Fully Automated Cell Cryopreservation Using Controlled Rate Freezing
Cryopreservation has transformed how scientists preserve and work with living systems. By freezing cells, tissues, and other biological materials for long-term storage, we can safeguard valuable resources, maintain consistency across experiments, and accelerate innovation in biotechnology. Nearly every research or manufacturing biosciences lab relies on this vital technology to keep progress moving forward.
However, the process of cryopreservation can impact the health, also known as viability, of cells after thawing due to the physical and biochemical stresses that occur during the freezing and thawing processes.
Controlling the Cooling Rate During Cryopreservation
If the freezing of cells is not performed at a specific rate, the ice crystal formation, especially intracellularly, can rupture cell membranes and damage organelles. Additionally, the use of cryoprotectants - such as dimethyl sulfoxide (DMSO) - is essential to reduce ice formation, these substances can be toxic for the cells if not handled properly. Additionally, the rate of freezing plays a critical role in the cryopreservation process; too rapid or too slow can both negatively affect cell integrity.
Controlled rate freezing helps maintain post-thaw cell viability by gradually lowering the temperature at an optimal rate, typically around 1℃ per minute, by allowing water to exit the cells slowly before ice crystals can form intracellularly. This controlled dehydration reduces the risk of intracellular ice damage and minimizes osmotic shock, preserving membrane integrity and increasing the likelihood of successful recovery and function after thawing.
In order to perform controlled rate freezing during cryopreservation, cells should be homogeneously resuspended in cell culture media supplemented with cryoprotective agents at a certain concentration, typically 1 million cells per millilitre. Following that, the cell resuspension is transferred, millilitre by millilitre, into cryovials - specially designed vials that can withstand ultra-low temperatures. These vials are then moved into a freezing container that is made of a plastic shell and an internal foam insert, and filled up with isopropyl alcohol to deliver a cooling rate for the contents of the cryovials at the desired -1℃ per minute in a -80℃ freezer.
While the principle of this process is strongly founded and proven to work, in practice, manual cell cryopreservation is prone to high degrees of inter-operator variability and adds unnecessary time pressure on staff during the process. A great way to increase the consistency and reliability of this crucial lab process and at the same time alleviate the pressure on staff, is workflow automation.
Automating Cell Cryopreservation
Automating cell cryopreservation is something that Automata is familiar with. By integrating already available third party instrumentation onto our LINQ platform, we can deploy fully integrated systems that use live growing cells as input material to deliver fully counted and viability checked cells, controlled-rate-frozen in cryovials, at -80℃, ready to be transferred to liquid nitrogen.
Our approach when designing fully integrated systems that wash, detach, count and cryopreserve cells is to bring together instrumentation that is reliable, has proven functionality on the market and, crucially, can be integrated into automated systems from a hardware and software perspective. We bring these instruments physically together using our LINQ benches as infrastructure and connect them using plate handling robots and plate transfer stations such as the LINQ Bridge. In terms of software, we enable all the instruments to communicate with each other and orchestrate entire workflows using our LINQ Cloud software. In this way, we obtain a fully integrated, totally open system, that enables staff to make full use of all instrumentation available.
An example of the input for such a system are live cells growing adherently on SBS format labware such as single-well plates (Nunc™ OmniTray™). These cells are manually loaded onto the system via an automated cell culture incubator that keeps the environmental conditions adequate, at 5% CO₂ and 37℃ - one such incubator is the LiCONiC STX110.
The next step in this process is to remove the used cell growth media and wash the cells with PBS, subsequently adding a cell dissociation reagent such as Trypsin to the cells - these steps can all be performed on a plate washer/dispenser such as Agilent’s Biotek MultiFlo FX. Once the cell dissociation reagent is added, the plate is then transferred to a small table-top cell incubator that maintains the cells at 37℃ for efficient cell dissociation.
As soon as the cells have been incubated with the cell dissociation reagent, they are transferred to a liquid handler, equipped with accessories such as a plate tilter and heat/cool modules. This ensures that all the cells are washed off the plate with fresh media and transferred to a deep-well centrifuge plate, which in-turn is transferred to the automated centrifuge available on the system, such as an SBS300 from Hettich.
Once the cells have been pelleted, they are transferred to a liquid handler. This removes the cell dissociation reagent containing media off the cell pellets, and resuspends and pools them together in fresh media, subsequently aliquoting a triplicate of the cell suspension into cell counting plates containing AO/PI dye.
This step enables the transfer of the cell counting plate to an automated cell counter available on the system, such as the Cellaca MX from Revvity. While the cells are being counted, an SBS format rack containing cryovials is being transferred from a storage position such as a stacker, to the SBS format labware barcode scanner that captures the unique ID of each tube being used in this workflow. This is further transferred to the cryovial capper/decapper that is capable of taking and storing the caps of an entire rack of cryovials at the same time, one such instrument is the IntelliXcap from Azenta. Eventually, the cryorack with uncapped cryovials is transferred to the liquid handler, ready to receive the cells for cryopreservation.
As the cell counter gathers the data on the number of cells and the viability of these, it communicates via the LINQ Cloud software with the liquid handler and informs it how much cell culture media containing cryopreservation reagent it should add to each cell pool in order to achieve the desired concentration per millilitre (i.e. 1 million cells/millilitre). Once the cells are homogeneously resuspended at the adequate concentration in cryopreservation media, they are reformatted into the cryovials available on the deck of the liquid handler. As soon as the reformatting is complete, the cryovial rack is transferred back to the capper/decapper which now puts the caps back on the cryovials.
Lastly, the cryovial rack with capped cryovials is transferred to an automated Controlled Rate Freezer - such as the ViaFreeze Quad from Cytiva - and the contents are cooled to -80℃ at -1℃ per minute., Tthe cryovials are then ready to be transferred to liquid nitrogen for long term storage.
Less Manual Interaction, More Reproducibility
As described in detail in the previous paragraphs, there is no human interaction between loading the live cells in the incubator and unloading the cryoracks with cryovials containing the characterised cells at -80℃. Everything is monitored through plate and vial barcode scanners and the data is captured for reference. This aspect highly increases the reproducibility of the entire process by ensuring that every labware containing cells and every cryovial containing cells is treated in the same manner with stringent consistency dictated by the automated workflow. The contents of these cryovials are recorded and the corresponding cell health and number data can be referenced.
In terms of throughput, this can be adjusted on the system by designing the work-cell to the scientific project specific needs. It is not unusual for systems that we design to process hundreds of cryovials, completely hands-free, within a working day.
Ready to Automate Your Cell Cryopreservation?
Cell cryopreservation is a delicate laboratory process that requires consistency, focus and stamina. While manual cell cryopreservation has its place in the lab, the long hours of monotonous lab work is not only boring, but introduces errors, increases variability, and is a waste of highly skilled scientists' time.
The best way to overcome manual cell cryopreservation is through end-to-end automation workflows such as the one described in this article. If your lab is considering taking a step closer to automating such processes, do not hesitate to get in touch with us. We have experience designing such systems of all shapes and sizes for a wide range of throughputs and cell types - let’s automate science together!
