Extracapillary Space Research Articles

Expansion of activated lymphocytes obtained from renal cell carcinoma in an automated hollow fiber bioreactor.

Immunotherapy using IL-2 alone or combined with activated lymphocytes has been promising for metastatic renal cell carcinoma. Cytotoxic lymphocytes can be isolated from tumors, expanded in vitro with IL-2, and adoptively transferred back into the tumor-bearing host. These cells can also be transduced with the genes coding for cytokines for local delivery to tumor sites. A major drawback in adoptive immunotherapy is the cumbersome and expensive culture technology associated with the growth of large numbers of cells required for their therapeutic effect. To reduce the cost, resources, and manpower, we have developed the methodology for lymphocyte activation and expansion in the automated hollow fiber bioreactor IMMUNO*STAR Cell Expander (ACT BIOMEDICAL, INC). Tumor Infiltrating Lymphocytes (TIL) isolated from human renal cell carcinoma tumor specimens were inoculated at a number of 10(8) cells in a small bioreactor of 30 ml extracapillary space volume. We have determined the medium flow rates and culture conditions to obtain a significant and repeated expansion of TIL at weekly intervals. The lymphocytes cultured in the bioreactor demonstrated the same phenotype and cytotoxic activity as those expanded in parallel in tissue culture plates. Lymphocyte expansion in the hollow fiber bioreactor required lower volumes of medium, human serum, IL-2 and minimal labor. This technology may facilitate the use of adoptive immunotherapy for the treatment of refractory malignancies.

Protein polarization in isotropic membrane hollow‐fiber bioreactors

AbstractA mathematical model has been developed to predict the coupled hydrodynamics and high‐molecular‐weight protein transport in mammalian‐cell hollow‐fiber bioreactors (HFBRs). The analysis applies to reactors with isotropic ultrafiltration membranes under startup conditions when the extracapillary space (ECS) is essentially unobstructed by cells. The model confirms the experimental finding that secondary ECS flows, engendered by the primary flow in the fiber lumens, can cause significant downstream polarization of ECS proteins at typical mammalian‐cell HFBR operating conditions. It also reveals that the osmotic activity of the proteins, by curtailing transmembrane fluid fluxes, can influence strongly the outcome of the polarization process. In fact, at order‐of‐magnitude higher protein concentrations and/or lower recycle flow rates, the secondary flow velocities can be reduced by as much as six orders‐of‐magnitude throughout the ECS, thereby virtually eliminating the polarization problem. This result has important implications for improved reactor startup procedures.

Protein adsorption in polysulfone hollow fiber bioreactors used for serum‐free mammalian cell culture

The recovery of serum-free medium proteins from poly-sulfone hollow fiber bioreactors (HFBRs) was investigated. More than 99% of the initial transferrin was adsorbed to the hydrophobic hollow fibers within 2 h of HFBR operation. A methodology to minimize transferrin adsorption by pre-adsorption of bovine serum albumin (BSA) was developed. BSA adsorption on suspended cut fibers was virtually complete within 1 h. BSA-coated fibers adsorbed only 5% of the transferrin within 10 days, whereas uncoated cut fibers adsorbed more than 99% of the transferrin within 1 h. An improved HFBR startup procedure, using a BSA-coating step before inoculation, resulted in substantially higher transferrin recovery. Additional factors influenced extracapillary space (ECS) transferrin concentrations. Pronounced downstream polarization of transferrin was observed in the ECS. In addition, the 30-kDa nominal molecular weight cutoff ultrafiltration membranes rapidly leaked transferrin from the ECS to the lumen.

Evaluation of a hepatocyte‐entrapment hollow fiber bioreactor: A potential bioartificial liver

We have developed a hepatocyte entrapment hollow fiber bioreactor for potential use as a bioartificial liver. Hepatocytes were entrapped in collagen gel inside the lumen of the hollow fibers. Medium was perfused through the intraluminal region after contraction of the hepatocyte-entrapment gel. Another medium stream, comparable to the patient's blood during clinical application, passed through the extracapillary space. Viability of hepatocytes remained high after 5 days as judged by the rate of oxygen uptake and viability staining. Urea and albumin synthetic activities were also sustained. Transmission electron microscopic examination demonstrated normal ultrastructural integrity of hepatocytes in such a bioreactor. With its sort-term, extracorporeal support of acute liver failure, the current bioreactor warrants further investigation.

Design and application of NMR-compatible bioreactor circuits for extended perfusion of high-density mammalian cell cultures.

MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions. This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS. In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability. This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit. The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes. The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space. This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products. In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment. Hence, this circuit can be applied to a variety of bioreactor configurations. The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet. Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution. At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.

An investigation of the diffusion‐limited growth of animal cells around single hollow fibers

To compare modeling with experimental data of cell growth surrounding individual fibers, the growth profiles of hybridoma cells in the extracapillary space of single hollow fiber bioreactors were examined. Agarose was provided in the extracapillary space to provide support and minimize convection. By sacrificing bioreactors at various time intervals, the growth profiles of cells surrounding a single hollow fiber could be monitored with increasing time. Using photomicroscopy and viable staining, areas of viable and nonviable cell growth were examined at various stages of development ranging from initial seeding to stable growth conditions. Cells were found to act as nucleation sites for the growth of individual colonies within the agarose. Profiles at stable growth conditions resulted in a thick cell mass near the surface of the fiber wall followed by cell colonies of decreasing size with increasing radial distance. A simplified theoretical model for cell growth was developed using mass balance equations for substrate penetration into individual cell colonies as well as away from the wall of a single fiber. The resulting profiles derived from theory were compared with experiments and found to be in good agreement for entering oxygen concentrations of 5% and 20%.

High density culture of hybridoma cells in a dual hollow fiber bioreactor

Hybridoma cells were cultured for two months in the dual hollow fiber bioreactor (DHFBR) which had been successfully used for high cell density cultures of various microbial cells. In batch suspension culture the concentration of monoclonal antibody (Mab) against human Chorionic Gonadotropin (hCG) and the cell density of Alps 25-3 hybridoma cells were obtained in 30 μg/mL and 2.35×106 cells/mL, respectively. The continuous culture with DHFBR produced Mab of 100–130 μg/mL for 30 days and the estimated cell density in the extracapillary space of DHFBR was 1.87×108 cells/mL based on the antibody production rate. The productivity of Mab was 205 mg/day per litre of the total reactor volume while that of the batch suspension culture was only 10 mg/L day.

Tumor-targeted chemotherapy with lipid contrast medium and macromolecular anticancer drug (SMANCS) for renal cell carcinoma

Twenty-five patients with renal cell carcinoma were treated with a lipophilic macromolecular drug, poly(stylene-co-maleic acid)-conjugated neocarzinostatin (SMANCS) dissolved in lipid contrast medium (Lipiodol). The drug was injected by catheterizing the renal artery and another feeding artery in 24 patients, and in the common hepatic artery in 1 patient with metastases to the liver after a radical nephrectomy. The procedure of selective arterial administration of 3-20 mg/mL of SMANCS/Lipiodol was simple to perform and was required once every two to three weeks. Total dose of SMANCS for each patient varied from 3 to 57 mg. Both SMANCS and Lipiodol accumulated more selectively in tumor than in any other tissue and remained in the neovasculature and extracapillary space for a long time. CT pattern of the remaining oil contrast medium in the tumor was characterized by the high-density area localized mainly in the periphery of the tumor around the central necrosis. When hyperviscosity Lipiodol (Lipiodol HV) was used as lipid contrast medium, it remained more persistently in the tumor and disappeared more slowly than Lipiodol. Moreover, the pronounced anticancer effect was recognized when SMANCS/Lipiodol HV was administered compared with only SMANCS/Lipiodol. Severe side effects, such as myelosuppression, unendurable pain, paralytic ileus, etc., were not observed. This targeting chemotherapy may be of great significance for advanced renal cell carcinoma.

Immobilization of Anabaena azollae in hallow fibre photobioreactors for ammonia production

Hollow fibres made from polysulphone and polypropylene were used for testing the growth of Anabaena azollae. The surface properties of the fibres for attachment of cyanobacteria and the morphological features of A. azollae growing on the surfaces and in the extra capillary space of the fibres were examined by scanning electron microscopy. Three types of photobioreactors for the photosynthetic production of ammonia by A. azollae were constructed. The reactors were made of hollow fibres and fibres with a calcium alginate gel matrix in the interfibre region. Dilute ammonia produced in the medium was concentrated with a membrane separator by absorption with HCl. Ammonia production was studied with and without the inhibitor MSX. The ammonia concentration in the effluent reached 600–700 μ m with MSX and 120–350 μ m without MSX in the medium.

Magnetic resonance imaging and modeling of flow in hollow‐fiber bioreactors

AbstractNuclear magnetic resonance flow imaging (MRFI) was used to measure fluid flow noninvasively in the extracapillary space (ECS) of a hollow‐fiber bioreactor without cells. Agreement between these axial flow measurements in a single hollow‐fiber module and predicted axial velocity contour plots at various image planes along the path length was good.Flow in a solid‐wall tube (phantom) was first used to calibrate pixel intensities with axial velocities. Flow images at several locations along the permeable hollow fiber length were then obtained in order to observe the well‐known leakage or Starling flow in the ECS. These quantitative spatially dependent velocity measurements were then compared to theoretically derived velocities obtained from a solution of Poisson's equation with a constant pressure gradient and no slip at the solid surfaces. A mathematical transformation was used to simplify the numerical methods. Leakage flow through the ECS of a multifiber bioreactor (40 fibers) was also measured by MRFI, illustrating the applicability of this method for optimizing operational procedures and design of membrane bioreactors and filtration devices.

Modeling analysis of an intercalated‐spiral alternate‐dead‐ended hollow fiber bioreactor for mammalian cell cultures

The model analysis of a bioreactor with two intercalated-spiral sets of hollow fibers with alternated dead ends is presented. Design equations are derived based on a model with two jumped fibers and an approximation of the fluid mechanics with a small fiber radius-to-length ratio. The performance of the bioreactor is simulated with and without cell growth utilizing a segregated radial flow model in the extracapillary space. The pressure modulus, the wall Peclet number, the Thiele modulus, and the Monod constant are used as model parameters. The results can be used to assess the proper choice of fiber permeability, fiber length, fiber spacing, and flow velocity.

Optimum fiber spacing in a hollow fiber bioreactor

A high surface area hollow fiber reactor was developed for mammalian cell culture. The reactor employs an interfiber gel matrix of agar or collagen for cell support. A model was developed to predict cell density as a function of fiber spacing. Optimum spacings are calculated for two sizes of Celgard hollow fibers. Ehrlich Ascites Tumor (EAT) cells were grown to an estimated density of 1.1 x 10(8) viable cells/mL in the extracapillary space-corresponding to an overall reactor density of 7 x 10(7) cells/mL. On the basis of available kinetic and diffusivity data, the model predicts that lactate accumulation may limit cell growth in the early stage of medium utilization, while oxygen delivery becomes limiting at later stages.

An Analysis of Intra‐Versus Extracapillary Growth In a Hollow Fiber Reactor

AbstractMammalian cell culture in hollow fiber reactors is hampered by oxygen limitation. Diffusive oxygen transport from the fiber wall to the cells can be enhanced by increasing the surface area for oxygenation and/or decreasing the penetration depth. One strategy for improving of surface area to penetration depth ratio is to increase the number of fibers in the reactor. An alternative way to manipulate this ratio is to grow the cells inside the fibers rather than on the outside. The theoretical analysis presented indicates that this is favorable when the volume of the extracapillary space exceeds that of the fiber lumen by a factor four or more. The impact of growing the cells on the inside of the fibers on other aspects of mammalian cell culture in hollow fiber reactors, including mass transport from the bulk of the medium flowing through the reactor to the fiber wall, is discussed.

Design and Fabrication of a Capillary Cell Culture Chamber for the Study of Convective Flow

The use of capillary culture chambers as artificial pancreas and artificial liver devices would be aided by an improved ability to control the movement of molecules through the capillary walls. The modeling and analysis of the flow and mass transfer in capillary cell culture chambers in which cultured mammalian cells are grown to form masses with tissue density in the extra-capillary spaces is desirable as a basis for scaleup and optimization of the particular microenvironment. The relative roles of diffusion and ultrafiltration with convection in enhancing mass transfer across the capillary membranes are poorly understood in real cell cultures and the effects on culture viability of flow conditions chosen to promote convective flow across the capillary membranes and through the cultured cell masses are also of interest. In this report, experience with materials and techniques for fabricating capillary culture chambers with a more readily analyzed fully defined regular geometric relationship between 90 to 100 capillaries in a parallel bundle is described. High and low pressure capillaries were interspersed in a regular array. Evidence is presented for retention of cell viability during flow conditions which were chosen to induce convective flow through the cultured cells in such a chamber.

X-Ray Responses of Human Colon Tumor Cells Grown in Artificial Capillary Culture&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Clone A human colon adenocarcinoma cells were grown in three-dimensional artificial capillary culture (ACC) to determine responses of capillaries treated 3 weeks after tumor cell inoculation with a specific, easily quantifiable cytotoxic agent, ionizing radiation. The high-density growth of tumor cells in ACC can be considered to be an in vitro analogue of a solid tumor. Changes in extracapillary space (ECS) fluid concentrations of lactate dehydrogenase (LDH) and aspartate aminotransferase (GOT) and the utilization of glucose in circulating medium were monitored after a supralethal radiation dose (90 Gy) of X-rays. Immediately after irradiation, increased levels of LDH and GOT were found that reached maximum levels about four to five times those found in nonirradiated control capillaries at 10-13 days post irradiation and then declined. Patterns of enzyme production appeared to correlate with the numbers of nonviable tumor cells collected from the ECS of the artificial capillaries. In contrast, glucose utilization showed little correlation with either enzyme concentration or dead cell production. It was determined that, while capillaries grown and treated in this manner appear to respond in a dose-dependent manner to ionizing radiation (as indicated by changes in LDH and GOT levels), these particular end points are relatively insensitive and are not suitable for studies in which therapeutic levels of X-radiation might be given. In other studies, tumor cells were removed from unirradiated capillaries by trypsinization and used to obtain complete survival curves after graded doses of X-radiation. The dose-response curves obtained indicate that clone A colon tumor cells grown in ACC show a marked decrease in their ability to accumulate sublethal radiation injury as compared to responses of these cells growing exponentially in asynchronous monolayer cultures, to synchronized mid-G1 tumor cells, or to tumor cells in stationary growth phase. These data suggest that ACC is a potentially useful model to study the effects of cytotoxic agents on human tumor cells.

A modified matrix perfusion–microcarrier bead cell culture system. I. Adaptation of the matrix perfusion system for growth of human foreskin fibroblasts

Two strains of human foreskin fibroblast cells were incapable of sustained growth in a matrix perfusion culture system, possibly because of their inability to attach to the fiber surfaces. Addition of microcarrier beads to the extracapillary space allowed attaining high cell densities in excess of 10(7) cells per culture unit. Microcarrier beads were tested in hollow fiber culture devices containing membranes of 10(4) or 10(5) D nominal porosities. Best results were obtained when initial cell densities of at least (2-3) x 10(6) cells were used in units with 10(5) D pore size membranes and DEAE-Sephadex or polyacryl-amide microcarrier beads in the extracapillary space. This extension of the matrix perfusion system should be useful for growing other anchorage dependent cells while retaining the advantages of perfusion culture.

A modified matrix perfusion–microcarrier bead cell culture system. II. Production of human (β) interferon in a matrix perfusion system

Addition of microcarrier beads to a matrix perfusion cell culture system allowed growth of anchorage dependent human foreskin fibroblasts which would not grow in the culture units alone. The utility of the system for collection of cellular products was demonstrated by the induction and harvesting of human (beta) interferon. Interferon production was highest in perfusion cultures when medium was circulated throughout the induction and when inducer containing 100 mug/mL polyriboinosinic: polyribocytidylic acid was placed directly in contact with cells in the extracapillary space. These conditions provided 4-to-10-fold greater interferon yields per cell, and approximately 12-fold increases per vessel, than monolayer cultures. Perfusion grown cells produced interferon at a maximal level for 20 h postinduction compared to approximately 2 h for monolayer grown cells, thus giving a higher total yield of interferon. Other procedures increasing the efficiency of the system included priming with 50 U/mL interferon standard, reinduction of cells, use of antibiotic free medium, reduced serum concentrations, and in vitro aging of the cells.

Continuous production of carcinoembryonic antigen in hollow fiber cell culture units: brief communication.

A line of cultured cells derived from a primary human adenocarcinoma of the colon was grown in the extracapillary spaces of hollow fiber tissue culture units in the absence of serum components of high molecular weight (greater than 10,000). The cells were capable of producing more than 30 microgram of carcinoembryonic antigen (CEA) per day, provided that the extracapillary fluid was changed frequently. The concanavalin-A binding and molecular sieve chromatography properties of the tissue culture-derived CEA were similar to those of CEA isolated from metastatic colon cancer tissue.

Autoregulatory escape and post-escape hyperaemia in a capillaron model.

Autoregulatory escape and post-escape hyperaemia were studied in a passive hydrodynamic model of the capillaron (ie, one or more soft-walled permeable capillaries enclosed together with extracapillary filtrate in a compliant capsule). Constriction was simulated in the model by reduction of the calibre of an orifice in the arteriole through which flow entered the ‘vascular bed’ of the capillaron. Constriction lowered the intracapillary pressure, and the capillary collapsed partially, with a resultant decrease in flow rate. The rate of filtration across the capillary wall into the extracapillary space of the capsule also declined. The falling extracapillary pressure increased the transmural pressure and reopened the capillary, returning flow to preconstriction values (autoregulatory escape). Reopening of the arterial constrition increased intravascular pressure, distending the capillary, with a transient increase in flow above the control value (post-escape hyperaemia). The time rate of constriction of the arterioles determined the magnitude of the fall in flow. The rate of removal of the arterial constriction determined the magnitude of the post-escape hyperaemia. These results show that the phenomena of autoregulatory escape and of post-escape hyperaemia resemble effects generated by passive changes in transmural capillary pressure. Such passive changes are affected by the rates of change of arteriolar constriction or relaxation. The effects of downstream capillary permeability and of capsular compliance on these phenomena were also examined.

Kräfte und Kraftwirkungen im Gebiet der Blutkapillaren

The equations of state for the forces acting between the intra- and extracapillary spaces have been developed, and their significance in the filtration and reabsorption properties of the blood capillaries are shown.