Coating Materials for Neural Stem/Progenitor Cell Culture and Differentiation

Abstract

Neural stem/progenitor cells (NSPCs) have a potential to treat various neurological diseases, such as Parkinson's Disease, Alzheimer's Disease, and Spinal Cord Injury. However, the limitation of NSPC sources and the difficulty to maintain their stemness or to differentiate them into specific therapeutic cells are the main hurdles for clinical research and application. Thus, for obtaining a therapeutically relevant number of NSPCs in vitro, it is important to understand factors regulating their behaviors and to establish a protocol for stable NSPC proliferation and differentiation. Coating materials for cell culture, such as Matrigel, laminin, collagen, and other coating materials, can significantly affect NSPC characteristics. This article provides a review of coating materials for NSPC culturing in both two dimensions and three dimensions, and their functions in NSPC proliferation and differentiation, and presents a useful guide to select coating materials for researchers.

Introduction

Neural stem/progenitor cells (NSPCs) are characterized by their potencies of self-renewal and differentiation into neurons, astrocytes, and oligodendrocytes. Based on these characteristics, NSPCs hold an immense promise for regenerative therapy of neurological diseases, such as Parkinson's disease [1], Alzheimer's disease, [2], and spinal cord injury [3].

However, the limited sources of NSPCs always remain an obstacle for clinical research and application, although some reprogramming studies have shown many promising results by transforming somatic cells into NSPCs by transcription factors [4,5]. Besides, the stemness maintenance of stem cells and efficient differentiation are also difficult to manipulate, making it hard to obtain sufficient amounts of therapeutic cells for clinical transplantation [6,7]. Therefore, it is necessary to understand the relevant factors associated with NSPC proliferation and differentiation in vitro.

NSPCs can be cultured in suspension or adhesion conditions. Compared to suspension culture, adherent cells with coating materials are easier to be nourished by plentiful nutrients from the culture medium [8], thus proliferating faster [9], although they have more tendency to differentiate when they are in monolayer [10,11].

Coating materials are generally components from extracellular matrix (ECM), such as collagen, laminin, fibronectin, or ECM analog like Matrigel, or poly-amino acids. They resemble cell microenvironment in vitro that deeply interferes with cell behaviors, acting mainly through the integrin family of receptors. It has been shown that coating materials can significantly influence cell proliferation, differentiation, viability, mobility, and many other behaviors [12 –14]. Besides two-dimensional (2D) culture, coating such materials onto scaffold or using them to form hydrogel for three-dimensional (3D) culture has become an alternative and prominent method to improve cell culture in vitro [15 –17].

However, it is important to point out that different coating materials normally have different effects on cell culture [12,18,19]. In contrast, the same coating material may have diverse influences on different cell types [20,21]. Therefore, we provide this review to summarize the different effects of coating materials on NSPC proliferation and differentiation, which may help researchers to establish practical protocols to obtain enough therapeutic cells for clinical application.

Laminin

Laminin (Table 1, No. 1.1) is one of the major components of ECM proteins that have shown significant functions in neural development [22]. Based on its importance for neural cells, it has been widely used for NSPC culture. Hall et al. added laminin into defined neural stem cell (NSC) medium with a concentration of 10 μg/mL and found that laminin promoted the proliferation/survival of human NSCs in an integrin β1-dependent manner, rather than improving the undifferentiated state [23]. In other studies, laminin has also been shown to accelerate NSPC proliferation as a coating material on 2D surface [24,25]. When coprecipitated within biomimetic apatite, laminin supports the proliferation and adhesion of NSCs without cytotoxicity for up to 28 days [26]. Three-dimensional cultures can present better physiological relevance than 2D cultures. Ortinau et al. have shown that a laminin-functionalized scaffold is essential for setting up 3D-growth patterns of NSPCs, and it has shown better neuronal differentiation and survival rate than 2D cultures [27]. Khayyatan et al. have also proved that laminin coating of collagen scaffolds enhanced the proliferation and infiltration of human induced pluripotent stem cell-derived neural progenitors in a dose-dependent manner [28].

Table 1.

Coating Materials for Neural Stem/Progenitor Cell Culture and Differentiation

No.	Material	Source	2D/3D culture	Function	Other features	Ref.
1.1	Laminin	Mouse sarcoma or human placenta	2D/3D	Promotes NSPC adhesion, proliferation, and neuronal oligodendrocyte differentiation	More effective than others in many cases; comparatively expensive	[24,25, 28 –34]
1.2	Collagen	Human body/recombinant	3D	Provides a compatible environment for NSPC culture in 3D	Usually bound with other materials like growth factors	[39,42 –57]
1.3	Fibronectin	Plasma/human fibroblasts/recombinant	2D/3D	Maintain the attachment, viability, migration, and expansion of NSPCs, Enhances differentiation into neurons and astrocytes	Used as transplantation matrix, can be added to culture media to increase cell survival	[33,58 –68]
1.4	Matrigel	Mouse sarcoma	2D/3D	Promotes neural generation and improves NSPC expansion and differentiation	Not well-defined components; similar effects with laminin on NSPCs regarding proliferation, differentiation, and migration	[25,69 –80]
1.5	Fibrin	Human plasma	3D	Supports proliferation, differentiation, and integration of NSPCs	Widely used as a vehicle for NSPC transplantation, degrades rapidly in vivo	[34,81 –87]
1.6	α-Polylysine	Synthetic amino acid	2D/3D	Improves cell attachment and enhances the adsorption of ECM to culture substrates	Positively charged, may be toxic to cells	[60,61,90 –94]
1.7	Poly-l-Ornithine	Synthetic amino acid	2D/3D	Promotes proliferation and differentiation into neuron and oligodendrocyte of NSPCs	Positively charged; may be toxic to cells; better than PLL and fibronectin	[25,56,93 –99,137]
1.8	Agarose	Seaweed	2D/3D	Maintains NSCs or neurosphere structures unattached	Supports micro patterning, shape, and structure control of NSCs	[10,11,100 –102]
1.9	PHA	Microorganism-like bacteria on fermentation	3D	Enhances proliferation and differentiation of NSCs	Biological polyester, can be used for spine fusion	[103 –105]
1.10	Hyaluronic acid	Human body (present in neural and epithelial tissues)	3D	Enhances survival differentiation into neurons and oligodendrocytes of NSCs	Usually combined with other materials when forming scaffold	[106 –113]
1.11	Graphene	Rocks or graphite	3D	keeps NSC proliferation state with good electrical properties; enables a wide neural network after the differentiation	An ideal scaffold for functional neuronal differentiation and NSC preservation	[34,114 –117]
1.12	Alginate	Brown microalgae	3D	Favors the viability, expansion, and differentiation of NSC	Injectable biomaterial; Usually combined with other materials when forming scaffold	[10,118 –126]
1.13	Chitosan	Shells of shrimp and other sea crustaceans	3D	Promotes adhesion, expansion, and neuronal or astrocyte differentiation of NSC	Preserves the electrical properties of cells	[56,113,127 –129]
1.14	Dextran	Microbial origin	3D	Facilitates neurogenesis, proliferation, and cell adhesion of NSPCs	A good coating material for nanoparticles in research	[130 –134]

2D, two-dimensional; 3D, three-dimensional; ECM, extracellular matrix; NSC, neural stem cell; NSPCs, neural stem/progenitor cells; PHA, polyhydroxyalkanoates; PLL, poly-l-lysine.

Moreover, laminin has been shown to enhance NSPC differentiation. Balasubramaniyan et al. found that ∼20% of embryonic mouse NSCs differentiated into neurons in laminin-coated wells even without neuronal cell inducers like brain derived neurotrophic factor (BDNF) or glial cell line–derived neurotrophic factor [29]. In addition, laminin-coated polymeric substrates also promoted differentiation of adult NSCs into functional neurons and glia [30] and enhanced adult rat hippocampal progenitor cell neuronal differentiation in 3D when cocultured with astrocytes [31]. Specifically, coimmobilizing laminin with oligohistidine-fused neurotrophic factor on a culture surface increased the efficacy of NSPC differentiation into dopaminergic neurons. Interestingly this scaffold even eliminated the differentiation to oligodendrocytes and glial cells [32].

Furthermore, NSCs could give more neuronal cells and oligodendrocyte precursors when cultured in a methylcellulose scaffold functionalized with laminin than methylcellulose alone as a control [33]. In another study, a scaffold formed by a combination of fibrin, hyaluronic acid (HA), and laminin gave the most robust neurite outgrowth from differentiated neurons of human NSPCs compared to scaffolds without laminin. Laminin can interact with the cells through several integrins (α7β1, α6β1, α6β4, and α3β1) [34]. It has also been reported that laminin leads to more oligogliogenesis from human umbilical cord blood cell–derived neural stem like cells (HUCB-NSCs) than other coating materials like collagen, fibronectin, or poly-l-lysine (PLL) [35].

Meanwhile, with the presence of astrocyte-derived soluble factors, a combination of entactin, collagen, and laminin has led to much more differentiated oligodendrocytes derived from adult rat hippocampal progenitor cells compared to laminin alone, but with a concomitant decrease of proliferating cells [36]. However, another study showed that the generation of oligodendrocytes was reduced on laminin likely through integrin α6 when NSPCs were provided with 10% static equibiaxial stretch, whereas no such effects were found on fibronectin [37], indicating the complicated mechanisms for NSPC lineage selection. Indeed, it has been reported that laminin-containing matrices can improve NSPC proliferation and differentiation better than other substrates like poly-l-ornithine (PLO) or fibronectin [25].

Interestingly, the effects of enhancing NSPC proliferation and differentiation of full-length laminin could also be achieved by a designed short laminin peptide, which may be used for further experiments to avoid purification from many ECM molecules [38]. Besides these functions, laminin has also been shown to promote neural cell induction from human embryonic stem cells (ESCs) and bone mesenchymal stromal cells [39,40]. Moreover, Tate et al. have reported that a laminin-based scaffold enhances NSC transplantation into the injured brain [41].

Collagen

Collagen (Table 1, No. 1.2) is the most abundant protein in ECM and our body, which plays many important roles, such as improving skin health [42] and relieving joint pain [43]. In addition, collagen (mostly collagen type I) has been wildly used in culturing NSPCs. Although collagen has been used for coating substrates in 2D cell culture in some research, it has mostly been used for building a stable cell carrier, which provides a cell-compatible environment. Han et al. showed that culturing NSCs in 3D collagen scaffolds gave much higher clone formation efficiency and less differentiation than on 2D surfaces by activating the expression of REDD1 thereby blocking the activity of mTOR [44]. Wang et al. also found that NPCs cultured on collagen nanofibers yielded a 30% increase in proliferation, compared to collagen-coated 2D culture [45].

Moreover, when it was compared with suspension culture, NSCs cultured in 3D collagen scaffolds showed better proliferation, differentiation, and process outgrowth [46]. Interestingly, NSPCs cultured in a cell–collagen–bioreactor culture system containing a rotating wall vessel can be maintained for up to 9 weeks and show enhanced growth and differentiation, compared to static cultures [47]. Although the suitability of 3D collagen culture system for NSPC proliferation and differentiation has been demonstrated [48,49], collagen in some cases needs to be bound with other materials like growth factors or other proteins to further promote its functions.

When collagen was fused with basic fibroblast growth factor (bFGF), NSPCs cultured in such a microenvironment showed a significantly higher number than those of bFGF without collagen as immobilizer [50]. Similar to bFGF, NSCs cultured in an epidermal growth factor (EGF)-bound collagen hydrogel also presented more living cells than in EGF-immobilized collagen-coated 2D culture [51]. Thinking of the function of BDNF and neurotrophin-3 (NT-3), a BDNF-NT-3-incorporated collagen scaffold has also shown to enhance the proliferation and differentiation of NSCs, and the growth factors were steadily released from collagen gels for 10 days [52]. Likewise, similar results have been found with BDNF-loaded plasma-collagen matrix [53].

When collagen hydrogel was combined with a laminin-derived peptide by recombinant DNA technology, the adhesion and viability of the cultured neurosphere-forming cells were promoted, indicating the suitable environments for neural cell culture [54]. Native collagen from animal sources is normally with minimal controlled cell–material interactions. Luckily, it has been shown that recombinant collagen also has similar effects to native collagen regarding NSPC adhesion, proliferation, and differentiation, which indicates its potential to replace native collagen as a coating material or a cell carrier [55]. In contrast, gelatin, which is also called hydrolyzed collagen, has also been used as a coating material for 2D cell culture [39] or a component to form scaffolds to promote neural generation [56,57].

Fibronectin

Fibronectin (Table 1, No. 1.3) is also a component of ECM that is involved in many biological processes, such as cell adhesion, differentiation, and migration. It was found that medium containing insulin, selenium, transferrin, and fibronectin preferentially supported neural cell generation from mouse ESCs [58]. Fibronectin could slightly improve the attachment of neurospheres onto substrate surface when added into the culture medium, and a combination of fibronectin and EGF could maintain NSPC viability and enhance their proliferation compared to serum-containing medium [59].

However, it was reported that HUCB-NSCs were attached more strongly when fibronectin was used to coat the subtract, while cell morphology was more flattened but cells proliferated less than on PLL-coated surfaces [60,61]. Fibronectin increases NSC adhesion and migration mainly through its arginine-glycine-aspartic acid (RGD) peptide, which binds to the α5β1 integrin receptor on the stem cells [62]. Although it has been shown that human NSPCs can grow as adherent culture on fibronectin-coated flasks [63], NSCs grow faster on recombinant fibronectin than as neurospheres and maintain their stem cell nature or multipotentiality [64].

Fibronectin has also been reported to trigger NPC migration and differentiation [65]. Li et al. found that a combination of serum fraction, poly (ethylene-co-vinyl alcohol) biomaterial, and fibronectin could increase neuronal differentiation rate over 85% of migrated cells from NSPCs [66], while another study showed that NSCs plated on fibronectin exhibited a less mature neuronal profile than laminin-1 [33]. Moreover, in a nanotopographical manipulation study, Yang et al. reported that fibronectin-coated polymer substrates with diverse nanoscale shapes and dimensions enhanced human NSC differentiation into neurons and astrocytes [67]. Interestingly, like laminin, the RGD peptide contained in fibronectin has also been proved equally effective in improving rat and human NSC adhesion, viability, and growth [62].

Furthermore, it was shown that fibronectin could also be used to form a biocompatible scaffold when bound to collagen to promote NSC transplantation into injured brain and increase NSC survival and migration, although it was not as good as laminin-based scaffolds [41,68].

Matrigel

Matrigel (Table 1, No. 1.4) is a gelatinous protein mixture extracted from Engelbreth-Holm-Swarm mouse sarcoma cells. The major components of Matrigel are laminin, collagen IV, heparan sulfate proteoglycans, and entactin. In addition, it contains growth factors like bFGF, EGF, and transforming growth factor beta. Matrigel has been commonly used for stem cell culture because it can retain their stemness [69]. Due to its richness in laminin, Matrigel normally shows similar effects with laminin on NSCs on cellular proliferation, differentiation, and migration, which is usually better than fibronectin, PLO, and gelatin [25,70,71]. Interestingly, it has been shown that 0.02% Matrigel was sufficient to promote NSC expansion and differentiation, which had no notable difference with 20 μg/mL laminin and higher Matrigel concentrations (0.05%–1%) [70].

Moreover, Ma et al. reported that human ESCs gave more neural progenitor and neuronal generation and better neurite outgrowth on laminin or Matrigel substrates than on Poly-D-Lysine (PDL), fibronectin, or type I collagen [72]. Furthermore, Matrigel is compatible with 3D culture as a scaffold with high concentration and is generally used to support the growth of organoids, including brain organoids [73,74]. It has been shown that NSCs proliferate and differentiate better in Matrigel scaffold than collagen I scaffold and peptide hydrogels for culture periods of the first 1–2 weeks. Nevertheless, cell viability was decreased under long-term studies, which might be due to the consumption of the growth factors [75].

Remarkably, a gelated (1%–50%) Matrigel has been reported to prevent neuronal differentiation and promote astrocytic differentiation of NSCs. This effect was noticed when 50% growth factor-reduced Matrigel (GFR-Matrigel) was used, while 100% GFR-Matrigel mostly enhanced neural differentiation [76,77]. GFR-Matrigel is a variant of normal Matrigel that has been modified to reduce the level of growth factors. GFR-Matrigel has also been reported to be more supportive to NPC survival than collagen IV, PLO/laminin, and PuraMatrix (a fully synthetic resorbable hydrogel) and promote the survival and neuronal differentiation of the grafted NPCs [78]. GFR-Matrigel also has more robust effects on neural differentiation than collagen-1, gelatin, and HA hydrogel [77].

Surprisingly, it has been reported that when retinoic acid and/or sonic hedgehog are provided, Matrigel stimulated dopaminergic neuron differentiation from ESCs, while collagen-1 scaffolds significantly enhance motor neuron formation [77]. Besides, microscale 3D Matrigel has also been used for high-throughput identification of factors promoting neuronal differentiation of hNPCs [79]. In contrast, Geltrex, which is considered to be an analog of Matrigel, has also been used recently for NSC expansion and astrocyte differentiation [80 –82], although the number of citations of Geltrex is much less compared with Matrigel.

Fibrin

Fibrin (Table 1, No. 1.5) is an insoluble protein formed from fibrinogen during blood clotting. It has been seldom used as a coating material for NSPC culture in 2D condition, binding integrins, αVβ1, and α5β1 on the cells [34]. Li et al. found that neurospheres but not single NSCs could adhere to fibrin and show better attachment and process extension than those on alveolate collagen and HA, although cells died off following the rapid degradation of fibrin [83].

In contrast, fibrin has been widely used as a vehicle for NSPC transplantation due to its mechanical properties, inherent biocompatibility, and susceptibility to proteolytic degradation [84 –89]. It is usually embedded with growth factors to support cell proliferation, differentiation, and integration [88 –90]. It is worthy to note that fibrin degrades rapidly in vivo [91]. Therefore, a serine protease inhibitor, aprotinin, or combination with other materials like HA has been used to delay gel degradation and to improve cell viability and proliferation [34,84].

α-Polylysine

α-Polylysine (Table 1, No. 1.6) is a positively charged synthetic lysine polymer that is classically used to improve cell attachment and survival in vitro. PDL and PLL are two types of α-polylysine as a coating material. Different from ECM proteins, it promotes cell adhesion in a nonreceptor mediated and nonspecific manner by binding to negatively charged cell membrane [92]. It has been reported that HUCB-NSCs on microarray platforms coated with PLL were rounder and more stable and showed more nondifferentiated cells than that with fibronectin [60,61]. Besides, the deprivation of serum could significantly enhance cell attachment to PLL pattern but not fibronectin [60].

However, it is worth to note that α-polylysine is only biocompatible at low concentrations and may be toxic to the cells as it could enhance the host inflammatory responses [93,94]. It can also enhance the adsorption of ECM to culture substrates and therefore improve their bioactivity as a coating material. The combination of α-polylysine and ECM proteins like laminin and fibronectin is widely used for culturing or studying NSPCs [72,95]. Interestingly, α-polylysine can also be incorporated into poly(ethylene glycol) or poly(ethylene glycol) diacrylate to form 3D hydrogels to improve cell attachment and promote their bioactivity, acting as delivery vehicles for NSPCs [96,97].

Poly-l-Ornithine

Similar to α-polylysine, PLO (Table 1, No. 1.7) is also a positively charged synthetic polymer of L-ornithine, which is widely used as a culturing substrate for studying NSPCs [25,95,98]. Moreover, it can be used with ECM proteins like laminin and fibronectin to support their function [99,100]. Interestingly, it has been reported that PLO can significantly promote NSPC proliferation and induce preferred differentiation of neurons and oligodendrocytes from NSPCs, compared with PLL and fibronectin, although there is not much difference on NSPC viability [101]. Furthermore, PLO improves NSPC migration through activating α-Actinins 4 to enhance filopodia formation, presenting it as a superior candidate for cell transplantation than PLL and fibronectin [56].

Agarose

Agarose (Table 1, No. 1.8) is a popular polysaccharide derived from seaweeds used largely in electrophoresis systems [102]. This purified form of agar has served its purpose in terms of bioengineering by micropatterning shape and structure control of NSCs with efficient cortical neuronal differentiation. Moreover, one of the biggest challenges encountered by the stem cell researchers is to maintain the neurosphere structures unattached as it has a genuine tendency for adherence resulting in differentiation [10,11]. Interestingly, different from other coating materials, agarose coatings can help in the anti-attachment of NSCs and neural spheres and also assist with long-term cultures for scientific studies [103].

Custom-made biomaterial of agarose with chitosan and alginate facilitates a 3D model of neural tissue to explore the expansion and functional differentiation into neurons [10,104]. Nevertheless, a similar scaffold of agarose has even demonstrated the critical cortical layer differentiation with micropatterning for basic optical research and as a good drug-screening platform [10].

Polyhydroxyalkanoates

As NSCs offer unlimited sources for differentiated brain cells, maintaining its multipotency and stemness is critically important. Compared to 2D coatings and extracellular matrices, 3D scaffolds are ideal in supporting the maintenance and differentiation of NSCs [105]. For that reason, polyhydroxyalkanoates (PHA) (Table 1, No. 1.9) evolve as a scaffold for NSCs and neurospheres.

It was first tested on NSC in 2010 by Xu et al. in which they screened different PHA based copolymers. They discovered that a copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate can mimic the ECM supporting rat-NSC expansion [106]. The material resulted in good in situ attachment and differentiation with synaptogenesis. Three years later, Xie et al. hypothesized that PHA, including preserved cell-binding motifs, such as RGD or IKVAV, could further improve the growth and differentiation. However, results indicated that this material could only improve the growth and differentiation temporarily [107].

Hyaluronic Acid

HA (Table 1, No. 1.10) is a biomaterial highly used in composite for NSPC research. Research conducted by Jose and Krishnan in 2010 revealed that a niche consisting of HA considerably promoted the survival of NSC, although its mechanisms are not well known [108]. HA was widely used with hydrogel for the homing and differentiation studies for NSPCs. Ventral brain precursor cells photo encapsulated on HA hydrogels remained stable and differentiated into mature dopaminergic neurons, suggesting its application in dopaminergic neuron transplants in Parkinson's disease [109].

To extend the preservation of NSC and to enhance angiogenesis after transplantation, mechanical properties of the HA hydrogels were optimized with recombinant BDNF and vascular endothelial growth factor (VEGF) embedded [110]. The modified scaffold released the cytokines in a controlled way ensuring the prolonged survival of NSCs [110]. Similar biomaterial was manufactured by incorporating HA into self-polymerizing hydrogels, which served as ideal scaffolds for transplanted cells for stroke treatment [111]. The modified HA hydrogel with MMP cross-linkers served as a platform for the adhesion of structural motifs as promoted vascularization [111,112]. Moreover, HA enriched materials enhanced the differentiation into neurons and oligodendrocytes over adherent cultures [113].

Furthermore, PLL combined HA hydrogels inhibited the differentiation of NSPCs and provided an ECM similar for brain niche [112]. Clinical compliant scaffolds that mimic the properties of brain tissue were developed by combining HA with laminin and salmon fibrin, which provided extensive preservation of NSCs [34]. Conductive scaffolds with chitosan and gelatin bridged by HA nanoparticles can support NSC expansion without compromising the quality of stemness [114].

Graphene

Graphene (Table 1, No. 1.11) was a not-so-popular material experimented for neural tissue engineering until 2013. Li et al. reported that 3D graphene foams could be used to keep the NSC proliferation state with good electrical properties from their neuronal differentiation [115]. Nanoparticles structured to graphene supported strong adhesive properties for NSC enabling a wide neural network after the differentiation [116]. A later study identified that graphene interferes with the stromal cell-derived factor 1 alpha or CXC chemokine receptor 4 pathway to enhance cell adhesion [117]. However, 2D graphene structures failed to favor NSC differentiation [118]. Furthermore, the stiffness of graphene also contributed widely, with the soft graphene reporting less capability for NSC expansion [119]. Overall, graphene demonstrates to be a potential scaffold for functional neuronal differentiation and NSC preservation.

Alginate

Alginate (Table 1, No. 1.12), a compound found within the cell walls of brown algae, is another biomaterial that can support NSPCs. Unmodified alginate can be ionically cross-linked to divalent cations, which makes scaffold fabrication and cell encapsulation simple, nontoxic, and efficient [120]. Encapsulation of alginate, with and without composite materials, favored the viability, expansion, and differentiation of NSC [120]. Manipulating the elasticity of alginate hydrogels could alter the proliferation and neural differentiation of NSC [121].

Similar to different brain cortical layers, 3D micropatterning of alginate-agarose mixture demonstrated excellent scaffolds mimicking physiological conditions, serving as a model for in vitro studies [10]. Such scaffolds on small platforms can not only be utilized for basic research but also to support high-throughput screening. Meli et al. have developed a 3D microarray platform for human NSC growth and differentiation, enabling the high-throughput screening of small molecules and nanomaterials for brain research [122]. Similar to HA-based extracellular matrices, alginate scaffolds permit a controlled release of growth factors and cytokine, preserving the stemness for longer time with a potential to facilitate plasticity and regeneration of brain cells to damaged areas, including spinal cord [123].

Moreover, alginate-based material encapsulated with NSCs has been tested in a spinal cord injury model [124]. The neurological outcomes on the subjects were considerably improved after a few weeks of transplantation with reduced apoptosis and inflammation at the site of the transplantation. Furthermore, a chitosan-based alginate mixture on hydrogel platform was formulated and tested as injectable biomaterials for NSC delivery [125]. The material mimicked in vivo brain conditions and possessed the ability to self-heal under normal physiological environment.

Moreover, the mixture can be fine-tuned to support the proliferation, migration, and differentiation of NSC with controlled release of cytokines [125]. Furthermore, the material provided an excellent scalable platform for pluripotent stem cell–derived NSPCs than 2D cultures. NSCs cultured on the platform yielded 250 times more than the conventional method followed for expansion [126]. In addition, alginate composites with fibronectin or HA and hydrogels can promote neuronal and oligodendrocyte differentiation, respectively [127,128].

Chitosan

Chitosan (Table 1, No. 1.13), which is a versatile polysaccharide made from the chitin shells of crustaceans, is yet another promising biomaterial for the NSCs and neuronal differentiation, although the interaction of chitosan with NSCs is unclear. The pioneering research conducted by Leipzig et al. in 2010 demonstrated that NSPCs have the best expandability and differentiation capability on chitosan materials with immobilized interferon-gamma, supplemented with neural growth factors [129]. Later, a composite film of chitosan containing PLL was identified to exhibit excellent adhesive and regenerative properties for NSCs, neurons, and glial cells [130]. However, the aforementioned custom scaffolds supported short-term survival only. Furthermore, chitosan combined with hydrogels and immobilized azide supported long-term survival of NSCs and neurons without compromising the functionality of the cells [131].

Three-dimensional matrix of poly 3,4-ethylenedioxythiophene covered with HA nanoparticles cross-linked onto gelatin containing chitosan scaffolds maintained the NSC expandability with robust Ki-67 expression [114]. In addition, the matrix also preserved the electrical properties and conducting nature of the NSC-derived neurons, with robust expression of neuronal markers [56]. Nevertheless, the method also supported efficient astrocyte differentiation in the scaffold. Briefly, conductive scaffolds are required to provide a stable microenvironment to maintain the functionality of NSCs in vivo.

Dextran

Nanomaterials are widely used as tracers and cell trackers in biomedical research [132]. An interesting aspect in tissue engineering is to coat the nanoparticles with biomaterials to increase their potential. The most widely and the first coating material used for nanoparticles is dextran (Table 1, No. 1.14), which promotes internalization [133,134]. Moreover, the properties of nanomaterials change with different coating materials. To ensure cell treatments are effective and successful, it is crucial to track the survival, migration, and differentiation of transplanted cells for their capabilities of reconstructing brain function [135].

In terms of NSCs, coatings like PDL and laminin are important and can serve as important coating materials for neuro-nanoparticles. Dextran coating of laminin and PDL improved the properties of the material to facilitate neurogenesis [136]. Another study involving the cryogelation of dextran has demonstrated cell adhesion and proliferation in niche-like structures. The cells differentiated to give rise to mature neuronal networks with minimal rejection and inflammatory response in rat brains after transplantation [136].

Discussion

NSPCs are a promising candidate for treating a range of neurological diseases. Coating materials that are a component of cell microenvironment in vitro play significant roles in determining NSPC behaviors. Therefore, it is necessary to establish a stable and effective cell culture protocol with suitable coating materials that promote NSPC proliferation and differentiation. Among the materials mentioned above, laminin, Matrigel, α-polylysine, and PLO are more commonly used for culturing NSPCs than the others, especially in 2D culture condition. Moreover, laminin seems to be the most effective coating material in terms of improving NSPC proliferation and differentiation in many cases followed by Matrigel and then α-polylysine and PLO. However, it still needs to be noted that each material has different features that may make it more or less favorable in some conditions. Although laminin is quite effective, it normally needs to combine with other materials such as PDL to enhance its adsorption to the culture substrates. Moreover, laminin made from mouse sarcomas is not suitable for clinical application and recombinant laminin or the one from the human is comparatively expensive. Matrigel is also generated from mouse sarcoma cells. More importantly, the components of Matrigel are not well defined, which may cause variable experimental results [69]. α-polylysine and PLO can be synthesized by basic polycondensation reactions, which make them comparatively cheap and easy to obtain. However, it is important to note that α-polylysine and PLO can be toxic to the cells [93,94,137]; therefore, it is necessary to find a suitable concentration for coating.

In contrast, 3D cell culture systems are increasingly being used for cell research as they present a better cell microenvironment mimicking in vivo condition compared to 2D culture [138]. Most of 3D cell culture systems are based on scaffolds, which are normally formed by ECM proteins or polymers that support cell behaviors (attachment, proliferation, migration, etc.). In addition to the biomaterials discussed in this review, some other polymers have also been reported to enhance NSPC proliferation or/and differentiation such as poly(lactide-co-glycolide), polyurea [138,139]. Moreover, scaffolds can also be formed by combinations of different materials to improve their biological functions [34,54,96].

Another type of 3D cell culture system is based on scaffold-free methods such as rotating bioreactors or micropatterned surfaces in which coating materials have also shown significant roles [47,60]. However, it is worthy to note that, although 3D culture condition has shown significant benefits to cells compared to 2D culture, there are still some disadvantages. For example, cells in 3D conditions are not as homogeneous as in 2D culture as they are not equally exposed to media components like growth factors. Moreover, the complexity of 3D material properties such as physical, chemical, and mechanical properties may lead to unintended results. Therefore, it is important to have a better understanding of the properties of these materials and to build up a suitable condition for culturing cells to get the desired consequences in each case.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by D&R Pharmaceutics China, UniSA Venture, UniSA International Research Tuition Scholarship (IRTS) and China Scholarship Council (CSC) Scholarship to Donghui Liu.

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