Tips for adaptation when transitioning hESCs to StemPro® hESC SFM + Geltrex™ from feeder-dependent or feeder-free conditions

  • Starter cultures should be of high quality, 70-80% confluent and contain no differentiated hESC.
  • Different hESC lines may behave differently when transitioning to a different culture medium until they have fully adapted.
  • Timing of passage is critical. For best results, hESC should be nearing confluence (70-80%) at the time of passage. If passaged when too “light” or when overgrown, hESC will differentiate.
  • Seeding density at passage is also critical. If seeded too low, hESC will differentiate.
  • hESC cultures must be fluid-changed daily for optimal performance.
  • Morphology – edges of colonies may be less compact than the centers of the colonies in StemPro® hESC SFM. However, these cells are not differentiated but exhibit normal stem cellcharacteristics.
  • Some differentiation may occur and should be removed manually prior to passaging.
  • There is no need to change the dissociation enzyme protocol during the adaptation process.

Other Feeder-Free Medium → StemPro® hESC SFM Feeder-Dependent → StemPro® hESC SFM
When adapting cells to StemPro® hESC SFM from feeder-free parent culture such as MEF conditioned medium or other feeder-free medium:
  • Make a frozen stock of cells in control medium (MEF conditioned medium or other feeder-free medium) prior to adaptation.
  • Maintain a stock culture in control medium throughout the hESC adaptation to StemPro® hESC SFM, as a “backup.”
  • Follow the adaptation protocols described below:
 When adapting cells to StemPro® hESC SFM from a feeder-dependent parent culture:
  • The best results will be obtained when the parent hESC culture has been maintained on either murine embryonic fibroblast (MEF) or Human Fibroblast Feeder (HFF) cells.
  • We recommend 100% MEF conditioned medium on Geltrex™ as a control for the adaptation protocols described below.
  • Some residual feeders may be observed in the cultures during the adaptation process but in subsequent passages the inactivated feeder cells will be passaged out.

Adaptation protocols for transitioning to StemPro® hESC SFM + Geltrex™

Sequential adaptation – Better results may be obtained by gradually adapting hESC to
StemPro® hESC SFM + Geltrex™:
  • Passage 1: 75% control medium + 25% StemPro® hESC SFM Complete Medium +Geltrex™
  • Passage 2: 50% control medium + 50% StemPro® hESC SFM Complete Medium +Geltrex™
  • Passage 3: 25% control medium + 75% StemPro® hESC SFM Complete Medium +Geltrex™
  • Passage 4 & thereafter: 100% StemPro® hESC SFM Complete Medium + Geltrex™

  • If hESC are extremely finicky, a further level of caution can be taken by maintaining a culture in each prior passage medium while starting the next level of adaptation. For example, when passaging the 25/75 control medium / StemPro® hESC SFM culture (as described above), hESC can be passaged into both 100% StemPro® hESC SFM AND 25/75 medium. If the 100% culture does poorly, adaptation can be resumed using the backup 25/75 culture.

Direct adaptation - If hESC are passaged directly into StemPro® hESC SFM Complete Medium
+ Geltrex™, a 1:2 split ratio is suggested for the first 3 passages.

Direct and partial sequential strategy:
  1. Split cells into three plates (Plate 1, 2 & 3)
  2. At the first passage, seed Plate 1 directly into StemPro® hESC SFM Complete Medium +Geltrex™, and Plates 2 & 3 into the feeder-free control medium + Geltrex™.
  3. On the next day, fluid-change Plate 2 with StemPro® hESC SFM Complete Medium +Geltrex™ that day, and every day thereafter.
  4. For Plate 3 at the second passage, try plating these cells directly into StemPro® hESC SFM Complete Medium + Geltrex™ at 1:2 split ratio (as above).

Using Geltrex™ hESC Qualified in various dilutions

Thin Gel Method (non-gelling) for hESC Applications:
  1. Thaw Geltrex™ as described on the product insert
  2. Mix Geltrex™ by slowly pipetting solution up and down; be careful not to introduce air bubbles.
  3. Dilute 1mL of Geltrex™ into 29mL of pre-chilled (2 to 8°C) DMEM/F12 medium (SKU:10565). Empirical determination of the optimal coating concentration for your application may be required. Volumes can be adjusted accordingly.
  4. Most customers have seen that a dilution higher than 1:30 may be appropriate for their hESC line. Try anywhere from 1:30 to 1:100.
  5. Add a sufficient amount of diluted Geltrex™ solution to cover the entire growth surface area (1.5 mL for 35 mm dish, 3 mL for 60 mm dish).
  6. Coat the dish and place at 37°C for a minimum of 60 minutes.
  7. The coated dish is stable for two weeks when stored at 2 to 8°C and sealed with Parafilm. Do not allow coated surface to dry out and is critical to maintain a storage temperature of 2 to 8°C to avoid premature gelling.
  8. At time of use, it is recommended to place plates at room temperature for an hour before aspirating. Aspirate Geltrex™ coating and immediately plate cells in preequilibrated cell culture medium.

For more information please visit: www.invitrogen.com/stempro/hesc
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Using Geltrex™ hESC Qualified in various dilutions

Thin Gel Method (non-gelling) for hESC Applications:
  1. Thaw Geltrex™ as described on the product insert
  2. Mix Geltrex™ by slowly pipetting solution up and down; be careful not to introduce air bubbles.
  3. Dilute 1mL of Geltrex™ into 29mL of pre-chilled (2 to 8°C) DMEM/F12 medium (SKU:10565). Empirical determination of the optimal coating concentration for your application may be required. Volumes can be adjusted accordingly.
  4. Most customers have seen that a dilution higher than 1:30 may be appropriate for their hESC line. Try anywhere from 1:30 to 1:100.
  5. Add a sufficient amount of diluted Geltrex™ solution to cover the entire growth surface area (1.5 mL for 35 mm dish, 3 mL for 60 mm dish).
  6. Coat the dish and place at 37°C for a minimum of 60 minutes.
  7. The coated dish is stable for two weeks when stored at 2 to 8°C and sealed with Parafilm. Do not allow coated surface to dry out and is critical to maintain a storage temperature of 2 to 8°C to avoid premature gelling.
  8. At time of use, it is recommended to place plates at room temperature for an hour before aspirating. Aspirate Geltrex™ coating and immediately plate cells in preequilibrated cell culture medium.

For more information please visit: www.invitrogen.com/stempro/hesc
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