Due to biannual updates of our product instruction manual, the following procedures are for reference only.
The most recent version included with the product shall prevail.
II Reagents and Materials
-Serum-free medium
-Vortex mixer
-Pre-chilled centrifugal tube
-Culture plate
-Pipette
-Pre-chilled tip
-Pipettor
-Biosafety cabinet
-CO2 incubator (37℃/5%)
III Methods
Precautions: For your safety and health, please wear a lab coat, a disposable mask and gloves, and take necessary precautions.
Thawing
Transfer the ECM-Gel product from the -20 °C refrigerator to the 4 °C refrigerator (on ice) for overnight thawing (it may take more time when the protein concentration is high), and shake the vial with vortex after liquefaction to ensure that the ECM-Gel is evenly dispersed. If you can't use it all at a time, you can divide it on ice once and freeze-thaw no more than 5 times. If the ECM-Gel has some bubbles, they can be removed by centrifugation of 400g for 15 seconds at a low temperature.
Gelling
1. Thin layer gel method (thickness 0.5 mm)
1）Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has some bubbles, they can be removed by centrifugation of 400g for 15 seconds at a low temperature.
2）Place the culture plate on ice, and add ECM-Gel to the culture plate according to 50μl of ECM-Gel/cm2 to ensure that the colloid is evenly coated at the bottom of the culture well.
3）Place the culture plate in a 37℃ incubator for 30 minutes to make the ECM-Gel completely gelatinous.
4）Gently wash the culture wells with serum-free medium to remove the unsolidified matrix proteins. Make sure that the tip of the pipette does not scratch the glue. The plates are now ready to use.
2. Thin layer coating method
1）Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has some bubbles, they can be removed by centrifugation of 400g for 15 seconds at a low temperature.
2）Coating: Dilute the ECM-Gel with pre-chilled serum-free medium in the ratio of 1:6-1:8 (protein coating amount of 100-400μg/cm2). Then, add the diluted ECM-Gel to the Transwell chamber. Taking the 24-well plate as an example, 50-100 μl diluted ECM-Gel is added to each chamber. After that, place it in a 37℃ incubator for 30 minutes to make the protein to be fully coated.
3）The coated Transwell or culture plate should be hydrated with serum-free culture in a 37℃ incubator for 30 minutes before use, followed by subsequent cell culture experiments.
4）The protein coating amount should be tested from 50μg/cm2 to 500μg/cm2. The optimal amount of coating needs to be optimized according to the experience of the researcher or the individual needs.
3. Thick layer gel method (1.0 mm, mainly used for 3D cell, organoid culture)
1）Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has some bubbles, they can be removed by centrifugation of 400g for 15 seconds at a low temperature.
2）Place the culture plate on ice and use a pre-chilled tip to add ECM-Gel to the pre-chilled cell suspension (ECM-Gel: the cell suspension volume is 4:1-0.5:1, the optimal volume ratio is 2:1). Then, mix gently.
3）Add cell-containing ECM-Gel to the culture plate at 150-200μl/cm2. Place the plate in a 37℃ incubator for 30 minutes to gel fully.
4）After gelatinization, add fresh medium and incubate in a 37℃ incubator.
Recommendation: Add medium to wash the uncoagulated protein and incubate in a 37℃ incubator for 10 minutes, to achieve better results.
4. Arch formation method (mainly used for 3D cell and organoid culture)
1）Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has some bubbles, they can be removed by centrifugation of 400g for 15 seconds at a low temperature.
2）Add ECM-Gel to pre-chilled cells/tissues, organoid suspensions (ECM-Gel: the cell volume ratio of suspension is 4:1-0.5:1, the optimal volume ratio is 2:1). Mix gently.
3）Aspirate the matrix-cell suspension with the pre-cooled tip and gently add it into the culture well. In order to improve the arching effect, the culture plate can be preheated at 37℃ for 30 minutes.
4）Place at room temperature for 1-2 minutes, quickly upside down the culture plate. Then, place the culture plate in a 37℃ incubator for 30 minutes.
5）After gelatinization, organoid medium will be added to continue culture.
5. PDX modelling
1）Due to the relatively conservative protein between species, the pig-origin ECM-Gel has low immunogenicity. In addition, PDX mice are usually immunodeficient. Therefore, the species differences between ECM-Gel and Matrixgel have little effect on the establishment of PDX models.
2）Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has some bubbles in the pre-gel solution, they can be removed by centrifugation of 400g for 15 seconds at a low temperature.
3）Add ECM-Gel to pre-chilled cells/tissues, organoid suspensions, and mix gently. This product can be rapidly gelatinized in mice. The minimum tested gel-forming concentration is 3mg/ml, and the gelling time is about 10 minutes. The higher concentration of the ECM-gel, the shorter gelling time and the stronger stiffness achieve.
4）Use the pre-chilled tip or needle to aspirate the ECM-cell suspension. Then quickly transplant it into subcutaneous and other sites of mice under anesthesia to avoid colloidal outflow. The injection process can be practiced, which confirms whether the ECM-Gel has been well gelatinized and the gelatinized time during the process.
IV The digestive passage/cryopreservation
When organoids need to be passed, organoids in the ECM-Gel can be recovered using the organoid recovery solution (LV-ORS001/2) produced by our company.
1. Thawed digestive solution
Thawed digestive solution could be temporarily stored at 4℃ for 1 week or refrozen back to -80℃ or -20℃ (freeze-thaw no more than 3 times). It is recommended to dispense according to the usage of digestive solution when opening a new one, to avoid repeated freeze-thawing. Repeated freeze-thaw or long-term storage at 4℃ will reduce the effect of the digestive solution.
2. Digestive process
1) Remove the medium, gently wash with PBS for 1-2 times. Add 200μl 37℃-preheated organoid recovery solution to 50μl ECM-Gel.
2) Destroy the ECM-Gel with a tip in the shape of“米”, so that the ECM-Gel will be suspended in the recovery solution.
3) Transfer the plate to 37℃ incubator for 20-30 minutes, during which the plate can be gently shake to speed up the digestion process.
4) Determine the digestion degree under a microscope. Excessive digestion may break down organoids into single cells.
Note: The digest time should be carefully tested, e.g. the excessive digestion of intestinal organoids may affect the structure and function of organoids. In some cases, the digestion process can be carried out at RT or on ice.
5) Terminate the digestion by adding the culture medium and transfer the organoid suspension to the centrifugal tube.
6) Clean the culture well once with medium, to fully collect the cells into the centrifugal tube.
7) Organoid precipitation will be obtained by centrifugating at 50-400g (adjust it according to personal requirements) at low temperature for 5 minutes.
8) Sub-culture can be carried out according to the step of “III”.
9) Cryopreservation can be improved by using our organoid cryoprotectant (LV-OCS001/2) and miniaturized programmed freezers (LV-SCryo001/2).
V Recommended products
ECM-Gel Products, relevant products and their applications:
Type | ECM-Gel | Cat.no. | Concentration | Applications |
General | ECM-Gel with phenol red | LV-6828-1-10ml | 8-10mg/ml | Primary cell growth and differentiation, cell adhesion, organoid maturation differentiation, and in vivo tumorigenesis experiments a |
ECM-Gel phenol red-free | LV-6828-2-10ml | 8-10mg/ml |
Tissue/Organ-specific Low GF phenol red-free | Liver | LV-6833-10ml | 8-10mg/ml | Organ/tissue primary cell maintenance and differentiation, stem cell and organoid maintenance and differentiation |
Kidney | LV-6834-10ml | 8-10mg/ml |
Lung | LV-6835-10ml | 8-10mg/ml |
Heart | LV-6836-10ml | 8-10mg/ml |
Small intestine | LV-6841-10ml | 8-10mg/ml |
Colon | LV-6842-10ml | 8-10mg/ml |
Bone | LV-6843-10ml | 8-10mg/ml |
Skin | LV-6844-10ml | 8-10mg/ml |
Customize | LV-6845-10ml | 8-10mg/ml |
Supporting reagents | Preservation solution | LV-TSM001/2 | 10/100ml | Tissue preservation & transport at RT |
Recovery medium | LV-ORS001/2 | 10/100ml | ECM-gel digestion and cell recovery |
Cryopreservation | LV-OCS001/2 | 10/100ml | Cryopreservation of organoid |
Consumables | ULA plate/diss/Flask | LV-ULA002X | Various | Culture of spheroid and organoid |
ECM coated | LV-Mg-X | Various | 2D cell culture |
a Poor angiogenesis, cell migration
