[The role of poloxamer 188 for cord blood mononuclear cells into megakaryocytes cultivation and induction in three-dimensional WAVE Bioreactor].

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.

[Mononuclear cells of umbilical cord blood differentiation to granulocyte cell in vitro].

Objective: To explore an optimal method for granulocyte cell production from umbilical cord blood mononuclear cells. Methods: Erythrocytes were precipitated by hydroxyethyl starch. Mononuclear cells were isolated through Ficoll density gradient centrifugation. Different media, additives and cultivation model were chosen for granulocyte induction. Cell morphology was observed by microscopy, and cell phenotype was detected by flow cytometry. The CD18 expression of granulocytes was tested by immunofluorescence assay, and phagocytosis test was executed as well. Results: Compared to fetal bovine serum (FBS) treatment group, cell viability, counts and differentiation rate of granulocytes induced by X-VIVO(TM) 15 combined with TPO, SCF, G-CSF but without FBS were superior. And X-VIVO(TM)15 medium was better than SCGM medium at effectiveness and cost. Using two-stage mode of hematopoietic stem cell expansion followed by granulocyte induction with X-VIVO(TM)15 combining TPO, SCF and G-CSF, cell proliferation was nearly 132 times at day 21. Flow cytometry showed that the differentiation was lagged in 2-stage mode than in direct induction mode, CD15 expression was (69.60± 1.06) % vs (97.73±0.39) %; Wright-Giemsa staining demonstrated mature granulocytes; immunofluorescence showed the expression of lysosomal proteins CD18. A strong phagocytic function of mature granulocytes was demonstrated by phagotrophic efficiency of (51.43±0.05) %. And granulocyte had chemotaxis ability under the role of chemotactic factor IL-8. Conclusion: Optimized culture media and cultivation mode are achieved for functional granulocytes induction in vitro.

[A clinical analysis of 123 cases of primary empty sella].

Objective: This study was conducted to analyze the clinical characteristics and pituitary function of patients with primary empty sella (PES). Methods: The clinical data from 123 hospitalized adult patients with PES from January 2010 to May 2016 were retrospectively studied. Results: (1) The average age of the 123 (male 43, female 80) PES patients was (59.2±13.6) years (ranging 24-92 years), among whom 61% patients were in the age group between 50-69 years. (2) The symptoms of the patients included fatigue (56.1%), headache (34.1%), nausea and vomiting (17.9%), gonadal dysfunction (17.1%), visual disturbance (5.7%) and hypopituitarism crisis (3.3%). (3) Hypopituitarism was found in 66 of the 123 patients. Among them, 36.6%, 31.7% and 17.1% were central hypoadrenalism, hypogonadism, and hypothyroidism, respectively.The percentage of hypopituitarism in complete PES was significantly higher than that in partial PES (P<0.05). (4) Sixteen patients were concomitant with other autoimmune diseases including 11 patients with Graves' disease and 2 with Cushing's syndrome due to adrenal adenoma. Conclusions: The incidence of hypopituitarism in PES was 53.7%, in which the pituitary-adrenal axis hypofunction was more common. An overall evaluation of the pituitary function was essential for the patients who had headache and fatigue, or with suspected PES. The patients with hypopituitarism should be given hormone replacement therapy in time and followed up afterword.