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Tunable hydrogels have gained significant attention in the bioengineering field due to their designer preparation approach. Towards this end, gelatine stands out as a promising candidate owing to its desirable attributes, such as biocompatibility, ability to support cell adhesion and proliferation, biodegradability, and cost-effectiveness. This study presents the preparation of a robust gelatine hydrogel employing sacran aldehyde (SDA) as a natural cross-linker. The resulting SDA-cross-linked gelatine hydrogels (GSDA) display an optimal compressive stress of 0.15 MPa at 50% strain, five times higher than pure gelatine hydrogel. As SDA cross-linking concentration is increased, the swelling capacity of GSDA declines. This decline in swelling capacity, from 80% to 40%, is a result of strong crosslinking of gelatin with SDA. Probing further with FT-IR spectroscopy and SEM at the micron scale unveiled a dual-cross-linking mechanism within the hydrogels. This mechanism encompasses both short- and long-range covalent cross-linking, along with thermo-induced physical cross-linking, resulting in a significant enhancement of the load-bearing capacity of the fabricated hydrogels.

Abstract Intracellular ice formation during cryopreservation is lethal to the cell, including during warming. Here, we examined the effect of sample volume and warming rate on the cryopreservation success of 1-cell rat embryos based on successful development into blastocysts in vitro and to term in vivo following embryo transfer. Embryos were equilibrated in 5% propylene glycol solution for 10 min, held for 40 s at 0 °C in cryopreservation solution (5%PG + PEPeS), and cooled by immersion in liquid nitrogen. When 1-cell embryos were cryopreserved in a volume of 30–100 μL at a cooling rate of 5830–7160 °C/min and warmed at 35,480–49,400 °C/min by adding 1 mL of 0.3 M sucrose solution at 50 °C, 17.3–28.8% developed into blastocysts, compared with 57.0% of untreated embryos. However, when 1-cell embryos were cryopreserved in a smaller volume of 15 μl at 7950 °C/min and warmed at 68,850 °C/min, 58.8 ± 10.6% developed into blastocysts and 50.0 ± 7.4% developed to term, comparable to that of non-treated embryos (57.0 ± 5.4% and 51.4 ± 3.1%, respectively). Cryopreserved embryos at other developmental stages also showed high in vitro culture potential similar to that of the control. Using a conventional cryotube and a small-volume vitrification procedure with rapid warming, we achieved high levels of subsequent rat embryonic development at all developmental stages.

The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.

LYDEX, which is a hydrogel composed of aldehyde-functionalized dextran and succinic anhydride-treated ε-poly-L-lysine, was previously developed by our group as a self-degradable adhesive with an excellent functionality. As a result, this hydrogel has been investigated for a wide range of medical applications, including hemostatic agents, sealants, wound dressings for endoscopes, and anti-adhesion materials. Previously, the unique potential of LYDEX to control the in vivo degradation rate depending on its composition was verified for use in anti-adhesion applications. Although the gel thickness also plays a significant role in determining the degradation rate, this issue was not sufficiently investigated in previous studies. Thus, in the current study, the optimal LYDEX dose (film thickness) for maximizing the anti-adhesion material performance was verified using a rabbit colon model and it was confirmed that the gel film thickness affects the degradation period and anti-adhesion effect. The obtained results suggest the potential for designing LYDEX materials suitable for application at a wider range of in vivo treatment sites, which could be extremely valuable for the development and use of novel anti-adhesion materials with adequate hemostatic and sealant performances.

Cellular cryopreservation is an important tool in modern biology. Here, previously reported polyampholyte cryoprotectants are studied by solid-state NMR, revealing the molecular mechanisms at play.

Introduction: Regenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinical application of chondrocyte sheet therapy is expected to be further advanced by the use of a feasible cryopreservation technique. Previously, we developed a chondrocyte sheet vitrification method; however, it was too complex to be used for routine clinical application. Here, we aimed to develop a prototype method for vitrifying chondrocyte sheets for clinical practice. Methods: We developed a “circulating vitrification bag” as a container to process cell sheets for vitrification in an efficient and sanitary fashion. Moreover, we invented the “vitrification storage box”, which is useful for the vitrification of cell sheets, long-term preservation, and transportation. These devices were used to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristics and the viability of the component cells after rewarming. Results: In all cell sheet samples (n = 7) vitrified by the circulating vitrification bag method, the integrity of the sheet structure was maintained, and the cell survival rate was similar to that of non-vitrified samples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major components of cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitrification similarly to that observed in the non-vitrified sheet. After long-term storage using the vitrification storage box, the cell sheets maintained normal structure and cell viability (survival rate: 81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitrified sheet. Conclusion: Our results indicate that the circulating vitrification bag method is an effective approach for realizing the clinical application of vitrified chondrocyte sheets. The vitrification storage box is also useful for the long-term preservation of vitrified cell sheets, further enhancing the feasibility of the clinical application of cryopreserved chondrocyte sheets.

In recent years, regenerative medicine research using human somatic and induced pluripotent stem cells has advanced considerably, promoting clinical applications. However, it is essential that these cells are cryopreserved safely and effectively. Most cryopreservation solution agents contain dimethyl sulfoxide (DMSO), which exhibits strong toxicity and can potentially promote cell differentiation. Hence, it is important to explore substitutes for DMSO in cryoprotectant solutions. One such alternative is StemCell Keep (SCK), a DMSO-free solution that has been reported to effectively cryopreserve human induced pluripotent stem cells (hiPS cells). To clarify the effect of cryopreservation agents on cells, DNA microarray analysis is useful, as it can identify a large number of gene expression differences in cryopreserved cells, as well as functional increases in gene groups. In this study, we performed gene expression analysis of SCK-cryopreserved hiPS cells using a DNA microarray gene chip. The hiPS cells vitrified with SCK or DMSO-based vitrification solutions were thawed and cultured on Matrigel under feeder-free conditions, and RNA was extracted for DNA microarray analysis. Genes obtained from DNA microarray data were classified by the keywords of Gene Ontology Biological Process Term, and their relationships were analyzed using DAVID or the GeneMANIA database.SCK-cryopreserved hiPS cells expressed several anti-apoptotic genes, as well as genes related to cell adhesion or proliferation at levels that were nearly equivalent to those of non-frozen hiPS cells. Gene enrichment analysis with selected genes of SCK-cryopreserved hiPS cells whose expression differences were superior to those of DAP-cryopreserved showed strong interactions of negative regulation of apoptotic process, cell adhesion and positive regulation of cell proliferation in DAVID analysis. We demonstrated that SCK successfully maintained the key functions of hiPS cells, including anti-apoptosis, cell adhesion, and cell proliferation, during cryopreservation.

A hydrogel consists of a three-dimensional network of polymer chains, with water as a solvent in the system [...]

Safe and stable cryopreservation is critical for research involving human embryonic stem cells (hESCs). Dimethyl sulfoxide (DMSO) is a popular cryoprotective agent; however, its cytotoxicity cannot be ignored. Thus, there is a need for an alternate cryoprotectant. We reported previously that a novel cryopreservation reagent, StemCell Keep™ (SCK), was effective for cryopreserving human induced pluripotent stem cells (hiPSCs) by vitrification. Because hESCs and hiPSCs are not identical, the current study examined the use of SCK on hESCs. hESCs cryopreserved with SCK were thawed and cultured on SNL 76/7 cells, which were derived from a mouse fibroblast STO cell line transformed with neomycin resistance and murine LIF genes. After cryopreservation, cultured hESCs were assessed for their attachment ability and characterized by alkaline phosphatase (AP) and immunocytochemical (ICC) staining, fluorescence-activated cell sorting (FACS), reverse transcription polymerase chain reaction (RT-PCR), and karyotyping. The proliferation of SCK-cryopreserved hESCs cultured on SNL cells, or in feeder-free conditions, was higher than that of cells preserved in a solution of 2 M DMSO, 1 M acetamide, and 3 M propylene glycol (DAP). The cell number with SCK-cryopreserved hESCs was about twice that of hESCs cryopreserved in DAP. The pluripotency of SCK-cryopreserved hESCs was similar to that of DAP-cryopreserved hESCs based on AP staining. Data from ICC, FACS, and RT-PCR analyses showed that stem cell markers were continually expressed on SCK-cryopreserved hESCs. The teratoma assay showed that SCK-cryopreserved hESCs differentiated into three germ layers. Furthermore, SCK-cryopreserved hESCs had normal karyotypes. These data indicate that SCK was effective for cryopreservation of hESCs by vitrification.

Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p

Poly (vinyl alcohol) hydrogel (PVA-H) is expected to be a suitable artificial articular cartilage material because of its high biocompatibility. However, it is difficult to affix to the surface of a living joint because it is bioinert and its mechanical strength needs to be improved. In this study, graphene oxide (GO) subjected to two oxidation rounds was used to form a nanocomposite material and the composite hydrogel PVA-GO-H was prepared by low-temperature crystallization. Scanning electron microscope (SEM) images showed that the addition of GO can increase roughness of the hydrogel surface. Contact angle measurements showed that the surface of PVA-GO-H exhibited hydrophobicity that increased with GO concentration and not with that of PVA-H, indicating that the hydrophilic parts of PVA and GO form hydrogen bonds and the hydrophobic part of GO was exposed on the surface. Tensile tests demonstrated that Young’s modulus was enhanced on the addition of GO. Osteoblast cells showed more affinity for PVA-GO-H than PVA-H, which much more cells adhere to than to PVA-GO-H after a certain period of culturing, suggesting GO can improve the cell attachment of PVA-H. Further studies on the influence of the oxidation time of GO are still required.

In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA), called as carboxylated ε-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.

Since donor T-cells' allorecognition of host antigens is a prerequisite for the onset of graft-versus-host disease (GVHD), blocking their cellular signaling pathways can decrease the severity of GVHD. We hypothesized that epigallocatechin-3-gallate (EGCG), due to its strong affinity to macromolecules, would adhere to surface molecules of donor T cells, inhibit their allorecognition, and attenuate GVHD in the recipient. We tested the hypothesis by treating donor splenocytes with EGCG in both in vitro and in vivo murine GVHD models. EGCG treatment decreased the proliferation of donor cells in MLR cultures and secretion of IL-2 and INF-γ. It also reduced the epitope detection of CD3∊, CD4, and CD28 but did not downregulate the protein expression of these molecules, suggesting blockage of cell surface stimulatory signals. Similarly, EGCG treatment did not decrease mRNA expression for some of these molecules but decreased mitogen-induced cell proliferation, indicating that EGCG did not interfere the transcription of these genes but affected cell proliferation pathways. Furthermore, EGCG-treated donor splenocytes, when transplanted into immunocompromized recipient mice, decreased of proliferation, and the treatment extended the recipients' survival at least during the early stage of GVHD. These results strongly suggest that EGCG attenuates GVHD by both blocking specific cell surface molecules and affecting the donor T-cell proliferation pathways.

Cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol have been used for the cryopreservation of cells and tissues. DMSO is the most effective CPA but shows high cytotoxicity and can effect differentiation. ∈-Poly-l-lysine (PLL) derivatives show higher cryopreservation efficiency than the conventional CPAs. Culture medium solutions with 7.5 w/w% of PLL whose amino groups of more than 50 mol% were converted to carboxyl groups by succinic anhydride showed higher postthaw survival efficiency of L929 cells than those of current CPAs without the addition of any proteins. In addition, rat mesenchymal stem cells were cryopreserved more effectively than with DMSO and fully retained the potential for proliferation and differentiation. Furthermore, many kinds of cells could be cryopreserved with PLL having the appropriate ratio of COOH groups, regardless of the cell types, including adhesive and floating cells, human- and mouse-derived cells, primary cells, and established cell lines. The properties might be associated with the antifreeze protein properties. These results indicate that these polymeric extracellular CPAs may replace current CPAs and the high viability after thawing and nonnecessity of serum ensure that these CPAs may be used in various preservation fields.

A fresh osteochondral allograft is one of the most effective treatments for cartilage defects of the knee. Despite the clinical success, fresh osteochondral allografts have great limitations in relation to the short storage time that cartilage tissues can be well-preserved. Fresh osteochondral grafts are generally stored in culture medium at 4°C. While the viability of articular cartilage stored in culture medium is significantly diminished within 1 week, appropriate serology testing to minimize the chances for the disease transmission requires a minimum of 2 weeks. (–)-Epigallocatechin-3- O -gallate (EGCG) has differential effects on the proliferation of cancer and normal cells, thus a cytotoxic effect on various cancer cells, but a cytopreservative effect on normal cells. Therefore, a storage solution containing EGCG might extend the storage duration of articular cartilages. Rabbit osteochondral allografts were performed with osteochondral grafts stored at 4°C in culture medium containing EGCG for 2 weeks and then the clinical effects were examined with macroscopic and histological assessment after 4 weeks. The cartilaginous structure of an osteochondral graft stored with EGCG was well-preserved with high cell viability and glycosaminoglycan (GAG) content of the extracellular matrix (ECM). After an osteochondral allograft, the implanted osteochondral grafts stored with EGCG also provided a significantly better retention of the articular cartilage with viability and metabolic activity. These data suggest that EGCG can be an effective storage agent that allows long-term preservation of articular cartilage under cold storage conditions.

Skin grafts can be preserved by cryopreservation and refrigerated storage at 4°C. Epigallocatechin-3- O -gallate (EGCG) enhances the viability of stored skin grafts and also extends the storage time up to 7 weeks at 4°C. EGCG, the major polyphenolic constituent present in green tea, has potent antioxidant, antimicrobial, antiproliferative, and free radical scavenging effects. This study examined the effects of EGCG on skin cryopreservation. Skin sample biopsy specimens from GFP rats were previously treated with/without EGCG then moved to −196°C. Skin samples were transplanted to nude mice after 2, 8, and 24 weeks of preservation. Glucose consumption was measured after thawing to assess the metabolic activity. Two weeks later the transplanted skin grafts were excised and histologically analyzed. Histological examinations revealed the degeneration of the epidermal and dermal layers in all groups. In the EGCG groups, the grafts showed higher integrity in the epidermal layer and dermal matrix. The present findings suggest the future clinical usefulness of EGCG for skin preservation; however, the mechanism by which EGCG promotes skin preservation still remains unclear.

The effect of epigallocatechin-3- O -gallate (EGCG), a major component of green tea, on platelet preservation was evaluated. Single donor platelets ( N = 10) were collected and preserved by the standard method. EGCG was added to the platelet concentrates before preservation and then the functional and biochemical parameters were monitored throughout the storage period. After 6 days of preservation, the aggregability of the platelets was significantly maintained by addition of 50 and 100 μg/ml of EGCG. Platelet prothrombinase activity was also significantly retained by the addition of EGCG. The accumulation of P-selectin and RANTES in the plasma preserved with EGCG was less than those preserved without EGCG, which indicated that EGCG might inhibit platelet activation. Furthermore, EGCG reduced the increase of LDH in plasma during preservation and inhibited the activation of caspase-3 and cleavage of gelsolin, thereby showing that EGCG could inhibit the apoptosis of platelets. These results suggest that EGCG may play an effective role in preserving platelets by inhibiting the activation and apoptosis of platelets.

We investigated the hibernation effect of epigallocatechin-3- O -gallate (EGCG) on neonatal human tarsal fibroblasts (nHTFs) by analyzing the expression of cell cycle-related genes. EGCG application to culture media moderately inhibited the growth of nHTFs, and the removal of EGCG from culture media led to complete recovery of cell growth. EGCG resulted in a slight decrease in the cell population of the S and G 2 /M phases of cell cycle with concomitant increase in that of the G 0 /G 1 phase, but this cell cycle profile was restored to the initial level after EGCG removal. The expression of cyclin D1 (CCND1), CCNE2, CCN-dependent kinase 6 (CDK6), and CDK2 was restored, whereas that of CCNA, CCNB1, and CDK1 was irreversibly attenuated. The expression of a substantial number of genes analyzed by cDNA microarray was affected by EGCG application, and these affected expression levels were restored to the normal levels after EGCG removal. We also found the incorporation of FITC-EGCG into the cytosol of nHTFs and its further nuclear translocation, which might lead to the regulation of the exogenous signals directed to genes for cellular responses including proliferation and cell cycle progression. These results suggest that EGCG temporarily affects not only genes related to the cell cycle but also various other cellular functions.

Green tea polyphenols are known to protect allogenic donor tissues from acute rejection by their recipients. This immunosuppressive effect may be generated by a unique chemical property of the major component, epigallocatechin-o-gallate (EGCG), which can block specific cell surface molecules of the donor tissues. To test this hypothesis, we examined the effects of EGCG on the murine mixed lymphocyte reactions. EGCG treatment of stimulator cells significantly attenuated the proliferation of responder T cells. The proliferation did not recover upon the secondary stimulations by fresh untreated cells or exogenous IL-2. Flow cytometric analyses showed that EGCG treatment decreased the staining intensities of various cell surface molecules including MHC II, which plays a major role in antigen presentation, and B7.1, B7.2, and their ligand, CD28, which are required for costimulatory signals in T-cell activation. These results suggest that an anergic state of alloreactive T cells may be induced by either weakening of antigen signaling or blockage of costimulatory signals with EGCG. Other possible mechanisms behind the immunosuppressive effect and a potential use of EGCG treatment of donor tissues in transplantation medicine are discussed.