The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The influence of the cryoprotectant prescriptions and the frozen methods on the cryopreservative effectiveness was little. It is concluded that the human bone marrow cells with DMSO-AuP or DMSO-HES-HuA as cryoprotectant, frozen by a programmed freezer or -80 degrees C refrigerator, could be then preserved in liquid nitrogen for long time. When the preserving time was as long as 21 to 25 years, the morphology, the recovery rate and the activity of various kinds of cells were still good. The method of freezing by -80 degrees C refrigerator with 5% DMSO-6% HES-4% HuA and preserving in liquid nitrogen would be convenient, cheap and easily-manipulated for preservation of the human bone marrow cells.
Bone Marrow Cells ; cytology ; Cell Survival ; Colony-Forming Units Assay ; Cryopreservation ; methods ; Cryoprotective Agents ; Humans ; Nitrogen ; Time Factors

The aim of this study was to detect DNA damage during expansion ex vivo of umbilical cord blood (UCB) hematopoietic cells and explore the optimal harvest time for culture of CB hematopoietic cells. Mononuclear cells (MNCs) separated from UCB were cultured in a serum-free system supplemented with cytokines and colony forming units were assessed by semisolid culture at the same time. On day 0, 7, 14 and 21 cells were collected for single cell gel electrophoresis (SCGE) analysis and CFUs were also assayed by SCGE, CD34+ cells and CD133+ cells were quantitated by fluorescence-activated cell sorting (FACS). The results showed that the percentage of CD34+ and CD133+ cells was found to be highest after short-term culture (<14 days) and the cord blood DNA damage rate was observed to be less than 5.0% at earlier time points, but at day 21 the DNA damage rate was 28.2%, which was higher than that at day 0 (p=0.000), the tail length of the DNA comet was longer than that at day 0 (p=0.000). The tail lengths of DNA damage on other time points were not significantly different from that at day 0. It is concluded that the DNA damage rate is less than 5.0% after short-term (<14 days) culture of UCB cells ex vivo by using this method. After 14 days DNA damage rate increases significantly. The optimal harvest time of cord blood cells after culture ex vivo would be within 14 days.
Cell Division ; Cells, Cultured ; Colony-Forming Units Assay ; DNA Damage ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVEThough the rabbit is one of most widely used experimental animals for medical regenerative research, it remains difficult to culture mesenchymal stem cells (MSC) on a in large scale due to the extremely lower number and hematopoietic cell contamination. This study was aimed to establish a novel protocol to generate rabbit MSC by culturing bone marrow plugs instead of bone marrow cells so as to obtain a large amount of MSC with higher proliferation and self-renewal properties.

METHODSThe primary MSC were generated from collagenase digested bone marrow plugs and bone marrow cells, respectively. The surface antigen profile of MSC was analyzed with flow cytometry and the cells were induced to differentiate into osteoblasts and adipocytes. The proliferation capacity of MSC were assessed by CCK-8 method. To test their self-renewal property, the colony forming unit-fibroblast assay was performed. Moreover, the cell yields of passage 1, 2, 3 and 4 were calculated.

RESULTSThe bone marrow plug-derived MSC shared the typical fibroblast-like morphology same as bone marrow cells derived MSC. Moreover, the ratio of CD45 positive hematopoietic cells in bone marrow plug-derived MSCs was significantly lower than that of bone marrow cell-derived MSC. The results of multi-differentiation experiments showed that bone marrow-plug-derived MSC exhibited similar multi-potent property to their bone marrow counterparts. In addition, the results of CCK-8 and CFU-F assay demonstrated that bone marrow plug-derived MSC grew more robustly and more CFU-F were formed in the culture plates, which indicated that the cells possessed higher proliferation and self-renewal capacities. Promisingly, a larger amount of cells were harvested via using the new protocol.

CONCLUSIONThe purity and yields of the bone marrow plug-derived MSC are satisfactory compared with previous rabbit MSC isolation methods. The findings may be helpful for the research of regenerative medicine.

Adipocytes ; Animals ; Bone Marrow ; Bone Marrow Cells ; Cell Differentiation ; Cell Separation ; Colony-Forming Units Assay ; Mesenchymal Stromal Cells ; Osteoblasts ; Rabbits

Cell Proliferation ; Colony-Forming Units Assay ; Glioma ; pathology ; Hematopoietic Stem Cells ; pathology ; Humans ; Stem Cells ; cytology ; metabolism

In order to investigate the influence of angiotensin II on hematopoietic system, CD34+ cells in cord blood were purified, and the effects of angiotensin II in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin II in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin II concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3 +/- 0.8 times to 7.8 +/- 2.3 times when angiotensin II was added in the presence of SCF+G-CSF+GM-CSF+IL3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1 +/- 1.8 times to 9. 2 +/- 2.3 times with angiotensin II in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin II could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin II had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.
Angiotensin II/*pharmacology ; Antigens, CD34/*metabolism ; Cells, Cultured ; Colony-Forming Units Assay ; Fetal Blood/*cytology ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoietic Stem Cells/*cytology

OBJECTIVE: To investigate the effect of hematopoietic stimulating factors on the expansion of mature megakaryocytes. METHODS: (2, 4, 6, 8, 10) x 10(5)/mL bone marrow single nucleus cells (BMNC) were added in the culture system of colony forming unit-megkaryocyte (CFU-Meg) to find out the relationship of the cultured BMNC with the output of CFU-Meg. rmSCF + rmTPO + rmIL-3 (3HSFs) and rmSCF + rmTPO + rmIL-3 + rmIL-6 (4HSFs) or F-CM were added in the liquid culture system of megkaryocytes respectively. The number of mature megakaryocytes were counted every other day. RESULTS: The number of CFU-Meg increased with the increase of the cultured BMNC. The CFU-Meg productivity of 1 x 10(6) BMNC/mL culture system was more than that of 2 x 10(5) BMNC/mL culture system. 3HSFs and 4HSFs or F-CM significantly promoted the expansion of mature megakaryocytes in the liquid culture system, but the effect was different. The peak time of the number of mature megakaryocytes in 3HSFs and 4HSFs or F-CM were 7 d, 7 d and 5 d respectively. CONCLUSION 3HSFs and 4 HSFs or F-CM had positive effect on the expansion of mature megakaryocytes. 4HSFs was better than 3HSFs and F-CM. 3HSFs was better than F-CM. The peak time of the number of mature megakaryocytes in different culture systems was different.
Animals ; Cells, Cultured ; Colony-Forming Units Assay ; Female ; Hematopoietic Cell Growth Factors ; pharmacology ; Interleukin-3 ; pharmacology ; Interleukin-6 ; pharmacology ; Macrophage Colony-Stimulating Factor ; pharmacology ; Male ; Megakaryocytes ; cytology ; Mice

The aim of the present paper is to better understand the mechanism of hematopoietic development through studying the biological characteristics of hematopoietic progenitor cells at different stages of development. Firstly, the c-kit expression levels of the mononuclear cells from murine embryonic aorta-gonad-mesonephros (AGM) region at embryonic day (E)10.5 and E11.5, fetal liver (FL) at E12.5, E14.5, E16.5, E18 and bone marrow (BM) were assayed with fluorescence activated cell sorting (FACS). Secondly, hematopoietic progenitor cells derived from AGM at E10.5, FL at E14.5 and BM were isolated by using c-kit microbeads. Isolated c-kit(+) population cells from AGM, FL and BM were then co-cultured with E14.5 FL-derived stromal cells in transwell co-culture system in vitro. After 3, 7, 10 days of co-culture, numerous floating cells were generated. The floating cells generated in transwell inserts were collected for FACS cell count, migration activity detection and colony forming unit (CFU) formation assay. The results showed that the c-kit was highly expressed in E10.5 AGM, with the percentage of c-kit(+) cells declining during AGM development. c-kit expression was highly expressed again in E12.5 FL, declining along with the progressive development of the FL region. Co-cultured with FL-derived stromal cells, E10.5 AGM-derived c-kit(+) cells produced the highest number of hematopoietic cells, while BM-derived c-kit(+) cells produced the lowest number of hematopoietic cells. Compared with E10.5 AGM-derived c-kit(+) cells, E14.5 FL- and BM- derived c-kit(+) cells inclined to differentiate after 7 to 10 days of culture in vitro. E10.5 AGM and E14.5 FL-derived c-kit(+) cells exhibited a higher migration activity than BM-derived c-kit(+) cells. Moreover, E10.5 AGM-derived c-kit(+) cells showed a higher ability to form mixed colony-forming unit (CFU-Mix) colony. In conclusion, compared with FL- and BM-derived c-kit(+) cells, E10.5 AGM-derived c-kit(+) hematopoietic progenitor cells exhibit better proliferation, migration potential, and have a higher ability to maintain the undifferentiation state in vitro, providing an insight into their clinical manipulation.
Animals ; Aorta ; embryology ; Coculture Techniques ; Colony-Forming Units Assay ; Gonads ; embryology ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Mesonephros ; embryology ; Mice ; Proto-Oncogene Proteins c-kit ; metabolism ; Stromal Cells ; cytology

OBJECTIVESTo investigate the stroma-independent growth ability, multilineage differentiation and expansion of the single hematopoietic stem/progenitor cell from patients with paroxysmal nocturnal hematoglobinuria (PNH).

METHODThe CD(34)(+) CD(59)(+) and CD(34)(+) CD(59)(-) cells from PNH patients and CD(34)(+) CD(59)(+) cells from normal volunteers were sorted as each single cell into a well of 96 well culture plates containing culture medium supplemented with SCF, IL-3, Epo, GM-CSF, G-CSF, IL-6, Tpo and Flt-3 ligand.

RESULTS(1) Single PNH CD(34)(+) CD(59)(-) cell had a higher capacities for plating efficiency, colony (>/= 50 cells) formation and cell expansion than that of the PNH CD(34)(+) CD(59)(+) cells (P < 0.05); (2) Both the single CD(34)(+) CD(59)(-) cells from PNH patients and the single CD(34)(+) CD(59)(+) cells from normal controls had similar capacities for cell plating efficiency and colony and large colony formation. The PNH CD(34)(+) CD(59)(-) cells had a lower average cell production and cell expansion capacity. (3) The single CD(34)(+) CD(59)(+) cells from both PNH patients and normal controls showed the same capacities for cell plating efficiency and colony formation. The PNH CD(34)(+) CD(59)(+) cells exhibited much lower capacity for large colony formation, average cell production and total cell expansion. (4) A diminished secondary colony formation ability was also observed in the PNH CD(34)(+) CD(59)(+) and CD(34)(-) CD(59)(-) clones.

CONCLUSIONThe single PNH CD(34)(+) CD(59)(-) cells had growth advantage over the single PNH CD(34)(+) CD(59)(+) cells to some extent, but they both had impaired growth abilities as compared with CD(34)(+) cells from normal volunteers.

Antigens, CD34 ; immunology ; CD59 Antigens ; immunology ; Cell Culture Techniques ; Cell Division ; physiology ; Colony-Forming Units Assay ; Hematopoietic Stem Cells ; immunology ; pathology ; Hemoglobinuria, Paroxysmal ; physiopathology ; Humans

To study the regulation of TGF-beta(1) on the development of hemangioblast, embryonic stem cell-derived blast forming cells (BL-CFC) were used as the model of hemangioblast in vitro. TGF-beta(1) or anti-TGF-beta(1) neutralization antibody was added in the medium of embryoid body (EB) generation for observating influence of TGF-B(1) addition in different culture stages on number of BL-CFC and differentiation of BL-CFC to endothelial and hematopoietic cells. The results showed that antagonizing TGF-beta(1) in the course of EB growth could significantly reduce the number of BL-CFC (P<0.01), and the frequency of Flk-1(+) cells was also decreased consistently. Furthermore, the BL-CFC derived from EB pretreated with TGF-beta(1) demonstrated remarkably elevated hematopoietic and endothelial potential, whereas such bi-potential was impaired in the group with neutralizing antibody. It is concluded that TGF-beta(1), a conventional negative regulator in hematopoiesis and angiogenesis exert positive effects on the development and differentiation capacities of BL-CFC in vitro.
Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Colony-Forming Units Assay ; Embryonic Stem Cells ; cytology ; Hematopoiesis ; physiology ; Mice ; Neovascularization, Physiologic ; physiology ; Transforming Growth Factor beta1 ; antagonists & inhibitors

Colony-Forming Units Assay ; Cryopreservation ; methods ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunotoxins ; immunology ; Leukocytes, Mononuclear ; cytology ; Ricin ; immunology ; T-Lymphocytes ; cytology ; immunology ; Time Factors