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.

Project Progress

Summary of the study:
We focused on using only sodium chloride (NaCl) solution for freezing platelets. Here are the key findings and methods from this study:

1. Freezing protocol:
   • Platelets were frozen in a high volume of NaCl solution without traditional cryoprotectants like DMSO. In addition, we also investigated a specific freezing protocol involving gradual cooling and controlled freezing.
2. Protective mechanism:
   • We propose that NaCl despite its isotonic properties may induce osmotic protection by extracting water from platelets before freezing. This can reduce the amount of water that freezes intracellularly and thereby decrease damage from ice crystal formation.
3. Results and observations:
   • After thawing, platelets frozen in NaCl solution showed impressive, improved recovery of cells as compared to DMSO frozen and thawed platelets.
   • NaCl-platelets demonstrate hemostatic function in vitro
   • Such platelets also retained all important phenotypic structures, crucial for their role in the coagulation process.
   • All platelets, regardless of freezing technique, release a significantly increased amount of platelet-specific microparticles.
   • Both DMSO and NaCl platelets exhibit elements of pre-apoptotic events in symbiosis with a highly activated and procoagulant phenotype
4. Potential for clinical use:
   • The study indicates that freezing with only NaCl solution may be a feasible alternative for cryopreservation of platelets, potentially simplifying and reducing costs of the process.
   • This could lead to less toxic methods of cryopreservation and possibly better compatibility for transfusions.

Mechanisms and hypotheses:

The following mechanisms were discussed as possible explanations for the success of using NaCl solution:

• Osmotic dehydration: NaCl solution can create an osmotic gradient that pulls water out of platelets before freezing, reducing intracellular water volume and thus minimizing ice crystal formation and cell damage.
• Minimal volume change: By reducing the amount of intracellular water, it also decreases the volume change that cells undergo when water freezes and expands, protecting cell membranes from mechanical damage.
• Controlled freezing rate: Controlled freezing can prevent the formation of large ice crystals, which is often problematic with rapid or to slow freezing.

Significance and future research:

This study represents a significant advancement in cryobiology and may lead to new, simpler methods for preserving platelets for clinical use. However, further research is needed to:

• Optimize freezing and thawing protocols: To ensure the method can be adapted to various clinical needs and volumes of platelets.
• Understand the exact mechanisms: To gain a deeper understanding of the underlying biological and physical processes enabling the survival and function of platelets frozen in NaCl solution.
• Verify long-term effects: To investigate how storage and long-term preservation affect platelets when frozen using this method.

References:

•  Ehn, K.; Wikman, A.; Uhlin, M.; Sandgren, P. Cryopreserved Platelets in a Non-Toxic DMSO-Free Solution Maintain Hemostatic Function In Vitro. Int. J. Mol. Sci. 2023, 24, 13097. https://doi.org/ 10.3390/ijms241713097

Conclusion:
This new method challenges previous understandings of cryopreservation and may offer a less complex and less toxic alternative to traditional cryopreservation methods, potentially improving the management and use of platelets in clinical settings. It represents a significant step forward in the field and could lead to better and more accessible solutions for blood storage and transfusion.
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Future directions / 3 ongoing follow-up studies:

Potential advantages of using hypertonic NaCl solution for freezing platelets:

1. Reduced risk of cell swelling: Hypertonic solutions help maintain osmotic balance, reducing the risk of cell swelling and preserving cellular structure and function during the freezing process.
2. Improved preservation of functional integrity: By using a hypertonic solution, it’s possible to better preserve the functional properties of platelets, which is crucial for transfusion or research purposes.
3. Extended shelf life: Platelets frozen in hypertonic NaCl solution may have a longer shelf life and better preservation over time compared to those stored in isotonic solution, which is important for long-term storage and distribution in clinical settings.
4. Enhanced survival post-thaw: Platelets stored in hypertonic NaCl solution may exhibit better post-thaw recovery and functionality compared to those stored in isotonic solution, which is particularly important for clinical scenarios requiring platelet transfusions.

These advantages highlight the potential benefits of using hypertonic NaCl solution for freezing platelets, emphasizing improved preservation of structure, function, and overall efficacy in clinical applications.

 However, potential Disadvantages of using hypertonic NaCl solution for freezing platelets may occur:

1. Initial cell exposure to osmotic stress: Platelets may undergo osmotic stress upon exposure to hypertonic solution, potentially causing initial cell damage or dysfunction.
2. Requires careful handling: Use of hypertonic solutions necessitates careful dosing and mixing to avoid excessive cell damage or unwanted effects on platelet biological functions.
3. Risk of increased aggregation: Higher osmotic pressure can increase the risk of platelet aggregation, affecting their ability to function properly upon thawing and use.

Follow-up study I aim to gain deeper insights into these issues.

Follow-up study II, also ongoing, aim to characterize, in depth, and find out the biological significance of the different EVs/microparticles that are released after freezing and thawing. These studies concern DMSO platelets as well as NaCl platelets.

Follow-up study III; both DMSO platelets as well as NaCl platelets have elements of apoptosis, which is rarely good. However, this is complex and apoptotic features can occur in parallel with the fact that the platelet is also highly activated and procoagulant, meaning that they are still a contributing factor in hemostasis. However, knowledge about this is limited and pilot studies have, therefore, been carried out during the summer with the intention of finding an approved and non-toxic counteracting factor for mitochondrial induced apoptosis in frozen DMSO-free platelets. These studies have begun, and preliminary data are expected to be presented at next year’s ISBT, Milan June 2025 the follow-up studies are necessary for a final optimization of the DMSO-free platelet protocol.

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