MimEX GI Starter Kit
MimEX GI Starter Kit Summary
Kit Summary
Reagents for the expansion and differentiation of adult gastrointestinal epithelial stem cells.
Key Benefits
- Generates 3-dimensional, multi-cellular gastrointestinal tissue
- Includes specially formulated media that promotes adult gastrointestinal stem cell expansion differentiation
- Contains enough reagents to generate tissue in 12 transwell inserts of a 24-well plate
- Includes transwell insert plate
- Quality control tested for consistent media performance
The Benefits of MimEX™ GI Tissue Model System for Generating 3-D Gastrointestinal Tissue In Vitro
Adult stem cell populations are found in most tissues of the human body and provide a long-lived source of self-renewing cells for tissue homeostasis under normal conditions and regeneration in disease or injury. In the adult, tissue specific stem cells can be unipotent or multipotent depending on the nature of the tissue, but they all exhibit an epigenetic memory conferring tissue specificity and regional identity. Moreover, adult stem cells may hold the keys to understanding aging, disease progression, and the development and spread of cancer.
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 (3-D) tissues in vitro and provide a valuable tool for biomarker discovery, disease modeling, drug screening*, and developmental biology studies. The MimEX™ GI Starter Kit will expand adult stem cell populations of the gastrointestinal tract under clonal conditions while maintaining regional identity, genome stability, and disease state. It will also differentiate gastrointestinal adult stem cells into 3-D epithelial in vitro tissue that mimics the regional cytoarchitecture or disease state of the stem cell’s origin tissue.
The MimEX™ GI Starter Kit contains specially formulated reagents and plates for the expansion and differentiation of adult gastrointestinal epithelial stem cells. The quantity of each component is sufficient to expand stem cells to generate enough cells for differentiation into 12 transwell inserts in a 24-well tissue culture plate.
This kit contains the following reagents to isolate and expand adult gastrointestinal stem cells:
- 250 mL of MimEX™ GI Expansion Media
- 100 mL of MimEX™ GI Differentiation Media
- 12 vials of MimEX™ Irradiated Fibroblasts
- 100 mL of MimEX™ Fibroblast Media
- 1.5 mL of Cultrex® Stem Cell Qualified RGF Basement Membrane Extract, Pathclear®
- 1 24-well plate containing 12 transwell inserts
Note: The components for this kit require different storage/shipping temperatures and will arrive in separate packaging.
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.
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Human Ileum Adult Stem Cell Colonies on Irradiated Fibroblasts. Adult stem cells were isolated from human ileum tissue using the MimEX™ Tissue Processing Kit. Ileum adult stem cells were cultured for 7 days in MimEX™ GI Expansion Media and MimEX™ Irradiated Fibroblasts. This phase contrast image shows colonies of ileum adult stem cells. These colonies show good distribution and are the ideal size for passaging. |
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Isolation of Adult Stem Cells from Specific Regions of the Gastrointestinal Tract. Tissue biopsies from the human ileum, duodenum, jejunum, and ascending colon were procured and processed using the MimEX™ Tissue Processing Kit (R&D Systems®; Catalog # MIM001). Images show adult stem cell colonies isolated from the region specific gastrointestinal tissue following expansion and passaging using MimEX™ GI Expansion Media and MimEX™ Irradiated Fibroblasts, which are included in this MimEX™ GI Starter Kit. |
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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 cultured for 9 days 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 the that the media should be switched from MimEX™ GI Expansion Media to MimEX™ GI Differentiation Media under ALI conditions. 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 on MimEX™ Irradiated Fibroblasts in MimEX™ GI Expansion Media. They were then differentiated in MimEX™ GI Differentiation Media for 9 days under air-liquid 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 MUC2 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 # NL004) and counterstained with DAPI (blue; Tocris Bioscience®; Catalog # 5748). Cadherin-17 staining was detected in cellular membranes throughout the epithelial layer while MUC2 staining was localized specifically to goblet cells. |
* 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 Starter Kit can be used to generate 3-D gastrointestinal tissue using the following procedure:
- Thaw or harvest adult gastrointestinal stem cells
- Expand stem cells using MimEX™ GI Expansion Media and MimEX™ Irradiated Fibroblasts
- Differentiate stem cells in an transwell insert in combination with MimEX™ GI Differentiation Media
Reagents provided in the MimEX™ GI Starter Kit (Catalog # MIM002):
- 250 mL of MimEX™ GI Expansion Media
- 100 mL of MimEX™ GI Differentiation Media
- 12 vials of MimEX™ Irradiated Fibroblasts
- 100 mL of MimEX™ Fibroblast Media
- 1.5 mL of Cultrex® Stem Cell Qualified RGF Basement Membrane Extract, Pathclear®
- 1 24-well plate containing 12 transwell inserts
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)
- DMEM/F12
- Penicillin-Streptomycin (100X), optional
- 0.05% Trypsin/EDTA
- Sterile Phosphate-Buffered Saline (PBS) (Tocris®; Catalog # 3156)
- Mouse Feeder Removal Microbeads (Miltenyi; Catalog # 130-095-531)
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 protocols below describe the expansion and differentiation of human adult gastrointestinal stem cells using the reagents provided in the MimEX™ GI Starter Kit.
Note: The detailed protocol must be read in its entirety before using this product.
Protocol to Prepare 6-well Plates for GI Stem Cell Expansion
Protocol to Expand Gastrointestinal Stem Cells
Protocol to Prepare Transwell Inserts for GI Stem Cell Differentiation
Protocol to Differentiate GI Stem Cells into Tissue
Protocol for Plating MimEX™ Irradiated Fibroblasts for Stem Cell Expansion in a 6-well Plate
The following protocol is to plate one vial of MimEX™ Irradiated Fibroblasts into a 6-well plate. If an alternate plate size is desired, adjust volumes and cell numbers accordingly.
Place a 6-well plate at 2-8 °C for 10 minutes.
Dilute thawed Cultrex® Stem Cell Qualified RGF BME 1:10 in ice cold DMEM/F12. Keep on ice.
Coat desired number of wells with diluted Cultrex® BME mixture by pipetting 1 mL into the center of a well, swirl to coat the entire well, and remove by pipette while tilting the plate. Repeat for all wells need.
Remove excess Cultrex® BME in the wells.
Incubate the plate at 37 °C/7.5% CO2 for 10 minutes.
Thaw MimEX™ Irradiated Fibroblasts in a 37 °C water bath.
Add 0.5 mL of MimEX™ Fibroblast Media to the cryovial of fibroblasts and gently pipette to re-suspend.
Add the resuspended cells dropwise to 2 mL of MimEX™ Fibroblast Media in a 50 mL centrifuge tube while swirling gently.
Add the fibroblast suspension to the coated wells, 2 mL/well. Ensure there are no air bubbles in the wells.
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™ Fibroblast Media daily. Plated fibroblasts should be used within 1-3 days of plating.
Expansion of Adult Gastrointestinal Stem Cells
Prepare a 6-well plate with MimEX™ Irradiated Fibroblasts 1–3 days before culturing Adult GI stem cells. Follow protocol for Plating MimEX™ Irradiated Fibroblasts.
Warm MimEX™ GI Expansion Media to 37 °C in a water bath.
Warm frozen vial of Adult GI Stem Cells until just thawed.
Transfer the cells to a 15 mL centrifuge tube containing at least 5 mL of prewarmed MimEX™ GI Expansion Media.
Centrifuge at 200 × g for 5 minutes.
Resuspend the pellet in 4 mL of MimEX™ GI Expansion Media.
Remove media from irradiated fibroblast-coated plates.
Add 2 mL of the Adult GI Stem Cell suspension to each well.
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 every other day and monitor for colony outgrowth.
Plating MimEX™ Irradiated Fibroblasts for Stem Cell Differentiation in a 24-well Transwell Insert Plate
The following protocol is for 12 transwell inserts of a 24-well plate. If an alternate plate size is desired, adjust volumes and cell numbers accordingly.
Place a 24-well plate with the desired number of transwell inserts at 2-8 °C for 10 minutes.
Dilute thawed Cultrex® Stem Cell Qualified RGF BME 1:5 in ice cold DMEM/F12. Keep on ice.
Coat desired number of wells with diluted Cultrex® BME mixture by pipetting 100 µL into the center of a transwell insert, swirl to coat the entire transwell insert, and remove by pipette while tilting the plate. Repeat for all wells need.
Remove excess Cultrex® BME in the transwell inserts.
Incubate the plate at 37 °C/7.5% CO2 for 10 minutes.
Thaw MimEX™ Irradiated Fibroblats in a 37°C water bath.
Add 0.5 mL of MimEX™ Fibroblast Media to the cryovial of fibroblasts and gently pipette to re-suspend.
Add the cells dropwise to a 50 mL centrifuge containing MimEX™ Fibroblast Media (250 µL for each transwell used) while swirling gently.
Add the fibroblast suspension to the coated wells, 250 µL/well. Ensure there are no air bubbles in the wells.
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™ Fibroblast Media daily. Plated fibroblasts should be used within 1-3 days of plating.
Differentiation of MimEX™ GI Human Descending Colon 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
Warm MimEX™ GI Differentiation Media to 37 °C in a water bath.
Initiate an air-liquid interface culture.
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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