44 SKIN CARE Signaling via exosomes plays a major role


in many cellular processes, such as signal transduction and immune response. In the skin, for instance, mesenchymal stem cells, precursors of different cell types, are essential for regeneration processes, such as wound healing. Interestingly, mesenchymal stem cells themselves are not needed for this process, but rather the exosomes that they secrete to communicate with other cells such as fibroblasts.


The potential of plant-derived exosomes As is the case with mammalian cells, plant cells use extracellular vesicles to transfer RNAs, proteins, lipids, and secondary metabolites. Such plant-derived exosome-like nanovesicles (‘plant exosomes’) function in intercellular communication, the plant’s defence system or cell wall organization. Interestingly, plant-derived exosomes can be taken up by mammalian cells and have a beneficial effect on human health through cross-kingdom cell communication. Exosomes from different plants, such as


apples, grapes, aloe vera, lemon, and ginger, showed anti-inflammatory, antioxidant, anti-tumour, or wound healing potential.1-3 Considering these effects and their high stability and safety, plant-derived exosomes are being investigated for medical and cosmetic use. Due to their size and composition, plant


exosomes, similar to mammalian exosomes, can be taken up by the skin where they can unleash their full potential. As the use of human-derived or animal-derived exosomes in cosmetics is not permitted in most countries, biomimetic or plant-derived exosomes are a promising alternative. Despite the advantages of plant-derived exosomes and the intensive research that is currently being performed, it remains difficult to extract pure plant-derived exosomes due to the intricate structure and cell walls of plant cells. Complex extraction and purification protocols, as well as impurities, are the challenges faced.


Plant stem cells as optimal source for plant exosomes To benefit from the great potential of plant exosomes but circumvent the challenges of isolation, cultivated plant stem cells can be used as source of exosomes. The PhytoCellTec technology developed by Mibelle Biochemistry relies on the wound healing mechanism of a plant, whereupon part of a plant is wounded to induce the formation of callus cells. This healing tissue consists of dedifferentiated


cells, which have stem cell properties. Such plant stem cells do not have rigid cell walls, contain no chlorophyll, and have a high number of exosomes inside and around the cells. In this study, an extract from a plant stem


cell culture originally derived from a goji seedling naturally containing exosomes was analyzed for its beneficial effect on the skin. Goji (Lycium barbarum) plants are native to Southeast Europe and Asia and their fruits, the goji berries, are one of the most famous superfruits. Recent studies specifically examining the exosomes derived from goji


PERSONAL CARE January 2025


plants demonstrated that these exosomes provide health benefits in the context of muscle atrophy and spinal cord injuries.4,5


Materials and methods Characterization of plant stem cell-derived exosomes Goji stem cells were cultivated at 25°C in the dark on a wave reactor. For sample preparation, the cell culture was homogenized using a microfluidizer (Microfluidics Corporation Newton) and afterwards centrifuged. The supernatant was lyophilized and filtered


in several steps. The particle size of the resulting sample was determined using a particle size analyzer (Malvern Panalytical Ltd.). For freeze-fracture transmission electron


microcopy, a sample was placed onto a gold sample holder between copper plates before jet-freezing with liquid propane as cryogen. Samples were fractured and freeze-etched (Leica Microsystems) and replicas were made by platinum-carbon shadowing, cleaned and dried before being examined by a transmission electron microscope at 80 kV (TEM CM 10, Philips). Images were taken with a CCD camera.


Gene expression in keratinocytes Normal human epidermal keratinocytes (NHEK) were seeded in 24-well plates and grown for 24 hours before treatment with 0.1% goji stem cell extract for 24 hours. Cells were harvested and washed in phosphate buffered saline (PBS), replicates were pooled and total RNA was extracted from cells of each condition. RNA was reverse transcribed and qRT-PCR


analysis was performed on genes selected for their importance in skin physiology. Normalization was performed using house- keeping genes and the resulting gene expression data was compared to the control.


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Quantification of exosome production by mesenchymal stem cells Dermal derived human mesenchymal stem cells (MSCs) were either treated or not (control) with 0.1% goji stem cell extract for a period of 24 hours. The exosomes that were released from the cells were isolated using EXOPrep kit (Hansabiomed) and quantified in two different ways: quantification of total protein amount and quantification of the activity of acetylcholinesterase, which is a known exosomal protein using the Pierce BCA Assay kit (Thermo Fisher Scientific) and Fluorocet exosome quantitation assay kit (System Bioscienes), respectively.


Gene expression in fibroblasts after treatment with MSC-conditioned medium Human mesenchymal stem cells (MSCs) were seeded in 6-well plates and cultured for 24 hours. The medium was then removed and replaced with culture medium containing or not (control conditioned medium) 1% goji stem cell extract and cells were incubated for 72 hours. All experimental conditions were performed in n=2. At the end of incubation, the supernatants


were collected, and the replicates of each condition were pooled to be used for the treatment of fibroblasts (conditioned medium). Fibroblasts were previously seeded in 12-


well plates and cultured in culture medium for 72 hours and then in assay medium for further four hours. Afterwards, for one series, medium was removed and replaced with assay medium containing or not (control) goji stem cell extract. For another series, medium was removed and replaced with the conditioned medium from MSCs (control or goji stem cell extract treated MSCs). Cells were then incubated for 24 hours. All experimental conditions were performed in n=3. At the end of incubation, cells were washed


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Figure 2: Structure and size of plant exosomes in the goji stem cell extract www.personalcaremagazine.com


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