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

Stirred culture offers a number of advantages over static systems as it maintains a stable, homogeneous culture environment and is easy to scale-up. This paper focused on the development and application of stirred tank bioreactor to culture hematopoietic cells. Preliminary study of stirred culture of hematopoietic cells was carried out in cord blood mononuclear cells culture in spinner flask. The results showed that the amplification rates of total cell, CFU-GM and BFU-E, with the exception of CFU-Mk, were greater in spinner flask than T-flask. The number of total cells increased 20 fold after 14 days incubation in spinner flask. The amplification rates of CFU-GM, CFU-Mk and BFU-E reached maximum at 10th day, 10th day and 7th day respectively, and the maximal amplification rates were 9.2-fold, 5.5-fold and 2.4-fold respectively, whereas the rate of CD34+ cells in spinner flask was (6.7 +/- 4.0)-fold at day 10. These results indicated that the stirred culture system is better than the static culture systems for hematopoietic cell proliferation. The biocompatibility of cord blood MNC to different types of materials used in bioreactors was also tested. The results showed that glass, stainless steel 316L and polytetraflouroethylene (PTFE) supported the growth of hematopoietic cells well. A higher cell density was reached in stirred bioreactors with controlled pH and DO than static culture. These findings suggested that the controlled large-scale culture could be used to overcome the clinical shortage of hematopoietic cells.
Antigens, CD34 ; metabolism ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Humans ; Polytetrafluoroethylene ; Stainless Steel

OBJECTIVETo investigate the properties of screw-bone interface of expansive pedicle screw (EPS) in osteoporotic sheep by micro-CT and histological observation.

METHODSSix female sheep with bilateral ovariectomy-induced osteoporosis were employed in this experiment. After EPS insertion in each femoral condyle, the sheep were randomly divided into two groups: 3 sheep were bred for 3 months (Group A), while the other 3 were bred for 6 months (Group B). After the animals being killed, the femoral condyles with EPS were obtained, which were three-dimensionally-imaged and reconstructed by micro-CT. Histological evaluation was made thereafter.

RESULTSThe trabecular microstructure was denser at the screw-bone interface than in the distant parts in expansive section, especially within the spiral marking. In the non-expansive section, however, there was no significant difference between the interface and the distant parts. The regions of interest (ROI) adjacent to EPS were reconstructed and analyzed by micro-CT with the same thresholds. The three-dimensional (3-D) parameters, including tissue mineral density (TMD), bone volume fraction (BVF, BV/TV), bone surface/bone volume (BS/BV) ratio, trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), were significantly better in expansive sections than non-expansive sections (P less than 0.05). Histologically, newly-formed bony trabeculae crawled along the expansive fissures and into the center of EPS. The newly-formed bones, as well as the bones at the bone-screw interface, closely contacted with the EPS and constructed four compartments.

CONCLUSIONSThe findings of the current study, based on micro-CT and histological evaluation, suggest that EPS can significantly provide stabilization in osteoporotic cancellous bones.

Animals ; Bone Screws ; Bone and Bones ; diagnostic imaging ; pathology ; Female ; Osteoporosis ; diagnostic imaging ; pathology ; Sheep ; Tomography, X-Ray Computed

OBJECTIVETo investigate the properties of screw-bone interface of expansive pedicle screw (EPS) in osteoporotic sheep using micro-CT and histology.

METHODSSix female sheep with bilateral ovariectomy-induced osteoporosis were employed in this experiment and were randomly assigned into 2 groups: A and B. After EPS insertion in each femoral condyles, sheep in group A were bred for 3 months, while those in group B 6 months. Femoral condyles with EPS were 3D-imaged and reconstructed by micro-CT. Histology was evaluated thereafter.

RESULTSThe trabecular microstructure was denser at the screw-bone interface than in the distant parts in expansive section, especially within spiral marking. In the non-expansive section, however, there was no significant difference between the interface and the distant parts. The regions of interest (ROI) adjacent to EPS were reconstructed and analyzed by micro-CT using the same thresholds. The 3D-parameters generated, including tissue mineral density (TMD), bone volume fraction (BVF, BV/TV), bone surface/bone volume (BS/BV) ratio, trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), were significantly better in expansive than in non-expansive sections (P < 0.05). Histologically, newly formed bone trabeculae crawled along the expansive fissures and into the center of EPS. The newly formed bones, as well as the bone at the bone-screw interface, closely contacted the EPS and constructed four compartments.

CONCLUSIONSBased on micro-CT and histological evaluation, the study suggest that EPS can significantly provide stabilization in osteoporotic cancellous bone.

Animals ; Bone Density ; Bone Screws ; Disease Models, Animal ; Female ; Femur ; diagnostic imaging ; metabolism ; surgery ; Osteoporosis ; etiology ; pathology ; surgery ; Ovariectomy ; adverse effects ; Reproducibility of Results ; Sheep ; Tomography, X-Ray Computed ; methods

Objective:To investigate the effects of induced pluripotent stem cell-derived exosome (iPSC-Exo) on releasing inflammatory factors from microglia induced by lipopolysaccharide (LPS).Methods:iPSC derived from the tubular epithelial cells of sepsis encephalopathy patients were resuscitated and cultured. The iPSC-Exo was isolated by low-temperature ultracentrifugation and analyzed by transmission electron microscopy, Western blot and high sensitivity flow cytometry (HSFCM). Based on the concentration of iPSC-Exo, human microglia line HMO6 cells activated by LPS (100 ng/mL) were divided into four groups randomly: LPS+ phosphate buffer solution (PBS) group, LPS+iPSC-Exo 10 5 group, LPS+iPSC-Exo 10 6 group and LPS+iPSC-Exo 10 7 group. The control group was added equal PBS but not LPS. After culture for 24 h, the concentrations of malondialdehyde in cells were detected. Quantitative RT-PCR was used to measure the mRNA expression levels of macrophage inflammatory protein 2 (MIP2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in the cells and enzyme-linked immunosorbent assay (ELISA) was used to assess the concentration of these cytokines in the supernatant. Under the same concentration of iPSC-Exo, one-way ANOVA and SNK- q test were used for comparison between groups. Results:The extracts showed spherical membrane structure by transmission electron microscopy. HSFCM showed the mean diameter of the extracts was (74.66±15.60) nm and the concentration around 2.98×10 10/mL. Western blot analysis showed high expression of exosome markers CD63, Alix and TSG101, but not GM130. Intracellular MDA concentration and mRNA expression levels and protein concentration of MIP2, TNF-α, IL-1β and IL-6 in the LPS+PBS group were significantly higher than those in the control group (all P<0.01). With the increase of iPSC-Exo concentration, the intracellular MDA concentration decreased gradually ( P<0.01), the mRNA expression levels of inflammatory factors showed a gradual downward trend (all P<0.05). Each inflammatory cytokine in the supernatant declined in a manner that was almost consistent with mRNA. Concentrations of MDA remained constant in the control group. Conclusions:iPSC-Exo derived from the tubular epithelial cells of sepsis encephalopathy patients alleviate oxidative stress and inflammation effect of microglia induced by LPS, and the modulatory effect is dose-dependent.