Geltrex from Life Technologies

May 09, 2013

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McLean Hospital/Harvard Medical School
Molecular Neurobiology Lab
Post-doc

hES (H9) cells has been cultivated under feeder free condition with StemPro media. Cell culture dished were coated with Geltrex before seeding.

Company:

Life Technologies

Product Name:

Geltrex LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix

Catalog Number:

A1413301
Image
  • Company: Life Technologies
  • Product Name: Geltrex LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix
  • Catalog Number: A1413301

Geltrex™ is a reduced growth factor basement membrane extract used for attachment and maintenance of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Therefore, Geltrex has been used for StemPro media which is a fully-defined, serum- and feeder-free medium (SFM) specially formulated for the growth and expansion of hES and hiPSCs. Especially when cells are grown with StemPro media, cell culture dishes should be coated with Geltrex. Since we are culturing our hESCs and hiPSCs in StemPro media (feeder-free culture), Geltrex was an obvious choice.

Experimental Design and Results Summary

Application

Growth and expansion of hESCs and hiPSCs.

Starting Material

Cell culture dishes (60mm or 100mm) for hESCs hiPSCs.

Protocol Overview

1. Thaw Geltrex in the cold room (2-8 °C) overnight. 2. Gently mix Geltrex by pipetting (make sure no bubbles form). 3. Dilute 1mL of Geltrex into 99mL of pre-chilled DMEM/F-12. 4. Add 3mL (or 9mL) of Geltrex solution into a 60mm (or 100mm) dish for coating. 5. Incubate coated dishes at 37°C incubator for 1 hr. 6. Transfer dishes to cell culture hood and allow them to equilibrate to room temperature for 1 hr before cell seeding. 7. Aspirate Geltrex solution (no washing) and seed cells.

Tips

Avoid multiple rounds of freeze-thawing. While preparing Geltrex solution, work asceptically on ice and use cold media to prevent polymerization.

Results Summary

After several passages under feeder-free culture, the pluripotency of hESC and hiPSCs was checked by alkaline phosphate staining (AP) and immunostaining with anti-Oct4, Sox2, SSEA-4, and Tra1-60 antibodies. All the hESC/hiPSCs stained positive for the pluripotent markers, which implied hES and iPSCs were maintained without feeder cells.

Additional Notes

None

Image Gallery

hES (H9) cells has been cultivated under feeder free condition with StemPro media. Cell culture dished were coated with Geltrex before seeding.

Summary

The Good

A good matrix for culturing hESC and hiPSC under feeder-free conditions.

The Bad

Coating plates with Geltrex is a little tricky so the protocol should be followed exactly.

The Bottom Line

Geltrex is necessary for feeder-free hES/hiPSC culture that does not change the cells’ pluripotency.

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