VESSEL WELL

PRIORITY CLAIM

[0001] This application claims priority of U.S. Provisional Patent Application Serial No. 63/399,764, filed August 22, 2022, the entire contents of which patent application is hereby incorporated herein by reference.

BACKGROUND

[0002] The present application relates to cell culturing and to a method and apparatus for determining the depth of cell culturing media in a cell culture vessel well.

[0003] Cell culture incubators are used to grow and maintain cells from a cell culture, which is the process by which cells are grown under controlled conditions. Cell culture vessels containing cells are stored within the incubator, which maintains conditions such as temperature and gas mixture that are suitable for cell growth. The environment inside an incubator is controlled by a control system that may be configured to control the temperature, humidity, carbon dioxide, oxygen and other gaseous components (e.g., sterilization gases, such as, ozone, and hydrogen peroxide) inside the incubator cell culture vessels (e.g. flasks, suspension culture flasks, spinner flasks, plates, petri dishes and bags).

[0004] Cell culturing of cells involves the addition of media to cells in the well of a cell culture vessel generally to promote growth. The cells are cultured in the media and observed to determine their growth rate, death rate, or other morphological attributes.

[0005] When running culture processes or experiments involving cell culturing, it is important to keep accurate records to enable others to reproduce the results. Aside from the type of cells, the type of media and the environmental conditions of the cell culturing, it is important to keep track of the amount of media in a well. Since the volume of the well is constant for a particular cell culture vessel well, the determination of the depth of the media will be a function of the volume of the media.

SUMMARY

[0006] One object of the present invention is to improve the reproducibility of cell culture processes and experiments. Another object is to improve recordkeeping of cell culture processes and experiments. A further object of the present invention is to improve the determination of the depth and/or the volume of cell culture vessel wells that are used for cell culture processes and experiments.

[0007] These and other objects of the present invention are achieved in accordance with the various embodiments of the present invention disclosed herein.

[0008] When the well is imaged by an imager, the depth of the media in a well will affect the image that is observed and therefore the depth is an important factor in processing the image to obtain details about the cell culture.

[0009] In accordance with the present invention, when a cell culture plate is used for cell culturing, each well with media therein is exposed to a light source to produce an illumination pattern. The resulting illumination pattern was previously correlated to a predetermined media depth and a method and apparatus in accordance with the invention records the depth and/or volume of the media in the well.

[0010] Those of skill in the art will understand that an illumination pattern resulting from exposing light onto a well varies with depth of the well, the depth of the media, the type of light, the shape of the light, and the size of well. Therefore, a database of depths correlated to images of illumination patterns of media in a particular size well plate is preferably generated for a given model of imager that is used to obtain images of illumination patterns for comparison to calibration images.

[0011] In some embodiments, a method for determining a depth of media in a cell culture vessel well comprises the steps of providing at least one calibration cell culture vessel with different predetermined depths of media in each well, imaging each of the calibration wells and storing the calibration images thereof, correlating the calibration images to the different predetermined media depths, imaging a cell culture vessel with at least one unknown media depth, comparing the image of the at least one unknown media depth to the calibration images and determining the at least one unknown media depth from the correlation to the different predetermined media depths.

[0012] In some embodiments, the step of correlating comprises using differences in image patterns of the calibration images to identify depths. In some embodiments, the differences in image patterns includes at least one of the number of rings, the width of rings, or the shading of rings alone or in combination.

[0013] In some embodiments, an apparatus for determining a depth of media in a cell culture vessel well comprises at least one calibration cell culture vessel with different predetermined depths of media in each well, an imager for imaging each of the calibration wells and storing the calibration images thereof, a processor correlating the calibration images to the different predetermined media depths and wherein the imager images a cell culture vessel with at least one unknown media depth and wherein an image processor compares the image of the at least one unknown media depth to the calibration images and determines the at least one unknown media depth from the correlation to the different predetermined media depths. [0014] Tn some embodiments the correlating comprises using differences in image patterns of the calibration images to identify depths. In some embodiments, the differences in image patterns includes at least one of the number of rings, the width of rings, or the shading of rings alone or in combination.

[0015] In some embodiments, the calibration images for a particular size cell culture plate and a particular imaging instrument are obtained by first putting a metered amount of media in each well of the plate and then determine the illumination pattern for each well for that instrument.

The illumination pattern for each depth is recorded in a database under the control of a processor or microcontroller. When a cell culture plate of that size is used in a cell culture process or experiment, the plate is imaged by that instrument and the illumination pattern is captured and an image processor compares the captured illumination pattern to the database of illumination patterns and the depth of the media is obtained.

[0016] These and other objects and advantages of the present invention are described in more detail with reference to the following drawings and specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a top view of a 96 well cell culture plate;

[0018] Figure 2 is a three-dimensional view of the meniscus of media in a well of the plate of

Figure 1;

[0019] Figure 3 is a view of the exposure of an imager light on the media in the well of Figure 2;

[0020] Figure 4 is an image of the light reflected from the well of Figure 3; and

[0021] Figure 5 is a block diagram of an apparatus for carrying the one embodiment of the method and apparatus according to the present invention. DETAILED DESCRIPTION

[0022] Referring now to Figure 1, a 96-well plate 10 is show with wells 11. When each well 11 is filled with liquid media, a meniscus 12 is formed, as shown in Figure 2. A plate 10 is placed in an imager wherein a light source 13 shines light on the media in the well, as shown in Figure 3, resulting in an illumination pattern that is a function of many factors, such as the type of light, the type of media, the size of the well and the depth of the well.

[0023] One example of a type of a resulting illumination pattern is shown in Figure 4. This pattern is of concentric rings of varying widths, and shades. For example, the outermost ring 14 is the darkest, whereas the inner rings 15, 16 and 17 are progressively lighter in shading. Other non-concentric and/or irregular patterns are produced depending on the factors listed above, but it is expected that for a particular size plate in a particular imaging instrument with the same media, the results are reproduced.

[0024] Accordingly, calibration plates can be provided wherein each well of the calibration plate has a predetermined volume of media and the predetermined volumes are stepped within a range, for example, 100 pL to 200 pL in desired increments of resolution. In a 96-well plate there can be steps of 1 pL if a single calibration plate is used or finer resolutions can be obtained by using 2 or more calibration plates. For plates with less wells, desired resolution steps will be achieved with multiple plates, for example, 6-well plates will likely require 15-50 calibration plates.

However, after the calibration is completed and the image results are stored in a database for that instrument, the plates can be reused.

[0025] Figure 5 shows an example of circuitry for carrying out an embodiment of the method and apparatus described herein. An incubator 21 is associated with an imager 22. The imager can be built into the incubator or located in the same laboratory as the incubator so that cell culture plates are removed from the incubator and placed in the imager. An example of an automated cell culture incubator with an integrated image is disclosed in U.S. Patent 11,319,523 issued May 3, 2022 and entitled Automated Cell Culture Incubator, the disclosure of which is hereby incorporated by reference herein. Examples of an imager separated from an imager and an imager integrated into an incubator and a method and apparatus for analyzing images generated by the imagers is disclosed in U.S. Application S.N. 63/252,671 filed October 6, 2021, the disclosure of which is hereby incorporated by reference herein.

[0026] The imager and incubator are under the control of one or more controllers 23 which stores the calibration images in database 25. The controller 23 also controls one or more image processors 24 which are used to compare images of wells with the calibration images to determine the depth of the media in the well.

[0027] The volume of a cylinder can be expressed by the following formula: V=7tr ² h, where r is the radius of the cylinder, and h is the height. Thus the height or depth of the media in a well is hm=V/ 7tr ² . If the processor or microcontroller knows the volume of media in a calibration well and the radius of the well, then the depth of the calibration well is determined.

[0028] For example, in order to calibrate a particular instrument with a 96 well plate, one can place 100 pL of media in a first well and then provide increments of pL in each successive well. This would provide a range of 100 pL to 196 pL in a calibration microplate. Alternatively, if greater precision is needed, then one can use two calibration microplates, wherein the increments of volume are 0.5 pL in each successive well. Since the depth of the media in the well is a function of the volume thereof, the depth of each well in the calibration microplates can be determined. For even greater precision, more than two, e.g., 3-5, 3-7 or 3-10 calibration plates can be used. [0029] The image processor compares the image of the unknown depth well with the previously stored illumination images. The comparison uses differences in image patterns of the calibration images to identify depths. The differences in image patterns includes at least one of number of rings, width of rings, or shading of rings. The image processor can use any one of the illumination image attributes or combinations thereof to match an unknown depth image to one of the calibration images.

[0030] In some embodiments, the image processor compares images of the same plate taken at different times to quantify differences in media height over time, without reference to a calibration image. The relative amount of media from time to time is useful for determining whether differences in media height have a negative or positive effect on cell growth, death, reactions to viruses, reactions to drugs, etc.

[0031] In some embodiments a method and apparatus for determining a depth of media in a cell culture vessel well comprises providing at least one cell culture vessel with media in at least one well, imaging the at least one well and storing images thereof reflecting a height thereof, imaging the at least one well at time intervals and storing images reflecting a height thereof at each time, comparing images of the at least one well at different times; and determining differences in the media height over time.

[0032] As used herein, cell culture refers to a procedure for maintaining and/or growing cells under controlled conditions (e.g., ex vivo). In some embodiments, cells are cultured under conditions to promote cell growth and replication, conditions to promote expression of a recombinant product, conditions to promote differentiation (e.g., into one or more tissue specific cell types), or a combination of two or more thereof. [0033] Tn some embodiments, cells are cultured in one of any suitable culture media. Different culture media having different ranges of pH, glucose concentration, growth factors, and other supplements can be used for different cell types or for different applications. In some embodiments, custom cell culture media or commercially available cell culture media such as Dulbecco’s Modified Eagle Medium, Minimum Essential Medium, RPMI medium, HA or HAT medium, or other media available from Life Technologies or other commercial sources can be used. In some embodiments, cell culture media include serum (e.g., fetal bovine serum, bovine calf serum, equine serum, porcine serum, or other serum). In some embodiments, cell culture media are serum-free. In some embodiments, cell culture media include human platelet lysate (hPL). In some embodiments, cell culture media include one or more antibiotics (e.g., actinomycin D, ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamycin, kanamycin, neomycin, penicillin, penicillin streptomycin, polymyxin B, streptomycin, tetracycline, or any other suitable antibiotic or any combination of two or more thereof). In some embodiments, cell culture media include one or more salts (e g., balanced salts, calcium chloride, sodium chloride, potassium chloride, magnesium chloride, etc.). In some embodiments, cell culture media include sodium bicarbonate. In some embodiments, cell culture media include one or more buffers (e.g., HEPES or other suitable buffer). In some embodiments, one or more supplements are included. Non-limiting examples of supplements include reducing agents (e.g., 2-mercaptoethanol), amino acids, cholesterol supplements, vitamins, transferrin, surfactants (e.g., non-ionic surfactants), CHO supplements, primary cell supplements, yeast solutions, or any combination of two or more thereof.

[0034] In some embodiments, one or more growth or differentiation factors are added to cell culture media. Growth or differentiation factors (e.g., WNT- family proteins, BMP-family proteins, TGF-family proteins, etc.) can be added individually or in combination, e g , as a differentiation cocktail comprising different factors that bring about differentiation toward a particular lineage. Growth or differentiation factors and other aspects of a liquid media can be added using automated liquid handlers integrated as part of an incubator provided herein.

[0035] In some embodiments, cell culture vessels are configured for culturing cells in suspension. In some embodiments, cell culture vessels are configured for culturing adherent cells. In some embodiments, cell culture vessels are configured for 2D or 3D cell culture.

[0036] In some embodiments, cell culture vessels include one or more surfaces or micro-carriers to support cell growth. In some embodiments, these are coated with extracellular matrix components (e.g., collagen, fibrin, and/or laminin components) to increase adhesion properties and provide other signals needed for growth and differentiation. In some embodiments, cell culture vessels include one or more synthetic hydrogels such as polyacrylamide or polyethylene glycol (PEG) gels to support cell growth.

[0037] In some embodiments, cell culture vessels include a solid support with embedded nutrients (e.g., a gel or agar, for example, for certain bacterial or yeast cultures). In some embodiments, cell culture vessels include a liquid culture medium.

[0038] As used herein, an “imager” refers to an imaging device for measuring light (e.g., transmitted or scattered light), color, morphology, or other detectable parameters such as a number of elements or a combination thereof. An imager may also be referred to as an imaging device. Tn certain embodiments, an imager includes one or more lenses, fibers, cameras (e.g., a charge-coupled device or CMOS camera), apertures, mirrors, light sources (e.g., a laser or lamp), or other optical elements. An imager may be a microscope. In some embodiments, the imager is a bright- field microscope. Tn other embodiments, the imager is a holographic imager or microscope. In other embodiments, the imager is a fluorescence microscope.

[0039] Cell culture plates come in a variety of sizes and number of wells. Typically, there are 6-well, 12-well, 24-well, 48-well and 96-well plates. These plates have a well volume of approximately 1-3 mL, 1-2 mL, 0.5-1.0 mL, 0.2-0.4 mb, and 0.1-0.2 m. respectively.

[0040] A 96 well standard microplate has 12 columns and 8 rows of wells and the dimensions are 127.71 mm x 85.43 mm with a height of 14.10 mm. Each well has a diameter of 7mm. The capacity of each well is .3 ml. The minimum required volume is recommended as 100 pL. The depth of the well depends on the type of bottom it has, i.e., whether it is U-shaped, V-shaped, flat or C-shaped. The respective depths are as follows:

[0041] U-Shaped: 10.85 mm, V- Shaped: 11.65 mm, Flat: 10.65 mm and C- Shaped: 10.85 mm. [0042] In some embodiments, before containers or vessels are brought into an incubator cabinet, a user can select an automation system protocol-based for the particular containers, vessels, ingredients, or cells that are being inserted into the incubator cabinet. Relevant information related to the incubator media or one or more incubator components, and the cells being grown can be entered into the database. For example, one or more identifiers such as barcodes (e.g. ID or 2D) barcodes) can be placed on the container or vessel and other significant information, such as, the type of container, e.g., the number of wells, the contents of the container, what assays or manipulations are to be performed on the sample in the container can be specified.

[0043] In some embodiments, information related to the incubator system and/or cells can be contained in one or more barcodes, in a separate database, or a combination thereof. The user may also enter information that identifies the dimensionality (e. g., height, diameter) of the vessel or other container, or the system itself may determine measure the height of the vessel or other container. Using this information, robotics can be requested to transport the vessels.

[0044] The incubator systems, robotics, etc., which may operate together at the direction of a computer, processor, microcontroller or other controller such as controller 23. The components may include, for example, a transfer device (e.g., robotic arm), a liquid handling device, a delivery system conveying culture vessels, or other components to or from the incubator cabinet, an environmental control system for controlling the temperature and other environmental aspects of the incubator cabinet, a door operation system, an imaging or detection system, and a cell culture assay system.

[0045] In some cases, operations such as controlling, operations of a cell culture incubator and/or components provided therein or interfacing therewith, controlling the imager and components thereof, controlling a database and image processing, may be implemented using hardware, software or a combination thereof; When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single component or distributed among multiple components. Such processors may be implemented as integrated circuits, with one or more processors in an integrated circuit component. The processor may be implemented using circuitry in any suitable format.

[0046] In some embodiments, a component (e.g., a controller) controls various processes performed inside the incubator, imager and image processing apparatus. For example, a controller may direct control equipment (e.g, a manipulator, an imager, a fluid handling, system, etc.). In some embodiments, the controller controls imaging of cell cultures, picking of cells, weeding of cells (e.g., removal of cell clumps) monitoring of cell culture conditions, adjustment of cell culture conditions, tracking of cell culture vessel movement within the incubator, comparing images, storing images maintaining a database and/or scheduling of any of the foregoing processes.

[0047] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

[0295] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one.”

[0048] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0049] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (e.g. "one or the other but not both") when preceded by terms exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law. [0050] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0051] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, e.g., to mean including but not limited to.

[0052] Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0053] Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0054] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.