A Protocol For Culture Of Microvascular Endothelial Cells From Different Tissues And Comparison Of Their Biological Characteristics

Objective:Isolating adipose microvascular endothelial cells(AMECs)and pulmonary microvascular endothelial cells(PMECs).Establishing a culture scheme for microvascular endothelial cells.Comparing the characteristics of two cells.Laying the foundation for our further study on amplification of HSCs in vitro.Providing the evidence for experimental study and clinical application about MECs.Methods:1.The cultivation of microvascular endothelial cells was established:(1)Tissues were collected from epididymal fat and lung in mice,and digested with type I collagenase.(2)To find the optimal age of mice for draw materials from epididymal adipose tissue,grouping:①4 w,②6w③8w,④ 10w,criterion:The number of seed cells from 1ml adipose tissue.(3)To explore the best time to change medium firstly,grouping:①24h,②48h,③72h,criterion:whether or not the MECs were obtained.(4)To search the best seeding density for primary culture,grouping:①5 ×105/cm2,② 1 × 106/Crcm2,③ 2×106/cm2,criterion:the growth of MECs.(5)To explore the best concentration of gelatin which was used to coat culture dishes.grouping:①0,②0.1%,③0.5%,④/%,criterion:the cell number of MECs.2.The biological characteristics of AMECs and PMECs were compared:(1)To compare the number of AMECs and PMECs from one mouse.(2)The growth and morphology of cells were observed under phase-contrast microscope.(3)The expression of CD31,CD34,CD45 and vWF in AMECs and PMECs were detected by immunofluorescence and flow cytometry.(4)To compare the passage cells from AMECs and PMECs.(5)Cell growth curve was drawn by counting method.(6)10ng/ml VEGF was used to culture two kinds of MECs.Results:1.The best method for isolation and culture of microvascular endothelial cells:(1)Two kinds of tissues were isolated successfully by enzyme method.(2)The number of seed cells in 1ml adipose tissue from 4w mice was 4.67±0.58(106),6w mice was10.18±0.28(106),8w mice was 9.94±0.35(106),10w mice was 6.72±0.39(106);6w and 8w were more than 4w、10w(P<0.05),There was no difference between 6w and 8w(P>0.05)。(3)AMECs and PMECs could be got by changing medium firstly after 24h,could not be got by changing medium firstly after 48h and 72h.(4)1×106/cm2 was optimal inoculum density,MECs was poor proliferation with a density of 5×105/cm2,ECs emerged contact inhibition with a density of 2×106/cm2.(5)In 8th day:the AMECs number of uncoated gelatin group was 148.33±13.54,0.1%group was 201.83 ±12,97,0.5%group was 191,50 ±11.52,1%group was193.00±8.67,The AMECs number of gelatin coated groups were more than uncoated gelatin group(P<0.05),There was no difference between the 3 concentrations(0.1%,0.5%,l%)(P>0.05).In 10th day:The PMECs number of uncoated gelatin group was 182.33±11.03,0.1%group was 219.50 ±11.4,0.5%group was212.00±12.39,1%group was213.00±12.12;The PMECs number of gelatin coated groups were more than uncoated gelatin group(P<0.05),There was no difference between the 3 concentrations(0.1%,0.5%,1%)(P>0.05).2.Comparison of biological characteristics between AMECs and PMECs:(1)In primary culture of AMECs and PMECs:colonies formed in 48h-72h,cells became polygonal,oval in 4th day,radiating cell mass formed in 7th day,AMECs arrayed into cobblestone-like structure in 10th day while PMECs was 14th day,cells surface covered with microvilli.(2).Seed cells of AMECs harvested from one mouse were 2.08±0.57(×106),PMECs werel9.07±0.72(×106)(P<0.05).Primary cells of AMECs harvested from one mouse were 0.26±0.05(×106),PMECs were 3.37 × 0.18(×106)(P<0.05).(3)AMECs and PMECs expressed CD31,CD34,vWF,did not express CD45.(4)The two kinds of passage cells grew faster than primary cells and their morphological characters were similar.(5)The growth curve showed that the growth peak of AMECs was 3-6 days after inoculation,and the growth peak of PMECs was 4-7 days after inoculation.(6)Cells extend,migrate,fuse,form cell cords,and the cords connect and communicate with each other.Conclusion:1.In this study,a simple,effective and economical method for isolate and culture AMECs and PMECs is established:changing medium firstly after 24h,primary density is 106/cm2,2ng/mlVEGF,0.1%gelatin,mice in 6-8 week.2.The morphology,the markers,the growth curve,the passage cells of AMECs are similar to PMECs.The number of AMECs from one mouse are different from PMECs.