Ex-vivo porcine organs with a circulation pump are effective for teaching hemostatic skills

Surgical residents have insufficient opportunites to learn basic hemostatic skills from clinical experience alone. We designed an ex-vivo training system using porcine organs and a circulation pump to teach hemostatic skills. Residents were surveyed before and after the training and showed significant improvement in their self-confidence (1.83 ± 1.05 vs 3.33 ± 0.87, P < 0.01) on a 5 point Likert scale. This training may be effective to educate residents in basic hemostatic skills

Arthroscopic, histological and MRI analyses of cartilage repair after a minimally invasive method of transplantation of allogeneic synovial mesenchymal stromal cells into cartilage defects in pigs

AbstractBackground aimsTransplantation of synovial mesenchymal stromal cells (MSCs) may induce repair of cartilage defects. We transplanted synovial MSCs into cartilage defects using a simple method and investigated its usefulness and repair process in a pig modelMethodsThe chondrogenic potential of the porcine MSCs was compared in vitro. Cartilage defects were created in both knees of seven pigs, and divided into MSCs treated and non-treated control knees. Synovial MSCs were injected into the defect, and the knee was kept immobilized for 10min before wound closure. To visualize the actual delivery and adhesion of the cells, fluorescence-labeled synovial MSCs from transgenic green fluorescent protein (GFP) pig were injected into the defect in a subgroup of two pigs. In these two animals, the wounds were closed before MSCs were injected and observed for 10min under arthroscopic control. The defects were analyzed sequentially arthroscopically, histologically and by magnetic resonance imaging (MRI) for 3 monthsResultsSynovial MSCs had a higher chondrogenic potential in vitro than the other MSCs examined. Arthroscopic observations showed adhesion of synovial MSCs and membrane formation on the cartilage defects before cartilage repair. Quantification analyses for arthroscopy, histology and MRI revealed a better outcome in the MSC-treated knees than in the non-treated control kneesConclusionsLeaving a synovial MSC suspension in cartilage defects for 10min made it possible for cells to adhere in the defect in a porcine cartilage defect model. The cartilage defect was first covered with membrane, then the cartilage matrix emerged after transplantation of synovial MSCs

Additional file 1: of Ex-vivo and live animal models are equally effective training for the management of a penetrating cardiac injury

Raw Data Ex vivo training objective for submission. (XLSX 31 kb

MHC-Matched Induced Pluripotent Stem Cells Can Attenuate Cellular and Humoral Immune Responses but Are Still Susceptible to Innate Immunity in Pigs

<div><p>Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (<i>n</i> = 4) and ovary (<i>n</i> = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (<i>n</i> = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (<i>n</i> = 8) (<i>p</i><0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.</p></div

Complement-mediated cytotoxicity to C1 iPS cells.

<p>(A) Cytochemical assay for sialic acids using lectins. The expression of sialic acids was examined with SNA lectin and MAL lectin, both of which specifically bind to sialic acids. C1 iPS cells exhibited very low levels of sialic acids compared to fibroblasts. C1 iPS cells, but not PEFs, expressed Oct3/4. (B) Cell injury by complement was assessed by measuring LDH released in the supernatants described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098319#s2" target="_blank">Materials and Methods</a>. Triton-X (2%) was used as a positive control, and 30% of heat-inactivated (complement-inactivated) serum was used as a negative control. The percent cytotoxicity was indicated as average values of triplicate (^*<i>p</i><0.01, ^**<i>p</i><0.05). Three independent experiments were conducted and similar results were obtained.</p

Susceptibility of C1 iPS cells to NK cells.

<p>(A) Immunocytochemical staining with an anti-porcine SLA class I antibody. Porcine PEFs showed positive staining for SLA class I, but C1 iPS cells were negative. (B) Reverse-transcription polymerase chain reaction (RT-PCR) analysis of the expression of <i>SLA class I</i> and ligands for NK cells. Lane 1, PEF; 2, C1 iPS cells; 3, STO feeder cells; 4, no reverse transcriptase; and 5, RT-PCR buffer alone. <i>MICA</i> and <i>ULBP1</i>, ligands for NK cells, were expressed on C1 iPS cells. <i>GAPDH</i> was used as a loading control. (C) Cell injury by NK cells was assessed by measuring LDH released in the supernatants as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098319#s2" target="_blank">Materials and Methods</a>. Triton-X (2%) was used as a positive control. The percent cytotoxicity was quantified and shown as mean of triplicate. Two independent experiments were performed and similar results were obtained. (D) Estimated volumes of the teratomas in immunodeficient mice and SLA-matched pigs. C1 iPS cells were transplanted into NK-competent NOD/SCID (<i>n</i> = 14) and NK-deficient NOG (<i>n</i> = 14) mice, and C1 pigs (<i>n</i> = 5). After transplantation of C1 iPS cells (1×10⁶ cells/site) into immunodeficient mice, the teratomas were dissected and their size (diameter) was measured (^*<i>p</i><0.01). After transplantation of C1 iPS cells (3×10⁷ cells/site) into C1 pigs, no teratomas were observed.</p

Cellular and humoral immune responses in an SLA-matched setting.

<p>(A) Mixed lymphocyte reactions (MLRs) against allogeneic (C2) porcine embryonic fibroblasts (PEFs), C1 iPS cells and STO feeder cells. Peripheral blood mononuclear cells (PBMCs) supplemented with concanavalin A (Con A) and C2 PEFs were used as positive controls, and autologous PBMCs (C1) were used as a negative control. The stimulation index of C1 iPS cells was significantly lower than that of allogeneic cells (^*<i>p</i><0.01), but significantly higher than that of autologous cells (^*<i>p</i><0.01). MLR was performed in triplicate and repeated three times and a typical result was shown. (B) Immunohistochemical staining with anti-CD3 and anti-CD79 antibodies. Pig spleen was stained with the anti-CD3 antibody as a positive control. Slight infiltration of CD3+ T-cells and CD79+ B cells was detected at the transplantation site in the SLA-matched pig CT19. (C) Porcine IgG antibodies against C1 iPS cells were determined by flow cytometry. As SLA-mismatched recipients, miniature pigs that were not C1 or C2 were used. The porcine IgG against C1 iPS cells was detected at much lower levels in the SLA-matched C1 pigs (CT19 and CU65) than in the SLA-mismatched allogeneic pigs. Cells labeled with secondary antibody without porcine serum as a negative control.</p