Medium Perfusion Rate Research Articles

A novel approach for perfusion process design based on a "Grey-Box" kinetic model.

Perfusion cell-culture mode has caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, perfusion-culture processes can support higher cell densities, higher productivities and longer process times. However, due to the inherent operational complexity and high running costs, the development and design of perfusion-culture processes remain challenging. Here, we present a model-based approach to design optimized perfusion cultures of Chinese Hamster Ovary cells. Initially, four batches of bench-top reactor continuous-perfusion-culture data were used to fit the model parameters. Then, we proposed the model-based process design approach, aiming to quickly find out the "theoretically optimal" operational parameters combinations (perfusion rate and the proportion of feed medium in perfusion medium) which could achieve the target steady-state VCD while minimizing both medium cost and perfusion rate during steady state. Meanwhile, we proposed a model-based dynamic operational parameters-adjustment strategy to address the issue of cell-growth inhibition due to the high osmolality of concentrated perfusion medium. In addition, we employed a dynamic feedback control method to aid this strategy in preventing potential nutrient depletion scenarios. Finally, we test the feasibility of the model-based process design approach in both shake flask semi-perfusion culture (targeted at 5 × 107 cells/ml) and bench-top reactor continuous perfusion culture (targeted at 1.1 × 108 cells/ml). This approach significantly reduces the number of experiments needed for process design and development, thereby accelerating the advancement of perfusion-mode cell-culture processes.

Experimental Study on Evaluation of Blood Supply Level and Embolization Ratio of Liver Cancer Based on I-Flow Software.

Objective:To confirm the feasibility and accuracy of the method for evaluating blood supply and embolization rate of liver cancer based on I-flow software through animal experiments and clinical study.Methods:Rabbits underwent selective angiography under different perfusion conditions in the same kidney. The blood supply level was evaluated by I-flow software method. The results were analyzed for coefficient of variation. Thirty patients with liver cancer who underwent selective hepatic artery embolization were enrolled. The mathematical methods and 3 diagnostic specialists were used to evaluate the preoperative blood supply level and embolization rate. The results were recorded and the results were tested for consistency.Results:Animal experiments confirmed that the blood supply level analysis method designed by the research team was consistent under different contrast conditions (including total contrast agent, contrast medium perfusion rate, and limiting pressure) (coefficient of variation: 8.55%). The mathematical calculation results of preoperative blood supply level and embolization ratio of liver cancer are consistent with the average value of visual judgment results of diagnostic experts. (Preoperative blood supply level: concordance coefficient = 0.284, P = 0.003; embolization ratio: concordance coefficient = 0.218, P = 0.011).Conclusion:Based on I-flow software, the mathematical calculation method designed by this research group can effectively estimate the preoperative blood supply level of liver cancer and the embolization rate of single vascular embolization treatment, which can provide reliable data support for embolization treatment of liver cancer.

Effects of different medium perfusion rates on the growth and metabolism of composite chitosan-based tissue-engineered dermis

Objective To evaluate effects of different medium perfusion rates on the growth and metabolism of composite chitosan-based tissue-engineered dermis in a dynamic three-dimensional culture system. Methods Human dermal fibroblasts (HDFs) at a density of 2 × 104/ml were inoculated onto chitosan-collagen composite hydrogels, and then cultured in a self-made perfusion culture device with the medium perfusion rates being 50, 100 or 200 ml/min for 12 days (perfusion culture groups), and statically cultivated HDFs served as the control group. An inverted microscope was used to observe morphological changes of HDFs, and methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate proliferative activity of HDFs. Test kits were used to determine glucose and lactate levels to evaluate cellular metabolic activity, and hydroxyproline content was measured to estimate collagen synthesis. An improved highly sensitive mechanical testing device was used to measure the tensile strength of the tissue-engineered dermis, and hematoxylin and eosin (HE) staining was performed to observe the morphology and distribution of HDFs. Statistical analysis was carried out by repeated-measures analysis of variance, one-way analysis of variance and the least significant difference (LSD) -t test. Results Compared with the control group, HDFs were distributed more densely in the perfusion culture groups. After 8-day culture, HDFs, which tended to grow along the direction of medium flow, grew to almost complete confluence in the 100-ml/min perfusion culture group, and to 80% confluence in the 200-ml/min group. As MTT assay showed, the proliferative activity of HDFs (expressed as absorbance [A] value) increased initially, but then gradually decreased over time in all the above 4 groups (all P < 0.05). Moreover, perfusion culture groups all showed significantly higher cellular proliferative activity compared with the control group, and the maximum cellular proliferative activity in the 100-ml/min group was 1.67 times that in the control group, and significantly higher than that in the other perfusion culture groups (both P < 0.05). The levels of glucose in the 4 groups all decreased over time (all P < 0.05), and the 100-ml/min group (1.604 ± 0.038 mmol/L) showed significantly lower levels of glucose compared with the control group (2.205 ± 0.020 mmol/L, P < 0.05), 50-ml/min group (1.939 ± 0.037 mmol/L, P < 0.05) and 200-ml/min group (2.047 ± 0.039 mmol/L, P < 0.05) after 12-day culture. However, in contrast to glucose, the levels of lactate changed in the opposite direction, and increased gradually over time in all the 4 groups (P < 0.05), but even the maximum level of lactate had no obvious effect on HDFs in the dermis. On day 12, the 100-ml/min group showed significantly higher hydroxyproline levels and maximum tensile strength compared with the other three groups (F = 61.512, 694.216, respectively, both P < 0.05). On days 6 and 10, all the perfusion culture groups showed increased quantities (all P < 0.05) of more evenly distributed HDFs with larger and longer nuclei in scaffolds compared with the control group. Moreover, the amounts of cells were significantly larger in the 100-ml/min group than in the other perfusion culture groups (both P < 0.05). Conclusions The medium perfusion rate of 100 ml/min is optimal for the culture of composite chitosan-based tissue-engineered dermis. At this medium perfusion rate, cellular proliferative activity, cell distribution, metabolic rates, collagen synthesis and the maximum tensile strength were more favourable compared with those at other perfusion rates. Key words: Chitin; Tissue engineering; Culture techniques; Fibroblasts

Modulation of 4-aminopyridine-induced neuronal activity and local pO(2)in rat hippocampal slices by changing the flow rate of the superfusion medium.

The brain slice preparation is the most frequently used tool for testing of pharmacological agents on the neuronal excitability. However in the absence of blood circulation in vitro, the tissue oxygenation strongly depends on the experimental conditions. It is well established that both hypoxia as well as hyperoxia can modulate the neuronal network activity. Thereby changes in tissue oxygen level during experiment may affect the final result. In the present study we investigated the effect of oxygenation on seizure susceptibility in the hippocampal slice preparation using 4-aminopyridine (4-AP) model of ictogenesis in inmature rats. We found that changing the medium perfusion rate in the range of 1-5 ml/min greatly affects the tissue oxygenation, amplitude and frequency of 4-AP-induced synchronous neuronal activity. The decrease in the flow rate as well as substitution of the oxygen in the extracellular medium with nitrogen causes a strong reduction of 4-AP-induced synchronous neuronal discharges. Our results demonstrate a significant linear correlation between the power of 4-AP-induced neuronal activity and the oxygen level in slice tissue. Also we demonstrated that the presence of medium flow is a necessary condition to support the constant level of the slice oxygenation. These data suggest that the oxygen supply of the brain slice strongly depends on experimental protocol and could modulate in vitro neuronal network excitability which should be taken into consideration when planning epilepsy-related studies.

Dependence of synchronized bursting activity on medium stirring and the perfusion rate in a cultured network of neurons

A cultured network of neurons coupled with a multi-electrode-array (MEA) recording system has been a useful platform for investigating various issues in neuroscience and engineering. The neural activity supported by the system can be sensitive to environmental fluctuations, for example, in the medium’s nutrient composition, ph, and temperature, and to mechanical disturbances, yet this issue has not been the subject. Especially, a normal practice in maintaining neuronal cell cultures involves an intermittent sequence of medium exchanges, typically at a time interval of a few days, and one such sudden medium exchange is unavoidably accompanied by many unintended disturbances. Here, based on a quantitative time-series analysis of synchronized bursting events, we explicitly demonstrate that such a medium exchange can, indeed, bring a huge change in the existing neural activity. Subsequently, we develop a medium perfusion-stirring system and an ideal protocol that can be used in conjunction with a MEA recording system, providing long-term stability. Specifically, we systematically evaluate the effects of medium stirring and perfusion rates. Unexpectedly, even some vigorous mechanical agitations do not have any impacts on neural activity. On the other hand, too much replenishment (e.g., 1.8 ml/day for a 1.8-ml dish) of neurobasal medium results in an excitotoxicity.

Timescale analysis for estimating upper limit perfusion rate in a microfluidic perfusion cell culture platform

Proliferation characteristics of CHO-K1 cells were investigated under a variety of medium perfusion rate conditions in a microfluidic perfusion cell culture platform. Five microcavities of \(800\,\upmu \hbox {m}~(w)\,\times \,800\,\upmu \hbox {m}~(l)\,\times \,400\,\upmu \hbox {m}~(h)\) were adopted in order to minimize or isolate the shear effects on cell surfaces. Microchannels of \(800\,\upmu \hbox {m}~(w)\,\times \,3.5\,\hbox {mm}~(l)\,\times \,100\,\upmu \hbox {m}~(h)\) serially connecting these microcavities created flow contractions and expansions repeatedly, resulting in two different diffusion and convection timescales through the platform. Average shear stresses on the bottom of microcavity were both numerically and analytically estimated, and medium flow was operated at rates where shear stress is below \(\sim\)2 mPa. Proliferation rates of CHO-K1 cells were investigated based both on population groups derived from the number of initially seeded cells and on the microcavity locations. Population groups showed minimal influences on proliferation rates, while proliferation rates increased clearly with medium perfusion rates. Strong effects of microcavity locations were observed on proliferation at \(\hbox {Pe}\,\ge \,45\). Such effects were analyzed by investigating the relationships of reaction, diffusion, and convection timescales associated with perfusion conditions. The ratio of diffusion timescale and convection timescale was suggested as a guideline to estimate the upper limit of perfusion rate in microfluidic perfusion cell culture platform.

액적의 균형공급에 의해 관류유량이 일정한 펌프 없는 세포배양 칩

본 논문에서는 액적의 균형공급에 의해 관류유량이 일정하게 유지되는 펌프 없는 세포배양 칩을 제안하였다. 기존의 펌프 없는 세포배양 칩은 유체 수위차가 시간에 따라 점차 감소하여 일정한 관류유량 유지가 어려웠다. 반면, 제안된 칩은 액적의 균형공급으로 유체 수위차를 일정하게 유지하여 일정한 관류유량의 세포배양이 가능하다. 제작된 세포배양 칩의 성능분석 결과, 펌프 없이 최대 9.96%와 6.92%의 편차 및 오차 내에서 0.1~0.3μl/min 의 관류유량, Q, 을 얻었다. H358 폐암 세포주 배양결과, Q=0.2μl/min 의 관류유량에서 최대 57.8±21.1%/일의 증식률을 보여, Q=0μl/min 의 정치배양보다 1.9 배 높은 값을 얻었으며 활성도 또한 정치배양보다 관류배양이 더 높은 값을 보였다. 제안된 펌프 없는 세포배양BR칩은 높은 증식률과 활성도의 좋은 배양환경을 제공하여 세포기반 바이오 분석에 응용 가능하다.

Optimizing the medium perfusion rate in bone tissue engineering bioreactors

There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone-like tissues in three-dimensional engineered constructs. We utilized custom-designed perfusion bioreactors to culture bone constructs for 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell-cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 µm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow velocity (80 µm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow velocity did not adequately support the development of bone-like tissue. The complexity of the cellular responses found at different flow velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered bone.

Effects of Initial Seeding Density and Fluid Perfusion Rate on Formation of Tissue-Engineered Bone

We describe a novel bioreactor system for tissue engineering of bone that enables cultivation of up to six tissue constructs simultaneously, with direct perfusion and imaging capability. The bioreactor was used to investigate the relative effects of initial seeding density and medium perfusion rate on the growth and osteogenic differentiation patterns of bone marrow-derived human mesenchymal stem cells (hMSCs) cultured on three-dimensional scaffolds. Fully decellularized bovine trabecular bone was used as a scaffold because it provided suitable "biomimetic" topography, biochemical composition, and mechanical properties for osteogenic differentiation of hMSCs. Trabecular bone plugs were completely denuded of cellular material using a serial treatment with hypotonic buffers and detergents, seeded with hMSCs, and cultured for 5 weeks. Increasing seeding density from 30 x 10(6) cells/mL to 60 x 10(6) cells/mL did not measurably influence the characteristics of tissue-engineered bone, in contrast to an increase in the perfusion rate from 100 microms(-1) to 400 microms(-1), which radically improved final cell numbers, cell distributions throughout the constructs, and the amounts of bone proteins and minerals. Taken together, these findings suggest that the rate of medium perfusion during cultivation has a significant effect on the characteristics of engineered bone.

Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells

In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel((R)) disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clinical and early clinical trials. It was originally operated with a perfusion of 2.6 vvd during production phase in order to supply the high cell density (2.5x10(7) cell ml(-1) of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a reduction of the medium perfusion rate by -25% and -50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clinical trials with an improved second-generation process. With a -25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a -30% loss of productivity was obtained when the medium perfusion rate was further reduced to -50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of -25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the production phase - provided that the product is not sensitive to residence time - with the benefits of reduced cost of goods and to simplify manufacturing operations.

Effects of oxygen on engineered cardiac muscle.

Concentration gradients associated with the in vitro cultivation of engineered tissues that are vascularized in vivo result in the formation of only a thin peripheral tissue-like region (e.g., approximately 100 microm for engineered cardiac muscle) around a relatively cell-free interior. We previously demonstrated that diffusional gradients within engineered cardiac constructs can be minimized by direct perfusion of culture medium through the construct. In the present study, we measured the effects of medium perfusion rate and local oxygen concentration (p(O2)) on the in vitro reconstruction of engineered cardiac muscle. Neonatal rat cardiomyocytes were seeded onto biodegradable polymer scaffolds (fibrous discs, 1.1 cm diameter x 2 mm thick, made of polyglycolic acid, 24 x 10(6) cells per scaffold). The resulting cell-polymer constructs were cultured for a total of 12 days in serially connected cartridges (n = 1-8), each containing one construct directly perfused with culture medium at a flow rate of 0.2-3.0 mL/min. In all groups, oxygen concentration decreased due to cell respiration, and depended on construct position in the series and medium flow rate. Higher perfusion rates and higher p(O2) correlated with more aerobic cell metabolism, and higher DNA and protein contents. Constructs cultured at p(O2) of 160 mm Hg had 50% higher DNA and protein contents, markedly higher expression of sarcomeric alpha-actin, better organized sarcomeres and cell junctions, and 4.5-fold higher rate of cell respiration as compared to constructs cultured at p(O2) of 60 mm Hg. Contraction rates of the corresponding cardiac cell monolayers were 40% higher at p(O2) of 160 than 60 mm Hg. The control of oxygen concentration in cell microenvironment can thus improve the structure and function of engineered cardiac muscle. Experiments of this kind can form a basis for controlled studies of the effects of oxygen on the in vitro development of engineered tissues.

Dynamics of Oxygen Evolution and Biomass Production during Cultivation of Agardhiella subulata Microplantlets in a Bubble‐Column Photobioreactor under Medium Perfusion

Cell and tissue cultures derived from macrophytic marine red algae are potential platforms for unique secondary metabolites. This work presents the first successful bioreactor cultivation study of an in vitro tissue culture derived from a macrophytic marine red alga. Specifically, the photosynthetic growth characteristics of a novel microplantlet suspension culture established from the macrophytic marine red alga Agardhiella subulata were studied. A bubble-column bioreactor with external illumination (43 microE m(-2) s(-1), 10:14 LD photoperiod), liquid medium perfusion, and 3800 ppm CO(2) in the aeration gas provided sufficient light and nutrient delivery for sustained growth of the microplantlet suspension at 24 degrees C and pH 8. Microplantlets, which consisted of shoot tissues of 3-5 mm length branching out from a common center, were not friable in a bubble-aerated suspension of about 1100 plantlets per liter. Since the microplantlet tissues were not friable, only batch and fed-batch cultivation modes were considered. Batch cultivation was phosphate-limited in ASP12 artificial seawater medium. However, cultivation at a medium perfusion rate of 20% per day avoided phosphate limitation and extended the growth phase to provide plantlet mass densities exceeding 14 g FW L(-1) (3.7 g DW L(-1)) after 50 days of cultivation if the suspension was not sampled. The specific oxygen evolution rate vs cultivation time profile possessed a significant pulse within the 14 days following inoculation and then leveled off at longer times. In recognition of this nonexponential growth pattern, a new photobioreactor growth model was developed that used the oxygen evolution rate vs time profile to predict the biomass growth curve in perfusion culture. Model predictions agreed reasonably with the measured growth curves.

Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression

Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, a reduction of the time span of estradiol exposure is required. Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48–72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day). Cell growth inhibition was successfully achieved for three consecutive cycles. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used. These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM. Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied. This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent. This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes.

Secreted enzyme production by fungal pellets in a perfusion bioreactor

In this study, extracellular enzyme production by fungal pellets cultured in a novel continuous perfusion bioreactor is investigated. Cell retention during perfusion culture is achieved by incorporating an internal settling zone into an external-loop air-lift bioreactor. Production of an extracellular enzyme, acid phosphatase, by the filamentous fungus Neurospora crassa was chosen as a model system. In order to control culture morphology to allow effective long-term perfusion culture, an anionic polymer Carbopol (carboxypolymethylene) at 0.1% was added to the culture medium to promote growth in a more dispersed form. The bioreactor has shown a high pellet retention efficiency over a wide range of medium perfusion rates. The fungal pellets were successfully cultivated in the bioreactor for over 30 days. By operating the bioreactor under phosphate limitation, and by a step-wise increase of the perfusion rate from 0.5 to 1 d −1, a steady state phosphatase volumetric productivity of ca. 900 U l −1 d −1 was reached while cell dry weight was maintained at over 4 g l −1.

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.

Long‐term perfusion culture of hybridoma: A “grow or die” cell cycle system

In order to elucidate the hybridoma life cycle and the limiting factors in perfusion systems, we performed cultures in a stirred tank bioreactor, coupled to an external tangential flow filtration unit. Cell density and antibody production in perfusion were consistent with previous studies. The average life span of the cells (2.1-2.2 days), antibody, productivity per cell produced (30-38 mg/10(9) cells) and cell size diameter evolution appeared similar to values observed in batch cultures. These observations highly suggest a similar "grow or die" life cycle. Cell and antibody production, strictly related to the medium perfusion rate, seem to be under the control of the nutrient availability. A hypothesis to explain such a life cycle of hybridoma cells in perfusion systems and a model for viable and dead cell density is proposed.

Rapid medium perfusion rate significantly increases the productivity and longevity of human bone marrow cultures.

Long-term in vitro human hematopoietic (Dexter) cultures are limited both in their longevity (8-12 weeks) and in their cell production over time. Hematopoiesis in vivo continues unabated throughout life, suggesting that failure to obtain continuous hematopoiesis in vitro is probably due to the failure of culture conditions to accurately reproduce the in vivo state. Since bone marrow cells in vivo exist at high density and are rapidly perfused by plasma components, we examined the effects of the culture medium perfusion rate and inoculum density on longevity and productivity of human bone marrow cultures. Culture efficiency and longevity were relatively independent of the variation in inoculum density from 10(6) to 5 x 10(6) cells per ml but were significantly altered by the medium perfusion rate. Increased culture perfusion was superior to traditional Dexter schedules, with 0.5 medium volume exchange per day (3.5 volume per week) being optimal. The cultures under these conditions demonstrated an increasing rate of cell production from weeks 4 to 10, with the cell production rate doubling approximately every 2 weeks. Following weeks 10-12 in culture, the cell production rate of all cultures decayed. Production of nonadherent progenitor cells was also highest in cultures perfused at 3.5 medium volume exchanges per week, where progenitor cell production was stable from weeks 6 to 18. The nonadherent cells collected were predominantly macrophages by week 19, except for the cultures perfused at a rate of 3.5 volumes per week and seeded at 5 x 10(6) cells per ml, in which production of granulocytes remained high through week 19. Establishment of more optimal perfusion conditions supports the continuous stable generation of progenitor cells over 5 months in culture, suggesting that primitive stem cells are surviving and proliferating in these cultures.

Influence of Medium Exchange Schedules on Metabolic, Growth, and GM‐CSF Secretion Rates of Genetically Engineered NIH‐3T3 Cells

The metabolic and secretory characteristics of NIH-3T3 fibroblasts transfected with a cDNA encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were examined as a function of the culture medium exchange schedule. The rates of glucose and glutamine consumption and of lactate and ammonia production were measured over exchange schedules ranging from complete medium replacement weekly (1/week) to complete medium replacement daily (7/week). All measured metabolic rates increased with increased medium exchange rates and accelerated sharply between exchange rates of 3.5/week and 7/week. The lactate/glucose and ammonia/glutamine yield coefficients, however, remained invariant at about 1.9 and 1.0 mol/mol, respectively, under all medium perfusion conditions. A shift-up in medium perfusion rates from 3.5/week to 7/week resulted in increased metabolic rates that resembled those observed in the cultures that were exchanged at the 7/week rate throughout, showing that the metabolic rates could be directly controlled by the perfusion rate. Differential regulation of medium versus serum perfusion demonstrated that increased NIH-3T3 cell metabolism was directly proportional to the serum flux to which the cells were exposed. Thus a limiting serum component is responsible for the altered metabolic and growth rates. The GM-CSF production by the transfected 3T3 cells was stable but exhibited substantial transient increases during periods of cell proliferation, demonstrating that the secretion of transfected gene products can be highly modulated even when the cDNA is driven from a constitutive promoter. These studies show that the metabolic and secretory behavior of genetically engineered cells is influenced by the medium exchange schedule.

Spin Filter Perfusion System for High Density Cell Culture: Production of Recombinant Urinary Type Plasminogen Activator in CHO Cells

We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

An Experimental Study of Extracorporeal Circulation for the Treatment of Postoperative Acute Pulmonary Edema.

The author investigated the application of extracorporeal circulation as treatment for the circulatory failure in the experimentally induced acute pulmonary edema.Method : 33 dogs were used for the present study. After pneumonectomy, the dogs were made to breath room air and were given plasma expander (50 cc/kg) intravenously to induce acute pulmonary edema, Jordan's grade IV. Then one of the 4 types of extracorporeal circulasion, i.e. 1) cardiopulmonary by-pass method (Fig. 1), 2) pulmonary by-pass method (Fig. 2), 3) simple oxygenation of the venous blood (Fig. 3), and 4) oxygenation of the venous blood of the inferior vena cave (Fig. 4) was started to treat the edema. Simultaneously positive pressure breathing with oxygen was started. Among these four methods, satisfactory result was obtained only by the cardiopulmonary by-pass method.The cardiopulmonary by-pass method was done by the following procedure. The venesection on the superior- and inferior venae cavae were performed. The venous blood was oxygenated by the bubble oxygenator of Lillehei-DeWall type, and then, the oxygenated venous blood was infused into both femoral arteries by the Sigma motor-pump.Results : In each method, results of treatment of pulmonary edema were as follows (Table I). 1) Cardiopulmonary by-pass method-Among 19 dogs treated 20-90 minutes by this method, 11 dogs recovered, 6 dogs improved and 2 dogs did not improve. 2) The pulmonary by-pass method-In this type of circulation one improved out of 5 dogs, 2 were not improved and died during the perfusion. None was cured by this method. 3) Simple oxygenation of the venous blood-Among 5 dogs, one cured, one improved, one not improved and 2 died immediately after the onset of the perfusion.4) Simple oxygenation of the venous blood of the inferior vena cava-Among 4 dogs, one improved, one not improved and 2 died during the course of the perfusion.To determine the optimum rate of perfusion in cardiopulmonary by-pass method, the clinical effectiveness of the treatment, changes in the arterial oxygen saturation and changes in the pulmonary arterial pressure were studied under various flow rates. Of the 4 dogs using the low flow rate, 35 cc/kg/min, 2 were improved and the other 2 were unchanged. In contrast, with the medium rate of perfusion, 40-65 cc/kg/min, 6 were recovered from the pulmonary edema and 2 were improved. Good results were obtained with the rate of perfusion over 70 cc/kg/min ; among 7 dogs, 5 were cured and 2 were improved.Immediate improvement of the arterial oxygen saturation was observed with any rate of perfusion (Fig. 5). Under the low perfusion rate the depressed arterial oxygen saturation showed steep elevation immediately after the beginning of the extracorporeal circulation, however, in this rate of perfusion, no significant improvement was obtained and the oxygen saturation remained below 90% even after 60 minutes' perfusion. The oxygen saturation rate in the 2 dogs not cured was 85 and 87% respectively, and it tended to decrease after 30 minutes' perfusion. Under the medium or the high rate of perfusion the arterial oxygen saturation was elevated up to the normal range (over 90%) within 5 minutes of perfusion. The maximum arterial oxygen saturation was observed 30 minutes after the onset of the extracorporeal circulation ; 91-96% in the medium rate of perfusion and 94-98% in the high rate of perfusion.The changes in the pulmonary arterial systolic pressure under various rates of perfusion were as follows (Fig. 6); During the established pulmonary edema the pulmonary arterial pressure was 40 mmHg or higher. Under the low perfusion rate 20-30 minutes elapsed before the pulmonary arterial pressure came down to the normal value (20 mmHg). It was 28 mmHg in one instance. It had also a tendency to begin to elevate after 30 minutes of perfusion. Under both the high and the medium perfusion rates the pulmonary arterial pressure fall to the normal range within 5 minutes perfusion. The de