Assessment of albumin and aspartate levels as a simple indicator of the efficacy of cryopreservation
Keywords:Albumin, Cryopreservation, Culture
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.
Terry C , Mitry RR, Lehec SC, Muiesan P, Rela M, Heaton ND, Hughes RD, Dhawan A. The effects of cryopreservation on human hepatocytes obtained from different sources of liver tissue. Cell Transplant. 2005;14(8):585-94.
Li AP, Gorycki PD, Hengstler JG, Kedderis GL, Koebe, HG, Rahmani R, de Sousas G, Silva JM, Skett P. Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel. Chem Biol Interact. 1999;121:117-23.
Katenz E, Vondran FWR, Schwartlander R, Pless G, Gong X, Cheng X, et al. Cryopreservation of Primary Human Hepatocytes: The Benefit of Trehalose as an Additional Cryoprotective Agent. liver transplantation. 2007;13:38-45.
Vries RJ, Banik PD, Nagpal, Weng, Ozer S, van Gulik TM. Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome. Langmuir. 2019;35(23):7354-63.
Dhawan A, Chaijitraruch N, Fitzpatrick E, Bansal S, Filippi C, Lehec SC, et al. Alginate microencapsulated human hepatocytes for the treatment of acute liver failure in children J Hepatol. 2019;S0168-8278(19):30714-7.
Donato T, Bolonio M, Cabezas E, Pelechá M, Pareja E, Domènech A, et al. Improved in vivo efficacy of clinical-grade cryopreserved human hepatocytes in mice with acute liver failure. Cytotherapy. 2020;22(2):114-21.
Lee SM, Schelcher C, Demmel M, Hauner M, Thasler WE. Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure. J. Vis. Exp. 2013;(79):e50615.
Green CJ. Charlton CA. Wang LM. Silva M. Morten KJ. Hodson L. The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus. Cell Tissue Bank. 2017;18:597-604.
Bhogal RH, Hodson J, Bartlett DC, Weston CJ, Curbishley SM. Isolation of Primary Human Hepatocytes from Normal and Diseased Liver Tissue: A One Hundred Liver Experience. PLoS ONE. 2011;6(3):e18222.
Terry C, Dhawan A, Mitry RR, Lehec SC, Hughes RD. Optimization of the Cryopreservation and Thawing Protocol for Human Hepatocytes for Use in Cell Transplantation. Liver transplantation. 2010;16:229-37.
Pless-Petig G, Walter, Bienholz A, Rauen U. Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions. Cell Transplantation. 2017;26(12):1855-67.
Vondran FWR, Katenz E, Schwartlander R, Morgul MH, Raschzok N, Gong X, et al. Isolation of Primary Human Hepatocytes After Partial Hepatectomy: Criteria for Identification of the Most Promising Liver Specimen. Artificial Organs. 2007: 32(3):205-13.
Baccarani U, Donini A, Risaliti A, Piccolo G, Dialti V, Cautero N, et al. Steatotic versus cirrhotic livers as a source for human hepatocyte isolation. Transplant Proc. 2001;33:664-65.
Alexandre E, Viollon-Abadie C, David P, Gandillet A, Coassolo P, et al. Cryopreservation of adult human hepatocytes obtained from resected liver biopsies. Cryobiol. 2002;44(2):103-13.
Bachmann A, Moll M, Gottwald E, Nies C, Zantl R, Wagner H. 3D Cultivation Techniques for Primary Human Hepatocytes. Microarrays. 2015;4:64-83.
Selden C, Roberts E, Stamp G, Parker K, love PW, Ryder T. Comparison of three solid phase supports for supporting three-dimensional growth and function of human liver cell lines. Artificial organs. 1998;22:308-19.
Moman RN, Gupta N, Varacallo M. Star Pearls Publishing; Treasure Island (FL): Physiology, Albumin. 2020.
Wroblewski KA, Ladue F. "Transaminase activity in human blood". J Clinic Invest. 1955;34(1):126-31.