How To Extract Smooth Muscle Cells

The Price Of Rat Aortic Smooth Muscle Cells Was 3500. The Technical Bone Glanced At It And Said That It Was Only Tens Of Dollars To Buy A 90-110g Healthy Male Rat, And The Domestic Antibody Was So Cheap. Why Not Mention It? The Next Day, When The Rats Got Home, The Small Bones Began To Use The Fancy Technique Of Aortic Smooth Muscle Cell Separation. The Methods Of VSMC Isolation And Culture Mainly Include Tissue Block Adherence Method And Enzyme Digestion Method. In The Former, The Adventitia Is Mechanically Stripped, The Intima Is Scraped, And The Blood Vessels Are Cut Into 1mm ³ The Small Pieces Of. This Method Requires High Operating Skills. It Is Easy To Peel Off The Outer Membrane, Scrape Off The Inner Membrane With Too Little Force, There Will Be Residual Endothelial Cells, And Scrape The Middle Membrane With Too Much Force. Therefore, When Beginners Use This Method To Separate VSMC, They Often Have The Problems Of Tissue Block Floating And Long Cell Culture Time. The Principle Of Enzyme Digestion Is To Use Protease To Directly Digest The Blood Vessels That Have Removed The Clean Surrounding Tissues To Separate VSMC. The VSMC Is Separated By Single Digestion Of Collagenase And Pancreatin Or Double Enzyme Digestion. However, The Digestion Time Of Collagenase Generally Takes 6-12 Hours. However, The Pancreatin Has Strong Damage To VSMC, And The Digestion Time Is Not Easy To Master. It Is Easy To Cause Great Damage To Smooth Muscle Cells Due To Uneven Digestion, And It Is Not Easy To Obtain Smooth Muscle Cells With Good Activity And Large Quantity. So At Present, We Usually Use The Method Of Combined Enzyme Digestion And Tissue Block Adhesion To Extract Smooth Muscle Cells; The Rats Were Anesthetized With Ether, And The Thoracic Aorta Was Quickly Cut Off; Take It Out And Place It In A Container At 1-4 ° C. 2. Use A 2ml Syringe To Take PBS Or Autoclaved Normal Saline. Rinse The Thoracic Aorta Repeatedly, Remove The Clotting And Other Tissue Blocks Attached To Its Surface, And Remove The Surrounding Tissues Of The Blood Vessels. When The Blood Vessels Become Transparent And The Surrounding Tissues Become Flocculent During Digestion, Digestion Can Be Terminated And Then The Vascular Adventitia, Intima And Tunica Media Can Be Peeled Off; 4. The HBSS Containing Ca2+and Mg2+rinses The Separated Middle Film Layer And Places It In A Clean Culture Dish For Quick Cutting (1-2mm ³)， Then It Is Transferred To A 6-well Plate With Compound Enzyme Solution For Secondary Digestion. Digestion Is Carried Out In The Cell Incubator For About I Hour. Take It Out And Shake It Slightly Every 20 Minutes. After 50 Minutes Of Digestion, It Is Generally Observed That A Large Number Of Cells Are Exposed In Grape Shape On The Surface Of The Tissue Block And Begin To Dissociate. At This Time, Add A Smooth Muscle Cell Culture Medium To Terminate Digestion. Note: The Serum Content Of The Medium To Terminate Digestion Needs To Be Increased To 10%. 5. Use A 1ml Syringe To Beat The Tissue Digestion Solution Until The Cells Are Basically Separated From The Tissue Fragments, And Then Filter It With A Cell Sieve (the Aperture Of The Cell Sieve Is 40 μ π ι) Obtain Suspension; Take The Tissue From The Filter Screen, Repeatedly Beat And Filter, Transfer The Cell Suspension After Multiple Filters Into A 15ml Centrifuge Tube, And Collect The Cells After Centrifugation For 5 Minutes At 1000 Rpm; 6. Take Smooth Muscle Cell Suspension Medium Containing 10% FBS, And Plant It Into 6-well Plate For Culture. 7. The Freshly Separated P0 Generation VSMC Was Suspended In The Smooth Muscle Cell Culture Medium And Incubated In The Incubator For 2 Days At 37 ° C And 5% CO2. 8.48 Hours Later, It Was Observed That The Cells Had Adhered To The Wall And Began To Spread Out And Replace With A New Smooth Muscle Cell Culture Medium. After About 7 Days Of Culture, The Cells Could Be Subcultured. After Subculture, The Serum Dropped To 2% Of The Normal Serum& Nbsp; & Nbsp;