Unprecedented skin regeneration of euglena gracilis-derived extracellular microvesicles fabricated through microalgae extrusion
Podium 1
Presented by: Jin Woong Kim
Introduction
Extracellular vesicles (EVs) are cell-derived lipid-bound vesicles which can be secreted from almost all types of cells. The EVs are subdivided into exosomes, microvesicles, and apoptotic bodies. Since EVs contain biomolecules of parent cells, their therapeutic efficacy is of great interest in biomedical treatments. However, the isolation of EVs from the cells is time-consuming process with low yields. These shortcomings have been tried to be overcome by employing the cell extrusion method, in which the cells are extruded through membrane pores, thus making the lipid bilayer membrane self-assembled to form the EV-mimetic vesicles. Thanks to the mass productivity as well as high yields of this method, there have been various attempts in the cosmetic industry to fabricate the EVs from non-animal-originated cells, including plant cells and microalgae. In this study, we introduce euglena gracilis-derived microvesicle (EMVEG) system which was established with aid of the modified-cell extrusion method. Taking advantage of abundant biomolecules, including proteins, vitamins, minerals, and amino acids, encapsulated in the EMVEG, we demonstrate that the EMVEG has practical dermatological applicability for skin regeneration.
Methods
We serially extruded 106 cells/ml euglena gracilis from 5 μm to 2 μm pore-sized polycarbonate (PC) membranes, which allowed us to produce a stable dispersion of EMVEG. We then observed the particle morphology of EMVEG through transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM), and measured the concentration EMVEG through nanoparticle tracking analysis (NTA). We also measured the concentration of β-glucan in EMVEG through aniline blue fluorescence assay. We observed skin cell proliferation of EMVEG in HaCaT (human keratinocyte) via cell viability assay and bromodeoxyuridine (BrdU) incorporation assay and skin cell migration via in vitro scratch assay. To confirm the skin regeneration effect more specifically, we measured the amount of synthesized collagen in CCD-986sk (human fibroblast) through the Sirius red staining method. Furthermore, we observed skin regeneration-related proteins in HaCaT cells by western blot analysis and ex vivo skin regeneration via keraskin model and porcine skin model.
Results
The serial extrusion of euglena gracilis through PC membranes enabled production of uniform EMVEG with the average diameter of ~1 μm and in the number of ~1.64x1010 particles/ml. The particle size and size distribution were determined by the clogging level of euglena gracilis compared to the pressure applied in the process of extrusion through the membrane pores. A vesicle-like structure of EMVEG was directly confirmed from TEM and CLSM. The encapsulated β-glucan in EMVEG took ~14% compared with that in euglena gracilis. We showed that the EMVEG induced ~150% higher cell proliferation and ~60% higher cell migration than bare β-glucan, respectively. Treating with EMVEG enhanced the collagen synthesis of fibroblast about 150% higher than the case treating with β-glucan. From the analyses of cytokeratin 10, cytokeratin 14, Slug and Src, we could figure out that the EMVEG treatment significantly enhanced all the protein expressions, thus proving the effective skin regeneration. Finally, it was confirmed through ex vivo keraskin and porcine skin model experiments that the EMVEG was directly involved in skin regeneration and significantly improved its performance.
Discussion and conclusion
In this study, we first propose a microalgae-derived EV system. For this purpose, we extruded euglena gracilis as a representative of microalgae to fabricate EMVEG. We showed that the EMVEG has a vesicle structure, thus effectively encapsulating β-glucan, the main bioactive of euglena gracilis. Through in vitro and ex vivo experiments, we could demonstrate that the use of EMVEG led to unpresented skin regeneration performance. These results highlight that our microalgae-derived EV system will pave a new way in the field of skin therapy and cosmetics.
Extracellular vesicles (EVs) are cell-derived lipid-bound vesicles which can be secreted from almost all types of cells. The EVs are subdivided into exosomes, microvesicles, and apoptotic bodies. Since EVs contain biomolecules of parent cells, their therapeutic efficacy is of great interest in biomedical treatments. However, the isolation of EVs from the cells is time-consuming process with low yields. These shortcomings have been tried to be overcome by employing the cell extrusion method, in which the cells are extruded through membrane pores, thus making the lipid bilayer membrane self-assembled to form the EV-mimetic vesicles. Thanks to the mass productivity as well as high yields of this method, there have been various attempts in the cosmetic industry to fabricate the EVs from non-animal-originated cells, including plant cells and microalgae. In this study, we introduce euglena gracilis-derived microvesicle (EMVEG) system which was established with aid of the modified-cell extrusion method. Taking advantage of abundant biomolecules, including proteins, vitamins, minerals, and amino acids, encapsulated in the EMVEG, we demonstrate that the EMVEG has practical dermatological applicability for skin regeneration.
Methods
We serially extruded 106 cells/ml euglena gracilis from 5 μm to 2 μm pore-sized polycarbonate (PC) membranes, which allowed us to produce a stable dispersion of EMVEG. We then observed the particle morphology of EMVEG through transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM), and measured the concentration EMVEG through nanoparticle tracking analysis (NTA). We also measured the concentration of β-glucan in EMVEG through aniline blue fluorescence assay. We observed skin cell proliferation of EMVEG in HaCaT (human keratinocyte) via cell viability assay and bromodeoxyuridine (BrdU) incorporation assay and skin cell migration via in vitro scratch assay. To confirm the skin regeneration effect more specifically, we measured the amount of synthesized collagen in CCD-986sk (human fibroblast) through the Sirius red staining method. Furthermore, we observed skin regeneration-related proteins in HaCaT cells by western blot analysis and ex vivo skin regeneration via keraskin model and porcine skin model.
Results
The serial extrusion of euglena gracilis through PC membranes enabled production of uniform EMVEG with the average diameter of ~1 μm and in the number of ~1.64x1010 particles/ml. The particle size and size distribution were determined by the clogging level of euglena gracilis compared to the pressure applied in the process of extrusion through the membrane pores. A vesicle-like structure of EMVEG was directly confirmed from TEM and CLSM. The encapsulated β-glucan in EMVEG took ~14% compared with that in euglena gracilis. We showed that the EMVEG induced ~150% higher cell proliferation and ~60% higher cell migration than bare β-glucan, respectively. Treating with EMVEG enhanced the collagen synthesis of fibroblast about 150% higher than the case treating with β-glucan. From the analyses of cytokeratin 10, cytokeratin 14, Slug and Src, we could figure out that the EMVEG treatment significantly enhanced all the protein expressions, thus proving the effective skin regeneration. Finally, it was confirmed through ex vivo keraskin and porcine skin model experiments that the EMVEG was directly involved in skin regeneration and significantly improved its performance.
Discussion and conclusion
In this study, we first propose a microalgae-derived EV system. For this purpose, we extruded euglena gracilis as a representative of microalgae to fabricate EMVEG. We showed that the EMVEG has a vesicle structure, thus effectively encapsulating β-glucan, the main bioactive of euglena gracilis. Through in vitro and ex vivo experiments, we could demonstrate that the use of EMVEG led to unpresented skin regeneration performance. These results highlight that our microalgae-derived EV system will pave a new way in the field of skin therapy and cosmetics.