Clin Transplant Res 2024; 38(4): 377-403
Published online December 31, 2024
https://doi.org/10.4285/ctr.24.0039
© The Korean Society for Transplantation
Kidus Haile Yemaneberhan1,2,* , Minseok Kang1,*
, Jun Hwan Jang3
, Jin Hee Kim3
, Kyeong Sik Kim1
, Ho Bum Park3
, Dongho Choi1,2,4,5
1Department of Surgery, Hanyang University College of Medicine, Seoul, Korea
2Hanyang Institute of Bioscience and Biotechnology, Hanyang University, Seoul, Korea
3Department of Energy Engineering, Hanyang University, Seoul, Korea
4Research Institute of Regenerative Medicine and Stem Cells, Hanyang University, Seoul, Korea
5Department of HY-KIST Bio-convergence, Hanyang University, Seoul, Korea
Correspondence to: Kyeong Sik Kim
Department of Surgery, Hanyang University College of Medicine, 222-1 Wangsimni-ro, Seongdong-gu, Seoul 04763, Korea
E-mail: crane87@hanyang.ac.kr
Dongho Choi
Department of Surgery, Hanyang University College of Medicine, 222-1 Wangsimni-ro, Seongdong-gu, Seoul 04763, Korea
E-mail: crane87@hanyang.ac.kr
*These authors contributed equally to this study as co-first authors.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Organ transplantation, a critical treatment for end-stage organ failure, has witnessed significant advancements due to the integration of improved surgical techniques, immunosuppressive therapies, and donor-recipient matching. This review explores the progress of organ preservation, focusing on the shift from static cold storage (SCS) to advanced machine perfusion techniques such as hypothermic (HMP) and normothermic machine perfusion (NMP). Although SCS has been the standard approach, its limitations in preserving marginal organs and preventing ischemia-reperfusion injury (IRI) have led to the adoption of HMP and NMP. HMP, which is now the gold standard for high-risk donor kidneys, reduces metabolic activity and improves posttransplant outcomes. NMP allows real-time organ viability assessment and reconditioning, especially for liver transplants. Controlled oxygenated rewarming further minimizes IRI by addressing mitochondrial dysfunction. The review also highlights the potential of cryopreservation for long-term organ storage, despite challenges with ice formation. These advances are crucial for expanding the donor pool, improving transplant success rates, and addressing organ shortages. Continued innovation is necessary to meet the growing demands of transplantation and save more lives.
Keywords: Organ transplantation, Machine perfusion, Cryopreservation, Nanobiotechnology
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The advent of organ transplantation has significantly improved the quality of life for many individuals suffering from end-stage organ failure. The first successful kidney transplant, performed between identical twins, took place in 1954. This was followed by liver, pancreas, and heart transplants in the late 1960s [1]. However, the success of these transplants was limited by immunological rejection until the development of effective immunosuppressive medications in the 1980s [2].
A variety of solutions with properties similar to those of the human body have been developed for organ preservation. These include Euro-Collins (EC), University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate solutions [3]. These solutions enable the flushing, storage in an icebox, and transportation of organs. This method, known as static cold storage (SCS), is currently the standard approach for organ preservation [4,5].
Despite ongoing efforts, the current approach is insufficient to meet the growing demand for organ transplants. According to the United Network for Organ Sharing, the number of people currently on the waiting list for organ transplants has reached 103,991, more than double the 46,630 transplants performed in 2023 [6]. The Health Resources and Services Administration notes that 17 individuals die each day while waiting for an organ transplant [7]. Additionally, the waiting list for organ transplants in South Korea has grown by 10,000 in the past 5 years [8]. In response to this discrepancy, healthcare professionals and researchers are exploring the use of extended criteria donor (ECD) organs, which are typically considered unsuitable for transplantation [9].
The development of improved organ preservation techniques and equipment has been driven by research into the cellular and molecular mechanisms of ischemia-reperfusion injury (IRI), which significantly contributes to organ damage during preservation and transplantation [10]. These innovations aim to sustain organs in an ideal environment, thus extending the preservation duration or employing advanced machines to simulate conditions akin to physiological states (Fig. 1). This approach facilitates the evaluation, preservation, and possible treatment of organs before transplantation [11].
Furthermore, novel subzero organ preservation techniques are currently under investigation with the goal of improving existing preservation methods and ultimately enabling organ banking. This review paper explores the current organ preservation strategies, including SCS, hypothermic machine perfusion (HMP), subnormothermic machine perfusion (SNMP), and normothermic machine perfusion (NMP). Additionally, this review examines the latest advancements and current status of subzero organ preservation techniques such as partial freezing, supercooling, isochoric preservation, vitrification, and nanowarming.
IRI poses a significant challenge in organ transplantation. It is unavoidable for transplant organs to undergo a period of reduced blood flow (ischemia) followed by the restoration of blood flow (reperfusion) upon transplantation. During the ischemic phase, anaerobic metabolism predominates, leading to adenosine triphosphate (ATP) depletion, cellular damage, and the accumulation of metabolic waste products. This process results in the generation of danger-associated molecular patterns (DAMPs) due to cellular debris and metabolic stress. DAMPs activate pattern recognition receptors, triggering a signaling pathway that leads to the release of proinflammatory cytokines and intensifies the inflammatory response. Upon reperfusion, the restored blood flow activates the immune system, and the sudden influx of oxygen promotes the production of mitochondrial reactive oxygen species (ROS), further exacerbating inflammation and tissue damage [63,64]. This cascade continues and ultimately leads to organ injury, potentially increasing the risk of graft dysfunction and rejection [65].
IRI has been shown to negatively impact organs in various transplantation scenarios. In liver transplants, IRI leads to the early demise of hepatocytes and sinusoidal cells. At the same time, it activates hepatic stellate cells, which affects long-term recovery and can lead to clinical complications such as early allograft dysfunction (EAD) and nonanastomotic biliary strictures (NAS) [66,67]. Similarly, in kidney transplantation, IRI is crucial in the development of conditions like delayed graft function (DGF) or primary nonfunction. The associated proinflammatory response may also promote progressive interstitial fibrosis, potentially leading to chronic graft dysfunction, which is characterized by interstitial fibrosis and tubular atrophy [68]. In heart transplantation, IRI increases the risk of primary nonfunction, graft rejection, and cardiac edema. It also worsens myocardial and endothelial damage, which in turn promotes allograft fibrosis [69]. Lastly, in lung transplantation, IRI causes increased permeability of the pulmonary endothelium, leading to pulmonary edema and a subsequent reduction in function. Furthermore, IRI heightens the likelihood of prolonged mechanical ventilation and extended hospital stays for lung transplant recipients, thereby increasing the risk of late graft failure [70].
SCS is currently the most prevalent method used for organ preservation. This process involves flushing the organs with a preservation solution and then storing them at low temperatures between 2 °C and 4 °C [71]. The fundamental principle of SCS is that low temperatures reduce the rate of cellular metabolism, thereby decreasing the energy demand. Additionally, the preservation solution helps optimize the cellular environment and mitigate cellular damage by reducing osmotic and reperfusion injury [72].
A variety of solutions have been introduced in clinical practice; however, the UW solution remains the gold standard for multiorgan retrieval [73]. The effectiveness of the UW solution depends on its unique composition, which includes multiple cell-impermeant agents such as lactobionic acid, raffinose, and hydroxyethyl starch. These agents help prevent cellular swelling during cold ischemic storage. Additionally, the solution contains glutathione and adenosine, which support the restoration of normal metabolism during reperfusion. They do this by enhancing the antioxidant capacity of the organs and promoting the generation of high-energy phosphates, respectively [72].
Despite the simplicity and cost-effectiveness of SCS compared to more complex preservation techniques, it has several significant drawbacks. Prolonged hypothermic preservation can damage tissue, reducing the viability of donor organs [74,75]. Additionally, assessing organ function and predicting viability during cold storage pose significant challenges. Furthermore, marginal organs, such as those from cardiac-death donors and ECDs, are more susceptible to IRI, leading to the discarding of many nonideal organs due to the high risk of unfavorable outcomes [76].
Although SCS presents inherent challenges, the primary limitation in organ transplantation is the scarcity of available organs. To broaden the donor pool and improve transplant outcomes from marginal organs such as ECD or donors after circulatory death (DCD), current research is shifting focus from SCS to
Perfusion protocols for organ preservation are primarily categorized based on the temperature of the perfusate. These categories include HMP (2 °C–10 °C), SNMP (20 °C–33 °C), and NMP (36 °C–38 °C) [77,78]. Table 1 summarizes the experiments and outcomes of machine perfusion for liver and kidney preservation, organized by temperature. The machine perfusion technique offers a promising approach to mitigate IRI. It is crucial, however, to understand the distinct advantages and limitations of each technique, as they are not interchangeable (Table 2) [79]. For example, HMP is primarily employed to reduce IRI, whereas NMP is utilized for assessing organ viability [80].
Table 1. Summary of machine perfusion experiments and outcomes according to the temperature used
Study | Type | Organ | Temperature (°C) | Warm ischemia time (min) | Cold ischemia time (hr) | Perfusion time (hr) | Perfusate solution | Result |
---|---|---|---|---|---|---|---|---|
Hypothermic machine perfusion (HMP) | ||||||||
Guarrera et al. (2010) [24] | Human | Liver | 4–6 | 40–50 | 7–11 | 3–5 | Vasosol | • The HMP group had a 5% early allograft dysfunction rate versus 25% in the control group, with no vascular complications and lower serum injury markers • They also had a shorter mean hospital stay |
Henry et al. (2012) [27] | Human | Liver | 4–6 | 40–50 | 7–11 | 3–5 | Hextend | • The HMP group had lower inflammatory marker expression and no ultrastructural damage, unlike the cold storage group |
Zhang et al. (2016) [35] | Rabbit | Kidney | 4–8 | 35 | NA | 4 | Human carbonic anhydrase II | • The HMP group had significantly lower apoptosis rates and cleaved caspase-3 expression, with higher ezrin and phosphorylated AKT levels than the cold storage group • HMP reduced renal cell apoptosis during ischemia-reperfusion injury via the ezrin/AKT pathway |
Faucher et al. (2022) [52] | Human | Kidney | 2–5 | NA | 16 | 10 | Kidney preservation solution-1 | • With a longer perfusion time, 23 metabolites increased and 8 decreased in the solution • Extended perfusion did not affect transporter mRNA expression, and neither were predictive of graft outcomes |
Hypothermic oxygenated perfusion (HOPE) | ||||||||
Jochmans et al. (2020) [48] | Human | Kidney | 4 | NA | NA | 24 | University of Wisconsin Machine Perfusion Solution + O2 (100%) | • In the study, the HOPE group had fewer severe complications (11% vs. 16%), a lower rate of graft failure (3% vs. 10%), and a mean estimated glomerular filtration rate difference of 3.7 mL/min per 1.73 m2 at 12 months compared to the HMP group |
Panayotova et al. (2024) [60] | Human | Liver | 5–7 | 20–30 | 2–5 | 2–4 | Vasosol + O2 (100%) | • Early allograft dysfunction rates were 11.1% with HOPE and 16.4% with static cold storage. Biliary strictures occurred in 16.4% of static cold storage cases and 6.3% of HOPE cases • The predicted 7-day graft failure risk was lower with HOPE (3.4%) than with static cold storage (4.5%) |
Subnormothermic machine perfusion (SNMP) | ||||||||
Tolboom et al. (2012) [26] | Rat | Liver | 20–30 | 60 | NA | 5 | Williams medium E, insulin erythrocytes, L-glutamine, heparin | • After 4 weeks posttransplantation, livers preserved with SNMP had a 100% survival rate, while those preserved with cold storage died within 24 hours • Hematoxylin and eosin analysis showed that machine perfusion-preserved livers maintained a fresh liver-like architecture • At 28 days, livers preserved at 20 °C and 30 °C had the highest total bilirubin values |
Hoyer et al. (2014) [28] | Pig | Kidney | 20 | 30 | NA | 7 | Custodiol-N, pyrogen-free dextran 40 | • Kidneys preserved by SNMP had significantly higher blood flow, urine output, and creatinine-clearance—10 times higher than cold storage and 2 times higher than hypothermic machine perfusion • SNMP also best-preserved structural integrity histologically |
Bruinsma et al. (2014) [29] | Human | Liver | 21 | 20–50 | 5–19 | 3 | Phenol-red Williams medium E, insulin, penicillin/streptomycin, nutrients, hydrocortisone | • After perfusion, liver oxygen uptake and ATP levels improved, bile production increased, and bile composition became more favorable • Liver function, indicated by urea and albumin production, was also observed during perfusion |
Abraham et al. (2024) [62] | Pig & human | Kidney | 22–25 | 30 | NA | 24 | Albumin, dexamethasone, heparin, insulin, multivitamins | • The human kidney maintained stable functional and biological parameters after 24 hours of SNMP perfusion • Pig kidneys perfused with SNMP for 24 hours and then autotransplanted showed reduced creatinine and blood urea nitrogen levels after 7 days compared to the cold storage group |
Normothermic machine perfusion (NMP) | ||||||||
Schön et al. (2001) [15] | Pig | Liver | 37 | 60–100 | NA | 4 | Whole pig blood, electrolyte solutions, heparin | • All animals receiving livers treated with normothermic extracorporeal liver perfusion survived more than 7 days posttransplant • In contrast, all animals in the 4-hour static cold storage group experienced primary graft nonfunction within 24 hours |
Hosgood et al. (2018) [39] | Human | Kidney | 35–36 | 14 | 27 | 1 | Red blood cell-based solution, supplements | • In Phase 1, 28 kidneys were deemed suitable for transplantation with a quality assessment score of 1–3 • In Phase 2, 10 kidneys were assessed by NMP, leading to 5 transplants, of which 4 showed initial graft function |
Nasralla et al. (2018) [40] | Human | Liver | 37 | 17–25 | 1–2 | 5–12 | Packed red blood cells, antibiotics, Gelofusine, insulin, heparin, nutrition | • Compared to cold storage, NMP resulted in a 50% reduction in graft injury, a 50% lower organ discard rate, and a 54% longer preservation time • However, there was no significant difference in bile duct complications or patient survival |
Thompson et al. (2022) [54] | Human | Kidney | 37 | NA | 20 | 6 | Packed red blood cells, heparin, dexamethasone, mannitol, sodium bicarbonate, 1 mg miRNA-24-3p antagomir | • During NMP, using a perfused antagomir targeting miR-24-3p increased the expression of genes controlled by this microRNA, such as heme oxygenase-1 • This effect was absent in the cold perfusion group, and other gene expressions remained unchanged, highlighting the antagomirʼs specificity |
Controlled oxygenated rewarming (COR) | ||||||||
Minor et al. (2013) [91] | Pig | Liver | Increase from 4 to 20 | NA | 18 | 1.5 | Custodiol-N | • COR significantly reduced cellular enzyme loss, gene expression, and tumor necrosis factor-alpha perfusate activities • It also led to higher bile production, lower histological injury scores, and decreased caspase 3 activation compared to cold storage |
Schopp et al. (2015) [31] | Pig | Kidney | Increase from 4 to 20 | NA | 18 | 3 | Custodiol-N | • COR led to a 2-fold increase in creatinine and urea clearances during warm reperfusion compared to controls, along with significant mitigation of postischemic mitochondrial dys-homeostasis • COR also improved renal oxygen consumption, preserved total nicotinamide adenine dinucleotide tissue content, and largely prevented mitochondrial initiation of apoptosis, as indicated by reduced caspase 9 activation |
Hoyer et al. (2016) [32] | Human | Liver | Increase from 4 to 20 | 18–33 | 6–15 | 1.5 | Custodiol-N | • COR reduced peak serum transaminases by approximately 50% posttransplantation compared to untreated controls • Graft survival after 6 months was 100% in the COR group versus 80.9% in controls, with patient survival at 100% in the COR group and 84.7% in controls |
Minor et al. (2022) [44] | Human | Kidney | Increase from 8 to 35 | NA | 12 | 2 | Steen solution, sodium bicarbonate, calcium gluconate, ampicillin | • The kidney was transplanted without complications and showed good immediate function • Serum creatinine decreased from 720 µmol/L preoperatively to 506 µmol/L within the first 24 hours, with a clearance of 43.1 mL/min after 1 week |
NA, not available; ATP, adenosine triphosphate.
Table 2. Comparison of perfusion methods for organ preservation
Method | Advantages | Disadvantages | Potential indications | Costs |
---|---|---|---|---|
SCS | • Widely adopted with well-defined protocols • No complex machinery required; simple and inexpensive • Reduced cellular metabolism | • Limited preservation time (12 hours in liver, 24 hours in kidney) • Higher risk of ischemia-reperfusion injury and posttransplant complications | • Low-risk grafts with shorter transport times | Low |
HMP | • Reduces IRI • Reduces cellular metabolism • Oxygenation increases ATP generation | • High complexity • Requires expertise and specialized ambulance for transportation | • High-risk grafts with longer transport times • Ideal for DCD and ECD organs needing better preservation | Moderate |
NMP | • Reduces IRI • Simulates physiological conditions • Potential for metabolic recovery • Allows functional evaluation of organs | • High complexity • High cost of consumables and machinery • Requires expertise and specialized ambulance for transportation | • Optimal for evaluating marginal organs • Ideal for DCD and ECD organs needing better preservation | High |
COR | • Reduces IRI • Minimizes reperfusion injury during temperature transition • Allows functional evaluation of organs | • High complexity • High cost of consumables and machinery • Limited clinical data | • Emerging technique to optimize hypothermic preservation strategies | Moderate |
SNMP | • Reduces IRI • Potential for metabolic recovery • Allows functional evaluation of organs • Simple and cost-effective with no need for temperature control or whole blood | • Not as ideal as NMP for evaluating graft function • Limited clinical data | • Emerging technique combining the advantages of HMP and NMP | Low-moderate |
SCS, static cold storage; HMP, hypothermic machine perfusion; IRI, ischemia-reperfusion injury; DCD, donation after circulatory death; ECD, extended criteria donor; NMP, normothermic machine perfusion; COR, controlled oxygenated rewarming; SNMP, subnormothermic machine perfusion.
HMP is a technique that involves the continuous circulation of a cold perfusate through the organ's vasculature, typically within a temperature range of 2 °C to 10 °C (Fig. 2). HMP provides a multifaceted strategy for mitigating IRI [51]. During IRI, ischemia-induced cellular hypoxia activates hypoxia-inducible factor (HIF). Studies have shown that HMP can reduce HIF expression, which is associated with improved transplant outcomes [27,37]. Additionally, HMP has been demonstrated to mitigate IRI by reducing cell apoptosis in comparison to SCS [35,41,52]. This results in a reduction in the production of DAMPs, which in turn attenuates the proinflammatory cascade. Furthermore, studies have demonstrated that HMP improves endothelial function, which is linked to a reduction in immune cell transmigration [23,41]. The microvascular injury and endothelial dysfunction caused by IRI make the organ vulnerable to alloimmune damage; HMP helps counteract these effects by reducing their severity [81]. Furthermore, hypothermic oxygenated machine perfusion (HOPE) has shown additional benefits. HOPE helps restore cellular functions by rejuvenating mitochondria and restoring ATP levels [34]. Furthermore, it modifies the redox state of mitochondria by temporarily inhibiting oxidative metabolism, thereby reducing the initial release of ROS, and limiting tissue damage upon reperfusion [82,83].
HMP has become the gold standard for high-risk donor kidney transplantation in many countries, supported by high-quality randomized clinical trials (RCTs) and meta-analyses [84–86]. Currently, a prospective study is evaluating the potential of HOPE in kidney transplantation, which has shown promise in reducing posttransplant complications without significant costs [48]. Additionally, the noninferiority of end-ischemic HMP compared to full HMP is being investigated, due to the cost-effectiveness and simplicity of the former. However, the existing evidence does not support this hypothesis, and the studies are statistically underpowered [50,84].
The use of HMP in liver transplantation has only recently started to gain popularity, with the first successful application in a human liver reported in 2010 [24]. Unlike in kidney transplantation, end-ischemic HOPE has become the primary focus in liver transplantation studies. This focus is due to technological constraints that allow oxygenation only through a fixed, continuous source for a period of 1–2 hours during the recipient's hepatectomy [60,87]. A meta-analysis has shown that end-ischemic HOPE, compared to SCS, can reduce complications and improve graft survival in DCD and ECD liver transplantations [61,88]. However, the routine use of end-ischemic HOPE in donation after brain death (DBD) organs has not been recommended [59].
The existing literature on HMP in human heart transplantation is limited, with no RCTs currently available. In a study conducted by Nilsson et al. [47], the 6-month survival rates were 100% for the HOPE group and 72% for the SCS group, despite the latter experiencing longer preservation times. In another study, Andrijauskaite et al. [89] found that a 6-hour HOPE procedure significantly improved left ventricular relaxation compared to SCS, and was also associated with reduced inflammation markers. McGiffin et al. [90] reported that HOPE allows for the extension of preservation times to nearly 9 hours.
SNMP is conducted at approximately 20 ºC with oxygenated perfusate. Unlike NMP, SNMP simplifies the protocol by eliminating the need for temperature control and the use of donor blood or oxygen carriers, since the demand for tissue oxygenation is lower [26]. Additionally, reoxygenation without neutrophils and blood-derived inflammatory factors allows for a reduction in proinflammatory responses during reperfusion [58,92]. In this setting, with a moderate metabolic rate, it becomes feasible to conduct viability testing while controlling adverse cellular events.
A study comparing SNMP and NMP in porcine kidneys found that kidneys treated with SNMP were more susceptible to tubular and renal injuries, highlighting the need for an oxygen-carrier [93]. However, animal studies that compared SNMP with SCS for liver or kidney transplantation showed that SNMP could increase tissue ATP levels through mitochondrial resuscitation, reduce markers of tissue injury, and improve graft function after surgery [28,30,46,94]. When applied to discarded human livers, SNMP enhanced oxygen uptake, decreased lactate levels, and increased ATP following perfusion [29]. During the perfusion process, liver function was preserved, with an increase in bile production and improvements in bile composition noted. In research conducted by Abraham et al. [62] a discarded human kidney was perfused with SNMP for 24 hours, resulting in stable functional and biological parameters.
SNMP has been explored as an effective platform for delivering cytoprotective drugs, such as the hydrogen sulfide (H2S) donor AP29, when added to UW solution, compared to normothermic or hypothermic conditions [38,49]. In another study focusing on liver supercooling, SNMP was used to introduce 3-O-methylglucose (3-OMG) into hepatocytes [29]. This method was selected because 3-OMG is absorbed by hepatocytes through glucose transporter protein 1 (GLUT-1) and GLUT-2 transporters, requiring the use of SNMP while the hepatocytes are still metabolically active.
NMP enables an organ to sustain its physiological state by perfusing essential metabolic substrates with oxygen at body temperature (Fig. 2). This process supports cellular metabolism and ATP production, while also preventing ischemic changes. Numerous studies have demonstrated the safety and efficacy of this approach, which reduces IRI by mitigating inflammation and cell death, and by promoting tissue regeneration [15,36,42].
As NMP simulates physiological conditions, it enables the evaluation of graft viability and functionality in high risk, ECD organs of uncertain quality by assessing various markers [39,57,95]. If the organ meets the viability criteria, it is approved for transplantation; if not, it is either discarded or kept on NMP for further therapeutic interventions. Several regenerative strategies have been suggested as potential methods for repairing damaged livers during NMP. These include the use of senolytics, defatting cocktails, RNA interference, and stem cell therapy [54,55]. In conclusion, NMP serves as an effective platform for the reconditioning and enhancement of high-risk allografts, potentially rescuing organs that might otherwise be discarded.
For the kidney, Hosgood et al. [39] conducted the first RCT to compare 1-hour NMP before implantation with SCS in DCD organ transplantation. This multicenter RCT found no statistically significant differences in the incidence of DGF or complications between the two groups. The evaluation of the allocated kidneys was based on a scoring system that included urine output, renal blood flow, and macroscopic appearance. Nonrandomized and experimental studies have shown that kidneys subjected to end-ischemic NMP exhibit a lower incidence of DGF, minimal tubular injury, and enhanced metabolic function [35,41]. However, other studies on end-ischemic NMP have found no significant differences in outcomes compared to SCS [23,52].
The first report of NMP in human liver transplantation appeared in 2016 [33]. RCTs have shown that NMP leads to a 50% decrease in discard rates and a 20% increase in liver transplantations compared to SCS, by incorporating high-risk donors previously considered unsuitable [40,45]. A meta-analysis of NMP-RCTs demonstrated a statistically significant reduction in the incidence of NAS, major complications, and EAD [56,88,96]. However, NMP has not shown any significant benefits in terms of 1-year graft or patient survival.
A meta-analysis comparing NMP-DBD and NMP-DCD with SCS-DBD heart transplantation showed no significant differences in short- and long-term survival outcomes [53], indicating that NMP is a safe and effective strategy. The efficacy of NMP for DCD hearts and high-risk cases is further supported by the results of studies that have explored these issues [97–99].
It has been demonstrated that exposing an organ stored at low temperatures to a sudden increase in temperature, either through NMP or reperfusion after transplantation, can cause mitochondrial dysfunction [80,91]. A potential solution to this problem is controlled oxygenated rewarming (COR) [43,91,100]. The COR technique involves oxygenated machine perfusion, where the temperature of the perfusate is gradually raised from hypothermia to subnormothermia or normothermia, facilitating a gentle and controlled rewarming process.
Minor et al. [91] compared COR with SCS, simple HMP, and SNMP in porcine livers. Following a viability assessment using NMP, COR significantly increased cellular ATP storage and decreased ROS production, hepatocellular necrosis, proinflammatory gene expression, and vascular resistance. These changes led to a substantial reduction in IRI. Another study on porcine kidneys showed that COR, compared to HMP, doubled the clearance rates of creatinine and urea, and improved renal oxygen consumption and mitochondrial functions [31]. The first clinical application of COR on a liver took place in 2014 [32]. The transplantation outcomes included a 50% reduction in peak serum transaminases compared to a historical cohort and showed 100% graft and patient survival. A case report on the first human kidney transplantation using COR to rewarm to 35 ºC indicated optimal immediate function and no complications, supporting its clinical use [44]. The first RCT focusing on COR in liver grafts demonstrated that COR significantly lowered 3-day postsurgery peak aspartate aminotransferase levels and major complications compared to SCS [100].
To minimize the duration of ischemia for organs, novel approaches such as ischemia-free liver transplantation are under investigation. This technique integrates normothermic regional perfusion with NMP, enabling the procurement, preservation, and implantation of livers while continuously providing normothermic, oxygenated blood. Studies have shown that this method offers significant advantages over SCS, including reductions in EAD, postreperfusion syndrome, NAS, and the comprehensive complication index at 1 year [101].
Cryopreservation is a biopreservation technique that involves storing biological samples at subzero temperatures. This method utilizes cryoprotective agents (CPAs) and controlled cooling techniques to preserve cellular viability and functionality [102]. The history of cryopreservation dates back to ancient civilizations, which utilized cold temperatures to preserve various items [103]. However, the modern era of cryopreservation began in 1948 when Polge and his colleagues discovered the cryoprotective properties of glycerol on fowl sperm [104]—a breakthrough that paved the way for preserving human red blood cells by 1950 [105]. This foundational discovery catalyzed the development of other effective cryoprotectants and significantly advanced biopreservation methods [106].
Unlike NMP, which is designed to maintain organ function by supporting metabolic activity, subzero preservation methods lower temperatures to induce metabolic depression, thereby extending the preservation period [45,107]. A 10 °C reduction in temperature has been shown to decrease metabolic activity by 50%, thus prolonging the preservation time [108]. Cryopreservation offers the possibility of storing biological samples indefinitely, depending on the techniques and conditions used [109]. Nonetheless, the process is intrinsically linked to the formation of ice, which can damage cells through extracellular or intracellular nucleation [110]. Typically, ice formation begins outside the cell, creating a hypertonic environment that draws water out and leads to dehydration (Fig. 3). Rapid cooling, in contrast, may cause intracellular ice formation, which is often fatal due to its potential to disrupt cell membranes and internal structures [111,112].
To mitigate ice-related cellular damage, researchers have developed two primary strategies based on the survival mechanisms of certain animals adapted to frigid climates: ice tolerance and ice avoidance [113,114]. Ice tolerance restricts ice formation to the extracellular space, thereby minimizing cellular dehydration, while ice avoidance aims to prevent ice formation altogether [115]. Both strategies extensively utilize CPAs, substances that protect biological specimens from the adverse effects of freezing and thawing [116]. CPAs are generally classified into two categories: permeable CPAs and nonpermeable CPAs [115]. Permeable CPAs, such as dimethyl sulfoxide, ethylene glycol, glycerol, and propylene glycol, can penetrate cell membranes, thus preventing excessive dehydration and reducing osmotic stress (Fig. 4). Nonpermeable CPAs, including sucrose, polyethylene glycol, glucan, albumin, and polyvinylpyrrolidone, remain outside the cells, minimizing ice formation in the extracellular space and reducing osmotic imbalances [117–119].
Cryopreservation is utilized across a broad spectrum of medical fields, including blood transfusion, regenerative medicine, research, and organ transplantation [120–123]. It is particularly critical in organ preservation, where it facilitates long-distance transport and can extend the timeframe for organ matching and recovery, potentially allowing transplantation to become an elective procedure [124,125]. However, applying cryopreservation to larger, more complex structures like whole organs presents several challenges. One major issue is achieving uniform cooling and rewarming, especially in large tissues where uneven temperatures can cause localized ice formation. Additionally, reducing CPA toxicity and cryoinjury is crucial for maintaining cellular integrity [126–128].
The categorization of subzero preservation techniques is a complex process. These techniques are divided into high subzero storage and low subzero storage based on temperature [129]. Additionally, cryopreservation techniques can be grouped into two categories according to their underlying mechanisms. These categories include ice-avoidance methods, such as supercooling, isochoric preservation, vitrification, and nanowarming, and ice-tolerance methods, such as partial freezing [130]. Fig. 5 illustrates the interrelationship between these two classification systems.
In the field of translational medicine, methods for avoiding ice formation have shown significant progress. These techniques have been successfully scaled up to human organs, and certain animal organs have been preserved for more than 3 months, yielding promising outcomes. The subsequent section offers a detailed analysis of the different preservation techniques (Table 3).
Table 3. Summary of subzero organ preservation methods and outcomes
Study | Type | Organ | Temperature (°C) | Perfusate solution | CPA/vitrification solution | Storage time (day) | Result |
---|---|---|---|---|---|---|---|
Partial freezing | |||||||
Tessier et al. (2022) [133] | Rat | Liver | –10 to –15 | UW, insulin, trehalose, dexamethasone, sodium bicarbonate | Glycerol, antifreeze glycoprotein, polyvinyl alcohol/propylene glycol, X/Z-1000 | 1–5 | • Livers preserved with glycerol showed minimal DNA damage on TUNEL but had the lowest ATP levels, while those preserved with antifreeze glycoprotein and X/Z-1000 had high ATP levels but experienced endothelial cell damage |
Tessier et al. (2022) [135] | Rat | Liver | –10 to –15 | UW | Propylene glycol, glycerol, ethylene glycol, 3-OMG | 1–5 | • Livers preserved for 5 days with partial freezing had better outcomes than controls, despite lower bile and higher enzyme levels • Propylene glycol also outperformed glycerol in preservation |
Supercooling | |||||||
Bruinsma et al. (2015) [152] | Rat | Liver | –6 | UW, insulin, dexamethasone, penicillin | 3-OMG, polyethylene glycol | 2–4 | • 3-month recipient survival was 100% for livers supercooled for 72 hours and 58% for those supercooled for 96 hours, tripling the 24-hour storage limit • However, postoperative recovery took longer, with liver function normalizing within a month |
de Vries et al. (2019) [153] | Human | Liver | –4 | UW, Trolox, insulin, dexamethasone, penicillin | Glycerol, polyethylene glycol, trehalose | 1 | • Livers remain viable post-supercooling with no change in function or stress tolerance during simulated transplantation • Key metrics, including bile levels, blood vessel resistance, oxygen uptake, and apoptotic cell levels, showed no significant differences from pre-supercooled livers |
Isochoric | |||||||
Wan et al. (2018) [154] | Rat | Heart | 0 to –8 | UW | No CPA was used, constant volume was maintained in an isochoric chamber at 0.1–78 MPa | 1 hr | • Hearts at –4 °C (41 MPa) showed similar histological injury to cold storage-preserved hearts but less interstitial edema, indicating better protection against vascular permeability • At –6 °C (60 MPa), heartbeat activity decreased, and at –8 °C (78 MPa), hearts failed to beat |
Botea et al. (2023) [155] | Pig | Liver | –2 | Custodial, normal saline | No CPA was used, constant volume was maintained in an isochoric chamber at –0.02 MPa | 1–2 | • Pig liver was successfully preserved ice-free for 2 days, and postthawing hematoxylin and eosin analysis showed normal appearance • In contrast, livers frozen to –2 °C showed severe tissue disruption after 24 hours |
Vitrification & nanowarming | |||||||
Sharma et al. (2021) [156] | Rat | Kidney | –150 | Euro-Collins | 100% VS55, 10 mg Fe/mL sIONP | NA | • Kidneys were vitrified without ice formation and successfully nanowarmed without devitrification or cracking • Histology, confocal imaging, and viability assessments indicated superior outcomes compared to convective rewarming controls |
Gao et al. (2022) [157] | Rat | Heart | –150 | Euro-Collins | 100% VS55, 10 mg Fe/mL sIONP | NA | • Successfully vitrified hearts were nanowarmed, displaying improved histology and endothelial integrity compared to convective rewarming, along with promising cardiac electrical activity |
Sharma et al. (2023) [158] | Rat | Liver | –150 | Euro-Collins | Ethylene glycol, sucrose, 10 mg Fe/mL sIONP | NA | • Liver retained intact architecture, normal bile ducts, and continuous epithelial layers, with preserved hepatocyte function and homogeneous perfusion • Bile effluent volumes were comparable to the control group |
Han et al. (2023) [159] | Rat | Kidney | –150 | LM5, X-1000, Z-1000 | VMP, 10 mg Fe/mL sIONP | Up to 100 | • Nanowarmed kidneys produced urine within 40–45 minutes following reperfusion • A 30-day posttransplant follow-up showed normal creatinine, urea, and estimated glomerular filtration rate levels • Histology revealed some focal tubular necrosis and hyaline changes, but basement membranes and vasculature remained intact |
CPA, cryoprotective agent; UW, University of Wisconsin solution; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; ATP, adenosine triphosphate; 3-OMG, 3-O-methyl-d-glucose; sIONP, silica-coated iron oxide nanoparticles; NA, not available.
In the extreme cold of nature, where survival is a challenge for humans, the wood frog (
In a recent study, researchers utilized cryopreservation techniques, incorporating CPAs and ice nucleators, to store rat livers in a partially frozen state. Ice nucleators play a crucial role in controlling the freezing process, which significantly reduces the risk of lethal ice formation [134]. This method successfully extended the preservation time by a factor of five. Subsequent analysis after thawing indicated that propylene glycol led to the most favorable outcomes, compared to ethylene glycol and glycerol [135]. Although still in the early stages of development, partial freezing demonstrates significant potential as a method that aligns with natural processes rather than contradicting them.
Supercooling is a technique used in subzero organ preservation that cools biological materials below their freezing point without forming ice crystals [136,137]. This process typically occurs between –4 °C and –6 °C, although temperatures can drop to as low as –20 °C [138]. Supercooling reduces cryoinjury and allows for the use of lower concentrations of CPAs, thereby decreasing toxicity [139]. The method primarily aims to prevent heterogeneous ice nucleation, which necessitates nucleation sites such as impurities. Despite its benefits, supercooling is still susceptible to spontaneous ice crystallization due to stochastic nucleation events [140].
Two primary mechanisms of ice nucleation are observed in supercooling: heterogeneous and homogeneous [141]. Heterogeneous nucleation occurs in the presence of surfaces or particles, whereas homogeneous nucleation results from the spontaneous clustering of water molecules [142]. The main objective of the technique is to inhibit heterogeneous nucleation to broaden the supercooling range [143]. However, maintaining supercooling poses a challenge due to its inherent instability. To stabilize supercooling, researchers have introduced supercooling-promoting substances and have sealed the gas-liquid interface with oil. This approach allows for the storage of water at –20 °C for extended periods without the formation of ice [144,145].
Recent studies have highlighted the potential of supercooling in organ preservation. For example, rat hepatocytes demonstrated improved viability following storage through supercooling [146–148], and this method preserved cortical bone allografts more effectively than conventional techniques [149]. Notably, supercooling also extended the storage time and enhanced posttransplant outcomes in organs such as porcine hindlimbs and murine hearts, compared to traditional preservation at 4 °C [150,151].
Pioneering work by Bruinsma et al. [152] demonstrated that rat livers could survive up to 4 days of supercooling, surpassing the results of HMP. The researchers used CPAs such as 3-O-methyl glucose and polyethylene glycol, along with a multistep perfusion strategy, achieving remarkable outcomes, including a 100% survival rate, posttransplant after 3 days [152]. Extending this technique to human livers, they successfully preserved organs for 27 hours at subzero temperatures by using additional CPAs like glycerol and trehalose, and by gradually introducing these agents to minimize osmotic injury [153].
Innovative supercooling methods, including the application of electric voltage, have shown promising results. Kuro et al. [160] successfully preserved rat femoral arteries at –2 °C using 1,000 V for 3 days, achieving favorable outcomes posttransplant. The use of voltage has also been explored in other organs, demonstrating reduced biomolecular markers and enhanced preservation outcomes compared to conventional cold storage [161]. These methods could potentially eliminate the need for CPAs, positioning supercooling as a viable alternative to existing preservation techniques.
While supercooling effectively prevents heterogeneous ice formation, it cannot inhibit homogeneous nucleation, which occurs around –20 °C [144,162]. Consequently, supercooling is not well-suited for long-term preservation but shows potential for facilitating global organ transport and short-term storage in transplantation settings.
Isochoric preservation is a technique used to protect biological specimens in an ice-free environment by applying pressure [163]. Also known as constant volume preservation, this method involves the use of a pressure chamber to monitor and apply pressure, effectively lowering the freezing point of water [164]. Ice formation causes an expansion that increases the pressure of the liquid inside the chamber, as ice has a larger molar volume than liquid water. This increased pressure further depresses the freezing point of the remaining liquid, allowing the specimen to be stored at lower temperatures without freezing [165,166].
This approach shares similarities with supercooling but distinguishes itself by using pressure instead of CPAs to prevent the formation of ice nuclei, thus avoiding CPA toxicity [167]. Wan et al. [154] found that rat hearts preserved at –4 °C (41 MPa) without CPAs had reduced interstitial edema, indicating better protection against increased vascular permeability compared to SCS. Pig livers were effectively preserved in a nonfreezing state for up to 48 hours in a pressure chamber, and subsequent analysis after warming showed that the tissue architecture remained intact [155]. Additionally, human cardiac tissue was successfully preserved at –3 °C without CPAs, with spontaneous contractility restored upon rewarming [168].
Isochoric preservation may be a highly effective method for organ preservation. However, Ueno et al. [169] demonstrated that hepatocytes experience pressure-dependent degeneration. Conversely, other experiments have shown that higher pressures are necessary to achieve further reductions in temperature [170]. Therefore, additional research is needed to find a balance between sustainable pressure levels and optimal temperature conditions.
Vitrification is a preservation technique in which biological samples are rapidly cooled, bypassing the crystallization phase to enter a glass-like, amorphous state [171]. In this state, the sample becomes highly viscous and molecular motion is nearly halted, effectively mimicking the properties of a solid [172]. Two critical parameters ensure successful vitrification: the critical cooling rate (CCR), which prevents ice formation during cooling, and the critical warming rate (CWR), which avoids devitrification during warming [173]. For water, the CCR is exceptionally high (greater than 107 °C/min), posing a challenge for biological specimens due to the practical limitations of achieving such rates [108]. To address this, CPAs are used to lower the CCR and help reach the glass transition temperature [174]. However, high concentrations of CPAs can be toxic, leading researchers to develop CPA cocktails that combine agents with different ice-inhibiting mechanisms to reduce toxicity [115].
Vitrification enables the long-term preservation of organs in liquid nitrogen by halting metabolic activity at extremely low temperatures. The foundational studies by Rall and Fahy [175] set the stage for this field, and Rall's [176] subsequent research marked a significant advancement, successfully vitrifying mouse embryos with an 80% survival rate upon rewarming. Since then, the scope of vitrification has broadened to include applications in reproductive medicine and organ preservation [177–180]. For example, rat livers have been vitrified and demonstrated preserved tissue architecture after thawing [158], and small animal hearts and kidneys have also been successfully vitrified [156,181]. A notable achievement by Fahy’s team was the successful vitrification of a rabbit kidney, which functioned for 48 days posttransplant, despite challenges such as persistently elevated creatinine levels and lethargy [182]. This highlights the potential of vitrification for organ preservation.
The primary challenge in vitrification is the rewarming process, which necessitates precise control to prevent devitrification and ensure uniform heating [183]. Early methods, such as water baths, were ineffective, as they could not meet the required CWR and often induced thermal stress due to uneven heating. Additionally, the high CPA concentrations needed for both cooling and warming often led to cytotoxicity [184]. These issues have led researchers to investigate alternative rewarming techniques, including the use of radiation [185,186]. Electromagnetic fields, for instance, can provide a more uniform heat distribution [187]. Research in this field, including studies on microwave heating, has demonstrated potential [188–190]. For instance, a study that involved rewarming dog kidneys frozen at –80 °C with microwaves reported a 50% survival rate over a period ranging from 2 to 14 months, though challenges related to organ shape and material properties persist [191].
Despite these advances, vitrification still faces technical challenges, particularly when applied to complex and irregularly shaped organs [156]. Achieving uniform rewarming and minimizing CPA toxicity remain key areas of ongoing research. As technology advances, vitrification shows promise in transforming organ preservation, potentially allowing for long-term storage and alleviating the time constraints that currently limit transplantation logistics.
Nanowarming is a novel rewarming technology that employs radiofrequency (RF) coils to generate an alternating electromagnetic field, thereby causing preloaded nanomagnetic particles within the organ to oscillate [192]. The oscillation produces heat, ensuring the organ warms uniformly and reducing the thermomechanical stress that typically accompanies nonuniform warming. Additionally, this method reduces the risk of devitrification, as the frequency and strength of the field can be adjusted to surpass the CWR required by the organ [193]. The nanoparticles used in this process are made of elements with magnetic properties, such as iron, and are thus known as iron oxide nanoparticles (IONPs) [194]. Their colloidally stable nature facilitates efficient loading into the organ and subsequent removal through machine perfusion [195].
The successful nanowarming of tissues [196] and small animal organs [157,197] has yielded encouraging results. In a recent study, Han et al. [159] successfully transplanted a nanowarmed, vitrified rat kidney that had been preserved for 100 days. Before vitrification, the kidney was infused with 10 mg Fe/mL IONPs and then uniformly rewarmed using a 15 kW RF coil. After rewarming, the nanoparticles were removed through NMP. Following transplantation, the rats demonstrated adequate urine output and creatinine levels comparable to those of the control group, with no significant histological changes [159]. In comparison to the previously discussed vitrified rabbit kidney, the posttransplant outcomes were superior [182]. Given that vitrification is an isovolumetric process [108], it is theoretically scalable to human organs. Additionally, nanowarming technology is engineered to uniformly rewarm organs regardless of size. Successful integration of this system could significantly advance the long-sought goal of organ banking.
Subzero preservation methods are continuously evolving, and researchers are striving to make a clinical impact, yet significant advancements are still necessary. Currently, supercooling is at the forefront, having successfully preserved human livers. The primary advantage of supercooling is that it necessitates only minor modifications to existing hypothermic equipment, such as adding a chiller with an independent cooling system [152]. Additionally, the use of lower concentrations of CPAs diminishes the risk of CPA toxicity, a common issue with vitrification. However, supercooling presents challenges; it is unstable and requires meticulous monitoring to avoid lethal ice formation. While partial freezing is a strong contender, other methods lag significantly behind. Table 4 offers a detailed comparison of the advantages, disadvantages, and potential applications of each cryopreservation method.
Table 4. Comprehensive overview of cryopreservation methods: advantages, disadvantages, and potential indications for organ preservation
Cryopreservation method | Advantages | Disadvantages | Potential indications |
---|---|---|---|
Partial freezing | • Allows controlled ice formation to avoid lethal cellular damage • Uses natural ice tolerance strategies • Low cryoprotective agent toxicity • Can be applied using currently available technology | • Risk of cellular dehydration and osmotic stress if not monitored correctly properly • Not ideal for organs with complex vascular structures | • For short-term organ storage • Useful for experimental studies on the effects of ice on organ preservation |
Supercooling | • Minimizes structural damage by avoiding ice and reduces the risk of ice formation from impurities • Low cryoprotective agent toxicity • Extends storage time compared to cold storage without additional sophisticated equipment • Promising results have been shown in human organ research | • Prone to spontaneous ice formation • Limited temperature range (–4 °C to –20 °C) • Stability issues require constant monitoring • Not suitable for long-term preservation | • Short-term organ storage • Transport organs over moderate distances (intracontinental) • This method has the potential to enable organ cryopreservation without the need for cryoprotective agents |
Isochoric preservation | • Utilizes pressure rather than cryoprotective agents, reducing toxicity risks • Effective at maintaining ice-free conditions • May be scalable with appropriate pressure control | • Specialized equipment is needed for constant pressure maintenance • High-pressure requirements limit scalability to larger organs | • This method has the potential to enable organ cryopreservation without the need for cryoprotective agents |
Vitrification & nanowarming | • Bypasses ice formation • Enables long-term storage • Nanowarming provides uniform, rapid rewarming, reducing devitrification risk • Supports preservation of complex organs with intricate structures • Can be scaled to human-size organs | • High cryoprotective agent concentrations needed, risking cytotoxicity • Requires loading of magnetic nanoparticles for nanowarming • Specialized equipment with significant energy requirements • Requires precise temperature control during both cooling and warming | • Long-term organ storage for potential organ banking • Transport organs over long distances (intercontinental) • Ideal for large organs and tissues needing uniform rewarming |
Despite being one of the most significant advancements in healthcare over the past century, organ transplantation still fails to meet the needs of a considerable number of individuals. In 2022, the World Health Organization reported that only 10% of the global demand for organ transplants was met, highlighting the urgent need for advanced preservation strategies to improve transplant outcomes and increase the donor pool.
Recent advancements in organ preservation techniques, particularly in machine perfusion, have significantly advanced the field. Currently, the trend in organ preservation involves maintaining the organ close to its physiological temperature and supplying oxygen during perfusion, as illustrated in Fig. 1. Machine perfusion offers notable advantages over traditional SCS. HMP has now become the gold standard for preserving high-risk kidneys. It reduces IRI by maintaining cellular metabolism at lower temperatures and facilitating the removal of waste products. Additionally, the benefits of HOPE include rejuvenating mitochondrial function and minimizing oxidative stress, which together optimize preservation.
NMP provides the benefit of preserving organs at body temperature, which allows for real-time evaluation of organ function and viability. This method could potentially recondition organs from ECD or DCD, potentially reducing discard rates and expanding the donor pool. While more complex and expensive than SCS, NMP has shown significant benefits in liver transplantation, such as improved early graft function and decreased discard rates.
SNMP provides a balanced solution that reduces metabolic rates while maintaining cellular activity and repair mechanisms. This method streamlines the preservation process, extends preservation times, and enhances organ viability. By integrating COR into preservation protocols, it addresses temperature-induced mitochondrial dysfunction, thus minimizing IRI and improving posttransplant outcomes.
While the necessity for preservation techniques in living donors remains a topic of debate [198], there is some evidence supporting the potential benefits of machine perfusion in this context. Machine perfusion has demonstrated promising results, including improved preservation quality and enhanced transplant success rates. However, this approach is still under-researched, especially for living donors [199]. The current literature is limited, with much of the existing research focusing on organs from deceased donors. Consequently, a more comprehensive investigation into the applications and long-term effects of machine perfusion for living donor organs is crucial to fully realize its potential and optimize organ preservation methods.
Despite current limitations, such as ice formation and cellular damage, cryopreservation holds considerable promise for long-term organ storage. If cryopreservation techniques are successfully implemented, they could help mitigate the organ shortage crisis by allowing organs to be stored indefinitely and enabling more equitable distribution across various regions and over time.
Notwithstanding these advances, several challenges remain. Many conservative societies continue to overlook basic innovations like machine perfusion, often due to financial constraints, limited awareness, and a lack of respect for organ donation [200]. To overcome these obstacles, it is crucial for researchers, clinicians, industry stakeholders, and healthcare administrators to engage in enhanced collaboration.
This review has certain limitations, notably the omission of particular solid organs and vascularized composite allografts. Future research should aim to fill these gaps, thereby offering a more thorough understanding of the advancements and challenges in organ preservation. It is crucial to continue innovating and researching to broaden the donor pool, enhance transplant success rates, and ultimately save more lives.
Conflict of Interest
Kyeong Sik Kim is an associate editor, and Dongho Choi is the editor-in-chief of the journal. They were not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflict of interest relevant to this article was reported.
Funding/Support
This study was supported by the National Research Foundation of Korea (NRF) grants funded by the Ministry of Science and ICT (MSIT) of the Korean government. Numbers (2022R1A2C2004593, 2023R1A2C1005279).