ATCC-DYS0100 Human Induced Pluripotent Stem (IPS) Cells (ATCC® ACS-1019)

Organism: Homo sapiens, human  /  Cell Type: sendai virus reprogrammed hiPSC  /  Tissue: derived from foreskin fibroblast  /  Disease: normal

Permits and Restrictions

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Organism Homo sapiens, human
Tissue derived from foreskin fibroblast
Cell Type sendai virus 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 newborn
Gender male
Ethnicity unknown
Storage Conditions Liquid Nitrogen Vapor Phase (-130°C or colder)
Derivation ATCC SCRC-1041
Complete Growth 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. 

Subculturing

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; 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 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.
Culture Conditions
Pluripotent Stem Cell SFM XF/FF, ATCC ACS-3002
CellMatrix Basement Membrane Gel, ATCC ACS-3035
Name of Depositor ATCC
Year of Origin 2012
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
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.  In addition, this product was generated using the proprietary technology of the DNAVEC Corporation, including, without limitation, recombinant Sendai virus vectors. For information on obtaining additional rights, please contact licensing@atcc.org