Assessment of albumin and aspartate levels as a simple indicator of the efficacy of cryopreservation

Sushrutha C. S., Sandhya K., Savitha Karlwad, Elango E. M.


Background: The process of hepatocytes cryopreservation is standardised by most of the laboratories. However there is a variation with respect to the Protocols, media and equipments used amongst the laboratories. Similarly, the tests available to evaluate the efficacy also varies. They are expensive and sometimes might not measure the parameter required for a particular research study. Hence we propose a methodology to study the few basic parameters like cell viability, synthetic function of the cell and cell stability. We have also used a simple percentile calculation to know the efficacy of cryopreservation. This shall help in functional validation of the cell after cryopreservation. The same can also be used to compare the quality of hepatocytes between different batches. The objective of the study was to characterisation of the cells to determine the efficacy of cryopreservation.

Methods: Two step collagen isolation method was used to isolate the hepatocytes. Initial cell viability was calculated. A sample of cells were taken for characterisation and the remaining cells cryopreserved. The sample cells were divided into two batches one for pre cryopreservation culture and the other for post cryopreservation. The pre cryopreservation culture was done on monolayer using collagen coated 6 well plate. The other sample was placed in the cryovials for cryopreservation for 1week. After 1 week the cryopreserved cells were thawed and the post cryopreservation viability calculated, followed by post cryopreservation culture. During the process of culture (both pre cryopreservation and post cryopreservation) for 5days Albumin was measured daily and average calculated, peak Aspartate (AST) at 24 hours was recorded. The percentile difference of the obtained values between the pre cryopreservation and post cryopreservation culture was calculated.

Results: A total of 12 specimen were enrolled for the study. The mean pre cryopreservation viability of the cells was 66.58%. The post cryopreservation, viability of the cells was 36.43%. The mean difference was -30.170%. The pre cryopreservation albumin values had a mean of 150ng/ml. The post cryopreservation albumin values had a mean of 135.83ng/ml. The mean difference was -14.170ng/ml. The pre cryopreservation peak Aspartate values had a mean of 234.17 IU/ml. The post cryopreservation peak aspartate values had a mean of 230 IU/ml. The mean difference was -4.176 IU/ml.

Conclusions: This simple method can validate the cells after cryopreservation by measurement of cell viability, synthetic function of the cell and cell stability.



Albumin, Cryopreservation, Culture

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