Patent Application
20220025314
The present invention relates to devices and methods for changing media for cells in cell culture, more particularly to methods and systems featuring a cell culture dish that allows for aspiration of media without contact with the cells in culture.
In order to grow cells of all types, including embryos, media that contains nutrients and growth factors is usually regularly changed, e.g., the old media is removed and new media (e.g., new type of media, same type of media) replaces the old. Typically, media exchange involves pipetting cells out of the dish and placing them in a new dish with the new media. This process of cell handling is stressful for cells, and it is always possible to damage the cells during the transfer. Alternatively, media may be aspirated and new media may be added. With this method, there is always a danger of aspirating the cells along with the media.
The present invention features methods and systems for cell media exchange, wherein media may be safely aspirated from the dish. For example, the present invention features a cell culture apparatus (e.g., cell culture dish, well, etc.) wherein media can be aspirated therefrom via a gap or slot that is small enough to retain cells therein and also sized to prevent media from leaking (e.g., via surface tension). Since media can be aspirated from the gap, there is typically not a need to handle the cells in a similar fashion as described above for previous methods; thus, the methods and systems of the present invention can help reduce stress and damage to cells.
The present invention features a cell culture sieve system that allows media exchange without having to remove the cells. In some embodiments, the cell culture sieve system comprises a cell culture container adapted to grow or sustain cells in culture, wherein a gap is disposed on a side wall of the container. The gap is sized and adapted to retain media in the container in the absence of suction and to allow aspiration of the media in the presence of suction.
For example, the present invention features a cell culture sieve system comprising a well atop a culture substrate forming a container for holding media and cells in culture, wherein at least one gap is disposed on a side wall of the well, the at least gap is sized to retain media in the well when suction is not applied to the gap and to allow aspiration of said media from the well when suction is applied to the gap. The gap is sized to retain cells in the well when suction is applied to the gap.
The size of the gap may be chosen based on the size of the cells to be contained. For example, the gap may be smaller than the size of the cells. Surface tension may help prevent media from leaking through the gap. In some embodiments, the gap is from 0.05 microns to 100 microns at its largest dimension. In some embodiments, the gap is 0.05, 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 microns at its largest dimension. In some embodiments, the gap is from 0.05 to 10 microns, 10 to 50 microns, or 50 to 100 microns at its largest dimension. In some embodiments, the gap is greater than 100 microns at its largest dimension. (In some embodiments, the largest dimension is diameter.)
In some embodiments, the system further comprises an outer wall surrounding the well and spaced a distance apart from the well. In some embodiments, the system further comprises a pipette positioning area adjacent to a gap. An unobstructed pipette positioning area allows for alignment of a pipette tip with the gap such that the pipette tip may seal against the side wall. In some embodiments, the culture substrate is a part of a slide or plate. In some embodiments, the cell culture sieve system is part of a multi-well cell culture plate. In some embodiments, the side wall that forms the well is attached or connected to the culture substrate. In some embodiments, the slot is a cut or an indentation in the culture substrate.
In some embodiments, the system comprises a slot disposed in the culture substrate intersecting with the gap and fluidly connected to the gap. In some embodiments, the slot is a cut or an indentation connecting the well to the culture substrate.
In some embodiments, the system comprises a plurality of gaps extending from at or near a bottom edge of the well and upwardly toward a top edge (e.g., to the top edge, to the middle, to a place near the top edge, etc.) of the well.
The present invention also features a method of aspirating cell culture media (or a method of changing cell culture media). In some embodiments, the method comprises applying suction to a gap in a cell culture sieve system of the present invention, wherein suction applied to the gap removes media but not cells from the well of the cell culture sieve system. In some embodiments, applying suction to the gap comprises using a pipette to aspirate media through the gap. In some embodiments, the method is manual. In some embodiments, the method is automated. In some embodiments, the method further comprises adding new media to the well of the cell culture sieve system.
Without wishing to limit the present invention to any theory or mechanism, it is believed that the methods and systems of the present invention are advantageous because they allow the exchange of media in the same cell reservoir yet there is little or no aspiration of cells during the aspiration process. Further, the media exchange process can be accomplished without physically contacting the cells, thereby avoiding the physical stress associated with re-plating of cells. Surface tension may prevent the media from leaking from the wells through the gaps. When suction is applied to the gaps, the suction breaks the surface tension of the media and allows the media to be aspirated from the well.
Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.
The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:
Following is a list of elements corresponding to a particular element referred to herein:
The present invention cell culture sieve systems and methods for media exchange featuring a cell culture dish that allows for aspiration of media without contact with the cells in culture.
The present invention features a cell culture sieve system (100). The cell culture sieve system may comprise: a) a culture substrate (105); b) a side wall (110) disposed atop the culture substrate (105) to form a container for holding media and cells in culture; c) a gap (122) disposed in the side wall (110) to directly connect an interior of the container with an exterior of the cell culture sieve system; and d) a slot (128) disposed between a bottom of the side wall (110) and the culture substrate (105) such that the slot (128) is fluidly connected with the gap (122). In some embodiments, the gap (122) is configured to retain media via surface tension and to prevent media from leaking from the container. In other embodiments, the slot (128) is sized and configured to receive a pipette tip (132). Without wishing to limit the present invention to any theory or mechanism, when suction is applied to the gap (122) via a pipette tip (132) positioned within the gap (122) media is aspirated from the container.
In some embodiments, the cell culture sieve system (100) may comprise: a) a culture substrate (105); b) a side wall (110) disposed atop the culture substrate (105) to form a container for holding media and cells in culture; c) a gap (122) disposed in the side wall (110) to directly connect an interior of the container with an exterior of the cell culture sieve system; and d) an unobstructed pipette positioning area (136), such that a pipette (130) may be positioned with its tip (132) in contact with the side wall (110) at an angle such that the tip (132) and the side wall (110) form a seal. In some embodiments, the cell culture sieve system (100) may further comprise a pipette (130) within the pipette positioning area (136), positioned with its tip (132) in contact with the side wall (110) at an angle such that the tip (132) and the side wall (110) form a seal.
As used herein, the term “pipette positioning area” refers to a three-dimensional area adjacent to a gap (122) in a side wall (110) where a pipette may be positioned such that the pipette is fluidly coupled with the gap (122) and the pipette tip (132) seals against the side wall (110). The pipette positioning area (136) may surround a pipette positioning axis, along which the pipette is positioned for best alignment with the gap (122). As a non-limiting example, the pipette positioning axis may be orthogonal to the surface of the side wall (110). A radius of the pipette positioning area (136) may be about equal to or slightly larger than the length of a pipette. In some embodiments, the pipette positioning area (136) may be cone-shaped, with boundaries offset by an angle from a pipette positioning axis. In some embodiments, the pipette positioning area (136) may be defined by a radius, a vertical angle, and a horizontal angle. In preferred embodiments, the pipette positioning area (136) is unobstructed, such that a pipette may be positioned along the pipette positioning axis, with the tip (132) in contact with the side wall (110).
The cell culture sieve system (100) of the present invention may comprise: a) a culture substrate (105); b) a side wall (110) disposed atop the culture substrate (105) to form a container for holding media and cells in culture; c) a gap (122) disposed in the side wall (110) to directly connect an interior of the container with an exterior of the cell culture sieve system; and d) an aspiration socket (134) disposed in the side wall (110) around the gap so as to guide positioning of a pipette tip (132) to form a seal between the tip (132) and the side wall (110) such that media may be aspirated through the gap (122) via the pipette tip (132).
The present invention also features a cell culture sieve system (100) comprising: a) a culture substrate (105); b) a side wall (110) disposed atop the culture substrate (105) to form a container for holding media and cells in culture; and c) at least one gap (122) disposed on the side wall (110). In some embodiments, the at least one gap (122) is configured to retain media via surface tension and to prevent media from leaking from the container. Without wishing to limit the present invention to any theory or mechanism, when suction is applied to the at least one gap (122), media is aspirated from the container via the at least one gap (122). In other embodiments, the at least one gap (122) is sized and configured to prevent cells within the container from exiting the container.
The present invention features a cell culture sieve system (100) comprising: a) a well (110) atop a culture substrate (105); and b) at least one gap (122) directly coupled to the single side wall of the well (110) between a bottom edge of the well and a top edge of the well. In preferred embodiments, the at least one gap (122) prevents media from leaking from the well (110) when suction is not applied to the gap (122), and when suction is applied to the at least one gap (122), the at least one gap (122) allows aspiration of said media directly from the well (110) but is sized to prevent cells from being suctioned out of the well (110). In some embodiments, the well (110) forms a container for holding media and cells in culture. In other embodiments, the well (110) comprises a single side wall.
The present invention also features a cell culture sieve system (100) comprising: a) a culture substrate (105); b) a well (110) atop the culture substrate (105); c) at least one gap (122) directly coupled to the single side wall of the well (110) between a bottom edge of the well and a top edge of the well; and d) a pipette. Without wishing to limit the present invention to any theory or mechanism, when the pipette is aligned with the at least one gap (122), suction is applied to the at least one gap (122) via the pipette (130), allowing for aspiration of media directly from the well (110). In some embodiments, the well (110) forms a container for holding media and cells in culture. In other embodiments, the well (110) comprises a single side wall.
Referring to
In some embodiments, the gap (122) is a hole disposed in the side wall (110). In other embodiments, the gap (122) may be disposed between a bottom edge of the container and a top edge of the container. In preferred embodiments, the gap (122) prevents media from leaking from the container via surface tension. In one embodiment, the gap (122) prevents media from leaking from the container when suction is not applied to the gap (122), and when suction is applied to the gap (122), the gap (122) allows aspiration of said media directly from the container.
In other embodiments, the gap (122) is directly connected to an exterior of the cell culture system. In other embodiments, the gap (122) may provide size selection. For example, the gap (122) may be sized to retain cells in the container when media is aspirated from the container. Thus, a gap size may be chosen based on the size of a cell of interest.
As a non-limiting example, if the cell of interest were approximately 100 microns in diameter, a gap of less than 100 microns (e.g., 70 microns) would be selected. If the cell was 10 microns in diameter, a gap of less than 10 microns (e.g., 5 microns) may be chosen.
In some embodiments, the gap (122) is sized between about 0.5 microns to 100 microns at its largest dimension (e.g., diameter). In some embodiments, the gap (122) is sized to be about 0.05 microns, 1 micron, 5 microns, 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, or 100 microns at its largest dimension (e.g., diameter). In some embodiments, the gap (122) is sized between about 0.05 to 10 microns at its largest dimension (e.g., diameter). In some embodiments, the gap (122) is sized between about 1 to 10 microns at its largest dimension (e.g., diameter). In some embodiments, the gap (122) is sized between about 10 to 50 microns at its largest dimension (e.g., diameter). In some embodiments, the gap (122) is sized between about 50 to 100 microns (e.g., 70-75 microns) at its largest dimension (e.g., diameter). In some embodiments, the gap (122) is smaller than about 100 microns at its largest dimension (e.g., diameter). In some embodiments, the gap (122) is larger than about 100 microns at its largest dimension (e.g., diameter).
In yet another embodiment, the cell culture sieve system comprises a plurality of gaps (122) extending from at or near a bottom edge of the container upwardly toward a top edge of the container. For example, as shown in
In other embodiments, the cell culture sieve system of the present invention comprises a plurality of gaps (122) disposed in the side wall. In one embodiment, the plurality of gaps (122) are disposed between a top edge and a bottom edge of the side wall. In some embodiments, each gap in the plurality of gaps is centrally aligned on a same vertical axis with one another. In another embodiment, each gap in the plurality of gaps are centrally aligned on a same horizontal axis with one another. In yet another embodiment, the plurality of gaps form a gap pattern. The gap pattern may be a plurality of gaps (122) concentrated in one area of the side wall. In some embodiments, each gap in the plurality of gaps is the same size. In other embodiments, each gap in the plurality of gaps is a different size. In further embodiments, each gap in the plurality of gaps is circular, ovular, rectangular, square-shaped or a combination thereof. In yet another embodiment, the gap (122) may be disposed at a bottom edge of the container, such that all of the media may be aspirated from the container. In some embodiments, the gap (122) is disposed between a bottom edge and a top edge of the side wall. In this configuration, media is aspirated from the gap (122) to lower a volume of media in the container without completely emptying the container.
In some embodiments, the cell culture sieve system (100) is a part of a multi-well plate. In other embodiments, the side wall is a single side wall. The single side wall may be one wall that forms the container. The single side wall may form a barrier between the media in the container and the exterior of the container. In this embodiment, the container is independent from any other containers that may be disposed on the culture substrate. Each container has its own side wall such that each container is independent from other containers of the multi-well plate. The container is not fluidly connected to other containers on the culture substrate. The gap disposed on the side wall connects an interior of the container to an exterior of the container such that when media is in the container, the media is in contact with air through the gap.
In some embodiments, an exterior of the container is not configured to contain fluid. In other embodiments, the cell culture sieve system does not have an exterior wall. A pipette may be able to access the gap from a top or from a side of the system. In yet another embodiment, a mark may be disposed on the side wall to indicate where the gap is located on the wall. The mark may help a user find the gap to aspirate media from the container.
In preferred embodiments, a pipette is able to access the gap (122) with no obstructions. The unobstructed pipette positioning area may allow positioning of a pipette such that its tip (132) is in contact with the side wall (110) at an angle such that the tip (132) and the side wall (110) form a seal. The pipette positioning area (136) may also be a spacing around the container that allows a pipette to access the gap (122). In a multi-well plate, the pipette positioning area (136) may be an unobstructed area between the containers on the plate that allows the pipette to access the gap (122). In the pipette positioning area (136), a micropipette may be positioned at an angle between about 0° to 90° from the line from a top of the container to the gap (122). A transfer pipette may have access to the gap (122) at an angle between about 0° to 180°, from the line from a top of the container to the bottom of the container.
In some embodiments, the gap (122) may be configured to receive a pipette tip (132) for suctioning media from the container. In the configuration where the system is a part of a multi-well plate, the containers may be spaced to allow a pipette tip (132) to access the one gap (122). The pipette tip (132) may interact with the one gap (122) at an angle (θ) between about 0° to 90°. In some embodiments, the pipette tip (132) may interact with the gap (122) at an angle between about 0° to 10°, 10° to 20°, 20° to 30°, 30° to 40°, 40° to 50°, 50° to 60°, 60° to 70°, 70° to 80°, or 80° to 90°.
In some embodiments, the tip (132) of the pipette forms a seal with the gap (122). The tip (132) of the pipette may comprise a flexible material that forms a seal with the gap (122). As a non-limiting example, the pipette tip (132) may have a rubber attachment (138) at the end to form a seal with the gap (122). In another embodiment, a flexible material may be disposed around the gap (122) to form a seal with a pipette tip (132). In yet another embodiment, the pipette tip (132) may be L-shaped.
The size of the gap (122) may require different pipette shapes or materials in order to create a seal to suction the media out of the well. If the gap (122) is large, this does not require a perfect seal because it takes less suction to break the surface tension of the media. A large gap (122) may be about 50 microns or larger. If the gap (122) is small, a pipette tip (132) that is pliable may be needed in order to make a better seal with the gap (122) to suction out the media from the well. A small gap (122) may be sized between about 0.5 microns to 50 microns. As a non-limiting example, the pipette tip (132) may be made out of rubber to create a seal with the gap (122). Alternatively, to create a better seal with the gap (122), an indentation can be molded into the side wall around the gap (122) such that the pipette tip (132) fits into the indentation. In some embodiments, the pipette tip (132) may be L-shaped to contact the indentation. An L-shaped pipette tip (132) may allow a user to align the pipette tip (132) orthogonal to the well to suction out the media from the well. This type of pipette tip (132) may be useful when the well is a part of a multi-well plate where access to gaps in wells in the middle of the multi-well plate may be obstructed by neighboring wells.
The cell culture system of the present invention may further comprise a slot fluidly connected to the gap (122). In some embodiments, the slot is a cut or an indentation in the culture substrate. In one embodiment, the slot is an indentation in the culture substrate that surrounds the gap (122). The slot may be used to guide a pipette tip (132) to the gap (122). A pipette tip (132) may fit into the slot. In preferred embodiments, the slot is configured to receive a pipette tip (132) for aspirating media from the gap (122).
As shown in
In further embodiments, the cell culture sieve system of the present invention comprises an aspiration socket (134). The aspiration socket (134) may be an indentation in the cell culture substrate (105) or in the side wall (110). In other embodiments, the aspiration socket (134) may be formed via a protrusion from the cell culture substrate (105) or the side wall (110). In some embodiments, the aspiration socket (134) is configured to receive a pipette tip (132) for aspirating media from the gap (122). The aspiration socket (134) may be a slot (128). In other embodiments, the aspiration socket (134) is fluidly connected to the gap (122). In one embodiment, an interior diameter of the aspiration socket (134) is sized to seal around an exterior diameter of the pipette tip (132).
In other embodiments, the container is filled with media (102) using an opening at a top end of the well. The gaps (122) may not be used to fill the container with media (102). In some embodiments, the gap (122) directly connects the container with an exterior of the cell culture system. As a non-limiting example, direct connection of the interior of the container with the exterior of the cell culture system via a gap (122) means that fluid which passes from the container through the gap would no longer be contained by the cell culture system. The container may only have a single side wall (110); with no additional walls surrounding the container such that the container is fluidly connected to another container or any other vessel or chamber that may be used to hold media. The container is independent from any other containers that may be disposed on the culture substrate. In this configuration, the gap (122) forms an interface between the media (102) and air.
In some embodiments, the container is mounted, attached or connected to the cell culture substrate (105). As a non-limiting example, the container may be connected to the cell culture substrate (105) via an adhesive, e.g., a double-sided adhesive. The present invention is not limited to this construction. For example, the container may be constructed as a single unit (e.g., via injection molding or other processes), e.g., the well has a base floor or is directly constructed on the cell culture dish).
In some embodiments, an inner wall is disposed in the container (not shown), e.g., an inner wall forming an enclosure. The inner wall may be very short in height, e.g., about the height of a cell (e.g., 10-15 um, 15-20 um, 20-30 um, etc. The inner wall may be for helping to enclose the cells or sequester the cells in a particular area on the cell culture substrate (105) within the container.
The present invention described herein uses specially designed cell culture wells or containers (e.g., wells, beakers, etc.) that allow for media exchange to take place with cells remaining in the same container and where these cells are not contacted during this media exchange. Surface tension keeps media from leaking from the gaps (122) of the systems described herein. In some embodiments, when the media needs to be exchanged, a pipette tip (132) is placed outside the gap (122) and suction is applied to the gap (122) to aspirate the media from the well. The suction that is applied to the gap (122) breaks the surface tension of the media and allows the media to be aspirated from the well. Once the media is aspirated, new media may be added to the well from the top of the well. In other embodiments, the pipette tip (132) may be aligned with the gap (122) using a slot or an aspiration socket. In this embodiment, the pipette tip (132) is aligned with the gap prior to aspiration of the media.
In one embodiment, a double sided adhesive is used to connect the well to the cell culture substrate. In some embodiments, the gap (122) is disposed in the double-sided adhesive (120). The present invention is not limited to the user of double sided adhesive. For example, the gap (122) may be disposed in a side wall of the well or in other components of the system. The suction force is stronger than the capillary surface tension holding the media in the well and the media can be evacuated, while the cells stay in the well due to size exclusion principle, as their size is larger than the gap size.
The system of the present invention may feature a slightly hydrophobic surface that will not allow fluid (e.g., media) to escape through the gap. For example, in some embodiments, the material used to form the system or components thereof (e.g., the gap) may be chosen to be slightly hydrophobic (e.g., a plastic). In some embodiments, the material is hydrophilic but is coated (or a portion is coated, e.g., the gap) to be slightly hydrophobic. In some embodiments, the gap may be slightly hydrophilic so as to allow fluid to go through but only to form a droplet just outside of the gap (so fluid doesn't escape further).
In some embodiments, the media exchange is manual. In some embodiments, the media exchange is automated.
The present invention features methods for aspirating media from a cell culture sieve system without contacting the cells in culture. The method may comprise providing any of the cell culture sieve systems described herein, adding media and cells to the container, incubating the cells, aspirating the media, and replacing the media. To aspirate the media from the container, a pipette may be used to suction the media from the container. In some embodiments, the pipette may be aligned with the gap to form a seal with the gap to suction the media from the container. The gap of the cell culture sieve system is sized such that media is retained in the container when suction is not applied to the gap, but when suction is applied to the gap, only media is aspirated and the cells stay in the container. In other embodiments, the pipette may be aligned with the obstructed pipette positioning area prior to aspirating the media.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.
Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting of”, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting of” is met.
The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.
This application is a continuation-in-part and claims benefit of U.S. patent application Ser. No. 15/719,180, filed Sep. 28, 2017, which is a non-provisional and claims benefit of U.S. Provisional Patent Application No. 62/412,722 filed Oct. 25, 2016, the specifications of which are incorporated herein in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62412722 | Oct 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15719180 | Sep 2017 | US |
| Child | 17450494 | US |
