Blood stream infection (BSI),a blood-borne disease caused by microorganisms such as bacteria,fungi,and viruses,can lead to bacteremia,sepsis,and infectious shock,posing a serious threat to human life and health.Identifying the pathogen is central to the precise treatment of BSI.Traditional blood culture is the gold standard for pathogen identification,while it has limitations in clinical practice due to the long time consumption,production of false negative results,etc.Nanopore sequencing,as a new generation of sequencing technology,can rapidly detect pathogens,drug resistance genes,and virulence genes for the optimization of clinical treatment.This paper reviews the current status of nanopore sequencing technology in the diagnosis of BSI.
Humans ; Nanopore Sequencing ; Sepsis/diagnosis* ; Bacteremia/microbiology* ; Bacteria ; Blood Culture/methods*

BACKGROUND AND OBJECTIVES: The imperative role of dental pulp stem cells (DPSCs) in regenerative therapy demands an in-vitro expansion which must deal with the safety and ethical problems associated with fetal bovine serum (FBS). The primary aim of this study was to compare the effects of human platelet rich fibrin (hPRF) exudate Vs FBS on proliferation and osteodifferentiation of human dental pulp stem cells (hDPSCs). The secondary one was to determine the optimum concentration of hPRF exudate inducing hDPSCs proliferation and osteodifferentiation. METHODS: The direct method was used to prepare hPRF exudate. hDPSCs were isolated from impacted mandibular third molars of twelve donors by the outgrowth method. For cell viability and proliferation rate testing, 96 well plates were used and the assay was done in duplicate and the trial repeated four times under the same conditions. Six wells were used to contain 10% FBS, serum free media, 1%, 5%, 10% and 20% concentrations of hPRF exudates, respectively. The proliferation assay was carried out by MTS tetrazolium cell proliferation assay kit and Elisa reader. The study design for osteodifferentiation protocol was exactly as the proliferation one and instead the assay was carried out by alizarin red with Elisa reader. RESULTS: Compared to 10% FBS, 10% hPRF exudate was the optimum concentration for hDPSCs proliferation, while 1% hPRF exudate was the optimum concentration for osteodifferentiation of hDPSCs. CONCLUSIONS: Avoiding the risk of zoonosis which may be occurred with FBS, it is recommended to use 10% hPRF exudate for proliferation and 1% for osteodifferentiation.
Blood Platelets* ; Cell Proliferation ; Cell Survival ; Culture Media, Serum-Free ; Dental Pulp* ; Enzyme-Linked Immunosorbent Assay ; Exudates and Transudates* ; Fibrin* ; Humans* ; Methods ; Molar, Third ; Stem Cells* ; Tissue Donors

OBJECTIVETo discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).

METHODSUCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.

RESULTSWith the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.

CONCLUSIONSThis non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Erythroblasts ; cytology ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Umbilical Cord

OBJCETIVETo investigate the method of separation of culture of bone microvascular endothelial cells (BMECs) of human femoral head in vitro.

METHODSFrom October 2013 to January 2014,15 femoral heads without pathologic change from patients resected during hip replacement were selected involving 2 males and 13 females with a mean age of 71.2 years old ranging from 38 to 92. Cancellous bone in femoral head was bited into broken bone grain and transfered into medium in aseptic contidion. Cells were isolated by the methods of enzymic digestion and density gradient centrifugation,purified by differiential attachment. The characteristics of cells was observed by inverted microscope. vWF and CD31 immunofluorescence analysis was applied for identification of cells.

RESULTSThe number of cells was positively correlated with patients' age after 24 hours in primary culture. The older patients had the less cells numbered. After 4 to 5 days' culture, primary cells appeared short spindle,polygon shaped and cobblestone-like morphology. After 7 to 10 days' culture, primary cells proliferated densely, became fusion, arranged in swirl, and contact inhibition appeared significantly. Immunofluorescence staining revealed the cells were 100% positive for vWF and CD31, and it showed that the cultured cells were BMECs.

CONCLUSIONIt was a simple, steady, effective method with good reproducibility, by which highly purified human BMECs can be obtained.

Adult ; Aged ; Aged, 80 and over ; Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Femur Head ; blood supply ; Humans ; Male ; Microvessels ; cytology ; Middle Aged

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Amniotic Fluid ; cytology ; Blood Coagulation ; Cell Culture Techniques ; Cell Separation ; methods ; DNA, Complementary ; Factor IX ; biosynthesis ; Genetic Engineering ; Genetic Vectors ; Humans ; Stem Cells ; cytology ; metabolism ; Transfection

OBJECTIVETo build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells.

METHODSRed blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed.

RESULTSThe best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture.

CONCLUSION1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.

Cell Culture Techniques ; Cell Differentiation ; Cell Division ; Cell Separation ; methods ; Cells, Cultured ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; Humans ; Megakaryocyte Progenitor Cells ; cytology

OBJECTIVETo isolate CD133(+) hematopoietic progenitor cells from human umbilical cord blood and optimize the culture condition for maintaining their stem cell characteristics.

METHODSCD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system, and the cells were detected by flow cytometry. Four methods were used for culturing cells. After 8 weeks' culture, cytomorphology, flow cytometry, immunocytochemistry and immunofluorescence assay were used to identify the characteristics of the stem cells.

RESULTSOver 80% of CD133(+) hematopoietic progenitor cells were isolated from human umbilical cord blood using magnetic cell sorting system. The cells were effectively expanded using optimized serum-free medium after 8 weeks of cell culture, whereas the cells in other media differentiated into adherent cells in a poor state.

CONCLUSIONThe optimized serum-free medium allows effective expansion of CD133(+) hematopoietic progenitor cells that maintain stem cell characteristics after a long-term culture.

AC133 Antigen ; Antigens, CD ; metabolism ; Cell Separation ; methods ; Cells, Cultured ; Culture Media, Serum-Free ; Culture Techniques ; methods ; Female ; Fetal Blood ; cytology ; Glycoproteins ; metabolism ; Hematopoietic Stem Cells ; cytology ; Humans ; Infant, Newborn ; Male ; Peptides ; metabolism

OBJECTIVETo establish an improved method for culturing primary mouse pulmonary microvascular endothelial cells (PMVECs).

METHODSAn improved tissue block adherent culture method was used to isolate and culture the PMVECs from C57 mice. The cultured cells were identified by factor VIII-related antigen and CD31 antigen, and the growth of cells cultured using the improved method and the conventional method was compared.

RESULTSThe cultured primary pulmonary microvascular endothelial cells showed a short fusiform or round morphology, and the cell monolayer displayed a cobble stone-like appearance. The cultured cells were positive for VIII-related antigen and CD31 antigen. The cell growth was accelerated in the cell cultures with the improved method compared with that in conventional cell cultures.

CONCLUSIONThe improved culture method allows more efficient acquisition of primary mouse PMVECs of a greater purity.

Animals ; Cells, Cultured ; Endothelial Cells ; cytology ; Endothelium, Vascular ; cytology ; Lung ; blood supply ; Mice ; Mice, Inbred C57BL ; Primary Cell Culture ; methods

This study was aimed to look for the potential application of human umbilical plasma (HUP) in the culture of human umbilical vein endothelial cells (HUVECs). The effect of HUP concentration in cell culture medium on cell proliferation activity and cell cycle was studied. HUVECs were obtained by digesting the umbilical cord with 0.25% trypsin mixed with the equal volume of 0.1% collagenase II, then were identified by morphology and factor VIII immunohistochemistry under phase contrast microscopy. The cells cultured after 7 days showed the typical cobblestone morphology with factor VIII immunohistochemical staining positive. The study showed that the groups of 20% (HUP), 15% HUP+20% Fetal Bovine Serum (FBS) and 20% HUP+20%FBS enhanced cell proliferation activity significantly when compared with the control group (20% FBS without HUP). On the contrary, 30% HUP+20% FBS caused cell cycle arrest, which significantly hindered the proliferation of HUVECs. The study proved that although HUP might not be able to completely replace the role of vascular endothelial growth factor in cell culture, as a supplement ingredient, it was an ideal candidate to replace FBS in culture medium.
Cell Culture Techniques ; methods ; Cell Proliferation ; Cells, Cultured ; Culture Media ; chemistry ; Fetal Blood ; chemistry ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Plasma ; physiology

OBJECTIVESet up a method to isolate and identify the small pulmonary arterial smooth muscle cells (PASMCs) in vitro.

METHODSIn sterile conditions, separated the male SD rat pulmonary artery, digested by collagenase I and cultured primary PASMCs. Measured cell viability; observed by phase contrast microscope; identified by immunocytochemistry and immunofluorescence staining as a label for smooth muscle alpha-actin.

RESULTSPASMCs were identified by morphology and immunocytochemistry, immunofluorescence staining, with the cell viability is over 96.5%. The primary culture could be subcultured after 4-7 days and successfully passaged without change in morphology and growth characteristic.

CONCLUSIONThis technique has advantage of the method is simple, short cultivate, good reproducibility, the primary cultured PASMCs quantity and the rapid growth.

Animals ; Arterioles ; cytology ; Cell Separation ; methods ; Lung ; blood supply ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Primary Cell Culture ; methods ; Pulmonary Artery ; cytology ; Rats ; Rats, Sprague-Dawley