MX2008009913A - Method of characterizing a biologically active compound - Google Patents

Abstract

A method of characterizing a biologically active compound by placing a cell mixture into a rotatable bioreactor to initiate a three-dimensional culture comprising a biological component and at least one cell, controllably expanding the cells in the rotatable bioreactor and testing the biological component to characterize the biologically active compound. The present invention may also preferably comprise exposing the cells to a time varying electromagnetic force.

Description

METHOD FOR CHARACTERIZING A BIOLOGICALLY ACTIVE COMPOUND FIELD OF THE INVENTION The present invention is related, in general terms, to the field of characterization of a biologically active compound. More specifically, the present invention relates to a method for controllingly expanding a three-dimensional culture in a rotary bioreactor to characterize a biologically active compound.

BACKGROUND OF THE INVENTION Most biologically active compounds are directed to specific tissue functions that are based on detailed structures and chemical processes that occur at all levels of the biological processes of tissue structure from molecular to large-scale. The efficacy tests and determination of the mechanism of action of these biologically active compounds require highly reliable cells and tissue and are usually carried out in conventional in vitro culture (for ordinary purposes), in animals, and finally in clinical trials. in humans Each of these methods has limitations, however, they are introduced either by low fidelity and / or ethics. In addition, the ability to investigate the specific detailed mechanism or physical site of an action of the active biologically active compounds Ref .: 195266 is limited by these conventional methods of analysis. A similar case is to understand the mechanism and degree of toxicity for toxic chemicals and materials or to understand or characterize the biological activity of a reagent. In the case of the use of animals for testing, the biological environment is too complex, not controllable, rich in confusing factors, it often poorly represents the human condition and suffers from ethical limits. Conventional cultures, such as two-dimensional cultures, or those that require agitation, movement, and other modes of culture mixing, are not capable of reproducing biologically active interactions with cells that would interact in the tissue microenvironment in vivo. Other methods of cultivation that use fixed matrices in conventional non-rotating systems, that is to say absent from any component of the freely suspended material, also introduce limitations of fidelity, accuracy, analytical capacity, and feasibility to conduct these studies. One-man tests introduce obvious severe ethical constraints along with many of those inherent in animal tests. The structural relationships of the primary functional cells with each other, with support cells, and with mechanical support substrate allow specific cellular and exact tissue and natural behavior. Traits, such as binding complexes, gland formation, cell polarity, and geometric relationships in general to support cells and acellular components, mediate such specific cellular and tissue behavior. In addition, the function of individual tissues and cells in a certain way depends on these, and other characteristics. Other features also contribute to the relationships between cells and the three-dimensional interactions between cells in the larger tissue structure including mucin, secreted hormones (insulin from pancreatic Beta cells), soluble intercellular signals, cell membrane surface markers, membrane bound enzymes , immune identity markers, adhesion molecules, vacuoles, neurotransmitters stored and released, and specialized cellular internal machinery, such as myosin contractile fibers in the case of muscle, glycogen and conjugate toxic elimination that is processed in the case of hepatocytes. Individual cell functions and cell-cell interactions are dependent on these and other characteristics. The efficacy and toxicity of biologically active compounds are analyzed and quantified by the determination of the effect that the biologically active compound has on the cell, weaving, and / or these characteristics. These measurable responses include gene expression, karyotype, growth rate characteristics, multicellular and individual cell morphology, metabolic measures, and intercellular relationships. These and other responses are known but the difficulty has been that traditional culture methods are unable to cultivate a sufficient amount of cells and tissue so that cells and cell interactions can substantially mimic the situation in vivo and any response to them. Biologically active compounds would be an accurate reflection of the cellular response in vivo to the biologically active compound. Therefore, traditional culture systems, which do not support enormous and accelerated cell and tissue growth over long periods of time, do not provide an accurate in vitro model for characterizing biologically active compounds by testing their effects. The growth of a variety of both normal and neoplastic mammal tissues both in monoculture and complementary culture has been established in both batch-fed and perfused rotating wall containers, Schwarz et al., US Patent Number 4 988 623, ( 1991) and Schwarz et al., U.S. Patent Number 5 026 590, (1991), and in conventional culture systems based on plate or flask. In some applications, the growth of the three-dimensional structure, eg, tissues, in these culture systems has been facilitated by the support of a solid matrix in the form of biocompatible polymers and microcarrier. In case of spheroid growth, the three-dimensional structure has been achieved without matrix support, Goodwin, et al., In Vitro Cell Dev. Biol., 28 A: 47-60 (1992), Goodwin, et al., Proc. Soc. Exp. Biol. Med., 202: 181-192 (1993), Goodwin, et al., J. Cell Biochem., 51: 301-311 (1993), Goodwin, et al., In Vitro Cell Dev. Biol. Anim. , 33: 366-374 (1997). However, human tissue has been largely refractory, in terms of controlled growth induction and three-dimensional organization, under conventional culture conditions. The current microgravity and, to a lesser degree, microgravity, rotationally simulated, has allowed enhanced cellular growth. Also, attempts have been made to employ static electric fields to enhance the growth of the nerve of a culture. The embryonic development has been successfully altered, and the axonal directional growth of the isolated nerve has been successfully achieved. However, the actual acceleration of the growth enhancement or genetic activity that causes this has not been achieved. Mechanical devices that aim to help grow and orient the three-dimensional tissue of mammalian neurons are currently available. Fukuda et al., U.S. Patent Number 5 328 843 used the zones formed between stainless steel razor blades to orient neuronal cells or axons. In addition, charged electrodes with electrical potential were used to improve the response of the axon. Aebischer, U.S. Patent Number 5 030 225, described an implantable and electrically charged tubular membrane that is used in the regeneration of severed nerves within the human body. Wolf, et al., US Patent Number 6 485 963, used the electromagnetic force to increase cell growth, but in many cases cell growth, or expansion, did not occur fast enough for necessary tests or treatment of a patient. There is still a need, therefore, for an in vitro culture system that essentially mimics the microenvironment in vivo to analyze the effects of biologically active compounds on cells and tissues, thus providing responses that are highly representative of the in vivo situation.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a method for characterizing a biologically active compound comprising the placement of a cellular mixture in a rotary bioreactor to initiate a three-dimensional culture where the three-dimensional culture comprises cells and a biological component, to controlly expand the cells in the three-dimensional culture while at the same time maintaining the three-dimensional geometry of the cell and the cell-to-cell support and the geometry by rotating the rotating bioreactor, introducing a biologically active compound into the three-dimensional culture, and analyzing the biological component using a test to characterize the biologically active compound. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a side elevational view of a preferred embodiment of a rotary bioreactor; Figure 2 is a side perspective of a preferred embodiment of the rotary bioreactor; Figure 3 schematically illustrates a preferred embodiment of a culture carrier flow circuit of a rotary bioreactor; Figure 4 is the orbital trajectory of a typical cell in a non-rotating reference frame; Figure 5 is a graph of the amount of deviation of one cell per revolution; NOTE: Centrifugal deviation accumulates over time, gravity-induced deviation varies sinusoidally; and Figure 6 is a representative cell trajectory as observed in a rotational reference frame of the culture medium. DETAILED DESCRIPTION OF THE INVENTION In the simplest terms, a rotary bioreactor comprises a culture chamber which, during operation, can be rotated about a substantially horizontal axis, and has an inner portion and an outer portion. The inner portion of the culture chamber defines a space that can be detachably coupled to a mixture of biological component. Preferably, the culture chamber is substantially cylindrical. In a preferred embodiment of the rotary bioreactor, an electrically conductive coil is wrapped around the outer portion of the culture chamber preferably attached to the culture chamber, more preferably in detachable form attached to the culture chamber. A source of TVEMF (electromagnetic field with temporal variation) is operatively connected to the electrically conductive coil so that, during operation, the TVEMF source supplies a TVEMF to the inner portion of the culture chamber and to the mixture of biological component to expand the biological component in it. The culture chamber has at least one opening so that, during operation, the biological component mixture can be placed in the inner portion of the culture chamber. The opening can also be preferably used for changing the culture medium and / or a biologically active compound, and removing samples of the biological component for testing, and preferably the opening is adjusted for use with a syringe.

In the figures, Figure 1 is a transverse elevated side view of a preferred embodiment of a rotary bioreactor 10. In this preferred embodiment a motor cover 12 is supported by a base 14. A motor 16 is enclosed within the cover 12 of motor and connected by a first cable 18 and a second cable 20 to a control box 22 that houses a control device by means of which the speed of the motor 16 can be increased in a controlled manner by turning the start button 24. Extending from the engine cover 12 is a motor shaft 26. A rotating assembly 28 is detachably coupled to a rotary bioreactor support unit 30 which removably engages a culture chamber 32 preferably disposable and also preferably substantially cylindrical, which is attached, preferably in detachable form, inside the rotary bioreactor support unit 30, preferably by a screw 34. The culture chamber 32 is mounted, preferably in removable form, to the rotary assembly 28. The rotary assembly 28 is received by the motor shaft 26. During operation, when the start button 24 is turned on, the culture chamber 32 rotates. The term "rotate" and similar terms is intended to mean that during operation, the rotation of the culture chamber prevents the collision of cells, tissue, or cell mass, with the inner portion of the rotating TVEMF bioreactor. The culture chamber can also preferably be perfused. The culture chamber of the rotary bioreactor 10 of the present invention can be preferably disposable which means that a new one can be removed and used in subsequent cultures when necessary. The rotary bioreactor 10 can also preferably be sterilized, for example in an autoclave, after each use and reused for subsequent cultures. A disposable culture chamber 32 could be manufactured and packaged in a sterile environment thus facilitating its use by physicians or researchers in much the same way as other disposable medical devices are used. Figure 2 is a side perspective view of a rotary bioreactor 10. Figure 2 illustrates the cover 42 of the engine which retains a start button 54 and is supported by a base 44. Extending from the engine cover 42 is an engine shaft 56. A rotary assembly 58 is removably coupled to a rotary bioreactor support unit 60 that removably engages a culture chamber. An electrically conductive coil 59 is wrapped around the outer portion of the culture chamber. The electrically conductive coil 59 may preferably be made from any electrically conductive material that conducts electricity including, but not limited to, the conductive materials that follow; silver, gold, copper, aluminum, iron, lead, titanium, uranium, a ferromagnetic metal, and zinc, or a combination thereof. The electrically conductive coil 59 may also preferably comprise salt water. The electrically conductive coil 59 can also preferably be a solenoid. In addition, the electrically conductive coil 59 may be preferably contained in an electrical insulator comprising, but not limited to, rubber, plastic, silicone, glass, and ceramic. The electrically conductive coil 59 may be wrapped around the outer portion of the culture chamber, and thus, the culture chamber maintains a shape of the electrically conductive coil 59, which preferably has a substantially oval cross section, more preferably a substantially elliptical cross section, and more preferably a substantially circular cross section. The electrically conductive coil 59 which is incorporated with a culture chamber which is preferably disposable is installed in the rotary bioreactor 10 together with the disposable culture chamber and is operatively connected to a TVEMF 64 source. When the chamber is removed of disposable culture, the electrically conductive coil 59 is discarded therewith. At a first end a first lead wire 62 and a second lead 66, both are incorporated with the electrically conductive coil 59, are operatively connected to a TVEMF source 64 having a knob of the source 65 which, during the operation, it can be turned on to generate a TVEMF. At a second end the cables 62, 66 are connected to at least one ring to facilitate the rotation of the electrically conductive coil 59. When the start button 54 is turned on, the culture chamber and the electrically conductive coil 59 rotate simultaneously . In addition, the electrically conductive coil 59 remains attached and spans the culture chamber, so that during operation, it provides a TVEMF to the cells in the culture chamber. The culture chamber of a rotary bioreactor may preferably be equipped with a flow path of culture medium 100 for support of respiratory gas exchange, nutrient supply, and metabolic waste disposal of a three-dimensional culture. A preferred embodiment of a culture medium flow circuit 100 is illustrated in Figure 3, has a culture chamber 119, an oxygenator 121, a device to facilitate the directional flow of the culture medium, preferably by the use of a pump main 115, and a feeder head 117 for selective entry of culture medium requirements such as, but not limited to, nutrients 106, buffers 105, freshly prepared medium 107, cytokines 109, growth factors 111, and hormones 113 In this preferred embodiment, the main pump 115 provides the fresh culture medium from the feeder head 117 to the oxygenator 121 where the culture medium is oxygenated and passes through the culture chamber 119. The waste is eliminated in the culture medium. spent from the culture chamber 119, preferably by the main pump 115, and is administered to the waste 118 and the remaining volume of the culture medium not removed to the residue 118 is regr This is preferably fed to the feed head 117 where this may preferably receive a fresh load of requirements from the culture medium before being recycled by the pump 115 through the oxygenator 121 to the culture chamber 119. In this preferred embodiment of a medium flow circuit 100 culture, adjustments are made in response to chemical sensors (not shown) that maintain constant conditions within the culture chamber 119. The control of carbon dioxide pressures and the introduction of acids or bases correct the pH. Oxygen, nitrogen, and carbon dioxide are dissolved in a gas exchange system (not shown) in order to support cellular respiration. The flow circuit of the culture medium 100 adds oxygen and removes the carbon dioxide from a circulating gas capacitance. Although Figure 3 is a preferred embodiment of a culture medium flow circuit that can be used in the present invention, the invention is not intended to be so limited. The entry of requirements of culture medium such as, among others, oxygen, nutrients, buffers, freshly prepared medium, cytokines, growth factors, and hormones in a rotary TVE F bioreactor can also be carried out manually, automatically , or by other means of control, such as the control and disposal of waste and carbon dioxide. Since various changes could be made in rotating TVEMF bioreactors such as those contemplated in the present invention, without departing from the scope of the invention, it is intended that all the matter contained herein be construed as illustrative and not limiting. The definitions that follow are proposed to aid in the description and understanding of the terms defined in the context of the present invention. The definitions are not intended to limit these terms unless described throughout this application. In addition, several definitions that are included are related to the TVEMF - all the definitions in this aspect should be considered as complementing each other and not contradicting each other. As used throughout the application, the term "TVEMF" refers to "the electromagnetic field with temporal variation".

As mentioned above, the TVEMF of this invention is in a delta wave, more preferably a differential square wave, and more preferably a square wave (which follows a Fourier curve). The TVEMF is preferably selected from one of the following: (1) a TVEMF with a force amplitude less than 100 gauss and rotational speed greater than 1000 gauss per second, (2) a TVEMF with substantially bipolar square wave of low amplitude force with a frequency of less than 100 Hz, (3) a TVEMF with a square wave with substantially low power amplitude with less than 100% of the duty cycle, (4) a TVEMF with rotation speed greater than 1000 gauss per second for pulses of duration less than 1 millisecond, (5) a TVEMF with pulses similar to the bipolar delta function of rotation speed with a duty cycle of less than 1%, (6) a TVEMF with a power amplitude less than 100 gauss peak-to-peak and pulse-like bipolar delta-function rotation speed and where the duty cycle is less than 1%, (7) a TVEMF is applied using a solenoid coil to form the uniform force resistance in all parts of the crop three-dimensional, (8) and a TVEMF is applied using a flow concentrator to provide spatial gradients of magnetic flux and magnetic flux that is concentrated within the three-dimensional culture. The frequency range in the oscillating electromagnetic force resistance is a parameter that can be selected to achieve the desired stimulation of the cells in the three-dimensional culture. However, these parameters are not intended to limit the TVEMF of the present invention, when such may vary based on other aspects of this invention. The TVEMF can be quantified for example by conventional equipment, such as an EN131 Cellular Sensor Gaussometer. As used throughout the Application, the term "electrically conductive coil," refers to any electrically conductive material that conducts electricity including, among other things, the conductive materials that follow; silver, gold, copper, aluminum, iron, lead, titanium, uranium, a ferromagnetic metal, and zinc, or a combination thereof. The electrically conductive coil may also preferably comprise salt water. The electrically conductive coil may also preferably be a solenoid. In addition, the electrically conductive coil may preferably contain an electrical insulator or comprising, inter alia, rubber, plastic, silicones, glass, and ceramic. The electrically conductive coil can be wrapped around the outer portion of the rotating TVEMF bioreactor culture chamber, and therefore, preferably the culture chamber bears a shape of the electrically conductive coil, preferably having a substantially cross section oval, more preferably a substantially elliptical cross section, and most preferably a substantially circular cross section. The culture chamber supports a shape of the electricity conducting coil preferably because the shape of the culture chamber and the shape of the electrical conductive coil are substantially similar. By "wrapped around," it is intended that the electrically conductive coil encompass the culture chamber so that preferably, during operation, a substantially uniform TVEMF is administered to the inner portion of the culture chamber and the cells thereof. "Encompassing" means that the electrically conductive coil surrounds the culture chamber, and during operation, preferably supplies a substantially uniform TVEMF to the inner portion of the culture chamber. As used throughout the application, the term "biological component" refers to a portion of the three-dimensional culture in the rotary bioreactor during the controllable expansion step of the method of the present invention. The biological component can be preferably analyzed during cultivation or by other means after the culture is completed or even inactivated by special analytical methods, like electron microscopy. The biological component is analyzed to characterize a biologically active compound. The biological component can preferably be cells in any form, T-cells for example activated, and any part of the cell that includes the membrane, the cell wall (in case of plants), and / or the internal cellular organelles that include the mitochondria . The biological component to be analyzed may also preferably include the secreted material, for example mucin, collagen, and matrix, secreted hormones (insulin from pancreatic Beta cells), secreted intercellular structural components, introduced structural matrices, adhesion matrices, growth substrates , intercellular soluble signals, cell membrane surface markers, membrane-bound enzymes, immune identity markers, adhesion molecules, vacuoles, neurotransmitters stored and released, and specialized cellular internal machinery, such as the contractile fibers of myosin in the case of muscle, glycogen, culture media, compounds under test, suspicious toxins in the test, test reagents, fungus, and complexes conjugated in the case of hepatocytes. A biological component may also preferably be a virus that is contained in the three-dimensional culture in the rotary bioreactor during expansion. Such viruses may include, but are not limited to, HIV, Avian Influenza, SIV, Hepatitis, HPV, Herpes Virus, which may contain viral DNA, or in the case of retroviruses, viral RNA and particles. The biological component can also preferably be bacterial cells. The biological component can also preferably be any other nuclease, DNA, RNA, protein, artificial bioactive particles, such as nanoparticles, and / or genes, but is not limited thereto. The biological component may be preferably contained in the cell mixture, or added to the three-dimensional culture, or placed in the rotary bioreactor before the addition of the cell mixture. The biological component is to focus a test to characterize a biologically active compound. As used throughout the application, the term "biologically active compound" refers to any biological substance, synthetic or non-synthetic, which must be characterized by the method of the present invention. The biologically active compound may preferably be in any form including, among other things, powder, liquid, vapor, and gas. The biologically active compound may also be preferably, inter alia, protein, cells, chemicals, gases, metals, growth factors, radiation, nanoparticles, viruses, bacteria, and / or water, and / or any combination thereof. The biologically active compound may also preferably be any toxic material in any portion of a three-dimensional culture, which comprises cells and a biological component. As used in this application the term the toxin refers to any material or physical process, which is suspected or known to negatively affect a cellular or tissue function or cause it to deviate from normal function. The toxins may preferably be heavy metals, and also preferably be thermal, radioactive, or even electrical exposures. In addition, a biologically active compound can also preferably be a reagent having an effect on a three-dimensional culture, comprising cells and a biological component. As used throughout the application, the term "reactive" refers to any material or physical process that is used to cause a change in cellular or tissue function, architecture, structure, growth, lifespan, genetic make-up, growth characteristics, secreted material, state of differentiation, predisposition of differentiation line, or expression of surface marker, metabolic state, internal cellular organelle structure, membrane structure, or tumorogenicity. In addition to the preferred biologically active compound, such as insulin that is transported in a cell and causes internal transport of glucose, a biologically active compound can preferably refer to reagent steps, such as electroporation, chemoporation, and nanoparticle interactions. Poration methods are particularly useful for the production of hybridomas for the production of monoclonal antibodies. Not being limited by theory, the well-distributed three-dimensional culture content allowed by rotating wall cultures is ideal for maximizing genetic change between transformed and immunological cells in the hybrid formation. This can improve the successful production of desired hybrids by producing desirable antibodies. Some additional preferred examples of a biologically active compound include, but are not limited to, insulin, interleukins, growth factors, differentiation modulators, chemotactic agents, inhibitors. According to the present invention, a biologically active compound can be characterized by testing its effects on a biological component. As used throughout the application, the term "cells" refers to a cell in any form, for example, individual cells, tissue, cell pools, hybrid cells, cells previously bound to cell-binding substrates, eg microcarrier beads, structures tissue-like, or intact tissue resections. The cells in this invention may also be preferably eukaryotic, more preferably prokaryotic. Cells that can be used in this invention are preferably mammalian, more preferably human, stem cells even more preferably adult, most preferably adult peripheral blood stem cells, and even more preferably mesenchymal cells. Other mammalian cells that can be used in the method of the present invention preferably include, but are not limited to, heart, liver, hematopoietic, skin, muscle, intestinal, pancreatic, central nervous system, cartilage, connective lung, spleen, bone, and kidney. As used throughout the application, the term "Rotary bioreactor" means comprising an engine connected to a cultivation chamber with an inner portion and an outer portion and which can rotate at a speed. Preferably, the rotary bioreactor is substantially cylindrical. The rotary bioreactor may also preferably have an electrically conductive coil wrapped around the outer portion of the culture chamber. In addition, the rotary bioreactor may also have a culture medium flow circuit attached thereto to help facilitate the flow of culture medium and three-dimensional culture therein. The flow of the culture medium through the culture chamber can be by means of perfusion. A source of TVEMF can preferably be connected operatively with the electrically conductive coil. During operation, a rotating bioreactor can be rotated and, without being limited by theory, rotation should be controlled to breed, support, and maintain a three-dimensional culture, as described for example in the Description of the Invention. In a preferred embodiment having an electrically conductive coil, a TVEMF can be generated by the TVEMF source, and an appropriate gauss level, can preferably be supplied to the inner portion of the culture chamber via the electrically conductive coil. The volume of the rotary bioreactor is preferably from about 15 ml to about 2 L. See for example Figures 1 and 2 herein for examples (without the purpose of limiting) of a rotary bioreactor. The culture chamber of a rotary bioreactor has rotating chamber walls in the inner portion so that, during operation, the chamber walls are set in motion relative to the culture medium, and therefore, the three-dimensional culture , so that there is essentially no sheer fluid tension in the culture medium. The culture chamber also has at least one opening for the addition and / or removal of culture medium, cells, and / or the biological component or portions thereof, and also for introducing a biologically active compound. The culture chamber of the rotary bioreactor is disposed substantially horizontally. The culture chamber is also preferably substantially cylindrical with two ends, and is capable of rotating about a substantially horizontal axis. The culture chamber is preferably partly transparent, so that the biological component, the culture medium, and / or the three-dimensional culture can be evaluated when necessary. In addition, the culture chamber may also be preferably adjusted with a microscope to evaluate the biological component, three-dimensional culture, and / or cells. Unrestricted by theory, the rotation of cells in a rotating bioreactor provides the controllable expansion of cells over time, while at the same time adapting, supporting, and maintaining intricate three-dimensional geometry, cell-to-cell support and geometry of the cells. As used throughout the application, the term "cell mixture" and similar terms refers to a mixture of cells, preferably with another substance including, among other things, culture medium (with and without additives), plasma, buffer, and conservatives. The cell mixture can also comprise the biological component. As used throughout the application, the term "three-dimensional culture," refers to the cells and the biological component in the culture chamber of the rotationally expanded bioreactor controlled by the method of the present invention. The cells in the three-dimensional culture have a three-dimensional geometry and cell-to-cell support and geometry bred, supported, and maintained in the culture chamber. The cells in the three-dimensional culture have essentially the same three-dimensional geometry and cell-to-cell support and geometry as the cells in vivo. The three-dimensional fabric, the non-necrotic cell mass, and / or the tissue-like structures can also develop from the cells and be prolonged and further expanded in the three-dimensional culture and at the same time mimic the microenvironment in vivo. The three-dimensional culture can be expanded (grown in quantity), prolonged, or degenerated according to the objective of the experiment. In other words, depending on the effects of the biologically active compound and / or the preferred microenvironment had to characterize the biologically active compound, the three-dimensional culture will be controlledly expanded which could preferably mean expanding, maintaining, or degenerating the three-dimensional culture, or portions thereof. As used throughout the application, the term "operatively connected" and similar terms, is intended to mean that the TVEMF source may be connected, preferably in detachable form, to the culture chamber in such a way that, during operation , the TVEMF source confers a TVEMF to the inner portion of the culture chamber of a rotating bioreactor and the three-dimensional culture contained therein. The TVEMF source is operatively connected if, during operation, it can confer a TVEMF to the inner portion of the culture chamber, preferably substantially uniform. As used throughout the application, the term "exposure", and similar terms, refers to the process of providing a TVEMF to the three-dimensional culture contained in the interior portion of the culture chamber of a rotating bioreactor. During operation, a TVEMF source is turned on and set with a preferred gauss range and a preferred waveform so that it is supplied via the TVEMF source to an electrically conductive coil, wrapped around the outer portion of the rotary bioreactor culture chamber. The TVEMF is then supplied to the three-dimensional culture containing cells in the culture chamber which thus exposing the cells to the TVEMF, preferably a substantially uniform TVEMF. As used throughout the application, the term "culture medium" and similar terms, refers to a liquid comprising, but not limited to, growth medium and nutrients, which means the sustenance of cells over time. The culture medium can be enriched by any of the following, but is not limited to; growth medium, buffers, growth factors, hormones, and cytokines. The culture medium is supplied to the cell mixture for suspension within the culture chamber of the rotary bioreactor and to support expansion. The culture medium may be preferably mixed with the cell mixture before being added to the culture chamber of the rotary bioreactor, or more preferably it may be added to the culture chamber before the cell mixture is added thereby mixing the culture medium. and cells in the rotary bioreactor. The culture medium can preferably be enriched and / or cooled during expansion when necessary. The residue contained in the culture medium, as well as the culture medium itself, can preferably be removed from the three-dimensional culture in the culture chamber during expansion when necessary. The residue contained in the culture medium may be, but not be limited to, metabolic waste, dead cells, and other toxic waste. The culture medium can preferably be enriched by oxygen and preferably has oxygen, carbon dioxide, and nitrogen transport capacities. As used throughout this application, the term, "placement," and similar terms refer to the mixing process, the cell mixture and the culture medium before adding the cells to the rotary bioreactor. The term "placement" can also preferably refer to the addition of the cell mixture to the culture medium which is already present in the rotary bioreactor. The cells can preferably be placed in the rotary bioreactor together with cellular fixation substrates, such as microcarrier beads. As used throughout the application, the term "controlled expansion," and similar terms, refers to the process of increasing, conserving, or reducing the number of cells in a rotating bioreactor by rotating the culture chamber. In a preferred embodiment, controllingly expanding cells also comprises exposing the three-dimensional culture to a TVEMF. Preferably, the cells are expanded without differentiation. If an increase in the number of cells is preferred, then the increase in the number of cells per volume is not expressly due to a simple reduction, in the volume of liquid, for example, by reducing the volume of the culture medium from 70 ml to 10 mi and so increasing the number of cells per my. By controllingly expanding the cells by preferably expanding (increasing the amount) of cells in a rotary bioreactor provides cells that have substantially the same three-dimensional geometry as the cells before expansion, preferably the same geometry and cell-cell interactions while the cells show ral sedimentation or tissue where these rally exist, the microenvironment in vivo. Also preferably, the controlled expansion can be referred to as supporting a three-dimensional culture where, for example, the effect of a preferred biologically active compound is to prevent the increase in the number of cells. More preferably, the three-dimensional culture can also be prolonged to characterize the biologically active compound. Controllably expanding the cells in a three-dimensional culture of a rotary bioreactor may also preferably refer to a degenerative culture where, for example, the effect of a preferred biologically active compound is to degenerate the three-dimensional culture. More preferably, the three-dimensional culture can be intentionally degenerated to characterize a preferred biological compound. Other aspects of the expansion may also provide the exceptional characteristics of the cells of the present invention. Preferably, the cells and / or tissue undergo expansion while it is necessary to analyze a biological component to characterize a biologically active compound. The three-dimensional culture may preferably undergo expansion for at least 160 days in a rotary bioreactor. As used throughout the application, the term "turn", and similar terms, refers to the rotation of the culture chamber of the rotating bioreactor, which is preferably substantially cylindrical and is rotated substantially on a horizontal plane. Preferably, the rotation speeds range from about 1 revolutions per minute (RPM) to about 120 RPM, and more preferably from about 2 RPM to about 30 RPM. The rotary bioreactor can preferably be rotated automatically or manually. In addition, the rotation speed can preferably be adjusted, started, or stopped manually, or more preferably adjusted, started, or stopped automatically when using a sensor. As used throughout the application, the term "introduction of a biologically active compound" refers to the process of adding a biologically active compound in the culture chamber before, during, and / or after the controlled expansion step. The biologically active compound may be preferably added when necessary during the method of the present invention and before and / or after various steps. According to the preferred test carried out, the biologically active compound can be added in different concentrations and at various times throughout the method of the present invention. The biologically active compound may also be intrinsically contained in the three-dimensional culture. As used throughout the application, the term "test" refers to the process of characterizing a biologically active compound by analyzing the effect of the biologically active compound or having no effect on a biological component. Depending on the biological component to be analyzed, the tests will vary. For example, if the biologically active compound is expected to perform an effect on the DNA or RNA of a biological component then the biologically active compound can be characterized by testing the effect on the DNA by polymerase chain reaction or RNA by reaction in reverse transcriptase polymerase chain. Other tests and methods of analysis include, but are not limited to, the following instruments and techniques including: mass spectroscopy, flow cytometry, immunofluorescence, chromatography, and mono and biclonal antibodies, viability tests, toxicity tests, species tests , bioassays, effective dilution and concentration tests, dose response tests, hazardous waste tests, lethal concentration tests, screening tests, static renewal tests, cell quantity and tissue growth tests, and radiolabelling. Preferably, the present invention provides a method for characterizing a biologically active compound by testing its effect on tumorigenicity and genetic abnormalities. Other examples of tests that can be carried out by the method of the present invention to characterize a biologically active compound preferably include, but are not limited to, tests related to gene expression, karyotype, growth rate characteristics, multicellular cell morphology and individual, metabolic measures, and intercellular relationships. The tests would preferably be directed to the measurement of binding complexes, gland formation, cell polarity, and geometric relationships between cells (cell-to-cell geometry), and acellular components. The present invention provides a method comprising the step of controlling cells in a controlled manner so that the three-dimensional geometry of the cells remains the same as it is in natural sedimentation, thus providing a biological component for characterizing a biologically active compound through testing its effects of a biologically active compound on a biological component in a mixed environment that is essentially the same as it is discovered in the in vivo situation. The biological component can be analyzed to characterize mechanisms of action of the biologically active compound, biological effects, efficacy, administration, utility, and / or toxicity. As used throughout the application, the term "characterize" refers to the process of determining the effect that a preferred biologically active compound has on a biological component when testing the biological component. The tests can be carried out preferably by analyzing the efficacy and toxicity of the biologically active compound. The biological component can be analyzed to characterize the action mechanisms of biologically active compound, biological effects, efficacy, administration, utility, and / or toxicity. As used throughout the application, the term "cell-to-cell geometry" refers to the geometry of cells that include the spacing, distance between these and the relative physical relationship between them. For example, in a preferred embodiment of the present invention, when cells are used, expanded cells, including those from tissues, cell assemblies, and tissue-like structures, cells remain in relation to each other as in the microenvironment in vivo. The expanded cells are within the limits of the natural spacing between cells, in contrast to expansion chambers such as two-dimensional, where such spacing is not conserved over time and expansion. As used throughout the application, the term "cell-to-cell support" refers to the support that a cell provides to an adjacent cell. For example, tissues, cellular assemblies, tissue-like structures, and cells maintain interactions such as chemical, hormonal, neural (where applicable / appropriate) with other cells. In addition, the cells provide the structural support among these. It is not necessary for the cells to be physically palpable to provide cell-to-cell support. In the present invention, these interactions are maintained within normal parameters that work, meaning that they do not begin, for example, to send toxic or harmful signals to other cells (unless it is carried out in the natural cellular and tissue environment). As used throughout the application, the term "three-dimensional geometry" refers to the geometry of cells in a three-dimensional state (equal to or very similar to their natural state), unlike two-dimensional geometry for example as it was discovered in cells cultured in a Petri dish, where the cells are flattened and / or stretched. Not being limited by theory, the three-dimensional geometry of the cells is maintained, supported, and conserved in such a way that the cell can develop into three-dimensional cellular assemblies, tissues and / or tissue-like structures in the three-dimensional culture of the rotary bioreactor. , while at the same time, the three-dimensional geometry is maintained, and support cell by cell and geometry. By rotating the three-dimensional culture in the culture chamber, cells can maintain three-dimensional geometry, cell-to-cell geometry and support, unlike cells grown in moving environments, such as agitation, bubble utilization, and agitation. In addition, the rotation of the rotating bioreactor keeps the cells in close proximity to each other so that they can establish and maintain the three-dimensionality that is found in the microenvironment of cells in vivo. For each of the three above definitions, in relation to the conservation of "cell-to-cell support" and "cell-to-cell geometry" and "three-dimensional geometry" of the cells of the present invention, the term "essentially the same" " and substantially the same, "means that the natural geometry and the support are provided in the expansion, so that the cells are not changed in such a way as to be, for example, dysfunctional, toxic or harmful to the three-dimensional culture. Rather, the cells of the present invention, during and after the expansion, they mimic the situation in vivo. During operation, a cell mixture is placed in the culture chamber of the rotating bioreactor. In a preferred embodiment, the culture chamber is rotated for the period of time, while at the same time a TVEMF is generated in the culture chamber by the TVEMF source. By "while at the same time," it attempts that the beginning of the administration of the TVEMF may be earlier, concurrent with, or after the rotation of the culture chamber begins. In a more complex rotary bioreactor, a culture medium enriched for culture medium requirements preferably including, but not limited to, growth medium, buffer, nutrients, hormones, cytokines, and growth factors, which provides sustenance to the cells, it can be periodically refreshed and eliminated. The biological component contained in the three-dimensional culture of the rotating bioreactor can be analyzed at any time throughout the expansion process. In addition, the characterized biologically active compound can be introduced into the three-dimensional culture at any time before, during, or after the start of the three-dimensional culture in the rotary bioreactor. By the tests of the biological component, the biologically active compound can be characterized. During operation, a rotating bioreactor provides a stabilized culture environment into which cells can be introduced, suspended, assembled, cultured, and maintained with enhanced retention or development of delicate three-dimensional structural integrity by simultaneously reducing to the minimum the shear stress of the liquid , to provide three-dimensional freedom to the cell and to the spatial orientation of the substrate, and to increase the location of cells in a particular spatial region for the duration of the expansion. In a preferred embodiment these three criteria are provided in the controlled expansion of the cells in a rotary bioreactor (hereinafter referred to as "the three above criteria"), and at the same time, the cells are exposed to a TVEMF. Of particular interest of the present invention is the size of the culture chamber, the rate of sedimentation of the cells, the speed of rotation, the external gravitational field, the TVEMF, and interaction of the biological component and the biologically active compound.

The present invention shows that even a cellular degradative process in response to a biologically active compound will represent the degradative process in vivo. For example, characterizing a biologically active compound, such as a chemotherapeutic agent, by determining whether there is any reduction in the size and number of tumorigenic cells and tissue, and determining mechanisms of action may involve analyzing a biological component associated with it. . Any successful tumor reduction in response to a chemotherapeutic agent would characterize the efficacy of the chemotherapeutic agent. In this case, administration of the biologically active compound in the tumor can be analyzed for penetration into or around cells and methods by which such administration can be enhanced by other manipulations or drugs. The identification of drug distribution in the construction of tissue culture, within the internal cellular sub-volumes, or on the cell surface is critical for the precise understanding of effective drug administration (analysis of toxic compounds, or reagent actions) . The tumor of the cultivated model is then analyzed for the response to potential treatments (chemotherapy, radiation regime, nanoparticle function, or combinations thereof) by ultrastructural, molecular, immunological, and physical tissue and cellular analysis. In this preferred embodiment, the biologically active compound comprises immunoactive elements, such as compounds that contain antibodies or even living immune cells (which can be modified such as by adoptive immunotherapeutic means - killer T cell activation). As such, the biologically active compound can preferably contain a living cellular component which can be particularly well analyzed by the present invention given the freedom of movement for these elements then these can freely interact with the target tissue (tumor in this case). The stabilized culture environment referred to in the operation of the rotary bioreactor is to say the condition in the culture medium, particularly the fluid velocity gradients, before the introduction of cells, which will support an almost uniform suspension of cells at the time of their introduction of this form initiates a three-dimensional culture at the time of the addition of the cell mixture. In a preferred embodiment, the culture medium is initially stabilized in a horizontal rotation of the nearly solid body around an axis within the limits of a rotating chamber wall in a similar manner to a rotary bioreactor. In this condition the culture chamber walls are mobile with the same angular velocity as the culture chamber content because the transient start processes and the associated transient fluid velocity gradients are dissipated. The walls of the culture chamber are set in motion in relation to the culture medium to initially introduce the rotation in the contents of the culture chamber. During this transient process, which also occurs during the decrease in the rotational speed of the culture chamber, significant fluid velocity gradients and the associated liquid shear stresses are present. After the culture chamber and the content reach a steady state, these gradients are considerably reduced and the field of shear stress of the fluid is minimal. The cells are introduced into, and move through, the culture medium in the stabilized culture environment which thus initiates and maintains a three-dimensional culture. The three-dimensional culture moves under the influence of gravity, centrifugal forces, and Coriolis, and the presence of cells, particles, or any other element, within the culture medium, of the three-dimensional culture induces secondary effects to the culture medium. The term "elements" means that it includes anything present in the culture medium of the three-dimensional culture including, but not limited to, viruses, nanoparticles, waste cells, dead cells and any other object in the present. The significant movement of the culture medium with respect to the culture chamber, shear stress of the significant liquid, and other fluid movements, are due to the presence of these cells, particles, and / or elements within the culture medium. It is also preferred that some of these elements may be fixed with the rotation of the culture chamber wall for convenience or advantage, with other elements free to move within the liquid compartment within the culture chamber. Such "fixed" elements can be objects (such as substrates) that would otherwise be too heavy to be suspended by the rotating liquid alone, the elements that are damaged by even the low sedimentation induced by the cutting of the residual fluid within the culture chamber , negatively affected by unavoidable wall impacts experienced by freely suspended elements, for closer observation, or simply for the convenience of the operator (for example, locating a particular element later.) It is notable that the introduction of such "fixed" elements represents an improvement of the culture process itself, independent of the tests of the ogically active compound that are the main objective of this document, for example, an example would be to "hang" a substrate in the form of a heart valve inside a chamber rotating culture while introducing more cells for fixation in the substrate in order to build a to an improved heart valve. In most cases the cells with which the stabilized culture environment is the sediment primed at a slow speed preferably less than 0.5 centimeters per second. It is therefore possible, at this early stage of three-dimensional cultivation, to select from a wide range of rotary speeds (preferably from about 1 to about 120 RPM, more preferably from about 2 to about 30 RPM) and chamber diameters (preferably from approximately 1.27 to approximately 91.4 cm (0.5 to approximately 36 inches)). Preferably, the slower rotary speed is advantageous because it minimizes the wear of equipment and other logistics associated with the handling of the three-dimensional crop. Not being limited by theory, rotation on a substantially horizontal axis with respect to the external gravity vector at an angular velocity optimizes the orbital trajectory of suspended cells within the three-dimensional culture. During operation, the cells expand to form a mass of cellular assemblies, three-dimensional tissues, non-necrotic cell masses, and / or tissue-like structures that increase in size as the three-dimensional culture progresses. Interactions between cells, such as three-dimensional geometry and cell-to-cell geometry, support and essentially and substantially mimic what is found in natural sedimentation cells, the microenvironment in vivo. The progress of the three-dimensional culture is preferably evaluated by a visual, manual, or automatic determination of an increase in the diameter of the three-dimensional cell mass in the three-dimensional culture. An increase or decrease in the size and / or amount of the cell set, tissue, non-necrotic cell mass, or tissue-like structure in the three-dimensional culture may require appropriate adjustment of the rotation speed in order to optimize the trajectories particular. The rotation of the culture chamber optimally controls collision frequencies, collision intensities, and location of the cells in relation to other cells and also the boundary limits of the culture chamber of the rotary TVEMF eactor. In order to control the rotation, if it is observed that the cells deform excessively inward on the downward side and outwardly on the upward side then the revolutions per minute ("RPM") may be increased. If it is observed that the cells centrifuge in excess to the outer walls then the RPM can preferably be reduced. Not being limited by theory, when operating limits are reached, in terms of elevated cellular sedimentation rates or high gravity resistances, the operator may be unable to satisfy both conditions and may be forced to accept degradation in performance that is quantified against all three criteria previous The cellular sedimentation rate and the external gravitational field place a lower limit on the shear stress of the available liquid, even within the operating range of the rotary bioreactor, due to the gravitationally induced movement of the cells and / or elements by the medium of Cultivation of three-dimensional culture. The calculations and measurements place this shearing stress of the minimum liquid very close to that resulting from the terminal sedimentation rate of the cells and / or elements (by the culture medium) for the resistance of the external gravity field. Centrifugal and Coriolis induced motion [classical angular kinematics provides the equation that follows and relates the Coriolis force to the target mass (m), its velocity in a rotating frame (vr) and the angular velocity of the reference rotating frame ( Fc): FCorioiis = ~2 m (wx vr)] along with side effects due to cell and culture medium interactions, which act to further degrade the level of shear stress of the fluid as the cells expand. Not being limited by theory, as the field of external gravity is reduced, three dimensional structures much denser and larger can be obtained. In order to obtain the minimum level of shear stress of the liquid it is preferable that the culture chamber rotate substantially at the same speed as the culture medium. By not being limited by theory, this minimizes the velocity gradient of the fluid induced at the time of the three-dimensional culture. It is advantageous to control the speed and size of the tissue formation in order to maintain the cell size (and associated sedimentation rate) within a range for which the rate of expansion is able to satisfy the three above criteria. However, preferably, the velocity gradient and shear stress of the resulting liquid can be intentionally introduced and controlled for specific research purposes, such as studying the effects of shear stress on the three-dimensional cell assemblies. In addition, transient interruptions of the expansion process are allowed and are tolerated for, among other reasons, logistic purposes during the priming of the initial system, sample acquisition, system preservation, and three-dimensional cultivation termination. The rotation of cells around an axis substantially perpendicular to gravity can produce a variety of sedimentation rates, all of which according to the present invention remain spatially localized in different regions for extended periods of time ranging from seconds (when the sedimentation characteristics). they are large) at hours (when the differences in sedimentation are small). By not being limited by theory, this allows this sufficient time of cells to be related if necessary to form multicellular structures and associate with each other in a three-dimensional culture. The cells can preferably expand in the rotary bioreactor when necessary. The cells can preferably continue to expand in the rotary bioreactor for at least 160 days. The dimensions of the culture chamber also influence the path of cells in the three-dimensional culture of the present invention. A diameter of culture chamber is preferably chosen having the appropriate volume, preferably from about 15 ml to about 2 L for intentional three-dimensional cultivation and which will allow a sufficient plant density of cells. Not being limited by theory, the movement of external cells due to the centrifugal force is exaggerated with larger radii of the culture chamber and for fast sedimentation cells. Thus, it is preferable to limit the maximum radius of the culture chamber as a function of the sedimentation properties of the tissues expected in the final three-dimensional culture stages (when larger cell assemblies are formed with high rates of sedimentation). The trajectory of the cells in the three-dimensional culture has been analytically calculated incorporating the cellular movement that originates with gravity, centrifugation, and Coriolis effects. A computer simulation of these governing equations allows the operator to model the process and selected parameters acceptable (or optimal) for the particular three-dimensional crop. Figure 4 shows the typical shape of the cell orbit that is observed from the external (non-rotating) reference frame. Figure 5 is a graph of the radial deviation of a cell of the ideal circular aerodynamic line plotted as a function of the RPM (for a typical cell that sediments at a terminal velocity of 0.5 cm per second). This graph (Figure 5) shows the decrease in the amplitude of the deviation of radial cells of sinusoidal variation that is induced by gravitational sedimentation. Figure 5 also shows the increase in radial cell deviation (per revolution) due to centrifugation when the RPM is increased. These contrary constraints influence the choice of optimal RPMs to preferably minimize the cellular impact with, or accumulation in, the chamber walls. A family of curves is generated which is increasingly restrictive, in terms of selectable RPM selections, while increasing the external gravity field strength or increasing the rate of cellular sedimentation. This family of curves, or preferably the computer model that solves these governing orbiting equations, is preferably used to select the optimum RPM and chamber dimensions for the expansion of cells with a given sedimentation velocity in an external gravity field resistance Dadaist. Not being limited by theory, while expanding a typical three-dimensional culture of tissues, cell assemblies, and tissue-like structures, the size and velocity of sedimentation increases, and therefore, rotation, the velocity can preferably be adjusted to optimize it In the three-dimensional culture, it is observed that the cellular orbit (Figure 4) of the rotational reference frame of the culture medium moves to an almost circular path under the influence of the rotary gravity vector (Figure 6). When not being limited by theory, the two pseudo-forces, of Coriolis and centrifuge, originates the (accelerated) rotation of the frame of reference and the deformation of the almost circular trajectory. Higher gravity levels and higher cellular sedimentation rates produce circular trajectories with larger radii that equate to larger trajectory deviations of the ideal circular orbit as seen in the non-rotating reference frame.

In the rotating frame of reference it is thought, not being limited by theory, that the cells with different sedimentation velocities will remain spatially located close to each other for long periods of time with the net cumulative separation greatly reduced as if the gravity vector did not turn; the cells settle, but in a small circle (as observed in the rotating reference frame). Thus, during operation the rotary bioreactor provides cells with sedimentation properties that differentiate with sufficient time to interact mechanically and by soluble chemical signals that thus substantially mimic the same cell-to-cell support and the geometry found in vivo. , the present invention provides for sedimentation rates preferably from about 0 cm / sec to 10 cm / sec. In addition, during operation, the culture chamber of the present invention has at least one opening preferably for the entry of fresh culture medium, a cell mixture, a biological component, and a biologically active compound, and also the elimination of a volume of the spent culture medium containing metabolic waste and samples of the biological component, but not limited thereto. Preferably, the change of the culture medium can also be via a circuit of the culture medium where the fresh or recycled culture medium can be moved into the culture chamber preferably at a sufficient speed to support the change of metabolic gas, the administration of nutrients, and the elimination of metabolic waste. This may slightly degrade inactive three-dimensional culture. It is preferable, therefore, to introduce a mechanism for supporting preferred components including, but not limited to, respiratory gas exchange, nutrient administration, administration of growth factor to the culture medium of the three-dimensional culture, and also a mechanism for the elimination of the metabolic waste in order to provide a long-term three-dimensional culture, capable of supporting significant metabolic loads during periods of hours to months. The present invention preferably exposes the three-dimensional culture, and therefore the biological component and cells, to a TVBMF that not only provides for the accelerated expansion of cells that maintain their three-dimensional geometry and cell-to-cell support and geometry, but also may affect some cell properties during expansion, for example-regulation of genes that promote growth, or down-regulation of genes that prevent growth. The electromagnetic field is generated by a source of TVE F. During operation, an electrically conductive coil of a rotating bioreactor is preferably rotatable with the culture chamber, which means on the same axis as the culture chamber and in the same address. Also, the electricity conducting coil may preferably be fixed in relation to a culture chamber of a rotary perfused TVEMF-bioreactor. The electrically conductive coil may preferably be integral with, which means attached to and surrounding the outer portion of the culture chamber of the electrically conductive coil of the culture chamber of the rotating TVEMF bioreactor. The TVEMF source is operatively connected to the rotating TVEMF bioreactor. The method of the present invention provides these three prior criteria in a previously untapped manner and optimizes a three-dimensional culture, and at the same time, facilitates and supports the expansion such that sufficient expansion (increase in the amount by volume, diameter in reference to the tissue, or concentration) is detected in a sufficient amount of time. In addition to the qualitatively exclusive cells that are produced by the operation of the rotary bioreactor, as it is not limited by theory, greater efficiency with respect to the use of the total culture chamber volume for cell and tissue culture can be obtained due to the homogeneous substantially uniform suspension achieved. Advantageously, therefore, a rotating bioreactor, during operation, provides an increased number of cells in the same rotating bioreactor with lower human resources. Most cell types can be used in this method. The fundamental cell and tissue biology research as well as the clinical applications that require accurate in vitro models of cell behavior in vivo are applications in which the present invention and the method of using same provide an improvement because, as indicated above and in all parts of this application, the expanded cells and the tissue of the present invention have essentially the same three-dimensional geometry and cell-to-cell support and cell-to-cell geometry as natural, non-expanded, and tissue cells. It is useful to test a biologically active compound in an environment that is so closely mimicking the situation in vivo. The toxicity and efficacy of a biologically active compound can be analyzed to characterize the biologically active compound. To analyze a biologically active compound, the formation of an exact in vitro tissue model is highly desirable. A rotary bioreactor is capable of providing unique and useful in vitro conditions that include an essentially inactive three-dimensional culture in which cells can respond to biologically active compounds in a manner that closely represents the microenvironment in vivo.

The different classes of drugs have clearly different mechanisms of action but there are general features shared by the drug development process to which the present invention is directed. For example, in the case of antiviral drugs, the complete life cycle of the viral particle offers the opportunity for intervention. The viral life cycle at least includes the initial transport of the viral particle and localization near the target cell, cell membrane penetration, genetic incorporation, manufacture of the viral subparticle, viral particle unit, and viral release. These particular steps in the viral life cycle are stages in which biologically active compounds, such as antiviral drugs are targeted and analyzed for their effectiveness. Therefore, such tests require high fidelity and behavior at the cellular and multicellular tissue level that closely mimic the in vivo microenvironment that are provided by the expansion in a rotary bioreactor, as in the present invention. Key examples of viruses that can be preferably analyzed by the method of the present invention include HTV, Avian influenza, Hepatitis, herpes viruses, and include conventional RNA-based DNA as well as retroviral (reverse transcriptase-dependent) infectious viral particles. . A similar program can be deduced to preferentially analyze the effects of a biologically active compound preferably an antibacterial agent on a bacterial infection that can be analyzed for toxic syndromes with respect to toxin exposure and provide methods of corrective intervention (e.g. , exposure of heavy metals). Other biologically active compounds can be preferably analyzed for their effects on normal cellular and tissue functions and morphology, including whether biologically active compounds can adjust these normal tissue and cellular functions to potentially restore normal cellular and tissue function or inhibit disease states. Without being limited by theory, in diseased states normal cellular and tissue functions are over or under expressed, often due to problems of regulation and feedback mechanisms. For example, in the case of Adult onset Diabetes, the cellular response to insulin (to admit glucose) is inadequate due to rare cell membrane insulin receptors, ineffective receptors, or due to blocked receptors (antibodies). The steady flow of drugs analyzed that target Adult onset Diabetes is aimed at restoring these cellular functions and structures to normal. An in vitro model that substantially mimics the situation in vivo, where cells having substantially the same three-dimensional geometry and cell-to-cell support and geometry as the microenvironment in vivo, and which prolongs cellular and tissue functions that mimic the in vivo situation, are provided in the present invention to analyze the effects of biologically active compounds, and therefore, characterize it.

Preferably, at the same time this efficiency of a biologically active compound is being analyzed, the toxicity to the cells and / or target tissue as well as other unrelated tissues, which may also be exposed to the drug and which may also be cultured to improve the fidelity, in vitro, can also be analyzed by the methods of the present invention. Preferably, tissue and abnormally functioning cells can be cultured in a similar manner in a three-dimensional culture of a rotary bioreactor for evaluation of the efficacy of the potential biologically active compound against the pathogenic target such as, but not limited to, tissue (carcinogenic) malignly transformed. Some preferred biologically active compounds that can be analyzed against malignant tissue include, but not limited to, chemotherapy, radiotherapy, antimetastatic, deprivation of tumor vasculature, and nanoparticle agents. Also, preferably, the hybrid cell lines can be analyzed by the methods of the present invention. It is notable that the future has great hope in the nanoparticle (the term "nanoparticles" means artificial bioactive particles) the treatment modalities (for cancer as well as the improvement in a disease state that has not undergone changes) and the development of these will be dependent on exact tissue culture in in vitro models (both affected and normal tissues). Preferably, therefore, the biologically active compounds that are directed to tests of the functions of the nanoparticle including, among other things, the directed movement of functions that follows, point to the identification, adhesion, entry, direct particle intervention , secondary particle functions, such as drug release, life / particle cycle, failure, and elimination can be analyzed by the expansion process of the present invention in a rotary bioreactor. Preferably, the hybrids comprising in the altered virus to meet therapeutic objectives can be similarly analyzed. The present invention also provides a method preferably of analysis of the mechanisms of modulation, change, or correction of stem cell renewal and pharmacologically differentiation development along routes directed both to renewal of the stem cell (or progenitor cell) congregation and producing the desired tissue (or lineage of intended parent) of stem cells. The present invention, in a preferred embodiment, provides a method to accurately expand the stem cells in vitro while at the same time substantially maintaining the same three-dimensional geometry, cell-to-cell support and geometry as discovered in vivo. In a preferred embodiment, a three-dimensional culture having stem cells that can be analyzed for their response in direct and direct contact control mechanisms can also be preferably analyzed without direct clinical use, such as evaluation of graft quality. To clarify the broad applicability of the present invention, some additional preferred embodiments include the diuretic test performance and kidney toxicity in the kidney tubule and matrix complexes; the responses to blood pressure control treatments in the smooth muscle were expanded in a rotary bioreactor; and the tests of biologically active compounds in autoimmune models. The present invention is well adapted to realize objectives and obtain the purposes and advantages mentioned herein, as well as those inherent therein. Without departing from the scope of the invention, it is intended that all the matter contained herein be construed as illustrative and not limiting. It will be apparent to those skilled in the art that various changes can be made in the invention without departing from the spirit and scope thereof and therefore the invention is not limited by what is included in the figures and specifications, but only as indicated in the appended claims.

METHOD OF OPERATION During operation, a rotary bioreactor that preferably has a culture chamber of 15 ml to approximately 2 l, is completely filled with the appropriate culture medium, preferably supplemented with albumin (5%) and also preferably G-CSF for human cells to be expanded, with the fourth only for any additional intentional volume of the culture medium, cells, biologically active compounds, and / or other preferred components of the culture medium of the intentional three-dimensional culture. Preferably a controlled room incubator that completely surrounds the rotary bioreactor and is preferably determined for approximately 5% C02 and approximately 21% oxygen, and the temperature is preferably from about 26 ° C to about 41 ° C, and more preferably about 37 ° C ± 2 ° C. Preferably the rotary bioreactor may also have an integral thermometer, heater, and air control (including control of C02, 02, and / or nitrogen). At the beginning, a stabilized culture environment is formed in the culture medium. The rotation may preferably begin at approximately 10 RP. 10 RPM is the preferred speed that produces an orbital path of the microcarrier bead in which the beads do not accumulate appreciably to the chamber walls by gravitationally induced sedimentation or by rotationally induced centrifugation. In this way, the rotary bioreactor produces the minimum gradients of fluid velocity and shear forces of the liquid in the three-dimensional culture. If the cellular binding substrates are to be used, the cellular binding substrates are preferably introduced simultaneously or sequentially with cells in the culture chamber to provide an appropriate density, preferably 5 mg of the cellular binding substrate per ml of the culture medium, and preferably the cell-binding substrate for anchoring the dependent cells are microcarrier beads. The cell mixture is preferably injected into the stabilized culture environment to initiate a three-dimensional culture through an opening in the culture chamber, preferably with respect to a short period of time, preferably 2 minutes, to minimize cell damage while passing through the system. of administration. Preferably, the cell mixture and / or the cell binding substrate, if used, are administered via a syringe.

After the injection of the cells is completed, the culture chamber is quickly returned to the initial rotation on a substantially horizontal axis, preferably in less than one (1) minute, preferably 10 RPM, thus returning the shear stress of the liquid to the level minimum affordable for cells. During the initial charge and fixation phase, the cells are allowed to reach equilibrium for a short period of time, preferably from 2 hours to 4 hours, more preferably for a sufficient period for transient fluxes to be impregnated. The biologically active compound to be analyzed in the present invention is introduced into the three-dimensional culture before, during, and / or after the expansion of the cells. The method of introducing the biologically active compound will depend on the state that the biologically active compound (ie, gas, liquid, or solid) obtains, and also the opening with the biologically active compound should be introduced into the culture chamber. In addition, the concentration will also depend on the preferred test to be finally carried out and the desired result of the biologically active compound. While the expansion of the three-dimensional culture progresses, the increase in the size and the sedimentation rate of the assembled cells is expected, according to the effect of a biologically active compound, and the system of rotary speeds can be increased (increasing in increments preferably of approximately 1). to 2 RP) in order to reduce the gravitationally induced orbital deformation of the ideal circular flow lines of the current pieces of tissue with larger diameter. According to the effects of the biologically active compound, the assembled cells, or cell mass, can increase or decrease in size. In one way or another, the rotational speed of the three-dimensional culture may necessarily be adjusted to prevent collision with the inner portion of the rotating bioreactor. The impacts of the wall are not preferred, however, these are possible. A rotary bioreactor, however, foresees any impact, in all, it will be sufficiently under the energetic impact so that this does not interrupt the inactive three-dimensional culture. During the expansion, the rotational speed of the three-dimensional culture in the culture chamber can be evaluated and adjusted so that the cells in the three-dimensional culture remain substantially at or on the horizontal axis. The increase of the rotary speed is guaranteed to prevent the excessive impact with the wall, which is detrimental to the three-dimensional growth even more of the delicate structure. For example, an increase in rotation is preferred if the cells in the three-dimensional culture fall excessively inward and downward on the downward side of the rotation cycle and in externally and insufficiently upward excess on the upstream side of the rotation cycle . Optimally, the user is advised to select preferably a rotating speed that causes the minimum frequency of collision with the wall and the intensity to maintain the three-dimensional geometry and cell-to-cell support and cell-to-cell geometry of the cells. The preferred speed of the present invention is from about 2 to about 30 RPM, and more preferably from about 10 to about 30 RPM. The three-dimensional culture can preferably be visually evaluated by the preferably transparent culture chamber and adjusted manually. The evaluation and adjustment of the three-dimensional culture can also be automated by a sensor (eg, a laser), which monitors the position of the cells within the culture chamber. A sensor reading that indicates too much cell movement will automatically cause a mechanism to adjust the rotational speed accordingly. After initial loading of the cell mixture and preferably the fixation phase if the cellular binding substrates are used (2 to 4 hours), in a preferred embodiment of the present invention, the TVEMF source is turned on and adjusted so that the output of TVEMF generates the desired electromagnetic field in the three-dimensional culture in the culture chamber. The TVEMF can also be preferably applied to the three-dimensional culture during the initial charge and fixation phase. It is preferable that the TVEMF be provided to the three-dimensional culture for the length of the expansion time until it is finished. The size of the electrically conductive coil, and the number of times it is entangled around the culture chamber of the rotating TVEMF bioreactor, are such that when a TVEMF is provided to the electrically conductive coil a TVEMF is generated within the culture. three-dimensional in the culture chamber of the rotating TVEMF bioreactor. The TVEMF is preferably selected from one of the following: (1) a TVEMF with a force amplitude less than 100 gauss and rotational speed greater than 1000 gauss per second, (2) a TVEMF with a bipolar square wave of low amplitude force at a frequency lower than 100 Hz, (3) a TVEMF with a square wave of low force amplitude with less than 100% duty cycle, (4) a TVEMF with rotation speed greater than 1000 gauss per second for pulses of duration less than 1 millisecond, (5) a TVEMF with pulses similar to bipolar delta function of rotation speed with a duty cycle less than 1%, (6) a TVEMF with a force amplitude less than 100 gauss pulses similar to peak-to-peak bipolar delta function and rotation speed and where the duty cycle is less than 1%, (7) a TVEMF applied using a solenoid coil to form uniform force resistance throughout the cell mixture, (8) and a TVEMF applied using a flow concentrator to provide spatial gradients of magnetic flux and magnetic flux that are concentrate inside the cell mixture. The frequency range in the oscillating electromagnetic force resistance is a parameter that can be selected to achieve the desired stimulation of the cells in the three-dimensional culture. However, these parameters do not mean that they are limited to the TVEMF of the present invention, and as such may vary based on other aspects of this invention. The TVEMF can be quantified for example by conventional equipment, such as an EN1 31 Cellular Sensor Gaussometer. Rapid cell expansion and increased total metabolic demand may require the intermittent addition of preferable components that enrich the culture medium in three-dimensional culture including, among other things, nutrients, fresh growth medium, growth factors, hormones, and cytokines This addition is preferably increased if necessary to maintain glucose and other nutritional levels. During rapid tissue and cell expansion in the rotary bioreactor, the culture medium comprising the residue can preferably be removed if necessary. Samples of the biological component can also be removed from the three-dimensional culture to be analyzed and crop chamber rotation can be temporarily stopped to allow practical management. The three-dimensional culture can be preferably allowed to progress beyond the point at which it is possible to select excellent orbits of cells; to a point when gravity has introduced coercions that somewhat degrade performance in terms of low effort of three-dimensional cultivation. In addition, after expansion, the cells can be used for therapeutic purposes to include for tissue regeneration, research, and disease treatment. The following examples are preferred illustrations of the invention, but these are not intended to limit the invention thereto.

EXAMPLE 1-EXPANSION OF ADULT STEM CELLS AND A BIOLOGICALLY ACTIVE COMPOUND Preparation A 75 ml culture chamber of a rotary bioreactor, illustrated in the preferred embodiment of Figures 1 and 2, may preferably be prepared by washing with the disinfectant solution. detergent and germicide (Roccal II) with the recommended concentration for disinfection and cleaning followed by copious rinsing and soaking with high quality deionized water. The rotary bioreactor can be sterilized by autoclaving then rinsed once with the culture medium. If a disposable culture chamber of a rotary bioreactor is used then preferably the disposable culture chamber is already sterilized and simply has to be removed from any container and assembled in the motor. For the preferred embodiment having an electrically conductive coil, the electrically conductive coil is connected to the TVEMF source. rotating bioreactor.

Peripheral Blood Stem Cell Expansion The rotary bioreactor may be preferentially filled with the culture medium comprising the modified Dulcocco's Isocove medium (IMDM) (GIBCO, Grand Island NY.) Supplemented with 5% human albumin, 100 ng / ml of recombinant human G-CSF (Amgen Inc., Thousand Oaks, CA), and 100 ng / ml recombinant human stem cell (SCF) factor (Amgen). In addition, D-penicillamine (D (-) -2-amino-3-mercapto-3-methylbutonic acid) (Sigma-Aldrich) a copper chelating agent, dissolved in DMSO, can be preferentially introduced into the culture medium in the rotary bioreactor in an amount of 10 ppm. Adult blood stem cells from the peripheral blood (CD34 + / CD38-) can be preferably placed in the culture chamber of the rotary bioreactor at a concentration of 0.75 x 106 cells / Ml. Preferably, the culture chamber is equilibrated before the cell mixture is placed. If a flow path of culture medium is used, as depicted in the preferred embodiment in Figure 3, then the equilibrium of the culture medium is also preferable, to form a stabilized culture environment. The stabilized culture environment provides substantially low levels of shear stress for the addition of the cell mixture. The engine should be turned on, preferably at a speed of approximately 30 RPM. If the culture chamber and culture medium have been balanced, the rotation speed should return slowly to the preferred speed of rotation. The rotation of the rotary bioreactor can preferably be evaluated every day, and adjusted to maintain rotation at a speed to prevent impact with the wall and the cells of the three-dimensional culture are maintained substantially in the middle of the culture chamber. The TVEMF source can also preferably be turned on at a preferred gauss range and oscillation, preferably from about 1 mA to about 1000 mA. The expansion should preferably be allowed to continue for seven days and then completed, at this time, the cells were analyzed.

Samples and Results At least two samples of peripheral blood stem cells should preferably be expanded in the rotary bioreactor under prior conditions as stated. Sample 1 should be expanded for seven days and then evaluated for viability under a microscope. It is expected that the cells in the first sample remain healthy and multiply. A biologically active compound should preferably be introduced into Sample 2 at the start of the three-dimensional culture. The biologically active compound may preferably be 3 ppm of Pseudomonas aeruginosa bacteria. Expansion conditions between Samples 1 and 2, regardless of the bacteria, should preferably be the same. After seven days, the experimental cultures should be terminated and the viability of the cells evaluated under a microscope. The microscopic examination is expected to reveal that the cells of Sample 2, containing a bacterial biologically active compound, will be dead while the cells in Sample 1 will remain viable and healthy. Such results predict that this preferred that the bacteria are likely to be toxic if allowed to enter the peripheral blood stream. The viability of the cells can be determined by any known and accepted method known in the art.

EXAMPLE 2-EXPANSION OF RENAL RAT CELLS AND A BIOLOGICALLY ACTIVE COMPOUND Preparation The rotary bioreactor should be prepared as in Example 1 above.

Expansion of Rat Kidney Cells Rat kidney cells may be preferentially isolated from the renal cortex collected from Sprague Dawley rats subjected to euthanasia (Harían Sprague Dawley, Cleveland Ohio). In summary, the renal cortex can be preferably dissected with scissors, finely and subtly cut in kidney cell buffer, 137 mmol NaCl, 5.4 mmol KC1, 2.8 mmol CaC12, 1.2 mmol MgC12, 10 HEPES-Tris mmol, pH 7.4. The finely cut tissue can be preferably placed in 10 ml of a 0.1% solution of type IV collagenase and 0.1% trypsin in normal saline. The solution containing the tissue can be preferentially incubated at 37 ° C, stirring the water bath for 45 minutes with intermittent titration. The cells can preferably be placed in a centrifuge and centrifuged gently (800 revolutions per minute for 5 minutes), the supernatant aspirated, the cells resuspended in 5-cell kidney buffer with 0.1% bovine serum, and passed fine mesh ( 70 mm). The fraction passing through the mesh can preferably be formed in a layer above a discontinuous gradient of 5% bovine serum albumin and gently centrifuged (800 revolutions per minute for 5 minutes). The supernatant should again be discarded leaving a cell pellet of rat kidney cells. At this point, the cells can be preferably frozen (preferably at -80 ° C, more preferably in liquid nitrogen) when necessary for future use. The granular pellet of rat kidney cells can preferably be suspended again in the DMEM / F-12 medium (ciprofloxacin and fungizone subjected to treatment), at a concentration which is approximately 1 x 10 6 cells / ml. At least two cell samples (1 x 10 6 cells / ml) are preferably expanded in the culture chamber of a rotary bioreactor. The rotary bioreactor should be placed in 5% C02, incubation in 02 to 95%, or have an integral air and temperatures adjusted to it. Rat kidney cells should preferably be expanded for 7 days.

Samples and Results Sample 1 of rat renal cells should be expanded without any additives that include any biologically active compound. A biologically active compound, 10 ppm of the diisoctyl phthalate plasticizer, should be introduced into Sample 2 preferably at the start of the three-dimensional culture. Both Samples, 1 and 2, should have the viability of the cells evaluated, preferably after 7 days, by methods known in the art such by microscopic determination. It is expected that the cells in Sample 1 will expand by at least seven times as many as were placed in the rotary bioreactor. On the other hand, it is expected that most of the cells in Sample 2 die. Such results predict that diisoctyl phthalate is toxic if allowed to be introduced into the body and accumulate in the renal cells. Other than the biologically active compound, all other conditions are preferably the same as in Samples 1 and 2. In addition, culture conditions and rotation of the rotary bioreactor should preferably be the same as in Example 1. However, the three-dimensional culture does not it is preferably exposed to a TVEMF in this Example 2.

EXAMPLE 3-EXPANSION OF RENAL RAT CELLS AND A BIOLOGICALLY ACTIVE COMPOUND Preparation The rotary bioreactor should be prepared as in Example 1 above.

Expansion of Rat Kidney Cells, Samples and Results In Example 3, Example 2 should be repeated except that in Sample 2, the diisoctyl phthalate plasticizer is preferably substituted by 10 ppm cisplatin. The test is preferably repeated 10 times under the same conditions as in Example 2. In almost all cases, it is expected that the cells in Sample 1 remain viable. It is expected that the cells in Sample 2, and in most cases have 10 ppm of cisplatin, the rat kidney cells will remain healthy and viable. Such results predict, therefore, that adding 10 ppm of cisplatin to the rat kidney cells and expanding them in a rotary bioreactor do not produce any adverse effects, finally it is suggested that cisplatin may result, in fact, helpful in the prevention of kidney failure. Additional studies should be conducted for the prevention of renal failure by the use of cisplatin before using cisplatin in humans.

EXAMPLE 4 EXPRESSION OF PERIPHERAL BLOOD STEM CELLS AND A BIOLOGICALLY ACTIVE COMPOUND Preparation The rotary bioreactor should be prepared as in Example 1 above.

Expansion of Peripheral Blood Stem Cells The rotary bioreactor can preferably be filled with the culture medium comprising the modified Dulcocco's Isocove's medium (IMDM) (GIBCO, Grand Island NY), supplemented with 5% human albumin, 100 ng / ml of recombinant human G-CSF (Amgen Inc., Thousand Oaks, CA), and 100 ng / ml of recombinant human stem cell factor (SCF) (Amgen). In addition, D-penicillamine (D (-) -2-amino-3-mercapto-3-methylbutonic acid) (Sigma-Aldrich) a copper chelating agent, dissolved in DMSO, can be preferentially introduced into the culture medium in the rotary bioreactor in an amount of 10 ppm. Adult peripheral blood stem cells (CD34 + / CD38-) can be preferably placed in the culture chamber of the rotary bioreactor at a concentration of 0.75 x 106 cells / ml. Two samples of peripheral blood stem cells should preferably be prepared by this method. Sample 1 should preferably be from a patient without known liver damage. Sample 2 should preferably be from a patient with known liver damage. The samples should be prepared as above and placed in two different rotary bioreactors under conditions observed in Example 1 and at the same concentrations. The biologically active compound, 20 ppm of acetaminophen, should be added to Sample 2 at the start of the three-dimensional culture. Both Samples 1 and 2 should preferably be exposed to a TVEMF of about 1 mA to about 1000 mA as in Example 1 for the duration of the expansion process.

Results Preferably, at the end of 14 days the viability of each sample should be evaluated and the number of cells counted, for example under a microscope with a hematocytometer. It is expected that the cells in Sample 1 expand to at least ten times the amount that were placed in the rotary bioreactor. It is also expected that the cells in Sample 2 neither die nor grow, but remain unchanged. Such results predict a potential problem of regenerating liver tissue in the presence of 20 ppm acetaminophen. More tests should be carried out to determine the effects of exposing liver cells to acetaminophen. It is expected, therefore, that rapid and significant cell expansion be carried out by the expansion in the rotary bioreactor of the present invention, as described herein. It is also expected that the rapid and significant expansion is accompanied by a three-dimensionality and cell-cell interactions that is substantially similar to the in vivo microenvironment. Various changes can be made without departing from the spirit and scope of the invention, and therefore, the invention is not limited by whatever is included in the figures and specification, including the examples. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (22)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Method for characterizing a biologically active compound, characterized in that it comprises: • placing a cell mixture in a rotating bioreactor to initiate a three-dimensional culture where the three-dimensional culture comprises cells and a biological component; • controlled expansion of cells in the three-dimensional culture while at the same time maintaining cells with three-dimensional geometry and support and cell-to-cell geometry by rotating the rotating bioreactor; • introduction of a biologically active compound to the three-dimensional culture; and • testing the biological component using a test to characterize the biologically active compound. Method according to claim 1, characterized in that the biological component is selected from the group comprising a cell, a portion of a cell, secreted materials (mucin, collagen, matrix), secreted hormones, secreted intercellular structural components, structural matrices introduced, adhesion matrices, growth substrates, nanoparticles, soluble intercellular signals, cell membrane surface markers, membrane binding enzymes, immune identity markers, adhesion molecules, vacuoles, neurotransmitters stored and released, specialized cellular internal machinery , glycogen, culture media, test compounds, suspicious toxins in the test, test reagents, fungus, conjugate complexes, tissue, enzymes, DNA, RNA, virus, protein, artificial bioactive particles, and a gene. Method according to claim 1, characterized in that the rotary bioreactor comprises a rotating culture chamber wall and where a portion of the three-dimensional culture is fixed with respect to the wall of the rotary culture chamber. 4. Method according to claim 1, characterized in that the step of controlling the cells further comprises the exposure of the cells to an electromagnetic field with temporal variation ("TVEMF"). Method according to claim 1, characterized in that the cells are expanded at least seven times the amount that was placed in the rotary bioreactor. Method according to claim 1 or 4, characterized in that the cells are selected from the group comprising eukaryotes, prokaryotes, animal, fungus, plant, abnormally functioning cells, cells containing nanoparticles, hybrid cells, altered viruses containing hybrids cell phones. Method according to claim 6, characterized in that the animal cells are adult mammalian stem cells. Method according to claim 1, characterized in that the test is for at least one of the group comprising graft quality, toxicity, efficacy, pathology, tumorogenicity, genetic expression, karyotype, characteristics of the growth rate, morphology multicellular, individual cell morphology, intercellular relationships, metabolic measures, a portion of a viral life cycle, diuretic performance, renal toxicity, blood pressure control and functions of nanoparticles. Method according to claim 1, characterized in that the biologically active compound is in a form selected from the group comprising powder, liquid, vapor, and gas. Method according to claim 1, characterized in that the biologically active compound is at least selected from the group comprising a protein, at least one cell, toxin, reagent, chemical substance, gas, metal, metal compound, radiation, less a nanoparticle, at least one virus, an antibacterial protein, electroporation, chemical poration, an activated derivative of an immune cell, and water. 11. Method of compliance with claim 9, characterized in that the test is to characterize at least one mechanism selected from the group comprising pharmacologically modulating the renewal of stem cells, the alteration of the renewal of stem cells, the correction of the renewal of stem cells, pharmacologically modulating the differentiation of stem cells , the change of differentiation of stem cells, and correction of stem cell differentiation. Method according to claim 1, characterized in that the rotary bioreactor is rotated at a speed of approximately 1 revolutions per minute up to approximately 120 revolutions per minute. Method according to claim 1, characterized in that the rotary bioreactor is rotated at a speed of approximately 2 revolutions per minute up to approximately 30 revolutions per minute. 14. Method according to claim 1, characterized in that the biologically active compound is introduced before the stage of controlled expansion of the cells. 15. Method according to claim 1, characterized in that the biologically active compound is introduced during the stage of controlled expansion of the cells. Method according to claim 1, characterized in that the biological component is analyzed before placing the cell mixture in the rotary bioreactor. Method according to claim 1, characterized in that the biological component is analyzed during the stage of controlled expansion of the cells. Method according to claim 1, characterized in that the biological component is analyzed after the stage of controlled expansion of the cells. 19. Method according to claim 1, characterized in that the biological component is analyzed during and after the stage of controlled expansion of the cells. Method according to claim 1, characterized in that the biological component is analyzed before, during, and after the stage of controlled expansion of the cells. 21. Method according to claim 1, characterized in that it further comprises using the biological component for a tissue graft in a mammal. 22. Method according to claim 4, characterized in that the TVEMF is selected from the group comprising a TVEMF with a force amplitude less than 100 gauss and rotation speed greater than 1000 gauss per second, a TVEMF with a bipolar square wave of amplitude substantially of low force with a frequency of less than 100 Hz, a TVEMF with a square wave of amplitude of substantially low force with less than 100% of the duty cycle, a TVEMF with rotation speeds greater than 1000 gauss per second for pulses of duration less than 1 millisecond, a TVEMF with pulses similar to a bipolar delta function of rotation speed with a duty cycle of less than 1%, a TVEMF with a force amplitude less than 100 gauss, and pulses similar to a bipolar peak-to-peak delta function and of rotation speed and where the duty cycle is less than 1%, a TVEMF applied using a solenoid coil to form the uniform force resistance in all p three-dimensional culture arts, and a TVEMF applied using a flow concentrator to provide spatial gradients of magnetic flux and the magnetic flux is concentrated within the three-dimensional culture.