OBJECTIVETo establish a method for isolation, cryopreservation and recovery of the highly viable human peripheral blood monomuclear cells (PBMNCs) so as to achieve the long-term preservation of PBMNCs.

METHODSA total of 80-100 ml peripheral blood were collected from the healthy volumteers aged over 50 years old. The PBMNCs were isolated by the Ficoll density gradient technique and cryopreserved gradually by program control method in liquid nitrogen freezer of -196 °C. The serum-free medium and autoloqous plasma medium were test for preservation of PBMNCs. The cell viability was assessed at time point of 1, 2, 4, 8, 12 and 24 months after thawing. Finally, the proliferation ability, purity and cytotoxicity were compared between the autologous immune lymphocytes (AIL) induced from cryopreserved PBMNCs and AIL as control from fresh PBMNCs.

RESULTSAfter separating, the cell viability was 99.6%±0.4%, and the recovery rate of lymphocytes was 58.4%±6.52%. The cell recovery rate of lymphocyte was 89.7%±3.82% at 24 months. The quality assurance program was reliable within 2 years of running. The AIL cells induced with cryopreserved PBMNCs were not significantly different from those induced from fresh PBMNCs in terms of proliferative action, purity and cytotoxicity(CD3(+)CD8(+) ≥45%,CD3(+)CD56(+) NKT≥10%,CD4(+)CD25(+) NKT≤10%).

CONCLUSIONManual separation of lymphocytes in vitro can get enough high-quality PBMNCs. The long-term cryopreserved PBMNC still maintain their high viability. The reinfusion of the clinical autologous immune cells would be advantageous for early tumor immunotherapy. Human AIL induced from cryopreserved PBMNC maintain their anti-tumor ability. These findings have the important implications for the application of these cells to adoptive cellular therapy.

OBJECTIVE: The aim of this study was to investigate the effect of saliva of patients with chronic periodontitis (CPD) on the differentiation, activation, and secretion of osteoclast-maturing mediators of macrophages. METHODS: A total of 40 saliva samples were collected from healthy donors (n=20) and severe periodontitis patients (n=20). Peripheral blood mononuclear cells (PBMCs) and THP-1 monocyte line cells were challenged with 15% saliva for 5 days. The phenotype, surface marker, and phagocytosis of macrophages were analyzed by flow cytometry and microscopy. Osteoclast-maturing mediators were assayed by using enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: When PBMCs were treated with CPD saliva for 5 days, 61.25%±11.33% of cells were transformed into large granular cells; 86.78%±13.69% of large granular cells were identified as CD14⁺⁺CD16⁺ macrophages. When THP-1 cells were treated with CPD saliva, most cells attached to the bottom of cell culture plates, thereby exhibiting macrophage morphology and releasing additional osteoclast-maturing mediators. Furthermore, the phagocytosis of THP-1 cells considerably increased in the presence of CPD saliva (66.35%±9.67%) compared with medium control (33.33%±7.52%), or healthy saliva (40.71%±3.52%). CONCLUSIONS Saliva from patients with CPD can induce macrophage differentiation, activate phagocytose microorganisms, and secrete osteoclast-maturing mediators.
Cell Differentiation ; Humans ; Leukocytes, Mononuclear ; Macrophages ; Monocytes ; Periodontitis ; immunology ; Saliva

Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.
Animals ; Bone Marrow ; Cell Differentiation ; Cell Line ; Cytokines ; Erythrocytes ; cytology ; Erythroid Cells ; Leukocytes, Mononuclear ; cytology ; Mice

OBJECTIVE: To explored whether adipose derived stem cells (ASCs) could inhibit the pro-liferation of peripheral blood mononuclear cells (PBMCs) and whether inflammatory priming could enhance this property of ASCs. METHODS: We isolated ASCs using collagenase from adipose tissue and expanded them in vitro. Cells were induced to differentiate into adipogenic and osteogenic lineages. The cells at passage 3 to passage 5 were used for the experiments. After carboxy fluoresce in succinimidyl ester (CFSE) staining, PBMCs were co-cultured with inflammatory priming ASCs. The PBMCs cultured without ASCs or with non-treated ASCs defined as control groups. Then we used flow cytometry to detect the proliferation of PBMCs. RESULTS: ASCs had fibroblast-like phenotype and were spindle shaped. They were able to differentiate into cells of adipogenic and osteogenic lineages in specific induction media. ASCs had the CD expression profile consistant with the International Federation for Adipose Therapeutics statement. The percentage of parent cells in PBMC after co-cultured with ASCs increased, though there was no statistical significance. However, when co-cultured with inflammatory priming ASCs, the percentage of parent cells significantly increased (with inflammatory priming ASCs group vs. without ASCs group, 38.7%±10.0% vs. 28.4%±8.9%, P<0.05). This indicated that inflammatory priming ASCs could significantly inhibit the proliferation of PBMCs. CONCLUSION Inflammatory cytokines can enhance the immunosuppressive ability of ASCs. Our findings may help the application of ASCs in tissue repairment with better results.
Adipocytes ; Adipose Tissue ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Inflammation ; Leukocytes, Mononuclear ; Stem Cells

The purpose of study was to investigate the in vitro proliferation ability of PHA-induced CIK cells and traditionally prepared CIK cells, the effector cell level and its influence on killing activity to K562 cells, and to analyze the difference between them. The peripheral blood mononuclear cells(PBMNC) of healthy persons were isolated and divided into A and B group. The CIK cells in A group were obtained by using traditional culture method, the CIK cells in B group were prepared by PHA induction. During the cultivation, the cell survival rate and cell absolute value in the cell culture system were counted every 3 days. On day 15 of culture, the cell immunophenotype of 2 groups were detected by flow cytometry, and the ratios of CD3(+)CD56(+), CD3(+)CD8(+) and CD3(+)CD4(+) cells in total cell amount of culture system were accounted. Meantime, the killing activity to K562 cells in different effector-target ratios was detected by using CCK-8 kit between the 2 groups. The results showed that the method of preparing CIK by PHA induction promoted the cell proliferation more than that of the traditional method (P < 0.05), moreover, both the survival rate of cells in 2 groups was more than 90%. The CD3(+)CD8(+), CD3(+)CD56(+) cell ratio in 2 groups obviously increased. As compared with traditional method, the CD3(+)CD8(+) cell level in B group was enhanced (P < 0.05); but there were no statistical differences in increase of CD3(+)CD56(+) cell level and decrease of CD3(+)CD4(+) cell level between 2 groups. while the effector-target ratio is 5:1, 10:1, 20:1 and 40:1, the killing activity of PHA-induced CIK cells to K562 cells was more stronger than traditionally-prepared CIK cells (P < 0.05), moreover, along with increase of effector-target ratio, the difference of killing activity to K562 cells in 2 groups significantly increased. It is concluded that compared with traditional method for preparing CIK cells, the new way by PHA induction can increase the proliferation of CIK cells obviously, enhance the ratio of CD3(+)CD8(+) cells and strengthen the killing activity to the K562 cells. This new way provides a new source of CIK cells and reliable evidence for cyto-immune therapy of leukemia and other tumors.
Cell Proliferation ; Cytokine-Induced Killer Cells ; cytology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; Phytohemagglutinins ; pharmacology

OBJECTIVETo detect the B cell activating factor (BAFF) and explore its significance in patients with warm autoimmune hemolytic anemia (WAIHA).

METHODSThe levels of serum soluble BAFF (sBAFF) and BAFF mRNA in peripheral blood mononuclear cells (PBMCs) in 30 healthy volunteers (control group) and 43 patients with WAIHA were measured by ELISA and real-time quantitative polymerase chain reaction (RT-qPCR) respectively.

RESULTSThe levels of serum sBAFF and BAFF mRNA in PBMCs in pretreatment group \[2311 (825 approximately 6523) ng/L and 884 (463 approximately 2346) ng/L\] was significanly higher than those in posttreatment group\[1205(358 approximately 5014) ng/L and 446(138 approximately 2699) ng/L\] and control group\[1128 (590 approximately 3201) ng/L and 341 (102 approximately 965) ng/L\] (both P < 0.01), the difference between the posttreatment group and control group was not statistically significant. There was no significant difference between therapy responsive and nonresponsive groups before treatment. There was a significant difference between the pre- and post-treatment resuets in responsive group (P < 0.01), but not in nonresponsive group (P > 0.05). The serum levels of sBAFF was positively correlated with the levels of the BAFF mRNA in PBMCs both in pre- and post therapy group (both P < 0.05).

CONCLUSIONThe levels of serum sBAFF and BAFF mRNA in PBMCs are increased in patients with WAIHA, their dynamic alterations may contribute to the development of WAIHA.

OBJECTIVETo investigate the expression of miR-21 and miR-30b in multiple myeloma (MM).

METHODSPeripheral blood mononuclear cells from patients with MM were cultured at 2.5 x 10(6) cells/ml in alpha-MEM supplemented with 10% of fetal bovine serum, antibiotics, RANKL (50 ng/ml), and macrophage colony-stimulating factor (25 ng/ml) for 10 to 14 days to obtain osteoclasts with bone-resorbing activity. Primary myeloma cells were purified from 12 MM patients. Of them, 8 samples were cocultured with osteoclasts and 4 as noncocultured control. The expression of miR-21 and miR-30b was detected by real-time PCR.

RESULTSThe viability of MM cells recovered from cocultures was higher than those of noncocultured control. After cocultured with osteoclasts, primary myeloma cells from eight patients exhibited a 1.3- 5.9-fold increase in miR-21 expression and 1.38- 4.32-fold decrease in miR-30b expression compared with controls. In highly purified plasma cells from 3 healthy subjects, 12 MM patients and 11 MM cell lines, the expression of miR-21 was 1.9 +/- 0.8, 6.5 +/- 4.9 and 35.1 +/- 36.2, respectively; the expression of miR-30b was 13.6 +/- 1.8, 7.2 +/- 6.3 and 4.5 +/- 1.9, respectively.

CONCLUSIONSmiR-21 acts as an oncogene and miR-30b a tumor suppressor gene in MM.

The influencing factors on cord blood storage after collection and mononuclear cell separation as well as cryopreservation were studied. The mononuclear cell are separated from blood after blood collection, then cryopreserved and washed after thawed. Results showed that the cord blood kept at 4 degrees C or room temperature less than 24 hours after blood collection, mononuclear cell separated by hydroxyethylstarch and 2 centrifugations, mononuclear cell cryopreserved with 50% DMSO and autoplasma from cord blood as protectives and washing the cells after thawing. In conclusion, the optimal project in this study can effectively preserve cord blood mononuclear cells.
Blood Preservation ; Cell Separation ; methods ; Cryopreservation ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; physiology

Objective: To establish a method for the induction of peripheral blood mononuclear cells to hepatocyte-like cells, and preliminarily investigate cell response to injury under the effect of acetaminophen (APAP). Methods: The surface marker CD45 of peripheral blood mononuclear cells wase detected cells by using flow cytometry and immunofluorescence methods. The cellular morphology of induced hepatocyte-like cells was observed under an inverted microscope. Real-time fluorescent quantitative PCR (RT-PCR) was used to detect the expression level of hepatocyte-specific genes, such as cytochrome (CY) P1A2, CYP3A4, CYP2C9, albumin (ALB), alpha-fetoprotein (AFP), and hepatocyte nuclear factor (HNF)4α mRNA. Immunofluorescence method was used to detect intracellular hepatocyte markers AFP, HNF4α, and ALB expression at the protein level. Biochemical analyzer was used to detect hepatocyte-specific secretory functions of AFP, ALB, and urea. Luciferase chemiluminescence method was used to detect the activity of key drug metabolizing enzyme CYP3A4. Colorimetric assay was used to detect the effect of the drug acetaminophen on hepatocyte-like cells, and alanine aminotransferase (ALT) was used as an indicator of liver cell injury. The statistical differences between the data were compared with t-test and rank-sum test. Results: The positive expression rate of CD45 cell surface markers isolated from peripheral blood mononuclear cells was about 98%, and hepatocyte-like cell morphology changes appeared on 15th day of induction. Compared with isolated mononuclear cells, CYP1A2, CYP3A4, CYP2C9, ALB, AFP and HNF4α mRNA was markedly elevated. The expression level of AFP, ALB and HNF4α protein were equally increased, and the secretory function of AFP, ALB and urea were enhanced. Compared with primary hepatocytes, CYP1A2, CYP2C9, AFP, HNF4α mRNA, and CYP3A4 mRNA did not decrease. The expression levels of AFP, ALB, and HNF4α proteins in the cells did not decrease, and the secretory function of AFP, ALB, and urea did not decrease. In addition, the CYP3A4 enzyme activity produced by hepatocyte-like cells was similar to that of primary hepatocytes. Compared with hepatocyte-like cells incubated without APAP, hepatocyte-like cells incubated with APAP had higher ALT level. Under the effect of APAP, the ALT level of hepatocyte-like cells was higher than isolated mononuclear cells. Conclusion: Peripheral blood mononuclear cells can be induced into hepatocyte-like cells with partial characteristics of hepatocytes, including the activity of CYP3A4, a key enzyme of hepatocyte drug metabolism. Additionally, preliminarily ALT secretory features reflect the hepatocytes injury under the effect of acetaminophen.
Acetaminophen/pharmacology* ; Cell Differentiation ; Cells, Cultured ; Hepatocytes ; Leukocytes, Mononuclear ; RNA, Messenger

OBJECTIVE: To study the role of gamma-delta T (γδ T) cells and its subsets in the immunopathogenesis of Henoch-Schönlein purpura (HSP) in children, and to provide new ideas for the treatment of HSP in children from the aspect of γδ T cell regulation. METHODS: A total of 33 children with HSP were enrolled as the HSP group, and 21 healthy children were enrolled as the healthy control group. The percentages of γδ T cells and its subsets Vδ1 T and Vδ2 T cells among peripheral blood mononuclear cells (PBMCs) were measured, as well as the apoptosis rate of γδ T cell and plasma level of interleukin-17 (IL-17). RESULTS: Compared with the healthy control group, the HSP group had significantly lower percentages of lymphocytes in PBMCs and Vδ2 T cells in γδ T cells (P<0.05). The HSP group had significantly higher percentage of Vδ1 T cells in γδ T cells and plasma level of IL-17 than the healthy control group. The HSP group had a significantly higher overall apoptosis rate of γδ T cells than the healthy control group (P<0.05), especially early apoptosis. The percentage of Vδ2 T cells was positively correlated with overall apoptosis rate (r=0.615, P<0.05) and was negatively correlated with IL-17 level (r=-0.398, P<0.05). CONCLUSIONS Vδ1/Vδ2 T cell immune imbalance mediated by γδ T cells and over-activation of IL-17 may be involved in the development of HSP, among which the disturbance of immune tolerance induced by Vδ2 T cells plays an important role in the pathophysiology of the disease.
Child ; Humans ; Leukocytes, Mononuclear ; Purpura, Schoenlein-Henoch ; Receptors, Antigen, T-Cell, gamma-delta ; T-Lymphocytes