Comparision Of Different Cryoprotective Solution - Albumin Vs Autologous Plasma - Its Effect On Cell Recovery and Clonogenic Potential Of Cryopreserved Peripheral Blood Hematopoietic Stem and Progenitor Cells
Authors: Smagur Andrzej , Mitrus Iwona , Giebel Sebastian , Ciomber Agnieszka , Gliwinska Joanna , Panczyniak Katarzyna , Sadus-Wojciechowska Maria , Holowiecki Jerzy 
Published: October 2019

Background
Peripheral blood is a preferable source of hematopoietic stem and progenitor cells (HSPCs) used for autologous transplantation. HSPCs are mobilized to peripheral blood and collected by leukapheresis. Prior to cryopreservation the cells need to be processed including the addition of cryoprotective mixture as dimethyl sulfoxide (DMSO) prediluted in human serum albumin solution (HSAS). In Europe there is no commercially available albumin manufactured in packs with tubing which would enable the use of sterile tubing welder. Alternatively cryoprotective solution can be prepared using autologous plasma (AP) obtained during the same leukapheresis, allowing for the preparation of HSPCs in a completely closed system and hence to reduce the risk of contamination. The goal of our study was to test if the HSAS may be replaced by autologous plasma without negative impact on cell recovery and clonogenicity.

Methods
Samples were prospectively collected from 18 patients with multiple myeloma (n=13) and lymphomas (n=5) mobilized with chemotherapy combined with G-CSF. Small volumes (1.5 ml) of cell suspensions obtained from the leukapheresis products were divided into 2 parts (0,5ml) placed in separate small vials, each containing different cryoprotective mixture - 5% HSAS or AP with a final 7.5% DMSO concentration. The final volume of cell suspensions equaling 1 ml, the cell concentration (0.7–1.5 × 108 /ml). The cells were frozen in IceCube, using a computer controlled cooling program and stored in liquid nitrogen for 2 - 4 months. Concentration of total protein and individual electrophoretic fractions of plasma proteins were measured. The quality of cryoprotective mixtures was evaluated by cell recovery and clonogenic potential. The recovery was determined by comparing number of living cells before and after cryopreservation, using trypan blue staining. Clonogenic potential was carried out by colony forming unit (CFU) assays. Depending on CD34+ percentage, 5-40 × 103 living cells were plated (in triplicates) in MethoCult medium and cultured for 14 days.

Results
The median recovery of nucleated cells for AP was 68.3% (range 40.6-96.1) and was similar to HSAS 68.5%, (41.7-100); (p=0.3; Wilcoxon matched pairs test). The number of CFUs calculated per 100 000 cryopreserved cells did not differ significantly between tested cryoprotective mixture: 187.3 (11.3-806.3) for albumin, 130.5 (15-924.2) for autologous plasma (p=0.5; Wilcoxon matched pairs test). No significant differences were observed when the number of specific types of CFUs were compared. Neither total protein nor albumin concentration of plasma correlated with the clonogenic potential of the leukapheresis product cryopreserved in AP when samples from patients with higher concentration than median were compared with the other (Mann-Whitney U test). Between January and July 2013 more than 50 successful transplants of autologous HSPCs cryopreserved with 7.5% DMSO prediluted in autologous plasma were performed in our Department.

Conclusions
Commercially available human serum albumin can be replaced by autologous plasma in procedure of HSPCs cryopreservation. The use of autologous plasma for cryoprotective mixture preparation does not appear to negatively affect cell recovery and clonogenic potential of leukapheresis product. The advantage of such solution is possibility of HSPCs preparation in closed system to reduce risk of auto-HSCT product contamination to the minimum.

Disclosures:
No relevant conflicts of interest to declare.