>Organoid Growth and Harvesting
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
Cultrex® Organoid Progenitor Cells
Organoid cultures are extracted directly from living tissues similar to primary cultures. When subjected to differentiating conditions, these organoids exhibit expression of tissue-specific genes and differentiation of stem cells into tissue-specific architecture (Figure 1) and cell types. Cultrex® Organoid Progenitor Cells from Mouse Small Intestine are derived from normal, healthy mouse small intestine tissue and are continuously cultured using Reduced Growth Factor BME-R1 and BME 2. They are never cultured directly on tissue-culture treated plastic surfaces. These organoid progenitor cells can be expanded on BME and may be induced to express tissue specific markers under differentiating conditions.
Figure 1. Differentiation of Mouse Small Intestine Organoids Cultured on RGF BME R1. During expansion, Mouse Small Intestine Organoids grow as spherical structures but under differentiating conditions, crypt-like structures bud from the organoid, mimicking intestinal epithelium. Nuclei were stained using DAPI (blue), and E-cadherin was visualized via immunofluorescence (green). Scale bar, 50 µM.
BME 2 RGF “Organoid Matrix”:
BME 2 provides a proprietary formulation that is higher in tensile strength than our original BME 1. In competitive beta tests,
Reduced Growth Factor BME 2 consistently outperforms competitor products for organoid
culture and has been described by Dr. Meritxell Huch (Gurdon Institute, University of Cambridge, UK) as her
“ECM of choice for these experiments”. Each BME 2 lot is qualified on Human Small Intestine Organoids.
Figure 2. Human Colorectal Cancer (CRC) organoids grown from single cells on Cultrex® BME 2 RGF. 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
BME R1 RGF Matrix
Cultrex® BME R1 provides a proprietary formulation that has higher tensile strength when compared to our other products Cultrex® BME, BME 2 and BME 3. It has a higher concentration of entactin, one of the BME components that connects laminins and collagens, reinforcing the hydrogel structure. Cultrex® BME R1 has been specifically designed to culture tissue organoids and is recommended for “difficult to grow” organoid cultures.
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.
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.
Cultrex® 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.
Cultrex® 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.