Long-term durability test of axial-flow ventricular assist device under pulsatile flow.

A long-term durability test was conducted on a newly developed axial-flow ventricular assist device (VAD) with hydrodynamic bearings. The mock circulatory loop consisted of a diaphragm pump with a mechanical heart valve, a reservoir, a compliance tank, a resistance valve, and flow paths made of polymer or titanium. The VAD was installed behind the diaphragm pump. The blood analog fluid was a saline solution with added glycerin at a temperature of 37 °C. A pulsatile flow was introduced into the VAD over a range of flow rates to realize a positive flow rate and a positive pressure head at a given impeller rotational speed, yielding a flow rate of 5 L/min and a pressure of 100 mmHg. Pulsatile flow conditions were achieved with the diastolic and systolic flow rates of ~0 and 9.5 L/min, respectively, and an average flow rate of ~5 L/min at a pulse rate of 72 bpm. The VAD operation was judged by not only the rotational speed of the impeller, but also the diastolic, systolic, and average flow rates and the average pressure head of the VAD. The conditions of the mock circulatory loop, including the pulse rate of the diaphragm pump, the fluid temperature, and the fluid viscosity were maintained. Eight VADs were tested with testing periods of 2 years, during which they were continuously in operation. The VAD performance factors, including the power consumption and the vibration characteristics, were kept almost constant. The long-term durability of the developed VAD was successfully demonstrated.

Hollow fiber vitrification provides a novel method for cryopreserving in vitro maturation/fertilization-derived porcine embryos.

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.

Hollow fiber vitrification: a novel method for vitrifying multiple embryos in a single device.

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.

In vitro development and postvitrification survival of cloned feline embryos derived from preadipocytes.

The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.

Effect of tandem forms of DAF(CD55) on complement-mediated xenogeneic cell lysis.

BACKGROUND: It is difficult to produce a transgenic animal with high expression of decay-accelerating factor (CD55: DAF) or other molecules. The purpose of this study was to assess the effect of tandem forms of DAF on a xenogeneic cell membrane against human complement. METHODS: cDNAs of the delta-Short Consensus Repeat (SCR) 1-DAF, the double-DAF, the triple-DAF, and the tetra-DAF with a FLAG-tag were established. Chinese hamster ovary (CHO) cell lines and a pig endothelial cell (PEC) line expressing these molecules were established. The amelioration of complement-mediated lysis by the transfectant molecules on these cells was examined. The CHO cell transfectants were also incubated with normal human serum, and the amount of C3 deposited was determined by FACS analysis. RESULTS: Stable CHO cells and PEC transfectants, in which each molecule was clearly expressed, and Western blots showed that each band corresponded to the expected molecular weight. The extent of amelioration of complement-mediated lysis by these four molecules was then examined. A clear tendency was found, as follows: The higher the tandem number of DAF, the greater was the effect on cytotoxicity. Additional experiments focusing on triple-DAF and tetra-DAF did not indicate any significant difference in complement-mediated lysis. Consistent with the complement-regulatory ability, the inhibitory effect of the deposition of C3 fragments by these molecules was closely related to the degree of amelioration. CONCLUSION: These data indicate that tandem DAF, especially a triple-DAF, is a very effective form for protecting against complement activation.

Only the light chain is sufficient for the serine protease function of the membrane bound form of factor I on a xeno-surface.

The cell membrane-bound forms of whole factor I (fI-PI), the light chain of the serine protease (SP) domain (SP-PI), and the light chain plus the COOH-terminal 45 amino acid (AA) of the heavy chain (SP+45-PI) were constructed. Chinese hamster ovary (CHO) cells, expressing these molecules were established by transfection of cDNA and confirmed by flow cytometry. Amelioration of complement-mediated cell lysis and complement fragment deposition on the cell surface by the transfectant molecules was tested in each CHO cell by means of a lactate dehydrogenase (LDH) assay and flow cytometry, respectively. A highly expressed fI-PI blocked human complement-mediated cell lysis by approximately 84% of the cells. CHO cell transfectants with SP-PI also showed a clear inhibition in cell lysis by human serum, whereas CHO cell transfectants with SP+45-PI showed no inhibition. In addition, fI-PI and SP-PI, but not SP+45-PI, suppressed C5b-9 deposition on CHO cell surface. These data indicate that the last 45 amino acid of the heavy chain, including a disulfide bridge area, did not participate in the serin protease function of factor I. The results suggest that SP-PI has potential for use in clinical xenotransplantation.

Effect of hybrid complement regulatory proteins on xenogeneic cells.

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2-4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8-11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1-3 of the C4b-binding protein (C4bp-PI) was found to be effective.

Effect of various forms of the C1 esterase inhibitor (C1-INH) and DAF on complement mediated xenogeneic cell lysis.

The purpose of the present study was to assess the effect of various forms of the surface-bound form of the C1 esterase inhibitor (C1-INH-PI) and decay accelerating factor (DAF) on xenogenic cells. cDNAs of various deletion mutants of the C1-INH-PI, such as delta-1-99 amino acid (AA), delta-108-183AA loop, delta-whole loop, delta-exon5, delta-exon6 + 7, and delta-exon5 + 6 + 7, and that of DAF, the delta-short consensus repeat (SCR) 1-DAF were established. While all deletion mutants of C1-INH-PI except the delta-1-99AA were expressed in the cytoplasm but not on the cell surface, the delta-1-99AA was clearly expressed on the xenogeneic cell surface. Amelioration of complement-mediated xenogeneic cell lysis by delta-1-99AA was next tested, and compared with delta-SCR1 DAF. Both molecules blocked human complement-mediated cell lysis by approximately 57 to 90 and 93 to 98%, respectively, in Chinese hamster ovarian tumor (CHO) cells and pig endothelial cells (PECs). The CHO cell transfectants were incubated with 20% normal human serum, and the amounts of C4 and C3 deposition on the cell surface were analysed by flow cytometry. The DAF transfectant showed a large amount of C4-deposition and much less C3-deposition than the controls (approximately 85% suppression), whereas the delta-1-99AA showed approximately a 40% suppression in both C4- and C3-deposition. Consequently, both the delta-1-99AA C1-INH-PI and delta-SCR1 DAF molecules are quite effective in down-regulating the xenogeneic cell lysis, but accomplished this in different manners.