Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants

Abstract

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization.

Cryopreservation of feline red blood cells in liquid nitrogen using glycerol and hydroxyethyl starch

“…[6][7][8][9][10][11][12] Canine cryopreservation techniques have also been successfully described in three previous reports using modifications of both automated and manual human protocols. [19][20][21][22] However, our findings are consistent with a recent study 23 that evaluated glycerol and HES as cryoprotectants for both canine and feline pRBCs. This study reported that neither HES nor glycerol were able to cryopreserve and recover feline RBCs successfully.…”

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes

“…Cryopreservation with 10% DMSO showed statistically similar survival compared to cryopreservation with 5% DMSO (data not shown). The lower concentration was selected for the further experiments since it is generally preferred to reduce the DMSO concentration in cryopreservation media due to cytotoxicity [ 31 , 32 , 33 , 34 , 35 , 36 , 56 ]. Importantly, it was observed, that even the native (non-cryopreserved) cells harvested from the MK differentiation cultures had no more than 74.25% ± 6.95% of viable cells.…”

“…Among the obligatory requirements to clinically relevant biobanking the key role belongs to the compatibility with good manufacturing practice and good laboratory practice standards, which assumes application of serum- and xeno-free protocols throughout the process. Besides, serious clinical safety concerns are currently being associated with the use of DMSO during cryopreservation [ 31 , 32 , 33 , 34 , 35 , 36 ]. Therefore, these issues were particularly addressed as the core of this study.…”

“…Previously, we developed efficient biobanking technologies for mesenchymal stem cells, blood cells, as well as tissues and tissue engineered constructs [ 26 , 32 , 35 , 63 , 64 ]. Results of these studies show substantial differences in cryopreservation outcomes and necessity to optimize the process for each particular object.…”

New cryoprotective media for cryopreservation of mammal erythrocytes

“…Concerning the cryopreservation of erythrocytes of domestic mammals however, the development of new techniques is still required. For example, the standard cryoprotectants for human erythrocytes, based on glycerol or 1.2-propanediol (1,2-PD) (3,4), are found to be ineffective for erythrocytes of many animals (5,6). Compared to glycerol and __________________________ *Correspondence to: Olena M. Bobrova, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Department of Cryobiophysics, 23 Pereyaslavskaya str., 61016 Kharkiv, Ukraine; е-mail: helen.bobrova.77@gmail.com 1,2-PD, dimethyl sulfoxide (DMSO) provides better results in the cryopreservation of equine, bovine, feline and canine erythrocytes, however, the level of hemolysis after all stages of cryopreservation is still quite high (5,6).…”