Description & Advantages

MCE Human Mammary Epithelial Organoid Kit includes a basic culture medium for human Mammary Epithelial organoids, as well as culture supplements, and is designed for the establishment and long-term maintenance of human Mammary Epithelial organoids.

Protocol

1. Preparation of Complete Culture Medium for Human Mammary Epithelial

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 Mammary Epithelial Organoid Basal Medium A	9.76 mL	19.52 mL	48.8 mL	1×
Human Mammary Epithelial Organoid Culture Supplement B (50×)	200 μL	400 μL	1 mL	1×
Human Mammary Epithelial 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 mammary epithelial tissue in pre-cooled tissue storage solution and process as soon as possible.

b. Wash thoroughly with Human Mammary Epithelial 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 Mammary Epithelial 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-K6130 -100 mL	HY-K6130 -500 mL	Storage
HY-K6130-A	Human Mammary Epithelial Organoid Basal Medium A	100 mL	500 mL	4°C, 1 year.
HY-K6130-B	Human Mammary Epithelial Organoid Culture Supplement B (50×)	1 mL × 2	10 mL	-20°C, 1 year. Avoid repeated freeze/thaw cycles.
HY-K6130-C	Human Mammary Epithelial Organoid Culture Supplement C (250×)	0.4 mL	1 mL × 2	-20°C, 1 year. Avoid repeated freeze/thaw cycles.

Documentation

Manual (4840 KB)

Help & FAQs

How should lyophilized recombinant proteins be reconstituted and stored?

1. Before opening the cap, centrifuge the vial at 13000 rpm for 20-30 seconds. This step will ensure that any lyophilized powder that may have adhered to the cap or walls is collected at the bottom of the vial, minimizing the risk of product loss.

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Human Mammary Epithelial Organoid Kit