OBJECTIVECurrently, thrombocytopenia is typically observed in patients undergoing hematopioetic stem cell transplantation (HSCT), high-dose chemotherapy or irradiation. Severe thrombocytopenia can cause intestinal and intracranial hemorrhage. To transfuse ex vivo-expanded megakaryocytes (MK) into patients can reinforce the ability of platelet formation and shorten the time of platelet recovery. Therefore it is one of the effective approaches to reduce the danger. The purpose of the present study was to explore the differences in MK expansion between CD(34)(+) stem cells derived from umbilical cord blood (CB) and peripheral blood (PB) and to establish the most optimal culture system.

METHODSMononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque gradient solution. CD(34)(+) cells were isolated by positive selection using an immunomagnetic separation system and the selected CD(34)(+) cells were seeded in Iscove's modified Dulbecco's medium (IMDM) supplemented with fetal calf serum (FCS) and certain kinds of cytokines. After 15 - 17 days of culture, the cells were counted and the content of CD(41)(+) cells was determined by using flow cytometry, and the number of megakaryocyte colony-forming unit (CFU-MK) was simultaneously measured.

RESULTSAfter the defined days of culture, the cytokine combination of thrombopoietin (TPO) + fetal liver tyrosine kinase ligand (FL) + IL-6 + IL-3 showed to be the most suitable for both PB and CB to obtain high numbers of MK, and to be better than any of the other three groups (P < 0.05). The CD(41)(+) cells from CB were expanded by193 +/- 25 fold on day 14, and those from PB were expanded by 131 +/- 18 fold on day 10. The number of CD(41)(+) cells from both CB and PB decreased.

CONCLUSIONFor PB and CB, the cytokine combination of TPO + FL + IL-6 + IL-3 is most suitable for obtaining large number of MK and is the best combination for ex vivo MK expansion. MKs from CB seemed to have higher proliferation potential than that from PB, and the optimal culture duration of MK from PB is shorter than that of MK from CB.

Antigens, CD34 ; Cell Culture Techniques ; methods ; Cell Proliferation ; Cell Separation ; Culture Media ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Megakaryocytes ; cytology

Observation of microcirculation plays an important role on the basic research and clinical diagnosis. However, an observation as such on the anesthetized patient will cause stress reaction, thus it will affect normal physiological state and interfere experimental results. At present, a method adopting dorsal microcirculatory chamber (DMC) to do in vivo observation in an unanesthetized state can eliminate the influence of anesthesia. Based on the research reports and practical applications of this method abroad, we summarize, in here, the configuration, function, observation techniques; the application of DMC; and the research states of microcirculation observation.
Animals ; Back ; blood supply ; Blood Flow Velocity ; Diffusion Chambers, Culture ; Humans ; Microcirculation ; physiology ; Monitoring, Physiologic ; methods ; Skin ; blood supply

This study was aimed to analyze the results of false positive reaction in bacterial detection of blood samples with BacT/ALERT 3D system, to evaluate the specificity of this system, and to decrease the false positive reaction. Each reaction flasks in past five years were processed for bacteria isolation and identification. When the initial cultures were positive, the remaining samples and the corresponding units were recultured if still available. 11395 blood samples were detected. It is worthy of note that the incubator temperature should be stabilized, avoiding fluctuation; when the cultures were alarmed, the reaction flasks showed be kept some hours for further incubation so as to trace a sharply increasing signal to support the judgement of true bacterial growth. The results indicated that 122 samples (1.07%) wee positive at initial culture, out of them 107 samples (88.7%) were found bacterial, and 15 samples (12.3%) were found nothing. The detection curves of positive samples resulted from bacterial growth showed ascent. In conclusion, maintenance of temperature stability and avoidance of temperature fluctuation in incubator could decrease the occurrence of false-positive reaction in detection process. The reaction flasks with positive results at initial culture should be recultured, and whether existence of a sharply ascending logarilhimic growth phase in bacterial growth curve should be further detected, which are helpful to distinguish false-positive reactions from true positive, and thus increase the specificity of the BacT/ALERT system.
Automation, Laboratory ; Bacteria ; isolation & purification ; Bacteriological Techniques ; methods ; Blood ; microbiology ; Culture Media ; False Positive Reactions ; Humans

OBJECTIVETo explore a new method for in vitro expansion of human mesenchymal stem cells (MSC) by using platelet-rich plasma (PRP) in place of fetal calf serum (FCS).

METHODSBone marrow(BM) samples were obtained from the proximal femurs of patients with normal haematopoietic function undergoing total hip arthroplasty. 2 x 10(5)/cm2 BM nucleated cells were seeded in 25 cm2 flasks for MSC cultivation containing one of the 3 mediums: complete Dexter medium with 12.5% FBS and 12.5% horse serum (medium1), alpha-MEM with 10% FCS (medium2) and alpha-MEM with 5% PRP(medium3). At the same time, 1 x 10(6) nucleated BM cells and same amount of nucleated cells from iliac aspirate were seeded in 25 cm2 tissue flasks, colony forming unit-fibroblast (CFU-F) assay.

RESULTSCulture-expanded cells from proximal femurs presented a typical fibroblast-like morphology. Cells were positive for SH2 (CD 105), SH3 (CD73), Thy-1 (CD90), while negative for CD34 and CD45. On induction, these cells could differentiate into osteoblasts. A significantly higher proliferative capacity of MSCs expanded in medium3 was observed in comparison to those in mediuml or 2 without alteration of the phenotype and the differentiation property. CFU-F assays indicated that bone marrow from the proximal femoral contained significantly more CFU-F than that from iliac aspirate.

CONCLUSIONPlatelet-rich plasma can be used in place of FCS to provide a safer and more effective culture condition to expand MSCs for clinical purpose. The proximal femur BM cells can be obtained in hip surgeries.

Blood Platelets ; Cell Culture Techniques ; methods ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Plasma

To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34+ cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 10(6) CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen (DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.
Bioreactors ; Cell Culture Techniques ; methods ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

This study was aimed to investigate a beneficial approach for resolving the deficiency of blood source, preventing the infection resulting from blood transfusion and overcoming the knotty match of patients with rare blood group by using massive expansion of erythroid cells from cord blood CD34(+) cells in vitro. The CD34(+) cells from human cord blood were cultured in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO) for 1 week, then expansion and differentiation of CD34(+) cells into erythroid cells were supported by co-culture with human mesenchymal stem cells (MSCs) derived from bone marrow for 2 weeks. The results indicated that after culture for 23 days, the expansion multiple of total cell number reached 2.52 x 10(5), and over 95% of these cells were erythroid cells as compared with less than 1% of myelomonocytic (CD14(+) or CD15(+)) cells and megakaryocytic (CD41(+)) cells. However, the culture system without MSC support was significantly disadvantaged both in expansion ability and ratio of erythroid cells when compared with MSC supporting system. It is concluded that the erythroid cells can be produced from CD34(+) cells in large scale by culturing in the system comprised of cytokine sets and MSC feeders, in which MSCs can support the proliferation and differentiation of erythroid cells.
Antigens, CD34 ; Cell Culture Techniques ; methods ; Cell Differentiation ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

Vascular tissue engineering is a novel approach by which an ideal vascualr graft constructed in vitro that will not be obstructed for a long time without immunological reaction after implantation. This article reviewed the definition of the tissue engineering blood vessel (TEBV), cellular resourses, the selection of biocompatible materials, the devising methods and the research achievements. Furthermore, it also discussed the current problems of TEBV and looked forward to future clinical application.
Biocompatible Materials ; Bioprosthesis ; Blood Vessel Prosthesis ; Cell Culture Techniques ; Tissue Engineering ; methods

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*

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

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