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ATCC-DYS0100 Human Induced Pluripotent Stem (IPS) Cells

ACS-1019

IMPORTANT: ATCC strongly recommends that  users download and read the ATCC® Stem Cell Culture Guide: Tips and Techniques for Culturing Stem Cells (www.atcc-guides.org/stemcell) before initiating their cultures.

ATCC-DYS0100 Human Induced Pluripotent Stem Cells (iPSCs) were derived from ATCC SCRC-1041 HFF-1, a human foreskin fibroblast cell line. The neonatal dermal fibroblasts were reprogrammed by the expression of OCT4, SOX2, KLF4 and MYC gene sequence using Sendai viral transduction. This cell line provides a “normal” control when designing iPSC experiments.

Product category
Human cells
Organism
Homo sapiens
Cell type
iPSC
Tissue
Skin; Foreskin
Disease
Normal
Applications
Stem cell research
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

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.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Stem Cell Dissociation Reagent

ACS-3010

Price: $98.00 ea

Dulbecco's Phosphate Buffered Saline (D-PBS), 1X

30-2200

Price: $16.00 ea

ROCK Inhibitor Y27632

ACS-3030

Price: $211.00 ea

DMEM: F-12 Medium

30-2006

Price: $24.00 ea

Fetal Bovine Serum (FBS)

30-2020

Price: $645.00 ea

CellMatrix Basement Membrane Gel

ACS-3035

Price: $221.00 ea

Pluripotent Stem Cell SFM XF/FF

ACS-3002

Price: $249.00 ea

Detailed product information

Characteristics

Derivation
ATCC SCRC-1041
Age
neonate
Ethnicity
Unknown
Gender
Male
Reprogramming method
Sendai viral expression of OCT4, SOX2, KLF4, and MYC genes

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
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® No. ACS-3002) which is a ready-to-use medium for serum-free and feeder-free iPSC culture.

Reagents for subculture
Stem Cell Dissociation Reagent (ATCC ACS-3010)
D-PBS (ATCC 30-2200)
ROCK Inhibitor Y27632 (ATCC ACS-3030)
Handling procedure

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

  1. Night before thawing iPSC cells – Thaw CellMatrix™ Basement Membrane Gel on ice in refrigerator or cold room (2°C to 8°C)
  2. One Hour Prior to Thawing the iPS Cells – Prepare coated plates as described.
  3. 30 Minutes Prior to Handling Cells – Pre-warm Pluripotent Stem Cell SFM XF/FF (stem cell culture medium) at 37°C for at least 30 minutes before adding to cells. If using ROCK Inhibitor Y27632, prepare stem cell culture medium supplemented with final concentration of 10 mM ROCK Inhibitor Y27632. Stem cell culture medium with ROCK inhibitor must be used immediately.

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: CellMatrix™ Gel will solidify in 15 to 30 minutes above 15° C. Keep CellMatrix™ Gel and labware on ice at all times to prevent the matrix from gelling prematurely. Calculate the appropriate CellMatrix™ volume per plate based on concentration and usage. The concentration of CellMatrix™ is found on the product label.

Example: 2 mL of Cell Matrix™ at 150 µg/mL is required to coat one 6 cm dish. To coat two 6 cm dishes, prepare as follows:
Dilute CellMatrix™ in DMEM:F12 at a working concentration of 150 µg/mL:
Protein concentration of CellMatrix™ (on product label): 14 mg/mL.
[(4 mL)(0.15 mg/mL)]/(14 mg/mL)= 0.043 mL
Add 43 µL CellMatrix™ directly into 4 mL DMEM: F12

  1. Thaw CellMatrix™ Gel in the refrigerator (2°C to 8°C), on ice, overnight.
  2. Dilute the thawed CellMatrix™ Gel to 150 µg/mL in cold DMEM: F-12 Medium (ATCC 30-2006) by directly adding the CellMatrix™ Gel to the medium on ice and mix well. 
  3. Immediately coat each 6-cm dish with 2 mL diluted CellMatrix Gel.
  4. Swirl dish gently to ensure that the entire dish is evenly covered.
Leave the coated dishes at 37°C for one hour. The plates are now ready for use.

Initiation of Cultures

  1. Rapidly thaw the cells by placing the cryovial in a 37°C water bath, swirling gently. Remove the cryovial from the water bath when only a few ice crystals are remaining.
  2. Sterilize the cryovial by rinsing with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  3. Using a 1 mL or 5 mL pipette, gently transfer the cell suspension to a 15 mL conical tube.
  4. Slowly add 4 mL stem cell culture medium drop-wise, to the conical tube. Rinse the cryovial by adding and removing an additional 1 mL of medium and transfer the liquid to the 15mL conical tube. Shake the conical tube gently to mix the cells while adding media. Do not break apart the aggregates into a single-cell suspension, as it is crucial to maintain the cells in aggregates.
  5. Centrifuge the cells at 200 x g for 5 minutes.
  6. Aspirate the supernatant and discard. Gently tap on the bottom of the tube to loosen the cell pellet.
  7. Add 1 mL of stem cell culture medium with ROCK Inhibitor Y27632. Gently resuspend the pellet by pipetting up and down 2 to 3 times with a 1 mL tip. Do not over pipette, as it is crucial to maintain the cells in aggregates.
  8. Aspirate the coating solution from the plates prepared in step 4 of the Protocol for Coating Plates section. Add 4 mL of stem cell culture medium with ROCK Inhibitor Y27632 to each of two 6 cm dishes.
  9. Seed 0.5 mL of cell aggregates onto the dishes prepared in step 8.
  10. Incubate the culture at 37°C in a suitable incubator. A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.    

Subculturing procedure

Cell culture dishes are coated with CellMatrix Basement Membrane Gel (ATCC® No. ACS-3035) to provide a surface for the attachment of iPSCs.

Coating Procedure:

  1. Thaw CellMatrix Gel on ice and swirl gently to mix. Important: CellMatrix Gel will solidify in 15 to 30 minutes above 15°C. Keep CellMatrix Gel, vials and pipette tips on ice at all times to prevent CellMatrix Gel from solidifying. If air bubbles form, they may be eliminated by centrifuging CellMatrix Gel at 300 x g for 10 minutes at 2°C to 8°C.
  2. Determine the appropriate volume per aliquot based on concentration and usage.
  3. Example: 2 mL of CellMatrix at 150 μg/mL is required to coat one 6-cm dish. To coat two 6-cm dishes, prepare as follows:

    Dilute CellMatrix in DMEM:F12 to a working concentration of 150 μg/mL. For instance, if the protein concentration of CellMatrix (on certificate of analysis) is 14 mg/mL, then: (4 mL) x (0.15 mg/mL)/(14 mg/mL) = 0.043 mL. Therefore, add 43 μL CellMatrix directly in 4 mL cold DMEM: F-12 Medium

  4. Cell culture dishes coated with CellMatrix Basement Membrane Gel should be incubated at 37°C for one hour. Aspirate coating solution and immediately plate the cells. It is critical that the coating does not dry out.
Volumes used in this protocol are for a 75 cm2 flask.

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.

ROCK Inhibitor Y27632 is not necessary each time the culture medium is changed. It is required when cells are recovering from thaw on CellMatrix Gel-coated dishes containing 5 mL Pluripotent Stem Cell XF/FF medium/6-cm dish.

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. 


Reconstitution of Stem Cell Dissociation Reagent

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.

  1. Dissolve the appropriate amount of Stem Cell Dissociation Reagent in DMEM: F-12 Medium to prepare a 0.5 U/mL working solution. Example: To prepare 40 mL of a 0.5 U/mL working solution: Specific activity of Stem Cell Dissociation Reagent (on certificate of analysis) =1.46 U/mg (40 mL) x (0.5 U/mL)/(1.46 U/mg) = 13.7 mg Dissolve 13.7 mg Stem Cell Dissociation Reagent in 40 mL DMEM: F-12 Medium. 
  2. Filter sterilize through a 0.22 μm filter membrane. 
  3. Aliquot into working volumes according to routine usage. 
  4. Store aliquots at -20°C for up to three months. Avoid repeated freezing and thawing. Thawed aliquots may be kept at 2°C to 8°C for up to two weeks. 

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.

  1. Warm an aliquot of Stem Cell Dissociation Reagent working solution to room temperature.
  2. Aspirate and discard the stem cell culture medium.
  3. Rinse the cells twice by adding and discarding 4 mL of DMEM:F12.
  4. Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish.
  5. Incubate at 37°C for 2 to 5 minutes.
  6. Aspirate the Stem Cell Dissociation Reagent and gently rinse the colonies with 4 mL of DMEM: F-12 Medium, taking care not to dislodge the cells during manipulation.  Aspirate the DMEM: F12 rinse and discard.
  7. Add 2 mL of stem cell culture medium to the dish, and detach the cells by pipetting up and down 2 to 3 times with a 1 mL tip. Take care not to over-pipette the culture into a single-cell suspension as single cells will not establish colonies after seeding
  8. Transfer the cell aggregates to a 15 mL conical tube.
  9. Add an additional 3 mL of stem cell culture medium to the dish to collect any remaining cells. Transfer this rinse to the 15 mL conical tube containing the cell aggregates.
  10. Centrifuge the cell aggregates at 200 x g for 5 minutes.
  11. Aspirate the supernatant and discard.
  12. Add 1 mL of stem cell culture medium. Gently resuspend the pellet by pipetting up and down 2 to 3 times with a 1 mL tip, maintaining the small cell aggregates. Take care not to over-pipette the culture into a single-cell suspension as single cells will not establish colonies after seeding.
  13. Plate the cells on CellMatrix Gel-coated dishes containing 5 mL Pluripotent Stem Cell XF/FF medium/6-cm dish.
  14. Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day. Passage the cells every 4 to 5 days (80% confluent).
Cryopreservation

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.

  1. Detach stem cell colonies from the dish as described in the recommended subculturing protocol (steps 1-11). Gently tap the bottom of the tube to loosen the cell pellet.
  2. Take the Stem Cell Freezing Media from storage and swirl to mix. Keep cold. Decontaminate by dipping in or spraying with 70% alcohol.
  3. Add 2 mL of cold Stem Cell Freezing Media to the tube. Gently resuspend the pellet by pipetting up and down 2 to 3 times with a 1 mL tip, maintaining the cell aggregates.
  4. Immediately transfer 1 mL each of the cell suspension into two labeled cryovials.
  5. Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. A cryopreservation container (e.g., CoolCell® freezing container) may also be used.
  6. The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.

Quality control specifications

Mycoplasma contamination
Not detected

History

Depositors
ATCC
Year of origin
2012

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

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.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

This material is subject to the following restrictions in addition to those outlined in the ATCC Material Transfer Agreement:

  1. iPS Label License 
  2. This product was generated using Sendai virus vectors from ID Pharma

For information on obtaining additional rights, please contact:

ATCC Licensing
Email: [email protected]

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Wren MC, et al. Frontotemporal dementia-associated N279K tau mutant disrupts subcellular vesicle traffickingand induces cellular stress in iPSC-derived neural stem cells. Mol Neurodegener 10:46, 2015. PubMed: 26373282

Frequently Asked Questions

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