共查询到20条相似文献,搜索用时 15 毫秒
1.
Eutimio Gustavo Fernández Núñez Jaci Leme Letícia de Almeida Parizotto Alexandre Gonçalves de Rezende Bruno Labate Vale da Costa Vera Lucia Lopes Boldorini Soraia Attie Calil Jorge Renato Mancini Astray Carlos Augusto Pereira Celso Pereira Caricati Aldo Tonso 《Cytotechnology》2016,68(1):95-104
Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 109 BHK-21 cells from 4 × 106 cells in 13 day with 1,051 mL culture medium. 相似文献
2.
Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 mum, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 mum, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (=2-3 h) attachment of inoculated cells (>/=95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate approximately 0.03-0.04 h(-1), maximum cell density of 8 x 10(6)-9 x 10(6) cells . mL(-1), and yields of 0.4 x 10(8) cells . mM(-1) on glucose and 2 x 10(8)-3 x 10(8) cells . mM(-1) on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 x 10(6) cells . mL(-1). Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems. 相似文献
3.
Porous microspherical carriers have great promise for cell culture and tissue engineering. Dynamic cultures enable more uniform cell population and effective differentiation than static cultures. Here we applied dynamic spinner flask culture for the loading and multiplication of cells onto porous biopolymer microcarriers. The abilities of the microcarriers to populate cells and to induce osteogenic differentiation were examined and the feasibility of in vivo delivery of the constructs was addressed. Over time, the porous microcarriers enabled cell adhesion and expansion under proper dynamic culture conditions. Osteogenic markers were substantially expressed by the dynamic cell cultures. The cell-cultured microcarriers implanted in the mouse subcutaneous tissue for 4 weeks showed excellent tissue compatibility, with minimal inflammatory signs and significant induction of bone tissues. This first report on dynamic culture of porous biopolymer microcarriers providing an effective tool for bone tissue engineering. 相似文献
4.
Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity 总被引:13,自引:0,他引:13
We developed a bioreactor for automated cell seeding of three-dimensional scaffolds by continuous perfusion of a cell suspension through the scaffold pores in oscillating directions. Using quantitative biochemical and image analysis techniques, we then evaluated the efficiency and uniformity of perfusion seeding of Polyactive foams as compared to conventional static and spinner flask methods. Finally, we assessed the efficacy of the perfusion seeding technique for different scaffolds and cell types. Perfusion seeding of chondrocytes into Polyactive foams resulted in "viable cell seeding efficiencies," defined as the percentages of initially loaded cells that were seeded and remained viable, that were significantly higher (75 +/- 6%) than those by static (57% +/- 5%) and spinner flask seeding (55% +/- 8%). In addition, as compared to static and spinner flask methods, cells seeded by perfusion were respectively 2.6-fold and 3.8-fold more uniformly distributed and formed more homogeneously sized cell clusters. Chondrocytes seeded by perfusion into Hyaff-11 nonwoven meshes were 26% and 63%, respectively, more uniformly distributed than following static and spinner flask seeding. Bone marrow stromal cells seeded by perfusion into ChronOS porous ceramics were homogeneously distributed throughout the scaffold volume, while following the static method, cells were found only near the top surface of the ceramic. In summary, we demonstrated that our cell seeding perfusion bioreactor generated constructs with remarkably uniform cell distributions at high efficiencies, and was effective for a variety of scaffolds and different mesenchymal cell types. 相似文献
5.
Culture of 293 cells in different culture systems: Cell growth and recombinant adenovirus production
Growth of 293 cells (human embryonic kidney) was compared in various cell culture systems including static flasks, cell aggregates and a variety of porous microcarriers. The best results were achieved with Fibra-Cel carriers and cell aggregates (1.1–1.4 × 106 cells/ml). Virus production was compared using a recombinant replication-deficient adenovirus as a model. Virus yields of lysates from cells grown on Fibra-Cel carriers, as cell aggregates and in static flasks were comparable (1.7–1.9 × 108 pfu/ml). 相似文献
6.
A new type porous carrier was fabricated from a mixture of sodium alginate, bovine serum albumin and sodium bicarbonate. The porous space of the carrier is an assembly of void spaces. The carrier was successfully applied to the cultivation of suspension animal cells. In the culture, while both cells and carriers were held in suspension, the cells were entrapped hydrodynamically into the void spaces in the carriers. A culture of hybridoma cells using this carrier resulted in a cell density up to 5.7×107 cells per ml-carrier. 相似文献
7.
Murine hybridoma cells were grown in a bubble column that was inclined up to 45 degrees from vertical. Inclining the column by a few degrees separated the rising bubbles against the upper surface, leaving the bulk of the liquid bubble free. The liquid was circulated well by the rising bubbles, but collection of cells by rising bubbles and exposure of cells to bursting bubbles were minimized. Maximum viable cell count and exponential growth of the cells were not affected by inclination, but an inclination of 30 degrees gave an antibody titer of 42 mg/L, which more than doubled the yield of 17 mg/L in the vertical position. By comparison, the culture gave yields of 30 mg/L when grown in spinner flasks. The enhanced antibody production in the inclined bioreactor corresponded to a prolonged stationary phase of 45 h. (c) 1995 John Wiley & Sons, Inc. 相似文献
8.
The spatial development of the cultivation of a bone marrow stromal cell line (SR-4987) in porous carriers was investigated
in order to construct a three-dimensional hematopoietic culture system. Low-rate continuous agitation, 20 rpm, was an appropriate
method to achieve initial adhesion of cells onto a cellulose porous beads (CPB, 100 μm pore diameter) in a spinner bottle,
compared with other methods such as centrifugation and intermittent agitation. Cell growth with continuous agitation at 70
rpm after initial cell adhesion was not inferior to that at 20 rpm. A 2- and 10-fold increase in the inoculum cell concentration
for CPB and another type of porous cellulose beads (Micro-cube (MC), 500 μm pore diameter) resulted in a 1.2- and 2-fold increase
in final cell concentrationm, respectively. Cells attached to the MC beads and a polyester nonwoven dic (Fibra-cell (FC))
could grow and spread well on the carriers and a fibroblast-like shape was observed under scanning electron microscopy while
the cells on CPB were globular. The flatness and inner surface area of these carriers may be the reason for the differences
in cell morphology.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
Summary We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability
to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic
center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGU-U1 cells
placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and consistently than cells
derived from monolayer culture. Spheroid diameter increased at a rapid rate of ∼100 μm/d in multiwell dishes, and necrosis
occurred only in spheroids of diameter >1 mm. Spheroids placed in spinner culture at a higher concentration (∼1.5 spheroids/ml)
grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture
was maintained at ∼190 μm over a wide range of spheroid diameters (400 to 1000 μm). Sequential trypsinization of spheroids,
which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from
varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids.
This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and
grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models
to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human
bladder biopsies to form spheroids.
Supported in part by a grant from the National Cancer Institute of Canada and by grant CA29526 NCI through the National Bladder
Cancer Project, U.S.A. 相似文献
10.
《Journal of Fermentation and Bioengineering》1993,75(2):138-144
A photo-crosslinkable resin, BIX12, was selected from among various photo-crosslinkable resins for the immobilization of animal cells. BIX12 had no cytotoxic effect on the growth of hybridoma cells and the production of monoclonal antibody, although other photo-crosslinkable resins had significant inhibitory effects. Using BIX12-alginate hybrid gel particles, hybridoma cells could grow in the resins and produce monoclonal antibody. For the continuous production of monoclonal antibody, perfusion culture using a fluidized-bed bioreactor with direct air bubbling was carried out. By this cultivation, monoclonal antibody could be produced stably for more than 50 d. A high viable cell density of more than 107 cells/ml-gel was attained, and the antibody productivity was improved 8.5-fold compared with conventional suspension culture using a spinner flask. Anchorage-dependent cells were also immobilized in the resin particles by three immobilization procedures. Among these procedures, porous BIX12 formed by adding gelatin powder provided good support strength and allowed the cells to grow on the surface inside of the support. 相似文献
11.
造血细胞体外悬浮培养和生物反应器开发 总被引:1,自引:0,他引:1
为解决造血细胞的静态培养中由浓度梯度引起的培养不稳定、环境不均一、难放大等问题,首先采用转瓶对脐血单个核细胞进行了悬浮培养研究,结果表明,悬浮培养中总细胞、集落和CD34+细胞的扩增都高于静态的方瓶培养。在测试了所用材料生物相容性的基础上,开发了可以控制溶氧和pH的生物反应器,并将其应用到造血细胞的批培养中,结果表明反应器的培养环境均一,可实现较高密度的培养,而且总细胞、集落和CD34+细胞的扩增都优于静态培养。大规模的反应器培养有利于解决临床应用中细胞数量不足的问题。 相似文献
12.
C K?sehagen F Linz G Kretzmer T Scheper K Schügerl 《Enzyme and microbial technology》1991,13(11):873-881
The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter [10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness] inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation. The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily. The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks. The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions. Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities. In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
培养方式对真皮组织体外构建的影响 总被引:2,自引:0,他引:2
采用静态培养和转瓶培养方式分别构建真皮组织,考察培养方式和搅拌转速对细胞在三维支架材料中增殖、代谢、分布的影响。结果表明,由转瓶培养方式构建的细胞-材料复合物,其最终细胞密度和细胞比生长速率均明显高于静态培养(14.2~27.6×106 cells/cm3 vs 10.1×106 cells/cm3和0.145~0.262 d-1 vs 0.111 d-1),而转速达80 r/min的转瓶尤其突出;静态培养的细胞-材料复合物内部细胞稀少,且分布不均匀,转瓶培养的细胞-材料复合物在材料表面和内部细胞密度都有所提高,分布情况也得到改善,且80 r/min转瓶培养的组织其细胞密度和分布均优于10 r/min和40 r/min转瓶培养。转瓶培养在其转速达到一定强度时能明显提高细胞在支架中的增殖速率,缩短培养时间,并有效改善细胞在支架内的分布,是一种理想的培养方式。 相似文献
14.
Summary N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) added to bicarbonate buffer in cell culture medium was found to
promote continuous logarithmic growth of human lymphocytoid cell lines. Consistent high yields of viable cells were obtained
in the combined buffer system. Storage of lymphocytoid cells in the combined buffer system at room temperature allowed successful
reinitiation of growth of the cultures. 相似文献
15.
Summary Growth and death of anchorage-independent animal cells entrapped within porous biomass support particles (BSPs) in static or shake-flask cultures were evaluated by comparison of enzyme activity with non-immobilized cells grown under static culture using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and release of lactate dehydrogenase into the culture medium. Mouse myeloma MPC-11 (ATCC CCL 167) cells inoculated within porous polyvinyl formal resin BSPs (3 × 3 × 3 or 2 × 2 × 2 mm; mean pore diameter, 60 ) grew exponentially at a specific growth rate comparable to that of non-immobilized cells in the initial period of incubation. Entrapped cells then reached the stationary phase with a cell density over 107 cells/cm3 BSP. The death rate of entrapped cells increased in response to the rise in viable cell density in the BSPs. Observation of viable cell distribution within the BSPs using MTT staining indicated that the cells concentrated within a thin outer shell of the BSPs with time. After the immobilized cells reached the stationary phase, penetration of cells into the outer shell ceased and heterogeneous distribution of cell density occurred in the viable cell layer in the shake-flask culture. 相似文献
16.
Spodoptera frugiperda (Sf-9) insect cells have been grown in serum-free medium in 250-ml spinner flasks. The maximum cell density obtained in these cultures was dependent on the aeration rate of the culture. Similar yields of uninfected cells were obtained when cultures were stirred in spinner flasks at 80 rev min-1 and in a 4-1 stirred-tank bioreactor and the dissolved oxygen in the bioreactor was controlled at 20% of air saturation. Cells were infected with a recombinant baculovirus at different multiplicities of infection: the timing and maximum level of expression of the recombinant protein were dependent on the multiplicity of infection, the cell density at infection, and on the aeration rate of the culture. Oxygen-limited growth resulted in undetectable levels of recombinant protein (< 6 ng recombinant protein 10(-7) cells). Compared with the maximum yields observed in spinner flask cultures, higher levels of recombinant protein were produced when cells were grown and infected in the bioreactor. The level of dissolved oxygen in the bioreactor was controlled at 50% of air saturation. 相似文献
17.
Evaluation of different formulations of potential biocontrol yeast isolates efficacy on apple blue mold at storage condition 总被引:1,自引:0,他引:1
L. Mokhtarnejad H. R. Etebarian M. R. Fazeli H. Jamalifar 《Archives Of Phytopathology And Plant Protection》2013,46(10):970-980
Post-harvest pathogens cause major losses in apple production. Biological control by using epiphytic yeasts against Penicillium expansum has been considered as an alternative method for controlling the post-harvest decays. The yeast isolates Rhodotorula mucilaginosa, Pichia guilliermondii, which showed high biocontrol efficacy against P. expansum, were selected for formulation tests. Formulation is an important step in developing a biocontrol product. The successful delivery of biocontrol agents, shelf life, stability and effectiveness in commercial conditions depend on the formulation. In the formulation, the carrier is the primary material used to allow a bioproduct to be dispersed effectively. Yeast isolates were grown in a cane molasses-based medium. Harvested yeast cells were combined with inorganic (talk, kaolin) and organic (Rice bran, wheat bran) carriers. Viability of the yeast cells in formulations stored at 4°C and 24°C was determined each month during 6 months storage. After 6 months storage to evaluate efficacy of formulations, all formulations were tested on apple to control blue mold in storage condition. High stability of antagonistic yeasts was achieved by using organic and inorganic carriers. Rice bran and wheat bran stimulated the germination of the yeasts cells during storage period. Both of the yeasts had significantly highest viable yeast cell content over 6 months in formulation containing wheat bran as a carrier. P.guillermondii in all formulations had significantly higher shelf life and was effective than R. mucilaginosa. 相似文献
18.
Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity 总被引:1,自引:0,他引:1
Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×106 to 7.9×105 cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 106 viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins. 相似文献
19.
Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor 总被引:4,自引:0,他引:4
Ex vivo expansion of T cells is a key step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant or virally infected cells. The use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems. Primary human T cells as well as the transformed TALL103/2 T cell line were cultured in 100-mL spinner flasks as well as 2-L bioreactors to investigate the effects of shear forces produced by agitation and sparging-based aeration on the expansion of T cells. Primary T cells could be successfully grown at agitation rates of up to 120 rpm in the spinner flasks and to 180 rpm in the bioreactors with no immediate detrimental effects on proliferation. Exposure to agitation and sparging did, however, cause a significantly increased rate of downregulation of the interleukin-2 receptor (IL-2R), resulting in lower overall expansion potential from a single stimulation as compared to static controls, with faster IL-2R downregulation occurring at higher agitation rates. For the primary T cells, no significant effects of agitation were found on expression levels of other key surface receptors (CD3, CD28, or CD62L) examined. No significant effects of agitation were observed on primary T cell metabolism or levels of cellular apoptosis in the cultures. The TALL103/2 T cell line was found to be extremely sensitive to agitation, showing severely reduced growth at speeds above 30 rpm in 100-mL spinner flasks. This unexpected increased fragility in the transformed T cell line as compared to primary T cells points out the importance of carefully selecting a model cell line which will accurately represent the characteristics of the cell system of interest. 相似文献
20.
多孔载体是一种新型的用于动物细胞培养的优秀的细胞支持物,其内部网状结构的小孔具有固定细胞和保护细胞免受机械损伤的功能,适合于贴壁细胞和悬浮细胞的培养,能提高培养密度,可应用于大规模培养系统。本文综述了多孔载体的物化性质、制作材料和制备方法。 相似文献