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Poly[N-pvinylbenzyl-O-D-galactopyranosyl-(1-4)-D-glucoamide], poly[N-pvinylbenzyl-O-D-glucopyranosyl-(1-4)-D-glucoamide], and poly[N-p-vinylbenzyl-O-mannopyranosyl-(1-4)-D-gluconamide] (referred to as PVLA, PVMA, and PV-Man) are polystyrene derivatives that contain galactose, glucose, and mannose moieties, which interact with hematopoietic cells (HCs). To clarify the specific interaction between the glucopolymers and hematopoietic cells, glycopolymers labeled with fluorescent isothiocyanate (FITC) were used to follow the specific interaction, which was visualized by confocal laser microscopy. We found that PV-Man binds strongly to HCs, probably because of a specific interaction mediated by specific receptors present on the cell membrane, while some cytotoxicity when was observed when PV-Man interacted with the cell membrane. The fluorescence intensity between PV-Man and HCs was up to four-fold (0.14 +/- 0.04) that of PVMA and PVLA with hematopoietic HCs (0.033 +/- 0.01). Moreover, cellular fluorescence increased significantly with increasing incubation time and increasing polymer concentration. Using hematopoietic lineage-specific antibodies, cells were stained and analyzed by flow cytometry to confirm which HCs showed specific binding with glycopolymers, especially hematopoietic stem cells and progenitor cells (c-kit+), B-lymphocyte progenitor cells (B220+), monocyte cells (CD11b+), and erythrocytes (Ter119+).

Polystyrene derivatives, poly[N-pvinylbenzyl-O-D-glucopyranosyl-(1-4)-D-glucoamide] (PV Maltose) and poly[N-p-vinylbenzyl-O-mannopyranosyl-(1-4)-D-glucoamide] (PV Mannose), which contain glucose and mannose moieties, respectively, have the specific binding ability with murine hematopoietic cells. In this study, we confirm the ability of these glycopolymers to interact specifically with human hematopoietic stem cells (HSCs) and mature cells derived from human cord blood (CB) and peripheral blood (PB). Using fluorescence isothiocyanate (FITC)-labeled glycopolymers, we observed that 98% to 93% of hematopoietic cells interacted very strongly with PV Mannose, and 63% of CB and 29% PB interacted with PV Maltose. Both glycopolymers bound better to cells from CB than from PB. Cytotoxic studies revealed that a 0.1 mM dose of PV Mannose induced apoptosis in 20% CB cells, in contrast to 3-5% PB cells. Furthermore, we demonstrated that all of CD34(+) HSCs of both origins bound specifically to PV Mannose, whereas 33-47% bound to PV Maltose. In addition, the majority of B cells (CD19(+)), T cells (CD3(+)), monocytes (CD14(+)), and erythrocytes (CD235a(+)) bound to PV Mannose, but a lower percentage interacted with PV Maltose. In vivo study, bone marrow, spleen, and liver tissues in NOD-SCID mice injected with PV Mannose conjugated CB, were detected PV Mannose positive hematopoietic cells. These data suggest that the use of PV Mannose and PV Maltose might be used for gene and drug delivery for hematopoietic cells and thus, may be useful in therapeutic settings.

Vitrification of bovine immature oocytes has been reported using an open pulled straw, but with limited success. In a previous report, we developed an alternative material (nylon mesh) for vitrification of large quantities of oocytes and embryos. This study was conducted to demonstrate effects of components of a cryoprotectant and a protocol of exposure for bovine immature oocytes on their subsequent in vitro maturation, fertilization and development after cryopreservation by vitrification using a nylon mesh. Bovine oocytes at the germinal vesicle stage were collected from 2-5 mm follicles in ovaries, and cumulus-oocytes complexes (COCs) were randomly assigned to treatment groups. Before vitrification, COCs were exposed to the cryoprotectant, which was composed of 40% ethylene glycol, 18% ficoll and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40) by single step or stepwise exposure. Forty COCs were transferred onto a nylon mesh (0.5 cm 2 ), which was then plunged directly into liquid nitrogen. After thawing in warm medium, vitrified COCs were in vitro-matured, fertilized and cultured. After culture for in vitro maturation, the rates in the oocytes reaching to metaphase II were 64.1% and 63.1% in the stepwise exposure to EFS40 or EFT40, respectively, which was significantly higher (P < 0.05) than the corresponding rates after a single step (22.6% and 10.0%, respectively). There was no significant effect of the two sugars on in vitro maturation after single or step-wise equilibration. Transmission electron microscopy revealed that the cytoplasm of oocytes equilibrated in a single step had many vacuoles and broken mitochondria, while oocytes equilibrated in a step-wise manner had significantly fewer abnormalities and were similar to untreated controls. Cleavage rate of thawed oocytes after IVMFC was significantly higher after stepwise exposure to EFS40 or EFT40 than that after single step exposure (37.7% and 22.2% v. 20.8% and 0%, respectively, P < 0.05).Step-wise equilibration of oocytes in EFT40 was dramatically detrimental: no cleaved embryos developed to blastocysts after a single step exposure to either vitrification solution, or stepwise exposure to EFT40. However, blastocysts were obtained following stepwise exposure to EFS40 (8%). These results suggest that stepwise equilibration and vitrification on a nylon mesh minimizes structural damage to the organelles of immature oocytes and facilitates successful cryopreservation.Various sample containers have been developed for ultra-rapid vitrification of oocytes and embryos to minimize chilling and other injuries. In this study, the survival rates of in vitro-derived porcine oocytes were compared after cryopreservation using open pulled straw (OPS), electron microscope © IETS 2004 10.1071/RDv16n1Ab89 ABSTRACTS FOR POSTER PRESENTATION Cryopreservation/CryobiologyReproduction, Fertility and Development 163 grid (EMG), and nylon loop system (NLS). In addition, the post-thaw survival of porcine morulae and blastocysts was assessed after vitrification by the OPS metho...

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