hESC BG01V (ATCC® SCRC-2002)

Organism: Homo sapiens, human  /  Cell Type: embryonic stem cell  /  Tissue: inner cell mass  / 

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Organism Homo sapiens, human
Tissue inner cell mass
Cell Type embryonic stem cell
Product Format frozen
Morphology spherical colony
Biosafety Level 1

[This cell line is not known to harbor an agent known to cause disease in healthy adult humans. Handle as a potentially biohazardous material under at least Biosafety Level 1 containment. This cell line has been screened and found negative for Hepatitis B and C, human immunodeficiency virus 1 and 2, Herpes Simplex Virus 1 and 2, Epstein Barr Virus, Human T-cell Lymphotrophic Virus I/II, and Cytomegalovirus. ATCC recommends that appropriate safety procedures be used when handling all cell lines, especially those derived from human or other primate material.  Detailed discussions of laboratory safety procedures are provided in Laboratory Safety: Principles and Practice (Fleming et al., 1995) the ATCC manual on quality control (Hay et al., 1992), the Journal of Tissue Culture Methods (Caputo, 1988), and in the U.S. Government Publication, Biosafety in Microbiological and Biomedical Laboratories , 4th ed. HHS Publication No. (CDC) 93-8395. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 1999. The entire text is available online at http://www.cdc.gov/biosafety/publications/bmbl5/index.htm.

This cell line is sent with the condition that you are responsible for its safe storage, handling and use.  ATCC is not liable for damages or injuries resulting from receipt and/or use of an ATCC culture.]

Age embryo, blastocyst
Applications
BG01V cells are pluripotent and can differentiate to representatives of the three primary germ layers.
Storage Conditions liquid nitrogen vapor phase
Karyotype 49, XXY, +12, +17
Derivation
BG01V is a human embryonic stem cell line with an abnormal karyotype. BG01V was derived from the wild-type, parental hESC line BG01. (Mitalipova M, Calhoun J, Shin S, Wininger D, Schulz T, Noggle S, Venable A, Lyons I, Robins A, Stice S. (2003). Human embryonic stem cell lines derived from discarded embryos. Stem Cells, 21(5), 521-6. [PMID 12968106]) (Zeng X, et al. Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells 22: 292-312, 2004. PubMed: 15153607)
Antigen Expression

The cells stain positive for pluripotency markers and alkaline phosphatase activity. 

Comments
BG01V is a human embryonic stem cell line with an abnormal karyotype.  Despite the abnormal karyotype, when grown on murine embryonic feeders (MEFs) these colonies exhibit uniform morphology, a predictable growth rate, and are easy to maintain in culture. The cells stain positive for pluripotency markers and alkaline phosphatase activity.
Complete Growth Medium 1:1 Mixture of Dulbecco's Modified Eagles Medium and Ham's F-12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate (ATCC 30-2006) supplemented with 2.0 mM L-Alanyl-L-Glutamine (ATCC 30-2115), 0.1 mM Non-essential amino acids (ATCC 30-2116), 0.1 mM 2-mercaptoethanol (Sigma Catalog No. M-7522) and 4 ng/ml bFGF (R& D Systems Catalog No. 233-FB), 80%; Knockout serum replacement (Invitrogen Catalog No. 10828), 5%; fetal bovine serum (ATCC SCRR-30-2020), 15%
Subculturing

To insure the highest level of viability, be sure to warm media to 37°C before using it on the cells.  The passaging ratio depends on the density/confluency of the colonies.  It ranges between 1:3 and 1:6.  Note: If the colonies are close to or touching each other the culture is overgrown. Overgrowth will result in differentiation.

  1. At least 24 hours prior to each passage, plate treated MEFs onto the culture vessels to be used. Base the number of dishs/flasks to be used on the passaging ratio.  Refer to Table 1 to determine the correct plating density for the feeders.
  2. Prepare 0.5 mg/mL or ~200 units/mL Collagenase IV solution (Invitrogen 17104-019) in DMEM/F12 and sterile filter using 0.22 µm low-protein binding filter. Check the units/mg for each lot of powder.
  3. Remove medium from cells. Add appropriate volume of Collagenase IV solution. Refer to Table 2 to determine the correct amount.
  4. Incubate at 37°C for up to 2 hours.
  5. Check the cells after the first 30 minutes and then every 15 minutes. When the majority of the hESC colonies have completely detached or the edges of the colonies have rounded up, add appropriate amount of DMEM/F12 (Table 2) and wash gently using a pipette.  Under optimal conditions, all the colonies can be washed off with feeder cells left behind.  If some colonies are still attached, gently scrape the surface area with the tip of a 5 mL pipette if necessary.
  6. Collect cell suspensions into a 50 mL conical tube.
  7. Centrifuge for 5 minutes at 200 x g at 25°C.
  8. Remove the supernatant and resuspend in complete growth medium.  Pipette up and down to break the colonies to smaller clumps and evenly distribute cells to feeder-covered dishes/flasks.
  9. Add complete growth medium to each tissue culture vessel to achieve the appropriate final volume. Refer to Table #2 to find the appropriate volume based on surface area.

Table 2. Reagent Quantities

Flask/Plate

Growth Area (cm²)

Collagenase (ml)

DMEM/F12  (ml)

Growth Medium (ml)

T225

225

10

10

30

T75

75

3.0

5

12

T25

25

2

5

6

6 well

9.5

0.5

1

3

Medium Renewal

Every day after the first 48 hours

Complete Growth Medium for Feeder Cells

The feeder cells are grown in DMEM (ATCC #30-2002) supplemented with 15% FBS (ATCC # 30-2020).

Cryopreservation
To freeze the cells:
  1. Follow the Subculturing Procedure above and use Collagenase IV to dissociate the cells.
  2. Centrifuge the cell suspension for 5 minutes at 200xg at 25°C.
  3. Resuspend the pellet in a 1:1 solution of 50% complete growth medium and 50% FBS. The total volume should be 0.5 ml times the number of vials to be frozen. Determine the number of vials using Table 3.
  4. Pipette up and down to break the colonies into small clumps. P1000 tips are used to efficiently break up the colonies.
  5. Slowly add an equal volume of complete growth medium with 20% DMSO. Mix gently.
  6. Evenly distribute 1 mL of the cell suspension into each cryovial.
  7. Store the vials in Styrofoam boxes at -80°C. Transfer the vials to liquid nitrogen 24 hours later.

 Table 3. # of Vials at 80-90% confluency

Flask/Plate

Growth Area (cm²)

# of vials

T225

225

16

T75

75

5

T25

25

2

6 well

9.5

1


Cryoprotectant Medium: Complete growth medium supplemented with 20% FBS and 10% DMSO. Follow the two step process in the Cryopreservation protocol. Cell-culture tested DMSO is available as ATCC Catalog No. 4-X.
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Name of Depositor BresaGen, Inc.
References

Zeng X, et al. Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells 22: 292-312, 2004. PubMed: 15153607

Brimble S, Zeng X, Weiler D, Luo Y, Liu Y, Lyons I, Freed W, Robins A, Rao MS and Schulz TC (2004). Karyotypic stability, genotyping, differentiation, feeder free maintenance and gene expression sampling in three human embryonic stem cell lines derived prior to August 9th 2001. Stem Cells and Development, 13(6), PMID 15684826

Mitalipova M, Calhoun J, Shin S, Wininger D, Schulz T, Noggle S, Venable A, Lyons I, Robins A, Stice S. (2003). Human embryonic stem cell lines derived from discarded embryos. Stem Cells, 21(5), 521-6. [PMID 12968106]

Schulz TC, Noggle S, Palmarini G, Weiler D, Lyons I, Pensa K, Meedeniya A, Davidson B, Lambert N, Condie B (2004). Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum Free Suspension Culture. Stem Cells, 22(7), PMID: 15579641

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988

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
Restrictions

Prior to purchase, for-profit commercial institutions must obtain a license agreement. For instructions on how to proceed, please contact the ATCC Office of Licensing and Business Development at licensing@atcc.org or 703 365 2773.

References

Zeng X, et al. Properties of pluripotent human embryonic stem cells BG01 and BG02. Stem Cells 22: 292-312, 2004. PubMed: 15153607

Brimble S, Zeng X, Weiler D, Luo Y, Liu Y, Lyons I, Freed W, Robins A, Rao MS and Schulz TC (2004). Karyotypic stability, genotyping, differentiation, feeder free maintenance and gene expression sampling in three human embryonic stem cell lines derived prior to August 9th 2001. Stem Cells and Development, 13(6), PMID 15684826

Mitalipova M, Calhoun J, Shin S, Wininger D, Schulz T, Noggle S, Venable A, Lyons I, Robins A, Stice S. (2003). Human embryonic stem cell lines derived from discarded embryos. Stem Cells, 21(5), 521-6. [PMID 12968106]

Schulz TC, Noggle S, Palmarini G, Weiler D, Lyons I, Pensa K, Meedeniya A, Davidson B, Lambert N, Condie B (2004). Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum Free Suspension Culture. Stem Cells, 22(7), PMID: 15579641

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988