Abstract:
An apparatus for the culturing of oocytes, embryos, stem cells and cells allows the culturing of the specimens by communally growing or grouping of the specimens and maintains identification of the specimens and allows for the ease in use and location of the specimens in the apparatus. The ability to group the embryos increases cleavage rates, embryos scores and increases the likelihood of better embryos and pregnancy results. This will increase the chances of the survival and future growth or use of the specimens. The apparatus takes the form of a dish which is formed with a plurality of integral wells in which the specimens are placed. The wells or chambers may be subdivided into separate compartments which contain individual embryos or other specimens that are being cultured. The compartments will be interconnected so that the culturing media and culturing byproducts from each specimen can be shared by all of the specimens in any one well, but so that the specimens cannot migrate from one compartment to another.

Description:
TECHNICAL FIELD 
     This invention relates to an apparatus and method for culturing or growing individually identifiable cells, stem cells, immature oocytes, embryos or any portions of each in a communal environment, which allowing the interaction of fluids, nutrients or growth enhancers among the individual specimens. This invention relates to an apparatus and methods wherein individual specimens in the apparatus are kept physically separated from each other, but are permitted to share a common growth-enhancing nutrient, or share common growth byproducts, thereby resulting in an increased proficiency of the growth process. The apparatus on a larger scale will allow the progressive culturing of specimens such as embryos, stem cells and cells. 
     BACKGROUND ART 
     Human, and other animal cells, are presently cultured in suitable nutrients such as culture media for hours or days for growth, in the case of embryos, and for reproduction purposes, in the case of stem cells. 
     The following relates to the growth culturing of individual embryos. There are several generally practiced embryo growth procedures which are presently in use. One such growth technique involves the use of a culturing container such as a Petrie dish in which individual embryos are placed in spaced-apart locations in the culturing dish. This technique involves the initial placement of individual embryos on a growth-enhancing nutrient, and subsequently immersing each of the individual embryos in a drop of a growth-enhancing nutrient, such as culture media. Several common media are HTF, Earl&#39;s salt solution and GLOBAL culture media. The individual specimens are kept separate from each other, can be individually identified, and separately examined. Thus, the advantage of this procedure is the ability to monitor each individual embryo throughout the growth period so that there is a degree of selectivity available at the uterine-implanting stage of the process. One drawback in using this procedure relates to the fact that embryonic growth seems to be improved when the several embryos are grown in a common droplet or vial of growth-enhancing nutrient and are thus able to share each other&#39;s growth induced byproducts and this procedure does not allow the transfer of nutrients or byproducts. 
     Another of the generally practiced embryo growth techniques involves clustering a plurality of embryos together on a Petrie dish and immersing the cluster with a common drop of the growth-enhancing nutrient, such as culture media. Using this technique, all of the embryos in a duster are exposed to the same growth-enhancing nutrient drop and are able to share that growth-enhancing nutrient and also share their respective byproducts of the growth process. The drawback with the second technique is that one cannot distinguish one embryo from another in the cluster, in other words, each individual embryo cannot be separately monitored during the growth process. Thus, the ability to select a preferred one of the grown embryos for implanting is somewhat impaired by use of the second growth technique. 
     A desirable supplement for the embryo growth media is one or more growth enhancers or hormones for providing increased embryo growth, or for supplying factors that may be missing from the specimen or from the culture media. Currently, the enhancers may be obtained through the use of natural or synthetic growth hormones or from the use of stem cells to produce or give off growth hormones. 
     U.S. Pat. No. 6,448,069 Cecchi et al describes the separation of embryos or other specimens using what could be described as picket fences to form separate specimen compartments in a media culturing dish. One problem that is not addressed by the &#39;069 patent is the migration of the specimens due to their size and/or weight and the possibility of them moving between the compartments. Additionally, they may not be readily discernible in the compartments by reason of the shape or configuration of the floor of the compartments. 
     Current products used for similar purposes are made with walls that are as vertical as practical. They do not address the fact that the embryos may measure less than ninety microns in size. This makes finding the embryos difficult in the dishes and also makes it difficult to locate an embryo f it has attached itself to a wall at some distance upwardly from the floor of the dish. The problem that arises is that the embryo is now at a different focal plane when compared to the top of the wall and the floor of the dish, and therefore is difficult to locate. 
     Current products of this type do not include alignment indices, which will allow the user to establish an orientation of the apparatus for viewing purposes or for the possible use with a mechanized viewing or work system. 
     It would be highly desirable to provide an growing method and apparatus, which would provide the ability to segregate the individual specimens, one from another, allow the addition of items such as growth factors to the apparatus while also providing the ability to allow the segregated specimens to share a common growth-enhancing cell culture media, nutrients such as growth factors and share each other&#39;s growth byproducts. It would be also highly desirable to provide an apparatus that will allow the ease in monitoring and visibility of the specimens, while keeping the specimens from migrating between compartments. It would be highly desirable to provide an apparatus that makes locating the specimens, such as embryos easier and therefore saves time and effort. 
     DISCLOSURE OF THE INVENTION 
     This invention relates to an improved method and apparatus for growing or replicating biological specimens. The method and apparatus may be used for many and various specimens which may include uterine tissues, immature oocytes, oocytes, embryos and stem cells or embryonic stem cells. The method and apparatus provide for the tracking and identification of each of the specimens as they grow or replicate. Several specimens can share a common space or configuration of wells or culturing compartment in the apparatus, which culturing compartment may include the specimens, growth-enhancing nutrient solution such as culture media, adjunct growth enhancers such as hormones and the specimen&#39;s growth byproducts. Thus the specimens may be individually monitored as to they grow, develop or replicate. The growth process is performed in a container, similar to a Petrie dish. 
     A plurality of compartmentalized structures may be molded in the apparatus in predetermined patterns. Each of the compartmentalized structures contains a plurality of separate compartments or sections which are sized to hold one specimen or may hold a plurality of specimens, or cell units. The compartments or sections may be separated from each other by one or more channels which form canal-like openings between each of the compartments or sections in each of the compartmentalized structures. Each of the compartmentalized structures thus allows inter compartmental migration of the growth-enhancing nutrient, culture media, fluids, growth hormones and growth byproducts that may be produced by each of the specimens disposed in one of the structures. The compartmentalized structures may be of any size necessary to hold the required number of specimens. The compartmentalized structures are preferably molded into an overall structure similar to a Petrie dish. The apparatus is molded from a suitable plastic material. The plastics preferred are plastics which exhibit minimal off gassing, low endotoxins of their structural compounds and are a material which may promote or enhance cell growth, or may be coated with cell growth materials or enhancers. The compartmentalized structures may also be structured so that the channels and connectivity is external to the compartments or sections and the compartments or sections are not directly connected by canals, channels or openings between the compartments or sections. 
     The following is a general description of one manner of using the apparatus of this invention for in vitro growing of fertilized embryos prior to implantation of an embryo into a female recipient&#39;s reproductive tract. As noted above, the compartmentalized specimen containing structures are preferably positioned in a Petrie dish or are an integral part of the dish itself. In such an arrangement there will be a plurality of the compartmentalized structures in the dish. These compartmentalized structures may be visibly designated by a series of identifiers such as numbers or letters, and of course their locations on the dish will be known and fixed. An area on the dish may be utilized to display information for monitoring of the specimens in the dish. Each of the compartmentalized structures will include a plurality of compartments, as, for example, two, three or four compartments. Each of the compartments will contain a separate specimen, or may contain a nutrient component such as growth hormone, growth enhancer or stem cell. 
     The dish can include a plurality of imbedded or molded in indices which will allow the user to orient the dish for use and also allow the user to establish fixed points of reference in the relationship to the user&#39;s workspace, microscope base. The user can thus orient the dish and then be able to locate specific points within the dish. The indices can be imbedded or molded in the bottom, or floor of the dish or in the external surface of the outer wall of the dish. This will allow greater ease in use, time savings and possible mechanical orientation for more difficult or exacting procedures. This orientation may allow the dish to be placed in an additional holding apparatus, and or placed within a controlled environment where it can be observed or worked remotely. 
     In the description, larger units of the apparatus will be referred to as structures, any divisions of the structures will be referred to as compartments, and the independent units will be referred to as wells. 
     Individual compartments in the dish will contain a raised or multiple level portions of the bottom of each compartment creating depressions or high points to cause the specimens to migrate toward the depressions of the compartments containing the specimens. Canals between the raised bottom compartments maintain the flow of fluids and material between adjacent compartments. Walls between the compartments will keep the specimens from migrating between adjacent compartments. One method to accomplish this is to make the separating walls in such a fashion that they extend above the outer walls of the compartments and extend into a layer of paraffin oil which has been dispersed in the dish. The viscosity of the paraffin oil keeps specimens, such as embryos, from moving between compartments. 
     It is therefore an object of this invention to provide an improved specimen growth-supporting method and apparatus which enables one to monitor the growth of individual specimens which are disposed in a growth-enhancing nutrient. 
     It is another object of this invention to provide a method and apparatus of the character described which enables individual embryos in a growth-enhancing nutrient to share growth byproducts of other embryos in the growth-enhancing nutrient. 
     It is a further object of this invention to provide a method and apparatus of the character described which will enable the user to readily locate individual embryos that are within the apparatus. 
     It is yet another object of this invention to provide a method and apparatus of the character described which will enable the user to establish a directional orientation of the apparatus which will allow the apparatus to be used with mechanical instruments and to readily find specific points of reference and therefore be able to find the specimens that are within the apparatus. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other objects and advantages of the invention will become more readily apparent from the following detailed description of the invention, when taken in conjunction with the accompanying drawings in which: 
         FIG. 1  is a top plan view of one embodiment of a specimen culturing dish formed in accordance with this invention; 
         FIG. 2  is a top plan view of one of the embryo growth compartments contained in the specimen sample culturing dish of  FIG. 1  which includes several internal compartmentalized embryo growth structures; 
         FIG. 3  is a top plan view similar to  FIG. 2  but showing another variation of an internally compartmentalized embryo growth compartment; 
         FIG. 4  is a sectional view of the compartment shown in  FIG. 3  taken along line  4 - 4  of  FIG. 3 ; 
         FIG. 5  is a top plan view similar to  FIG. 3  but showing yet another variation of an internally compartmentalized embryo growth compartment; 
         FIG. 6  is a top plan view of an embryo growth compartment formed in accordance with this invention and showing how an individual embryo will settle to a predetermined location in the compartment which position is dictated by the configuration of the bottom wall of the compartment; 
         FIG. 7  is a top plan view of yet another configuration of an embryo growth compartment showing how an embryo settles to a predetermined location on the bottom wall of the compartment; and 
         FIG. 8  is a sectional view of the compartment taken along line  8 - 8  of  FIG. 7 . 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Referring now to  FIGS. 1 and 2  there is shown an oocyte, embryo and stem cell specimen growth container, denoted generally by the numeral  2  which is formed in accordance with this invention. The container  2  has an outer wall  4  and a bottom wall  6 . The bottom wall  6  is flat, and has a small raised portion below the outer wall  4  which will keep the bottom wall  6  slightly elevated above a surface it may be placed on, thereby keeping the bottom wall  6  from being scratched by the surface it is placed on. Thus, in the areas where the user needs to maintain clear ocular views, they will be maintained. Molded into the container  2  are a plurality of structures  8  that form compartments  10  which are used for the culturing of the specimens which can be, for example, embryos. The structures  8  include an outer wall  12  and a recessed formed bottom wall or floor  14 , and can include inner walls or separators  16  that create individual chambers  18  in each compartment  10 . The recessed bottom walls  14  allow the user to more readily locate the embryos under magnification in the compartments  10  and chambers  18 . The container  2  measures typically fifty five cm. in diameter and has an outer wall  4  which is typically one cm in height The multiple culturing compartments  10  measure typically 1.2 cm in diameter and their exterior walls  12  typically measure two mm in height. The interior walls or separators  16  are slightly taller and typically measure 2.2 mm in height. 
     As shown in  FIG. 2 , within these separators or walls  16  are spaces or channels  20  which will allow the fluids to flow between the culturing compartments  18  and  19 . The interior walls or separators  16  are taller than the outer wall  12  of the culturing compartments  10  so that when the dish  2  is flooded with mineral oil or paraffin oil which overlays the media in the compartments, the taller walls  16  will penetrate the oil layer and, due to the viscosity of the oil, the embryos will not migrate through this layer and thus cannot migrate into a different compartment  18 . 
     The dish  2  may contain a plurality of smaller structures  8 ′ which form additional compartments  10 ′. These structures  8 ′ may be evenly placed about the dish  2  and are typically eight mm in diameter and have walls which are typically two mm in height. These structures  8 ′ take the form of wells which can be used as reservoirs of culture media, for the holding or washing of embryos or specimens, or for the individual culturing of embryos or specimens. The floor  14  of the wells  8 ′ is contoured so as to create a low point and/or depression  22  in the floor  14 . The depressions  22  allow the embryos or other specimens to settle into known locations in the wells  8 ′ to simplify locating of the specimens when viewed under magnification. The depressions  22  in the culturing wells  8 ′ are all the same viewing distance and at the same ocular setting of the microscope so as to allow the user to move among the culturing compartment wells  8 ′ and floor  6  of the dish  2  without changing the setting of the microscope. During use, the culturing compartments  18  and wells  8 ′ will be immersed with culture media. The entire dish  2  can then be flooded with paraffin oil, or mineral oil to keep the medium from evaporating, prevent absorption of air contaminants, and to utilize the structural height of the inner compartment walls or separators  16  to keep embryos or other specimens from migrating among compartments  18 . 
     Embedded or molded into floor  6  of the dish  2  are orientation indices  24 . These indices  24  allow the user to orient the dish  2  and simplify locating of the specimens. The indices  24  will also allow the dish  2  to be set up on the microscope stage in the same orientation and may be used to establish the same orientation each time the specimens are viewed. The dish  2  can also have external indices  26  imbedded in or on the outer wall  4  of the dish  2  for this purpose. 
     As noted above,  FIG. 2  is a top plan view of an oocyte, embryo or stem cell growth compartment  12 . The walls  16  separate the compartment  12  into a central chamber  18  for containing the specimen being cultured which is surrounded by an inner annular trough  19 . The walls  16  may be continuous and uninterrupted, or they may be segmented with the segments being separated by gaps in the walls  16 . The trough  19  may hold addition fluids for use in culturing the specimens. Within the walls  16  that separate the chambers  18  and  19  are openings  20  which will allow the flow of liquids between and among the chambers  18  and  19 . The areas of chambers  18  and  19  may be used to hold specimens, and in this example, will allow fluid flow between the four separate chambers. 
     Referring now to  FIGS. 3 and 4 , there is shown a modification of one of the specimen compartments  8 . The compartment  8  contains an outer wall  12  and an inner wall  28 . The inner wall  28  forms a chamber  30  which is segregated into two halves  31  and  33  by a wall  32 . An opening  34  separates the wall  32  into two spaced-apart sections. The opening  34  allows fluids to move between the two halves  31  and  33  of the chamber  30 . The opening  34  is small enough to prevent embryos from moving from one half  31  to the other half  33  of the chamber  30 , thereby allowing the chamber  30  to be used to culture two embryos which can share the culturing fluids that are put in the chamber  30 , and also share growth byproducts which are produced by each of the embryos during the culturing period. The opening  34  may also contain a membrane to allow fluids but not specimens to move. Specimens may also be placed in areas  18 ,  19 ,  24  and  26 . This will enable these specimens to share nutrients and byproducts in all four of these areas. Specimens may also be placed in areas  19 ,  24  and  26  with special ingredients, such as growth factors, being placed in area  18 . This will allow specimens in these three areas to share amongst each other. 
     As shown in  FIG. 4 , the bottom wall of the chamber  30  is a compound structure which has a central lowest flat horizontal portion  36  and outer flat downwardly angled portions  38 . Embryos which are cultured in the chamber sections  31  and  33  will thus settle down onto the lowest portion  36  of the bottom wall of the chamber  30  where they can be easily located for visual inspection during the culturing period.  FIG. 4  also shows the use of taller walls  32  and opening  34  to keep the embryos contained in the individual chamber sections  31  and  33  and prevent them from migrating from one chamber section to the other. As shown in  FIG. 4 , a culture media  40  is placed in the chamber  30 . The compartment  8  is then flooded with paraffin oil or mineral oil  42 . The oil  42  overlays or sits on top of the media  40 . The walls  32  extend up into the layer of oil  42 . Due to the viscosity of the oil  42 , embryos or other specimens cannot migrate or move from one chamber section  31  to the other chamber section  33 . Thus, the individual specimens which are being cultured are “trapped” in their respective culturing chamber sections  31  and  33  during the culturing procedure. The upper section of walls  32  are immersed in the oil layer  42 , above the wall  28 . This embodiment illustrates the ability of media, nutrients, fluids and growth hormones or growth byproducts to flow through the opening  34  to be shared by specimens in chamber sections  31  and  32 . The opening  34  is of a size which will not allow the specimens to move through the opening  34 . 
       FIG. 5  shows a modification of a culturing compartment  8  which includes a culturing chamber  30  having chamber sections  44  that are separated from each other by walls  46  that block direct media flow between the culturing chamber sections  44 . Each of the sections  44  is bounded by an outer wall  48  which has openings  50  for each of the sections  44 . An outer chamber  52  bounded by an outer wall  54  surrounds the sections  44 . The culturing media can be placed in the outer chamber  52  and will be able to migrate into the sections  44  through the openings  50 . The specimens to be cultured will be placed in the sections  44  and will be able to share the culturing media and culturing byproducts indirectly by reason of the openings  50 . The openings  50  will be too small to allow any of the specimens to move from one of the sections  44  to another or may utilize a membrane to keep the specimens from migrating. Thus the specimens will be restricted to their respective sections  44  but will still be able to share the culturing media and culturing byproducts that each produces via the outer chamber  52 . Not shown in this figure is the raised walls or depression of the floor of the compartments, but these items can be used in this configuration also. 
       FIG. 6  is a plan view of a culturing compartment  8  or  8 ′ showing how one version of the contoured floor  14  of the compartment serves to place an embryo  56 , or other specimen, in a predetermined location on the compartment floor  14 . In this embodiment, the floor  14  has three segments  58  which are planar and slope at the same angle downwardly to a central point  60 . This configuration will cause the embryo  56  to migrate toward the central point  60  where it can be readily located visually with a microscope or other optical instrument so that the stages of development of the embryo  56  can be monitored during the culturing cycle. The slope of the floor segments  58  creates a known depth of the point  60  so that the focal point of the viewing instrument can be preset so that each of the compartments  8  or  8 ′ in a culturing dish can be monitored for embryo stage development without having to refocus the viewing instrument. 
     Referring now to  FIGS. 7 and 8  there is shown therein a culturing compartment  8  or  8 ′ which is similar to that shown in  FIG. 6 , but which has a different bottom floor  14  contour configuration than that shown in  FIG. 6 . The bottom floor  14  has four planar segments  62  which slope downwardly to form a linear central low location  64  into which an embryo  56  or other specimen being cultured will settle. This configuration will cause the embryo  56  to migrate toward the central location  64  where it can be readily located visually with a microscope or other optical instrument so that the stages of development of the embryo  56  can be monitored during the culturing cycle. The slope of the floor segments  62  creates a known depth of the location  64  so that the focal point of the viewing instrument can be preset so that each of the compartments  8  or  8 ′ in a culturing dish can be monitored for embryo stage development without having to refocus the viewing instrument. The sloped floor segments  62  also will result in the dispersal of debris along the line  64  so as to allow the specimen to be debris-free. As shown in  FIG. 8 , the embryo  56  will be covered by a culturing media and/or oil layer  66  during the culturing procedure. 
     It will be readily appreciated that the cell culturing apparatus of this invention will allow individual specimens being cultured in the apparatus to share nutrients, and perhaps even more importantly, to share growth byproducts produced by each of the specimens. This result is accomplished while physically isolating each specimen from each of the other specimens in the apparatus. Additionally, each specimen in the apparatus can be monitored visually or with an appropriate monitoring instrument at a predetermined focal plane in the apparatus. The apparatus can also include visible indicia which enable a technician to spatially orient the apparatus and identify individual specimens in the apparatus. The apparatus preferably takes the form of a specimen culturing dish which has individual chambers that can be subdivided into separate compartments. 
     Since many changes and variations of the disclosed embodiment of the invention may be made without departing from the inventive concept, it is not intended to limit the invention except as required by the appended claims.