Cryopreservation is the gold standard method to store biospecimens in biobanking organizations. Cryopreservation usually involves flash freezing samples and storing them at ultra-low temperatures (-160°C to -190°C) to prevent chemical degradation. If biobanking scientists follow strict freezing and thawing protocols, cryopreservation can maintain sample quality and allow recovery of functional cells.
However, cryopreservation is expensive, requiring staff training and complex infrastructure. Biobanking scientists must optimize freeze-thaw protocols for each cell and tissue type, and all staff must be trained in these protocols to maintain sample quality. Furthermore, temperature fluctuations can lead to sample degradation. Therefore, biobanks should monitor the temperature of all their samples, and minimize sample handling times. The International Society for Biological and Environmental Biorepositories (ISBER) recommends that biobanking institutions have 24/7 temperature monitoring systems and backup systems in the case of equipment failure.
Biobanking Alternatives to Cryopreservation
Due to the cost and complexity of cryopreservation, there is a lot of interest in developing ways to store biomaterials at room temperature. Room temperature storage lowers biobanking costs, making it easier to store more samples in less space and to transport samples.
A common way to store biospecimens at room temperature is to fix them in formalin and embed them in paraffin blocks. This is an effective way to preserve tissue structure. However, the process of fixation fragments DNA and RNA molecules. We cannot currently isolate high quality DNA and RNA from paraffin blocks.
Lyophilization in Biobanking Organizations
Lyophilization is also called freeze-drying or cryodessication. It is a common method to preserve pharmaceutical products. Lyophilized products are frozen and then reheated in low pressure chambers to remove water content. Most chemical reactions that degrade biomaterials require water. Therefore, removing water allows products to be stored at room temperature with minimal degradation.
Researchers from the University of Debrecen in Hungary recently freeze-dried whole human cells and stored them at room temperature for up to two months (1). They recovered high-quality RNA from the freeze-dried samples, showing that lyophilization may be a cost-effective way to store RNA and DNA samples. However, the researchers could not recover intact cells. Therefore, their method is not appropriate for storing cells for cell therapy applications.
The researchers loaded cultured human B-lymphoblastoid cells into a 0.1M trehalose/PBS protectant solution. They then flash froze the cells in liquid nitrogen and dried them in a manifold freeze dryer over 6 hours. This method is faster and more cost-effective than standard industry freeze drying methods.
The researchers stored their freeze-dried samples in air-tight dark boxes with a dessicate compound to minimize sample degradation due to light, oxygen or water absorption. Then the researchers tested the integrity of RNA isolated from the freeze-dried samples, and from paired untreated control samples.
Surprisingly, they found similar RNA yield and integrity in lyophilized and untreated cells. The researchers found that freeze-dried samples stored at room temperature for up to 2 months had RNA Integrity Numbers (RIN) of 9.8. This was very close to the control sample RIN of 10. The researchers also tested the integrity of the common housekeeper gene GAPDH by measuring the expression of gene sections at both ends (the 3’ and 5’ ends). They found the same ratio in control and lyophilized samples, suggesting that freeze-drying did not affect the integrity of this abundant gene.
The researchers also tested expression of lowly expressed genes and found no difference in freeze-dried samples compared with controls.
They went on to perform RNA sequencing on lyophilized samples stored at room temperature for 2 weeks, and on untreated controls. They found similar RNA quality in all samples: similar unique reads; duplication rates; gene coverage; library complexity and GC content.
However, they did find that freeze drying changed the expression of 28 genes. 21 of these genes were protein coding – and half of these encoded transcriptional regulators, including POLR2A, CIC, INTS1, KDM6B and KMT2D. Therefore, the authors propose that their freeze-drying method may have left some residual water and allowed degradation of the RNA encoding these genes.
Conclusions
Freeze-drying is a more cost effective way for biobanking organizations to store biomaterials than cryopreservation. This study shows that freeze-drying may be an effective way to store RNA samples. However, the authors were unable to recover intact cells using their protocol. This study also looked at a small sample size and used cultured cell lines. It would be very interesting to expand on these results by testing freshly isolated human cells, and testing the effect of lyophilization on DNA and protein integrity
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