Objective To investigate the effects of different culture conditions on the isolation and expansion of stem cells from second-trimester amniotic fluids.Methods Amniotic fluids were obtained from 15 pregnant women undergone amniocenteses for medical indications between 16-24 gestation weeks by transabdominal amniocenteses from September 2007 to June 2008.Amniotic fluids(10-20 ml)samples were collected and each WaS cultured under different conditions or groups.(1)Low-glucose DMEM(LD) medium supplemented with 10%of fetal bovine serum(group of 10% FBS);(2)LD medium with 20%of FBS(group of 20%FBS);(3)LD medium with 15%of FBS and 4 ng/ml of basic fibroblast growth factor (group of bFGF);(4)LD medium with 10%of FBS as well ag the culture plate coated with gelatin(group of gelatin).The effects of different conditions were evaluated by comparing the number of primary colonies,the cell morphology and the ability of expansion.The isolated stem cells were identified by flow cytometry,RT-PCR and differentiation ability to edipocyte.Resuits (1)The success rates of primary culture of the group of 10%FBS,20%FBS,bFGF and gelatin were 60%,73%,73%and 60% respectively(P>0.05).The numbers of colonies were 0.9±0.5,2.6±1.5,2.9±1.5,1.1±0.8(P<0.01 when group of 10%FBS and gelatin compared with group of 20%FBS and bFGF);among the primary colonies,fibroblast-like colonies accounted for 46%,49%,64%.44%respectively(P>0.05).(2)The second passage cells obtained from all of these four groups could difierentiate into adipocyte after induction.(3)In the group of bFGF,stem cells were isolated from 5 samples and expanded to nearly 107 cells after 5 passages(P<0.01 compared with other groups).(4)Karyotype were normal in all samples.(5)Stem cells from bFGF group showed positive expression of SSEA-4.Oct-4 and Nanog gene detected by flow cytometry and RT-PCR.Conclusion Stem cells can be isolated from second-trimester amniotic fluids;moderate serum concentration and supplementation of bFGF can improve the efficiency of isolation and expansion of amniotic fluid of stem cells.

Objective:To examine the effects of in-house made heat preservation socks on body temperature maintenance in elderly patients undergoing posterior approach spinal surgery.Methods:This was a randomized, controlled trial.A total of 84 patients aged 65-75 years treated with posterior approach spinal surgery under general anesthesia were enrolled.Patients were randomly divided into two groups: the experimental group(n=42)and the control group(n=42). The two groups were treated with the same anesthesia procedure.The control group was given routine temperature management, while the experimental group used in-house made heat preservation socks in addition to routine temperature management during the entire surgical process.The anal temperature of patients was dynamically monitored with a disposable body temperature probe, and body temperature, heart rate, mean arterial pressure and oxygen saturation were recorded at the time of anesthesia induction and intubation(T 0), skin incision(T 1), 1 hour into surgery(T 2), 2 hours into surgery(T 3), the end of surgery(T 4), arrival at the post-anesthesia care unit(T 5), immediately after extubation(T 6)and 1 hour after extubation(T 7). The occurrence of intraoperative body temperature lower than 36℃, postoperative extubation time, incidence of shivering, postoperative incision infection rate and average length of stay were recorded.Changes in C-reactive protein and procalctonin levels were recorded. Results:There was no significant difference in sex composition, age, height, weight, body mass index and operative time between the two groups(all P>0.05). From T 2 to T 7, the body temperature of the experimental group was higher than that of the control group[T 2: (36.5±0.5)℃ vs.(36.3±0.3)℃, (36.6±0.6)℃ vs.(36.2±0.4)℃, (36.6±0.6)℃ vs.(36.2±0.4)℃, (36.6±0.6)℃ vs.(36.2±0.4)℃, (36.6±0.6)℃ vs.(36.2±0.4)℃, (36.6±0.5)℃ vs.(36.2±0.3)℃, t=2.229, 3.514, 3.823, 3.790, 3.722, 4.408, P=0.029, 0.001, 0.001, 0.001, 0.001, 0.000]. The incidence of intraoperative body temperature lower than 36 ℃ in the control group was higher than that in the experimental group(47.6% vs.21.4%, χ2=6.372, P=0.012). The incidence of postoperative shivering in the experimental group was lower than that in the control group(21.4% vs.59.5%, χ2=12.649, P<0.001). There was no significant difference between the two groups in postoperative incision infection rate, average length of stay and postoperative inflammatory infection indicators(all P>0.05). Conclusions:For elderly patients undergoing posterior spinal surgery, the in-house made heat preservation socks have favorable effects on body temperature maintenance and help reduce the occurrence of hypothermia and postoperative shivering.

Objective: To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC). Methods: The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14. Results: In the two-dimensional (2D) culture, CD41⁺, CD41⁺/CD61⁺, CD61⁺ megakaryocytic numbers increased significantly after adding P188 (all P<0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group (P<0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P>0.05). Conclusion 3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.

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

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