KYOU-DXR0109B Human Induced Pluripotent Stem (IPS) Cells [201B7] (ATCC® ACS-1023)

Organism: Homo sapiens, human  /  Cell Type: Yamanaka retrovirus reprogrammed hiPSC  /  Tissue: Derived from dermal fibroblasts  /  Disease: Normal

Permits and Restrictions

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Organism Homo sapiens, human
Tissue Derived from dermal fibroblasts
Cell Type Yamanaka retrovirus reprogrammed hiPSC
Product Format frozen
Biosafety Level 2 [It is the responsibility of the investigator to determine appropriate safety procedures for use with this material. As a reference, laboratory safety is discussed in the publication Biosafety in Microbiological and Biomedical Laboratories and can be accessed by searching "BMBL" at www.cdc.gov.]
Disease Normal
Age 36 years
Gender Female
Ethnicity Non-Hispanic/Latino White
Intended Use This product is intended for research use only. It is not intended for any animal or human therapeutic or diagnostic use.
Shipping Information Frozen
Storage Conditions liquid nitrogen vapor phase (-130°C or colder)
Derivation KYOU-DXR0109B Human Induced Pluripotent Stem Cells (iPSCs) were derived at Kyoto University from dermal fibroblasts obtained from a healthy donor.
Clinical Data 36 years
Female
Non-Hispanic/Latino White
Comments Depositor cell line designation: 201B7
Subculturing

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; it is recommended when the cells are being passaged.

This protocol is designed to passage stem cell colonies cultured in a 6 cm dish, using EDTA Dissociation Reagent 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. 

EDTA Dissociation Reagent
500ul 0.5M EDTA
0.9g NaCl
in 500ml Calcium/Magnesium free PBS
Sterile filter and store at 4°C

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.

  1. Warm an aliquot of EDTA 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 D-PBS.
  4. Add 2 mL of EDTA Dissociation Reagent working solution to the dish.
  5. Incubate at 37°C for 2 to 5 minutes.
  6. Aspirate the EDTA 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 with ROCK Inhibitor Y27632 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 with ROCK Inhibitor Y27632 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 with ROCK inhibitor Y27632. 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 as desired on feeder or feeder-free cultures.
  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 5-6 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. An isopropanol freezing container also may 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.
Culture Conditions Pluripotent Stem Cell SFM XF/FF, ATCC ACS-3002
CellMatrix™ Basement Membrane Gel, ATCC ACS-3035
Cells per Vial ≥ 30 colonies after 5 days when seeded as directed
STR Profile confirmed by  STR analysis and karyotype by G-banding
Sterility Tests

Negative for bacterial, fungal, and mycoplasma

Viability passes
Name of Depositor Shinya Yamanaka, Ph.D.
Year of Origin 2007
References

Takahashi K, et.al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5): 861-872, 2007. PubMed: 18035408

Chen G, et.al. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7(2): 240-248, 2010. PubMed:20682449

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
FAQ's
  1. ACS-1023 iPS cells following passage


    Date Updated: 1/23/2014

  2. iPSC - ROCK inhibitor


    Date Updated: 1/23/2014

  3. iPSC - Karyotypic instability


    Date Updated: 1/23/2014

  4. iPSC - Long recovery time of iPSC


    Date Updated: 1/23/2014

  5. Derivation of KYOU_DXR0109B iPSC line


    Date Updated: 1/23/2014

Restrictions

This product may be used by investigator for research purposes subject to the Limited Use Label License and any additional third party terms. This product is subject to claims under U.S. Patent Nos. 8,058,065 and 8,048,999, pending patent applications, and foreign counterparts thereof. For information on obtaining additional rights, please contact licensing@atcc.org

References

Takahashi K, et.al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5): 861-872, 2007. PubMed: 18035408

Chen G, et.al. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7(2): 240-248, 2010. PubMed:20682449