In physiological conditions, cells interact in three-dimensional (3D) environments with other cells and with the extracellular matrix (ECM). Realizing complex structures able to mimic physiological conditions is a goal of primary importance for understanding how cells behave in tissues and organs, and requires a strongly interdisciplinary work between biologists, physicists and engineers. Among other techniques, 3D direct laser writing, also known as two-photon lithography (DLW-2PL), has emerged as a promising technology for fabricating tailored 3D scaffolds for cell biology studies. DLW-2PL consists in polymerizing a photosensitive material at the focal spot of a laser beam (often called voxel, i.e. volume pixel), by exploiting the nonlinear effect of two-photon absorption. The relative movement between the laser and the substrate on which the photopolymer is drop-cast allows for obtaining 3D structures in the micrometer range, with resolution of approximately 100nm (Fig.1a).

In recent years, a variety of materials have been synthesized allowing for expanding the versatility of the 2PL technique, e.g. for biological systems [1] and stimuli-responsive devices [2]. In particular, the different mechanical properties and the possibility of selective protein surface functionalization of the used materials for 2PL can be exploited to control adhesion of cells [3] on soft 3D structures to induce the bending of parts of them [4].

Here we realized squared structures via 2PL using a passivating (i.e. cell-repulsive) photopolymer with four fibronectin-adhesive spots (Fig.1b) to induce selective adhesion of mouse 3T3 fibroblasts in 3D. After 3h culture, single cells were found to have colonized the scaffolds, by contacting the structure only in correspondence to the fibronectin-coated spots (Fig.1c). Moreover, actomyosin contractility induced bending of the four flexible beams of the scaffolds, which was measured via confocal microscopy (Fig.1d) and used to estimate the force exerted by the cell in the scaffold. In particular, a finite element method (FEM) simulation system was implemented, which replicated the geometry of the scaffold and the structure materials stiffness (known via AFM measurements). It was thus possible to iteratively estimate the force exerted by the cell in correspondence to the four adhesive spots, which induced the respective measured beam deflection. Interestingly, 3T3 fibroblasts exerted force in the range of 20−100nN, in partial agreement with some previous literature reports [4].

Given the possibility to tune the stiffness of the materials used in 2PL [5], the aforementioned scaffolds may be modified in order to obtain structures with one stiffer or softer beams. Deflection measurements and consequent FEM simulations in this case showed that even on stiffer or softer beams force is kept constant to 5−25nN.

Taken together, these results show that 2PL can be successfully used to investigate properties of biological systems on single-cell scale in different experimental conditions. The scaffold here shown may also lead to further observations on similar systems, e.g. regarding the actin cytoskeleton organization under stress conditions [6], or the protein expression involved in mechanotransduction pathways in 3D.

Figure 1. (a): sketch of DLW-2PL; (b): 3D scaffold of passivated material (blue) with selective adhesive spots for cells, functionalized with fibronectin (red); (c): immunostaining (yellow:fibronectin, green:actin, blue:DAPI, red:paxillin) of a fibroblast on adhesive spots; (d): deflection of flexible beams; (e):FEM simulation of structure deflections; (f): beam displacement (left) and force values (right) distribution of cells on 3D scaffolds.

References

[1] M.Hippler et al., Adv.Mater.,1808110,2019. [2] C.A.Spiegel et al., Adv.Funct.Mater.,1907615,2019. [3] F. Klein et al., Adv. Mater.,23,11,2011. [4] F.Klein et al., Adv.Mater.,22,8,2010. [5] E.D.Lemma et al., IEEE Trans. on Nanotechn.,16,1,2016. [6] Brand et al., Biophys.J.,113,4,2017.