MimEX GI Differentiation Media
MimEX GI Differentiation Media Summary
Kit Summary
Media for the differentiation of adult human gastrointestinal stem cells.
Key Benefits
- Ready-to-use media that promotes the differentiation of adult gastrointestinal stem cells
- Preserves ability of adult stem cells to differentiate into organ tissue
- Generates 3-dimensional, multi-cellular gastrointestinal tissue
- Differentiates on transwell insert providing access to epithelial GI surface
- Quality control tested for consistent media performance
The Benefits of MimEX™ GI Expansion Media for Generating 3-D Gastrointestinal In Vitro
Three-dimensional (3-D) cell culture models of the gastrointestinal epithelium are quickly being adopted for toxicology, drug discovery, and disease modeling. These more complex models provide a tremendous benefit over cell-line and primary cell-based methods, including the mimicking of native intestinal cytoarchitecture and importantly, the recapitulation of physiological attributes of the tissue.
The MimEX™ GI Tissue Model System allows for the expansion and differentiation of ground-state, adult stem cell populations from the gastrointestinal tract. These cells create sustainable and accessible 3-dimensional gastrointestinal tissues in vitro and provide a valuable tool for biomarker discovery, disease modeling, drug screening*, and developmental biology studies
The MimEX™ GI Differentiation Media component of this system will differentiate adult stem cell populations of the gastrointestinal tract into 3-dimensional epithelial in vitro tissue that mimics the regional identity and disease state of the stem cell’s origin tissue. MimEX™ GI Expansion Media has been tested for its ability to support differentiation of adult human intestinal stem cells into 3-D intestinal tissue in vitro when cultured in an air-liquid interface.
This kit contains the following reagents to differentiate adult gastrointestinal stem cells:
- 100 mL of MimEX™ GI Differentiation Media
Precaution
When handling biohazardous materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.
Limitations
- FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
- The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
- These reagents should not be used beyond the expiration date indicated on the label.
- Results may vary due to variations among adult stem cell lines originating from different donors.
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Human Descending Colon Adult Stem Cell Differentiation with Air Liquid Interface (ALI) Cultures. Adult Human GI Stem Cells from the descending colon were expanded in MimEX™ GI Expansion Media (R&D Systems®; Catalog # MIM003) and differentiated with MimEX™ GI Differentiation Media under ALI conditions. These phase contrast images show the progression of 3-dimensional tissue differentiation over time. On Day 4, tissue invaginations begin to form (arrows) indicating that the media should be switched from MimEX™ GI Expansion Media to MimEX™ GI Differentiation Media. With time, the tissue continues to differentiate forming increasingly more intricate, in vivo-like structures. |
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Human Ascending Colon Adult Stem Cells Differentiate into Ascending Colon Tissue. Adult Human GI Stem Cells from the ascending colon were expanded in MimEX™ GI Expansion Media (R&D Systems®; Catalog # MIM003) and then differentiated in MimEX™interface conditions. To evaluate differentiation, adult stem cell-derived ascending colon tissue was immersion-fixed, paraffin-embedded, and sections stained with Mouse Anti-Human Cadherin-17 Monoclonal Antibody (green; R&D Systems®; Catalog # MAB1032) and Rabbit Anti- Human/Mouse/Rat/Bovine Villin 1 Antibody (red; Novus Biologicals®; Catalog # NBP1-31231). Tissue was then stained using the NorthernLights™ (NL)493-conjugated Anti-Mouse IgG Secondary Antibody (R&D Systems®; Catalog # NL009) and NL557-conjugated Anti-Rabbit IgG Secondary Antibody (R&D Systems®; Catalog # NL0044) and counterstained with DAPI (blue; Tocris®; 5748). Cadherin-17 staining was detected in cellular membranes throughout the epithelial layer while Villin 1 staining was localized specifically to the apical surface of the polarized epithelial layer. |
* Portions of MimEX Tissue Model Systems are covered by a license to intellectual property owned by, or licensed to, Multiclonal Therapeutics, Inc. The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right under these intellectual property rights to use this product for research purposes only. The purchase of this product does not include any right to use this product or components of this product for resale, for drug discovery intended for commercial gain, for the generation of cells or tissues intended for transplantation in human patients, or for any other commercial purposes, including without limitation, provision of services to a third party, generation of commercial databases, or as a diagnostic or therapeutic. Multiclonal Therapeutics, Inc., reserves all other rights under these rights. For information on purchasing a license to the intellectual property for uses other than in conjunction with this product or to use this product for purposes other than research, please contact Multiclonal Therapeutics, Inc. at licensing@multiclonaltherpaeutics.com.
All trademarks and registered trademarks are the property of their respective owners.
Specifications
Product Datasheets
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, MimEX™ GI Expansion Media is prepared using the following procedure:
- Thaw MimEX™ GI Expansion Media at 2-8 °C or room temperature.
- Store completed media at 2–8 °C for up to 2 weeks
Reagents provided in the MimEX™ GI Expansion Media (Catalog # MIM004):
- 100 mL of MimEX™ GI Expansion Media
Reagents
- GI Adult Stem Cells (purchased directly from R&D Systems or isolated from tissue using the MimEX™ Tissue Processing Kit (R&D Systems®; Catalog # MIM001))
- MimEX™ Irradiated Fibroblast Kit (R&D Systems®; Catalog # MIM005)
- MimEX™ GI Expansion Media (R&D Systems®; Catalog # MIM003)
- Transwell inserts/plates (Corning; Catalog # 3470 or equivalent).
- DMEM/F12
- Penicillin-Streptomycin (100X), optional
- 0.05% Trypsin/EDTA
- Sterile Phosphate-Buffered Saline (PBS) (Tocris®; Catalog # 3156)
Materials
- 6-well tissue culture treated plates
- 15 and 50 mL centrifuge tubes
- Serological pipettes
- Pipette and pipette tips
Equipment
- 37 °C and 7.5% CO2 incubator
- Centrifuge (low speed clinical or equivalent)
- Hemocytometer
- Inverted microscope
- Water bath
The protocol below describes the expansion of human adult gastrointestinal (GI) stem cells in MimEX™ GI Differentiation Media.
Note: The detailed protocol must be read in its entirety before using this product.
Differentiation of Adult Gastrointestinal Stem Cells
Determine the number of wells needed for the experiment.
Prepare an appropriate number of transwell inserts in a 24-well plate with MimEX™ Irradiated Fibroblasts 1–3 days before adding adult GI stem cells for differentiation. Follow protocol for Plating MimEX™ Irradiated Fibroblasts.
Warm PBS to 37 °C in a water bath.
Warm MimEX™ GI Expansion Media to 37 °C in a water bath.
Rinse expanded Adult GI Stem Cells with prewarmed PBS to dislodge mucus produced from the stem cells.
Incubate expanded Adult GI Stem Cells in 1 mL of 0.05% Trypsin/EDTA.
Pipette the cell suspension up and down to ensure full dissociation of cells. Avoid bubbles.
Add 1 mL of MimEX™ GI Expansion Media to inhibit the trypsin, triturate gently.
Transfer to a 15 mL centrifuge tube.
Centrifuge at 200 × g for 5 minutes.
Resuspend the cell pellet in 80 µL of DMEM/F12 media.
Perform a viable cell count using a hemocytometer.
Re-suspend the cells in MimEX™ GI Expansion Media at a concentration of 1.8 x 106 cells/mL.
Remove media from the upper and lower chambers of the 24-well plate.
Add 700 µL of MimEX™ GI Expansion Media to the lower chamber.
Add 250 µL of the Adult Stem Cell suspension to the transwell insert.
Distribute cells in the well using the MimEX™ Cell Plating Procedure. (Critical Step)
Incubate the plate in a 37 °C/7.5% CO2 incubator. (Critical Step) without disturbing the plate.
Replace MimEX™ GI Expansion Media in the transwell insert and the bottom well daily.
After approximately 4-5 days initiate an air-liquid interface culture.
Warm MimEX™ GI Differentiation Media to 37 °C in a water bath.
Remove all media from the transwell insert.
Replace the media in the bottom of the well with 700 µL of MimEX™ GI Differentiation Media.
Exchange media in the bottom well daily with 700 µL of fresh MimEX™ Differentiation Media. Adult GI Colon tissue should be fully differentiated in 9-12 days after initially plating Adult GI Stem Cells.
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