Human Airway Organoid Kit
MCE Human Airway Organoid Kit includes a basic culture medium for Human Airway organoids, as well as culture supplements, and is designed for the effective construction of human airway organoids (hAOs) from human airway stem cells.
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Storage :
Basal Medium A: 4°C, 1 year. Shipping with blue ice. Supplement B (50×) & Supplement C (250×): -20°C, 1 year. Avoid repeated freeze/thaw cycles. Shipping with dry a ice.
- Manual (4623KB)
- COA
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Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
MCE Human Airway Organoid Kit includes a basic culture medium for Human Airway organoids, as well as culture supplements, and is designed for the effective construction of human airway organoids (hAOs) from human airway stem cells.
The self-renewal of the airway epithelium is driven by the proliferation of basal cells. Human airway organoids comprise basal cells, ciliated cells, secretory cells, and a small number of neuroendocrine cells. As such, these organoids recapitulate all key features of the airway epithelium, including its architecture, cellular composition, and self-renewal dynamics. Therefore, they hold great promise for advancing studies on airway homeostasis and related diseases.
Protocol
1. Preparation of Complete Culture Medium for Human Airway
Prepare the complete organoid medium according to the table below, mix thoroughly, and keep on ice. Perform all steps under sterile conditions.
| Reagent | 10 mL | 20 mL | 50 mL | Final concentration |
|---|---|---|---|---|
| Human Airway Organoid Basal Medium A | 9.76 mL | 19.52 mL | 48.8 mL | 1× |
| Human Airway Organoid Culture Supplement B (50×) | 200 μL | 400 μL | 1 mL | 1× |
| Human Airway Organoid Culture Supplement C (250×) | 40 μL | 80 μL | 200 μL | 1× |
2. Primary Tissue Processing and Isolation of Epithelial Structures
a. Place fresh human airway epithelial tissue in pre-cooled tissue storage solution and process as soon as possible.
b. Wash thoroughly with Human Airway Organoid Basal Medium A or PBS to remove blood, fat, connective tissue, and other non-target components.
c. Cut the tissue into small pieces suitable for dissociation and use the tissue-appropriate dissociation solution to loosen epithelial cell clusters, glands, or ductal structures.
d. Release the target epithelial structures by gentle pipetting or mechanical agitation and remove large debris using an appropriate cell strainer.
e. Centrifuge at 250-300 × g for 3-5 min at 4°C, collect the target cell/epithelial-structure pellet, and wash 1-2 times with Basal Medium A.
3. Construction of Organoid
a. Keep the procedure on ice and resuspend isolated epithelial clusters, glands, or ductal structures in MCE Basement Membrane Matrix. Use 100% Basement Membrane Matrix when possible; if dilution is required, maintain a Basement Membrane Matrix-to-organoid-medium ratio > 2 : 1.
b. Using a pre-wetted 200 μL pipette tip, dispense approximately 25-35 μL per well into a 24-well plate, avoiding bubbles. Incubate at 37°C with 5% CO2 for 15-30 min until gelled.
c. After gelation, slowly add 500 μL Human Airway Organoid Complete Medium along the wall of each well.
d. Replace 500 μL pre-warmed complete medium every 3-4 days and monitor organoid growth and differentiation according to tissue source.
4. Organoid Passaging
a. Aspirate the culture medium, add an appropriate amount of Basal Medium A, detach the matrix/organoids with a cell scraper or pipette tip, and transfer to a centrifuge tube.
b. Gently pipette to separate organoids from the matrix and centrifuge at 250-300 × g for 3 min at room temperature.
c. Resuspend in Basal Medium A and disperse organoids into smaller clusters. If mechanical dissociation is insufficient, briefly use organoid dissociation solution and stop when clusters of approximately 10-50 cells are observed to avoid over-digestion.
d. Centrifuge and wash 1-2 times with Basal Medium A or PBS.
e. Re-embed on ice in Basement Membrane Matrix, dispense 25-35 μL per well, allow the matrix to gel, add 500 μL complete medium, and continue culture.
Storage
Basal Medium A: 4°C, 1 year. Shipping with blue ice.
Supplement B (50×) & Supplement C (250×): -20°C, 1 year. Avoid repeated freeze/thaw cycles. Shipping with dry a ice.
Attention
1. Cells need to be kept sterile when extracted from primary tissue to avoid contamination from subsequent experiments.
2. Observe the fragmentation status of the organoid during passaged dissociation, and terminate the dissociation when small cell clusters (10-50 cells) appear to avoid prolonging the subsequent growth viability of the organoid.
3. Operations involving MCE Basement Membrane Matrix need to be kept at low temperature throughout. MCE Basement Membrane Matrix should be injected rapidly into the cell culture wells after resuspension with the cells, while avoiding air bubbles.
4. This product is for R&D use only, not for drug, household, or other uses.
5. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Cat. No. | Components | HY-K6127 -100 mL |
HY-K6127 -500 mL |
Storage |
|---|---|---|---|---|
| HY-K6127-A | Human Airway Organoid Basal Medium A |
100 mL | 500 mL | 4°C, 1 year. |
| HY-K6127-B | Human Airway Organoid Culture Supplement B (50×) |
1 mL × 2 | 10 mL | -20°C, 1 year. Avoid repeated freeze/thaw cycles. |
| HY-K6127-C | Human Airway Organoid Culture Supplement C (250×) |
0.4 mL | 1 mL × 2 | -20°C, 1 year. Avoid repeated freeze/thaw cycles. |
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