Comparison between slow and rapid freezing for adrenal gland cryopreservation and xenotransplantation.

The aim of our study was to examine whether slow or rapid cryopreservation of adrenal xenografts affected xenotransplant outcome. Adrenal xenografts were got from newborn piglets (<24 h after birth). Receptor rats were randomly divided into four groups: a bilateral adrenalectomy group, fresh xenotransplantation group, rapid cryopreservation xenotransplantation group, and a slow cryopreservation xenotransplantation group. 30 days after xenotransplantation, the survival rates of rats in the fresh xenotransplantation group, rapid cryopreservation xenotransplantation group and slow cryopreservation xenotransplantation group were 80 %, 60 % and 60 %, respectively, which were significantly higher than 40 % of the bilateral adrenalectomy group. In addition, the survival rate of rats in the slow cryopreservation group was consistently significantly higher than that in the rapid cryopreservation group at 29 days after xenotransplantation. Morphological observation showed that there were a few medulla cells existed in the adrenal glands in the slow cryopreservation group after 30 days of xenotransplantation, but no medulla cells were found in the rapid cryopreservation group. The plasma cortisol level of rats in the fresh xenotransplantation group and the slow xenotransplantation group 30 days after xenotransplantation was significantly higher than that of the rapid cryopreservation group and bilateral adrenalectomy group (P < 0.05). The levels of liver glycogen and cholesterol in the xenotransplantation rats were increased relative to those of the bilateral adrenalectomy rats, and close to normal level. In conclusion, compared with rapidly frozen preserved grafts, slowly frozen preserved grafts not only ensure the structural integrity of adrenal tissues, but also have corresponding physiological functions, which provid a basic research opportunities for the preservation of xenografts and the treatment of adrenal corticosteroid deficiency. Moreover, these findings can provide evidence for xenotransplantation in the treatment of Addison's disease (adrenal cortex hormone deficiency).

Change in the Content of Immunoproteasomes and Macrophages in Rat Liver At the Induction of Donor-Specific Tolerance.

Induction of donor specific tolerance (DST) by the introduction of donor cells into a recipient's portal vein is one of the approaches used to solve the problem of transplant engraftment. However, the mechanism of DST development remains unclear to this moment. In the present work, we first studied the change in the content of immunoproteasomes and macrophages of the liver at early stages of the development of allospecific portal tolerance in rats by Western blotting and flow cytofluorimetry. On the basis of the data obtained, we can conclude that the induction of DST is an active process characterized by two phases during which the level of the proteasome immune subunits LMP2 and LMP7 in liver mononuclear cells, including Kupffer cells, and the number of Kupffer cells change. The first phase lasts up to 5 days after the beginning of DST induction; the second phase - from 5 to 14 days. In both phases, the level of the subunits LMP2 and LMP7 in the total pool of mononuclear cells and Kupffer cells increases, with maximum values on days 1 and 7. In addition, the total number of Kupffer cells increases in both phases with a shift in several days. The most noticeable changes take place in the second phase. The third day is characterized by a lower content of mononuclear cells expressing immunoproteasomes compared to the control value in native animals. Presumably, at this time point a "window of opportunity" appears for subsequent filling of an empty niche with cells of different subpopulations and, depending on this fact, the development of tolerance or rejection. The results obtained raise the new tasks of finding ways to influence the cellular composition in the liver and the expression of immunoproteasomes on the third day after the beginning of DST induction to block the development of rejection.

Neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands.

Stem/progenitor cells are thought to have the potential in the treatment of severe neurodegenerative diseases. Recently, sympathoadrenal progenitors expressing specific markers of neural crest derivatives and capable to differentiate into neurons were discovered in adult bovine and human adrenal glands, but there was no reported data on cryopreservation of sympathoadrenal progenitors. The aim of the present study was to examine the neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands. Considering impact of various initial state of frozen biomaterial on cell recovery, we carried out a comparative estimation of cryopreservation outcome both for adrenal tissue fragments and isolated primary cells. The estimation consisted of determining cell yield, viability, ability to adhere, proliferate and differentiate in vitro. Cells isolated from the fresh adrenal glands were cultured until confluence. A formation of sympathoadrenal progenitors-embedded spherical cell colonies, whose cells are differentiated then into ßIII-tubulin-positive cells with neuron-like morphology, was observed on the monolayer. The colonies were well preserved after cryopreservation of cell culture with a cooling rate of 1 °C/min in the cryoprotectant media containing 5-15% of dimethylsulfoxide. Adrenal tissue fragments were cryopreserved in the presence of 10% dimethylsulfoxide at the cooling rates of 0.3; 1: 5; 40 and > 100 °C/min. Sympathoadrenal progenitors were recovered after cryopreservation with 0.3 °C/min cooling rate but not higher.

[Proteasomes on thyroid tissue allotransplantation under induction of donor specific tolerance in rats].

The proteasomes in the liver of August rats (RT1C) were investigated 30 days after the allotransplantation of Wistar rat (RT1u) thyroid tissue under renal capsule with/without induction of donor specific tolerance by donor splenocyte intraportal administration. The level of the total proteasome pool, immune proteasomes containing the LMP2 and/or LMP7 subunits, proteasome 19S- and 11S-regulators was defined. The intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups that indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of non-tolerated rats compared to tolerated animals. In the liver of tolerated rats with survived transplants, the quantity of mononuclear cells, expressing the immune subunit LMP2, greatly increased in comparison with control and non-tolerated animals. Study of the survived transplants showed the increase of the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them compared to the tissue replacing the rejected transplants. In the control intact thyroid tissue, the immune proteasomes were almost not revealed, while 19S/11S ratio was maximal. Thus, the development of the immune reaction or its suppression is accompanied by change of the balance between different proteasome forms. The immune subunit LMP7 and 11S regulator are connected with the response against donor tissue. On the contrary, the immune subunit LMP2 and 19S regulator are likely to be important for the immune tolerance development and survived tissue functioning. The low content of the immune proteasomes in the follicle cells was found by immunofluorescence assay. The formation of antigens for major histocompatibility complex class I molecules was impaired by low immune proteasome content that led to immunological tolerance to hormone-producing follicle cells.

Outcome of adrenal tissue fragments allotransplantation: the impact of cryopreservation.

Cryopreservation is thought to have the potential to preserve tissue for transplantation. In addition, it can also be used for decreasing tissue immunogenicity, which might be important for prolonging allograft survival. In the present study we examined the impact of cryopreservation at various cooling rates on the outcome of allotransplantation of murine adrenal tissue fragments (ATFr). ATFr were cryopreserved with a cooling rate at 1; 10; 40 and more than 100 °C/min. After thawing it was found that the number of the cells expressing markers of dendritic cells (CD11c) and macrophages (CD11b) in the suspension obtained from ATFr decreased with increasing cooling rate. After allotransplantation the survival rates of adrenalectomized mice and the blood serum levels of corticosterone were higher in recipients of cryopreserved ATFr. By immunohistochemistry, cryopreserved allografts displayed a decreased infiltration by CD4+ and CD8+ T-lymphocytes as compared to fresh grafts. These findings suggest that cryopreserved allografts cause a less severe rejection by decreasing graft immunogenicity.