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1.
The capability to image real time cell/material interactions in a three-dimensional (3D) culture environment will aid in the advancement of tissue engineering. This paper describes a perfusion flow bioreactor designed to hold tissue engineering scaffolds and allow for in situ imaging using an upright microscope. The bioreactor can hold a scaffold of desirable thickness for implantation (>2 mm). Coupling 3D culture and perfusion flow leads to the creation of a more biomimetic environment. We examined the ability of the bioreactor to maintain cell viability outside of an incubator environment (temperature and pH stability), investigated the flow features of the system (flow induced shear stress), and determined the image quality in order to perform time-lapsed imaging of two-dimensional (2D) and 3D cell culture. In situ imaging was performed on 2D and 3D, culture samples and cell viability was measured under perfusion flow (2.5 mL/min, 0.016 Pa). The visualization of cell response to their environment, in real time, will help to further elucidate the influences of biomaterial surface features, scaffold architectures, and the influence of flow induced shear on cell response and growth of new tissue. 相似文献
2.
The combination of hMSCs with 3D scaffolds has become an important approach to creating functional bone constructs. Bioreactors are important tools to mitigate mass transfer limitations and to provide controlled physiochemical and biomechanical environments for the 3D bone construct development. Media flow in the bioreactor systems is generally controlled either parallel or transverse with respect to the 3D construct, creating different cellular and biomechanical microenvironments in the 3D constructs. In this study, a custom designed modular perfusion bioreactor system was operated under either the parallel or transverse flow. The influence of the flow patterns on the characteristics of the hMSCs' cellular microenvironment and subsequent construct development was investigated. The parallel flow configuration retained ECM proteins and mitogenic growth factors within the scaffold, effectively preserving hMSC progenicity and proliferation potential (e.g., CFU-F, proliferation, and OCT-4), whereas the transverse flow induced hMSC osteogenic differentiation with higher ALP activity and calcium deposition and up-regulation of osteogenic bone markers (e.g., BMP-2, ALP, RUNX2, OSX, and OC). These results demonstrate the regulatory role of the macroscopic flow on the cellular microenvironment of the 3D hMSC construct, and suggest configuring media flow as a strategy for directing hMSC fate and 3D bone construct development in the perfusion bioreactor. 相似文献
3.
Geert A.A. Nibourg Justin D. Boer Tessa V. van der Hoeven Mariëtte T. Ackermans Thomas M. van Gulik Robert A.F.M. Chamuleau Ruurdtje Hoekstra 《Biotechnology and bioengineering》2012,109(12):3182-3188
Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver‐failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC‐BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β‐hydroxylation of testosterone showed 2–20‐fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG‐BAL); and (3) in the current set‐up the mass balance of substrate, metabolite, or cell damage markers between in‐and out‐flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG‐BAL). Biotechnol. Bioeng. 2012; 109: 3182–3188. © 2012 Wiley Periodicals, Inc. 相似文献
4.
Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask. 相似文献
5.
A literature review: the effects of magnetic field exposure on blood flow and blood vessels in the microvasculature 总被引:2,自引:0,他引:2
The effect of magnetic field (MF) exposure on microcirculation and microvasculature is not clear or widely explored. In the limited body of data that exists, there are contradictions as to the effects of MFs on blood perfusion and pressure. Approximately half of the cited studies indicate a vasodilatory effect of MFs; the remaining half indicate that MFs could trigger either vasodilation or vasoconstriction depending on initial vessel tone. Few studies indicate that MFs cause a decrease in perfusion or no effect. There is a further lack of investigation into the cellular effects of MFs on microcirculation and microvasculature. The role of nitric oxide (NO) in mediating microcirculatory MF effects has been minimally explored and results are mixed, with four studies supporting an increase in NO activity, one supporting a biphasic effect, and five indicating no effect. MF effects on angiogenesis are also reported: seven studies supporting an increase and two a decrease. Possible reasons for these contradictions are explored. This review also considers the effects of magnetic resonance imaging (MRI) and anesthetics on microcirculation. Recommendations for future work include studies aimed at the cellular/mechanistic level, studies involving perfusion measurements both during and post-exposure, studies testing the effect of MFs on anesthetics, and investigation into the microcirculatory effects of MRI. 相似文献
6.
In tissue engineering, bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. This study examined the effect of short term flow perfusion bioreactor culture, prior to long‐term static culture, on human osteoblast cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold for bone tissue engineering. Human fetal osteoblasts (hFOB 1.19) were seeded onto CG scaffolds and pre‐cultured for 6 days. Constructs were then placed into the bioreactor and exposed to 3 × 1 h bouts of steady flow (1 mL/min) separated by 7 h of no flow over a 24‐h period. The constructs were then cultured under static osteogenic conditions for up to 28 days. Results show that the bioreactor and static culture control groups displayed similar cell numbers and metabolic activity. Histologically, however, peripheral cell‐encapsulation was observed in the static controls, whereas, improved migration and homogenous cell distribution was seen in the bioreactor groups. Gene expression analysis showed that all osteogenic markers investigated displayed greater levels of expression in the bioreactor groups compared to static controls. While static groups showed increased mineral deposition; mechanical testing revealed that there was no difference in the compressive modulus between bioreactor and static groups. In conclusion, a flow perfusion bioreactor improved construct homogeneity by preventing peripheral encapsulation whilst also providing an enhanced osteogenic phenotype over static controls. Bioeng. 2011; 108:1203–1210. © 2010 Wiley Periodicals, Inc. 相似文献
7.
Understanding and modeling alternating tangential flow filtration for perfusion cell culture
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William Kelly Jennifer Scully Di Zhang Gang Feng Mathew Lavengood Jason Condon John Knighton Ravinder Bhatia 《Biotechnology progress》2014,30(6):1291-1300
Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014 相似文献
8.
S. A. J. Chamuleau B. L. F. van Eck-Smit M. Meuwissen K. T. Koch M. G. W. Dijkgraaf H. J. Verberne J. G. P. Tijssen J. J. Piek 《Netherlands heart journal》2007,15(11):369-374
ObjectiveIn this multicentre study, we investigated the long-term prognostic value of intracoronary derived haemodynamic parameters compared with the results of myocardial perfusion scintigraphy (MPS). MethodsPatients (n=191) who were referred for angioplasty of a severe lesion in the presence of an intermediate lesion in another coronary artery were included. MPS was performed to determine the presence of reversible perfusion defects in the area of the intermediate lesion. Coronary flow velocity reserve (CFVR), and additionally fractional flow reserve (FFR; n=129), were determined distal to the intermediate lesion; CFVR ≥2.0 and FFR ≥0.75 were considered negative. ResultsIn total 67 events occurred in 49 patients (3 deaths, 9 MI, 9 CABG, 46 PTCA) during a mean of 793 days follow-up. Event-free survival was 63% for MPS, 79% for CFVR, and 79% for FFR if a negative test result was obtained. The relative risk was 1.2 (not significant) for MPS, 2.2 (p=0.001) for CFVR, and 2.4 (p=0.004) for FFR. ConclusionSelective evaluation of an intermediate lesion using CFVR or FFR allows more adequate risk stratification in patients with multivessel disease than MPS. A CFVR <2.0 or a FFR <0.75 was associated with a significant increase of the occurrence of cardiac events during long-term follow-up, predominantly associated with revascularisation. (Neth Heart J 2007;15:369-74.) 相似文献
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Daniel J. Karst Kevin Ramer Erik H. Hughes Canping Jiang Pieter J. Jacobs Fernie G. Mitchelson 《Biotechnology progress》2020,36(6):e3040
Mammalian cells were grown to high density in a 3,000 L culture using perfusion with hollow fibers operated in a tangential flow filtration mode. The high-density culture was used to inoculate the production stage of a biomanufacturing process. At constant permeate flux operation, increased transmembrane pressures (TMPs) were observed on the final day of the manufacturing batches. Small scale studies suggested that the filters were not irreversibly fouled, but rather exposed to membrane concentration polarization that could be relieved by tangential sweeping of the hollow fibers. Studies were undertaken to analyze parameters that influence the hydrodynamic profile within hollow fibers; including filter area, cell density, recirculation flow rate, and permeate flow rate. Results indicated that permeate flow rate had the greatest influence on modulating TMP. Further evaluation showed a significant decrease in TMP when permeate flow was reduced, and this occurred without any negative effect on cell growth or viability. Hence, a 30% reduction of permeate flow rate was implemented at manufacturing scale. A stable operation was achieved as TMP was successfully reduced by 75% while preserving all critical factors for performance in the perfusion bioreactor. 相似文献
11.
M. Reiter N. Borth G. Blüml K. Wimmer H. Harant N. Zach T. Gaida C. Schmatz H. Katinger 《Cytotechnology》1992,9(1-3):247-253
Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is
demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 108 cells/ml a stable growth rate of 0.004 h−1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE
in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression. 相似文献
12.
The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development. 相似文献
13.
Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture
The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc. 相似文献
14.
半边结灌注培养中杂交瘤细胞的生长和代谢 总被引:1,自引:0,他引:1
考察了半连续灌注培养中WuT3杂交瘤细胞在不同灌注速率下细胞生长的动态变化,培养其中主要基质的消耗和代谢物的生成。当灌注速率D从1.0/升高到2.0/d升高到2.0/d时,乳酸得率系数Ylac/glu降低18%,氨得率系数Yamm/gln降低40%,丙氨酸得率系数Yala/gln升高58%,甘氨酸得率系数Ygly/gln基本恒定。说明在灌注速率升高的条件下,细胞会调整代谢机制,丙酮酸和过量的谷氨酸 相似文献
15.
Marie‐Françoise Clincke Carin Mölleryd Ye Zhang Eva Lindskog Kieron Walsh Véronique Chotteau 《Biotechnology progress》2013,29(3):754-767
High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 106 cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 108 cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 108 cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 108 cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013 相似文献
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The importance of cell cycle analysis in cell culture development has been widely recognised. Whether such analysis is useful
in indicating future performance of high cell density culture is uncertain. Using flow cytometric approach to address this
question, we utilised the fraction of cells in the S phase to control specific growth rate and productivity in spin filter
perfusion cultures and found a significant increase in the accumulated interferon-γ over that obtained from the nutrient-based
controlled fed culture. While a general decrease with time exists in both percentage of S phase cells and specific growth
rate, a clear oscillatory behaviour of both parameters is found in perfusion cultures.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
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目的:探讨16层螺旋CT灌注成像对肝硬化血流状态的评估价值及其与肝硬化程度的相关性。方法:选取2014年1月至2016年1月于我院接受诊治的肝硬化患者126例作为肝硬化组,根据Child-Pugh分级分为A组(Child A级,n=35例)、B组(Child B级,n=50例)、C组(Child C级,n=41例)。另选取同期于我院接受体检的健康人员100例作为对照组。应用16层螺旋CT对受试者肝脏、脾脏、主动脉以及门静脉的层面进行CT动态增强扫描,对比CT灌注参数,采用Pearson相关性分析分析CT灌注参数与肝硬化病情严重程度的关系。结果:肝硬化组肝动脉灌注量(HAP)、肝动脉灌注指数(HPI)、肝脏血流量(TBV)以及平均通过时间(MTT)均明显高于对照组,而门静脉灌注量(PVP)、总肝灌注量(TLP)均明显低于对照组(P0.05)。A组患者HAP、HPI均明显高于C组,而PVP与TLP均明显低于C组,差异有统计学意义(P0.05);两组TBV、MTT比较无统计学差异(P0.05);而A组与B组相比以及B组与C组相比,各项CT灌注参数均无统计学差异(P0.05)。肝硬化患者病情严重程度与HAP、HPI均呈正相关关系(P0.05),而与PVP、TLP均呈负相关关系(P0.05)。结论:16层螺旋CT灌注成像对肝硬化血流状态具有一定的评估价值,且CT灌注参数的水平变化与肝硬化患者病情严重程度存在密切相关。 相似文献
20.
A 15.6 frames per second 1‐megapixel multiple exposure laser speckle contrast imaging setup
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Martin Hultman Ingemar Fredriksson Marcus Larsson Atila Alvandpour Tomas Strömberg 《Journal of biophotonics》2018,11(2)
A multiple exposure laser speckle contrast imaging (MELSCI) setup for visualizing blood perfusion was developed using a field programmable gate array (FPGA), connected to a 1000 frames per second (fps) 1‐megapixel camera sensor. Multiple exposure time images at 1, 2, 4, 8, 16, 32 and 64 milliseconds were calculated by cumulative summation of 64 consecutive snapshot images. The local contrast was calculated for all exposure times using regions of 4 × 4 pixels. Averaging of multiple contrast images from the 64‐millisecond acquisition was done to improve the signal‐to‐noise ratio. The results show that with an effective implementation of the algorithm on an FPGA, contrast images at all exposure times can be calculated in only 28 milliseconds. The algorithm was applied to data recorded during a 5 minutes finger occlusion. Expected contrast changes were found during occlusion and the following hyperemia in the occluded finger, while unprovoked fingers showed constant contrast during the experiment. The developed setup is capable of massive data processing on an FPGA that enables processing of MELSCI data in 15.6 fps (1000/64 milliseconds). It also leads to improved frame rates, enhanced image quality and enables the calculation of improved microcirculatory perfusion estimates compared to single exposure time systems. 相似文献