Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges

Li, Rui; Walsh, Patrick J.; Truong, Vincent; Petersen, Ashley; Dutton, James R.; Hubel, Allison

doi:10.3389/fcell.2021.796960

Cited by 13 publications

(7 citation statements)

References 53 publications

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“…Firstly, the inclusion of ROCK inhibitor (ROCKi) has several benefits throughout the bioprinting process and during early culture. Cell thawing is a critical point in which the neurons can experience a stress response, and improper thawing protocols can decrease the chances of survival 22 . It is typically recommended to thaw cells, add media, and raise cells to incubator temperature as efficiently as possible 23 .…”

Section: Discussionmentioning

confidence: 99%

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

Whitehouse,

He,

Brownlees

et al. 2023

JoVE

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For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

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Section: Discussionmentioning

confidence: 99%

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

Whitehouse,

He,

Brownlees

et al. 2023

JoVE

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“…The studies in this investigation involved single hiPSCs. When attached to other hiPSCs, significant changes in freezing response were observed (Li et al, 2018;Li et al, 2019;Li et al, 2020;Li et al, 2021). It is possible that attachment to other cells changes the membrane permeability, the partitioning of DMSO and therefore the freezing response.…”

Section: Different Cell Types Have Different Partitioning Propertiesmentioning

confidence: 99%

Characterizing cellular membrane partitioning of DMSO using low-temperature Raman spectroscopy

Louwagie

Wagner²,

Li³

et al. 2023

Front. Mol. Biosci.

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Additives that help cells survive the stresses of freezing and thawing are known as cryoprotective agents (CPAs). Two different types of CPAs have been identified: penetrating and non-penetrating. Common penetrating CPAs include dimethylsulfoxide (DMSO) and glycerol. The location of a CPA (intracelluar or extracellular) is important for understanding the molecular mechanisms of action for the agent. Low-temperature Raman spectroscopy is a label-free method of detecting the location of CPAs at low temperature with high spatial resolution and chemical specificity. To this end, cells cryopreserved in DMSO using a variety of cooling rates and DMSO concentrations and imaged using Raman spectroscopy were analyzed using automated image analysis to determine the partitioning ratio (concentration of DMSO outside/concentration of DMSO inside the cell). The partitioning ratio was roughly 1 for Jurkat cells frozen at 1°C/min in varying concentrations of DMSO with the exception of 1% DMSO which had a partitioning ratio of 0.2. The partitioning ratio increased from 1 to 1.3 as the cooling rate increased from 1°C to 5°C/min. Different cell types, specifically sensory neurons cells and human induced pluripotent stem cells, exhibited differences in partitioning ratio when frozen in 10% DMSO and 1°C/min suggesting that differences in freezing response may result from differences in solute partitioning. The presence of intracellular ice changed the distribution of DMSO inside the cell and also the partitioning ratio.

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“…Induced pluripotent cells technology it is suitable for this purpose. Some research groups have previously reported on the use of Raman fingerprinting for identifying differentiating states of human induced pluripotent stem cells (hiPSCs) [16][17][18][19][20]. Naturally, the time course of differentiation of many single cells should be measured using multiple cell line types.…”

Section: Introductionmentioning

confidence: 99%

Yuragi biomarker concept for evaluating human induced pluripotent stem cells using heterogeneity-based Raman finger-printing

Fujita,

Haruki,

Sudo

et al. 2024

BIOPHYSICS

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Considering the fundamental mechanism causing singularity phenomena, we performed the following abduction: Assuming that a multicellular system is driven by spontaneous fluctuation of each cell and dynamic interaction of the cells, state transition of the system would be experimentally predictable from cellular heterogeneity. This study describes the assessment of this abductive hypothesis by investigating whether analysis of cellular heterogeneity to distinguish pre-state of state transition of differentiating cells by using Raman spectroscopy and human induced pluripotent stem cells (hiPSCs) technique. Herein, we investigated the time development of cellular heterogeneity in Raman spectra during cardiomyogenesis of six hiPSC lines and tested two types of analyses for heterogeneity. As expected, some spectral peaks, attributed to glycogen, correctively exhibited higher heterogeneity, prior to intensity changes of the spectrum in the both analyses in the all cell-lines tested. The combination of spectral data and heterogeneity-based analysis will be an approach to the arrival of biology that uses not only signal intensity but also heterogeneity as a biological index.

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Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges

Cited by 13 publications

References 53 publications

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

Characterizing cellular membrane partitioning of DMSO using low-temperature Raman spectroscopy

Yuragi biomarker concept for evaluating human induced pluripotent stem cells using heterogeneity-based Raman finger-printing

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