| OBJECTIVE Firstly our research was to find a goodresearch method on cell survival rate at the procedure of cryopreservation of seeding cells; Secondly to find how different factors affect the cells survival rate of seeding cells in the process of cryopreservation, so we can make use of them and find one of the best prescription to cryopreserve the seeding cells, optimize various kinds of factors in the process of cryopreservation to raise the cell survival rate. By that way, we can know some rules in cryopreservation and get ready to preserve of tissue engineered tendons; Lastly some preliminary study on cryopreservation of tissue engineered tendons have done. METHODS1. Cell suspension of tissue engineered tendon's Seeding cells collected and divided equally into two parts,One half of them freezed and defrost repeatly for 3 times to kill the cells,do nothing to the other half, then cell suspension of different living cells to dead cells ratio was made before fluorescence staining and flow cytometry to study the correlation of cell suspension volume of living cells and cell survival rate. Pictures have taken and image analysis have done. To find a good way in study Cell survival rate at the procedure of cryopreservation of seeding cells.2. Experimental study on preservation of tissue engineered tendon's seeding cells(1) Seeding cells cryopreserved with 3 prescriptions of vitrificationsolution, defrost them half month late, Stained with Ho and PIrespectively before flow cytometry to study which is the bestcryoprotectant.(2)Seeding cells cryopreserved with vitrification solution number 2, defrostthem half month late and transfer to 20% human albumin, calf serum,DMEM(including FCS) and PBS( phosptat buffe salt) respectively for 20minutes. Stained with Ho and PI respectively before flow cytometry tostudy which is the best treatment after they are defrost fromcryopreservation.(3) The seeding cells of different concentrations are preserved for twoweeks then defrost them and stained with Ho and PI respectively beforeflow cytometry to find the relationship between cells concentration and cellsurvival rate.(4)Before cryopreservation, the seeding cells cooled by ways of slowcooling procedure and rapid cooling(drop into liquid nitrogen directly)method respectively. 3 weeks late defrost them and marked with Ho and PIrespectively before flow cytometry to find if the cooling methods affect thecell survival rate.(5) Both seeding cells before cryopreservation and those defrost fromcryopreservation by 10%DMSO +15%FCS+75%DMEM cultured on slidesthen compare their collagen type I and type III byimmunohistochemical study to know their cells secretivefunction.(6)The cell growth curve, cell cycle and chromosome mode of seedingcells not cryopreserved and cryopreserved are detect to compare theirdifferences of the cells growth curve.cells cycle and cells chromosome mode.3. Seeding cells co-culture with human derived tendon scaffold for 4 days to make tissue engineered tendons, then cryopreserved them in 3 prescriptions of vitrification solution for 1 month, defrost them and digest with TNE for a few minutes to purge cells from the scaffold before stained with Ho and PI respectively and flow cytometry them. They are also scaned with scanning electron microscope, HE stain before and after cryopreservation for 1 month. Seeding cells marked by Brdu (5-bromo-3-deoxyuridine), we did immunohistochemical study before cryopreservation and 1 month, 2 months and 3 months after cryopreservation respectively to check the cells of tissue engineered tendons by microscape. RESULT1. Cell survival rate have much to do with that of cell suspension volume of cryopreserved and cell suspension volume of non-cryopreserved by flow cytometry ; same result also found in image analysis study, and both of the correlation is significant at the 0.05 level.2. Experimental study on cryopreservation of tissue engineered tendon's seeding cells(1) The group with cryopreservation solution DMSO have the hightest cell survival rate, the differences are significant at 0.05 level.(2) Those treated with Calf serum or DMEM(including FCS)after their cryopreservation have the highest cell survival rate,while those treated with human albumin have the lowest cell survival rate, the differences are significant at 0.05 level.(3)Cells concentration is important to cell survival rate in process ofcryopreservation, the difference is significant at 0.05 level while it above2.0X 106 /ml and less than 1.0X 106 /ml.(4)Cooling methods in cryopreservation is important to cell survival rate,the difference bewteen slow cooling method and rapid cooling method issignificant at 0.01 level.(5)Both seeding cells cryopreserved by 10%DMSO+15%FCS+75%DMEM and not cryopreserved secret type I and typeIII collagen, no difference observed by image analysis from theirnmunohistochemical study P>0.05.(6)No significant changes observed on the cells growth curve, cells cycleand chromosome mode between seeding cells not cryopreserved andwhose cryopreserved.3. Cryopreservation of tissue engineered tendons(l)Living cells can found on tissue engineered tendons before theircryopreservation by scanning electron microscopy, HE stain and Brduirnmunohistochemical study.(2) Cells decreaseed after cryopreservation, and it is not enough for flow cytomentry.(3) Some cells observed under scanning electron microscope, HE stain and Brdu immunohistochemical study in that of cryopreservation solution number 2 one month after cryopreservation. Brdu positive cells can observed under microscope even 3 months after their cryopreservation. (4)Living cells are hardly seen after cryopreserved by vitrification solution number 1 and solution number 3 by way of scanning electron microscope, HE stain and Brdu immunohistochemical study 1 month aftertheir cryopreservation. CONLUSION1. Flow cytometry and fluorescence image analysis after PI and Ho staining is a good way in study Cell survival rate at the procedure of cryopreservation of tissue engineered tendons seeding cells. But a number of cells are needed.2. Blood serum nourishment is very important in cells culture, preservation and management after cryopreservation, keep it the same will increase cell survival rate in the process of cryopreservation.3. MDSO is a good cryoprotectant in cryopreservation of tissue engineered tendons seeding cells.4. In the process of cryopreservation, cells concentration is important to cell survival rate, cell survival rate will decrease when it less than 1.0xl06/ml.5. In the process of cryopreservation, the cooling speed is important to cells survival rate, slow cooling method had a higher cells survival rate than that of rapid cooling method.6. Cryopreserved by 10%DMSO +15%FCS+75%DMEM does not affect Seeding cells collagen secretion function.7. Crypresered by 10%DMSO +15%FCS+75%DMEM does not affect seeding cells growth curve,cell cycle and chromosome mode.8. Seeding cells grown well on the scaffold before tissue engineered tendons' cryopreservation, but it is very difficult to have enough cells for flow cytometry.9. Group number 2 with cryoprotectant of DMSO is much better than that of group number 1 with cryoprotectant of ethylene glycol and group number3 with cryoprotectant of glycerol.10. To find a more sensitive way to detect cells viability on tissueengineered tendons is something important and impending .ll.Cryopreservation of tissue engineered tendons is an intricated issue,many factors will affect it,and more researches are needed.
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