Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium
July 15th, 2010
This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.
The overall goal of this procedure is to cryopreserve and recover IPS cells in feeder based or feeder free culture conditions. This is accomplished by first beginning packaging your IPCs as you normally would. The second step of the procedure is to transfer the cells into serum free freezing medium.
The third step of the procedure is to cryopreserve as you normally would. The final step of the procedure is to thaw the cells and begin maintenance. Ultimately, results can be obtained that show successful freezing and thawing of IPS cells in serum free freezing medium through healthy morphology and recovery post thaw.
Hi, I'm Kate Wagner at the GCO site at Life Technologies. Visual demonstration of this protocol is critical because IPCs can be very sensitive to freezing and T fine. What makes this protocol easier is that knockout serum replacement can be used throughout your entire workflow.
Today the KSR family of products includes a full list of media and reagents for feeder based feed free and xeno free culture of your embryonic stem cells and induced pluripotent stem cells. KSR can be used for derivation growth, cryo-preservation, expansion, and the first initial steps of differentiation. The following video will show you thaw and freezing yourselves using KSR in a Peter free machine.
To begin this procedure. First warm in an appropriate amount of complete feeder free medium to 37 degrees Celsius in a water bath aspirate spent medium from the culture vessel, which is a 60 millimeter dish. In this case, rinse the human induced pluripotent stem cells twice with DPBS.
Then add one milliliter of dispa to the dish. Incubate the dish at 37 degrees Celsius for three minutes. After that, aspirate the dispa solution and gently wash the cells with DPBS.
Next, add complete feeder free medium to cover the dish and use a cell scraper to gently scrape the cells transfer cells to a sterile 15 milliliter centrifuge tube. Rinse the dish with complete feed of free medium, gently spraying off any cells that have not detached. Transfer the rinse solution to the same 15 milliliter tube containing the cells.
Rinse the dish a second time with medium and pull that rinse solution with the cells pellet cells by centrifugation at 1000 RPM for three minutes at room temperature. After centrifugation, carefully aspirate and discard the supernatant without disturbing the cell pellet. Gently flick the tube to fully dislodge the cell pellet from the bottom of the tube.
Then add one milliliter of freezing medium A, using a five milliliter serological, pipette, and resuspend the cells by gently pipetting up and down. Following uniform suspension of clumps, add one milliliter of freezing medium B to the tube in a dropwise manner while gently swirling the cell suspension to mix. Allocate one milliliter of cell suspension into each cryo vile freeze cells at minus 80 degrees Celsius overnight in an isopropanol chamber.
On the following day, transfer the vials of cells into a liquid nitrogen tank for long-term storage. Rapidly thaw the frozen vial of cells by placing it in a 37 degree Celsius water bath for about three to four minutes until just a small frozen chunk remains in the vial. After removing the vial from the water bath spray with 70%isopropanol to decontaminate it, then use a five milliliter pipette to aseptically transfer the entire contents of the vial into a 15 milliliter conical tube.
The next step may be the most difficult aspect of the procedure. The feeder free medium must be added slowly and carefully to the cells. To reduce osmotic shock Slowly add four milliliters of complete feeder free medium dropwise to the cells in the 15 milliliter conical tube.
While adding the medium gently move the tube back and forth to mix the cells. This will reduce osmotic shock to the cells. Complete feeder free medium may be replaced with knockout serum replacement containing meth conditioned medium or knockout serum replacement medium for use with feeders, refer to the protocol text for media preparation instructions.
Once all four milliliters of medium have been added, centrifuge the cells at 1000 RPM for five minutes at room temperature. After centrifugation, carefully aspirate and discard the sane without disturbing the cell pellet. Gently resuspend the cell pellet in four milliliters of complete feeder free medium.
Using a five milliliter pipette, gently add the cell suspension dropwise to a gelt trucks coated 60 millimeter culture dish. Place the culture dish in a 36 to 38 degrees Celsius incubator with a humidified atmosphere of four to 6%carbon dioxide in air. Carefully swirl the dish in a north to south, east to west pattern to evenly distribute the cells.
24 hours post thaw. Replace the medium in the dish with fresh medium. Replace the spent medium daily thereafter until cells become approximately 70 to 80%confluent.
This contrast image shows human induced pluripotent stem cells grown on gelt TRX coated culture dishes using complete serum replacement, feeder free medium. The IPC's exhibit morphology similar to human embryonic stem cells characterized by large nuclei and scant cytoplasm. An example of cells post thaw and knockout serum replacement.
Feeder free medium on gelt TrackX is shown here. When following this procedure, it is important to remember to keep all your cell colonies the same size. When cryo preserving this improves cell recovery.
So that's it. Thanks for watching and good luck with your experiments.
Overview
This protocol outlines the procedure for cryopreserving human induced pluripotent stem (iPS) cells using KnockOut SR cryopreservation medium and recovering them in either complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium. The method emphasizes the importance of visual demonstration due to the sensitivity of iPS cells during freezing and thawing.
Key Study Components
Area of Science
- Cell Biology
- Stem Cell Research
- Cryopreservation Techniques
Background
- Human iPS cells are sensitive to freezing and thawing processes.
- KnockOut SR products facilitate various stages of stem cell culture.
- Proper cryopreservation is crucial for maintaining cell viability.
- Feeder-free and feeder-based culture conditions can be utilized for iPS cells.
Purpose of Study
- To provide a reliable protocol for cryopreserving iPS cells.
- To ensure successful recovery of cells post-thaw.
- To demonstrate the use of KSR products throughout the workflow.
Methods Used
- Preparation of iPS cells for cryopreservation.
- Use of serum-free freezing medium for cell preservation.
- Cryopreservation at -80 degrees Celsius followed by storage in liquid nitrogen.
- Thawing and resuspending cells in complete feeder-free medium.
Main Results
- Successful freezing and thawing of iPS cells demonstrated by healthy morphology.
- Cells maintained viability and growth in both feeder-free and feeder-based conditions.
- Protocol effectiveness confirmed through visual assessment of cell recovery.
- Maintaining uniform cell colony size improved recovery rates.
Conclusions
- The protocol provides a systematic approach to cryopreserving iPS cells.
- KnockOut SR products are effective for maintaining cell health during the process.
- Visual demonstrations enhance understanding and execution of the protocol.
Frequently Asked Questions
Executive Industry Relevance
Reliable cryopreservation and recovery of human iPS cells using feeder-free, defined media directly addresses scalability and standardization challenges in early discovery and translational research. This protocol enables consistent pluripotent cell banking, supporting robust assay development and downstream applications in drug discovery portfolios. Feeder-free workflows reduce biological variability, enhancing predictive confidence and portfolio triage at key inflection points.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables consistent banking of pluripotent cells for repeated hypothesis testing and pathway interrogation.
- Reduces biological variability by eliminating feeder layers, supporting functional target validation.
- Facilitates mechanistic de-risking by maintaining cell health and pluripotency post-thaw.
- Supports predictive confidence in early-stage screening and triage decisions.
Screening & Assay Development
- Provides a standardized, feeder-free system for preparing validated iPS cells for high-throughput assays.
- Improves reproducibility and scalability of cell-based assays by using defined media components.
- Enables reliable compound evaluation by ensuring healthy, undifferentiated cell recovery.
- Supports platform reuse and assay standardization across discovery teams.
Translational & Preclinical Research
- Maintains disease-relevant cell models for translational biomarker studies when using patient-derived iPS cells.
- Ensures continuity from discovery through preclinical validation by supporting robust cell recovery and expansion.
- Reduces risk of batch-to-batch variability in preclinical model development.
- Supports risk-adjusted advancement decisions by providing reliable cell sources.
Pipeline & Workflow Integration
This cryopreservation and recovery protocol fits from early discovery through preclinical research, enabling seamless transitions between banking, expansion, and assay deployment stages.
- Discovery Biology: Supports hypothesis testing and biological de-risking by providing healthy, pluripotent cells on demand.
- Screening: Delivers assay-ready iPS cells with reproducible morphology and viability for quantitative readouts.
- Analytics: Enables consistent measurement of cell health and recovery, supporting cross-condition comparisons.
- Translational Research: Maintains continuity for disease modeling and biomarker alignment when using patient-derived lines.
- Enterprise Reuse: Establishes a reusable, feeder-free workflow for pluripotent cell management across programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in cell-based studies.
- Operational Value: Standardizes cryopreservation and recovery, improving reproducibility and scalability.
- Strategic Value: Enables better go/no-go decisions and capital efficiency by reducing late-stage biological risk.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of discovery and translational programs.
Implementation Considerations
- Requires expertise in pluripotent stem cell handling and feeder-free culture techniques.
- Needs access to defined media, cryopreservation equipment, and controlled thawing infrastructure.
- Demands cross-team standardization of cell colony size and handling procedures for optimal recovery.
- Adaptation may be needed for different iPS cell lines or disease models.
- Careful osmotic shock management is critical for maintaining cell viability post-thaw.
