Recapitulating macro-scale tissue self-organization through organoid bioprinting

A 3D bioprinting approach has been developed to facilitate tissue morphogenesis by directly depositing organoid-forming stem cells in an extracellular matrix, with the ability to generate intestinal epithelia and branched vascular tissue constructs. Bioprinting promises enormous control over the spatial deposition of cells in three dimensions(1-7), but current approaches have had limited success at reproducing the intricate micro-architecture, cell-type diversity and function of native tissues formed through cellular self-organization. We introduce a three-dimensional bioprinting concept that uses organoid-forming stem cells as building blocks that can be deposited directly into extracellular matrices conducive to spontaneous self-organization. By controlling the geometry and cellular density, we generated centimetre-scale tissues that comprise self-organized features such as lumens, branched vasculature and tubular intestinal epithelia with in vivo-like crypts and villus domains. Supporting cells were deposited to modulate morphogenesis in space and time, and different epithelial cells were printed sequentially to mimic the organ boundaries present in the gastrointestinal tract. We thus show how biofabrication and organoid technology can be merged to control tissue self-organization from millimetre to centimetre scales, opening new avenues for drug discovery, diagnostics and regenerative medicine.

Homeostatic mini-intestines through scaffold-guided organoid morphogenesis

Nathalie Brandenberg, Nicolas Antoine Vincent Broguiere, Devanjali Dutta, Nikolche Gjorevski, Matthias Lütolf, Olga Mitrofanova, Mikhail Nikolaev, Yoji Tabata

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology(1-4). However, the approaches that are used at present to derive these organoids in three-dimensional matrices(5,6)result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.

NATURE PUBLISHING GROUP2020

Engineering next generation organoids through guided stem cell self-organisation

Mikhail Nikolaev

One of the long-standing goals in the field of tissue engineering is the fabrication of de novo tissues or even organs to provide better tissue models for basic research and drug discovery, and to alleviate a shortage of donor organs in the future. Research in recent decades to understand tissue biology has led to the discovery of stem cell niches and the development of innovative protocols for the generation of three-dimensional in vivo-like tissues called organoids. Organoids represent multiple histological and functional aspects of real organs and offer unprecedented perspectives for modelling tissue development, regeneration, and disease in vitro. However, relying so far almost exclusively on spontaneous self-organisation of stem cells, organoids develop uncontrollably into small structures of highly variable size and architecture. Moreover, the closed, cystic architecture of most epithelial organoids limits their lifespan and makes experimental manipulation difficult. In this thesis, three innovative engineering technologies were developed that offer the possibility of steering in vitro stem cell patterning and organoid formation into more controllable and physiologically relevant miniature tissues.	The aim of the first project was to study how predefined geometric constraints, as they exist in vivo, affect organoid development. This required the development of a novel fabrication process of the topographically micro-patterned hydrogel surfaces. This approach allowed the generation of large arrays of the identical organoids, having pre-defined shape and size to study cellular mechanisms involved in the proper, stereotypical cell type pattering of intestinal organoids.		The second project focused on replicating tissue dynamics and aspects of organ physiology by developing a novel three-dimensional organ-on-chip approach. Using stem cells derived from patient biopsies, this technology enables to production of miniature versions of patientâs organs that can be used in personalized diagnostics and medical treatment assays.	Bioprinting has long been considered as one of the most promising technologies for fabricating organs in vitro. However, despite encouraging advances made in recent years, the degree of multicellular spatial complexity and function of organoids remains unmatched by any existing bioprinting technology. Therefore, the third project was aimed to develop a new bioprinting strategy that preserves local stem cell self-organization potential and extends organoids growth to the macroscopic scale.	Overall, this thesis introduces several cutting-edge technologies that offer the possibility to control in vitro organoid development and should therefore become efficient research tools to facilitate the translation of organoid technology towards pharmaceutical and clinical applications. Hydrogel micro-pattering is an efficient technology that can be widely used to explore the mechanisms by which tissue geometry can regulate morphogenesis of different organs and can be readily applied for high-throughput screening in pharmacological assays. The microscope-based bioprinter can be set-up in any laboratory to guide stem cell self-organization and tissue morphogenesis from millimetre to centimetre scales. The microfluidic organoid-on-chip concept should enable the creation of various miniature organs with complex three-dimensional spatial organisation and multicellular complexity for basic research and drug discovery.

EPFL2021

New Tools for Hydrogel-Based 3D Bioprinting

Andrea Negro

The in vitro recapitulation of tissue and organ function represents one of the main objectives of tissue engineering. Developments in this field have numerous applications, from alleviating the shortage of donor organs to providingmore representative platforms for drug testing. As such, there is an increasingly significant body of literature devoted to the development of better strategies to reconstruct living matter outside of the body. The three-dimensional (3D) architecture of a tissue, its histoarchitecture, is a key aspect to consider when engineering a tissue. Indeed, tissue function not only arises from individual elements that compose the tissue, its different cell types and their extracellular matrices (ECM), but also fromtheir precise spatial arrangement and interactions. Bioprinting is considered as one of the most promising techniques to recapitulate the complexity of in vivo histoarchitectures. Early bioprinting examples have shown successful application of inkjet printing to geometrically arrange cells and biomolecules in 2D and, to a much lesser extent, in 3D. However, no group has shown yet a technological platformable to print a living tissue construct whose make-up could even closelymimic the histoarchitecture of a native tissue. In this thesis the most significant challenges in using inkjet printers for tissue engineering were tackled. Perhaps the most critical bottleneck of state-of-the-art bioprinting is the lack of suitable bioinks; precursor liquids with distinct physico-chemical properties that very rapidly transforminto solid hydrogels and also possess the necessary bioactive characteristics to guide cell development into a functional tissue. To this end, a novel double network hydrogel systemwas conceived which combines ultra-rapid cross-linking of alginate, a biologically inert polysaccharide network, with the slower enzymatic crosslinking of a synthetic but highly bioactive poly(ethylene glycol) (PEG) gel. The latter could be tailored virtually on demand tomimic a desired ECM in a tissue. Based on this bioink concept it was demonstrated that complex multicomponent 3D shapes could be printed. Importantly, using primary human fibroblasts as model system, the bioinks showed outstanding characteristics as cellular microenvironments, facilitating rapid 3D cell migration and self-organization into multicellular networks. To mimic the spatial complexity of living tissues, it is crucial to master the deposition of multiple, cell-containing bioinks into user-defined 3D structures. Each bioink could thus mimic a specific cell/ECMcomponent present in a native tissue. To be able to reproduce an appropriate histoarchitecture, bioinks have to be deposited precisely enough to approximate their natural 3D spatial arrangement down to a cellular scale. Technically, this implies that the dispensing units must be extremely well aligned and synchronized to one another in order to pattern according to a specified design. In order to address these challenges, several novel bioprinting concepts were developed, which allowed the patterning of multiple bioinks containing living cells into 3D tissue-like geometries. In the last part of this thesis, an important first step towards the engineering of macroscopic tissue models was taken. Accordingly, a fundamental requirement for the viability and maturation of a printed tissue construct is the supply of nutrients and growth factors through a microfluidic network, similar to the vascularization in a native tissue. To this end, a novel approach for the printing of 3D constructs with an interconnected channel network enabling rapid nutrient transport was devised. This was achieved by exploiting the multicomponent printing developed earlier. A channel network inside the construct was patterned using a degradable hydrogel that could be selectively removed to leave behind the perfusable microfluidic system. Moreover, it was possible to combine this sacrificial layer approach with cell-containing bioinks to generate a cellularized perfusable tissue model. Encouraging preliminary long-termculture experiments revealed that dynamic cell culture conditions could be maintained, leading to viable cell growth over more than one week under perfusion conditions. Taken together, this thesis presents several important steps toward an inkjet-based platform for in vitro tissue engineering. It is expected that the strategies developed here will provide a solid basis for future advances in the emerging bioprinting field.

EPFL2014