Matrigengel Matrix IPSC Level-082777

PRODUCT 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. Wintop Matrigengel Matrix IPSC Level is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumor.Mogengel Matrigengel Matrix IPSC Level at 37 °C to form a reconstituted basement membrane. The major components of Wintop Matrigengel Matrix IPSC Levelinclude laminin, collagen IV, entactin, and heparin sulfate proteoglycan. INTENDED USE It is a mature growth substrate for feeder-free hES culture. It’s used in combination with specific media to culturize stem cells (e.g, hESC, iPSC). The screened stem cell Matrigengel Matrix  provide the reproducibility and consistency required for trophoblast culture of human embryonic stem cells and induced pluripotent stem cells, and can also be used in vivo differentiation studies, such as teratoma formation. MATERIALS PROVIDED Art.No. Product Name Specification STORAGE/Shipment 082777 Matrigengel Matrix  IPSC Level 10mL ≤-20°C 0827775 Matrigengel Matrix  IPSC Level 5mL ≤-20°C 082777 T Matrigengel Matrix  IPSC Level 1mL ≤-20°C 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 l Routine screening of mouse colony pathogensby mouse antibody product (MAP) tests l Testing for bacteria, fungi and mycoplasmato ensure negative results l Extensive PCR testing for a variety of pathogens including LDEV to ensure strict control of raw materials used in the production process l Extraction from LDEV-free mouse tumor cells l Gel stability testing at 37℃ for 14 days l Detection of endotoxin levels using serological methods l Biological function verification of each lot (organoid culture and differentiation experiments; Subcutaneous tumor formation test; Stem cell culture; Angiogenesis experiment etc.) COATING PROCEDURES: Thaw Matrigengel Matrix overnight at 2-8 °C. Refrigerator temperatures may vary, therefore it is recommended to keep Matrigengel on ice in a refrigerator during the thawing process. Thawed Matrigengel solidifies quickly at temperatures above 15 °C; when working with Matrigengel, keep it on ice to prevent untimely gelling. There are many applications for Matrigengel 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: Thaw Matrigengelas stated above. Mix Matrigengel by slowly pipetting solution up and down; be careful not to introduce air bubbles. Pipette 200-300 μL per cm2onto the growth surface. Place coated object at 37 °C for 30 minutes. Coated objects are ready for use. Thin Layer Method (non-gelling): Thaw Matrigengelas stated above. Mix Matrigengelby slowly pipetting solution up and down; be careful not to introduce air bubbles. Dilute Matrigengelto 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. Add a sufficient amount of solution to cover the entire growth surface area. A volume of 300 µL per cm2is recommended. Incubate coated object at room temperature for one hour. Aspirate coating solution and immediately plate cells. Do not allow coated surface to dry out. iPSCs experiment PROCEDURES 1、Materials 1.1 Reagent: Matrigengel(Art no.082777/0827775/082777T); ROCK inhibitor (Y-27632); DMEM/F12; mTeSR1/E8 medium; Accutase Dissociating solution, PBS(D-PBS) 1.2 Consumables: sterile tips, 6-well plate（or other well number of plates，This protocol will use a six-well plate as an example）, sterile EP tube. 2、Preparation 2.1. Material pre-cooling 2.1.1 Put the Matrigengel in the ice box and put it in the refrigerator at 4℃ so that the Matrigengel can slowly melt overnight; (Do not allow this product to warm up above 4°C during manipulation. Keep the product on ice and dilute using ice-cold solutions or cell suspensions.)； 2.2.2 Supplies or reagents that come into contact with Matrigengel, such as sterile centrifuge tube, sterile tips and DMEM/F12, were be pre-cooled at 4℃ in advance. 2 Dilute the Matrigengel(Art no.0827775) at（1:80 –1:100）(Tip：1:80 – 1:100 are all appropriate,equivalent to Matrigengel concentration of 0.1mg/mL.For iPSC culture, Mogengel＆ABW recommends coating at a concentration of approximately 0.013 mg/cm², for example, after diluting 12.5 mg/mL of Matrigengel 1:100, the coating volume of each 6-well plate is 1 mL) 2.2.1. Transfer appropriate of DMEM/F12 at 4℃ into the cooled EP tube； 2.2.2. Use a pipette with the pre-cooled tips to transfer the DMEM/F12 to the aliquot of Matrigengel, mixed well and then transferred to the other EP tube(kept on ice). Repeat this step 2-3 times until the matrix gel is completely pipetted into the EP tube，replenish the dilution to a final concentration that meets the requirements of your own design after that； 2.2.3. The mixture is as a reserve for subsequent coating. 2.3.Plate coating procedure  2.3.1 Add the 1mL/ well Matrigengel mixture into the pre-cooled 6-well plate , and gently shake the plate to ensure that the mixture is evenly spread on the plate; 2.3.2 Transfer the 6-well plates to a 37℃ incubator for overnight incubation (the plates can be used after incubating for 1-2 hour, but the coating for overnight incubation is better for cell culture.Coated plates with coating solution can be stored at 4ºC and should be used within one week of coating. Coating solution should be aspirated just before using the plates); 2.3.3 Absorb the liquid above the coating before use. 2.4 Configure ROCK inhibitor (Y-27632) working solution: use sterile PBS to dissolve Y-27632 and configure it into 10mM solution (1000X) with working concentration of 10uM; 2.5 Preparation of medium containing ROCK inhibitor: ROCK inhibitor (Y-27632) with 10mM solution was added to mTeSR1/E8 medium until the final concentration was 10uM. Note :The Matrigengel at 4℃ will gradually polymerization into glue.Please strictly control the operating temperature and the operating time. Cell culture 3.1. iPSC thawing 3.1.1 Remove the ipsc from the liquid nitrogen or dry ice and thaw it in water at 37℃. The thawing should be completed quickly; 3.1.2 Disinfect the frozen tube with 75% alcohol and transfer it to bechtop; 3.1.3 Transfer the cell solution to a new 15ml EP tube and flush the primary tube twice with DMEM/F12/DMEM; 3.1.4 Centrifuge the 15ml EP tube at room temperature 300g for 5min(ipsc has good tolerance to 200-300g speed, and 300g is recommended to maximize cell capture, and 200g is recommended in the standard procedure) 3.1.5 Discard the supernatant, gently resuspend the iPSC with 2mL of medium containing ROCK inhibitor, and transfer it to the coated 6-well plate. Shake the plate evenly to distribute the cells (Cell density was adjusted to 1×106 cells per well) 3.1.6 Put the 6-well plate back into the 37℃ incubator (please do this immediately after the cells are transferred to avoid increased cell center density). 3.1.7 The ROCK inhibitor was removed the next day, and the cells were cultured with the non-inhibitor medium. Note: The use of antibiotics in cell culture is not recommended as they can interfere with cells and their differentiation potential. The culture environment should be isolated from other cells, and the mycoplasma should be detected after two passages; If the cryopreservation solution contains DMSO，It’s toxic to cells at room temperature and the cell thawing procedure should be completed quickly. 3.2 iPSC passaging 3.2.1 Discard the culture supernatant, rinse with 1mL PBS, and add 1mL Acuutase; 3.2.2 Transfer the plate to a 37℃ incubator for 3 minutes, or observe under a microscope until most cells fall off (if cells are still attached, place the culture plate in your hand and gently place the other hand on the flap which