Bioelectric Potential in Next-Generation Organoids: Electrical Stimulation to Enhance 3D Structures of the Central Nervous System

Surprisingly, studies translating these findings into neural differentiation of ESCs and iPSCs are very limited. Yamada et al. (2007) described the first study showing mild electrical stimulation of mouse ESCs resulted in increased propensity for neural differentiation. The ESC-derived NPCs generated from electrically stimulated cultures were reported to have a broad capacity for neuronal subtype specification, with greater plasticity than early ESC-derived 2D neuronal differentiations (Yamada et al., 2007). Furthermore, when transplanted into injured spinal cord, NPCs from stimulated mESC-derived cells were more capable of incorporating and forming neural cells than unstimulated controls (Yamada et al., 2007). Previous findings of EF-induced differentiation and fate determination in studies using primary or human NSC lines, were similarly replicated in iPSC-derived neuronal cultures (Stewart et al., 2015; Tomaskovic-Crook et al., 2019; Tomaskovic-Crook et al., 2020; Oh et al., 2021). Specifically, iPSCs were cultured in clusters in suspension within a polymer, followed by neuronal differentiation and electrical stimulation. The resultant neuronal cultures contained more MAP2+ neurons and fewer glial cells than unstimulated counterparts (Tomaskovic-Crook et al., 2020).

Since PSCs are prone to genomic and epigenetic instability, there may be hesitancy to apply exogenous stimuli which could adversely alter pluripotent capacity. However, exogenously stimulated human ESCs and iPSCs have been demonstrated to give rise to all three germ layers, with accelerated or enhanced differentiation capacity (Yamada et al., 2007; Tomaskovic-Crook et al., 2020). In vivo, endogenous EFs are present at stages overlapping with the division of pluripotent embryonic stem cells (Levin et al., 2002). Therefore, EF modulation has the potential to aid morphogenesis from the earliest stage in vitro and effects of EF-modulation prior to the NPC stage should not be overlooked. Further studies on electrical stimulation of PSCs and ESCs are required to understand EF-induced fate determination, which will aid optimisation of PSC differentiation protocols.

In summary of the above, EFs are capable of enhancing neurogenesis both in vitro and in vivo. Despite the overwhelming evidence that endogenous electrical stimulation promotes neurogenesis, researchers are yet to explore the potential of bioelectric cues in guiding PSC-derived CNS organoids. The proof-of-principle studies on mouse and human iPSCs and ESCs support the ability of electrical stimulation to be employed in neural cultures at the PSC stage without hampering differentiation capacity (Yamada et al., 2007; Tomaskovic-Crook et al., 2020). The biological processes regulated by EFs in NSC and NPC cultures, align closely with the processes involved in generating, and optimising, PSC-derived organoids. This includes increases in survival, proliferation, differentiation and maturation of neural cells (Figures 3, 5).

PSC-derived brain organoids represent one of the most advanced in vitro human PSC-derived models. However, several unpredictable variables continue to arise in culture. For example, in the case of unguided organoids, reliance on self-patterned differentiation results in variability from batch-to-batch differentiation and heterogeneity of organoids (Figure 5A). Non-neuroectodermal cells are also identifiable in cerebral organoids (Quadrato et al., 2017). Given that studies unanimously report EF modulation to have an inducing and enhancing effect on neurogenesis, exploiting EFs in organoid cultures could be a legitimate approach to improving the success rate of neurogenesis and reducing differentiation heterogeneity (Figure 5B). Electrical stimulation of ESCs in vitro is reported to bias fate towards NPCs (Yamada et al., 2007). Furthermore, PSCs stimulated at early phases of brain organoid differentiation may offer more robust induction of neuroectodermal, and subsequent neural lineage cells.

Guided protocols offer less batch-to-batch variability. However, the addition of extrinsic factors does not account for existing endogenous levels of molecular cues present in PSCs. PSC lines vary intrinsically and therefore each new protocol needs to be optimised to generate the desirable cytoarchitecture (Strano et al., 2020). This is also a more artificial style of differentiation, which may be suitable for high-throughput studies such as drug screening, but less appropriate for studies of development or fate determination. EF modulation is reported to upregulate neurotrophic factors, particularly in a 3D environment, therefore electrical stimulation of brain organoids could endogenously upregulate key neural-determinant signalling molecules (Figure 5C) (Song et al., 2019). This would offer a valuable opportunity to reduce addition of extrinsic factors in organoid differentiation, both limiting interference in the intrinsic differentiation process and associated costs for laboratories.

In the case of cerebral organoids with multiple regions, the 3D tissues grow to a considerable size, up to 4 mm in diameter, and transport of oxygen and nutrients becomes limited, restricting overall organoid growth and maturation (Sutcliffe and Lancaster, 2019). To counteract this, cultures are maintained on spinning bioreactors or shakers to improve oxygen diffusion in the media and thereby organoid survival (Lancaster et al., 2013; Lancaster and Knoblich, 2014; Qian et al., 2016). However, EF-induced effects on increased cell survival and reduced cell death hold promise for additional implementation in organoid cultures.

To enhance brain organoids functionality, the formation of mature neural networks is key. Both primary and PSC-derived neural cells respond to electrical stimulation with increased neurite outgrowth (Fields et al., 1990; Chang et al., 2011; Pires et al., 2015; Stewart et al., 2015; Tomaskovic-Crook et al., 2019). Electrical stimulation is able to rescue stunted neurite growth in neuregulin-1 knockout primary-derived cortical cells (Zhang et al., 2018). Moreover, when comparing electrical stimulation in a 2D versus 3D environment, whilst 2D cells exhibited increased neurite outgrowth from individual cells, in 3D cells formed aggregates which in turn formed neurite bundles (Zhang et al., 2018). These projected into neighbouring cell aggregates, creating more complex and dense neural networks that better resemble the in vivo brain (Zhang et al., 2018). Neurite density is tightly linked to pathology in psychiatric, neurodegenerative and neurological disease (Saad et al., 2015; Grussu et al., 2017; Kamagata et al., 2017; Rae et al., 2017; Song et al., 2018; Wang et al., 2019; Prem et al., 2020). The utility of EF modulation to increase neurite outgrowth and neural network density therefore provides a wealth of opportunity to model disease, therapeutics and enhanced functionality of brain organoids in vitro.

Retinal organoids have also been a thriving area of research. Diseases of the retina, such as inherited retinal diseases, or age-related macular degeneration are leading causes of blindness. Similarly to neurodegenerative diseases of the brain, retinal degenerations are amenable to therapeutic EF modulation. Electrical stimulation has been applied to the retina via several modes in vivo, either targeting the retina from the front or side of the eye, non-invasively, or alternatively directly contacting the retinal tissue (sub-retinal). Non-invasive EF modulation has been demonstrated to preserve vision in diseased retina in both animal models and clinical trials (Reviewed in Sehic et al., 2016; Sanie-Jahromi et al., 2021). As with deep-brain stimulation, further studies are required to elucidate full mechanisms of action. The human retina differs significantly from that of lower order animals, and human PSC-derived organoids therefore provide a better platform to study EF-induced therapeutic effects on the human retina. In vitro, primary eye-derived retinal glial cells (Müller Glia) were found to respond to electrical stimulation with increased proliferation and expression of cell fate determinant genes (Enayati et al., 2020). Specifically, this was linked to the action of Calcium signalling (Enayati et al., 2020). Several studies have also reported a regenerative response of the output neurons of the retina, the retinal ganglion cells, in response to biomimetic stimulation (Fu et al., 2015; Gordon, 2016). However, the retina contains multiple cell types which are likely to have differing responses to stimulation. Thereby, testing of stimulation within PSC-derived organoids that contain multiple retinal cell subtypes would allow better characterisation of cell-specific phenotypes.

Spinal cord injuries have a devastating impact on quality of life with a huge global disease burden. Therefore, regenerative medicine approaches, or modelling of diseased spinal cords is a compelling objective. Currently, there is no cure for spinal cord damage and injuries are irreversible. The spinal cord contains a niche of NSCs which do not demonstrate multipotency or undergo neurogenesis in vivo (Johansson et al., 1999; Horner et al., 2000; Shihabuddin et al., 2000). However, when isolated and injected into the brain, spinal cord derived-NSCs are capable of producing neurons (Shihabuddin et al., 2000). This led to the hypothesis that the spinal cord microenvironment is masking the stemness of these cells (Becker et al., 2018). Considering the established role of EFs in modifying the tissue microenvironment to induce neurogenesis, electrical stimulation of spinal cord tissue holds great promise. In vivo, clinical application of electrical stimulation for spinal cord injuries has been reported to restore motor activity in paralysed patients (Reviewed in Marquez-Chin and Popovic, 2020; Karamian et al., 2022). Primary spinal cord-derived NSCs, alike to brain-derived NSCs, respond to EFs with directed migratory behaviour (Babona-Pilipos et al., 2011; Meng et al., 2012). This could be harnessed in vivo, to activate and recruit NSCs to the area requiring regeneration. Moreover, electrical stimulation holds the potential to modulate neuroinflammation, which is a major aetiology of neuronal death following spinal cord injury (Rust and Kaiser, 2017; Hellenbrand et al., 2021). Application of EFs in vivo is demonstrated to reduce inflammatory response in the CNS, partly via microglia deactivation (Ayanwuyi et al., 2021; Park et al., 2021). However, studies of the effects of electrical stimulation on 3D spinal cord injury models are minimal. Meng et al.

(2012) describe a protocol to compare EF-induced effects on NPC migration in both a 2D format and 3D ex vivo spinal cord slice platform. Organ slice models present an in vitro system with a microenvironment more representative of in vivo. However, PSC-derived organoids would provide an unlimited resource of spinal cord tissue for study of the complex mechanisms of EF on this unique population of NPCs. Recently, an organ-on-a-chip model using hPSC-derived spinal cord organoids demonstrated electrophysiological function via microelectrode array (MEA) (Ao et al., 2022). Electrical stimulation could easily be implemented in this system to observe neurogenic responses, whilst co-culture with endothelial cells could probe effects on neuroinflammation.

As researchers in the field have recognised the limitations of 2D-based in vitro models, studies have focused on cultures of NPCs in a 2D or neurosphere format, mediated with biomaterials and scaffolds (Figure 4). Bioengineered polymers and scaffolds represent a unique opportunity to explore electrical field modulation in vitro, due to the ability to confer electro-conductive properties on polymer formulations. Organic polymers can be “doped” with anionic biomolecules to confer innate conductivity and biocompatibility. These electroconductive polymers, such as polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT) are readily manufactured with bioprinting. An increasing number of studies utilise conductive polymers to deliver electrical stimulation to NSCs and NPCs in vitro for tissue engineering purposes (Reviewed in Bierman-Duquette et al., 2021). In fact, culturing of NPCs on conductive polymers alone, without exogenous stimulation, may enhance the survival and proliferation, polarisation and axonal projections, or differentiation and maturation of NPCs (Luo et al., 2013; Shin et al., 2017; Wang et al., 2018; Garrudo et al., 2019). Promisingly, this has also been linked to improved electrophysiological functionality (Shin et al., 2017). When compared to bare hydrogels, the addition of PPy or carbon nanotube electroconductive motifs in 3D hydrogel-cultured NPCs caused an increase of calcium channel expression, depolarisation and intracellular calcium influx (Shin et al., 2017). Thus, providing an electroconductive environment alone for NPCs may promote endogenously-driven EF-mediated effects.

Combined with exogenous electrical stimulation, studies report the use of conductive polymer 2D-based platforms to enhance or accelerate neurogenesis, differentiation and neural maturation (Li et al., 2013a; Stewart et al., 2015; Kwon et al., 2021; Sordini et al., 2021). Transcriptomic analysis of PPy-stimulated human NPCs, originally derived from ESCs, identified differential expression of multiple genes implicated in survival and synaptic modelling, and the VEGF-A pathway which plays major roles in the cell cycle and neuroprotection (Georges et al., 2006). Advantageously, the use of biomaterials in in vitro cultures has facilitated transformation from 2D-based electrical stimulation to 3D-based studies by utilising biomaterial-based scaffolds (Reviewed in Bertucci et al., 2019). 3D biomaterial-mediated cultures of both brain-derived and iPSC-derived NPCs have been demonstrated to be numerously advantageous to 2D, increasing cell survival, proliferation and maturation (Cukierman et al., 2001; Georges et al., 2006; Banerjee et al., 2009; Chandrasekaran et al., 2017; Mauri et al., 2018). Therefore, emerging studies are now incorporating 3D-based conductive polymer electrical stimulation platforms for NPC culture (Li et al., 2013a; George et al., 2017; Xu et al., 2018; Zhu et al., 2018; Song et al., 2019; Tomaskovic-Crook et al., 2019). Direct comparison of primary mouse NSCs receiving stimulation on a 2D or 3D graphene-based foam identified increased proliferation and differentiation in the 3D-cultured NSCs (Li et al., 2013a). Human brain-derived NSCs, and then later colonies of iPCS, have been cultured and electrically stimulated in a 3D environment via encapsulation with PEDOT-based hydrogels. (Tomaskovic-Crook et al., 2019; Tomaskovic-Crook et al., 2020). In the encapsulated environment, both the iPSCs and NSC formed clusters and differentiated into neurons.

Culturing of the cells in the 3D environment re-affirmed findings previously established in 2D cultures of human brain-derived NSCs (Stewart et al., 2015). Specifically, in both 2D and 3D formats for NSCs or iPSCs, conductive polymer-mediated electrical stimulation resulted in increased neural fate induction, as demonstrated by increased expression of Beta-III-Actin + neurons (Stewart et al., 2015; Tomaskovic-Crook et al., 2019; Tomaskovic-Crook et al., 2020). In addition, the resultant neurons displayed longer and more branched neurites. However, the ability to study this phenotype in a 3D environment, and better examine the network of neurite extensions within the 3D micro-environment, is far more informative. Interestingly, Song et al. (2019) compared electrical stimulation of hiPSC-derived NPCs using a 2D and 3D platform. Using NPCs embedded in a 2D PPy hydrogel film or 3D PPy tube structures, the authors demonstrate the relation of dimensionality to EF-induced effects. In both a 2D and 3D setting, stimulated NPCs showed differential expression in neurotrophic factors when compared to controls, including heparin binding EGF like growth factor (HBEGF), heat shock protein family member 1 (HSPB1), glial cell derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), and neurotrophin 3 (NTF3). However, there were significant differences between the two. For example, GDNF and BDNF were far more upregulated in the 3D platform (Song et al., 2019). These are important factors regulating brain development, differentiation, survival and maturation (Allen et al., 2013; Popova et al., 2017).

Accordingly, BDNF and GDNF have been implemented in vitro to guide differentiation of brain organoids, including region-specific forebrain (Birey et al., 2017; Qian et al., 2018), midbrain (Kim et al., 2019), cerebellar (Holmes and Heine, 2017), telencephalic (Mariani et al., 2015, 1), as well as cerebral organoids (Quadrato et al., 2017; Watanabe et al., 2017). Specifically, addition of GDNF and BDNF is shown to enhance the maturation of neuronal networks and improve electrophysiological function in forebrain organoids (Qian et al., 2018).

Apart from improved development and maturation for modelling in vitro, another major utility of PSC-derived stimulated cells is for regenerative medicine. Promisingly, ESC-derived and stimulated NPCs cultured on a PPy scaffold were more able to contribute to neural regeneration following implantation in a stroke-model rat brain, than unstimulated NPCs (George et al., 2017). However, for the purpose of better mimicking in vivo morphology and function, organoids represent a far more advanced in vitro model than neurospheres or NPCs, and thus provide a more robust source of cells or tissue for transplantation.

In the move towards “next-generation” organoids, techniques such as micro-patterning of morphogen gradients, 3D bioprinting of substrates and microfluidic devices are being used to improve organoid cultures of various tissues, including lung, kidney and intestine (Reviewed in Garreta et al., 2021; Yi et al., 2021). For brain organoid differentiation, Matrigel was early implemented to aid biomechanical development. Commercially available decellularized matrices, such as Matrigel, are the most easily attained biomaterials for cell biology laboratories (McCrary et al., 2020). These matrices contain cell-derived ECM proteins along with biochemical factors that typically have wide-spread effects on the cell cycle, such as FGF and EGF (Vukicevic et al., 1992; Kleinman and Martin, 2005). However, concentrations of growth factors are unknown and undefined proteins may also be present, which may contribute to batch-to-batch heterogeneity in organoid differentiation. Whilst growth-factor reduced variants are now available, a chemically defined synthetic polymer composition offers an alternative approach to reduce this issue. Currently, few biopolymers have been explored to support PSC-derived brain organoids. Culturing brain organoids with a silk-fibroin based biopolymer offered tuneable biomechanical properties to aid 3D cytoarchitecture formation (Tang-Schomer et al., 2014). In another study, a calcium-alginate hydrogel, formed into hollow fibres, was combined with a microfluidic device to form brain organoids from iPSCs (Zhu et al., 2017). Thus, the device provided a physically controlled environment to sustain organoid development, with the ability to exchange oxygen and nutrients using microfluidics (Zhu et al., 2017).

An alternative to Matrigel is a chemically defined hyaluronan-based hydrogel which has been proven to support induction of human iPSCs into cerebral organoids, but not in long-term culture (Lindborg et al., 2016). Altering the properties of Matrigel, by tuning with an alginate polymer, has been explored for embedding of brain organoids (Cassel de Camps et al., 2022). Encapsulating brain organoids in alginate-tuned Matrigel, which increases stiffness, skewed cell populations towards a more mature neural fate, although in a trade-off for growth capacity. Here, the authors describe differing morphology of brain organoids in response to differing matrix stiffness (Cassel de Camps et al., 2022). This stresses the importance of the biomechanical microenvironment on the self-organisation program of PSC-derived neural organoids. Successful implementation of biopolymers that provide suitable culturing conditions for 3D PSC-derived brain organoids will require optimisation. To date, brain organoids have not yet been combined with conductive polymers such as PPy or PEDOT. However, the addition of alginate or calcium to hydrogels likely confers increased conductive properties, and electrical stimuli could more readily be applied to these formulations which have already been tested in brain organoid differentiations (Kaklamani et al., 2018; Zhu et al., 2018; Cassel de Camps et al., 2022).

Exploiting EF modulation via conductive polymers in brain organoids present several exciting opportunities to generate more advanced in vitro models of the brain and CNS. Markedly, brain-region specific organoid systems don’t offer the opportunity to model multiple regions and interactions between them. To address this, different brain-region organoids have been co-cultured or fused together in “assembloid” approaches (Bagley et al., 2017; Birey et al., 2017; Xiang et al., 2017; Sloan et al., 2018; Xiang et al., 2019; Miura et al., 2022). This can be achieved via several methods, including stepwise assembly of already formed organoids according to neurodevelopmental trajectory, or spontaneous fusion on a shaker. The fused organoids are demonstrated to functionally integrate and have transcriptomes representative of the foetal brain (Xiang et al., 2017; Miura et al., 2022). Application of exogenous EFs during the assembly process could enhance migratory behaviour between regions and promote regional fate determination. This would allow co-culturing at earlier timepoints in development to model early neurodevelopment. However, this is a forced co-culture, rather than spontaneous development from PSC stage. Ideally, to better mimic in vivo CNS development, brain, retina, spinal cord, and olfactory organoids would be generated spontaneously in the same dish together. Currently, the generation of multiple cell types of CNS origin may be achieved with the same media formulations, or within a single structure. For example, cerebral brain organoids contain retinal cells (Lancaster et al., 2013; Quadrato et al., 2017). Moreover, optimisation of culture conditions for cerebral organoids demonstrated development of bilateral optic vesicle structures (Gabriel et al., 2021). These were resemblant of the early embryonic retina in gene expression patterns (Gabriel et al., 2021).

Forebrain structures can also spontaneously arise in PSC-derived retinal organoids cultures (Meyer et al., 2011; Ohlemacher et al., 2015) and retinal-brain assembloids have been generated (Fligor et al., 2021). Therefore, suitable biochemical signals exist to substantiate development of both brain and retina, and development of optimised biomechanical and bioelectric signals could enhance more robust multi-organ organoid formation. Additionally, both brain region and whole-brain organoids lack broader structure, particularly the characteristic cortical folding, or gyrification, present in the human brain in vivo. Recently, a biomaterial scaffold was used to induce a flattened morphology in brain organoids (Rothenbücher et al., 2021). The resulting organoids were size and shape controlled due to a larger surface area for oxygenation, and primitive cortical folding. This could be a beneficial prospect for high throughput screening. However, these manipulations may sway development from the innate development trajectory we are trying to recapitulate in the dish.

In vivo, changes in bioelectric signalling of the brain can cause major structural changes (Smith et al., 2018). The mutation of a voltage-gated sodium channel, SCN3A, causes misfolding of the human cortical brain. Mutated SCN3A causes aberrant ionic current flow during early stages of neurodevelopment (Smith et al., 2018). Therefore, the effect of exogenous bioelectricity application alone to alter organoid structure should not be overlooked.

Another major goal in neural organoid engineering is overcoming the lack of blood supply. For brain organoids, efforts have been made to create vascularisation, via co-culture with endothelial cells, genetic manipulation to express endothelial cell transcription factor, human ETS variant 2 (ETV2), and transplantation of brain organoids into animals to permit in vivo vascularisation (Mansour et al., 2018; Pham et al., 2018; Cakir et al., 2019; Shi et al., 2020). PSC-derived organoid models of the blood-brain-barrier have also been established (Bergmann et al., 2018). Bioelectric signals are reported to influence angiogenesis, the formation of blood vessels within existing vasculature. Electric field-induced angiogenesis has been identified in endothelial cells receiving electrical stimulation (Wei et al., 2020). Blood flow increases in response to electrical fields, and blood flow alone causes a change in electric field potential (Petrofsky et al., 2007; Trivedi et al., 2013). Endothelial progenitor cells, which form vasculature tissue, respond to in vitro application of exogenous EFs with directional migration (Zhao et al., 2012). Moreover, signalling via vascular endothelial growth factor (VEGF), the master regulator of angiogenesis, was found to be upregulated by EF modulation (Zhao et al., 2012). The ability of bioelectric signals to guide neo-angiogenesis is also consistent with the established role of bioelectricity in wound healing. It may therefore be a worthwhile approach to test EF-induced effects on blood vessel formation within vascularised organoids.

The work described here highlights the likely complex interplay of bioelectric signals with biomechanical and biochemical development. Importantly, a major stall in the development of more advanced multi-region or organ CNS organoids is the requirement each tissue has for a specific microenvironment, including biochemical, biomechanical, and likely bioelectrical. Thus, an ultimate approach to generating CNS organoids would utilise multiple microenvironments in the dish. For example, providing regionalised condition suitable signalling factors, and uniquely tailored biomaterials for each tissue, with the application of bioelectricity to enhance endogenously-driven mechanisms of proliferation, migration, neurogenesis and maturation.

Optimisation of polymers and devices to enable organoid development and electrical stimulation needs to be tailored, taking experimental requirements into consideration. Many electrical stimulation devices and conductive polymers are not readily amenable to 3D culturing conditions. For example, varying polymer stiffness and dopant ratio has direct effects on NPC survival and proliferation (Ma et al., 2016; Garrudo et al., 2019). This may limit their applications in the field of cell biology and developmental biology. Culture surface area tends to be very limited, and rigid structures of scaffolds may interrupt the native biomechanical rules of organogenesis. One of the allures of PSC-derived brain organoid differentiation for cell and developmental biologists is the intrinsic patterning and organisation abilities. It would therefore also be worthwhile to consider the development of novel mechanisms to deliver EFs to 3D organoid cultures in situ, including without the use of advanced biomaterials and conductive polymers.

Establishment of suitable delivery platforms for organoids and 3D culture environments will enhance the utility of this technology and facilitate the use of stimulation devices for cell biology laboratories. Electrical charges can be delivered to cells using either direct stimulation, with conductive metal electrodes submerged in culture medium, or salt bridges, such as agar embedded in a glass capillary. This can be facilitated in any device suitable for cell culture. Both microfluidic devices, and multi-well plate electrical stimulation platforms have been developed, with the latter being more suitable for long-term organoid cultures (Figure 4D). Tomaskovic-Crook et al. (2019) developed a microelectrode array-based culture-stimulation platform, with vertical “pilar” electrodes, allowing for more high-throughput screening and generation of 3D neural tissue (Tomaskovic-Crook et al., 2019). Vertical electrodes have also been employed in a custom designed 96-well platform electrical stimulation plate, capable of providing several stimulation parameters instantaneously, permitting high-throughput screening of stimulated cells (Du et al., 2018). This design could aid the optimisation of suitable stimulation parameters for different tissues or developmental stages. Moreover, a pillar, or column-shaped vertical electrode may permit contact with organoid tissue whilst allowing maximal capacity for culture medium. Such approaches have greater utility in advancing guided differentiation protocols, whereby physical interference with cultures is of less concern. A microelectrode array design, with multiple electrodes in each well capable of generating an electric current, could also be utilised to have more precise control over the EFs generated (Figure 3D).

This could probe discrete regions of an organoid, or assembloid, to promote EF-induced effects at particular timepoints according to developmental stage. On the other hand, a simpler electrode circuit design in a standard 6, 12, or 24-well plate format would allow for easier implementation, catering for organoids of varying size and media volume requirements. For example, attachment of electrodes into a plate lid for direct immersion into cell culture media may offer maximal compatibility with in situ suspension culture (Mobini et al., 2016; Mobini et al., 2017). Unguided differentiation protocols may favour a stimulation device with minimal interference to structural composition of the organoid, delivering stimulation directly into the media to best utilise the ions and molecules present. For fabrication of electrodes, platinum offers excellent conductivity and charge injection capacity with minimal corrosive properties (Kim et al., 2007; Xiong et al., 2015; Mobini et al., 2016; Mobini et al., 2017; Leppik et al., 2019). Careful consideration should also be given to the uniformity of EF stimulation in order to generate reproducible data. Whilst cell culture plates are typically circular, many electrode configurations generate a rectangle or square-shaped EF. One study aimed to address this, by 3D printing a polymer into a circular insert to provide a uniform EF (Tsai et al., 2016). In addition, computational modelling of EFs in vitro will be required, specifically to understand electrical current distribution in the 3D organoid environment and with varying media compositions. (Zimmermann et al., 2021).

Ultimately, the choice of electrode material and electrochemical characterisation of the set up will be critical to determine a biologically safe stimulation regime, which delivers the required amount of charge without splitting the water or producing toxic faradaic by-products (Boehler et al., 2020; Mobini et al., 2022).

Recently, a novel 6-well plate device was generated using 3D printing to sustain combinatorial mechanical and electrical stimulation (Cortes et al., 2020). This device was optimised to consider tissue culture conditions, with FDA-approved autoclavable materials able to sustain high humidity incubator environments. Vacuum-controlled flexible membranes provided a programmable mechanic stimulation alongside electrical pulses (Cortes et al., 2020). Whilst this was used to demonstrate improved iPSC-derived cardiac differentiation, a device providing multi-modal stimulation could be similarly implemented for culture of CNS tissues to provide suitable biomechanical cues for in vitro organotypic culture. In another interesting example, in vitro electrical stimulation combined with heat-shock treatment, at 42°C, induces ESC differentiation into definitive endoderm (Koga et al., 2017).

Ideally, the prospect of delivering multiple forms of stimuli, such as thermal, mechanical, optical and electrical, suitably optimised for your tissue of interest, would be an ultimate goal in bioengineering “next-generation” CNS organoids.

Altogether, these studies provide proof-of-concept that devices can be customised for specific applications and tissue culture setups. Regardless, the optimisation of parameters, including signal shape, current, frequency and duration, is a large tissue-specific task which may present a bottleneck. Pre-clinical and clinical studies have indicated parameters capable of generating therapeutic effects in a safe manner (Merrill et al., 2005; Boehler et al., 2020; Krauss et al., 2021; Neudorfer et al., 2021). For example, for deep-brain stimulation, a square shape signal, with biphasic pulses (with an anodal and cathodal phase), seems most suitable (Reviewed in Krauss et al., 2021). Biphasic stimulation is charge-balanced and may also be preferable for in vitro application to NPCs, due to minimising the build-up of oxidation by-products (Iwasa et al., 2019). Secondly, in vivo physiological measurements of EFs in cells and systems of interest should also guide stimulation paradigms. Thirdly, computational modelling and experimental measuring of stimulation experiments should be undertaken. Several analytical or theoretical methods have been developed to aid the calculation of EFs generated by different delivery platforms within a range of culture conditions (Schopf et al., 2016; Abasi et al., 2020; Boehler et al., 2020; Guette-Marquet et al., 2021; Zimmermann et al., 2021). One study aimed to address this by developing a “digital twin” of an electrical stimulation device (Zimmermann et al., 2021). This is a digital representation of the device, (a computational model) which is also able to compute a real set of data, and accordingly respond or adapt the model based on the data (i.e., machine learning). This provides the ability to measure complex electrochemical reactions at the electrode-electrolyte interface.

The capability to record or predict the movement of EFs in vitro would be ideal for applications to organoid cultures, where the complexity of electrical current patterns is likely enhanced by the additional dimensionality of 3D cultures. Alternatively, to offer real-time measurements of experimental conditions, another study developed a dual-function electrical stimulation and recording apparatus. This monitors the electrochemical status of the culture conditions using impedance spectroscopy (Abasi et al., 2020).