Application and Assessment of Optical Clearing Methods for Imaging of Tissue-Engineered Neural Stem Cell Spheres

Boutin, Molly E.; Hoffman–Kim, Diane

doi:10.1089/ten.tec.2014.0296

Cited by 45 publications

(73 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…24,25 Briefly, spheroids were fixed in 4% v/v paraformaldehyde and 8% w/v sucrose in phosphatebuffered saline (PBS) overnight, followed by three 1-h PBS washes. All of the following steps were performed on a shaker at room temperature.…”

Section: Whole Spheroid Immunostaining and Optical Clearingmentioning

confidence: 99%

“…24,25 Briefly, spheroids were fixed and washed as described (see Three-dimensional self-assembled cortical spheroid fabrication) and subjected to a sucrose gradient of 15% and 30% w/v sucrose in PBS, for 3 h per step. Whole agarose gels containing spheroids were embedded in O.C.T.…”

Section: Spheroid Cryosectioning and Immunostainingmentioning

confidence: 99%

“…This culture method has the potential to be applied to a wide range of neural cell types. 24 Here we have characterized rat postnatal cortical spheroids, and additionally, to demonstrate that other types of neural cells can self-assemble, we fabricated spheroids using rat hippocampal cells. Hippocampal cells from a commercially available tissue source formed spheroids, and the spheroid sizes were dependent on the seeding densities ( Supplementary Fig.…”

Section: Versatile Culturementioning

confidence: 99%

See 2 more Smart Citations

Three-Dimensional Neural Spheroid Culture: AnIn VitroModel for Cortical Studies

Dingle

Boutin

Chirila

et al. 2015

Tissue Engineering Part C: Methods

Self Cite

122

117

View full text Add to dashboard Cite

There is a high demand for in vitro models of the central nervous system (CNS) to study neurological disorders, injuries, toxicity, and drug efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional two-dimensional culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry through both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the CNS in vitro.

show abstract

Section: Whole Spheroid Immunostaining and Optical Clearingmentioning

confidence: 99%

Section: Spheroid Cryosectioning and Immunostainingmentioning

confidence: 99%

Section: Versatile Culturementioning

confidence: 99%

See 1 more Smart Citation

Three-Dimensional Neural Spheroid Culture: AnIn VitroModel for Cortical Studies

Dingle

Boutin

Chirila

et al. 2015

Tissue Engineering Part C: Methods

Self Cite

122

117

View full text Add to dashboard Cite

show abstract

“…For example, Boutin and Hoffman-Kim recently compared Clear T2 , Scale, and SeeDB on tissue-engineered neural sphere samples. 37 Although the original demonstration of SeeDB on murine neonatal brain and adult hemi-brain samples showed minimal sample volume change, Boutin and Hoffman-Kim found significant shrinkage in their SeeDB-treated neural sphere samples. It is thus crucial to test different clearing protocols on the tissue of interest, while monitoring the sample during the process could give greater insight as to the effects of each protocol.…”

Section: Introductionmentioning

confidence: 98%

“…39 Although Boutin et al have studied optical clearing in neural stem cell spheroids and glioma, 37 to the best of our knowledge, no study has been performed on optical clearing of ECM-containing tumour spheroids. Most of the clearing protocols were developed for millimetre-scale tissue samples and require successive incubations in different solutions; this can be difficult to implement, requiring centrifugation for solution exchange around tumour spheroid samples.…”

Section: Introductionmentioning

confidence: 99%

On-chip clearing of arrays of 3-D cell cultures and micro-tissues

et al. 2016

View full text Add to dashboard Cite

Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 lm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear T2 , and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear T2 ; however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This onchip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. Published by AIP Publishing. [http://dx

show abstract

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Costa

Silva

Moreira

et al. 2019

Biotech & Bioengineering

View full text Add to dashboard Cite

Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a lightscattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI;(b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted. K E Y W O R D Simaging, light scattering, optical clearing, optical microscopy, spheroids.

show abstract

Application and Assessment of Optical Clearing Methods for Imaging of Tissue-Engineered Neural Stem Cell Spheres

Cited by 45 publications

References 35 publications

Three-Dimensional Neural Spheroid Culture: AnIn VitroModel for Cortical Studies

Three-Dimensional Neural Spheroid Culture: AnIn VitroModel for Cortical Studies

On-chip clearing of arrays of 3-D cell cultures and micro-tissues

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Contact Info

Product

Resources

About