Abstract:
The invention provides an apparatus and method to grow tissues in a three-dimensional mass, in a fashion similar to the way tissues grow in the mammalian body. A bioreactor according to the invention permits the growth and/or maintenance of biological tissue having a normal histotype, unlike prior art or methodology. The bioreactor further permits viewing of the tissues without disturbing the tissues.

Description:
RELATED APPLICATION DATA  
       [0001]     This application is based on and claims the benefit of U.S. Provisional Patent Application No. 60/360,918 filed on Feb. 28, 2002. 
     
    
     FIELD OF THE INVENTION  
       [0002]     This invention relates to a device and method for culturing and studying cells, tissues and organs.  
         [0003]     The study of cells and tissues has long been performed via the static growth of tissue and cells in a petri dish or plate. However, static cultures lack many of the in vivo intricacies that effect the growth, maturation, and sustenance of living tissue. Therefore, tissues and cells grown in petri dishes do not tend to exhibit identical characteristics to those found in a living body. In a dynamic system, as is normally found in living systems, nutrients and vital gases are constantly delivered while harmful toxins and waste are continually removed.  
         [0004]     In contrast, the static environment of a petri dish depends largely on the diffusion of nutrients and waste. The pitfall of a diffusion dependent environment is that a significant concentration gradient must exist in order to drive the diffusion Process. In the absence of a concentration gradient, the tissue is nutritionally starved and surrounded by potentially toxic waste, until there is a significant-concentration difference for the macromolecules to move. Tissue grown in petri dish is also forced to conform to a flat two-dimensional environment, in contrast to the three-dimensional environment from which it originated.  
       DESCRIPTION OF THE RELATED ART  
       [0005]     There have been a number of approaches for growing tissue/cells in three-dimensional mass. The factors that limit the growth of cells in a three-dimensional mass include the ability to obtain nutrients and oxygen, and the ability to remove waste and carbon dioxide. In mammalian bodies, including human bodies, the circulatory system normally provides these exchanges (see Tortora, Gerard J., et al.,  Principles of Anatomy and Physiology,  7 th  Edition, Harper Collins Publishing, 1992; incorporated herein by reference). Examples of prior approaches range from the relatively simple approach of using a dish referred to as an organ-culture dish (see Freshney, R. I., et al.,  Culture of Animal Cells: A Manual of Basic Technique,  3rd Edition, Wiley-Liss Inc., 1994; incorporated herein by reference), to more complex systems such as making a two-dimensional slice through an organ (see Harris and Stewart, “Propagation of Synchronous Epileptiform Events From Subiculum Backward Into Area CA1 of Rat Brain Slices”,  Brain Res.  895, pp. 41-49, 2001; Victorov, I., et al., “A Modified Roller Bottle Method for Organotypic Brain Cultures: Free-Floating Slices of Postnatal Rat Hippocampus”,  Brain Res. Protocols  7, pp. 30-37,2001; both incorporated herein by reference). In the organ culture dish, small chunks of tissue are collected into the center of the dish by a conically sloped surface. While the cells in aggregates or chunks placed in such a dish retain their histotype for longer time intervals than when placed directly onto the surface of the traditional cell culture plate, the organization of the tissue is disrupted over time.  
         [0006]     In order to circumvent the aforementioned disruption of tissue, some investigators have taken the approach of using slices of tissue from an organ that retain tissue organization best in two-dimensions, that is length and width, while the depth of the tissue slice is limited to only a few cell layers to permit good nutrient/waste/gas exchange. This latter approach is exemplified by experiments with brain slices. However, brain slices that are 300-450 micrometers thick are limited in viability by the amount of oxygen reaching cells in the center of the tissue slice, although the viability of such tissue is increased when the culture is incubated in hyperbaric oxygen (see Mulkey, et al., “Oxygen Measurements in Brain Stem Slices Exposed to Normobaric Hyperoxia and Hyperbaric Oxygen”,  J. App. Physiol.  90, pp. 1887-1899, 2001, incorporated herein by reference).  
         [0007]     U.S. Pat. No. 6,001,585 discloses a hollow fiber bioreactor of the prior art. Prior technology provides a means of using bioreactors for generating biological compounds, harvesting cells, producing biopharmaceutical by cells, and expanding cell populations rapidly. The bioreactor chamber of the prior art provides a growth surface for cells, which multiplies with the number of fibers potted in the chamber. However, inoculation in previous bioreactor designs requires cells to be trypsinized, pelleted by centrifugation, and resuspended before injection into the extra-capillary space.  
         [0008]     The prior art bioreactors are also not capable of allowing the placement and removal of large aggregates of cells (neither tissues nor organs) from among the fibers because there is no means of inserting the three-dimensional mass of tissue. One of the features of the present invention is an access door allowing the placement and removal of tissue in any desired orientation from among the fibers. The present invention permits sections of tissue to be inserted into the bioreactor, with minimal or no disruption to the tissue structure organization. In contrast to the limitation of the prior art, the present invention provides a means for the user to grow tissues, open the bioreactor door to biopsy the tissue and close the door to continue the experiment aseptically and without interruptions.  
         [0009]     Previous commercially available bioreactors are constructed of plastic cylindrical casings that are too large for proper microscope staging. Moreover, the round shape lacks stability on microscope stages. The present invention, however, features a stable, flat design with optically clear glass viewing windows for optimal photographic quality under a microscope.  
         [0010]     Yet another important device feature according to the present invention is a replaceable fiber cartridge design. This enables a single device to be used multiple times by simply changing the microcapillary fiber with a new fiber cartridge with similar or different fiber characteristics. The design&#39;s replaceable cartridge, tissue access door, as well as its viewing and photography capabilities are expected to enable future studies to document unobserved biological phenomena in conditions that mimic the in vivo environment.  
         [0011]     The present invention overcomes the aforementioned drawbacks of culturing in a petri dish or plate, via a bioreactor for growing three-dimensional aggregates of tissue in a fashion that closely mimics the in vivo environment. The invention provides a novel way to grow tissues in a three-dimensional mass, in a fashion similar to the way tissues grow in the mammalian body. The invention permits the growth and/or maintenance of histotype, unlike prior art methodology. The bioreactor has been shown to permit the successful maintenance of viable mouse ovary explants, the observation of continual oocyte maturation and release, and also provides for the three-dimensional adherence of tissue to a microcapillary scaffold mimicking the circulating system. (This was in stark contrast to control mouse ovary explants observed in a petri dish, which exhibited minimal viability of superficial cells, lacked any sort of oocyte maturation or release, and failed to adhere to the flat two-dimensional surface of the petri dish.)  
       BRIEF SUMMARY OF THE INVENTION  
       [0012]     The present invention provides a bioreactor that employs semi-permeable microcapillary fibers to replace the blood vessels normally found in tissue, for the purpose of growing cells in an ex vivo matrix/scaffold environment. Specifically, the bioreactor uses microcapillary fibers to continuously carry nutrients (oxygen, glucose, etc.) to the tissue, and to continuously carry away waste products (carbon dioxide, urea, etc.) from the tissue, whilst encouraging the growth of “normal” tissue histotype. The bioreactor of the present invention includes the following components: a chamber for growing tissue of interest, an access door for easy placement of the tissue onto the inner microcapillary fiber scaffolding, a structure which allows for easy viewing of cells while on standard microscope stages, enables circulating medium to pass through the inner capillary fibers, and enables easy syringe injection of fluids, such as hormones, growth factors, pharmaceuticals, toxins, etc., into the chamber&#39;s extra-capillary space. The device also allows easy replacement of microcapillary fibers and sterilization of the whole assembly for reuse.  
         [0013]     The device is made of material selected for easy sterilization and for bioinertness to cells. Tissues are grown on and amongst the microcapillary fibers, and are able to retain a normal or almost normal three-dimensional structure. Reagents or substances such as hormones, growth factors, pharmaceuticals, toxins, etc. may be introduced into the bioreactor via an extra-capillary input port, and the effects of these substances on viability, tissue morphology, growth patterns and rates, and other biological functions of the tissue can be measured and observed. Those reagents may then be removed from the bioreactor via an extra-capillary output port. The tissues in the bioreactor may be continuously or intermittently viewed such as via a microscope, and pictures or other data recorded. Using the reactor of this invention, the environment, including reagents, to which the tissues are subjected can be closely controlled, and the effect on the tissues closely monitored. The extra-capillary port may also be used to sample the extra-capillary medium, which is analogous to the extra-cellular environment of the body, for changes in medium constituents or cellular metabolical excretions.  
         [0014]     The bioreactor&#39;s ability to maintain healthy tissue was tested using tissue from the mouse ovary. For these experiments adult ovaries were removed from 7-week-old female mice and sliced into three thick segments. One segment from each ovary was place in the microcapillary bioreactor while another segment from each ovary was placed in a standard cell culture dish and both preparations were covered with cell culture medium. Comparisons were made concerning the health of the thick tissue slices over time. One of the objects of the invention is to provide a means for nutrient/gas exchange in order to promote maintenance of healthy tissue better than the tissue slice in the cell culture dish.  
         [0015]     This invention mimics the body&#39;s circulatory system environment and allows precise ex vivo control. This invention provides a means for tissue placement and removal access from among microcapillary fibers, microscope observations, assembly sterilization for reuse, and easy replacement of microcapillary fibers.  
         [0000]     Problem Definition and Solution.  
         [0016]     The purpose of this device is to grow tissues in a three-dimensional mass, in a fashion similar to the way tissues grow in the body. While aggregates of cells placed in an organ-culture dish retain their histotype for longer time intervals than when placed directly onto the surface of the traditional cell culture plate, the organization of the tissue is disrupted over time.  
         [0017]     The factors that limit the growth of cells in a three-dimension mass are the ability to obtain nutrients and oxygen in addition to the ability to remove waste and carbon dioxide. The circulatory system normally provides these exchanges (see Tortora, Gerard J., et al.,  Principles of Anatomy and Physiology,  7 th  Edition, Harper Collins Publishing, 1992; incorporated herein by reference). The bioreactor of the present invention utilizes semi-permeable microcapillary fibers to replace the blood vessels. This design solution maintains an optimal physiological environment for tissue to remain viable ex vivo and carry on functions that are otherwise difficult or impossible when undergoing static growth in a petri dish. 
     
    
     DESCRIPTION OF THE DRAWINGS  
       [0018]     The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate the presently preferred embodiments and methods of the invention. Together with the general description given above and the detailed description of the preferred embodiments and methods given below, they serve to explain the principles of the invention.  
         [0019]      FIG. 1  is a front perspective view of one embodiment of a bioreactor according to the invention.  
         [0020]      FIG. 2  is a rear perspective view of the bioreactor of  FIG. 1   
         [0021]      FIG. 3  is an exploded view of the bioreactor of  FIG. 1 .  
         [0022]      FIG. 4  is a partial sectional perspective view of the base of the bioreactor of  FIG. 1 .  
         [0023]      FIG. 5  is a front elevation cross-section of the base of the bioreactor of showing the microcapillary fiber cartridge held within the bioreactor with the fibers in a taut position within the chamber.  
         [0024]      FIG. 6  is a front elevation cross-section of the base of the bioreactor of showing the microcapillary fiber cartridge held within the bioreactor with the fibers in a loose position within the chamber.  
         [0025]      FIG. 7  is a schematic drawing of a bioreactor system utilizing a bioreactor in accordance with the invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0026]     Reference will now be made in detail to the presently preferred embodiments and methods of the invention as illustrated in the accompanying drawings, in which like reference characters designate like or corresponding parts throughout the drawings.  
         [0027]     Referring to  FIGS. 1 through 6 , an illustrative embodiment of a bioreactor  10  according to the present invention is shown. The bioreactor  10  includes a body  11  having a chamber  12  therein for holding the cells or tissue to be grown in the bioreactor. Microcapillary fibers  13  are disposed within and traverse the chamber  12 . A circulating medium, typically comprising a nutritive composition, is circulated through the microcapillary fibers  13  (in the intra-capillary space of the fibers, i.e. the space within the bores of the fibers).  
         [0028]     The body  11  is generally plate-shaped and includes a base  16  and a cover  18 . The base  16  has a generally flat rear face  14  and an opposing, generally flat front face  15 . The chamber  12  includes an access opening  17  in the base front face  15 . The cover  18  is generally flat and covers the chamber access opening  17  to provide a generally flat front face  19  for the body  11 . The cover  18  is removably secured to the base  16  using fasteners  20 , such as screws, made of a suitable material. Suitable materials for the fasteners  20  include any material that will not corrode and that will adequately permit fastening of the cover  18  and the base  16 . In a preferred embodiment, the fastener is a screw that can be unscrewed to permit the cover  18  and the base  16  to be separated. Preferred fasteners are stainless steel screws. Most preferably the screw heads or other fasteners are flush with or recessed below the cover front face  19 . By removing the cover  18  from the base  16 , one can load cells or tissue into, or remove cells or tissue from, the chamber  12  of the bioreactor  10 .  
         [0029]     A seal  22  between the cover  18  and base  16  prevents fluids from leaking into or out of the bioreactor chamber  12  and maintains a sterile environment within the chamber  12 . In a preferred embodiment, the seal  22  includes a polymeric O-ring  22   a  that is seated in a circular groove  24   a  in the base front face  15 . In yet a still more preferred embodiment, the seal  22  also includes a second outer O-ring  22   b  seated in a second circular groove  24   b  that is concentric with the inner circular groove  24   a ; in this configuration, the inner O-ring  22   a  provides the primary seal, and the outer O-ring  22   b  provides a secondary seal in the event that the primary seal of the inner O-ring  22   a  leaks and also prevents ambient air from reaching the inner O-ring  22   a , thus keeping it aseptic. The O-rings  22  should be of a polymeric material that is able to withstand autoclaving and be bioinert, and must be sufficiently deformable when the cover  18  and the base  16  are secured together to provide a tight seal.  
         [0030]     In this configuration, the bioreactor can be produced to have a thickness that is within the focal length of a microscope so that it can be placed on the stage of a standard microscope to examine the contents of the chamber  12 . The bioreactor base  16  and cover  18  are preferably made from a polymeric material having sufficient durability to permit sterilization by autoclaving, ethylene oxide, or other methods. It is preferred that at least a portion of the body  11  of the bioreactor is translucent. In a preferred embodiment, the body  11  is made of a polycarbonate material such as Lexan™, which permits the bioreactor  10  to be autoclaved, and thus reused, about twenty times, or to be sterilized via ethylene oxide and reused indefinitely. In another preferred embodiment, the body is made of acrylic, which cannot be autoclaved but which may be sterilized using ethylene oxide.  
         [0031]     The cover  18  includes a viewing window  26  for viewing into the chamber  12  through the cover  18 . In addition, the base  16  includes a viewing window  28  for viewing into the chamber  12  through the base  16 . In a preferred embodiment both viewing windows  26 ,  28  are included. It will be understood, however, that either or both of the viewing windows can be included. The windows  26 ,  28  are preferably of an optically clear material, so as to permit viewing into the chamber  12  using a microscope. It is also preferred that the windows  26 ,  28  be made of a material which does not lose its visibility due to sterilization procedures. In one advantageous embodiment, the viewing windows  26 ,  28  are an optically clear glass such as a cover slip for slides. This permits the user to clearly view the contents of the chamber  12  using a microscope, and/or enables clear photographs to be taken of the contents. The front glass viewing window  26  is inserted into a recess  27  in cover  18  and is secured to the cover  18  using an adhesive, such as a medical grade silicone adhesive. Similarly, the rear glass viewing window  28  is inserted into a recess  29  in the base  16  of the bioreactor  10  using such an adhesive. The adhesive must be autoclavable and/or able to withstand ethylene oxide or other chemical sterilization, and be bioinert.  
         [0032]     Referring to  FIGS. 4-6 , a circulating medium passageway  30  extends through the base  16  and has an input port  32  and an output port  34 . The circulating medium passageway  30  intersects and is in fluid communication with the chamber  12 . Also, an extra-capillary input port  36  is in fluid in communication with the chamber  12  via an extra-capillary input passageway  39 , and an extra-capillary output port  38  is in communication with the chamber  12  via an extra-capillary output passageway  40 .  
         [0033]     As mentioned above, the semi-permeable microcapillary fibers  13  are disposed within the circulating medium passageway  30  and traverse the chamber  12 . Each of the of microcapillary fibers  13  has an input end  42  that is in fluid communication with the circulating medium passage input port  32  and an output end  44  in fluid communication with the circulating medium passage output port  34 . The interior bores of the microcapillary fibers  13  collectively define an intra-capillary space (not shown). The chamber  12  and the microcapillary fibers  13  define an extra-capillary space  46  that is within the chamber  12  and on the exterior of the fibers  13 . The chamber  12 , which defines the extra-capillary space  46 , can be of virtually any shape or size (volume). In most instances, it is preferable that the volume of the chamber  12  be relatively small, in order to reduce the amount of reagent that need be added to effect the tissue in the chamber. This is often due to cost concerns, because reagents such as hormones and growth factors tend to be quite expensive, and therefore it is desirable to use as small amount of such reagents as is possible. The smaller the volume of the chamber  12 , the less reagent that needs to be introduced into the chamber  12  via the extra-capillary input port  36 . The larger the portion of tissue desired to be loaded into the bioreactor  10 , the larger one will want the chamber  12  to be. The size of the chamber  12  may also be chosen based upon the type of tissue to be grown therein. Thus, if one is growing or maintaining embryos in the bioreactor  10  (for example, in conjunction with human fertility treatments), a bioreactor with a larger chamber  12  may be desirable.  
         [0034]     Referring again to  FIGS. 3, 5  and  6 , in one preferred embodiment, the bioreactor includes a removable microcapillary fiber cartridge  50  that carries the microcapillary fibers  13  in a bundle  52  and is inserted into the circulating medium passageway  30 . The cartridge  50  has an end tube  54  disposed over each end of the fiber bundle  52  with ends of the microcapillary fibers  13  extending out of the end tubes  54 . The fiber bundle  52  is rigidly held in position within each of the end tubes  54  with a potting material in the end tube  54 . In one preferred embodiment, the end tubes  54  are glass tubes and the potting material is a biocompatible silicon adhesive. The end tubes  54  have a diameter that is less than the diameter of the circulating medium passage input port  32  and output port  34 , so that the cartridge  50  can be inserted through either of the circulating medium passage input port  32  and output port  34  and positioned with one end tube  54   a  resting in the circulating medium passage input port  32  and the opposing end tube  54   b  resting in the circulating medium passage output port  34  with the microcapillary fibers  14  traversing the length of the chamber  12 . A polymeric O-ring  56  is disposed around each of the end tubes  54  and is pressed into the circulating medium passage input port  32  and output port  34  so as to provide a seal between the bioreactor chamber  12  and the exterior of the bioreactor  10 . In this configuration, the extra-capillary space includes the space within the chamber  12  on the exterior of the fibers  13  as well as the space in the circulating medium passageway  30  that is between the O-rings  56  and the chamber  12  and that is not occupied by the fiber cartridge  50 . The seal prevents fluid within the chamber  12  from leaking out, and also prevents circulating fluid from entering the chamber  12 . Preferably, the O-rings  56  are composed of a material that is autoclavable and/or able to withstand ethylene oxide or other chemical sterilization, and be bioinert. When it is desired to re-use the bioreactor  10 , the microcapillary fiber cartridge  50  can be removed, and a cartridge containing new fibers can be inserted, and the entire assembly is then sterilized. Thus, the cartridge provides a convenient way to re-load the bioreactor  10  for subsequent use.  
         [0035]     The fiber cartridge  50  also permits the user to adjust the tautness of the microcapillary fibers  13  within the bioreactor  10  by adjusting the position of the end tubes  54  within the circulating medium passage input port  32  and output port  34 . Thus, the fibers  13  can be held tautly, as shown in  FIG. 5 , which is preferred for loose cells, and thus is better for cellular systems not necessarily at the tissue level. Alternatively, the position of the end tubes  54  can be adjusted so that the fibers  13  are held more loosely, as shown in  FIG. 6 , which permits tissue or pieces of organs to be inserted amongst the fibers  13 .  
         [0036]     The microcapillary fibers  13  are semi-permeable, permitting the passage through their walls of a range of biological nutrients and waste products. Examples of biological nutrients are glucose and oxygen, although the term biological nutrient is intended to encompass any type of material that assists in sustaining the life of cells and cellular tissue. Examples of biological waste products include carbon dioxide and urea, but this term is intended to encompass any type of byproduct of the biological activities of cells and cellular tissue. Examples of various types of biological nutrients and waste products are set forth in Table 1. The microcapillary fibers  13  bring nutrients to the tissues in the bioreactor  10 , and transport out of the tissue waste products, thereby simulating in vivo conditions. This results in the ability to maintain and/or grow tissues in the bioreactor  10  that are identical to or highly similar to in vivo tissues.  
         [0037]     In choosing which microcapillary fibers  13  to employ in the bioreactor  10 , the user may consider what types and sizes of biological nutrients and waste products are intended to be conveyed into and out of the bioreactor  10 . Fibers  13  can then be chosen based on their particular upper and lower ranges of molecular weight permeability. Thus, in most cases it is desired to introduce glucose through the fibers  13 , and so fibers having a permeability large enough to permit glucose molecules through must be chosen.  
         [0038]     In the case wherein a reagent, such as a hormone, a growth factor, a pharmaceutical, a toxin, etc., is to be added to the bioreactor chamber  12  via the extra-capillary input port  36 , yet another consideration in choosing fibers is to choose fibers  13  which are not permeable to the reagent, to avoid the reagent from being carried away via diffusion into the intracapillary circulating medium.  
         [0039]     Also with respect to the microcapillary fibers  13 , the choice of the material itself of which the fibers are made is important. Depending upon the choice of the material, the fibers  13  can be adherent or nonadherent to cells and tissue. Generally speaking, adherent microcapillary fibers will over the short term tend to slightly disrupt the histotype of the tissue, but will provide the advantage that the tissue will become immobilized on the fibers. An example of an adherent microcapillary fiber is cellulosic fiber, which tends to permit cells to “crawl” or “walk” along the fiber. In contrast, nonadherent fibers will tend not to distort or disrupt the histotype of the tissue, and therefore provide an advantage wherein it is important to retain the histotype of the tissue grown in the bioreactor  10 . An example of a nonadherent microcapillary fiber is polysulfone, which tends to not permit cells to “crawl” or “walk” along the fiber, and thus promotes the growth of the tissue in a structure more similar to nature.  
         [0040]     Adherent fibers such as cellulosics are thought to be useful wherein one is studying cancer cells within the bioreactor. This is because cancer cells are not anchorage dependent; thus, unlike normal cells which will tend not to go through mitosis unless they are anchored, cancer cells will go through mitosis and proliferate even if they have not adhered to a surface.  
         [0041]     Referring to  FIG. 7 , the bioreactor  10  is used in a system including a pump  60  for circulating media through the microcapillary fibers  13 . The circulation of the media through the fibers  13  insures that the cells or tissue within the chamber  12  of the bioreactor  10  are supplied with a constant stream of nutrients, and that waste products are constantly removed, as occurs in vivo. Peristaltic pumps are preferred, because they simulate the natural pulsitile flow of blood through capillaries. However, in certain situations a non-peristaltic pump may be used. For example, non-peristaltic pumps may be used in cases where the cells and/or tissues are sensitive to the pressure change brought about by the peristaltic pump.  
         [0042]     The bioreactor system also utilizes tubing  62 , preferably silicone tubing, to transport media from a reservoir  64  containing the nutrients to the bioreactor  10 , and back to a gas exchange device  66  for removing waste materials from the bioreactor. The reservoir  64  can be easily changed, either to replenish the nutrients or to change the nutrient balance. The gas exchange device  66  can be lengths of silicone tubing wrapped around a stand, which provides large areas for gas exchange, or alternatively can be a radiator-style device in a Teflon bag.  
         [0043]     The number of microcapillary fibers  13  to be selected for a particular use in the bioreactor depends in part upon the size of the biological sample to be inserted in to the bioreactor. For example, the larger the piece of tissue, the more microcapillary fibers that will be required to transport nutrients and carry away waste products therefrom. However, the use of too many fibers may obstruct the view of the tissue. Therefore, the user of the bioreactor will have to weigh these factors in determining how many fibers to use in a particular application.  
         [0044]     The bioreactor system is placed inside a cell culture incubator  68 , wherein atmospheric conditions such as temperature, humidity and gas content are controlled. Typically, the cell culture incubator  68  atmosphere is maintained at about 37 degrees Celsius, at the highest humidity (water saturation) level possible, and contains about 5% carbon dioxide. However, the ratios of gases, humidity levels and temperature can be manipulated as desired. For example, different levels of oxygen may be used, and even hyperbaric oxygen may be used. The bioreactor system utilizes a gas exchange device or mechanism to achieve proper gaseous exchange between the atmosphere in the incubator and the bioreactor. It is preferred to use a gas permeable tubing, more preferably silicone tubing, to transport the circulating media from the reservoir containing the nutrients to the bioreactor and to perform gaseous exchange. Any method for accomplishing gas exchange may be used, but in a preferred embodiment a length of tubing is used. The length of tubing may be wrapped around a stand, provided that sufficient amounts of the tubing are exposed to the atmosphere inside the incubator. Other types of gas exchangers that may be used include but are not limited to gas permeable Teflon bags. Description of an Embodiment of the Invention.  
         [0045]     The bioreactor was built with 50 microcapillary fibers potted into a milled plastic assembly. The assembly had a 1-cm 3  extra-capillary space, which was connected to two extra-capillary ports. These extra-capillary ports were present in the design in order to test the effect of the addition of reagents, such as hormones, growth factors or other biologically influential molecules, directly to the cells while the bioreactor is operating. Reagents introduced into these ports have the potential of remaining concentrated in the extra-capillary space and not being diluted into the circulating media depending on the microcapillary fiber molecular weight cutoff. The top and the bottom of the extra-capillary chamber were sealed with a #2 cover slip.  
         [0046]     The assembly was connected in series to a medium reservoir bottle, a coiled up length of silicone tubing, and a pump fixture. The reservoir bottle had a capacity of 100-milliliters of medium, which was sufficient to sustain the 1-millimeter ovary fragment for a long period of time. The ovary fragment, or any other type of tissue, remains suspended among the bed of microcapillary fibers, which mimics the mass transfer of nutrients/wastes achieved by capillaries in the circulatory system. The long length of coiled silicone tubing was necessary to allow adequate time for gas exchange with the incubator&#39;s environment. Silicone tubing is specifically preferred because of the material&#39;s high permeability to physiological gases that are readily exchanged by the body to maintain homeostasis.  
         [0047]     A pump fixture designed to fit a CellMax peristaltic pump or another commercially available peristaltic pump is needed to circulate medium around the closed circuit and through microcapillary fibers. A Cellmax Quad [Spectrum Laboratories Inc., Cat: CMQUAD-B] with the capability of simultaneously pumping four bioreactors was used.  
         [0000]     Benefits of the Bioreactor.  
         [0048]     The bioreactor of this invention is expected to allow scientist to do research at much reduced costs. Often research projects are dependent on observing time progressive phenomenon. This results in the need for hundreds of replicates in an experiment, with experimentation halting on a few replicates at a time in order to obtain time course data. For example, in a small experiment a hundred mice with liver tumors might be injected with a pharmaceutical drug and ten mice would be anesthetized and sampled each month for ten months. Biopsies of the liver tissues would reveal the time course effects of the pharmaceutical drug. Alternatively, liver tumor tissue could be placed in the bioreactor with the pharmaceutical drug and the histological changes could be photographed under a microscope as frequently as desired. Thus, there is a reduction in the number of animals being sacrificed and a reduction of general experimentation costs for initial screening.  
         [0049]     This invention has potential societal benefits as well. Women that are scheduled to undergo chemotherapy or radiation treatments for cancer often have ovulated eggs removed, or part or all of their ovaries removed, and cryopreserved. This is done in order to provide eggs that can be fertilized and used to produce children should the women survive the cancer treatments. Radiation therapy and chemotherapy can cause mutations in the chromosomes of the egg, and therefore removal of the egg prior to cancer or other medical treatment circumvents this potential problem. In the past, ovarian tissue fragments have been cultured after cryopreservation (Hovatta, 1999), but such fragments had to be cultured in the kidney capsule (Oktay et al., 1998; Posada et al., 2001). The present invention would be a viable solution to enabling these women to have children.  
         [0000]     Test Data And Analysis.  
         [0050]     The tissue used to test the ability of the device to maintain tissue health was from the mouse ovary. For these experiments adult ovaries were removed from 7-week-old female mice and sliced into two thick segments. One segment from each ovary was place in the microcapillary bioreactor while the other segment from each ovary was placed in a standard cell culture dish and both preparations were covered with cell culture medium.  
         [0051]     Two types of fibers were tested and are designated as Fiber A, which is made of cellulosic with a molecular weight cutoff 30 kD and Fiber B, which is made of high flux polysulfone with a molecular weight cutoff of 20 kD.  
         [0052]     Ovary fragments grown with Fiber A appeared to spread along the fiber with increasing time of incubation. This spreading of cells along Fiber A may have occurred because the surface of Fiber A permitted cell attachment while the surface of Fiber B prevented cell attachment. In contrast fragments grown with Fiber B retained the original shape of the ovary fragment.  
         [0053]     The ovary fragments grown with Fiber A appeared to produce mature follicles and subsequently eggs ovulated out of the follicles. The ability of the ovary fragments to produce mature follicles and to subsequently ovulate eggs indicated that the tissue had not died, and that the tissue retained its normal function (i.e., the production of follicles and eggs).  
         [0054]     Ovary fragments in bioreactors made with Fiber A showed a darkening of some of the fibers over time when viewed under a microscope. The composition of this material was not determined. However since Fiber A allows adhesion of cells, the darkened portions of the fibers could represent sites where proteins from cells stuck to the fiber.  
         [0055]     The ovary fragments in the bioreactors appeared healthy for the entire period of the experiment, that is 28 days. In contrast, visual observation of the ovarian fragments grown in the culture plate appeared to deteriorate over time.  
         [0000]     Microcapillary Fiber Selection.  
         [0056]     Microcapillary fibers are available in numerous lengths, diameters, materials, and molecular weight cutoffs. Fiber length is an important consideration for larger bioreactor designs, which are suitable for large aggregates of tissue. The bioreactors of the present invention may be built relatively small for viewing under high power microscopes and so readily available 10-centimeter length fibers may be utilized.  
         [0057]     The material that the fibers are made from greatly affects the performance and function of the bioreactor. Depending on the surface properties of the material, use of a particular type of microcapillary fiber material will influence how the cells and tissue grow in the bioreactor. Some types of fibers will easily allow cell adhesion and encapsulations, and other types will prevent cell motility or scaffold incorporation. There are useful applications for particular fibers manufactured with material of any surface characteristic. Fiber material types conducive to cell motility could be used to increase an aggregate tissue mass by many folds whereas fiber material types prohibiting cell motility could be used as scaffolding to sustain an aggregate tissue for observations at increasing time intervals. The third fiber characteristic, molecular weight cutoff, has a highly significant influence on the function of the bioreactor. A proper fiber selection will allow easy diffusion of desired molecules to the cells while prohibiting molecules larger than the weight cutoff limit from reaching the cells. Table 1 lists molecules and their respective molecular weights for a comparison of molecular weight cutoff effects on a biological system.  
         [0058]     Two types of fibers have been tested. These are nonlimiting examples of the brands and types of fibers which may be used. Fiber A is from a Cellmax Artificial Capillary module [Spectrum Laboratories Inc., Cat: 400-022], is made of cellulosic material and has a molecular weight cutoff 30 kD. The 30 kD cutoff allows for the diffusion of electrolytes in but does not permit large enzymes and factors to pass through much like the capillaries in a human circulatory system. The cellulosic is a substrate onto which cells can adhere and move around. This is in contrast to fiber B, which is from a FiberCell™ module [FiberCell™ Systems Inc., Cat: 4300-C2011], is made of high flux polysulfone, and has a molecular weight cut off of 20 kD. Fiber B&#39;s material has surface properties that are not conducive to cell adhesion nor cell motility and has a lower molecular weight cutoff that essentially reduces the quantity of biological components that are able to diffuse freely. However, FiberCell™ Systems Inc. Claims that fiber B&#39;s naturally hydrophilic polymer can improve cell viability by providing 10 times the gross filtration rate of cellulosic hollow fibers. Tables 2 and 3 below contain information about microcapillary fibers and their characteristic from FiberCell™ Systems Inc. and Spectrum Laboratories Inc.  
                                             TABLE 1                           Molecular   Can diffuse   Can diffuse       Molecule Name   Weight   through Fiber A   through Fiber B                                Water   18   ♦   ♦       Albumin   ˜68000       Transferrin   78000       T3, Leu-4 (CD3γ)   25000-28000   ♦       T3, Leu-4 (CD3δ)   20000   ♦   ♦       T3, Leu-4 (CD3ε)   20000   ♦   ♦       T4, Leu-3   55000       Neutral Endopeptidase   100000       Aminopeptidase   150000       Fas Antigen   45000       Fibrinogen (Protein)   ˜341000       Urea (Waste)   60.06   ♦   ♦       Uric Acid (Waste)   168.11   ♦   ♦       Creatine (Waste) [C 4 H 9 N 3 O 2 ]   131.1   ♦   ♦       Creatinine (Waste) [C 4 H 7 N 3 O]   113.12   ♦   ♦       Bilirubin (Waste) [C 33 H 36 N 4 O 6 ]   584.68   ♦   ♦       Glucose (Nutrients) [C 6 H 12 O 6 ]   182.14   ♦   ♦       Glycerol (Nutrients) [C 3 H 8 O 3 ]   92.09   ♦   ♦       Enzymes (Regulatory Substance)   &gt;34000       Oxygen (Gases) [O 2 ]   32.00   ♦   ♦       Carbon Dioxide (Gases) [CO 2 ]   44.01   ♦   ♦       Nitrogen (Gases) [N 2 ]   28.01   ♦   ♦       Na +  (Electrolytes)   22.99   ♦   ♦       K +  (Electrolytes)   39.10   ♦   ♦       Ca +  (Electrolytes)   40.08   ♦   ♦       Mg +  (Electrolytes)   24.31   ♦   ♦       Cl −  (Electrolytes)   35.45   ♦   ♦       HPO 4   2−  (Electrolytes)   95.97   ♦   ♦       SO 4   2−  (Electrolytes)   96.06   ♦   ♦       HCO 3   −  (Electrolytes)   61.01   ♦   ♦                 Molecules, their molecular weight, and their ability to diffuse through fibers used in this experiment            (Fiber A = Cellmax module with 30 k MW cutoff, Fiber B = FiberCell module with 20 k MW cut off) (Abbas, 2000; Janeway, 1999)             
 
         [0059]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   
               
               
                 Microcapillary characteristics of cartridges available from 
               
               
                 FiberCell Systems Inc. 
               
             
          
           
               
                   
                   
                 Surface 
                   
                 Packing 
                 ECS 
                 MWCO 
                 Max. 
               
               
                 Stock No. 
                 Size 
                 Area 
                 Fiber Type 
                 Density 
                 Vol 
                 50% 
                 Cell# 
               
               
                   
               
             
          
           
               
                 4300-C2025 
                 Small 
                 75 
                 cm2 
                 activated PS 
                 30% 
                 2.5 
                 mL 
                 .2 
                 μm 
                 10 8   
               
               
                 4300-C2008 
                 Med. 
                 2100 
                 cm2 
                 low flux PS 
                 50% 
                 12 
                 mL 
                 5 
                 kd 
                 10 9   
               
               
                 4300-C2011 
                 Med. 
                 2100 
                 cm2 
                 high flux PS 
                 50% 
                 12 
                 mL 
                 20 
                 kd 
                 10 9   
               
               
                 4300-C2003 
                 Large 
                 1.2 
                 m2 
                 low flux PS 
                 50% 
                 70 
                 mL 
                 5 
                 kd 
                 5 × 10 10   
               
               
                 4300-C2018 
                 Large 
                 1.2 
                 m2 
                 high flux PS 
                 50% 
                 70 
                 mL 
                 20 
                 kd 
                 5 × 10 10   
               
               
                 4300-C2023 
                 Med. 
                 2100 
                 cm2 
                 cellulosic 
                 38% 
                 12 
                 mL 
                 10 
                 kd 
                 10 9   
               
               
                   
               
             
          
         
       
     
         [0060]                                                                                                    TABLE 3                           Microcapillary characteristics of cartridges available from       Spectrum Laboratories Inc.                        Wall   Lumenal   Outer                   Catalog       O.D.   Thickness   Area   Surface   ECS   95%   50%       Number   Material   μm   μm   cm 2     Area cm 2     Vol.   MWCO   MWCO                    400-022   Cellulosic   210   15   220   250   2.2   150   k   30 k       400-008   Cellulosic   216   8   780   847   7   20   k    4 k       400-023   Cellulosic   200   10   1866   2100   12   50   k   10 k       400-011   Cellulosic   210   15   1900   2200   12   150   k   30 k       400-012   Polyethylene   430   50   964   1500   12   0.3   μm   —       400-014   Polyethylene   430   50   109   123   2.3   0.3   μm   —       420-015   Polyethylene   430   50   108   123   2.3   0.3   μm   —           coated       400-007   Polypropylene   630   150   327   423   7   0.5   μm   —       420-007   Polypropylene   630   150   327   423   7   0.5   μm   —           coated       400-025   Polypropylene   630   150   70   100   1.4   0.5   μm   —       420-025   Polypropylene   630   150   70   100   1.4   0.5   μm   —           coated                    
 Bioreactor Sterilization. 
 
         [0061]     The bioreactors were gas sterilized with ethylene oxide, although other methods of sterilization may be utilized, provided that the structure and function of the bioreactor is not adversely affected. The sterilization procedure involved placing the bioreactor chambers inside gas permeable sterilization pouches and sealing the pouches with sterilization indicator tape. Inside each sterilization pouch were placed all other necessary components such as screws and luer lock caps. The complete pouches were then placed inside a gassing chamber. The chamber air was evacuated and ethylene oxide gas was introduced to replace the evacuated air. The bioreactors remained in contact with the ethylene oxide gas for 24-hours to insure that the sterilizing gas adequately diffused through all microcapillary tubes. This was followed by a 24-hour period of degassing where the ethylene oxide gas was evacuated and sterile air was continually introduced into the sterilization chamber. Prior to use the bioreactor&#39;s capillaries were given a final flush with sterile PBS.  
         [0000]     Medium Preparation.  
         [0062]     A laminar flow hood was prepared by spraying the inside with 70% ethanol while the hood was filtering the air through HEPA filters and by exposure to an ultraviolet germicidal lamp (253.7 nano-meter light) for 15 minutes. A 100-ml bottle of fetal bovine serum (FBS) [BioWhittaker, Cat: 14-502F] was thawed from freezer storage by using a warm water bath [Fisher Scientific, Model 220] at 37.0 degrees centigrade. After the 15-minute sterilization of the hood, the thawed FBS, a 1-liter bottle of Dulbecco&#39;s Modification of Eagle&#39;s Medium 1× (DMEM) [Mediatech Cellgro, Cat: 15-013-CM], and 10 steam autoclaved 100-ml bottles were placed in the hood aseptically. Following all aseptic techniques, the 100-ml of FBS was added to the 1 liter bottle of DMEM with 20,000 IU Pen-Strip antibiotics and 3 mM glutamate. The DMEM bottle cap was replaced snuggly and the bottle was inverted and reverted continually for approximately 1-minute to insure adequate mixing of the liquids. The bottle was gently inverted so that bubble formation was minimized. After this mixing, the prepared medium was aliquoted equally among the 10 steam autoclaved 100-ml bottles and these bottles were immediately placed in the refrigerator (8 degrees centigrade) for later use as needed.  
         [0000]     Bioreactor Preparation &amp; Ovary Fragment Inoculation.  
         [0063]     To insure that all of the sterilizing ethylene oxide gas had been eliminated, a 100-ml bottle of Dulbecco&#39;s phosphate buffer solution 1× (PBS) [Mediatech Cellgro, Cat: 21-030-CM] was attached in place of the medium reservoir bottle and continuously circulated for four hours. This washing phase took place in a HEPA laminar flow hood [Ferma Scientific, Class IIA/B3 Biological Safety Cabinet] with the blower and the UV lamp operating. Meanwhile, four whole ovaries were removed from mice, cut into smaller gragments about one half the original size, and placed into petri dishes with the same medium prepared for the bioreactor. Next the PBS was completely pumped out of the system before the introduction of the DMEM containing 9% FCS. Sterile forceps were unpackaged and used to spread the microcapillary fibers apart so the ovary slices would be nestled in the bioreactor&#39;s microcapillary fibers. Each bioreactor was successively placed into the incubator as ovary fragment inoculation was completed. Upon the inoculation of all bioreactors, time lapsed photography began.