Patent Publication Number: US-2010120136-A1

Title: Bioreactor for cell and tissue culture

Description:
FIELD OF THE INVENTION  
     The present invention relates to a bioreactor or a culture vessel for incubation of one or more cell cultures, tissue biopsies, cell clusters, tissue-like structures, “prototissues” or similar samples. 
     BACKGROUND OF THE INVENTION 
     During “classical” cell culture in an essentially flat culture vessel, cells in general and biopsies in particular tend to de-differentiate. Visibly, biopsies exhibit the ‘melting ice-cream effect’ as cells migrate from a block of tissue out onto the flat supporting surface of the culture vessel. Gene expression is altered in these “migrating” cells, which begin to behave biochemically as isolated cells rather than as cellular components of a differentiated tissue. De-differentiated cells express different biochemical pathways than those normally expressed by corresponding cells in an intact organism. 
     In contrast with “classical” cell culture conditions, “microgravity” conditions preserve the differentiation state of many types of cells in culture. Microgravity bioreactors maintain microgravity conditions by continuous rotation of a typically cylindrical or tubular incubation compartment. This rotation continuously forces cells towards the center of the incubation chamber, suspending the cells in a fluid environment using a minimum shear force. This induces them to interact and to aggregate into colonies. For microgravity culturing, cells are often initially sown out onto small (ca. 100 μm diameter) beads (this accelerates the formation of microtissue structures but is not essential). As prototissues are formed by cell growth around these beads, the beads are often ejected or become completely covered with cells. Prototissues formed in this manner become very highly differentiated so as to resemble adult tissue. 
     Microgravity bioreactors have been used in a variety of contexts. Early studies showed that microgravity bioreactor systems helped cells form three dimensional structures by reducing shear stress on the cells [Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. Goodwin T J, Prewett T L, Wolf D A, Spaulding G F. J Cell Biochem. 1993 March; 51(3):301-11. 
     Now a significant body of literature demonstrates increased differentiation of cells grown in a microgravity bioreactor system. For a review see: [Growing tissues in microgravity. Unsworth B R, Lelkes P I. Nat Med. 1998 August; 4(8):901-7.] For example, microgravity culturing induces neural precursor cells to form cellular clusters or “neurospheres”. These prototissues are characterized by a crude, but organized, architecture, having a surface layer of immature proliferating cells (nesting- and proliferating cell nuclear antigen-positive) that enclose strata of more differentiated cells (beta-tubulin III- and glial fibrillary acidic protein-positive). These “neurospheres” show promise for development of neurotransplantable tissue. See e.g. [Neural precursor cells form rudimentary tissue-like structures in a rotating-wall vessel bioreactor. Low HP, Savarese T M, Schwartz W J./n vitro Cell Dev Biol Anim. 2001 March; 37(3):141-7.] and see [Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor. Saporta S, Willing A E, Shamekh R, Bickford P, Paredes D, Cameron D F. Brain Res Bull. 2004 December 150;64(4):347-561. 
     Or for another example, microgravity culturing of a multipotential human retinal cell line induced expression of a nearly in vivo phenotype, which could not be achieved when the cells were grown under other conditions [Generation of 3D retina-like structures from a human retinal cell line in a NASA bioreactor. Dutt K, Harris-Hooker S, Ellerson D, Layne D, Kumar R, Hunt R. Cell Transplant. 2003;12(7)117-31.] 
     Some technical problems with microgravity bioreactors have been reported. For example, when temporomandibular joint (TMJ) disc tissues were engineered using either flat culture or a microgravity bioreactor, there were no significant differences in total matrix content and compressive stiffness, notwithstanding marked differences in gross appearance, histological structure, and distribution of collagen types I and II (with the bioreactor discs having more collagen type II). The authors concluded that improvements of the microgravity bioreactor culture system were needed [Detamore M S, Athanasiou K A. Use of a rotating bioreactor toward tissue engineering the temporomandibular joint disc. Tissue Eng. 2005 July-August; 11(7-8):1 188-97]. 
     Although well known and widely used, currently available microgravity bioreactors have significant limitations: 
     Currently, the minimum size of a microgravity bioreactor is about 10 ml. In many circumstances, a much smaller volume is desirable, in order to reduce consumption of precious materials and/or facilitate “high throughput” testing. For example, a smaller volume bioreactor would reduce amounts of both cells and compounds used in testing pharmaceutical compounds at different concentrations and times. Reduction of materials consumption is especially important in radioactive labelling experiments, where costs and hazards can be especially high. To give a practical example, during investigation of effects of new pharmaceutical compositions on gene expression or other biological responses of cultured cells, multiple concentrations would typically be tested over multiple time points. For statistical power such analyses are typically repeated multiple times. A single experiment would require as much as 2.5 litres of cells, where one composition is tested in 5 replications at 5 concentrations, for 10 time points, using a 10 ml bioreactor. If metabolic incorporation of a radioactive label was required at, for example, 1 mCi/ml, such an experiment would require 2.5 Ci of radioactivity. Accordingly, it would be advantageous to provide a microgravity bioreactor with much smaller incubation compartment volume. 
     Another significant limitation of microgravity bioreactors of the prior art is moisture loss, which affects cell growth. De-hydration (even only by 5-10%) during incubation can result in changes in pH and other concentration-dependent parameters, such as concentrations of salts, nutrient substances, and the like. Many cell types are highly sensitive to their environment. For such cells, even a small change in such environmental conditions can influence cell growth and gene expression. This problem is especially pronounced in a small volume bioreactor, where small changes in volume can cause relatively large changes in concentration-dependent parameters. Without some solution to this de-hydration problem, a small volume bioreactor would experience rapid loss of moisture, notwithstanding maintenance of humidified conditions (100% relative humidity) in the incubator where the bioreactor was used. This tendency for rapid de-hydration in a small volume bioreactor, that is, this tendency for rapid change in relative volume greatly increases the need for time-consuming manual monitoring and manipulation, for example to replenish or exchange culture medium. This tendency effectively renders long-term maintenance of cultures in a small volume bioreactor impractical or impossible. Accordingly, it would be advantageous to provide a microgravity bioreactor with very high relative water retention in the cell incubation compartment. 
     Still another limitation of microgravity bioreactors of the prior art is that access ports used for adding or removing cells and growth medium have typically relied on conventional “luer lock” closures. These and similar closures have a finite ‘dead’ volume and this becomes proportionally larger as the volume of the bioreactor is reduced. This disadvantage can be circumvented by using ports of essentially no dead volume. 
     Luer lock closures can also lead to presence of air bubbles in the incubation compartment. Bioreactors are preferably kept free of air bubbles in the incubation compartment which otherwise have detrimental effects, breaking up the prototissues. This air bubble problem is especially pronounced in a small volume bioreactor, where a single bubble can represent a relatively significant volume. Some solutions to the air bubble problem are known in the prior art. For example, WO 95/07344 provides a reservoir chamber for entrapping gas bubbles away from the incubation compartment. However, these solutions would be wholly unsuitable for a small volume bioreactor because of the volumes involved. A better solution is thus to provide a closure mechanism for access ports that excludes any possibility of introducing air bubbles into the incubation compartment. 
     Conventional “luer lock” and similar closures also increase fluid turbulence and this can lead to increased shear forces which will have a detrimental effect on the prototissues. Microgravity bioreactors require continuous rotation of the incubation compartment to maintain microgravity conditions for differentiated or differentiating cells and other tissues. If the incubation compartment inner surface is not suitably adapted, it may give rise to turbulence. Such turbulence may lead to tearing or “shearing” of prototissues. Accordingly, it is advantageous to provide microgravity bioreactors with an access port closure mechanism that avoids turbulence. 
     WO 95/07344, U.S. Pat. No. 5,153,131, U.S. Pat. No. 5,437,998, U.S. Pat. No. 5,665,594, U.S. Pat. No. 5,989,913, and U.S. Pat. No. 6,642,019 each disclose improvements of microgravity bioreactors. US 2005/0084965 discloses use of a conventional, commercially available microgravity bioreactor for incubating hepatocyte spheroids. However none of these patents or published applications addresses the problem of de-hydration or discloses a microgravity bioreactor having a small incubation compartment volume or having a zero volume access port closure. 
     Accordingly, it is advantageous to provide an improved microgravity bioreactor that addresses these problems. 
     SUMMARY OF THE INVENTION 
     In preferred embodiments, the invention provides a bioreactor that mitigates, alleviates eliminates or otherwise solves one or more of the above mentioned problems of the prior art. 
     This and several other objectives are obtained in a first aspect of the invention by providing a bioreactor adapted for rotation, the reactor comprising
         an incubation cavity, the incubation cavity providing, in conjunction with a first semipermeable filter, a substantially closed confinement, said semipermeable filter being permeable to molecules up to a predetermined molecular weight or size, allowing an inflow of nutrients and fluid culture medium in the incubation chamber and retainment of cells and cellular aggregates in the incubation cavity,   an equilibrium chamber, said equilibrium chamber providing a volume for a supply of nutrient factors and/or oxygen and optionally carbon dioxide and/or pH controlling substances for one or more cell cultures, tissue biopsies, or cell clusters resident in the incubation cavity, and   conduction means providing a substantially fluid tight conduit from the semipermeable filter to the equilibrium chamber, wherein the incubation cavity has an internal fluid volume of about 25 μl to about 1 ml.       

     In a second aspect, embodiments of the invention provide a bioreactor adapted for rotation, the bioreactor comprising
         an incubation cavity, the incubation cavity providing, in conjunction with a first semipermeable membrane, a substantially closed confinement,   a humidity chamber comprising an aqueous liquid, the chamber comprising a second semipermeable membrane and a third semipermeable membrane both arranged in fluid contact with the aqueous liquid, said third permeable membrane further being arranged for exchange of gases with an atmosphere surrounding the bioreactor, and   conduction means providing a substantially air tight conduit from the first semipermeable membrane to the second semipermeable membrane,       

     wherein the first, second and third semipermeable membranes are substantially impermeable to water and substantially permeable to oxygen and carbon dioxide so as to facilitate: 
     a) aeration of the incubation cavity through the first semipermeable membranes and the humidity chamber, and 
     b) substantial retainment of water in the incubation chamber. optionally wherein the incubation cavity has an internal fluid volume of about 25 μl to about 1 ml. 
     In a third aspect, embodiments of the invention provide a bioreactor adapted for rotation, the bioreactor comprising
         an incubation cavity, the cavity comprising an inner surface in conjunction with a flexible membrane or a flexible filter,   one or more through-going ports adapted for filling of the incubation cavity, said ports being arranged azimuthally at different angles as viewed from a central axis of the incubation cavity,   one or more inserts, each insert being adapted to fit in a corresponding through-going port to provide a substantially fluid tight lock of each port,       

     wherein one or more of the inserts has an end portion that is substantially aligned with the inner surface of the incubation cavity when inserted in the corresponding port thereby creating an essentially zero volume port, 
     optionally wherein the incubation cavity has an internal fluid volume of about 25 μl to about 1 ml. 
     In a fourth aspect, embodiments the invention provide a bioreactor adapted for rotation, the bioreactor comprising
         an incubation cavity, the incubation cavity providing, in conjunction with a semipermeable membrane, a substantially closed confinement,       

     wherein at atmospheric pressure the semipermeable membrane is almost completely impermeable to water while substantially permeable to oxygen and carbon dioxide so as to facilitate: 
     a) aeration of the incubation cavity through the first semipermeable membranes, and 
     b) substantial retainment of water in the incubation chamber. optionally wherein the incubation cavity has an internal fluid volume of about 25 μl to about 1 ml. 
     In a fifth aspect, embodiments of the invention provide use of the bioreactor for the incubation of one or more cell cultures, tissue biopsies, cell clusters, tissue-like structures, “prototissues” or similar samples. 
     In a sixth aspect, embodiments of the invention provide a method of incubating one or more cell types tissue biopsies, cell clusters, tissue-like structures, “prototissues” or similar samples. 
     In a seventh aspect, embodiments of the invention provide a method of creating molecular profiles of the biological effect of chemical compositions. 
     In an eighth aspect, embodiments of the invention provide a method of compiling a library of molecular profiles of the biological effect of chemical compositions having predetermined toxicities. 
     In a ninth aspect, embodiments of the invention provide a method of typing toxicity of a test chemical composition. 
     Preferred embodiments of the present invention are particularly, but not exclusively, advantageous for obtaining a microgravity bioreactor that provides:
         1) Reduced consumption of resources (chemicals, cells or tissues) with corresponding reduction of cost, due to a smaller incubation compartment volume.   2) Improved moisture retention in the incubation chamber with corresponding improved maintenance of culture conditions for long-term growth, due to introduction of a humidity chamber.   3) Increased duration of long-term cultures incubated in a small volume bioreactor, due to improved moisture retention in the incubation chamber.   4) Reduced fluid turbulence, due to introduction of essentially zero-volume access ports.   5) Additional benefits of smaller bioreactor volume including improved monitoring of cell growth, in some embodiments by automatic video/camera imaging techniques; improved attainment of biological threshold levels of growth factors and signal molecules using smaller samples of selected biopsies or cultures; increased efficiency of standard sized incubators, due to the increased number of individual bioreactors that can be operated simultaneously.       

     The first, second, third, fourth, fifth, sixth, seventh, eighth and ninth aspect of embodiments of the present invention may each be combined with any of the other aspects. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       Some embodiments of the present invention are illustrated by the accompanying Figures, where 
         FIG. 1  is a schematic cross-sectional drawing of a bioreactor according to the first aspect of the invention, 
         FIG. 2  is a more detailed cross-sectional drawing of a bioreactor according to the first aspect of the invention showing inter alia pumping means, 
         FIG. 3A  is a schematic cross-sectional drawing of a bioreactor according to the second aspect of the invention, 
         FIG. 3B  is a schematic cross-sectional drawing of an alternative embodiment of a bioreactor according to the second aspect of the invention (where the membrane used is totally impervious to salts but still previous to gasses), 
         FIG. 4  is a plane view (A) and a cross-sectional view (B) of a humidity chamber of a bioreactor according to the second aspect of the invention, 
         FIG. 5  is a more detailed cross-sectional drawing of a bioreactor according to the second aspect of the invention showing inter alia air inlets, 
         FIG. 6  is a schematic cross-sectional drawing of a bioreactor according to the fourth aspect of the invention 
         FIG. 7  is a graph showing experimental results of the liquid loss under long-term incubation with and without the humidity chamber of the bioreactor according to the second aspect of the invention, 
         FIG. 8  is a cross-sectional drawing of a bioreactor according to the third aspect of the invention, 
         FIG. 9  is another cross-sectional drawing of a bioreactor according to the third aspect of the invention, 
         FIG. 10  is a schematic drawing of a portion of the inner surface of the bioreactor and inserts for closing the bioreactor, 
         FIG. 11 , A to D, is a sequence of schematic drawings illustrating a method for operating a bioreactor according to the third aspect of the invention, and 
         FIG. 12  shows a cross-sectional side view (part A) and a cross-sectional front view (part B), respectively, of a mount for a bioreactor according to the third aspect of the invention. 
         FIG. 13  is another cross-sectional drawing of a bioreactor according to the second aspect of the invention. 
         FIG. 14  is another cross-sectional drawing of a bioreactor according to the third aspect of the invention. 
         FIG. 15  is a plane view (A) and cross-sectional view (B) and (C) of a bioreactor according to the second aspect of the invention. 
     
    
    
     DETAILED DESCRIPTION 
     Definitions 
     As used herein, the following terms have the following meanings: 
     The terms “semipermeable filter” and “semipermeable membrane” refer to a filter or membrane that can be penetrated by some, but not all, chemical or biological substances. The terms are used interchangeably except that, in general, “filter” is used where water is freely permeable while, in general, “membrane” is used both where water is not freely permeable and also where water is freely permeable. 
     The term “incubation cavity” refers to that portion of a bioreactor in which cell cultures, tissue biopsies, cell clusters, tissue-like structures, “prototissues” or similar samples are grown, differentiated, incubated, or otherwise cultured. The term “incubation cavity” is used interchangeably with “incubation chamber” and “incubation compartment.” 
     The term “substantially impermeable to water” is used to describe characteristics of membranes of the present invention and refers to a membrane that exhibits a high degree of repulsion of water and water-like molecules in gas and/or liquid phase. 
     The term “almost completely impermeable to water” is used to describe characteristics of membranes of the present invention and refers to a membrane across which the water flow rate at 1 bar is not greater than 0.1 mL/min/cm 2 . 
     The term “substantially permeable to oxygen and carbon dioxide” is used to describe characteristics of membranes of the present invention and refers to a membrane across which air will readily pass. 
     The term “relative retainment” is used to describe conditions arising from operation of a bioreactor of the invention with an aqueous solution or suspension in the incubation cavity and refers to the relative amount of residual substance initially present. For example, the relative retainment of water in the incubation cavity (with a flexible membrane) may be calculated as the volume of the cavity after operating the bioreactor divided by the volume of the cavity at the beginning of operating the bioreactor. 
     The term “toxic” has the usual meaning known in the art. A “toxic” substance is a substance that in the amount present in the chemical compositions as defined above can impair the functioning of, or cause structural damage to a cell, tissue or organism. 
     The term “predetermined toxicity” relates to both toxic and non-toxic substances. As Paracelsus stated in the 16 th  century, “All things are poison and nothing is without poison, only the dose permits something not to be poisonous”. The toxicity type of a substance may e.g. be determined according to the toxicity typing scheme of the Food and Drug Administration (FDA) of the United States of America. According to this scheme, the predetermined toxicity of a substance may belong to toxicity type A, B, etc. or may be non-toxic. 
     The term “cell cultures” refers to any kind of cells, tissue biopsies, cell clusters, tissue-like structures, “prototissues” or similar samples obtained or initially cultured by any method known in the art. 
     The term “microgravity bioreactor” refers to a bioreactor adapted for rotation. 
     The term “incubating under microgravity conditions” refers to growth of cell cultures in a bioreactor adapted for rotation while rotating said bioreactor about a substantially horizontal central axis at a rate that suspends one or more cell cultures in a liquid culture medium and continuing such rotation for a time period that permits growth of said one or more cell cultures. 
     The term “means of relative retainment of water” is used to describe features of a bioreactor and refers to any means other than perfusion that is used in combination with a membrane or filter that substantially confines the incubation chamber to achieve relative retainment of water in the incubation chamber or, in the alternative, to any single membrane that substantially confines the incubation chamber across which membrane the water flow rate at 1 bar is not greater than 0.1 mL/min/cm 2 . The term “chemical composition” has the usual meaning known in the art. It may include, but is not limited to, any mixture of one or more chemical or biological agents, such as small molecules, peptides, proteins, bases, nucleic acids, and lipids, wherein said chemical or biological agents result in alterations in gene expression or protein expression in one or more cell type selected from the group consisting of:
         Keratinizing epithelial cells (including Epidermal keratinocyte (differentiating epidermal cell); Epidermal basal cell (stem cell); Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell); Medullary hair shaft cell; Cortical hair shaft cell; Cuticular hair shaft cell; Cuticular hair root sheath cell; Hair root sheath cell of Huxley&#39;s layer; Hair root sheath cell of Henle&#39;s layer; External hair root sheath cell; Hair matrix cell (stem cell)).   Wet stratified barrier epithelial cells (including Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina; basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina; Urinary epithelium cell (lining urinary bladder and urinary ducts)).   Exocrine secretory epithelial cells (including Salivary gland mucous cell (polysaccharide-rich secretion); Salivary gland serous cell (glycoprotein enzyme-rich secretion); Von Ebner&#39;s gland cell in tongue (washes taste buds); Mammary gland cell (milk secretion); Lacrimal gland cell (tear secretion); Ceruminous gland cell in ear (wax secretion); Eccrine sweat gland dark cell (glycoprotein secretion); Eccrine sweat gland clear cell (small molecule secretion); Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive); Gland of Moll cell in eyelid (specialized sweat gland); Sebaceous gland cell (lipid-rich sebum secretion); Bowman&#39;s gland cell in nose (washes olfactory epithelium); Brunner&#39;s gland cell in duodenum (enzymes and alkaline mucus); Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm); Prostate gland cell (secretes seminal fluid components); Bulbourethral gland cell (mucus secretion); Bartholin&#39;s gland cell (vaginal lubricant secretion); Gland of Littre cell (mucus secretion); Uterus endometrium cell (carbohydrate secretion); Isolated goblet cell of respiratory and digestive tracts (mucus secretion); Stomach lining mucous cell (mucus secretion); Gastric gland zymogenic cell (pepsinogen secretion); Gastric gland oxyntic cell (hydrochloric acid secretion); Pancreatic acinar cell (bicarbonate and digestive enzyme secretion); Paneth cell of small intestine (lysozyme secretion); Type II pneumocyte of lung (surfactant secretion); Clara cell of lung).   Hormone secreting cells (including Anterior pituitary cells; Somatotropes; Lactotropes; Thyrotropes; Gonadotropes; Corticotropes; Intermediate pituitary cell, secreting melanocyte-stimulating hormone; Magnocellular neurosecretory cells secreting oxytocin or vasopressin; Gut and respiratory tract cells (secreting one or more of the following: serotonin, endorphin, somatostatin, gastrin, secretin, cholecystokinin, insulin, glucagon, bombesin); Thyroid gland cells; thyroid epithelial cell; parafollicular cell; Parathyroid gland cells; Parathyroid chief cell; oxyphil cell; Adrenal gland cells; chromaffin cells (secreting one or more steroid hormones mineralcorticoids or glucocorticoids); Leydig cell of testes (secreting testosterone); Theca interna cell of ovarian follicle (secreting oestrogen); Corpus luteum cell of ruptured ovarian follicle (secreting progesterone); Kidney juxtaglomerular apparatus cell (secreting renin); Macula densa cell of kidney; Peripolar cell of kidney; Mesangial cell of kidney).   Epithelial absorptive cells (Gut, Exocrine Glands and Urogenital Tract) (including Intestinal brush border cell (with microvilli); Exocrine gland striated duct cell; Gall bladder epithelial cell; Kidney proximal tubule brush border cell; Kidney distal tubule cell; Ductulus efferens nonciliated cell; Epididymal principal cell; Epididymal basal cell; Metabolism and storage cells; Hepatocyte (liver cell); Adipocyte (white or brown fat cell); Liver lipocyte).   Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract) (including Type I pneumocyte (lining air space of lung); Pancreatic duct cell (centroacinar cell); Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.); Kidney glomerulus parietal cell; Kidney glomerulus podocyte; Loop of Henle thin segment cell (in kidney); Kidney collecting duct cell; Duct cell (of seminal vesicle, prostate gland, etc.)).   Epithelial cells lining closed internal body cavities (including Blood vessel and lymphatic vascular endothelial fenestrated cell; Blood vessel and lymphatic vascular endothelial continuous cell; Blood vessel and lymphatic vascular endothelial splenic cell; Synovial cell (lining joint cavities, hyaluronic acid secretion); Serosal cell (lining peritoneal, pleural, and pericardial cavities); Squamous cell (lining perilymphatic space of ear); Squamous cell (lining endolymphatic space of ear); Columnar cell of endolymphatic sac with microvilli (lining endolymphatic space of ear); Columnar cell of endolymphatic sac without microvilli (lining endolymphatic space of ear); Dark cell (lining endolymphatic space of ear); Vestibular membrane cell (lining endolymphatic space of ear); Stria vascularis basal cell (lining endolymphatic space of ear); Stria vascularis marginal cell (lining endolymphatic space of ear); Cell of Claudius (lining endolymphatic space of ear); Cell of Boettcher (lining endolymphatic space of ear); Choroid plexus cell (cerebrospinal fluid secretion); Pia-arachnoid squamous cell; Pigmented ciliary epithelium cell of eye; Nonpigmented ciliary epithelium cell of eye; Corneal endothelial cell).   Ciliated cells with propulsive function (including Respiratory tract ciliated cell; Oviduct ciliated cell (in female); Uterine endometrial ciliated cell (in female); Rete testis cilated cell (in male); Ductulus efferens ciliated cell (in male); Ciliated ependymal cell of central nervous system (lining brain cavities)).   Extracellular matrix secretion cells (including Ameloblast epithelial cell (tooth enamel secretion); Planum semilunatum epithelial cell of vestibular apparatus of ear (proteoglycan secretion); Organ of Corti interdental epithelial cell (secreting tectorial membrane covering hair cells); Loose connective tissue fibroblasts; Corneal fibroblasts; Tendon fibroblasts; Bone marrow reticular tissue fibroblasts; Other nonepithelial fibroblasts; Blood capillary pericyte; Nucleus pulposus cell of intervertebral disc; Cementoblast/cementocyte (tooth root bonelike cementum secretion); Odontoblast/odontocyte (tooth dentin secretion); Hyaline cartilage chondrocyte; Fibrocartilage chondrocyte; Elastic cartilage chondrocyte; Osteoblast/osteocyte; Osteoprogenitor cell (stem cell of osteoblasts); Hyalocyte of vitreous body of eye; Stellate cell of perilymphatic space of ear).   Contractile cells (including Red skeletal muscle cell (slow); White skeletal muscle cell (fast); intermediate skeletal muscle cell; nuclear bag cell of Muscle spindle; nuclear chain cell of Muscle spindle; Satellite cell (stem cell); Ordinary heart muscle cell; Nodal heart muscle cell; Purkinje fiber cell; Smooth muscle cell (various types); Myoepithelial cell of iris; Myoepithelial cell of exocrine glands).   Blood and immune system cells (including Erythrocyte (red blood cell); Megakaryocyte (platelet precursor); Monocyte; Connective tissue macrophage (various types); Epidermal Langerhans cell; Osteoclast (in bone); Dendritic cell (in lymphoid tissues); Microglial cell (in central nervous system); Neutrophil granulocyte; Eosinophil granulocyte; Basophil granulocyte; Mast cell; Helper T cell; Suppressor T cell; Cytotoxic T cell; B cells; Natural killer cell; memory cell; Reticulocyte; Stem cells and committed progenitors for the blood and immune system (various types)).   Sensory transducer cells (including Photoreceptor rod cell of eye; Photoreceptor blue-sensitive cone cell of eye; Photoreceptor green-sensitive cone cell of eye; Photoreceptor red-sensitive cone cell of eye; Auditory inner hair cell of organ of Corti; Auditory outer hair cell of organ of Corti; Type I hair cell of vestibular apparatus of ear (acceleration and gravity); Type II hair cell of vestibular apparatus of ear (acceleration and gravity); Type I taste bud cell; Olfactory receptor neuron; Basal cell of olfactory epithelium (stem cell for olfactory neurons); Type I carotid body cell (blood pH sensor); Type II carotid body cell (blood pH sensor); Merkel cell of epidermis (touch sensor); Touch-sensitive primary sensory neurons (various types); Cold-sensitive primary sensory neurons; Heat-sensitive primary sensory neurons; Pain-sensitive primary sensory neurons (various types); Proprioceptive primary sensory neurons (various types)).   Autonomic neuron cells (including Cholinergic neural cell (various types); Adrenergic neural cell (various types); Peptidergic neural cell (various types)).   Sense organ and peripheral neuron supporting cells (including Inner pillar cell of organ of Corti; Outer pillar cell of organ of Corti; Inner phalangeal cell of organ of Corti; Outer phalangeal cell of organ of Corti; Border cell of organ of Corti; Hensen cell of organ of Corti; Vestibular apparatus supporting cell; Type I taste bud supporting cell; Olfactory epithelium supporting cell; Schwann cell; Satellite cell (encapsulating peripheral nerve cell bodies); Enteric glial cell).   Central nervous system neurons and glial cells (including Neuron cells (large variety of types, still poorly classified); Astrocyte (various types); Oligodendrocyte).   Lens cells (including Anterior lens epithelial cell; Crystallin-containing lens fiber cell).   Pigment cells (including Melanocyte; Retinal pigmented epithelial cell).   Germ cells (including Oogonium/Oocyte; Spermatid; Spermatocyte; Spermatogonium cell (stem cell for spermatocyte); Spermatozoon).   Nurse cells (including Ovarian follicle cell; Sertoli cell (in testis); Thymus epithelial cell).   Stem cells (omnipotent, totipotent, pleuripotent, unipotent stem cells or precursor or progenitor cells, of both embryonic and adult origin).   All cancer cells (including teratocarcinomas of indefinable origin) derived from any of the above mentioned cell types.       

     Preferred Embodiments 
     In preferred embodiments, the semipermeable filters utilised in the present invention allow passage of molecules up to a certain molecular weight or size. Semipermeable filters with a well-defined pore size are known to the person skilled in the art and are commercially available. In preferred embodiments of the invention, semipermeable filters may be permeable to molecules up to a predetermined molecular weight, such as 50 kDa, 100kDa, 150 kDa, 200 kDa or 250 kDa. Alternatively, the permeability of semipermeable filters may be determined by the pore sizes therein. The pore size of semipermeable filters may be less than or equal to 0.5 μm, such as less than or equal to 0.3 μm, preferably less than or equal to 0.2 μm, even more preferably less than or equal to 0.1 μm, and most preferably less than or equal to 0.05 μm. A wide variety of filters can be used. These could be made of materials selected from (but not limited to) the group consisting of polytetrafluroethylene (PTFE), Polyvinylidene fluoride (PVDF), silicon rubber, foam plastics, radiation treated plastic, and similar materials. In one preferred embodiment, a TE 35 filter from Whatman or a Zefluor filter (cat. no. 66142 from Pall Life Sciences can be used. 
     In preferred embodiments of the invention, the water flow rate at 1 bar accross membranes that are “substantially impermeable to water” and “substantially permeable to oxygen and carbon dioxide” is not greater than 50 ml/min/cm 2 , preferably not greater than 40 ml/min/cm 2 , more preferably not greater than 30 ml/min/cm 2 , even more preferably not greater than 20 ml/min/cm 2 , most preferably not greater than 10 ml/min/cm 2 . It will be readily understood by those skilled in the art that water permeability can be expressed in other units, which can be converted into ml/min/cm 2 . 
     In preferred embodiments of the invention, the air flow rate at 3 mbar accross membranes that are “substantially impermeable to water” and “substantially permeable to oxygen and carbon dioxide” is at least 5 ml/min/cm 2 , preferably at least 10 ml/min/cm 2 , more preferably at least 15 ml/min/cm 2 , even more preferably at least 20 ml/min/cm 2 , most preferably at least 25 ml/min/cm 2 . It will be readily understood by those skilled in the art air flow can be expressed in other units, which can be converted into ml/min/cm 2 . 
     Membranes comprised of a wide variety of materials can be used, that are “substantially impermeable to water” and “substantially permeable to oxygen and carbon dioxide,” including but not limited to membranes well known in the art comprised of polytetrafluoroethylene (PTFE), Polyvinylidene fluoride (PVDF), silicon rubber, foam plastics, radiation treated plastic or similar materials. One example of a suitable membrane is commercially available from Whatman under the trade mark “TE 35®,” a PTFE membrane with polyester support having characteristics (quoted by the manufacturer): pore size 0.2 μM, thickness 190 μM, water flow rate at 0.9 bar of 20 ml/min/cm 2  when measured with ethanol, air flow rate 15 ml/min/cm 2  at 3 mbar and bubble point 1.4 bar. Another example of a suitable membrane is commercially available from Millipore under the trade mark “SureVent®,” a PVDF membrane having characteristics (quoted by the manufacturer): pore size 0.22 μM, thickness 100-150 μM, water breakthrough 45 mbar, air flow rate &gt;1 slpm /cm 2  at 10 psi. In some embodiments, the membranes can be Millipore 0.22 μm “Durapel” membranes or Whatman TE 35 and TE36 membranes. 
     In preferred embodiments of the invention, the water flow rate at 1 bar accross membranes that are “almost completely impermeable to water” while “substantially permeable to oxygen and carbon dioxide” is not greater than 0.1 ml/min/cm 2 , even more preferably not greater than 0.05 mL/min/cm 2 , still more preferably not greater than 0.04 ml/min/cm 2 , even more preferably not greater than 0.03 ml/min/cm 2 , still more preferably not greater than 0.02 ml/min/cm 2 , most preferably not greater than 0.01 ml/min/cm 2 . It will be readily understood by those skilled in the art that water permeability can be expressed in other units, which can be converted into ml/min/cm 2 . 
     In preferred embodiments of the invention, the air flow rate at 3 mbar accross membranes that are “almost completely impermeable to water” while “substantially permeable to oxygen and carbon dioxide” is at least 5 ml/min/cm 2 , preferably at least 10 ml/min/cm 2 , more preferably at least 15 ml/min/cm 2 , even more preferably at least 20 ml/min/cm 2 , most preferably at least 25 ml/min/cm 2 . 
     Membranes comprised of a wide variety of materials can be used, that are “almost completely impermeable to water” while “substantially permeable to oxygen and carbon dioxide” including but not limited to membranes initially prepared for ultrafiltration purposes that have very low water permeabilities at atmospheric pressures, for example, due to low porosity and high hydrophobicity. Such membranes include ultrafiltration membranes commercially available from Amicon under the trademark “YM1®” and from Pall Corp. under the trademark “Omega 1K. ®”. Other suitable membranes include thermoplastic ultrafiltration membranes prepared by thermally induced phase inversion process of semi-crystalline materials such as poly(ether ether ketone)(PEEK) and poly(phenylene sulfide)(PPS), as described by [Micro- and ultrafiltration film membranes from poly(ether ether ketone)(PEEK). Sonnenschein M, Journal of Applied Polymer Science 1999 74:1146]. Immobilized, stable supported liquid membranes (SLM) can also be used comprising a suitable oligomeric or polymeric liquid membrane material immobilized within a solid, microporous, hydrophobic support, such as the system disclosed in U.S. Pat. No. 5,507,949. 
     In preferred embodiments, the internal fluid volume of the incubation cavity of a bioreactor according to the present invention may be less than 1.0 mL, less than 900 uL, less than 800 uL, less than 700 uL, less than 600 uL, less than 500 uL, less than 400 uL, less than 300 uL, less than 200 uL, less than 100 uL, less than 50 uL, or 25 uL. 
     Many different cell cultures, tissue biopsies, cell clusters, tissue-like structures, “prototissues or similar samples may be used in practising the present invention. Different methods of cell culture include (but are not restricted to) growth in glass and plastic vessels e.g. culture flasks, cell factories or cell cubes and soft plastic bags (like infusion bags) filled with a 3D cell-supporting matrix; roller bottles; spinner bottles; fermentors; or hollow fibres of various materials. The cell cultures may be in the form of cell clusters, such as spheroids on a microcarrier bead, or cells on a scaffold (e.g. biodegradable scaffolds, often made from polyglycolic acid (PGA) PGA, polylactic acid (PLA) or a mixture of the two [Characterization of knitted polymeric scaffolds for potential use in ligament tissue engineering. Ge Z, Goh J C, Wang L, Tan E P, Lee E H. J Biomater Sci Polym Ed. 2005;16(9):1179-92.] and [Synthesis and characterizations of biodegradable and crosslinkable poly(epsilon-caprolactone fumarate), poly(ethylene glycol fumarate), and their amphiphilic copolymer. Wang S, Lu L, Gruetzmacher J A, Currier B L, Yaszemski M J. Biomaterials. 2005 Aug. 12; [Epub ahead of print]], tissue or tissue biopsies, or tissue organoids. The invention may be practised using inter alia one or more of the following cell types:
         Keratinizing epithelial cells (including Epidermal keratinocyte (differentiating epidermal cell); Epidermal basal cell (stem cell); Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell); Medullary hair shaft cell; Cortical hair shaft cell; Cuticular hair shaft cell; Cuticular hair root sheath cell; Hair root sheath cell of Huxley&#39;s layer; Hair root sheath cell of Henle&#39;s layer; External hair root sheath cell; Hair matrix cell (stem cell)).   Wet stratified barrier epithelial cells (including Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina; basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina; Urinary epithelium cell (lining urinary bladder and urinary ducts)).   Exocrine secretory epithelial cells (including Salivary gland mucous cell (polysaccharide-rich secretion); Salivary gland serous cell (glycoprotein enzyme-rich secretion); Von Ebner&#39;s gland cell in tongue (washes taste buds); Mammary gland cell (milk secretion); Lacrimal gland cell (tear secretion); Ceruminous gland cell in ear (wax secretion); Eccrine sweat gland dark cell (glycoprotein secretion); Eccrine sweat gland clear cell (small molecule secretion); Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive); Gland of Moll cell in eyelid (specialized sweat gland); Sebaceous gland cell (lipid-rich sebum secretion); Bowman&#39;s gland cell in nose (washes olfactory epithelium); Brunner&#39;s gland cell in duodenum (enzymes and alkaline mucus); Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm); Prostate gland cell (secretes seminal fluid components); Bulbourethral gland cell (mucus secretion); Bartholin&#39;s gland cell (vaginal lubricant secretion); Gland of Littre cell (mucus secretion); Uterus endometrium cell (carbohydrate secretion); Isolated goblet cell of respiratory and digestive tracts (mucus secretion); Stomach lining mucous cell (mucus secretion); Gastric gland zymogenic cell (pepsinogen secretion); Gastric gland oxyntic cell (hydrochloric acid secretion); Pancreatic acinar cell (bicarbonate and digestive enzyme secretion); Paneth cell of small intestine (lysozyme secretion); Type II pneumocyte of lung (surfactant secretion); Clara cell of lung).   Hormone secreting cells (including Anterior pituitary cells; Somatotropes; Lactotropes; Thyrotropes; Gonadotropes; Corticotropes; Intermediate pituitary cell, secreting melanocyte-stimulating hormone; Magnocellular neurosecretory cells secreting oxytocin or vasopressin; Gut and respiratory tract cells (secreting one or more of the following: serotonin, endorphin, somatostatin, gastrin, secretin, cholecystokinin, insulin, glucagon, bombesin); Thyroid gland cells; thyroid epithelial cell; parafollicular cell; Parathyroid gland cells; Parathyroid chief cell; oxyphil cell; Adrenal gland cells; chromaffin cells (secreting one or more steroid hormones mineralcorticoids or glucocorticoids); Leydig cell of testes (secreting testosterone); Theca interna cell of ovarian follicle (secreting oestrogen); Corpus luteum cell of ruptured ovarian follicle (secreting progesterone); Kidney juxtaglomerular apparatus cell (secreting renin); Macula densa cell of kidney; Peripolar cell of kidney; Mesangial cell of kidney).   Epithelial absorptive cells (Gut, Exocrine Glands and Urogenital Tract) (including Intestinal brush border cell (with microvilli); Exocrine gland striated duct cell; Gall bladder epithelial cell; Kidney proximal tubule brush border cell; Kidney distal tubule cell; Ductulus efferens nonciliated cell; Epididymal principal cell; Epididymal basal cell; Metabolism and storage cells; Hepatocyte (liver cell); Adipocyte (white or brown fat cell); Liver lipocyte).   Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract) (including Type I pneumocyte (lining air space of lung); Pancreatic duct cell (centroacinar cell); Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.); Kidney glomerulus parietal cell; Kidney glomerulus podocyte; Loop of Henle thin segment cell (in kidney); Kidney collecting duct cell; Duct cell (of seminal vesicle, prostate gland, etc.)).   Epithelial cells lining closed internal body cavities (including Blood vessel and lymphatic vascular endothelial fenestrated cell; Blood vessel and lymphatic vascular endothelial continuous cell; Blood vessel and lymphatic vascular endothelial splenic cell; Synovial cell (lining joint cavities, hyaluronic acid secretion); Serosal cell (lining peritoneal, pleural, and pericardial cavities); Squamous cell (lining perilymphatic space of ear); Squamous cell (lining endolymphatic space of ear); Columnar cell of endolymphatic sac with microvilli (lining endolymphatic space of ear); Columnar cell of endolymphatic sac without microvilli (lining endolymphatic space of ear); Dark cell (lining endolymphatic space of ear); Vestibular membrane cell (lining endolymphatic space of ear); Stria vascularis basal cell (lining endolymphatic space of ear); Stria vascularis marginal cell (lining endolymphatic space of ear); Cell of Claudius (lining endolymphatic space of ear); Cell of Boettcher (lining endolymphatic space of ear); Choroid plexus cell (cerebrospinal fluid secretion); Pia-arachnoid squamous cell; Pigmented ciliary epithelium cell of eye; Nonpigmented ciliary epithelium cell of eye; Corneal endothelial cell).   Ciliated cells with propulsive function (including Respiratory tract ciliated cell; Oviduct ciliated cell (in female); Uterine endometrial ciliated cell (in female); Rete testis cilated cell (in male); Ductulus efferens ciliated cell (in male); Ciliated ependymal cell of central nervous system (lining brain cavities)).   Extracellular matrix secretion cells (including Ameloblast epithelial cell (tooth enamel secretion); Planum semilunatum epithelial cell of vestibular apparatus of ear (proteoglycan secretion); Organ of Corti interdental epithelial cell (secreting tectorial membrane covering hair cells); Loose connective tissue fibroblasts; Corneal fibroblasts; Tendon fibroblasts; Bone marrow reticular tissue fibroblasts; Other nonepithelial fibroblasts; Blood capillary pericyte; Nucleus pulposus cell of intervertebral disc; Cementoblast/cementocyte (tooth root bonelike cementum secretion); Odontoblast/odontocyte (tooth dentin secretion); Hyaline cartilage chondrocyte; Fibrocartilage chondrocyte; Elastic cartilage chondrocyte; Osteoblast/osteocyte; Osteoprogenitor cell (stem cell of osteoblasts); Hyalocyte of vitreous body of eye; Stellate cell of perilymphatic space of ear).   Contractile cells (including Red skeletal muscle cell (slow); White skeletal muscle cell (fast); Intermediate skeletal muscle cell; nuclear bag cell of Muscle spindle; nuclear chain cell of Muscle spindle; Satellite cell (stem cell); Ordinary heart muscle cell; Nodal heart muscle cell; Purkinje fiber cell; Smooth muscle cell (various types); Myoepithelial cell of iris; Myoepithelial cell of exocrine glands).   Blood and immune system cells (including Erythrocyte (red blood cell); Megakaryocyte (platelet precursor); Monocyte; Connective tissue macrophage (various types); Epidermal Langerhans cell; Osteoclast (in bone); Dendritic cell (in lymphoid tissues); Microglial cell (in central nervous system); Neutrophil granulocyte; Eosinophil granulocyte; Basophil granulocyte; Mast cell; Helper T cell; Suppressor T cell; Cytotoxic T cell; B cells; Natural killer cell; memory cell; Reticulocyte; Stem cells and committed progenitors for the blood and immune system (various types)).   Sensory transducer cells (including Photoreceptor rod cell of eye; Photoreceptor blue-sensitive cone cell of eye; Photoreceptor green-sensitive cone cell of eye; Photoreceptor red-sensitive cone cell of eye; Auditory inner hair cell of organ of Corti; Auditory outer hair cell of organ of Corti; Type I hair cell of vestibular apparatus of ear (acceleration and gravity); Type II hair cell of vestibular apparatus of ear (acceleration and gravity); Type I taste bud cell; Olfactory receptor neuron; Basal cell of olfactory epithelium (stem cell for olfactory neurons); Type I carotid body cell (blood pH sensor); Type II carotid body cell (blood pH sensor); Merkel cell of epidermis (touch sensor); Touch-sensitive primary sensory neurons (various types); Cold-sensitive primary sensory neurons; Heat-sensitive primary sensory neurons; Pain-sensitive primary sensory neurons (various types); Proprioceptive primary sensory neurons (various types)).   Autonomic neuron cells (including Cholinergic neural cell (various types); Adrenergic neural cell (various types); Peptidergic neural cell (various types)).   Sense organ and peripheral neuron supporting cells (including Inner pillar cell of organ of Corti; Outer pillar cell of organ of Corti; Inner phalangeal cell of organ of Corti; Outer phalangeal cell of organ of Corti; Border cell of organ of Corti; Hensen cell of organ of Corti; Vestibular apparatus supporting cell; Type I taste bud supporting cell; Olfactory epithelium supporting cell; Schwann cell; Satellite cell (encapsulating peripheral nerve cell bodies); Enteric glial cell).   Central nervous system neurons and glial cells (including Neuron cells (large variety of types, still poorly classified); Astrocyte (various types); Oligodendrocyte).   Lens cells (including Anterior lens epithelial cell; Crystallin-containing lens fiber cell).   Pigment cells (including Melanocyte; Retinal pigmented epithelial cell).   Germ cells (including Oogonium/Oocyte; Spermatid; Spermatocyte; Spermatogonium cell (stem cell for spermatocyte); Spermatozoon).   Nurse cells (including Ovarian follicle cell; Sertoli cell (in testis); Thymus epithelial cell).   Stem cells (omnipotent, totipotent, pleuripotent, unipotent stem cells or precursor or progenitor cells, of both embryonic and adult origin).   All cancer cells (including teratocarcinomas of indefinable origin) derived from any of the above mentioned cell types.       

     In a preferred embodiment of the invention, cells that can be applied in the context of the present invention are selected from the group consisting of hepatocytes, adipocytes, kidney cells, muscle cells, or similar cells, liver tissue, fat tissue (brown or white), liver biopsies, kidney biopsies, muscle biopsies, ovarian follicles, islets of Langerhans, and all cancer cells derived therefrom. 
     In a particularly preferred embodiment of the invention, cells that can be applied in the context of the present invention are hepatocytes, in particular human hepatocytes. 
       FIG. 1  is a schematic cross-sectional drawing of a bioreactor  10  according to the first aspect of the invention. The bioreactor  10  has a high degree of rotational symmetry around a horizontal axis as viewed in  FIG. 1 . The reactor comprises an incubation cavity  15  for incubation of cells, tissues etc. The incubation cavity  15  provides, in conjunction with a first semipermeable filter (also known as a sterile filter) F 1 , a substantially closed confinement for incubation of cells etc. In order to provide nutrients and/or fresh fluid culture medium, the semipermeable filter F 1  is permeable to molecules up to a predetermined molecular weight, such as 50 kDa, 100kDa, 150 kDa, 200 kDa or 250 kDa. Standard dialysis membranes can fulfil these requirements. Alternatively, the permeability of the semipermeable filter F 1  is determined by the pore sizes therein. The pore size of the semipermeable filter F 1  may be less than or equal to 0.5 μm, such as less than or equal to 0.3 μm, preferably less than or equal to 0.2 μm, even more preferably less than or equal to 0.1 μm, and most preferably less than or equal to 0.05 μm. Sizes less than or equal to 0.2 μm are preferable because of the need to prevent infections (e.g. bacteria, mycoplasma or yeasts) entering through the filter. In this preferred embodiment the main purpose of the semipermeable membrane is to allow exchange of nutrients and waste products while excluding cells and bacteria from entering the incubation cavity. Thus a wide variety of filters could be used for F 1 . These could be made of polytetrafluroethylene (PTFE), Polyvinylidene fluoride (PVDF), silicon rubber, foam plastics, radiation treated plastic or similar materials. Thereby an inflow of nutrients and fluid culture medium into the incubation chamber is provided while at the same time providing retainment of cells and cellular aggregates and their protection from external infection in the incubation cavity  15 . The incubation cavity  15  has an internal fluid volume of about 25 μl to about 1 ml. Preferably, the fluid volume of the incubation cavity  15  is about 50 μl to about 0.5 ml, more preferably about 0.1 to about 0.4 ml. The small size significantly reduces the cost of use and the amount of materials (both organic and inorganic) necessary for successful operation. The small size will facilitate close-up monitoring of the cells (e.g. by remote camera), and automated processing (e.g. medium exchange in the non-perfused versions) 
     An equilibrium chamber (not shown in  FIG. 1 ) provides a volume for an exchange of nutrient factors and/or oxygen and optionally carbon dioxide and/or pH controlling substances for one or more cell cultures, tissue biopsies, or cell clusters resident in the incubation cavity  15 . Conduction means  20 , e.g. tubes and tubular cavities within the back part  12  of the bioreactor  10 , provide a substantially fluid tight conduit from the semipermeable filter F 1  to the equilibrium chamber. The white arrow indicates fresh culture medium with nutrients and the black arrow indicates used fluid culture medium to be returned to the equilibrium chamber (or to waste disposal). 
     In the embodiment shown in  FIG. 1 , the conduction means  20  also comprises a media chamber  25  in front of the filter F 1  in order to enhance the area of fluid contact between the conduction means  20  and the filter F 1  and thereby increase the flow of nutrients and/or fresh fluid culture medium across the filter F 1 . The media chamber  25  may be provided by a specially designed annulus  26  surrounding the media chamber  25 . The annulus  26  could be a sealing O-ring clamped between the back part  12  and a front part  11  of the bioreactor  10 . Similarly, the filter F 1  is preferably clamped between the back part  12  and the front part  11  to provide a fluid tight and airtight conjunction from the media chamber  25  to the incubation cavity  15 . For reasons of clarity small air gaps are visible between the annulus  26  and the filter F 1 , and between the outer portions of the back part  12  and the outer portions of the front part  11 , but when assembled these air gaps are normally absent, because the bioreactor  10  should be as air tight as possible to eliminate fluid leakage. 
     In the front of the bioreactor  10 , a transparent section  14  is located so that the cultivation of cells etc. may be monitored and assessed visually, either manually or automatically with e.g. a camera, from outside of the bioreactor  10 . The transparent section  14  could be made of glass, plastic or any other suitable materials being both transparent and biologically and chemically inert with respect to the cultivation process. Preferred materials would include (but not be limited to) various types of glass, polystyrene, polycarbonate, polypropylene, polyethylene and Polymethyl methacrylate (PMMA). Suitable variants of polymethyl methacrylate (PMMA) are available commercially including products marketed under the trademarks/trade names Perspex®, Plexiglas®, Lucite®, Acrylite®, Rhoplex®, and Oroglas®. Any embodiment of the bioreactor could be made in whole or in part from such transparent materials. 
     The incubation cavity  15  preferably has a substantially cylindrical shape but other shapes are also possible, e.g. elliptical shapes, spherical shapes etc. Preferably, the bioreactor  10  is adapted for rotation around a horizontal, rotational axis by associated rotation means (not shown) to facilitate growth of the cells in the cavity  15 . The rate of rotation is adjusted to maintain the cells or prototissues in suspension and this rate has to be varied as the size of the prototissues increases. The person skilled in the art will know how to adjust the rotation speed in order to maintain the cells or prototissues in suspension. 
       FIG. 2  is a more detailed cross-sectional drawing of a bioreactor  10  according to the first aspect of the invention showing also pumping means  26 . 5  for flowing the fluid culture medium through the conduction means  20 , a equilibrium chamber  28  embedded in a constant environment chamber  29 , control valves  27  for controlling the flow of fluid culture medium to and from the media chamber  25 , to allow the introduction of fresh media (not necessarily the same as that already present, i.e. for the introduction of a compound for testing) and the voiding to waste of spent medium and out of the conduction means  20 . The constant environment chamber  29  comprising the equilibrium chamber  28  may be adapted for rotation synchronously with the bioreactor  10  or the container  29  may be fixed in a position next to the bioreactor  10 . In the latter case, twisting of the conduction means  20  has to be avoided by incorporating fully rotationally joint readily available and well known to the skilled person into the conduction means  20 . It may be preferred that the fluid conduction means  20  has a portion substantially parallel to the rotational axis of the bioreactor  10  to ease design of the overall set-up. 
     The bioreactor  10  comprising the front part  11  and the back part  12  is to be mounted on a base part  17  by appropriate fastening means, e.g. a through going assembly screw  28 . For easy mounting of the base part  17  and the bioreactor  10  on suitable rotation means (not shown), the base part  17  may advantageously have a threaded end section  17   a . Similarly, the front part  11  and the back part  12  are additionally held together by an assembly screw  19  in order to provide a fluid tight conjunction to the cavity  15  as explained above. For reasons of clarity small air gaps are visible between the annulus  26  and the filter F 1 , between the outer portions of the back part  12  and the outer portions of the front part  11 , and between the base part  17  and the back part  12 , but when assembled these air gaps are normally absent. 
     In an embodiment of the invention, the conduction means  20  has an internal fluid volume of about 25 μl to about 2 ml, preferably about 50 μl to about 1 ml, most preferably about 0.1-0.4 ml. The volume of conductions means  20  should be minimal and is thereby comparable to the volume of the cavity  15 . Otherwise, the reduction provided by the present invention in the usage of fluid culture medium and/or relatively expensive reagents (e.g. radioisotopes) may not be significant. Similarly, the equilibrium chamber  28  may advantageously have an internal fluid volume of about 25 μl to about 2 ml, preferably about 50 μl to about 1 ml, most preferably about 0.1-0.4 ml. 
       FIG. 3A  is a schematic cross-sectional drawing of a bioreactor  50  according to the second aspect of the invention. The bioreactor  50  has a high degree of rotational symmetry around a horizontal axis as viewed in  FIG. 1 . The bioreactor comprises an incubation cavity  55  positioned in conjunction with a first semipermeable membrane M 1 , thereby providing a substantially closed confinement, wherein incubation of cells, tissue and similar may be take place. Incubation of the cells may be may be monitored and assessed visually, either manually or automatically, from outside of the bioreactor  50 . The transparent section  58  could be made of glass, plastic or any other suitable materials being both transparent and biological and chemical inert relative to the cultivation process. Preferred materials would include (but not be limited to) various types of glass, polystyrene, polycarbonate, polypropylene, polyethylene and Polymethyl methacrylate (PMMA). Suitable variants of polymethyl methacrylate (PMMA) are available commercially including products marketed under the trademarks/trade names Perspex®, Plexiglas®, Lucite® Acrylite®, Rhoplex®, and Oroglas®. 
     Additionally, there is a provided a humidity chamber  60  that comprises an aqueous liquid, the chamber  60  further comprises a second semipermeable membrane M 2  and a third semipermeable membrane M 3 . The latter membranes M 2  and M 3  are both arranged in fluid contact with the aqueous liquid and confine the liquid from the right and left side, respectively, as shown in  FIG. 1 . The third permeable membrane M 3  is further arranged for exchange of gases with an atmosphere surrounding the bioreactor  50 . The rear part  52  of the bioreactor  50  has a recession fitted to receive the humidity chamber  60  so as to provide a substantially air tight closing of the intermediate cavity IC from the left as shown in  FIG. 1 . The rear part  52  is further arranged with sidewalls  52   a  and  52   b  that provide conduction means for a substantially airtight conduit from the first semipermeable membrane M 1  to the second semipermeable membrane M 2 . Thus, the front part  51  of the reactor  50  and the rear part  52  may be manufactured in inert plastic such as high-density poly propylene (HDPP) or similar. Preferred materials would include (but not be limited to) various types of glass, nylon, plastic, polyvinylchloride, polystyrene, polycarbonate, polypropylene, polyethylene and Polymethyl methacrylate. In combination with sealing means  53 , e.g. O-shaped sealing ring, the air flow through the intermediate cavity IC is effectively only possible through the first membrane M 1  (forming part of the incubation cavity  55 ) and the second and third membranes M 2  and M 3  (forming part of the humidity chamber  60 ). 
     The first M 1 , second M 2 , and third M 3  semipermeable membranes are particular in that these membranes are substantially impermeable to water and substantially permeable to oxygen and carbon dioxide. Thereby the membranes M 1 , M 2  and M 1  facilitate aeration of the incubation cavity  55  through the first semipermeable membranes M 1  and the humidity chamber  60  (traversing M 2 , the aqueous liquid, and M 3 ). Furthermore, there is achieved a substantial retainment of water in the incubation chamber due to the impermeability for water of the three membranes M 1 , M 2  and M 3 . The membranes M 1 , M 2  and M 3  can be manufactured from a variety of materials well known in the art including but not limited to polytetrafluoroethylene (PTFE), Polyvinylidene fluoride (PVDF), silicon rubber, foam plastics, radiation treated plastic or similar materials. One example of a suitable membrane is commercially available from Whatman under the trade mark “TE 35®,” a PTFE membrane with polyester support having characteristics (quoted by the manufacturer): pore size 0.2 μM, thickness 190 μM, water flow rate at 0.9 bar of 20 ml/min/cm 2  when measured with ethanol, air flow rate 15 ml/min/cm 2  at 3 mbar and bubble point 1.4 bar. Another example of a suitable membrane is commercially available from Millipore under the trade mark “SureVent®,” a PVDF membrane having characteristics (quoted by the manufacturer): pore size 0.22 μM, thickness 100-150 μM, water breakthrough 45 mbar, air flow rate &gt;1 slpm/cm 2  at 10 psi. Other examples are Zefluor filters (cat. no. 66142 from Pall Life Sciences 
     Due to the highly hydrophobic nature of these materials water and water-like molecules will to a high degree be repelled from the surface of the membranes M 1 , M 2  and M 3 . However, some water will inevitably penetrate through the membranes M 1 , M 2  and M 3  and this evaporated water from the humidity chamber  60  and/or the incubation chamber  55  will provide a relative humidity of at least 50%, preferably at least 70%, or even more preferably at least 90%, such as at least 95%, 96%, 97%, 98% or 99%, in the air tight conduit formed by the intermediate cavity IC. 
       FIG. 3B  shows an alternative embodiment of a bioreactor according to the second aspect of the invention, wherein the membranes M 1  and M 2  and the intermediate cavity IC may be replaced by a membrane M 12  that is impervious to salts and pervious to gasses and liquids. Examples of this type of film membrane are commercially available including for example products available from Dupont such as polytetrafluoroethylene marketed under the trade mark “Teflon®,” FEP membrane or polyvinyl fluoride film marketed under the trade mark “Tedlar®.” 
     This embodiment is simpler in construction than the embodiment shown in  FIG. 5  and the humidity chamber is in direct contact with the incubation chamber. One or more cell cultures that may be cultivated in the incubation chamber are very sensitive to changes in the conditions in the incubation chamber, including the amount of salts present. It is therefore important that the membrane M 12  is impervious to salts so that the salt level of the incubation chamber remains substantially constant while retaining the amount of liquid in the incubation chamber. 
     The bioreactor  50  is typically operated at 37° C. (for human cells) with an aqueous solution or suspension in the incubation cavity  55 , the bioreactor  50  being heated by one or more thermostat controlled heating elements, typically in an incubator for cell culture (not shown). As shown in  FIG. 7 , initial experiments demonstrate that the relative retainment of water in the incubation cavity  55  may be at least 80%, preferably at least 90%, or even more preferably at least 95%, such as at least 96%, 97%, 98% or 99%, after 3 days, more preferably after 5 days, or even more preferably after 10 days. Preliminary results also indicate that cell cultures, tissue biopsies, cell clusters, tissue-like structures, “prototissues or similar samples may be incubated for an extended period of time, preferably at least 1 month, such as 2 months, most preferably at least 10 months. Accordingly, by practice of the present invention, cell cultures can be maintained in a bioreactor for prolonged periods of time with minimal change of concentration-dependent aspects of the aqueous growth medium arising from evaporation. 
       FIG. 4  is a plan view (A) and a cross-sectional view (B) of a humidity chamber  60  of a bioreactor  50  according to the second aspect of the invention. In the cross-sectional view in part A, the semipermeable membranes M 2  and M 3  are shown by dotted lines closing the chamber  60  from above and below as viewed in the cross-sectional view A. The diameter c of the humidity chamber is preferably similar to the diameter of the incubation cavity and thus in the range from 4 to 20 mm, such as such as 6, 8, 10, 12, 14, 16, or 18 mm. The side portions of the chamber  60  are constituted by an annulus  62  manufactured in an inert, stable material such as nylon, plastic, polyvinylchloride, polystyrene, polycarbonate, polypropylene, polyethylene and Polymethyl methacrylate. The annulus  62  may be manufactured in a disposable material so that renewal of the liquid in the humidity chamber  60  during long-term incubation may simply be done by discharging the old humidity chamber  60  and inserting a new humidity chamber  60  in the bioreactor  50 . Alternatively or additionally, the humidity chamber  60  may be re-filled with liquid through a filling port  61  closed by e.g. a grub-screw or similar. 
       FIG. 5  is a more detailed cross-sectional drawing of a bioreactor  50  according to the second aspect of the invention showing inter alia air inlets  65  and a base part  56  whereupon the bioreactor is mountable. The air inlet  65  supplies a rear cavity R with fresh air. Optionally, the air inlet  65  is connected to or embedded in a gas supply unit (not shown) for providing a controlled atmosphere in the rear cavity R. Thereby the aeration of the cells in the incubation cavity  55  will also be controlled via the air flow passing through the humidity chamber  60 . The rear cavity R is constituted by a recession in the base part  56  and sealing means, e.g. a suitable O-ring clamped onto the membrane M 3 . In order to hold the various parts of the bioreactor  50  firmly together, appropriate fastening means are provided. As shown in  FIG. 5 , a through-going assembly screw  70   a  keeps the front part  51  and the rear part  52  (and the intermediate positioned sealing  53  and filter M 1 ) together. Similarly, the front part  51  and the rear part  52  are held onto the base part  56  by a through-going screw  70   b . Again for reasons of clarity small air gaps are visible between the various parts, e.g.  57  and M 3 ,  52  and  56 ,  51  and  52 ,  53  and M 1 , M 1  and  51  and so forth, but when assembled these air gaps are normally absent in order to provide a fluid and air tight closure of the bioreactor  50 . 
     The incubation cavity  55  of  FIG. 5  has a substantially cylindrical shape having a diameter of the cavity  55  in the range from 4 to 20 mm, such as 6, 8, 10, 12, 14, 16, or 18 mm. The depth of the cavity  55  may be in the range from 2 to 6 mm, such as 2.5, 3, 3.5, 4, 4.5 or 5 mm. Thus, the volume of the cavity  55  is in the range from about 0.03 to 2 ml. Some preferred values of the depth and the diameter, respectively, are 4 mm and 18 mm (resulting in a fluid volume of about 1 ml), 3 mm and 10 mm (resulting in a fluid volume of about 0.24 ml), and 3 mm and 7.5 mm (resulting in a fluid volume of about 0.15 ml). 
     The bioreactor  55  is adapted for rotation around a horizontal, rotational axis by associated rotation means (not shown). The base part  56  has a threaded portion  56   a  in order to facilitate easy and flexible mounting on such rotation means. Typically, the rotational axis is substantially coincident with a central axis through the incubation cavity  55 . 
       FIG. 6  is a schematic drawing of a bioreactor according to the fourth aspect of the invention. 
       FIG. 7  shows liquid loss from the incubation chamber of a small volume bioreactor equipped with (lower curve) and without (upper curve) the humidity chamber  60  of the bioreactor  50  according to the second aspect of the invention. In the example, five 1 ml bioreactors were filled with DMEM culture medium (without cells) and maintained rotating in a standard, humidified incubator at 37° C. with 5% CO 2 . The humidity chamber was filled with pure water. The incubator was in normal use (i.e. the door was opened and closed for the routine cultivation of cells). Loss of water from the incubation chamber was measured in two different ways: firstly as a change in the total amount of water inside the bioreactor (left axis) and secondly in separate experiments as percentage change in the absorption of the indicator phenol red (absorption maximum at 559 nM in DMEM, no change in pH of the DMEM was observed, right axis). As shown in  FIG. 7  (which presents averaged data), there is significant loss of water from the incubation chamber of small volume bioreactors, notwithstanding humidification of the incubator. Without the humidity chamber  60  in front of the incubation cavity  55  there is a significant loss of liquid from the cavity  55  even after ½ day, reaching nearly 40% after 5 days. Such a high loss of liquid will concentrate the growth medium, thereby affecting concentration-dependent parameters that can have critical effects on gene expression. The loss of liquid is so rapid that long term growth of cells and/or long-term pharmaceutical or toxicological experiments are rendered impossible to perform without nearly constant manual manipulation (i.e., adding growth medium). In contrast, using the humidity chamber, relative liquid retainment by the incubation cavity  55  is above 95% for 2 days, and above 90% even after 5 days. Accordingly, by practice of the present invention, cell cultures can be maintained in a differentiated state for indefinitely long term periods—cell culture medium can be exchanged every few days and cells occasionally sub-cultured without need for nearly constant efforts to counteract rapid water loss from the incubation chamber. 
       FIG. 8  is a cross-sectional drawing of a bioreactor  100  according to the third aspect of the invention with an incubation cavity  105  for incubation of cells. The cavity  105  has an inner surface  110 . In the embodiment shown in  FIG. 8 , the inner surface  110  has a cylindrical symmetry as viewed from a central axis of the incubation cavity  105 . However, application of the present invention is not limited to this geometry but may readily be applied to other shapes as it has recently been observed that improved cultivation may result from spherical, oblate spherical, extended spherical shapes and combinations thereof. See in particular U.S. Pat. No. 6,642,019, which is hereby incorporated by reference in its entirety. 
     During cultivation of cells the bioreactor  100  will be rotated slowly around a central axis, typically at a rotational frequency of about 5-15 revolutions per minute (RPM). For mounting and clamping of the bioreactor parts the bioreactor comprises a plurality of holes  140 , such as three, four or five holes, for mounting on a base member (not shown) whereupon the bioreactor  100  can be mounted and rotated under cultivation. 
     The bioreactor  100  also comprises a plurality of through-going ports  130   a  and  130   b  adapted for filling of the incubation cavity  105 . In the embodiment shown in  FIG. 8 , the reactor  100  comprises two ports  130   a  and  130   b . The two ports  130   a  and  130   b  are arranged azimuthally at different angles as viewed from a central axis of the incubation cavity  105 , the first port  130   a  and the second port  130   b  being positioned in the shown embodiment with an azimuthal angle difference of about 65 degrees. There is no particular restriction on the azimuthal angle difference, but preferably the angle is less than 90 degrees, more preferably between 70 and 20 degrees and most preferably between 35 and 55 degrees. The skilled person may however readily implement other angles and numbers of ports  130  once the principle of the present invention is recognised. These ports need not be of equal size and one port could be thus larger to provide easier access to the incubation chamber  105  for example for the insertion of tissue biopsies. As a further example, an embodiment of the bioreactor having only one port is illustrated in  FIG. 14 . 
     In order to close the ports  130 , a plurality of inserts  120  are provided. Each insert  120  is adapted to fit in the corresponding through-going port  130  to provide a substantially fluid tight lock of each port  130  during operation of the bioreactor  100 . Inserts  120  may be manufactured in nylon, hard rubber, polyethylene, POM, metals or any other suitable non-cell toxic material for providing a fluid tight closure of the ports  130 . Preferably, the inserts  120  has an outer portion being threaded and similarly the ports  130  are adapted for receiving such threaded inserts  120  as indicated in  FIG. 8 . The inserts  120  are further characterized by having an end portion  121  that is substantially aligned with the inner surface  110  of the incubation cavity  105  when inserted in the corresponding port. This will be explained in further details below, see  FIG. 10 . The purpose of the alignment of the end portion  121  and the inner surface  110  is to create an essentially zero volume port, i.e. fluid from the cavity  105  cannot flow into the port  130 . Additionally, the alignment of the end portion  121  and the inner surface  110  will minimize or eliminate the turbulence that might otherwise be created in the cavity  105  due to the flow of fluid in and out of the ports  130  and most importantly during its rotation. The ports  130  have outer recessions  150  to facilitate effective filling of the bioreactor  110 , eliminating spillage of fluids see  FIG. 11 . 
       FIG. 9  is another cross-sectional drawing of a bioreactor  100  according to the third aspect of the invention along the line X-X shown in  FIGS. 8 and 14 . In the cross section of  FIG. 9 , only one port  130  is shown. The port  130  is connected to the cavity  105  for easy filling of the reactor  100 . In  FIG. 8 , the flexible filter F 1  or the flexible membrane M 1  is mounted so as to close the rear part of the cavity  105 . The bioreactor  100  preferably has a recession in the rear to facilitate easy and fluid tight fastening of the flexible filter F 1  or the flexible membrane M 1 . Typically, additional fastening means (not shown) are provided. If easy access to the incubation cavity is provided via one of the ports  130 , then the membrane M 1  or filter F 1  can be attached (e.g. welded to the bioreactor  100 ). The function of this flexible part M 1 /F 1  is to almost equalize the overpressure in the cavity  105  that results from overfilling the cavity  105  according to the present invention. Preferred materials for the flexible membrane M 1  are polytetrafluroethylene (PTFE), polyvinylidene fluoride (PVDF), silicon rubber, radiation treated plastic or similar materials which are highly water impervious but gas permeable. Preferred materials for the flexible filter F 1  are polytetrafluroethylene (PTFE), polyvinylidene fluoride (PVDF), silicon rubber, foam plastics, radiation treated plastic or similar materials which are water and gas permeable. 
     In the front of the bioreactor  100 , a transparent section  111  is located so that the cultivation of cells etc. may be monitored and assessed visually, either manually or automatically, from outside of the bioreactor  100 . The transparent section  111  could be made of glass, plastic or any other suitable materials being both transparent and biologically and chemically inert relative to the cultivation process. Preferred materials may include (but are not limited to) various types of glass, polystyrene, polycarbonate, polypropylene, polyethylene and polymethyl methacrylate. 
     The width “b” of the cavity  105  may be in the range from 4 to 20 mm, such as 6, 8, 10, 12, 14, 16, or 18 mm. The depth “a” of the cavity  105  may be in the range from 2 to 6 mm, such as 2.5, 3, 3.5, 4, 4.5 or 5 mm. Thus, the volume of the cavity  105  is in the range from about 0.03 to 2 ml. Some preferred values of “a” and “b” are 4 mm and 18 mm (resulting in a fluid volume of about 1 ml), 3 mm and 10 mm (resulting in a fluid volume of about 0.24 ml), and 3 mm and 7.5 mm (resulting in a fluid volume of about 0.15 ml). The said dimensions of the inner cavity  105  may be applicable for a bioreactor  10 ,  50  and/or  100  according to the first, second or third aspect of the invention of any combinations thereof. 
       FIG. 10  is a schematic drawing of a portion of the inner surface  110  of the bioreactor  100  and inserts  120  for closing the cavity  105 .  FIG. 10  illustrates how various widths of the insert  120  can influence the alignment of the inner surface  110  with the end portion  121  of the insert  120 . In part A, the width w 1  is sufficiently small compared to the radius of curvature of the inner surface section  110  with the result that effectively there is still substantially alignment between the inner surface  110  and the end portion  121 . However, in  FIG. 10  part B the width w 2  of the insert  120  is of comparable magnitude to the radius of curvature for the inner surface section  110  and therefore the alignment of the inner surface  110  and the end portion  121  is lower relative to the situation illustrated in part A. Nevertheless, the alignment is still sufficiently high enough for the inner surface  110  and the end portion  121  to be aligned within the context of the present application 
     In a mathematical approach, a measure for circularity, e.g. radius of curvature or similar, may be introduced and coupled with a fluid dynamic model to assess the turbulence limit so as to avoid tearing of the cell culture or tissue. Thus, a Reynold&#39;s number may be estimated to predict the operating conditions for the onset of turbulence with a certain inner surface  110  and end portion  121  configuration. The onset of turbulence is given by a transitional Reynold&#39;s number, which is calculated using a characteristic length of the perturbation introduction by the insert  120 . For the configuration of the present invention, a transitional Reynold&#39;s number may be estimated by use of the diameter of the insert  120  and/or a level difference between the end portion  121  and the inner surface  110 . 
     In a practical approach, the edges of the end portion  121  abutting the inner surface  110  may be flushed or levelled with the inner surface section  110 . Irregularities on the edges will induce turbulence and are therefore to be avoided. Hence, the edges of the end portion  121  may be flush to within 3 mm, preferably 2 mm and most preferably within 1 mm of the inner surface  110 . Note that the insert  120  could be either too short or too long—both options are undesirable. 
     In order to improve the alignment of the inner surface  110  and the end portion  121  the end portion  121  may itself be designed with a curvature, either inwards or outwards, so as to increase the alignment when inserted in the ports  130 . For threaded inserts  120  the curvature should of course be co-ordinated with the threading arrangement so as to ensure correct alignment of the end portion  121  with the inner surface  110  when the inserts  120  are threaded all the way into the fluid locking position. 
       FIG. 11 , A to D, is a sequence of schematic drawings illustrating a method for operating a bioreactor  100  according to the third aspect of the invention. The bioreactor comprises at least a first  130   a  and a second  130   b  through-going port. A syringe or pipette  200  for filling the reactor  100  with liquid  210  is also shown in part A and part B. The syringe  200  in part D is used for removing excess liquid from the outer recess  150  of the ports  130   
     Initially, the syringe  200  is applied for filling the cavity  105  with liquid 210. The ports  130   a  and  130   b  should of course be arranged so that the liquid  210  cannot flow out of the reactor  100  during filling. 
     As shown in part B, the bioreactor  100  is overfilled with liquid  210  through the first port  130   a  so that the bioreactor  100  contains liquid  210  at least in the entire incubation cavity  105  and in at least part of the first port  130   a  and/or in at least part of the second port  130   b . This approach for filling the bioreactor may facilitate the removal of effectively all air bubbles from the bioreactor. The corresponding insert  120   a  is inserted in the first port  130   a  to lock the first port  130   a  completely. Thereby all the fluid  210  previously situated in the first port  130   a  is forced out of the port  130   a.    
     In part C, a corresponding insert  120   b  is inserting in the second port  130   b  to lock the second port. Thus, there is provided a modest overpressure (Δp) in the liquid  210  in the incubation cavity  105  but by the functioning of the flexible membrane M 1  or flexible filter F 1  (not shown) the overpressure will be equalized by an outwards bending of the membrane or the filter away from cavity  105 , see  FIG. 8 . Effectively, the cavity  105  has a variable fluid volume. Preferably, the relative increase of the fluid volume in the cavity  105  may be from about 2 to 10%. The overpressure (Δp) may be in the range from 2 to 10%. 
       FIG. 12  shows a cross-sectional side view (part A) and a cross-sectional front view (part B) of a mount  220  for a bioreactor  100  according to the third aspect of the invention. In part B, the bioreactor  100  is resting on the mount  220 , the mount 220 and the reactor  100  being arranged so as to allow for relatively small rotations of the bioreactor  100 , preferably by manual operation. Thereby it is easier for the user to arrange the bioreactor  100  for filling and/or refilling with liquid, cells, nutrients, removal of air bubbles etc. 
     A variant of the present invention may include only one inlet port  130  and one corresponding insert  120  (see  FIG. 14 ). 
       FIG. 13  shows an alternative embodiment of a bioreactor shown in  FIG. 5  according to the second aspect of the invention. Part  52  is adapted so as to be screwed directly into part  56 . This modification eliminates 3 screws that were used to hold parts  52  and  56  together and thus simplifies and reduces the hands on time working with the bioreactor. The humidity chamber is held in place using two loosely fitting O rings  57  inside of part  56  and can be rapidly and simply exchanged when needed. 
       FIG. 14  shows an alternative construction of the bioreactor  100  and illustrating a single through going port. Filling this type of construction can be achieved using a syringe with a narrow needle to inject growth medium, forcing the air out of the bioreactor past the needle. 
       FIG. 15  shows an alternative construction of the humidity chamber (shown in  FIG. 4 ) where the filters have been recessed to protect them from mechanical wear. This design also lends itself to snap closure or ultrasounic welding of the annulus ring  63  to  62  to hold the filters securely in place. 
     According to a fifth aspect of the present invention, the bioreactor of the invention may be used for the incubation of one or more cell cultures, tissue biopsies, cell clusters, tissue-like structures, “prototissues” or similar samples. 
     A particular embodiment concerns the use of the bioreactor herein for the incubation of one or more cell cultures, wherein the one or more cell cultures are incubated for an extended period of time, such as 1 week, 2 weeks, or 3 weeks, preferably at least 1 month, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, most preferably at least 12 months. 
     One advantage of using the bioreactor according to the present invention for the incubation of one or more cell cultures is that the cells remain in or achieve a highly differentiated state for an extended period of time. The use of the bioreactor according to the present invention furthermore avoids the use of trypsinisation to obtain the cells from the bioreactor. 
     According to a sixth aspect of the invention, a method of incubating one or more cell cultures is provided. In one preferred embodiment, the method comprises the steps of:
     a) obtaining the one or more cell cultures to be inoculated,   b) transferring the one or more cell cultures to the incubation cavity of a bioreactor according to the invention,   c) filling the incubation cavity with growth medium,   d) optionally eliminating air (bubbles) from the incubation cavity,   e) incubating said cell cultures under microgravity conditions,   f) changing growth medium whenever necessary to sustain incubation for the desired time period, and   g) optionally letting the culture differentiate and form tight junctions or other forms of close cell contacts (e.g. by the secretion of intercellular matrix proteins) between the cells.   

     In another embodiment, the method comprises the steps of:
     a) obtaining the one or more cell cultures to be inoculated,   b) transferring the one or more cell cultures to the incubation cavity of a microgravity bioreactor equipped with means of relative retainment of water in the incubation cavity wherein the incubation cavity has an internal fluid volume of less than 1 ml   c) filling the incubation cavity with growth medium,   d) optionally eliminating air (bubbles) from the incubation cavity,   e) incubating said cell cultures under microgravity conditions,   f) changing growth medium whenever necessary to sustain incubation for the desired time period, and   g) optionally letting the culture differentiate and form tight junctions or other forms of close cell contacts (e.g. by the secretion of intercellular matrix proteins) between the cells,   

     wherein the relative retainment of water in the incubation cavity is greater than 95% after 2 days or, alternatively, wherein the relative retainment of water in the incubation cavity is greater than 95% after 5 days. 
     Alternatively, the incubation cavity of the bioreactor used in step b) has an internal fluid volume of less than 900 uL, less than 800 uL, less than 700 uL, less than 600 uL, less than 500 uL, less than 400 uL, less than 300 uL, less than  200  uL, less than 100 uL, less than 50 uL, or 25 uL. 
     It will be readily understood by one skilled in the art that steps b) and c) could be performed in whole or in part in any order and sequence. 
     In a particular embodiment, the one or more cell cultures are spheroids or are on microcarrier beads. 
     In another embodiment, the method of incubating one or more cell cultures comprises replenishing the humidity chamber of the bioreactor of the invention with an aqueous liquid whenever it is necessary to maintain a relative humidity of at least 50%, preferably at least 70%, or even more preferably at least 90%, such as at least 95%, 96%, 97%, 98% or 99%, in the air tight conduit of the bioreactor according to the invention. 
     In order to reduce the amount of air bubbles in the incubation cavity of the bioreactor according to the invention, it may be an advantage to wet the incubation cavity prior to transferring the cells and growth medium to the incubation cavity. Hence, another embodiment of the method of incubating one or more cell cultures according to the invention further comprises wetting the incubation cavity of the bioreactor according to the invention prior to transferring the cells and growth medium to the incubation cavity. 
     The cells incubated according to the method of the invention maintain a high level of differentiation. In the case of e.g. hepatocyte cells this means that the cells incubated according to the method of the invention will mimic the behaviour of a real liver. This is an advantage in e.g. toxicology studies, wherein the highly differentiated state of the cells is necessary to achieve a toxicological response matching the response of a real, functioning liver. A further advantage of using small bioreactors according to the present invention for toxicology studies, which often require testing at many different concentrations and at many different time points, is that fewer cells and less growth medium are needed to obtain useful results. This advantage is even more significant when human hepatocytes are used so that the toxicology can be determined in a human tissue microstructure liver piece. 
     Accordingly, a sixth aspect of the invention concerns a method of creating a molecular profile of the biological effect of a chemical composition, comprising the steps of:
         a) contacting an isolated population of one or more cell cultures with a chemical composition; and   b) recording alterations in gene expression or protein expression in the one or more cell cultures in response to the chemical composition to create a molecular profile of the biological effect of the chemical composition;       

     wherein the one or more cell cultures have been incubated according to the method described above. 
     The toxicity of a chemical composition may express itself very soon after contacting with one or more cell cultures (acute toxicity). Accordingly, a particular embodiment of the invention concerns a method of creating a molecular profile of the biological effect of a chemical composition as described above, wherein the one or more cell cultures is contacted with the chemical composition for a short period of time, such as at the most seven days, preferably not greater than three days, or even more preferably not greater than one day. 
     Alternatively, the toxicity of a chemical composition may also express itself after contacting with one or more cell cultures for an extended period of time (chronic toxicity). Therefore, a particular embodiment of the invention concerns a method of creating a molecular profile of the biological effect of a chemical composition as described above, wherein the one or more cell cultures is contacted with the chemical composition for at least one month, preferably at least two months, or even more preferably at least three months. 
     As mentioned above, it is an advantage to utilize the bioreactor according to the present invention for incubating one or more cell cultures. Similarly, contacting a chemical composition with one or more cell cultures may be carried out by incubating the one or more cell cultures together with the chemical composition. Accordingly, a preferred embodiment of the invention concerns a method of creating a molecular profile of the biological effect of a chemical composition as described above, wherein said contacting is performed by incubating the chemical composition and the one or more cell cultures together according to the method described herein. 
     The method according to the invention of creating molecular profiles may be repeated for several compounds. In particular, it may be repeated for several chemical compositions of known toxicity thus creating a library of molecular profiles. Therefore, a seventh aspect of the invention concerns a method of compiling a library of molecular profiles of the biological effect of chemical compositions having predetermined toxicities, comprising the steps of:
         a) contacting an isolated population of one or more cell cultures with a chemical composition having predetermined toxicities;   b) recording alterations in gene expression or protein expression in the one or more cell cultures in response to the chemical composition to create a molecular profile of the biological effect of the chemical composition; and   c) compiling a library or database of molecular profiles by repeating steps a) and b) with at least two chemical compositions having predetermined toxicities;       

     wherein the one or more cell cultures have been incubated according to the method described above. 
     Said method of compiling a library or database of molecular profiles may comprise contacting the one or more cell cultures with a chemical composition for an extended period of time (chronic toxicity) or a short period of time (acute toxicity), such as at least one month, preferably at least two months, or even more preferably at least three months, or not greater than seven days, preferably not greater than three days, or even more preferably not greater than one day. In a preferred embodiment, said contacting is performed by incubating the chemical composition and the one or more cell cultures together according to the method of the invention for incubating one or more cell cultures. 
     Once a library or database of molecular profiles has been compiled for chemical compositions of predetermined toxicities, said library or database may be compared to the profile of a chemical composition of unknown toxicity. This provides for a method of determining the toxicity of chemical compositions with a reliability that was previously only achieved with samples of adult human tissue, such as liver biopsies. Of course, the availability of such samples of adult human tissue is not very high. Hence, a ninth aspect of the present invention concerns a method of typing toxicity of a test chemical composition, comprising the steps of:
         a) creating a molecular profile of the biological effect of the test chemical composition as described in above; and   b) comparing the molecular profile in step a) with a library of molecular profiles of the biological effect of chemical compositions having predetermined toxicities compiled according to the method described above;       

     wherein the type of toxicity of the test chemical composition is determined by the comparison in step b). 
     By using the bioreactor of the invention for typing toxicity, the toxicity of a chemical composition of unknown toxicity can be determined with only a small amount of cells, growth medium and chemical composition. Hence, with the method of typing toxicity according to the present invention, it is possible to type the toxicity of a chemical composition with the reliability achieved when using adult human tissue, but at a much lower cost. The method according to the invention for typing toxicity may also be applied in instances where a toxicological profile is normally not created. For instance, a chemical composition of two or more compounds that are each individually non-toxic may turn out to be toxic when administered together (this phenomenon is known as ‘drug-drug’ interactions (even though there need be no chemical reaction between the two drugs). 
     Although the present invention has been described in connection with the specified embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the scope of the present invention is limited only by the accompanying claims. In the claims, the term comprising does not exclude the presence of other elements or steps. Additionally, although individual features may be included in different claims, these may possibly be advantageously combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. Thus, references to “a”, “an”, “first”, “second” etc. do not preclude a plurality. Furthermore, reference signs in the claims shall not be construed as limiting the scope. 
     Each of the references cited herein is hereby incorporated by reference in its entirety. 
     Examples  
     Example 1  
     Maintenance of Differentiation State of Human Liver Cells in Long-Term Culture: Expression of Cellular Proteins 
     Specific proteins expressed by fully differentiated human liver and not typically found in hepatocytes grown under normal—flat-culture conditions are found in cells cultured using a bioreactor of the present invention. The fully differentiated liver cell proteins can still be found after 302 days in culture. 
     Human hepatocytes were grown in a bioreactor of the present invention for 302 days and then labelled with [ 35 S}-methionine for 20 hrs. 
     The one or more cell cultures are cultivated as described above under normal culture conditions (e.g. 37° C. for human cells), using culture media like Eagles, DMEM or RPMI 1640. Culture media needs to be changed every second or third day depending on the particular cell culture in question. Liver cells require fresh media every second day. The media is changed by stopping the rotation of the bioreactor, waiting for the cells or prototissues to sink towards the bottom and then exchanging approximately 50% of the medium for fresh medium. 
     Initially, depending on the cell type cells will grow rapidly with a doubling time of 2-3 days but as they differentiate, the doubling time increases so that it can reach weeks or even months. Correspondingly, the frequency with which the cultures need to be subcultivated falls dramatically. Initially subcultivation should be carried out after the first week or two and thereafter at considerably longer intervals (e.g. bimonthly or longer). Subcultivation in these bioreactors is carried out by simply diluting the contents of the bioreactor by a factor of 2 to 4. Trypsin or other enzymes are not used. 
     Proteins were extracted and analysed by two dimensional gel electrophoresis and mass spectrometry. 
     The proteins are identified by protein, accession number and protein description in Table 1. The number of isoforms identified relates to the number of gel spots that were positively identified as the protein in question. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Cellular proteins expressed by fully differentiated liver cells found in 
               
               
                 bioreactor cultures after 302 days in culture and not typically 
               
               
                 secreted by hepatocytes grown under normal - flat-culture conditions. 
               
            
           
           
               
               
               
            
               
                 Protein 
                 Accession No. 
                 Protein description (No. of identified isoforms) 
               
               
                   
               
               
                 GTM3 
                 P21266 
                 Glutathione S-transferase Mu 3 (1) 
               
               
                 HS7H 
                 P34931 
                 Heat shock 70 kDa protein HOM (1) 
               
               
                 HS71 
                 P08107 
                 Heat shock 70 kDa protein 1 (1) 
               
               
                 HS74 
                 P34932 
                 Heat shock 70 kDa protein 4 (5) 
               
               
                 PDX2 
                 P32119 
                 Peroxiredoxin 2 (1) 
               
               
                 PYC 
                 P11498 
                 Pyruvate carboxylase (1) 
               
               
                   
               
            
           
         
       
     
     Example 2  
     Maintenance of Differentiation State of Human Liver Cells in Long-Term Culture: Viability of Secretory Mechanisms 
     Specific proteins secreted by fully differentiated normal human adult liver and not typically secreted by hepatocytes grown under normal—flat-culture conditions are secreted by hepatocytes cultured under microgravity conditions using a bioreactor of the present invention. 
     Proteins were precipitated from aliquots of growth medium from the tissue cultures of example 1 and analysed by two dimensional gel electrophoresis and mass spectrometry. The proteins are identified by protein, accession number and protein description in Table 2. The number of isoforms identified relates to the number of gel spots that were positively identified as the protein in question. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Proteins secreted by fully differentiated liver cells found in bioreactor 
               
               
                 culture media and not typically secreted by hepatocytes grown under 
               
               
                 normal - flat-culture conditions. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Protein description 
               
               
                   
                 Protein 
                 Accession No. 
                 (No. of identified forms) 
               
               
                   
                   
               
               
                   
                 ALBU 
                 P02768 
                 Serum albumin (8) 
               
               
                   
                 APA1 
                 P02647 
                 Apolipoprotein A-I (3) 
               
               
                   
                 CO3 
                 P01024 
                 Complement C3 alpha chain (1) 
               
               
                   
                 FGL1 
                 Q08830 
                 Fibrinogen-like protein 1 (1) 
               
               
                   
                 FIBB 
                 P02675 
                 Fibrinogen beta chain (1) 
               
               
                   
                 MTN3 
                 O15232 
                 Matrilin-3 (1) 
               
               
                   
                 THRB 
                 P00734 
                 Prothrombin (1) 
               
               
                   
                 TRFE 
                 P02787 
                 Serotransferrin (5)