As the field of regenerative medicine advances, biologic therapies continue to diversify in composition and application. At Free to Feed and Nova Vita Labs, we specialize in acellular amniotic fluid-derived products that contain exosomes, signaling proteins, hyaluronic acid (HA), and other powerful regenerative biomolecules. One of the most frequent questions we receive is: "Why do you store your product at -80°C?"
The answer lies in both scientific evidence and our product's unique composition. We’ve conducted comprehensive internal studies to assess shelf stability and bioactivity under various storage conditions—and -80°C emerged as the clear winner.
What Makes Our Product Different?
Unlike cellular therapies that depend on the preservation of viable cells, our product is acellular. This means it does not contain live cells that require ultra-cryopreservation. Instead, it is rich in:
- Exosomes
- Signaling proteins (e.g., cytokines, growth factors)
- Hyaluronic acid (HA)
- Nucleic acid fragments
- Other regenerative biomolecules
This acellular nature provides flexibility in storage, but careful temperature control is still essential to preserve the structural integrity and bioactivity of these components.
Comparing Storage Methods for Biologics
There are several common strategies used to store exosome- and protein-rich biologics. Each comes with its own pros and cons, depending on the product’s formulation and clinical goals.
Cryopreservation (-196°C / Liquid Nitrogen)
Cryopreservation is the standard for cell-based therapies, preserving cellular viability and function long-term. However, for acellular products like ours, this level of preservation is unnecessary and impractical. Liquid nitrogen storage demands specialized infrastructure and carries significant cost, which is not justified when viable cells are not present.
Ultra-Low Freezing (-80°C)
Our products are stored at -80°C to maintain the native structure and function of exosomes and proteins. This temperature offers excellent preservation of:
- Exosomal membrane integrity
- Surface markers (CD63, CD81, etc.)
- Protein conformation and activity
- HA molecular weight stability
Peer-reviewed research supports the efficacy of -80°C for long-term exosome storage [1-2]. Our own shelf stability studies corroborate these findings, showing minimal degradation over time at this temperature. This internal data is available on request. To ensure our product remains in this ideal storage condition from our facility to yours, we ship all units on dry ice via overnight delivery. This protects the structural and functional integrity of exosomes, proteins, HA, and more until they arrive, preserving utility and product reliability.
Lyophilization (Freeze-Drying)
Lyophilization enables room-temperature storage, eliminating cold chain logistics. While appealing in theory, this method can be detrimental to exosomes. Studies have shown that lyophilization can compromise the lipid bilayer, leading to reduced vesicle integrity, altered uptake, and loss of functional surface proteins [3-4].
Furthermore, improper reconstitution may result in aggregation or loss of biological activity, posing risks to maintaining the structural and functional characteristics typically associated with these types of biologic materials. Though lyophilization is viable with advanced stabilizers, it’s not currently suitable for maintaining the native structure of our acellular product.
Standard Freezing (-20°C to -30°C)
While more accessible, standard freezing is suboptimal for long-term storage of exosomes and proteins. Our testing revealed accelerated degradation and loss of key surface markers at these temperatures. Over time, this leads to functional instability and potential variability in therapeutic outcomes.
Refrigeration (2–8°C)
Some biologic products are shipped or stored refrigerated in aqueous solutions. Unfortunately, this method exposes exosomes and proteins to risks such as bacterial growth, protein denaturation, and rapid aggregation. For high-performance biologics, refrigeration is only suitable for short-term use.
Ambient Shelf-Stable Formulations
New technologies aim to stabilize exosomes at ambient temperature using methods like sugar matrices, polymer encapsulation, or lipid-based carriers. While promising, these approaches are still under validation. Current peer-reviewed evidence does not yet support their use in clinical-grade exosome formulations [5]. Additionally, some stabilizers can alter bioavailability or uptake, potentially reducing the product’s intended effects.
Many preservation approaches, such as lyophilization and room-temperature stabilization, often require the addition of excipients like sugars (e.g., trehalose), polymers, or other additives to maintain exosome structure. While these agents can help mitigate damage during drying or storage, they may also alter the final product’s behavior, uptake, or compatibility depending on formulation. In some cases, individuals may also react negatively to these additives, particularly in sensitive patient populations or when introduced in higher concentrations. By contrast, our -80°C storage strategy preserves the product’s native form without the need for external stabilizers or modification, supporting a cleaner, more biologically representative product.
Our Shelf Stability Testing Program
We’ve conducted extensive in-house and 3rd-party studies to monitor our product’s behavior across various temperatures. These include nanoparticle tracking analysis, surface marker retention, protein degradation, HA stability assessment, sterility, endotoxin, mycoplasma, and more.
The data clearly demonstrate that -80°C provides optimal preservation of our product’s bioactive components. These findings support our goal of providing a biologic that maintains consistency from manufacturing through application when handled and stored under validated conditions.
Regulatory Compliance
We maintain full compliance with FDA regulations for HCT/P products under 21 CFR Part 1271. Our commitment to rigorous storage standards not only protects product quality, but also aligns with key regulatory requirements for traceability, sterility, and structural integrity.
Best Practices for Thawing Biologics
Proper thawing of biologic products, especially those derived from acellular components like amniotic fluid, is essential to preserve their integrity and therapeutic potential. While it might be tempting to speed up the thawing process using heat or agitation, doing so can damage sensitive structures like extracellular vesicles or degrade critical signaling proteins.
For best results, the product should be removed from cold storage and allowed to come to temperature gradually. The ideal approach is to place the vial upright on a clean surface at ambient room temperature—around 20 to 25°C—and allow it to thaw gently over 5 to 10 minutes. Avoid any agitation or exposure to direct heat sources like warm water or heating blocks, as these can compromise biologic activity.
Once thawed, the vial should be gently inverted a few times to ensure the contents are mixed evenly. It’s important not to vigorously shake or vortex the vial, as this may shear vesicles or disrupt molecular components. After confirming the product is fully thawed, it should be used promptly—ideally within 30 minutes of thawing. Re-freezing or prolonged refrigeration post-thaw is not recommended, as these conditions can lead to eventual degradation or reduced clinical efficacy.
Maintaining aseptic technique during this process is also key. Using a sterile field and proper handling practices ensures the product remains free from contamination and ready for safe patient administration.
By taking a few extra minutes to follow a thoughtful thawing process, clinicians can help ensure they’re delivering the full regenerative potential of these advanced biologic therapies.
Conclusion
Not all storage methods are created equal, and for biologic therapies containing fragile exosomes and proteins, the choice of preservation method is critical. While lyophilization and room temperature formulations offer convenience, they introduce risks to biological function that are not acceptable in high-performance clinical applications.
By selecting -80°C as our standard, we prioritize scientific rigor, regulatory compliance, and the consistent performance of our acellular regenerative biologic. For clinicians and researchers who demand reliability, this approach supports the preservation of native bioactive components and structural integrity for downstream application. These efforts are further supported by certified third-party laboratory partners who verify key quality attributes such as bioactive content, sterility, endotoxin levels, mycoplasma, and more—ensuring consistency and accountability at every step.
To request detailed shelf stability data or to learn more about our preservation protocols, please feel free to contact us at info@novavitalabs.com.
References
[1] Lötvall, J., et al. (2014). "Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles." Journal of Extracellular Vesicles, 3: 26913.
[2] Théry, C., et al. (2018). "Methods for the isolation and characterization of extracellular vesicles." Nature Reviews Molecular Cell Biology, 20(6), 307–320.
[3] Mazzeo, C. (2024). "Exosome Preservation Challenges in Aesthetic Regenerative Medicine." Presented at MEIDAM International Congress.
[4] Charoenviriyakul, C., et al. (2018). "Preservation of exosomes at room temperature using lyophilization." International Journal of Pharmaceutics, 552(1-2), 115–123.
[5] Bosch, S., et al. (2016). "Trehalose prevents aggregation of exosomes and cryodamage." Scientific Reports, 6: 36162.
