REVIEW ARTICLE

Open Access

Cryoprotectants for red blood cells: Evaluate safety and effectiveness by in vitro measures

Yuying Hu,

Yuying Hu

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - original draft (equal)

Search for more papers by this author

Xiangjian Liu,

Xiangjian Liu

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - original draft (equal)

Search for more papers by this author

AKhlaq Ahmad,

AKhlaq Ahmad

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Jiangming Chen,

Jiangming Chen

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Xiaoxiao Chen,

Xiaoxiao Chen

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Wenqian Zhang,

Wenqian Zhang

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Songwen Tan,

Corresponding Author

Songwen Tan

[email protected]

orcid.org/0000-0003-0698-4410

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Correspondence Songwen Tan, Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, China.

Email: [email protected]

Search for more papers by this author

Yuying Hu,

Yuying Hu

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - original draft (equal)

Search for more papers by this author

Xiangjian Liu,

Xiangjian Liu

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - original draft (equal)

Search for more papers by this author

AKhlaq Ahmad,

AKhlaq Ahmad

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Jiangming Chen,

Jiangming Chen

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Xiaoxiao Chen,

Xiaoxiao Chen

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Wenqian Zhang,

Wenqian Zhang

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Contribution: Writing - review & editing (supporting)

Search for more papers by this author

Songwen Tan,

Corresponding Author

Songwen Tan

[email protected]

orcid.org/0000-0003-0698-4410

Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China

Correspondence Songwen Tan, Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, China.

Email: [email protected]

Search for more papers by this author

First published: 06 December 2023

https://doi.org/10.1002/mef2.67

Yuying Hu and Xiangjian Liu contributed equally to this study.

Share a link

Email
Wechat
Bluesky

Abstract

The transfusion of red blood cells (RBCs) is crucial in current medicinal research. The shelf-life of donated RBCs preserved by the normal method is very short, limiting their clinical application. Cryopreservation is a reliable and effective technology for the long-term storage of RBCs. During the process, ice formation will cause irreversible injuries to RBCs. Glycerol is used as a cryoprotectant (CPA) for RBCs to mitigate cryoinjuries. But it severely induces RBC hemolysis and deformation. This review introduces some biocompatible and effective CPAs used for RBC cryopreservation, outlining recent advances. Currently, there is no uniform standard to determine whether a CPA is suitable for RBCs. According to previous studies, we summarize for the first time comprehensive methods to evaluate the performance of CPAs by ensuring their safety and effectiveness. The safety of CPAs is defined as the degree of damage to RBCs, while the effectiveness is demonstrated by the properties of thawed RBCs, including membrane properties, protein activities, rheological properties, and metabolites levels. A novel CPA that is confirmed suitable for RBCs by these methods may be applied to other cells. We believe comprehensive methods can thoroughly evaluate the performance of CPAs and promote the development of transfusion therapy in clinic.

1 INTRODUCTION

As early as the 17th century, experiments with blood transfusion were conducted. Transfusion medicine was greatly advanced by the discovery of the ABO blood groups in the early 1900s.¹ Since then, the safety of the transfusion of blood products has significantly increased, and refrigerated stored red blood cells (RBCs) are now frequently infused to make up for excessive blood loss or to treat moderate-to-severe anemia, traumas, leukemia, and so forth.² According to the American Red Cross, 29,000 units of RBCs are needed each day in the United States.³ However, like with any medical procedure, the patients may be at risk for the adverse reactions of RBC transfusion, including immunological and nonimmune-mediated complications.⁴ Effective methods for the preservation of RBCs are important to maintain the quality and safety of RBCs provided to the patient as well as the overall clinical usage of blood products.

The use of RBCs during hypothermic storage in transfusion medicine has been extensively studied and evaluated. To date, RBCs are routinely stored at 2–6°C ranging from 21 to 42 days, depending on the used preservation solutions, including acid-citrate-dextrose, citrate-phosphate-dextrose-adenine, saline-adenine-glucose-mannitol (SAGM), and so forth.^{5, 6} Although RBC biochemical processes are slowed down at 4°C, cellular metabolism is not completely suppressed during storage.⁷ A variety of metabolic, biochemical, and morphologic changes are observed, known as the storage lesion, which could affect the RBC quality after infusion.⁸ Cryopreservation is an advanced technology for the long-term storage of cells.⁹ Similar to hypothermic storage, cryopreservation employs the advantage of low temperature to reduce molecular motion and halt metabolic and biochemical processes. Under ultra-low temperatures (−80 or −196°C), biological samples would maintain a state of “suspended animation” and the physiological activity would almost stop.¹⁰ Compared to hypothermic storage, RBC physiology, including hemoglobin (Hb) structure and membrane and cellular energetics, is not affected by long-term storage in the frozen state.⁶ Therefore, RBCs can be stored even for decades under a suitable cryopreservation protocol.¹¹

However, the cells inevitably suffer from cryoinjuries during cryopreservation. First, the ice formation inside the extracellular matrix increases the concentration of solutes and leads to severe cell osmotic dehydration. Second, the ice can directly cause mechanical injury to cells.^{12, 13} Furthermore, the overproduction of reactive oxygen species (ROS) induced by low temperature can cause lipid peroxidation, DNA damage, and protein oxidation.^14-17 To decrease cryoinjuries, cryoprotectants (CPAs) should be used in the cryopreservation process.¹⁸

For RBCs, glycerol is regarded as the current state-of-the-art CPA in many clinical settings.^{6, 19, 20} During cryopreservation, glycerol can permeate through RBCs to reduce intracellular ice formation and osmotic shock.²¹ Currently, two methods for the cryopreservation of RBCs are developed accordingly for clinical use, which employ glycerol at concentrations of 20% and 40% (w/v) termed the Low Glycerol Method (LGM) and the High Glycerol Method (HGM), respectively. In the first one (used in Europe), RBCs are cryopreserved and stored at −196°C using 20% w/v glycerol with rapid freezing rates (~100°C/min).^22-24 The HGM protocol (used in the United States) is based on the slow freezing (~1°C/min) of RBCs at −80°C in 40% w/v glycerol.^25-27 However, the optimal concentration of glycerol is up to 40%w/v, which may lead to hemolysis and RBC morphology alteration.^{28, 29} Therefore, the search for safe and effective CPAs is ongoing to promote cell cryopreservation. Many CPAs, such as hydroxyethyl starch (HES),³⁰ trehalose,³¹ block copolymer worms,³² proline,³³ and so forth, have been explored as CPAs for RBC cryopreservation.

However, there is no uniform standard to evaluate the performance of CPAs for RBCs. In earlier articles, the protective effects of CPAs were evaluated only by RBC recovery due to the limitations of technologies.^{34, 35} But it is insufficient and inaccurate. Although some researchers have added functional indicators of thawed RBCs to evaluate CPAs, it is still not enough to fully determine their performance and may be an obstacle for the cryopreservation of RBCs and transfusion therapy. According to previous studies, we have summarized for the first time comprehensive methods to evaluate the performance of CPAs based on their safety and effectiveness. First, the toxicity of conventional CPAs is a main challenge for cell cryopreservation. Therefore, it is necessary to use biocompatible CPAs to ensure the safety. Moreover, the effectiveness of CPAs can be determined by the properties of thawed RBCs, which are elucidated from membrane properties, protein activities, rheological properties, and metabolites levels.

In this review, we summarize the current development of CPAs for RBC cryopreservation, describing their advantages and disadvantages and the impact on the RBCs being frozen, which can help researchers fully understand the latest advances. We highlight the safety and effectiveness of CPAs evaluated by comprehensive methods. Further, we discuss specific parameters that need to be considered before CPAs progress to clinical transfusion therapy. Consequently, this review provides a new perspective to comprehensively evaluate CPAs for RBC cryopreservation, and novel CPAs confirmed by the methods may also be applied to other cells.

2 DEVELOPMENT OF CPAS FOR RBC CRYOPRESERVATION

Based on the cryoinjuries during cryopreservation, it is obvious that the addition of CPAs can weaken the effects of cryoinjuries and improve the efficiency of RBC cryopreservation. With the rapid development of materials and chemistry, a series of CPAs have been explored and used in the process of cell cryopreservation for a long time. Herein, we briefly sum up the CPAs for RBC cryopreservation, which can be broadly classified into two categories: small molecule- and macromolecule-based CPAs (Figure 1). Additionally, more details about various CPAs used for the cryopreservation of RBC, including RBC species, the volumes of the RBC suspensions, CPA concentration, the recovery of cells after thawing or washing, and the advantages and disadvantages of different CPAs have been further summarized in Table 1.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

History of the development of small molecule- and macromolecule-based cryoprotectants for red blood cell cryopreservation. DMSO, dimethyl sulfoxide.

Table 1. Summary of various cryoprotectants (CPAs) used for the cryopreservation of red blood cells (RBCs).

	CPAs	RBC species	CPA concentrations	Outcomes	The advantages of CPAs	The disadvantages of CPAs	References
Small molecule-based CPAs	Glycerol	Human RBCs	20% (w/v) (LGM) 40% (w/v) (HGM)	Postdeglycerolized recovery: ≥80%a	High-thawed RBC recovery, the only clinically licensed CPA for cryopreserving RBCs	Time-consuming and expensive deglycerolization process	[6, 36]
	DMSO	Human RBCs	10 wt %	Postthaw recovery: ~95%	Great membrane permeability, high thawed RBC recovery	Cytotoxicity	[37, 38]
	Trehalose	0.5–1 mL human RBCs	1 M	Postthaw recovery: ~81%	Good biocompatibility, high thawed RBC recovery	Trehalose cannot provide sufficient intracellular protection due to its impermeability	[31, 39]
	l-carnitine	Sheep RBCs	8 wt %	Postthaw recovery: 83.99%	Good biocompatibility, high thawed RBC recovery	More experiments are needed to prove the effectiveness and safety of l-carnitine on a large volume of human RBCs	[40]
	Proline	100 μL sheep RBCs	4.5 wt %	Postthaw recovery: ~72%	Good biocompatibility	Low efficiency when used alone	[33]
	Betain	40 μL sheep RBCs	6 wt %	Postthaw recovery: ~80%; postthaw-wash recovery: ~70%	Good biocompatibility, easy removal via membrane transport proteins	More experiments are needed to prove the effectiveness of betain on a large volume of human RBCs	[41]
	Tricine	100 μL sheep RBCs	6 wt %	Postthaw recovery: 81.2 ± 8.5%	Low toxicity, high thawed RBC recovery	Hard to be removed from cells	[42]
Macromolecule-based CPAs	PVP	Sheep RBCs (1 × 10⁹/mL)	10% PVP Kb 15 MWc 10000	Postthaw-wash recovery: 83.7%	Great biocompatibility, directly transfusable CPA	Increased hemolysis after transfusion	[43-45]
	Dextran	0.5-1 mL human RBCs	30% (w/v)	Postthaw recovery: ~90%	Great biocompatibility, directly transfusable CPA	Increased hemolysis after transfusion	[31, 43, 46]
	HES	450 mL Human RBCs	11.5 wt %	Postthaw viability in terms of saline stabilityd: 92 ± 1%	Great biocompatibility, directly transfusable CPA	Increased hemolysis after transfusion	[43, 47, 48]
	AFP	Human RBCs	30% (w/v) HES; 62 μg mL⁻¹ type I AFP + 30% (w/v) HES	Postthaw recovery (slow warminge): ~55%; postthaw recovery (slow warming): ~87.5%	The low concentration of AFPs, a much higher IRI activity	Toxicological and immunological issues, high cost, and scale-up restrictions	[34, 49, 50]
	PVA	500 μL ovine and human RBCs	0.1 wt % 9 kDa PVA	Postthaw recovery: >40%	Good biocompatibility, low concentration	Low efficiency when used alone	[51]
	Block copolymer worms	250 μL ovine RBCs	5 wt % worms; 5 wt % worms +1 mg mL⁻¹ PVA	Postthaw recovery: 20%; postthaw recovery: 68%	Worms enable postthaw gelation by warming to 20°C, thus providing a new approach for RBC storage and transport and a convenient matrix for 3D cell cultures.	Low efficiency when used alone	[32]
	QDs	50 μL sheep RBCs	10.0 mg mL⁻¹ OQCNs-180-3	Postthaw recovery: ∼55%	Good biocompatibility, simple and up-scalable synthetization compared to AFPs	Low efficiency when used alone	[52]
	MOF NPs	human RBCs	0.5 mg mL^–1 UiO-66-OH MOF NPs	Postthaw recovery: ∼40%	Good biocompatibility	Low efficiency when used alone	[53]
	Polyampholytes	500 μL sheep RBCs	100 mg mL^–1	Post-thaw recovery: >60%	Less processing challenges and easy removal	Low efficiency when used alone	[51]
	SH	100 μL sheep RBCs	1 mg/mL MSH	Post-thaw recovery: 63.2 ± 3.5%; post-thaw-wash recovery: 60.7 ± 2.3%	Good biocompatibility, easy removal by direct washing	Low efficiency when used alone	[54]

^a AABB guidelines: American Association of Blood Banks.
^b K = relative viscosities.
^c MW = average molecular weights.
^d Saline stability [%]=(1–Hb_S/Hb_T) × 100, where Hb_T corresponds to the total hemoglobin and Hb_S to the hemoglobin in the supernatant.
^e Slow warming: the sample was thawed in the air at room temperature.

2.1 Small molecule-based CPAs

Small molecule-based CPAs comprise glycerol,¹⁹ dimethyl sulfoxide (DMSO),³⁷ trehalose,³¹ and so forth, which have been assessed for their cryoprotective activities in RBCs.

CPAs have received many researchers' attention since glycerol was first discovered in 1949.⁵⁵ Freezing RBCs with glycerol as a CPA dates back to 1950. Smith¹⁹ reported an approach for freezing whole blood to −79°C with the addition of glycerol, which prevented hemolysis and did not alter the normal morphology of RBCs. At present, glycerol is the only CPA used for RBC transfusion products.⁶ Although glycerol is often regarded as having low toxicity, the working concentrations during cryopreservation are up to 20%–40%, which require the washing process after thawing to prevent RBC death or subsequent side effects.⁵⁶ Moreover, cryopreserved RBCs in glycerol must meet certain guidelines (Table 2). After cryopreservation, thawed RBCs require a deglycerolization process to reduce the residual glycerol concentration to below 1%. According to international guidelines, thawed deglycerolized RBC recovery needs to be at least 80%, and the hemolysis in the RBC units should not exceed 0.8% in Europe and 1% in the United States. Additionally, at least 75% of transfused RBCs should still circulate 24 h after infusion.^{36, 57, 58}

Table 2. Requirements of cryopreserved RBCs.

Variable	European guidelines⁵⁷	AABB guidelines³⁶
Hemolysisa	< 0.8%	<1.0%
Postthaw recovery	-	≥80%
In vivo RBC viability after 24 h transfusion	-	≥75%
Volume	>185 mL	-
Hb content	>36 g/unit	-
Hematocrit	0.65%–0.75%	-
Leukocytes	<1 × 10⁶/U	-
Osmolarity	<340 mOsm/L	-

Abbreviations: AABB guidelines, American Association of Blood Banks; Hb, hemoglobin; RBCs, red blood cells.
^a Maximum allowable hemolysis at the time of infusion.

Subsequently, Lovelock and Bishop³⁷ reported the use of DMSO for RBC cryopreservation in 1959. The advantage of DMSO is its more rapid penetration into cells as a CPA, but it is more toxic compared to glycerol.⁵⁹ Other CPAs have been researched as substitutes for glycerol. Trehalose, a nonreducing disaccharide, could protect RBCs against cryoinjuries during cryopreservation owing to its exceptional cryoprotective properties.³¹ The nontoxicity of trehalose allows it to not be removed from cells after thawing. However, trehalose cannot be produced by mammalian cells and cannot penetrate the cell membrane, thus its cryoprotective activity is limited.⁶⁰ Currently, a variety of methods have been researched to transport trehalose into cells for effective cryopreservation, including phospholipid phase transition, fluid-phase endocytosis, microinjection, and so forth, which could significantly promote the application of trehalose in the cryopreservation of cells.³⁹ Moreover, Yang et al.³³ compared the abilities of three biocompatible osmoprotectants (proline, glycine, and taurine) to inhibit osmotic injury and reduce ice formation and explored their potential as CPAs. Proline demonstrated the strongest capacity to prevent cryoinjuries. As a result, it also achieved the highest RBC recovery during the freezing and thawing process. Another study by Zhai et al.⁴⁰ showed that natural zwitterionic l-carnitine could effectively cryopreserve RBCs by reducing osmotic and mechanical injury. The thawed sheep RBC recovery was up to 83.99% at 8% l-carnitine, demonstrating an obvious advantage over the other CPAs. Additionally, betaine⁴¹ and N-[Tri(hydroxymethyl)methyl]glycine (tricine)⁴² have also been explored as novel CPAs for RBC cryopreservation.

2.2 Macromolecule-based CPAs

The main benefit of using macromolecular CPAs like polyvinyl pyrrolidone (PVP),⁶¹ HES,³⁰ antifreeze protein (AFP),³⁴ and so forth is that they do not penetrate the cells. This property greatly facilitates their removal after thawing.⁶² Moreover, some nonpermeating CPAs that are biodegradable and well tolerated by the patient do not even need to be removed after thawing.⁶³

In 1955, Bricka and Bessis⁶¹ demonstrated that RBCs suspended in high concentrations of dextran or PVP would survive at an extremely low temperature. In 1967, human RBCs were successfully cryopreserved using HES and liquid nitrogen (LN₂) for the first time by Knorpp et al.³⁰ Compared to dextran, HES is more stable during the freezing and thawing processes and long-term storage and is inexpensive to produce. HES is also as effective as PVP in the cryopreservation of RBCs, but it has the advantage over PVP of being metabolized and consequently not retained in the recipient, which would eliminate the need for laborious blood processing before transfusion.

AFPs have potent ice recrystallization inhibition (IRI) activity in frozen solutions.^{64, 65} Carpenter et al.³⁴ found that the addition of relatively low concentrations (5–160 μg/mL) of (Pseudopleuronectes americanus) AFP greatly improved the survival of RBCs that were cryopreserved in HES. However, clinical use of AFPs in cryopreservation is challenging because of toxicological and immunological issues, high cost, and scale-up restrictions.⁴⁹ Thus, synthetic AFP mimics were developed. Polyvinyl alcohol (PVA) has been explored as an AFP mimic and possesses the strong ability to inhibit ice recrystallization.^66-68 Deller et al.⁶⁹ reported that the RBC recovery in a 0.1 wt% PVA solution was more than 40%, indicating PVA provided protection to the RBCs by inhibiting ice crystal growth during thawing.

Nanomaterials with ice-regulation function show great potential in the cryopreservation of RBCs. Zhu et al.⁵³ used zirconium (Zr)-based metal-organic framework (MOF) nanoparticles (NPs) for the cryopreservation of RBCs. The thawed human RBC recovery could reach ~40%, which was higher than that obtained using HES. Bai et al.⁵² found that oxidized quasi-carbon nitride quantum dots (OQCNs), which exhibited thermal-hysteresis activity, an ice-crystal shaping effect, and IRI activity, had good protective effects on sheep RBCs and could improve RBC recovery to 55% during cryopreservation.

Polymeric CPAs, such as block copolymer worms and polyampholytes, have demonstrated promising applications as cell cryopreservation enhancers.^{32, 49} Highly hydroxylated block copolymer worms are demonstrated to be a viable alternative to HES as an extracellular matrix for RBCs. When used alone, the worms are not an especially efficient preservative. However, when complemented by poly(vinyl alcohol), an ice-inhibiting polymer, the thawed RBC recovery was significantly increased from 20% to 68%.³² For polyampholytes, there is a study conducted by Bailey et al.⁵¹ wherein a polyampholyte synergistically enhanced sheep RBC cryopreservation and achieved >60% thawed RBC recovery. The polymer has the advantages of fewer processing challenges and easy removal due to its nonpermeability compared to glycerol.

Moreover, the natural macromolecule sodium hyaluronate (SH) has been reported as a biocompatible CPA for RBC cryopreservation. The addition of SH greatly improved RBC survival, up to 63%, which was higher than in the control group (33%). And the properties of thawed RBCs were well maintained.⁵⁴

3 THE SAFETY OF CPAS

The safety of CPAs can be represented by their biocompatibility to RBCs, which refers to interactions occurring between them and RBCs.⁷⁰ Most of the organic solvent CPAs show poor biocompatibility, which can lead to severe side effects.^{71, 72} Therefore, it is necessary to evaluate the biocompatibility of CPAs, which can be determined by the hemolysis test and RBC morphology observation.

MOF NPs could be excellent candidates for cryopreservation applications due to their good biocompatibility. Hemolysis of RBCs after incubation with MOF NPs at different concentrations was lower than 8%. To understand the underlying reason for low hemolysis, the interaction between the RBC membrane and UiO-66 MOF NPs that showed the highest hemolytic activity was evaluated through scanning electron microscopy (SEM) analysis. The results displayed that UiO-66 MOF NPs were flatly orientated on the surface of the RBCs along their planes with no evident membrane deformation, which explained the reason for their good biocompatibility.⁵³ Besides this, Stefanic et al.²⁹ first reported that colloidal bioinspired apatite NP could assist in delivering nonpermeable trehalose into RBCs through improved membrane permeation, thus increasing thawed RBC recovery, up to 91% (42% improvement over the control group without NP assistance). Incubation of RBCs with apatite NP at pH 6.5 and 7.4 for 7 h resulted in low levels of hemolysis (<8%), indicating its good biocompatibility.

After cryopreservation, the residual CPAs pose a risk for thawed RBCs. If CPAs exhibit toxicity or poor biocompatibility, they must be completely removed before clinical use. One-step and multistep methods are usually utilized to remove CPAs from cryopreserved cells, whose processes often require cell washing and centrifugation.^73-75 For glycerol-based freezing methods, intracellular glycerol must be washed by the multi-step method to avoid posttransfusion intravascular hemolysis.⁷⁶ After the deglycerolization process, ~15% of the RBCs are hemolyzed, the RBC morphology alters, and there is ~1% Gly residue in the cells.^{11, 77, 78} The residual concentration of glycerol can be determined by measuring the osmolarity of the final resuspension fluid.⁵⁷ However, CPAs with excellent biocompatibility are usually well-tolerated and do not need to be removed. Janis et al.⁷⁹ delivered trehalose into RBCs by sonoporation without removal steps, and the thawed RBCs can be directly used for clinical applications without any adverse transfusion reactions. Some previous studies have declared that HES is somewhat special. Due to its low toxicity, it is unnecessary to be removed under certain circumstances.⁸⁰ Therefore, nontoxic CPAs are more attractive because they are not removed in some clinical applications, but whether they can fully replace glycerol as a CPA has to be further explored.

Moreover, it must be noted that free hemoglobin released from damaged RBC needs to be removed before transfusion. Due to the toxicity of free hemoglobin, the final washing steps restored a hemolysis level below 1% (United States) or 0.8% (Europe).⁸¹

As a result, it is important to overcome the biocompatibility problem when designing CPAs. For intracellular CPA, it should be nontoxic to the cryopreserved cells at the concentration used in the study and be easily removed from cells. The zwitterionic molecule, betaine, can provide both intracellular and extracellular protection to cells as a natural osmoprotectant. During cryopreservation and the removal of betain, RBC integrity was well maintained and hemolysis was avoided, indicating that it had good biocompatibility and could be easily removed from cells via transport proteins.⁴¹ Similarly, it is equally crucial to assess the cytotoxicity of extracellular CPAs for cells. Cryoprotective extracellular macromolecules should be biocompatible, biodegradable, and well tolerated by the patient, like some polymers, sugars, and starches. Because they do not penetrate the cell membrane, the osmotic problems would be avoided, and the removal process of CPA would be easier than that of glycerol.⁶

4 THE EFFECTIVENESS OF CPAS

The effectiveness of CPAs is traditionally evaluated by thawed cell recovery because it is the key indicator of in vitro quality required by current regulatory bodies in blood transfusion therapy.^{82, 83} But the protective effects of CPAs need to be estimated further by considering thawed cell functional properties. Therefore, the four aspects of thawed RBCs: membrane properties, protein activities, rheological properties, and metabolites levels are chosen to evaluate the effectiveness of CPAs. First, the integrity of the RBC membrane, which maintains normal cell shape, deformability, and mechanical stability, is crucial for RBC functionality and viability.⁸⁴ Second, proteins in RBCs are involved in most biochemical reactions. For example, the ATPases can maintain intracellular gradients of ions.⁸⁵ Third, the rheological properties of RBCs play a vital role in ensuring oxygen transport in the microcirculation.⁸⁶ Fourth, the metabolites levels can indicate the physiological properties of RBCs.⁸⁷ These must be taken seriously because transfusion of impaired RBCs can cause infection, multiple organ dysfunction, and delirium.⁸⁸

Herein, Figure 2 clearly shows the protective effects of different CPAs on RBCs, which are reflected by thawed RBC properties. Additionally, all the detailed parameters and detection methods for cell properties are summarized in Table 3. It should be noted that the detection methods are a summary of techniques that have been used by other researchers and are not necessarily the gold standard.

Table 3. The effectiveness of CPAs evaluated by the properties of thawed RBCs.

Properties of RBCs	Parameters	Detection methods	References
Membrane properties	Osmotic fragility	Spectrophotometry	[85]
	Morphology	SEM, flow cytometry	[11, 76]
	Membrane lipid peroxidation	MDA level, mass spectrometry	[89, 90]
	Membrane stiffness	AFM	[91]
	PS exposure and CD47 expression	Flow cytometry	[92]
Protein activities	Hb content	HiCN, proteomics, and metabolomics	[93, 94]
	Band-3 phosphorylation	Immunofluorescence staining, proteomics, and metabolomics	[95, 96]
	ATPase activitya	Inorganic phosphorus method	[54]
	Protein oxidation	Carbonylation assay	[89]
	Antioxidant enzyme activity	SOD: xanthine oxidase method	[97]
		CAT: ammonium molybdate colorimetry
		GSH-PX: DTNB chromogenic reaction
Rheological properties	Aggregation	Automated hemorheology analyzer	[98]
Rheological properties	Deformability	Ektacytometry, AFM, microfluidics	[99-101]
Metabolites levels	ATP level	Firefly luciferase method	[102]
	2,3-DPG level	NADH oxidation method	[102]
	NO content	Photolysis/Chemiluminescence	[103]

Abbreviations: 2,3-DPG, 2,3-diphosphoglycerate; AFM, atomic force microscopy; ATP, adenosine triphosphate; CAT, catalase; CPAs; cryoprotectants; DTNB, 5′-dithiobis (2-nitrobenzoic acid); GSH-PX, glutathione peroxidase; Hb, hemoglobin; HiCN, hemoglobin cyanide method; MDA, malondialdehyde; NADH, nicotinamide adenine dinucleotide; NO, nitric oxide; PS, phosphatidylserine; RBCs, red blood cells, SEM, scanning electron microscopy; SOD, superoxide dismutase.
^a Including Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase.

4.1 Membrane properties

Maintaining the integrity of the membrane is the basis for the functions of RBCs. If there is not a selectively permeable barrier to stop the loss of its contents, the cell will die.^{104, 105} Osmotic fragility, morphology, membrane lipid peroxidation, membrane stiffness, phosphatidylserine (PS) exposure, and CD47 expression can be evaluation criteria for the membrane properties of thawed RBCs.

4.1.1 Osmotic fragility

Osmotic fragility is the resistance of RBC hemolysis to osmotic changes.¹⁰⁶ It is an efficient way of showing how susceptible RBCs are to osmotic stress.^{107, 108} The osmotic fragility is measured by stepwise diluting graded NaCl solutions ranging from 0.1% to 1.0%.⁸⁵ The osmolarity at half-maximal hemolysis is defined as the osmotic fragility index. According to this approach, a higher osmotic fragility index indicates that the cellular ability to resist osmotic stress is weaker.¹⁰⁹ Dou et al.⁸⁵ found that RBCs cryopreserved in l-proline and trehalose showed a higher osmotic fragility index compared to fresh RBCs, which indicated they could not maintain normal membrane stability. Dextran was evaluated as a glycerol substitute for human RBC cryopreservation by Pellerin-Mendes et al.³¹ 90% cryopreserved RBCs in 30% (w/v) dextran in LN₂ were recovered. The osmotic fragility of RBCs was not changed.

4.1.2 Morphology

The normal morphology of RBCs plays a key role in their survival and oxygen-carrying capacity.¹¹⁰ Healthy RBCs are unique biconcave discocytes, which makes RBCs more flexible to move across the microvasculature and aids in enhancing their oxygen-delivering ability.^{111, 112} The mature RBC is about 7.2 μm in diameter, 1.5–2.5 μm thick, and has a 90 fL mean volume.¹¹³ SEM analysis is utilized to investigate the morphology of the RBCs.¹¹ Graham et al.¹¹⁴ compared the effectiveness of HES and DMSO solutions for cryopreservation of avian RBCs. SEM revealed that RBCs cryopreserved in a freezing solution containing 10% DMSO remained in their regular shape with no observable cell deformation. Conversely, many RBCs that were frozen in a 20% HES solution presented severe membrane changes, including deformation and hemispheric protrusions. Another type of analysis of cell morphology is flow cytometry. The size of the forward scatter would increase in swollen RBCs, while the size of the side scatter would increase in shrunken cells. These analyses would demonstrate if cells suffer damage in a manner that alters the shape of their surface membrane.³⁸ Yao et al.⁷⁶ explored the hydrogel microencapsulation method combined with 0.7 M trehalose to cryopreserve RBCs. Compared to fresh RBCs, cryopreserved RBCs using the above method displayed similar characteristics after freeze-thaw-wash, with no significant alterations in either forward scatter or side scatter.

4.1.3 Membrane lipid peroxidation

When RBCs are subjected to extreme environmental stress, such as freezing, oxidative injury leads to a level of membrane lipid peroxidation, measured as malondialdehyde (MDA) content. Alotaibi et al.⁸⁹ investigated a protective agent called salidroside (Sal) to promote the cryopreservation of RBCs. The addition of Sal to the cryo-solutions containing glycerol or trehalose improved RBC survival. Sal is also an antioxidant, which could reduce oxidative injury during cryopreservation. The supplementation of Sal decreased the lipid peroxidation of thawed RBCs that were cryopreserved using glycerol or trehalose.

4.1.4 PS exposure and CD47 expression

PS exposure onto the outer leaflet of the RBC membrane and loss of the cell adhesion molecule glycoprotein CD47 expression, which leads to RBCs being recognized and phagocytosed by macrophages, can act as potentially important indicators of RBC membrane damage.^{94, 112} Flow cytometry was used to measure RBC membrane PS externalization and CD47 expression.⁹² Holovati et al.¹¹⁵ studied the use of liposomes, which are regarded as microscopic vesicles, to deliver trehalose to mammalian cells. This study demonstrated that trehalose-containing liposomes greatly enhance human RBC recovery and membrane quality after cryopreservation. Flow cytometry analysis of RBC PS externalization showed that freezing greatly damaged the membrane asymmetry of both l-RBCs (liposome-treated RBCs) and c-RBCs (control RBCs, not treated by liposomes). But the PS expression of l-RBCs was much lower than that of c-RBCs after cryopreservation in terms of RBC mean fluorescence intensities. For RBC expression of CD47 antigen, thawed l-RBCs produced tremendously more CD47 antigen on the membranes than thawed c-RBCs.

4.2 Protein activities

Proteins in RBCs serve an important role in most biochemical reactions, such as Hb, Band-3, ATPases, and so forth. Therefore, being aware of their changes could be used as indicators to evaluate thawed RBC properties.

4.2.1 Hb content

Hb is the main component of RBCs and constitutes up to 95% of the cell's total protein.¹¹⁶ This protein comprises four globular subunits, each having a heme group with a central molecule of iron in the ferrous state. Each heme group can bind one oxygen molecule.¹¹⁷ The main function of Hb is to carry O₂ from the lungs to the tissues, where it returns CO₂ to the lungs.¹¹⁸ Hb can be estimated by the hemoglobincyanide method (HiCN). The number of RBCs per milliliter is determined using a hemocytometer.^{93, 119} Consequently, the Hb content of RBCs could be obtained. Liu et al.⁴² explored the potential of tricine as a CPA for RBC cryopreservation. With the aid of tricine, the Hb of thawed RBCs is comparable to that of fresh RBCs during the freezing and thawing process.

4.2.2 Band 3 phosphorylation

Band 3 is a crucial protein that maintains the flexibility and stability of the RBC membrane, as it is a transporter that mediates electroneutral anion exchange across the plasma membrane and a structural protein for the cytoskeleton network.^{112, 120} Changes in band 3 phosphorylation levels have been related to the increased rigidity of RBCs, which reduces their perfusion to host organs and their lifespan in the circulation.¹²¹ The band 3 proteins on the RBC membrane can be measured by immunofluorescence staining.¹²² Shen et al.⁹⁵ demonstrated a practical and simple approach for improving the efficacy of RBC cryopreservation and examined their functional expressions, including RBC band 3 proteins after cryopreservation. By choosing 5 × 6 mm polytetrafluoroethylene (PTFE) tubes with superior cooling capabilities and using the trehalose dehydration technique, the results showed that most band 3 proteins were evenly distributed and clearly outlined on the RBC membrane in the 5% Gly and 0.4 M trehalose as well as the fresh group.

4.2.3 ATPase activity

ATPases can maintain intracellular gradients of ions.⁸⁵ And the stability of ion concentration in cells plays a key role in maintaining signal transduction and regulating cell metabolism.¹²³ Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase are two ATP-hydrolyzing enzymes that are determined by the inorganic phosphorus method.⁵⁴ Dou et al.⁸⁵ explored the combination of l-proline and trehalose as a cryoprotective protocol in the cryopreservation of RBCs. As a result, the postthawing hemolysis rate decreased, and the activities of Na⁺/K⁺-ATPase and Ca²⁺/Mg²⁺-ATPase were not altered by using this protocol.

4.2.4 Protein oxidation

Protein oxidation may occur in RBCs due to excessive ROS, which can exacerbate metabolic impairments.^{88, 124} For proteins, the measurement of protein carbonyl content (PCC) is the most reliable and widely used marker for protein oxidation.¹²⁵ Protein carbonylation occurs when protein molecules are attacked by ROS, altering the side chains of amine groups such as lysine, arginine, and proline into carbonyl groups.¹²⁶ There are several methods used to detect and quantify protein carbonylation, and the most common one is based on the derivatization of carbonylated proteins with 2,4-dinitrophenylhydrazine (2,4-DNPH).¹²⁷ RBCs cryopreserved with glycerol or trehalose were discovered to exhibit increased ROS accumulation and protein oxidation by immunodetection of 2,4-DNPH derivatized proteins. Supplementation of the antioxidant Sal mitigated this effect.^{89, 128}

4.2.5 Antioxidant enzyme activity

To prevent and alleviate intracellular oxidative stress, RBCs express several potent and efficient enzymatic antioxidant defense systems. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) are part of them to counteract free radicals.¹²⁹ SOD removes excess superoxide radicals (O₂⁻) by transforming them into oxygen (O₂) and hydrogen peroxide (H₂O₂). The latter is in turn converted by CAT to water and oxygen. Therefore, SOD and CAT in RBCs can defend Hb and other cellular components from free radicals to some extent. And GHS-PX eliminates peroxide metabolites of macromolecules.^{97, 130} Liu et al.⁴² assessed the antioxidant enzyme activities of RBCs cryopreserved in tricine after cryopreservation. With the aid of tricine, the activities of antioxidant enzymes were maintained well, indicating tricine has the ability to mitigate antioxidant damage.

4.3 Rheological properties

The rheological properties of RBCs (i.e., aggregation and deformability) are crucial for oxygen delivery in microcirculation.^{86, 131} Changes to the rheologic properties of RBCs may restrict or impede blood flow in the microcirculation, which can affect tissue perfusion and cause ischemia or even infarction.^{132, 133}

4.3.1 Aggregation

RBC aggregation is an essential factor in the flow behavior of blood, which refers to the tendency of RBCs to stick together.^{110, 134} It is influenced by numerous factors, including shear stress, the tonicity of suspension solutions, and the presence of potentiators such as dextran that can induce RBC aggregation.^{135, 136} The aggregation ability of RBCs was measured by the aggregation index (AI) depending on the kinetics and extent of aggregation, where a larger AI indicates an enhanced ability to aggregate.¹⁰⁹ Gao et al.⁹⁸ synthesized a dynamic membrane-active glycopeptide called PL-g-(MT/BA) (PMB) to improve RBC cryopreservation, which could efficiently help nonpermeable trehalose enter human RBCs with low hemolysis. In addition to high trehalose uptake, maintaining the biofunctions of human RBCs is crucial during incubation and freeze-thaw. The aggregation index of PMB-treated RBCs after cryopreservation, determined by an automated hemorheology analyzer, was comparable to that of the fresh RBCs, suggesting the aggregability of thawed RBCs was maintained normally.

4.3.2 Deformability

The deformability of RBCs is a result of their unique dynamic cell membrane shape, which also enables them to effectively transport oxygen to tissues and organs through microcirculation.^{110, 137} RBC deformability, which is expressed by the RBC elongation index (EI), is analyzed by ektacytometry (LORCA, a laser-assisted optical rotational red cell analyzer). The EI is positively associated with RBC deformability at a given shear stress (SS), where higher EI values at any given shear stress correspond to greater cell deformation. After ektacytometry, the data are linearized by Eadie-Hofstee analysis, which obtains the maximum theoretical elongation index (EI_max) and shear stress at half EI_max (K_EI) parameters (Equation 1).^{138, 139} Stoll et al.⁹⁹ studied the synergistic effects of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes combined with trehalose and HES in the cryopreservation of RBCs. After freezing and thawing, DMPC-treated RBCs showed an extremely low EI_max. The sharp decrease in EI_max indicated that the RBCs were not capable of deforming even under high shear stresses. Although the EI_max of RBCs treated with DPPC was unaffected, K_EI increased at higher DPPC concentrations, implying the membrane of RBCs was more rigid. In RBCs treated with DOPC, EI_max, and K_EI increased, indicating that the RBCs are more flexible.

\text{EI}\,=-\,{{\rm{K}}}_{\text{EI}}\frac{\text{EI}}{\text{SS}}+{\text{EI}}_{\max }.

()

The deformability of RBCs is mostly influenced by the membrane mechanical properties. Variations in mechanical properties, particularly stiffness and viscosity, often impair RBC deformability, which might restrict blood flow through the microvascular system¹⁴⁰ and result in pathophysiological effects like anemia¹⁴¹ and sepsis.¹⁴²

RBCs must maintain the proper stiffness range to go through capillaries in the microcirculation in the human body.¹⁴³ Cell stiffness is assessed by determining the cells' elastic moduli using atomic force microscopy (AFM).¹⁴⁴ The higher the cell's elastic modulus, the stiffer it is.¹⁴⁵ El Assal et al.⁹¹ used a novel cryo-ink integrated with a bioprinting approach to preserve RBCs during nanoliter vitrification. Vitrification is the solidification process from liquid to glass.¹⁴⁶ This cryo-ink includes ectoine, trehalose, and polyethylene glycol, which functions as a CPA to aid the cells in preventing freeze-thaw injury during cryopreservation. The results showed that the recovered RBCs after ectoine-based vitrification maintained their comparable stiffness to fresh RBCs.

Blood viscosity is affected by plasma viscosity, haematocrit, and the mechanical properties of the RBC.¹⁴⁷ The effects of cryopreservation on RBC hemorheological properties and related metabolic parameters were explored by Bizjak et al.⁸⁶ A sample of 10 healthy participants' blood was collected, glycerolized, and frozen at −80°C. Following 4, 8, and 12 weeks, aliquots were thawed and processed further, respectively. At these points in time, fresh blood samples were also taken from each participant. The influence on blood viscosity after cryopreservation was evaluated. The data demonstrated that the sample viscosity of stored blood was lower than the corresponding values for fresh blood, which was most likely caused by residual cellular glycerol. The possible consequences of the viscosity change need to be further studied.

4.4 Metabolites levels

Strong evidence suggests that the metabolites levels are crucial for determining post-transfusion RBC viability.^{86, 148} It includes adenosine triphosphate (ATP),⁸⁴ 2,3-diphosphoglycerate (2,3-DPG),¹⁴⁹ and nitric oxide (NO).¹⁵⁰

4.4.1 ATP level

ATP levels in RBCs are critical for maintaining the overall functioning of the RBC. ATP is the energy source for numerous biochemical reactions in RBCs, such as actively holding PS to the inner layer of the RBC membrane.¹⁴³ A reduction in ATP levels of RBCs triggered by storage will impair all RBC metabolic activities, including glycolysis, the formation of cytosolic antioxidants, and the maintenance of membrane integrity.⁸⁴

4.4.2 2,3-DPG level

The main function of RBCs is to carry oxygen to tissues and collect carbon dioxide. In this process, 2,3-DPG plays an essential role in regulating hemoglobin oxygen affinity.¹⁵⁰ A decrease in 2,3-DPG during storage impairs RBCs ability to release oxygen and enhances rigid RBC adhesion to endothelial cells.⁸⁶ As a result, stored RBC cannot provide the tissues with oxygen as well as fresh cells. After 6 to 8 h in circulation, this impairment is repaired.¹⁵¹ However, patients who require urgent oxygen transport to organs and tissues are nearly put at risk when using low 2,3-DPG blood.⁸³ Moreover, many transfusion complications are related to the low 2,3-DPG levels of transfused RBCs.¹⁵²

In the cryopreservation of RBCs, CPAs are added to help reduce the loss of ATP and 2,3-DPG.¹⁵³ Shen et al.¹⁰² showed that when RBCs were cryopreserved with trehalose and glycerol, ATP and 2,3-DPG levels were not visibly affected after cryopreservation and met clinical standards for the safety of blood transfusion.

4.4.3 NO content

NO is a crucial signaling molecule that contributes to hypoxic vasodilation by acting as a strong vasodilator.¹⁵⁴ Cellular NO, which is produced in RBCs by RBC-NO synthase activity, is essential to advantageously promote RBC deformability.¹⁵⁵ Rogers et al.¹⁰³ refined the glycerolization process by minimizing front-end processing time and optimizing a deglycerolization protocol. To assess the effect of RBC cryopreservation in the research-optimized process, the NO content of cryopreserved in 40% v/v glycerol or deglycerolized RBCs was compared to that of fresh RBCs. NO content was measured by photolysis/chemiluminescence. Total NO content was not significantly different between cryopreserved or deglycerolized RBCs and fresh RBCs, though freezing seemed to somewhat increase NO levels. The iron nitrosyl hemoglobin (HbFeNO) content stayed unchanged under all circumstances. Independent of the presence of glycerol, the content of S-nitrosohemoglobin (SNO-Hb) significantly increased after freezing. These findings suggest that the research-optimized process for RBC cryopreservation and deglycerolization in 40% v/v glycerol has no significant effect on the NO content.

In short, the effectiveness of CPAs can be evaluated by membrane properties, protein activities, rheological properties, and metabolites levels. Apart from these aspects, some indicators on RBCs that are not used in the cryopreservation field can be carried out to evaluate the quality of cryopreserved RBCs. For example, RBC microparticles (RMPs) that can be used as biomarkers of RBC quality are potentially immunogenic and inhibitory to NO regulation. The organization and integrity of the RBC membrane, which is made up of asymmetrically distributed phospholipids (PLs), cholesterol (C), and proteins, are crucial for in vivo RBC circulation and function. RBC quality can also be determined by the composition and quantity of PLs and C.⁷

5 DISCUSSION AND FUTURE PERSPECTIVES

There is an enormous demand for blood products in clinical medicine, particularly RBCs, which can significantly enhance survival. To reduce hypothermic storage lesions and extend the shelf life of RBCs, cryopreservation is a promising method to preserve cells. During the process, cells inevitably suffer from cryoinjuries. Therefore, many CPAs have been found and used in cryopreservation processes to protect cells. The recent development of CPAs for RBC cryopreservation has been outlined in this review. Currently, glycerol is the only CPA that is used clinically for cryopreservation of RBCs. Meanwhile, there are many more biomaterials that are worthy of further study, such as sugar derivatives, amino acids, AFP mimics, nanomaterials, and so forth.

Moreover, we have pointed out that biocompatible CPAs may have advantages in the cryopreservation of RBCs. Some articles have declared that the use of trehalose and HES in RBCs does not require removal steps because of their good biocompatibility, which can decrease the cost and the risk of hemolysis in the cryopreservation of RBCs. Conversely, CPAs with poor biocompatibility must be removed after thawing. For example, when a high concentration of glycerol is used as the CPA, the thawed RBCs need to be washed with a deglycerolization process before transfusion. The addition of other biocompatible CPAs can reduce the concentration of glycerol, which may be a more effective and less toxic method for RBC cryopreservation.

We have also discussed the effectiveness of CPAs based on RBC properties. Membrane properties, protein activities, rheological properties, and metabolites levels of RBCs can be used to judge the effectiveness of CPAs. Osmotic fragility, morphology, membrane lipid peroxidation, membrane stiffness, PS exposure, and CD47 expression are employed as indicators of RBC membrane properties. Protein activities of RBCs are elucidated by different aspects: Hb leakage, Band-3 phosphorylation, ATPase activity, protein oxidation, and antioxidant enzyme activity. The rheological properties of RBCs include aggregation and deformability. ATP, 2,3-DPG, and NO are several important metabolites of RBCs. It must be noted that all studies have only been done at the laboratory level, and more experiments are needed for the clinical applications of thawed RBCs.

However, it is unrealistic to measure all the in vitro tests mentioned for the evaluation of CPAs. The important parameters include the biocompatibility test of CPAs and postfreeze-thaw/wash cell recovery used for evaluating the safety and effectiveness of CPAs, respectively, which are really needed. In contrast, thawed cell functional properties, including membrane properties, protein activities, rheological properties, and metabolites levels, would be nice to know.

We believe that CPAs confirmed by comprehensive methods have the potential to be used for clinical transfusion therapy. In Figure 3, we provide a schematic figure to illustrate how to screen novel CPAs for the cryopreservation of RBCs. To further ensure CPA effectiveness in cryopreserving RBCs for clinical applications, more experiments are needed before transfusion. Currently, the RBCs cryopreserved in CPAs are from sheep or avian, which cannot simply be extrapolated to human RBCs. Apart from the RBC species, a volume of at least 150 mL of packed RBCs for transfusion is needed,¹⁵⁶ while most CPAs are tested on very small volumes of RBCs. Therefore, further studies are required to prove the safety and effectiveness of CPAs on a large volume of human RBCs. Apart from the species and volume of RBCs, serologic compatibility between the recipient and the donor should be tested before transfusion. Pretransfusion testing of patients receiving allogeneic blood includes ABO and rhesus D (RhD) typing and antibody screens to detect unexpected antibodies to RBC antigens.⁵

6 CONCLUSION

In conclusion, this paper reviewed the recent advances in CPAs for RBC cryopreservation and summarized the methods for determining the safety and effectiveness of CPAs comprehensively. More importantly, RBC cryopreservation can be used as a simple model for novel CPA screening, and research results from in vitro measures can initially ensure the biocompatibility and effectiveness of CPAs before progressing to nucleated cell cryopreservation. We believe that the review can provide a new perspective for cryopreservation and promote the future development of biomedical research.

AUTHOR CONTRIBUTIONS

Yuying Hu: Writing—original draft (equal). Xiangjian Liu: Writing—original draft (equal). AKhlaq Ahmad: Writing—review & editing (supporting). Jiangming Chen: Writing—review & editing (supporting). Xiaoxiao Chen: Writing—review & editing (supporting). Wenqian Zhang: Writing—review & editing (supporting). All authors have read and agreed to the published version of the manuscript.

ACKNOWLEDGMENTS

Figures are created with BioRender.com.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

The authors have nothing to report.

Open Research

DATA AVAILABILITY STATEMENT

The authors have nothing to report.

REFERENCES

1Giangrande PLF. The history of blood transfusion. Br J Haematol. 2000; 110(4): 758-767.
10.1046/j.1365-2141.2000.02139.x
CAS PubMed Web of Science® Google Scholar
2Tzounakas VL, Seghatchian J, Grouzi E, Kokoris S, Antonelou MH. Red blood cell transfusion in surgical cancer patients: targets, risks, mechanistic understanding and further therapeutic opportunities. Transfus Apher Sci. 2017; 56(3): 291-304.
10.1016/j.transci.2017.05.015
PubMed Web of Science® Google Scholar
3https://www.redcrossblood.org/donate-blood/how-to-donate/how-blood-donations-help/blood-needs-blood-supply.html, T.A.R. Cross, Facts about blood needs, 2022.
Google Scholar
4Guzzetta NA. Benefits and risks of red blood cell transfusion in pediatric patients undergoing cardiac surgery. Pediatric Anesthesia. 2011; 21(5): 504-511.
10.1111/j.1460-9592.2010.03464.x
PubMed Web of Science® Google Scholar
5Storch EK, Custer BS, Jacobs MR, Menitove JE, Mintz PD. Review of current transfusion therapy and blood banking practices. Blood Rev. 2019; 38:100593.
10.1016/j.blre.2019.100593
PubMed Web of Science® Google Scholar
6Scott KL, Lecak J, Acker JP. Biopreservation of red blood cells: past, present, and future. Transfus Med Rev. 2005; 19(2): 127-142.
10.1016/j.tmrv.2004.11.004
PubMed Web of Science® Google Scholar
7Almizraq R, Tchir JDR, Holovati JL, Acker JP. Storage of red blood cells affects membrane composition, microvesiculation, and in vitro quality. Transfusion. 2013; 53(10): 2258-2267.
10.1111/trf.12080
CAS PubMed Web of Science® Google Scholar
8Bosman GJCGM, Lasonder E, Luten M, et al. The proteome of red cell membranes and vesicles during storage in blood bank conditions. Transfusion. 2008; 48(5): 827-835.
10.1111/j.1537-2995.2007.01630.x-i2
CAS PubMed Web of Science® Google Scholar
9Chang T, Moses OA, Tian C, Wang H, Song L, Zhao G. Synergistic ice inhibition effect enhances rapid freezing cryopreservation with low concentration of cryoprotectants. Adv Sci. 2021; 8(6):2003387.
10.1002/advs.202003387
CAS Google Scholar
10Jang TH, Park SC, Yang JH, et al. Cryopreservation and its clinical applications. Integr Med Res. 2017; 6(1): 12-18.
10.1016/j.imr.2016.12.001
PubMed Web of Science® Google Scholar
11Pallotta V, D'Amici GM, D'Alessandro A, Rossetti R, Zolla L. Red blood cell processing for cryopreservation: from fresh blood to deglycerolization. Blood Cells Mol Dis. 2012; 48(4): 226-232.
10.1016/j.bcmd.2012.02.004
CAS PubMed Web of Science® Google Scholar
12Ishibe T, Congdon T, Stubbs C, Hasan M, Sosso GC, Gibson MI. Enhancement of macromolecular ice recrystallization inhibition activity by exploiting depletion forces. ACS Macro Lett. 2019; 8(8): 1063-1067.
10.1021/acsmacrolett.9b00386
CAS PubMed Web of Science® Google Scholar
13Yang J, Gao L, Liu M, et al. Advanced biotechnology for cell cryopreservation. Trans Tianjin Univer. 2020; 26(6): 409-423.
10.1007/s12209-019-00227-6
Google Scholar
14Evangelista-Vargas S, Santiani A. Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa. Reprod Domestic Anim. 2017; 52(5): 819-824.
10.1111/rda.12984
CAS PubMed Web of Science® Google Scholar
15Chen W, Li D. Reactive oxygen species (ROS)-responsive nanomedicine for solving ischemia-reperfusion injury. Front Chem. 2020; 8: 732.
10.3389/fchem.2020.00732
CAS PubMed Web of Science® Google Scholar
16Len JS, Koh WSD, Tan S-X. The roles of reactive oxygen species and antioxidants in cryopreservation. Biosci Rep. 2019; 39(8):BSR20191601.
10.1042/BSR20191601
CAS PubMed Web of Science® Google Scholar
17Li P, Li Z-H, Dzyuba B, Hulak M, Rodina M, Linhart O. Evaluating the impacts of osmotic and oxidative stress on common carp (Cyprinus carpio, L.) sperm caused by cryopreservation Techniques1. Biol Reprod. 2010; 83(5): 852-858.
10.1095/biolreprod.110.085852
CAS PubMed Web of Science® Google Scholar
18Dou M, Lu C, Rao W. Bioinspired materials and technology for advanced cryopreservation. Trends Biotechnol. 2022; 40(1): 93-106.
10.1016/j.tibtech.2021.06.004
CAS PubMed Web of Science® Google Scholar
19Smith A. Prevention of haemolysis during freezing and thawing of red blood cells. Lancet. 1950; 256(6644): 910-911.
10.1016/S0140-6736(50)91861-7
Google Scholar
20Rowe AW, Eyster E, Kellner A. Liquid nitrogen preservation of red blood cells for transfusion. Cryobiology. 1968; 5(2): 119-128.
10.1016/S0011-2240(68)80154-3
CAS PubMed Web of Science® Google Scholar
21Murray A, Congdon TR, Tomás RMF, Kilbride P, Gibson MI. Red blood cell cryopreservation with minimal post-thaw lysis enabled by a synergistic combination of a cryoprotecting polyampholyte with DMSO/trehalose. Biomacromolecules. 2022; 23(2): 467-477.
10.1021/acs.biomac.1c00599
CAS PubMed Web of Science® Google Scholar
22Pert J, Schork P, Lundberg A, Mourad N, Moore R. In vitro and in vivo studies on frozen red cells processed by low glycerol-rapid freeze method. Amer Assoc Blood Banks. 1967; 2749: 382.
Google Scholar
23Pert J, Schork P, Moore R. A new method of low-temperature blood preservation using liquid nitrogen and glycerol sucrose additive. Clin Res. 1963; 11(197): 34.
Google Scholar
24Lelkens CCM, Noorman F, Koning JG, et al. Stability after thawing of RBCs frozen with the high-and low-glycerol method. Transfusion. 2003; 43(2): 157-164.
10.1046/j.1537-2995.2003.00293.x
PubMed Web of Science® Google Scholar
25Valeri CR, McCallum L, Fowler KR, Sobucki JP. In vivo survival and supernatant hemoglobin of autologous, deglycerolized, resuspended erythrocytes processed using centrifugation: III. The effect of the length of storage at −80°C. Transfusion. 1966; 6(2): 112-115.
10.1111/j.1537-2995.1966.tb04707.x
CAS PubMed Web of Science® Google Scholar
26Valeri CR. Simplification of the methods for adding and removing glycerol during freeze-preservation of human red blood cells with the high or low glycerol methods: biochemical modification prior to freezing. Transfusion. 1975; 15(3): 195-218.
10.1046/j.1537-2995.1975.15375160354.x
CAS PubMed Web of Science® Google Scholar
27Tullis JL. Studies on the in vivo survival of glycerolized and frozen human red blood cells. J Am Med Assoc. 1958; 168(4): 399-404.
10.1001/jama.1958.03000040035008
CAS PubMed Web of Science® Google Scholar
28Murray KA, Gibson MI. Chemical approaches to cryopreservation. Nat Rev Chem. 2022; 6: 579-593.
10.1038/s41570-022-00407-4
CAS PubMed Web of Science® Google Scholar
29Stefanic M, Ward K, Tawfik H, et al. Apatite nanoparticles strongly improve red blood cell cryopreservation by mediating trehalose delivery via enhanced membrane permeation. Biomaterials. 2017; 140: 138-149.
10.1016/j.biomaterials.2017.06.018
CAS PubMed Web of Science® Google Scholar
30Knorpp CT, Merchant WR, Gikas PW, Spencer HH, Thompson NW. Hydroxyethyl starch: extracellular cryophylactic agent for erythrocytes. Science. 1967; 157(3794): 1312-1313.
10.1126/science.157.3794.1312
CAS PubMed Web of Science® Google Scholar
31Pellerin-Mendes C, Million L, Marchand-Arvier M, Labrude P, Vigneron C. In vitro study of the protective effect of trehalose and dextran during freezing of human red blood cells in liquid nitrogen. Cryobiology. 1997; 35(2): 173-186.
10.1006/cryo.1997.2038
CAS PubMed Web of Science® Google Scholar
32Mitchell DE, Lovett JR, Armes SP, Gibson MI. Combining biomimetic block copolymer worms with an Ice-Inhibiting polymer for the solvent-free cryopreservation of red blood cells. Angew Chem Int Ed. 2016; 55(8): 2801-2804.
10.1002/anie.201511454
CAS PubMed Web of Science® Google Scholar
33Yang J, Pan C, Zhang J, et al. Exploring the potential of biocompatible osmoprotectants as highly efficient cryoprotectants. ACS Appl Mater Interfaces. 2017; 9(49): 42516-42524.
10.1021/acsami.7b12189
CAS PubMed Web of Science® Google Scholar
34Carpenter JF, Hansen TN. Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth. Proc Natl Acad Sci USA. 1992; 89(19): 8953-8957.
10.1073/pnas.89.19.8953
CAS PubMed Web of Science® Google Scholar
35Capicciotti CJ, Kurach JDR, Turner TR, Mancini RS, Acker JP, Ben RN. Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations. Sci Rep. 2015; 5(1): 9692.
10.1038/srep09692
PubMed Google Scholar
36Kakaiya K, Aronson CJ. Whole blood collection and component processing. Techn Manual. 16th ed. AABB; 2008.
Google Scholar
37Lovelock JE, Bishop MWH. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature. 1959; 183: 1394-1395.
10.1038/1831394a0
CAS PubMed Web of Science® Google Scholar
38Murray A, Congdon TR, Tomás RMF, Kilbride P, Gibson MI. Red blood cell cryopreservation with minimal post-thaw lysis enabled by a synergistic combination of a cryoprotecting polyampholyte with DMSO/trehalose. Biomacromolecules. 2021; 23(2): 467-477.
10.1021/acs.biomac.1c00599
PubMed Web of Science® Google Scholar
39Hu Y, Liu X, Liu F, Xie J, Zhu Q, Tan S. Trehalose in biomedical cryopreservation–properties, mechanisms, delivery methods, applications, benefits, and problems. ACS Biomater Sci Eng. 2023; 9(3): 1190-1204.
10.1021/acsbiomaterials.2c01225
CAS PubMed Web of Science® Google Scholar
40Zhai H, Yang J, Zhang J, et al. Natural zwitterionic l-Carnitine as efficient cryoprotectant for solvent-free cell cryopreservation. Biochem Biophys Res Commun. 2017; 489(1): 76-82.
10.1016/j.bbrc.2017.05.045
CAS PubMed Web of Science® Google Scholar
41Yang J, Sui X, Wen C, et al. A hemocompatible cryoprotectant inspired by freezing-tolerant plants. Colloids Surfaces B. 2019; 176: 106-114.
10.1016/j.colsurfb.2018.12.053
CAS PubMed Web of Science® Google Scholar
42Liu X, Hu Y, Zhang W, et al. Tricine as a novel cryoprotectant with osmotic regulation, ice recrystallization inhibition and antioxidant properties for cryopreservation of red blood cells. Int J Mol Sci. 2022; 23(15): 8462.
10.3390/ijms23158462
CAS PubMed Web of Science® Google Scholar
43Wagner CT, Martowicz ML, Livesey SA, Connor J. Biochemical stabilization enhances red blood cell recovery and stability following cryopreservation. Cryobiology. 2002; 45(2): 153-166.
10.1016/S0011-2240(02)00124-4
CAS PubMed Web of Science® Google Scholar
44Hess JR. Red cell freezing and its impact on the supply chain. Transfus Med. 2004; 14(1): 1-8.
10.1111/j.0958-7578.2004.00472.x
PubMed Web of Science® Google Scholar
45Myhrvold V. Cryopreservation of sheep red blood cells: 1. Experiments with polyvinylpyrrolidone and other protective agents. Acta Vet Scand. 1979; 20(4): 525-530.
10.1186/BF03546579
CAS PubMed Web of Science® Google Scholar
46Banerjee A, Bandopadhyay R. Use of dextran nanoparticle: a paradigm shift in bacterial exopolysaccharide based biomedical applications. Int J Biiol Macromol. 2016; 87: 295-301.
10.1016/j.ijbiomac.2016.02.059
CAS PubMed Web of Science® Google Scholar
47Wang H, Hu H, Yang H, Li Z. Hydroxyethyl starch based smart nanomedicine. RSC Adv. 2021; 11(6): 3226-3240.
10.1039/D0RA09663F
CAS PubMed Web of Science® Google Scholar
48Sputtek A, Singbartl G, Langer R, Schleinzer W, Henrich HA, Kühnl P. Cryopreservation of red blood cells with the nonpenetrating cryoprotectant hydroxyethyl starch. Cryo Lett. 1995; 16: 283-288.
CAS Web of Science® Google Scholar
49Gore M, Narvekar A, Bhagwat A, Jain R, Dandekar P. Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives. J Mater Chem B. 2022; 10(2): 143-169.
10.1039/D1TB01449H
CAS PubMed Web of Science® Google Scholar
50Wu X, Yao F, Zhang H, Li J. Antifreeze proteins and their biomimetics for cell cryopreservation: mechanism, function and application—a review. Int J Biiol Macromol. 2021; 192: 1276-1291.
10.1016/j.ijbiomac.2021.09.211
CAS PubMed Web of Science® Google Scholar
51Bailey TL, Stubbs C, Murray K, Tomás RMF, Otten L, Gibson MI. Synthetically scalable poly(ampholyte) which dramatically enhances cellular cryopreservation. Biomacromolecules. 2019; 20(8): 3104-3114.
10.1021/acs.biomac.9b00681
CAS PubMed Web of Science® Google Scholar
52Bai G, Song Z, Geng H, et al. Oxidized quasi-carbon nitride quantum dots inhibit ice growth. Adv Mater. 2017; 29(28):1606843.
10.1002/adma.201606843
CAS Web of Science® Google Scholar
53Zhu W, Guo J, Agola JO, et al. Metal–organic framework nanoparticle-assisted cryopreservation of red blood cells. J Am Chem Soc. 2019; 141(19): 7789-7796.
10.1021/jacs.9b00992
CAS PubMed Web of Science® Google Scholar
54Liu X, Hu Y, Pan Y, et al. Exploring the application and mechanism of sodium hyaluronate in cryopreservation of red blood cells. Mater Today Bio. 2021; 12:100156.
10.1016/j.mtbio.2021.100156
PubMed Google Scholar
55Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949; 164(4172): 666.
10.1038/164666a0
CAS PubMed Web of Science® Google Scholar
56Sputtek A, Sputtek R. Cryopreservation in transfusion medicine and hematology. Life in the frozen state. CRC Press; 2004: 509-530.
10.1201/9780203647073.ch17
Google Scholar
57 European Committee (Partial Agreement) on Blood Transfusion. Guide to the preparation, use and quality assurance of blood components. Strasbourg, France: Council of Europe Recommendation No. 2015.
Google Scholar
58Henkelman S. Rheologic Changes of Hypothermic Preserved Red Blood Cells. Henkelman S; 2012.
Google Scholar
59Meryman HT. Cryopreservation of living cells: principles and practice. Transfusion. 2007; 47(5): 935-945.
10.1111/j.1537-2995.2007.01212.x
CAS PubMed Web of Science® Google Scholar
60Huang J, Guo J, Zhou L, et al. Advanced nanomaterials-assisted cell cryopreservation: a mini review. ACS Appl Bio Mater. 2021; 4(4): 2996-3014.
10.1021/acsabm.1c00105
CAS PubMed Google Scholar
61Bricka M, Bessis M. Preservation of erythrocytes by freezing in the presence of polyvinylpyrrolidone and dextran. C R Seances Soc Biol Fil. 1955; 149(9-10): 875-877.
CAS PubMed Google Scholar
62Holovati JL, Hannon JL, Gyongyossy-Issa MIC, Acker JP. Blood preservation workshop: new and emerging trends in research and clinical practice. Transfus Med Rev. 2009; 23(1): 25-41.
10.1016/j.tmrv.2008.09.003
PubMed Web of Science® Google Scholar
63Rowe AW. Cryopreservation of red blood cells. Vox Sang. 1994; 67: 201-206.
10.1111/j.1423-0410.1994.tb04576.x
PubMed Web of Science® Google Scholar
64Knight CA, Duman JG. Inhibition of recrystallization of ice by insect thermal hysteresis proteins: a possible cryoprotective role. Cryobiology. 1986; 23(3): 256-262.
10.1016/0011-2240(86)90051-9
CAS Web of Science® Google Scholar
65Liu K, Wang C, Ma J, et al. Janus effect of antifreeze proteins on ice nucleation. Proc Natl Acad Sci USA. 2016; 113(51): 14739-14744.
10.1073/pnas.1614379114
CAS PubMed Web of Science® Google Scholar
66Congdon T, Notman R, Gibson MI. Antifreeze (glyco) protein mimetic behavior of poly (vinyl alcohol): detailed structure ice recrystallization inhibition activity study. Biomacromolecules. 2013; 14(5): 1578-1586.
10.1021/bm400217j
CAS PubMed Web of Science® Google Scholar
67Richards SJ, Jones MW, Hunaban M, Haddleton DM, Gibson MI. Probing bacterial-toxin inhibition with synthetic glycopolymers prepared by tandem post-polymerization modification: role of linker length and carbohydrate density. Angew Chem Int Ed. 2012; 51(31): 7812-7816.
10.1002/anie.201202945
CAS PubMed Web of Science® Google Scholar
68Deller RC, Congdon T, Sahid MA, et al. Ice recrystallisation inhibition by polyols: comparison of molecular and macromolecular inhibitors and role of hydrophobic units. Biomater Sci. 2013; 1(5): 478-485.
10.1039/c3bm00194f
CAS PubMed Web of Science® Google Scholar
69Deller RC, Vatish M, Mitchell DA, Gibson MI. Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing. Nat Commun. 2014; 5(1): 3244.
10.1038/ncomms4244
PubMed Google Scholar
70Podsiedlik M, Markowicz-Piasecka M, Sikora J. Erythrocytes as model cells for biocompatibility assessment, cytotoxicity screening of xenobiotics and drug delivery. Chem Biol Interact. 2020; 332:109305.
10.1016/j.cbi.2020.109305
CAS PubMed Web of Science® Google Scholar
71Pal R, Mamidi MK, Das AK, Bhonde R. Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells. Arch Toxicol. 2012; 86(4): 651-661.
10.1007/s00204-011-0782-2
CAS PubMed Web of Science® Google Scholar
72Shu Z, Heimfeld S, Gao D. Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplant. 2014; 49(4): 469-476.
10.1038/bmt.2013.152
CAS PubMed Web of Science® Google Scholar
73Levin RL, Miller TW. An optimum method for the introduction or removal of permeable cryoprotectants: isolated cells. Cryobiology. 1981; 18(1): 32-48.
10.1016/0011-2240(81)90004-3
CAS PubMed Web of Science® Google Scholar
74Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol Cell Physiol. 1984; 247(3): C125-C142.
10.1152/ajpcell.1984.247.3.C125
CAS Web of Science® Google Scholar
75Gao DY, Liu J, Liu C, et al. Andrology: prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod. 1995; 10(5): 1109-1122.
10.1093/oxfordjournals.humrep.a136103
CAS PubMed Web of Science® Google Scholar
76Yao J, Shen L, Chen Z, Zhang B, Zhao G. Hydrogel microencapsulation enhances cryopreservation of red blood cells with trehalose. ACS Biomater Sci Eng. 2022; 8(5): 2066-2075.
10.1021/acsbiomaterials.2c00051
CAS PubMed Web of Science® Google Scholar
77Asghar W, El Assal R, Shafiee H, Anchan RM, Demirci U. Preserving human cells for regenerative, reproductive, and transfusion medicine. Biotechnol J. 2014; 9(7): 895-903.
10.1002/biot.201300074
CAS PubMed Web of Science® Google Scholar
78Bakaltcheva IB, Odeyale CO, Spargo BJ. Effects of alkanols, alkanediols and glycerol on red blood cell shape and hemolysis. Biochim Biophys Acta (BBA). 1996; 1280(1): 73-80.
10.1016/0005-2736(95)00279-0
PubMed Web of Science® Google Scholar
79Janis BR, Priddy MC, Otto MR, Kopechek JA, Menze MA. Sonoporation enables high-throughput loading of trehalose into red blood cells. Cryobiology. 2021; 98: 73-79.
10.1016/j.cryobiol.2020.12.005
CAS PubMed Web of Science® Google Scholar
80Stolzing A, Naaldijk Y, Fedorova V, Sethe S. Hydroxyethylstarch in cryopreservation–mechanisms, benefits and problems. Transfus Apher Sci. 2012; 46(2): 137-147.
10.1016/j.transci.2012.01.007
CAS PubMed Web of Science® Google Scholar
81Vardaki MZ, Schulze HG, Serrano K, Blades MW, Devine DV, Turner RFB. Non-invasive monitoring of red blood cells during cold storage using handheld Raman spectroscopy. Transfusion. 2021; 61(7): 2159-2168.
10.1111/trf.16417
CAS PubMed Web of Science® Google Scholar
82Högman CF, Meryman HT. Red blood cells intended for transfusion: quality criteria revisited. Transfusion. 2006; 46(1): 137-142.
10.1111/j.1537-2995.2006.00681.x
PubMed Web of Science® Google Scholar
83Holme S. Current issues related to the quality of stored RBCs. Transfus Apher Sci. 2005; 33(1): 55-61.
10.1016/j.transci.2005.02.004
PubMed Web of Science® Google Scholar
84Orlov D, Karkouti K. The pathophysiology and consequences of red blood cell storage. Anaesthesia. 2015; 70(s1): 29-e12.
10.1111/anae.12891
PubMed Web of Science® Google Scholar
85Dou M, Lu C, Sun Z, Rao W. Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation. Cryobiology. 2019; 91: 23-29.
10.1016/j.cryobiol.2019.11.002
CAS PubMed Web of Science® Google Scholar
86Bizjak DA, Jungen P, Bloch W, Grau M. Cryopreservation of red blood cells: effect on rheologic properties and associated metabolic and nitric oxide related parameters. Cryobiology. 2018; 84: 59-68.
10.1016/j.cryobiol.2018.08.001
CAS PubMed Web of Science® Google Scholar
87Shen L, Guo X, Ouyang X, Huang Y, Gao D, Zhao G. Fine-tuned dehydration by trehalose enables the cryopreservation of RBCs with unusually low concentrations of glycerol. J Mater Chem B. 2021; 9(2): 295-306.
10.1039/D0TB02426K
CAS PubMed Web of Science® Google Scholar
88Yoshida T, Prudent M, D'Alessandro A. Red blood cell storage lesion: causes and potential clinical consequences. Blood Transfus = Trasfusione del sangue. 2019; 17(1): 27-52.
PubMed Web of Science® Google Scholar
89Alotaibi NAS, Slater NKH, Rahmoune H. Salidroside as a novel protective agent to improve red blood cell cryopreservation. PLoS One. 2016; 11(9):e0162748.
10.1371/journal.pone.0162748
PubMed Web of Science® Google Scholar
90Seghatchian J, Putter JS. Pathogen inactivation of whole blood and red cell components: an overview of concept, design, developments, criteria of acceptability and storage lesion. Transfus Apher Sci. 2013; 49(2): 357-363.
10.1016/j.transci.2013.07.023
PubMed Web of Science® Google Scholar
91El Assal R, Guven S, Gurkan UA, et al. Bio-inspired cryo-ink preserves red blood cell phenotype and function during nanoliter vitrification. Adv Mater. 2014; 26(33): 5815-5822.
10.1002/adma.201400941
CAS PubMed Web of Science® Google Scholar
92Holovati JL, Wong KA, Webster JM, Acker JP. The effects of cryopreservation on red blood cell microvesiculation, phosphatidylserine externalization, and CD47 expression. Transfusion. 2008; 48(8): 1658-1668.
10.1111/j.1537-2995.2008.01735.x
PubMed Web of Science® Google Scholar
93Eruotor HO, Asiwe JN, Eruotor TM. Cymbopogon citratus protect against lead-induced suppression of haematological and tubuloglomerular functions as well as disruption of hepatocellular membranes in male Wistar rats. J Trace Elements Miner. 2023; 3:100045.
10.1016/j.jtemin.2022.100045
Google Scholar
94Thomas T, Stefanoni D, Dzieciatkowska M, et al. Evidence of structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients. J Proteome Res. 2020; 19(11): 4455-4469.
10.1021/acs.jproteome.0c00606
CAS PubMed Web of Science® Google Scholar
95Shen L, Qin X, Wang M, Gao D, Ouyang X, Zhao G. Combining cooling enhancement and trehalose dehydration to enable scalable volume cryopreservation of red blood cells with low concentration of glycerol. Adv Eng Mater. 2022; 24(11):2200835.
10.1002/adem.202200835
CAS Web of Science® Google Scholar
96Satchwell TJ, Toye AM. Band 3, an essential red blood cell hub of activity. Haematologica. 2021; 106(11): 2792-2793.
10.3324/haematol.2021.278643
PubMed Web of Science® Google Scholar
97Zhou X-L, He H, Liu B-L, Hua T-C, Chen Y. Effects of glycerol pretreatment on recovery and antioxidant enzyme activities of lyophilized red blood cells. CryoLetters. 2008; 29(4): 285-292.
CAS Web of Science® Google Scholar
98Gao S, Niu Q, Wang Y, et al. A dynamic membrane-active glycopeptide for enhanced protection of human red blood cells against freeze-stress. Adv Healthcare Mater. 2022; 12(10):2202516.
10.1002/adhm.202202516
Web of Science® Google Scholar
99Stoll C, Holovati JL, Acker JP, Wolkers WF. Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes. Biotechnol Prog. 2012; 28(2): 364-371.
10.1002/btpr.1519
CAS PubMed Web of Science® Google Scholar
100Yeow N, Tabor RF, Garnier G. Atomic force microscopy: from red blood cells to immunohaematology. Adv Colloid Interface Sci. 2017; 249: 149-162.
10.1016/j.cis.2017.05.011
CAS PubMed Web of Science® Google Scholar
101Cluitmans JCA, Chokkalingam V, Janssen AM, Brock R, Huck WTS, Bosman GJCGM. Alterations in red blood cell deformability during storage: a microfluidic approach. BioMed Res Int. 2014; 2014: 1-9.
10.1155/2014/764268
Web of Science® Google Scholar
102Shen L, Guo X, Ouyang X, Huang Y, Gao D, Zhao G. Fine-tuned dehydration by trehalose enables cryopreservation of RBCs with unusually low concentrations of glycerol. J Mater Chem B. 2020; 9(2): 295-306.
10.1039/D0TB02426K
Web of Science® Google Scholar
103Rogers SC, Dosier LB, McMahon TJ, et al. Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes. PLoS One. 2018; 13(12):e0209201.
10.1371/journal.pone.0209201
CAS PubMed Web of Science® Google Scholar
104Da Costa L, Suner L, Galimand J, et al. Diagnostic tool for red blood cell membrane disorders: assessment of a new generation ektacytometer. Blood Cells Mol Dis. 2016; 56(1): 9-22.
10.1016/j.bcmd.2015.09.001
PubMed Web of Science® Google Scholar
105Raju R, Bryant SJ, Wilkinson BL, Bryant G. The need for novel cryoprotectants and cryopreservation protocols: insights into the importance of biophysical investigation and cell permeability. Biochim Biophys Acta. 2021; 1865(1):129749.
10.1016/j.bbagen.2020.129749
CAS Web of Science® Google Scholar
106Wu SG, Jeng FR, Wei SY, et al. Red blood cell osmotic fragility in chronically hemodialyzed patients. Nephron. 1998; 78(1): 28-32.
10.1159/000044878
CAS PubMed Web of Science® Google Scholar
107Beutler E, Kuhl W, West C. The osmotic fragility of erythrocytes after prolonged liquid storage and after reinfusion. Blood. 1982; 59(6): 1141-1147.
10.1182/blood.V59.6.1141.1141
CAS PubMed Web of Science® Google Scholar
108Henkelman S, Noorman F, Badloe JF, Lagerberg JWM. Utilization and quality of cryopreserved red blood cells in transfusion medicine. Vox Sang. 2015; 108(2): 103-112.
10.1111/vox.12218
CAS PubMed Web of Science® Google Scholar
109Henkelman S, Lagerberg JWM, Graaff R, Rakhorst G, Van Oeveren W. The effects of cryopreservation on red blood cell rheologic properties. Transfusion. 2010; 50(11): 2393-2401.
10.1111/j.1537-2995.2010.02730.x
PubMed Web of Science® Google Scholar
110Wang Y, Yang P, Yan Z, et al. The relationship between erythrocytes and diabetes mellitus. J Diabetes Res. 2021; 2021:6656062.
10.1155/2021/6656062
PubMed Web of Science® Google Scholar
111Guest MM, Bond TP, Cooper RG, Derrick JR. Red blood cells: change in shape in capillaries. Science. 1963; 142(3597): 1319-1321.
10.1126/science.142.3597.1319
CAS PubMed Web of Science® Google Scholar
112Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood. 2008; 112(10): 3939-3948.
10.1182/blood-2008-07-161166
CAS PubMed Web of Science® Google Scholar
113Schwabbauer M. Normal erythrocyte production, physiology and destruction. In: M Schwabbauer, ed. Clinical Hematology: Principles, Procedures and Correlations. Vol 61, 2nd ed. Lippincott; 1998.
Google Scholar
114Graham JE, Meola DM, Kini NR, Hoffman AM. Comparison of the effects of glycerol, dimethyl sulfoxide, and hydroxyethyl starch solutions for cryopreservation of avian red blood cells. Am J Vet Res. 2015; 76(6): 487-493.
10.2460/ajvr.76.6.487
CAS PubMed Web of Science® Google Scholar
115Holovati JL, Gyongyossy-Issa MIC, Acker JP. Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality. Cryobiology. 2009; 58(1): 75-83.
10.1016/j.cryobiol.2008.11.002
CAS PubMed Web of Science® Google Scholar
116Dybas J, Bokamper MJ, Marzec KM, Mak PJ. Probing the structure-function relationship of hemoglobin in living human red blood cells. Spectrochim Acta, Part A. 2020; 239:118530.
10.1016/j.saa.2020.118530
CAS PubMed Web of Science® Google Scholar
117Greene DN, Eckel A, Lockwood CM. Hemoglobin variant detection. In: DN Greene, A Eckel, CM Lockwood, eds. Contemporary Practice in Clinical Chemistry. Elsevier; 2020: 413-427.
10.1016/B978-0-12-815499-1.00024-7
Google Scholar
118Zeidat A, Ebeido I. Design of Non-Invasive Methemoglobin Concentration Measuring System; 2018.
Google Scholar
119Mieczkowska E, Koncki R, Tymecki Ł. Hemoglobin determination with paired emitter detector diode. Anal Bioanal Chem. 2011; 399: 3293-3297.
10.1007/s00216-010-4358-4
CAS PubMed Web of Science® Google Scholar
120Li J, Lykotrafitis G, Dao M, Suresh S. Cytoskeletal dynamics of human erythrocyte. Proc Natl Acad Sci USA. 2007; 104(12): 4937-4942.
10.1073/pnas.0700257104
CAS PubMed Web of Science® Google Scholar
121Zimrin AB, Hess JR. Current issues relating to the transfusion of stored red blood cells. Vox Sang. 2009; 96(2): 93-103.
10.1111/j.1423-0410.2008.01117.x
CAS PubMed Web of Science® Google Scholar
122Caielli S, Cardenas J, de Jesus AA, et al. Erythroid mitochondrial retention triggers myeloid-dependent type I interferon in human SLE. Cell. 2021; 184(17): 4464-4479.
10.1016/j.cell.2021.07.021
CAS PubMed Web of Science® Google Scholar
123Ji L, Chauhan A, Brown WT, Chauhan V. Increased activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in the frontal cortex and cerebellum of autistic individuals. Life Sci. 2009; 85(23): 788-793.
10.1016/j.lfs.2009.10.008
CAS PubMed Web of Science® Google Scholar
124Cecarini V, Gee J, Fioretti E, et al. Protein oxidation and cellular homeostasis: emphasis on metabolism. Biochim Biophys Acta. 2007; 1773(2): 93-104.
10.1016/j.bbamcr.2006.08.039
CAS PubMed Web of Science® Google Scholar
125Xu M-Y, Wang P, Sun Y-J, et al. Redox status in liver of rats following subchronic exposure to the combination of low dose dichlorvos and deltamethrin. Pest Biochem Physiol. 2015; 124: 60-65.
10.1016/j.pestbp.2015.04.005
CAS PubMed Web of Science® Google Scholar
126Butterfield DA, Dalle-Donne I. Redox proteomics: from protein modifications to cellular dysfunction and disease. Mass Spectrom Rev. 2014; 33(1): 1-6.
10.1002/mas.21404
CAS PubMed Web of Science® Google Scholar
127Delobel J, Prudent M, Rubin O, Crettaz D, Tissot J-D, Lion N. Subcellular fractionation of stored red blood cells reveals a compartment-based protein carbonylation evolution. J Proteomics. 2012; 76: 181-193.
10.1016/j.jprot.2012.05.004
CAS PubMed Web of Science® Google Scholar
128Han J, Luo L, Wang Y, Wu S, Kasim V. Therapeutic potential and molecular mechanisms of salidroside in ischemic diseases. Front Pharmacol. 2022; 13:974775.
10.3389/fphar.2022.974775
CAS PubMed Web of Science® Google Scholar
129Skrzep-Poloczek B, Poloczek J, Chełmecka E, et al. The oxidative stress markers in the erythrocytes and heart muscle of obese rats: relate to a high-fat diet but not to DJOS bariatric surgery. Antioxidants. 2020; 9(2):183.
10.3390/antiox9020183
CAS PubMed Web of Science® Google Scholar
130Gu J, Chang TMS. Extraction of erythrocyte enzymes for the preparation of polyhemoglobin-catalase-superoxide dismutase. Artif Cells Blood Substitutes Biotechnol. 2009; 37(2): 69-77.
10.1080/10731190902742240
CAS PubMed Google Scholar
131Marossy A, Švorc P, Kron I, Grešová S. Hemorheology and circulation. Clin Hemorheol Microcirc. 2009; 42: 239-258.
10.3233/CH-2009-1192
CAS PubMed Web of Science® Google Scholar
132Machiedo GW, Zaets SB, Berezina TL, et al. Trauma-hemorrhagic shock-induced red blood cell damage leads to decreased microcirculatory blood flow. Crit Care Med. 2009; 37(3): 1000-1010.
10.1097/CCM.0b013e3181962d39
PubMed Web of Science® Google Scholar
133Piagnerelli M. The red blood cell: an underestimated actor in alterations of the microcirculation. Crit Care Med. 2009; 37(3): 1158-1160.
10.1097/CCM.0b013e318196fd86
PubMed Web of Science® Google Scholar
134Baskurt OK, Meiselman HJ. Erythrocyte aggregation: basic aspects and clinical importance. Clin Hemorheol Microcirc. 2013; 53(1-2): 23-37.
10.3233/CH-2012-1573
PubMed Web of Science® Google Scholar
135Razavian SM, Del Pino M, Simon A, Levenson J. Increase in erythrocyte disaggregation shear stress in hypertension. Hypertension. 1992; 20(2): 247-252.
10.1161/01.HYP.20.2.247
CAS PubMed Web of Science® Google Scholar
136Fernandes HP, Cesar CL, Barjas-Castro ML. Electrical properties of the red blood cell membrane and immunohematological investigation. Rev Bras Hematol Hemoter. 2011; 33: 297-301.
10.5581/1516-8484.20110080
PubMed Google Scholar
137Huisjes R, Bogdanova A, Van Solinge WW, Schiffelers RM, Kaestner L, Van Wijk R. Squeezing for life–properties of red blood cell deformability. Front Physiol. 2018; 9: 656.
10.3389/fphys.2018.00656
PubMed Web of Science® Google Scholar
138Stadnick H, Onell R, Acker JP, Holovati JL. Eadie-Hofstee analysis of red blood cell deformability. Clin Hemorheol Microcirc. 2011; 47(3): 229-239.
10.3233/CH-2010-1384
CAS PubMed Web of Science® Google Scholar
139Poisson JS, Briard JG, Turner TR, Acker JP, Ben RN. Hydroxyethyl starch supplemented with ice recrystallization inhibitors greatly improves cryopreservation of human red blood cells. Bioprocess J. 2016; 15.
Google Scholar
140Xiao LL, Chen S, Lin CS, Liu Y. Simulation of a single red blood cell flowing through a microvessel stenosis using dissipative particle dynamics. Mol Cell Biomech. 2014; 11(1): 67-85.
CAS PubMed Web of Science® Google Scholar
141Miller LH, Baruch DI, Marsh K, Doumbo OK. The pathogenic basis of malaria. Nature. 2002; 415(6872): 673-679.
10.1038/415673a
CAS PubMed Web of Science® Google Scholar
142Drost EM, Kassabian G, Meiselman HJ, Gelmont D, Fisher TC. Increased rigidity and priming of polymorphonuclear leukocytes in sepsis. Am J Respir Crit Care Med. 1999; 159(6): 1696-1702.
10.1164/ajrccm.159.6.9803061
CAS PubMed Web of Science® Google Scholar
143Xu Z, Dou W, Wang C, Sun Y. Stiffness and ATP recovery of stored red blood cells in serum. Microsyst Nanoeng. 2019; 5(1): 51.
10.1038/s41378-019-0097-7
CAS PubMed Google Scholar
144Thomas G, Burnham NA, Camesano TA, Wen Q. Measuring the mechanical properties of living cells using atomic force microscopy. J Vis Exp. 2013;(76):e50497.
PubMed Web of Science® Google Scholar
145Katiyar VK, Fisseha D. Analysis of mechanical behavior of red blood cell membrane with malaria infection. World J Mech. 2011; 01(03): 100-108.
10.4236/wjm.2011.13014
Google Scholar
146Richards AB, Krakowka S, Dexter LB, et al. Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food Chem Toxicol. 2002; 40(7): 871-898.
10.1016/S0278-6915(02)00011-X
CAS PubMed Web of Science® Google Scholar
147El-Sayed MS, Ali N, El-Sayed ali Z. Haemorheology in exercise and training. Sports Med. 2005; 35: 649-670.
10.2165/00007256-200535080-00001
PubMed Web of Science® Google Scholar
148Hess JR. An update on solutions for red cell storage. Vox Sang. 2006; 91(1): 13-19.
10.1111/j.1423-0410.2006.00778.x
CAS PubMed Web of Science® Google Scholar
149Hess JR. Measures of stored red blood cell quality. Vox Sang. 2014; 107(1): 1-9.
10.1111/vox.12130
CAS PubMed Web of Science® Google Scholar
150Almac E, Ince C. The impact of storage on red cell function in blood transfusion. Best Pract Res Clin Anaesthesiol. 2007; 21(2): 195-208.
10.1016/j.bpa.2007.01.004
CAS PubMed Google Scholar
151Heaton A, Keegan T, Holme S. In vivo regeneration of red cell 2, 3-diphosphoglycerate following transfusion of DPG-depleted AS-1, AS-3 and CPDA-1 red cells. Br J Haematol. 1989; 71(1): 131-136.
10.1111/j.1365-2141.1989.tb06286.x
CAS PubMed Web of Science® Google Scholar
152Sohmer PR, Dawson RB, Huggins C. The significance of 2, 3–DPG in red blood cell transfusions. CRC Crit Rev Clin Lab Sci. 1979; 11(2): 107-174.
10.3109/10408367909105855
CAS PubMed Google Scholar
153McMahon TJ. Red blood cell deformability, vasoactive mediators, and adhesion. Front Physiol. 2019; 10: 1417.
10.3389/fphys.2019.01417
PubMed Web of Science® Google Scholar
154Guo J, Yu Y, Zhu W, et al. Modular assembly of red blood cell superstructures from metal–organic framework nanoparticle-based building blocks. Adv Funct Mater. 2021; 31(10):2005935.
10.1002/adfm.202005935
CAS Web of Science® Google Scholar
155Grau M, Pauly S, Ali J, et al. RBC-NOS-dependent S-nitrosylation of cytoskeletal proteins improves RBC deformability. PLoS One. 2013; 8(2):e56759.
10.1371/journal.pone.0056759
CAS PubMed Web of Science® Google Scholar
156Akahat J, Sripara P, Thanukarn T, Jaroonsirimaneekul T, Mongkunkhamchaw N, Punikom K. Quality of blood components for transfusion in blood transfusion centre, faculty of medicine, Khon Kaen University, Thailand. Srinagarind Med J. 2016; 31(5): 118-121.
Google Scholar

Volume2, Issue4

December 2023

e67

Cryoprotectants for red blood cells: Evaluate safety and effectiveness by in vitro measures

Abstract

1 INTRODUCTION

2 DEVELOPMENT OF CPAS FOR RBC CRYOPRESERVATION

2.1 Small molecule-based CPAs

2.2 Macromolecule-based CPAs

3 THE SAFETY OF CPAS

4 THE EFFECTIVENESS OF CPAS

4.1 Membrane properties

4.1.1 Osmotic fragility

4.1.2 Morphology

4.1.3 Membrane lipid peroxidation

4.1.4 PS exposure and CD47 expression

4.2 Protein activities

4.2.1 Hb content

4.2.2 Band 3 phosphorylation

4.2.3 ATPase activity

4.2.4 Protein oxidation

4.2.5 Antioxidant enzyme activity

4.3 Rheological properties

4.3.1 Aggregation

4.3.2 Deformability

4.4 Metabolites levels

4.4.1 ATP level

4.4.2 2,3-DPG level

4.4.3 NO content

5 DISCUSSION AND FUTURE PERSPECTIVES

6 CONCLUSION

AUTHOR CONTRIBUTIONS

ACKNOWLEDGMENTS

CONFLICT OF INTEREST STATEMENT

ETHICS STATEMENT

Open Research

DATA AVAILABILITY STATEMENT

REFERENCES

Figures

References

Related

Information