Preparation Of High Purity Umbilical Cord Mesenchymal Stem Cells And Umbilical Cord Blood CIK Cells

With the continuous innovation of biological targeted drugs and medical technology,cellular medicine is also constantly changing the therapeutic environment of traditional medicine,opening the door to regulate sub-health and treat malignant diseases.New technological update will bring new challenges and opportunities.Therefore,how to prepare qualified cell products is the primary task in front of us at present.In order to improve the cell process system,this study has done related research.1.Optimization of the preparation method of high-purity umbilical cord mesenchymal stem cells:the Wharton’s Jelly of neonatal umbilical cord was cut into pieces,evenly arranged at the bottom of the culture flask,subcultured to the third generation,harvested and analyzed,and different levels of parameters were set from the extraction site,lotion type,centrifugal force and centrifugation time.The results showed that the best preparation parameters were:The optimal preparation process was using complete medium as detergent,centrifugal force 500g and centrifugation time of 7min.Under these conditions,the number of umbilical cord mesenchymal stem cells P3 could reach 4.83×10¹⁰,the mean positive immunophenotype was 99.29%,the mean negative immunoexpression was 0.92%,and the proliferative ability parameter was 1.71.From the morphological microscopic observation of the cell,it was spindle-shaped tightly vortex growth.The experimental conditions of this process optimization are practical and the results are satisfactory.2.Construction of CIK cell technology system derived from umbilical cord blood:to study the proliferation efficiency and cytotoxicity of the cells,and to provide high-quality CIK cells for clinical application to do basic research.The umbilical cord blood of healthy full-term puerpera was collected,and the umbilical cord blood PBMC was obtained by density gradient centrifugation method and cultured into mature CIK.The results of the four aspects of Ficoll dosage ratio,lotion,centrifugal force and centrifugation time were analyzed by single factor test and orthogonal test.The experimental results showed that the optimal extraction process of CIK immune cells from cord blood was as follows:Ficoll and blood mixture ratio was 1:2,300g PBS lotion,centrifugal force,the centrifugal time for 7 min for the optimal preparation process,and the process of cell proliferation,most powerful from the cell morphology of microscopic observation,there was no significant difference of each group,are all full bright,suspended mass of proliferation,under the condition of the process,the cord blood CIK cells can reach 5.267×10⁹.The mean value of positive immunophenotype was 42.07%,in which CD3+CD56+double positive reached17.98%.In this in vitro activity experiment,it was concluded that cord blood CIK cells had an efficent-target ratio of 20:1,and the strongest killing activity reached a high efficiency of more than 60%.3.Research on microbial safety testing for clinical cell applications:To the requirement of clinical application level cells,the experiment of the above two kinds of cells in batches to strict microbial safety testing analysis,the sample is the donor infectious disease progressive immune colloidal gold labeling technique showed negative,germiculture negative,mycoplasma kits negatie results of rapid identification,endotoxin test results were negative limulus reagent gel method,Setting in detail the quality control standards do optimization,are important,to avoid pollution diffusion in the laboratory and clinical application of safety is essential,with the development of basic research,review the latest methods of the standard quality control system and establish the dimensional variety of detection index is very necessary,this study for cell therapy provides certain guiding value in the future.Aiming at establishing standard cell preparation system,this study provides all aspects of the process parameters,the source of the difficulty lies in the cells due to individual differences may not be exactly the same,there is a quality control standard cell products different from chemical drugs,can’t do content and forms of the same,and cell products in the human body without a stable blood drug concentration testing standard,In order to avoid the existence of these unstable factors as much as possible,it is extremely important and urgent to cultivate high quality cells.In this experiment,the preparation process with strong feasibility,simple operation,high efficiency and standard was determined,which provided a reliable preparation quality document system for the establishment of standard cell bank and subsequent cell application research.