Long-term Perfusion Culture Research Articles

3D bioprinting of Gelatin-Alginate bioinks for biofabrication of in vitro liver sinusoid model

In vitro liver models are pivotal in early drug development as they serve in assessing drug toxicity. Currently, most of the liver models include static cultures of liver derived cell spheroids. Mimicking dynamic conditions (similar to native liver – such as blood flow, glucose gradients etc.) in in vitro liver models would be essential for studying drug induced liver injury (DILI). The current work investigated and developed a simple bioink composed of gelatin and alginate blends (GA) that can be used to 3D bioprint hepatocyte laden scaffolds, serving as an in vitro liver model for drug toxicity assays.Rheological evaluation (using a rotary rheometer) of various compositions of GA blends was performed to assess their printability with an extrusion bioprinter. Suitable GA compositions with human liver carcinoma cells (HepG2) were 3D bioprinted. The scaffolds were stabilized by crosslinking the alginate (using calcium chloride) and gelatin (using microbial transglutaminase). Cell viability and proliferation of HepG2 in bioprinted scaffolds was assessed for 28 days. Further work includes fabrication of HepG2 laden hollow tubes and colonization of inner lumen with human umbilical vein endothelial cells (HUVEC), followed by DILI studies in long-term perfusion culture.

On-chip long-term perfusable microvascular network culture

Long-term perfusion culture of lumens constructed using spontaneous vasculogenesis in vitro has attracted attention in elucidating angiogenesis and subsequent remodeling phenomena. A Braille-based integrated microfluidic system for reconfigurable perfusion culture of a spontaneous 3D microvascular network was developed to transition from spontaneous vasculogenesis to long-term lumen perfusion using conventional methods. The combination of Braille microfluidics and the On-chip Incubation system allowed the elimination of the need for CO2 incubators and external tubing and pumps, as well as adjusted the interstitial flow rate and direction following the visual feedback morphology of the lumens easily. Using this device, lumens constructed by human umbilical vein endothelial cells with dynamic interstitial flow conditions were stimulated. Consequently, the lumen structure was maintained over 40 d and exhibited the possibility of long-term maintenance of perfusable capillary network, adjusting the magnitude of interstitial flow, and switching the flow direction.

Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.

A glycosaminoglycan based, modular tissue scaffold system for rapid assembly of perfusable, high cell density, engineered tissues.

The limited ability to vascularize and perfuse thick, cell-laden tissue constructs has hindered efforts to engineer complex tissues and organs, including liver, heart and kidney. The emerging field of modular tissue engineering aims to address this limitation by fabricating constructs from the bottom up, with the objective of recreating native tissue architecture and promoting extensive vascularization. In this paper, we report the elements of a simple yet efficient method for fabricating vascularized tissue constructs by fusing biodegradable microcapsules with tunable interior environments. Parenchymal cells of various types, (i.e. trophoblasts, vascular smooth muscle cells, hepatocytes) were suspended in glycosaminoglycan (GAG) solutions (4%/1.5% chondroitin sulfate/carboxymethyl cellulose, or 1.5 wt% hyaluronan) and encapsulated by forming chitosan-GAG polyelectrolyte complex membranes around droplets of the cell suspension. The interior capsule environment could be further tuned by blending collagen with or suspending microcarriers in the GAG solution These capsule modules were seeded externally with vascular endothelial cells (VEC), and subsequently fused into tissue constructs possessing VEC-lined, inter-capsule channels. The microcapsules supported high density growth achieving clinically significant cell densities. Fusion of the endothelialized, capsules generated three dimensional constructs with an embedded network of interconnected channels that enabled long-term perfusion culture of the construct. A prototype, engineered liver tissue, formed by fusion of hepatocyte-containing capsules exhibited urea synthesis rates and albumin synthesis rates comparable to standard collagen sandwich hepatocyte cultures. The capsule based, modular approach described here has the potential to allow rapid assembly of tissue constructs with clinically significant cell densities, uniform cell distribution, and endothelialized, perfusable channels.

Robust on-line sampling and analysis during long-term perfusion cultivation of mammalian cells

In an attempt to support robust automated sampling and analysis of mammalian cell bioreactors, an integrated platform, BaychroMAT®, was developed which includes an innovative sterile sampling device, automated sample transport, a sample preparation module, online analyzers, and communication interfaces to process automation systems. The robustness of this platform was verified by applying it to a laboratory-scale perfusion bioreactor that was operated for over 100 days. Both manual and automated samples were collected over the course of the run and a comparison was made for cell density, viability, glucose, and lactate concentrations. The highest variability (14.4%) was seen for cell density estimates while those for viability, glucose, and lactate were 0.7, 12.9, and 8.2%, respectively. In addition, cell density set-point changes were made towards the end of the perfusion culture and the high frequency automated samples provided a higher resolution description of the dynamics of cell density change compared to less frequent manual sampling. Overall, our results indicate stable and robust operation of the BaychroMAT® platform in a long-term perfusion culture. This success should readily translate to shorter duration fed-batch cultures thereby enabling feed-back control based on real-time nutrient measurements.

Fabrication of viable centimeter-sized 3D tissue constructs with microchannel conduits for improved tissue properties through assembly of cell-laden microbeads

Bottom-up approaches have emerged as a new philosophy in tissue engineering, enabling precise control over tissue morphogenesis at the cellular level. We previously prepared large bone-like tissues using cell-laden microbeads (microtissues) by following a modular approach to ensure cell viability. However, a long-term culture of such avascular macroscopic tissues (macrotissues) has not been evaluated. In the present study, microtissues were fabricated by cultivating human fibroblasts on Cytopore-2 microbeads in spinner flasks for 16 days. We then examined the long-term perfusion culture for macrotissues. Specifically, following assembly in a perfusion chamber for 15 days, cell death was found to be prominent at a depth of 500 µm from the surface of macrotissues towards the interior, suggesting that there was a new mass transfer limit leading to cell death instead of tissue maturation. Subsequently, we developed a strategy by incorporating microchannel structures in centimeter-sized tissue constructs to promote mass transport. By installing glass rods (1 mm diameter, 1 mm wall-to-wall spacing) in the perfusion chamber, stable microchannel architectures were introduced during the microtissue assembly process. Based on live/dead assay and scanning electron microscopy (SEM), these channelled macrotissues (length × diameter, 1.6 × 2.0 cm) demonstrated high cell viability and compact packing of microbeads. Comparative biochemical analysis further suggested a more homogeneous spatial distribution of cells and extracellular matrix (ECM) in the channelled macrotissues than in solid ones. Viable 3D large tissues can therefore be prepared by assembling cell-laden microbeads in conjunction with microchannel carving, meeting clinical needs in tissue repair.

A simple apparatus for measuring cell settling velocity

Accurate cell settling velocity determination is critical for perfusion culture using a gravity settler for cell retention. We have developed a simple apparatus (a "settling column") for measuring settling velocity and have validated the procedure with 15-μm polystyrene particles with known physical properties. The measured settling velocity of the polystyrene particles is within 4% of the value obtained using the traditional Stokes' law approach. The settling velocities of three hybridoma cell lines were measured, resulting in up to twofold variation among cell lines, and the values decreased as the cell culture aged. The settling velocities of the nonviable cells were 33-50% less than the corresponding viable cells. The significant variation of settling velocities among cell populations and growth phases confirms the necessity of routine measurement of this property during long-term perfusion culture.

An integrated microfluidic system for long-term perfusion culture and on-line monitoring of intestinal tissue models

Conventional cell-based assays in life science and medical applications can be difficult to maintain functionally over long periods. Microfluidics is an emerging technology with potential to provide integrated environments for cell maintenance, continuous perfusion, and monitoring. In this study, we developed an integrated microfluidic device with on-chip pumping and detection functionalities. The microfluidic structure in the device is divided into two independent channels separated by a semipermeable membrane on which cells are inoculated and cultured. Perfusion and fluorescence measurements of culture media for each channel can be conducted by the on-chip pumping system and optical fiber detection system. Performance of the device was examined through long-term culture and monitoring of polarized transport activity of intestinal tissue models (Caco-2 cells). The cells could be cultured for more than two weeks, and monolayer transport of rhodamine 123 was successfully monitored by on-line fluorescent measurement. This device may have applications in toxicity testing and drug screening.

Application of a Cell-Once-Through Perfusion Strategy for Production of Recombinant Antibody from rCHO Cells in a Centritech Lab II Centrifuge System

Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.

Dynamic seeding and perfusion culture of hepatocytes with galactosylated vegetable sponge in packed-bed bioreactor

A galactose moiety was introduced into the fiber surface of a vegetable sponge by the covalent binding of lactobionic acid. The galactosylated sponge was used as scaffold for the culture of rat hepatocytes in a packed-bed bioreactor. Hepatocytes could be dynamically seeded into and uniformly distributed throughout the scaffold, and the immobilized cells maintained high albumin and urea production rates during long-term perfusion culture. The hepatocytes showed an increasing albumin production rate from 49 to 109 microg/10(6) cells/d over the 7-d culture.

Establishment of long-term perfusion cultures of recombinant moss in a pilot tubular photobioreactor

The moss Physcomitrella patens has been successfully used for the expression of interesting heterologous proteins. Humanization of native glycosylation patterns in Physcomitrella has opened the possibility to express complex glycoproteins in cultures with simplified down stream processing. In order to characterize this expression system, the transgenic moss tWT11.51 VEGF that produces the vascular endothelial growth factor VEGF 121 has been cultured in a pilot 30-l tubular photoreactor. Long-term perfusion cultures with total cell recovery were performed by means of cross-flow filtration (CFF). Morphology, concentration and debris of moss showed to be determinant for the performance of CFF. Controlled cell disruption by means of rotor–stator devices and medium perfusion of 0.2 day −1, helped to delay cell differentiation, maintain low the proportion of leafy gametophores and postpone the decay of rhVEGF 121 secretion that has been observed in standard batches. However, only through continuous cultures ( D = 0.2–0.3 day −1), it was possible to stabilize the concentration of recombinant protein in the biosuspension. Here, perfusion of 18 and 30 day −1 showed to increase the specific production rate of rhVEGF 121.

Three-Dimensional Microfluidic Tissue-Engineering Scaffolds Using a Flexible Biodegradable Polymer.

Three-dimensional microfluidic networks using a flexible biodegradable polymer have been fabricated using modified microfabrication processes tailored specifically for poly(glycerol-co-sebacate). A model hepatocyte cell line (HepG2) is seeded and perfused in the microfluidic networks to demonstrate cell viability and function, which is maintained in long-term perfusion culture (see Figure; scale bar is 50 μm). The seeded, fully degradable device can potentially be integrated into a patient's existing vasculature in order to restore organ function.

Characterization and optimization of acoustic filter performance by experimental design methodology

Acoustic cell filters operate at high separation efficiencies with minimal fouling and have provided a practical alternative for up to 200 L/d perfusion cultures. However, the operation of cell retention systems depends on several settings that should be adjusted depending on the cell concentration and perfusion rate. The impact of operating variables on the separation efficiency performance of a 10-L acoustic separator was characterized using a factorial design of experiments. For the recirculation mode of separator operation, bioreactor cell concentration, perfusion rate, power input, stop time and recirculation ratio were studied using a fractional factorial 2(5-1) design, augmented with axial and center point runs. One complete replicate of the experiment was carried out, consisting of 32 more runs, at 8 runs per day. Separation efficiency was the primary response and it was fitted by a second-order model using restricted maximum likelihood estimation. By backward elimination, the model equation for both experiments was reduced to 14 significant terms. The response surface model for the separation efficiency was tested using additional independent data to check the accuracy of its predictions, to explore robust operation ranges and to optimize separator performance. A recirculation ratio of 1.5 and a stop time of 2 s improved the separator performance over a wide range of separator operation. At power input of 5 W the broad range of robust high SE performance (95% or higher) was raised to over 8 L/d. The reproducible model testing results over a total period of 3 months illustrate both the stable separator performance and the applicability of the model developed to long-term perfusion cultures.

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor.

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.

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.

Long‐Term continuous culture of hepatocytes in a packed‐bed reactor utilizing porous resin

As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.

Application of a rotating ceramic membrane to dense cell culture

The effects of fine bubbles on the separation performance of a rotating ceramic membrane were examined using aerated and unaerated yeast cell suspensions. The permeation flux of the aerated cell suspension was much higher compared with that of the unaerated cell suspension. Fine bubbles that accumulated around the ceramic membrane were found to prevent the compression of a cake layer, thereby resulting in a reduction of the filtration resistance. To investigate the possibility of long-term operation of the rotating ceramic membrane, dense cell perfusion cultures of wild strains of Corynebacterium glutamicum and Propionibacterium freudenreichli were carried out in a jar fermentor coupled with the rotating ceramic membrane module. During these long-term perfusion cultures no serious membrane-fouling problem was observed. The final cell concentrations attained with C. glutamicum and P. freudenreichii were 120 g-dry cell/dm 3 and 53 g-dry cell/dm 3, respectively.

Hybridoma perfusion systems: A comparison study

The efficiency of lgM production by hybridoma cells (1) cultured in suspension; (2) entrapped in alginate beads; or (3) packed in hollowfiber cartridge bioreactors, were compared in long-term perfusion cultures. The results showed that steady-state cell concentration and antibody production, per liter of perfused medium per day, were similar when cells were either entrapped in alginate beads of maintained in suspension. These values were also similar whether cells were maintained at high density in a hollowfiber cartridge bioreactor, or at low density in suspension. This work points out that cell behavior and antibody yield are comparable overall in the various perfusion systems currently used. However, a significant reduction of antibody production appeared whenever a part of the viable cells was lost in the filtrate. The reduction was due both to a decrease of viable cell yield and a decline of lgM productivity on a per-cell basis. This result is well in agreement with the previously presented model of "grow or die" cell cycle system of hybridoma, which proposes that the ratio of arrested to proliferating cells in perfusion cultures, should be increased in proportion to cell retention in the bioreactor, with a concomitant increase of lgM productivity.

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10(7) cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.

Characterisation of somatostatin and TRH release by rat hypothalamic and cerebral cortical neurons maintained on a capillary membrane perfusion system.

We have investigated the nature of the immunoreactive somatostatin (SS) and thyrotropin-releasing hormone (TRH) produced by long-term capillary perfusion cultures of rat fetal cortical and hypothalamic cells. Dispersed cortical and hypothalamic cells from 17-day-old fetal rats were injected on to the outer surfaces of separate capillary membrane perfusion systems. Recirculating nutrient medium (Minimum Essential Medium with added glucose, antibiotics and 10% fetal calf serum) was then perfused via the capillary lumen at a rate of 1.5 ml/min and was changed three times weekly. Medium reservoirs, gaseous exchange coils and capillary columns were maintained in a 95% air/5% CO2 environment with 100% humidity. After 6 and 12 days in continuous perfusion, both cortical and hypothalamic cells demonstrated immuno-reactive SS release following 60 mMK+ depolarization (5- to 7-fold increase from basal secretion levels of 15-20 pg/3 X 10(7) cells/10 min). This response was clearly calcium dependent since it was abolished during washes with Ca2+-free Krebs-Ringer bicarbonate solution. Affinity purified material from pooled neuronal perfusates showed three distinct peaks of somatotropin release-inhibiting factor (SRIF) immunoreactivity following polyacrylamide gel chromatography on Biogel-P10. The dominant form coeluted with synthetic tetradecapeptide-somatostatin (SS-14) and a smaller amount (c 30%) coeluted with synthetic SS-28. (The SS-14 antibody used showed equimolar cross reactivity with SS-28). A larger form of immunoreactive material was also detected with an apparent molecular weight of about 11,500 daltons. Hypothalamic and cortical perfusates produced similar electrophoretic patterns of immunoreactive SS (ISS).(ABSTRACT TRUNCATED AT 250 WORDS)