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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 µL 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.

Introduction

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

Protocol

NOTE: See the Table of Materials for details related to all materials, reagents, and software used in this protocol. The protocol has been graphically summarized in Figure 1.

1. Two- dimensional (2D) cell culture

NOTE: Immortalised ADSCs (1 x106 cells) are stored at -195.8 °C in liquid nitrogen in a cryopreservation vial containing 1 mL of cell freezing media.

  1. Preparing the 2.......

Representative Results

To assess the morphology and visually inspect the cell density of the hydrogels, inverse microscopy was used (Figure 2). The ADSCs retained a rounded morphology 24 h after seeding and PBM exposure. The cells were scattered throughout the gel as single cells or in grape-like clusters. The morphology was unchanged after 10 days in 3D culture. No definitive difference in morphology was noted between the experimental groups and controls or between the different experimental groups.

Discussion

ADSCs are an ideal cell type to use for regenerative medicine as they stimulate various processes to aid in wound healing3,4. However, there are several challenges that need to be circumvented, e.g., poor survival rates and ineffective engraftment of the cells in an injury site9. Immortalized cells were used as a commercially available cell line, as they can be passaged for more generations compared to primary cells, they do not need to be.......

Acknowledgements

This research was funded by the National Research Foundation of South Africa Thuthuka Instrument, grant number TTK2205035996; the Department of Science and Innovation (DSI) funded African Laser Centre (ALC), grant number HLHA23X task ALC-R007; the University Research Council, grant number 2022URC00513; the Department of Science and Technology's South African Research Chairs Initiative (DST-NRF/SARChI), grant number 98337. The funding bodies played no role in the design of the study, collection, analysis, interpretation of the data or writing the manuscript. The authors thank the University of Johannesburg (UJ) and Laser Research Centre (LRC) for their use of the f....

Materials

NameCompanyCatalog NumberComments
525 nm diode laserNational Laser Centre of South AfricaEN 60825-1:2007
825 nm diode laserNational Laser Centre of South AfricaSN 101080908ADR-1800
96 Well Strip PlatesSigma-AldrichBR782301
Amphotericin BSigma-AldrichA2942Antibiotic (0.5%; 0.5 mL)
CellTiter-Glo 3D Cell Viability AssayPromegaG9681ATP reagent, Proliferation assay Kit
Corning 2 mL External Threaded Polypropylene Cryogenic VialCorning430659cryovial
CryoSOfreeSigma-AldrichC9249Cell freezing media
CytoTox96 Non-Radioactive Cytotoxicity AssayPromegaG1780Cytotoxicity reagent
Dulbecco’s Modified Eagle MediaSigma-AldrichD5796Basal medium (39 mL/44 mL)
FieldMate Laser Power MeterCoherent1098297
Flat-bottomed Corning 96 well clear polystyrene plateSigma-AldrichCLS3370
Foetal bovine serumBiochromS0615Culture medium enrichment (5 mL; 10% / 10 mL; 20%)
Hanks Balanced Salt Solution (HBSS)Sigma-AldrichH9394Rinse solution
Heracell 150i CO2 incubatorThermo Scientific51026280
Heraeus Labofuge 400Thermo Scientific75008371Plate spinner for 96 well plates
Heraeus Megafuge 16R centrifugeThermoFisher75004270
Immortalized ADSCsATCCASC52Telo hTERT, ATCC SCRC-4000Passage 37
Invitrogen Countess 3InvitrogenAMQAX2000Automated cell counter for Trypan Blue
Julabo TW20 waterbathSigma-AldrichZ615501Waterbath used to warm media to 37 °C
Olympus CellSens EntryOlympusVersion 3.2 (23706) Imaging software: digital image acquisition
Olympus CKX41OlympusSN9B02019Inverted light microscope
Olympus SC30 cameraOlympusSN57000530Camera attached to inverted light microscope
Opaque-walled Corning 96 well solid polystyrene microplatesSigma-AldrichCLS3912Opaque well used for ATP luminescence
Penicillin-StreptomycinSigma-AldrichP4333Antibiotic (0.5%; 0.5 mL)
SigmaPlot 12.0Systat Software Incorporated
TrueGel3D – True3Sigma-AldrichTRUE3-1KT10 µL
TrueGel3D Enzymatic Cell Recovery SolutionSigma-AldrichTRUEENZ01:20
Trypan Blue StainThermo Fisher - InvitrogenT102820.4% solution
TrypLE Select Enzyme (1x)Gibco12563029Cell detachment solution
Victor Nivo Plate ReaderPerkin ElmerHH3522019094Spectrophotometric plate reader

References

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3D Cell CultureAdipose derived Stem CellsPhotobiomodulationHydrogelStem Cell Regenerative TherapyProliferationDifferentiation

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