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Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

ABSTRACT: The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bottom milliwells composed of biomimetic hydrogels, combined with an optimized medium formulation, promoted the rapid generation of retina-like tissue from mESC aggregates in a highly efficient and stereotypical manner: ?93% of the aggregates contained retinal organoid structures. 26 day-old retinal organoids were composed of ?80% of photoreceptors, of which ?22% are GNAT2-positive cones, an important and rare sensory cell type that is difficult to study in rodent models. The compartmentalization of retinal organoids into predefined locations on a two-dimensional array not only allowed us to derive almost all aggregates into retinal organoids, but also to reliably capture the dynamics of individual organoids, an advantageous requirement for high-throughput experimentation. Our improved retinal organoid culture system should be useful for applications that require scalability and single-organoid traceability.

SUBMITTER: Decembrini S

PROVIDER: S-EPMC7314858 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

Decembrini S S Hoehnel S S Brandenberg N N Arsenijevic Y Y Lutolf M P MP

Scientific reports 20200624 1

The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bot ...[more]

PMID: 32581233

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Project description:BACKGROUND:Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. METHODS:Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis. RESULTS:This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7?days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33?±?8.37%), acinar (2.80?±?1.25%) cells, and pancreatic progenitors (5.81?±?2.65%). CONCLUSION:This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

Dataset Information

Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

Publications

Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

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