Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes†.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Effects of macromolecular crowding on the folding and aggregation of glycosylated MUC5AC.

OBJECTIVE: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. METHODS: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. RESULTS: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. CONCLUSION: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.

Effects of anticoagulants and Ficoll on human serum antibody reactivities and functions against Mycobacterium tuberculosis.

The ability to utilize leftover samples containing anticoagulants or Ficoll would provide substantial opportunities for future antibody and biomarker studies. Some anticoagulants might influence antibody reactivity against pathogens, but comprehensive studies investigating effects in the context of TB are lacking. We enrolled 24 individuals with and without history of M. tuberculosis and/or HIV-infection and investigated TB antibody reactivities, function, and other host protein biomarkers in simultaneously obtained serum and plasma from serum separation, EDTA, heparin, acid citrate dextrose (ACD), or mononuclear cell preparation (CPT™) tubes which contain heparin and Ficoll. Antibody isotype reactivities to two mycobacterial antigens, as well as phagocytosis of M. tuberculosis, correlated strongly and significantly between serum and plasma, irrespective of type of anticoagulant or Ficoll present (r ≥ 0.85, p < 0.0001). However, the presence of ACD resulted in slightly lower values than those obtained with serum in both indirect (antibody reactivities to mycobacterial antigens) and Sandwich ELISAs (soluble CD14 measurements). Our data demonstrate that leftover plasma, regardless of containing anticoagulants or Ficoll, can be used in TB antibody or other host protein biomarker studies but suggest the value of a correction factor when using ACD plasma interchangeably with serum in antibody binding studies.

Macromolecular Crowding Modulates Actomyosin Kinetics.

Actomyosin kinetics is usually studied in dilute solutions, which do not reflect conditions in the cytoplasm. In cells, myosin and actin work in a dense macromolecular environment. High concentrations of macromolecules dramatically reduce the amount of free space available for all solutes, which results in an effective increase of the solutes' chemical potential and protein stabilization. Moreover, in a crowded solution, the chemical potential depends on the size of the solute, with larger molecules experiencing a larger excluded volume than smaller ones. Therefore, since myosin interacts with two ligands of different sizes (actin and ATP), macromolecular crowding can modulate the kinetics of individual steps of the actomyosin ATPase cycle. To emulate the effect of crowding in cells, we studied actomyosin cycle reactions in the presence of a high-molecular-weight polymer, Ficoll70. We observed an increase in the maximum velocity of the actomyosin ATPase cycle, and our transient-kinetics experiments showed that virtually all individual steps of the actomyosin cycle were affected by the addition of Ficoll70. The observed effects of macromolecular crowding on the myosin-ligand interaction cannot be explained by the increase of a solute's chemical potential. A time-resolved Förster resonance energy transfer experiment confirmed that the myosin head assumes a more compact conformation in the presence of Ficoll70 than in a dilute solution. We conclude that the crowding-induced myosin conformational change plays a major role in the changed kinetics of actomyosin ATPase.

Quadruplex forming promoter region of c-myc oncogene as a potential target for a telomerase inhibitory plant alkaloid, chelerythrine.

Guanine rich sequences present in the promoter region of oncogenes could fold into G-quadruplexes and modulate transcription. Equilibrium between folding and unfolding of the quadruplexes in these regions play important role in disease processes. We have studied the effect of a putative anticancer agent chelerythrine on G-rich NHE III1 present in the promoter region of c-myc oncogene. We have demonstrated the ability of chelerythrine, a telomerase inhibitor, to block the hybridization of Pu27 with its complementary strand via folding it into a quadruplex structure. Calorimetry shows that the association of Pu27 with chelerythrine is primarily enthalpy driven with high binding affinity (∼10(5) M(-1)). The association does not lead to any major structural perturbation of Pu27. The resulting 2:1 complex has enhanced stability as compared to free Pu27. Another notable feature is that the presence of molecular crowding agent like ficoll 70 does not change the mode of recognition though the binding affinity decreases. We suggest that the anticancer activity of chelerythrine could be ascribed to its ability to stabilize the quadruplex structure in the c-myc promoter region thereby downregulating its transcription.

Novel use for polyvinylpyrrolidone as a macromolecular crowder for enhanced extracellular matrix deposition and cell proliferation.

Macromolecular crowding (MMC) is a biophysical effect that governs biochemical processes inside and outside of cells. Since standard cell culture media lack this effect, the physiological performance of differentiated and progenitor cells, including extracellular matrix (ECM) deposition, is impaired in vitro. To bring back physiological crowdedness to in vitro systems, we have previously introduced carbohydrate-based macromolecules to culture media and have achieved marked improvements with mixed MMC in terms of ECM deposition and differentiation of mesenchymal stem cells (MSCs). We show here that although this system is successful, it is limited, due to viscosity, to only 33% of the fractional volume occupancy (FVO) of full serum, which we calculated to have an FVO of approximately 54% v/v. We show here that full-serum FVO can be achieved using polyvinylpyrrolidone (PVP) 360 kDa. Under these conditions, ECM deposition in human fibroblasts and MSCs is on par, if not stronger than, with original MMC protocols using carbohydrates, but with a viscosity that is not significantly changed. In addition, we have found that the proliferation rate for bone marrow-derived MSCs and fibroblasts increases slightly in the presence of PVP360, similar to that observed with carbohydrate-based crowders. A palette of MMC compounds is now emerging that enables us to tune the crowdedness of culture media seamlessly from interstitial fluid (9% FVO), in which the majority of tissue cells might be based, to serum environments mimicking intravascular conditions. Despite identical FVO's, individual crowder size effects play a role and different cell types appear to have preferences in terms of FVO and the crowder that this is achieved with. However, in the quest of crowders that we have predicted to have a smoother regulatory approval path, PVP is a highly interesting compound, as it has been widely used in the medical and food industries and shows a novel promising use in cell culture and tissue engineering.

Optimization of culture conditions for bone marrow stromal cells in RPMI-1640 medium.

UNLABELLED: Bone marrow mesenchymal stem cells are important for both research and clinical purpose. A number of culture methods for these cells are available on the market, many of them consisting of specialized growing media in combination with growth factors. Our goal was to optimize a less expensive culture method for bone marrow mesenchymal cells. MATERIAL AND METHODS: Eight samples of bone marrow aspirates from patients were used. Out these 8 samples 2 were from healthy people, 3 from chronic granulocytic leukemia patients, 2 from multiple myeloma patients and 2 from patients with myelodysplastic syndrome. Bone aspirates from healthy people were used to optimize the culture method and the rest were used for testing the optimized method. Two methods were tried: 1. Cell culture starting from whole bone marrow, 2) cell culture after bone marrow separation in density gradient with Histopaque. RESULTS: Cell culture starting from whole bone marrow gives better yields for mesenchymal stem cells than methods which include gradient density separation of mononuclear cells with Ficoll-Histopaque. CONCLUSIONS: We have optimised a less expensive cell culture method for bone marrow mesenchymal cells.

Structure of metaphase chromosomes: a role for effects of macromolecular crowding.

In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ~30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100-200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ~30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.

Characteristic differences among osteogenic cell populations of rat bone marrow stromal cells isolated from untreated, hemolyzed or Ficoll-treated marrow.

BACKGROUND AIMS: Although bone marrow (BM) stromal cells (SC; BMSC) isolated from adherent cultures of untreated BM are known to contain both committed and uncommitted osteogenic cells, it remains unknown whether BMSC isolated either by hemolysis or Ficoll centrifugation also contain both of these populations. METHODS: Differences in the osteogenic cell populations of rat BMSC isolated from untreated, hemolyzed or Ficoll-treated BM were analyzed by in vivo transplantation, flow cytometry, alkaline phosphatase (ALP) assay, real-time polymerase chain reaction (PCR) and alizarin red staining. RESULTS: Transplantation of non-cultured samples indicated that the Ficolled BMSC contained the lowest number of committed osteogenic cells. Flow cytometric analysis of cultured, non-induced samples showed that the percentage of ALP-positive cells was significantly lower in Ficolled BMSC. Quantitative ALP assays confirmed that the lowest ALP activity was in the Ficolled BMSC. Hemolyzed BMSC also contained lower numbers of committed osteogenic cells than untreated BMSC, but still more than Ficolled BMSC. Interestingly, the Ficolled BMSC showed the greatest levels of osteogenic ability when cultured in osteogenic induction medium. CONCLUSIONS: These findings suggest that, although Ficolled BMSC rarely contain committed osteogenic cells, they are able to show comparable or even greater levels of osteogenic ability after induction, possibly because they contain a greater proportion of uncommitted stem cells. In contrast, induction is optional but recommended for both untreated and hemolyzed BMSC before use, because both these groups contain both committed and uncommitted osteogenic cells. These findings are of significant importance when isolating BMSC for use in bone tissue engineering.

Wiskott-Aldrich syndrome protein (WASP) and N-WASP are critical for peripheral B-cell development and function.

The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)-Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell-independent antigen TNP-Ficoll and to the T cell-dependent antigen TNP-keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.

Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens.

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.

An acidic pH and activation of phosphoinositide 3-kinase stimulate differentiation of pancreatic progenitors into insulin-producing cells.

Adult pancreatic nonendocrine cells represent a potential alternative source of insulin-producing tissue for the treatment of diabetes. Differentiation of these cells is regulated by various signaling pathways including the phosphoinositide 3-kinase (PI3K) pathway. Therefore, we evaluated the effect of PI3K on this process. Compared with untreated cells the differentiation of human nonendocrine pancreatic cells into insulin-producing elements was increased after treatment with IGF-1, EGF, and Exendin-4, growth factors known to be activators of the PI3K pathway (12.2 +/- 3.2% vs 9.1 +/- 3.2%). Treatment with PI3K pathway inhibitor wortmannin reduced the number of differentiated beta cells from 9.1 +/- 3.2 to 0.7 +/- 0.4%. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and Exendin-4 significantly increased the expression of the transcription factor neurogenin-3, whereas the expressions of pancreatic and duodenal homeobox 1 (PDX-1), neurogenic differentiation 1 (NeuroD) were increased only among samples treated with ZnCl2 and not significantly affected by treatment with the tested growth factors. Successful differentiation of IGF-1, EGF-, and Exendin-4-treated cells into functional beta cells was confirmed by C-peptide secretion in response to 5 versus 20 mmol glucose stimulation (0.24 vs 0.91 pmol C-peptide/microg DNA). These results showed that activation of the PI3K signaling pathway might be used to stimulate the differentiation of nonendocrine pancreatic cells into insulin-producing elements.

Differential effects of polymers PVP90 and Ficoll400 on storage stability and viability of Lactobacillus coryniformis Si3 freeze-dried in sucrose.

AIMS: To investigate the effect of freeze-dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose-based formulations and to examine the relationship between amorphous matrix stability and cell viability. METHODS AND RESULTS: The resistance to moisture-induced sucrose crystallization and effects on the glass transition temperature (Tg) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. CONCLUSIONS: Polymers can be used to enhance the stability of freeze-dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50 degrees below its Tg for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will aid in the rational formulation design and proper determination of storage conditions for freeze-dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.

In vivo comparison of hard tissue regeneration with human mesenchymal stem cells processed with either the FICOLL method or the BMAC method.

OBJECTIVE: To compare new bone formation in maxillary sinus augmentation procedures using biomaterial associated with mesenchymal stem cells (MSCs) separated by two different isolation methods. BACKGROUND: In regenerative medicine open cell concentration systems are only allowed for clinical application under good manufacturing practice conditions. METHODS: Mononuclear cells, including MSCs, were concentrated with either the synthetic polysaccharide (FICOLL) method (classic open system--control group, n = 6 sinus) or the bone marrow aspirate concentrate (BMAC) method (closed system--test group, n = 12 sinus) and transplanted in combination with biomaterial. A sample of the cells was characterized by their ability to differentiate. After 4.1 months (SD +/- 1.0) bone biopsies were obtained and analyzed. RESULTS: The new bone formation in the BMAC group was 19.9% (90% confidence interval [CI], 10.9-29), and in the FICOLL group was 15.5% (90% CI, 8.6-22.4). The 4.4% difference was not significant (90% CI, -4.6-13.5; p = 0.39). MSCs could be differentiated into osteogenic, chondrogenic, and adipogenic lineages. CONCLUSION: MSCs harvested from bone marrow aspirate in combination with bovine bone matrix particles can form lamellar bone and provide a reliable base for dental implants. The closed BMAC system is suited to substitute the open FICOLL system in bone regeneration procedures.

The murine equivalent of the A181E TACI mutation associated with common variable immunodeficiency severely impairs B-cell function.

TNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NF kappaB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI(-/-) background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions.

[Blood mesenchymal stem cell culture from the umbilical cord with and without Ficoll-Paque density gradient method]. / Cultivo de células mesenquimais do sangue de cordão umbilical com e sem uso do gradiente de densidade Ficoll-Paque.

OBJECTIVES: Implantation of cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood with and without using the Ficoll-Paque gradient density method (d=1.077 g/ml). METHODS: Ten samples of the umbilical cord blood obtained from full-term deliveries were submitted to two different procedures of mesenchymal stem cell culture: a) Method without the Ficoll-Paque density gradient, which concentrates all nucleated cells; b) Method with the Ficoll-Paque density gradient, which selects only low-density mononuclear cells. Cells were initially plated into 25 cm(2) cultures flasks at a density of 1 x 10(7) nucleated cells/cm(2) and 1 x 10(6) mononuclear cells/cm(2). RESULTS: It was obtained 2-13 x 10(7) (median = 2.35 x 10(7)) nucleated cells/cm(2) by the method without the Ficoll-Paque gradient density, and 3.7-15.7 x 10(6) (median = 7.2 x 10(6)) mononuclear cells/cm(2) by the method with the Ficoll-Paque gradient density. In all cultures adherent cells were observed 24 hours after being cultured. Cells presented fibroblastoid and epithelioid morphology. In most of the cultures, cell proliferation occurred in the first week, but after the second week only some cultures - derived from the method without the Ficoll-Paque gradient density-maintained the growth rate reaching confluence. Those cultures were submitted to trypsinization with 0.25% trypsin/EDTA solution and cultured for two to three months. CONCLUSION: In the samples analyzed, cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood by the method without the Ficoll-Paque density gradient was more efficient than the method with the Ficoll-Paque density gradient.

Cultivo de células mesenquimais do sangue de cordão umbilical com e sem uso do gradiente de densidade Ficoll-Paque / Blood mesenchymal stem cell culture from the umbilical cord with and without Ficoll-Paque density gradient method

OBJETIVOS: Implantação de técnicas de isolamento e cultivo de células-tronco mesenquimais do sangue de cordão umbilical humano, com e sem uso de gradiente de densidade Ficoll-Paque (d=1,077g/ml). MÉTODOS: Dez amostras de sangue de cordão umbilical humano de gestação a termo foram submetidas a dois procedimentos de cultivo de células-tronco mesenquimais: sem gradiente de densidade Ficoll-Paque e com gradiente de densidade. As células foram semeadas em frascos de 25cm² a uma densidade de 1x10(7)células nucleadas/cm² (sem Ficoll) e 1,0x10(6) células mononucleares/cm² (com Ficoll). As células aderentes foram submetidas a marcação citoquímica com fosfatase ácida e reativo de Schiff. RESULTADOS: No procedimento sem gradiente de densidade Ficoll, foram obtidas 2,0-13,0x10(7) células nucleadas (mediana=2,35x10(7)) e, no procedimento com gradiente de densidade Ficoll, foram obtidas 3,7-15,7x10(6) células mononucleares (mediana=7,2x10(6)). Em todas as culturas foram observadas células aderentes 24 horas após o início de cultivo. As células apresentaram morfologias fibroblastóides ou epitelióides. Na maioria das culturas houve proliferação celular nas primeiras semanas de cultivo, mas após a segunda semana, somente três culturas provenientes do método sem gradiente de densidade Ficoll-Paque mantiveram crescimento celular, formando focos confluentes de células. Essas culturas foram submetidas a várias etapas de tripsinização para espalhamento ou subdivisão e permaneceram em cultivo por períodos que variaram de dois a três meses. CONCLUSÃO: Nas amostras estudadas, o isolamento e cultivo de células-tronco mesenquimais do sangue de cordão umbilical humano pelo método sem gradiente de densidade Ficoll-Paque foi mais eficiente do que o método com gradiente de densidade Ficoll-Paque.

OBJECTIVES: Implantation of cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood with and without using the Ficoll-Paque gradient density method (d=1.077g/ml). METHODS: Ten samples of the umbilical cord blood obtained from full-term deliveries were submitted to two different procedures of mesenchymal stem cell culture: a) Method without the Ficoll-Paque density gradient, which concentrates all nucleated cells; b) Method with the Ficoll-Paque density gradient, which selects only low-density mononuclear cells. Cells were initially plated into 25 cm² cultures flasks at a density of 1x10(7) nucleated cells/cm² and 1x10(6) mononuclear cells/cm². RESULTS: It was obtained 2-13x10(7) (median = 2.35x10(7)) nucleated cells/cm² by the method without the Ficoll-Paque gradient density, and 3.7-15.7x10(6) (median = 7.2x10(6)) mononuclear cells/cm² by the method with the Ficoll-Paque gradient density. In all cultures adherent cells were observed 24 hours after being cultured. Cells presented fibroblastoid and epithelioid morphology. In most of the cultures, cell proliferation occurred in the first week, but after the second week only some cultures - derived from the method without the Ficoll-Paque gradient density - maintained the growth rate reaching confluence. Those cultures were submitted to trypsinization with 0.25 percent trypsin/EDTA solution and cultured for two to three months. CONCLUSION: In the samples analyzed, cell separation and mesenchymal stem cell culture techniques from human umbilical cord blood by the method without the Ficoll-Paque density gradient was more efficient than the method with the Ficoll-Paque density gradient.

Molecular crowding enhances native structure and stability of alpha/beta protein flavodoxin.

To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue single-domain alpha/beta protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (DeltaT(m) of 20 degrees C at 400 mg/ml; pH 7). As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble (pH 7, 20 degrees C) but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model and hard-sphere particles, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25% volume occupancy of spheres, native flavodoxin is thermally stabilized, and the free energy landscape shifts to favor more compact structures in both native and denatured states. The difference contact map reveals that the native-state compaction originates in stronger interactions between the helices and the central beta-sheet, as well as by less fraying in the terminal helices. This study demonstrates that macromolecular crowding has structural effects on the folded ensemble of polypeptides.

TACI is required for efficient plasma cell differentiation in response to T-independent type 2 antigens.

The control of systemic infection by encapsulated microorganisms requires T-independent type II (TI-2) Ab responses to bacterial polysaccharides. To understand how such responses evolve, we explored the function of transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI), a member of the TNFR family, required for TI-2 Ab production. Quasimonoclonal (QM) mice produce robust TI-2 responses to 4-hydroxy-3-nitrophenylacetate (NP)-Ficoll, owing to the high precursor frequency of NP-specific B cells in the marginal zone of the spleen. QM mice that lack TACI produce decreased numbers of IgM (2-fold) and IgG (1.6-fold) NP-specific ASCs, compared with TACI-positive QM mice in response to immunization with NP-Ficoll. Our studies indicate that TACI acts at a remote time from activation because TACI is not necessary for activation and proliferation of B cells both in vitro and in vivo. Instead, TACI-deficient QM B cells remained in the cell cycle longer than TACI-proficient QM cells and had impaired plasma cell differentiation in response to NP-Ficoll. We conclude that TACI has dual B cell-autonomous functions, inhibiting prolonged B cell proliferation and stimulating plasma cell differentiation, thus resolving the longstanding paradox that TACI may have both B cell-inhibitory and -stimulatory functions. By promoting plasma cell differentiation earlier during clonal expansion, TACI may decrease the chances of autoantibody production by somatic hypermutation of Ig genes in response to T-independent Ags.

Dendritic cell culture: a simple closed culture system using ficoll, monocytes, and a table-top centrifuge.

Dendritic cells (DCs) are potent antigen-presenting cells involved in the induction of T cell-mediated immune responses and as such have emerged as important candidates for cellular-based therapies. Critical to safe clinical use is the easy manipulation of DCs and their precursors in a closed system. We have developed a serum-free, closed culture system applying a simple wash-Ficoll centrifugation method to reduce platelet and red blood cell (RBC) contamination. This procedure optimized adherence of monocytes (44 +/- 10.9% recovery, >85% expressed CD14(+)/CD163(+)) for the generation of DCs from mononuclear cell (MNC) apheresis units. Most RBCs and up to 98% of platelets were removed. Following density sedimentation, cell viability remained high (98 +/- 2%) with only minimal loss of monocytes (3 +/- 3%). Importantly, Ficoll-treated monocytes retained their ability to differentiate to mature DCs demonstrated by morphology, phenotype (MHC class II(+), CD1a(+), CD80(+), CD86(+), and CD83(+)), ability to stimulate mixed lymphocyte responses (MLR), present antigen, and produce interleukin-12 (IL-12). Nonadherent CD3(+) (80 +/- 4%) were also isolated for functional assays. Ficoll can be easily incorporated into a simple adherence-based closed system for collection of lymphocytes and adherent monocytes for DC culture. The procedure is relatively fast (effective working time 5-6 h), does not impair monocyte function or induce substantial cell activation, and can be performed economically using equipment found in a typical blood banking environment.