This article first discusses some fundamentals of cryobiology and challenges for cell and tissue cryopreservation. Then, the results of cryopreservation of adipose cells and tissues, including adipose-derived stem cells, in the last decade are reviewed. In addition, from the viewpoint of cryobiology, some desired future work in fat cryopreservation is proposed that would benefit the optimization, standardization, and better application of such techniques.
Key points
- •
Controlled slow freezing (1–2°C/min) and fast thawing together with a combination of cryoprotective agents (CPAs) shows optimal results.
- •
Adipose-derived stem cells (ADSCs), either isolated from fresh adipose tissues before ADSC cryopreservation or isolated from cryopreserved adipose tissue, can retain their proliferation and differentiation capabilities after cryopreservation. This property ensures the probability of ADSCs serving as an important source for cell-based therapy and tissue engineering.
- •
Future work for cryopreservation of adipose tissue includes development of novel cryoprotection media (DMSO free, serum free, and even xeno free), fundamental cryobiological studies of adipose tissue and ADSCs, optimization and standardization of fat cryopreservation protocols toward good manufacturing practice products.
Introduction
One main obstacle to achieve long-term favorable results of soft tissue filling with autologous fat transplantation is the high absorption rate of the injected fat in the grafted site, reaching up to 70% of its volume. Some dead materials and insufficient revascularization in the grafts are probably responsible for this problem over time. This high absorption rate necessitates overcorrection and reinjection procedures in the desired area, which are associated with repeated suction-assisted lipectomy operations to obtain the fat, as well as higher cost, unfavorable appearance, increasing patient morbidity, and discomfort. In order to solve this problem, long-term cryopreservation of the obtained fat is indispensable for both surgeons and patients. Adipose cells and tissues obtained from only a single harvest can be stored and used for the multiple grafting sessions in the future, therefore significantly reducing the patient discomfort, morbidity, cost, and time. Some researchers have also reported that transplantation of frozen fat caused less swelling and discoloration than fresh fat in the operation area.
Another fact that necessitates cryopreservation of adipose tissues is that adipose tissue is an ideal source of many cell types, including adipocytes, preadipocytes, vascular endothelial cells, vascular smooth muscle cells, and adipose tissue–derived stem cells. These stem cells can be differentiated with specific growth factors into multiple lineages, such as fat, bone, cartilage, skeletal, muscle, endothelium, hematopoietic cells, hepatocytes, and neuronal cells. Hence, they have immeasurable applications in regenerative medicine and tissue engineering. When a cellular therapy is needed, previously cryopreserved adipose tissues can be processed in vitro to obtain such cell types for patients. Cryopreservation of adipose tissues can provide an abundant source for the ready availability of such cell types. It also serves several other purposes, including sample shipment, quality assay, donor-recipient matching, disease screening, and pooling of samples obtained from multiple procedures to get a large enough dose for clinical treatment.
Despite its advantages and significance, cryopreservation can also be a notable variable in fat processing, which may lead to viability and functionality loss of cells and tissues after freezing and thawing. There has been a lot of work conducted in the last decade to investigate and optimize the cryopreservation protocols for fat cells, tissues, and adipose tissue–derived stem cells (ADSCs). Inconsistent or even opposite results can be found in the literature. Therefore, cryopreservation of adipose cells and tissues is updated in this article, starting with some fundamentals of cryobiology and cryopreservation. Then, progress in cryopreservation of adipose tissues and ADSCs is reviewed, followed by discussion of future work.
Introduction
One main obstacle to achieve long-term favorable results of soft tissue filling with autologous fat transplantation is the high absorption rate of the injected fat in the grafted site, reaching up to 70% of its volume. Some dead materials and insufficient revascularization in the grafts are probably responsible for this problem over time. This high absorption rate necessitates overcorrection and reinjection procedures in the desired area, which are associated with repeated suction-assisted lipectomy operations to obtain the fat, as well as higher cost, unfavorable appearance, increasing patient morbidity, and discomfort. In order to solve this problem, long-term cryopreservation of the obtained fat is indispensable for both surgeons and patients. Adipose cells and tissues obtained from only a single harvest can be stored and used for the multiple grafting sessions in the future, therefore significantly reducing the patient discomfort, morbidity, cost, and time. Some researchers have also reported that transplantation of frozen fat caused less swelling and discoloration than fresh fat in the operation area.
Another fact that necessitates cryopreservation of adipose tissues is that adipose tissue is an ideal source of many cell types, including adipocytes, preadipocytes, vascular endothelial cells, vascular smooth muscle cells, and adipose tissue–derived stem cells. These stem cells can be differentiated with specific growth factors into multiple lineages, such as fat, bone, cartilage, skeletal, muscle, endothelium, hematopoietic cells, hepatocytes, and neuronal cells. Hence, they have immeasurable applications in regenerative medicine and tissue engineering. When a cellular therapy is needed, previously cryopreserved adipose tissues can be processed in vitro to obtain such cell types for patients. Cryopreservation of adipose tissues can provide an abundant source for the ready availability of such cell types. It also serves several other purposes, including sample shipment, quality assay, donor-recipient matching, disease screening, and pooling of samples obtained from multiple procedures to get a large enough dose for clinical treatment.
Despite its advantages and significance, cryopreservation can also be a notable variable in fat processing, which may lead to viability and functionality loss of cells and tissues after freezing and thawing. There has been a lot of work conducted in the last decade to investigate and optimize the cryopreservation protocols for fat cells, tissues, and adipose tissue–derived stem cells (ADSCs). Inconsistent or even opposite results can be found in the literature. Therefore, cryopreservation of adipose cells and tissues is updated in this article, starting with some fundamentals of cryobiology and cryopreservation. Then, progress in cryopreservation of adipose tissues and ADSCs is reviewed, followed by discussion of future work.
Fundamental cryobiology and cryopreservation processes
Freezing of Cells
Optimization of isolated cell cryopreservation requires a quantitative understanding of the biophysical response of cells during the freezing process. As cells are cooled to a subzero temperature, such as about −5°C, both the cells and surrounding medium usually remain unfrozen despite the temperature having fallen below the freezing point of water (a supercooled state). Between −5°C and about −15°C, ice forms in the external medium but the cell contents remain unfrozen and supercooled, presumably because the plasma membrane blocks the growth of ice crystals into the cytoplasm. The supercooled water in the cells has, by definition, a higher chemical potential than that of water in the partially frozen extracellular solution, and thus water flows out of the cell and freezes externally.
The subsequent physical events in the cell depend on the cooling rate. These cell responses to freezing were first expressed quantitatively by Mazur and directly linked with cell cryoinjury by Mazur’s 2-factor hypothesis: (1) at slow cooling rates, cryoinjury occurs because of a solution effect (ie, the intracellular solute/electrolyte concentration increases as water leaves the cell, to a point at which severe cell dehydration occurs); and (2) at high cooling rates, water is not lost fast enough and cryoinjury occurs because of intracellular ice formation (IIF), which ruptures the cell membrane. The optimal cooling rate for cell survival should be slow enough to reduce IIF but fast enough to minimize the solution effects. The freezing behavior of the cells can be modified by the addition of cryoprotective agents (CPAs), which affect the rates of water transport, ice nucleation, and ice crystal growth.
More detailed information about cryobiology can be found in a review published in 1970 by Mazur in Science . Important milestones in cryobiology since then have been the development of cryomicroscopy, allowing the observation of cell behavior during freezing and thawing ; devices to model and measure cell membrane permeabilities ; and mathematical modeling to describe the probability of IIF as a function of cooling rate, temperature, and cell type. Karlsson and colleagues incorporated into these models the effect of CPA addition on IIF formation and successfully predicted IIF formation as a function of cooling rate, temperature and CPA concentration, leading to optimal cooling protocols preventing IIF.
Thawing of Cells
Cells that have survived cooling to low temperatures still face the challenges of thawing, which can exert effects on survival comparable with those of cooling. The effects depend on whether the prior rate of cooling has induced intracellular freezing or cell dehydration. In the former case, rapid thawing can rescue many cells, possibly because it can prevent the harmful growth of small intracellular ice crystals into larger crystals by recrystallization.
Addition and Removal of Cryoprotective Agents
Cells require equilibration with molar concentrations of CPAs to survive freezing. However, these CPAs have dramatic osmotic effects on cells. Cells exposed to molar concentrations of permeating CPAs undergo extensive initial dehydration followed by rehydration and potential gross swelling when the CPAs are removed. Unless precautions are taken, this shrinkage and/or swelling can be extensively enough to cause cell damage and death. Knowledge of cell membrane permeability to water and CPAs allows the prediction of the minimal and maximal cell volume excursions during addition and removal of CPAs, providing a quantitative optimization approach (eg, stepwise increase or decrease of CPA concentration in cells or tissues) to avoid osmotic damage.
Fig. 1 shows the general process, cellular injury mechanisms, and optimization work for cell cryopreservation.
Cryopreservation of Whole Tissue
It is important to emphasize that successful cryopreservation of tissues is not a simple matter of extrapolating the well-established principles of cell cryopreservation to more complex tissues. Multicellular tissues are more complex than single cells, both structurally and functionally, and this is reflected in their requirements for cryopreservation. Successful cryopreservation of individual cells in the tissues is necessary, but not sufficient, for the successful cryopreservation of tissues. More than cell survival, complete structural integrity and function retention are vital for tissue cryopreservation.
Cryopreservation of complex tissues adds a set of additional problems to the known mechanisms of cryoinjury that apply to single cells in suspensions :
- 1.
Extracellular ice formation presents a major hazard for cryopreservation of multicellular tissues. The amount and location of extracellular ice affects the postthaw function of tissues. As a result, ice formation needs to be limited, restricted to harmless sites, or even totally prevented (which leads to the significance of vitrification).
- 2.
Vascular damage or rupture is caused by ice formation/expansion in capillaries and blood vessels. The rupture of capillaries by accumulating ice explains the deleterious effect of extracellular ice in severely damaged tissues. To minimize this cryodestructive effect, a better understanding of the fundamental mechanisms of ice formation and cell dehydration in tissues is required. Although these biophysical events have been extensively studied in single cells using cryomicroscopy, similar experimental data in whole tissues are still lacking.
- 3.
Thermal stress during the warming process causes frozen tissues to crack or fracture. Thermal stress is one type of mechanical stress caused by nonuniform heating in a frozen body, which can be reduced by uniform heating. However, because biological tissues have low thermal conductivities, high specific heats, and large volumes, conventional heating methods (eg, heating in a stirred water bath) cause large temperature gradients within the tissue, leading to high thermal stress and tissue fracture, especially for tissue with large volumes.
Cryopreservation of adipose tissue
Prior studies on the viability of cryopreserved fat tissues have shown a wide range of results, which were even contradictory and continuously in debate. For example, it was reported that no or very few harmful effects were generated on the fat samples cryopreserved at −20°C without controlling of the cooling process and even without CPA, and live adipocytes were found after preservation at this temperature. However, this opinion was questioned by many researchers. The adipocytes were destroyed after short-term freezing at −20°C and the transplantation of adipose tissues cryopreserved at −20°C provided an injection of mostly dead cells, which had no advantage compared with inert fillers and should be avoided. This may be one reason for the higher rate of volume adsorption after surgery when cryopreserved fat is transplanted. For another example, the procedures for fat cryopreservation were not well documented, or varied significantly in the literature. In most reports, scientific details of the cooling and thawing processes (eg, cooling/thawing rates, temperature, and processing time) were not provided. Some researchers provided different protocols from those that were used in their studies. Butterwick and colleagues found that fast freezing to −40°C and slow thawing over a couple of hours at room temperature for fat resulted in comparable effects with fresh fat transplantation for hand surgery. After a series of comparison experiments, Pu and Cui and colleagues concluded that controlled slow cooling (1–2°C/min from 22°C to −30°C) followed by transferring into liquid nitrogen, and fast thawing by stirring in a 37°C water bath until thoroughly thawed, led to the best results.
These results seem contradicting and confusing. However, they may be explained by a few uncertainties in the research reports. First, the viability and functionality of adipose cells and tissues after cryopreservation and transplantation can be affected by many variables, such as the methods of fat harvest, processing, storage, and injection; recipient and donor sites; pretreatment of fat tissues with cell culture medium or growth factors before transplantation; correction volumes; grafting intervals; and the instrument used. For cryopreservation, variables include CPA type and concentration, addition and removal of CPA (operation procedure, temperature, and duration), cooling and thawing protocols (temperatures, cooling, and thawing rates), storage temperature, and duration. Any change in these variables can contribute to the difference in results and make it difficult to compare studies. However, most reports failed to provide scientific details of these parameters. For example, cooling rate is the vital factor in cryopreservation and can lead to life or death for the cells and tissues. The cooling rate of biosamples depends on the cooling device, protocol, sample container, and sample volume. The best way to investigate the freezing process is to directly measure the temperature change profile in dummy samples during the whole process with temperature meters, which is also necessary for better quality control toward good manufacturing practice (GMP) products. However, this information was omitted in most studies.
The second uncertainty in the different studies is the methods used for the cell/tissue viability and functionality assessments. So far, several methods have been applied in the literature to assess adipose cells and tissues, which can be classified by cell membrane integrity assays; cell functional assays and tissue assessments, including extracellular glycerol-3-phosphate dehydrogenase assessment; trypan blue and flavin adenine dinucleotide/ethidium bromide for cell membrane integrity; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,3-bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2 H -tetrazolium (XTT) for mitochondrial activity, using cell surface markers to identify survived transplanted cells; histology; and weight and volume study for tissue evaluation. Different methods and different personnel may generate different results and conclusions. Some staining techniques like Trypan blue are recognized as inaccurate because mature adipocytes contain scant cytoplasm. Dead cells without nuclei or mitochondrial activity may be interpreted as live cells when the shapes of the cells appear normal under the microscope. All these factors also make it difficult to compare different studies.
Another significant variable in the literature for adipose cryopreservation is the CPA used. Besides the most widely used CPA, DMSO, many other kinds of CPAs were investigated for adipose cryopreservation in the last decade, including permeable and nonpermeable ones, such as glycerol, trehalose, sucrose, hydroxyethyl starch (HES), polyvinyl pyridine (PVP), and dextran. In spite of few reports claiming no or even negative effects of CPAs, most studies agreed that CPAs were needed for optimal cryopreservation of fat. Permeable and nonpermeable CPAs play their roles in cryoprotection with different mechanisms. A permeable CPA, like DMSO, is thought to protect cells against freezing injury by reducing ice formation inside and outside the cells. It can also penetrate into cells, fill the intracellular space, and prevent severe cell shrinkage during cell dehydration (water transport across cell membrane). Nonpermeable CPAs, like trehalose and HES, may provide protection in several ways. They dehydrate cells, thus reducing the amount of intracellular water before freezing. They also enhance the vitrification tendency of the solution, and stabilize cell membranes and proteins during freezing and drying. It was found that trehalose alone or together with DMSO at reduced concentration provided comparable cryoprotection in adipose cryopreservation. Besides the mechanisms mentioned earlier, the special cryoprotection function of trehalose may be related to its unique properties. As a type of disaccharide, trehalose has a large hydration radius (about 2.5 times that of other common sugars) and distinctly higher glass transition temperature than other sugars, and so can function better to keep the cell membrane intact after dehydration, facilitate the ion transport through the membrane, decrease the melting point of the membrane lipids, maximize the stability of proteins, and prevent injury to membranes caused by membrane phase transition. Therefore, more extensive studies are needed on the cryoprotection function of trehalose for adipose cells and tissues.
DMSO can protect cells from cryoinjury; however, it is also cytotoxic, especially at high temperatures (such as room temperature or human body temperature). Infusion of frozen-thawed cellular products together with DMSO has been associated with several types of adverse reaction, ranging from mild events like nausea/vomiting, hypotension or hypertension, abdominal cramps, diarrhea, and flushing and chills to more severe life-threatening events like cardiac arrhythmia, encephalopathy, acute renal failure, and respiratory depression. Hence, it is preferable for DMSO to be removed before the infusion. Removal of DMSO can cause cell loss caused by osmotic injury and clumping; in addition, it is time consuming, needs special equipment and expertise, and may introduce contamination during processing. Thus, cryopreservation media with reduced DMSO concentration, or even a DMSO-free alternative CPA, are desirable. Pu and colleagues found that, by using optimized freezing and thawing protocols, trehalose plus DMSO at reduced concentration, or even trehalose alone, could provide comparable results for adipose tissue cryopreservation ( Fig. 2 ). Further progress will increase the clinical applications of fat transplantation.
