293T-HA-Rspo1-Fc稳定表达细胞株BioVector® R-Spondin1 stable cell line BioVector NTCC质粒载体菌种细胞基因保藏中心

BioVector® NTCC® 293T-HA-Rspo1-Fc稳定表达细胞株BioVector® R-Spondin1 stable cell line

R-Spondin1 expressing 293T Cell Line
The R-Spondin1-Expressing 293T cell line produces RSPO1, a critical protein used to establish 3D organoid cultures from stomach, small intestine, colon, pancreas, and liver sources.

General description
3D organoid culture systems are increasingly employed as powerful tools for the study of human diseases. Organoids are thought to better represent the tissue-specific niche microenvironment. R-spondin-1 (RSPO1) is one of the most extensively used niche factors for culturing 3D organoids. The first published report of an organoid culture system able to promote long-term survival and multilineage differentiation of intestinal stem cells (ISCs) employed mouse Rspo1 as one of the critical factors . Since then, R-Spondin1 has been used to establish organoid cultures from the stomach, small intestine, colon, pancreas and liver from both mouse and human sources .
RSPO1 is an intestinal growth factor that works as a potent activator of the Wnt/beta-catenin signaling pathway by binding to leucine-rich repeat–containing G protein–coupled receptors (LGRs), which are expressed in tissue stem cells. RSPO1 also binds to the transmembrane E3 ubiquitin ligases RNF43 and ZNRF3 and triggers their internalization, thereby potentiating Wnt signaling.

The 293T cell line is stably transfected to express mouse R-spondin1 protein tagged with N-terminus HA epitope tag and C-terminus mouse IgG2α Fc region. R-Spondin1 expressing 293T cell line can be used to produce either purified Rspo1 or Rspo1 conditioned media. The FC region and HA tags enable ease of purification and characterization. Cell line is not the published cell line from the Kuo lab, but rather a related cell line developed in the same lab using the reference protocol.
Cell Line Description
Epithelial Cells

PROTOCOL
These procedures should be performed in a biological hood utilizing aseptic technique
to prevent contamination. Vessels should be sprayed down with 70% Ethanol before
placing in tissue culture hood.
THAWING CULTREX HA-R-SPONDIN1-FC 293 T CELLS:
1. Pre-warm Basal Growth Medium by placing in a 37 °C water bath or in a tissue culture
incubator.
2. Immediately before use, remove the vial of cryopreserved cells from liquid nitrogen and
thaw quickly (3-4 minutes) in a 37 °C water bath. Ensure cells are completely thawed before
proceeding. Do not leave cells at 37 °C past thawing.
3. Immediately transfer the thawed cells into an empty 15 mL conical tube. Wash cryovial
with 1.0 mL of warm Basal Growth Medium and add to the 15 mL conical tube. Add 1.0 mL
of warm Basal Growth Medium to 15 mL conical tube containing cells, gently swirling to
mix between drops. Total volume in the conical tube should be 3.0 mL.
4. Centrifuge cells at 200 x g for 3 minutes.
5. Gently remove supernatant resuspend cell pellet in 6.0 mL of fresh Basal Growth Medium.
6. Transfer cell suspension to a sterile T25 (25 cm2
) cell culture flask.
7. Place cell culture flask in 5% CO2
incubator at 37 °C.
8. The next day, remove Basal Growth Medium and add 6.0 mL of freshly prepared and
pre-warmed Selection Growth Medium.
9. Cells should be maintained in Selection Growth Medium, which should be refreshed every
2-3 days.
PASSAGING CULTREX HA-R-SPONDIN-1 FC293 T CELLS:
Cultrex HA-R-Spondin1-Fc 293T Cells may be sequentially passaged under selection into
larger culture vessels to expand cell numbers. We recommend that cultures be
maintained at densities between 40 – 90% confluent for optimal growth and survival.
Note: Cells should be passaged when 80-90% confluent for optimal growth rate/efficiency.
We recommend splitting cells at a density of 1:4 to 1:6. Cells have a doubling time of
approximately 24 hours.
1. Prepare the appropriate amount of Selection Growth Medium on day of use
(10-12 mL/T75 flask).
2. Remove medium from the T25 flask containing the Cultrex HA-R-Spondin1-Fc 293T Cells.
3. Gently wash flask with 5.0 mL of sterile 1X PBS. Remove PBS.
4. Add 1.0 mL of warmed Trypsin to the T25 flask and place at 37 °C for 3-5 minutes until cells
are no longer attached.
5. Inactivate Trypsin by add 3.0 mL of Basal Growth Medium to the flask. Transfer full volume
to a 15 mL conical tube.
6. Centrifuge cells at 200 x g for 3 minutes.
7. Remove supernatant gently to avoid disturbing cell pellet and resuspend cell pellet in
2.0 mL of fresh Selection Growth Medium.
8. Add 1.0 mL of cell suspension to 11.0 mL of Selection Growth Medium and transfer to one
T75 (75 cm2
) flask.
Note: After at least five days in Selection Growth Medium, HA-R-Spondin1-Fc cells may be
expanded using Basal Growth Medium until they reach 80% confluence in the desired
culture vessel.

CULTREX HA-R-SPONDIN 1 FC EXPRESSION AND PURIFICATION
A 3-layer flask is recommended to express and purify HA-R-Spondin 1-Fc. A 3-layer flask
has a surface area of 525 cm2
, accommodates 150-200 mL of medium, and can produce
up to 300 µg of HA-R-Spondin 1-Fc.
Note: After at least five days in Selection Growth Medium, Cultrex HA-R-Spondin1-Fc Cells may be
expanded in Basal Growth Medium until they reach 80% confluence.
1. Once the cells have been passaged to the 3-layer flask and reach 80% confluence, change
the medium to Expression Medium and continue to culture for 7-10 days.
Note: The cells will detach from the cell culture vessel and grow in suspension after several
days.
2. After 7-10 days in culture, collect the supernatant, and centrifuge at 3,000 x g for
15 minutes at 2-8 °C.
3. Filter the supernatant through 0.22 µm filter at 2-8 °C.
4. Purify HA-R-Spondin1-Fc using the Fc tag via Protein A Agarose Purification.
CULTREX HA-R-SPONDIN 1-FC CONDITIONED MEDIUM
A 3-layer flask is recommended to produce HA-R-Spondin 1-Fc conditioned medium. A
3-layer flask has a surface area of 525 cm2
and can produce 150-200 mL of conditioned
medium.
Note: After at least five days in Selection Growth Medium, HA-R-Spondin1-Fc cells may be
expanded in Basal Growth Medium until they reach 80% confluence.
1. Once the cells have been passaged to the 3-layer flask and reach 80% confluence, add
freshly prepared Conditioned Medium and continue to culture for 7-10 days.
Note: The cells will detach from the cell culture vessel and grow in suspension after several
days.
2. Collect the supernatant, and centrifuge at 3,000 x g for 15 minutes at 2-8 °C.
3. Filter the supernatant through 0.22 µm filter at 2-8 °C.
4. Verify the presence of HA-R-Spondin 1-Fc in the medium by Western Blot using an
anti-R-Spondin 1 antibody or an anti-HA tag antibody. The expected size should be
approximately 70-75 kDa

Supplier来源:BioVector NTCC Inc.
Email: biovector@163.com
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