# A Review on Microfluidic Platforms Applied to Nerve Regeneration
## 2 Design of Microfluidic Devices
### Structures
#### 2.2.2 3D Culture Structure  
Most existing microfluidic devices for nerve regeneration studies are constructed in 2D culture structure. However, with increasing requirements to mimic in vivo environment, 2D culture devices have revealed some drawbacks. The 2D culture devices neither accurately recapitulate the structures, functions or physiology of living tissues, nor the highly complex and dynamic 3D environments in vivo [50]. Recent years, to better represent the intricate human physiology and relevant conditions, 3D cell culture platforms have gradually gained attentions. In 3D culture, cells are grown on a scaffold-based matrix that mimics the extracellular matrix (ECM) [51]. Compared with 2D culture, 3D culture can realize better simulations of actual complex environment in vivo, including cell matrix components, intercellular and cell-substrate interactions. The 3D culture is able to reproduce more physiological equilibration and transport of soluble factors [52]. In some experiments, such as drug screening, the results of 3D culture are closer to clinical trials than experiments with 2D culture [53]. The 3D culture structure can improve the simulation of the in vivo setting of cells inside organs and tissues. This helps to simulate the complex environment within the vertebral cavity of post-SCI, which has significant implications for SCI research. Generally, 3D cell culture is formed on pore plates or the biocompatible extracellular scaffolds (hydrogels) of trans-well membranes. Figure 3A,B show two typical structures for 3D culture microfluidic devices chemical gradients-based 3D culture and 3D scaffolds culture using hydrogels. By using hydrogels containing natural molecules of extra-cellular matrix (ECM) or synthetic polymers, cells are induced to polarize and interact with adjacent cells, randomly interspersed in ECM. [PERSON] [54] developed a microfluidic device to investigate regeneration of CNS neurons in response to neurotoxic natural small molecules. The device consists of three parallel gradient units, and each unit has a central main channel interconnected by two asymmetric peripheral channels through 3D microgrooves. As shown in Figure 3C, this structure drives the flow direction of the fluid, which constructs a stable 3D concentration gradient in the central channel. [PERSON], [PERSON] [55] presented a platform that combines a gradient-generated microfluidic device with 3D protein-engineered hydrogels to study the effects of RGD ligand density on neurite outgrowth and neuronal 3D pathways, shown as Figure 3D. [PERSON] [56] proposed a gel-free 3D microfluidic cell culture system in order to improve neurogenic potency. The structure is consisted of a fluidic channel with a size of 1000 mm \(\times\) 0.6 mm. An array of ellipticals micro-pillars with size of 30 mm \(\times\) 50 mm and interval of 20 mm, is fixed on the center of the microfluidic channel. The central part of cell culture compartment is connected to a reservoir inlet for loading cells. [PERSON] [57] presented a four layers microfluidic device to investigate neuronal cell culture in 3D hydrogel scaffolds. The hydrogel is structured in parallel layers to reconstruct cell layers that close to the natural environment. [PERSON] [58] designed a 3D layered hydrogel scaffold microfluidic device using an agarose-alginate mixture that enables the primary cortical cell culture in micro-patterned multi-layered scaffold. As shown in Figure 3E,