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This is a preprint.

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The National Library of Medicine isrunning a pilot to include preprints that result from research funded by NIH in PMC and PubMed.
[Preprint].2024 Jul 25:2024.07.25.605147.
doi: 10.1101/2024.07.25.605147.

Cryopreservation of neuroectoderm on a pillar plate andin situ differentiation into human brain organoids

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Cryopreservation of neuroectoderm on a pillar plate andin situ differentiation into human brain organoids

Mona Zolfaghar et al. bioRxiv..

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Abstract

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80°C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 μm. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

Keywords: Cryopreservation; human brain organoids; in situ tests; pillar plate.

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Figures

Figure 1.
Figure 1.
Neuroectoderms (NEs) formed in an ultralow attachment (ULA) 384-well plate at the seeding densities of 1,000 and 3,000 cells/well were transferred to a 36PillarPlate with Matrigel on day 7.
Figure 2.
Figure 2.
Relative viability of NEs after 2 hours of incubation at room temperature with cryoprotective agents (CPAs) including(I) Control (no CPA exposure),(II) PSC kit,(III) CryoStor,(IV) 3dGRO, and (V) 10% DMSO:(A) 1,000 and(B) 3,000 cell seeding density/well. The basal cytotoxicity of CPAs was compared with the control. The PSC kit and 10% DMSO maintained relatively high cell viability after 2 hours of incubation with the CPAs. n > 15. Statistical difference was analyzed with the control using one-way ANOVA. **** for p < 0.0001, *** for p < 0.001, ** for p < 0.01, * for p < 0.05, and ns = non-significant (p > 0.05).
Figure 3.
Figure 3.
The staining of NEs with calcein AM in the presence of various CPAs, including(I) PSC kit,(II) CryoStor,(III) 3dGRO,(IV) 10% DMSO, and(V) Control (no CPA exposure). This was conducted for 2 hours at room temperature to evaluate the rate of molecule diffusion into the core of NEs and to assess the basal toxicity of the CPAs. The cell seeding density per well was(A) 1,000 and(B) 3,000. The diffusion of calcein AM into the core of NEs took approximately 1 hour, suggesting that the diffusion of CPAs may require at least the same amount of time.
Figure 4.
Figure 4.
The changes in relative viability of NEs after a 2-hour incubation with the PSC kit at room temperature. Cell viability was subsequently assessed using the CellTiter-Glo luminescent cell viability assay kit following a 24-hour incubation period in a CO2 incubator. The number of replicates (n) was greater than 8 (n > 8).
Figure 5.
Figure 5.
The changes in relative viability of NEs following cryopreservation using the PSC kit. The conditions compared were:(I) NE control (without cryopreservation) and NE samples after cryopreservation with the PSC kit at the seeding densities of(II) 1,000 cells and(III) 3,000 cells per well. The NEs were initially preserved at − 80°C overnight, followed by storage in liquid nitrogen for 3 days. Subsequently, they were thawed and incubated at 37°C for 24 hours. Post-incubation, cell viability was assessed using the CellTiter-Glo luminescent cell viability assay kit. n > 6.
Figure 6.
Figure 6.
Optimum protocol of cryopreservation and thawing of NEs on the pillar plate.
Figure 7.
Figure 7.
(A) The viability of NEs on the pillar plate before cryopreservation and after thawing:(I) Before cryopreservation,(II) 1 Day after thawing, and(III) 8 Days after thawing. The viability of the NEs was measured with calcein AM and ethidium homodimer 1 (EthD-1). Scale bars: 500 μm.(B) The changes in the morphology of NEs by differentiation on the pillar plate for 1, 4, 8, 21, and 30 days after cryopreservation and thawing. The cryopreserved NEs were properly recovered and showed the expansion of neuroepithelium on day 4 after differentiation. Scale bars: 500 μm.
Figure 8.
Figure 8.
Characterization of gene expression in(I) iPSCs (control) and brain organoids differentiated from(II) cryopreserved and(III) non-cryopreserved NEs. Gene expression ofPAX6 forebrain neuroprogenitor marker,SOX2 proliferating neuroprogenitor marker,TBR2 intermediate progenitor marker,TUBB3 neuronal cytoplasm marker,CTIP2 deep cortical neuronal marker,FOXG1 forebrain marker,MAP2 mature neuronal marker, andOCT4 pluripotency marker were analyzed by qPCR. Statistical significance was performed using one-way ANOVA. **** for p < 0.0001, *** for p < 0.001, ** for p < 0.01, * for p < 0.05, and ns = non-significance (p > 0.05). n = 10–12 per qPCR run.
Figure 9.
Figure 9.
Immunofluorescence (IF) staining of brain organoids 30 days after thawing. SOX2 proliferating neuroprogenitor marker, MAP2 mature neuronal marker, PAX6 forebrain neuroprogenitor marker, and TBR2 intermediate progenitor marker were characterized in day 30 brain organoids differentiated from cryopreserved and thawed NEs. Scale bars: 50 μm.
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