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Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.

Abstract. Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 µl of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/ group). The HFV method was also proven to be effective for vitrifying porcine in vitro-and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs. Key words: Cryopreservation, Hollow fiber, Mouse embryos, Pig embryos, Vitrification (J. Reprod. Dev. 58: [599][600][601][602][603][604][605][606][607][608] 2012) E mbryo cryopreservation is widely and routinely used both for research in reproductive biology and for the development of practical applications in animal industries and human reproductive medicine. The cryopreservation of oocytes and embryos is essential for the long-term preservation of valuable genetic resources in experimental and livestock animals [1][2][3][4][5], and the cryopreservation of embryos improves pregnancy rates in assisted reproductive technology applications [6][7][8]. Various protocols have been developed for the cryopreservation of animal embryos, and many are used in research and clinical applications [1,5,9].In recent years, embryo vitrification has been applied to an increasing number of animal species, and high post-cryopreservation embryo viability has been achieved [10,11]. The basic concept of vitrification is as follows: a solution containing a high concentration of a cryoprotective agent (CPA) is rapidly cooled, causing it to transform from the liquid phase to the solid phase without forming ice crystals. As a result, cells suspended in the solution are preserved in an ultra-low temperature glassy (amorphous) material [10,12]. To create this glassy state, a solution containing a high concentration of CPA (normally 4-6 M) needs to be cooled ultrarapidly. Howeve...

Osteoarthritis of the knee (OAK) is a chronic degenerative disease and progresses with an imbalance of cytokines and macrophages in the joint. Studies regarding the use of platelet-rich plasma (PRP) as a point-of-care treatment for OAK have reported on its effect on tissue repair and suppression of inflammation but few have reported on its effect on macrophages and macrophage polarization. Based on our clinical experience with two types of PRP kits Cellaid Serum Collection Set P type kit (leukocyte-poor-PRP) and an Autologous Protein Solution kit (APS leukocyte-rich-PRP), we investigated the concentrations of humoral factors in PRPs prepared from the two kits and the effect of humoral factors on macrophage phenotypes. We found that the concentrations of cell components and humoral factors differed between PRPs purified using the two kits; APS had a higher concentration of M1 and M2 macrophage related factors. The addition of PRP supernatants to the culture media of monocyte-derived macrophages and M1 polarized macrophages revealed that PRPs suppressed M1 macrophage polarization and promoted M2 macrophage polarization. This research is the first to report the effect of PRPs purified using commercial kits on macrophage polarization.

BackgroundThere is considerable interest in using cell sheets for the treatment of various lesions as part of regenerative medicine therapy. Cell sheets can be prepared in temperature-responsive culture dishes and applied to injured tissue. For example, cartilage-derived cell sheets are currently under preclinical testing for use in treatment of knee cartilage injuries. The additional use of cryopreservation technology could increase the range and practicality of cell sheet therapies. To date, however, cryopreservation of cell sheets has proved impractical.ResultsHere we have developed a novel and effective method for cryopreserving fragile chondrocyte sheets. We modified the vitrification method previously developed for cryopreservation of mammalian embryos to vitrify a cell sheet through use of a minimum volume of vitrification solution containing 20% dimethyl sulfoxide, 20% ethylene glycol, 0.5 M sucrose, and 10% carboxylated poly-L-lysine. The principal feature of our method is the coating of the cell sheet with a viscous vitrification solution containing permeable and non-permeable cryoprotectants prior to vitrification in liquid nitrogen vapor. This method prevented fracturing of the fragile cell sheet even after vitrification and rewarming. Both the macro- and microstructures of the vitrified cell sheets were maintained without damage or loss of major components. Cell survival in the vitrified sheets was comparable to that in non-vitrified samples.ConclusionsWe have shown here that it is feasible to vitrify chondrocyte cell sheets and that these sheets retain their normal characteristics upon thawing. The availability of a practical cryopreservation method should make a significant contribution to the effectiveness and range of applications of cell sheet therapy.

In vitro matured (IVM) oocytes have been used to create genetically modified pigs for various biomedical purposes. However, porcine embryos derived from IVM oocytes are very cryosensitive. Developing improved cryopreservation methods would facilitate the production of genetically modified pigs and also accelerate the conservation of genetic resources. We recently developed a novel hollow fiber vitrification (HFV) method; the present study was initiated to determine whether this new method permits the cryopreservation of IVM oocyte-derived porcine embryos. Embryos were created from the in vitro fertilization of IVM oocytes with frozen-thawed sperm derived from a transgenic pig carrying a humanized Kusabira-Orange (huKO) gene. Morula-stage embryos were assigned to vitrification and nonvitrification groups to compare their in vitro and in vivo developmental abilities. Vitrified morulae developed to the blastocyst stage at a rate similar to that of nonvitrified embryos (66/85, 77.6% vs. 67/84, 79.8%). Eighty-eight blastocysts that developed from vitrified morulae were transferred into the uteri of three recipient gilts. All three became pregnant and produced a total of 17 piglets (19.3%). This piglet production was slightly lower, albeit not significantly, than that of the nonvitrification group (27/88, 30.7%). Approximately half of the piglets in the vitrification (10/17, 58.8%) and nonvitrification (15/27, 55.6%) groups were transgenic. There was no significant difference in the growth rates among the piglets in the two groups. These results indicate that the HFV method is an extremely effective method for preserving cryosensitive embryos such as porcine in vitro maturation/fertilization-derived morulae.

BackgroundWe previously conducted a first-in-human clinical study of articular cartilage repair using autologous chondrocyte sheets and confirmed the regeneration of hyaline-like cartilage in all eight patients. However, regenerative medicine with autologous chondrocyte sheets requires the harvesting of tissue from healthy regions, and the quality of this tissue varies between individuals. To overcome such limitations, allogeneic transplantation is a promising treatment method, particularly for articular cartilage repair. In this study, we investigated the characteristics of polydactyly-derived chondrocyte sheets fabricated from the chondrocytes of young polydactyly donors.MethodsPolydactyly-derived chondrocyte (PD) sheets were fabricated from the tissue obtained from eight polydactyly donors (average age = 13.4 months). To create these PD sheets, chondrocytes at passage 2 or 3 were seeded on temperature-responsive culture inserts and cultured for 2 weeks. For comparison, adult chondrocyte sheets were fabricated from tissue obtained from 11 patients who underwent total knee arthroplasty (TKA; average age = 74 years). To create these TKA sheets, chondrocytes and synovial cells were cocultured, and the chondrocyte sheets were triple-layered according to the protocol from our previous clinical study. Cell count, cell viability, cell surface markers, cell histology, and humoral factors secreted by the sheets were characterized and compared between the PD sheets and TKA sheets.ResultsPolydactyly-derived chondrocytes proliferated rapidly to establish a layered structure with sufficient extracellular matrix and formed sheets that could be easily manipulated without tearing. Similar to TKA sheets, PD sheets expressed aggrecan and fibronectin at the protein level and the surface markers CD44, CD81, and CD90, which are characteristic of mesenchymal cells. PD sheets also produced significantly higher levels of transforming growth factor beta-1 and lower levels of matrix metalloproteinase-3 than those produced by TKA sheets, suggesting that young polydactyly-derived chondrocytes have advantages as a potential cell source.ConclusionsPD sheets exhibited characteristics thought to be important to chondrocyte sheets as well as proliferative capacity that may facilitate provision of a stable supply in the future.

The effect of using vitrified-thawed chondrocyte sheets on articular cartilage repair was examined because the methods for storing chondrocyte sheets are essential for allogeneic chondrocyte sheet transplantation. Six Japanese white rabbits were used as sources of articular chondrocytes and synovial cells. Chondrocytes were harvested from the femur, and synovial cells were harvested from inside the knee joints. After coculture of the chondrocytes with synovial cells, triple-layered chondrocyte sheets were fabricated. Eighteen rabbits were used, with six rabbits in each of three groups: osteochondral defect only (control, group A); chondrocyte sheets (group B); and vitrified-thawed chondrocyte sheets (group C). An osteochondral defect was created on the femur. After transplantation, the weight distribution ratio of the undamaged and damaged limbs was measured as a pain-alleviating effect. The rabbits were euthanized at 12 weeks, and the transplanted tissues were evaluated for histology (Safranin O staining and immunostaining) using the International Cartilage Repair Society grading system. For both evaluations, significant differences were observed between groups A and B, and between groups A and C (p < 0.05). No significant differences were observed between groups B and C. Thus, pain-alleviating effects and tissue repair were achieved using vitrified-thawed chondrocyte sheets. Copyright © 2017 John Wiley & Sons, Ltd.