| 產品名稱 |
ATCC-BYS0111 Human [Hispanic Male] Induced Pluripotent Stem (IPS) Cells |
| 商品貨號 |
B230721 |
| Organism |
Homo sapiens, human |
| Tissue |
bone marrow CD34+ cells |
| Cell Type |
sendai virus reprogrammed hiPSC |
| Product Format |
frozen |
| Biosafety Level |
2 [Cells contain Sendai viral DNA sequences]
[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.]
Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. |
| Disease |
Normal |
| Age |
24 |
| Gender |
Male |
| Ethnicity |
Hispanic |
| Storage Conditions |
Liquid Nitrogen Vapor Phase (-130°C or colder) |
| Karyotype |
Normal karyotype, 46 XY |
| Derivation |
ATCC-BYS0111 Human Induced Pluripotent Stem Cells (iPSCs) were derived from bone marrow CD34+ cells obtained from a healthy Hispanic male donor. |
| Antigen Expression |
SSEA4, Tra-1-60 (expressed on undifferentiated hiPSCs) >85%; SSEA1 (expressed on differentiated hiPSCs) < 15%. |
| 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:
- 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.
- Determine the appropriate volume per aliquot based on concentration and usage.
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
- 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 cm 2 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.
- 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.
- Filter sterilize through a 0.22 μm filter membrane.
- Aliquot into working volumes according to routine usage.
- 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.
- Warm an aliquot of Stem Cell Dissociation Reagent working solution to room temperature.
- Aspirate and discard the stem cell culture medium.
- Rinse the cells twice by adding and discarding 4 mL of DMEM:F12.
- Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish.
- Incubate at 37°C for 2 to 5 minutes.
- 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.
- 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.
- Transfer the cell aggregates to a 15 mL conical tube.
- 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.
- Centrifuge the cell aggregates at 200 x g for 5 minutes.
- Aspirate the supernatant and discard.
- 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.
- Plate the cells on CellMatrix Gel-coated dishes containing 5 mL Pluripotent Stem Cell XF/FF medium/6-cm dish.
- 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 |
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:
- 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.
- Determine the appropriate volume per aliquot based on concentration and usage.
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
- 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 cm 2 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.
- Warm an aliquot of EDTA Dissociation Reagent working solution to room temperature.
- Aspirate and discard the stem cell culture medium.
- Rinse the cells twice by adding and discarding 4 mL of D-PBS.
- Add 2 mL of EDTA Dissociation Reagent working solution to the dish.
- Incubate at 37°C for 2 to 5 minutes.
- 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.
- 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.
- Transfer the cell aggregates to a 15 mL conical tube.
- 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.
- Centrifuge the cell aggregates at 200 x g for 5 minutes.
- Aspirate the supernatant and discard.
- 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.
- Plate the cells as desired on feeder or feeder-free cultures.
- 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).
|
| Cells per Vial |
≥ 30 colonies after 5 days when seeded as directed |
| STR Profile |
Amelogenin: X,Y
CSF1PO: 10:12
D13S317: 8,12
D16S539: 12
D5S818: 11,12
D7S820: 9,11
THO1: 7,8
TPOX: 8,11
vWA: 15,16 |
| Sterility Tests |
No growth after 21 days
Mycoplasma - None Detected
Zero Footprint Confirmation - residual Sendai virus not detected |
| Viral Testing |
Hepatitis B: Negative
HPV: Negative
HIV1: Negative
CMV: Negative
EBV: Negative |
| Functional Tests |
Pluritest - Pluripotency Score >20, Novelty Score < 1.67 |
| Name of Depositor |
ATCC |
| Year of Deposit |
2013 |
