OBJECTIVE: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. MATERIALS AND METHODS: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. RESULTS: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. CONCLUSION: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.
Animals ; Blastocyst ; Embryonic Structures* ; Ethylene Glycol ; Ficoll ; Humans ; Mice* ; Morula ; Sucrose ; Survival Rate ; Vitrification*

OBJECTIVE: The aim of this study was to compare the survival and developmental rate of two vitrification solutions for the vitrification of mouse expanded blastocysts. METHODS: Mouse embryos were obtained at 2 cell stage and cultured to expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% serum substitute supplement (SSS). The vitrification solutions used were EFS40 and VS. EFS40 consisted of 40% ethylene glycol, 18% ficoll, and 0.5 M sucrose while VS consisted of 20% ethylene glycol, 20% DMSO, and 10% 1,3-butanediol diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% calf serum (CS). Toxicity was tested by exposing expanded blastocysts to vitrification solution. The vitrification procedure used for EFS40 was performed in three steps, after which they were warmed in 5 steps with 0.5 M sucrose. VS was performed in two steps, after which they were warmed with 1.0 M trehalose. Recovery, survival and hatching rate per expanded blastocysts recovered were compared between two groups. RESULTS: In toxicity test, survival and hatching rate of EFS40 group were 95% and 100%, respectively. In contrast, survival and hatching rate of VS group were 100% and 87.5%, respectively. After vitrification and warming in solution, recovery rate for EFS40 group was 73.7% whereas recovery rate for VS group was 66.5%. After 24 h culture, survival and hatching rate were 80.5% and 20.7% for EFS40 and 66% and 0% for VS group, respectively. After 48 h culture, survival and hatching rate were 69% and 33.3% for EFS40 and 58.3% and 1.9% for VS group, respectively. Survival and hatching rate in EFS40 group were significantly higher than those found in VS group. CONCLUSION: The EFS40 solution was better than VS solution for vitrification of mouse expanded blastocysts.
Animals ; Blastocyst* ; Dimethyl Sulfoxide ; Embryonic Structures ; Ethylene Glycol ; Ficoll ; Humans ; Mice* ; Sucrose ; Toxicity Tests ; Trehalose ; Vitrification*

PURPOSE: The purpose of the present study is to determine the relation between in vitro resistance to 9 drugs, measured with colorimetric methylthiazol tetrazolium(MTT) assay and prognostic factors. METHODS: Thirty children with leukemia were evaluated at the pediatric department of Dongsan Medical Center. All samples tested with the MTT assay contained 80% or more leukemic cells, which were isolated by Ficoll density gradient centrifugation, and were incubated with 9 drugs for 4 days. The optical density(OD) of the wells was measured with microplate spectrophotometer. Leukemic cell survival(LCS) was calculated by OD treated well/OD control wellsx100(%). LD50 was calculated from the dose-response curve and used as a measure of resistance. RESULTS: Among the 30 children with leukemia, 16 were ALL, 14 were AML. Seventeen boys and thirteen girls ranged in age from 9 months to 16 years. Comparing LD50 values according to leukemic type, AML revealed relatively high LD50 values for all drugs, except VCR. But there were no significant differences between ALL and AML(P>0.05). Male showed high LD50 values for ASP, DET, ARA-C, VP16, ADR and 6TG. Age<2 and >10 years children showed high LD50 values for all drugs, except 6TG. Patients with a leukocyte count>100,000/mm3 at diagnosis showed high LD50 values for VCR, ASP, DET, MTX, ARA-C, ADR, and 6TG. Patients with normal chromosome showed higher LD50 values. CONCLUSION: Our study showed higher LD50 values at AML, male, age<2 and 10>years old, leukocyte count>100,000/mm3, and normal karyotype. The MTT test may contribute to the selection of effective chemotherapeutic agent for children with acute leukemia.
Centrifugation, Density Gradient ; Child ; Cytarabine ; DEET ; Diagnosis ; Etoposide ; Female ; Ficoll ; Humans ; Karyotype ; Lethal Dose 50 ; Leukemia ; Leukocytes ; Male ; Viperidae

PURPOSE: Considering the complications of nonspecific immunosuppression, as well as the availability of insulin, heavy immunosuppressive treatment to pancreatic islet transplantation patients is not justified. Antigen administration via the portal vein has been demonstrated to induce immunosuppression, and may present a possible mechanism for the induction of tolerance. Using a mouse model, without any immunosuppressive treatment, the islet allograft survivals were compared between portal venous transfusion and portal venous saline injection groups. METHODS: Six C57BL/6J mice were used as pancreatic islet donors per Balb/c recipient mouse. Islets were harvested by digestion of the pancreata with collagenase, with subsequent Ficoll density gradient separation. Recipient mice were divided into two groups: seven mice received a portal venous injection of 0.1 cc saline (PVS) and eight a portal venous transfusion of 0.1 cc donor blood (PVT). Islets were transplanted into the subcapsular space of the left kidney. Transplantation failure was determined if the transplanted mouse failed to show a blood glucose level less than 200 mg/dl at 24 hours after the transplantation; these mice were not included in the statistics. Rejection was determined when the normalized blood glucose level (<200 mg/dl) returned to above 300 mg/dl. RESULTS: The mean islet equivalent numbers (IEQ) of the seven PVS and eight PVT mice were 893+/-262 and 911+/-288, respectively. The islet allograft survival of the PVS group ranged between 1 to 9 days; whereas, that of the PVT group ranged between 6 to 16 days. The PVT group showed significantly higher islet allograft survival than the PVS group (P=0.0443). CONCLUSION: A portal venous transfusion prolonged the islet allograft survival.
Allografts* ; Animals ; Blood Glucose ; Collagenases ; Digestion ; Ficoll ; Humans ; Immunosuppression ; Insulin ; Islets of Langerhans* ; Kidney ; Mice* ; Portal Vein* ; Tissue Donors ; Transplantation

OBJECTIVE: The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates. METHODS: Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 microg/mL Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups. RESULTS: Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05). CONCLUSION: The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation.
Animals ; Apoptosis ; Blastocyst ; Dimethyl Sulfoxide ; Embryonic Structures ; Ethylene Glycol ; Ethylenes ; Ficoll ; Mental Competency ; Mice ; Microscopy, Electron ; Plastics ; Sucrose ; Vitrification

PURPOSE: A high yield of viable pancreatic islets is an essential prerequisite for the study of pancreatic islet transplantation. The purpose of this study is to compare the yield between intra-gall bladder (intra-GB) and intra-common bile duct (intra-CBD) injection of collagenase solution for isolation of mouse pancreatic islets. METHODS: The mice were divided into two groups, the intra-GB and intra-CBD groups, and each group included twelve mice, respectively. Collagenase solution was injected via the gallbladder in the intra-GB group mice, while this was done via the common bile duct in the intra-CBD group. After removal and digestion of the mouse pancreases, the pancreatic islets were isolated by Ficoll density gradient centrifugation and hand picking. RESULTS: The intra-GB group yielded 121.67+/-39.86 IEQs, and the intra-CBD group reveled 168.17+/-29.23 IEQs. There was a statistically significant difference in islet yield between the two groups (P=0.005, Mann-Whitney Test). The purities of the isolated islets were 86.42+/-3.99% for the intra-GB group and 87.17+/-4.47% for the intra-CBD group, and there was no difference between the two groups (P=0.755, Mann- Whitney Test). CONCLUSION: Both the intra-GB and intra-CBD groups yielded an average of >120 IEQs. However, the intra-CBD group revealed a higher yield than the intra-GB group for isolating mouse pancreatic islets.
Animals ; Bile Ducts* ; Bile* ; Centrifugation, Density Gradient ; Collagenases* ; Common Bile Duct ; Digestion ; Ficoll ; Gallbladder ; Hand ; Islets of Langerhans* ; Mice* ; Pancreas ; Urinary Bladder*

PURPOSE: To evaluate the function of Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) in human neonatal monocytes. METHODS: The peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), was synthesized, purified, and prepared in the Peptide Library Support Facility at Pohang University of Science and Technology. Female Sprague-Dawley rats (200+/-10 g) were preinfected with S. aureus and treated with WKYMVm through femoral vein. At various time points, blood samples were obtained by puncture of femoral artery and the serum was plated on the nutrient agar plate. The number of viable bacteria was determined by counting the number of bacterial colonies. In addition, using S. aureus and C. albicans, we evaluated the bactericidal and fungicidal activities of neonatal monocytes, which were separated from umbilical cord blood by Ficoll gradient. RESULTS: The numbers of bacteria in the blood of WKYMVm-treated rats were rapidly decreased with time, as compared with those of the untreated rats. The peptide treatment enhanced the bactericidal activity in vivo within 10 minutes. In neonatal monocytes, WKYMVm stimulated the intracellular killing of S. aureus in a dose dependent manner, showing the maximum effect at 100 nM. WKYMVm stimulated the phagocytic and fungicidal activities against C. albicans in a dose dependent manner, with the maximum effect at the 100 nM. CONCLUSION: These results suggest that WKYMVm may be an effective agent against the neonatal infections.
Agar ; Animals ; Bacteria ; Female ; Femoral Artery ; Femoral Vein ; Fetal Blood ; Ficoll ; Gyeongsangbuk-do ; Homicide ; Humans ; Monocytes* ; Peptide Library ; Punctures ; Rats ; Rats, Sprague-Dawley

Bacterial lipopolysaccharide (LPS) plays a major role in stimulating the synthesis and release of the principal osteoclast-activating cytokines, namely, interleukin 1 and tumor necrosis factor-alpha from immune cells. Although monocytes/macrophages are the main producers of these cytokines, recent evidence has indicated that polymorphonuclear leukocytes (PMN) have the ability to release IL-1 and TNF-alpha. Calcium hydroxide has been shown to be an effective medicament in root canal infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The purpose of this study was to investigate whether treatment of Porphyromonas endodontalis LPS with calcium hydroxide alters its biological action as measured by human PMN secretion of IL-1 and TNF-alpha, and it was compared with Escherichia coli LPS. P. endodontalis ATCC 35406 was cultured in anaerobic condition, and LPS was extracted using the hot-phenol water extraction method and purified. Purchased E. coli LPS was also purified. 100 microg/ml of each LPS in pyrogen free water were incubated with 25mg/ml Ca(OH)2 at 37degrees C for 7 days. The supernatants were subjected to ultrafiltration, and the isolates were lyophilized and weighed. PMNs were obtained from peripheral blood by centrifugation layered over Lymphoprep. The cells were resuspended (4x106 cells/ml) in RPMI 1640 followed by treatment with various concentrations of LPS (0, 0.1, 1, 10microg/ml) for 24 hours at 37degrees C in 5% CO2 incubator. The supernatants of cells were collected and the levels of IL-1alpha, IL-1beta and TNF-alpha were measured by enzyme-linked immunosorbent assay. The results were as follows; 1. The levels of IL-1alpha, IL-1beta, TNF-alpha from PMN treated with each LPS were significantly higher than those released from unstimulated PMN of the control group (p<0.05). 2. The levels of all three cytokines released from PMN stimulated with each calcium hydroxide treated LPS were significantly lower than those released from PMN stimulated with each untreated LPS (p<0.05), while they were not significantly different from those released from unstimulated PMN of the control group (p>0.05). 3. The levels of secretion for all three cytokines were affected in a dose-dependent manner in PMN stimulated with each LPS (p<0.05), but not in PMN stimulated with each calcium hydroxide treated LPS (p>0.05). 4. The levels of all three cytokines released from PMN stimulated with P. endodontalis LPS were significantly lower than those released from PMN stimulated with E. coli LPS (p<0.05).
Calcium ; Calcium Hydroxide ; Centrifugation ; Cytokines ; Dental Pulp Cavity ; Escherichia coli ; Ficoll ; Humans ; Hydroxides ; Incubators ; Interleukin-1 ; Metrizoate ; Neutrophils ; Porphyromonas ; Porphyromonas endodontalis ; Tumor Necrosis Factor-alpha ; Ultrafiltration ; Water

It is difficult to immunize human lymphocytes in vitro by conventional cell culture methods. Activation of lymphocytes requires not only specific antigen stimulation but also delicate cell to cell interaction. If the cellular organization could be maintained in culture system, lymphocytes could be immunized in vitro with higher frequency. For the purpose of in vitro immunization of human lymphocytes, we used slice culture system which could maintain morphological and functional organization. Human tonsils resected from eleven -year old boy were evenly divided into two pieces, and one was cultivated in conventional cell culture and the other in slice culture system. In the former system, tonsilar mononuclear cells, separated by Ficoll -Hypaque density gradient centrifugation, were cultivated in RPMI 1640 supplemented with 10% human type AB serum in the cell density of 5 x10 6 /ml. In the latter, tonsillar tissues were sliced into small pieces of 8 mm 3 , and were cultivated in Waymouth MB 752/1 medium supplemented with 10% Human type AB serum, gassed under 5% CO2 and 95% O2 at 37 C. After stabilized for one hour, each system wasw challenged with 50 microgram/ml of KLH or 100 microgram/ml of LPS. At 3, 6, 12 and 24 hours after antigen challenge, culture supernatants were assayed for the specific antibody by ELISA, and cells or tissues were analyzed for the expression of CD23 by flow cytometry. The result showed that tonsilar B lymphocytes in slice culture system expressed CD23 as early as 3 hours after antigen challenge, while those in cell culture system expressed CD23 from 6 hours after challenge. Specific antibodies were detected only in supernatants of slice culture system from 6 hours after challenge. These results suggested thathuman lymphocytes could be immunized in vitro if the cellular organization was maintained.
Antibodies ; B-Lymphocytes ; Cell Communication ; Cell Count ; Cell Culture Techniques ; Centrifugation, Density Gradient ; Enzyme-Linked Immunosorbent Assay ; Ficoll ; Flow Cytometry ; Humans* ; Immunization* ; Lymphocytes* ; Male ; Palatine Tonsil

PURPOSE: Umbilical cord blood (CB) transplantation is an alternative method instead of allogeneic bone marrow (BM) transplantation. CB transplantation has used grafts with red blood cell (RBC) depletion but previous reports in CB investigated the immune reaction about mononulcear cell separated by density gradient. To study the real immune response in CB transplantation, the experimental group designed the total nuclear cell (TNC) groups by RBC depletion and the mononuclear cell (MNC) groups by Ficoll Hypaque in CB and BM. METHODS: We evaluated the various cytokine gene expression by semiquantitative RT- PCR method after immune stimulation with phytohemagglutinin in cord blood and bone marrow according to cell separation method. RESULTS: 1) All samples of CB and BM expressed IL-2 mRNA. There was no difference in amounts of IL-2 mRNA between CBTNC and CBMNC, between BMTNC and BMMNC. 2) All samples of CB and BM expressed IL-10 mRNA. There was no difference in amounts of IL-10 mRNA between the CBTNC and CBMNC but significantly different between the BMTNC and BMMNC (P<0.05). The amounts of IL-10 mRNA in the BMMNC and CBTNC group showed larger than in the BMTNC group (P<0.05). 3) The expression of IL-4, IFN-gamma was not shown in this study. These results suggest that no difference of IL-2 mRNA between CB and BM may reveal IL-2 as major cytokine gene in CB after immune stimulation. The expression of IL-10 mRNA in CBTNC showed more than in BMTNC group. Conclusion: Our results suggest that CBTNC may contain a lot of cells producing IL-10, as a cytokine of immunoregulatory function, and therefore the immune reaction in CB transplantation may be less apparent than in BM transplantation. The cell component producing IL-10 in CB may be T regulator cell. There were not showed the IFN-gamma and IL-4 mRNA due to short duration of immune stimulation. Therefore, further studies will identify the IL-4 and IFN-gamma mRNA expression after long time stimulation and various cytokine gene expression to certificate the precise immune response after flow cytometry to composed of immune cells in CB and BM.
Bone Marrow* ; Cell Separation* ; Cellular Structures ; Diatrizoate ; Erythrocytes ; Fetal Blood* ; Ficoll ; Flow Cytometry ; Gene Expression* ; Interleukin-10 ; Interleukin-2 ; Interleukin-4 ; Polymerase Chain Reaction ; RNA, Messenger ; Transplants