ATCC-BXS0114 Human [African American Female] Induced Pluripotent Stem Cells (ATCC® ACS-1028)

Organism: Homo sapiens, human  /  Cell Type: sendai virus reprogammed hiPSC  /  Tissue: Derived from Bone Marrow CD34+ cells  /  Disease: Normal

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Organism Homo sapiens, human
Tissue Derived from Bone Marrow CD34+ cells
Cell Type sendai virus reprogammed 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 31
Gender Female
Ethnicity African American
Storage Conditions Liquid Nitrogen Vapor Phase (-130°C or colder)
Karyotype Normal karyotype, 46 XY
Derivation

ATCC-BXS0114 Human Induced Pluripotent Stem Cells (iPSCs) were derived from bone marrow CD34+ cells obtained from a healthy African American Female donor.

Antigen Expression SSEA4, Tra-1-60 (expressed on undifferentiated hiPSCs) >85%; SSEA1 (expressed on differentiated hiPSCs) < 15%.
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 recommended when the cells are recovering from thaw or being passaged.

This protocol is designed to passage stem cell colonies cultured in a 6 cm dish, using Stem Cell Dissociation Reagent to detach the cell colonies. Stem Cell Dissociation Reagent is stored as a 0.5 U/mL working solution at -20°C. 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. 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 CoA): 1.46 U/mg

(40 mL)(0.5 U/mL) = 13.7 mg.

(1.46 U/mg)

Dissolve 13.7 mg Stem Cell Dissociation Reagent in 40 mL DMEM: F-12.

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 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 D-PBS.
  4. Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish.
  5. Incubate at 37°C for 10 to 15 minutes or until the edges of the individual colonies begin to loosen and fold back. View the dish under the microscope starting at 5 minutes as incubation time will vary depending on the cell line and colony size.
  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 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. If cells are not detaching, use a cell scraper to detach the cells.
  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 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 1mL 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 (ATCC ACS-3020) 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

ROCK Inhibitor Y27632, ATCC ACS-3030

Stem Cell Dissociation Reagent, ATCC ACS-3010


Cells per Vial ≥ 30 colonies after 5 days when seeded as directed
STR Profile Consistent with expected
Sterility Tests

No growth after 21 days

Mycoplasma - None Detected

Zero Footprint Confirmation - None Detected

Viral Testing
Hepatitis B: Negative
HPV: Negative
HIV1: Negative
CMV: Negative
EBV: Negative
Functional Tests Pluripotency Score >20, Novelty Score < 1.67
Name of Depositor ATCC
Year of Deposit 2013
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. iPSC - ROCK inhibitor


    Date Updated: 1/23/2014

  2. iPSC - Karyotypic instability


    Date Updated: 1/23/2014

  3. iPSC - iPS cells following passage


    Date Updated: 1/23/2014

  4. iPSC - Long recovery time of iPSC


    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.  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 contactlicensing@atcc.org