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2 citations
Background: Embryonic Stem Cells

Kit Summary

Conditioned media to support growth and maintain the pluripotency of embryonic and induced pluripotent stem cells.

  • Eliminates the need for a MEF feeder layer
  • Lot-to-lot consistency decreases experimental variability
  • Addition of FGF basic generates complete media

Why Culture Stem Cells Using MEF Conditioned Media?

Feeder layers of mouse embryonic fibroblasts (MEF) support stem cell growth and the maintenance of pluripotency by secreting growth factors, cytokines, and nutrients. However, the use of MEFs as a feeder layer can be laborious and MEF contamination is a significant concern.   See Details

MEF conditioned media represents an efficient alternative to MEF feeder cells and is one of the mostly widely used feeder-free systems for embryonic stem cell culture. MEF Conditioned Media includes the soluble factors required to support stem cell growth and pluripotency.

Mouse Embryonic Fibroblast (MEF) Conditioned Media:

  • Widely used feeder-free system for culturing embryonic and induced pluripotent stem cells.
  • Lot-to-lot consistency decreases experimental variability.
  • Eliminates the need for a MEF feeder layer.
  • Free of mycoplasma and microbial contamination.
  • Generate complete media by adding FGF basic (4ng/mL) (Catalog # 233-FB or 4114-TC).

Kit Contents

  • 100 mL MEF Conditioned Media

Stability and Storage

Upon receipt, store the conditioned media at < -20 °C in a manual defrost freezer. When ready to use, thaw the solution overnight at 2 °C to 8 °C in the dark. Aliquot and store the unused portions at < -20 °C in a manual defrost freezer. Long-term storage at 2 °C to 8 °C is not recommended. Avoid repeated freeze-thaw cycles, and do not use past the expiration date.

 

Data Examples


View Larger Image

Induced Pluripotent Stem Cells Grown in MEF Conditioned Media Express Pluripotent Stem Cell Markers SSEA-4. Oct-3/4, Oct-4A, and E-Cadherin. Human iPS2 stem cells were cultured in MEF Conditioned Media (Catalog # AR005) supplemented with 4 ng/mL Recombinant Human FGF basic (Catalog # 233-FB or Catalog # 4114-TC). A. Expression of SSEA-4 was detected using a Mouse Anti-Human/Mouse SSEA-4 Monoclonal Antibody (Catalog # MAB1435) followed by a NorthernLights™ (NL) 493-conjugated Donkey Anti-Mouse Secondary Antibody (Catalog # NL009, green). Expression of Oct-3/4 was detected using a Goat Anti-Human Oct-3/4 Antigen Affinity Purified Polyclonal Antibody (Catalog # AF1759) followed by NL557-conjugated Donkey Anti-Goat IgG (Catalog NL001, red). The nuclei were counterstained with DAPI. B. Expression of Oct-4A was detected using Mouse Anti-Human Oct-4A Monoclonal Antibody (Catalog # MAB17591) followed by a NorthernLights™ 493-conjugated Donkey Anti-mouse Polyclonal Antibody (Catalog # NL009, green). Expression of E-Cadherin was detected using a Goat Anti-Human E-Cadherin Antigen Affinity Purified Polyclonal Antibody (Catalog # AF648) followed by a NL557-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL001, red). The nuclei were counterstained with DAPI. View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips.


View Larger Image

Human Embryonic Stem Cells Cultured in Mouse Embryonic Fibroblast Conditioned Media Express Pluripotent Markers SSEA-4 and Oct-3/4. A. Human embryonic stem cells were cultured in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog #AR005) supplemented with 4 ng/mL Recombinant Human FGF basic (Catalog # 233-FB). SSEA-4 and Oct-3/4 were detected using a Mouse Anti-Human/Mouse SSEA-4 Monoclonal Antibody (Catalog # MAB1435, red) and a Goat Anti-Human Oct-3/4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1759, green). The nuclei were counterstained with DAPI (blue). B. Human embryonic stem cells were cultured in MEF Condition Media supplemented with 4 ng/mL Recombinant Human FGF basic. SSEA-4 and Oct-3/4 were detected as described above. The nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips.

Image courtesy of Dr. Frank Soldner of the National Institutes of Health.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, MEF Conditioned Media can be used to culture human pluripotent stem cells using the following procedure:

  • Supplement MEF Conditioned Media with 4 ng/mL FGF basic
  • Culture cells in supplemented media
  • Replace media daily and passage cells as needed

Reagents Provided

Reagents Supplied in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005)

  • 100 mL MEF Conditioned Media

Other Supplies Required

Reagents

  • Recombinant Human FGF basic (Catalog # 233-FB) or tissue culture grade Recombinant Human FGF basic (Catalog # 4114-TC)
  • Accutase® (Innovative Cell Technologies)
  • Cultrex® Reduced Growth Factor Basement Membrane Extract (BME) (Catalog # 3433-005-01)
  • StemXVivo Culture Matrix (Catalog # CCM013)
  • DMEM/F12

Materials

  • BG01V human embryonic stem cells or equivalent
  • 60 or 100 mm tissue culture plates
  • 15 mL centrifuge tubes
  • Pipettes and pipette tips

Equipment

  • 37 °C and 5% CO2 incubator
  • Centrifuge (low speed clinical or equivalent)
  • Hemocytometer
  • Microscope

Procedure Overview

Prepare Either A) Cultrex BME-coated plates or B) StemXVivo Culture Matrix-coated Plates

    A. Preparation of BME-coated Plates

  1. Thaw Cultrex BME on ice at 2 °C to 8 °C overnight.
  2. Aliquot thawed Cultrex BME into pre-cooled tubes and store at -20° C.
  3. Thaw the aliquot on ice at 2 °C to 8 °C.
  4. Dilute Cultrex BME 1:40 in DMEM/F12. This can be stored at 4 °C for up to 2 weeks.
  5. Coat the desired number of plates with diluted Cultrex BME (approximately 2.5 mL/60 mm plate) and incubate for 1-2 hours at room temperature.
  6. Remove the Cultrex BME solution immediately prior to plating the cells.

  7. B. Preparation of StemXVivo Culture Matrix-coated Plates

  8. Thaw StemXVivo Culture Matrix at 2 °C to 8 °C before use.
  9. Determine the volume of diluted culture matrix required. For example, 1.5 mL is needed for each well of a 6-well tissue culture plate.
  10. Dilute the culture matrix 1:100 in sterile 1X PBS. Do not vortex.
  11. Note: The concentration of culture matrix used may need to be optimized for some cells.
  12. Immediately add diluted culture matrix to the plates and incubate at 37 °C for 2-3 hours.
  13. Immediately prior to plating the cells, remove the diluted culture matrix and rinse once with sterile 1X PBS.

  14. I. Preparation and plating of BG01V Human Embryonic Stem Cells

  15. Gently transfer thawed BG01V human embryonic stemc cells to a 15 mL centrifuge tube containing pre-warmed MEF Conditioned Media.
  16. Centrifuge at 200 x g for 4 minutes.

  17. Resuspend the cell pellet in MEF Conditioned Media supplemented with 4 ng/mL FGF basic.

  18. Add the BG01V human embryonic stem cells to the Cultrex BME- or StemXVivo-Culture Matrix-coated plates.

  19. Culture the cells at 37 °C and 5% CO2 and change the media daily.

  20. II. Passaging BG01V Human Embryonic Stem Cells

  21. Coat plates with Cultrex BME or StemXVivo Culture Matrix as described.
  22. Remove the media from cells and add 1 mL of Accutase solution to each 60 mm plate.
  23. Pipette gently over the plate until all of the cells have detached.

  24. Transfer the cell suspension to a 15 mL centrifuge tube containing < 5 mL of MEF Conditioned Media.
  25. Centrifuge at 200 x g for 4 minutes.

  26. Resuspend the cell pellet in MEF Conditioned Media.
  27. Perform a cell count.

  28. Plate the desired number of cells (approximately 1.0 x 106 cells/60 mm plate) on Culture BME- or StemXVivo Culture Matrix-coated plates in MEF Conditioned Media containing 4 ng/mL FGF basic.

  29. Culture the cells at 37 °C and 5% CO2 and change the media daily.

Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

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Species
  1. Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny.
    Authors: Cheli Y, Guiliano S, Botton T, Rocchi S, Hofman V, Hofman P, Bahadoran P, Bertolotto C, Ballotti R
    Oncogene, 2011;30(20):2307-18.
    Species: Mouse
  2. Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells.
    Authors: Mandal S, Lindgren AG, Srivastava AS, Clark AT, Banerjee U
    Stem Cells, 2011;29(3):486-95.
    Species: Human

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    AR005

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