Embodiments of a method for the production of human urokinase are disclosed. Also disclosed are embodiments of a cell culture well-suited for use with the disclosed method. The method involves culturing urokinase-producing cells, such as immortalized human renal cells, in a cell culture. The cell culture comprises microcarrier structures and a tissue culture medium. The urokinase production is allowed to occur while the cell culture remains relatively static, i.e., the cell culture is not substantially mixed.

Description of the Invention

SUMMARY

Embodiments of a method are disclosed for the production of human urokinase by culturing cells. Embodiments of a specialized cell culture for use with this method also are disclosed. The method can include culturing urokinase-producing cells in a cell culture comprising a plurality of microcarrier structures and a tissue culture medium. In contrast to conventional methods, the cells can be cultured without substantial mixing for a period, such as a period greater than about three hours. In one example, the cells are cultured under conditions with minimal shear stress, such as shear stress that is less than about 0.1 dynes/cm.sup.2. Cells cultured according to the disclosed method produce urokinase, which can be isolated from the cell cultures and purified.

The urokinase-producing cells can be, for example, human renal cells or immortalized human renal cells. The plurality of microcarrier structures can include a variety of three-dimensional structures, such as beads and/or rods. In some embodiments, the plurality of microcarrier structures includes glass beads and/or microcarrier structures comprising a dextran core coated with collagen. The ratio of cells to microcarrier structures in the cell culture can be, for example, greater than about 25:1 and less than about 500:1.

In some embodiments of the disclosed method, the cell culture is contained within a vessel. The vessel, for example, can be a flexible bag, such as a TEFLON.RTM. bag. Some suitable vessels have interior surfaces that are resistant to cell attachment. To promote optimal cell culture conditions without the need to supply gases from an external source, some vessels used with embodiments of the disclosed method are substantially CO.sub.2-retaining and substantially O.sub.2-permeable. For example, some vessels allow a sufficient amount of oxygen to diffuse into the cell culture to replace the oxygen consumed by the urokinase-producing cells and also retain at least a portion of the carbon dioxide produced by the urokinase-producing cells, so as to maintain an elevated concentration of carbon dioxide (relative to air) within the cell culture during the production of urokinase.

DETAILED DESCRIPTION

Production of Urokinase in a Three-Dimensional Cell Culture

Described herein are embodiments of a method for the production of human urokinase, as well as embodiments of a cell-culture device. The disclosed embodiments are based, in part, on the surprising discovery that urokinase production is enhanced when urokinase-producing cells are cultured without substantial mixing, at normal gravity and with microcarrier structures.

A. Culture without Substantial Mixing

Conventional cell-culture techniques typically involve some mixing of the cell culture. The mixing is intended to distribute nutrients and oxygen and thereby enhance the productivity of the cells. Mixing can be accomplished, for example, by introducing a magnetic stir bar into the cell culture. However, excessive mixing can damage cells, especially animal cells, which lack a cell wall (see, for example, R. S. Cherry, Animal Cells in Turbulent Fluids: Details of the Physical Stimulus and the Biological Response, 11(2) Biotechnol Adv. 279-99 (1993)). Somewhat gentler mixing can be accomplished by slowly rotating the cell culture, for example, in a roller bottle rotated at a rate of about 5 to 60 rotations per hour. Another approach to mixing the cell culture is to use a continuously flowing media. In a perfusion culture, new media is continuously introduced as used media is removed. All of these culture systems include conditions wherein substantial mixing of the medium components occurs. Generally, it is believed that substantial mixing increases the production of biological components, such as proteins, by cultured cells.

Prior to the experiments associated with this disclosure, there was no reason to expect that urokinase production would differ from the production of other biological agents. It is disclosed herein that urokinase production is inhibited by substantial mixing of the cell culture. The embodiments of the method described herein for urokinase production involve allowing the urokinase-producing cells to produce urokinase during a static period characterized by a lack of substantial mixing. The urokinase-producing cells can be mixed before and/or after the static period without compromising the enhanced urokinase production achieved during the static period. During the static period, the cells generally are immobilized or stationary, and an intentional effort is made to avoid mixing. Thus, substantial mixing does not occur, wherein the absence of substantial mixing is sufficient to enhance urokinase production in the culture medium.

One way to quantify the degree of mixing is to measure the shear stress of the cell-culture media. Shear stress can be measured easily with a rheometer (see, for example, U.S. Pat. No. 6,655,194). To achieve enhanced urokinase production, the shear stress of the cell-culture media during substantially all of the static period typically is less than the shear stress of the cell-culture media in conventional cell cultures used for the production of urokinase. Shear stress data for conventional cell cultures can be found, for example, in N. L. Cowger et al., Expression of Renal Cell Protein Markers is Dependent on Initial Mechanical Culture Conditions, 92 J. APPL. PHYSIOL. 691-700 (2002); T. G. Hammond and J. M Hammond, Optimized Suspension Culture--The Rotating Wall Vessel, 281 .mu.M. J. RENAL PHYSIOL. at F12 (2001); M. A. Gimbrone Jr., Vascular Endothelium, Hemodynamic Forces, and Atherogenesis, 155 AM. J. PATHOL 1-5 (1999); and P. Guo et al., A Hydrodynamic Mechanosensory Hypothesis for Brush Border Microvilli, 280 AM. J. PHYSIOL. at C962-9 (2001), each of which is incorporated herein by reference. In some embodiments of the disclosed method, the shear stress of the cell-culture media during substantially all of the static period is less than about 0.4 dyne/cm.sup.2, such as less than about 0.1 dynes/cm.sup.2 or less than about 0.05 dynes/cm.sup.2.

Although generally it is important to maintain the shear stress of the cell-culture media at a low level, periodic spikes in the shear stress will not necessarily inhibit the enhanced production of urokinase. These spikes may be caused, for example, by periodic handling of the cell culture (such as to feed the cells or to supplement the medium with additives), ambient vibration (such as air movement), convection, or other incidental mixing. Aside from incidental movement, mixing is avoided and, in particular, intentional agitation or mixing of the medium is not performed. Therefore, any incidental spikes of movement are very brief and transient. Generally the culture is maintained as a stationary system, without substantial mixing.

The static period can be any period during which the urokinase-producing cells in a cell culture exhibit enhanced urokinase production. For example, the static period can be a period greater than about 1 hour, such as a period greater than about 2 hours and less than about 72 hours or a period greater than about 10 hours and less than about 48 hours.

B. Culture at Normal Gravity

In the examples detailed below, several cell culture conditions were investigated for their effect on gene expression. Among these cell culture conditions was actual microgravity achieved in space and simulated microgravity achieved in a rotating wall vessel. Actual and simulated microgravity were not found to enhance the production of urokinase. Thus, in one example, the static period is characterized by normal gravity forces acting on the cell culture in addition to a lack of substantial mixing.

C. Culture with Microcarrier Structures

Some embodiments of the disclosed method involve the use of microcarrier structures in the cell cultures. Microcarrier structures can be any solid, physical structures used to modify cell cultures to promote cell growth, viability, and/or activity. For example, these structures can be used to increase the surface area available to cells in a cell culture and thereby increase the number of cells that can be cultured in a specified volume. In some embodiments, the presence of microcarrier structures makes the cell culture more closely resemble the native environment of the cells being cultured. For example, some cells prefer to adhere to surfaces. These cells often respond positively to the presence of microcarrier structures because microcarrier structures create a three-dimensional surface in the cell culture and thereby provide greater surface area within the cell culture. In some embodiments, microcarrier structures also promote the optimum spacing of the cells. Certain cells exhibit advantageous characteristics when they are situated a certain distance from other cells in the cell culture. Without being bound by theory, this distance can be manipulated by modifying the size of the microcarrier structures and/or by modifying the ratio of cells to microcarrier structures. The influence of cell spacing may be related to the diverse interactions between cells, including the chemical signals passed between cells. Spacing also can promote the distribution of oxygen and nutrients. Some or all of the above factors, among others, may be responsible for the positive effect of microcarrier structures on the production of urokinase, as observed in the experiments associated with this disclosure.

In general, microcarrier structures can be any structures introduced into a cell culture that facilitate the growth, viability, and/or activity of cells in that culture. Microcarrier structures can take any form, but typically are spherical or cylindrical. Other examples of microcarrier structures include conical, frustoconical, pyramidal, square, rectangular, and ovoid structures. Some microcarrier structures also have internal pores or surface indentations that further increase the surface-area-to-volume ratio.

Microcarrier structures of various sizes are useful for the enhanced production of urokinase. The individual microcarrier structures in a batch of microcarrier structures typically are not of uniform size. A batch of microcarrier structures is best characterized by the median size of the microcarrier structures within the batch. The actual sizes of the microcarrier structures within the batch typically vary within a normal distribution around the median size. This distribution can be, for example, a distribution in which at least about 90% of the microcarrier structures have a size (such as a diameter or maximum dimension) within about 50 .mu.m to about 300 .mu.m of the median size.

In some embodiments, the median microcarrier structure size is small enough to provide sufficiently enhanced surface area to promote cell activity and large enough to accommodate an optimum number of urokinase-producing cells per microcarrier structure, such as about 100 urokinase-producing cells per microcarrier structure. In several examples, batches of microcarrier structures have median diameters of between about 50 .mu.m and about 300 .mu.m, such as between about 100 .mu.m and about 250 .mu.m or between about 150 .mu.m and about 200 .mu.m. Some microcarrier structures often swell in different media such that their diameter is increased. A batch of swelling microcarrier structures can be selected based on the median size of the microcarrier structures in an environment resembling a typical tissue culture medium, such as a 0.9% NaCl solution.

The effect of microcarrier structures can be enhanced when a greater fraction of the cells attach themselves to the microcarrier structures. Several forces promote this attachment process, including electrostatic forces. Cells typically have a slightly negative surface charge. It therefore is possible to promote attachment by providing microcarrier structures with a slightly positive surface charge.

The microcarrier structures useful for embodiments of the disclosed method can comprise a variety of materials, including, but not limited to, collagen (for example, types I or V), dextran, silica, glass, gelatin, fibrin, fibronectin, laminin, urokinase receptor antibodies, poly-L-lysine, scaffolds (for example, GELFOAM.RTM.), and combinations thereof. Some microcarrier structures have a core comprising a first material and a surface comprising a second material. For example, some microcarrier structures are coated with a matrix, such as a matrix comprising collagen (for example, types I or V), and/or urokinase receptor antibodies. Since cells typically attach themselves to an extracellular matrix deposited by other cells, some microcarrier structures mimic the extracellular matrix to promote cell attachment and viability. In one embodiment, the presence of an extracellular matrix facilitates removal of attached cells from the microcarrier structures, which can be accomplished, for example, by introducing proteases.

An example of a microcarrier structure that is well-suited for use with embodiments of the disclosed method and is commercially available is CYTODEX-3, manufactured by Amersham Biosciences (Piscataway, N.J.). CYTODEX-3 microcarrier structures comprise a dextran core and a layer of denatured collagen coupled to the dextran core. CYTODEX-3 microcarrier structures have a density of 1.04 grams/ml, an approximate area of 2700 cm.sup.2/gram dry weight, and a swelling factor of 15 ml/gram dry weight. A gram of CYTODEX-3 microcarrier structures contains approximately 3.0.times.10.sup.6 individual microcarrier structures. The median diameter of the microcarrier structures in 0.9% NaCl is 175 .mu.m. About 90% of the microcarrier structures have a diameter in 0.9% NaCl between 141 .mu.m and 211 .mu.m.

As described in the examples below, the ratio of urokinase-producing cells to microcarrier structures in the cell culture can effect the production of urokinase. In some of the disclosed examples, enhanced production of urokinase occurred when the cell cultures contained more than 25 urokinase-producing cells per microcarrier structure. In one example, the ratio of urokinase-producing cells to microcarrier structures was between about 75:1 and about 200:1. In another example, the ratio of urokinase-producing cells to microcarrier structures was about 100:1.

D. Cells

A variety of urokinase-producing cells can be used with embodiments of the disclosed method. For example, human renal cells are capable of producing urokinase. Human renal cells can be isolated, for example, from kidneys unsuitable for transplantation. As noted, human cells can be used. Alternatively, non-human animal cells can be used, such as mouse, rat, rabbit, monkey, dog, sheep or goat cells. The cells can be primary cells, such as primary human renal cells. In some embodiments, the cells are immortalized. The cells can be renal cells, but are not limited to renal cells. In one example, non-renal cells are transfected with nucleic acid that encodes urokinase, such as human urokinase. In several embodiments, cells of a cell line can be utilized. These cells include commercially available cells, including both primate and human cells, or cells from other species. In one example, the cells are fibroblasts. Specific non-limiting examples of cells of use include NIH3T3 cells, CHO cells, PC-12 cells, BN cells, and combinations thereof. In the examples below, immortalized human renal cells and immortalized CHO cells transfected with a nucleic acid that encodes urokinase were used. However, the embodiments of the method disclosed herein are not limited to the use of these specific cell types.

E. Other Considerations

In addition to microcarrier structures, the disclosed cell cultures also typically utilize a tissue culture medium. This can be any medium for sustaining the urokinase-producing cells. Suitable components of the tissue culture medium include vitamins, minerals, amino acids, sugars, salts, urokinase receptor antibodies, inhibitors to prevent the breakdown of urokinase (for example, amiloride), and combinations thereof. Such media are well known in the art and are commercially available. One example of a medium well-suited for the growth of urokinase-producing cells is Dulbecco's Modified Eagle's Medium (DMEM), or a mixture of DMEM and F12 (Sigma, St. Louis, Mo.) (such as DMEM/F12 at 1:1). The medium can supplemented with serum, such as fetal calf serum (Life Technologies, Grand Island, N.Y.). Antibiotics also can be added to inhibit the growth of competing microorganisms. A suitable antibiotic cocktail includes ciprofloxacin and fungizone (Life Technologies, Grand Island, N.Y.). Other suitable antibiotics include amoxicillin, penicillin, streptomycin, and combinations thereof.

The components of the disclosed cell cultures can be contained in a variety of vessels. One well-suited vessel is a bag, such as a flexible bag or a flexible bag comprising a fluoropolymer resin, such as TEFLON.RTM.. In some embodiments, the vessel includes a material that is resistant to cell attachment. For example, some suitable bags have inner surfaces that resist cell attachment.

Some vessels have gas permeabilities that are advantageous for the production of urokinase. Urokinase-producing cells, like other cells, consume oxygen and release carbon dioxide. Urokinase-producing cells benefit from a continuous supply of oxygen and an elevated concentration of carbon dioxide relative to the surrounding environment, which typically is air. A suitable oxygen concentration in the cell culture is, for example, from about 80% to about 110% of the oxygen concentration of the surrounding environment, or about 20%. A suitable carbon dioxide concentration in the cell culture is, for example, greater than about ten times the carbon dioxide concentration of the surrounding environment, or about 5%.

Some vessels achieve an optimum oxygen concentration by allowing enough oxygen to diffuse into the vessel's interior to replace the oxygen consumed by the urokinase-producing cells. These vessels can be, for example, substantially O.sub.2-permeable. Similarly, some vessels achieve an optimum carbon dioxide concentration by retaining some or all of the carbon dioxide produced by the urokinase-producing cells. These vessels can be, for example, substantially CO.sub.2-retaining. Vessels that have a low permeability to carbon dioxide and a high permeability to oxygen are advantageous because they facilitate optimal culture conditions without the need for carbon dioxide sensors, oxygen sensors, carbon dioxide supplies, or oxygen supplies.

In several examples, optimum carbon dioxide and/or oxygen concentrations are maintained in the cell culture, such as during the static period, without the use of carbon dioxide and/or oxygen supplies, such as external tanks. In these examples, however, the cell culture can be situated in controlled environments with elevated or decreased carbon dioxide or oxygen concentrations relative to air. Alternatively, the cell culture can be situated in air. In one example, the cell culture is not directly supplied with carbon dioxide from a carbon dioxide supply and the carbon dioxide concentration in the cell culture is greater than about ten times the carbon dioxide concentration in the environment during the static period. In another example, the cell culture is not directly supplied with oxygen from an oxygen supply and the oxygen concentration in the cell culture is from about 80% to about 110% of the oxygen concentration in the environment during the static period.

A suitable vessel is the VUELIFE 30 ml TEFLON.RTM. bag #3P-0027 (American Fluoroseal Corp., Gaithersburg, Md.). However, additional vessels can be utilized with the disclosed method, including flasks and bottles. These vessels can be made, for example, from tissue culture plastic, such as a polypropylene or polystyrene, and are commercially available from a variety of sources.
 

Claim 1 of 22 Claims

1. A method for the production of human urokinase, comprising: culturing urokinase-producing cells in a cell culture comprising a plurality of microcarrier structures and a tissue culture medium for a culture period, where the urokinase-producing cells are cultured in static culture conditions without substantial mixing for a portion of the culture period sufficient to cause enhanced urokinase production; and isolating human urokinase from the cell culture.

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