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1.
This paper describes a flexible, metallic multielectrode array, made on kapton to fit in a recording chamber for interface-type organotypic cultures. This multisite recording system is designed for continuous multisite monitoring of electrophysiological activity in rat brain organotypic slice cultures. The system is composed of a signal conditioning set-up, which also masters electrical stimulation paradigms and a card containing the microelectrode array. The card comprises a perfusion chamber closed by a rigid and permeable membrane on which the pierced microelectrode array supporting the slice culture is placed. Once closed with a gaseous chamber, the inside of the card remained sterile and free of contamination and could be maintained inside or outside the incubator for electrophysiological analyses. Dimensions of each 28-plated gold microelectrode recording site are 50 microns x 100 microns. The design of the chambers and the card makes it possible to modify both the perfusion medium and the gaseous atmosphere in sterile conditions, allowing thus analyses of long-term effects of pharmacological compounds. Using this array one can perform stimulation and recordings of the electrical activity of the slice. Signals obtained with this reusable system exhibit a good signal-to-noise ratio. This device was tested to follow the evolution and modifications of the evoked and/or spontaneous electrical activity of the same groups of neurones during several days. 相似文献
2.
Steffen Kreye Rainer Stahn Karina Nawrath Vicky Goralczyk Barney Zoro Steffen Goletz 《Biotechnology progress》2019,35(5):e2832
Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 107 cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale. 相似文献
3.
Sandstrom CE Bender JG Miller WM Papoutsakis ET 《Biotechnology and bioengineering》1996,50(5):493-504
Perfusion and static cultures of peripheral blood (PB) mononuclear cells (MNCs), obtained from patients following stem cell mobilization, were supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal layer. Perfusion cultures without a stromal layer effectively retained nonadherent cells through the use of a novel "grooved" perfusion chamber, which was designed with minimal mass transfer barriers in order to achieve a well-defined culture environment. The grooved chamber allowed easy and efficient culture inoculation and cell recovery. Average maximum expansion of CFU-GM (colony-forming unit granulocyte-macrophage) cells was observed on day 10 for all cultures. Perfusion cultures had a maximum CFU-GM expansion of 17- and 19-fold with and without a stromal layer, respectively. In contrast, static cultures had a maximum CFU-GM expansion of 18- and 13-fold with and without a stromal layer, respectively. Average long-term-culture initiating cell (LTC-IC) numbers on day 15 were 34% and 64% of input in stroma-containing and stroma-free perfusion cultures and 12% and 11% of input in stroma-containing and stroma-free static cultures, respectively. Thus, perfusion enhanced CFU-GM expansion and LTC-IC maintenance more for the stroma-free cultures than for stroma-containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma-containing cultures were metabolically more active than the stroma-free cultures. In view of their equivalent, if not superior, performance compared to stroma-containing cultures, stroma-free perfusion cultures may offer significant advantages for potential clinical applications. (c) 1996 John Wiley & Sons, Inc. 相似文献
4.
Myoung Keun Shin Kyungmoon Park Gyu Heon Cho 《Biotechnology and Bioprocess Engineering》1999,4(4):264-268
A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator)
and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the
efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow
rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher
than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell
density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming
both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the
chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic
cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L. 相似文献
5.
Development of a novel microperfusion chamber for determination of cell membrane transport properties. 总被引:3,自引:0,他引:3
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D Y Gao C T Benson C Liu J J McGrath E S Critser J K Critser 《Biophysical journal》1996,71(1):443-450
A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties. 相似文献
6.
The metabolic pattern and cell culture kinetics of high-cell-density perfusion cultures were compared under two different oxygen transfer conditions: oxygen limiting and not limiting. When oxygen was a limiting factor during perfusion culture, both specific glucose uptake and lactate production rates increased, compared to non-oxygen-limited condition, by about 60% and 30%, respectively. The specific glutamine uptake rate under oxygen-limited conditions was almost 4.0 times higher than that under non-oxygen-limited conditions. The activity of lactate dehydrogenase (LDH) released into the medium by the dead cells can be used as an indicator for the metabolic and physiological conditions related to oxygen limitation. There was a 3.2 times higher specific rate of LDH activity released by dead cells in oxygen-limited cultures than those in non-oxygen-limited cultures. The specific production rate of monoclonal antibody was not significantly affected by the oxygen transfer conditions during the rapid cell growth period, but it rapidly increased toward the end of perfusion cultures. The higher perfusion rate may have limited further cell growth during high-cell-density perfusion culture, because cell damage was caused by the hydrodynamic shear within a hollow fiber microfiltration cartridge installed to withdraw the spent medium and the waste metabolites. (c) 1993 John Wiley & Sons, Inc. 相似文献
7.
《ImmunoMethods》1994,4(2):163-178
Nuclear magnetic resonance (NMR) can be used in a nondestructive manner to study cellular metabolism in intact cell samples such as a suspension of cells. However, many different cell types require a well-regulated medium that includes a buffered pH, as well as a continuous supply of oxygen. A series of methods that have been used for the maintenance of the extracellular conditions involves the immobilization of cells, followed by perfusion of the immobilized cell sample. NMR studies can then be performed for extended periods of time, as well as under sterile conditions. We discuss methodology, with perfused erythrocytes and thymocytes as specific examples. 相似文献
8.
This article is a review highlighting the application of the acoustic filter as a reliable cell retention device during the long-term perfusion of animal cell cultures. Critical operating parameters such as duty cycle, perfusion and re-circulation flow rates, acoustic power and backflush frequency are discussed with regard to influence on the separation efficiency and optimal operating ranges have been identified. Perfusion data gathered from the literature have been complemented with original data from a series of perfusion experiments carried out in the context of industrial projects for industrially relevant cell lines including NS0, HEK-293, SP2-derived hybridoma and insect cells in different serum-supplemented and serum-free media at different perfusion rates and acoustic chamber volumes. Finally, scale-up potential of the acoustic filter for large-scale industrial applications is discussed. 相似文献
9.
The construction of a modified perfusion chamber is presented, which can be used for a prolonged cultivation of mammalian cells and tissues, for observation of the behavior of living cells as well as for the study of different effects on these cells. The chamber is made as non-demountable of optical glass, with a diffusive barrier separating the pericellular zone from that with a perfusion medium. The scheme of the equipment for cultivation of cells and tissues in this diffusion chamber on controlling the composition of nutrient medium and gas phase is given. 相似文献
10.
Frankia菌种保藏 总被引:5,自引:0,他引:5
对用4种方法保藏的Frankia菌,进行了培养物存活、形态及其固氮活性的检测.发现在无氮液体培养基中保藏6年的Frankia.菌丝断裂,孢囊不完整.同期经有氮液体保藏的Frankia菌孢囊较完整.冷冻干燥保藏3.5年和砂管保藏8年,孢囊和菌丝均较完整.上述方法保藏的菌种,经活化后均能生长,且具有典型的Frankia菌形态特征和固氮活性.4种方法比较,无氮液体保藏法的菌体细胞生长速度快,固氮活性强,有侵染结瘤能力. 相似文献
11.
Development of an automated plant culture system 总被引:11,自引:0,他引:11
An apparatus was constructed that could be used to grow plant tissues, organs, and whole plantlets under sterile conditions. This system accommodated independent or multiple concurrent growth of cultures. Growth of plants either equalled or exceeded that observed using the manual transfer procedure. The automated plant culture system (APCS) consists of silicone tubing, 2 impeller pumps, 2 glass medium reservoir bottles, a 3-way stainless steel valve, a plant culture chamber, and an interface module containing relay boards. Control of the APCS is through interfacing with a microcomputer (e.g. Apple IIe or Atari 400). The computer controlled medium introduction, evacuation, and replenishment in a sterile environment. The APCS was inexpensively constructed and provides a labor-saving, long-term method to culture plants in vitro.Mention of a trade name or proprietary product in the paper does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. 相似文献
12.
Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12 总被引:2,自引:0,他引:2
We examined the ability of transformed Escherichia coli cells in fermentor cultures to accumulate interleukin-2 (IL-2) intracellularly under temperature-regulated control of the phage lambda pL promoter. Induction of expression was undertaken at different culture optical densities, and specific IL-2 accumulation was found to decrease with increasing cell density at induction. Induction at higher culture optical densities was also accompanied by decreased growth during induction and increased acetate accumulation in the culture medium. Experiments were undertaken to study the effect of replacing spent medium by perfusion with fresh medium both before induction and during IL-2 expression at high cell density. Improved IL-2 expression was seen only when perfusion was continued past 1.6 h after the start of induction, and it was accompanied by a significant reduction in acetate buildup. Further improvements were not seen when perfusion was continued beyond hour 3 of induction. Replenishing medium components and decreasing the concentration of diffusible inhibitors before induction did not alleviate acetate buildup, growth limitation, or limitation of IL-2 synthesis. These results suggested that accumulation of diffusible inhibitors such as acetate during induction may be a significant factor limiting IL-2 expression in high-density cultures, but other factors intrinsic to the organism or the protein also played a major role. 相似文献
13.
Effects of medium quality on the expression of human interleukin-2 at high cell density in fermentor cultures of Escherichia coli K-12. 总被引:4,自引:4,他引:0
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We examined the ability of transformed Escherichia coli cells in fermentor cultures to accumulate interleukin-2 (IL-2) intracellularly under temperature-regulated control of the phage lambda pL promoter. Induction of expression was undertaken at different culture optical densities, and specific IL-2 accumulation was found to decrease with increasing cell density at induction. Induction at higher culture optical densities was also accompanied by decreased growth during induction and increased acetate accumulation in the culture medium. Experiments were undertaken to study the effect of replacing spent medium by perfusion with fresh medium both before induction and during IL-2 expression at high cell density. Improved IL-2 expression was seen only when perfusion was continued past 1.6 h after the start of induction, and it was accompanied by a significant reduction in acetate buildup. Further improvements were not seen when perfusion was continued beyond hour 3 of induction. Replenishing medium components and decreasing the concentration of diffusible inhibitors before induction did not alleviate acetate buildup, growth limitation, or limitation of IL-2 synthesis. These results suggested that accumulation of diffusible inhibitors such as acetate during induction may be a significant factor limiting IL-2 expression in high-density cultures, but other factors intrinsic to the organism or the protein also played a major role. 相似文献
14.
The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×106 to 6–12×106 cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed. 相似文献
15.
P. F. Greenfield J. -M. Guillaume D. H. Randerson C. G. Smith 《Bioprocess and biosystems engineering》1991,6(5):213-219
A perfusion system for production of monoclonal antibodies was developed using an externally-mounted, hollow-fibre cartridge. The experimental apparatus was operated for 420 h and demonstrated increased steady-state viable cell concentration with increase in perfusion rate. Antibody titres were up to three times those measured for batch cultures and specific antibody productivity was doubled.The procedure was successfully scaled to a 10 dm3 system which produced antibody under conditions of Good Manufacturing Practice (GMP). A calculation of productivity between the scaled perfusion system and 260 dm3 batch cultures resulted in comparable antibody production, whereas the perfusion allowed a halving in medium utilisation. Reactivity assays conducted on the purified antibody from both batch and perfusion cultures showed no evidence of proteolysis or altered antibody activity in the final perfusion product. This study provides additional support for the use of homogeneous perfusion cultures in production of monoclonal antibodies under GMP conditions. 相似文献
16.
Trifunctional bispecific antibodies open up new immunological possibilities in tumour treatment. Prior to clinical application, comprehensive investigations using animal models and in vitro examinations need to be done. To investigate long-term interactions between various immunologically active blood cells and individual tumour cells in the presence of antibodies, we developed an incubation system for experimental cell cultures on an inverted microscope. The system consists of a perspex box with a central moisture chamber with integrated water reservoir, external air circulation heating, and a CO2 supply. The sterile cell cultures are located in the wells of a slide positioned within a depression in the water reservoir. The newly developed incubation system enables continuous observation over the long term of experiments under optimal cell cultures conditions in combination with modern video techniques. 相似文献
17.
Sehgal LR Wong J He J Wood T Takagi I Eldibany M Caprini J Mousa SA 《Journal of cellular biochemistry》2005,96(4):700-708
The association between cancer and hemostasis has long been studied in cell culture, animal models, and cancer patients developing thrombosis. The variety of biologic mechanisms involved in malignancy and metastasis makes the understanding of the relative importance of each mechanism difficult. We have developed a novel in vitro perfusion model that allows for the isolated study of the interactions between tumor cells and components of the hemostatic system under normal physiologic conditions. Segments of denuded umbilical cord or saphenous vein are cut longitudinally and mounted in a perfusion chamber under sterile conditions. Human breast cancer cells are perfused for 24 h under venous flow conditions with either whole blood (WB), platelet-rich plasma (PRP), platelet-poor plasma (PPP), or serum. Tissue samples are fixed and stained with hematoxylin and eosin as well as with pan-cytokeratin. Morphometric analysis is performed to quantify cancer cell adhesion. With PRP, this model maintains normal human physiologic conditions for the duration of the experiment. It differentiates between previously characterized high and low metastatic breast cancer cell lines. In addition, different vein tissue types do not alter tumor cell attachment. This model appears to be an accurate representation of the pathophysiology of in vivo metastasis. This model may serve as a useful bridge between cell culture studies and animal models. It may be a useful tool to elucidate the role of selected hemostatic systems in blood-borne metastasis and may potentially serve as a screening tool for the development of antimetastatic pharmaceutical agents. 相似文献
18.
Hubert Schwarz Kevin Lee Andreas Castan Veronique Chotteau 《Biotechnology and bioengineering》2023,120(9):2523-2541
A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex-centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N-glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred-tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N-glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 106 and 120 × 106 cells/mL and a low cell-specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry. 相似文献
19.
G N Pluzhnikova N V Sadovnikova Ia Iu Kondrat'ev E I Lezhnev V P Lavrovskaia 《Biulleten' eksperimental'no? biologii i meditsiny》1986,102(7):86-88
The data are reported on albumin secretion by rat hepatocytes and insulin secretion by pancreatic beta-cells of newborn rats during cell cultivation on flat synthetic membrane in conditions of continuous medium perfusion. Albumin and insulin secretion by the appropriate cultures was higher in continuous medium perfusion than in the control. Enhanced sensitivity of pancreatic beta-cells to glucose, as compared to the control was revealed. It is concluded that continuous medium perfusion of hepatocytes and pancreatic beta-cells in the primary culture had a favourable effect on albumin and insulin secretion by the appropriate cultures. 相似文献
20.
A flow diffusion chamber designed for studying cells and tissues in culture is described. The chamber contains a plate with a great number of isolated holes, which enables one to perform the cultivation of cells at different distances from the porous membrane separating the cells from the perfused medium. An individual porous membrane can be placed above each hole. Evidence for the selective permeability of domestic membranes under the conditions of cell culture in chamber is presented. The chamber makes possible a simultaneous cultivation of a great number of various cultures with different conditions of mass exchange with common perfused medium, which contributes to intensification of studies. 相似文献