Tissue slices are viable tissue sections used as in vitro representatives of their originating tissues or organs for metabolic studies, including live-cell imaging, electrophysiological analysis, and pharmacological studies.
Theoretically, slices of tissue or organ are immediately used for their intended examinations. However, this is not usually realistic. Tissue slices are prepared once but often examined in batches because analyses can consume substantial time. In other cases, tissue slices from rare or unique specimens may be stored long-term for potential future studies.
In either case, tissue slices must be preserved after sectioning. Good preservation methods retain the tissue slices’ viability without destroying any functional structure in the tissue.[1]
The followings are methods used to preserve the viability of tissue slices:
Tissues, organs, and tissue slices can be cryopreserved at -196°C, the temperature of liquid nitrogen. At this temperature point, all metabolic activities are halted and are resurrected when the temperature rises to the physiological level.
In practice, successful cryopreservation lies in the ability to prevent damage to tissue slices during freezing and rewarming to the physiological temperature.[1]
The followings are contemporary cryopreservation methods:
Also called equilibrium freezing, tissue slices in slow freezing are immersed in a preservation solution containing cryoprotectants. Tissue slices in the preservation solution are cooled below the water freezing point, causing the liquid to become supercooled.
Initially, the supercool fluid remains liquid because of the cryoprotectants. However, prolonged freezing will cause pure water to crystallize into ice in the extracellular space.
Ice formation increases the solute concentration, leading to exosmosis where cellular water starts to leave the cell. At equilibrium, cells are completely dehydrated while the intercellular components remain unfrozen.[1,2]
The slow freezing method is the traditional cryopreservation method. It is simple and does not require any specialized equipment. However, the ice crystals can inadvertently damage the cells and their organelles during freezing and rewarming. Slow freezing can be effective for preserving liver tissue slices, but it’s less effective for tissue slices from renal and brain tissues.[1-2]
In this method, tissue slices are preincubated in a buffer or medium containing a relatively high concentration of cryoprotectants like dimethyl sulfoxide (DMSO) or glycerol.
The tissue slices rapidly freeze at 100-1000°C per minute, usually by liquid nitrogen. Frozen tissue slices are immediately stored at -20°C, -80°C, or in liquid nitrogen after fast freezing until they are ready for examination.
Like slow freezing, the cryoprotectants in the solution hinder ice crystallization. However, the rate of temperature decrease reduces the size of the forming ice crystals.
Hence, rapid freezing is more effective and applicable to many tissue types than slow freezing, including tissue slices from kidneys. It is also applicable for preserving brain tissues for imaging and electrochemical analysis.[1,3]
Literally translated to glass formation, vitrification uses a vicious preservation solution that contains an extremely high concentration of cryoprotectants. Tissue slices are immersed in the solution, and the temperature is slowly reduced, as seen in slow freezing.
As the temperature decreases, the solution becomes solidified, called vitrified. But unlike slow freezing, ice crystals are not formed in vitrified tissue slices.
In vitrification, solidification occurs because of an extreme elevation of the solute viscosity during cooling. In other words, tissue slices become glass-like during vitrification because the preservation solution is too vicious to form ice crystals.
Vitrification is a more effective tissue slice preservation method than slow and fast freezing. It has been used to preserve many tissue slices from various organs, including kidneys, livers, and brains.
Nevertheless, a high concentration of cryoprotectants can be toxic to cells and tissues, thus reducing tissue slice viability. Therefore, the concentration of cryoprotectants and vitrification procedures must be tested and optimized for each cell and tissue type before use. [1-2,4]
Theoretically, cryopreservation of tissue slices can maintain the viability of the tissue slices indefinitely, provided that they are stored in liquid nitrogen. In reality, the longevity and viability of cryopreserved tissue slices depend on the storage temperature and method used to rewarm and resurrect the slices.[1]
Cryopreservation is the go-to technique for the long-term storage of cells and tissue, including tissue slices. Nonetheless, cryopreservation methods need to be adapted and optimized for each tissue type, which can be time- and labor-consuming.
For short-term preservation, tissues, organs, and tissue slices can be preserved at low temperatures (0-4°C).
In general, tissue slices prepared while the tissue is submerged can be preserved, if they are sliced in a pre-chilled slice buffer, culture medium, or a preservation solution. After preparation, the slices can be stored at low temperatures for a duration. Similarly, tissues and organs that cannot be sliced in submerged conditions can be preserved as a whole in a cold buffer or preservation solution.[1,2]
Typical slice buffer, media, or preservation solution are often supplemented with glucose or other sugar at a concentration of 25mM or higher. Glucose and other sugars can act like a cryoprotectant and protect the tissue slices from low temperature-induced damage.
After tissue slices are immersed in the cold solution, they can be stored at similar temperatures from 12 hours up to 21 days after preparation, depending on the originating organs and tissues.[1,5]
The choice of slice buffers or preservation solutions depends on the characteristics of the originating tissues or organs.
Intestinal slices, for example, are frequently preserved in the Krebs-Henseleit buffer that is constantly supplied with oxygen.
Brain tissue slices are preserved in a cold artificial cerebrospinal fluid and must be supplied with continuous carbonation (95% O2 and 5% CO2). The artificial fluid is a specialized preservation solution that contains high glucose, high calcium, and low magnesium. The fluid is also supplemented with antioxidants, which suppress oxidizing damages to the synapses, prolonging the longevity of the neurons.[1,5]
To ease the logistics of metabolic studies, equipment combining the workflow is available. It enables users to integrate the workflows in tissue slice preparation and preservation into one device.
For example, the Dual Cell Slice Chamber enables users to prepare tissue slices in a submerged or non-submerged condition. After slicing, the slices can be transferred and preserved in holding compartments filled with preservation solutions and are continuously carbonated (a combination of 95% O2 and 5% CO2). With four holding compartments, the Dual Cell Slice Chamber is for intricate tissues like brain tissues, where each section may require different preservation methods.
In some devices, the routine analysis workflow is also integrated therein. For instance, the Visual Patching Imaging Chamber combines the slicing, preservation, and imaging of brain tissues and neurons into one device. It allows users to prepare the tissue slices and maintain them in a feedback sensor-controlled bath. Here, the volume of the solution and temperature is monitored and maintained to the physiological values. A microscope can be mounted over the bath to examine and acquire images of the tissue slices.
Tissue slice preservation in cold buffer and cold storage only slows down cellular activities and extends tissue viability. In other words, the metabolic activities in tissue slices stored in a cold buffer and cold storage do not come to a complete stop, and they will begin to deteriorate the longer they are stored. Nevertheless, it is a simple method with low risks of losing the viability of tissue slices to freezing-thawing damage and cell toxicity.[1-2]
Viable tissue slices can represent the larger intact tissues and organs and provide insights into their workings in vitro. For viability preservation, the tissue slices can be cryopreserved in a suitable cold buffer at low temperatures.
Cryopreservation is suitable for long-term storage, but there’s a risk of losing viability to freezing-thawing damage and toxicity to cryoprotectants.
The second technique discussed above reduces cellular activity by storing the tissue slices in a cold buffer at low temperatures. While it is not fit for long-term storage, the tissue slices do not need to be resurrected and can be instantly used for metabolic studies.
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