’s instructions (Reverse transcription kit).
   3. Amplify cDNA according to the manufacturer’s instructions (qPCR master mix). Perform qPCR under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Primers used are listed in Table 1.

Gene	Forward sequence	Reverse sequence
CDKN2A	5’-GGCATTGTGAGCAACCACTG-3’	5’-CCTGTAGGACCTTCGGTGAC-3’
CDKN1A	5'-CTGGAGACTCTCAGGGTCGAA-3'	5'-CCAGGACTGCAGGCTTCCT-3'
SCF	5'-AATCCTCTCGTCAAAACTGAAGG-3'	5'-CCATCTCGCTTATCCAACAATGA-3'
IL-18	5'-TGCAGTCTACACAGCTTCGG-3'	5'-GTTTGTTGCGAGAGGAAGCG-3'
ELN	5’-GCAGGAGTTAAGCCCAAGG-3'	5’-TGTAGGGCAGTCCATAGCCA-3'
FBN1	5’-GGTGAATGTACAAACACAGTCAGCAG-3'	5’-ATAGGAACAGAGCACAGCTTGTTGA-3'
IL-6	5’-AGCCCTGAGAAAGGAGACATGTA-3'	5’-TCTGCCAGTGCCTCTTTGC-3'
IL-8	5’-ATTTCTGCAGCTCTGTGTGAAGGT-3'	5’-TTTTTTATGAATTCTCAGCCCTCT-3'
CTGF	5’-GGAAATGCTGTGAGGAGTGGGTGT-3´	5´-TGTCTTCCAGTCGGTAGGCAGCTA-3
IGF1	5’-CAGAGCAGATAGAGCCTGCG-3'	5’-CAGGTAACTCGTGCAGAGCA-3'

Table 1: List of primers used for qPCR assays. Forward and reverse sequences are provided for each primer corresponding to the genes studied and quantified in this model.

Results

The present protocol describes a method to simulate the altered intercellular communication hallmark of aging in vitro. Figure 1 presents a graphical schematic illustrating the main steps of the protocol, including senescence induction, marker quantification, and the analysis of altered intercellular communication in skin cells in vitro.

Discussion

Understanding the aging process at the molecular level is key to designing novel approaches and interventions that delay aging-related features and prolong the longevity and functionality of cells and tissues. The definition and identification of the hallmarks of aging have been essential in establishing a scientific framework for the development of these interventions^2,16. Among the recognized hallmarks of aging, altered intercellular communication constitutes a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well plates	Fisher Scientific	10578911	Cell culture multiwell plates to use during the assay (senescence induction and compound treatment)
Bio-LINK Crosslinker BLX-312/36 (UVB irradiation device)	Vilber Lourmat	VL-6111-1090-1	Device used to produce damage by UVB irradiation and consequently induce senescence
Dulbecco's Modified Eagle Medium (DMEM)	Merck Life science	D6046-500ML	Standard cell culture medium for dermal fibroblasts
Fetal bovine serum (FBS)	Fisher Scientific	17593595	Nutrient supplement used a a source of growth factors, hormones, proteins and essential micronutrients for cell growth
H3K27me3 ELISA kit	Epigentek	P-3014T-096	A specific immunoassay to detect the epigenetic modification of H3K27me3 in biological simples
Human dermal fibroblasts	Promocell	C-12302	Main skin cell in the dermis and regulator of skin photoaging
Phosphate buffered saline (PBS)	Merck Life science	D8537-500ML	Buffer solution that mimicks physiological environment. Used to wash cells without inducing strss or reducing cell viability
PrimeScript RT reagent kit (reverse transcription kit)	Takara Bio	RR037A	The kit to obtain cDNA from RNA isolated from cells. 2nd step for qPCR assay after RNA purification.
TB Green Premix Ex Taq  (qPCR master mix)	Takara Bio	RR420A	The kit to produce the polymerase chain reaction from cDNA to quantify gene expression levels. 3rd step for qPCR assay after reverse transcription.
Total RNA Purification Kit (RNA extraction kit)	Norgen	17200	The kit to obtain RNA from harvested cells. 1st step for qPCR assay after cell harvesting.
Trypsin/EDTA 0.05%	Merck Life science	T2601-100ML	Enzyme solution used in harvesting/passaging steps for cell detachment from cell culture plates