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Creating Scaffolds for 3D Neuronal Tissue Models
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ScienceDirect www.sciencedirect.com IRBM 39 (2018) 4–8
General Review
Creating Scaffolds for 3D Neuronal Tissue Models C. Bouyer ∗ , F. Padilla LabTAU, INSERM, Centre Léon Bérard, Université Lyon 1, Univ Lyon, F-69003, Lyon, France Received 5 January 2017; received in revised form 8 November 2017; accepted 9 November 2017 Available online 22 November 2017
Graphical abstract
Abstract Background: Many human tissues are comprised of multilayered tissue structures in which spatial organization is essential to provide biological tissue functions. Methods: Recently, strategies such as 3D bioprinting, photolithography, 3D auto-assembly, molding or bulk acoustic cells manipulation have been developed to fabricate layered tissue mimics. These methods have broad applications in tissue engineering for the bioengineering of multilayered structures, and for the fundamental understanding of many microphysiological and pathological process like cell differentiation. Each method relies on the use of a special scaffold structure made of natural or artificially created biopolymers, and of specific cell types. In the field of neuronal 3D constructs fabrication, where ex-vivo samples are difficult to get, different strategies have been developed going from rat neurons culture to embryonic stem cells culture and differentiation into neurons after their encapsulation in 3D scaffolds. Conclusion: All those possibilities open new perspectives for the future, aiming to the development of different types of tissues composed of different multilayer structures. © 2017 AGBM. Published by Elsevier Masson SAS. All rights reserved. Keywords: Tissue engineering; Scaffolds; Neural constructs 3 dimensional tissue structure
1. Introduction
* Corresponding author.
E-mail address: [email protected] (C. Bouyer). https://doi.org/10.1016/j.irbm.2017.11.001 1959-0318/© 2017 AGBM. Published by Elsevier Masson SAS. All rights reserved.
Many human tissues are comprised of multilayered tissue structures in which spatial organization is essential to provide biological tissues functions. Cell culture in 2 dimensions cannot provide all information about a native organ, as 2D cells
C. Bouyer, F. Padilla / IRBM 39 (2018) 4–8
patterning cannot represent the complex cells behavior in a 3D environment such as 3D cell–cell interactions. In many situations, establishment of artificial 3D tissue structures is necessary. For example, after large bone fracture, tissue graft may be needed to bring substrate and growth substances necessary to trigger bone fracture healing. Tissue engineering was born in this context, to provide replacement tissues, such as bone graft, where resources are limited or unavailable. During the last decades tissue engineering was expanded to many applications as many improvements were achieved in the available technologies [1]. Internal organs such as bladder [2] or pancreas [3], and more superficial tissues such as skin [4,5], bone/cartilage [6], or corneal [7] can now be engineered. Tissue engineering was then expanded to obtain 3D tissue model, to study tissue functions or development. In that aim, controlling cell–cell interaction, by controlling their patterning and their 3D microenvironment is required. Several technologies are being proposed to control cell patterning within scaffolds, such as micro-scale hydrogels (microgel) assembly, scaffold seeding or bioprinting. To control cell fate in 3 dimensions, a 3D microenvironment has to be chosen to fit with the desired application. In order to obtain construct with similar properties than native tissues and within which cells can grow and/or differentiate, scaffold properties such as porosity, size, geometry or elasticity are important parameters to control. In that aim, new biomaterials were developed to support cells viability and functions [8–10], with the possibility to obtain specific mechanical properties similar to those of native tissues. Such developments have led to the development of neuronal tissue engineering. The needs arose from the lack of preclinical models mimicking the architectural and developmental complexity of native tissue. Post-mortem functional tissues are essential ex-vivo models to study mental illnesses [11, 12], neurotransmitters mechanism of action [13], the dynamic of proteins/ions signaling [14], or to screen novel therapeutics agents [12], but they are of limited access. At the premise of neuronal tissue engineering, rat neurons were used and embedded in 3D scaffolds. More recently, scaffolds were developed to support Embryonic Stem Cells (ESC) cultures and the use of stem cells became more and more popular [4,7] as it allows bioengineering with cells from different origins, such as blastocyst, skin or muscle, according to the applications [15]. The aim of this review is to provide a rapid overview of methods developed to model neuronal tissue, and in particular cortical tissue. The generation of such cortical tissue models must respect the spatial organization of native tissue, which in first approximation can be viewed as a multilayered structure for which spatial organization and inter-layers cellular communications are essential to sustain their biological functions. To model this complex multilayers structures, the bioengineering of multilayered constructs with tunable layers thickness and cell types is necessary. Four different strategies can be implemented to generate 3D neuronal tissue structures. Each one is based on the use of specific cell types, physical strategies to spatially organized cells
5
and different scaffold types to embed them. We will discuss in this review the advantages and limits of those methods. 2. Methods 2.1. Two different approaches to engineer complex tissues To obtain 3D tissue models, bioengineering tools are needed to offer the ability to control the complex patterns and high cell density of native tissue, such as brain, liver or cardiac tissues [16]. In this context two different approaches can be used: “top-down” and “bottom-up” approaches [17]. These two approaches have advantages and shortcomings, which prevent them yet to be widely used and commercialized. In the top-down approach, cells are directly seeded into a fully made porous scaffold of predefined shape to form a tissue construct. Cells are expected to populate the scaffold and create the appropriate extracellular matrix and microarchitecture often with the aid of a bioreactor that furnish the set of stimuli required for an optimal cellular viability [18]. This approach is associated with some limitations such as the difficulty to shape scaffolds, difficulty to build large tissue constructs and low cell diffusion, which result in a low cell density and non-uniform distribution within the scaffold [17]. In the bottom-up approach, small cell laden modules such as spheroids or cell sheets are used as building blocks to create a larger construct [19]. In that case, the final construct organization and composition is tunable by the organization of the micro-architectural units. With this approach, the cell distribution is more likely controlled, but an external and often complex technology is needed to assemble the building units. New challenges in tissue engineering are to combine both approaches to enable a better cell seeding within a complex scaffold organization to create native-like tissue patterns. 2.2. Cell culture methods The random distribution of neurons in culture dishes does not sufficiently recapitulate the 3D aspects of neural connectivity or the microenvironment of the brain. Initially, hanging drop method was used to create 3D structures, but couldn’t represent cerebral tissue structure. Now, new technologies are trying to find a way to obtain specific scaffolds shape. 2.2.1. Conventional 3D culture Hanging drop is a technology for the fabrication of cells spheroids to create cells aggregation in 3D, to mimic in vivo tumor models on the lab-on-chip scale [20], for different applications like drug screening [21]. The methods are based on the use of either specific well plates or of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. The advantage of this technology is that it is possible to control the spheroid sizes, it is relatively easy to implement and it is low cost. 2.2.2. Technologies to obtain specific 3D structure shapes New techniques were developed to individually create single gels and to assemble blocks together [2,9,17,22,23]. Conven-
6
C. Bouyer, F. Padilla / IRBM 39 (2018) 4–8
tional 3D bioengineering technologies to create specific patterns, such as micro-scale hydrogels (microgel) assembly [9, 24–26], scaffold seeding [27] or bioprinting [28] cannot control cell–cell interactions or cell-packing density, which is essential in cerebral tissue modeling. In fact, spatial organization and interconnection of neural cells in and between these layers determine functions of cerebral cortex such as memory, language and consciousness. To address these issues, new scaffold-free strategies have been developed [29–32], mostly using cell spheroids to build highly-packed tissue construct elements. These methods suffer from long assembly time that prohibits building large, millimeter-scale constructs, and cannot reach an assembly precision of a few hundred micrometers required to pattern constructs such as neural constructs. These assembly methods rely on different physical methods such as magnetic [9,24], Faraday waves at liquid–air interface [33], acoustic or 3D printer. Organ printing [28,31], using printer, enables to build large scale scaffold with cells. This allows a precise scaffold patterns droplet by droplet but cannot go down the micro-scale size precision. In the other hand, magnetic assembly [9,24] allows spatial positioning of building blocks with cells. This technique is a contactless strategy but requires multistep procedure and a paramagnetic solution, which is not fully cell-friendly. On the other hand, acoustic strategy [33] enables a contactless, biocompatible manipulation to assemble cells following different patterns simply by tuning the acoustic frequency. Commonly used techniques generate large construct that mostly need to be cultivated in a bioreactor for proper media and CO2 diffusion within the sample [34,35]. 2.3. Stem-cell self-assembly to create cerebral organoids Several multistep strategies have been developed to create 6-layered tissue surrogates of the cerebral cortex, either by selfassembly or bioprinting approaches. In 2013, Lancaster et al. [34] generated a cerebral cortex-like tissue by first generating spheroids by self-assembly of embryonic stem cells (ESCs). Human pluripotent stem cells (hPSCs) spheroids were obtain by culture in hESC media and then in neural induction media for 11 days. Then, droplets were cultured in matrigel droplet and transferred into a spinning bioreactor [34]. In each step of the process, a specific media was attributed. When cultured in a spinning bioreactor in the presence of a differentiation medium and retinoic acid, cells were spontaneously organized to form a cortex-like structure. Neuronal differentiation of cerebral organoids revealed heterogeneous neuron subtypes production and auto-organization, with the presence of abundant radial glial stem cells during the development. Histological analysis of the differentiated structure revealed an important degree of molecular heterogeneity and regionalization that recapitulates the different brain regions. In 2015, in a study using human cortical spheroids obtained from pluripotent stem cells, a laminated cerebral cortex-like
structure could be generated and used for the study of normal and abnormal corticogenesis [36]. Cerebral organoids were formed from deep and superficial cortical layers neurons that were characterized by immunostaining observation. Different neuronal subtypes were found and located at different depths of the organoids, characteristic of both deep and superficial cerebral cortex. After complete neuronal maturation (after 180 days of culture), a synaptogenesis analysis was conducted to show the visualized individual synapses activity. The developed system provides a platform where deep and superficial neurons layers are present, surrounded by astrocytes to support neurons activity. 2.4. Scaffold-based bioengineering techniques of brain models Another alternative technique to bioengineer 3D neuronal multilayer structures is bioprinting approaches. For instance, primary rodent neurons were encapsulated in a layer-by-layer assembly approach in concentric 3D donut-shaped constructs, made from composite hydrogel and silk protein [37]. Micromolding has also been employed to embed primary neurons in 3D networks in collagen [38]. The generated 3D neural surrogate tissues displayed the characteristic cell density, interlayer neurite connections and layer thickness of the cortex, as well as biochemical and electrophysiological functional responses. Although these methods recapitulate some features of native neuronal tissues, such as 3D neuronal network design with interlayer neurite formation, neural cell density and layer thickness, and have shown biochemical and electrophysiological outcomes, some limitations hamper their wide applications in biological laboratories. Multistep operations required in both layer-by-layer assembly and micro-molding approaches may require special expertise and are time consuming. To address critical challenges encountered in self-assembly and bioprinting approaches in brain bioengineering, we developed a unique, single step bio-acoustic levitational (BAL) assembly technology [39]. With this cell-friendly technology, based on near field standing waves, direct assembly of multilayer 3D geometries within one single hydrogel construct was performed. Cells are trapped in a stable polymer network by controlling the gelation dynamics of fibrin, which can be achieved through optimization of the prepolymer concentrations. A final cohesive multilayer gel made of ESC was formed with cortex-like mechanical properties. The made gel was then cultured in neuronal differentiation media to obtain neuronal cell–cell interactions. Combination of available bioengineering methods could also be used to create different type of tissues. As an example, we are now combining bulk acoustic levitation and layer-by-layer assembly to bioengineer multilayered tissues with micrometerscale control over biological and structural features of native layered tissues. We can assemble, within minutes and with minimal acoustic pressure, multilayered constructs with multiple cell types in fibrin hydrogels (Fig. 1) [40,41].
C. Bouyer, F. Padilla / IRBM 39 (2018) 4–8
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Fig. 1. Hydrogel construct formed of multi-layered heterogeneous cell types. A layer-by-layer assembly was performed to assembly live cells expressing eGFP (green), mCherry (red) and non-transfected HeLa cell lines. mCherry cells are seen on bottom of the left picture and on the right picture. eGFP cells are on the top of left picture and on the middle picture. Scale bar is 250 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3. Conclusion and perspectives
Acknowledgements
Novel techniques were recently developed to enable 3D cell culture such as hanging drop (self-assembly) or bio-printing of building blocks (controlled assembly). Compared to the previous studies where rat primary neurons were used [37,38] to create a cortex-like structure with microfluidic chip [38] or hydrogels [37], the use of ESC is very promising but very challenging [34]. Recent development in scaffolds biochemistry and manipulation now enables the encapsulation of ESC in 3D structures, their culture and their differentiation into neuronal lineage. Cerebral organoids are to date the method reproducing with the most accuracy the complexity of the biological processes during brain development, but require a lot of expertise. Controlled assembly represent an additional set of methods that, while limited in biological relevance compared to cerebral organoids, can provide easy and rapid way to generate model systems tailored to answer specific biological questions, such as cell migration during development or neurotoxicology of novel drugs. We are confident that these novel methods that can be carried out in a simple, rapid and biocompatible way in a fluidic environment, will find their place as widespread strategy for bottom-up tissue engineering and regenerative medicine where multilayered cell organization is required, to assemble cells alone in high densities to mimic the natural density and cell– cell interactions observed in the physiological complexity, such cortical tissue, but also for skin, cardiac or breast tissue. Last years, many developments in the field were made, developing new cell-friendly methods for cells manipulation. Moreover new technologies tend to allow the use of multiple cell types allowing to create multilayered tissue structures more and more representative of human tissues. In a near future, we can anticipate that novel technologies will be developed for fabrication of many types of organized structures.
This work was supported by the LabEx DEVweCAN (ANR-10-LABX-0061) of the University of Lyon, within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). The authors would also like to acknowledge the Fulbright program and the “Commission Franco-Américaine” for their support to C. Bouyer through a Fulbright award supporting her work at the BAMM Labs as a visiting Ph.D. student.
Conflict of interest statement No conflict of interest has to be declared.
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[13] Calabresia P, Centonzea D, Gubellinib P, Marfia GA, Pisania A, Sancesarioa G, et al. Synaptic transmission in the striatum: from plasticity to neurodegeneration. Prog Neurobiol 2000;61(3):231–65. [14] Müller W, Connor JA. Ca2+ signalling in postsynaptic dendrites and spines of mammalian neurons in brain slice. J Physiol 1992;86:57–66. [15] Lutolf MP, Gilbert PM, Blau HM. Designing materials to direct stem-cell fate. Nature 2009;462:433–41. [16] Guven S, Chen P, Inci F, Tasoglu S, Erkmen B, Demirci U. Multiscale assembly for tissue engineering and regenerative medicine. Trends Biotechnol 2015;33(5):269–79. [17] Tiruvannamalai-Annamalai R, Randall Armant D, Matthew HWT. A glycosaminoglycan based, modular tissue scaffold system for rapid assembly of perfusable, high cell density, engineered tissues. PLoS ONE 2014;9(1):1–15. [18] Urciuolo F, Imparato G, Guaccio A, Mele B, Netti PA. Novel strategies to engineering biological tissue in vitro. Methods Mol Biol 2012;811:223–44. [19] Nichol JW, Khademhosseini A. Modular tissue engineering: engineering biological tissues from the bottom up. Soft Matter 2009;5:1312–9. [20] Wu Huei-Wen, Hsiao Yi-Hsing, Chen Chih-Chen, Yet Shaw-Fang, Hsu Chia-Hsien. A PDMS-based microfluidic hanging drop chip for embryoid body formation. Molecules 2016;21(7):882–93. [21] Neto AI, Correia CR, Oliveira MB, Rial-Hermida MI, Alvarez-Lorenzo C, Reis RL, et al. A novel hanging spherical drop system for the generation of cellular spheroids and high throughput combinatorial drug screening. Biomater Sci 2015;3(4):581–5. [22] Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol 2014;32:773–85. [23] Tasoglu S, Diller E, Guven S, Sitti M, Demirci U. Untethered microrobotic coding of three-dimensional material composition. Nat Commun 2014;5:3124. [24] Xu F, Wu CA, Rengarajan V, Finley TD, Keles HO, Sung Y, et al. Threedimensional magnetic assembly of microscale hydrogels. Adv Mater 2011;23(37):4254–60. [25] Gurkan UA, Fan Y, Xu F, Erkmen B, Urkac ES, Parlakgul G, et al. Simple precision creation of digitally specified, spatially heterogeneous, engineered tissue architectures. Adv Mater 2013;25(8):1192–8. [26] Xu F, Finley TD, Turkaydin M, Sung YR, Gurkan UA, Yavuz AS, et al. The assembly of cell-encapsulating microscale hydrogels using acoustic waves. Biomaterials 2011:32. [27] Yeong WY, Chua CK, Leong KF, Chandrasekaran M. Rapid prototyping in tissue engineering: challenges and potential. Trends Biotechnol 2004:22.
[28] Tasoglu S, Demirci U. Bioprinting for stem cell research. Trends Biotechnol 2013;31(1):10–9. [29] Mironov V, Kasyanov V, Markwald RR. Organ printing: from bioprinter to organ biofabrication line. Curr Opin Biotechnol 2011;2(5):667–732. [30] Chen P, Guven S, Usta B, Yarmush M, Demirci U. Biotunable acoustic node assembly of organoids. Adv Healthc Mater 2015. https://doi.org/ 10.1002/adhm.201500279. [31] Mironov V, Boland T, Trusk T, Forgacs G, Markwald RR. Organ printing: computer-aided jet-based 3D tissue engineering. Trends Biotechnol 2003;21(4):157–61. [32] Bratt-Leal AM, Kepple KL, Carpenedo RL, Cooke MT, McDevitt TC. Magnetic manipulation and spatial patterning of multi-cellular stem cell aggregates. Integr Biol 2011;3(12):1224–32. [33] Chen P, Luo Z, Guven S, Tasoglu S, Ganesan AV, Weng A, et al. Microscale assembly directed by liquid-based template. Adv Mater 2014;26:5936–41. [34] Lancaster MA, et al. Cerebral organoids model human brain development and microcephaly. Nature 2013;501(7467):373–9. [35] Garvin KA, Dalecki D, Hocking DC. Vascularization of three-dimensional collagen hydrogels using ultrasound standing wave fields. Ultrasound Med Biol 2011;37(11):1853–64. [36] Pa¸sca AM, Sloan SA, Clarke LE, Tian Y, Makinson CD, Huber N, et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat Methods 2015;12(7):671–8. [37] Tang-Schomer MD, White JD, Tien LW, Schmitt LI, Valentin TM, Graziano DJ, et al. Bioengineered functional brain-like cortical tissue. Proc Natl Acad Sci USA 2014;111(38):13811–6. [38] Odawara A, Gotoh M, Suzuki I. A three-dimensional neuronal culture technique that controls the direction of neurite elongation and the position of soma to mimic the layered structure of the brain. RSC Adv 2013;3(45):23620–30. [39] Bouyer C, Chen P, Güven S, Demirta¸s TT, Nieland TJ, Demirci F, et al. A bio-acoustic levitational (BAL) assembly method for engineering of multilayered, 3D brain-like constructs, using human embryonic stem cell derived neuro-progenitors. Adv Mater 2016;28(1):161–7. [40] Bouyer C, Chen P, Nieland TJ, Demirci U, Padilla F. Bio-acoustic levitational assembly of heterocellular multilayer constructs for tissue engineering. eCells Mater J 2016;31(Suppl. 1). [41] Padilla F, Miskic T, Bouyer C, Wianny F, Dehay C. Bioengineering of cortex-like constructs by bio-acoustic levitational assembly of primate neural stem cells. eCells Mater J 2017 [eCM meeting abstracts, Collection 2, TERMIS EU (0080)].
ScienceDirect www.sciencedirect.com IRBM 39 (2018) 4–8
General Review
Creating Scaffolds for 3D Neuronal Tissue Models C. Bouyer ∗ , F. Padilla LabTAU, INSERM, Centre Léon Bérard, Université Lyon 1, Univ Lyon, F-69003, Lyon, France Received 5 January 2017; received in revised form 8 November 2017; accepted 9 November 2017 Available online 22 November 2017
Graphical abstract
Abstract Background: Many human tissues are comprised of multilayered tissue structures in which spatial organization is essential to provide biological tissue functions. Methods: Recently, strategies such as 3D bioprinting, photolithography, 3D auto-assembly, molding or bulk acoustic cells manipulation have been developed to fabricate layered tissue mimics. These methods have broad applications in tissue engineering for the bioengineering of multilayered structures, and for the fundamental understanding of many microphysiological and pathological process like cell differentiation. Each method relies on the use of a special scaffold structure made of natural or artificially created biopolymers, and of specific cell types. In the field of neuronal 3D constructs fabrication, where ex-vivo samples are difficult to get, different strategies have been developed going from rat neurons culture to embryonic stem cells culture and differentiation into neurons after their encapsulation in 3D scaffolds. Conclusion: All those possibilities open new perspectives for the future, aiming to the development of different types of tissues composed of different multilayer structures. © 2017 AGBM. Published by Elsevier Masson SAS. All rights reserved. Keywords: Tissue engineering; Scaffolds; Neural constructs 3 dimensional tissue structure
1. Introduction
* Corresponding author.
E-mail address: [email protected] (C. Bouyer). https://doi.org/10.1016/j.irbm.2017.11.001 1959-0318/© 2017 AGBM. Published by Elsevier Masson SAS. All rights reserved.
Many human tissues are comprised of multilayered tissue structures in which spatial organization is essential to provide biological tissues functions. Cell culture in 2 dimensions cannot provide all information about a native organ, as 2D cells
C. Bouyer, F. Padilla / IRBM 39 (2018) 4–8
patterning cannot represent the complex cells behavior in a 3D environment such as 3D cell–cell interactions. In many situations, establishment of artificial 3D tissue structures is necessary. For example, after large bone fracture, tissue graft may be needed to bring substrate and growth substances necessary to trigger bone fracture healing. Tissue engineering was born in this context, to provide replacement tissues, such as bone graft, where resources are limited or unavailable. During the last decades tissue engineering was expanded to many applications as many improvements were achieved in the available technologies [1]. Internal organs such as bladder [2] or pancreas [3], and more superficial tissues such as skin [4,5], bone/cartilage [6], or corneal [7] can now be engineered. Tissue engineering was then expanded to obtain 3D tissue model, to study tissue functions or development. In that aim, controlling cell–cell interaction, by controlling their patterning and their 3D microenvironment is required. Several technologies are being proposed to control cell patterning within scaffolds, such as micro-scale hydrogels (microgel) assembly, scaffold seeding or bioprinting. To control cell fate in 3 dimensions, a 3D microenvironment has to be chosen to fit with the desired application. In order to obtain construct with similar properties than native tissues and within which cells can grow and/or differentiate, scaffold properties such as porosity, size, geometry or elasticity are important parameters to control. In that aim, new biomaterials were developed to support cells viability and functions [8–10], with the possibility to obtain specific mechanical properties similar to those of native tissues. Such developments have led to the development of neuronal tissue engineering. The needs arose from the lack of preclinical models mimicking the architectural and developmental complexity of native tissue. Post-mortem functional tissues are essential ex-vivo models to study mental illnesses [11, 12], neurotransmitters mechanism of action [13], the dynamic of proteins/ions signaling [14], or to screen novel therapeutics agents [12], but they are of limited access. At the premise of neuronal tissue engineering, rat neurons were used and embedded in 3D scaffolds. More recently, scaffolds were developed to support Embryonic Stem Cells (ESC) cultures and the use of stem cells became more and more popular [4,7] as it allows bioengineering with cells from different origins, such as blastocyst, skin or muscle, according to the applications [15]. The aim of this review is to provide a rapid overview of methods developed to model neuronal tissue, and in particular cortical tissue. The generation of such cortical tissue models must respect the spatial organization of native tissue, which in first approximation can be viewed as a multilayered structure for which spatial organization and inter-layers cellular communications are essential to sustain their biological functions. To model this complex multilayers structures, the bioengineering of multilayered constructs with tunable layers thickness and cell types is necessary. Four different strategies can be implemented to generate 3D neuronal tissue structures. Each one is based on the use of specific cell types, physical strategies to spatially organized cells
5
and different scaffold types to embed them. We will discuss in this review the advantages and limits of those methods. 2. Methods 2.1. Two different approaches to engineer complex tissues To obtain 3D tissue models, bioengineering tools are needed to offer the ability to control the complex patterns and high cell density of native tissue, such as brain, liver or cardiac tissues [16]. In this context two different approaches can be used: “top-down” and “bottom-up” approaches [17]. These two approaches have advantages and shortcomings, which prevent them yet to be widely used and commercialized. In the top-down approach, cells are directly seeded into a fully made porous scaffold of predefined shape to form a tissue construct. Cells are expected to populate the scaffold and create the appropriate extracellular matrix and microarchitecture often with the aid of a bioreactor that furnish the set of stimuli required for an optimal cellular viability [18]. This approach is associated with some limitations such as the difficulty to shape scaffolds, difficulty to build large tissue constructs and low cell diffusion, which result in a low cell density and non-uniform distribution within the scaffold [17]. In the bottom-up approach, small cell laden modules such as spheroids or cell sheets are used as building blocks to create a larger construct [19]. In that case, the final construct organization and composition is tunable by the organization of the micro-architectural units. With this approach, the cell distribution is more likely controlled, but an external and often complex technology is needed to assemble the building units. New challenges in tissue engineering are to combine both approaches to enable a better cell seeding within a complex scaffold organization to create native-like tissue patterns. 2.2. Cell culture methods The random distribution of neurons in culture dishes does not sufficiently recapitulate the 3D aspects of neural connectivity or the microenvironment of the brain. Initially, hanging drop method was used to create 3D structures, but couldn’t represent cerebral tissue structure. Now, new technologies are trying to find a way to obtain specific scaffolds shape. 2.2.1. Conventional 3D culture Hanging drop is a technology for the fabrication of cells spheroids to create cells aggregation in 3D, to mimic in vivo tumor models on the lab-on-chip scale [20], for different applications like drug screening [21]. The methods are based on the use of either specific well plates or of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. The advantage of this technology is that it is possible to control the spheroid sizes, it is relatively easy to implement and it is low cost. 2.2.2. Technologies to obtain specific 3D structure shapes New techniques were developed to individually create single gels and to assemble blocks together [2,9,17,22,23]. Conven-
6
C. Bouyer, F. Padilla / IRBM 39 (2018) 4–8
tional 3D bioengineering technologies to create specific patterns, such as micro-scale hydrogels (microgel) assembly [9, 24–26], scaffold seeding [27] or bioprinting [28] cannot control cell–cell interactions or cell-packing density, which is essential in cerebral tissue modeling. In fact, spatial organization and interconnection of neural cells in and between these layers determine functions of cerebral cortex such as memory, language and consciousness. To address these issues, new scaffold-free strategies have been developed [29–32], mostly using cell spheroids to build highly-packed tissue construct elements. These methods suffer from long assembly time that prohibits building large, millimeter-scale constructs, and cannot reach an assembly precision of a few hundred micrometers required to pattern constructs such as neural constructs. These assembly methods rely on different physical methods such as magnetic [9,24], Faraday waves at liquid–air interface [33], acoustic or 3D printer. Organ printing [28,31], using printer, enables to build large scale scaffold with cells. This allows a precise scaffold patterns droplet by droplet but cannot go down the micro-scale size precision. In the other hand, magnetic assembly [9,24] allows spatial positioning of building blocks with cells. This technique is a contactless strategy but requires multistep procedure and a paramagnetic solution, which is not fully cell-friendly. On the other hand, acoustic strategy [33] enables a contactless, biocompatible manipulation to assemble cells following different patterns simply by tuning the acoustic frequency. Commonly used techniques generate large construct that mostly need to be cultivated in a bioreactor for proper media and CO2 diffusion within the sample [34,35]. 2.3. Stem-cell self-assembly to create cerebral organoids Several multistep strategies have been developed to create 6-layered tissue surrogates of the cerebral cortex, either by selfassembly or bioprinting approaches. In 2013, Lancaster et al. [34] generated a cerebral cortex-like tissue by first generating spheroids by self-assembly of embryonic stem cells (ESCs). Human pluripotent stem cells (hPSCs) spheroids were obtain by culture in hESC media and then in neural induction media for 11 days. Then, droplets were cultured in matrigel droplet and transferred into a spinning bioreactor [34]. In each step of the process, a specific media was attributed. When cultured in a spinning bioreactor in the presence of a differentiation medium and retinoic acid, cells were spontaneously organized to form a cortex-like structure. Neuronal differentiation of cerebral organoids revealed heterogeneous neuron subtypes production and auto-organization, with the presence of abundant radial glial stem cells during the development. Histological analysis of the differentiated structure revealed an important degree of molecular heterogeneity and regionalization that recapitulates the different brain regions. In 2015, in a study using human cortical spheroids obtained from pluripotent stem cells, a laminated cerebral cortex-like
structure could be generated and used for the study of normal and abnormal corticogenesis [36]. Cerebral organoids were formed from deep and superficial cortical layers neurons that were characterized by immunostaining observation. Different neuronal subtypes were found and located at different depths of the organoids, characteristic of both deep and superficial cerebral cortex. After complete neuronal maturation (after 180 days of culture), a synaptogenesis analysis was conducted to show the visualized individual synapses activity. The developed system provides a platform where deep and superficial neurons layers are present, surrounded by astrocytes to support neurons activity. 2.4. Scaffold-based bioengineering techniques of brain models Another alternative technique to bioengineer 3D neuronal multilayer structures is bioprinting approaches. For instance, primary rodent neurons were encapsulated in a layer-by-layer assembly approach in concentric 3D donut-shaped constructs, made from composite hydrogel and silk protein [37]. Micromolding has also been employed to embed primary neurons in 3D networks in collagen [38]. The generated 3D neural surrogate tissues displayed the characteristic cell density, interlayer neurite connections and layer thickness of the cortex, as well as biochemical and electrophysiological functional responses. Although these methods recapitulate some features of native neuronal tissues, such as 3D neuronal network design with interlayer neurite formation, neural cell density and layer thickness, and have shown biochemical and electrophysiological outcomes, some limitations hamper their wide applications in biological laboratories. Multistep operations required in both layer-by-layer assembly and micro-molding approaches may require special expertise and are time consuming. To address critical challenges encountered in self-assembly and bioprinting approaches in brain bioengineering, we developed a unique, single step bio-acoustic levitational (BAL) assembly technology [39]. With this cell-friendly technology, based on near field standing waves, direct assembly of multilayer 3D geometries within one single hydrogel construct was performed. Cells are trapped in a stable polymer network by controlling the gelation dynamics of fibrin, which can be achieved through optimization of the prepolymer concentrations. A final cohesive multilayer gel made of ESC was formed with cortex-like mechanical properties. The made gel was then cultured in neuronal differentiation media to obtain neuronal cell–cell interactions. Combination of available bioengineering methods could also be used to create different type of tissues. As an example, we are now combining bulk acoustic levitation and layer-by-layer assembly to bioengineer multilayered tissues with micrometerscale control over biological and structural features of native layered tissues. We can assemble, within minutes and with minimal acoustic pressure, multilayered constructs with multiple cell types in fibrin hydrogels (Fig. 1) [40,41].
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Fig. 1. Hydrogel construct formed of multi-layered heterogeneous cell types. A layer-by-layer assembly was performed to assembly live cells expressing eGFP (green), mCherry (red) and non-transfected HeLa cell lines. mCherry cells are seen on bottom of the left picture and on the right picture. eGFP cells are on the top of left picture and on the middle picture. Scale bar is 250 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3. Conclusion and perspectives
Acknowledgements
Novel techniques were recently developed to enable 3D cell culture such as hanging drop (self-assembly) or bio-printing of building blocks (controlled assembly). Compared to the previous studies where rat primary neurons were used [37,38] to create a cortex-like structure with microfluidic chip [38] or hydrogels [37], the use of ESC is very promising but very challenging [34]. Recent development in scaffolds biochemistry and manipulation now enables the encapsulation of ESC in 3D structures, their culture and their differentiation into neuronal lineage. Cerebral organoids are to date the method reproducing with the most accuracy the complexity of the biological processes during brain development, but require a lot of expertise. Controlled assembly represent an additional set of methods that, while limited in biological relevance compared to cerebral organoids, can provide easy and rapid way to generate model systems tailored to answer specific biological questions, such as cell migration during development or neurotoxicology of novel drugs. We are confident that these novel methods that can be carried out in a simple, rapid and biocompatible way in a fluidic environment, will find their place as widespread strategy for bottom-up tissue engineering and regenerative medicine where multilayered cell organization is required, to assemble cells alone in high densities to mimic the natural density and cell– cell interactions observed in the physiological complexity, such cortical tissue, but also for skin, cardiac or breast tissue. Last years, many developments in the field were made, developing new cell-friendly methods for cells manipulation. Moreover new technologies tend to allow the use of multiple cell types allowing to create multilayered tissue structures more and more representative of human tissues. In a near future, we can anticipate that novel technologies will be developed for fabrication of many types of organized structures.
This work was supported by the LabEx DEVweCAN (ANR-10-LABX-0061) of the University of Lyon, within the program “Investissements d’Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). The authors would also like to acknowledge the Fulbright program and the “Commission Franco-Américaine” for their support to C. Bouyer through a Fulbright award supporting her work at the BAMM Labs as a visiting Ph.D. student.
Conflict of interest statement No conflict of interest has to be declared.
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