Hi,

Generally, Geltrex and Cultrex is the best-known alternative to Matrigel. You can try: ECM Gel from Engelbreth-Holm-Swarm murine sarcoma (Sigma-Aldrich), but unfortunately, it requires experimental verification.

Geltrex, Cultrex, Matrigel and ECM are hydrogels from Engelbreth-Holm-Swarm murine sarcoma. The most important thing in them is laminin.

The composition may vary between manufacturers. The products can have also different parameters between one manufacturer lots.

It is best to plan experiments that will use one ECM from the same producer with the same Lot number. This is especially important for brain organoids.

For small intestine organoids, collagen may treat as an alternative. Potencjalnie laminin can be used also as an alternative for small intesine organoids.

But multi-component ECMs from Engelbreth-Holm-Swarm murine sarcoma are used most often and have an advantage over other hydrogels. Their biggest disadvantage is the variability of the composition.

It all depends on the type of organoids. Organoids derived from PSC cells have greater requirements than tissue-derived organoids.

If the product is protected by a patent, it cannot be identical between manufacturers. The composition of ECM hydrogels also depends on the extraction technology.

Justyna Augustyniak

Justyna Augustyniak

Thank you so much for your comprehensive answer. Do you know a cultrex, geltrex and ECM based protocol for polymerization to generate monolayers?

I need a matrigel-free protocol based on cultrex, geltrex and ECM to generate monolayers.

Hi,

The protocol for preparing plates for monolayer culture is the same for Matrigel, Geltrex, Cultrex, and ECM.

1. Dissolve Matrigel, Geltrex, Cultrex or ECM on ice

2. Add on ice to DMEM F12 in the ratio (1 (Matrigel, Cultrex, ECM, or Geltrex): 30 (DMEM F12). Dilution depends on cells types.

3. Add 1 ml of the solution to each well of a 6-well plate. (Plates can be stored at 4C for up to 2 weeks).

4. Directly before use incubate plates for 1h at RT or 37C.

5. After 1 hour of incubation, collect the solution. Add fresh cell culture medium with cells.

6. Passage cells with Tryple or Accutase when they reach confluence.

Justyna Augustyniak

Justyna Augustyniak

Thank you very much for your valuable guides. Is there a reference to this procedure that I can present to my supervisor?

Hi,

You can find a reference on the manufacturers' websites. Product manuals also often contained references. The preparation of the plates is based on the ratio (V/V or m/V) of Geltrex, Matrigel, Cultrex, ECM or proteins concentration which will be added to the DMEM F12. You should remembered that the recommended protein concentration may be different for different cell types.

What type of cells do you want to grow?

Justyna Augustyniak

Justyna Augustyniak

Small intestine and biliary organoids.

Thanks for all your kindly helps.

Hi,

3D culture

Organoids must be grown in the undiluted Extra Cellular Matrix (ECM) drops.

2D culture

Organoids should be dissociated into single cells and transferred on plates covered with ECM with the addition of ROCK inhibitor (plates should be prepared according to the protocol mentioned above).

If a specific cell type is needed then cell enrichment should be performed (after about 2-3 passages).

Justyna Augustyniak

Justyna Augustyniak

Thank you very much. I am grateful for your kind help.