ATCC-CYS0105 Human Induced Pluripotent Stem (IPS) Cells
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Cells contain Sendai virus (SeV) DNA sequences
ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
ATCC iPSCs have been adapted to feeder- and serum-free culture conditions.
The base medium for this cell line is Pluripotent Stem Cell SFM XF/FF (ATCC ACS-3002) which is a ready-to-use medium for serum-free and feeder-free iPSC culture.
To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If, upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –80°C. Storage at –80°C will result in loss of viability.
Preparation for Culture
Note: Addition of ROCK inhibitor has been shown to increase the survival rate during subcultivation and thawing of human iPSCs. The use of ROCK inhibitor may cause a transient spindle-like morphology effect on the cells. However, the colony morphology will recover after subsequent media change without ROCK inhibitor.
Protocol for Coating Plates
Important: Cell Basement Membrane Gel will solidify in 15 to 30 minutes above 15° C. Keep Cell Basement Membrane Gel and labware on ice at all times to prevent the matrix from gelling prematurely.
Calculate the appropriate Cell Basement Membrane Gel volume per plate based on concentration and usage. The concentration of Cell Basement Membrane Gel is found on the product label.
Example: 2 mL of Cell Basement Membrane Gel at 150 µg/mL is required to coat one 6 cm dish. To coat two 6 cm dishes, prepare as follows:
Dilute Cell Basement Membrane Gel in DMEM:F12 at a working concentration of 150 µg/mL:
Protein concentration of Cell Basement Membrane Gel (on product label): 14 mg/mL.
(4 mL) x (0.15 mg/mL) = 0.043 mL
Add 43 µL Cell Basement Membrane Gel directly in 4ml DMEM: F12.1. Thaw Cell Basement Membrane Gel in the refrigerator (2°C to 8°C), on ice, overnight.
Initiation of Cultures
Cell culture dishes are coated with Cell Basement Membrane Gel (ATCC ACS-3035) to provide a surface for the attachment of iPSCs.
Post thaw day 1, perform a 100% medium change and remove all cells that did not attach. Perform a 100% medium change every day. Passage the cells every 4 to 5 days (80% confluent) at an appropriate split ratio (a 1:4 split ratio is recommended). If the colonies are close to, or touching each other, the culture is overgrown. Overgrowth will result in differentiation.
This protocol is designed to passage stem cell colonies cultured in a 6 cm dish, using Stem Cell Dissociation Reagent (ATCC ACS-3010) to detach the cell colonies. The recommended spilt ratio is 1:4. Volumes should be adjusted according to the size and number of the tissue culture vessels to be processed.
Lyophilized proteins tend to be hygroscopic. Bring the vial of Stem Cell Dissociation Reagent to room temperature before opening. The vial should not be cool to the touch. Once opened, the lyophilized material should be stored desiccated. The specific activity of the reagent is found on the certificate of analysis. Dissolve the appropriate amount of Stem Cell Dissociation Reagent in DMEM: F-12 Medium to prepare a 0.5 U/mL working solution.
Note: Addition of ROCK inhibitor has been shown to increase the survival rate. The use of ROCK inhibitor may cause a transient spindle-like morphology effect on the cells. However, the colony morphology will recover after subsequent media change without ROCK inhibitor.
Stem Cell Freezing Medium (ATCC ACS-3020)
This product sheet describes the use of ATCC's system for serum-free/feeder free maintenance and expansion of human iPS cells. For detailed protocols covering all aspects of this culture system as well as related methods for serum-free, feeder-dependent culture, refer to the ATCC Stem Cell Culture Guide.
For optimal results, cryopreserve stem cell colonies when the cell cultures are 80% confluent. This protocol is designed to cryopreserve stem cell colonies cultured in a 6 cm dish.
The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.
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Edwards SL. A Multiwell Cardiac μGMEA Platform for Action Potential Recordings from Human iPSC-Derived Cardiomyocyte Constructs. Stem Cell Reports 11:1-15, 2018. PubMed: 30033088