Collagens are the main components of the ECM of most soft tissues, including the retina, where they form a fibrillar net in charge of maintaining its structural strength, its attachment to the vitreous and the retinal vasculature (43). The main collagens in the retina secreted by Müller glia are collagen types I-VII and XVIII.

Collagen I is the most common fibril-forming collagen in vertebrates. It is both synthesised by retinal astrocytes in direct contact with RGCs and Müller glia (44), thus being an integral part of the ganglion cell layer (GCL) and the inner limiting membrane (ILM), a membrane mostly comprise of Müller glia end-feet that separates the vitreous from the retina. Upregulation of collagen I has been linked as a response to IOP elevation (45) and as a major component of the glial scar in the wound healing process (46).

Collagen II has been found mainly around retinal blood vessels, as part of the ECM sheath that encapsulates retinal vasculature (47) but also as isolated deposits in the retina, which have been suggested to intervene in the remodelling of the ILM and the collagen intraretinal network by associating with Müller glia via integrin α1β1 and α2β1 (48, 49). Although in the ILM collagen II is mainly generated by the vitreous-originated hyalocytes (50), Müller glia express the transcription factor Sox9, which directly regulates the gene encoding collagen II (51).

Collagen IV is secreted by Müller glia and is the principal ECM component of the ILM of the retina, comprising approximately 60% of its total proteins (45, 52). Due to its structural role, it has been reported to participate in RGCs survival by maintaining ILM integrity, as well as in retinal angiogenesis, where it is part of the collagen network surrounding new vessels (31, 47, 53). It has also been found in the Bruch’s membrane, anchoring the RPE and intervening in its differentiation and wound healing (54, 55).

Collagens VI, VII, and XVIII are secreted by Müller glia in the ILM of the retina which suggest a role in vitreoretinal attachment due to their matrix anchoring function (56). Collagen VI has also been described in retinal blood vessels and collagen VII is found in most retina layers (NFL, GCL, IPL, INL, OPL, and ONL), especially at the optic nerve head. Furthermore, endostatin, proteolytically derived from Collagen XVIII, has been associated with a protection against neovascularization and maintaining RPE integrity (57).

Proteoglycans are composed of a core protein on which long glycosaminoglycan (GAG) chains are attached (58). Depending on which disaccharide conforms said chains, proteoglycans are classified as: hyaluronan (HPGs), chondroitin sulfate (CSPGs), dermatan sulfate (DSPGs), heparan sulfate (HSPGs) and keratan sulfate (KSPGs).

In retinal tissues, CSPGs are secreted primarily from the retinal cells per se, and constitute the ECM, whereas other types of proteoglycans including HSPGs, that also predominantly expressed in the retina, remain bound to the cell membrane and do not contribute to the formation of the ECM (42). Especially during retinal development, CSPGs represent major constituents of the ECM, being found in all plexiform layers of the retina and contributing to neural network formation by creating inhibitory boundaries that direct RGC axons. In the adult CNS, CSPGs are typically upregulated in response to injury or neurodegeneration, blocking axonal and cell migration and regeneration as part of the glial scar, created by Müller glia in the retina (59, 60). In the CNS aggrecan, versican, brevican, neurocan and phosphacan are the most common CSPGs (58).

Versican and aggrecan are released by both neurons and Müller glia and are located in the inner plexiform layer (IPL) and the GCL of the retina (61), as well as the photoreceptor segment (PRS) and the outer plexiform layer (OPL) (62). Versican plays an important role in the attachment of the neural retina to the RPE (63). The main role of aggrecan is to maintain RGC structure in the IPL and to provide support to photoreceptors (64), which is compromised in gliotic conditions, where Müller glia expression of aggrecan is downregulated (65). Both aggrecan and versican are reported to be more prevalent in ageing and degeneration models, probably due to a higher content of GAG chains compared to other CSPGs that may make up an inhibitory barrier (62).

Brevican is secreted by Müller glia and proliferating astrocytes in development and gliosis in the plexiform layers of the retina, where it stabilizes neural synapsis, and in association with RGC somata (61, 66). In physiological conditions its expression is tightly controlled by Müller glia via the miRNA miR-9, which appears to be essential to maintaining retinal integrity. Indeed, in a genetic model where miR-9 regulation was lost, brevican overexpressing Müller glia aggregated, migrated to abnormal locations in the OPL, the outer nuclear layer (ONL), and the IPL and lost cell polarity, leading to a loss of retinal tensile strength and overall retinal disorganization (67).

Neurocan and phosphacan have been suggested to being involved in the establishment and upkeep of retinal lamination (62). During retinal development, both CSPGs are highly associated with the IPL and nerve fiber layer (NFL) (42), where they display an inhibitory effect on RGCs neurite outgrowth (68). In the adult retina, phosphacan expression is restricted to Müller glia (13), where it has been linked to playing a role in the formation of the glial scar during reactive gliosis as a consequence of RGC damage in a genetic glaucomatous model (33).

Fibronectin is a glycoprotein, which N-terminal portion contains a self-assembly domain that allow the formation of a fibronectin matrix. As such, fibronectin functions as a regulator of cellular processes, and directs and maintains tissue organization and ECM composition and remodelling. For example, type I, III, and IV collagens depend on fibronectin for their incorporation into the ECM; it has been observed that although integrins α₁₁β₁ and α₂β₁ are able to promote fibrillogenesis of type I and type III collagens, a collagen network cannot be formed in the abscence of a preconstituted fibronectin matrix and, more so fibronectin is required to maintain the composition of cell–matrix adhesion sites (19). During retinal development, Müller glia and astrocyte-secreted fibronectin has been found in the interstitial matrix of the retina acting as scaffolding, and its disruption has been linked to the dissolution of retinal layers, neuronal cell death and Müller glia activation (69). Fibronectin has also been shown to be key in endothelial radial migration during mouse retinal angiogenesis (70). However, in the adult retina fibronectin seems to be restricted to a structural component of the RPE and retinal vessels (19), as well as act as anchors of Müller glia end-feet to the ILM (71, 72). Interestingly in in vitro studies the fibronectin receptor α5β1 integrin, has been linked to neurite outgrowth of RGCs grown on various ECM substrates as a model for RGC regeneration in the mature retina (73).

Laminins are a family of heterotrimeric glycoproteins, composed of one α, one β and one γ chain, which play an important role in multiple biological processes such as adhesion, differentiation, migration, and resistance to apoptosis via cell membrane receptor signaling (74, 75). Members of the laminin family, mainly γ3, α4 and α5 are major components of the retinal vascular basement membrane and play a functional role in its differentiation and maintenance (76). Laminin has been observed in the ganglion cell layer (GCL) and in close association with Müller glia end-feet in the ILM (73, 77). Müller glia is considered the main producer of laminin in the developing rat retina, and specifically the β2 and γ3 laminin chains are key to the formation and stability of the ILM and retinal lamination during development, while their disruption has been linked to Müller glia disorganization, and consequently RGC apoptosis (53, 77). These two laminin chains have also been connected to photoreceptor synapsis formation and the organization of the outer nuclear layer (ONL) (78). Furthermore, it has been observed that laminin stimulates the motility of Müller glia through the activation of its receptor dystroglycan (79). Additionally, in cultures of both mature RGCs and Müller glia, laminin (plus poli-L-lysine) yielded the greatest survival rate in different substrates (73, 80).

Tenascin-C (tnc) is a glycoprotein expressed by horizontal and amacrine cells in the plexiform layers of the retina, as well as astrocytes in the optic nerve, exhibiting both adhesive and anti-adhesive properties (13, 81). During development of the CNS, tenascin-C regulates neurite outgrowth and glial cell differentiation (82, 83), and it also acts as a barrier in the optic nerve preventing oligodendrocyte precursors from migrating to the retina (84). However, upon retinal damage the expression of tenascin-C by Müller glia is upregulated, which has been linked to contribute to ECM deposition, retinal mechanical stiffness and a gliotic environment, photoreceptor degeneration and neuroinflammation (40, 85–87). This molecule can interact with other ECM components such as fibronectin and proteoglycans such as aggrecan and neurocan (88), and it has been proposed to promote the generation of stable focal adhesions between Müller glia and the ECM (89), being implicated in the morphology of Müller glia. Interestingly, the effect of tenascin-C on Müller glia mediated neurite outgrowth depends on which Tnc-derived fibronectin type III (TNfn) domains they express, with TNfn domain D being required to promote retinal outgrowth by Müller glia (90, 91). Additionally, tenascin-c has been found to increase Müller glia sensitivity to fibroblast growth factor 2 (FGF2), a factor capable of inducing the dedifferentiation of Müller glia in vitro, and tenascin-c knock-out has been reported to impair FGF2 effect on the dedifferentiation state of Müller glia (89).

Matrix metalloproteinases (MMPs) are calcium-dependent endopeptidases that cleave structural motifs of their substrates (e.g., collagens, CSPGs) and have major roles in ECM remodelling, neovascularization and wound healing (94). Depending on their substrate and structural domains, MMPs are classified into: collagenases (MMP-1, −8, −13, −18); gelatinases (MMP-2 and MMP-9); stromelysins (MMP-3, −10, −11); matrilysins (MMP-7, −26); membrane-type matrix metalloproteinases (MT1/2/3/4/5/6-MMP); and unclassified MMPs (MMP-12, −19, −20, −21, −23, −27, −28) (95). Their expression levels in healthy tissue are generally low, and are characterized by an autoinhibitory prodomain that keeps the MMP in an inactive state (96). They are also tightly regulated by the expression of endogenoues tissue inhibitors of metalloproteinases (TIMPs). In the retina, the main MMPs expressed are MMP-2, −3 and-9 (97).

MMP-2 is the main gelatinase in the retina, where it is constitutively expressed by Müller glia (98) and is activated by the release of MT1-MMP (also known as MMP-14) by RGCs. Different functional studies have identified MMP-2 as a key player in axonal regeneration both via resolution of the glial scar and/or proteolysis of molecular cues that guide axon outgrowth. Indeed, in a retinal progenitor cell (RPC) transplantation study, it was observed that neurite outgrowth was dependent on the activation of MMP-2, which cleaves the CSPG neurocan, thus disrupting its inhibitory effect (99). This was further confirmed in a mouse retinal explant model, where MMP-2 was found to act at the growth cone via a β1-integrin-dependent pathway (100). Furthermore, in a mouse optic nerve crush model, it has been proposed that MMP-2 produced by inflammatory myeloid cells activates Müller glia, which in turn produce anti-inflammatory molecules, such as ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6), as well as increased levels of MMP-2 that could activate certain intrinsic growth-inducing pathways in the RGCs (101), although the specific mechanisms are yet to be elucidated. MMP-2 is also capable of degrading collagen IV (102), thus altering the integrity of the ILM.

MMP-3, mainly expressed by the microglia and in loaded vesicles across Müller glia (97). Its expression is tightly connected to retinal vasculature homeostasis, although its involvement is controversial. While some studies report that MMP-3 contributes to blood-retinal barrier (BRB) disruption and neuroinflammation by degrading tight junction proteins and stimulating the activation of cytokines (103, 104), a more recent study showed that upon inflammation MMP-3 expression at the glia limitans (a barrier formed by the end feet of astrocytes and Müller glia that surround the BRB) is upregulated by Müller glia and promotes the expression of astrocyte-specific tight junction proteins, which in turn tighten the glia limitans reducing leukocyte infiltration and adhesion to the retina (105). Moreover, MMP-3 appears to cleave and inactivate the chemokine CCL2 further restricting leukocyte infiltration.

MMP-9, as well as its endogenous inhibitor TIMP-1, is expressed in the nuclei of Müller glia. In healthy retinas, MMP-9 and TIMP-1 form an axis in which TIMP-1 tightly regulates the expression of active MMP-9 (97). MMP-9 primarily cleaves collagen IV and the MMP-9/TIMP-1 axis appears to be key in maintaining normal retinal blood vessel structure and basement membrane integrity (97, 106). However, it has been reported that upon inflammation TIMP-1 tends to aggregate in the cytoplasm of Müller glia, impairing its secretion and reducing TIMP-1 abundance to bind to and inhibit MMP-9 in vitro (107). In this context, the overactivation of MMP-9 plays an important role in the promotion of detachment-induced RGC death by excessive degradation of collagen IV, and indeed, its expression has been observed in apoptosing RGCs (97). Furthermore, MMP-9 have been found to cleave both RPE tight junction proteins (108) and blood vessel tight junction protein ZO-2 (106), contributing to the disruption of the BRB integrity and the progression of chronic inflammation. Interestingly, upon photoreceptor damage in a zebrafish model, MMP-9 has been shown to be required for the survival of regenerated cones by modulating the inflammatory response and negatively regulating Müller glia-derived progenitors overproliferation (109).

Adamalysins are a family of zinc-dependent metallopeptidases conformed by transmembrane-A-disintegrin and metalloproteinases (ADAMs), and secreted A-disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTSs) that play a key role in ECM remodelling (110).

Regarding membrane bound ADAMs, ADAM-10 and ADAM-17 are the best characterized members of this proteinase family due to their ubiquitous expression and involvement in tissue development (111). Both proteinases have been associated with early retinal development via NOTCH1 receptor cleavage, which facilitates neurogenesis by maintaining RPCs in an undifferentiated state, and N-cadherin cleavage, which allows RPC migration and proper retinal lamination (112). Although they are membrane-bound proteases, ADAM-10 and-17 can be released to the ECM by ADAM-15 and-8, respectively, where they are capable of cleaving collagen IV and fibronectin (111). In the developed retina, they are mainly expressed by Müller glia and have been reported to be upregulated in a neovascularization and pro-inflammatory context (113). In the outer limiting membrane (OLM), a tight regulation of ADAM-10 by the Wnt signaling modulator SFRP1, also expressed by Müller glia helps maintain OLM integrity and photoreceptor homeostasis (114). ADAM-10 is also expressed by RGCs and is required for axon guidance and formation of the optic projection (115).

ADAMTSs can be classified by their substrate as: procollagen N-propeptidases, proteoglycanases, fibrillin/fribronectin-associated peptidases and angiogenesis regulators (116). In the retina, Müller glia constitutively express ADAMTS-1, −2, −4, −5, and −13 (117). Especially ADAMTS-1 and-4, and to a lesser extent also ADAMTS-5, have been found to promote synaptogenesis by cleaving the CSPGs neurocan and phosphacan, which inhibit RGCs neurite outgrowth (68, 118, 119), while ADAMTS-4 thrombospondin repeats may also induce neurite extension independent of CSPG cleavage (120). Meanwhile, ADAMTS-2 has been reported to cleave fibrillar collagen precursos (type I-III), thus contributing to the maturation and formation of collagen fibers within the ECM (121). As for ADAMTS-13, it cleaves the von Willebrand factor, a proteoglycan that participates in platelet aggregation (122); interestingly, while by itself promotes angiogenesis, in the presence of vascular endothelial growth factor (VEGF), a potent vascular permeability and angiogenesis factor, ADAMTS-13 become anti-angiogenic (123).

Collagen is the main component of the ECM of most soft tissues. Thanks to its natural origin, collagen gels are biocompatible and biodegradable and present low cytotoxicity, while promoting cell adhesion, proliferation and motility (187) via α₁β₁ and α₂β₁ integrin-binding sites (188). Furthermore, collagen hydrogels are highly porous allowing free diffusion of ions and molecules to support cell growth through the polymer matrix (189).

Since collagen type I comprises 90% of fibrillar collagens (190), it is used preferably to prepare hydrogels, although collagen type IV can also be used. Collagen used as polymer material is usually derived from solubilized type I collagen obtained from rat tail tendon and gelation of these solutions is achieved by raising the temperature and the pH to initiate collagen fibril self-assembly (191). Interestingly collagen hydrogel stiffness can be tuned by increasing the pH (above pH 10.0) while decreasing polymerization temperature; this changes augment fibril packaging thus increasing overall matrix stiffness (192, 193).

Collagen hydrogels have been sparsely used for culturing retinal cells such as RPE cells in an AMD model (194), while their use in Müller glia culture has been mostly restricted to contractility essays (195). In cultures of cerebellar granule neuron-glial spheroids, collagen type I, but not type IV, hydrogels have been shown to stimulate astrocyte proliferation to a certain extent but fail to induce neuritogenesis and Calcium signaling activity (196), which suggest that collagen hydrogels might be a poor candidate for CNS tissue mimicking and thus to culture retinal cells. Indeed, previous studies in which the influence of ECM in the neurite branching and regrowth of RGCs was addressed and only a discrete effect was found (73).

In general, collagen hydrogels present some important drawbacks including limited long-term stability and batch-to-batch variability. These hydrogels display low stiffness, requiring extensive chemical crosslinking to achieve stiffnesses higher than 1 kPa (191). This crosslinking leads to significant matrix contraction and altered degradability, restricting the culturing time. Furthermore, cells cultured may alter the intrinsic mechanical properties of the hydrogel (197).

Matrigel is a commercial preparation of ECM components obtained from Engelbreth–Holm–Swarm (EHS) mouse sarcoma tumours; it is composed primarily of laminin, collagen type IV, and entactin, with various other constituents including proteoglycans, such as perlecan, metalloproteinases and growth factors (including the TGFβ family) (198). Matrigel is considered a reconstituted basement membrane that undergoes gelation at temperatures in the range 22–37°C, when entactin acts as a crosslinker between the laminin and collagen IV to create the hydrogel (199).

Matrigel ability to maintain self-renewal and pluripotency due to its tumorigenic origin makes it a good candidate for culturing stem cells. Indeed, as a thin gel coating, Matrigel has been used to culture and expand human pluripotent stem cells (hPSCs) (200) and neuronal stem cells (201), while thicker coatings have been used to investigate angiogenesis (202).

Matrigel has been used as substrate to develop retinal organoids, where different groups have achieved the differentiation of human induced Pluripotent Stem Cells into Müller glia and further expand the obtained cells; these cells were functional and expressed typical Müller glia markers (203, 204), which suggested that Matrigel’s enriched ECM composition favoured Müller glia differentiation. Likewise, Matrigel was also used to differentiate mouse Embryonic Stem Cells into RGC-like cells (205) and human Embryonic Stem Cells into RGCs and Müller glia precursors (206).

Due to its origin, Matrigel is not well-defined and present high batch-to-batch variability and xenogenic contaminants (191, 199), which leads to a significant level of uncertainty to experimental results. Furthermore, Matrigel stiffness is relatively low (around 400 Pa) and due to its temperature sensitivity drops to lower stiffnesses if it is not kept at 37°C during experimentation (207); although it can be tuned by increasing overall protein concentration, this leads to alterations in its biological functionality.

Alginate is a polysaccharide typically obtained from brown algae that presents biocompatibility and low toxicity for biomedical applications. It is formed by α-L-guluronate (G) and β-D-mannuronate (M) residues, and hydrogels are formed by the interaction of G residues with different divalent cations (mainly calcium and barium) (11).

The stiffness of the gel depends on the overall G section (208) and the cation used for gelation (209). Since mammalian cells lack receptors for alginate, and alginate gels show low protein adsorption, their coupling to specific peptides is required for proper cellular adhesion (210). The most commonly used peptide is arginine-glycine-aspartic acid (RGD), which concentration required for cell adhesion and growth is cell-type dependent.

RGD-alginate gels have been found to display compatibility with CNS tissue, promoting the outgrowth of regenerating axons and the elongation of astrocytic processes of the spinal cord (211) and favouring the expansion of hipoccampus neural progenitor cells in vitro (212). These hydrogels have also been shown to promote the generation of retinal progenitor cells from iPSC-and hESC-derived embryoid bodies (213). Although there are no studies of Müller glia grown on alginate hydrogels, this biomaterial has been used in brain astrocyte activation studies where different alginate concentrations elicited a different stiffness-mediated response (214, 215); as Müller glia take up the mechanosensor function in the retina that astrocyte carry out in the brain and both cell types are glial cells of the CNS these results suggest that alginate hydrogels could be a good alternative to study the effect of substrate stiffness on Müller glia.

However, as in other natural hydrogels, alginate shows poor long-term stability, and batch-to-batch variability in their degradation and mechanical properties (216). Furthermore, native alginate gels display low mechanical integrity, as a result of their swelling behaviour in the aqueous culture environment, requiring the incorporation of a compatible reinforcing agent (e.g., cellulose, polylactic acid) within the alginate structure (217).

Polyacrylamide is a cyto-compatible, bioinert material, formed by the synthetic polymer acrylamide and its crosslinker (bis-acrylamide), gelation being activated by ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). Polyacrylamide gels exhibit a strong homogeneity in surface topography, mechanical properties, and coating density, displaying high reproducibility (219).

Polyacrylamide hydrogels stiffness can be easily tuned over several orders of magnitude within the physiological range, by varying the concentration of the constituents (219, 220). Although the gel elastic modulus increases with increasing cross-link concentration, the formation of highly cross-linked clusters add heterogeneity to the network structure, leading to an inflection point, after which gel stiffness decreases under high cross-linking conditions (221). Therefore, crosslinking should be tightly controlled when fabricating the hydrogel. Due to their bioinert characteristics, polyacrylamide hydrogels need to be functionalised by the tethering of ECM components in order to support cell adhesion, including poly-L-lysine (222), laminin (14) or collagen I (223), among others. This tethering is UV-dependent.

One of the major disadvantages observed in polyacrylamide gels is the change in porosity dependent on the gel formulation (224). However, Wen et al. (225) have shown that porosity and stiffness can be independently tuned. Furthermore, when they cultured mesenchymal stem cells on polyacrylamide gels of different stiffnesses and porosities they observed that differentiation into either adipocytes or osteocytes did not depend on porosity or protein tethering but only on the substrate stiffness, which makes polyacrylamide gels good candidates for studying substrate stiffness as an isolated parameter.

Polyacrylamide gels are ideal for CNS mimicking. Cultures of adult neural stem cells on soft polyacrylamide gels promote neural expansion while stiffer ones favoured the appearance of glial cells of astrocytic phenotype (226, 227). Likewise, the survival and proliferation of oligodendrocyte progenitor cells has been shown to be modulated by substrate stiffness (228).

Thanks to polyacrylamide capacity to tune its rigidity over a wide range of stiffnesses, they can be used to model pathologic contexts, where stiffness is heightened. For example, a stiff polyacrylamide matrix led to impaired myosin activity and branching inhibition in an oligodendrocyte differentiation study (229).

In the study of retinal fibrotic diseases, Müller glia cultured on polyacrylamide gels showed that on stiff substrates there is an upregulation of stress fibers and reorganization of the cytoeskeletal integrity, changes in gene regulation and a morphologic transition to a myofibroblast-like phenotype (39, 40). This is due to stiffening-stimulated YAP activation by the TGFβ1-PI3K/Akt pathway (15) and the expression of CTFG, implicated in the gliotic process (17).

Polyacrylamide gels manufacturing parameters such as UV activation intensity and exposure time, gel thickness, and acrylamide/bis-acrylamide concentration should be tightly controlled to achieve accurate, reproducible results (219).

Polydimethylsiloxane (PDMS) is a silicon-containing polymeric material. PDMS hydrogels are formed by an elastomer base (PDMS oligomers with vinyl-terminated groups, a platinum catalyst and dimethylvinylated/trimethylated silica) and a curing agent (dimethyl methylhydrogen siloxane and tetramethyl tetravinyl cyclotetrasiloxane) (11). PDMS hydrogels display biocompatibility, high oxygen permeability, thermal stability, a high protein binding capability for cell expansion and tunable mechanical properties (230, 231). Substrate stiffness can be easily tuned by curing temperature and time and changing the base-to-curing agent ratio (232), where an increased ratio correlates to augmented stiffness.

The elastic modulus of PDMS is typically in the order of megapascals (233). However different strategies, i.e., altering the geometrical organization by fabricating microposts of different heights (234), have been developed to decrease the effective stiffness of PDMS hydrogels, making them apt for mimicking soft tissues such as the CNS.

PDMS hydrogels have also been used to study mechanotransduction in different glial cells of the CNS. Thanks to their elastic properties they have been applied as a stretchable material to test the effects of tensile forces on oligodendrocyte progenitor cells (235), which have been shown to favour cell expansion and differentiation. In other studies, tuning their rigidity have been used to study the effect of mechanical stiffness on cortical astrocyte activation and progression of astrogliosis (236), which could be extrapolated to Müller glia. Also for Schwann cell proliferation and transcription of basal lamina receptor genes in axonal sorting and myelination which is promoted by the substrate stiffness activation of the YAP/TAZ complex (237). Likewise, PDMS have been used as substrate for the developing of retinal organoids, where mESCs were differentiated mainly into photoreceptors, but also RGCs, amacrine cells, horizontal cells, bipolar cells and Müller glia (238, 239).

However, PDMS being a synthetic material, is bioinert and require the adsorption of charge enhancers, e.g., poly-L-lysine (236), and/or the binding of adhesive ECM proteins, e.g., laminin, fibronectin (240, 241), for proper cell attachment and integrin signaling. Thus, the nature of the generated interactions and the coating density are critical for cell adhesion and spreading.

PDMS hydrogels are preferentially used when looking for larger structural stiffness, while in starting studies of compliant substrates polyacrylamide gels are usually chosen (219).

Polyethylene glycol (PEG) is a hydrophilic polymer of ethylene oxide that display a variety of end groups (e.g., alcohol, methyl ether, amine, N-hydroxysuccinimidyl (NHS) ester) which are cross-linked for hydrogel formation (242).

PEG is hydrophilic, present biocompatibility and low toxicity, is non-adhesive toward proteins and cells and its stiffness can be tuned by varying the polymer concentration. As with other synthetic hydrogels PEG is bioinert and as such requires functionalization by ECM protein (e.g., vitronectin, fibronectin) binding for cell attachment (243–245). PEG hydrogels can mimic soft substrates; however, softer gels (~1 kPa) are somewhat limited in the amount of protein that can be coupled to the gel substrate, which can pose a problem for cells with weak adhesion forces (246). Alternatively, PEG cross-linking to natural-based materials, such as gelatin, and natural-based gelatin cross-linking has been shown to enhance the biophysical properties, including gelation time, and biocompatibility of PEG hydrogels improving cell long-term viability (247).

Although less frequently than polyacrylamide or PDMS gels, PEG hydrogels have been utilized to study neural cell biomechanical behaviour, increasing PEG composition and consequential increase in stiffness has been associated with the expansion of neural progenitor cell cultures and differentiation to glial cells (248). PEG hydrogels have also been fabricated for retinal organoid culturing, their intrinsic inertness and hydrophilicity allowing for a good diffusion of metabolites as well as factors, favouring organoid development and colony formation in this type of essay (238). Although PEG hydrogels (fused with hyaluronan acid) have been proved to be non-citotoxic to Müller glia in vitro (249), no further studies have been carried out.

Interestingly, RGCs grown on PEG hydrogels of different stiffnesses, coupled with poly-L-lysine, attached better in hydrogels with an stiffness range of 3.8–5.7 kPa than on expected optimal stiffness hydrogels (0.9–1.8 kPa), due to the ratio of free amines to hydroxyls (250), which suggests that stiffness cannot be measured as an isolated parameter in PEG hydrogels.