Application of Microfluidic Systems for Neural Studies

Application of Microfluidic Systems for Neural Studies: History

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Whereas the axons of the peripheral nervous system (PNS) spontaneously regenerate after an injury, the occurring regeneration is rarely successful because axons are usually directed by inappropriate cues. Therefore, finding successful ways to guide neurite outgrowth, in vitro, is essential for neurogenesis. Microfluidic systems reflect more appropriately the in vivo environment of cells in tissues such as the normal fluid flow within the body, consistent nutrient delivery, effective waste removal, and mechanical stimulation due to fluid shear forces. At the same time, it has been well reported that topography affects neuronal outgrowth, orientation, and differentiation.

neural cells
neural tissue engineering
microfluidics
microfluidic flow
shear stress
topography

1. Introduction

Taking into consideration the fact that neurological injuries are hardly self-recoverable, the development of successful methods to guide neurite outgrowth, in vitro, is of high significance. Indeed, although the peripheral nervous system (PNS) exhibits a higher rate of regeneration than that of the central nervous system (CNS) through spontaneous regeneration after injury, functional recovery is fairly infrequent and misdirected ^[1].

Neural tissue engineering has emerged as a promising alternative field for the development of new nerve graft substitutes to overcome the limitations of the current grafts ^[2]. Indeed, the ultimate goal of a tissue-engineered construct is to sufficiently mimic the topographic features of the extracellular matrix and the surrounding environment of cells (e.g., mechanical properties, soluble factors, shear stress) so that cells will respond in their artificial environment in the same way they would in vivo.

Several approaches have been developed to create cellular substrates at the micro- and/or nano-scales that aim to reconstruct the architecture of the extracellular matrix in vitro ^[3]^[4]^[5]^[6]. However, apart from topography, it has recently become increasingly evident that neurogenesis may be also driven by mechanical factors ^[7]^[8]. Indeed, shear stress is a significant component of the host environment of regenerating axons ^[9]. Flow-induced shear stress can be applied to cells, in vitro, using specially designed microfluidic systems. In such systems, microfluidic flow replicates the physiological fluid flow inside the body, facilitates mass transport of solutes, and supplies consistent nutrient delivery and effective waste removal resulting in a more in vivo-like environment ^[10]. Previous research and review studies have described the main fabrication techniques, the produced types of microfluidic systems, as well as their respective advantages and disadvantages ^[10]^[11]^[12]^[13]^[14]^[15]^[16].

2. Application of Microfluidic Systems for Neural Studies

The positive effect of dynamic cultures on the response of neuronal and/or glial cells (such as adhesion, proliferation, directionality, and differentiation) has been demonstrated by many studies. Studies on the (i) gradient and chemical stimulation of neural cells, (ii) perfusion and shear stress on neural cells, and (iii) co-culture of neural cells via microfluidic systems were reported below.

2.1. Gradient Generation—Chemical Stimulation

Chung et al. ^[17] developed a polydimethylsiloxane (PDMS) gradient-generating microfluidic platform that exposed human neural stem cells (hNSCs) to a concentration gradient of known growth factors (GFs), under continuous flow (5 × 10⁻⁵ Pa). As a result, a minimization of autocrine and paracrine signaling was observed. Additionally, they found that the differentiation of hNSCs into astrocytes was inversely proportional to GF concentration while proliferation was directly proportional. Kim et al. ^[18] performed a similar study by using a microfluidic chip-generated growth factor gradient system and neural stem cells (NSCs). Results showed that NSCs proliferation and differentiation were directly dependent on the concentration gradient of GF. Additionally, Nakashima and Yasuda ^[19] fabricated a microfluidic device to investigate the effect of GF on the differentiation and axon elongation guidance of adrenal pheochromocytoma (PC12) cells. The microfluidic device was composed of a cell culture chamber, a micro-channel, a nano-hole array (containing GF), and a micro-valve allowing the precise release of chemicals from the nano-hole. Nerve growth factor (NGF) was used to stimulate the differentiation of PC12 cells. They showed that the cell growth, differentiation, and axon elongation were dependent on micro-valve switching and release gradient of NGF. Futai et al. ^[20] developed a one-layer H-shaped microfluidic channel, for concentration gradient generation, consisting of a thin (~2 μm) but high-aspect-ratio (0.5–1) microchannel. Using a long-lasting concentration gradient of NGF, they examined the axon elongation of primary dorsal root ganglion (DRG) neuronal cells. The results revealed a directional elongation of axons following the NGF concentration gradient during cultivation for 96 h. Furthermore, Park et al. ^[21] designed a microfluidic platform to expose human embryonic stem cells (ESCs)-derived neural progenitor cells to stable concentration gradients of extracellular signaling molecules. Human ESC-derived neural progenitor cells were cultured in the microfluidic system under continuous cytokine gradients (0.15 μL/h) for 8 days. Neural progenitor cells proliferated and differentiated, into neurons, in a controlled manner, and the cell properties reflected the different concentrations of extracellular signaling molecules. Bhattacharjee and Folch ^[22] fabricated a microfluidic chip containing 1024 biochemical gradient generators, with each generator entrapping a single neuron, to investigate the axon guidance and growth dynamics of primary hippocampal neurons in response to biochemical cues. The microfluidic chip produced a reproducible, stable gradient with negligible shear stress on the culture surface (100 μL/h). Using this platform, it was demonstrated that the hippocampal axon guidance was dependent on the concentration and incidence angle of the netrin-1 gradient. In particular, regarding the concentration, it was found that hippocampal growth cones close to the source of netrin-1 (high concentration) were strongly attracted, while those far from the source of netrin-1 (low concentration) were repelled. With regards to the angle of incidence, it was shown that hippocampal growth cones oriented away from the gradient axis (90–135°) turned toward the netrin-1 source, while those oriented toward the gradient (less than 45°) were strongly repelled. Finally, Cheng et al. ^[23] developed a microfluidic system that could analyze the effect of chemical and mechanical stimulation on the neuronal differentiation of placenta-derived multipotent stem cells (PDMCs). They investigated the effect of shear stress using different flow rates (1.4 × 10⁻⁴, 3.31 × 10⁻³, 4.97 × 10⁻³ Pa) on PDMCs beside 3-isobutyl-1-methylxanthine (IBMX), as the chemical stimulant, for 3 days. They showed that shear stress could not differentiate PDMCs into other cell types. Although chemical stimulation played a crucial role in the differentiation of PDMCs, shear stress enhanced PDMCs for earlier neuronal differentiation. In the 48-h condition, the maximum flow rate and IBMX showed the highest cell differentiation ratio of 42.4%.

The neuronal response to gradient and chemical stimulation through microfluidic systems is summarized in Table 1. The different approaches and the respective nerve responses are presented.

Table 1. Reported studies on the gradient and chemical stimulation through microfluidic systems on nerve cell morphology/response.

Gradient Generation—Chemical Stimulation
Cell Type	Circulating Flow?	Shear Stress	Findings	Ref.
Human neural stem cells (hNSCs)	Yes	5 × 10⁻⁵ Pa for 4 and 7 days	▪ Minimization of autocrine and paracrine signals ▪ Differentiation of hNSCs into astrocytes was inversely proportional to growth factors (GF) concentration ▪ Proliferation was directly proportional to GF concentration ▪ Simultaneous application of multiple gradients in a single experiment ▪ Low media requirements ▪ Low cell requirements	^[17]
Neural stem cells (NSCs)	Yes	-	▪ Proliferation and differentiation of NSCs were directly dependent on GF concentration	^[18]
Adrenal pheochromocytoma (PC12) cells	No	-	▪ Cell growth, differentiation and axon elongation were dependent on micro-valve switching and release gradient of nerve growth factor (NGF)	^[19]
Primary dorsal root ganglion (DRG) neuronal cells	No	-	▪ Directional elongation of axons following the NGF concentration gradient	^[20]
Human embryonic stem cells (ESCs)-derived neural progenitor cells	Yes	-	▪ Proliferation and differentiation of neural progenitor cells in a controlled manner ▪ Cell properties reflected the different concentrations of extracellular signaling molecules	^[21]
Primary hippocampal neurons	Yes	Lower than 1.2 × 10⁻³ Pa	▪ Hippocampal axon guidance was dependent on the concentration and incidence angle of netrin-1 gradient	^[22]
Placenta-derived multipotent stem cells (PDMCs)	Yes	▪ 1.4 × 10⁻⁴ Pa ▪ 3.31 × 10⁻³ Pa ▪ 4.97 × 10⁻³ Pa for 1, 2, 24, 48, 72 h	▪ No differentiation of PDMCs into other cell types with shear stress ▪ Shear stress combined with 3-isobutyl-1-methylxanthine (IBMX) enhanced the PDMCs for earlier neuronal differentiation ▪ Highest cell differentiation in the maximum flow rate and IBMX in 48 h condition	^[23]

2.2. Perfusion—Shear Stress

Chafik et al. ^[9] developed a custom-designed flow chamber that applied shear stress (1.33 Pa for 2 h), through laminar fluid flow, to Schwann cells. They showed that mechanical stimuli enhanced the proliferation of Schwann cells and caused a slight movement from their original positions. Gupta et al. ^[24]^[25] used an in vitro model to apply shear stress on primary Schwann cells in the form of laminar fluid flow (3.1 Pa for 2 h). They observed increased proliferation and downregulation of two pro-myelinating proteins, myelin-associated glycoprotein (MAG) and myelin basic protein (MBP). These results implied that a low level of mechanical stimulus may directly trigger Schwann cell proliferation. Moreover, Millet et al. ^[26], considering that cell-to-cell signaling is local, developed a specific culture system that sustained small numbers of primary hippocampal neurons and enabled analysis of the microenvironment. They observed that cultured neurons inside perfused channels (by gravity flow) composed of native, autoclave, or extracted PDMS showed increased viability and channel-length capacity (increasing 2-fold for all but native PDMS). Park et al. ^[27] designed a two-dimensional microfluidic system to study the effect of continuous flow shear stress (10⁻⁴ and 10⁻³ Pa) on radial glial cells (RGCs). They found that flow shear stress possibly activated mechanosensitive Ca⁺² channels that significantly enhanced the proliferative capacity of RGCs in response to increased shear stress. Furthermore, Park et al. ^[28] developed a microfluidic chip to apply a continuous flow of fluid that is readily observed in the brain’s interstitial space, on three-dimensional (3D) micro-spheroidal neural tissue (neurospheroids). The slow interstitial level of flow (0.15 μL/min) was maintained using an osmotic micropump system and the uniform neurospheroids were formed in concave microwell arrays. Using this system, the effect of flow on the size of neurospheroids, neural networks, and neural differentiation was investigated. Under flow conditions, the results indicated that the size of neurospheroids was larger and formed more complex and robust neural networks (increased levels of synapsin IIa and β-ΙΙΙ tubulin, decreased levels of nestin) compared to static conditions. This phenomenon was attributed to the continuous nutrient, cytokine, and oxygen transport, as well as the removal of metabolic wastes provided by the presence of slow interstitial flow. Additionally, this system was utilized to investigate the toxic effects of amyloid-β, which is thought to be the main cause of Alzheimer’s disease. Under flow conditions, the results revealed a decreased viability of neurospheroids, as well as increased neural destruction and synaptic dysfunction compared to static conditions. Conclusively, this system had significant potential as an in vitro brain model since it offered an environment that was similar to that found in vivo.

The neuronal response to perfusion and shear stress through microfluidic systems is summarized in Table 2.

Table 2. Reported studies on the perfusion and shear stress through microfluidic systems on nerve cell morphology/response.

Perfusion—Shear stress
Cell Type	Circulating Flow?	Shear Stress	Findings	Ref.
Schwann cells	Yes	1.33 Pa for 2 h	▪ Increased proliferation ▪ Slight cells’ movement from their original positions	^[9]
Schwann cells	Yes	3.1 Pa for 2 h	▪ Increased proliferation	^[24]
Schwann cells	Yes	3.1 Pa for 2 h	▪ Increased proliferation ▪ Down-regulation of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP)	^[25]
Primary hippocampal neurons	No	-	▪ Increased viability and channel-length capacity	^[26]
Radial glial cells (RGCs)	Yes	▪ 10⁻⁴ Pa ▪ 10⁻³ Pa for 5 days	▪ Increased proliferation of RGCs in response to increased shear stress	^[27]
Neural progenitor cells	Yes	-	▪ Larger size of neurospheroids ▪ Formation of more complex and robust neural networks ▪ Increased levels of synapsin IIa and β-ΙΙΙ tubulin ▪ Decreased levels of nestin ▪ Investigation of the toxic effects of amyloid-β: decreased viability of neurospheroids; increased neural destruction and synaptic dysfunction	^[28]

2.3. Microfluidic Cell Co-Culture Platforms

Majumdar et al. ^[29] designed a PDMS microfluidic cell co-culture platform that allowed individual manipulation of various cell types with the placement of a microfabricated valve that served as a reversible barrier between the chambers. As a result, healthy co-cultures of hippocampal neurons and glia were maintained for several weeks under optimal conditions. In particular, co-culture with glia provided nutrient media for maintaining healthy neural cultures, eliminating the need to supply neurons with pre-conditioned glia media, thereby enhancing the transfection efficiency of neurons in the platform. Similarly, Shi et al. ^[30] fabricated two PDMS microfluidic cell culture systems, a vertically-layered set-up, and a four-chamber set-up for studying communication between neurons and glia in close proximity. The chambers were separated by pressure-enabled valve barriers that allowed them to control communication between the two cell types. In this study, the number and stability of synaptic contacts, as well as the secreted levels of soluble factors, were increased in the co-culture system, thus confirming the importance of communication between neurons and glia for the development of stable synapses in microfluidic platforms. Robertson et al. ^[31] developed an in vitro system to examine the synaptic interaction between two interconnected populations of mixed primary hippocampal co-cultures by integrating microfluidics with calcium imaging techniques. Moreover, a computational model was verified to characterize the fluidic characteristics of the system and improve the experimental protocols (prevent substance cross-contamination between co-cultures). The results revealed that neurons and glia, in each of the separated chambers, grew within the microchannels where they physically interacted and formed synapses. In addition to this, the function of the neuron-glia synapse was confirmed by calcium imaging. Yang et al. ^[32] fabricated a microfluidic array platform to modulate hNSC differentiation in a 3D ECM microenvironment using recapitulation of paracrine action of genetically engineered human mesenchymal stem cells (hMSCs). hMSCs were genetically engineered to increase the expression of glial cell-derived neurotrophic factor (GDNF) utilizing cationic polymer nanoparticles. A simulation study produced by mathematical modeling of accumulated GDNF secreted from engineered hMSCs confirmed that in vivo-like signaling of secreted factors could be created in a 3D ECM hydrogel in the microfluidic system. Specifically, in the central channels of the microfluidic system, which were filled with a 3D ECM hydrogel, hNSCs were cultivated, and GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in the channels on each side of the central channel. Reduced differentiation of hNSCs into glial cells and increased differentiation of hNSCs into neuronal cells, including dopaminergic neurons, were observed in the co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system. Moreover, neuronal cells demonstrated functional neuron-like electrophysiological characteristics. Finally, an animal model of hypoxic-ischemic brain injury was used to confirm the improved paracrine capacity of GDNF-hMSCs. Park et al. ^[33] fabricated a circular microfluidic co-culture platform where embryonic CNS neurons and postnatal oligodendrocytes (OLs) were co-cultured in two separated compartments which were connected by arrays of axon-guiding microchannels. These microfluidic channels allowed physical isolation for cell bodies, but not for axons, and maintained fluidic isolation. The embryonic CNS neurons and postnatal OL progenitors were co-cultured in the platform for up to four weeks to study the axon-glia interaction and myelination. The circular design demonstrated excellent cell loading characteristics where a significant number of cells were positioned near the axon-guiding microchannels. Moreover, it showed enhanced axonal growth characterized by the significantly increased axon coverage ratio in the axon-glia compartment. The co-culture capability of the platform was confirmed by successfully co-culturing OL progenitors with axons in the axon/glia compartment resulting in the maturation of OLs. In a related study, Park et al. ^[34] developed a multi-compartment microfluidic co-culture platform for studying axon-glia interaction at a higher throughput. The platform allowed for conducting parallel localized biomolecule and drug treatments while carrying out various co-culture conditions in a single device. Using this platform, they were able to simultaneously study the axon-glia communication, the development and differentiation of oligodendrocytes, as well as the axonal-specific response to different stimuli. The results revealed that mature oligodendrocytes were needed in order to obtain a robust myelin sheath instead of oligodendrocyte progenitor cells. Additionally, it was shown that astrocytes stimulated the development of oligodendrocytes and were detrimental when added to an already existing axonal layer. Finally, Ristola et al. ^[35] developed a novel PDMS-based microfluidic cell culture device, with open compartments, for neuron-oligodendrocyte in vitro myelin studies. It was shown that the primary rat DRG neurons were successfully co-cultured with the oligodendrocytes in the device, which could also be used for time-lapse imaging. The results also demonstrated successful interactions and contacts between neurites and oligodendrocytes, as well as the deposition of myelin segments in an aligned distribution in the device.

Table 3 summarizes the co-culture studies of neural cells using microfluidic systems.

Table 3. Reported studies on the co-culture of neural cells using microfluidic systems on nerve cell morphology/response.

Co-Culture
Cell Type	Circulating Flow?	Shear Stress	Findings	Ref.
Co-culture of hippocampal neurons and glia	No	-	▪ Maintenance of healthy co-cultures for several weeks under optimal conditions ▪ Elimination of the need to supply neurons with pre-conditioned glia media ▪ Enhancement of the transfection efficiency of neurons	^[29]
Co-culture of hippocampal neurons and glia	No	-	▪ Increased number of synaptic contacts ▪ Increased stability of synaptic contacts ▪ Increased secreted levels of soluble factors	^[30]
Primary hippocampal co-culture of neurons and glia	No	-	▪ Growth of neurons and glia within the microchannels ▪ Formation of synapses ▪ Confirmation of the function of neuron-glia synapse via calcium imaging	^[31]
Co-culture of human neural stem cells (hNSCs) with glial cell-derived neurotrophic factor (GDNF)-overexpressing human mesenchymal stem cells (hMSCs)	No	-	▪ Reduced glial differentiation of hNSCs ▪ Enhanced differentiation of hNSCs into neuronal cells including dopaminergic neurons ▪ Functional neuron-like electrophysiological features of neuronal cells ▪ Confirmation of the enhanced paracrine ability of GDNF-hMSCs via an animal model of hypoxic-ischemic brain injury	^[32]
Co-culture of embryonic central nervous system (CNS) neurons and postnatal oligodendrocytes (OLs)	No	-	▪ Enhanced axonal growth; increased axon coverage ratio in the axon-glia compartment ▪ Maturation of OLs	^[33]
Co-culture of CNS neuron and glia	No	-	▪ To obtain a robust myelin sheath mature oligodendrocytes were needed instead of oligodendrocyte progenitor cells ▪ Stimulation of the development of oligodendrocytes by astrocytes ▪ Detrimental effect of astrocytes when added to a pre-existing axonal layer	^[34]
Co-culture of rat dorsal root ganglion (DRG) neurons and oligodendrocytes	No	-	▪ Successful interactions and contacts between neurites and oligodendrocytes ▪ Deposition of myelin segments in an aligned distribution	^[35]

3. Application of Microfluidic Systems in Combination with Topography for Neural Studies

It has been well reported that surface topography significantly affects the adhesion, orientation, proliferation, and differentiation of cells. Therefore, many studies have focused on the controlled modification of materials’ surfaces, with the ultimate aim of guiding neuronal outgrowth, which is considered crucial for the development of functional neuronal interfaces ^[3]^[4]^[5]^[6]^[36]^[37]^[38]^[39]^[40]^[41]^[42]^[43]^[44]^[45]^[46]^[47]^[48]^[49]^[50]^[51]^[52]^[53]^[54]^[55]^[56]^[57]^[58]^[59]^[60]^[61]^[62]^[63]^[64]^[65]^[66]^[67]^[68]^[69]^[70]^[71]^[72]. Nevertheless, the combined effect of microfluidic flow and topography on neuronal outgrowth has been rarely reported.

Specifically, Hesari et al. ^[73] fabricated a hybrid microfluidic system composed of a PDMS microchip and poly(lactic-co-glycolic acid) (PLGA) nanofiber-based substrate for differentiation of human induced pluripotent stem cells (hiPSCs) into neurons. The results revealed an increase in β-tubulin III (a specific neuronal marker) gene expression and a decrease in glial fibrillary acidic protein (GFAP) (a classic astrocyte marker) gene expression. Thus, this hybrid microfluidic system could be optimum for neuronal differentiation. Afterward, for further in vivo evaluation of this system, the cell-loaded scaffold was implanted in a spinal cord model of rats. During the 28 days of study, animals who received this implant displayed improved functional outcomes. However, the difference with the control group was not statistically significant.

Kim et al. ^[1] developed a fluid flow system to study the effect of mechanical stimulation on PC12 cells cultured in microfiber-based substrates. They observed that the shear stress affected the length and orientation of neurons along the microfibers. Furthermore, Jeon et al. ^[74] investigated the combined effects of topography and flow-induced shear stress on the neuronal differentiation of hMSCs. They applied different shear stresses in a PDMS substrate with micrometric grooves. The results demonstrated that shear stresses affected the expression of synaptophysin, β-tubulin III, and microtubule-associated protein 2 (MAP2), as well as the intracellular calcium concentration. The alignment was also confirmed. In addition to this, an increased neurite length was noticed on the seventh day. However, a significant decrease in neurite length was observed on the tenth day. It should be noted that in both studies ^[1]^[74], the flow was not continuous but was rather applied for only a few hours per culture day.

Babaliari et al. ^[75] have studied the combined effect of shear stress and topography on Schwann (SW10) cells’ behavior under dynamic culture conditions attained via continuous flow. For this purpose, a precise flow-controlled microfluidic system with specific custom-designed chambers incorporating laser-microstructured polyethylene terephthalate (PET) substrates comprising microgrooves ^[76] was developed. The microgrooves were positioned either parallel or perpendicular to the direction of the flow and the response of SW10 cells was evaluated in terms of growth, orientation, and elongation. The cell culture results were also combined with computational flow simulation studies employed to accurately calculate the shear stress values. It was revealed that, depending on the relation of the direction of the flow with respect to the topographical features, wall shear stress gradients were acting in a synergistic or antagonistic manner to topography in promoting guided morphologic cell response.

Table 4 summarizes the reported studies on the combined effect of microfluidic flow and topography on neuronal response.

Table 4. Reported studies on the combined effect of microfluidic flow and topography on nerve cell morphology/response.

Substrate	Fabrication Technique	Cell Type	Circulating Flow?	Shear Stress	Findings	Ref.
Poly(lactic-co-glycolic acid) (PLGA) nanofiber-based substrate	Electrospinning	Human induced pluripotent stem cells (hiPSCs)	No	-	▪ Increase in β-tubulin III gene expression ▪ Decrease in glial fibrillary acidic protein (GFAP) gene expression ▪ Animals receiving this implant showed functional improvement but no significant difference with the control group	^[73]
PLGA microfiber-based substrate	Electrospinning	Adrenal pheochromocytoma (PC12) cells	Yes	0.1–1.5 Pa for 2 h, 3 times per day for 2 days	▪ 0.25 Pa: increased neurite length ▪ 0.5 Pa: increased cellular alignment	^[1]
Polydimethylsiloxane (PDMS) substrate with micrometric grooves	Photolithography	Human mesenchymal stem cells (hMSCs)	Yes	0.1 and 0.25 Pa for 3 h per day for 2 days	▪ Shear stresses affected the expression of synaptophysin, β-tubulin III, and microtubule-associated protein 2 (MAP2) ▪ Increased neurite length at day 7; decrease in neurite length at day 10 ▪ Higher calcium concentration under 0.1 Pa ▪ Shear stress of 0.1 Pa most effective	^[74]
Polyethylene terephthalate (PET) microgrooved substrate	Ultrafast laser direct writing	Schwann (SW10) cells	Yes	0.04 and 0.15 Pa, continuous flow for 2 days	▪ Flow parallel to microgrooves’ length: enhancement of cells’ alignment; increased cell length by 11.7% (0.04 Pa) and 12.3% (0.15 Pa) compared to static culture conditions ▪ Flow perpendicular to microgrooves’ length: cells retained their orientation along the direction of microgrooves; decrease in cell length	^[75]

This entry is adapted from the peer-reviewed paper 10.3390/bioengineering10080902

References

Kim, I.A.; Park, S.A.; Kim, Y.J.; Kim, S.-H.; Shin, H.J.; Lee, Y.J.; Kang, S.G.; Shin, J.-W. Effects of mechanical stimuli and microfiber-based substrate on neurite outgrowth and guidance. J. Biosci. Bioeng. 2006, 101, 120–126.
Lanza, R.; Langer, R.; Vacanti, J.P. Principles of Tissue Engineering, 4th ed.; Elsevier: Amsterdam, The Netherlands, 2013; ISBN 9780123983589.
Chow, W.N.; Simpson, D.G.; Bigbee, J.W.; Colello, R.J. Evaluating neuronal and glial growth on electrospun polarized matrices: Bridging the gap in percussive spinal cord injuries. Neuron Glia Biol. 2007, 3, 119–126.
Schnell, E.; Klinkhammer, K.; Balzer, S.; Brook, G.; Klee, D.; Dalton, P.; Mey, J. Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-ε-caprolactone and a collagen/poly-ε-caprolactone blend. Biomaterials 2007, 28, 3012–3025.
Yao, L.; Wang, S.; Cui, W.; Sherlock, R.; O’connell, C.; Damodaran, G.; Gorman, A.; Windebank, A.; Pandit, A. Effect of functionalized micropatterned PLGA on guided neurite growth. Acta Biomater. 2009, 5, 580–588.
Simitzi, C.; Efstathopoulos, P.; Kourgiantaki, A.; Ranella, A.; Charalampopoulos, I.; Fotakis, C.; Athanassakis, I.; Stratakis, E.; Gravanis, A. Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth. Biomaterials 2015, 67, 115–128.
Lim, J.; Stoll, H.; Kwon, I. Material and mechanical factors: New strategy in cellular neurogenesis. Neural Regen. Res. 2014, 9, 1810–1813.
Franze, K. The mechanical control of nervous system development. Development 2013, 140, 3069–3077.
Chafik, D.; Bear, D.; Bui, P.; Patel, A.; Jones, N.F.; Kim, B.T.; Hung, C.T.; Gupta, R. Optimization of Schwann Cell Adhesion in Response to Shear Stress in an in vitro Model for Peripheral Nerve Tissue Engineering. Tissue Eng. 2003, 9, 233–241.
Karimi, M.; Bahrami, S.; Mirshekari, H.; Basri, S.M.M.; Nik, A.B.; Aref, A.R.; Akbari, M.; Hamblin, M.R. Microfluidic systems for stem cell-based neural tissue engineering. Lab Chip 2016, 16, 2551–2571.
Kim, L.; Toh, Y.-C.; Voldman, J.; Yu, H. A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab Chip 2007, 7, 681–694.
Young, E.W.K.; Beebe, D.J. Fundamentals of microfluidic cell culture in controlled microenvironments. Chem. Soc. Rev. 2010, 39, 1036–1048.
Zhang, C.; van Noort, D. Cells in Microfluidics. Top. Curr. Chem. 2011, 304, 295–321.
Mehling, M.; Tay, S. Microfluidic cell culture. Curr. Opin. Biotechnol. 2014, 25, 95–102.
Coluccio, M.L.; Perozziello, G.; Malara, N.; Parrotta, E.; Zhang, P.; Gentile, F.; Limongi, T.; Raj, P.M.; Cuda, G.; Candeloro, P.; et al. Microfluidic platforms for cell cultures and investigations. Microelectron. Eng. 2019, 208, 14–28.
Iakovlev, A.P.; Erofeev, A.S.; Gorelkin, P.V. Novel Pumping Methods for Microfluidic Devices: A Comprehensive Review. Biosensors 2022, 12, 956.
Chung, B.G.; Flanagan, L.A.; Rhee, S.W.; Schwartz, P.H.; Lee, A.P.; Monuki, E.S.; Jeon, N.L. Human neural stem cell growth and differentiation in a gradient-generating microfluidic device. Lab Chip 2005, 5, 401–406.
Kim, J.H.; Sim, J.; Kim, H.-J. Neural Stem Cell Differentiation Using Microfluidic Device-Generated Growth Factor Gradient. Biomol. Ther. 2018, 26, 380–388.
Nakashima, Y.; Yasuda, T. Cell differentiation guidance using chemical stimulation controlled by a microfluidic device. Sens. Actuators A Phys. 2007, 139, 252–258.
Futai, N.; Tamura, M.; Ogawa, T.; Tanaka, M. Microfluidic Long-Term Gradient Generator with Axon Separation Prototyped by 185 nm Diffused Light Photolithography of SU-8 Photoresist. Micromachines 2018, 10, 9.
Park, J.Y.; Kim, S.-K.; Woo, D.-H.; Lee, E.-J.; Kim, J.-H.; Lee, S.-H. Differentiation of Neural Progenitor Cells in a Microfluidic Chip-Generated Cytokine Gradient. STEM CELLS 2009, 27, 2646–2654.
Bhattacharjee, N.; Folch, A. Large-scale microfluidic gradient arrays reveal axon guidance behaviors in hippocampal neurons. Microsyst. Nanoeng. 2017, 3, 17003.
Cheng, Y.-C.; Tsao, C.-W.; Chiang, M.-Z.; Chung, C.-A.; Chien, C.-C.; Hu, W.-W.; Ruaan, R.-C.; Li, C. Microfluidic platform for human placenta-derived multipotent stem cells culture and applied for enhanced neuronal differentiation. Microfluid. Nanofluidics 2014, 18, 587–598.
Gupta, R.; Steward, O. Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells. J. Comp. Neurol. 2003, 461, 174–186.
Gupta, R.; Truong, L.; Bear, D.; Chafik, D.; Modafferi, E.; Hung, C.T. Shear stress alters the expression of myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) in Schwann cells. J. Orthop. Res. 2005, 23, 1232–1239.
Millet, L.J.; Stewart, M.E.; Sweedler, J.V.; Nuzzo, R.G.; Gillette, M.U. Microfluidic devices for culturing primary mammalian neurons at low densities. Lab Chip 2007, 7, 987–994.
Park, M.G.; Jang, H.; Lee, S.-H.L.A.C.J.; Lee, C.J. Flow Shear Stress Enhances the Proliferative Potential of Cultured Radial Glial Cells Possibly Via an Activation of Mechanosensitive Calcium Channel. Exp. Neurobiol. 2017, 26, 71–81.
Park, J.; Lee, B.K.; Jeong, G.S.; Hyun, J.K.; Lee, C.J.; Lee, S.-H. Three-dimensional brain-on-a-chip with an interstitial level of flow and its application as an in vitro model of Alzheimer’s disease. Lab Chip 2014, 15, 141–150.
Majumdar, D.; Gao, Y.; Li, D.; Webb, D.J. Co-culture of neurons and glia in a novel microfluidic platform. J. Neurosci. Methods 2011, 196, 38–44.
Shi, M.; Majumdar, D.; Gao, Y.; Brewer, B.M.; Goodwin, C.R.; McLean, J.A.; Li, D.; Webb, D.J. Glia co-culture with neurons in microfluidic platforms promotes the formation and stabilization of synaptic contacts. Lab Chip 2013, 13, 3008–3021.
Robertson, G.; Bushell, T.J.; Zagnoni, M. Chemically induced synaptic activity between mixed primary hippocampal co-cultures in a microfluidic system. Integr. Biol. 2014, 6, 636–644.
Yang, K.; Park, H.-J.; Han, S.; Lee, J.; Ko, E.; Kim, J.; Lee, J.S.; Yu, J.H.; Song, K.Y.; Cheong, E.; et al. Recapitulation of in vivo-like paracrine signals of human mesenchymal stem cells for functional neuronal differentiation of human neural stem cells in a 3D microfluidic system. Biomaterials 2015, 63, 177–188.
Park, J.; Koito, H.; Li, J.; Han, A. Microfluidic compartmentalized co-culture platform for CNS axon myelination research. Biomed. Microdevices 2009, 11, 1145–1153.
Park, J.; Koito, H.; Li, J.; Han, A. Multi-compartment neuron–glia co-culture platform for localized CNS axon–glia interaction study. Lab Chip 2012, 12, 3296–3304.
Ristola, M.; Sukki, L.; Azevedo, M.M.; Seixas, A.I.; Relvas, J.B.; Narkilahti, S.; Kallio, P. A compartmentalized neuron-oligodendrocyte co-culture device for myelin research: Design, fabrication and functionality testing. J. Micromech. Microeng. 2019, 29, 065009.
Yiannakou, C.; Simitzi, C.; Manousaki, A.; Fotakis, C.; Ranella, A.; Stratakis, E. Cell patterning via laser micro/nano structured silicon surfaces. Biofabrication 2017, 9, 025024.
Ranella, A.; Barberoglou, M.; Bakogianni, S.; Fotakis, C.; Stratakis, E. Tuning cell adhesion by controlling the roughness and wettability of 3D micro/nano silicon structures. Acta Biomater. 2010, 6, 2711–2720.
Angelaki, D.; Kavatzikidou, P.; Fotakis, C.; Stratakis, E.; Ranella, A. Laser-induced topographies enable the spatial patterning of co-cultured peripheral nervous system cells. Mater. Sci. Eng. C 2020, 115, 111144.
Simitzi, C.; Karali, K.; Ranella, A.; Stratakis, E. Controlling the Outgrowth and Functions of Neural Stem Cells: The Effect of Surface Topography. ChemPhysChem 2018, 19, 1143–1163.
Simitzi, C.; Ranella, A.; Stratakis, E. Controlling the morphology and outgrowth of nerve and neuroglial cells: The effect of surface topography. Acta Biomater. 2017, 51, 21–52.
Chen, W.; Shao, Y.; Li, X.; Zhao, G.; Fu, J. Nanotopographical surfaces for stem cell fate control: Engineering mechanobiology from the bottom. Nano Today 2014, 9, 759–784.
Stratakis, E.; Ranella, A.; Fotakis, C. Biomimetic micro/nanostructured functional surfaces for microfluidic and tissue engineering applications. Biomicrofluidics 2011, 5, 013411.
Hoffman-Kim, D.; Mitchel, J.A.; Bellamkonda, R.V. Topography, Cell Response, and Nerve Regeneration. Annu. Rev. Biomed. Eng. 2010, 12, 203–231.
Stevens, M.M.; George, J.H. Exploring and Engineering the Cell Surface Interface. Science 2005, 310, 1135–1138.
Zhu, M.; Zhou, L.; Li, B.; Dawood, M.; Wan, G.; Lai, C.; Cheng, H.; Leong, K.; Rajagopalan, R.; Too, H.; et al. Creation of nanostructures by interference lithography for modulation of cell behavior. Nanoscale 2011, 3, 2723–2729.
Mitra, J.; Jain, S.; Sharma, A.; Basu, B. Patterned growth and differentiation of neural cells on polymer derived carbon substrates with micro/nano structures in vitro. Carbon 2013, 65, 140–155.
Gnavi, S.; Fornasari, B.E.; Tonda-Turo, C.; Laurano, R.; Zanetti, M.; Ciardelli, G.; Geuna, S. The Effect of Electrospun Gelatin Fibers Alignment on Schwann Cell and Axon Behavior and Organization in the Perspective of Artificial Nerve Design. Int. J. Mol. Sci. 2015, 16, 12925–12942.
Radhakrishnan, J.; Kuppuswamy, A.A.; Sethuraman, S.; Subramanian, A. Topographic Cue from Electrospun Scaffolds Regulate Myelin-Related Gene Expressions in Schwann Cells. J. Biomed. Nanotechnol. 2015, 11, 512–521.
Kim, Y.-T.; Haftel, V.K.; Kumar, S.; Bellamkonda, R.V. The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps. Biomaterials 2008, 29, 3117–3127.
Corey, J.M.; Lin, D.Y.; Mycek, K.B.; Chen, Q.; Samuel, S.; Feldman, E.L.; Martin, D.C. Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth. J. Biomed. Mater. Res. Part A 2007, 83A, 636–645.
Cao, H.; Marcy, G.; Goh, E.L.K.; Wang, F.; Wang, J.; Chew, S.Y. The Effects of Nanofiber Topography on Astrocyte Behavior and Gene Silencing Efficiency. Macromol. Biosci. 2012, 12, 666–674.
Mukhatyar, V.J.; Salmerón-Sánchez, M.; Rudra, S.; Mukhopadaya, S.; Barker, T.H.; García, A.J.; Bellamkonda, R.V. Role of fibronectin in topographical guidance of neurite extension on electrospun fibers. Biomaterials 2011, 32, 3958–3968.
Chew, S.Y.; Mi, R.; Hoke, A.; Leong, K.W. The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation. Biomaterials 2008, 29, 653–661.
Xia, H.; Sun, X.; Liu, D.; Zhou, Y.; Zhong, D. Oriented growth of rat Schwann cells on aligned electrospun poly(methyl methacrylate) nanofibers. J. Neurol. Sci. 2016, 369, 88–95.
Johansson, F.; Carlberg, P.; Danielsen, N.; Montelius, L.; Kanje, M. Axonal outgrowth on nano-imprinted patterns. Biomaterials 2006, 27, 1251–1258.
Cecchini, M.; Bumma, G.; Serresi, M.; Beltram, F. PC12 differentiation on biopolymer nanostructures. Nanotechnology 2007, 18, 505103.
Ferrari, A.; Cecchini, M.; Dhawan, A.; Micera, S.; Tonazzini, I.; Stabile, R.; Pisignano, D.; Beltram, F. Nanotopographic Control of Neuronal Polarity. Nano Lett. 2011, 11, 505–511.
Gomez, N.; Lu, Y.; Chen, S.; Schmidt, C.E. Immobilized nerve growth factor and microtopography have distinct effects on polarization versus axon elongation in hippocampal cells in culture. Biomaterials 2007, 28, 271–284.
Goldner, J.S.; Bruder, J.M.; Li, G.; Gazzola, D.; Hoffman-Kim, D. Neurite bridging across micropatterned grooves. Biomaterials 2006, 27, 460–472.
Hsu, S.-H.; Lu, P.S.; Lai, C.-S.; Chen, C.-J. Oriented Schwann cell growth on microgrooved surfaces. Biotechnol. Bioeng. 2005, 92, 579–588.
Miller, C.; Jeftinija, S.; Mallapragada, S. Micropatterned Schwann Cell–Seeded Biodegradable Polymer Substrates Significantly Enhance Neurite Alignment and Outgrowth. Tissue Eng. 2001, 7, 705–715.
Mitchel, J.A.; Hoffman-Kim, D. Cellular Scale Anisotropic Topography Guides Schwann Cell Motility. PLoS ONE 2011, 6, e24316.
Béduer, A.; Vaysse, L.; Flahaut, E.; Seichepine, F.; Loubinoux, I.; Vieu, C. Multi-scale engineering for neuronal cell growth and differentiation. In Microelectronic Engineering; Elsevier: Amsterdam, The Netherlands, 2011; Volume 88, pp. 1668–1671.
Li, G.; Zhao, X.; Zhang, L.; Yang, J.; Cui, W.; Yang, Y.; Zhang, H. Anisotropic ridge/groove microstructure for regulating morphology and biological function of Schwann cells. Appl. Mater. Today 2019, 18, 100468.
Zhang, D.; Wu, S.; Feng, J.; Duan, Y.; Xing, D.; Gao, C. Micropatterned biodegradable polyesters clicked with CQAASIKVAV promote cell alignment, directional migration, and neurite outgrowth. Acta Biomater. 2018, 74, 143–155.
Li, G.; Zhao, X.; Zhao, W.; Zhang, L.; Wang, C.; Jiang, M.; Gu, X.; Yang, Y. Porous chitosan scaffolds with surface micropatterning and inner porosity and their effects on Schwann cells. Biomaterials 2014, 35, 8503–8513.
Schulte, C.; Rodighiero, S.; Cappelluti, M.A.; Puricelli, L.; Maffioli, E.; Borghi, F.; Negri, A.; Sogne, E.; Galluzzi, M.; Piazzoni, C.; et al. Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation. J. Nanobiotechnol. 2016, 14, 18.
Maffioli, E.; Schulte, C.; Nonnis, S.; Scalvini, F.G.; Piazzoni, C.; Lenardi, C.; Negri, A.; Milani, P.; Tedeschi, G. Proteomic Dissection of Nanotopography-Sensitive Mechanotransductive Signaling Hubs that Foster Neuronal Differentiation in PC12 Cells. Front. Cell. Neurosci. 2018, 11, 417.
Schulte, C.; Ripamonti, M.; Maffioli, E.; Cappelluti, M.A.; Nonnis, S.; Puricelli, L.; Lamanna, J.; Piazzoni, C.; Podestà, A.; Lenardi, C.; et al. Scale Invariant Disordered Nanotopography Promotes Hippocampal Neuron Development and Maturation with Involvement of Mechanotransductive Pathways. Front. Cell. Neurosci. 2016, 10, 267.
Terakawa, M. Femtosecond Laser Processing of Biodegradable Polymers. Appl. Sci. 2018, 8, 1123.
Fadeeva, E.; Deiwick, A.; Chichkov, B.; Schlie-Wolter, S. Impact of laser-structured biomaterial interfaces on guided cell responses. Interface Focus 2014, 4, 20130048.
Simitzi, C.; Stratakis, E.; Fotakis, C.; Athanassakis, I.; Ranella, A. Microconical silicon structures influence NGF-induced PC12 cell morphology. J. Tissue Eng. Regen. Med. 2013, 9, 424–434.
Hesari, Z.; Soleimani, M.; Atyabi, F.; Sharifdini, M.; Nadri, S.; Warkiani, M.E.; Zare, M.; Dinarvand, R. A hybrid microfluidic system for regulation of neural differentiation in induced pluripotent stem cells. J. Biomed. Mater. Res. Part A 2016, 104, 1534–1543.
Jeon, K.J.; Park, S.H.; Shin, J.W.; Kang, Y.G.; Hyun, J.-S.; Oh, M.J.; Kim, S.Y.; Shin, J.-W. Combined effects of flow-induced shear stress and micropatterned surface morphology on neuronal differentiation of human mesenchymal stem cells. J. Biosci. Bioeng. 2014, 117, 242–247.
Babaliari, E.; Kavatzikidou, P.; Mitraki, A.; Papaharilaou, Y.; Ranella, A.; Stratakis, E. Combined effect of shear stress and laser-patterned topography on Schwann cell outgrowth: Synergistic or antagonistic? Biomater. Sci. 2020, 9, 1334–1344.
Babaliari, E.; Kavatzikidou, P.; Angelaki, D.; Chaniotaki, L.; Manousaki, A.; Siakouli-Galanopoulou, A.; Ranella, A.; Stratakis, E. Engineering Cell Adhesion and Orientation via Ultrafast Laser Fabricated Microstructured Substrates. Int. J. Mol. Sci. 2018, 19, 2053.

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