Extraction Of HUVEC Cells

Endothelial Cells Have Many Biological Functions, Which Can Be Used In The Remodeling Of Atherosclerotic Blood Vessels To Reduce Vascular Damage And Prevent Cardiovascular Disease; Relieve The Symptoms Of Lower Limb Ischemia In Patients With Furosemide Disease, And Prevent The Occurrence Of Non Traumatic Amputation; In Addition, Endothelial Cells Have Positive Reports On Drug Research Targeting Angiogenesis, Tissue Regeneration And Wound Healing. The Isolation Methods Of Umbilical Vein Endothelial Cells Include Mechanical Scraping, Tissue Block Transplantation, Magnetic Bead Sorting After Tissue Digestion And Collagenase Perfusion. The First Two Methods Have Low Cell Positive Rate, And The Third Method Has High Cost. At Present, The Fourth Method Is The Main Method. Preparation Materials: Fresh Umbilical Vein 15-20cm, ECGM Medium, DMEM Basis, Type II Collagenase, 0.25% Trypsin EDTA, Fetal Bovine Serum, Penicillin, Streptomycin, Gelatin, Rabbit Anti Human Factor VIII Antibody, FITC Labeled Goat Anti Rabbit IgG& Nbsp; The Operation Methods Are As Follows: 1. Aseptically Take About 15 Cm Of Fresh Umbilical Cord And Put It Into Sterile PBS Solution. In The Ultra Clean Workbench, Take Out The Umbilical Cord, Clamp Both Ends Of The Umbilical Cord With Hemostatic Forceps, Immerse It In 75% Alcohol For About 60s, And Take It Out. 2. Place The Umbilical Cord In A Large Plate, Squeeze Out The Blood, Suck It With A Pap Straw, And Cut Off The Umbilical Cord Segments With Many Hematomas And Clip Marks. One End Of The Umbilical Cord Was Cut Off, Exposing Two Umbilical Arteries And One Umbilical Vein (the Umbilical Artery Wall Is Thick, The Lumen Is Small, The Vein Wall Is Thin, And The Lumen Is Large). The Transfusion Extension Tube Shall Be Cut To An Appropriate Length, Inserted Into The Umbilical Vein For More Than 3cm, And Fixed With Hemostatic Forceps. Use A 20mL Syringe To Draw PBS From One End Of The Extension Tube To Wash The Umbilical Vein, And Suck The Washing Solution. 3. Transfer The Umbilical Cord To A New Plate, Ligate The Umbilical Cord At The Other End, Inject 1mL/1cm Collagenase Solution (concentration Of Collagenase Solution: 1mg/ml, Prepared With DMEM) Into The Umbilical Cord, Fill The Umbilical Vein, Seal The Port With Hemostatic Forceps, Digest At Room Temperature For 15-20 Minutes, And Shake The Umbilical Cord Up And Down From Time To Time& Nbsp; 4. After Digestion, Loosen The Hemostatic Forceps Ligated At The Lower End, And The Digestive Solution Flows Into A 50 Ml Sterile Centrifuge Tube. Rinse The Umbilical Cord With Sterile PBS Solution For 2-3 Times. 5. Centrifuge The Collected Suspension At 1000 Rpm For 5 Minutes To Obtain Cell Suspension; 6. Discard The Supernatant, Use ECGM Endothelial Cell Culture Medium With An Additional Increase Of Serum To 15% To Re Suspend The Cells, Inoculate Them In A T25 Culture Bottle Pre Coated With 0.02% Gelatin, And Culture Them In A 37 ℃, 5% CO2 Incubator. 7. Induce Adherence For 24-48 Hours, Change The Solution In Full Amount, And Reduce The Serum To The Concentration Of 5% Of The Normal Culture, Remove The Non Adherent Cells, Add The Normal Fresh ECGM Endothelial Cell Culture Medium, Solve The Problem Of Cell Purification, And Change The Solution In Full Amount The Next Day, When The Cell Fusion Degree Reaches 80-90%, Human Vein Endothelial Cells (HUVEC) Are Obtained By Digestion And Passage With 0.05-0.1% EDTA Containing Trypsin. 8. Using VWF Antibody Immunofluorescence Method To Identify HUVEC Cells, The Specific Procedures Are As Follows: Take Out The Cell Slides From The Culture Plate, Wash Them With PBS For Three Times, And Fix Them With 4% Paraformaldehyde For 15 Minutes; 0.2% Triton X-100 Film Breaking; 10% Goat Serum Was Sealed At Room Temperature For 30 Min; Add Rabbit Anti Human VWF Antibody (1 ∶ 100 Dilution) And Put It Into The Wet Box At 37 ℃ For 60 Minutes; Add FITC Labeled Goat Anti Rabbit IgG, And Incubate At 37 ℃ In Dark For 60 Minutes; ５ μ The Nuclei Were Stained With G · ML - 1 DAPI At Room Temperature For 2 Min And Observed Under Fluorescence Microscope. As Shown Below:  Reference Studies Showed That The Endothelial Cells Did Not Completely Fall Off And The Number Of Cells Was Small When Incubated For 10 Minutes; When Incubated For 20 Min, Not Only Endothelial Cells But Also Smooth Muscle Cells Fell Off, Which Made It Difficult For Later Purification And Culture. The Best Incubation Time Is 15 Minutes, At This Time, The Endothelial Cells Are Completely Digested, And There Is No Contamination Of Foreign Cells. References: [1] Establishment And Optimization Of The Method Of Isolating And Culturing Human Umbilical VeinEndo - The LialCells In Vitro 1671 － 8151 (2015) 03 － 0285 － 05 [2] SiowRC. Culture Of Human Endodermal Cells From Umbilicals [J]. Methods MolBiol, 2012806:265 － 274. [3] Yan Fengying, Yu Ping, Guan Linbo, Et Al. Study On The Method Of Isolation And Culture Of Primary Human Umbilical Vein Endothelial Cells And Its Identification ［ J ］. West China Medical Journal, 2010 (4): 727-729;