Making your own biopreservation media may be costing you more than you realize!
Biopreservation of cells, tissues, and organs is a frequently applied and required practice used to extend the stability and viability in both short-term and long-term storage of samples for research and clinical applications.
Hypothermic biopreservation is typically performed at temperatures above 0°C and usually between 2-10°C, but certainly below ambient (around 22°C) or normothermic conditions. It is used for short-term transport and storage, during which metabolic activity is greatly reduced but still present at some level.
Cryopreservation, or frozen storage, involves long-term storage of biologics at or below -80°C (typically below -140°C) in conditions of metabolic arrest.
A wide array of biopreservation media solutions, biologic packaging products, and methods are available to the user, which can be selected based on the specific requirements of the biologic of interest.
Biopreservation is an important step for transport and storage of biologics including the multitude of products being developed for cellular therapy indications. But biopreservation is often regarded as a standard process with minimal optimization applied to the procedure or the reagents used.
For non-clinical applications, most if not all of the attention is focused on the simplest and least costly method to ensure some level of post-preservation recovery.
Unfortunately, a similar thought process is often used for potential clinical cellular applications. Again, a means to reduce the cost and time to develop optimal preservation methods are the focus.
- By doing so, how will cutting these corners impact the post-preservation recovery, viability, and the anticipated function of the biologic?
- When considering clinical cell therapy products, what are the additional quality and regulatory components one must consider?
- What is the true cost?
Traditional biopreservation media typically fall into two broad groups:
- Home-brew (solutions made up in-house)
- Commercial, pre-formulated products.
Generally, home-brew media solutions, are prepared in-house by combining a number of components of varying quality and under varying levels of cleanliness, while commercially available solutions consist of ready-to-use media solutions that do not require the addition or mixing of components prior to use.
Home-brew solutions are often the first choice of practitioners for their biopreservation needs, since these solutions are assumed to be both effective and more cost effective compared to commercial solutions.
Home-brew preservation media can consist of any number of components, but are generally comprised of cell growth (culture) medium or other physiologic buffers, serum and/or protein, and a cryoprotectant such as DMSO if cryopreservation is required. Since cell culture media and other physiologic buffers (extracellular-like) are designed for growing or maintaining biologics rather than preservation, additional components including serum and protein are frequently added to the culture media to enhance preservation efficacy.
As demonstrated in Examples 1A and 1B below, normal human dermal fibroblasts (NHDF) and human mesenchymal stem cells (hMSC) were cryopreserved in a variety of preservation media and assessed for post-thaw viability following one day of recovery at 37°C.
A selection of home-brew cryopreservation solutions were prepared with and without the addition of serum, and compared to CryoStor, the commercially available cGMP, completely defined, serum-free and protein-free freeze media from BioLife.
The results indicate the inclusion of serum in each of the home-brew solutions improved post-thaw viability, compared to the respective solutions without serum; but none of these solutions were found to be as effective as CryoStor, which has ionic concentrations that effectively balance the intra-cellular environment at hypothermic and cryopreservation temperatures.
Finally, as compared to the other serum-free solutions, CryoStor enabled considerable improvement in viability of both the NHDF and hMSC, which is an indication of the preservation efficacy provided by the other components in this proprietary formulation.
Hypothermic transport and storage of biologics, like cryopreservation, incorporates an array of home-brew and commercial preservation solutions. Biologics are harvested and frequently transported or stored at refrigerated temperatures in culture media (with/without serum) or other commercially available isotonic and electrolyte solutions (i.e. saline, Normosol®-R, Plasma-Lyte®, Celsior®).
Again, these solutions are ideal for maintenance of cells at normothermic temperature in cell culture conditions that mimic native environments. However, they do not balance cells when subjected to low temperature preservation, as opposed to commercially available intracellular-like solutions (i.e. HypoThermosol-FRS, Viaspan).
One day of storage for overnight shipping is often considered to be sufficient, but what happens if extended stability options are required for instances when shipments get delayed or tissues for procurement arrive outside of normal hours?
A critical issue affecting commercialization of new cell therapy and regenerative medicine products is minimal storage and transport stability that limits geographic clinical distribution and may result in costly redundant manufacturing facilities to support worldwide product availability.
In Examples 2A and 2B, NHDF and hMSC cultures were exposed to hypothermic conditions for one to five days in a selection of home-brew and commercially available preservation solutions. Fluorescent micrographs were obtained following one day of recovery at 37°C to assess cell recovery and preservation solution efficacy.
At first glance, each of the solutions tested provide complete protection to NHDF cells during one day of hypothermic storage (Example 2A).
However, when the storage time was extended to three days, little to no protection was afforded to NHDF cells stored in culture media (with serum), Celsior, or Viaspan; while a complete recovery of cells was observed in cells stored in HypoThermosol-FRS. In comparison, no viable hMSC cells were recovered following one-day storage in culture media or Celsior. Viaspan did provide a minimal improvement in recovery, while HypoThermosol-FRS enabled a complete recovery and a signifi cant extension of stability, compared to each of the other tested solutions (Example 2B).
Viability, function, overall recovery, and the stability footprint of the preserved biologic are of critical importance, but the following aspects should also be considered when preservation solutions are evaluated and considered: component quality (USP, serum, proteins, WFI quality water), manufacturing environment (GMP, sterile filtered), release testing (sterility, endotoxin, particulates).
The estimated cost of home-brew solutions is usually based only on the cost of components used, and typically excludes labor costs. Home-brew solutions appear to offer a quick, simple, and relatively inexpensive alternative for short-term and long-term storage of cells and tissues.
Unfortunately, home-brew preservation media formulations may expose the practitioner to several compromises and areas of risk related to lower than expected cell viability and recovery, limited stability, and quality concerns. Compared to home-brew solutions, many commercially available solutions offer improvements in specific quality aspects (i.e. sterility, grade of components), but extended stability options are limited and post-preservation viability and recovery remain sub-optimal.
While a number of biopreservation solution options are available, it’s clear that not all preservation solutions are created equally.