Container for culturing, micro manipulation and identification of small specimens

Abstract

A disposable polymer container for manipulation of small specimens adapted to optimize heat transfer between an external heating element and specimens herein contained. In particular, this invention relates to the field of containers for Assisted Reproductive Technology hereunder In-vitro fertilization (IVF).

Claims

1. A specimen container comprising: at least one well configured to receive a specimen comprising a liquid, the at least one well comprising a bottom wall with an externally flat bottom surface, and an open hollow wall structure surrounding the at least one well, the open hollow wall structure comprising: an inner wall and an outer wall, an upper wall connecting a top portion of the inner wall to a top portion of the outer wall, and at least one ridge extending downwardly from a bottom surface of the upper wall and positioned between the inner wall and the outer wall, wherein the externally flat bottom surface of the bottom wall of the at least one well is at a lower position than any portion of the open wall structure such that the externally flat bottom surface is configured to rest directly on and fully contact a heated plate when the specimen container is placed thereon; and a lid configured to cover at least the at least one well of the specimen container, the lid comprising: a rim extending downwardly from a bottom surface of the lid, the rim configured to be at least partially received within a recess on the upper wall of the open wall structure when the lid is placed thereon, and one or more protrusions extending upwardly from a top surface of the lid, the one or more protrusions configured to contact and support an additional specimen container when stacked thereon without contacting an externally flat bottom surface of the additional specimen container.

2. The specimen container of claim 1, wherein the open hollow wall structure further comprises an identification area portion positioned on the upper wall of the open wall structure, wherein the identification area portion is not covered by the lid when the lid is placed thereon.

3. The specimen container of claim 2, wherein the identification area portion comprises a barcode, an inscription, an embossed text, a writing or an RFID tag.

4. The specimen container of claim 1, wherein an upper surface of the bottom wall of the at least one well comprises one or more sloped surfaces configured such that the specimen is directed toward a bottom of the at least one well.

5. The specimen container of claim 4, wherein the one or more sloped surfaces comprise an angle of 45 degrees or less measured with respect to the externally flat bottom surface.

6. The specimen container of claim 1, wherein the at least one well of the specimen container comprises a plurality of wells surrounded by the open hollow wall structure, each of the plurality of wells separated by walls and comprising bottom walls with externally flat bottom surfaces.

7. The specimen container of claim 1, wherein the at least one well comprises a circular or oval shape.

8. The specimen container of claim 7, wherein the specimen container comprises a square or rectangular shape.

9. The specimen container of claim 7, wherein the specimen container is formed from a polymer material.

10. The specimen container of claim 9, wherein the open hollow wall structure is configured to prevent twisting of the bottom wall of the at least one well during solidification of the polymer during manufacturing, thereby maintaining the externally flat bottom surface.

11. The specimen container of claim 1, wherein the specimen comprises spermatozoa, sperm precursor cells, oocytes, pre-implantation embryos, zygotes, blastocysts, embryonic cells or stem cells.

12. The specimen container of claim 11, wherein the specimen container is configured for an in vitro fertilization (IVF) procedure, wherein the specimen container is positioned on the heated plate such that the externally flat bottom surface fully contact the heated plate and the specimen is held at approximately 37 degrees Celsius.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The polymer specimen container according to the invention will now be described in more detail with regard to the accompanying figures. The figures show one way of implementing the present invention and is not to be construed as being limiting to other possible embodiments falling within the scope of the attached claim set.

(2) FIG. 1a shows a top view of an exemplary container and FIG. 1b is the corresponding lid and FIG. 1c is the container with lid not covering the identification area in accordance with embodiments of the invention.

(3) FIG. 2a shows a top view and FIG. 2b shows a cross-sectional view of an exemplary container having a secondary appearance with an innermost wall and an outer most wall and an area for identification here between in accordance with embodiments of the invention.

(4) FIG. 3 shows a side view of an exemplary container without lid having a bottom surface being downwardly flat and having a wall base level higher than the bottom base level in accordance with embodiments of this invention.

(5) FIG. 4 shows a cross-sectional view of the lid having protrusions in accordance with embodiments of the invention.

(6) FIG. 5 shows a back-side view of to exemplary containers with lid stacked on top of each other. The lid having protrusions in accordance with embodiments of the invention making contact with reassesses on the open wall structure allowing stacking of containers or carriers without scratching the bottom surface.

(7) FIG. 6 shows a top view of an exemplary container having a third appearance with separated wells in accordance with embodiments of the invention.

(8) FIG. 7 shows a top view of an exemplary container having a forth appearance with a slope in each separated well in accordance with embodiments of the invention.

(9) FIG. 8 shows a top view on an exemplary container having a fifth appearance with a centred well in accordance with embodiments of the invention.

(10) FIG. 9a shows a top view and FIG. 9b the corresponding lid and FIG. 9c the cross-sectional view of an exemplary container having a sixth appearance in accordance with some aspect of the invention.

(11) FIG. 10 shows a top view of an exemplary container in accordance with embodiments of this invention alongside two commercially available containers, all placed on an external surface heated to 37 degrees Celsius, illustrating temperature transfer and homogeneity in the different containers.

(12) FIG. 11 shows a top view of a commercially available Petri dish alongside an exemplary container in accordance with embodiments of this invention, both placed on an external surface heated to 37 degrees Celsius, illustrating temperature transfer and homogeneity in the different containers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(13) With reference to the drawings for purpose of illustration, the present invention is embodied in a dish (FIG. 1a) with a separate lid (FIG. 1b) not covering the dedicated identification area (FIG. 1c) securing constant visibility during use.

(14) On FIG. 2 the lid fits into structures (4,9). A writing area (3) is positioned between the innermost wall (5) and the outermost wall (2) being part of the open wall structure on one side of the bottom surface, just like the innermost wall (8) and the outermost wall (10) also being part of the open wall structure. The open wall structure has reinforcements by ridges and recesses (11,12,15,16). The reinforced open wall structure prevents distortion of the polymer container during polymer solidification. The open wall structure is interconnected to a bottom base (13) having a surface being downwardly flat. The bottom base (13) has a base level (18) lower than the lowest level (1) of any other structures on the article securing full contact of the bottom surface to the underlying often heated working surface and thereby securing optimal temperature transfer to the specimens to be contained inside the container e.g. in a well (14). The well could be separated by a wall structure having a slope (6,7) between the actual wall structure and the bottom structure, securing positioning of the specimen away from the wall structure of the well. If a label, barcode or RFID tag is to be placed below the container for imaging purpose it may be placed on the outermost wall structure (2) and bend underneath the slightly elevated wall structure and be supported by a protrusion (17).

(15) The lid in FIG. 4 has a rim (19) to secure stability when positioned on the container. The lid also has a protrusion (20) which makes contact on ridges and recesses on the open wall structure of an adjacent container during stacking, making sure the bottom of the container is not scratched during stacking as well as to facilitate easy gripping or grasping. Stacking is illustrated on FIG. 5.

(16) The lid, as shown in FIG. 1b, may further have small protrusions (21) securing gas exchange between the outside of the container and the inside of the container during use.

Definitions

(17) Sterile means free from living bacteria and other microorganisms. Sterilisation is the process by which living organisms are removed or killed to the extent that they are no longer detectable in standard cell culture media in which they had previously proliferated. Sterility Assurance Level of six-log reduction (also termed log of minus 6 reduction or ten to the minus six reductions) is considered acceptable for many medical devices.

(18) Open wall structure means a wall structure consisting of a non-closed wall structure persisting in more than one plane.

(19) Innermost wall structure means the wall nearest to the inside of the container or carrier.

(20) Outermost wall structure means the wall nearest to the outside of the container or carrier.

(21) Downwardly flat bottom base and externally flat bottom base means that the bottom area facing downwards, towards the outside of the container, is flat. The part of the bottom area facing upwards, towards the inside of the container could be flat, but could also contain well structures.

(22) Square with or without rounded corners means a substantially square geometry with corner angle of 90 degrees or rounded.

(23) Center of the dish means the center of the dish confined by the outermost wall.

(24) Individually separated wells means wells separated by a wall structure or embedded in the bottom structure in a way so liquid in one well is separated from liquid in another well.

(25) Slope means a ramp with an inclination between 0 and 90 degrees. A slope in the bottom of a well may interconnect the bottom area with the wall structure of the well.

(26) Functionalized by surface modification means modifying the surface properties e.g. by use of electric discharge, plasma, biological molecules, irradiation, structuring, material embedding, chemical or molecular vapor deposition or other means.

(27) Identification marking means marking with text, print, labels, inscription, transmitter or other means in order trace container and content

(28) RFID means Radio-frequency Identification. RFID is a technology that incorporates the use of electromagnetic spectrum to uniquely identify an object.

(29) Assisted Reproductive Technology (ART) is a general term referring to methods used to achieve Pregnancy by artificial or partially artificial means. It is a reproductive technology used primarily in Infertility treatments, such as In Vitro Fertilisation.

(30) In Vitro Fertilization (IVF) is a laboratory procedure in which sperm are placed with an unfertilized egg in a dish to achieve fertilization.

(31) XY-stage station means automated or manual positioning table delivering smooth and accurate two-dimensional motion.

(32) Collapsing drops means liquid drops placed on a solid support, e.g. bottom surface of a polymer container, and not having a measurable contact angle with the solid support, a phenomenon occurring when liquid is placed on hydrophilic surfaces and a well defined drop with measurable contact angle cannot be maintained.

(33) Protrusion means something that bulges out or is protuberant and thereby raises above the surrounding support.

(34) Meiosis activation means activating cell division necessary for sexual reproduction

(35) Chemical vapor deposition (CVD) means a chemical process used to produce high-purity, high-performance solid materials. The process is generally used in the semiconductor industry to produce thin films. In a typical CVD process, the substrate is exposed to one or more volatile precursors which react and precipitate or decompose on the substrate surface to produce the desired deposit

(36) Molecular vapor deposition (MVD) means an enhancement of the conventional vapor deposition of ultra-thin layers. It is a non-plasma vacuum process where volatile components in gas phase react to form a solid deposit and gaseous by-products. In MVD the reagents concentrations control the deposition rate. Reagents are distributed uniformly in the system by means of pressure and MVD is performed as a batch process so that no new reagent is added to the process during the deposition. MVD is not an invasive technique, the low temperature, the absence of plasma and the introduction of reagents as gases do not damage the substrate.

(37) Base height means the height of the supporting base of any structure on the article.

(38) Bottom base means the lowest base of the bottom surface.

(39) Wall base means the lowest surface of the open wall structure. When the wall is at one end connected to the bottom surface and stretching only onto one side of the bottom surface, the wall base is defined as being above the level of the bottom base.

(40) Flat is herein defined as substantial parallel to the surface on which the container is placed, e.g. zero degree of inclination in respect to the surface on which the container is placed.

(41) Working surface is defined as the surface onto which the container or carrier in placed during use. E.g. this working surface may be a heated or non-heated surface, a structured surface within an incubator, a surface within a laminar airflow hood, the surface of the lid of another container or carrier and more.

EXAMPLES

(42) Polymer specimen container with open wall structure, flat bottom base in contact with working surface, area for identification, and optionally slope between wall structure of the well and bottom of the well

(43) The polymer container of the present invention may have any suitable size for the intended use.

(44) In particular polymer container being substantially square or square with rounded corners having a side length of 25 mm or less, such as 20 mm or less, 15 mm or less, 10 mm or less such as approximately 7.5 mm. A substantially square geometry is useful during microscopy using an XY stage moving the container in X- and Y directions.

(45) In particular polymer container being substantially circular having a diameter of 120 mm or less, such as 90 mm or less, such as 60 mm or less, such as 50 mm or less, such as 40 mm or less. A substantially circular geometry is useful during microscopy rotating the container by hand.

(46) In particular polymer container being substantially square or square with rounded corners containing circular well(s) having any suitable size for the intended use, in particular well diameter of 90 mm or less, 60 mm or less, 45 mm or less, 35 mm or less, 10 mm or less, 5 mm or less. A substantially square or square dish with rounded corners containing a circular well with center at the center of the container is useful during microscopy rotating the container by hand. Centered or a non-centered well is useful for micro drop culture as well as collection and transferring specimens. Several wells within the same container are useful for culturing embryos separately and securing no cross contamination between embryo cultures, also in the event of collapsing drops.

(47) The well(s) may have any shape suitable for intended use. When used during procedures where tools are applied from left and right hand side at the specimens within the well, a circular, oval or rectangular well shape may be preferred. When used during embryo culture a circular well may be preferred. The individual wells may optionally have a slope between the flat bottom of the well and the well structure, useful for preventing small specimens to hide in the otherwise sharp angles. The slope ensures that the small specimens such as embryos are transferred to the bottom of the well by gravity and easily accessible at a known distance from the wall of the well. The slope of the present invention may have suitable size for intended use, in particular slope angle of 89 degrees or less, 45 degrees or less, 30 degrees or less, such as 25 degrees.

(48) Some procedures require larger amounts of media within the container, some requires smaller amount of media, and some requires easy access with tools approaching the containers from the sides. The height of the container may have any shape suitable for intended use, in particular 20 mm or less, 15 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less.

(49) In contrast to a traditional Petri dish the invention provides an open wall structure

(50) One benefit being that part of the area on the open wall structure provides an identification area suitable for marking, engraving, labeling, barcode, RFID tag or the like. The Identification area of the present invention may have suitable size for the intended use. In particular area of 100 cm.sup.2 or less, 50 cm.sup.2 or less, 25 cm.sup.2 or less, 10 cm.sup.2 or less, 8 cm.sup.2 or less.

(51) Another benefit being that the open wall structure interconnected by a bottom structure being downwardly flat and said bottom base having a lower base level than the base of the wall structure is that the bottom surface is being in close contact with the external working surface below, which may be a heated table. The open wall structure adapted with recesses and ridges prevents the injection molded flat bottom structure to twist during solidification of the heated polymer. The bottom structure may have suitable size for the intended use. In particular thickness of 12 mm or less, 10 mm or less, 8 mm or less, 6 mm or less, 4 mm or less. The bottom structure may also embed well structures. The temperature transfer from the external heated surface to the specimens is thus optimized for the individual container, as well as ease of procedure is optimized when several various containers using same principle are used during an IVF procedure.

(52) A third benefit being that the lid may be fitted to cover the area constraint by the innermost wall structure, while allowing the identification area to not be covered by the lid for easy identification.

(53) The lid has protrusions on the upper side contacting recesses and ridges on the open wall structure during stacking of the containers on top of each other. This reduces risk of scratching the bottom, which can lead to reduced visibility of the specimens. This feature also ensures that only a container with its lid is gripped when reaching out towards the stacked products.

(54) The lid having protrusions on the lower side ensuring that gas exchange can take place between the content of the container and the outside of the container with lid, e.g. during placement in gas controlled incubator.

(55) The polymer container of the present invention is formed from a heated polymer, in particular a thermoplastic compound. Non-limiting examples of thermoplastic compounds that may be used are acrylonitrile butadiene styrene (ABS), acrylic, celluloid, cellulose acetate, Ethylene-Vinyl Acetate (EVA), Ethylene vinyl alcohol (EVAL), Flouroplastics, Liquid Crystal polymer (LCP), polyacetal, polyacrylate, polyacrylonitrile, polyamide, polyamide-omide (PAI polychlorotrifluoroethylene (PCTFE), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester, polyethylene (PE), polyetherketone (PEEK), polyetherimide (PEI), polyesthersulfone (PES), polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polypropulene (PP), polystyrene (PS), polysulfone (PSU), polyurethane (PU), polyvinyl acetate (PVA), polyvinyl chloride (PVC), polyvinylidenechloride (PVDC) and styrene-acrylonitrile (SAM), or mixes or copolymers thereof.

(56) In a particularly preferred embodiment the polymer container is formed from a polymeric material which is biocompatible.

(57) The polymeric material may be selected such that it has a primarily hydrophobic or primarily hydrophilic surface or primarily in-between. In a particular embodiment the surface of the polymer article may be modified to make it in-between, e.g. by coating or by use of corona or plasma treatment. The polymers are generally hydrophobic by nature and the strength and time of the coating process determines the degree of hydrophilicity obtained.

(58) Process for Producing a Polymer Container Optionally Functionalized by Surface Modification

(59) Typical methods used in the mass production of polymer articles are injection molding. Injection molding is performed by heating a suitable polymer until molten, injecting the molten polymer into a mold, allowing the polymer to cool and harden, and removing the molded article from the mold after solidification.

(60) The surface of the polymer container is optionally made more hydrophilic to facilitate the formation of drops of media for culturing embryos, as well as made sterile to prevent contamination. Typically the surface of the polymer container is modified by corona or plasma treatment to increase surface wettability through electric discharge.

(61) Sterilisation by irradiation (beta or gamma), steam autoclave, ethylene oxide, chemical disinfectants or dry heat are the typical sterilization methods used.

(62) The sterile polymer container may further be functionalized by structuring the surface during the actual injection molding step and/or simply by wet chemical coating or vapor coating following the injection molding.

(63) A functionalisation by adding structure to the surface of the polymer container during molding may be in the form of molded nano or micro structures within the polymer or by integrating a porous scaffold into the surface of the polymer container during injection molding. The structures allows specimens in contact with the structures within the container to obtain more 3D like morphology as well as intercommunicate with other specimens through the medium travelling unhindered through the structures retaining the specimens.

(64) Coating using wet chemical process covalently bonds biological molecules to the polymer surface to facilitate specific functionality in relation to interaction with the surroundings being it meiosis activation, cell attachment, molecule capture or separation and/or purification processes.

(65) Coating using vapor process deposits oxide layers onto the surface of the polymer container. Examples of oxide layers may be Al.sub.2O.sub.3, SiO.sub.2, TiO.sub.2, ZrO.sub.2, CeO.sub.2 or a mixture or a multilayer structure thereof. Oxide layers may be deposited by Chemical Vapor deposition (CVD) or preferably by Molecular vapor deposition (MVD).

Experimental Example

(66) The following examples serve to more fully describe the functionality of the externally flat bottom in contact with external heated surface compared to traditional containers for IVF. These examples do not limit the scope of the disclosure, but rather are presented for illustrative purposes.

Example 1 Temperature Transfer to Polymer Container

(67) Two conventionally polymer containers with wells and a polymer container according to this invention are placed on a metal plate heated to 37.2 Degrees Celsius.

(68) In FIG. 10 the two conventional polymer containers (B and C) have a bottom situated approx 0.5 mm above the heated plate surface. The container (A) according to this invention has full contact between the bottom of the container and the heated plate.

(69) Each well is filled with liquid to obtain equal liquid height.

(70) Allow 5 minutes for the heated plate to transfer heat to the containers.

(71) Use a thermo sensitive camera to take an image of the heat distribution within the containers.

(72) FIG. 10 illustrates the more optimal heat transfer from the heated plate to the liquid within the 5 well container (A) according to this example and this invention compared to commercially available 5 well (B) and 4 well (C) plates.

(73) One conventionally polymer Petri dish and a polymer container according to this invention are placed on a metal plate heated to 37.2 Degrees Celsius.

(74) In FIG. 11 the one conventional Petri dish (D) has a bottom situated approx 0.5 mm above the heated plate surface. The container (E) according to this invention has full contact between the bottom of the container and the heated plate.

(75) Each container is filled with liquid to obtain equal liquid height.

(76) Allow 5 minutes for the heated plate to transfer heat to the containers.

(77) Use a thermo sensitive camera to take an image of the heat distribution within the containers.

(78) FIG. 11 illustrates the more optimal heat transfer from the heated plate to the liquid within the container (E) according to this example and this invention compared to commercially available Petri dish (D).

(79) Clearly, the polymer specimen of the invention has an improved ability of transferring heat to the liquid therein contained. In particular, the polymer specimen of the invention transports heat better than commercially available containers. The polymer specimen structure comprising ridges and recesses allows for a better contact between the heated plate and the bottom of the polymer specimen.

(80) Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is set out by the accompanying claim set. In the context of the claims, the terms “comprising” or “comprises” do not exclude other possible elements or steps. Also, the mentioning of references such as “a” or “an” etc. should not be construed as excluding a plurality. The use of reference signs in the claims with respect to elements indicated in the figures shall also not be construed as limiting the scope of the invention. Furthermore, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

(81) The following numbered items provide in term of conceptual statements further disclosure of the present subject matter.

(82) 1. A polymer specimen container comprising:

(83) a specimen carrier, said carrier comprising a bottom base externally flat connected to a wall, said wall comprising an open hollow structure adapted to avoid twisting of said bottom base during the polymer specimen container production process.

(84) 2. A polymer specimen carrier according to item 1, wherein said open hollow structure comprises recesses and ridges adapted to provide stable support to said specimen carrier.

(85) 3. A polymer specimen carrier according to any of the preceding items wherein said open wall structure has its lowest level above the lowest level of said bottom base externally flat.

(86) 4. A polymer specimen container according to any of the preceding items further comprising a lid, said lid comprising a top and a bottom surface, wherein said top surface comprises protrusions complementary to at least one of said recesses or ridges in said open hollow wall structure.

(87) 5. A polymer specimen carrier according to any of the preceding items wherein said open hollow structure comprises area for identification means.

(88) 6. A polymer specimen carrier according to any of the preceding items, wherein said identification means is in the form of barcode, inscription, embossed text, writing or RFID tag.

(89) 7. A polymer specimen carrier according to any of the preceding items, wherein at least part of the internal surface of said bottom base externally flat is treated so as to modify its surface properties.

(90) 8. A polymer specimen carrier according to any of the preceding items, wherein said open wall structure forms substantially a square with or without rounded corners or being substantially circular.

(91) 9. A polymer specimen carrier according to any of the preceding items, wherein said open wall structure having an innermost wall structure forming a well substantially square with or without rounded corners, or being substantially circular.

(92) 10. A polymer specimen carrier according to any of the preceding items, wherein said innermost wall structure forms a circular well, wherein the centre of said circular well is substantially identical to the centre of the said specimen carrier.

(93) 11. A polymer specimen carrier according to any of the preceding items, wherein said flat bottom base externally flat comprises one or more individually separated wells for holding liquid medium sustaining the specimens during procedure.

(94) 12. A polymer specimen carrier according to item 11, wherein the internal surface of the bottom base of said individually separated wells has an inclination.

(95) 13. A polymer specimen carrier according to any of the preceding items comprising at least an innermost and an outermost wall structure, said innermost wall structure interconnected by a sterile bottom base being externally flat and having equal or lower base height than any other carrier structures, optimizing bottom base contact to a heated working surface and thereby improving heat transfer to the inside of the carrier.

(96) 14. A polymer specimen carrier according to any of the preceding items wherein the specimens are oocytes, embryos, blastocysts, spermatozoa or stem cells.

(97) 15. The use of a polymer specimen container according to any of the preceding items within the area of Assisted Reproductive Technology.

(98) 16. A method for producing a polymer specimen container according to items 2-15 wherein the presence of an open hollow structure comprising recesses and ridges prevents distortion of said flat bottom base during said polymer specimen carrier solidification.