Organoid Growth and Harvesting

Organoid Culture:

Organoids are organ-like structures that can be formed by 3D cell culture and differentiation of stem cells or organ progenitors; and are capable of recapitulating aspects of organ function in vitro. AMSBIO is launching a suite of products to assist the increasing number of scientists adopting this model for use in:

• Organogenesis Models
• Tumor / Disease Models
• Drug Testing
• Toxicity Screening
• Personalised Medicine
• Regenerative Medicine / Organ Replacement

Figure 2. Human Colorectal Cancer (CRC) organoids grown from single cells on ECM from AMSBIO. Immunofluorescence for Phalloidin (red) to mark actin filaments and E-cadherin (green) as epithelial marker; nuclei counterstained with DAPI (blue). Images courtesy of the Batlle Lab, IRB Barcelona

R-spondin1 (RSPO1) Cell Line:

This cell line is used to produce either purified RSPO1 or RSPO1 conditioned media for use in organoid culture.

Roof plate-specific Spondin-1 (R-Spondin 1 or RSPO1), also known as CRISTIN3, is a 27 kDa secreted activator protein that belongs to the R-Spondin family. R-Spondins positively regulate Wnt/beta-catenin signaling, most likely by acting as a ligand for LGR4-6 receptors and an inhibitor for ZNRF3. R-Spondin-1 induces proliferation of intestinal crypt epithelial cells, increases intestinal epithelial healing, and supports intestinal epithelial stem cell renewal. This 293T cell line is stably transfected to express murine RSPO1 with an N-terminal HA epitope tag and fused to a C-terminal murine IgG2a Fc fragment.

Figure 3. Production of R-Spondin1 for organoid culture. The 293T cell line is stably transfected to express murine RSPO1 with an N-terminal HA epitope tag and fused to a C-terminal murine IgG2a Fc fragment. A) The HA-R-Spondin1-Fc 293T cell line is cultured with zeocin to select for stably transfected cells. B) Production of HA-R-Spondin1-Fc is characterized using Western Blot for R-Spondin1 protein (arrow). C) HA-R-Spondin1-Fc induces activation of Wnt/ß-catenin response when evaluated using the Top-Flash Luciferase assay.

Description	Datasheet	Pack Size	Order
HA-R-Spondin1-Fc 293T Cells		1 x vial (10e6 cells)

Recombinant Wnt3A

Recombinant human and Mouse Wnt3A are both available from AMSBIO. The combination of Wnt3A and the Wnt amplifier R-spondin1 is essential to grow organoids from normal epithelium.

Organoid Harvesting Solution:

Organoid cultures exhibit cellular behaviors and morphologies similar to those found in vivo, but the adaptation of such models to study biochemical processes has been impeded by the challenge of separating intact organoids from extracellular proteins in the hydrogel. Commonly, proteases have been employed to degrade these extracellular proteins, but proteases also degrade proteins on the cell surface and protease activity may carry over into subsequent cultures or lysate preparations.

Our Organoid Harvesting Solution provides a non-enzymatic method for depolymerizing extracellular matrix proteins to allow harvesting of intact organoids for passaging, cryopreservation, or biochemical analysis; and to facilitate PDX xenograft models.

Description	Datasheet	Pack Size	Order
Organoid Harvesting Solution		100 ml