Optimization of preparation of rat kidney decellularized scaffold by combining freeze-thawing with perfusion / 生物工程学报

We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS (group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate (SDS) (group S), Triton X-100 combined with SDS (group TS), and Triton X-100 combined with SDS after repeated freezing and thawing (group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.

Breast Milk-Transmitted Cytomegalovirus Infection in Preterm Infants

PURPOSE: The purpose of this study is to describe the rate of cytomegalovirus (CMV) virolactia, and the prevalence of breast milk (BM)-transmitted postnatal CMV infection among premature infants after freeze-thawing (FT) and Holder pasteurization (HP) of breast milk. METHODS: This is a single-center, retrospective study of 312 infants born at less than 32 weeks of gestation, or with a birth weight less than 1,500 g from January 2013 to June 2017. All infants were screened for CMV-specific immunoglobulin (Ig) G and IgM at birth. Initial CMV specific polymerase chain reaction (PCR) and CMV culture were performed on mothers' BM and babies' urine within the first 21 days of life. FT and HP of BM was used to prevent the transmission of CMV. For the surveillance of postnatal CMV infection, CMV culture and CMV specific PCR of urine from babies were repeated one to two months after the initial screening. Screening for viremia and viruria was performed if postnatal CMV infection was suspected. RESULTS: Among 178 BM samples obtained from mothers of CMV-IgG-seropositive infants, 80 (44.9%) were CMV PCR positive. CMV deoxyribonucleic acid (DNA) was detected in five of the 22 BM samples (22.7%) obtained from the mothers of CMV-IgG seronegative infants. When CMV DNA load in BM was measured before and after HP, various results were shown. Sixty-three infants out of 232 (27.2%) were evaluated for postnatal CMV infection and four infants out of 63 (6.3%) were infected. CONCLUSION: Interventions to prevent BM-transmitted CMV infection can reduce the chance of postnatal CMV infection, but not completely eliminate it.

Physical, Mechanical and Antimicrobial Evaluations of Physically Crosslinked PVA/Chitosan Hydrogels Containing Nanoparticles.

Chitosan/montmorillonite (MMT) and Poly vinyl Alcohol (PVA) / Silver nanoparticles (AgNPs) nanocomposites were prepared and formed hydrogel membranes using freeze thawing technique. The properties of the prepared hydrogels were investigated and compared to hydrogel membranes in presence and absence of nanometals. The physical behavior, mechanical properties and antibacterial activity was examined. Also the surface morphology monitored using scanning electron microscope. Antimicrobial activity against bacteria and yeast was also examined. The obtained results showed positive effect of nanometals especially AgNPs on swelling percent on the other hand tensile strength were combined by presence of MMT nanoparticles. The surface morphology showed homogenous images for all samples except samples containing MMT. All prepared samples containing nanoparticles showed antibacterial activity especially hydrogel membranes containing AgNPs.

Cellular immunity of influenza vaccine lyophilized liposome produced by freeze-thawing / 中国药科大学学报

@#To investigate the cellular immunogenicity of influenza vaccine liposomes dry powder using the film-dispersion and freeze-thawing. Mice were divided into the non-liposomal influenza vaccine group, film-dispersion prepared liposomal influenza vaccine group, freeze-thawing lyophilized influenza vaccine liposome group, positive control group and negative control group(n=5). 6 μg of hemagglutinin of H1N1 subtype per mouse was delivered tracheally to the mice of lyophilized liposomes groups, with the same dose for non-liposomes intraperitoneally delivered groups as the positive control, and PBS intraperotoneal injection group as the negative control. After 28-day of immunization, the proliferationofsplenic lymphocytes was detected by MTT assay; CD4+cell and CD8+cell were analyzed by flow cytometry. The dry powder of influenza vaccine liposomes prepared by the above two methods, both induce cellular immunity, with no significant difference in the induced on immunogenicity between the prepared products(P< 0. 05). The results showed that freeze-thawing method is feasible in the preparation of vaccine liposomes, leading to the attenuated live vaccine liposome preparation.

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

The purpose of this work was to study a rapid yeast DNA extraction by boiling and freeze-thawing processes without using chemical reagents or any purification procedures, to obtain a high grade PCR-product. A specific DNA fragment of the 18S region of Dekkera bruxellensis and Saccharomyces cerevisiae was chosen. The described boiling and freeze-thawing protocols generated the PCR-grade product preparations and could be used to process many samples. The amplification of the fragments could be observed after 30 and 35 cycles. These processes of extraction without using any kind of chemical reagents, especial water, and purification procedures proved to be efficient, reproducible, simple, fast, and inexpensive.

Study on Inhibition Effects of Freeze-thawing Leech Extract on HepG2 Cells / 中国中医药信息杂志

Objective To study the inhibition effect of leech extract on HepG2 cells. Methods Human hepatocellular cancer cell line HepG2 were treated with different concentrations of leech extracts which were extracted by method of freeze-thawing with liquid nitrogen and contrasted with that by method of water extracting and ethanol precipitating. The inhibition effects and cell morphous were examined by MTT assay and Acridine orange (AO) fluorescent staining method respectively. Result The 6～15 mg/mL drug concentrations of leech extract by method of freeze-thawing with liquid nitrogen had an obvious inhibition on proliferation of HepG2 cells in a dose-dependent manner, and the effect was better than that by method of water extracting and ethanol precipitating (P