Mogengel Matrix Organoid Culture -0827555-Mogengel

Description

Basement membranes are continuous sheets of specialized extracellular matrix that form an interface between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma. Basement membranes are degraded and regenerated during development and wound healing. They not only support cells and cell layers, but they also play an essential role in tissue organization that affects cell adhesion, migration, proliferation, and differentiation. Basement membranes provide major barriers to invasion by metastatic tumor cells.

Mogengel Matrix Organoid Culture is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor. Mogengel Matrix Organoid Culture at 37 °C to form a reconstituted basement membrane. The major components of Mogengel Matrix Organoid Culture include laminin, collagen IV, entactin, and heparin sulfate proteoglycan.

Intended Use

This is the selected Mogengel Matrix to prove organoid culture, if you encounter organoids that cannot be cultured with other types of substrate glue, the use of this series of matrigel can ensure the effectiveness and reproducibility of the experiment.For example, mice intestinal organoids, lung  organoids, human brain organoids and so on.

Materials Provided

Product Parameter

Source: Mouse Tumor

Appearance：

① Color: phenol-containing red matrigengel is yellow-pink, and phenol-free red matrix gum is translucent light yellow;

② Form: standard matrigengel dissolved at 4℃, a transparent liquid state; High concentration matrigengel after 0℃ solution, transparent liquid state, 4℃ for a long time to show a semi-gel.

Concentration: Protein concentration ranges from 8 to 26mg/mL

Endotoxin:≤ 4.5EU/ mL

Gel time: 5-30min gel at room temperature, the speed of gel formation is accelerated when the temperature is from 22°C to 37°C.

Material Qualifications

•  Routine screening of mouse colony pathogensby mouse antibody product (MAP) tests
•  Testing for bacteria, fungi and mycoplasmato ensure negative results
•  Extensive PCR testing for a variety of pathogens including LDEV to ensure strict control of raw materials used in the production process
•  Extraction from LDEV-free mouse tumor cells
•  Gel stability testing at 37℃ for 14 days
•  Detection of endotoxin levels using serological methods
•  Biological function verification of each lot (organoid culture and differentiation experiments; Subcutaneous tumor formation test; Stem cell culture; Angiogenesis experiment etc.)

Coating Procedures

Thaw Mogengel Matrix overnight at 2-8 °C. Refrigerator temperatures may vary, therefore it is recommended to keep Mogengel on ice in a refrigerator during the thawing process. Thawed Mogengel solidifies quickly at temperatures above 15 °C; when working with Mogengel,

keep it on ice to prevent untimely gelling.

There are many applications for Mogengel which require different thicknesses and concentrations. A thick gel is needed for applications such as endothelial cell formation of capillary-like structures (Tube Formation Assay), the differentiation of rat aorta tissue into capillary-like structures (Aortic Ring Assay), epithelial organoid formation, or tumor organoid formation. Some applications, such as propagation of primary cells, require a thin layer coating and not a thick gel; therefore, the thin layer method should be used.

Thick Gel Method:

1. Thaw Mogengel as stated above.

2. Mix Mogengel by slowly pipetting solution up and down; be careful not to introduce air bubbles.

3. Pipette 200-300 μL per cm² onto the growth surface.

4. Place coated object at 37 °C for 30 minutes.

5. Coated objects are ready for use.

Thin Layer Method (non-gelling):

1. Thaw Mogengel as stated above.

2. Mix Mogengel by slowly pipetting solution up and down; be careful not to introduce air bubbles.

3. Dilute Mogengel to desired concentration in cold serum-free medium. A 1:100 dilution is recommended for the propagation of primary cells. Empirical determination of the optimal coating concentration for your application may be required.

4. Add a sufficient amount of solution to cover the entire growth surface area. A volume of 300 µL per cm² is recommended.

5. Incubate coated object at room temperature for one hour.

6. Aspirate coating solution and immediately plate cells. Do not allow coated surface to dry out.

Protocol for Establishment of Organoids(Mouse Intestinal Organoids is an example) 

A.Establishment of Organoids from Primary Mouse Intestinal

1. Equipment, reagents and consumables

1.1 Equipment: Biosafety cabinet, pipette, carbon dioxide incubator, inverted microscope, centrifuge(Low-speed).

1.2 Reagent:Mogengel(Cat:082703/082755) Mouse Intestinal Organoid Kit（Mogengel Cat：MA-0817H006L）,DPBS,Anti-Adherence Rinsing Solution(Mogengel Cat：MB-0818L03L)，Penicillin-Streptomycin Solution，DPBS containing 0.1%BSA，0.5M EDTA Solution（pH=8.0）

1.3 Consumables: sterile pipette tips; cell culture plate（48-well for example in this protocol）;Cell culture dish (diameter:3.5 cm, 6 cm and 10 cm),Sterile forceps, sterile tissue scissors, 70 μm strainer, and surgical blade, Sterile EP tube and other consumables. (Or be adjusted according to the experimental design).

2. Preparation before Experiment:

2.1 Put the Mogengel in the ice box and put it in the refrigerator at 4℃ so that the Mogengel can slowly melt overnight; (Do not allow this product to warm up above 4℃ during manipulation. Keep the product on ice and dilute using ice-cold solutions or cell suspensions.)

2.2 Prepare Mouse Intestinal Organoid Complete Medium as directed.

2.3 Prepare plenty of DPBS containing 0.1%BSA.

3.Experimental operating procedures

3.1Sacrifice mice according to the experimental animal ethics and operating norms approved by the unit.

3.2 Prepare 6 cm dishes and add ice-cold DPBS containing 0.1%BSA for later use.(kept on ice)

3.3 Under sterile conditions,remove 3~5cm intestinal tissue near the gastric end and put into the“3.2”pre-cold DPBS ..

3.4 Cut the intestinal cavity lengthwise, gently scrape off the surface villi, wash twice，cut to a 2mm wide intestinal segment, wash twice, and transfer to pre-cooled DPBS containing 5mM EDTA，wait 20min for digestion（kept on ice）.

3.5 After digestion, transfer the tissue fragments to a new dish containing DPBS to wash, and repeat twice to remove EDTA.

3.6 Rinse the 5 mL pipette tip by Anti-Adherence Rinsing Solution.Resuspend intestinal fragment with DPBS containing  0.1% BSA，pipetting the mixture up and down 3~4times， collect the suspension and filter it with a 70 μm strainer. and repeat this step once more time.

3.7 Centrifuge 300 g for 3 min to collect crypts, resuspend using 1mL of DPBS containing 0.1% BSA, take 20 μL of suspension for microscopic examination and crypt counting.

3.8 Counting is completed, aspirate the suspension containing the required amount of crypts, centrifuge 300g for 3 min 。

3.9 Aspirate the supernatant and resuspend the crypts in Mogengel. The Maogengel should be kept on ice to prevent it from solidifying. Perform the process as quickly as possible. The volume of Mogengel used depends on the size of the pellet. Approximately 70~100 crypts should be plated in 10 μL of Mogengel.

CRITICAL: Do not overly dilute the Mogengel (Mogengel ratio should be >70% (Mogengel vol/Total vol)), as this may inhibit the proper formation of the solid droplets.

3.10 Plate the Mogengel containing crypts at the bottom of 48-well cell culture plates in droplets of 12 ~20μL each around the center of the well.proceed with plating as quickly as possible, as the Mogengel may solidify in the tube or pipette tip. Do not let the Mogengel touch the wall of wells.

The amount of crypts-Mogengel mixture added for different plates is shown in the table below:

3.11 Place the culture plate into carbon dioxide incubator at 37°C for 15 min to let the Mogengel solidify.

3.12 After Mogengel was completely solidified, the prepared complete medium of mouse  intestine organoid was added to a 48-well plate at 250uL per well.

Note: Please add slowly along the wall to avoid damaging the solidified structure.

3.13 The 48-well plate was incubated in a carbon dioxide incubator at 37℃.

3.14 The medium should be changed every 3 days to avoid damaging Mogengel when changing the medium.

3.15 Closely monitor the growth status of organoids. Ideally, mouse intestinal organoids should be established within 5 to 7 days.（Changes in mouse intestinal organoids can be observed by taking pictures daily.）

B.Splitting and Passaging of Mouse Intestinal Organoids

4. Equipment, reagents and consumables

4.1 Equipment: Same as 1.1.

4.2 Reagent:Melted Mogengel, Mouse Intestinal Organoid Kit, Organoid Dissociation Solution (Mogengel Cat：MB-0818L01L), Epithelial Organoid Basal medium(Mogengel Cat：MB-0818L07)

4.3 Consumables: Sterile pipette tips; 48-well cell culture plate;Sterile EP tube (Or be adjusted according to the experimental design).

5. Experimental operating procedures

5.1 Keep the original culture medium, gently scrape off the extracellular Mogengel and organoid mixture with a pipette tip, transfer to a 1.5mL，Pipette the organoid suspension up and down to mix thoroughly by pipetting against the bottom of the tube to create pressure, which will aid the removal of Mogengel。

5.2 Centrifuge with 300g at 4℃ for 3min, discard the supernatant,Aspirate the supernatant and split organoids using either Organoid Dissociation Solution or by mechanical disruption. For Organoid Dissociation Solution-based cell dissociation, resuspend the pellet in 0.2 mL of Organoid Dissociation Solution, pipette up and down and incubate at 37 °C until organoids fall apart（Do not dissociate in Organoid Dissociation Solution for >7 min）. For mechanical disruption-based resuspended the bottom organoid precipitation with Epithelial Organoid Basal medium, Pipette the organoid suspension up and down 5~10 times，centrifuge again with 300g at 4℃ for 3min,Clean and place on ice.

5.3 After shearing is complete, wash twice with 1 mL of Epithelial Organoid Basal medium, and centrifuge at 300g for 3 min.

5.4. Organoid precipitation was resuspended with appropriate amount of Mogengel, then placed on ice after resuspended, and the resuspended time should not be more than 30s to avoid premature solidification of Mogengel.

Note: The dilution ratio of Mogengel should be above 70% to ensure the structural stability of Mogengel during culture.

5.5. Place the mixed suspension of Mogengel and organoid into the center of the bottom of the 48-well cell culture plate, avoiding the suspension from contacting the side wall of the plate, with about 15uL for each well.

Note: This step should be completed as soon as possible to prevent Mogengel from solidifying at room temperature.

5.6. Put the inoculated culture plate into 37℃ carbon dioxide incubator and incubate for about 15min until Mogengel solidified.

5.7. After Mogengel was completely solidified, the prepared complete medium of mouse small intestine organoid was added to a 48-well cell culture plate of 250uL per well.

5.8. The 48-well cell culture plate was placed in a humidified incubator at 37 °C and 5% (vol/vol) CO₂.