Experimental Study On Vitreous Cryopreservation Of Tenocyte-PDMS Scaffold Composite

Objective For meeting the needs of tissue-engineered tendon industry,we want to establish a experimental model for studying the cryoperservationof tissue-engineered tendon, and to find a better CPA cocktail withappropriate protocol. The final object is achieving a higher recovery andviability of post-crypreservation tissue-engineered tendon.Methods Primary tenocytes were isolated from tail of young SD rat andwere sub-cultured for collagen typeⅠ,Ⅲimmunocytochemistry identificationand subsequent experiments. A surface microgrooved PDMS substrate and3D PDMS foam was coated by collagen typeⅠfor the tenocytes adhesion.The cells and PDMS were co-cultured for 9 day for study. The vitrificationsolution VS55 and 21%DMSO was added by continuous instillation within30min at 0℃. VS55 group was immersed directly in liquid nitrogen and the21%DMSO group was cooled at 1℃/min to -60℃and then was immerseddirectly in liquid nitrogen. All samples stayed at liquid nitrogen temperatureat least for 24 hours then were thawed in a 37℃water bath. CPA was removed by continuous instillation at 0℃too. After 1 hour's re-culture, thepost-thaw cells adhesion and morphology on substrate were observed byscanning electron microscopy and fluorescence staining. The post-thawviability was measured by double fluorescence staining flow cytometricmeasurement.Results In cells isolated from SD rat tail, collagen typeⅠshowedpositive immunocytochemistry staining and collagen typeⅢnegative. Itmeets the biological characteristic of tenocyte. Tenocyte can grow as adefinite shape and direction on collagen coated surface microgrooved PDMSsubstrate. This can act as a model for studying the relationship betweenshapes of cells adhesion on substrate and cryopreservation output ofcell-material composite. Post-thaw viability of tenocytes adhesion oncollagen typeⅠcoated 3D PDMS foam were achieve by VS55 (mean 64.9%)and 21%DMSO (mean 76.2%) (P＜0.05).Conclusions Vitreous cryopreservation needs a complicated operation foraddition and removing of high concentration CPA. As lacking of effectivetemperature control equipment, my experimental results are not satisfactory.Vitreous cryopreservation needs a standard machine controlled protocol forstable results output. 21%DMSO as a relation high concentration freezingCPA which only needs simple temperature control and CPAaddition/removing protocol is better than VS55 for tenocytes croperservatonin current conditions. The shape of adhesion cell may affect the results of cryopreservation, it needs a further research.