A survey of the impact of COVID-19 on the management of animal experiments and laboratory animal facilities in Korea.

The pandemic has affected the lives of people all over the world. The effects of the pandemic on laboratory animal facilities and their operations through this unusual global event are poorly understood. Here, we have applied a methodological framework of qualitative approach including semi-structured interviews to investigate laboratory animal operations in Korea and how it has shaped the on-going management and laboratory operations of such facilities. A total of fifty-two individuals, including members and administrators of the Institutional Animal Care and Use Committee (IACUC), researchers, and animal facility managers and staff, were surveyed through purposeful sampling. Survey questions explored how the pandemic impacted the IACUC and the functioning of animal facilities, and what steps to take in preparation of a future pandemic-like crisis. Our survey found evidence of an increase in animal experiments in Korea during the pandemic that correlated with increases in research funding during that period, such as for vaccine development. Also, operational challenges due to pandemic-related health issues in personnel were resolved through overtime, rather than by reducing facility operations. Moreover, a refinement of post-approval monitoring (PAM) practices was also discussed by respondents. Taken together, our study offers insights into animal facility operations during the pandemic and outlines recommendations for safeguarding operations in such future scenarios.

Double Emulsion-Mediated Delivery of Polyphenol Mixture Alleviates Atopic Dermatitis.

Although the polyphenols have been studied to alleviate inflammation, there are still challenges to delivering the polyphenols with stabilized formulation due to their low water solubility and susceptibility to oxidation. Herein, the transdermal delivery system of polyphenol mixture (PM), including quercetin (Q), phloretin (P), and ellagic acid (E), is developed using double emulsion for applying to atopic dermatitis (AD). Through the in vitro anti-degranulation assay, the optimal molar ratio of each polyphenol (Q:P:E = 5:1:1) is obtained, and the PM shows at most a 43.6% reduction of degranulation of immune cells, which is the primary factor of AD. Moreover, the water-in-oil-in-water double emulsion (W/O/W) enhances the PM's stability and has a higher anti-degranulation effect than the oil-in-water emulsion (O/W). In the in vivo 1-chloro-2,4-dinitrobenzene (DNCB)-induced mice AD model, PM reduces more AD symptoms than every single polyphenol. The PM-encapsulated W/O/W (PM_W/O/W) shows the most effectiveness in AD by decreasing dermatitis score, i.e., skin/ear thickness, mast cells, and serum IgE level. Finally, this suggests that the findings on the optimal ratio of PM and double emulsion-based delivery would be beneficial in treating AD and can be applied to other allergic diseases.

Use of laboratory animals and issues regarding the procurement of animals for research in Korea.

BACKGROUND: Laboratory animals remain critical to biomedical research, despite the increasing availability of alternative approaches. Indeed, scientists strive to reduce and refine and replace the use of laboratory animals, even in the face of public calls for ever-more stringent regulation for the protection and care of animals in research. This report outlines the current status and legal regulatory issues with regard to the procurement and use of animals for research in Korea. RESULTS: The number of animals used for education and research purposes was increased nationwide, from 2.5 to 4.9 million in 2015 and 2021, respectively. When compared with figures from the UK, institutions in Korea were found to use more mammals such as mice and dogs. In our research, we identified three major issues concerning recent animal supply in Korea, particularly: (1) Purchase of dogs from unregistered animal supplier for a dog cloning project; (2) Purchase of dogs from an unclear source for veterinary education and training; (3) Illegal cat experiments using cats obtained from unauthorized routes. CONCLUSIONS: Our findings support the notion that alternatives to laboratory animal research should be implemented. We conclude that improvements in the regulations and guidelines for animal suppliers, together with the recent introduction of legislation will improve animal safety and wellbeing of animals in laboratory research in Korea.

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway.

BACKGROUND: Bone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms. METHODS: hDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria. RESULTS: The calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture. CONCLUSIONS: The stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.

Improvement plans on the operation of the Institutional Animal Care and Use Committee: focusing on the case of Seoul National University.

BACKGROUND: The Institutional Animal Care and Use Committee (IACUC) became compulsory in 2008 by the Animal Protection Act in Korea. Seoul National University (SNU), which conducts 5% of Korea's total animal protocol reviews and uses 10% of national laboratory animal usage, has been influential in the review of animal protocols and management of animal facilities. This study was undertaken to suggest the operational improvement of the IACUC. It focused on the case of SNU. RESULTS: The methodological framework consists of a qualitative approach. In particular, this study is focused on the grounded theory approach and sixty people were surveyed through purposeful sampling. Through this study, we found that various practical educations are necessary such as: (1) education for researchers on how to write a protocol, (2) standardization of screening criteria for various animal experiments by presenting various cases, (3) training on a detailed understanding of relevant laws and policies. In particular, an integrated management system, making it possible to share information among the related committees, would be essential for smoother operation of the IACUC. CONCLUSIONS: If various levels of education and the integrated management system are established, it will be possible to enhance the excellence of researchers and to better manage the operation of the IACUC.

Operational issues of the Institutional Animal Care and Use Committee in Korea.

Korean Institutional Animal Care and Use Committee (IACUC) is currently facing some operational pressing issues. 1) Review of the animal protocol containing controversial technology. 2) Review of the multi-institution animal protocol. 3) Review of veterinary clinical trials for client-owned animals. 4) Delay the review process in large institutions with a single IACUC. Here, the following three solutions are proposed to address the above issues. 1) Establishment of public IACUC. 2) Establishment of the Veterinary Clinical Study Committee as an advisory body to the IACUC. 3) Operating multiple committees rather than increasing the number of committee members on a single committee.

Effect of LOXL2 on metastasis through remodeling of the cell surface matrix in non-small cell lung cancer cells.

Lung cancer is the prominent cause of cancer-associated death primarily because of distant metastatic disease. The metastatic potential of non-small cell lung cancer (NSCLC) is associated with tumor cell aggregation. However, the systemic mechanotransduction mechanism by which tumor cells dynamically aggregate and disseminate is poorly understood, especially in NSCLC. In this study, we examine whether the cell surface matrix plays an important role in metastasis. We used poly-2-hydroxyethyl methacrylate-based 3D spheroid formation methods to mimic in vivo metastatic lesions. Supra-structural analysis of human NSCLC A549 cells stained with ruthenium red for transmission electron microscopy (TEM) showed that glycocalyx surrounding the cell surface in 2D culture decreases in 3D culture. Comprehensive gene expression analysis revealed that the genes associated with cell adhesion were distinctly enriched in A549 cell spheroids. Of these, downregulation of the tumor metastatic microenvironment facilitator LOXL2, a copper-dependent enzyme catalyzing posttranslational oxidative deamination of peptidyl lysine, was of special interest. Knockdown of LOXL2 thickened the cell surface matrix in 2D culture and impaired compact aggregate formation in 3D culture. Moreover, A549 cell spheroids with endogenous overexpression of LOXL2 increased their dissemination on basement extracellular matrix Matrigel. Overall, these data imply that cell detachment-downregulated LOXL2 contributes to cell surface matrix remodeling, leading to collective dissemination of free-floating aggregates.

The status and issues of the Institutional Animal Care and Use Committee of Seoul National University: from its establishment to the present day.

The Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU) plays a key role in monitoring and managing the humane use of animals in scientific research. Here, as one of the pioneers of the IACUC in Korea, we reported SNU-IACUC operations and activities including committee establishment and legal formulation, protocol review, and post-approval monitoring of protocols, which the IACUC has undertaken in the last decade. In addition, legal regulations and improvements were also discussed, and encompassed the limited number of committee members and the single IACUC policy in Korea. As of December, 2020, amendments are on the table at the National Assembly. We also emphasized the independent nature of the IACUC in protecting activities, including approval and monitoring animal experiments, and its public role in narrowing the knowledge gap between society and scientists. Thus, the aim of this report is to help society and scientists understand the operations of the SNU-IACUC and its role in animal welfare.

Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle.

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.

A Rho Kinase (ROCK) Inhibitor, Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells.

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.

Comparison of Cell-Free Extracts from Three Newly Identified Lactobacillus plantarum Strains on the Inhibitory Effect of Adipogenic Differentiation and Insulin Resistance in 3T3-L1 Adipocytes.

Obesity and associated metabolic disorders, including cardiovascular disease and diabetes, are rapidly becoming serious global health problems. It has been reported that Lactobacillus plantarum (L. plantarum) extracts have the beneficial activities of antiobesity and antidiabetes, although few studies have compared the beneficial effects among various L. plantarum extracts. In this study, three new L. plantarum (named LP, LS, and L14) strains were identified, and the antiobesogenic and diabetic effects of their extracts were investigated and compared using 3T3-L1 cells in vitro. Lipid accumulation in maturing 3T3-L1 cells was significantly decreased by the addition of LS and L14 extracts. The mRNA expression levels of Pparγ, C/ebpα, Fabp4, Fas, and Dgat1 were significantly decreased by the addition of LP, LS, and L14 extracts. Interestingly, the protein expression levels of PPARγ, C/EBPα, FABP4, and FAS were downregulated in mature 3T3-L1 cells with the addition of the L14 extract. Moreover, the LS and L14 extract treatments stimulated glucose uptake in maturing adipocytes. The L14 extract treatments exhibited a significant reduction in TNF-α protein expression, which is a key factor of insulin resistance in adipocytes. Of the three extracts, L14 extract markedly reduced adipogenic differentiation and insulin resistance in vitro, suggesting that the L14 extract may be used as a therapeutic agent for obesity-associated metabolic disorders.

AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions.

Tumor cell aggregation is critical for cell survival following the loss of extracellular matrix attachment and dissemination. However, the underlying mechanotransduction of clustering solitary tumor cells is poorly understood, especially in non-small cell lung cancers (NSCLC). Here, we examined whether cell surface protrusions played an important role in facilitating the physical contact between floating cells detached from a substrate. We employed poly-2-hydroxyethyl methacrylate-based 3D culture methods to mimic in vivo tumor cell cluster formation. The suprastructural analysis of human NSCLC A549 cell spheroids showed that finger-like protrusions clung together via the actin cytoskeleton. Time-lapse holotomography demonstrated that the finger-like protrusions of free-floating cells in 3D culture displayed exploratory coalescence. Global gene expression analysis demonstrated that the genes in the organic hydroxyl transport were particularly enriched in the A549 cell spheroids. Particularly, the knockdown of the water channel aquaporin 3 gene (AQP3) impaired multicellular aggregate formation in 3D culture through the rearrangement of the actomyosin cytoskeleton. Moreover, the cells with reduced levels of AQP3 decreased their transmigration. Overall, these data indicate that cell detachment-upregulated AQP3 contributes to cell surface protrusions through actomyosin cytoskeleton remodeling, causing the aggressive aggregation of free-floating cells dependent on the property of the substratum and collective metastasis.

Oral intake of Lactobacillus plantarum L-14 extract alleviates TLR2- and AMPK-mediated obesity-associated disorders in high-fat-diet-induced obese C57BL/6J mice.

OBJECTIVES: Whether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity-associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L-14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high-fat-diet (HFD) mouse model. MATERIALS AND METHODS: Mouse pre-adipocyte 3T3-L1 cells and human bone marrow mesenchymal stem cells (hBM-MSC) were treated with L-14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti-adipogenic effects was analysed at cellular and molecular levels. L-14 extract was orally administrated to HFD-feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti-adipogenic mechanism of exopolysaccharide (EPS) isolated from L-14 extract was also analysed using Toll-like receptor 2 (TLR2) inhibitor C29. RESULTS: L-14 extract inhibited 3T3-L1 and hBM-MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L-14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L-14 extract also significantly decreased the serum triacylglycerol/high-density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis. CONCLUSIONS: EPS from L-14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L-14 extract improves obesity and obesity-associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.

Exopolysaccharide Isolated from Lactobacillus plantarum L-14 Has Anti-Inflammatory Effects via the Toll-Like Receptor 4 Pathway in LPS-Induced RAW 264.7 Cells.

Inflammation is a biological response of the immune system to defend the body from negative stimulation. However, the excessive inflammatory response can damage host tissues and pose serious threats. Exopolysaccharide (EPS), one of the postbiotics, is secreted from lactic acid bacteria. Although many studies have described the beneficial effects of EPS, such as its anti-inflammatory and anti-oxidant effects, its underlying mechanisms have remained to be poorly understood. Thus, we identified that EPS obtained from Lactobacillus plantarum L-14 was a homogeneous polysaccharide primarily comprised of glucose. To examine these anti-inflammatory effects, an inflammatory response was induced by lipopolysaccharide (LPS) administration to mouse macrophage RAW 264.7 cells that were pretreated with EPS. The anti-inflammatory effects of EPS were identified by analyzing the changes within inflammatory markers at the molecular level. We demonstrate here that EPS suppressed proinflammatory mediators, such as cyclooxygenase-2, interleukin-6, tumor necrosis factor-α, and interleukin-1ß, and downregulated the expression of an inducible nitric oxide synthase known to lead to oxidative stress. It was also confirmed that EPS had anti-inflammatory effects by blocking the interaction of LPS with Toll-like receptor 4 (TLR4), as demonstrated by using the known TLR4 inhibitor TAK-242. In addition, we found that EPS itself could suppress the expression of TLR4. Consequently, our data suggest that EPS can be a potential target for the development of natural product-derived medicine for treating inflammatory diseases related to TLR4.

GRIM-19 Ameliorates Multiple Sclerosis in a Mouse Model of Experimental Autoimmune Encephalomyelitis with Reciprocal Regulation of IFNγ/Th1 and IL-17A/Th17 Cells.

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM19 OVN. GRIM-19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

Anti-Cancer Effects of Lactobacillus plantarum L-14 Cell-Free Extract on Human Malignant Melanoma A375 Cells.

Human malignant melanoma is the most aggressive type of skin cancer with high metastatic ability. Despite several traditional therapies, the mortality rate remains high. Lactobacillus plantarum (L. plantarum), a species of lactic acid bacteria (LAB), is being studied for human health, including cancer treatment. However, few studies have elucidated the relationship between L. plantarum extract and human malignant melanoma. To investigate the effects of L. plantarum on human melanoma cells, A375 human melanoma cells were used and treated with L. plantarum L-14 extract. After the treatment, viability, migration ability, molecular changes of migration- and apoptosis-related genes, and the location of cytochrome c was evaluated. The L-14 extract inhibited the viability, migration of A375 cells as well as reduced expression of migration-related genes. In addition, it was confirmed that the L-14 extract induced intrinsic apoptosis in A375 cells. This study demonstrated that the L-14 extract exerted anticancer effects on A375 cells. Therefore, these data suggest that the L-14 extract is worth studying for the development of melanoma drugs using LAB.

Brown adipose tissue ameliorates autoimmune arthritis via inhibition of Th17 cells.

The functions of adipose tissue are associated with autoimmune diseases, such as rheumatoid arthritis (RA). Some studies have shown that the three compositions of adipose tissue (white, brown, and beige) have different functions. Brown adipose tissue (BAT) is known to secrete several factors that differ from those in white adipose tissue. This suggests that BAT might have potential positive advantages in the physiology of autoimmune diseases. We compared the functions of collagen-induced arthritis mice-derived BAT (CIA BAT) with normal mice-derived BAT. DBA/1J mice (6-7 weeks of age) were immunized by intradermal injection at the base of the tail with 100 µg of bovine type II collagen (CII) emulsified in complete Freund's adjuvant. Immunized mice then received booster immunizations by intraperitoneal injection with 100 µg of CII in incomplete Freund's adjuvant. We transplanted CIA BAT and normal BAT into CIA recipient mice. After transplantation, we measured the functions of CIA BAT and normal BAT in mice. Normal BAT-transplanted mice showed significantly lower scores of bone damage, inflammation, and cartilage damage. The proinflammatory cytokines in normal BAT-transplanted mice, such as IL-12, IL-17, IL-6, and tumor necrosis factor-α (TNF-α), tended to decrease. Microarray analysis showed that the PI3K-AKT signaling pathway and IL-17 levels of CIA BAT tissues were significantly higher than those of normal BAT tissues. These results suggest that the transplantation of normal brown fat may have a therapeutic effect in RA patients.

Newly synthesized peptide, Ara-27, exhibits significant improvement in cell-penetrating ability compared to conventional peptides.

Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 µM Ara-27 in both HDF and hDPSC. In contrast, 2 µM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 µM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.

Cell-Penetrating Peptides Enhance the Activity of Human Fibroblast Growth Factor 2 by Prolonging the Retention Time: A New Vision for Drug-Delivery Systems.

Cell-penetrating peptides (CPPs) are defined by their ability to deliver cargo into cells and have been studied and developed as a promising drug-delivery system (DDS). However, the issue of whether the CPPs that have already entered the cells can be re-released or reused has not been studied. The purpose of this research was to construct CPP-conjugated human fibroblast growth factor 2 (hFGF2) and investigate whether they can be re-released from the cell membrane for reuse. This study combined hFGF2 with Tat or Ara27, a newly developed CPP derived from the zinc knuckle (CCHC-type) family protein of Arabidopsis. Human dermal fibroblast (HDF) was treated with Tat-conjugated hFGF2 (tFGF2) and Ara27-conjugated hFGF2 (NR-FGF2) for both long and short durations, and the effects on cell growth were compared. Furthermore, tFGF2 and NR-FGF2 re-released from the cells were quantified and the effects were evaluated by culturing HDF in a conditioned medium. Interestingly, the proliferation of HDF increased only when NR-FGF2 was treated for 1 h in endocytosis-independent manner. After 1 h, NR-FGF2 was significantly re-released, reaching a maximum concentration at 5 h. Furthermore, increased proliferation of HDF cultured in the conditioned medium containing re-released NR-FGF2 was discovered. While previous studies have focused on the delivery of cargo and its associated applications, this study has revealed that combinations of superior CPPs and therapeutics can be expected to prolong both the retention time and the cell-penetrating capacity, even in the presence of external factors. Therefore, CPPs can be applied in the context of topical drugs and cosmetics as a new DDS approach.