A radial flow hollow fiber bioreactor for the large‐scale culture of mammalian cells

A Radial Flow Hollow Fiber Bioreactor for the Large-Scale Culture of Mammalian Cells

John P. Tharakan and Pao C. Chau Depaflment of AMES (Chemical Engineering), University of California, San Diego, California, 92093

Accepted for publication May 20, 1985

A radial flow hollow fiber bioreactor has been developed that maximizes the utilization offibersurface for cell growth while eliminating nutrient and metabolic gradients in- herent in conventional hollow fiber cartridges. The reactor consists of a central flow distributor tube sur- rounded by an annular bed of hollow fibers. The central flow distributor tube ensures an axially uniform radial convective flow of nutrients across the fiber bed. Cells attach and proliferate on the outer surface of the fibers. The fibers are pretreated with polylysine to facilitate cell attachment and long-term maintenance of tissuelike densities of cell mass. A mixture of air and COz is fed through the tube side of the hollow fibers, ensuring direct oxygenation of the cells and maintenance of pH. Spent medium diffuses across the cell layer into the tube side of the fibers and is convected away along with the spent gas stream. The bioreactor was run as a recycle reactor to permit maximum utilization of nutrient medium. A bioreactor with a membrane surface area of 11 50 cm2 was developed and H I cells were grown to a density of 7.3 x lo6 cells/cm2.

INTRODUCTION

The large-scale culture of mammalian cells is re- garded as a viable method for the production of many clinically and medically important biochemicals. These include vaccines, interferons, hormones, growth factors, and monoclonal antibodies. In addition to biochemicals, certain cells are important as end products. Artificial pancreas‘ and artificial livers’ are two such examples.

There are two types of mammalian cells, anchorage dependent and suspension cells. Anchorage-dependent cells require a surface for propagation, while suspension cells grow in suspension, much like most microbial cells. The suspension-type mammalian cells may be cultured in modified microbial fermenters, though in some cases (e.g., human hybridomas) this may be difficult because of the extreme delicacy and shear sen- sitivity of the cell line.

For the successful and economic production of anchorage-dependent cells, the surface attachment re-

quirement demands a large surface-to-volume ratio in the culture unit. Many surface configurations have been proposed to meet this requirement. These include the multiplate p r ~ p a g a t o r , ~ the spiral film,4 plastic bags,s rotating tube bundle: hollow fiber^,^ tubular spiral film,8 and microcarrier beads.9 Of all these methods, only the hollow fiber systems, microcarrier beads, and spiral film have surface-to-volume ratios high enough to warrant further research.I0

In the early generation of hollow fiber ~ y s t e m s , ~ J ~ both liquid nutrients and oxygen were supplied from the tube side of different hollow fibers. These systems have been used to study the production of prolactin and human gonadotropin” and i n ~ u l i n . ’ ~ In addition, they have been used to study the formation of solid mammary carcinoma in vitroI4 and for the continuous production of carcinoembryonic antigen. 1 5 ~ 1 6 Ehrlich et al.” used various capillary culture units to study metabolism of different cell lines. Both a hybrid artificial pan~reas ’ . ’~ and an artificial liver2 have been developed by growing cells to tissue-like densities. Tze et a1.I8 studied the long-term culture of rat pancreatic islets and found that they could be maintained viable and secreting insulin for as long as 90 days in culture. Weiman et al.I9 used an artificial capillary cell culture unit to grow AML-2-23 and PM-81 murine hybridomas. They also reported growth of a human hybridoma cell line, with good yields of monoclonal antibody.

For efficient functioning of these systems, the number of fibers supplying oxygen has to be 3 times the number of fibers supplying liquid medium8 The nutrients are consumed relatively rapidly as they flow down the axis of the fiber, and thus cells at the distal end of the fiber become nonviable due to starvation. Cell growth is nonuniform and Jensen8 suggested that such systems would only work with a small culture volume, as witnessed by available commercial units. Waterland et al.’O demonstrated through theoretical calculations of an enzymatic hollow fiber reactor that substrate concentrations can decrease dramatically along

Biotechnology and Bioengineering, Vol. XVIH, Pp. 329-342 (1986) 0 1986 J o h n Wiley & Sons, Inc. CCC 0006-3592/86/030329- 1 G04.00

the length of a fiber. In a more recent development Ku et al.zl fed the cells with nutrient from the shell side in a flat-bed reactor configuration, which served to overcome many of the earlier problems. Their system was limited to a maximum fiber bed depth of six fibers. It is likely that the system geometry and operating parameters have not been optimized.

Swabb et a1.2z and Davis et al.23 found that, in vivo, convection as well as diffusion were important to nutrient delivery, depending on the solute molecular size. All the older generation and in fact all the presently commercially available devices essentially rely on diffusion as the sole mode of nutrient transport. Wei and R ~ s s ~ ~ showed that in vivo conditions could be simu- lated through the use of a dual-circuit hollow fiber unit, each circuit being held at a different pressure to sim- ulate the arterial and venous side of the vasculature. Knazek et al.25 utilized this concept in a dual-circuit capillary culture unit where convection was induced via a pressure differential between the “arterial” and “venous” sides of the unit.

Though the device of Knazek et al.25 was a major improvement, it still has certain drawbacks. In vivo, no tissue mass is more than 100 p m removed from a source of nutrients or a sink where metabolic waste products are removed.l0 With Knazek’s method of dual circuits, a source fiber would have to be adjacent to a sink fiber. When working with this constraint, the con-

struction of the device becomes difficult. The present construction procedurez5 does not guarantee perfect distribution of source and sink fibers. Cells may pref- erentially grow on the source fibers, and thus the effective surface area would be reduced. Medium would have to be oxygenated externally. An axial gradient would still persist and may be important in large systems since the nutrient is supplied through the lumen of the fibers.

It was the objective of this project to develop a system that would closely emulate in vivo conditions and still be amenable to scale-up. The system would have to be easy to construct, would incorporate a large surface area within a small volume, and would be able to maintain high densities of anchorage-dependent cells for extended periods of time. In addition, cells would not be exposed to excessive shear fields or to nutrient or waste product gradients. A bioreactor was developed and successfully operated to fulfill these objectives.

Conceptually, it offers an excellent blueprint for de- veloping large-scale cell culture bioreactors. The design is depicted in Figure 1. Nutrients flow radially across an annular bed of hollow fibers from a central feed distributor tube while an air-C02 mixture flows through the tube side of the hollow fibers serving to oxygenate the cells that are growing on the outside of the hollow fibers. Since there is a uniform delivery of nutrients along the entire length of the fiber system,

Liquid nutrient

Central radial flow distributc tr

- Hollow fibei immobilized

.Periodic bleed- concentrated pi

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r with cells

-off of -oduct

4 Liquid nutrient

Figure 1. Conceptual design of a radial flow hollow fiber bioreactor.

330 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 28, MARCH 1986

cells proliferate evenly. The culture can support high- density cell growth for the production of clinically important biochemicals.

DESIGN OF BIOREACTOR

A schematic of the radial flow hollow fiber bioreactor is shown in Figure 2. The core of the bioreactor consists of a central feed distributor tube that ensures a uniform supply of nutrients along the bioreactor axis. Surrounding this central feed distributor tube is an annular bed of hollow fibers that is held in place with silicone rubber. This assembly is pulled into a polycarbonate shell and potted at both ends with epoxy, resulting in a unit that consists of two primary spaces, the extracapillary space (ECS) and the intralumen space (ILS). Liquid nutrient is fed continuously through the central feed distributor tube with a Manostat cassette peristaltic pump. Uniformly along the entire length of the distributor, the medium flows radially outward across the fiber bed, diffuses across the wall of the hollow fiber into the ILS, and is convected away by the gaseous flow.

Gaseous nutrients are fed through the lumen of the hollow fibers. Gaseous exchange occurs via diffusion through the fiber wall. In addition to oxygenating the cells in situ, pH is also maintained by C02 exchange. Cell inoculum is introduced into the ECS through the inoculum ports on the outer shell.

The fibers are pretreated with poly-d-lysine, facili- tating attachment of anchorage-dependent cells. Poly- d-lysine is a polycation that adsorbs onto the fiber surface as a monolayer26 and facilitates cell adhesion.27

Another distinct feature of the present system is that the reactor is run as a continuous-flow recycle reactor with a very high recycle ratio to conserve expensive medium and reduce internal gradients. This is important as the cost of nutrient media is often the deciding factor whether a system is economically feasible. Thus, even though the conversion of substrate per pass is quite low, the overall utilization of nutrient can be high.

This reactor configuration possesses several advantages over conventional cartridge-type hollow fiber units. Since the feed is uniform along the length of the fiber, there are no axial gradients and the unit is not limited in length, as earlier generation units were. Oxygenation is carried out in situ, obviating the need for external oxygenation procedures. Since the nutrient diffuses radially through the cells growing on the fiber surface and through the wall of the fiber, spent medium is convected away by the gas stream, avoiding accu- mulation of metabolic waste products.

CELL LINE AND CULTURE MEDIUM

The cells used were HI cells, a fibroblast cell line derived from Chinese hamster lung connective tissue

U u -ll

Figure 2. Cross-sectional view of the radial flow hollow fiber bioreactor. The unit is symmetrical about the horizontal axis. A, In- oculum ports; B, hollow fiber bed; C, gas inlet (ILS); D, spent gaseous and liquid nutrient; E, central radial flow distributor; F, stainless steel mesh; G, O-ring seals; H, epoxied fiber ends; I, concentric stainless steel tubes in central distributor; J , reactor outer shell.

and donated to us by Dr. Immo Scheffler, Biology Department, University of California, San Diego. All cell culture work was conducted in a sterile laminar flow hood. H1 cells are anchorage-dependent cells that grow to confluent monolayers in conventional tissue culture.

Cells were grown in Dulbecco’s modified Eagle medium (1 g/L glucose, Irvine Scientific) supplemented

THARAKAN AND CHAU: RADIAL FLOW HOLLOW FIBER BIOREACTOR 331

with 2% fetal calf serum, 4% newborn calf serum, 0.01% nonessential amino acids, 0.01% fungizone, and 0.004% gentamycin. Medium was filter sterilized prior to use by passage through a 0.2-pm filter module.

MATERIAL AND METHODS

Batch Culture Experiments

Batch cultures do not provide a homeostatic environment, and without adequate oxygenation, anaerobiosis sets in quite early after inoculation. In fact, withn an hour, cells in a 100-mm petri dish with a 2-mm layer of medium over them will enter anaerobiosis.28 Nu- trients are consumed very fast, and toxic metabolic waste products build up quickly. In many circum- stances, batch cultures do not necessarily provide the correct physiological and kinetic information regarding continuous cultures. However, as a first approximation, useful information about cell growth, maximum specific growth rate, rate of consumption of nutrients, and rate of production of wastes can be obtained from a simple batch culture study. Batch culture experiments were performed to estimate these parameters.

Cells were allowed to grow to confluency in a 100- mm petri dish and then washed repeatedly with sterile buffer to remove all the debris and dead cells. The cells were trypsinized, centrifuged, resuspended in complete medium, and counted in a Coulter counter (Coul- ter Electronics, Florida). This cell suspension was di- vided into 20 aliquots of 1-mL each and inoculated onto 20 previously prepared LUX dishes with 10 mL complete medium. Two dishes were removed from the incubator at designated time intervals. The medium was collected, spun down, and refrigerated to be assayed later. The pH was recorded with a digital pH meter. The dishes were then washed to remove dead cells and trypsinized, and the viable cells counted in a Coulter counter. At the end of the experiment, assays were conducted for glucose using glucose dehydrogenase and for lactic acid using lactate dehydrogenase.

Cell Culture on Different Fibers

Cells can be cultured on different surfaces.29 Poly- sulfone, polyacrylamide, and cellulose acetate fibers are available commercially. The following experiments were conducted to establish the best support and the most efficient coating material for the anchorage-dependent HI cells. Jensen et al.29 and McLimans et a1.28 showed that cells grew better on a gas-permeable surface. Brunner e t al.30 showed that coating conventional supports with polylysine or collagen allowed for a more stable culture and also permitted anchorage-dependent cells to proliferate as a multilayer. For ease of steri- lization, polysulfone 3S100 fibers (Amicon Corp., MA) with a 100,000-dalton molecular weight cutoff and Te-

flon microtubules were used. Both poly-d-lysine (75,000 daltons, Sigma Chemicals) and collagen (Collagen Corp., CA) were tested as attachment enhancement factors.

Fibers and microtubules were autoclaved and washed with sterile buffer. The fibers to be coated with polylysine were placed in a dish and submerged in a 0.1- mg/mL polylysine solution. They were then incubated for 30 s after which 1 mL phosphate buffered saline and 0.1 mL newborn calf serum were added. Fibers were subsequently incubated overnight. Similar procedures were followed with collagen coating, using 0.34 mg/mL collagen solution. On the following day the buffer was replaced by fresh complete medium, and the fibers were inoculated with lo6 cells/mL. Cells were allowed to attach overnight. After 1 day, fibers were removed and placed in fresh dishes with fresh medium. Uncoated fibers were used as control.

Oxygen Mass Transfer and Uptake Rate

The oxygen transfer rate of the hollow fiber system was determined under conditions analogous to the radial flow cell culture system. The dynamic method of Bandyopadhyay et al.3' was used. To facilitate data analysis, the radial cross-flow configuration was sim- ulated utilizing a U-loop of 3S100 hollow fibers im- mersed in a beaker that was agitated with a magnetic stirrer. Because of the high permeability of the fibers, the setup emulates a transmembrane counterdiffusion situation, and thus the oxygen flux measured from such an experiment will be a good approximation of the actual culture system. The apparatus is illustrated in Figure 3.

Initially, nitrogen was fed through the fibers until the bulk oxygen concentration was zero, as determined by an Orion 97-08 oxygen electrode (Orion Research Inc., MA). An air-CO, mixture was then passed through the fibers, and the oxygen concentration was recorded as a function of time.

The oxygen uptake rate of the H1 cells was determined in a separate experiment utilizing cells growing on a culture dish. Cells were inoculated onto 100-mm petri dishes and allowed to grow to various densities. The cover of the dish was then removed, and a cap that held an oxygen electrode was placed over the dish, with the electrode membrane dipping into the medium. Fresh medium was added to the dish, and the oxygen concentration was monitored as a function of time.

Radial Flow Hollow Fiber Bioreactor

The system contained a stainless steel central distributor of concentric tubes that was wrapped with stainless steel meshes. A schematic of the flow distributor is included in Figure 2. The flow distributor was designed to ensure a uniform radial cross-flow of liquid medium over an annular fiber bed of thickness


Flowmeter ,$

Figure 3.

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Humidifier

Schematic of oxygen apparatus for transmembrane coun- tekow mass transfer estimation. A, U-loop of hollow fibers; B, oxygen electrode; C , peristaltic pump.

1.5 cm and along the entire length of the fiber bed (15 cm). The design was established through a series of hydrodynamic experiments utilizing fluorescent dye for flow visualization. Adjacent to the central feeder tube and in a tight concentric annulus was the fiber bed, consisting of packed bundles of four fibers each. The fibers were held in place by silicone rubber. The total lumen surface area available in the unit was 1150 cm2. This complete unit was pulled into a polycarbonate shell with flanged caps and sealed with O-rings. The ECS volume was 145 cm3, with the fiber bed occupying

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46 cm3 of the ECS. All connectors were either 316 stainless steel or autoclavable polypropylene. The medium was fed to the central feeder with a Manostat cassette peristaltic pump (Manostat Co., New York, NY). The lumen of the fibers was connected in another circuit, through which an air-C02 mixture (10% C02, 90% air) was supplied. The air-C02 mixture ensured that the cells would be adequately oxygenated and that the pH would be maintained between 7.2 and 7.3.

The reactor was autoclaved, and poly-d-lysine in 0.1 m g h L solution was introduced into the reactor through the inoculum ports. The reactor was then placed on a roller table in a 37°C environmental room for 1 h. Sub- sequently, sterile phosphate-buffered saline and newborn calf serum were added, and the reactor was left on the roller table overnight.

H1 cells were grown to confluency in 100-mm LUX tissue culture dishes. An inoculum of 4.0 x lo5 cells cmP3 of reactor volume was used. The cells were washed, trypsinized, centrifuged? and resuspended in medium and inoculated into the reactor through the inoculum ports. The reactor was then placed on the roller table to allow the cells to attach for 24 h.

After 1 day on the roller table the unit was placed on-line in an incubator and hooked up to a flow system, as shown in Figure 4. The system was operated as a recycle reactor, with a recycle ratio of 90 to increase utilization of media and minimize the nutrient and metabolic gradients. It also enabled us to perform design

i- - - I I I I I I I L-

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BJ I -- J

Figure 4. Flow schematic of the continuous radial flow hollow fiber reactor. A, Bioreactor; B, incubator; C, quick release couplers; D, humidifier; E, air cylinder; F, CO, cylinder; G , air flow meter (10%); H , CO, flow meter (90%); I, sterile air vents; J, nutrient carboy; K , spent medium carboy; L, recycle vessel; M, Manostat pump; N, Masterflex recycle pump; 0, 0.2-pm air filter.


and operation calculations assuming that the reactor was a well-mixed tank.

Samples were withdrawn at 12-24-h intervals from the reactor and the effluent stream and assayed for glucose and lactic acid. The reactor was run for a pe- riod of 2 weeks. At shutdown, fiber samples were snipped off at different locations in the reactor for cell mass estimation and scanning electron microscopy.

Since cell mass keeps increasing, the system will not reach steady-state operation. To minimize nutrient consumption and maximize cell mass, the high-recycle- ratio reactor is modeled as an unsteady-state CSTR with simple Monod growth kinetics. Specific growth rate and the Monod constant are approximated from the batch kinetic data. Information based on batch kinetics will not truly reflect the continuous hollow fiber culture, especially with multilayer cell growth. How- ever, with no other information, the approximation does help to preprogram the nutrient feed rate as a function of time on stream.*]

The resulting immobilized cell mass and limiting substrate equations were integrated numerically using a fourth-order Runge-Kutta method.32 Using the dilution rate as the parameter, cell mass and substrate variation with time were calculated. The results for various flowrates are shown in Figure 5 . For the first few days of operation, when the cell mass remains

relatively low, the choice of dilution rate is not important. However, to maintain cell growth, subsequent increases in the feed flowrate are required. The curves in Figure 5 were used to schedule the necessary flowrate increase to compensate for the proliferating cell mass. As the calculations were based on a fairly crude kinetic model, we felt that manual step changes would be appropriate. In addition to the schedule provided by the computations, daily assays of the lactic acid and glucose concentrations in the reactor were used to ensure that the dilution rate employed was adequate to provide the cells with sufficient nutrients.

Analytical Methods

Glucose and lactic acid from both the batch and continuous culture experiments were assayed enzy- matically. Glucose was estimated using glucose dehydrogenase. Lactic acid was assayed for using lactate dehydrogenase and NADH. The pH was measured using a Beckman phi-70 pH meter.

Cell number was measured using a Coulter counter for all the batch experiments where cells were grown on conventional tissue culture surfaces. Trypsinization was not effective for the cells growing in the reactor on poly-D-lysine-coated polysulfone fibers. Cell number was estimated using ethidium bromide and flu-

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Results of numerical integration of unsteady-state well-mixed tank model with medium flowrate Q (in mUh)


orescent spectrophotometry. The details of the method can be found elsewhere.))

Electron Microscopy

Cells growing on hollow fibers were fixed for scanning electron microscopy. Primary fixation was done using a 2% formaldehyde, 4% gluteraldehyde mixture in 0.1M phosphate buffer. After washing with buffer, fibers and cells were fixed for 1 h in 1% osmium te- troxide followed by extensive washing with distilled water. Subsequently, fibers and cells were dehydrated through a graded series of ethanol washings, finally ending in a 100% EtOH soak. The samples were then cut off and placed in a critical point dryer and coated with a thin film of gold for observation. Observations and photomicrographs were made using a Hitachi SEM.

RESULTS

Batch Culture

The results of the lactic acid and glucose assays are plotted in Figure 6 along with the cell growth data and the pH fluctuation. With reference to cell growth, there

was a lag phase during which cell number actually decreased. This is because cells do not attach to the dish surface immediately. Following this was the ex- ponential growth phase that ended as glucose was com- pletely consumed. Then the cell number started to decrease as cells began to die and slough off the surface of the dish. The data for glucose consumption and lactic acid production indicate that nutrient and metabolic gradients will develop extremely quickly in batch culture. There were also fluctuations in pH even though the dishes were maintained in a humidified air-C02 environment. The fluctuations were most severe during the lag and death phase of the culture.

The data closely resembled Monod kinetics with glucose as the limiting substrate. The maximum specific growth rate and the Monod constant were evaluated to be 0.036 h- ' and 0.099 mg/mL, respectively. The yield coefficient was 4 x lo6 cell/mg glucose. Using these parameters, the Monod curves for glucose and cell mass were computed and are plotted in Figure 6. The fit to the batch culture is reasonably good. The maximum specific glucose uptake rate calculated for batch culture is presented in Table I. The kinetic parameters were also used in preliminary design calculations and in the unsteady-state calculations in Fig- ure 5.

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Figure 6. lines of the cell mass and glucose concentration are obtained from Monod model calculations.

Batch culture data for HI cells. Glucose (A); lactic acid (0); cell number density (m); pH fluctuation (+). The solid


Table I. in batch culture.

Comparison of glucose uptake rate in the bioreactor and

Relative specific glucose uptake rate in bioreactop Time (h)

21 0.80 55 1 .OO

114 1.09 1 60 0.70 262 0.29

"The maximum specific uptake rate in batch culture was 1.1 pmoV106 cells min and the specific uptake rate in the bioreactor was estimated with a projected cell mass from calculations using the initial and final cell mass measurement.

Cell Culture on Different Fibers

Cell attachment and proliferation as observed under phase contrast microscopy were much faster on the coated fibers than on the coated microtubules and the uncoated fibers. After 2 weeks in culture with medium replaced daily, cells had grown to form multilayers on the coated fibers. On the uncoated fibers cells even- tually grew to multilayers, but after week 2 sloughing off of cells was observed. After 3 weeks of growth, only a few cells remained attached to the uncoated fibers, but one could still observe multilayers of cells on the fibers coated with collagen and polylysine. At the end of 6 weeks of culture, cells on the collagen- coated fibers had begun to slough off, but cells on the polylysine coated fibers remained viable and the cell mass remained intact. Figure 7 is a scanning electron micrograph of H1 cells growing on a polysulfone fiber that has been coated with poly-d-lysine. The cells have been in culture for 4 weeks. The micrograph shows that a uniform multilayer of cells is proliferating on the fiber surface, and the morphology is identical to dish- grown cell. On the basis of these observations, it was decided that polysulfone fibers coated with polylysine should be employed in our reactor and in subsequent studies.

Oxygen Mass Transfer and Uptake Rate

The oxygen mass transfer rate across polysulfone hollow fibers was determined using the dynamic method. The data are presented in Figure 8. The oxygen transfer coefficient is calculated to be 1.8 h- ' , and the oxygen flux across the fibers is 0.21 mmol 02/cm2 h.

The oxygen uptake rate was calculated using the fact that, in the absence of an oxygen source, the oxygen concentration in a culture will depend only on the utilization of oxygen by the cells. Thus a direct plot of the oxygen concentration against time will yield a straight line with a slope equal to the oxygen uptake rate of the organism. The data are shown in Figure 9. As can be seen from the plot, oxygen consumption is initially

Figure 7. Scanning electron micrograph of HI cells growing on polylysine-coated polysulfone hollow fibers (200 x ). The exposed surface of the fiber is an artifact of the fixing procedure.

linear, and after the dissolved oxygen concentration falls to 0.2 mmol 02/L, the cells turn to anaerobic metabolism.

However, as long as oxygen is supplied above a critical concentration, H1 cells grow as aerobes. The initial portion of the data is representative of aerobic metabolism. An oxygen uptake rate is calculated from the initial slope to be 0.14 mmol 02/L h per lo6 cells/mL.

Radial Flow Hollow Fiber Bioreactor

After 2 weeks of culture the radial flow hollow fiber bioreactor was dismantled, and samples of fibers at various locations were removed for cell mass estimation and for scanning electron microscopy. Macro- scopic observations showed that there was a multilayer of cells growing uniformly on all the fibers in the reactor. In addition to this uniform multilayer, which was visible to the naked eye as a milky tissue, were clumps of cells. The clumps were 3-4 times thicker than the surrounding multilayer. Figure 10 shows scanning electron micrographs of fiber samples from different points on the reactor. The fibroblast morphology of the H1 cells is clearly visible, and the cells were clearly propagating in a uniform multilayer.

The results of the glucose and lactic acid assays of the samples taken from the reactor along with the pH data are shown in Figure 11. Glucose concentration decreased until the dilution rate was increased. The times of increase in the dilution rate are indicated in Figure 11 by arrows. At each increase in dilution rate, the glucose concentration would increase for a few hours and the lactic acid concentration would corre- spondingly decrease. The data shows that the pH remained fairly constant in the reactor throughout the experimental run.


0 10 20 30

Tirnc ( m i n u t tLs)

Figure 8. Measurement of oxygen mass transfer coefficient for 3S100 polysulfone hollow fibers in nutrient medium. KLa = 1.8 h-I. Using a mixture of 90% air and 10% COz, an oxygen flux of 0.21mM OJh crnz was measured in a transmembrane counterflow configuration.

5 10 15 20 25 0

Time ( h r s )

Figure 9. negligible as the dissolved oxygen concentration decreases below a critical value.

Oxygen uptake data for HI cells. The oxygen concentration initially drops, but oxygen uptake becomes


Representative samples of fibers were also removed for analysis of cell mass. From an inoculum of 4 x lo5 cells/mL, the number of cells increased to 5.8 x lo7 cells/mL in 12 days.

DISCUSSION

The premise of this approach to large-scale mammalian cell culture using hollow fibers was based on the idea that an axial tube-fed system was not easily amenable to scale-up. In fact, as indicated by previous workers,8,20 systems were limited in length from 2 to 3 in. This has been supported by experiments we conducted on feed distribution in single- and double-fiber reactors similar to those of Inloes et a1.34,35 Feed en- tered through the lumen and flowed down the length of the fiber. Based on observations with methyl red dye, it was found that the larger percentage of feed exited through the distal end of the fiber. The data (Fig.

Figure 10. Scanning electron micrographs of H1 cells growing on fibers at various points in the reactor. (a) Uniform multilayer of cells propagating on the fiber surface (200 x ). (b) Close-up showing the preserved morphology of HI cells (ZOOOX).

12) revealed that 90-95% of the feed does not reach the shell side of the unit in which the cells are growing. In addition, pressure drop measurements showed that the flow was from the shell into the tube side for the latter axial half of the fibers. It is unlikely that this configuration will provide an even distribution of nutrients or utilize the expensive nutrient medium effi- ciently in a large-scale system. A conclusion of these considerations is that a shell side cross-flow nutrient delivery system is a more effective method of supplying nutrient to the cells. The work of Ku et aL2' supported this observation. Our results confirm their find- ings and at the same time present a system that is potentially amenable to scale-up.

The most useful indication of a successful cell culture technique is the final cell mass distribution and density. This information enables us to determine if nutrients are being supplied at sufficient rates, if toxic metabolic wastes are adequately removed, and if the culture configuration contributes any inhibitory effects. Macroscopic observation of the fiber unit after termination of the experiment showed that there was a uniform tissue-like distribution of cell mass that was visible to the naked eye and thus obviously several layers thick. In addition to this, there was a uniform distribution of clumps of cells that was up to 2 times the diameter of the rest of the uniform multilayer, as depicted in Figure 13. Samples removed from various parts of the unit and observed under scanning electron microscopy showed that there were no local variations of cell mass (Fig. 10). This was also confirmed by the cell numbers estimated through fluorescence measurements. Representative samples of fibers removed from various locations in the reactor unit yielded cell numbers that did not differ by more than 5%.

The reactor (145 cm3) enclosed a fiber bed of volume 46 cm3 and a surface area of 1150 cm2. The inoculum was 5 x lo4 cells/cm2, and after 12 days in culture the average cell density per unit surface area was 7.3 x lo6 cells/cm2. This compares very well with the average values of 1-4 x lo5 cells/cm2 for roller-bottle-grown cells. Densities of 1.35 x lo6 cell/cm2 and 2.6 x lo6 cells/cm2 have been reported. The former was for normal-tissue-derived cells2' and the latter was reported for sarcoma cells.36 The cell line we used was a normal- tissue-derived fibroblast cell line, and our results show that confluent multilayers of these cells can be obtained in our system.

The data for the glucose and lactic acid concentration variation in the bioreactor with time are shown in Figure 11. From the figure it can be seen that the level of nutrient and metabolic waste reach a value about which they fluctuate as a result of the increases in feed flowrate. The system is in an unsteady state as cell mass continues to increase. Thus the nutrient require- ments increase continuously, and ideally a feedback control mechanism should be implemented. With a


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Figure 11. the times when the medium flow rate (mL/h) was increased in a step change to the value indicated.

Glucose (m), lactic acid (O), and pH variation in the bioreactor plotted against time. The arrows indicate

0.922 0 . 0 4 4 0.366 0.088 0.11 0 .13

T o t a l f l o w rate (mls / sec )

Figure 12. (A) and permeate flow (0).

Comparison of tube and permeate flow in a conventional hollow fiber unit. Tube side flow


Figure 13. of H1 cells ( 7 5 ~ ) .

Scanning electron micrograph showing turnour-like clump

feedback control loop, the amplitude and duration of the fluctuations would be minimized, and a more constant environment could be achieved.

In our case we were seeking to demonstrate a viable reactor system where high densities of anchorage-dependent cells could be grown in vitro. Our observations indicated that information from monolayer batch cultures would not be adequate for long-term, high-density continuous cultures. The kinetic constants obtained from batch cultures are likely to overestimate the cell growth. The specific glucose uptake rate is used to illustrate this point. The specific uptake rate in the bioreactor compared well with the batch data in the beginning (Table I). However, after day 5 the glucose uptake rate in the reactor fell below the predicted value. Initially, it is likely that the cells are well supplied with nutrient and no diffusional limitations exist. The pH remained essentially constant throughout the run, and thus the decrease in glucose uptake rate can be attrib- uted to diffusional limitations to the cells in the lower layers of the tissue mass. Additionally, though batch studies show very neatly that glucose is the limiting nutrient, the maximum specific growth rate, the Monod constant, and the yield coefficient may have signifi- cantly different values in a continuous culture. Also, glucose may no longer be limiting in a continuous per- fusion culture.

Cell crowding may be another factor in limiting cell growth and decreasing the predicted uptake of glucose. In conventional tissue culture H1 cells grow to a confluent monolayer and then cease growth. Inhibition apparently arises from cell surface properties that result in the transmission of growth-inhibiting signals between two cells.37 Normal tissue cells have been known to form multilayers when they are exposed to a continuous supply of nutrients.38 In addition, if the pH is closely controlled, monolayers can be induced to grow as r n ~ l t i l a y e r s . ~ ~ The data from our reactor shows that

the pH remained constant throughout the length of our run, and we observed multilayers of cells growing at tissue-like densities. However, in a three-dimensional culture configuration, inhibition may be brought about at a later stage or by other factors.

Cell crowding may present another problem. As multilayers form, there is an increasing diffusional resis- tance to nutrient delivery for the cells at the lower layers of the multilayer. After day 5 there are an estimated 1.8 x lo6 cells/cm2 in the reactor. This is al- ready an order of magnitude greater than that achieved with roller-bottle-grown cells, and serious diffusional limitations on nutrient delivery to cells in the lower layer may begin to be of significance. These mass transport effects will have to be accounted for in a more complete analysis.

A problem observed by many workers using hollow fibers for the culture of various cells has been that of cracking of the hollow fiber due to pressures exerted by an expanding cell mass. Inloes et al.34 reported damage to the integrity of the fiber wall when E . coli cells were cultured to high densities. This has also been the case with plant cells40 grown in hollow fiber culture units. Both microbial and plant cells possess cell walls that are stiff enough to collapse a delicate hollow fiber wall at high cell densities. Such a breach of the integrity of the fiber wall would render the unit nonoperational. We did not observe such ruptures in the membrane at the high surface densities achieved in our unit, and the integrity of the membrane was maintained.

As our experimental results indicate, H1 cells respire as facultative aerobes. This is the case with many established cell lines. However, when one is culturing primary cells and tissue, oxygen supply is very important. The present bioreactor design allows greatly enhanced rates of oxygen supply via in situ oxygenation. In conventional tissue culture, microcarrier culture, and conventional hollow fiber cartridges, external oxygenation of medium is required. This has mostly been achieved through passive diffusion of oxygen into a length of silicone tubing placed between the liquid nutrient reservoir and the culture nit.^,'^,'^ Such a method is unwieldly and inefficient. Glacken et al.” reported an oxygen mass transfer coefficient (KLa) for silicone tubing of 0.35mM 02/atm cm2 h. This value corresponds to an oxygen flux of 0.07mM 02/h cm2 if a 95% air, 5% C 0 2 humidified mixture is used as the gaseous nutrient supply. With such a flux, 100 ft of 1 .O-in. silicone tubing would be required’O to maintain a 7.5-L culture of lo6 cells/mL FS-4 cells. We obtain an oxygen flux across Amicon 3S100 polysulfone hollow fibers in a counterflow configuration of 0.21mM 02/cm2 h. With a surface area of 1150 cm2 enclosed in the reactor, this would be able to deliver an oxygen supply of 241mM 02/h and is a good choice for main- taining high densities of oxygen-sensitive cell lines.

The operation of the reactor system as a high-recycle


unit was successful in that there was a fairly high utilization of substrate while no significant nutrient and waste product gradients developed. The cross-flow velocity across the fiber bed with a recycle flowrate of 15 mL/min was on the order of c d s . This velocity is slow enough to ensure that the cells are not subjected to excessive shear fields. From the scanning electron micrographs it was clear that the HI cells maintained their morphology at these recycle rates and did not suffer shear damage.

This reactor configuration has been successfully utilized to grow H1 anchorage-dependent fibroblasts at high densities. It offers an excellent prototype for the large-scale culture of anchorage-dependent cells that produce useful biochemicals. The negligible axial and radial gradients of nutrient and metabolic wastes, in situ oxygenation, and low-shear fields provide an environment closely emulating the physiological environment in vivo.

In addition, there are several potential advantages that will need further research. For example, in most biochemical processes, product separation and puri- fication is usually the cost-determining step. Compli- cating the problem further, many important biochemicals are produced at extremely low concentrations, and their concentration in bioreactor effluent streams are often negligible. Thus, for practical reactor design, effluent stream product concentration should be high. In the bioreactor presented here the effluent from the reactor can be designed, in principle, to contain no product (complete retention) or all the product (zero retention). This is achieved through the choice of ex- clusion limit for the hollow fiber used in the bioreactor. The ultrafiltration properties of the hollow fibers can be taken advantage of to effect preliminary separation. A possible scenario is the concentration of product in the shell space accompanied by the removal of cell wastes in the effluent stream. Withdrawal of concentrated product could be effected by draining the shell space. The scenario seems promising, but further research is necessary to study problems such as clogging of the fiber membranes and gel polarization at the fiber membrane surface.

CONCLUSION

The aim of this project was to synthesize the high surface-to-volume ratios of hollow fiber units with the feed delivery system of a radial flow reactor, with the idea that such a system would be able to emulate in vivo conditions much more closely than presently available systems. This implies that the cells should be adequately oxygenated, there should be no nutrient or metabolic waste product gradients, and the cells should not be subject to high-shear fields and at the same time must be able to be maintained at high den-

sities, especially for the long-term production of bio- logicals. An efficient reactor design that incorporates these features has been presented. The reactor system developed here serves as an excellent prototype for further research and scale-up design.

The authors would like to thank Dr. Immo Scheffler and Elysa Waltzer of the Biology Department at the University of Cal- ifornia, San Diego, and the UCSD Biomedical Research Sup- port Fund for partial financial support of this project.

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A radial flow hollow fiber bioreactor for the large‐scale culture of mammalian cells

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