Three-Dimensional Cell Culture of Adipose-Derived Stem Cells in a Hydrogel with Photobiomodulation Augmentation
April 5th, 2024
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.
The laser research data are leading efforts to investigate phototherapy or laser radiation of biological tissue, focusing on stem cell photobiomodulation for regenerative therapy. In this study, we're investigating 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 advancing 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 coenocytes. To begin, transfer 44 milliliters of basal medium into a 50-milliliter centrifuge tube. Add five milliliters of 10%FBS to the media, then pipette 0.5 milliliters each of penicillin streptomycin and amphotericin B antibiotic solutions.
Centrifuge a vial containing fast dexstran solution at 1000 G for 30 seconds at 20 degrees Celsius. Add 175 microliters of water to the vial to obtain a final concentration of 30 millimoles per liter. Vortex the tube for 30 seconds on medium speed until the material is fully dissolved.
Incubate the dissolved material on ice for five minutes. Centrifuge the tube before vortexing its contents again. Then place the tube on ice until further use.
Next centrifuge the crosslinker to concentrate the material. Pipette 188 microliters of water to the crosslinker vial to achieve a final concentration of 20 millimoles per liter of thiol groups. Vortex the crosslinker solution until all material is dissolved.
Centrifuge the tube after incubation, then vortex briefly before placing it at room temperature until further use. Next, prepare the adipose-derived stem cell suspension for 10 microliters of hydrogel as given in the table to create a master mix. Transfer nine microliters of the master mix into a well in a 96-well strip plate.
After three minutes, add one microliter of the degradable crosslinker to the mix to solidify the gel. Pipette 170 microliters of pre-warmed complete culture media over the hydrogels. Replace the culture medium after incubation.
To begin, set up the diode laser system for the green wavelength. Switch on the lasers and allow them to warm up for the recommended duration. After the lasers have reached a steady state and stabilized, use a laser power meter to measure the power output of the lasers.
Record the power values for each wavelength. After replacing the complete media, place the well plate containing adipose-derived stem cells embedded in hydrogel under the diode laser system. Irradiate the hydrogels with the calculated laser irradiation time for the selected fluency at the chosen wavelength.
Ensure that each experimental group has a non-irradiated control. Incubate the culture plates at 37 degrees Celsius under 5%carbon dioxide supplementation and 85%humidity. Next, dilute the enzymatic cell recovery solution with PBS at a ratio of one to 20 to prepare a working solution.
Add 30 microliters of the working solution to each gel or well containing the cells needing recovery. Gently rock or swirl the plate to allow for even distribution of the recovery solution. Incubate the plate with the recovery solution for 45 minutes.
After incubation, carefully remove the plate from the incubator. Centrifuge the plates at 1, 409 G for five minutes at 20 degrees Celsius to pellet the cells. Next, carefully discard the supernatant without disturbing the cell pellet.
Pipette 220 microliters of sterile PBS into the pellet. Ensure thorough mixing to achieve a homogenous single cell suspension. The adipose-derived stem cells retained a rounded morphology 24 hours after seeding and photobiomodulation exposure.
The cells were scattered throughout the gel as single cells or in grape-like clusters. The morphology remained unchanged after 10 days in 3D culture.
Overview
This study presents a protocol for using hydrogel as a 3D cell culture framework for adipose-derived stem cells (ADSCs) and explores the effects of photobiomodulation (PBM) on their proliferation.
Key Study Components
Area of Science
- Stem Cell Biology
- Photobiomodulation
- 3D Cell Culture Techniques
Background
- Photobiomodulation has been shown to enhance stem cell viability and proliferation.
- 3D cell culture provides a more physiologically relevant environment for studying cell behavior.
- Adipose-derived stem cells are a promising source for regenerative therapies.
- Hydrogels are used to create a supportive matrix for cell growth.
Purpose of Study
- To demonstrate the effectiveness of hydrogel in supporting ADSC culture.
- To investigate the impact of PBM on the proliferation of ADSCs in a 3D culture setting.
- To assess the cytotoxic effects of hydrogel on ADSCs over a 10-day culture period.
Methods Used
- Utilization of hydrogel as a 3D culture matrix for ADSCs.
- Application of photobiomodulation to enhance cell proliferation.
- Assessment of cell viability and proliferation rates.
- Longitudinal study over a 10-day culture period.
Main Results
- Hydrogel showed no cytotoxic effects on ADSCs during the culture period.
- Photobiomodulation significantly increased the proliferation rates of ADSCs.
- 3D culture conditions were beneficial for studying the effects of PBM.
- The study supports the potential of PBM in regenerative therapies using ADSCs.
Conclusions
- Hydrogel is an effective 3D culture system for ADSCs.
- Photobiomodulation can enhance the proliferation of stem cells in 3D cultures.
- This approach may improve regenerative therapy outcomes.
Frequently Asked Questions
Executive Industry Relevance
Three-dimensional hydrogel culture of adipose-derived stem cells (ADSCs) with photobiomodulation (PBM) augmentation addresses a key challenge in regenerative medicine: enhancing cell proliferation and viability in physiologically relevant environments. This approach supports predictive confidence in early discovery by enabling robust assessment of stem cell behavior under controlled, quantifiable conditions. The method is positioned to inform portfolio decisions where stem cell-based therapies require validated, scalable, and reproducible preclinical models.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of ADSC proliferation and viability in a 3D matrix relevant to in vivo conditions.
- Supports biological de-risking by demonstrating non-cytotoxicity of both hydrogel and PBM over extended culture periods.
- Facilitates functional target validation for stem cell-based regenerative strategies.
Screening & Assay Development
- Establishes a standardized 3D culture system for reproducible ADSC assays.
- Provides quantitative proliferation and cytotoxicity readouts for downstream screening workflows.
- Enables scalability and platform reuse for compound or modality evaluation in stem cell contexts.
Translational & Preclinical Research
- Aligns with disease-relevant modeling by mimicking tissue-like environments for ADSCs.
- Supports continuity from discovery through preclinical validation by maintaining cell phenotype and viability over time.
- Reduces mechanistic ambiguity in evaluating PBM as an augmentation strategy for cell-based therapies.
Pipeline & Workflow Integration
This method integrates into the discovery-to-preclinical continuum by providing a physiologically relevant, quantifiable system for ADSC evaluation and PBM parameter optimization.
- Discovery Biology: Supports hypothesis testing on PBM effects and hydrogel compatibility with ADSCs.
- Screening: Delivers reproducible, quantitative proliferation and cytotoxicity outputs for assay development.
- Analytics: Enables statistical comparison of PBM fluencies, wavelengths, and timepoints on cell outcomes.
- Translational Research: Provides a bridge to preclinical studies by modeling cell behavior in 3D matrices.
- Enterprise Reuse: Offers a reusable workflow for evaluating other stem cell types or photobiomodulation parameters.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in stem cell viability and proliferation under physiologically relevant conditions.
- Operational Value: Standardizes 3D culture and PBM protocols for reproducibility and scalability.
- Strategic Value: Informs go/no-go decisions for stem cell therapy development by reducing early-stage biological risk.
- Portfolio Impact: Enables risk-adjusted prioritization of regenerative medicine assets based on robust preclinical data.
Implementation Considerations
- Requires expertise in 3D cell culture, hydrogel preparation, and PBM instrumentation.
- Demands access to diode laser systems with precise wavelength and fluency control.
- Necessitates standardized protocols for cross-team reproducibility and data comparability.
- May require adaptation for different stem cell sources or hydrogel compositions.
- Dependent on validated quantitative assays for proliferation and cytotoxicity measurement.
