Comparing a novel non-toxic freezing approach with conventional DMSO-freezing of cryopreserved platelets: development of a new frozen platelet product

Organisation: Karolinska Institutet (CLINTEC), Sweden

Lead researcher: Per Sandgren, Associate Professor

Grant amount: €27.200

Project description: 

Platelets have an essential role in hemostasis. During vascular injury platelets stop bleeding by aggregating into the damaged area.  Substances such as collagen fibres are exposed during damage thus leading to triggering and activation of platelets through interplay of adhesive receptors. Transfusion of platelets is needed when this critical hemostatic function is dislodged due to severe thrombocytopenia, cancer treatment or trauma. Platelets are currently stored at room temperature for limited time, five to seven days after preparation.  Therefore, clinical development of cryopreserved platelets in 5-6% dimethyl sulfoxide (DMSO) is currently in progress, bridging inventory shortages of conventionally stored platelets. From recent studies on cryopreserved platelets using DMSO as cryoprotectant (CPA) a reduced recovery and viability is demonstrated after thawing regardless of freezing the platelets in material with lower conductivity for temperature, as well as using different freezing rate protocols¹.

In contrast, data obtained from our pilot-studies indicate that using a novel freezing approach can reduce some of the negative impacts of DMSO-freezing and provide a near optimal non-toxic profile for the final reconstituted unit aimed for transfusion, which is a desirable. This novel freezing profile may also lend itself to further optimization of cryopreserved platelets.

Project duration: 01.2023 – 31.2025

Project Aims

• investigate and compare the cellular effects such as recovery, viability and functionality on platelets after cryopreservation comparing a novel freezing approach with conventional freezing of platelets by using DMSO as cryoprotectant.
• develop and finalize a clinical protocol generating a completely non-toxic frozen platelet product aimed for transfusion.

Outcomes

Initially, we hypothesized that saline could be used as a non-toxic medium for the cryopreservation of platelets, tested in a series of in vitro studies: Double-dose buffy coat platelets (n = 10) were divided and cryopreserved at −80 °C using 5–6% dimethyl sulfoxide (DMSO) or in NaCl (9 mg/mL). Paired testing was conducted pre-freeze, post-thaw (PT 1 h). Upon analysis, each bag was thawed and reconstituted in fresh plasma. Analyses included cell counts and the metabolic, phenotypic, and functional properties of the platelets together with thromboelastometry. The cryopreserved platelets showed several biochemical and ultrastructural changes compared to pre-freezing. Platelet recovery was approximately 17% higher in DMSO-free units (p < 0.001), but the platelet viability was reduced (p < 0.001). However, using controlled freezing, the platelet viability was improved. The clot formation time (CFT) was comparable, but DMSO-free platelets showed slightly decreased maximum clot firmness (MCF) (p = 0.034). By reducing the reconstituted plasma volume, a reduced CFT and increased MCF were obtained (p < 0.001). This study demonstrates that platelets can be cryopreserved in saline without the addition of DMSO, with high recovery and maintained hemostatic function. However, controlled freezing is required to optimize platelet quality. In summary, this study² has shown, for the first time, that it is possible to freeze platelets in saline in the absence of a CPA. Further studies are needed to fully understand the mechanisms involved. We have shown that DMSO-free cryopreserved platelets are viable and hemostatically active in vitro, which needs to be confirmed in future in vivo studies.

Related resources: 

¹A short review report including articles from Karolinska University Hospital and Karolinska Institutet referenced in PubMed and published in English between 2018 and 2023 studying the effects of cryopreservation on platelets in vitro and in vivo. LD03-L01.1 Cryopreservation of platelets: Karolinska experiences. Vox Sang. 2023. Volume 118. can be accessed here: https://doi.org/10.1111/vox.13433

²The study on DMSO-free cryopreserved platelets is published in Int. J. Mol. Sci. 2023, 24(17), 13097 and can be accessed here: https://www.mdpi.com/1422-0067/24/17/13097