A declaration for the study was approved by the IRB committee of the Korea Institute of Dermatological Sciences (approval number: 1-70005239-AB-N-01-201711-HR-120-01) in according to the Helsinki Declaration.
Cell lines and cell culture
Human adipose tissue-derived mesenchymal stem cells (hADMSCs) were obtained with the consent of the donors. They were first washed with phosphate buffered saline (PBS) and extracted using a cell extraction kit, SmartX® (DongKoo Bio & Pharma Co., Ltd.), which allows easy extraction of hADMSCs from tissues. Cells were cultured with CEFOgro™ ADMSC (CEFO Co., Ltd).
Human dermal fibroblasts (hDFs) were obtained from males of 20 years old or under and cultured with CEFOgro™ hDF (CEFO Co., Korea) supplemented with 1% penicillin/streptomycin (Gibco, UK) (100 IU/50 μg/mL). Serial culture was performed 3 days after cell inoculation.
All cell cultures were done in a 37 °C incubator with 5% CO2 and relative humidity of > 95% unless indicated otherwise.
Three-dimensional ADMSC culture
Three-dimensional (3D) gel matrixes with a series of concentrations for every 5% from 5 to 30% were prepared with a biodegradable synthetic biogel (BASF, Germany). Powder of the biogel was melted in sterilized deionized water. Approximately 250 μL/cm2 of the melted gel was spread on a polymer membrane of 0.4–1 μm thick (Corning, USA) and solidified at 37 °C for 90 min to produce 3D cell culture matrix. Next, hADMSCs from 2D culture were inoculated on the biogel matrix and cultured in a 37 °C incubator with 5% CO2 and relative humidity of > 95%. The medium was replaced 1 day after the cells stabilized.
Preparation of ADMSC-conditioned medium
A conditioned medium from 2D culture was produced by inoculating ADMSC at the cell density of 12,000 cells/cm2 on a standard culture plate. Next day, the cells were washed with PBS three times and fresh CEFOgro™XF ADMSC (CEFO Co., Korea) was added. The cells were cultured for one more day. The medium was collected and centrifuged at 1500 rpm for 5 min to obtain the supernatant. The supernatant was filtered using a 0.22-μm filter to obtain 2D cultured ADMSCs-CM.
To obtain 3D cultured ADMSCs-CM, ADMSC was inoculated at the density of 12,000 cells/cm2. Next day, the cells were washed with PBS three times and CEFOgro™XF ADMSC was added. The cells were cultured for one more day. The medium was harvested and centrifuged at 1500 rpm for 5 min to obtain the supernatant. The supernatant was filtered with a 0.22-μm filter to obtain a 3D cultured ADMSCs-CM.
Protein quantitation of procollagen and collagen
To assess the effects of the ADMSC-CM on the production of procollagen and collagen, human skin-derived fibroblasts were seeded into a 12-well plate (Corning, USA) at 2 × 104/cm2. The medium was replaced 24 h later. A negative control was cultured with a basal medium without serum, and a positive control was cultured in a basal medium supplemented with 0.04% adenosine. The experimental group was treated with stem cell–conditioned media derived from the 2D or 3D culture for 48 h. ELISA Kit (Takara, Japan) and human Type 1 Collagen ELISA kit (R&D systems, USA) were used to measure the protein amounts of procollagen and collagen. The experiment was conducted following the manual provided by the kit manufacturers.
RT-qPCR for procollagen, collagen and lysyl hydroxylase
To measure the gene expression of procollagen, collagen, and lysyl hydroxylase (LH), human skin-derived fibroblasts were seeded on a 6-well plate (Corning, USA) at 2 × 104/cm2, and the medium was replaced 24 h later. The negative control was cultured in a medium without serum. The positive control was cultured in a medium supplemented with 0.04% adenosine but no serum. The experimental group was grown in the stem cell–conditioned media from the 2D or 3D culture for 48 h. The cells were washed with PBS twice on the culture plates, and mRNA was extracted using Trizol (Invitrogen, USA).
cDNAs were synthesized using a cDNA synthesis kit (ReverTra ACE qPCR RT Master, Toyobo Co.). PCR was performed for 35 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. The PCR products were resolved and detected in gel electrophoresis and visualized using LAS4000 (GE healthcare Co.). The PCR primers for procollagen were TGC CGT GAC CTC AAG ATG TGC C and CAT CCA CAA GCG TGC TGT AGG TG for the forward and reverse primers, respectively. For collagen primers, the forward primer was TGC CGA TGT CGC TAT CCA and the reverse was TCT TGC AGT GAT AGG TGA TGT TCT G. For LH primers, the forward primer was GGA ACC TGG CCT ATG ACA CCC T and the reverse primer was TGC CAT GCT GTG CCA GGA ACT. A set of β-actin primers was used as a control. These were ATC TGG CAC ACC TTC TAC AAT GAG CTG CG and CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC for the forward and the reverse primers, respectively.
Production of a cream-based product containing 3D cultured ADMSCs-CM
A water phase was added in a main beaker, stirred and heated up to 80 °C. A liquid crystal emulsifier was then added to the heated water phase and hydrate at 6000 rpm using a homogenizer at 80 °C for 10 min. An oil phase in a separate container was heated up to 80 °C, added to the main beaker containing the water phase, and emulsified using a homogenizer at 6000 rpm for 5 min at 75 °C. The mixture was cooled to 45 °C. A liquid crystal capsule phase was stirred and encapsulated in another container. The liquid capsule phase was then added to the mixture and mixed by homogenizer at 45 °C and 6000 rpm for 3 min. It was cooled to 33 °C and de-aerated. A colorless, opaque, cream-based substance containing 1% 3D cultured ADMSCs-CM was prepared for the human skin test.
Human skin test
To assess the efficacy of cream-based cosmetic products containing 3D cultured ADMSCs-CM, it was performed with the approval of the Korea Institute of Dermatological Sciences.
Subjects
Volunteers were recruited for the human skin test of cream-based cosmetic products containing 3D cultured ADMSCs-CM. Female adults aged 30–60 years were selected. The participants were fully informed regarding the study and voluntarily signed the consent form.
Test substance usage and dose
The test substance was stored at room temperature (5–25 °C) away from the exposure of high temperature and direct light.
- (1)
Thirty microliters of the cream containing 3D cultured ADMSCs-CM was applied on the left lower forearm of the subjects (3.0 × 3.0 cm2) by a same researcher using a disposable syringe (SUNGSHIM MEDICAL CO., LTD, Korea). Absorption was induced using a latex finger coat.
- (2)
An equal amount of cream containing 3D cultured ADMSCs-CM was evenly applied on the left upper forearm after treated with sodium lauryl sulfate (SLS; BioShop, Canada) by the subjects every day during the 4-week research period. The cream was applied twice a day in the morning and evening after washing the face.
- (3)
The subjects applied an equal amount of cream containing 3D cultured ADMSCs-CM evenly on their face twice every day in the morning and evening after washing the face for 4 weeks.
- (4)
During the skin test period, the subjects were instructed not to use any functional cosmetic products that may affect the test results such as eye cream, skin whitening cream, antiaging cream, and body moisturizer. They were also restricted from using facial masks or massage.
Primary skin irritation assessment
The back of the subjects was washed with 70% ethanol and dried. An 8-mm diameter Finn Chamber (Smart Practice, USA) containing 20 μL of the test substance was applied on the back of the subjects for 24 h. Skin irritation was examined at 30 min, 24 h, and 48 h after the patch removal and graded by a dermatologist based on the guideline of the International Contact Dermatitis Research Group (Table 1).
Table 1 Criteria for human skin patch test by International Contact Dermatitis Research Group Assessment of skin texture damaged by external stimuli
A Finn Chamber containing SLS was applied on both forearms of the subjects to induce skin irritation. ANTERA 3D (Miravex, Ireland) was used to measure the effect of the test substances on the damaged skin. The entire measurements were performed by a single researcher who evaluated the sites of forearms treated with or without the test substances. The measurement was done at the same sties by overlapping the images obtained before the application of the test substances to ensure the accuracy of the measurement. The measurements were performed before using the test substance (after SLS application) and 3 weeks after the use of the test substances.
Dermal torque assessment
Dermal Torque Meter (Dermal Torque Meter DTM310, Dia-Storn Ltd., UK) was used to assess improvement in dermal elasticity. A single researcher fixed a probe on the right cheek of all subjects with an adhesive tape and applied a rotational force at a uniform angle and with a uniform force for 10 s to measure dermal elasticity. Measurement using a measuring device was performed before applying the test substance and after 2 and 4 weeks of use.
Wrinkle improvement assessment
ANTERA 3D was used to assess wrinkle improvement. A single researcher evaluated the creases around the right eye for all participants. For the sake of measurement reproducibility, images obtained prior to the use of the test substance were overlapped to obtain measurements from the same location. Device-assisted measurement was performed before applying the test substance and after 2 and 4 weeks of use.
Dermal density assessment
DUB-Skin Scanner (Taberna pro medicum, Luneburg, Germany), a device that produces high-resolution ultrasound images, was used to assess improvements in dermal density. Ultrasound gel was applied on the test site, and the probe of DUB-Skin Scanner was positioned perpendicular to the skin. A single researcher pressed on the skin 3 cm next to the outer corner of the left eye to measure dermal density for all subjects. Device-assisted measurement was performed before applying the test substance and after 2 and 4 weeks of use.
Assessment of skin moisture retention
Epsilon E100 (Biox Systems Ltd., UK) was used to assess skin moisture retention in an environment with artificial cold air. A single researcher applied an equal amount of the test substance on the lower left forearm and exposed it to a cool wind of 17 °C ± 2 °C from a misting fan in the natural wind mode at the distance of 50 cm for 20 min. Device-assisted measurement was performed before application of the test substance, immediately after the first application, and after 20 min of exposure to the artificial cool breeze.
Statistical analysis
All statistical analyses were performed using SPSS 17.0 for Windows (IBM, USA).
The subjects’ responses to the questionnaire were analyzed using mean, standard deviation, frequency, and percentage. A paired t test was used to identify any significant improvements in various parameters of the skin condition.