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

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.