Product Introduction
ECM-Gel
Store at -20℃, avoid repeated freezing. Please follow the instructions of the products. If you have any questions, please consult the technical personnel of the company. This instruction is applicable to all kinds of ECM-gel of our company.
Ⅰ. Introduction
ECM-Gel is a kind of transparent colloid made from the extracellular matrix of healthy pigs through elution, crushing and digestion. Compared to rodents, pigs are closer to humans in terms of genetic distance. In addition, there are a large number of application examples of pig-derived materials in clinical transplantation, medicine, cosmetic beauty and pharmaceutical auxiliary materials. At present, hundreds of documents have been published on Pubmed, indicating that pig-derived ECM-Gel has natural substitutability and clinical transformation potential. The major components of this product are laminin, type IV collagen, nestin and heparan sulfate proteoglycan. Different tissues-derived or organ-derived ECM-Gel has great differences in their composition with the advantages of low endotoxin and DNA content. It is mainly used to support cell growth and differentiation in vitro, metabolic/toxicology studies, cell adhesion, angiogenesis, cells migration/invasion and in vivo tumorigenesis experiments, etc.
ECM-Gel Products and their Applications:
Type |
ECM-Gel |
Cat.no. |
Size |
Applications |
Standard |
ECM-Gel |
LV-6828-1 |
5/10ml |
Cell growth, differentiation, invasion, morphology, function, gene expression, etc. Protein concentration is 8-10mg/ml. |
ECM-Gel phenol red-free |
LV-6828-2 |
5/10ml |
||
Growth Factor Reduced (GFR) |
ECM-Gel |
LV-6829-1 |
5/10ml |
Suitable for higher requirements of ECM-Gel experiments, reduce the interference of cytokines;Protein concentration is 8-10mg/ml. |
ECM-Gel phenol red-free |
LV-6829-2 |
5/10ml |
||
Standard, High Concentration |
ECM-Gel |
LV-6830-1 |
5/10ml |
In vivo experiments, angiogenesis, culture of tumor organoids, etc. Better support for 3D cell culture, cells grow better in 3D environment;Protein concentration is 16-20mg/ml. |
ECM-Gel phenol red-free |
LV-6830-2 |
5/10ml |
||
GFR, High Concentration |
ECM-Gel |
LV-6830-3 |
5/10ml |
|
ECM-Gel phenol red-free |
LV-6830-4 |
5/10ml |
Remarks: Our ECM-Gel products are derived from healthy pigs, not mouse EHS tumor tissue. The matrix collagen composition and proportion are slightly different, and the optimal gelatinization concentration is slightly lower than the Matrix-gel concentration.
Ⅱ. Reagents and Materials
-Serum-free medium
-Vortex mixer
-Refrigerated centrifuge
-Pre-chilled centrifugal tube
-Culture plate
-Pipette tube
-Pre-chilled
-Biosafety cabi tip
-Pipettenet
-CO2 incubator (37℃/5%)
Ⅲ. Method
Note:
For your safety and health, please wear experimental clothes, disposable masks and gloves, and take necessary protective measures.
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 it only once. If the ECM-Gel has more bubbles, they can be removed by centrifugation at a low temperature of 400g for 5 minutes. Place the ECM-Gel in a sterile area and set it aside on ice.
Gelling:
1. Thin layer gel method (thickness 0.5 mm, low growth factor, 8-10 mg/ml)
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 more bubbles, they can be removed by centrifugation at a low temperature of 400g for 15 seconds.
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 (culture plate or the Transwell chamber, standard/growth factor reduced 8-10mg/ml)
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 more bubbles, they can be removed by centrifugation at a low temperature of 400g for 15 seconds.
2)Dilute the ECM-Gel with pre-chilled serum-free medium in the ratio of 1:6-1:8 (protein coating amount of -500μg/cm2). Then, add the diluted ECM-Gel to the Transwell chamber, taking the 24-well plate as an example, 50-100μl can be 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 migration invasion experiments.
4)The amount of protein coating ranges from 50µg/cm2 to 500µg/cm2 (cm2 is the culture floor area). The optimal amount needs to be optimized according to the researcher's experience or the personalized needs of the researcher.
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 more bubbles, they can be removed by centrifugation at a low temperature of 400g for 15 seconds.
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, gently mix well.
3)Add cell-containing ECM-Gel to the culture plate at 150-200μl/cm2 (cm2 is the culture floor area).
4)After gelatinization, add fresh medium and incubate in a 37℃ incubator.
Recommendation: Add medium to wash the uncoagulated matrix protein and incubate in a 37℃ incubator for 10 minutes. Then switch to fresh medium to achieve excellent 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 more bubbles, they can be removed by centrifugation at a low temperature of 400g for 5 minutes.
2)Add ECM-Gel to pre-chilled cells/tissues, organoid suspensions (The optimal concentration of ECM-Gel is 4-6mg/ml). Mix gently;
3)Draw the matrix gel suspension and gently add it into the 37℃ preheated culture well;
4)Place at room temperature for 1-2 minutes, quickly upside down the culture plate (the strength needs to be mastered by oneself and the standard is to form a teardrop-like arch). Then, place it in a 37℃ incubator for 30 minutes.
5)After gelled, organoid medium will be added to continue culture.
5. PDX modelling
Pig-derived ECM-Gel is less immunogenic due to relative conservation among protein species, and PDX mice are usually immune-deficient mice, so differences between species have no effect on the establishment of PDX model. After a lot of tests, our company found pig-derived ECM-Gel can be used for tumor formation of various tumors, and the effect is similar to that of imported brands.
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 more bubbles in the pre-gel solution, they can be removed by centrifugation at a low temperature of 400g for 15 seconds.
2)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.
Use the pre-chilled tip or needle to aspirate the ECM-Gel suspension. Then quickly transplant it into subcutaneous and other sites of mice under anesthesia to avoid colloidal outflow. The injection process can be practiced, and it confirms whether the ECM-Gel has been gelatinized and the gelatinized time in the process.
Ⅳ. The digestive passage/cryopreservation
When the organoids need to be passaged, our supporting organoid recovery solution (LV-ORS001 /2) can be used to recycle the organoids in the ECM-Gel.
1. 1. Thawed digestive solution could be temporarily stored at 4℃ for 1 week or refrozen back to -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 freezing and thawing or long-term storage at 4℃ will reduce the effect of the digestive solution.
2. 2. Remove the old culture medium, gently wash with PBS for 1-2 times. Add 200μl of recovery solution per 50μl of the ECM-Gel to the amount of recovery solution pre-warmed at 37 °C. Then, draw the appropriate amount of recovery solution and blow it down from the top of the organoid. Destroy the ECM-Gel in the shape of“米” with a tip, so that the ECM-Gel will be suspended in the recovery solution. Transfer the plate to 37℃ incubator for 20-30 minutes, during which the plate can be gently shake to speed up the digestion process. Determine the digestion degree under a microscope. Excessive digestion may break down organoids into single cells; Note: If the gel disappears completely, there may be over digested. The digest time should be carefully tested, e.g. the excessive digestion of intestinal organoids may affect the occurrence and function of organoids. 3. Terminate the digestion by adding the culture medium and transfer the organoid suspension to the centrifugal tube. Clean the culture well once with medium and collect the cells into the centrifugal tube. Organoid precipitation will be obtained by centrifuging at 50-400g (adjust it according to personal requirements) at low temperature for 5 minutes. 4. Sub-culture can be carried out according to the step of “III”.
5. Cell cryopreservation can be improved by using our organoid cryopreservation solution (LV-OCS001/2) and Small-scale Controlled Rate Freezer (LV-SCryo001/2).
Ⅴ. ECM-Gel Products and their Applications