Research Report: A Paradigm Shift in Organ Preservation: How Suppressing Thermomechanical Stress Revolutionizes Viability and Cellular Integrity in Cryopreservation

Executive Summary

This report synthesizes extensive research into the field of vascularized organ cryopreservation, addressing how novel methods for suppressing thermal stress fractures fundamentally alter organ viability and post-thaw integrity compared to traditional vitrification. The findings indicate a paradigm shift from a strategy fraught with insurmountable physical and biological barriers to a viable, multi-disciplinary approach with the potential to revolutionize transplantation medicine.

Traditional vitrification, which aims to prevent ice formation by creating a glassy state, has historically failed for large organs due to a triad of interrelated challenges: the severe chemical toxicity and osmotic stress induced by high concentrations of cryoprotective agents (CPAs); chilling injury that triggers cell death pathways even without ice; and, most critically, catastrophic thermomechanical stress. The inability to uniformly cool and, especially, rewarm a large, complex organ leads to massive thermal gradients, causing differential expansion and contraction that result in fractures, destroying the delicate vascular architecture.

Novel methods directly and successfully confront these failure points. The most significant breakthrough is volumetric nanowarming, a technology that uses biocompatible iron oxide nanoparticles perfused into an organ's vasculature. When activated by an external radiofrequency field, these nanoparticles generate rapid, uniform heat throughout the entire organ volume simultaneously. This eliminates the thermal gradients responsible for stress fractures and allows for rewarming rates that far exceed the critical threshold needed to prevent lethal ice recrystallization (devitrification). This technique has been successfully scaled to porcine liver-sized volumes, demonstrating a clear path to human application.

The impact of these advancements is transformative and quantifiable:

1. Fundamentally Altered Viability Window: The potential organ storage window has been extended from the 4-24 hours of conventional hypothermic storage to demonstrated periods of months. Vitrified rat kidneys have been successfully stored for 100 days before being transplanted, where they provided life-sustaining function. Alternative methods like supercooling have extended viable rat liver storage to 96 hours with 100% post-transplant survival for 3-day stored organs, a dramatic improvement over the 0% survival seen with traditional cold storage over the same period. This extension makes the concept of long-term organ banking a tangible reality, with profound implications for logistics, donor-recipient matching, and global organ sharing.

2. Profoundly Enhanced Post-Thaw Cellular Integrity: By preventing macroscopic fractures and microscopic ice crystal damage, novel methods preserve the crucial structural integrity of the organ, particularly the vascular endothelium. Beyond structural preservation, these techniques incorporate a sophisticated biological approach. They utilize advanced CPA cocktails and molecular agents to mitigate CPA toxicity and actively suppress post-thaw cellular self-destruction cascades, such as apoptosis and necrosis (Delayed-Onset Cell Death). This includes protecting mitochondrial function, maintaining cellular energy (ATP) levels, and preserving critical endothelial barrier function. The result is not just a structurally intact organ, but a functionally robust one, as evidenced by restored blood vessel contractility, bile production in thawed livers, and even reinnervation in cryopreserved rat limbs.

In conclusion, the suppression of thermal stress fractures is not an incremental improvement but the pivotal innovation that unlocks the potential of cryopreservation for vascularized organs. By solving this core physical problem, a new frontier of biological optimization has opened, shifting the field from damage mitigation to functional restoration. This fundamentally alters the landscape of organ preservation, paving the way for the establishment of organ banks and a new era in transplant medicine.

Introduction

The ability to preserve viable human organs for extended periods remains one of the most significant challenges in modern medicine. Current clinical standards, primarily based on hypothermic (cold) storage, limit the viability of organs to a matter of hours—typically 4-6 for a heart and 12-24 for a kidney. This narrow window imposes severe logistical constraints, creating a frantic race against time that limits donor-recipient matching, prevents transportation over long distances, and results in the tragic discard of thousands of viable organs annually.

For decades, cryopreservation via vitrification has been heralded as the theoretical solution. By cooling biological systems to a glass-like, amorphous state at cryogenic temperatures (typically below -120°C), all metabolic and decay processes are effectively halted, promising a theoretically indefinite storage duration. While vitrification has been highly successful for small samples like embryos and oocytes, its application to large, complex, vascularized organs has been an almost universal failure.

The central obstacle has been the immense physical and biological stress imposed by the process. Traditional vitrification requires high concentrations of cryoprotective agents (CPAs) that are often toxic, and it demands ultra-rapid cooling and warming rates that are physically impossible to achieve uniformly across a large, thermally resistive mass like a kidney or liver. This non-uniform temperature change generates catastrophic thermomechanical stress, leading to fractures that shatter the organ's delicate architecture. This report addresses the central research query: How does the novel method of suppressing thermal stress fractures during cryopreservation fundamentally alter the viability window and post-thaw cellular integrity of vascularized organs compared to traditional vitrification techniques?

This comprehensive analysis synthesizes findings from extensive research to detail the paradigm shift occurring in the field. It first deconstructs the multifaceted failure mechanisms of traditional vitrification, then provides a detailed examination of the novel technologies—particularly volumetric nanowarming—that directly counteract these failures. Finally, it presents the quantifiable evidence demonstrating how these breakthroughs are fundamentally redefining the possibilities for organ viability and cellular integrity, moving the long-held dream of organ banking from theoretical concept to practical reality.

Key Findings

The research reveals a clear and profound distinction between the limitations of traditional vitrification and the successes of novel cryopreservation strategies. The findings are organized around the core challenges of traditional methods, the innovative solutions now being deployed, and the transformative impact on organ viability and health.

1. The Inherent and Catastrophic Failures of Traditional Vitrification

• Thermomechanical Stress is the Primary Barrier: The most significant and destructive failure point for large-scale vitrification is the generation of thermal stress fractures. These arise from differential thermal expansion and contraction during non-uniform cooling and, more critically, rewarming. This leads to catastrophic mechanical damage, severing blood vessels and destroying tissue architecture.
• Interrelated Stresses Create a Cascade of Injury: Traditional methods are constrained by a triad of interconnected challenges:
  • Chilling Injury: Damage, including the triggering of apoptosis, occurs from cooling itself, even without ice formation.
  • CPA Toxicity & Osmotic Stress: The high CPA concentrations needed to prevent ice are cytotoxic. They disrupt membranes, denature proteins, and cause extreme osmotic stress (the "shrink-swell" cycle) during perfusion and washout, leading to mechanical membrane damage and cell lysis.
  • Size-Dependent Perfusion & Thermal Control Failure: The sheer size and low thermal conductivity of organs make it physically impossible to achieve the uniform, rapid cooling and warming rates required. This exacerbates thermal stress and leads to a destructive heterogeneity where some regions suffer from ice damage due to insufficient CPA, while others are subjected to severe toxicity.
• Devitrification as a Consequence of Failure: Inadequate rewarming rates—a certainty in large organs using conventional surface heating—lead to devitrification, where the glassy matrix destabilizes and crystallizes. This secondary ice formation, often initiated at fracture sites, is lethal to cells and negates the primary purpose of vitrification.

2. Novel Fracture-Suppression Mechanisms Revolutionize the Process

• Volumetric Nanowarming Overcomes the Rewarming Barrier: The introduction of iron oxide nanoparticles into an organ's vasculature enables rapid, uniform heating via an external alternating magnetic field. This technology, known as nanowarming, directly eliminates the severe temperature gradients that cause thermal stress fractures. Rewarming rates exceeding 130°C/minute have been achieved in significant volumes, a feat impossible with conventional methods.
• A Multi-faceted Approach to Stress Mitigation: The success of novel methods stems from a combination of advanced techniques:
  • Optimized Thermal Protocols: Controlled, slower warming in specific low-temperature ranges (e.g., -180°C to -100°C), followed by rapid thawing, has been shown to reduce fracture rates. Annealing protocols (thermal cycling around the glass transition temperature) can also relax accumulated stresses.
  • Advanced Vitrification Solutions: CPA cocktails are being re-engineered to have higher glass transition temperatures (Tg), which inherently reduces the propensity for cracking. Methods like isochoric (constant-volume) vitrification leverage high pressure to allow for the use of lower, less toxic CPA concentrations.
  • Environmental Alterations: Techniques like "cryopreservation in air" (freezing without submersion in a liquid CPA medium) have demonstrated a substantial reduction in external thermal stress generation.

3. A Fundamental Alteration of the Viability Window: From Hours to Months

• Quantitative Extension of Viable Storage: Novel cryopreservation strategies have demonstrably extended viable storage times from the hours offered by cold storage to periods exceeding three months.
  • Long-Term Vitrification: Rat kidneys have been successfully cryopreserved for 100 days at -150°C using vitrification and nanowarming, followed by successful transplantation and life-sustaining function.
  • Medium-Term Supercooling: Rat livers have been preserved via supercooling at -6°C for up to 96 hours. Transplanted organs from 3-day storage showed a 100% 30-day survival rate, compared to 0% for livers in traditional cold storage for the same duration.

Preservation Method	Organ/Tissue	Demonstrated Viable Storage Duration	Post-Transplant Outcome
Static Cold Storage (Clinical Standard)	Kidney, Liver, Heart	4 - 24 Hours	Standard clinical success, declines with time
Traditional Vitrification	Vascularized Organs	Effectively 0 (failure due to fractures)	Uniform failure
Novel Vitrification + Nanowarming	Rat Kidney	100 Days	Life-sustaining function
Novel Supercooling Protocol	Rat Liver	96 Hours (4 Days)	58% survival; 100% survival for 3-day storage

4. Unprecedented Improvement in Post-Thaw Cellular and Functional Integrity

• Preservation of Structural and Vascular Integrity: By preventing macroscopic fractures and subsequent devitrification, the intricate architecture of the organ—from major vessels down to delicate capillary networks—is maintained. This is critical for successful reperfusion post-transplant.
• Mitigation of Delayed-Onset Cell Death (DOCD): Advanced protocols actively suppress post-thaw apoptosis and necrosis. This is achieved using cryoprotective solutions and targeted molecular agents, including caspase inhibitors, necroptosis inhibitors (necrostatin-1), and antioxidants.
• Maintenance of Mitochondrial Function and Bioenergetics: Novel approaches prioritize the health of mitochondria, the cellular powerhouses. Success is marked by the preservation of mitochondrial membrane potential, sustained ATP content, and reduced production of Reactive Oxygen Species (ROS), preventing the activation of cell death pathways.
• High Levels of Functional Recovery: The ultimate measure of success is function. Novel methods have demonstrated remarkable post-thaw functional recovery:
  • Tissue Contractility: Vitrified and nanowarmed tissue-engineered blood vessels retained over 82.7% of their contractility, compared to just 10.7% in frozen samples.
  • Metabolic Function: Nanowarmed rat livers maintained normal tissue architecture and demonstrated key functions like bile production.
  • Complex Tissue Regeneration: Cryopreserved rat hind limbs showed long-term survival (>30 days) and complex functional recovery, including hair regrowth, normal blood flow, and even reinnervation.

Detailed Analysis

The Pathophysiology of Cryo-Injury: Deconstructing Traditional Vitrification's Failure

The failure of traditional vitrification for vascularized organs is not a singular event but a cascade of biophysical and biochemical insults. Understanding this cascade is essential to appreciate the targeted nature of novel solutions.

1. The Chemical and Osmotic Assault of Cryoprotective Agents (CPAs) To achieve an ice-free glassy state, traditional vitrification requires extremely high molar concentrations of CPAs like dimethyl sulfoxide (DMSO) and ethylene glycol. This chemical necessity introduces severe biological consequences.

• Direct Cytotoxicity: At these concentrations, CPAs are potent chemical disruptors. They interfere with the delicate hydrogen bonding of water that stabilizes biological macromolecules, leading to the denaturation of proteins and enzymes. They can intercalate into lipid bilayers, altering membrane fluidity, permeability, and the function of crucial receptors and transport channels. This direct biochemical toxicity compromises cellular signaling, transport, and structural integrity.
• Osmotic Stress and Mechanical Damage: The process of perfusing an organ with high-concentration CPAs and subsequently washing them out subjects cells to extreme osmotic gradients. During CPA loading, water rapidly exits the cell, causing it to shrink dramatically. During washout, the reverse occurs, leading to rapid swelling that can exceed the membrane's elastic limit. This violent "shrink-swell" cycle exerts immense mechanical stress on the cell membrane and cytoskeleton, often causing irreversible damage, membrane blebbing, or outright lysis. This damage occurs before the organ is even cooled, fundamentally compromising its baseline health.

2. The Physics of Failure: Thermomechanical Stress and Devitrification The most dramatic and insurmountable challenge for traditional methods is managing heat transfer across a large biological mass. The laws of thermodynamics dictate that heating or cooling an object from its surface creates thermal gradients—the surface temperature changes far more rapidly than the core. In organ vitrification, this physical reality is catastrophic.

• Genesis of Thermal Stress Fractures: As an organ is cooled or rewarmed, different regions expand or contract at different rates due to these temperature gradients. This disparity generates immense internal tension. Once this tension exceeds the material strength of the vitrified (glassy) tissue, it shatters. This is not a microscopic event; it results in macroscopic fractures that can cleave the organ, severing major blood vessels and destroying the parenchymal architecture. The rewarming phase is particularly dangerous, as the material properties of the tissue change rapidly around the glass transition temperature, making it highly susceptible to cracking.
• The Rewarming Problem and Devitrification: Successful vitrification requires not just a critical cooling rate but also a critical warming rate to avoid devitrification—the process of ice crystallization from the glassy state. For any sample larger than a few milliliters, achieving this critical warming rate via external conduction or convection is physically impossible. The slow, non-uniform warming creates a prolonged window where the vitrified matrix is unstable, allowing lethal ice crystals to nucleate and grow. Fractures exacerbate this problem, acting as nucleation points for widespread devitrification. This secondary ice formation perforates cell membranes and destroys organelles, causing the very damage vitrification was designed to prevent.

3. Systemic Failure at the Organ Level These challenges are magnified in a complex, vascularized organ. The intricate network of blood vessels presents a formidable challenge for achieving uniform CPA perfusion. Inevitably, this leads to significant mass transport heterogeneity: regions near major vessels may receive toxic levels of CPA, while distal capillary beds receive insufficient protection and are vulnerable to freezing. This patchwork of fatally injured tissue renders the entire organ non-viable. The vascular endothelium, being the first point of contact with the CPA and critical for post-transplant reperfusion, is especially vulnerable and often catastrophically damaged.

Technological Breakthroughs: A Targeted Assault on Cryo-Injury

Novel methods succeed because they systematically dismantle the failure cascade of traditional vitrification. They represent a multi-disciplinary approach that integrates physics, chemistry, and molecular biology.

1. Revolutionizing Rewarming: Volumetric Nanowarming The most impactful innovation is the development of volumetric nanowarming. This technology directly solves the rewarming problem, the primary physical barrier to scaling up vitrification.

• Mechanism: The organ's vascular system is perfused with a solution containing biocompatible silica-coated iron oxide nanoparticles (sIONPs). After the organ is vitrified, it is placed within an alternating, high-frequency magnetic field. The nanoparticles couple with the field and act as millions of microscopic heat sources, rapidly and uniformly raising the temperature throughout the entire organ volume simultaneously.
• Significance: This approach decouples the rewarming rate from the tissue's poor thermal conductivity. By heating the core and surface at the same rate, it eliminates the thermal gradients that cause stress fractures. Furthermore, the achievable rewarming rates (e.g., >100 °C/min) are extremely high, allowing the organ to pass through the dangerous devitrification temperature range so quickly that ice crystals do not have time to form. This single technology neutralizes the two most destructive physical forces in traditional cryopreservation: thermomechanical stress and devitrification. Its demonstrated success in rewarming volumes up to 3 liters and a ~1-liter porcine liver confirms its scalability for human organs.

2. A Systems-Level Approach to Cryoprotection Beyond rewarming, novel strategies employ a more sophisticated approach to protecting the organ at every stage.

• Isochoric Vitrification: This technique performs vitrification in a constant-volume chamber. As the solution cools and tries to expand, pressure builds up (up to hundreds of atmospheres). This pressure itself suppresses ice formation, which allows for the use of significantly lower, less toxic CPA concentrations. This reduces both chemical and osmotic injury, leading to healthier cells post-thaw.
• Biological Intervention and Functional Rescue: The new paradigm recognizes that preventing physical damage is necessary but not sufficient. It incorporates a biological rescue strategy. Advanced cryopreservation solutions (e.g., CryoStor™) are supplemented with a cocktail of molecular agents designed to combat the cellular stress responses triggered by the process.
  • Inhibition of Delayed-Onset Cell Death (DOCD): Pan-caspase inhibitors like Z-VAD-FMK are used to block the execution pathways of apoptosis. Necrostatin-1 is used to inhibit programmed necrosis (necroptosis).
  • Preservation of Mitochondrial Health: Additives like mitochondria-targeted antioxidants and inhibitors of the mitochondrial permeability transition pore (mPTP) are used to maintain mitochondrial membrane potential (ΔΨm). This preserves the cell's ability to generate ATP, providing the energy needed for post-thaw repair and function, and prevents the release of pro-apoptotic factors like cytochrome c.
  • Protection of the Endothelium: The entire protocol, from slower, optimized cooling/warming profiles to less toxic CPA cocktails, is designed to protect the fragile endothelial lining. This is crucial for preventing the edema, inflammation, and thrombosis associated with ischemia-reperfusion injury upon transplantation.

The Quantifiable Impact: A New Reality for Organ Viability

The success of these targeted interventions is not theoretical but is demonstrated through quantifiable and dramatic improvements in organ viability and function.

• The Viability Window: From Logistical Constraint to Strategic Asset: The demonstrated 100-day storage of a life-sustaining rat kidney represents a fundamental transformation. It proves that stable, long-term storage is possible, moving the field from the short-term challenge of transportation logistics to the long-term potential of true organ banking. Similarly, the 96-hour preservation of rat livers via supercooling provides an immediate, clinically-relevant extension that could dramatically improve organ allocation and transplant outcomes today. This shift transforms the viability window from a narrow, high-risk constraint to a broad, reliable asset.
• Post-Thaw Integrity: From Survival to Full Functional Restoration: The endpoint of success has shifted from mere cell survival to comprehensive functional recovery. The preservation of over 82% of contractility in blood vessels shows that the delicate cellular machinery remains intact. The demonstration of metabolic function (bile production) in thawed livers and, most strikingly, the reinnervation and hair growth in a cryopreserved rat limb, indicates a profound level of preserved cellular and systems-level integrity that was previously unimaginable. This high-fidelity preservation is the direct result of mitigating the entire cascade of cryo-injury—from macroscopic fractures down to mitochondrial dysfunction.

Discussion

The synthesis of these research findings illuminates a fundamental shift in the science of organ cryopreservation. The transition is away from a brute-force, chemically-driven attempt to prevent ice at all costs, and towards a sophisticated, systems-level strategy that integrates physics, chemistry, and biology to manage the entire spectrum of cryo-injury.

The suppression of thermal stress fractures stands out as the linchpin of this new paradigm. By solving this core physical problem with technologies like nanowarming, researchers have unlocked the ability to address the more subtle but equally lethal biological challenges. In traditional vitrification, the certainty of catastrophic mechanical failure during rewarming rendered any effort to mitigate CPA toxicity or apoptosis almost moot. With the threat of fractures removed, optimizing the biological resilience of the organ becomes a meaningful and achievable goal. This explains the concurrent rise of research into DOCD inhibitors and mitochondrial protectants; these interventions are only valuable if the macroscopic structure of the organ can be reliably preserved.

This represents a change in the definition of the primary obstacle. The problem is no longer simply "ice," but the holistic management of thermal stress, chemical toxicity, osmotic shock, and post-thaw biological self-destruction cascades. Novel methods do not treat these as independent issues but as an interconnected web of injury. Isochoric vitrification reduces the need for toxic CPAs, which in turn lessens the osmotic stress and the baseline biochemical injury, making the cells more resilient to the stresses of cooling and warming. Nanowarming prevents the fractures that would otherwise trigger devitrification and compromise the vascular network needed to deliver post-thaw recovery solutions.

Consequently, the outcomes are not just incrementally better; they are qualitatively and quantitatively different. The alteration of the viability window is not a minor extension but a leap of orders of magnitude—from hours to months. This is a change that fundamentally restructures the entire field of transplant medicine, enabling the creation of organ banks, facilitating elective rather than emergency surgeries, and allowing for meticulous immunological matching that could reduce rejection rates and improve long-term graft survival.

Similarly, the improvement in post-thaw cellular integrity moves beyond basic metrics of viability (e.g., membrane integrity) to encompass high-level biological function. The preservation of endothelial barrier function, mitochondrial energy production, and complex tissue regeneration signifies the difference between an organ that is merely "not dead" and one that is "ready for function." This enhanced integrity is the prerequisite for successful transplantation and long-term graft success. The novel approach delivers an organ that is not just structurally intact but is functionally primed for recovery.

Conclusions

The novel methods for suppressing thermal stress fractures during the cryopreservation of vascularized organs represent a historic breakthrough that fundamentally alters the potential for long-term organ banking and transplantation. By directly addressing the catastrophic failure points of traditional vitrification, these new technologies have transformed a field long defined by its limitations into one characterized by quantifiable success and immense future potential.

1. Fundamental Alteration of the Viability Window: The suppression of thermomechanical stress, primarily through volumetric nanowarming, has shattered the long-standing time constraints of organ preservation. By eliminating the rewarming fractures that plagued traditional methods, it has unlocked the potential for stable, long-term storage. The demonstrated success of preserving a rat kidney for 100 days moves the viability window from a scale of hours to one of months, or potentially years. This is a fundamental change that makes the establishment of global organ banks, with all their attendant benefits for logistics and patient outcomes, a realistic and achievable goal.

2. Fundamental Alteration of Post-Thaw Cellular Integrity: The new paradigm shifts the focus from simple structural survival to comprehensive functional restoration. By preventing macroscopic fractures and microscopic devitrification, the crucial architecture of the organ's vascular network is preserved. Crucially, this physical protection is now coupled with a sophisticated biological strategy that reduces CPA toxicity and actively suppresses post-thaw cell death pathways while preserving mitochondrial function. The result is a post-thaw organ with vastly superior cellular integrity, evidenced by high levels of restored metabolic function, tissue contractility, and even complex regenerative processes like reinnervation.

In conclusion, the research demonstrates a definitive and positive answer to the core query. The systematic suppression of thermal stress is not merely an incremental improvement; it is the enabling technology that makes the successful vitrification of whole vascularized organs possible for the first time. This breakthrough directly leads to a massively expanded viability window and the preservation of a high degree of post-thaw cellular and functional integrity, heralding a potential revolution in transplantation medicine and regenerative biology.

References

Total unique sources: 139

ID	Source	ID	Source	ID	Source
[1]	frontiersin.org	[2]	news-medical.net	[3]	scitechdaily.com
[4]	nih.gov	[5]	nih.gov	[6]	nih.gov
[7]	nih.gov	[8]	nih.gov	[9]	nih.gov
[10]	tamu.edu	[11]	researchgate.net	[12]	nih.gov
[13]	nih.gov	[14]	frontierspartnerships.org	[15]	frontiersin.org
[16]	nih.gov	[17]	sci-hub.se	[18]	nih.gov
[19]	researchgate.net	[20]	nih.gov	[21]	nih.gov
[22]	umn.edu	[23]	researchgate.net	[24]	semanticscholar.org
[25]	nih.gov	[26]	nih.gov	[27]	cryonicsarchive.org
[28]	nih.gov	[29]	nih.gov	[30]	nih.gov
[31]	nih.gov	[32]	nih.gov	[33]	news-medical.net
[34]	tamu.edu	[35]	nih.gov	[36]	news-medical.net
[37]	frontiersin.org	[38]	nih.gov	[39]	nih.gov
[40]	nih.gov	[41]	nih.gov	[42]	nih.gov
[43]	nih.gov	[44]	researchgate.net	[45]	news-medical.net
[46]	tamu.edu	[47]	nih.gov	[48]	researchgate.net
[49]	researchgate.net	[50]	semanticscholar.org	[51]	nih.gov
[52]	nih.gov	[53]	nih.gov	[54]	nih.gov
[55]	nih.gov	[56]	nih.gov	[57]	nih.gov
[58]	nih.gov	[59]	umn.edu	[60]	nih.gov
[61]	nih.gov	[62]	researchgate.net	[63]	nih.gov
[64]	nih.gov	[65]	nih.gov	[66]	nih.gov
[67]	nih.gov	[68]	nih.gov	[69]	frontiersin.org
[70]	nih.gov	[71]	researchgate.net	[72]	blade.com
[73]	biolifesolutions.com	[74]	nih.gov	[75]	nih.gov
[76]	mdpi.com	[77]	nih.gov	[78]	escholarship.org
[79]	dntb.gov.ua	[80]	scilit.com	[81]	nih.gov
[82]	nih.gov	[83]	nih.gov	[84]	engineering.org.cn
[85]	nih.gov	[86]	creative-bioarray.com	[87]	nih.gov
[88]	nih.gov	[89]	nih.gov	[90]	arvojournals.org
[91]	nih.gov	[92]	nih.gov	[93]	mdpi.com
[94]	nih.gov	[95]	nih.gov	[96]	jyi.org
[97]	nih.gov	[98]	nih.gov	[99]	nih.gov
[100]	researchgate.net	[101]	imrpress.com	[102]	nih.gov
[103]	mdpi.com	[104]	nih.gov	[105]	researchgate.net
[106]	nih.gov	[107]	nih.gov	[108]	nih.gov
[109]	nih.gov	[110]	researchgate.net	[111]	nih.gov
[112]	nih.gov	[113]	nih.gov	[114]	nih.gov
[115]	taylorfrancis.com	[116]	nih.gov	[117]	nih.gov
[118]	nih.gov	[119]	scitube.io	[120]	cmu.edu
[121]	nih.gov	[122]	nih.gov	[123]	scitechdaily.com
[124]	nih.gov	[125]	nih.gov	[126]	nih.gov
[127]	nih.gov	[128]	nih.gov	[129]	nih.gov
[130]	nih.gov	[131]	bohrium.com	[132]	nsf.gov
[133]	researchgate.net	[134]	researchgate.net	[135]	nih.gov
[136]	researchgate.net	[137]	researchgate.net	[138]	nih.gov
[139]	nih.gov