3d cell culture of adipose&#45;derived stem cells in hydrogels for growth augmentation

Aumento do crescimento da fotobiomodulação de células-tronco incorporadas em hidrogel 

ADSCs are mesenchymal multipotent progenitor cells with the capacity to self-renew and differentiate into several cell lineages. These cells can be harvested from the stromal vascular fraction (SVF) of adipose tissue during a lipoaspiration procedure1. ADSCs have emerged as an ideal stem cell type to use in regenerative medicine because these cells are abundant, minimally invasive to harvest, easily accessible, and well characterized2. Stem cell therapy offers a possible avenue for wound healing by stimulating cell migration, proliferation, neovascularization, and reducing inflammation within wounds3,4. Roughly 80% of the regenerative capacity of ADSCs is attributable to paracrine signaling via their secretome5. Previously, it was suggested a direct local injection of stem cells or growth factors into damaged tissue could illicit sufficient in vivo repair mechanisms6,7,8. However, this approach faced several challenges, such as poor survival and reduced stem cell engraftment within damaged tissues as a result of the inflammatory environment 9. Furthermore, one of the reasons cited was a lack of an extracellular matrix to support the survival and functionality of the transplanted cells10. To overcome these challenges, emphasis is now being placed on the development of biomaterial carriers to support stem cell viability and function.
Three-dimensional (3D) cell culture enhances cell-to-cell and cell-to-matrix interaction in vitro to provide an environment that better resembles the in vivo environment11. Hydrogels have been extensively studied as a class of biomaterial carriers that provide a 3D environment for stem cell culture. These structures are made of water and crosslinked polymers12. Encapsulation of ADSCs in hydrogel has virtually no cytotoxic effect on the cells during culture while maintaining the viability of the cells6. Stem cells cultured in 3D demonstrate enhanced retention of their stemness and improved differentiation capacity13. Similarly, hydrogel-seeded ADSCs demonstrated increased viability and accelerated wound closure in animal models14. Furthermore, hydrogel encapsulation significantly increases the engraftment and retention of ADSCs in wounds15,16. TrueGel3D is made of a polymer, either polyvinyl alcohol or dextran, solidified by a crosslinker, either cyclodextrin or polyethylene glycol17. The gel is a synthetic hydrogel that does not contain any animal products that may interfere with the experiments or trigger an immune reaction during the transplantation of the gel into a patient while effectively mimicking an extracellular matrix18. The gel is fully customizable by altering the composition and individual components. It can house different stem cells and support the differentiation of several cell types by adjusting the stiffness of the gel19. Attachment sites can be created through the addition of peptides20. The gel is degradable by the secretion of metalloproteases, allowing for cell migration21. Lastly, it is clear and allows for imaging techniques.
PBM is a minimally invasive and easily performed form of low-level laser therapy used to stimulate intracellular chromophores. Different wavelengths elicit different effects on cells22. Light in the red to near-infrared range stimulates increased adenosine triphosphate (ATP) and reactive oxygen species (ROS) production by enhancing flux through the electron transport chain23. Light in the blue and green ranges stimulates light-gated ion channels, allowing for the non-specific influx of cations, such as calcium and magnesium, into cells, which is known to enhance differentiation24. The net effect is the generation of secondary messengers that stimulate the transcription of factors triggering downstream cellular processes such as migration, proliferation, and differentiation25. PBM can be used to pre-condition cells to proliferate or differentiate before transplanting the cells into an adverse environment, e.g., damaged tissue26. Pre- and post-transplant PBM (630 nm and 810 nm) exposure of ADSCs significantly enhanced the viability and function of these cells in vivo in a diabetic rat model27. Regenerative medicine requires an adequate number of cells for effective repair of tissues28. In 3D cell culture, ADSCs have been associated with slower proliferation rates compared to two-dimensional cell culture6. However, PBM can be used to augment the 3D cell culture process of ADSCs by enhancing viability, proliferation, migration, and differentiation29,30.

Autor em destaque: Avanços na terapia regenerativa com células-tronco por meio da fotobiomodulação

Cultura Celular Tridimensional de Células-Tronco Derivadas do Tecido Adiposo em Hidrogel com Aumento da Fotobiomodulação

The Laser Research Centre are leading efforts to investigate phototherapy or laser radiation of biological tissue, focusing on stem cell photobiomodulation for regenerative therapy. In this study, we investigated stem cell photobiomodulation in adipose-derived stem cells using three-dimensional cell culture. Recent research has been focusing on combining photobiomodulation and adipose-derived stem cells for stem cell regenerative therapy.Studies have indicated that photobiomodulation enhances stem cell viability, proliferation, and differentiation potential. 3D cell culture techniques are an advancement of photobiomodulation research. As 3D cell culture supplies us with a physiologically relevant environment to study cell behavior.The current study demonstrated that hydrogel has no cytotoxic effect on cells over a 10-day culture period. And that photobiomodulation has significant positive augmentative potential and can significantly up-regulate the proliferation rates of adipose-derived mesenchymal stem cells despite being in a three-dimensional cell culture. The results of the current study will pave the way for the development of standardized photobiomodulation parameters and culture conditions for the differentiation of adipose-derived mesenchymal stem cells into various specialized types of cells, especially tenocytes.

Cultura Celular 3D de Células-Tronco Derivadas do Tecido Adiposo em Hidrogéis para Aumento do Crescimento

Para começar, transfira 44 mililitros de meio basal para um tubo centrífugo de 50 mililitros. Adicione 5 mililitros de 10%FBS à mídia. Em seguida, pipetar 0,5 mililitros cada de penicilina, estreptomicina e anfotericina B soluções antibióticas.Centrifugar um frasco para injetáveis contendo solução rápida de Dexstran a 1000G durante 30 segundos a 20 graus Celsius. Adicione 175 microlitros de água ao frasco para injetáveis para obter uma concentração final de 30 milimoles por litro. Vórtice o tubo por 30 segundos em velocidade média até que o material esteja totalmente dissolvido.Incubar o material dissolvido no gelo durante cinco minutos. Centrifugar o tubo antes de vomitar seu conteúdo novamente. Em seguida, coloque o tubo sobre gelo até o uso posterior.Em seguida, centrifugar o reticulante para concentrar o material. Pipetar 188 microlitros de água para o frasco reticulante para atingir uma concentração final de 20 milimoles por litro de grupos tióis. Vórtice a solução do reticulador até que todo o material esteja dissolvido.Centrifugar o tubo após a incubação. Em seguida, vórtice brevemente antes de colocá-lo à temperatura ambiente até novo uso. Em seguida, prepare a suspensão de células-tronco derivadas do tecido adiposo para 10 microlitros de hidrogel, conforme indicado na tabela, para criar uma mistura mestre.Transfira nove microlitros da mistura master para um poço em uma placa de tira de 96 poços. Após três minutos, adicione um microlitro do reticulante degradável à mistura para solidificar o gel. Pipeta. 170 microlitros de meio de cultura completo pré-aquecido sobre os hidrogéis.Substitua o meio de cultura após a incubação.

3d-cell-culture-adipose-derived-stem-cells-hydrogels-for-growth

Research

JoVE Journal

Biology

3D Cell Culture of Adipose&#45;Derived Stem Cells in Hydrogels for Growth Augmentation

95 visualizações.  University of Johannesburg. Para começar, transfira 44 mililitros de meio basal para um tubo centrífugo de 50 mililitros. Adicione 5 mililitros de 10%FBS à mídia. Em seguida, pipetar 0,5 mililitros cada de penicilina, estreptomicina e anfotericina B soluções antibióticas.Centrifugar um frasco para injetáveis contendo solução rápida de Dexstran a 1000G durante 30 segundos a 20 graus Celsius. Adicione 175 microlitros de água ao frasco para injetáveis para obter uma concentração final de 30 milimoles ...

Vídeo: Cultura Celular 3D de Células-Tronco Derivadas do Tecido Adiposo em Hidrogéis para Aumento do Crescimento

Three&#45;Dimensional Cell Culture of Adipose-Derived Stem Cells in a Hydrogel with Photobiomodulation Augmentation

Adipose-derived stem cells (ADSCs), possessing multipotent mesenchymal characteristics akin to stem cells, are frequently employed in regenerative medicine due to their capacity for a diverse range of cell differentiation and their ability to enhance migration, proliferation, and mitigate inflammation. However, ADSCs often face challenges in survival and engraftment within wounds, primarily due to unfavorable inflammatory conditions. To address this issue, hydrogels have been developed to sustain ADSC viability in wounds and expedite the wound healing process. Here, we aimed to assess the synergistic impact of photobiomodulation (PBM) on ADSC proliferation and cytotoxicity within a 3D cell culture framework. Immortalized ADSCs were seeded into 10 &#181;L hydrogels at a density of 2.5 x 103 cells and subjected to irradiation using 525 nm and 825 nm diodes at fluencies of 5 J/cm2 and 10 J/cm2. Morphological changes, cytotoxicity, and proliferation were evaluated at 24 h and 10 days post-PBM exposure. The ADSCs exhibited a rounded morphology and were dispersed throughout the gel as individual cells or spheroid aggregates. Importantly, both PBM and 3D culture framework displayed no cytotoxic effects on the cells, while PBM significantly enhanced the proliferation rates of ADSCs. In conclusion, this study demonstrates the use of hydrogel as a suitable 3D environment for ADSC culture and introduces PBM as a significant augmentation strategy, particularly addressing the slow proliferation rates associated with 3D cell culture.

Here, we present a protocol demonstrating the use of hydrogel as a three-dimensional (3D) cell culture framework for adipose-derived stem cell (ADSC) culture and introducing photobiomodulation (PBM) to enhance the proliferation of ADSCs within the 3D culture setting.