The Dos and Don’ts of Cryopreservation

The two options are a matter of preference. Some researchers choose external thread designs since nothing gets inserted into the vial which may help minimize contamination. Some researchers choose internal thread designs because they fit better in their freezer boxes. If someone uses automation, they need to consider what thread can be used with their instrument grippers.

These homemade devices may work with many cell lines. However, they don’t always give controlled, reproducible, or uniform cooling. As a result, there may be serious differences in viability among the vials upon thawing. Therefore, we do not recommend using insulated cardboard or polystyrene foam for your culture.

Cryoprotective agents (CPAs) can be divided into two groups, intracellular and extracellular cryoprotectants. An intracellular CPA is a small molecule which can penetrate the cell membrane. DMSO, glycerol, ethylene glycol, propylene glycol and cell banker series belong to intracellular CPA. An extracellular CPA is a large molecule that is added to the cryoprotectant solution. Sucrose, dextrose, methylcellulose and polyvinylpyrrolidone (PVP) are examples ⁽¹⁾.

DMSO has been successfully used for cryopreservation in a variety of cell types, including stem cells and dendritic cells which are used for cell therapy. According to the literature^(2,3), many authors used freezing media containing FBS/HSA with 10% DMSO.

The use of PVP has been investigated as an alternative to cryopreservation with DMSO and fetal calf serum (FCS) in human adipose tissue-derived adult stem cells ⁽⁴⁾. The author observed that recovery of cells cryopreserved in 10% PVP with human serum was similar to cells cryopreserved in DMSO with animal serum. In addition, they used methylcellulose either alone or combined with reduced levels of DMSO which indicated that 1% methylcellulose produced comparable results with DMSO concentrations as low as 2% during an apoptosis assay.

(1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395684/

(2) https://stemcellsjournals.onlinelibrary.wiley.com/doi/full/10.1002/stem.2415

(3) https://www.nature.com/articles/srep39071

(4) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088734/

Cryogenic preservation of cell cultures is widely used to maintain backups or reserves of cells without the associated effort and expense of feeding and caring for them. The success of the freezing process depends on four critical areas:

1. Good cell condition. iPSC should be fed daily before cryopreservation to get the healthier condition. Cells should be frozen after being passaged for 2-4 days. Overgrowth might make poorly viability after thawing. Before the cryopreservation, cell clumps should be dissolved. The cryoprotectant may be hard to penetrate the cell cluster, which results in only a small part of cells surviving after thawing. Properly handle and gently harvest the cultures. When collecting iPSC, we recommend centrifuging at 200 -300 X g for 2min, and operating pipettors gently. 1-2 x 10⁶ cells/ml is the typical density of cryopreservation. Too high density might reduce the cell viability.
2. Correct use of the cryoprotective agent. The most common cryoprotectant is DMSO. The final concentration of around 10% is most often used. For iPSC cryopreservation, some researchers also tend to add FBS or Ficoll to the freezing media. There are also some commercial products available in the market. To gain high recovery efficiency, researchers should use fresh medium materials. The cryoprotectant mix should be prepared on the day of the experiment.
3. A controlled rate of freezing. The ideal cooling rate for cells is -1°C per minute. The best way to control cooling rates is by using electronic programmable freezing units. To control the cost, researchers can use the Corning® CoolCell™. When cells are stored into Corning cryogenic vials, the vials can be inserted into a room temperature CoolCell container. Place the CoolCell container upright into a -80°C freezer or dry ice locker.
4. Storage under proper cryogenic conditions. Liquid nitrogen freezers permit storage in the vapor phase above the liquid at a temperature between -140°C and -180°C. Using vapor phase storage greatly reduces the possibility of leaky vials or ampules exploding during removal.

When thawing the iPSC:

1. Cells should be thawed rapidly by placing the cryovials in a water bath set at 37°C.
2. Use pipettor to transfer the cell suspension into 10 X volume of medium, drop by drop. The operation should be gentle and slow.
3. The seeding density range for each 35mm well (6-well plate) is between 2x10⁵ - 1x10⁶ viable cells. Cells might attach to the Corning Matrigel® coated plate in 30 minutes after thawing. 70-80% confluence may be observed after 24-48 hours of plating.

I would suggest cryopreserving the iPCS in the vapor phase above the liquid nitrogen in the long run. The temperature of the vapor phase can reach between -140°C and -180°C. Although -80°C is another common temperature to store mammalian cells, the cells tend to reduce viability within months of storage. Research has reported that adding 10% Ficoll 70 to the 10% DMSO containing cryoprotectant makes cells frozen at -80°C for one year without loss of viability, compared with liquid nitrogen storage. You may find more details on the paper below.

https://www.nature.com/articles/srep34476

There are many reports regarding cryopreservation of hepatocytes from different species like mouse, monkey, dog, rat, and human. According to many literature, the authors used 10-20% of DMSO for cryopreservation of hepatocytes. 10% of DMSO is most common concentration as a minimum of cryoprotectant for hepatocytes. If you are worried about cell viability, you can attempt to add oligosaccharides as a supplement in the freezing media that contains 10% of DMSO⁽¹⁾. The authors observed use of oligosaccharides resulted in greatest improvement in cell viability using trypan blue (TB) exclusion method. In addition, a researcher team in Sweden evaluated cell viability of hepatocytes using STEM-CELLBANKER^TM (CB) which is a xeno-free cryopreservation solution containing 10% DMSO, glucose and anhydrous dextrose, comparing standard 12% DMSO-UW medium⁽²⁾. They observed higher cell viability in CB protocols than DMSO-UW in hepatocytes.

(1) https://journals.sagepub.com/doi/pdf/10.3727/000000006783981404

(2) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365437/

There are a variety of factors that are critical for successful cryopreservation such as cell harvesting, cryoprotective agents (CPAs), storage vessels, cooling rate, cryogenic storage and thawing. Unfortunately, we usually notice cell viability problems which are associated with cryopreservation after the thawing and plating steps. I would suggest four major check points that you need to consider for improving cell viability. First, cell health and cell density are critical when the cells are frozen. The healthier your cells are, the higher post thawing viability you may obtain. We recommend 2 x 10⁶ cells for a typical Corning® cryogenic vial (Corning Cat. No. 430661 or 430489). If cell density is too high, the nutrients or CPAs might not be sufficient to maintain the health of cells. In addition, before freezing, the cells need to avoid excessive exposure to cell dissociation reagents or CPAs and keeping too long at room temperature during cell harvesting.  Second, when you transfer the cryogenic vials containing resuspended cells with freezing media into a freezing container such as Corning CoolCell™ Container at -80°C, cooling rate (-1°C per minute) is an important factor for cell freezing. You need to use a suitable freezing container, type and concentration of CPAs during cell freezing. Third, you should avoid exposing the cells to warm temperatures during transfer to cryogenic storage (-196°C). Finally, thawing must be done quickly and CPAs removed properly in order to avoid toxicity or osmotic shock. I recently presented a webinar regarding a general guide to cryogenically storing animal cell cultures. Please refer to the webinar which is available on the Corning website, with particular attention to time mark 25:27 when I go over a troubleshooting methodology which may improve your viability.

(1) https://www.corning.com/worldwide/en/products/life-sciences/resources/webforms/we-get-you-guide-for-cryogenically-storing-animal-cell-cultures.html.

(2) webinar: https://www.corning.com/worldwide/en/products/life-sciences/resources/webinars.html

Despite our best efforts to optimize CPA and the other numerous variables within a freezing/thawing protocol, cryopreservation is a traumatic process for cells. Therefore, observing some loss of viability after the freezing/thawing process is to be expected, and repeated cycles may result in progressive cell loss. Generally speaking, there are several factors that you need to consider when observing low cell viability: cell health and density when freezing, avoiding prolonged exposure to CPA or room temperature during harvesting, controlled and appropriate cooling rate, avoiding exposure to warm temperatures during transfer to cryogenic storage, and quick thawing and proper removal of CPAs. We have outlined this in more detail in a previous question. I would also suggest that you also check cell viability under microscopy before freezing in order to check dead cell populations in your vessels. To remove the dead cells, you can spin the PBMC with low speed centrifugation during the washing step.
Specifically for PBMCs, progressive loss of cell viability can occur as storage time increases(1). In addition, the disease of the individual from whom the PBMCs come also affects cryopreservation. According to literature(2), PBMCs from pediatric patients who are naturally infected with Dengue virus showed reduction of cell viability after cryopreservation, compared to fresh PBMCs from healthy children using the TB exclusion method.
I have included some PBMC protocols for your review. I would pay particular attention to PBMC centrifugation, DMSO and serum concentration, and freezing medium temperature(3,4).
(1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1538356/pdf/clinexpimmunol00251-0174.pdf
(2) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860468/
(3) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920500/
(4) https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187440

The impact of storage at -80°C to cells varies depending on the cell type. Days to months’ storage will affect the post-thawing cell status, including viability, property, or gene expression. A standard 10% DMSO freezing medium is insufficient to protect the pluripotency of cells. Three days of storage at -80 °C might cause a 50% loss of live cells and a 90% loss of Oct-4 expression. If the cryopreservation lasts three months, the damage turns worse. There is still the opportunity to wait for the recovery of pluripotency after thawing. But even 14 days culture after thawing won’t lead to full recovery.
Researchers also make efforts to develop better protocols to improve the long-term cryopreservation at -80 °C. It was reported that 10% Ficoll 70 in the freezing medium might extend the iPSC storage at -80 °C to one year with comparable viability, plating efficiency, and pluripotent phenotype versus LN2 storage. The improvements have also been achieved in red blood cells and peripheral hematopoietic stem cells.
The two papers below may help you to learn more details about this topic.
https://www.sciencedirect.com/science/article/pii/S0011224006000897?via%3Dihub
https://www.nature.com/articles/srep34476

Although DMSO is an extensively applied cryoprotectant, there are still some disadvantages of this compound, such as neurological toxicity and genotoxicity. In cases of tissue freezing, researchers would like to avoid DMSO as the cryoprotectant, especially for downstream clinical applications. The selection of alternative compounds varies from tissue origin and the following application. Many DMSO alternatives have been proven on tissue freezing, including non-penetrating cryoprotectants (e.g., glucose, sucrose, galactose, or trehalose) and intracellular cryoprotectants (e.g., ethylene glycol, propylene glycol, glycerol, formamide, methanol, and butanediol). Some commercial products are also available in the market. You may find more details on the following references.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4554388/

https://link.springer.com/article/10.1007/s10517-018-4305-5

https://link.springer.com/article/10.1186/s13287-018-0992-0

Several cryopreservation protocols have been successfully reported for T-cells. However, the best protocol of cryopreservation for your T-cell needs to be modified by your own testing since there are a variety of factors for successful cell freezing (please refer to Question #7 and #8 for detailed information). For cryoprotectant, many authors have used 5-10% DMSO with Fetal Calf Serum (FCS) or Human Serum Albumin (HAS), and they emphasized an appropriate cooling rate (-1°C per minute or slower) in T-cells^(1,2,3). Please note that “There are reports on loss of response and reduced T-cell functions in frozen cells compared to freshly isolated cells and loss of antigen recognition of T-cells that increases with prolongation of freezing time”⁽⁴⁾.

(1) https://www.pparesearch.com/wp-content/uploads/2016/09/CPC__WhitePaper.pdf

(2) https://www.nature.com/articles/s41598-019-39957-x

(3) https://cvi.asm.org/content/16/8/1176

(4) https://www.tandfonline.com/doi/full/10.3109/0284186X.2012.737020

There are some principles you should pay attention to when freezing tissues. Firstly, tissues should be frozen or fixed promptly to avoid morphological distortions. Secondly, be sure to keep fresh tissues on ice and process them as quickly as possible. Thirdly, the smaller size of the sample is preferred. Fourthly, keep the sample dry and avoid too much liquid before freezing. Thawing of tissue samples should be done rapidly in a 37°C water bath. For some tissues, blood vessels, and bone, controlling the thawing speeds may reduce tissue fragility. Besides these, the cryoprotectant or process will vary according to the organ. You may find more particular methods on the reference below.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781091/

In general, cryovials can be transferred from -80°C to -196°C directly for long-term storage.