Abstract
Despite the remarkable advances of dermal fillers that reduce wrinkles caused by dermis thickness reduction, they still lack effective hydrogel systems that stimulate collagen generation along with injection convenience. Here, we develop a stem cell-derived extracellular vesicle (EV)-bearing thermosensitive hydrogel (EVTS-Gel) for effective in vivo collagen generation. The TS-Gel undergoes sol–gel transition at 32.6°C, as demonstrated by the storage and loss moduli crossover. Moreover, the TS-Gel and the EVTS-Gel have comparable rheological properties. Both hydrogels are injected in a sol state; hence, they require lower injection forces than conventional hydrogel-based dermal fillers. When locally administered to mouse skin, the TS-Gel extends the retention time of EVs by 2.23 times. Based on the nature of the controlled EV release, the EVTS-Gel significantly inhibits the dermis thickness reduction caused by aging compared to the bare EV treatment for 24 weeks. After a single treatment, the collagen layer thickness of the EVTS-Gel-treated dermis becomes 2.64-fold thicker than that of the bare EV-treated dermis. Notably, the collagen generation efficacy of the bare EV is poorer than that of the EVTS-Gel of a 10× lesser dose. Overall, the EVTS-Gel shows potential as an antiaging dermal filler for in vivo collagen generation.
Keywords: dermal filler, thermosensitive hydrogel, extracellular vesicles, collagen
Graphical Abstract
1. Introduction
Skin wrinkles associated with aging are characterized by fibroblast reduction, and the resulting attenuation of the extracellular matrix is considered to be an inevitable fate [1]. Over the past few decades, significant efforts have been devoted to overcoming unavoidable skin aging in humans by incorporating various biomedical engineering solutions [2]. Different types of dermal fillers that complement skin wrinkles have particularly been developed over the past three generations [3]. To represent each generation, collagen-, hyaluronic acid-, or synthetic polymer-based dermal fillers have been created. Some of which have successfully settled on the market [3-4]. Regarding biocompatibility and immediate volumizing performance, hyaluronic acid-based hydrogels are encroaching on the current market of skin fillers [5]. However, conventional hyaluronic acid-based dermal fillers have representative issues, such as the absence of a collagen production ability and injection inconvenience [6]. This has led to the tremendous need of developing a next-generation filler that can overcome the major drawback of conventional dermal fillers.
Mesenchymal stem cell-derived small extracellular vesicles (MSC-EVs) have recently been considered to be a powerful candidate for regenerating aged tissues [7]. Considering their antiinflammatory and tissue regenerative internal contents (e.g., cytokines and miRNAs), MSC-EVs can stimulate collagen synthesis in the dermis by augmenting the fibroblast and the macrophage [8]. The significant involvement of miRNAs (e.g., let-7b-5p and miR-24-3p) in collagen generation via antiinflammatory macrophage-mediated fibroblast activation has recently been reported [8b, 9]. Hyaluronic acid-based hydrogels containing MSC-EVs also show an antiaging performance by reprogramming the dermis microenvironment [8b]. In clinical settings, however, conventional dermal fillers often face practical challenges related to a clinician’s proficiency in injecting hydrogels.
Stimuli-responsive polymer-based hydrogels formed by physical interactions, such as temperature, pH, and ionic strength, assemble reversible polymer networks through sol–gel transitions [10]. This unique sol–gel transition mechanism in thermosensitive hydrogels allows the (i) minimization of the injection force below the lower critical solution temperature, (ii) adoption of biomolecule encapsulation (e.g., genes, proteins, and EVs), and (iii) controlled release of therapeutic contents after in situ gelation [10c]. This paper reports on injectable hydrogels that contain human adipose-derived stem cell (ADSC)-derived EVs for collagen generation in the dermis (Figure 1). With their antiaging performance, these EV-bearing thermosensitive hydrogels (EVTS-Gels) have considerable potential for development as nextgeneration dermal fillers.
Figure 1.
Schematic of the EVTS-Gels as injectable dermal fillers for collagen generation in the dermis.
2. Results and Discussion
2.1. Characterization of the TS-Gel
The concentrated aqueous solutions of the ABA- or BAB-type amphiphilic triblock copolymers exhibited a reversible sol–gel transition at a critical temperature [11]. Based on thermosensitive gelation behaviors, we prepared the poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) (PCL–PEG–PCL) copolymer as a main ingredient of our antiaging functional dermal filler. PCL–PEG–PCL was synthesized through the ring opening polymerization of ε-caprolactone using PEG as an initiator (Figure S1), followed by the determination of its chemical structure by 1H NMR spectroscopy (Figure 2a). The PCL–PEG–PCL had a molecular weight of 6564 Da (Mp), as measured using gel permeation chromatography. We investigated the in vitro gelation behavior by observing the flow or nonflow states of PCL–PEG–PCL through concentration variation (Figure S2). The sol–gel transition temperature tended to decrease as the copolymer concentration increased. When the thermosensitive polymer is injected into the body in a sol state, the rapid gelation behaviors are advantageous for the controlled release of therapeutic agents [12]. Thus, we chose PCL–PEG–PCL at 25 wt.% concentration for further experiments. The tube inversion technique confirmed that PCL–PEG–PCL existed in a sol state at 25°C and in a gel state at 37°C (Figure 2b). We also determined the sol–gel transition of the TS-Gel using a rheometer (Figure 2c). The TS-Gel exhibited a sol–gel transition at 32.6°C, as demonstrated by the storage (G′) and loss (G″) moduli crossover.
Figure 2.
Characterization of the TS-Gel. a) 1H NMR spectrum of the PCL–PEG–PCL triblock copolymer. b) Sol–gel transition images of the TS-Gel. c) Storage and loss moduli crossover.
2.2. Characterization of the EVTS-Gel
Tangential flow filtration has good batch-to-batch consistency and high yield [13]. Therefore, we used it to isolate the ADSC-EVs (see Supporting Information for details). We chose the ADSC-EVs as a main ingredient of our antiaging functional dermal filler because of their outstanding collagen generation performance [8]. The isolated EVs had a hydrodynamic size of 117.4 ± 8.6 nm and a spherical shape (Figures S3a and b). We then confirmed the representative surface proteins of these EVs (Figure S3c). The ADSC-EVs expressed significant CD9 and CD36 levels on their surface, as demonstrated via magnetic bead-based flow cytometry. Subsequently, we prepared the EVTS-Gel by mixing the TS-Gel with the EVs. We investigated the homogeneous distribution of the EVs in the TS-Gel by observing the fluorescent signals of the Flamma 496-labeled TS-Gel and the Flamma 675-labeled EVs through confocal microscopy (Figure 3a). The results showed that the EVs were evenly distributed in the TS-Gel. Consistent with the thermosensitive gelation behaviors of the TS-Gel, the EVTS-Gel also showed a successful gelation behavior at 37°C (Figure S4). These results suggest a successful EV encapsulation in the TS-Gel while keeping their in vitro gelation properties.
Figure 3.
Characterization of the EVTS-Gel. a) Confocal microscope images of the EV (red) distribution in the TS-Gel (green). b) Storage and c) loss moduli of the TS- and EVTS-Gels. d) Complex viscosities of the TS- and EVTS-Gels. e) Mean storage and loss moduli of the TS- and EVTS-Gels at 25°C or 37°C. The error bars represent the standard deviation (n = 3). The P values are analyzed via three-way ANOVA. f) Injection force of the TS- and EVTS-Gels, Restylane®, and Sculptra® as a function of time. g) Mean injection force of the TS- and EVTS-Gels. The dashed line represents the injection forces required by Restylane® and Sculptra®. The error bars represent the standard deviation (n = 5).
2.3. Rheological properties of the EVTS-Gel
Rheological properties, such as storage (G′) and loss (G″) moduli and complex viscosity, are important factors for evaluating the physical properties of dermal fillers. These properties also affect the injectability of hydrogels. We evaluated the rheological properties of the TS- and EVTS-Gels to verify whether or not the physical encapsulation of EVs affects their characterization. In Figures 3b and c, the aqueous solutions of both gels exhibited low G′ and G″ at 25°C. A remarkable increase of G′ and G″ was observed for both as a function of temperature (i.e., at least up to 37°C). Consistent with the storage and loss moduli results, both the TS- and EVTS-Gels showed low complex viscosities at 25°C. By contrast, both also exhibited a notable increase in viscosity as a function of temperature (Figure 3d). These results suggest the advantage of the TS-Gels mixed with various therapeutic agents (e.g., small molecular drugs, proteins, genes, and EVs). We, however, found no remarkable changes in the rheological properties of Restylane® and Sculptra® as a function of temperature (Figures 3e and S5). Overall, both TS- and EVTS-Gels showed comparable gelation behaviors as a function of temperature.
2.4. Injection force of the EVTS-Gel
The convenient use of dermal fillers by clinicians can be guaranteed when less force is required to elute the product from a syringe. For injection convenience, the force at which the dermal filler elutes from the syringe must remain constant. In this study, we compared the injection forces of the TS- and EVTS-Gels with that of conventional dermal fillers (Figure 3f). Although a 27-G needle was used for injection, Restylane® required a high injection force during elution because of its gel state at 25°C. Sculptra®, which is a microparticle-type dermal filler, required a very low injection force during elution from syringes with a 31-G needle. Interestingly, both TS- and EVTS-Gels had constant injection forces during elution despite being equipped with a 31-G needle. Figure 3g shows the mean injection force of the TS- and EVTS-Gels. Note that both required a lower injection force compared with Sculptra® (TS-Gel: 1.40 N; EVTS-Gels: 1.21 N; Sculptra®: 2.04 N). In terms of injectability, a thin needle can be used to inject the EVTS-Gel into the skin. In this manner, scabbing can also be minimized. Due to these advantages, we can expect patients to prefer the use of the EVTS-Gel.
2.5. Controlled EV release from the TS-Gel in the skin tissue
Bare EVs have a short biological half-life after administration [8b, 14]; therefore, the controlled release behaviors of the ADSC-EVs in the dermis area are essential in maximizing their collagen generation performance (Figure 4a). This prompted us to investigate the controlled release behaviors of the EVs from the TS-Gel after local administration into the skin of the SKH1 mice (Figure 4b). The controlled release profile of the Flamma 675-labeled EVs from the TS-Gel was investigated using an optical imaging system as a function of time. The bare EVs (1 × 108 particles/head) showed a strong fluorescence signal immediately after injection, but this rapidly vanished within 1 day and was not even detected after 3 days. By contrast, we were able to detect the fluorescence signal of the Flamma 675-labeled EVs (1 × 108 particles/head) from the TS-Gel for 7 days (Figure 4c). We examined the biological half-lives of the bare EVs and the EVs from the TS-Gel based on their relative fluorescence intensities (Figure 4d). The biological half-life of the EVs from the TS-Gel was extended by 2.23× compared with that of the bare EVs (bare EV: 1.32 days; EVTS-Gel: 2.95 days). In other words, the TS-Gel is a powerful carrier for the controlled delivery of ADSC-EVs as an antiaging agent.
Figure 4.
Release behaviors of the EVs from the TS-Gel in the skin tissue. a) Controlled EV release from the TS-Gel. b) In vivo fluorescence imaging of the Flamma 675-labeled bare EVs or the Flamma 675-labeled EVTS-Gel-treated SKH-1 mice, c) Heatmap graph of the fluorescence intensity of the EVs from the bare EVs or the EVTS-Gel-treated SKH-1 mice as a function of time (n = 5). d) Mean fluorescence intensity of c) and biological half-life of the EVs from the bare EVs or EVTS-Gel-treated SKH-1 mice.
2.6. Collagen generation performance of the TS-Gel in the dermis
With aging, the thickness of the dermis area in the skin tissues decreases due to reduced fibroblast-mediated collagen generation. Consequently, skin wrinkles are formed [1]. In this study, we examined herein the collagen generation efficacy of the EVTS-Gel after local injection into the skin tissues of the SKH-1 mice. We observed histological changes after a single dose of the EVTS-Gel through Masson’s trichrome for 24 weeks (Figure 5a). The TS- Gel and bare EV (1 × 108 particles/head)-treated groups showed comparable levels of collagen generation to the nontreated group (Figure 5b). Notably, the EVTS-Gel (1 × 107 particles/head)-treated mice showed superior collagen generation efficacy over the bare EV (1 × 108 particles/head)-treated mice for 24 weeks (Figure 5c, left). Note that the dermis thickness obtained with the bare EV was much thinner than that with the EVTS-Gel of a 10× lesser dose. The histological results also showed that the collagen generation efficacy was enhanced in an EV dose-dependent manner (Figures 5a and b). The EVTS-Gel (1 × 108 particles/head)-treated mice had the most outstanding collagen generation efficacy among the treatment groups (Figures 5d and e). Particularly, the dermis thickness of the EVTS-Gel-treated dermis was 2.64-fold thicker than that of the bare EV-treated dermis (Figures 5d). By contrast, the conventional dermal fillers, Restylane® and Sculptra®, showed poor collagen generation efficacies for 24 weeks (Figure 5c, right). Overall, as a potential dermal filler with an antiaging function, the EVTS-Gel enables wrinkle reduction through its collagen production performance.
Figure 5.
In vivo collagen generation efficacy of the EVTS-Gel in the SKH-1 mice. a) Masson’s trichrome staining of the skin layer of the SKH-1 mice. After the single-dose injection of samples into the mice, the skin tissues were dissected after 4, 8, or 24 weeks. b) Heatmap graph of the dermis thickness of the SKH-1 mice as a function of time (n = 5). c) Changes in the mean dermis thickness after the bare EV (1 × 108 particles/head), EVTS-Gel (1 × 107 particles/head), EVTS-Gel (1 × 108 particles/head), Restylane®, and Sculptra® treatments as a function of time (n = 5). The dashed lines represent the mean dermis thickness of the nontreated SKH-1 mice at Week 4. d) Dermis thickness and e) collagen layer area of the SKH-1 mice 24 weeks after administration. The error bars represent the standard deviation (n = 5). The P values are analyzed via one-way ANOVA.
3. Conclusion
In this study, we developed injectable EVTS-Gel as a potential dermal filler that produces collagen in the dermis. The EVTS-Gel provided on-demand injectability and gelation properties because of its temperature-sensitive characteristics. Moreover, it controlled the EV release in the dermis area when locally administered into the skin tissue of the mice. Based on the boosted nature of collagen generation, the EVTS-Gel effectively inhibited the thinning of the dermis thickness for 6 months. In conclusion, the EVTS-Gel has considerable potential as a next-generation dermal filler with antiaging functions beyond those of conventional dermal fillers.
4. Experimental Section
Materials:
PEG (Mn = 1650 g/mol) was purchased from Pharmicell (Seoul, Republic of Korea). ε-Caprolactone was purchased from Tokyo Chemical Industry (Tokyo, Japan). Tin-2-ethylhexanoate (Sn(Oct)2) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary human ADSCs (age: 38 years, female, 70E21-062) were obtained from Cefobio Inc. (Seoul, Republic of Korea). The Flamma dyes were purchased from GE Healthcare (IL, USA). Lastly, fetal bovine serum (FBS), antibiotic–antimycotic (AA) solution, trypsin–EDTA, and Dulbecco’s phosphate buffered saline were purchased from WelGENE (Gyeongsan, Republic of Korea).
Synthesis of the PCL–PEG– PCL triblock copolymer:
A PCL–PEG–PCL triblock copolymer was synthesized via ring opening polymerization. PEG (4 g, 2.42 mmol) and Sn(Oct)2 (0.04 g, 0.01 mmol) were briefly added to a flask and dried under vacuum at 110°C for 2 h to remove moisture. Subsequently, ε-caprolactone (9.89 mL, 84.1 mmol) was added, and the mixture was stirred for 24 h at 130°C under reflux and nitrogen atmosphere. The mixture was then dissolved in chloroform and precipitated in an excess amount of n-hexane/diethyl ether (50:50, v/v). After filtration, PCL–PEG–PCL was obtained by drying under vacuum conditions for 72 h at 25°C. The chemical structure of PCL–PEG–PCL was characterized via 1H NMR spectroscopy (500 MHz, Varian Unity INOVA, CA, USA), for which the samples were dissolved in chloroform-d. The molecular weight of PCL–PEG–PCL was measured via gel permeation chromatography (GPC, Waters, Milford, USA), for which the samples were dissolved in dimethylformamide with 15 mM LiBr as eluent.
Preparation of the TS- and EVTS-Gels:
The EVTS-Gel was prepared by mixing the TS-Gel with the EVs. We first prepared the TS-Gel solution (PBS, pH7.4) via stirring for 12 h at 25°C. Next, 3 mL syringes were separately filled with 2.8 mL TS-Gel and 200 μL EVs at 25°C. We gently mixed the TS-Gel and the EVs using a three-way cock to prepare a homogeneous EVTS-Gel. In this setup, the final TS-Gel and EV concentrations were set to 250 mg/mL and 1 × 108 particles/mL, respectively. We labeled the TS-Gel and the EVs as Flamma 496 hydrazide and Flamma 675 NHS ester, respectively, following the manufacturer’s instructions.
Characterization of TS- and EVTS-Gels:
We investigated the sol–gel transition properties by conducting an in vitro gelation of the TS- and EVTS-Gels (EV: 1 × 108 particles/mL; TS-Gel: 250 mg/mL) using the tube-inverting method. Gelation was confirmed by inverting the vials and observing the flow or nonflow states after 5 min incubation at 25°C or 37°C. We verified the EV distribution in the TS- and EVTS-Gels (EV labeling: Flamma 675; TS-Gel labeling: Flamma 496) through a confocal laser microscope observation (TCS SP8 HyVolution, Leica Microsystems CMS GmbH, Germany) with 405 diode (405 nm), Ar (458, 488, and 514 nm), and He–Ne (633 nm) lasers at the BIORP of the Korea Basic Science Institute.
Rheological properties of the TS- and EVTS-Gels:
We verified the rheological properties of the TS- and EVTS-Gels by observing 1 mL syringes (Ultra-Fine II, BD, NJ, USA) filled with the samples and equipped with a 31-G needle at 25°C. The bubbles in the syringes were removed by centrifugation for 3 min at 1500 rpm. We then placed the TS- and EVTS-Gels (EV: 1 × 108 particles/mL; TS-Gel: 250 mg/mL), Restylane®, and Sculptra® on the parallel plates (50 μm gap) of the ARES-G2 rheometer (TA Instruments, DE, USA). The upper and lower plates were coated with mineral oil and enclosed in a chamber to prevent the samples from drying. The measurement was performed in oscillation mode (frequency: 1 rad s−1; controlled shear stress: 0.4 Pa). The temperature was varied from 25°C to 37°C. Finally, we obtained the rheological properties of the samples, including their storage (G′) and loss (G″) moduli and complex viscosity.
Injection forces of the TS- and EVTS-Gels:
We investigated the injectability of the TS- and EVTS-Gels by measuring their injection forces. To do this, we filled a syringe (Ultra-Fine II, BD, NJ, USA), which was equipped with a 31-G needle, with 1 mL of the samples. To examine the injectability of Restylane® and Sculptra®, we used the syringes and the needles that came with their injection kits. The injection forces of the TS- and EVTS-Gels (EV: 1 × 108 particles/mL; TS-Gel: 250 mg/mL), Restylane®, and Sculptra® were measured using a universal testing machine (Model 3367, Instron Corporation, MA, US) under the predetermined experimental conditions of 30 mm/min flow rate and 25°C temperature.
In vivo biodistributions of the EVs and the EVTS-Gel:
All performed animal experiments complied with the relevant laws and institutional guidelines of Sungkyunkwan University and were approved by institutional committees (SKKUIACUC2021-07-56-1, SKKUIACUC2020-03-53-1, and SKKUIACUC2019-05-25-1). We used Flamma 675-labeled EVs for the in vivo biodistribution study. We observed the fluorescence signals of the EVs as a function of time after a local injection of 100 μL Flamma 675-labeled EVs or Flamma 675-labeled EVTS-Gel (EV: 1 × 108 particles/head; TS-Gel: 250 mg/mL) into 6-week-old male SKH1 mice. Fluorescence imaging was performed using the IVIS Lumina III in vivo imaging system (Caliper Life Sciences, MA, USA) with a 670 nm pulsed laser diode. The fluorescence signal quantification was measured using embedded software (Caliper Life Sciences, MA, USA).
Histological analysis:
The collagen generation efficacy of the EVTS-Gel was determined through Masson’s trichrome staining [8b]. We first divided the SKH1 mice (6 weeks old, male) into eight groups. After single-dose injections of 100 μL of the TS-Gel, EV (1 × 108 particles/head), EVTS-Gel (1 × 107 particles/head), EVTS-Gel (5 × 107 particles/head), EVTS-Gel (1 × 108 particles/head), Restylane®, and Sculptra® into the mice, the skin tissues were dissected after 4–24 weeks. For the histological analysis, we stained 5-μm-thick tissue sections using Masson’s trichrome method after fixation and paraffinization and observed the samples through a slide scanner (Axio Scan Z1, Carl Zeiss, Oberkochen, Germany). The dermis thickness was calculated using ZEN 3.1 software. The collagen area quantification was performed using Adobe Photoshop CS4.
Supplementary Material
Supporting Information is available from the Wiley Online Library or from the author.
Despite tremendous advances in biomedical engineering, a definitive countermeasure to meet the public’s demands for enduring healthy and youthful appearance remains elusive. Here, we report on the EVTS-Gel as a potential next-generation dermal filler for effective in vivo collagen generation. This innovative hydrogel, injectable with a fine needle to minimize scabbing, showcases efficacy in wrinkle reduction through EV-based collagen generation.
Acknowledgements
This research was supported by the Korean Fund for Regenerative Medicine (2021M3E5E5096677) of the National Research Foundation (NRF) and Korea Basic Science Institute (National Research Facilities and Equipment Center, 2020R1A6C101A191) grant from the Ministry of Education, Republic of Korea.
Footnotes
Conflict of Interest
The authors declare the following competing interests: Y. W. Cho is the chief executive officer of ExoStemTech Inc. D.-G. Jo and J. H. Park are stockholders of ExoStemTech Inc. The other authors declare no competing financial interests.
Contributor Information
Dong Gil You, Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA..
Jae Min Jung, School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
Chan Ho Kim, School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
Jae Yoon An, School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
Van Dat Bui, School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
Jungmi Lee, School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
Wooram Um, Department of Biotechnology, Pukyong National University, 45, Yongso-ro, Nam-gu, Busan 48513, Republic of Korea.
Dong-Gyu Jo, Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea ; School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea; ExoStemTech Inc., 55 Hanyangdaehak-ro, Sangnok-gu, Ansan 15588, Republic of Korea.
Yong Woo Cho, ExoStemTech Inc., 55 Hanyangdaehak-ro, Sangnok-gu, Ansan 15588, Republic of Korea; Department of Chemical Engineering, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan 15588, Republic of Korea.
Doo Sung Lee, School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
Leonora Balaj, Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA..
Hakho Lee, Center for Systems BiologyMassachusetts General HospitalBoston, MA 02114, USA.
Jae Hyung Park, School of Chemical Engineering, College of Engineering, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea; Biomedical Institute for Convergence at SKKU (BICS), Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea; ExoStemTech Inc., 55 Hanyangdaehak-ro, Sangnok-gu, Ansan 15588, Republic of Korea.
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