Nucleoside-Derived Low-Molecular-Weight Gelators as a Synthetic Microenvironment for 3D Cell Culture

Abstract

For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.

Keywords: 3D culture; extracellular matrix; glyco-nucleo-bola-amphiphiles; growth kinetics; low-molecular-weight gelators; scaffold; supramolecular gels; thermoreversibility.

Figure 1

Chemical structure of the two molecules studied, named Glycosyl-Nucleo-Bola-amphiphile (GNB). Color emphasizes the polar end (blue), the linear aliphatic linker (black), and the only subtle difference between these molecules: amide (orange) or urea (red). Abbreviations are BA and BU for amide (top) and urea (bottom) GNBs, respectively.

Figure 2

Stiffness characterization of GNB. Left: Concentration dependency of the GNB stiffness in PBS buffer (inset: power law of the G′–concentration relationship); right: stiffness of GNB at different concentrations in PBS and in cell culture medium. All experiments were performed at 37 °C.

Figure 3

Stress relaxation of GNB. Inset: Half time characteristics of the stress relaxation of GNB in PBS. Means and standard deviations were determined through three independent experiment replications. Stress relaxation experiments were performed with a controlled strain of 15% at 37 °C.

Figure 4

Cryo-SEM of LMWGs prepared in cell culture medium (DMEM + 10% FBS). Left: BU 0.2% w/v; right: BA 0.5% w/v. Scale bar: 1 μm.

Figure 5

Gelation kinetics of BU (left, circle) and BA (right, square) at two concentrations in DMEM + 10% FBS at 20 °C.

Figure 6

Migration of molecules with different molecular weights. Top: Diffusion of MTT, Cytochrome C (CytC) and Hemoglobin (Hb) from the hydrogel composed of BU 0.2% w/v (left) or BA 0.25% w/v (right) in PBS to the supernatant. Bottom: Diffusion of antibody R633 (left) and riboflavin noted RF (right) from the supernatant to hydrogels composed of BU 0.2% w/v (red circle) or BA 0.25% w/v (orange square) in PBS at 37 °C. [X]_gel and [X]_SN correspond to the concentrations of the compound in the gel and in the supernatant, respectively. Mean values and error bars for the antibody internalization experiment correspond to three independent experiments.

Figure 7

Cryo-SEM of U87-MG cells. Left: scanning electron micrograph of cells cultured in 2D. Right: cryo-scanning electron micrograph of hosted cells in BU 0.2% w/v gel. Samples were fixed after 35 days of culture.

Figure 8

3D culture of the U87-MG cell line in (A) BA 0.25% w/v and (B) BU 0.1% w/v after 5, 15, 25, and 35 days. Top panel represents imaging using bright field, and middle and bottom panels represent imaging using the live/dead kit to evaluate the live cells with calcein AM dye (in green) and dead cells using ethidium homodimer-1 dye (in red), respectively. Scale bar = 250 μm.

Figure 9

Rheological data showing the gel stability of BA 0.25% w/v with and without cells, at day 0 and day 30 of culture, measured using a rheometer. G′ values were taken at a frequency of 1 Hz (6.283 rad s^–1).