Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

Abstract

Background: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. Objective: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. Methods: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. Results: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. Conclusion: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

Distinct Effects of Aducanumab and Lecanemab on Intraneuronal Endogenous Aβ42 and Phosphorylated Tau in Alzheimer’s Disease Treatment

“…By optimizing conversion and culture conditions, we successfully generated BFCNs that rapidly develop Aβ42 and pTau pathologies within 13–21 days, a timeframe considerably shorter than the typical 50-day period 28 . Additionally, these BFCNs were freezable, with freezing/thawing viability comparable to or higher than the iPSC-derived fetal neurons 31 , which significantly facilitates their storage, transportation, and application. This accelerated onset of natural endogenous neuropathology enhances the efficiency and reliability of evaluating potential AD therapeutics, offering a more accessible and dependable platform for testing.…”

Robust Generation of Ready-to-Use Cryopreserved Motor Neurons from Human Pluripotent Stem Cells for Disease Modeling

“…However, few studies have reported protocols for cryopreserving near-mature PSC-derived neurons with fully identified neuronal properties after thawing. Most studies have demonstrated NSC or progenitor-stage cell storage for ~15 days of differentiation, which still require another 2–3 weeks for neural patterning or maturation [ 18 , 19 ]. Here, we demonstrate an efficient protocol to cryopreserve MN EBs for 32 days using Stem-CellBanker with a sphere size of <300 µM and ROCKi addition.…”

“…The cryopreservation of neuronal cells with high viability that retains their properties is difficult. Although there have been current studies that have reported the cryopreservation of iPSC-derived dopaminergic progenitors for Parkinson’s disease transplantation [ 18 , 19 , 20 ], only a few have achieved MN or MNP cryopreservation but without further functional analysis [ 21 ]. Moreover, most reports demonstrated that neuronal progenitors or stem cells are frozen within 10–20 days of differentiation instead of late-stage premature neurons (>30 days) [ 18 , 19 , 20 ], requiring additional maturation to recapitulate disease phenotypes for model applications.…”

Induced pluripotent stem cells in companion animals: how can we move the field forward?

“…Stem cell banks increase the availability and ensures the quality of the cell products, while reducing the time to administer the therapy ( 168 ). Not only iPSCs can be banked, but also their derivatives including iMSCs ( 169 ) and some types of differentiated cells ( 170 ), as well as their secretomes ( 171 ). However, it is important to note that allogeneic therapy also has its own limitations, highlighting the risk of immunological rejection ( 167 ).…”