2.1. Alginate Hydrogel Cryopreservation System
Alginate is a kind of natural polysaccharide extracted from seaweed and is a commonly used material for cell encapsulation because of its abundance and good biocompatibility. For cell cryopreservation based on microencapsulation technology, a variety of alginate capsules demonstrated a high efficiency in encapsulating living cells, aggregation and tissues, which could optimize the cryopreservation procedure, improve the survival rate and retain the normal functions of cells more than the traditional methods [31
The alginate hydrogels are obtained by the crosslinking between alginate and some divalent or trivalent cations. The crosslink can be achieved at room temperature, and the common cations, Ca2+
are dedicated to forming biocompatible alginate hydrogels for biomedical and biological applications [34
]. In the field of the cryopreservation of living cells, as mentioned above, the formation of ice crystals and the use of high-concentration CPAs lead to the loss of cell viability and function, and so it is necessary to reduce the addition of CPAs and synchronously decrease the amounts of ice crystals during the cooling and thawing steps. Compared with standard slow freezing or vitrification methods, the ice crystal growth was inhibited in a three-dimensional network of hydrogel, but not open as with the former, and the osmotic pressure was also minimized when CPAs permeated into cells, which guaranteed a low CPA as well as ice crystal formation and osmotic injury. With respect to the storage of living cells in hydrogels, the existing studies have indicated that alginate hydrogels can effectively reduce the dosage of CPAs and minimize the amounts of ice crystals for high levels of viability and the maintenance of normal function.
Huang and co-workers reported a low-CPA vitrification method to cryopreserve mouse embryonic stem cells and human adipose-derived stem cells in a conventional plastic straw [36
]. After cells were encapsulated in alginate hydrogel via a nonplanar microfluidic device, the cells microencapsulated in alginate hydrogel had an obvious impact on inhibiting intracellular ice formation when cells were thawed at physiological temperature (Figure 1
). The ice formation inside the microcapsule was inhibited throughout the whole thawed process. Compared with the traditional low-CPA vitrification using a quartz microcapillary, the alginate hydrogel cryopreservation system achieved a low-CPA concentration of 2 mol/L (approximately four times lower) and a sample volume of 250 µL (100 times larger). Obviously, the preferential vitrification of the bulk solution occurred outside the alginate microcapsules, which inhibited the ice crystal injuries of cells in alginate hydrogel microcapsules due to the devitrification during warming. The alginate hydrogel cryopreservation system can effectively confine the ice formation more than the common slow freezing method.
Compared with cells, tissues have more complex structures and inner interaction systems including the compact stroma tissue and cell types, and so their encapsulation and cryopreservation must be distinguished. Xu and co-workers investigated in follicle maturation (IFM) using a slow-freezing strategy in an alginate hydrogel cryopreservation system, which was an option for the fertility maintenance of special human [29
]. During the freeze–thaw process, partial follicles survived and grew into a mature oocyte. In this regard, the authors cryopreserved the ovarian tissue in both cortical strips and stroma in follicles (Cryo-Ov) and individually isolated follicles (Cryo-In). Subsequently, the three groups, Cryo-Ov, Cryo-In and non-cryopreserved control follicles, were cultured in an alginate hydrogel system with a unique three-dimensional structure for 12 days, which served as an assessment of follicle growth and oocyte maturation. The results of a culture period of 12 days showed that the Cryo-Ov group had lower androstenedione levels and the Cryo-Ov and Cryo-In group had a higher ratio of progesterone to estradiol. At the culture stage of 6 days, the cryopreservation group had a decreased Gja1 (Gap junction protein alpha 1, known as connexin 43) and Gja4 (Gap junction protein alpha 4 known as connexin 37) mRNA expression, and the Gja1 mRNA expression of three groups arrived at the same level after 12 days. Thus, the thawed follicles could maintain good viability and growth characteristics as fresh follicles, and could also be matured in eggs. The alginate hydrogel maintained follicular cell–cell and cell–matrix connections by the structure of a three-dimensional matrix, which was important for proper follicle development and oocyte maturation. Thus, the alginate hydrogel cryopreservation system can be applied in the cryopreservation of more complex cells or tissues.
In Perteghella’s work, buffalo (Bubalus bubalis
) spermatozoa and Holstein Friesian (Bos taurus
) semen were encapsulated and cryopreserved [37
], which served for the monitoring of buffalo’s ovarian activity affected by climate change. Besides this, the defects of artificial insemination (AI) were also covered. The basic freezing and thawing process included an equilibration of 3 h at 4 °C, with the sustained spermatozoa freezing steps being from +4 °C to −10 °C (rate: −5 °C/min in bovine, −3 °C/min in buffalo), −10 °C to −100 °C (rate: −30 °C/min in bovine, −40 °C/min in buffalo), and −100 °C to −140 °C (rate, −20 °C/min in bovine, −20 °C/min in buffalo) and subsequent thawing at 37 °C for 1 min. The results showed that the progressive motility, path average velocity and pregnancy rates in either species were not obviously decreased, and the detrimental effects in the encapsulation process had not been observed.
In the actual cell-based application, the survival and function of cells in short-storage could be retained effectively in alginate capsules. Chen and co-workers used alginate hydrogels to encapsulate human mesenchymal stem cells (hMSCs) and mouse embryonic stem cells (mESCs) for short-term storage of stem cells [38
]. After successful storage within alginate hydrogels for 5 days at 18–22 °C in sealed cryovials, the cell viability of hMSC and mESCs released from hydrogels achieved 74% and 80%, respectively. Moreover, the proliferation and expression of routine cell markers of hMSCs and mESCs showed no significant difference between alginate-encapsulated cells and cells cryopreserved in liquid nitrogen. The microcapsule membrane could cryopreserve the common cells, but this also limited and hindered some special functions of partial cell specials such as the adherence, migration, proliferation and differentiation of adherent cells.
The encapsulation-based cryopreservation technique is an effective method to monitor the activity and assess the quality of living cells and obtain relevant numbers of characterized cells prior to cell-based therapy. For example, Chen and co-workers proposed a novel islet quality assays method. The single rat pancreatic islets and fluorescent oxygen-sensitive dye (FOSD) were encapsulated in alginate hydrogel microcapsules via microchannels (Figure 2
], and single-islet capsules embedding FOSD were cryopreserved for islet function simulation and evaluation. Here, FOSD was embedded to label and characterize the real-time oxygen uptake of individual islets. In the cryopreservation system of islet capsules with FOSD, the whole process of encapsulation and the cooling/thawing procedure had little impact on the function of FOSD, which provided a reliable assessment. Correspondingly, adenosine triphosphate (ATP), static insulin release measurement, and the oxygen consumption rate were tested for the assessment of the functions of encapsulated single-islet microcapsules after storing for 1 days and 7 days at −80 °C (Figure 3
). These results implied that an individual-islet-based quality control method was proved simply and reliably for the fast and real-time assessment of the islet processing procedure in transplantation. A further result revealed that the alginate capsules benefitted the increased survival rate of thawed islets and the islets could release insulin in the alginate capsules. In addition, trehalose, added as a cryoprotectant during the freezing process, was suitable to improve the activity of the thawed islets. It is notable that the dye acted as a real-time single-islet oxygen sensor, which could analyze the viability and function of islets. This method offered a simple and real-time strategy to assess the viability of a single islet and enriched available islet source and also had a potential to face the current challenge in islet transplantion.
In clinical and industrial applications, the production of human embryonic stem cells (hESCs) with the feature of cell integrality and their long-term storage is a major challenge. An integrated expansion and cryopreservation strategy for hESCs was reported by Serra and co-workers [40
]. Single cells, aggregates and immobilized microcarriers were microencapsulated (Figure 4
). The microcarriers and aggregates were obtained after culturation for 13 and 14 days, and were pre-treated with 5 mM ROCKi (Rho kinase (ROCK) inhibitor) for 1 h for the following cryopreservation. In the freezing process, the cryopreservation equilibration was conducted for 20 min at 4 °C and subsequently frozen to −80 °C at a rate of 1 °C/min. The thawing process was conducted quickly on cryovials in a 37 °C water bath, diluted and cultured in certain conditions for a further assessment. The results showed that the hESCs lost viability quickly in single cell microcapsules and differentiated spontaneously in aggregate microcapsules. Furthermore, the results of the microencapsulation of hESC microcarriers showed that cell concentrations increased approximately twice and exhibited over 70% of cell recovery yield, and three times the survival rate of encapsulated hESCs compared with non-encapsulated cells, which was a prospective protocol for the scalable production and storage of pluripotent hESCs in a synergetic bioprocess. The microcapsulation of stem cells was a benefit to the culture of its aggregates, which could adjust the size of aggregates and maintain the integrality properties of cells for more than 2 weeks. As opposed to the traditional three-dimensional cell culture system, this research was promising to apply to clinical medicine, and combined cell proliferation with cell cryopreservation.
2.2. Chitosan/Alginate Hydrogel Cryopreservation System
The permeability and morphology of alginate capsules, and the biocompatibility of membrane materials, are related to cell cryopreservation. Thus, some biocompatible compounds, such as poly-l
], chitosan [44
], amino acid and derivatives [47
], gelatin [49
] and collagen [51
] are usually employed for the chemical modification of the alginate capsule membrane. Based on different cell encapsulation requirements, recently, these modified alginate hydrogels have been gradually designed for the protection of various cells in the corresponding cryopreservation systems. For the high survival rate of probiotic bacteria S. phocae
PI80, the frozen parameters were optimized with different cryopreservation methods and cryoprotectants by Kanmani and co-workers [52
]. In the common cryopreservation method, the relative viability of S. phocae
PI80 was retained at 74.6 ± 5.9% in an optimal trehalose system at −20 °C after storage for 6 months. When S. phocae
PI80 cells were encapsulated with alginate–chitosan hydrogels, a high survival rate of S. phocae
cells with high bacteriocin activity was obtained at −20 °C after storage for 6 months. Furthermore, the immobilization of cells was in a position to resist an acidic environment in simulated gastrointestinal conditions. The capsules were broken after 6 h in vivo treatment, and probiotic cells could enter the intestinal tract.
The results of Hardikar and his co-workers also showed that islets could perform with higher viability and functionality than the routine method in the chitosan–alginate encapsulation system [53
]. The islet was encapsulated and cryopreserved via a routine method. Here, an islet-suspended solution in sodium alginate at the ratio of 500 islets/mL to alginate was used for the formation of islet beads in a microfluidic method. The cryopreservation was conducted including equilibration for 5 min at 22 °C in a 0.1 mL of 2 mol/L dimethyl sulfoxide (Me2SO), 0.4 mL of 3 mol/L Me2SO for 25 min at 0 °C, and finally, 2 mol/L Me2SO for 5 min at −7.5 °C. The thawing process was at 37 °C in a water bath of cryovials. The authors indicated that the islets used trypan blue (0.4% w
, 0.4 g/100 mL solution) for the assessment of the percentage of islet viability. Different groups including encapsulated, nonencapsulated and freshly isolated groups were compared, and the results showed that the cell viability of the encapsulated group (value: 95.4 ± 1.3%) was much higher than that of the nonencapsulated group (value: 69.4 ± 3.5%), which maintained the same level as that of the freshly isolated group (value: 97.5 ± 0.8%). Insulin release in glucose at a concentration of 16 mmol/L revealed a similar effect, and the value of the encapsulated, nonencapsulated and freshly isolated groups were 247 ± 14 mU/10 islets, 110 ± 32 mU/10 islets and 255 ± 27 mU/10 islets, respectively.
To date, the development of alginate-based microencapsulation has been demonstrated to optimize freezing and thawing procedures and minimize cryo-injury; however, there are still some problems in developing cryopreservation protocols for microencapsulated cells. It is difficult for he alginate capsules used for cell protection to match the size of the encapsulated cell and simultaneously ensure specific biocompatibility [42
], which gives an opportunity for the extra space for ice formation to bring about ice crystal injury. Besides this, the poor uniformity and fragile semipermeable alginate membrane reduce the chance of CPAs entering the capsules, which causes a low efficiency of CPAs. During the freezing process, unpredictable thermal stress may induce the formation of cracks in the weaknesses of the capsule material, which dramatically changes the capsule permeability for the cell damage. The difficult removal of the capsule membrane also limits its application, and so a novel alginate modified method may be beneficial to extend the alginate-based hydrogel cryopreservation system.