MXPA99009265A - Production of functional proteins:balance of shear stress and gravity - Google Patents

Abstract

La presente invención provee método para producir proteínas funcionales que incluyen hormonas mediante células renales en un procedimiento de co-cultivo tridimensional sensible a tensión por esfuerzo cortante usando un recipiente de pared giratoria;la mezcla natural de células renales expresa la enzima 1-a-hidroxilasa que se puede tusar para generar la forma activa de la vitamina B:1,25-diOH de vitamina D3;los cultivos de fibroblastos y el co-cultivo de células corticales renales expresan el gen para eritropoyetina y secretan la misma en el sobrenadante de cultivo;otros genes de respuesta a tensión por esfuerzo cortante son también modulados mediante tensión por esfuerzo cortante, tales como los receptores de toxinas megalina y cubulina, a saber, gp280;se provee también un método para tratar a individuos con las proteínas funcionales producidas en un procedimiento de co-cultivo tridimensional sensible a tensión por esfuerzo cortante usando un recipiente de pared giratorio.

Description

PRODUCTION OF FUNCTIONAL PROTEINS: TENSION BALANCE BY CUTTING EFFORT AND GRAVITY ORIGIN OF THE INVENTION The jointly made invention described herein was made by an employee of the government of the United States, and may be elaborated and used by the same or by the government of the United States.

United of North America for governmental purposes without the payment of any royalty on it or for it. The invention described herein was also performed by inventors in the performance of their work under an agreement with the Tulane Educational Fund, and is subject to the provisions of section 305 of the National Aeronautics and Space Act of 1958, Public Law 85-568 (72 Stat 435, 42 U.S.C. 2457).

RELATED REQUESTS The present application claims the benefit of 35 U.S.C. §1 1 1 (b), provisional patent application 60/043205, filed on April 8, 1997.

BACKGROUND OF THE INVENTION Notification on the Federal Foundation The present invention was founded by NIH, Concession DK461 17, NIH R21 and NASA NRA, Concession 9-81 1. Accordingly, the government of the United States has certain rights to this invention.

FIELD OF THE INVENTION The present invention relates generally to the fields of protein chemistry, endocrinology and gene therapy. More specifically, the present invention relates to a method for producing functional proteins in culture in response to shear stress using a rotating wall vessel.

DESCRIPTION OF THE RELATED TECHNIQUE A successful and documented modality for inducing polarization and differentiation of cells in culture is the rotating wall vessel (1-4). In rotating wall containers, gravity is balanced by equal and opposite physical forces that include shear stresses. In terms of engineering, this has been claimed as simulated microgravity in boundary conditions [Wolf D. A. and R. P. Schwarz (1991) NASA Technical Paper 3143]. The rotating wall containers, including models with perfusion, are an advance in quantity. The rotating wall container is a cylindrical device for cell culture that is rotated horizontally with a tubular coaxial oxygenator (1, 5-7). The rotating wall vessel induces the expression of selected tissue-specific proteins in diverse cell cultures (1 -2, 8-9). Examples of the expression of tissue-specific proteins include the expression of carcinoembryonic antigen in MIP-101 colon carcinoma cells (2) and the induction of prostate-specific antigen in human prostate fibroblasts (7), through the induction of matrix material during the culture of chondrocytes (8). The resting environment of the cell culture of the rotating wall vessel, balances gravity with shear stress and other forces without obvious exchange of mass transfer (1-2, 4). The rotating wall container provides a suitable culture environment for co-cultures of diverse cell types, as well as formation of three-dimensional tissue structures. The technology of the rotating wall vessel is being recently used in clinical medical practice facilitating the implantation of pancreatic islets (4, 10). The pancreatic islets are prepared in rotating wall containers to maintain the production and regulation of insulin secretion. The islets are encapsulated alginate to create a noninflammatory immune refuge, and are implanted in the peritoneal cavity of type I diabetic patients. This pancreatic islet implantation has maintained normoglycemia for 18 months in diabetic patients and progressed to phase III clinical trials ( 4, 10). These containers have also been applied to, for example, skeletal muscle tissue, cartilage, salivary glands, ovarian tumor cells and crypt cells of the colon of mammals (1 1-13). Previous studies on the response to shear stress in endothelial cells, and culture in rotating wall containers, have been limited to structural genes (14-16). These studies did not highlight the subject of a procedure for the production of functional molecules, such as hormones. Elements of stress response by shear stress have not previously been demonstrated in epithelial cells, either for structural genes or the production of functional molecules. For centuries, vitamin D-dependent rickets has been a known disease in private farms and larger industries for animal conservation (17-18). The development of renal replacement therapy by dialysis in humans, expanded vitamin D deficient bone disease from an occasional warning in human clinic, to a common clinical problem. This led to the identification of the active form of vitamin D as 1, 25-diOH D, and to the development of a global market of billions of dollars per year, predominantly in patients with end-stage renal disease, to provide hormone clinically Replacement (18). The active form 1, 25-diOH of vitamin D, is used mainly to treat bone disease in dialysis patients, but has also been implicated as osteoporosis therapy and some forms of cancer. Recently, it has been recognized that the effects of vitamin D play a central role not only in other common bone lesions such as osteoporosis due to aging and osteoporosis induced by steroids, but also in the functioning and surveillance of the immune system, growth and development and functioning of the heart and skeletal muscle (19-22). Several active forms of vitamin D have been identified, vitamin D receptors have been cloned and nuclear binding proteins for the hormone have been identified and cloned (17-22). Studies on the regulation of 1-hydroxylase activity are limited by the lack of a renal cell line with regulated expression of the enzyme. The only reports on the activity of 1-a-hydroxylase in culture use freshly isolated chicken kidney cortical cells in which the activity decreases precipitously within 48 hours of deposition in culture plate (28). The importance of renal 1-hydroxylase is best understood by comparing the kinetics of the renal enzyme with other forms in the body (29-30).

The demonstration that nephrectomy in pregnant rats did not completely suppress the formation of 1, 25-diOH-D3, unleashed an intensive search for extrarenal sites of 1-α-hydroxylase activity (29). Although 1-α-hydroxylase activity has been reported in monocytes, liver, aortic endothelium and a variety of placental and fetal tissues, enzyme kinetics contrasts markedly with renal 1-α-hydroxylase. The extrarenal 1-a-hydroxylase has a much higher Km, indicating that much higher substrate levels are required for its activity (29). In the uraemic patient, the production of extrarenal 1, 25-diOH-D3 is very limited due to the relative lack of substrate. The administration of large amounts of 25-OH D substrate to aneroid patients, moderately increases the levels of 1,25-diOH-D3 in plasma (29). The lack of a differentiated polarized line of renal tubular epithelial cells for research purposes persists despite extensive searches by several laboratories (31 -38). Renally derived cell lines transformed with virus or tumor cells to produce immortality continue to be some of the most popular cellular biological tools for studying polarized delivery (31, 33, 35). However, these immortal renally derived cell lines such as MDCK or LLP-CK1 retain few, if any, of the characteristic features of renal epithelial cells. In the same way, the primary cultures were rapidly dedifferentiated, and modalities as diverse as base membrane matrices, growth complements or Millipore insertions achieve only moderate degrees of polarity (37-38). The pathognomonic structural characteristics of proximal renal tubular epithelial cells are the abundance of apically derived microvilli, the glycoprotein content of associated clefts between the microvilli and the highly distinctive arrangement of subapical endosomal elements (39, 40). The renal epithelial cells of the proximal tubule are characterized by thousands of long apical microvilli. The apical endosomal machinery begins in the clefts between the microvilli. The endosomal pathway is characterized by clathrin-coated vesicles and small spherical endosomal vesicles with larger and deeper endosomal vacuoles (33, 39). From the endosomal vacuoles, the proteins and lipids recirculate towards the apical surface in dense apical tubules, or they come and go towards the lysosomes to be degraded. A group of apical proteins with homologous sequence repeats is highly convenient for expression in cultured cells, since they are thought to be molecular mediators of renal injury (41-43). Two of these proteins, megalin (gp330) and cubulin (gp280) (Moestrup et al., J. Biol. Chem. B273 (9): 5325-5242 (1998)), are molecular mediators of tubular vacuolation and result from secondary damage. megalin (gp330) is a receptor present on the luminal surface of proximal tubular cells of the kidney.Megalin binds to several proteins and drugs that include aminoglycoside antibiotics and other polybasic drugs.Megalin is expressed in the kidney, lung, testicles The only cells that express the megalin in culture are the immortalized placental cells.There is no known renal cell culture that expresses the megalin.Gp280 is a receptor present on the luminal surface of the proximal tubular cells of the kidney. binds to several proteins and drugs that include cobalamin-intrinsic factor (vitamin B12 bound to its carrier protein) and light chains of myeloma. Cubulin (gp280) is expressed in the kidney, ear and placenta. The only cells that express cubulin (gp280) in culture are the immortalized placental cells. There is no known renal cell culture that expresses cubulin (gp280). Erythropoietin (EPO) is a hormone produced in the kidney and secreted in the blood. Erythropoietin controls the rate of production of erythrocytes by the bone marrow. Erythropoietin can be produced by interstitial cells between tubules or proximal tubular cells, or both. The production of erythropoitein is lost in all known renal cell culture systems. Erythropoitein is used primarily to treat anemia in dialysis patients, but it is also used to treat anemia in patients with AIDS and many other forms of cancer. The prior art lacks effective means to produce functional proteins that include hormones in response to shear stress. In addition, the prior art lacks the identification of shear stress stress elements in epithelial cell genes. The present invention satisfies this long-lasting desire and need in the art.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment of the present invention, there is provided a method for producing a functional protein, which comprises the steps of: isolating mammalian cells; placing said cells in a rotating wall vessel containing a cell culture comprising culture media and culture matrix; produce three-dimensional aggregates of cells under simulated microgravity conditions; and detecting the expression of the functional protein in the cell culture. In another embodiment of the present invention, there is provided a method for inducing the expression of at least one gene in a cell, which comprises the steps of: contacting said cell with an oligonucleotide sequence decoding transcription factor directed against a nucleotide sequence encoding a stress response element by shear stress; and determining the expression of said gene in said cell. In yet another embodiment of the present invention, a transcription factor decoy is provided, which comprises an oligonucleotide sequence directed against a nucleotide sequence encoding a shear stress response element. These and other aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments thereof given for purposes of description.

BRIEF DESCRIPTION OF THE DRAWINGS In order that the subject in which the characteristics, advantages and objectives of the invention described above, as well as others that will be clear, are attainable and can be understood in detail, one can have more particular descriptions of the invention summarized above in brief form in relation to certain modalities of the same, which are illustrated in the attached drawings. These drawings are part of the specification. However, it will be noted that the appended drawings illustrate preferred embodiments of the invention, and therefore should not be considered as limiting in their scope. Figure 1 shows the homogeneity and structure of human renal epithelial cells in culture. Flow cytometry frequency histograms demonstrate the number of cells positive for the proximal tubular marker? -glutamyl transferase. Figure 1A shows the number of cells with? -glutamyl transferase activity as the frequency of activity in 2000 cells, compared to a control not only stained with capture agent. This is the basic review of human kidney cells. Figure 1B shows that after differential trypsinization, the percentage of proximal tubular cells present can be increased up to 99 + 1%. Figures 1 C and 1 D show electron micrographs of transmission of human epithelial cells in culture. The intact renal cortex is compared in vivo (far left panel) with the culture of the natural mixture of human renal cortical cells in conventional two-dimensional culture (left panel, center), which completely lacks microvilli. The culture of pure proximal tubular cells in the rotating wall vessel shows some microvilli (right panel, center), but there are many more microvilli during the culture of the natural mixture of renal cortical cells in the rotating wall vessel (far right panel) . Comparatively with these representative images, some areas of the natural mixture of cells in the rotating wall vessel show much greater abundance of microvilli, as well as well-defined desmosomes (lower panel), which are lacking in the other cultures. Figure 2 shows the expression of proteins in the rotating wall vessel. Figure 2A shows the analysis of the expression and endosomal compartmentalization of megalin and cubulin in kidney cells after culture in the rotating wall vessel. The ability of flow cytometry to make simultaneous measurements of fluorescein-dextran trapped as an endosomal marker, and the antiquase junction, allows the construction of three-dimensional frequency histograms showing the fluorescence of fluorescein-dextran trapped against the antibody binding on horizontal axes. A control sample shows negative vesicles for fluorescein to the left, and endosomes containing fluorescein to the right (2000 vesicles shown, left panel). A control without trapped fluorescein shows only the population on the left (not shown). Colocalization of the anticubulin binding demonstrates that all fluorescein-positive endosomes are positive for cubulin, while non-endosomal membranes can be subdivided into positive and negative cubulin populations (central panel). This pattern is repeated for antimegaline binding in renal cortical cells (right panel). Figure 2B shows the quantification of the antibody binding of cubulin and megalin to the kidney cell membrane under different culture conditions. The analysis of protein expression in cells cultured by antibody binding, used classical logarithmic curves in series for antibody dilution. An increase in binding with a decrease in dilution is pathognomonic for the binding of specific antibody during flow cytometric analysis. The binding of anti-tubulin antisera to membrane vesicles prepared from renal cortical cells after 16 days in culture, detected by the fluorescence of a secondary antibody labeled with phycoerythrin, shows a nearly double logarithmic increase in the binding with antibody dilution (panel upper left, lower). This increased binding of cubulin antibody in cells grown in the rotating wall vessel (STLV) is more than 5 times the expression observed in stirred fermentors. Also, there was no detectable expression in conventional cultures, resulting in a constant line (not shown). The binding of normal serum and the minimum dilution of primary antisera was not detectably different. The binding curves for antimegaiin antiserum showed a similar pattern (not shown). Figure 2C shows the non-specific (minimum) and maximum binding of each antiserum after culture in the rotating wall vessel, as well as the two-dimensional SDS-PAGE analysis of the protein content of the cells, following the culture in the wall vessel rotary. The analysis of the protein content of the cultures of the natural mixture of rat renal cortical cells after culture for 16 days in gas permeable bags as control (left panel), or of the rotating wall container (right panel), shows changes in a select series of proteins. The molecular weight (14-220 kDa) is shown on the abscissa against the isoelectric point (pH 3-10) on the ordinate. Figure 3 shows the expression of genes in the rotating wall vessel. Figures 3A and 3B show the differential display of the gene expression of rat renal cortical cells grown in conventional culture or rotating wall vessels. The differential display of genes expressed in aliquots of the same cells cultured in a 55 ml rotating wall vessel (STLV) or conventional two-dimensional gas-permeable bag controls was compared. For differential display, copies of the expressed genes were obtained by polymerase chain reaction using random primers of 25 elements and separated on a 6% gel for determination of DNA sequence (figure 3a). Bands of different intensity between the control and STLV, representing differently expressed genes, were identified by visual inspection, cut and amplified again using the same primers. The differential expression and the size of the transcript were confirmed by Northern hybridization (Figure 3B). The PCR products were then subcloned into the vector pGEM-T, and its sequence was determined.

The sequences were compared to the gene bank sequences using the BLAST search engine. An expressed gene which decreased in the STLV (band D in the previous gel), was identified as rat superoxide dismutase containing manganese (98% coincidence, 142 of 144 nucleotides). Two genes were increased in the STLV. Band A was identified as the ß gene for interleukin-1 (100% match for 32 of 32 nucleotides), and band B, which corresponded to a 20 kB transcript in a Northern blot, appears to be an unidentified gene that has 76% homology with the GABA transporter gene in mice. Figures 3C and 3D show the RT-PCR of the change in genes dependent on time during culture in the rotating wall vessel. Semi-quantitative RT-PCR shows increases in the epithelial genes for megalin, vilin and extracellular calcium detector receptor (ECaR), stress response element genes for ICAM, VCAM and MnSOD (Figure 3C). There was no change in actin b or GADPH. Unlike what happens in endothelial cells, many of these changes are prolonged, since at 16 days, changes in megalin, ECaR, ICAM, VCAM and vilina persist (Figure 3D). Figure 4 shows the structure and effects of the antisense probe for the shear stress response element in rat renal cortical epithelial cells. Figure 4A shows the structure. The probe with the sequence CTGAGACCGATATCGGTCTCAG (SEQ ID NO: 1) has two possible conformations. As an individual chain, it would fold back on itself to form a binding element for the transcription factor. As a double strand, it would have two binding sites for the transcription factor, one in sense orientation and one in antisense orientation. Figure 4B shows the effects of the probe of the response element to stress by antisense shear stress on the time-dependent gene expression. The antisense probe added to conventional bidimensional cultures of rat renal cortical cells at 80 nm increases MnSOD in a time-dependent manner. The comparison is made with controls with the active binding site crammed. In contrast, the probe has no effect on the expression of the gene for villin. Figure 5 shows gene expression in the rotating wall vessel: automated gene analysis. The abundance of expression of more than 18,300 genes was tested by binding poly A RNA from human renal cortical epithelial cells developed in a rotating wall vessel for 8 days, with a filter robotically loaded with oligonucleotide primers. The poly A RNA from a non-sticky bag culture serves as a control. The filters are shown in the upper part of the diagram, as well as the analysis of stress response genes by shear stress, specific genes of the renal epithelium and other genes applicable to the usual analysis.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method for producing a functional protein, which comprises the steps of: isolating mammalian cells; placing said cells in a rotating wall vessel containing a cell culture comprising culture media and culture matrix; produce aggregates of three-dimensional cells under simulated microgravity conditions; and detecting the expression of the functional protein in the cell culture. In general, simulated microgravity conditions comprise a balance between gravity and oppositely directed physical forces. Representative examples of said physical forces include stress by sedimentation shear stress, centrifugal forces, viscosity and Coriolis forces. Preferably, the functional protein is selected from the group consisting of a hormone, a toxin receptor and a functional biomolecule dependent on shear stress. Representative examples of hormones that can be produced according to the method of the present invention include 1,25-dihydroxy-vitamin D3 and erythropoietin.

Representative examples of toxin receptors that can be produced in accordance with the method of the present invention include megalin and cubulin. Representative examples of the shear stress dependent functional biomolecule that can be produced according to the method of the present invention include that selected from the group consisting of villin, magnesium-dependent superoxide dismutase, nitric oxide synthetase, c-fos, c -jun, platelet-derived growth factor b, transforming growth factor b, tissue-type plasminogen activator and monocyte chemotactic protein 1, megalin, cubulin, erythropoietin and 1-a-hydroxylase. In general, any mammalian cell could be used in the methods of the present invention. Representative examples of mammalian cells include renal cortical cells, renal fibroblasts, hepatocytes, pancreatic islets, renal interstitial cells, parathyroid cells, thyroid cells, pituitary cells, ovarian cells and testicular cells. Generally, the cell is selected from the group consisting of epithelial cell and endothelial cell. Preferably, the cell contains stress response elements by shear stress. Representative examples of the shear stress response element include GAGACC and GGTCTC. In the methods of the present invention, the rotating wall vessel is started and maintained from about 6 rotations per minute to about 16 rotations per minute. Preferably, the tension by sedimentation shear is about 0.2 dynes per cm2 to about 1.0 dynes per cm2. The culture matrix may contain a central structure selected from the group consisting of aggregates of cells and microcarrier spheres, although other components such as a culture matrix are well known to those skilled in the art. The present invention is also directed to a method for inducing the expression of at least one gene in a cell, which comprises the steps of: contacting said cell with a sequence of oligonucleotide decoys of transcription factor directed against a sequence of nucleotides encoding a stress response element by shear stress; and determining the expression of said gene in said cell. Generally, the oligonucleotide comprises a terminal phosphothiorate moiety and a phosphodiester base structure and a structure that allows the oligonucleotide to traverse cell membranes and accumulate in the cell's nuclear compartment. In general, the cell is a cultured cell. Preferably, the cell is selected from the group consisting of an epithelial cell and an endothelial cell. Representative examples of cells that can be used in this method include renal cortical cell, renal fibroblast, hepatocyte, pancreatic islet, renal interstitial cell, parathyroid cell, thyroid cell, pituitary cell, ovarian cell and testicular cell. In one embodiment, the cell develops in a two-dimensional culture. Representative examples of stress response elements by shear stress include GAGACC and GGTCTC. Preferably, the gene codes for a protein selected from the group consisting of megalin, cubulin, erythropoietin and 1-a-hydroxylase. The concentration of the oligonucleotide useful in this method generally ranges from about 10 nm to about 10 mm. The present invention is also directed to a decoy of a transcription factor, which comprises an oligonucleotide sequence directed against a nucleotide sequence encoding a shear stress response element. Preferably, the nucleotide sequence encoding a shear stress response element has a sequence selected from the group consisting of GAGACC and GGTCTC. In a preferred technique, the rotating wall vessel is generally started and maintained at 10 rotations per minute. Preferably, the rotating wall container provides a balance of forces comprising gravity and tension by equal and opposite sedimentation shear stress. The stress velocities for sedimentation shear that are useful within the context of the claimed methods are from about 0.2 dynes per cm2 to about 1.0 dynes per cm2. As used herein, rotating wall containers refer to a horizontal cylindrical rotary culture vessel with a coaxial oxygenator.

As used herein, the shear stress response element refers to a sequence of a gene family in the cell nucleus that binds to one or more transcription factors in response to stress stress cutting on the cell. A representative example of a shear stress response element is GAGACC or its complementary sequence GGTCTC. As used herein, shear stress conditions refer to liquid flow or liquid flow over cells, which causes the genes to be activated or deactivated. As used herein, the slow rotating side container refers to the specific size and shape of a rotating wall container. As used herein, "differential display" refers to the deployment on a filter, gel or chip, of a defined series of genes activated or deactivated in a cell under two different conditions. As used herein, simulated microgravity refers to the balance of gravity by opposingly directed forces, including shear stresses during cultivation in the rotating wall vessel. As used herein, graduated gravitational sedimentation shear refers to the shear stress imparted to a particle or cell that falls through the fluid.

As used herein, the functional protein refers to a protein with biological effects. As used herein, three-dimensional coculture procedures refer to cells developed in a matrix or on spheres (or other three-dimensional structural support) in a three-dimensional arrangement, rather than on a flat plate. As used herein, the Coriolis force refers to an incidental flow field caused by the rotary gravity vector in the rotating wall vessel. As used herein, shear stress refers to the force felt at the surface of the particle as it moves through the fluid. As used herein, gravity-induced sedimentation refers to the force on a particle in the rotating wall vessel that causes it to fall through the fluid due to gravity. As used herein, the centrifugal force refers to the force on a particle in the rotating wall vessel that pulls it towards the wall due to the rotating speed. As used herein, the decoy of transcription factor refers to a folded oligonucleotide to form a double-stranded DNA that binds to a nuclear transcription factor. The decoy of the transcription factor prevents the transcription factor from binding to the promoter regions that regulate the expression of specific genes.

The following examples are given for the purpose of illustrating various embodiments of the invention, and are in no way intended to limit the same.

EXAMPLE 1 Human renal cortical cells Human renal cortical cells were isolated by Clonetics Inc. (San Diego, CA) of kidneys unsuitable for transplantation. Differential trypsinization resulted in highly purified cell fractions of proximal tubular cells, compared to the natural mixture of cells in the renal cortex. The co-culture of the natural mixture of cells, and highly purified proximal tubular cells, were cultured separately in a special growth medium with fetal calf serum at 2%.

EXAMPLE 2 Rat renal cortical cells Renal cortical rat kidney cells harvested from Sprague Dawley rats subjected to euthanasia (Harlan Sprague-Dawley, Cleveland OH) were isolated, as described (44). In summary, the renal cortex was dissected with scissors, finely shredded in a pH regulator for renal cells, 137 mMol of NaCl, 5.4 mmole of KCl, 2.8 mmole of CaCl2, 1.2 mmoles MgCI2 and 10 mmoles of HEPES-Tris, pH 7.4. The shredded tissue was placed in 10 ml of a collagenase solution of type IV to 0. 1% and trypsin 0.1% in normal saline. The solution was incubated in a water bath with shaking at 37 ° C for 45 minutes with intermittent titration. The cells were gently rotated (800 rpm for 5 minutes), the supernatant was aspirated, the cells were resuspended in 5 ml of pH buffer for renal cells with 0.1% bovine serum, and passed through the cells. a fine mesh of 70mm. The fraction that passed through the mesh was stratified on a discontinuous gradient of 5% bovine serum albumin, and gently rotated. The supernatant was discarded again. Cells were resuspended in DMEM / F-12 medium (treated with ciprofloxacin and fungizone), and placed in culture in several culture vessels in an incubator with 5% CO 2 and 95% O 2.

EXAMPLE 3 Cultivation Techniques: Rotating Wall Containers When cultured under con ventional conditions in DMEM / F12 supplemented with fetal calf serum and a cocktail of antibiotics such as ciprofloxacin and fungizone, the highly purified cells and the cell mixture form a monolayer. Fetal calf serum was used at optimal concentration: 2% for human cells and 10% for rat cells. To increase the differentiation of epithelial cells (1, 45), kidney cells were cultured in rotating wall containers known as 55 ml slow rotation side vessels (STLV) (1, Four. Five). To initiate cell culture, the slow-rotating side vessel was filled with medium and seeded by addition of cell suspension (2X106 cells / ml). The residual air was removed through the orifice of a syringe, and the rotation of the container started at 10 rotations per minute and was maintained for 10 to 16 days. The medium was changed every 2 to 3 days, depending on the use of glucose. Concomitant with the cells, microvehicle spheres and 5 mg / ml were added to promote the formation of aggregates in the slow-rotating lateral vessel. Without spheres, the cells were destroyed in the container in a few hours. Throughout the protocol, the spheres were Cytodex-3 except when electron microscopy was planned when much more costly but more easily sectioned GL cells were added to the containers.

EXAMPLE 4 Controls with shaking and static controls To provide agitated control, agitated fermenters were charged which mixed in the horizontal plane, with identical concentrations of cells and spheres of the same group added to the slow rotating side vessel (1, 31, 46). As conventional static controls, gas-permeable Fluoroseal bags were selected (Fluoroseal Inc, Urbana IL) of size from 7 to 55 mi. The culture spheres were added to the conventional controls at the same density as the cultures in lateral containers of slow rotation (1, 45).

EXAMPLE 5 Quantification of the number of microvillosities by electron microscopy Transmission electron micrographs of the cell aggregates of rotating wall vessels and conventional monolayers were obtained. The cells were washed with pH regulated saline solution cooled with ice-cold phosphate, and were then fixed for electron microscopy with 2.5% glutaraldehyde in pH-regulated saline with phosphate (9, 47). The samples were then transferred to 1% osmium tetroxide in 0.05 M sodium phosphate (pH 7.2) for several hours, and dehydrated in acetone series followed by inclusion in Epon. Thin sections stained with lead were examined and photographed using an EM / 200 Phillips electron microscope. For electron microscopy, the easily sectioned GL Cultisphere spheres replaced Cytodex-3, which is almost impossible to section.

EXAMPLE 6 Analysis of the epithelial marker of proximal tubules, g-glutamyl transpeptidase The renal cortical cells were proximal tubules of 75 + 4% (n = 4), as determined by aliquot flow cytometric analysis for the proximal marker g-glutamyl transferase using Schiff's base, capturing the Lg-cut products. giu-4-methoxy-4-b-naphthylamine (44) (figure 1).

EXAMPLE 7 Analysis of the endosomal distribution of megalin and cubulin by flow cytometry To quantify the endosomal and total cell expression of cubulin, megalin and aquaporin-2 in conventional culture, stirred fermentors and rotating wall vessels and slow-rotating side vessels, 0.3 mg / ml dextran-fluorescein 10S was added to each culture. of cells for 10 minutes at 37 ° C in the incubator with CO2. This charges a fluorescent dye trapped in the early endosomal pathway (9, 47). The cells were then immediately diluted in pH regulated saline with ice-cold phosphate, and washed once. Afterwards, the cells were homogenized with 6 steps of a Teflon-glass motor-driven homogenizer with hermetic adjustment. A post-nuclear supernatant was formed as the supernatant of 11,000 g, pellets of 180,000 g of membrane vesicles (Figure 2A). Aliquots of membrane vesicle were labeled with megalin or cubulin antiserum. The megalin and cubulin antisera were rabbit polyclonal produced for affinity purified and chromatographically pure receptor (43, 48). The membrane vesicles were previously incubated in normal goat serum at 50% for 2 hours to reduce the non-specific binding of secondary antisera produced in goats. After washing, the aliquots of the membrane vesicles were stained with serial logarithmic dilution of antisera, and incubated at 4 ° C overnight. After the additional washing, 1: 40 of secondary goat anti-rabbit antiserum conjugated with affinity purified rat pre-absorbed phycoerythrin was added and incubated for 4 hours at room temperature. Before flow cytometry, the membrane vesicles were washed and resuspended in 200 mM mannitol, 100 mM KCl, 10 mM HEPES, pH 8.0 with Tris, to which nigericin had been added. mM. In the presence of potassium, nigecirine collapses the pH ingredients, ensuring the optimum fluorescence of the emission of fluorescein-dextran highly dependent on pH. Fluorescein-dextran and antibody stained with phycoerythrin were then analyzed and colocalized on a vesicle base per vesicle by flow cytometry (Figure 2B).

EXAMPLE 8 Differential deployment The differential display of the expressed genes was compared in aliquots of the same cells cultured in 55 ml rotating wall containers (slow rotating side vessels) or conventional two-dimensional gas permeable bag controls (Figures 3A and 3B). The differential deployment was carried out using the Delta RNA fingerprint system (Clontech labs, Palo Alto CA). Copies of the expressed genes were obtained by polymerase chain reaction using random primers of 25 elements, and separated on a 6% gel for determination of DNA sequence. Bands of different intensity between the control and the lateral vessels of slow rotation, representing differentially expressed genes, were identified by visual inspection, separated and re-amplified using the same primers. The differential expression and the size of the transcript were confirmed by Northern hybridization. The PCR products were then subcloned into the vector pGEM-T (Promega, Madison Wl), and their sequence was determined using the cyclic sequence determination system fMOL (Promega, Madison Wl). The sequences were compared to the gene bank sequences using the BLAST search machine (National Center for Biotechnology Information). For the genes of interest, the bands were labeled with 32 P for confirmation of the changes by Northern blot analysis.

EXAMPLE 9 Detection of gene expression in cell cultures by RT-PCR The aggregates of cells from the rotary wall vessel culture were washed once in pH-regulated saline with ice-cooled phosphate., and they were frozen instantly at -70 ° C, until the RNA was isolated. The total RNA was isolated first, followed by the isolation of the poly A + RNA. After reverse transcription, 10 to 20% of each cDNA (Robocycler 40, Stratagene, La Jolla, CA) was amplified using denaturation temperatures at 95 ° C, pairing at 63 ° C and extension at 72 ° C. The amplification was carried out for a total of 30 cycles, the first three cycles having extended denaturing and coupling times. Positive and negative chain PCR primers were respectively derived from published sequences using the Generunner program. 20% of the PCR reaction was subjected to electrophoresis on agarose / ethidium bromide gels and visualized under UV light, so that a comparison of the amplified gene fragments with DNA standards could be made (DNA X174 digested with Haelll , Promega) and subjected to electrophoresis in the same gel (figures 3C and 3D). Representative amplified fragments were isolated from the gels for each gene in question, and their sequence was directly determined to ensure the identity of the PCR product. In addition, 5% of the same cDNA was subjected to PCR for the expression of the domestic messenger RNA, glyceraldehyde 3-phosphate dehydrogenase and actin b to make sure that similar amounts of protein were being compared.

Incoming RNA and similar efficiencies of reverse transcription. Each cDNA was run in at least three dilutions to ensure measurements were made on the initial linear portion of the response curve.

EXAMPLE 10 Genetic lures Double-stranded genetic decoys were synthesized by matching the sequence of a known stress response element by shear stress (Chemicon International Inc., La Jolla, CA) (its structure and sequence are shown in the upper part of Figure 4). These lures had a terminal portion of phosphothiorate to prevent intracellular lysis, and a phosphodiester base structure to facilitate its passage through cell membranes (49). The passage to, and the accumulation in, the nuclear compartment of the cultured cells, was confirmed by the confocal imaging of a fluorescein-labeled decoy. Three decoys were synthesized: the active decoy, a random sequence control in which the six bases of the stress response element were hauled by shear stress, and a decoy form conjugated with fluorescein. The decoys were placed in the culture medium of rat renal cortical cells developed as mentioned above in the two-dimensional culture. Aliquots of cells exposed to the control or decoy of active sequence were harvested at a concentration of 80 nm at 2, 6 and 24 hours after exposure.

EXAMPLE 11 Disposition of the genetic finding A sample of human renal cortical cells grown in conventional flask culture was trypsinized and separated into a gas-permeable bag control and a rotating wall vessel (slow rotation side vessel of 55 ml). After 8 days of cultivation in 5 mg / ml Cytodex-3 spheres, the cells were washed once with pH-regulated phosphate-cooled ice solution, then used, and messenger RNA was selected with biotinylated oligo (dT) , and then separated with paramagnetic streptavidin particles (system 1000 of PoIyATtract, Promega Madison, Wl). 32P-labeled cDNA probes were then obtained by reverse transcription with 32P dCTP. The cDNA probes were hybridized with identical Gene Discovery Array filters (Genome Systems Inc. St. Louis, MO). The Gene Discovery Array filters contain 18,394 unique human genes from the cDNA libraries of the consortium I.M.A.G.E. [LLNL] (15), which are arranged with the help of a robot on each of a pair of filter membranes. Then, gene expression was detected by phosphorus imaging, and analyzed using the Gene Discovery [Genome Systems] program (50).

EXAMPLE 12 A1-Hydroxylase Activity Test Because the 1-a-hydroxylase enzyme has never been isolated or cloned, it is functionally tested by the production of 1,25-dihydroxy-vitamin D3 from ultra-pure exogenous 25-hydroxy vitamin D3. For each measurement, the classical Michaelis Menten kinetics of the enzyme is determined by testing equal aliquots of renal cell aggregates on a 25-OH D3 substrate curve at concentrations of 0.1 to 10 mg / ml in 6 steps. All incubations are carried out in the presence of the DPED antioxidant at 10 mM to ensure that there is no contribution of non-enzymatic oxygenation (23-26). The 1, 25-diOH D3 generated in vitro was quantified as described (23-27). The in vitro incubations were terminated by adding a volume of acetonitrile equal to the incubation volume. Each incubation tube received 1, 000 cpm of 3H-1.25 dihydroxy D3 to estimate recovery losses during the extensive extraction and purification scheme. The 1.25 dihydroxy D3 is extracted from the incubation medium by extraction in C18 solid phase (24-25). After extraction, the samples are evaporated to dryness under N2 and dissolved in 2 ml of methylene chloride. Then, the samples are applied to Bond-Elut silica cartridges and the fraction containing 1, 25-dihydroxy D3 is isolated and collected (26). Individual fractions are contained at 1, 25-diOH D3 and then subjected to normal phase HPLC in a Beckman model 344 liquid chromatography system.

Normal phase HPLC was performed with a Zorbax-Sil (26) column (4 X 25 cm) developed in, and eluted with, methylene chloride / isopropanol (96: 4 v / v) with a flow rate of 2. ml / min. The 1, 25-dihydroxy D3 eluted from this system was dried under N2 and resuspended in ethanol and quantified by radioreceptor or radioimmunoassay (25-26). The 1 -25-dihydroxy vitamin D3 in plasma was tested in a similar manner, but since the product has been formed, the assay begins with extractions in acetonitrile (23-26). Therefore, all measurements of the 1-a-hydroxylase activity in the cells included the determination of the Km and Vmax of Michaelis Menten of the enzyme. The parameters of Michaelis Menten were determined by automated curve fitting.

EXAMPLE 13 Culture of renal fibroblasts and test for the production of erythropoietin Since renal fibroblasts are the source of erythropoietin secreted in the circulation, these cells were cultured. Freshly dissected rat kidney cortex was minced and digested with collagenase / trypsin before debris removal in a discontinuous individual gradient of 5% albumin. The mixture of rat renal cortical cells was placed in a culture in DMEM / F12 with 20% fetal bovine serum. After two weeks, the fibroblast growth factor was added to promote the overgrowth of fibroblasts in the enriched media. The resulting culture had fibroblast characteristics in the culture flask, and was inoculated into a high aspect rotating vessel (HARV) for culture under increased shear stress conditions. The cells aggregate on the spheres and slowly increase their number. After developing the fibroblasts for 3 weeks in a HARV, the erythropoietin was evaluated in the cell supernatant. The media was concentrated 15X and tested by RIA. The media alone also concentrated 15X as control.

EXAMPLE 14 Hepatocyte culture and test for the production of erythropoietin In view of the fact that the hepatocytes are a source of erythropoietin that is secreted in the circulation, immortalized human hepatocytes were cultured under control, and shear stress conditions were applied. Hep3B cells were placed in culture in DMEM with 10% fetal bovine serum in static flask cultures. The resulting culture was divided, one half remained in static flask culture, and the other half was inoculated into a HARV for cultivation under increased stress conditions by shear stress. The cells were added on the spheres. After 24 hours of developing the Hep3B cells in a HARV, the erythropoietin was evaluated in the cell supernatant. The means were analyzed by RIA. The media in the static flask was also analyzed as a control.

EXAMPLE 15 Shear stress response elements mediate changes in erythropoietin gene expression The Hep3B immortal liver cell line constitutively produces erythropoietin. The promoter and 3 'enhancer regions of the gene contain putative elements of response to shear stress conditions. The function of these elements in the increase of erythropoietin production in response to shear stress was tested, using cultures in perfused, rotating wall containers to reintroduce tension by graded shear stress. This protocol uses a library of promoters that control the luciferase reporter genes, with various constructions lacking the putative stress response response elements. It also allows the DNA fingerprint analysis of the histones that bind to the promoter and enhancer elements.

EXAMPLE 16 Results Proportion of proximal tubular cells in human renal cell fractions isolated by differential triptinization was tested, using a fluorogenic substrate trapped for the marker of the proximal enzyme g-glutamyl transferase (44.) Flow cytometry analysis on a cell-by-cell basis showed that the natural cellular mixture in the human renal cortex is 85 + 4%, n = 4 proximal tubular cells (Figure 1A, left panel). After differential trypsinization and selection of pure fractions, proximal tubular enrichments as high as 99 + 1% (right panel) could be achieved. As reported in other systems, rotating wall vessels led to vigorous cell growth, as evidenced by the high glucose consumption rates evaluated as 30 mg / dL of glucose / 100,000 cells / day. A cellular doubling time of 4 + 3 days was evaluated using Alamar blue in the rotating wall vessel, comparatively with 4 + 2 days in conventional culture (n = 4). The ultrastructures of cultures of pure proximal tubular cells or mixtures of renal cortical cells of human kidneys were developed in rotating wall vessels for 16 days, and examined by transmission electron microscopy (Figures 1 B and 1C). The quantification of the number of microvilli present by random plate count at the same magnification shows not only that the rotating wall vessel induces the formation of microvilli, but that co-culture with the normal mixture of renal cortical cells increases the effect (Table 1 ). The mixture of normal cortical cells in conventional two-dimensional culture has 21 microvilli per field; the "pure" proximal tubular culture in the rotating wall vessel has 104 microvilli per field; and the mixture of normal cortical cells in the rotating wall vessel has 3511 microvilli per field.

TABLE 1 Microvilli of human proximal tubular cells counted in electron microphlets of cell transmission developed for 16 days under various culture conditions There are advantages to using human cells instead of rat cells to examine the expression of megalin and cubulin in cultured kidney cells. Specifically, the megalin and rat cubulin sequences have been cloned, while the sequences in humans have not yet been determined, and the antisera recognize the rat but not human soformas of these proteins. Therefore, the natural mixture of cells in the rat renal cortex was placed in culture in rotating wall vessels, in stirred fermentors and in traditional culture to analyze the expression of proteins. Since it has been implicated that the endosomal pathway plays a central role in the functioning and pathophysiology of cubulin and megalin, trapped endosomal labels were co-localized with receptor antibody binding. The ability of flow cytometry to perform simultaneous measurements of trapped dextran-fluorescein as an endosomal marker and antibody binding allows the construction of three-dimensional frequency histograms showing the fluorescence of fluorescein-dextran trapped against the antibody binding on the horizontal axes and a number of vesicles in each channel to the outside of the page (figure 2A). A control sample shows negative vesicles for fluorescein on the left, and endosomes containing fluorescein on the right (200 vesicles shown, left panel). A control without trapped fluorescein shows only the left population (not shown). The co-localization of anticubulin binding shows that all fluorescein-positive endosomes were positive for cubulin, while non-endosomal membranes could be subdivided into positive and cubulin-negative populations (central panel). This pattern was repeated for the anti-megalin binding in renal cortical cells (right panel) in culture.

Then, the analysis of protein expression in cultured cells by antibody binding used classical serial logarithmic antibody dilution curves. An increase in binding with a decrease in dilution is pathognomonic for specific binding to antibody during flow cytometric analysis. The binding of anti-cubulin antisera to membrane vesicles prepared from renal cortical cells after 16 days in culture, detected by the fluorescence of a secondary antibody labeled with phycoerythrin, demonstrates an increase of almost two logarithms in the binding with the antibody dilution (Figure 2B). This increase in the cells growing in the rotating wall vessel (slow rotating side vessel) is more than 5 times the expression seen in the stirred fermenters. Similarly, there was no detectable expression in conventional cultures, resulting in a constant line (not shown). In Figure 2C the comparison of the maximum binding of the anti-cubulin antibody is shown by taking the minimum as the dilution of antibody in which there is no further decline in the signal with the dilution of primary antibody. The binding of normal serum and the minimum dilution of primary antisera were not detectably different. The binding curves for anti-megalin antiserum showed a similar pattern (not shown), but the maximum binding was a little lower (Figure 2C). Again, the stirred fermentor has a much lower expression than that of the rotating wall vessel (slow rotating side vessel), and conventional cell membranes do not have a detectable linkage (not shown).

To examine the proportion of proteins that change in the rotating wall vessel, two-dimensional gel SDS-PAGE analysis was performed on cultures developed in the rotating wall vessel and bag controls (Fig. 2d). The results shown in Figure 2D show that the changes were in a select group of proteins. To identify genes that change during culture in rotating wall vessel, differential deployment was made. The differential display of expressed genes was compared in aliquots of the same cells developed in a 55 ml rotating wall vessel (slow rotating side vessel), or in conventional two dimensional gas permeable bag controls. The differential display of copies of the expressed genes was generated by polymerase chain reaction using 25-element random primers and separated on a 6% gel for DNA sequence determination. The bands of different intensity between the control and the lateral vessel of slow rotation, which represents differentially expressed genes, were identified by visual inspection, separated and reamplified using the same primers. The differential expression and the size of the transcript were confirmed by Northern hybridization. The PCR products were then subcloned into the pGEM-T vector, and their sequence was determined. The sequences were compared to the gene bank sequences using the BLAST search engine. An expressed gene that decreased in the slow-rotating lateral vessel (band D in the gel, Figure 3A), was identified as mouse superoxide dismutase containing manganese (98% coincidence, 142 of 144 nucieotides). Two genes that were increased in the slow-rotating side vessel, band A, were identified as the beta gene of interleukin-1 (100% match for 32 of 32 nucleotides), and the B band that corresponded to a 20 kB transcript in a Northern blot, it appears to be an unidentified gene that has 76% homology to the mouse GABA transporter gene. To examine the genetic changes in specific genes, the expression of tissue-specific epithelial cell markers and classical genes dependent on stress response by shear stress was examined by RT-PCR (Figure 3c). Several genes specific for renal proximal tubular epithelial cells, including megalin, cubulin, the extracellular calcium detector receptor and the structural protein vilin of microvillosities, are increased early in culture in the rotating wall vessel. Likewise, there were dynamic time-dependent changes in classical genes dependent on shear stress, including intercellular adhesion molecule 1 (ICAM), vascular cell adhesion molecule (VCM) (increased) and manganese-dependent superoxide dismutase (deleted) Many of these changes were prolonged (but not all), since after 16 days in culture, the expression of the megalin genes, ICAM, VCAM and the extracellular calcium detector receptor was still high, while the vilin and superoxide Manganese-dependent dismutase were at control levels.

The expression of the control GADPH, actin b and 18S genes did not change over the course of time. To test the function of a putative endothelial element of response to shear stress in these renal cortical cell changes, an antisense probe was synthesized for the sequence (figure 4A). A control probe had the active motif crammed. Confocal imaging of a fluorescein-conjugated probe form confirmed the nuclear delivery of the probe (images not shown). Culture of rat renal cortical cells at 80 nm of the probe resulted in a time-dependent increase in the concentration of magnesium-dependent superoxide dimisutase, but no change in gene expression for villin (Figures 4B and 4C) . The control probe had no effect. To confirm the genetic responses to the culture in the rotating wall vessel and the analysis with human cells, automated gene deployment analysis of RNA expressed on human renal cortical cells developed in a gas-permeable control bag and the recipient was carried out. side of slow rotation for 8 days (50). Of the more than 18,000 genes tested, the change in a select group of genes was again observed (figure 5). In particular, the changes observed in the classic shear stress response genes subjected to test were confirmed by RT-PCR and differential display in rat cell culture. A group of tissue-specific genes was increased, including villin, angiotensin-converting enzyme, parathyroid hormone receptor, and sodium channels. Other genes dependent on physical forces, such as the heat shock proteins 27/28 KDa and 70-2, changed, as did the focal adhesion kinase, and changed a putative transcription factor for responses to shear stress, NF -kb. Fusion proteins such as sinabtobrevin 2, moderately decreased gene expression, and clathrin light chains greatly increased their expression. To determine whether the kidney cells developed in simulated microgravity showed 1-a-hydroxylase activity, the activity of 1-hydroxylase from cultures of cells grown in traditional two-dimensional culture in gas-permeable bags and rotary wall containers was compared. The NASA. Rat kidney cells (Table 2) and human embryonic kidney cells were tested (Table 3).

TABLE 2 Activity of 1-hydroxylase of the different cell cultures The results shown in Table 2 indicate that rat kidney cells show increased structural differentiation during culture under simulated microgravity conditions, and that they express much higher 1-a-hydroxylase activity than under conventional culture conditions.

TABLE 3 Activity of 1-hydroxylase from the different cultures of human embryonic kidney cells, detected as production of 1,25-diOH D3 Table 3 indicates that human embryonic kidney cells show increased structural differentiation during culture under simulated microgravity conditions, and that they express 10-fold greater 1-a-hydroxylase activity than under conventional culture conditions.

TABLE 4 Erythropoietin test in supernatant of renal fibroblasts TABLE 5 Erythropoietin test in hepatocyte supernatant Condition Erythropoietin (mu / ml) Culture under stress by exertion 141 .7 mu / l x 10 cells shear Static flask control Not detectable Tables 4 and 5 show respectively the results of the erythropoietin test in supernatants of fibroblasts and renal hapatocytes. The results shown in Tables 4 and 5 indicate that the production of erythropoietin increased in liver and kidney cells during Graduated gravitational sedimentation shear stress Erythropoietin has the classic stress response elements for shear stress in the promoter and enhancer regions that control the expression of its gene. The results shown in Tables 4 and 5 also indicate that the gene expression for erythropoietin was up-regulated by stress response elements by shear stress during graduated sedimentation gravitational shear in the vessel.

EXAMPLE 17 Discussion Rotating wall containers have been used by other researchers as "simulated microgravity". The present invention argues that gravity is still active, and that in a rotating wall vessel, gravity is balanced by tension by equal and opposite sedimentation shear stress. A centrifugal force due to the rotation of the cells, quantitatively much smaller than the gravity, is also present, and is compensated by the tension by equal and opposite sedimentation shear stress. In this way, the present invention presents the new concept that rotating wall vessels provide this new balance of forces, including the application of sedimentation shear, rather than microgravity. The bioreactor of the rotating wall vessel provides standing co-location of different cell types (1, 46), mass transfer rates that accommodate the molecular framework, and a microenvironment that includes growth factors (1, 46). The engineering analysis of the active forces in the container is complex (1, 5-7). This study provides the first evidence of cellular biological mechanisms by which the vessel induces changes in the expression of genes and tissue-specific proteins.

There are two possible explanations for why the rotating wall vessel induces more an order of magnitude expression of the renal toxin cubulin and megalin receptors than the agitated fermenter culture. First, there are significant differences in the degree of stress induced shear stress. The rotating wall vessel induces 0.5 to 1 .0 dynes / cm2 of shear stress (1), while agitated fermenters induce 2 to 40 dynes / cm2, depending on the design and the speed of rotation (1, 5, 46). This degree of stress damages or destroys most of the epithelial cells (1, 5, 46). Secondly, the trauma caused by the impeller in the stirred fermentor is absent in the rotating wall vessel. This explains why there was much more cubulin and megalin induced in kidney cultures in the culture in the rotating wall vessel, than in a stirred fermentor, and both receptors were not detectable in conventional two-dimensional culture. The culture in the rotating wall vessel induced changes in a select series of genes, as demonstrated by genetic analysis in gels for differential display and two-dimensional protein analysis in gels. For example, the production of erythropoietin is controlled by a shear stress element that mediates the changes observed during the graduated gravitational sedimentation shear stress. The activity of 1-a-hydroxylase is maintained and increased in renal cortical epithelial cells and human embryonic kidney cells, where the induction of the enzyme (1-a-hydroxylase) converts the 25-hydroxy-vitamin D3 to the active form 1 , 25-dihydroxy-vitamin D3. The present invention is the first demonstration of a process for the production of molecules that include hormones and other biomolecules induced by shear stress and other physical forces. Mechanistic information can be interpreted from knowledge of the response pattern and distribution of certain gene products. Megalin and cubulin represent the first pattern of change, since these proteins are restricted in distribution to tubular renal cortical epithelial cells. The increase in messenger RNA and megalin protein, and the expression of the cubulin protein, is therefore unequivocal evidence of changes in epithelial cells. This provides a new important tool for studies of nephrotoxicity. Considered for a long time to have a role in renal toxicity, giant glycoprotein receptors restricted to megalin and cubulin tissues, have recently been shown to be recipients of common nephrotoxins. Megalin is a receptor for polybasic drugs such as the aminoglycoside antibiotic gentamicin (48) and the vitamin D binding protein (51), and cubulin is the receptor for intrinsic vitamin B12 factor (52). Although these receptors are expressed by placental cells transformed in culture (9, 43), there is currently no renal model that expresses these markers for research in toxicology (53). The culture in the rotating wall container provides a new approach to the expression of specific renal markers in culture for studies in pharmacology, biochemistry and toxicology, which define the properties and unique sensitivities of renal epithelial cells. The second pattern of change is represented by the vilina. The message for the villin microvill protein is increased in the rotating wall vessel on the first day of culture, and microvilli reformation is soon observed. A decoy that matches the nuclear binding motif of a putative stress response element could not induce similar changes. Although the promoter for villin has not been cloned, this suggests that changes in villin were induced by other transcription factors which could be due to shear stress or other stimuli in the bioreactor. Vilin is also restricted to brush border membranes, such as proximal renal tubular cells, or colonic villi (54-55). The increments observed in the vilina message were resolved after 16 days of culture in the rotating wall vessel. Magnesium-dependent superoxide dismutase represents a third response pattern: a mitochondrial enzyme, ubiquitous in distribution, modulated by the classical element of response to shear stress in endothelial cells (56-57). The message for magnesium-dependent superoxide dismutase decreased early on the first day of culture in the rotating wall vessel, and this persisted after 16 days in culture. These changes were confirmed when it was determined that magnesium-dependent superoxide dismutase is suppressed in the differential display analysis of gene changes, and confirmation was carried out by Northern blot. A decoy for the classical element of shear stress response induced an increase in magnesium-dependent superoxide dismutase (MnSOD), which indicates that similar changes can be induced in the rotating wall vessel by the use of genetic decoys. In this way, the biological process of genetic induction by defined stress stress elements can be produced by multiple means including genetic decoys or the use of the rotating wall vessel. Other genes dependent on the shear stress response element, specifically, intercellular adhesion molecule 1 (ICAM) and vascular cell adhesion molecule (VCAM), showed changes in the rotating wall vessel, as opposed to superoxide magnesium-dependent dismutase, reflecting observations made during flow-induced stress in endothelial cells (56-57). This provides three lines of evidence consistent with the function for shear stress as a mediator of induced genetic changes in the rotating wall vessel. The differential display of the genes turned on and off under culture conditions in the rotating wall vessel showed that the culture therein was associated with decreased expression of the messenger RNA of manganese-dependent superoxide dismutase and increased expression of the messenger RNA of the interleukin-1 gene. This extends widely and brings with it several observations about the interactions of stress, the expression of manganese-dependent superoxide dismutase, and interleukin-1. Topper et al. Reported an oppositely directed effect, that is, the differential deployment of vascular endothelial cells exposed to high voltage demonstrates increased gene expression of manganese-dependent superoxide dismutase (57).

Other direct evidence links superoxide dismutase and interleukin-1 as increases in levels of manganese-dependent superoxide dismutase and decreases in interleukin-1 levels in HT-1080 fibrosarcoma cells (58). In more direct evidence, the overexpression of manganese-dependent mitochondrial superoxide dismutase promotes the survival of tumor cells exposed to interleukin-1 (59). The present study provides direct evidence that moderate shear stress decreases magnesium-dependent superoxide dismutase in association with an inverse effect on interleukin-1. The data here shows internal consistency. Changes in magnesium-dependent superoxide dismutase were observed on differential deployment confirmed by Northen blot analysis, and duplicate responses were detected by RT-PCR. Megalin showed equal changes in the expression of genes and proteins by RT-PCR. Changes in villin observed by RT-PCR were associated with dramatic microvilli reformation, in which villin is an important structural protein. Although semi-quantitative RT-PCR is subject to inherent variation due to massive signal amplification, the use of multiple controls that remained unchanged (actin b, GAPDH and 18S), and experimental confirmation that the reactions were linearly related At the concentration of cDNA, it minimizes these problems. The internally consistent findings by other methods strongly suggest that these RT-PCR data are valid. The study of the mechanisms of action of the rotating wall vessel to induce the expression of genes and proteins during cell culture has been hampered by nomenclature. In the first place, the fixation of the name "simulated microgravity", based on the engineering analysis of boundary conditions, tarnishes the intuitive analysis of cell biology, since there is no cellular equivalent for this term (1, 6-7). In the same way, the reduced shear stress in the rotating wall container, compared to stirred fermenters, leads to the term "crop under stress by reduced shear stress" (1), while there is increased shear stress compared to the crop. conventional two-dimensional (1, 5). While aggregates of cells remain suspended in rotating wall culture vessels, gravity is balanced by an equal and opposite force. Engineering arguments about the relative contributions of shear stress, drag, centrifugal force, Coriolis motion, and sedimentation induced by tangential gravity of fluids are themselves tangential to cellular biology. Several lines of evidence have been documented that shear stress responses are one of the components of the biological response. This lays the groundwork for the analysis of other mediators of the biological response in the vessels, and the investigation of whether the gravity discharge performs a function as large as the opposingly directed balancing forces. By using the rotating wall vessel as a tool, these data provide the first evidence that stress response elements by shear stress, which modulate gene expression in endothelial cells, are also active in epithelial cells, although Other researchers have not been able to see the effect of shear stress on epithelial cells. The present invention demonstrates that epithelial cells have shear stress response elements, and that they change the expression of genes in response to physical forces including, but not limited to, tension by shear stress. While the rotating wall vessel gains popularity as a clinical tool to produce hormonal implants, it is convenient to understand the mechanisms by which it induces genetic changes (10, 60), if it is necessary to prolong the useful life of the implants. Several lines of evidence are provided that stress response elements by shear stress are the first mechanism identified by which the rotating wall vessel induces genetic changes. Using as a decoy a binding site for stress response elements by endothelial cell shear stress, the function of this sequence in the regulation of selected genes in epithelial cells was validated. However, many of the changes observed in the rotating wall container are independent of said response element. It is necessary to define other genetic response elements modulated during the culture in the rotating wall vessel, and if the induced changes are secondary to the balance of forces, or mainly related to the gravity discharge.

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Any of the patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are incorporated herein by reference at the same level as if each individual publication was specifically and individually indicated to be incorporated herein by reference. The person skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the purposes and advantages mentioned., as well as those inherent in the present. The present examples together with the methods, procedures, treatments, molecules and specific compounds described herein, are currently representative of preferred embodiments, serve as examples, and are not intended to be limitations on the scope of the invention. Changes will occur in the present and other uses for those skilled in the art, and which are encompassed within the spirit of the present invention, as defined by the scope of the claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: Hammond et al. (I) TITLE OF THE INVENTION: Production of functional proteins: Balance of shear stress and gravity (iii) SEQUENCE NUMBER: 1 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Benjamin Aaron Adler, Ph.D. J.D. (B) STREET: 801 1 Candle Lane (C) CITY: Houston (D) STATE: Texas (E) ZIP CODE: 77071 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible Disk of 1.44 Mb (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: Macintosh (D) PROGRAM: Microsoft Word for Macintosh (vi) COMMON DATA OF THE APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (vii) PREVIOUS INFORMATION OF THE APPLICATION: (A) APPLICATION NUMBER: 60 / 043,205 (B) DATE OF SUBMISSION: 04/08/97 (viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: Benjamín Aaron Adler, Ph. D. (B) RECORD NUMBER: 35,423 (C) REFERENCE / CASE NUMBER: D6081 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (713) 777-2321 (B) TELEFAX: (713) 777-6908 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: (A) DESCRIPTION: cDNA for mRNA (iii) HYPOTHETIC: No (iv) ANTICIPATION: Yes (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (vii) IMMEDIATE SOURCE: (viii) POSITION IN THE GENOME: (ix) FEATURE: (x) INFORMATION ABOUT THE PUBLICATION: (xi) DESCRIPTION OF THE SEQUENCE: SEQIDNO: 1: CTGAGACCGA TATCGGTCTC AG 22

Claims (26)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for inducing the expression of at least one gene in a cell, characterized in that it comprises the steps of: contacting said cell with a sequence of oligonucleotide decoys of transcription factor directed against a nucleotide sequence coding for an element of stress response by shear stress; and determining the expression of said gene in said cell.

2. The method according to claim 1, further characterized in that said oligonucleotide comprises a terminal phosphoryorate moiety and a phosphodiester base structure.

3. The method according to claim 1, further characterized in that said oligonucleotide crosses the cell membranes and accumulates in the nuclear compartment of said cell.

4. The method according to claim 1, further characterized in that said cell is a cultured cell.

5. The method according to claim 1, further characterized in that said cell is selected from the group consisting of an epithelial cell and an endothelial cell.

6. The method according to claim 4, further characterized in that said cell is selected from the group consisting of renal cortical cell, renal fibroblast, hepatocyte, pancreatic islet, renal interstitial cell, parathyroid cell, thyroid cell, cell pituitary, ovarian cell and testicular cell.

7. The method according to claim 1, further characterized in that said cell is grown in two-dimensional culture.

8. The method according to claim 1, further characterized in that said shear stress response element is selected from the group consisting of GAGACC and GGTCTC.

9. The method according to claim 1, further characterized in that the gene codes for a protein selected from the group consisting of megalin, cubulin, erythropoietin and 1-a-hydroxyiase.

10. The method according to claim 1, further characterized in that the concentration of said oligonucleotide is from about 10 nm to about 10 mm. 1.

A decoy of a transcription factor, characterized in that it comprises a sequence of oligonucleotides directed against a nucleotide sequence that codes for a stress response element by shear stress.

12. The decoy of transcription factor according to claim 1, further characterized in that said nucleotide sequence encoding a shear stress response element has a sequence selected from the group consisting of GAGACC and GGTCTC.

13. A method for producing a functional protein, characterized in that it comprises the steps of: isolating mammalian cells; placing said cells in a rotating wall vessel containing a cell culture comprising culture media and culture matrix; produce three-dimensional aggregates of cells under simulated microgravity conditions; and detecting the expression of the functional protein in the cell culture.

14. The method according to claim 13, further characterized in that said simulated microgravity conditions comprise a balance between gravity forces and oppositely directed physical forces.

15. The method according to claim 14, further characterized in that said physical forces are selected from the group consisting of tension by sedimentation shear, centrifugal forces, viscosity and Coriolis forces.

16. The method according to claim 13, further characterized in that said functional protein is selected from the group consisting of a hormone, a toxin receptor and a functional biomolecule dependent on stress by shear stress.

17. - The method according to claim 16, further characterized in that said hormone is selected from the group consisting of 1, 25-dihydroxy-vitamin D3 and erythropoietin.

18. The method according to claim 16, further characterized in that said toxin receptor is selected from the group consisting of megalin and cubulin.

19. The method according to claim 16, further characterized in that said stress-dependent functional biomolecule by shear stress is selected from the group consisting of vilin, magnesium-dependent superoxide dismutase, nitric oxide synthetase, c-fos, c-jun , platelet-derived growth factor b, growth transforming factor b, tissue-type plasminogen activator and monoclonal protein 1, megalin, cubulin, erythropoietin and 1-a-hydroxylase.

20. The method according to claim 13, further characterized in that said cell is selected from the group consisting of renal cortical cells, renal fibroblasts, hepatocytes, pancreatic islets, renal interstitial cells, parathyroid cells, thyroid cells, pituitary, ovarian cells and testicular cells.

21. The method according to claim 13, further characterized in that said cell is selected from the group consisting of epithelial cell and endothelial cell.

22. - The method according to claim 13, further characterized in that said cell contains stress response elements by shear stress.

23. The method according to claim 22, further characterized in that said stress response element by shear stress is selected from the group consisting of GAGACC and GGTCTC.

24. The method according to claim 13, further characterized in that said rotating wall vessel is started and maintained from about 6 rotations per minute to about 16 rotations per minute.

25. The method according to claim 15, further characterized in that said tension by sedimentation shear is about 0.2 dynes / cm2 at about 1.0 dynes / cm2.

26. The method according to claim 13, further characterized in that said culture matrix contains a central structure selected from the group consisting of cellular aggregates and microcarrier spheres.