Microfluidic 3D cell culture: potential application of collagen hydrogels with an optimal dose of bioactive glasses

Abstract

We engineered a microfluidic platform to study the effects of bioactive glass nanoparticles (BGNs) on cell viability under static culture. We incorporated different concentrations of BGNs (1%, 2%, and 3% w/v) in collagen hydrogel (with a concentration of 3.0 mg/mL). The microfluidic chip's dimensions were optimized through fluid flow and mass transfer simulations. Collagen type I extracted from rat tail tendons was used as the main material, and BGNs synthesized by the sol-gel method were used to enhance the mechanical properties of the hydrogel. The extracted collagen was characterized using FTIR and SDS-PAGE, and BGNs were analyzed using XRD, FTIR, DLS, and FE-SEM/EDX. The structure of the collagen-BGNs hydrogels was examined using SEM, and their mechanical properties were determined using rheological analysis. The cytotoxicity of BGNs was assessed using the MTT assay, and the viability of fibroblast (L929) cells encapsulated in the collagen-BGNs hydrogel inside the microfluidic device was assessed using a live/dead assay. Based on all these test results, the L929 cells showed high cell viability in vitro and promising microenvironment mimicry in a microfluidic device. Collagen3-BGNs3 (Collagen 3 mg/mL + BGNs 3% (w/v)) was chosen as the most suitable sample for further research on a microfluidic platform.

Keywords: 3D cell culture; Bioactive glass nanoparticle; Collagen hydrogel; Microfluidic system.

Fig. 1

Schematic overview of the research.

Fig. 2

The schematic of the microfluidic system. Its dimensions are introduced as parameters, which are explained in the simulation section. This device is composed of two lateral media channels and one interposed gel channel.

Fig. 3

Schematic of microfluidic chip fabrication using photolithography, collagen solution loading process without any leakage, and cells encapsulated in the collagen3-BGNs3 hydrogel into the chip.

Fig. 4

Simulation of gel filling into the microfluidic device for different designs (a) Movement of collagen solution with leakage to media channel of the design number 1; (b) Movement of collagen solution without leakage along the gel channel of the design number 2; (c) Movement of collagen solution without leakage along the gel channel of the design number 3; (d) Movement of collagen solution with leakage to media channel of the design number 4; The dynamic viscosity of the collagen solution with a concentration of 2.0 mg/mL is 6 mPa × s .

Fig. 5

Simulation and experimental results of gel filling into the microfluidic device: (a) Simulation showing a viscosity of 9 mPa·s with low leakage of the collagen solution along the gel channel; (b) Simulation showing a viscosity of 11 mPa·s with leakage of the collagen solution into the media channel; (c) Experimental result of collagen solution filling the central channel at a concentration of 4 mg/mL; (d) Experimental result of collagen solution filling the central channel at a concentration of 9 mg/mL with high leakage.

Fig. 6

Mass transfer simulation within the gel channel of the designed chip (Design Number 2). (a) t = 2.5 h (b) t = 4 h.

Fig. 7

Characterizing collagen type I, derived from rat tail. (a) Freeze-dried collagen; (b) the collagen solution, pre-crosslinking at a concentration of 3 mg/mL; (c) self-assembled collagen under pH 7.4 and 37 °C conditions; (d) The FTIR spectrum indicating specific molecular vibrations such as amide A, amide B, amide I, amide II, and amide III, confirming the presence of the type 1 collagen structure; (e) SDS-PAGE data revealed collagen type 1 with two α₁ chains and one α₂ chain, as well as a β-dimer, further confirming the structure of collagen type I.

Fig. 8

The characterization results of the synthesized BGNs with the sol–gel method (a) XRD spectra; (b) General observation photographs; (c) FTIR spectra; (d) FESEM micrograph image of BGNs; (e) Particle size distributions.; (f) EDX analysis of BGNs.

Fig. 9

SEM images of collagen hydrogels with varying concentrations of BGNs components. (a). collagen3-BGNs0; (b, c). Collagen3-BGNs1; (d). Collagen3-BGNs2; (e, f). Collagen3-BGNs3; Yellow arrows indicate BGNs. (g) Rheological evaluation of storage and loss modulus of different hydrogels as a function of strain at the same temperature.

Fig. 10

Evaluation of cell viability encapsulated in hydrogels. (a) Comparison of the cell viability in different concentrations of BGNs by MTT assay; (NS, no significant difference; p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001); (b) Live/dead was utilized to assess the viability of the cells encapsulated in collagen3-BGNs3 hydrogel into the microfluidic on day 3, the cells were highly viable, the green color shows the living cells, and the red color indicates the dead cells (scale bar = 200 µm).