An On‐Chip Method for Long‐Term Growth and Real‐Time Imaging of Brain Organoids

Karzbrun, Eyal; Tshuva, Rami Yair; Reiner, Orly

doi:10.1002/cpcb.62

Cited by 19 publications

(12 citation statements)

References 15 publications

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“…This phenomenon has been observed in mathematical models, computer simulations, and swelling gels models [57,58,59,60]. Recently, we have observed folding of human brain organoids cultured in confined microdevices [7,61]. Despite the early onset of gyrification in the organoids, our results indicate that the organoid surface folding is driven by a combination of differential growth and tension at the apical surface.…”

Section: Recapitulation Of In Vivo Neurodevelopmentsupporting

confidence: 64%

“…Microfluidic devices have been previously designed for 3D spheroid cultures [123,124]. We have recently developed a microfabricated device to improve organoid culture conditions and enable long-term live-imaging (Figure 3a) [7,61]. The device consisted of a reaction chamber of 150μm height, confined between an imaging compatible coverslip and a semipermeable membrane, coupled to a media reservoir.…”

Section: Bioengineering Challenges and Opportunities In Brain Orgamentioning

confidence: 99%

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Brain Organoids—A Bottom-Up Approach for Studying Human Neurodevelopment

Karzbrun

Reiner

2019

Bioengineering

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Brain organoids have recently emerged as a three-dimensional tissue culture platform to study the principles of neurodevelopment and morphogenesis. Importantly, brain organoids can be derived from human stem cells, and thus offer a model system for early human brain development and human specific disorders. However, there are still major differences between the in vitro systems and in vivo development. This is in part due to the challenge of engineering a suitable culture platform that will support proper development. In this review, we discuss the similarities and differences of human brain organoid systems in comparison to embryonic development. We then describe how organoids are used to model neurodevelopmental diseases. Finally, we describe challenges in organoid systems and how to approach these challenges using complementary bioengineering techniques.

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Section: Recapitulation Of In Vivo Neurodevelopmentsupporting

confidence: 64%

Section: Bioengineering Challenges and Opportunities In Brain Orgamentioning

confidence: 99%

Brain Organoids—A Bottom-Up Approach for Studying Human Neurodevelopment

Karzbrun

Reiner

2019

Bioengineering

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“…Folding increased the 3D surface area size of the organoids, however, due to folding on the surface, the circularity decreased. Previous studies, using ECM embedding or confining EBs in laminated devices, demonstrated that wrinkling formation is induced and modulated by the mechanical micro-environment of the outer layer cells [ 11 , 65 , 66 ]. We believe our microwell device played a similar role by providing spatial confinement to the growing EBs.…”

Section: Resultsmentioning

confidence: 99%

A matrigel-free method to generate matured human cerebral organoids using 3D-Printed microwell arrays

Chen

Rengarajan

Kjar

et al. 2021

Bioactive Materials

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The current methods of generating human cerebral organoids rely excessively on the use of Matrigel or other external extracellular matrices (ECM) for cell micro-environmental modulation. Matrigel embedding is problematic for long-term culture and clinical applications due to high inconsistency and other limitations. In this study, we developed a novel microwell culture platform based on 3D printing. This platform, without using Matrigel or external signaling molecules (i.e., SMAD and Wnt inhibitors), successfully generated matured human cerebral organoids with robust formation of high-level features (i.e., wrinkling/folding, lumens, neuronal layers). The formation and timing were comparable or superior to the current Matrigel methods, yet with improved consistency. The effect of microwell geometries (curvature and resolution) and coating materials (i.e., mPEG, Lipidure, BSA) was studied, showing that mPEG outperformed all other coating materials, while curved-bottom microwells outperformed flat-bottom ones. In addition, high-resolution printing outperformed low-resolution printing by creating faithful, isotropically-shaped microwells. The trend of these effects was consistent across all developmental characteristics, including EB formation efficiency and sphericity, organoid size, wrinkling index, lumen size and thickness, and neuronal layer thickness. Overall, the microwell device that was mPEG-coated, high-resolution printed, and bottom curved demonstrated the highest efficacy in promoting organoid development. This platform provided a promising strategy for generating uniform and mature human cerebral organoids as an alternative to Matrigel/ECM-embedding methods.

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“…We used early passages of the NIH-approved embryonic stem cell line NIHhESC-10-0079 (WIBR3), which were stably labeled with a fluorescent H2B-mCherry reporter, labeling histones, and a LifeAct-GFP reporter labeling actin, as previously described [6] and maintained in NHSM media [33,34]. Cell transfection was carried out in a NEPA21 electroporation system according to the company's protocol (Nepa Gene, Chiba, Japan).…”

Section: Cell Linesmentioning

confidence: 99%

“…The NHSM media contained DMEM/F12 (21331-020 Gibco, ThermoFisher, Waltham, MA, USA), Albumax I (5gr, 11020-039, Invitrogen, Carlsbad, CA, USA), Pen-strep (5 mL, 03-031-1B, Biological Industries, Beit HaEmek, Israel), L-glutamine (5 mL, 02-022-1B, Biological Industries, Beit HaEmek, Israel), NEAA (5 mL, 01-340-1B, Biological Industries, Beit HaEmek, Israel), KSR (Knockout Serum Replacement, 50 mL, 10828-028, Invitrogen, Carlsbad, CA, USA), human insulin (12.5 µg/mL I-1882, Sigma, St. Louis, MO, USA), Apo-transferrin 100 µg/mL T-1147 Sigma, St. Louis, MO, USA), Progesterone 0.02 µg/mL P8783 Sigma, St. Louis, MO, USA), Putrescine 16 µg/mL P5780, Sigma, St. Louis, MO, USA), Sodium selenite 30 nM S5261 Sigma, St. Louis, MO, USA), 2-mercaptoethanol 50 mM (50 µL, 31350-10 Gibco, ThermoFisher, Waltham, MA, USA), L-ascorbic acid 2-phosphate (Vitamin C, 50 µg/mL, A92902, Sigma, St. Louis, MO, USA), Human LIF (20 ng/mL, Homemade), FGF2 Recombinant human (FGF-basic; 8 ng/mL, 100-18B, Rocky Hill, NJ, USA), TGFB1 (1 ng/mL, 100-21c, Peprotech, Rocky Hill, NJ, USA), IWR1 (5 µM, HY-12238, Medchem Express, Monmouth Junction, NJ, USA), Chir99021 (3 µM, HY-10182, Medchem Express, St. Louis, MO, USA), ERKi (PD0325901, 1 µM, HY-10254, Medchem Express, Monmouth Junction, NJ, USA), p38i (BIRB0796, 2 µM, HY-10320, Medchem Express, USA), JNKi (SP600125, 5 µM, 1496, Tocris, Bristol, UK), BMPi (LDN193189, 0.4 µM, HY-12071A, Medchem Express, Monmouth Junction, NJ, USA) and ROCKi (Y27632, 5 µM, HY-10583, Medchem Express, Monmouth Junction, NJ, USA). The Matrigel-coated plates were prepared as previously described [34]. Briefly, 10 cm tissue-culture plates were coated with 1:100 Matrigel diluted in DMEM/F12 (21331-020 Gibco, ThermoFisher, Waltham, MA, USA) and kept at 37 • C for at least 2 h before use.…”

Section: Human Esc Aggregatesmentioning

confidence: 99%

Toward Spatial Identities in Human Brain Organoids-on-Chip Induced by Morphogen-Soaked Beads

Ben-Reuven

Reiner

2020

Bioengineering

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Recent advances in stem-cell technologies include the differentiation of human embryonic stem cells (hESCs) into organ-like structures (organoids). These organoids exhibit remarkable self-organization that resembles key aspects of in vivo organ development. However, organoids have an unpredictable anatomy, and poorly reflect the topography of the dorsoventral, mediolateral, and anteroposterior axes. In vivo the temporal and the spatial patterning of the developing tissue is orchestrated by signaling molecules called morphogens. Here, we used morphogen-soaked beads to influence the spatial identities within hESC-derived brain organoids. The morphogen- and synthetic molecules-soaked beads were interpreted as local organizers, and key transcription factor expression levels within the organoids were affected as a function of the distance from the bead. We used an on-chip imaging device that we have developed, that allows live imaging of the developing hESC-derived organoids. This platform enabled studying the effect of changes in WNT/BMP gradients on the expression of key landmark genes in the on-chip human brain organoids. Titration of CHIR99201 (WNT agonist) and BMP4 directed the expression of telencephalon and medial pallium genes; dorsal and ventral midbrain markers; and isthmus-related genes. Overall, our protocol provides an opportunity to study phenotypes of altered regional specification and defected connectivity, which are found in neurodevelopmental diseases.

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An On‐Chip Method for Long‐Term Growth and Real‐Time Imaging of Brain Organoids

Cited by 19 publications

References 15 publications

Brain Organoids—A Bottom-Up Approach for Studying Human Neurodevelopment

Brain Organoids—A Bottom-Up Approach for Studying Human Neurodevelopment

A matrigel-free method to generate matured human cerebral organoids using 3D-Printed microwell arrays

Toward Spatial Identities in Human Brain Organoids-on-Chip Induced by Morphogen-Soaked Beads

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