Chapter 9.1

Cryopreservation and Banking of Tissues

Tissue Architecture

For the cryopreservation of isolated cells, we were just concerned with the response of individual cells that were suspended in an extracellular solution. The freezing process was assumed to follow the phase diagram of the solution since there were no structures to affect ice growth. Since we only had a single cell type, the response of the cells was assumed to be identical, with appropriate allowance for variation within a population (e.g. cells of different size, in different parts of the cell cycle, etc.). With tissues the situation is complicated by the presence of different cell types (that may respond quite differently to identical freezing and thawing conditions) and the presence of extracellular structure (matrix) that can affect the freezing process as well as the cellular response (the cells are attached to this matrix). Before looking more closely, perhaps we should define what we mean by a tissue.

A biological tissue is an association of cells of a multicellular organism, with a common embryological origin as well as a similar structure and function. Often, cells of a tissue are contiguous at the cell membranes but it can also be fluid, e.g. blood. There is often a particular extracellular matrix that is associated with a tissue. The cells of a tissue can all be of the same type or of several types.

This definition can be compared to that of an organ. An organ is the functional and anatomical unit of multicellular organisms (i.e. organisms are constructed out of organs). An organ is constructed out of at least two tissue types (often more) that are integrated in such a way as to perform at least one recognizable function in the physiology of the organism.

Penetration of Cryoprotectants

The first problem that has to be addressed is that the tissue has a volume associated with it. It is composed of cells, matrix, and an aqueous solution. Most tissues have a water content of about 80%, and this must be taken into consideration when designing a cryopreservation protocol.

The addition of a cryoprotectant is made more difficult by this extra volume, as it usually must penetrate, and equilibrate, with the cells to provide the maximal protective effect. This means that the microenvironment surrounding each cell must first equilibrate with the cryoprotective additive, and then the cell must become equilibrated (usually the former step is rate-limiting). For small tissues, this is usually accomplished by immersing the tissue in a solution containing the appropriate concentration of cryoprotectant, although with highly vasularized tissues, it may be preferable to use vascular perfusion to speed up the process. If the tissue is 80% water, and 1 ml of tissue is placed in 1 ml of a 1M DMSO solution, then the final concentration of DMSO in the tissue will be closer to 0.5M due to the dilution by tissue water. The volume of tissue must be taken into account when designing these procedures.

The kinetics of cryoprotectant addition need to be considered as well. One method that has been used to successfully measure the kinetics of CPA permeation is ¹H-NMR. The concentration of CPA in the tissue is measured by looking at the absolute magnitude of the NMR peak from protons on the CPA and comparing that with the absolute magnitude of the water peak.

An example of this methodology is the measurement of DMSO uptake by articular cartilage. This is the tissue that allows a near-frictionless surface in the articulating joints and also distributes loads between the bones viscoelastically. A simple cross-section shows the tissue architecture of articular cartilage:

Fig. 9.1.1

The cartilage was placed in a solution of DMSO and allowed to sit for a certain amount of time. A piece was then removed and placed in D₂O and left to equilibrate. An aliquot of the D₂O was then placed in an NMR machine to integrate the peaks from the methyl groups and water. Since there are 6 protons on DMSO and 2 protons on water (and the integral of an NMR peak is proportional to the number of protons contributing to the peak), then the ratio of DMSO molecules to water (X) can be calculated as:

(9.1.1)
where I is the integral of the peak and P is the number of protons in the molecule.

The concentration of DMSO within the sample is then given by:

(9.1.2)
where MW is the molecular weight and rho is the density.

The measured concentrations at various time intervals can then be fit to the diffusion equation with appropriate boundary conditions (an infinite plane sheet, for the purpose of articular cartilage). The following diagram shows the measured data and the fit.

Fig. 9.1.2

This gives a diffusion coefficient for DMSO in articular cartilage of 1x10⁵ cm²/s, approximately 1/2 the value that would be expected for DMSO in water, based on its molecular weight. This value can be used to design cryopreservative loading regimens for articular cartilage, using the same boundary conditions for the diffusion equation. For example, for cartilage that is 3 mm thick, the DMSO concentration as a function of time and distance from the outer surface is given by:

Fig. 9.1.3

It can be easily seen that the addition of cryoprotective additives to tissues and organs can become a problem in optimization; the lengthy time required for equilibration has to be balanced against the increasing likelihood of cryoprotectant toxicity. Removal of the cryoprotectant must be similarly considered, if it is a necessary step before transplantation of the tissue.

IIF propagation

A further problem that occurs during the freezing of tissues that is not an issue with cells is suspension has to do with the propagation of ice between cells that contact each other. Cells that are joined to other cells often have porous connections that connect their cytoplasms (e.g. gap junctions). Intracellular ice is more likely to form in these neighboring cells since the "flashing" of a single cell is enough to cause ice formation in all the cells that are connected. There is some preliminary evidence, however, that intracellular ice that forms by this mechanism is not lethal.

Mechanical Damage from Freezing

Planar vs. Dendritic Ice Growth

When liquid water turns to ice, the latent heat of fusion is liberated at the interface between ice and liquid. If the ice crystal is in contact with a heat sink, then heat flows through the crystal due to its higher thermal conductivity. This situation leads to a planar ice front, because any instability that advances in front of the interface (into the liquid) will be at a disadvantage for shedding the latent heat due to the greater distance between the surface and the heat sink and due to the greater surface area at which more heat is generated. Conversely, if nucleation occurs in bulk solution (i.e., the crystal is not in contact with a heat sink), then the latent heat must be shed into the liquid. This situation leads to an unstable planar interface because the instability that moves into the liquid, in front of the planar interface, will shed its heat to a greater volume of liquid.

A growing ice crystal will also exclude any solutes present in the solution. These will concentrate at the interface and lower the freezing point of the solution just in front of the interface. If the interface is planar, then the high solute concentration at the interface will stop ice growth at a temperature below the freezing point of the bulk solution, thus leaving the solution further away from the ice front in a supercooled state. This phenomenon is known as "constitutional supercooling" and is probably a significant consideration for the cryopreservation of tissues and organs. When it occurs, the ice front is even more unstable, as any protuberance will not only shed latent heat better, but will also grow into a supercooled solution, once it passes through the initial region of concentrated solute.

Ice growth in a normal physiological solution creates such a situation, and instabilities grow into the supercooled compartment via "dendritic growth". With dendritic growth, the ice crystal extends throughout the solution and encapsulates solute in unfrozen channels. There is no region of the solution that is shielded from the ice crystal, thus supercooling does not occur to any significant degree. Furthermore, ice reaches all regions of the solution, as the initial planar ice front is broken down and dendritic crystals extend throughout the container.

Ice Growth in Porous Media

With the exception of soil scientists and engineers, very few people realize that the damage caused by freezing to porous materials is not due to the expansion that accompanies the phase change from liquid water to ice. When water freezes, it increases its volume by about 10%, and this phenomenon can generate mechanical forces in closed vessels (as anybody who has put a coke bottle in the freezer and forgotten to remove it will attest). However, the water that freezes in rocks and soil, for example, is not enclosed. The openings that allow water to enter pores in the first place can also allow ice to expand without mechanically altering the medium. The mechanism that generates mechanical forces associated with frost heave is the thermodynamics of ice growth within capillaries, where the expansion comes from the addition of water molecules to the ice crystal rather than the phase change.

An ice crystal in a capillary will form a contact angle with the capillary wall that is dependent on the interfacial free energies between the solid, liquid, and substrate. When a liquid perfectly wets the substrate, the contact angle will be 180°. Under such a condition, the ice crystal will have a hemispherical interface with liquid water of radius r. The temperature at which such a crystal will be thermodynamically stable is a function of r. As the diameter of the capillary decreases, the freezing point of water in the capillary is lowered.

Consider a system in which ice is separated from water by a wetted porous material:

Fig. 9.1.4

With the temperature above the freezing point of liquid within the pores, but when the temperature goes below the freezing point, the water will move through the pores and join the ice for as long as the temperature and the supercooled state of the water can be maintained.

In a porous medium with heterogeneous interstitial spaces, ice will form initially at the surfaces (if ice is present externally) or in the larger aqueous cavities during freezing. Once an ice crystal forms in such a cavity, if it is connected to water sources through capillaries that are small enough so that freezing cannot occur within them, then water will flow to the ice crystal, leaving part of the sample in a dessicated state. The ice crystal in the cavity continues to grow, generating mechanical forces that are responsible for enlarging the cavity. In soils, where this phenomenon occurs, the ice cavities are usually shaped like a convex lens and are thus called "ice lenses". The resulting movement of the soil is referred to as "frost heave".

Ice Growth in Articular Cartilage

The freeze-substitution method replaces the ice in a sample with alcohol at low temperatures. This allows the morphology of ice to be visualized by looking at the vacancies in the tissue. When articular cartilage is frozen at a cooling rate of 1°C/min, stored in liquid nitrogen, and then freeze-substituted, the following structure is obtained:

Fig. 9.1.5

The outer surface (the joint surface) of the cartilage is visible at the top of the image. On the right, the surface is a cut from a scalpel blade that was made before freezing. The holes inside the matrix are lacunae, where the chondrocytes were situated. The two distinct regions visible in the figure are due to fundamentally different ice morphologies in these regions. Near the surface of the tissue, ice grew into the matrix as a single crystal, leaving the tissue without apparent disruptions. Although articular cartilage is about 78% water, the pore size is on the order of 5 nm, therefore we do not expect to see any aqueous vacancies in the tissue except for the lacunae, which house chondrocytes. In the figure, the matrix of the interior region is porous and open, suggesting mechanical damage to the matrix architecture. The growth of ice in this region must be of a completely different nature from that near the surface, where the ice did not alter the structure of the matrix. Of note, the recovery of cells correlated strongly with the presence of ice that grew into the tissue from the surrounding solution, whereas the destroyed cells were in the region with tissue disruption.

Ice growth in articular cartilage may be vastly different from ice growth in a bulk physiological solution. Ice moves into the cartilage as a planar front and remains planar even at very low temperatures. The salt concentrates at the ice front and lowers the freezing point so that ice growth slows dramatically. Apparently the only mechanism for continued ice growth is for the salt to diffuse away from the ice front so that the freezing point at the interface is raised, or by lowering the temperature. Lowering the temperature increases the supercooling in the interior of the tissue, but apparently dendritic growth cannot be initiated. If dendrites penetrated the region of high salt concentration at the planar interface, then ice could grow throughout the tissue and encapsulate the excluded solute as well as the cells, allowing cryopreservation in the manner that occurs with cells suspended in bulk solution.

The consequence of further lowering of the temperature is an increased supercooling of the interior of the tissue that leads to spontaneous nucleation at many sites within the matrix and the formation of ice lenses. Since these lenses draw water to them from every direction, they can expand against a resisting force and create mechanical effects (e.g., frost heave). Figure 9.1.5 above reveals a greatly disrupted matrix, with pores on the order of 1 um, at least 100 times larger than the pore size in normal cartilage. Presumably, cells in this region are crushed with the mechanical forces from the ice lenses, and lacunae are the most likely sites for nucleation to occur. A further consequence of ice lens formation would be that the distribution of water is altered. Although water and solute would be drawn through the capillaries to an ice lens, only pure water will join the crystal. Since the crystal can enlarge without forming a network of aqueous channels (as occurs with dendritic ice growth), upon warming the potential exists for cells to be subject to transient osmotic stresses, especially if the lacunae are the nucleation sites for ice lens formation. Thus, the formation of ice lenses could produce mechanical forces and osmotic stresses that lead to the injury of chondroctyes in intact articular cartilage, acting in parallel with the conventional mechanisms of cryoinjury.

Vascular Damage

The vasculature within a tissue will also be a primary site of ice growth. Since the vasculature is filled with aqueous solution, ice will readily form and grow throughout the interconnected tubes. The walls of the blood vessels may act like the matrix of articular cartilage, preventing ice from growing through them and into the interstitial space. Thus, the increased salt concentration within the unfrozen fraction of the blood vessels will draw water out of the rest of the tissue to join the ice crystals within the vascular channels. These channels may then expand beyond the elastic capabilities of the blood vessel walls causing mechanical damage. Thus it may be impossible to re- establish proper blood flow upon transplantation of the tissue.

Tissue Banking

Currently, there are several tissues that are banked in a cryopreserved state for clinical transplantation. Blood, bone, cartilage, heart valves, bone marrow, blood vessels, skin, corneas, islets of langerhans, and embryos (to name a few) are all routinely cryopreserved, kept in a tissue bank, and then used in clinical situations (with varying degrees of success). In addition, the growing field of tissue engineering is demanding the cryopreservation of biological and synthetic tissues (such as a bioreactor) for commercial distribution to be realized. Since the tissues that are cryopreserved are often not capable of regeneration, it is sometimes imperative that the majority of cells survive the cryopreservation process.

The cryopreservation procedures that are currently in use for these tissues can vary greatly between tissue banks. Some of the current practices are protected by patent or trade secret, others are known to result in low (or even no) recovery of cells. The American Association of Tissue Banks (AATB) publishes standards for tissue banking but does not recommend any specific cryopreservation procedures. Rather, they impose guidelines for establishing procedures and implementing them in a rigorous manner. Thus it is up to the individual tissue bank to determine an optimal cryopreservation protocol for a particular tissue experimentally, basing their optimization on a particular assay or series of assessments. The AATB guidelines then instruct the tissue bank how to implement the protocol with the necessary standards of quality control for clinical work.

A Short Catalog of Cryopreservation Protocols

These may be a little vague...

Small Elastic Arteries: Soak in CPTES medium with 15% w/w DMSO until equilibrated. Freeze at 1°C/min and store at -196°C. During thawing, the warming rate must be less that 50°C/min between the range of -140°C to -120°C (the glass transition temperature of the solution is -123°C); thaw in a 37°C water bath when above this temperature. Uncontrolled thawing leads to fractures which make the arteries useless.
Heart Valves: Place in Media 199 with 0.1 mg/ml cephapirin, 0.2 mg/ml azlocilin, 0.08 mg/ml tobramycin, and 0.1 mg/ml amphotericin B with 10% DMSO. Cool at 1°C/min and store at -196°C.
Osteochondral Dowels: Soak in 1M DMSO in MEM for 30 minutes before cooling at 1°C/min to -60°C. Plunge in liquid nitrogen. Thaw in a 37°C water bath.

Quality Control in Tissue Banking

A proper quality control program means that the tissue bank must constantly investigate, survey and monitor all processes, products, and environmental parameters that could affect the quality of the preserved material. Such a program will usually have the following components:

Standard operating procedures manual
- A procedures manual should clearly define the policies and procedures that are currently used as well as the organizational structure and the responsibilities of everyone involved. It is always good form if the standards comply with those set by regulatory agencies.
- The procedures manual must be written with clarity and be unambiguous in its meaning.
Donor files
- There must be a systematic and consistent methodology and documentation of the evaluation of donors. This documentation should cover donor consent, donor and tissue screening, tissue processing, and release of tissue.
- The donor files will document that the standards set out in the procedures manual have been met. Those responsible for ensuring that standards are met will vouch for their supervision by signing appropriate statements in the donor files.
Processing and storage parameters
- Equipment Qualification: Will a critical piece of equipment actually do what is stated in the procedures manual? This must be checked experimentally and documented; manufacturer's claims are not sufficient.
- Process Validation: Each process in the standars manual must be shown experimentally to produce the desired outcome. E.g. If a procedure is to result in uncontaminated, viable cells following cryopreservation, the cell identity must be established, the viability must be measured with a validated assay (including tolerance limits), the lack of contamination must be established, and the storage requirements must be detailed with tolerance limits.
- Process Control: The aim of process control is to provide consistency to a process, and confidence that the equipment and supplies are monitored for operation within tolerance limits, expiration dates, and manufacturer stability.
- Documentation: If it's not documented, it didn't happen.
Review and release requirements
- The requirements must be clearly defined along with the responsibilities for carrying them out. Signatures to hold people accountable.
Training and education program
- Technical staff must be kept educated regarding the procedures and responsibilities. They must be competent and proficient to ensure a quality product. Education regarding technical aspects can also motivate them to perform better and look for ways to further improve procedures.
Audits and investigations
- There should be periodic and responsive audits to ensure that the standard operating procedures are being followed. Any anomolies found during audits should be investigated fully.
- Investigations provide a mechanism for the identification of, collection and review of information about, and action taken on potentially significant events. There must be documentation of the details and resolution of each event.
Tracking requirements
- It must be possible to trace from donor and recipient and back. Any problems such as disease transmission or other adverse outcomes need to be discovered as soon as possible so that any suspect inventory can be tested or quarantined.