Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads
Abstract Long-term preservation of fully differentiated human neurons poses a longstanding challenge in neuroscience research. Numerous cellular disease models have been established using cultured human neuronal cells, including our three-dimensional (3D) human neural cell culture model of Alzheimer...
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Nature Portfolio
2025-04-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-025-94810-8 |
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| author | Jae Jung Kim Matthias Hebisch Sang Su Kwak Monica Zheng Shreya Nuli Jun-Seok Bae Emma Brand Rudolph E. Tanzi Daniel Irimia Doo Yeon Kim |
| author_facet | Jae Jung Kim Matthias Hebisch Sang Su Kwak Monica Zheng Shreya Nuli Jun-Seok Bae Emma Brand Rudolph E. Tanzi Daniel Irimia Doo Yeon Kim |
| author_sort | Jae Jung Kim |
| collection | DOAJ |
| description | Abstract Long-term preservation of fully differentiated human neurons poses a longstanding challenge in neuroscience research. Numerous cellular disease models have been established using cultured human neuronal cells, including our three-dimensional (3D) human neural cell culture model of Alzheimer’s disease (AD). However, the absence of a reliable method for preserving fully differentiated human neural cell cultures for a long time has hindered the sharing and standardization of these models. To address this critical limitation, we focused on cryopreservation, which is the gold standard for long-term preservation, and combined this with three key technological advancements. First, we employed parallelized microfluidic devices for the efficient generation of 3D cell cultures within uniform hydrogel microbeads (~ 220 μm), which facilitate the rapid exchange of media ingredients and cryoprotectants. Second, we implemented a cytophobic microwell system to safeguard neuron-encapsulated microbeads from fusion and aggregation. Third, we developed a novel inducible AD cell model optimized for cryopreservation and AD drug testing. We have successfully maintained encapsulated control and AD neural progenitor cells in microwells during differentiation for 12 days. Notably, fully differentiated human neural cells can be cryopreserved within Matrigel microbeads while retaining intact and mature neuronal processes, exhibiting no signs of damage to neurites following freeze/thaw cycles. Furthermore, we have demonstrated the successful cryopreservation, thawing, and induction of pathogenic Amyloid-β 42 (Aβ42) generation in fully differentiated AD neural progenitor cells. Our study offers a solution for one of the major challenges in neuroscience research, utilizing porous hydrogel microbead structures to facilitate rapid delivery of cryoprotectants and protect complex neuronal structures without undergoing damaging cell dissociation steps. The inducible "3D human microbead model of AD" enhances the speed, efficacy, and reproducibility of AD drug screening. |
| format | Article |
| id | doaj-art-73ce3494dbbb4f09bbdc706df889cc6c |
| institution | OA Journals |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-73ce3494dbbb4f09bbdc706df889cc6c2025-08-20T02:17:13ZengNature PortfolioScientific Reports2045-23222025-04-0115111510.1038/s41598-025-94810-8Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeadsJae Jung Kim0Matthias Hebisch1Sang Su Kwak2Monica Zheng3Shreya Nuli4Jun-Seok Bae5Emma Brand6Rudolph E. Tanzi7Daniel Irimia8Doo Yeon Kim9Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard School of Medicine, Shriners Children’s BostonGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolCenter for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard School of Medicine, Shriners Children’s BostonGenetics and Aging Research Unit, Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Harvard Medical SchoolAbstract Long-term preservation of fully differentiated human neurons poses a longstanding challenge in neuroscience research. Numerous cellular disease models have been established using cultured human neuronal cells, including our three-dimensional (3D) human neural cell culture model of Alzheimer’s disease (AD). However, the absence of a reliable method for preserving fully differentiated human neural cell cultures for a long time has hindered the sharing and standardization of these models. To address this critical limitation, we focused on cryopreservation, which is the gold standard for long-term preservation, and combined this with three key technological advancements. First, we employed parallelized microfluidic devices for the efficient generation of 3D cell cultures within uniform hydrogel microbeads (~ 220 μm), which facilitate the rapid exchange of media ingredients and cryoprotectants. Second, we implemented a cytophobic microwell system to safeguard neuron-encapsulated microbeads from fusion and aggregation. Third, we developed a novel inducible AD cell model optimized for cryopreservation and AD drug testing. We have successfully maintained encapsulated control and AD neural progenitor cells in microwells during differentiation for 12 days. Notably, fully differentiated human neural cells can be cryopreserved within Matrigel microbeads while retaining intact and mature neuronal processes, exhibiting no signs of damage to neurites following freeze/thaw cycles. Furthermore, we have demonstrated the successful cryopreservation, thawing, and induction of pathogenic Amyloid-β 42 (Aβ42) generation in fully differentiated AD neural progenitor cells. Our study offers a solution for one of the major challenges in neuroscience research, utilizing porous hydrogel microbead structures to facilitate rapid delivery of cryoprotectants and protect complex neuronal structures without undergoing damaging cell dissociation steps. The inducible "3D human microbead model of AD" enhances the speed, efficacy, and reproducibility of AD drug screening.https://doi.org/10.1038/s41598-025-94810-8HydrogelExtracellular matrixNeurodegenerationAlzheimer’s disease |
| spellingShingle | Jae Jung Kim Matthias Hebisch Sang Su Kwak Monica Zheng Shreya Nuli Jun-Seok Bae Emma Brand Rudolph E. Tanzi Daniel Irimia Doo Yeon Kim Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads Scientific Reports Hydrogel Extracellular matrix Neurodegeneration Alzheimer’s disease |
| title | Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads |
| title_full | Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads |
| title_fullStr | Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads |
| title_full_unstemmed | Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads |
| title_short | Cryopreserving 3D cell culture models of Alzheimer’s disease in hydrogel microbeads |
| title_sort | cryopreserving 3d cell culture models of alzheimer s disease in hydrogel microbeads |
| topic | Hydrogel Extracellular matrix Neurodegeneration Alzheimer’s disease |
| url | https://doi.org/10.1038/s41598-025-94810-8 |
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