Apr 08, 2025
Cryopreservation of iPSC-Derived Microglia
- Jae-Hyeon Park1,
- Aleksandra Beylina1,
- Hebao Yuan2,
- Michael Ward2,
- Mark Cookson1
- 1Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA.;
- 2Inherited Neurodegenerative Diseases Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Protocol Citation: Jae-Hyeon Park, Aleksandra Beylina, Hebao Yuan, Michael Ward, Mark Cookson 2025. Cryopreservation of iPSC-Derived Microglia. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwe567vmk/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: April 04, 2025
Last Modified: April 08, 2025
Protocol Integer ID: 126186
Keywords: Cryopreservation, iPSC-Derived Microglia, Freezing
Funders Acknowledgements:
Mark Cookson
Grant ID: ZIA AG000948-17
Abstract
This protocol outlines cryopreservation and thawing procedures for human induced pluripotent stem cell (iPSC)-derived microglia (iMicroglia). The described methods enable the long-term storage of mature iMicroglia in either -80 °C freezers or liquid nitrogen tanks, ensuring cell viability upon thawing. By utilizing appropriate cryoprotectants, this protocol facilitates the effective preservation of microglia for future studies, allowing for reproducible experiments and robust recovery after cryopreservation.
Guidelines
o Time Sensitivity:
Both freezing and thawing should be done as quickly as possible to minimize cellular damage. After thawing, immediately transfer the contents to 5 mL of prewarmed fresh media.
o Sterile Environment:
All work should be conducted in sterile laminar flow hoods (clean bench) designated for human iPSC work. Always use filter tips to minimize the risk of contamination.
Materials
DPBS no calcium, no magnesiumInvitrogen - Thermo FisherCatalog #14190136
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
Accutase Gibco - Thermo Fisher ScientificCatalog # A1110501
Knockout™ Serum Replacement Gibco - Thermo Fisher ScientificCatalog #10828028
Bambanker™ Serum Free Cell Freezing MediumBulldog BioCatalog #BB05
Advanced RPMI 1640 MediumThermo FisherCatalog #12633012
GlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061
Recombinant Human IL-34Thermo Fisher ScientificCatalog #200-34
Recombinant Human GM-CSFThermo Fisher ScientificCatalog #300-03 .
o Equipment
- Centrifuge
- P1000, P200, and P10 tips
- 50 mL falcon tubes (Avantor, cat #89039-658)
- Cryogenic storage vials (Corning, cat #431416)
- Cell counting equipment
- Corning CoolCell or Freezing container
- -80 °C freezer
- Liquid Nitrogen tanks
Before start
- Prepare and label the cryogenic vials with the following information: cell line, clone number, day of differentiation, cell number, and freezing date.
- The cell freezing media should be stored at 4 °C before use.
Freezing Procedure
Freezing Procedure
25m
25m
Collection of Cells:
- Aspirate the culture media from the T-75 culture flask. Add 5 mL of Accutase to the cells and incubate for 00:20:00 .
- Add 1X phosphate-buffered saline (PBS) to the cells, gently lift them off the surface, and transfer the suspension into a 50 mL centrifuge tube.
- Centrifuge the cells at 1000 rpm, 00:05:00 . Resuspend the cell pellet in 1xPBS and count the cell density with a cell counter.
- Centrifuge the cells at 1000 rpm, 00:05:00 . Carefully aspirate the supernatant and resuspend the cell pellet into either cryoprotective media below at appropriate cell density.
1) KnockOut™ Serum Replacement + 10% Dimethyl sulfoxide (DMSO)
2) Bambanker™ Serum-Free Cell Freezing Medium
25m
Storage:
- Aliquot the cell suspension into cryovials (typically 800 µL per vial) and place them in a Corning CoolCell or similar freezing container, then transfer the container to a -80 °C freezer.
- For long-term storage, the cryovials can then be transferred to liquid nitrogen tanks after 24:00:00 , once they have stabilized at the -80 °C temperature. An important feature of Bambanker Cell Freezing Media is its ability to enable the cryopreservation of cells for long-term storage at -80 °C freezer without stepwise freezing.
Thawing Procedure
Thawing Procedure
2d 0h 5m
2d 0h 5m
Preparation:
- Remove the cryovials from liquid nitrogen or the -80 °C freezer.
- Quickly thaw the cryovials by placing them in a 37 °C water bath, gently swirling to ensure uniform thawing.
Culture Recovery:
- After thawing, transfer the cell suspension to a tube containing prewarmed microglia maturation media.
- Centrifuge the cells at 1000 rpm, 00:05:00 to remove the cryoprotectant, which can be toxic to cells.
- Carefully aspirate the supernatant and resuspend the cell pellet in fresh maturation media. Transfer the cells to an appropriate plate or flask to allow them for recovery.
- To recover a cryopreserved culture, the cells need to be cultured for a few hours to 72:00:00 to recover their morphology and functionality.
2d 0h 5m
Post-Thaw Culture:
- Maintain the cells in a suitable microglia maturation media consisting of Advanced RPMI 1640 Medium, 2 mM GlutaMax, 100 ng/mL IL-34, and 10 ng/mL GM-CSF.
- Change the media every other day and check their viability regularly.
Protocol references
o Material References
Navarro et al., LRRK2 G2019S variant is associated with transcriptional changes in Parkinson's disease human myeloid cells under proinflammatory environment. bioRxiv. [Preprint]. 2024 May 31:2024.05.27.594821. doi: 10.1101/2024.05.27.594821).
Acknowledgements
This research was supported entirely by the Intramural research Program of the NIH, National institute on Aging and National Institute of Neurological Disorders and Stroke.