Automated cell culture systems and methods

Abstract

Systems and methods for automated cell culture are disclosed. In some embodiments, one or more cell culture vessels are fluidly connected to one or more multiport valves and one or more fluid pumps. The fluid pumps may pump various fluids into or out of the cell culture vessels as needed to support cell growth, which are routed by the one or more multiport valves. In some embodiments, one or more components may be detachable from other components so that some components may be prepared and sterilized independently before use.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 978,012, entitled "Systems and Methods for Automated Cell Culturing" (filed on February 18, 2020). This document is hereby incorporated by reference in its entirety. This application is also related to U.S. Patent Application No. 16 / 543,369, entitled "Systems and Methods for Automated Cell Culturing" (filed on August 16, 2019) (U.S. Patent Application Publication No. 2020 / 0056140), which claims priority to U.S. Provisional Patent Application No. 62 / 719,652, entitled "Automated Cell Culture" (filed on August 19, 2018). These disclosures are each hereby incorporated by reference in their entirety.

[0002] This specification generally relates to systems and methods for culturing cells.

Background Art

[0003] Cells can be grown or cultured under controlled conditions in a laboratory or industrial environment for various purposes. Typically, cells grow inside sealed containers, covered with a solution called cell culture medium, which provides essential nutrients and other supplements to promote cell growth. Examples of containers used for cell culture include flat, round dishes such as petri dishes and laboratory flasks. As cells grow and proliferate, they consume nutrients in the cell culture medium and produce waste byproducts. For this reason, the cell culture medium must be replaced periodically to ensure that the cells continue to proliferate. Furthermore, cell culture can be expanded by transferring some of the cells to a new container, providing an additional volume or area in which the cells can proliferate. This process of transferring some of the cells to a new container is sometimes called subculturing or subculturing. Additionally, cells can be removed from the container in preparation for their use. The process of separating cells from the container in which they are growing is sometimes called harvesting.

[0004] Cell cultures typically grow according to a standard growth pattern. The first stage of growth after seeding is a quiescent phase, a period of slow growth as the cells adapt to the culture environment. Following the quiescent phase is a logarithmic phase in which cells grow exponentially and consume nutrients in the growth medium. When the cell culture has consumed all the nutrients in the growth medium or reached the capacity of the environment by occupying all available space, growth slows down and the cells enter a quiescent or plateau phase, where growth decreases significantly or stops completely. Known cell culture treatments often involve passage the cells before they enter this quiescent phase to optimize growth.

[0005] Generally, adherent cells are more difficult to grow than suspension cells. Adherent cells grow by adhering to surfaces such as the bottom of culture flasks or dishes. The amount of cells in a flask is usually measured as the percentage of the growth surface covered by cells, and is called the percentage of confluence. Adherent cells need to be detached from the surface before they can be removed from the container. Cells can be detached by one of several methods, including mechanically scraping or using enzymes such as trypsin to break their adhesion to the container surface. The detached cells are then resuspended in fresh growth medium and returned to the growth surface to settle. These further steps increase the possibility of cell damage or contamination.

[0006] Furthermore, dissociation reagents used to promote the detachment of adherent cells can also be harmful to the cells and should therefore be completely removed before returning the cells to fresh growth medium. These processes—removing used medium from cell culture vessels, adding fresh medium, detaching adherent cells, and transferring cells from one vessel to another—are typically performed by cumbersome manual procedures. For example, known cell culture methods frequently involve repetitive operations, including moving cells (within cell culture vessels) between various work stations and / or opening cell culture vessels to move fluids in and out of the vessels. Specifically, known methods first involve loading cells and cell culture medium into a vessel in a sterile environment (e.g., a laminar flow hood). Once ready, the cell culture vessel is closed (to minimize contamination) and moved to an incubator to promote growth. Cell culture vessels are often manually monitored to determine the appropriate time to change the cell culture medium and are periodically manually monitored to examine parameters such as confluence and cell morphology by removing the vessels from the incubator and imaging them under a microscope. These manual monitoring steps typically require moving the culture to the lab to check on it and determine if any further operations need to be performed. When it's time to change the cell culture medium, the cell culture container is moved from the incubator to a sterile environment, opened (or connected to a source of waste and fresh cell culture medium), and fluids are moved into and / or removed from the cell culture container. The container is also moved and / or opened to complete other operations such as cell subculturing or cell harvesting.

[0007] Such known procedures are inefficient, costly, and susceptible to contamination. For example, repeatedly opening cell culture systems and moving cell culture containers between experimental stations can expose cells to contamination. Furthermore, all operations performed manually are costly and susceptible to contamination (or cell damage) if the operator does not follow proper procedures. In addition, determining when to change the culture medium or when to subculture cells is usually done according to a set schedule, which may not be optimal. Adhering to a set schedule may result in the additional (and potentially unnecessary) use of laminar flow hoods (which can be costly as the operation can consume a large amount of energy). Adhering to a set schedule may also reduce the efficiency of cell growth (for example, if cell growth reaches a plateau before the cell culture medium is changed).

[0008] Several known systems and methods for culturing adherent cells are used in various applications, including drug development and cell therapy. Different cell types may require different levels of environmental control (e.g., induced pluripotent stem cells (iPSCs)), and culturing them can be extremely difficult. In addition, known systems for monitoring cell health do not take into account the details of iPSCs, such as morphological changes that may indicate undesirable differentiation. Furthermore, known systems do not efficiently enable the passage of adherent cells. Many known systems require the use of centrifugation systems to remove cells from the culture system and separate them from dissociation reagents. Therefore, the cells are not maintained within a closed system.

[0009] As mentioned earlier, many existing cell culture systems are designed to grow large batches of one type of cell, and these cells are typically then used as a “factory” to produce the desired biological product (e.g., a protein). Usually, only the product is retained (not the cells). This use case, where cells produce the product for the user, is called “bioprocessing,” and such known systems are often called bioreactors. Since the product of interest is the biological product produced, not the cells, such known systems often use some suitable robust cells that can easily grow and produce the desired product (or such cells are seeded). Often, the type of cell that grows most easily is selected as the factory ("producer"), and such cells are often cultured in suspension. Known bioreactor systems are large and used for a single type of cell (to produce large quantities of product). Such systems are not suitable for cell culture for development or therapeutic purposes. In these cases, multiple different types of cells are required in smaller quantities.

[0010] However, there are times when it is desirable to obtain cells for use in drug testing or other therapeutic purposes. In other words, in certain situations, the cells (not the products the cells produce) are actually the desired products for the experiment. However, many known bioreactors are not suitable for culturing cells that are to be used as the final product. Specifically, when growing cells for drug testing, the cells are subjected to various requirements that differ from the requirements and functions of known bioprocessing systems. Firstly, it is necessary to grow the most realistic cells possible (i.e., cells related to the drug, therapeutic regulation, or condition that the drug addresses). In other words, cells are selected not simply based on their ability to produce products, but on their applicability to the desired test. Typically, cells used for drug testing do not grow in suspension, but instead adhere to surfaces as they grow (i.e., adherent cells). Handling adherent cells introduces technical challenges to their culture. Secondly, it is necessary to generate small batches of many different types of cells growing simultaneously so that different types of cells are always ready for testing. Known existing machines may not be configured to perform this type of cell harvesting.

[0011] The challenge lies in designing a system with an acceptable setup time, taking into account all the fluidics that are typically discarded with each batch. In bioprocessing, batches are usually very large, so long setup times are acceptable. Use cases involving growing multiple different cell types simultaneously for testing require very fast setup times, as there are many small batches to handle (at least one for each cell type). Known systems require long setup times, including extensive cleaning after use. Careful setup of piping to valve heads can further increase setup time. Therefore, there is a need for a cell culture system that can provide efficient and fast setup times.

[0012] Furthermore, many known cell culture systems (e.g., bioreactors) are not suitable for growing different types of cells within a single system. For example, certain test methods may involve using multiple different cell types. Known systems are typically not suitable for containing multiple different cell types because they contain a single reservoir to accommodate one type of cell (e.g., one intended for use as a factory). In addition, many known cell culture systems lack the ability to passage cells from one vessel to another when the culture becomes confluent, requiring operators to periodically intervene manually and replace consumables on the system each time the culture reaches confluence.

[0013] For effective cell culture, it is crucial that cells are uniformly dispersed on the surface of the culture plate. This is especially important for stem cell culture, because if cells are not uniformly dispersed, they may undergo undesirable differentiation during culture. Many known systems and methods for cell culture involve manually vibrating the cell culture vessel after the cells have been placed inside. Such vibrating methods are not reproducible, are not always effective, and can cause cells to begin adhering to the trophoblast on the cell surface; therefore, vibrating after seeding is not always effective.

[0014] Cell counting is also a crucial aspect of cell culture. Known methods for cell counting are difficult, requiring cells to be removed from the cell culture system and placed in an external counting device. More specifically, cell samples are removed from culture trays and placed in a separate cartridge outside the incubator. This process increases the possibility of contamination due to access to the closed system. In addition, known counting systems rely on having a homogeneous mixture of cells. If sedimentation or discrepancies exist when cell samples are manually removed and placed in the counting cartridge, the results may be inaccurate. Furthermore, the counted samples must be discarded as waste. Therefore, there is a need for a system and method for accurately counting cells in a closed system.

[0015] Stem cell cultures are prone to problems associated with undesirable differentiation. Often, if undesirable stem cell behavior is detected, the entire container must be discarded and the culture restarted. While several methods exist to selectively isolate desired cells and avoid discarding the entire culture, these methods are typically labor-intensive, requiring the opening of the system and manual separation of desired cells from undesirable cells / surfaces. Therefore, there is a need for systems and methods to isolate desired cells while avoiding these problems.

[0016] Further challenges may arise when the cells to be cultured are stem cells. Specifically, culturing pluripotent stem cells can be difficult because even slight changes in the environment can trigger unintended differentiation of stem cells. Known cell culture systems often fail to maintain the desirable environmental control necessary to preserve the pluripotency of stem cells. For example, differences in cell density and uneven seeding of cells within the cell culture vessel can increase the likelihood of induced pluripotent stem cells (iPSCs) undergoing undesirable spontaneous differentiation. Therefore, known methods for seeding stem cells typically involve manually moving the cell vessel after the cells have been loaded to promote a more homogeneous mixture of stem cells before they settle and adhere to the surface. However, such methods are inconsistent and do not reliably produce the desired spatial homogeneity. Specifically, such manual methods vary greatly depending on the experimental technician and the procedures followed. Furthermore, because the movement (or gentle vibration) is performed after the cells have been loaded (and the vessel has been closed), over time, some of the seeded cells may settle before the movement begins. Therefore, improvements to systems and methods for seeding stem cells are needed.

[0017] Another example is the periodic counting of cells in known cell culture methods. Known systems and methods for cell counting often involve opening the cell culture environment, removing a portion of the cells, and counting them via an external cell counting system. Because the counted cells are handled through multiple steps and are contaminated or damaged, they are usually discarded. Therefore, there is a need for improvements in cell counting methods that ensure the counting of a homogeneous mixture of cells within a closed system. There is also a need for improvements in cell counting methods and systems that allow for the recovery of counted cells for continued use.

[0018] Known cell culture systems commonly utilize a centrifugation process to wash cells or remove used culture medium and / or reagents. Such known methods often involve removing cell samples from the culture system and completing the filtration / washing process by centrifugation. Such methods can expose cells to potential contamination and damage. Therefore, improvements to systems and methods for filtering and / or washing cells are needed.

[0019] There is also a need for cell culture systems that improve efficiency and limit potential contamination during cell culture. Specifically, there is a need for systems and methods that automate cell culture procedures, maintain the cell culture system in a closed, sterile environment during culture, and enable efficient setup and use. There is also a need for automated cell culture systems that can be operated with existing, off-the-shelf cell culture vessels at will. [Overview of the project]

[0020] According to one implementation, this specification describes systems and methods for automatically culturing cells. The automated cell culture systems disclosed herein enable scientists to accelerate their research and development by automating manual cell culture. The systems and methods disclosed in various embodiments may provide automated cell growth medium replenishment, automated cell subculturing, and / or automated cell culture analysis. These automated cell culture systems and methods can increase efficiency and reduce errors compared to manual cell culture operations. Furthermore, these embodiments increase the quantity and quality of cell culture data points available to scientists through an integrated automated analysis mechanism.

[0021] An automated cell culture system according to an embodiment includes a housing in which a valve actuator and a fluid pump are arranged. The automated cell culture system also includes a removable tray configured to be removably fitted into the housing. Multiple cell culture container brackets mounted on the removable tray are configured to hold each of the multiple cell culture containers, each cell culture container covered with a sterile lid. A selector valve is configured to connect to the valve actuator of the housing when the removable tray is fitted into the housing. In some embodiments, multiple culture medium sources may be provided outside the housing and the removable tray. A multi-port selector valve is configured to fluidly connect a master port to a selected port of a plurality of selectable ports, the master port of the multi-port selector valve is fluidly connected to a fluid pump, and each of the multiple cell culture containers and culture medium sources is directly fluidly connected to one of the multiple selectable ports of the multi-port selector valve. In some embodiments, the multiple cell culture containers and their sterile lids, the multi-port selector valve, and the fluid connections between them form a first sterile sealing system mounted on the removable tray.

[0022] In some embodiments, a method for cell line maintenance using an automated cell culture system includes sending commands to a movable imaging system of the automated cell culture system to image cells in a selected vessel of the automated cell culture system, receiving an image of the cells in the selected vessel from the imaging system, measuring cell passage criteria based on the image of the cells in the selected vessel, comparing the cell passage criteria to a threshold cell passage criteria, and determining based on the comparison to initiate passage of the cells in the selected vessel to a passage vessel. The method for cell line maintenance also includes passage of a composed portion of the cells of the selected vessel to a secondary vessel, and the automated cell culture system sending a notification that the composed portion of the cells of the selected vessel has been passed to the secondary vessel. Other embodiments of this aspect include corresponding computer systems, devices, and computer programs recorded on one or more computer storage devices configured to perform the operations of the method, respectively.

[0023] Details of one or more implementations of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other potential features, aspects, and advantages of the subject matter will become apparent from the description, the drawings, and the claims.

Brief Description of the Drawings

[0024] [Figure 1A] It is a schematic diagram of an automated cell culture system according to an embodiment. [Figure 1B] It is a schematic diagram of an automated cell culture system according to an embodiment. [Figure 2] It is a top view of an automated cell culture system according to an embodiment. [Figure 3A] It is a top view of a base housing of an automated cell culture system according to an embodiment. [Figure 3B] It is a diagram showing a removable tray assembly of an automated cell culture system according to an embodiment. [Figure 4] It is a diagram showing an exemplary removable tray of an automated cell culture system fitted to an exemplary base housing according to an embodiment. [Figure 5] A cross-sectional view of an exemplary multiport valve according to an embodiment. [Figure 6A] A diagram showing an exemplary multiport valve according to an embodiment. [Figure 6B] A bottom view of an exemplary multiport valve. [Figure 7] A diagram showing a lid of a cell culture vessel according to an embodiment. [Figure 8] A cross-sectional view of a lid of a cell culture vessel according to an embodiment. [Figure 9] A diagram showing steps of a method for transferring liquid from a first vessel to a second vessel using an automated cell culture system equipped with a single-port pump according to an embodiment. [Figure 10] A diagram showing steps of a method for transferring liquid from a first vessel to a second vessel using an automated cell culture system equipped with a two-port pump according to an embodiment. [Figure 11] A diagram showing steps of a method for changing a cell culture medium during maintenance of an adherent cell line. [Figure 12] A diagram showing steps of a method for maintaining or proliferating an adherent cell line with passage to a new cell culture vessel. [Figure 13] A diagram showing steps of a method for maintaining a suspension cell line with optional passage. [Figure 14] A diagram showing steps of a method for suspension cell line proliferation. [Figure 15] A diagram showing an exemplary machine of a computer system in which a series of instructions for causing a machine to execute any one or more of the methodologies discussed herein may be executed. [Figure 16A] A schematic diagram of a tray assembly of a cell culture system according to an embodiment. [Figure 16B] A schematic diagram of a base unit of a cell culture system according to an embodiment. [Figure 16C]This is a schematic diagram of a cell culture system according to an embodiment, including the tray assembly shown in Figure 16A and the base unit shown in Figure 16B. [Figure 17] This is a schematic diagram of an electronic control system for a cell culture system according to an embodiment. [Figure 18] This figure shows an example of a screenshot illustrating various GUI elements generated in relation to the operation of an electronic control system. [Figure 19] This figure shows an example of a screenshot illustrating various GUI elements generated in relation to the operation of an electronic control system. [Figure 20] This figure shows an example of a screenshot illustrating various GUI elements generated in relation to the operation of an electronic control system. [Figure 21] This is a top view of a tray assembly of a cell culture system according to an embodiment. [Figure 22] This is a top view of the tray assembly in Figure 21, positioned within the protective overlap. [Figure 23] Figure 21 is a top view of the tray assembly, showing the fluid pump connected to the tray assembly. [Figure 24] Figure 21 is a perspective view of the tray assembly, showing the fluid pump (Figure 23) connected to the tray assembly. [Figure 25] Figure 21 is a perspective view of the tray assembly, showing cell culture containers connected to the tray assembly. [Figure 26] Figure 21 is a top view showing three cell culture containers connected to the fluid pump and tray assembly shown in Figure 23. [Figure 27] Figure 21 is a top view of the tray assembly, showing the connection to the base unit according to an embodiment. [Figure 28] Figure 27 is a perspective view of the multi-port valve connected to the base unit. [Figure 29] Figure 27 is a top view of a portion of the base unit. [Figure 30]This is a perspective view of the tray assembly shown in Figure 21, which is connected to the base unit shown in Figure 27. [Figure 31] A flowchart shows a method for preparing a cell culture system for use in cell culture treatment according to an embodiment. [Figure 32] This is a perspective view of an imaging device for the base unit of a cell culture system according to an embodiment. [Figure 33] Figure 32 is a top view of the imaging device. [Figure 34] Figure 32 is a side view of the imaging device. [Figure 35] This is a perspective view of a tray assembly of a cell culture system according to another embodiment. [Figure 36] Figure 35 is a perspective view of a portion of the tray assembly with its removable components removed. [Figure 37] Figure 35 is a perspective view of a portion of the tray assembly. It shows a multi-port valve, lid, and fluid pump connected to the tray. [Figure 38] Figure 35 is a perspective view of the base unit of a cell culture system that can be used with the tray assembly. [Figure 39] Figure 38 is a perspective view of the pump actuator of the base unit. [Figure 40] Figure 38 is a perspective view of the base unit, which includes a fluid pump and a multi-port valve connected to it. [Figure 41] Figure 38 is a partially exploded view of the base unit, showing the multiport valve before it is assembled into the base unit. [Figure 42] Figure 38 is a side view of the base unit. [Figure 43] Figure 38 is a side view of the base unit, showing the inside of the base unit. [Figure 44] Figure 38 shows the inside of the base unit, and these are opposing side views of the base unit. [Figure 45] This is a perspective view of a cell culture system according to another embodiment. [Figure 46] Figure 45 is a top view of the cell culture system. [Figure 47] This is a cross-sectional view along lines 47-47 in Figure 46. [Figure 48] This is a perspective view of a tray assembly according to an embodiment. [Figure 49] Figure 48 is a top view of the tray assembly. [Figure 50] This is a cross-sectional view obtained along line 50-50 in Figure 49. [Figure 51] This is a perspective view of the base unit according to the embodiment. [Figure 52] This is a diagram of a cell culture system according to another embodiment. [Figure 53] Figure 52 is a side view of the cell culture system, showing the imaging system located inside the base unit. [Figure 54] Figure 52 is a top view of the base unit of the cell culture system. [Figure 55] Figure 52 is a top view of the tray assembly of the cell culture system. [Figure 56] Figure 55 is a side view of the tray assembly. [Figure 57] Figure 55 is a top view of the tray in the tray assembly. [Figure 58] This is a front view of a pair of incubators equipped with multiple cell culture systems, arranged on shelves within the facility. [Figure 59] This is a system diagram showing an exemplary fluid setup within the system during cell culture treatment. [Figure 60] Figure 59 is a table showing the contents. [Figure 61A] This figure includes a table showing examples of cell subculturing procedures. [Figure 61B] This figure includes a table showing examples of cell subculturing procedures. [Figure 62] Figures A through C show the lids of containers according to the embodiments. [Figure 63A] This is a top view of a multi-port valve according to an embodiment. [Figure 63B]Figure 63A is a bottom view of the multi-port valve. [Figure 63C] Figure 63A is a side view of a multi-port valve. [Figure 63D] This is a cross-sectional view along the line segment 63D-63D in Figure 63C. [Figure 64A] Figures 63A to 63D are cross-sectional views of the valve body of a multiport valve. [Figure 64B] This is a side view of the valve body shown in Figure 64A. [Figure 64C] Figure 64A is a cross-sectional side view of the valve body. [Figure 65] A is a side view of the valve rotor of the multiport valve shown in Figure 63A. B is a cross-sectional view along the line segment 65B-65B in Figure 65A. C is a top view of the valve rotor. [Figure 66A] This is a schematic diagram of a tray assembly of a cell culture system according to an embodiment. [Figure 66B] This is a schematic diagram of the base unit of a cell culture system according to an embodiment. [Figure 66C] This is a schematic diagram of a cell culture system according to an embodiment, and includes the tray assembly shown in Figure 66A and the base unit shown in Figure 66B. [Figure 66D] Figure 66C is a schematic diagram of a part of the cell culture system according to an embodiment, showing a multi-port valve connected to various components. [Figure 67A] This is a side view of a portion of the tray assembly of a cell culture system according to an embodiment. [Figure 67B] Figure 67A is a plan view of the tray in the tray assembly. [Figure 68] This is a schematic diagram illustrating an exemplary fluid setup within a system during cell culture treatment. [Figure 69] This diagram illustrates an example of incident illumination for a fluorescence system. [Figure 70] This diagram illustrates different examples of software workflows and decision loops. [Figure 71]This diagram illustrates different examples of software workflows and decision loops. [Figure 72] This diagram illustrates different examples of software workflows and decision loops. [Figure 73] This diagram illustrates different examples of software workflows and decision loops. [Figure 74] This is a system diagram illustrating an exemplary fluid setup within a system during cell culture treatment, and includes a tangential flow filtration system. [Figure 75] This is a schematic diagram of a tray assembly of a cell culture system including a tangential flow filtration cartridge according to an embodiment. [Figure 76] This is a schematic diagram illustrating an exemplary fluid setup within a cell culture system according to an embodiment, and includes a tangential flow filtration system. [Figure 77] This is a schematic diagram illustrating an exemplary fluid setup within a cell culture system according to an embodiment, and includes a tangential flow filtration system. [Figure 78] This is a schematic diagram illustrating an exemplary fluid setup within a cell culture system according to an embodiment, and includes a tangential flow filtration system. [Figure 79] This is a schematic diagram illustrating an exemplary fluid setup within a cell culture system according to an embodiment, and includes a tangential flow filtration system. [Figure 80] This is a schematic diagram illustrating an exemplary fluid setup within a cell culture system according to an embodiment, and includes a tangential flow filtration system. [Figure 81] This is a schematic diagram of a tray assembly of a cell culture system according to an embodiment. [Figure 82] A and B are schematic diagrams of a portion of the tray assembly according to an embodiment in the first configuration. [Figure 83] A and B are schematic diagrams of parts of the tray assembly shown in Figures 82A and 82B in the second configuration. [Figure 84] This is a top perspective view of the tray assembly of a cell culture system according to an embodiment. [Figure 85] Figure 84 is a perspective view of the tray assembly. [Figure 86] Figure 84 is a partially exploded view of the tray assembly. [Figure 87] This is a perspective view of the tray assembly shown in Figure 84, with the valve assembly and two containers removed for illustrative purposes. [Figure 88] Figure 84 is a perspective view of the tray in the tray assembly. [Figure 89] Figure 84 is a perspective view of the cell counting chip in the tray assembly. [Figure 90A] Figure 84 is a perspective view showing the valve assembly of the tray assembly with two tubes positioned inside. [Figure 90B] Figure 84 is an exploded view of a portion of the valve assembly in the tray assembly. [Figure 90C] Figure 84 is a bottom perspective view of a portion of the valve assembly in the tray assembly. [Figure 90D] Figure 84 is a side view of a portion of the valve assembly in the tray assembly. [Figure 91] This is a perspective view of an input tray assembly according to an embodiment. [Figure 92] Figure 91 is a perspective view of the input tray assembly. [Figure 93] This is a front perspective view of the equipment for a cell culture system according to an embodiment. [Figure 94A] This is a magnified view of a part of the device shown in Figure 93. [Figure 94B] This is a magnified view of another part of the device shown in Figure 93. [Figure 95] A and B are perspective views of different agitator coupling elements of the apparatus shown in Figure 93, respectively. [Figure 96] Figure 93 is a magnified view of the valve connector and actuator of the device shown. [Figure 97]Figure 84 is an enlarged view showing that the alignment portion of the tray assembly is aligned with the protrusion on the fixture shown in Figure 93. [Figure 98] This is a perspective view showing a part of the device in Figure 93 connected to the tray assembly in Figure 84. [Figure 99] Figure 84 shows an example where the fluid pump assembly of the tray assembly is connected to the pump actuator of the fixture shown in Figure 93. [Figure 100] This is a front perspective view of the device in an open configuration, with the tray assembly shown in Figure 84 and the input tray assembly shown in Figure 91 connected. [Figure 101] This is a front perspective view of the device in a closed configuration, with the tray assembly shown in Figure 84 and the input tray assembly shown in Figure 91 connected. [Figure 102] This is a flowchart of a cell culture method according to an embodiment. [Figure 103] This is a flowchart illustrating a method for seeding a cell sample into a cell culture container according to the embodiment. [Figure 104] A is an image of a cell culture container containing a cell sample seeded using the method shown in Figure 103. B is a magnified view of a portion of the image in Figure 104A. [Figure 105] This is a flowchart illustrating a method for counting cells in a cell culture system according to an embodiment. [Figure 106] This is a flowchart illustrating a method for selectively removing cells from a cell culture system according to the embodiment. [Figure 107A] This is an image of cells in region R1 of a cell culture container, showing cells that have undergone spontaneous differentiation. [Figure 107B] This is a magnified view of a portion of region R1 in Figure 107A, showing the first part of the dissociated iPSC and the second part that remained in a predetermined position within the cell culture container. [Figure 108] This is a flowchart illustrating a method for selectively removing cells from a cell culture system according to the embodiment. [Modes for carrying out the invention]

[0025] As described herein, in some embodiments, cell culture systems and methods are configured to obtain cells to be used for testing drugs, such that the cells are actually the reagents required for the experiment. When growing cells for drug testing for this purpose, the cells are typically adherent cells. Furthermore, it is desirable that such cell cultures produce small batches of many different types of cells growing simultaneously, so that different types of cells are always ready for testing. In addition, since batches of different types of cells growing simultaneously means that each batch needs to be set up separately, a rapid setup time per batch is important when automating this process. Various embodiments of consumable tray assemblies and base units described herein are configured to perform this type of cell harvesting and provide a reduction in setup time.

[0026] In some embodiments described herein, the cell culture system or group of systems is designed to maintain or simultaneously increase (produce more cells) different types of cells during culture, while simultaneously maximizing the time that does not require manual intervention from the operator (i.e., maximizing the time extension when the user does not need to physically touch the system to replace consumables, etc.). For example, in some embodiments described herein, an imaging device (e.g., a microscope) is integrated into the system to enable remote monitoring without the need to physically remove the cell culture vessel from the system.

[0027] In some embodiments described herein, a consumable tray assembly is provided that houses an empty cell culture vessel into which the system subcultures cells when the current vessel becomes confluent (adherent cells). As described herein, in some embodiments, the cell culture system can sufficiently remove dissociation reagents from the cell culture vessel. In some embodiments, the cell culture system can harvest cells at a desired density. In some embodiments, the cell culture system described herein can rescue a stem cell culture in which cells have unintentionally begun to differentiate by detaching only the undifferentiated cells and transferring them to a new vessel.

[0028] In some embodiments described herein, cell culture systems can be used to grow cells and test batches of advanced therapeutic drugs (ATMPs). Typically, when producing batches of cells edited for cell therapy or batches of viruses for use in gene therapy, manual cell culture procedures are performed on small samples to demonstrate that the batch is safe for use and will function in patients. In the case of cell therapy, this may involve manually culturing a sample of cells for a period of time to demonstrate that they behave as intended in patients. The systems described herein enable manual cell culture capabilities combined with monitoring (i.e., limited manual interaction by the operator) and are therefore well-suited for culturing cells for ATMPs. In the case of virus batches, this testing may involve manually growing these cells over many passages after transfecting them with a virus, and checking for the absence of virus in the final passage to demonstrate that the virus is incapable of replication. The systems and methods described herein are also well-suited for such operations of virus batches.

[0029] In some embodiments, the apparatus includes a tray configured to be removably coupled within the apparatus. The tray includes a aligning portion configured to mate and engage with a corresponding aligning portion of the apparatus. The tray includes a shoulder portion that defines a sensor opening and surrounds at least a portion of the sensor opening. The apparatus includes a container having a top and a bottom surface. The top and bottom surfaces each have a transparent portion. The container is coupled to the tray, with the edge of the bottom surface supported by the shoulder portion, and the transparent portion of the bottom surface aligned with the sensor opening. A mounting bracket is coupled to the tray and to the edge of the top surface of the container, securing the container to the tray. A valve assembly is fluidly coupled to the container and a fluid pump. The valve assembly is removably coupled to the tray. The valve assembly and the fluid pump are each configured to drive to cause the transfer of fluid into and out of the container.

[0030] In some embodiments, the apparatus includes a tray configured to be detachably connected within the cell culture apparatus. A first container is connected to the tray and configured to receive cell samples inside. A second container is also connected to the tray. The apparatus includes a first tangential flow filter assembly having an inlet port, a first outlet port, and a second outlet port, and a second tangential flow filter assembly having an inlet port, a first outlet port, and a second outlet port. The second outlet port of the first tangential flow filter assembly is fluid-coupled to the inlet port of the second tangential flow filter assembly. The apparatus further includes a fluid pump assembly and a valve assembly operably connected to the first container, the second container, the inlet port of the first tangential flow filter assembly, and the fluid pump assembly. The valve assembly and the fluid pump assembly are each driven by the cell culture apparatus and configured to perform the following actions: A) transferring a cell sample from a first container into the inlet port of a first tangential flow filter assembly; B) transferring a first volume of unpermeated material from the cell sample from the first outlet port of the first tangential flow filter assembly to a second container; and C) transferring a first volume of permeated material from the second outlet port of the first tangential flow filter assembly to the inlet port of a second tangential flow filter assembly.

[0031] In some embodiments, the instrument includes a base unit having a lower housing, a support plate, and a first portion of a cell sensor assembly movably connected within the lower housing. The support plate is configured to be detachably connected to a cell culture tray assembly. The cell culture tray assembly has a tray and a container connected to the tray. The tray defines a sensor opening, and a portion of the container is transparent and connected to the tray, providing light access to the contents of the container through the sensor opening and the transparent portion of the container. The instrument includes an upper unit having an upper housing and a second portion of a cell sensor assembly connected within the upper housing. The upper unit is movably connected to the base unit and configured to move between an open position and a closed position. The support plate is accessible when the upper unit is in an open configuration. The support plate is at least partially enclosed when the upper unit is in a closed configuration. An electronic control system connected within at least one of the lower housing or the upper housing, the electronic control system is configured to control the movement of at least a first portion of the cell sensor assembly to align the first portion of the cell sensor assembly with the container.

[0032] In some embodiments, the apparatus includes a base unit having a lower housing, a support plate, and a stirrer assembly located within the lower housing. The support plate is configured to be detachably coupled to a cell culture tray assembly. The cell culture tray assembly has a tray and a container coupled to the tray. An upper unit having an upper housing is movably coupled to the base unit and configured to move between an open position and a closed position. The support plate is accessible when the upper unit is in the open configuration. The support plate is at least partially enclosed when the upper unit is in the closed configuration. The stirrer assembly is located within the lower housing of the base unit and operably coupled to the support plate via a plurality of coupling elements located at mounting locations around the support plate. At least one of the plurality of coupling elements is configured to maintain the position of the support plate in a first direction. At least one of the plurality of coupling elements is configured to maintain the position of the support plate in a second direction different from the first direction. The stirrer assembly is configured to move the support plate when driven and to stir the cell culture tray assembly when coupled to the support plate. An electronic control system is connected within at least one of the lower or upper housings and is configured to control the drive of the agitator assembly.

[0033] In some embodiments, the method includes removing a cell culture tray assembly from an external protective wrap. The cell culture tray assembly includes a tray, a container connected to the tray, and a pump and valve assembly detachably connected to the tray, wherein the tray includes a aligning portion, and the container is sterilely connected to the pump and valve assembly to form a closed system. The valve assembly and the fluid pump are each configured to drive to cause the transfer of fluid into and out of the container. The cell culture tray assembly is connected to an instrument by engaging the aligning portion of the tray with the corresponding aligning portion of the instrument, and the instrument includes a valve actuator and a pump actuator. With the container, pump, and valve assembly still connected within the closed system, the valve assembly is removed from the tray and connected to the valve actuator of the instrument. With the container, pump, and valve assembly still connected within the closed system, the pump is connected to the pump actuator of the instrument. By driving at least one of the valve assembly and the pump, one or more cell culture operations are performed on the cell sample in the container connected to the tray.

[0034] In some embodiments, a method for seeding a cell sample into a cell culture container includes connecting a cell culture tray assembly to a support plate of the instrument. The cell culture tray assembly includes a tray, connects the cell culture container to the tray, and removably connects a pump and valve assembly to the tray. The cell culture container is aseptically connected to the pump and valve assembly to form a closed system. The valve assembly and the fluid pump respectively enter and exit the cell culture container. The system is configured to be driven to cause the transfer of a fluid. The apparatus includes a support plate, a valve actuator, a pump actuator, and a stirrer assembly. The stirrer assembly is configured to agitate the support plate. Within the closed system, a seeding container is connected to the container, pump, and valve assembly. The seeding container contains the cell sample. At least one of the pump or valve assembly is driven to transport a portion of the cell sample from the seeding container to the cell culture container, thereby seeding the cell sample into the cell culture container. While transporting a portion of the cell sample from the seeding container into the cell culture container, the stirrer assembly is driven to agitate the support plate and the cell culture tray assembly.

[0035] In some embodiments, a method is provided for counting cells in a cell culture system. The cell culture system includes a tray, a cell culture container connected to the tray, a holding container, a counting chip connected to the tray, and a pump. The cell culture container, holding container, counting chip, and pump are each sterile and connected to each other to form a closed system. The method includes driving the pump to transport a cell sample from the cell culture container to the holding container. The cell sample is mixed in the holding container by further driving the pump to transport a certain volume of air into the holding container. After mixing, the cell sample is transported from the holding container into the counting chip. The cell sample is analyzed in the counting chip to generate a cell signal related to the amount of cells in the cell sample.

[0036] In some embodiments, a method is provided for selectively removing cells from a cell culture system. The cell culture system includes a tray assembly and an instrument. The tray assembly includes a tray, a cell culture container connected to the tray, a sample container, a holding container, and a pump. The cell culture container, sample container, holding container, and pump are each sterile and connected to each other to form a closed system. The instrument includes a support plate to which the tray is detachably connected, a pump actuator, a stirrer assembly configured to agitate the support plate, and a cell sensor. The method involves driving the pump to transport a dissociation reagent from the sample container to the cell culture container. Driving the stirrer assembly to agitate the support plate and the tray assembly to facilitate the dissociation of a first portion of cells in the cell culture container. Receiving a sensor output from the cell sensor. The sensor output is related to a cell sample in the cell culture container. Based on the sensor output, a cell signal is generated that is related to at least one of the dissociation states of a first portion of cells in the cell culture container or a second portion of cells in the cell culture container. Based on cellular signals, a pump is activated to transport the first portion of the cells from the cell culture container to the holding container.

[0037] In some embodiments, a method for processing cells in a cell culture system is provided. The cell culture system includes a tray assembly and an instrument. The tray assembly includes a tray, a first container connected to the tray, a second container connected to the tray, a tangential flow filtration assembly, and a pump. The first container, the second container, the tangential flow filtration assembly, and the pump are each sterile and connected to each other to form a closed system. The instrument includes a support plate to which the tray is detachably connected, a pump actuator, and a cell sensor. The method includes receiving a sensor output from the cell sensor. The sensor output is related to a cell sample in the first container. Based on the sensor output, a cell signal is generated that is related to the state of the cells in the first container. The pump is driven to transport the cell sample from the first container into the tangential flow filtration assembly to generate a permeate output and an impermeate output. One of the permeate output or impermeate output is transported to the second container.

[0038] In some embodiments, the apparatus includes a tray, a first lid, a second lid, and a multiport valve. The tray is configured to be removably connected to the housing of the base unit. The tray has a first coupler configured to connect a first container to the tray and a second coupler configured to connect a second container to the tray. The first lid is configured to connect to the first container and includes a first liquid exchange port and a first gas exchange port. The second lid is configured to connect to the second container and includes a second liquid exchange port and a second gas exchange port. The multiport valve is connected to the tray and includes a master port and a set of selectable ports. The multiport valve engages with a valve actuator of the base unit and is configured to connect to a fluid pump connected to the base unit. The first selectable port of the set of selectable ports is aseptically connected to the first liquid exchange port of the first lid. The second selectable port of the set of selectable ports is aseptically connected to the second liquid exchange port of the second lid.

[0039] In some embodiments, a first coupler maintains a first container in a fixed position on the tray during the operation of the device, and a second coupler maintains a second container in a fixed position on the tray. In some embodiments, the first container is a cell culture container configured to receive cell samples, and the second container is one of a waste container, a reagent container, or a cell collection container. In some embodiments, the first coupler is configured to removably connect the cell culture container to the tray. In some embodiments, the cell culture container and the tray each include a transparent portion. The first coupler is configured to connect the cell culture container to the tray such that the transparent portion of the cell culture container aligns with the transparent portion of the tray.

[0040] In some embodiments, a multiport valve and a fluid pump are configured to transfer fluid between a first and second container within a closed sterile system. In some embodiments, the multiport valve is configured to be detachably connected to a tray and also to a valve actuator on a base unit. In some embodiments, the pump includes a pump actuator and a pump body defining a pump chamber. The pump body is configured to be connected to the master port of the multiport valve.

[0041] In some embodiments, the tray is configured to engage with a stirrer connected to a base unit. The stirrer is configured to agitate the tray when in operation.

[0042] In some embodiments, the apparatus includes a counting chip connected to a tray and to a third selectable port of a multiport valve. The counting chip is configured to receive a portion of the cell sample mixture from a first container at periodic time intervals.

[0043] In some embodiments, the tray, first lid, second lid, and multiport valve are sealed within a wrap. In some embodiments, the tray, first lid, second lid, and multiport valve are sterilized within the wrap.

[0044] In some embodiments, the base unit of the cell culture system includes a housing, a pump actuator, and a valve actuator. The housing defines (or includes) a receiving portion configured to removably receive a cell culture tray assembly. The cell culture tray assembly includes a tray, a first lid connected to the tray which can be removably connected to a first container, and a second lid connected to the tray which can be removably connected to a second container. The first and second lids each include a fluid exchange port and a gas exchange port. The cell culture tray also includes a multiport valve connected to the tray, which includes a master port and a set of selectable ports. The pump actuator is connected to the housing and configured to operably connect to a fluid pump connected to the master port of the multiport valve. The valve actuator is connected to the housing and configured to connect to the multiport valve when the cell culture tray assembly is connected to the receiving portion of the housing. The valve actuator and the pump actuator are collectively configured to selectively move fluid in and out of a first container connected to the first lid and in and out of a second container connected to the second lid.

[0045] In some embodiments, the multiport valve is configured such that it is removed from a tray and connected to a valve actuator, while the first port of the multiport valve is sterile-connected to a first lid and the second port of the multiport valve is sterile-connected to a second lid. In some embodiments, the valve actuator includes a keyed actuator material configured to mate and engage with the multiport valve.

[0046] In some embodiments, the fluid pump is sterilely connected to the master port of a multiport valve via a pipe of a certain length. In some embodiments, the fluid pump is one of a piston pump, a peristaltic pump, or a vane pump.

[0047] In some embodiments, the base unit further includes a stirrer connected to the housing and configured to engage with the cell culture tray assembly when the cell culture assembly is connected to the housing. The stirrer is configured to agitate the cell culture tray assembly when in operation. In some embodiments, the receiving portion of the housing includes a support plate connected to the stirrer. The support plate includes a surface to which the cell culture tray assembly can be removably connected.

[0048] In some embodiments, the base unit further includes (or is connected to) an electronic (or computer) control system configured to control the movement of fluids entering and leaving a first container connected to a first lid and a second container connected to a second lid. In some embodiments, the base unit includes a sensor movably connected to the housing and configured to generate a cellular signal relating to a certain amount of cells in the first container. In some embodiments, the sensor is an imaging device connected to the housing and configured to image the contents of the first container so that at least one of the cellular confluence or density in the first container can be determined. In some embodiments, the sensor is configured to monitor the color of the contents of the first container. The first container may include a color-based pH indicator so that the pH of the contents of the first container can be determined.

[0049] In some embodiments, the base unit of the cell culture system includes a housing, a pump actuator, a valve actuator, and an electronic control system. The housing defines a receiving portion configured to removably receive a cell culture tray assembly. The cell culture tray assembly includes a tray, a first lid connected to the tray which can be removably connected to a first container, and a second lid connected to the tray which can be removably connected to a second container. The cell culture tray also includes a multiport valve connected to the tray, which includes a master port and a set of selectable ports. The pump actuator is connected to the housing and configured to be operably connected to a fluid pump. The valve actuator is connected to the housing and configured to be connected to the multiport valve when the cell culture tray assembly is connected to the receiving portion of the housing. The valve actuator and the pump actuator are collectively configured to selectively move the fluid in and out of a first container connected to the first lid and in and out of a second container connected to a second lid. The electronic control system includes a cell sensor, a cell sensor module, and an actuator module. A cell sensor is configured to generate an output associated with the contents of a first container. The cell sensor module is implemented in at least one of the memory or processing devices of the electronic control system and generates a cell signal associated with a certain amount of cells in the first container based on the output of the cell sensor. The actuator module is implemented in at least one of the memory or processing devices and receives the cell signal and generates at least one of the valve control signal or pump signal based on the cell signal to move the cells out of the first container.

[0050] In some embodiments, the actuator module is configured to control the movement of a first volume of fluid from a first container to a waste container, and the movement of a second volume of fluid from a reagent container to the first container. In some embodiments, the actuator module is configured to control the movement of a certain volume of enzyme into the first container to facilitate cell dissociation of adherent cells in the first container.

[0051] In some embodiments, the apparatus includes a stirrer connected to the housing and configured to engage with the tray assembly when the tray assembly is connected to the receiving portion. The stirrer is configured to agitate the tray assembly. An actuator module of the electronic control system is configured to control the operation of the stirrer (e.g., when to agitate and for how long to agitate).

[0052] In some embodiments, the cell sensor is movably coupled to the housing. The sensor module is configured to control the movement of the cell sensor relative to the housing so that the cell sensor can align with the first container.

[0053] In some embodiments, the base unit includes a valve sensor configured to generate a valve position signal related to the rotational position of a valve actuator. The valve position signal indicates that one of the selectable ports of a multiport valve has been selected. The actuator module is configured to generate a valve control signal, partially based on the valve position signal. In some embodiments, the base unit includes a pump sensor configured to generate a pump signal related to the position of a pump actuator during operation. The actuator module is configured to generate a pump control signal, partially based on the pump signal.

[0054] In some embodiments, the electronic control system further includes a radio configured to communicate electronically with a computing device. The radio is configured to transmit radio signals to the computing device related to measurements relating to the amount of cells in a first container.

[0055] In some embodiments, the base unit of the cell culture system includes a housing, a pump actuator, a valve actuator, and an electronic control system. The housing defines a receiving portion configured to removably receive a cell culture tray assembly. The cell culture tray assembly includes a tray, a first cell culture container, a second cell culture container, a reagent container, a waste container, and a multiport valve. The multiport valve includes a master port and a set of selectable ports. The first selectable port is connected to the first cell culture container, the second selectable port is connected to the second cell culture container, the third selectable port is connected to the reagent container, and the fourth selectable port is connected to the waste container. The pump actuator is connected to the housing and configured to be operably connected to a fluid pump connected to the master port of the multiport valve. The valve actuator is connected to the housing and configured to be connected to the multiport valve. The electronic control system is operably connected to the valve actuator and the pump actuator. The electronic control system includes an actuator module implemented in at least one of memory or processing devices and configured to generate a series of valve control signals and pump control signals. Specifically, the actuator module can generate a first valve control signal to activate a multiport valve in a valve actuator, and a first pump control signal to activate a fluid pump in a pump actuator to move cell culture medium from a first cell culture container to a waste container. The actuator module can generate a second valve control signal to activate a multiport valve in a valve actuator, and a second pump control signal to activate a fluid pump in a pump actuator to move reagents from a reagent container to a first cell culture container. The actuator module can generate a third valve control signal to activate a multiport valve in a valve actuator, and a third pump control signal to activate a fluid pump in a pump actuator to move multiple cells from a first cell culture container to a second cell culture container.

[0056] In some embodiments, the electronic control system includes a cell sensor module implemented in at least one of a memory or processing device. The cell sensor module receives the output from the cell sensor and generates a cell signal indicating the dissociation of cells in a first cell culture vessel. The actuator module is configured to generate at least one of a third valve control signal or a third pump control signal in response to the cell signal. In some embodiments, the cell sensor is a microscope, and the output from the microscope is an image. The cell sensor module is configured to generate a cell signal indicating the dissociation of cells based on the image. In some embodiments, the cell sensor module is configured to generate an alignment signal to move the cell sensor to alignment with the first cell culture vessel.

[0057] In some embodiments, the base unit includes a stirrer connected to the housing and configured to engage with the tray assembly. The stirrer is configured to agitate the tray assembly. An actuator module of the electronic control system is configured to generate agitation signals that cause agitation of the tray assembly.

[0058] In some embodiments, the computer implementation method includes an electronic control system for a cell culture assembly receiving sensor outputs from sensors in the cell culture assembly. The cell culture assembly includes a disposable cell culture tray assembly that can be coupled to a reusable base unit. The cell culture tray assembly includes a tray, a first lid coupled to a first container, a second lid coupled to a second container, and a multiport valve coupled to the tray. The multiport valve includes a plurality of selectable ports and a master port coupled to a fluid pump. At least one of the first or second containers contains a plurality of cells. A cell signal associated with a certain amount of a plurality of cells in the first container and one of the second containers is generated based on the sensor output. Based on the cell signal, the electronic control system generates at least one of either a valve control signal to activate the multiport valve or a pump control signal to activate the fluid pump, thereby initiating the flow of fluid from at least one of the first containers.

[0059] In some embodiments, the sensor is part of an optical measurement assembly configured to move the sensor, and this method further includes transmitting a position signal to the optical measurement assembly to move the sensor to a measurement position relative to at least one of a first or second container. In some embodiments, the cell sensor is a microscope, and the sensor output from the microscope is an image. An electronic control system can generate a cell signal indicating the dissociation of cells in the first or second container based on the image.

[0060] In some embodiments, the base unit includes a stirrer operably coupled to the tray of the tray assembly. This method optionally includes transmitting a stirrer signal from an electronic control system to the stirrer to activate the stirring of the tray assembly to maintain the cells suspended in at least one of the first or second containers. In some embodiments, after transmitting the stirrer signal, this method includes transmitting at least one of the actuator signal or pump signal to cause a flow of a fluid mixture from one of the first and second containers to a counting chip fluid-coupled to one of the first and second containers.

[0061] In some embodiments, a computer-implemented method can control the movement of fluids within a cell culture assembly, including a disposable cell culture tray assembly connected to a reusable base unit. The method includes generating a first valve control signal and a first pump control signal via an actuator module of the electronic control system of the cell culture assembly. The first valve control signal causes a valve actuator in the base unit to actuate a multiport valve, fluidizing a first selectable port of the multiport valve to the master port of the multiport valve. The master port is fluidizing a fluid pump, and each selectable port is fluidizing one of a first cell culture container, a second cell culture container, a reagent container, or a waste container. The first pump control signal causes a pump actuator in the base unit to actuate a fluid pump, moving cell culture medium from the first cell culture container to the waste container. A second valve control signal is generated, causing a valve actuator to actuate a multiport valve, fluidizing a second selectable port to the master port, and the second pump control signal causes a pump actuator to actuate a fluid pump, moving reagents from the reagent container to the first cell culture container. A third valve control signal is generated, causing the valve actuator to activate a multi-port valve to fluidly connect a third selectable port to the master port, and a third pump control signal causes the pump actuator to activate a fluid pump to move multiple cells from the first cell culture container to the second cell culture container.

[0062] In some embodiments, the method includes generating a fourth valve control signal via an actuator module to actuate a multiport valve to a valve actuator and fluidly connect a fourth selectable port to a master port, and a fourth pump control signal to actuate a fluid pump to a pump actuator to move a cleaning medium from a cleaning container to one of the multiport valve, a retainer, a tube connected to the multiport valve, or a cell culture container.

[0063] In some embodiments, the base unit includes a cell sensor, and this method includes receiving an output from the cell sensor. A cell signal is generated indicating the dissociation of cells in a first cell culture vessel. The actuator module responds to the cell signal by generating at least one of a third valve control signal or a third pump control signal. In some embodiments, this method includes generating an alignment signal to move the cell sensor to align with the first cell culture vessel.

[0064] In some embodiments, a computer-implemented method can control the movement of fluid within a cell culture assembly based on measured or calculated values ​​of the volume of fluid in one or more containers. The cell culture assembly includes a disposable cell culture tray assembly coupled to a reusable base unit. The method includes generating a first valve control signal and a first pump control signal via an actuator module of the electronic control system of the cell culture assembly. The first valve control signal causes a valve actuator in the base unit to actuate a multiport valve, fluidizing a first selectable port of the multiport valve to the master port of the multiport valve. The master port is fluidizing a fluid pump. Each selectable port is fluidizing one of a cell culture container, a second cell culture container, or a cell culture medium container. The first pump control signal causes a pump actuator in the base unit to actuate a fluid pump, moving a first volume of cell culture medium from the cell culture medium container to the first cell culture container. The volume of liquid in the first cell culture container is determined. This method includes generating a second valve control signal and a second pump control signal via the actuator module when the fluid volume falls below a threshold volume. The second valve control signal causes the valve actuator to actuate the valve or, otherwise, maintain the fluid connection between the first selectable port and the master port of the multiport valve. The second pump control signal causes the pump actuator of the base unit to actuate the fluid pump to move a second volume of cell culture medium from the cell culture medium container to the first cell culture container. This method includes generating a third valve control signal and a third pump control signal via the actuator module when the fluid volume exceeds a threshold volume. The third valve control signal causes the valve actuator to actuate the multiport valve to fluidly connect the second selectable port of the multiple selectable ports to the master port of the multiport valve. The third pump control signal causes the pump actuator of the base unit to actuate the fluid pump to move multiple cells from the first cell culture container to the second cell culture container.

[0065] In some embodiments, the method includes removing the cell culture tray assembly from an outer protective wrap. The tray assembly includes a tray, a first lid, a second lid, and a multiport valve. The first lid is connected to the tray and configured to be removably connected to a first container. The first lid includes a first fluid exchange port and a first gas exchange port. The second lid is connected to the tray and configured to be removably connected to a second container. The second lid includes a second fluid exchange port and a second gas exchange port. The multiport valve is connected to the tray and includes a master port and a plurality of selectable ports. The first selectable port of the plurality of selectable ports is aseptically connected to the first fluid exchange port of the first lid, and the second selectable port of the plurality of selectable ports is aseptically connected to the second fluid exchange port of the second lid. At least one cell is added to the first container through the opening of the first container. The first lid is secured to the first container and closes the opening. The tray assembly is connected to the base unit. The valve actuator of the base unit engages with the multi-port valve of the tray assembly after the tray assembly is connected, or simultaneously with the connection of the tray assembly to the base unit. The fluid pump is connected to the pump actuator of the base unit.

[0066] In some embodiments, this method includes connecting a tray assembly, connecting a fluid pump, and then moving the base unit together with the connected tray assembly into an incubation environment. In some embodiments, this method includes removing a multiport valve from the tray assembly and connecting the multiport valve to the base unit so that the valve actuator of the base unit mates and engages with the multiport valve. In some embodiments, removing the multiport valve is performed while the first selectable port of the multiport valve is aseptically connected to the first lid and the second selectable port of the multiport valve is aseptically connected to the second lid. In some embodiments, removal, addition, and fastening are performed in a sterile environment. In some embodiments, a certain volume of reagent and at least one cell are added to the first container before fastening the first lid to the first container. In some embodiments, after fastening the first lid to the first container, the first container is connected to the coupler of the tray assembly. In some embodiments, this method further includes connecting a fluid pump to the ports of the multiport valve via a tube. In some embodiments, connecting a fluid pump to a multiport valve includes connecting the master port of the multiport valve to the fluid pump via a pipe.

[0067] When used in relation to a referenced numerical representation, the term “approximately” means the referenced numerical representation plus or minus up to 10%. For example, “approximately 100” means between 90 and 110. When used in relation to geometric relationships, numbers, and / or ranges, for example, the term “substantially” is intended to carry that the geometric relationship (or the structure described thereby), number, and / or range thus defined is nominally the listed geometric relationship, number, and / or range. For example, two structures described herein as “substantially parallel” are intended to carry that a parallel geometric relationship is desirable, but some non-parallelism may occur in a “substantially parallel” arrangement. As another example, a structure that defines a volume that is “substantially 0.50 milliliters (mL)” is intended to carry that the stated volume is desirable, but some tolerance may occur if the volume is “substantially” the stated volume (e.g., 0.50 mL). Such tolerances may arise from manufacturing tolerances, measurement tolerances, and / or other practical considerations (e.g., minute defects, the age of the structure as defined, pressure or force applied within the system, etc.). As stated above, appropriate tolerances may be, for example, ±10% of the stated geometric structure, numerical values, and / or range.

[0068] As used herein, the term “reagent” includes any substance used in connection with any of the reactions described herein. For example, a reagent may include buffers, enzymes, cell culture media, washing solutions, etc. A reagent may include a mixture of one or more components. A reagent may include such components regardless of the state of matter (solid, liquid, gas, etc.). Furthermore, a reagent may include multiple components that may be present in a mixed state, an unmixed state, and / or a partially mixed state. A reagent may include both active and inactive components. Thus, as used herein, a reagent may include inactive and / or inactive components such as water, colorants, etc.

[0069] As used herein, the term “set” can refer to a single feature having multiple features or multiple parts. For example, when referring to a set of walls, the set of walls can be considered as one wall having multiple parts, or as multiple separate walls. Thus, an item constructed monolithically may include a set of walls. Such a set of walls may include multiple parts that are continuous or discontinuous with respect to each other, for example. A set of walls may also be manufactured from multiple items that are manufactured separately and then joined together (for example, by welding, gluing, or any suitable method).

[0070] Figure 1A shows a schematic diagram of an automated cell culture system according to an embodiment. This exemplary automated cell culture system 100 has three cell culture containers 111, 113, and 115. These containers may be, for example, laboratory flasks or dishes. The cell culture containers hold cell cultures, growth media, and any other additives or reagents related to cell culture. The cell cultures in the containers may be any kind of adherent or suspended cell cultures.

[0071] The fluid pumps 103 and 105 are one-port fluid pumps that include an internal fluid reservoir. An example of a one-port fluid pump is a syringe fitted to a syringe driver. A syringe fluid pump can draw fluid into its internal reservoir by creating an suction force on the reservoir by pulling out the plunger of the syringe. Similarly, a syringe pump can push liquid out of the reservoir by pushing the plunger back into the syringe. In other embodiments, one or both of the fluid pumps 103, 105 may include a bidirectional inline pump with a separate reservoir. A bidirectional pump may be, for example, a peristaltic pump or an impeller-based fluid pump that can pump fluid in two directions along a fluid channel. A bidirectional inline pump may have one end fitted to a dedicated reservoir and the other end used as an input and output port in a similar manner to a syringe pump. A dedicated reservoir fitted to the pump may be flexible and sealed to prevent the formation of air pockets within the reservoir when the fluid is pumped out of the reservoir, such as a bag or pouch.

[0072] Fluid pumps 103 and 105 are fluidly connected to multiport valves 107 and 109, respectively. Multiport valves 107 and 109 have one master port and a number of selectable ports. The multiport valve can selectively fluidize the master port to one of the selectable ports at a time. When the master port of the multiport valve is connected to the selected port, the other selectable ports are sealed and not fluidly connected to the master port. When the master port of the multiport valve is fluidly connected to a selectable port, the fluid can flow through the valve in either direction. That is, the fluid may flow into the multiport valve through the master port and out through the selected port, or the fluid may flow in the opposite direction, flowing into the multiport valve through the selected port and out through the master port. In some embodiments, the multiport valve may be a mechanical valve device, and in other embodiments, the multiport valve may consist of microfluidic chip components.

[0073] The fluid pumps 103 and 105, the multiport valves 107 and 109, and the cell culture vessels 111, 113, and 115 are all fluidly connected to each other by fluid channels. In some embodiments, the fluid channels consist of flexible tubes. In other embodiments, some or all of the fluid channels may be rigid tubes or channels in a substrate. In the example shown in Figure 1A, the fluid pump 103 is fluidly connected to the master port of the multiport valve 107 by a flexible tube. The multiport port 107 has several selectable ports 107a to d. Selectable port 107a is fluidly connected to the cell culture vessel 111, selectable port 107b is fluidly connected to the cell culture vessel 113, selectable port 107c is fluidly connected to the cell culture vessel 115, and selectable port 107d is fluidly connected to the vessel 119. Container 119 can be any type of fluid container for supplying fluid to or receiving fluid from an automated cell culture system. For example, container 119 could be a waste container for receiving waste from an automated cell culture system. In another example, container 119 could contain fresh cell culture medium for supplying fresh culture medium to a cell culture vessel.

[0074] The fluid pump 105, multiport valve 109, and container 117 are configured similarly to the fluid pump 103, multiport valve 107, and container 119. The multiport 109 has several selectable ports 109a to d. Selectable port 109a is fluidly connected to the cell culture vessel 111, selectable port 109b is fluidly connected to the cell culture vessel 113, selectable port 109c is fluidly connected to the cell culture vessel 115, and selectable port 109d is fluidly connected to the container 117.

[0075] During operation, the combination of fluid pumps, multiport valves, containers, and cell culture vessels in the example shown in Figure 1A can be used to transfer fluid between the cell culture vessels and containers. In some embodiments, a first fluid pump 103 is used to add culture medium from container 119 to the cell culture vessel, and a second fluid pump 105 is used to remove culture medium from the cell culture vessel to container 117. In another embodiment, a single fluid pump is used for both adding to and removing from the cell culture vessels and containers. In some embodiments, the components of group 101, including cell culture vessels 111, 113, 115 and multiport valves 107 and 109, may be separable from the fluid pumps 103 and 105 and containers 117 and 119. Fluid connections between components within group 101 may be established independently in a first stage of assembly, after which additional components may be connected in a later stage. The components of group 101 may be sterilized or treated independently in a first stage and then introduced into the remaining components in a second stage. Fluid connections between components of group 101 and other components can be made using sterile connections to prevent contaminants from being introduced into the sterilized components of group 101. Cell culture vessels 111, 113, and 115 can be connected to valves 107 and 109 using tubing and sterile connections, so that the vessels can be sterilely detached from the system when removing cells from the vessels for use or analysis.

[0076] Figure 1B shows a schematic diagram of an automated cell culture system according to an embodiment. The automated cell culture system 110 includes one bidirectional fluid pump 121. In this embodiment, the cell culture vessels 111, 113, 115, multiport valves 107 and 109, and containers 117 and 119 are the same as those described in relation to Figure 1A. In Figure 1B, the fluid pump 121 is a two-port fluid pump, such as a peristaltic pump. The first port 121a of the two-port fluid pump 121 is fluidly connected to the master port of the multiport valve 107, and the second port 121b of the fluid pump 121 is fluidly connected to the master port of the multiport valve 109. The fluid pump 121 can pump fluid in two directions. In the first operating mode, the fluid pump 121 pumps fluid from port 121a to port 121b, and in the second operating mode, the fluid pump 121 pumps fluid from port 121b to port 121a.

[0077] Figure 2 shows a top view of an automated cell culture system according to an embodiment. The automated cell culture system 200 has two fluid pumps, two multiport valves, and 12 cell culture containers. For simplicity, the illustrated example does not include fluid connections, but it should be understood that at least some of the various components of the automated cell culture system will be fluid-connected when in use. The removable tray 223 contains cell culture containers 201-212 and multiport valves 213 and 215. Each cell culture container is covered by a sterile lid, such as a sterile lid 237 that covers cell culture container 206. Each cell culture container is removablely mounted to the removable tray 223 by brackets such as brackets 217, 219, and 221 that hold the cell culture container 206. The removable tray 223 is removablely inserted into the base housing 235 and guided by guides 225a-f. The base housing 235 contains two syringe-style fluid pumps. The first fluid pump consists of a syringe 229 and a syringe actuator 227. The syringe actuator 227 pushes and pulls the plunger of the syringe 229 to produce a flow of fluid in and out of the syringe. In this embodiment, the syringe actuator is a linear actuator, but any other method of pushing and pulling the syringe plunger can be used. The second pump consists of a syringe 233 and a syringe actuator 231.

[0078] Figure 3A shows a top view of the base housing of an automated cell culture system according to an embodiment. The illustrated exemplary base housing 301 includes fluid pumps 305 and 307 and multiport valve actuators 309 and 311. The base housing 301 also includes a controller that controls the operation of the fluid pumps, multiport valves, and any other system, such as an automated cell counter system, hemocytometer, imaging system, microscope, or other measurement or analysis system for facilitating automated cell growth. The controller may include one or more processors configured to execute instructions contained in one or more memory systems in order to control the automated cell culture system and other corresponding systems. Furthermore, the controller may include one or more network interfaces from which various notifications or data transfers can be sent or received.

[0079] Figure 3B shows a removable tray assembly of an automated cell culture system according to an embodiment. The removable tray assembly 303 is configured to fit into the base housing 301. When the removable tray assembly 303 is positioned on top of the base housing 301, the multiport valve actuators 309 and 311 are mechanically connected to the multiport valves 319 and 321, respectively. For example, in the embodiment, the multiport valve actuator 309 rotates an internal component of the multiport valve 319 to align the master port of the multiport valve 319 with one of the selectable ports 319a to d. The multiport valves 319 and 321, as well as the cell culture containers 313, 315, and 317, are carried onto the removable tray 303. When the base housing 301 and the removable tray 303 are combined, fluid pumps 305 and 307 may be fluidly connected to the master ports of the multiport valves 319 and 321.

[0080] In some embodiments, the base housing 301 may also include a stirrer configured to agitate a removable tray assembly 303 in relation to the base housing. This stirrer can agitate the tray with rocking, vibrating motion, circular swirling motion, or other motion useful for cell culture. In some embodiments, individual cell culture containers may be agitated independently by a separate stirrer positioned between the cell culture container and the removable tray. The separate stirrer can be used in applications where agitating all cell culture containers on the tray is disadvantageous, such as when agitation is required for only a subset of cell culture containers. In some embodiments, the separate stirrer may be integrated into one or more brackets used to secure the cell culture container to the removable tray. In some embodiments, the stirrer may have an active component located within the base housing that mechanically connects to a passive component on the removable tray, similar to how a multiport valve on the removable tray mechanically engages with an actuator in the base housing.

[0081] During use, the removable tray 303 can be configured separately from the base housing 301 with any number or configuration of multiport valves, cell culture vessels, and fluid tubing, as needed. The removable tray 303 and its associated components can then be sealed and sterilized before being introduced into the base housing 301. In some embodiments, cell culture vessels can be added to the tray 303 in a sterile environment after sterilization. Since the base housing 301 can remain fixed and its components do not have fluid contact with the sterilization system on the removable tray 303, any electromechanical components, such as valve actuators or pump mechanisms, located within the base housing do not need to undergo transport or sterilization procedures. When using a syringe-style fluid pump, a sterile syringe can be placed in the syringe actuator and used so that the syringe actuator does not come into contact with any fluid in the sterile system. Similarly, a peristaltic pump can use the sterile portion of tubing so that the fixed components associated with the base housing do not come into fluid contact with the sterile system.

[0082] Figure 4 shows an exemplary removable tray fitted into an exemplary base housing according to an embodiment of the automated cell culture system. As shown in this example, the automated cell culture system 400 includes a removable tray 401 and a base housing 403. The removable tray 401 includes multiport valves 405 and 407 and cell culture containers 409, 411, and 413. The removable tray 401 is lowered onto the base housing 403, and the multiport valve actuators 415 and 417 are aligned with the multiport valves 405 and 407, respectively. Once the removable tray 401 is lowered onto the base housing 403, the multiport valve actuators 415 and 417 mechanically connect to the multiport valves 405 and 407. After the two components are connected, the fluid pumps 419 and 421 are fluidly connected to the multiport valves 405 and 407 mounted on the removable tray, such as by a manual connection step.

[0083] Figure 5 shows a cross-sectional view of an exemplary multiport valve according to an embodiment. In this embodiment, the multiport valve 500 comprises a valve body 503 having a master port 507 on its upper surface, and a plurality of selectable ports 505 and 509 distributed around it. Two selectable ports are shown in this cross-sectional view. However, it should be understood that various embodiments of the multiport valve may include any number of selectable ports.

[0084] The valve body 503 has a cylindrical cavity on its underside into which a rotatable cylindrical valve rotor 501 is inserted. Within the rotatable cylindrical valve rotor 501 is a fluid channel 517 that fluidly connects the axial master port of the rotatable cylindrical valve rotor 501 to the radial master port of the rotatable cylindrical valve rotor 501. Within the valve body 503 is a fluid channel 513 that fluidly connects the master port 507 to the fluid channel 517 of the rotatable cylindrical valve rotor 501. The connection between fluid channel 513 and fluid channel 517 remains constant while the rotatable cylindrical valve rotor 501 rotates because both fluid channels are centered on the axis of rotation of the rotatable cylindrical valve rotor 501 within the cylindrical cavity of the valve body 503.

[0085] In the configuration shown in Figure 5, the rotatable cylindrical valve rotor 501 is rotated so that fluid channel 511 aligns with fluid channel 517. Thus, the fluid circuit is established from the master port 507 through fluid channels 513, 517, and 511 to the selectable port 505. In this illustrated configuration, fluid channel 515 and then the selectable port 509 are sealed by the presence of the solid portion of the rotatable cylindrical valve rotor 501. During operation, the rotatable cylindrical valve rotor 501 can rotate to establish a fluid path from the master port 507 to the selectable port 509 while sealing the selectable port 505 and fluid channel 511.

[0086] The multiport valve 500 can be made of any suitable material, and the valve body 503 and valve rotor 501 can be made of the same or different materials. Examples of materials that can be used include plastics, TFE-based materials such as polytetrafluoroethylene (PTFE), metals, rubber, or similar materials. In some embodiments, the valve body 503 and valve rotor 501 may be machined to fit with very close tolerances, resulting in a liquid-tight seal between the two components. In some embodiments, additional gaskets, bearings, seals, and / or flanges may be incorporated into the multiport valve 500 to provide a liquid-tight connection between the valve body 503 and the valve rotor 501.

[0087] Figure 6A shows an exemplary example of a multiport valve according to an embodiment. In this example, the multiport valve 600 has an axial port 601 and eight selectable ports, four of which (ports 603, 605, 607, and 609) can be seen in the perspective view of Figure 6A. Figure 6B shows a bottom view of the multiport valve 600 showing a mechanical coupler 611 configured to mechanically connect to a multiport valve actuator. The corresponding multiport valve actuator has a cavity shaped to receive the mechanical coupler 611 and to transfer rotational mechanical energy to the multiport valve 600.

[0088] Figure 7 shows a lid for a sterile cell culture vessel according to an embodiment. In this exemplary embodiment, the cell culture vessel lid 703 is attached to the cell culture vessel 701. In this exemplary embodiment, the cell culture vessel lid 703 has three ports 705, 707, and 709. In this example, the three ports are arranged vertically. When the cell culture vessel 701 is filled with a liquid such as cell growth medium, the tube entering through the lowest port 709 may be submerged in the liquid, and the tube can be used to draw up the liquid through port 709. The tube entering through the central port, port 707, can be positioned so that the tube does not come into contact with the liquid contents of the cell culture vessel, and as a result, additional liquid can be added to the cell culture vessel without contaminating the fluid pathway to port 707. Port 705 may be configured to allow gas exchange in and out of the cell culture vessel 701. In some embodiments, port 705 includes a filter for filtering the gas on its way into the flask to sterilize it. In some applications, an automated cell culture system can be placed in an incubation chamber to regulate the environment adjacent to the cell culture vessel. In some embodiments, the incubation chamber can be integrated with the base housing of the automated cell culture system. In one embodiment, the regulated environmental characteristics include gas mixture, temperature, and humidity levels. In one embodiment, the incubation chamber regulates the gas mixture, temperature, and humidity levels depending on the cell line being grown. In some embodiments, port 705 can be attached to an environmental control device that manages the temperature, humidity, oxygenation, gas mixing, and other such parameters of the gas environment inside the cell culture vessel. A sterile lid can be fabricated to fit any cell culture vessel so that any incubator used in manual cell culture can be integrated with the system.

[0089] Figure 8 shows a cross-sectional view of the lid of a cell culture vessel according to an embodiment. The lid 803 of the cell culture vessel is screwed onto the opening of the cell culture vessel 801 such that the threads of the lid 803 engage with the threads of the opening of the cell culture vessel 801. In this exemplary embodiment, the cell culture lid 803 has a liquid port 807 and a gas port 811. The liquid channel 809 is screw-engaged with the liquid port 807. The gas filter 805 is screw-engaged with the gas port 811. The gas filter 805 can enable gas exchange inside and outside the cell culture vessel while preventing any microorganisms or pathogens from entering the cell culture vessel from the outside. In this embodiment, the gas filter 805 is a 0.22 micron filter.

[0090] Figure 9 illustrates the steps of a method for transferring liquid from a first container to a second container using an automated cell culture system equipped with a single-port pump, according to an embodiment. In this example, the automated cell culture system has a single-port pump, such as the syringe-type pump described above, or a two-port pump with a holder attached to one port. This method can be used to transfer liquid from any container to another. For example, the first container may be a cell culture container and the second container may be a waste container. In another example, the first container may be a container of fresh cell growth medium and the second container may be a cell culture container.

[0091] In Figure 9, in step 901, a multiport valve with a master port connected to a single-port pump or a two-port pump with a retainer is configured to select a selectable port to communicate fluid with the first vessel. In step 902, the single-port pump is operated so that fluid is drawn from the first vessel into the reservoir of the single-port pump, or similarly, the two-port pump is operated so that fluid is drawn into the retainer. Next, in step 903, the multiport valve is configured to select a selectable port to communicate fluid with the second vessel. Next, in step 904, the fluid is pumped from the reservoir of the single-port pump or similarly, from the retainer by the two-port pump through the configured multiport valve to the second vessel.

[0092] Some embodiments of automated cell culture systems can use a two-port pump with a multi-port valve to which fluid is connected at each port. The two-port pump may be unidirectional or bidirectional. A two-port pump does not need to transfer the fluid to a holding reservoir like a single-port pump, but can pump directly from one vessel to another. Figure 10 shows the steps of a method for transferring fluid from a first vessel to a second vessel using an automated cell culture system with a two-port pump, according to an embodiment. In this example, the first port of the two-port pump is fluid-connected to the master port of the multi-port valve, and the second port of the two-port pump is fluid-connected to the master port of the multi-port valve. In step 1001, the first multi-port valve is configured to select a selectable port to fluidize the first vessel. In step 1002, the second multi-port valve is configured to select a selectable port to fluidize the second vessel. Finally, in step 1003, the two-port pump is operated to pump in the direction of the first port toward the second port, so that the liquid from the first container is pumped into the second container.

[0093] In any embodiment disclosed herein, a simple reference to pumping from a first vessel to a second vessel may be referred to as an alternative to a suitable method, depending on whether the automated cell culture system consists of a one-port pump or a two-port pump. Some embodiments of the automated cell culture system may also combine a two-port pump and a single-port pump in one system, so that one step of pumping uses one type of pump and another step of pumping uses a different type of pump.

[0094] In some embodiments, for example, when signs of differentiation are observed in stem cells, culture media from different sources may be supplied to the cells depending on the observed state of the cells. In embodiments, the first step of the method is to observe the state of the cells, such as signs of differentiation in stem cells. The first step may be performed by a microscope, camera, or other measuring device. The second step of the method is to select an appropriate culture media source based on the state of the cells. The third step of the method is to activate a one-port or two-port pump system to transfer the culture media from the selected culture media source to a container containing the cells.

[0095] In some embodiments, the automated cell culture system includes a microscope that can be moved to image the contents of any cell culture vessel in the automated cell culture system. In some examples, the microscope may be mounted on a mechanical system that can move the microscope to the cell culture vessel, such as a two-dimensional or three-dimensional gantry mechanism or a hinged robotic arm mechanism. In some embodiments, the microscope may remain fixed while the automated cell culture system is moved to position individual cell culture vessels from the viewpoint of a fixed microscope. In some embodiments, the microscope and movable assembly may be contained within the base housing of the automated cell culture system, as a result, the cell culture vessels may be imaged from below. In such embodiments, a removable tray holding the cell culture vessels may have a transparent window or notch beneath the cell culture vessel to allow the microscope to image the cells contained therein. In some embodiments, an adjustable and controllable light source is positioned on the opposite side of the cell culture vessel as a microscope to supply light to the microscope. For example, the light source may be mounted on a mechanical system that can move the light source to any cell culture vessel as needed, as well as the microscope. In some embodiments, a fixed light source may be positioned on one side of the automated cell culture system so that each cell culture vessel is adequately illuminated.

[0096] The automated cell culture system may also include other imaging devices. For example, the automated cell culture system may include one or more cameras, or a pair of LEDs and light sensors, to image the contents of the cell culture containers. This type of imager may be useful for measuring and monitoring the macroscopic visual characteristics of the cell culture containers. For example, a color camera, or a pair of LEDs and light sensors, may be useful for monitoring the color of the contents of a cell culture container containing a color-based indicator, such as phenol red, from which the pH of the contents of the cell culture container can be determined. In an embodiment, each cell culture container bracket may include a camera for imaging the contents of the cell culture container. In another embodiment, a single camera may be mechanically movable to each cell culture container in the same or similar manner as a microscope can be moved to image each cell culture container. In an embodiment, LEDs and light sensors may be mechanically movable to each cell culture container in the same or similar manner as a microscope can be moved to monitor the color of the cell culture containers.

[0097] In some exemplary implementations, one or more tray-detached devices may interface with an automated cell culture system. For example, an automated cell counter may be fluidized to a selectable port of a multiport valve so that a sample of the contents of a cell culture container can be delivered to the automated cell counter. In some embodiments, the automated cell counter may be controlled by a controller so that the entire process of counting cells in the automated cell counter is automated by the automated cell culture system. As a further example, a cell counting chamber may be fluidized to a selectable port of a multiport valve so that a sample of the contents of a cell culture container can be delivered to the cell counting chamber. A microscope can image the cell counting chamber to count the cells within it. As a further example, an external chamber may be fluidized to a selectable port of a multiport valve so that a sample of the contents of a cell culture container can be delivered to the external chamber. An LED and a light sensor can be used to measure the condensation of the solution in the external chamber. As a further example, to collect a cell sample, a sampling device can be sterilely connected to the port of a multiport valve, a sample of the contents of a cell culture vessel can be transported to the vessel, and then the vessel can be sterilely cut open to collect the cells.

[0098] Some operations in automated cell culture systems may require various support methods or procedures. For example, liquid lines or pumps may need to be primed before pumping liquid through the line. For instance, a fluid line from a bottle of new growth medium to a multi-port valve may need to be primed before pumping the new growth medium into the cell culture vessel. To do this, a small amount of new growth medium can be pumped from the new growth medium bottle into the waste bottle, ensuring there are no air pockets in the line.

[0099] Similarly, lines, pumps, or valves may need to be cleaned or washed periodically to remove contaminants. This can be achieved by running a cleaning solution through the lines, pumps, or valves for a certain period of time, or by pumping until the lines, pumps, or valves are thoroughly flushed out.

[0100] Figure 11 shows the steps of a method for maintaining adherent cell lines. In step 1101, the used cell culture growth medium in the container is pumped out of the container into a waste container. In step 1102, a determined amount of new cell culture growth medium is pumped into the container.

[0101] Figure 12 shows the steps of a method for maintaining or growing adherent cell lines with passaging to a new cell culture vessel. In contrast to the method discussed in relation to Figure 11, here adherent cells in a cell culture vessel are transferred to a new vessel. In step 1201, the cell culture growth medium in the vessel is pumped from the vessel into a waste container. Next, in step 1202, a washing solution is pumped into the vessel, and in step 1203, the vessel may be optionally agitated. Then, in step 1204, the washing solution is pumped from the vessel into a waste container.

[0102] In step 1205, the dissociation reagent is pumped into the vessel. An example of a dissociation reagent is trypsin. The dissociation reagent is used to resuspend cells adhered to the wall of the cell culture vessel. Depending on the cells being cultured and the dissociation reagent used, the cell culture vessel may be gently agitated to help separate the adherent cells from the wall of the cell culture vessel. The automated cell culture system then waits for a time configurable in step 1206, depending on the cells being cultured and the dissociation reagent used. In an alternative embodiment, the automated cell culture system dynamically monitors the dissociation of cells from the vessel with a microscope to determine when the amount of dissociation reaches a threshold. The vessel may optionally be agitated while waiting in step 1206. In step 1207, optionally, the cells are imaged to observe the detachment of adherent cells. If the cells have not detached sufficiently, the automated cell culture system may wait for a further time. Once the adherent cells have sufficiently detached from the cell culture vessel walls, the action of the dissociation reagent can be stopped by pumping a dissociation reagent inhibitor or neutralizer into the cell culture vessel. In step 1208, the contents of the cell culture vessel may optionally be removed from the automated cell culture system and rotated inside a centrifuge to separate the cells from the liquid contents of the cell culture vessel, and then resuspended. In step 1209, the cells may be counted to determine the total number of cells or the cell density and viability. In step 1210, some of the cells are transferred to a new cell culture vessel. Next, in step 1211, the determined amount of new growth medium is pumped into the new vessel. If the automated cell culture system is configured only for cell line maintenance, the original cell culture vessel may be removed from the system and discarded, so that only the new vessel remains in the cell growth system. If the automated cell culture system is configured for cell line growth, the original vessel may be retained, and a proportional amount of new growth medium may be added to it so that both the original cell culture vessel and the new cell culture vessel remain in the cell growth system. Although this is explained in the context of using a single new container, it should be understood that this process can be extended to any number of containers, just as the single original container can be divided into any number of new containers.

[0103] Figure 13 shows the steps of a method for maintaining a suspension cell line by optional passaging. In step 1301, the cell culture vessel is gently agitated to uniformly disperse the cells within the growth medium. Next, in step 1302, the cells in the vessel are counted, and in step 1303, the optimal amount of new growth medium is determined based on the cell number or cell density. In step 1304, the final volume of fluid in the cell culture vessel is determined after adding the determined amount of new growth medium. Each time fluid is added to the cell culture vessel by the procedure, the amount of fluid added is recorded and tallied by the controller. In this way, the controller maintains the current value of the amount of fluid in each cell culture vessel. In step 1305, the estimated final volume of fluid in the cell culture vessel is compared to the maximum volume configured for the particular cell culture vessel being used. For example, the total volume of the vessel cannot exceed the total capacity of the vessel. In some embodiments, the threshold maximum volume may be significantly less than the total volume of the vessel. If the estimated final fluid volume is lower than the configured threshold, in step 1306, the determined amount of new culture medium is added to the container. If the estimated final fluid volume is greater than the configured threshold, the automated cell culture system can divide the contents of the cell culture container into two or more cell culture containers to accommodate the estimated final fluid volume. In this exemplary method, the contents of the cell culture container now called the first cell culture container are divided between the first cell culture container and an additional second cell culture container. In step 1307, a portion of the contents of the first cell culture container can be transferred to the second cell culture container. The proportion of the contents of the first and second cell culture containers is recorded by the controller. Next, in step 1308, a proportional amount of new cell culture growth medium is added to each of the first and second cell culture containers, proportional to the amount of final fluid volume each contains. For example, if the fluid contents of the first cell culture container are evenly divided between the first and second cell culture containers, the new culture medium will similarly be evenly divided between the first and second cell culture containers.

[0104] Figure 14 shows the steps of a method for growing a suspension cell line. The method for growing a suspension cell line reflects a method for maintaining a suspension cell line, but in step 1407, the contents of a cell culture container can be transferred to a new cell culture container even if the total volume remains below the total volume threshold of the cell culture container. That is, cells can be transferred to a new cell culture container not only in response to a lack of volume in the cell culture container, but also if it is appropriate to promote cell proliferation.

[0105] Figure 15 shows an exemplary machine of a computer system in which a set of instructions can be executed to cause the machine to perform one or more of the methodologies discussed herein. In alternative implementations, the machine may be connected to (e.g., networked) other machines in a LAN, intranet, extranet, and / or internet. The machine may operate as a server or client machine in a client-server network environment, as a peer machine in a peer-to-peer (or distributed) network environment, or as a server or client machine in a cloud computing infrastructure or environment.

[0106] A machine can be a personal computer (PC), tablet PC, set-top box (STB), personal digital assistant (PDA), mobile phone, web appliance, server, network router, switch, or bridge, or any machine capable of executing a set of instructions (sequential or otherwise) that specify the actions to be performed by such machine. Furthermore, although a single machine is shown, the term “machine” should also be interpreted to include any collection of machines that individually or collectively execute a set (or set) of instructions for performing one or more of the methodologies discussed herein.

[0107] An exemplary computer system 1500 includes a processing device 1502, main memory 1504 (e.g., read-only memory (ROM), flash memory, dynamic random access memory (DRAM), e.g., synchronous DRAM (SDRAM) or Rambus DRAM (RDRAM)), static memory 1506 (e.g., flash memory, static random access memory (SRAM)), and data storage device 1518, which communicate with each other via a bus 1530.

[0108] The processing device 1502 represents one or more general-purpose processing devices, such as a microprocessor or a central processing device. More specifically, the processing device may be a composite instruction set computing (CISC) microprocessor, a reduced instruction set computing (RISC) microprocessor, a very long instruction word (VLIW) microprocessor, or a processor implementing another instruction set, or a processor implementing a combination of instruction sets. The processing device 1502 may also be one or more application-specific processing devices, such as an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), a digital signal processor (DSP), or a network processor. The processing device 1502 is configured to execute instructions 1526 for performing the operations and steps discussed herein.

[0109] The computer system 1500 may further include a network interface device 1508 for communication via a network 1520. The computer system 1500 may also include a video display unit 1510 (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)), an alphanumeric input device 1512 (e.g., a keyboard), a cursor control device 1515 (e.g., a mouse), a graphics processing unit 1522, a signal generation device 1516 (e.g., a speaker), another graphics processing unit 1522, a video processing unit 1528, and an audio processing unit 1532.

[0110] The data storage device 1518 may include a machine-readable storage medium 1524 (also known as a computer-readable medium) in which one or more sets of instructions or software 1526 that embody any one or more of the methods or functions described herein are stored. The instructions 1526 may also be entirely or at least partially present in the main memory 1504 and / or the processing device 1502 during their execution by the computer system 1500, and the main memory 1504 and the processing device 1502 also constitute a machine-readable storage medium.

[0111] In one embodiment, instruction 1526 includes instructions for performing functions corresponding to components of a device for performing the disclosures herein. Although the machine-readable storage medium 1524 is shown to be a single medium in an exemplary implementation, the term “machine-readable storage medium” should be understood to include a single or multiple mediums (e.g., concentrated or distributed databases, and / or associated caches and servers) that store one or more sets of instructions. The term “machine-readable storage medium” should also be understood to include any medium that can store or encode a set of instructions for execution by a machine, causing the machine to perform one or more of the methodologies of this disclosure. Accordingly, the term “machine-readable storage medium” should be understood to include, but is not limited to, solid-state memory, optical media, and magnetic media.

[0112] Figures 16A–16C show schematic diagrams of an automated cell culture system according to another embodiment. This exemplary automated cell culture system 1600 includes a consumable or disposable cell culture tray assembly 1601 (also referred to herein as the “tray assembly,” see Figure 16A) and a reusable base unit 1620 (see Figure 16B). The disposable tray assembly 1601 includes various components described below, some of which are pre-assembled on (or together with) the tray assembly 1601 and sealed within a protective overlap to maintain the components in a sterile state. Some of the components of the tray assembly 1601 can be added to the tray assembly 1601 in a sterile environment (e.g., a laminar flow hood) before using the tray assembly 1601 in a cell culture procedure. Once the tray assembly 1601 is assembled and ready for use, it can be coupled to the base unit 1620, as described in more detail herein.

[0113] As shown in Figure 16A, the tray assembly 1601 includes a tray 1602 which can be removably coupled to a base unit 1620 as described herein. In some embodiments, the tray 1602 may include one or more transparent or notched portions, so that an object placed on the top surface of the tray 1602 can be seen from below. For example, as will be described in more detail below, the cell culture system 1600 may optionally include imaging devices and / or other sensors located in the base unit 1620 and under the tray 1602 when the tray assembly 1601 is coupled to the base unit 1620. The transparent portions or notches may allow images and / or other data to be acquired through the transparent portions or notches, such as the contents of a cell culture container coupled to the tray 1602, as will be described in more detail below. In some embodiments, the tray assembly 1601 may include a cell counting chip 1617 as shown in Figure 16A. The cell counting chip 1617 may also include a bottom transparent section which can be used to obtain information about the contents of a cell culture container, as described below. In some embodiments, the cell counting chip 1617 may be coupled to or mounted within a base unit 1620 instead of being pre-assembled on a tray assembly 1601.

[0114] The tray assembly 1601 also includes one or more couplers 1603 that can be used to hold cell culture containers or vessels. The tray 1602 optionally includes holders 1604 that can be used to removably connect reagent containers 1605 and waste containers 1606 to the tray 1602 (for example, to secure containers during transport, initial setup, etc.). Although two couplers 1603 are shown, in other embodiments only one or two couplers 1603 may be present. For example, in some embodiments the tray assembly may be configured to support only one cell culture container and therefore include only a single coupler 1603 to maintain the cell culture container in a fixed position on the tray. Similarly, although one waste container 1606 and one reagent container 1605 are shown, in alternative embodiments multiple waste and multiple reagent containers may be present. Furthermore, while Figure 16A shows the waste container 1606 and reagent container 1605 as part of the tray assembly 1601, in other embodiments the waste container 1606 and / or reagent container 1605 may be separate components within the automated cell culture system 1600 that are not connected to the tray 1602 in use. For example, in some embodiments the reagent container 1605 may be used to contain cell culture media and may be located in the refrigerated section (not shown) of the automated cell culture system 1600 or in another refrigerated location. The coupler 1603 and holder 1604 may be separate components attached to the tray 1602, or they may be components formed integrally or monolithically with the tray 1602. For example, in some embodiments the coupler 1603 and / or holder may include a deformable bracket, a movable pin, or any other suitable structure for connecting the container to the tray 1602. In some embodiments, the tray assembly 1602 may optionally include a handle 1614 that can be used by a user to move and carry the tray assembly 1602. The handle 1614 may be a separate component from the tray 1602, or it may be formed integrally with the tray 1602 or monolithically.In some embodiments, the tray assembly 1601 may not include the holder 1604. In some embodiments, although not shown, the tray assembly 1601 can be pre-assembled in one or more cell culture containers.

[0115] The tray assembly 1601 also includes a multiport valve 1607 and one or more container lids 1608 (Figure 16A shows two container lids 1608). The container lids 1608 can be attached to the tray 1602 using disposable packaging mounts (not shown in Figures 16A–16C). Each lid 1608 is configured to be attached to a different cell culture container, as described below. In this exemplary embodiment, two lids 1608 are present, but it should be understood that different numbers of lids 1608 can be provided to accommodate different numbers of cell culture containers. Each lid 1608 may include a fluid exchange port (also referred to herein as a “fluid port”) and a gas exchange port (not shown in Figures 16A–16C, respectively). As shown, each fluid port is connected to a select port of the multiport valve 1607 by piping (see piping A, B, C, and D in Figure 16A). A gas exchange port can allow the transfer of gas from the cell culture vessel to which it is connected. For example, in some embodiments, lid 1608 may be similar to lid 803 or lid 2408 of the cell culture vessel shown and described herein. For example, lid 1608 may include a gas filter to prevent microorganisms and / or contaminants from entering the cell culture vessel, thereby allowing cell culture and fluid transfer through lid 1608 while maintaining a closed (and / or sterile) system with other containers in the system (e.g., reagent container 1605, waste container 1606, or other containers). In some embodiments, the tray assembly may optionally include lids 1609 and 1610 connected to reagent container 1603 and waste container 1606, respectively. Lids 1609 and / or lid 1610 may be similar in structure and function to lid 1608 and / or lid 803 of the cell culture vessel.

[0116] The multiport valve 1607 may include the same or similar components and functions in the same or similar manner as the multiport valves described above for previous embodiments (for example, the multiport valve 600 or multiport valve 2407 described herein). The multiport valve 1607 may include a master port (described below and shown in Figures 16B and 16C) configured to connect to the fluid pump 1613 of the base unit, and a plurality of selectable ports that can be fluid-connected to the fluid exchange ports of lids 1608, 1609, 1610 and / or other components of the cell culture assembly 1600, as described herein. For example, one of the selectable ports may be sterile and / or fluid-connected to a first fluid exchange port of a first lid 1608, and a second selectable port may be sterile and / or fluid-connected to a second fluid exchange port of a second lid 1608. In some embodiments, the third port of the multiport valve 1607 can be connected to the liquid exchange port of the reagent container 1605, the fourth port can be connected to the liquid exchange port of the waste container 1606, and the fifth port can be connected to the liquid exchange port of the cell collection container (not shown in Figures 16A-16C). The multiport valve 1607 can be connected to various other components, such as cell counting chips, cell collection containers, and various reagent and enzyme containers. A schematic diagram of an exemplary system showing some exemplary connections of the multiport valve is presented in Figure 59. Thus, when driven, the multiport valve 1607 can facilitate the exchange of fluids between various containers in the automated cell culture system 1600. For example, as described herein, the multiport valve 1607 can be driven to facilitate the addition of cell culture medium or reagents to the cell culture container, the removal of cells from the cell culture container (e.g., cell subculturing or cell collection), or any other fluid movement related to cell culture.

[0117] The multiport valve 1607 can be pre-assembled and connected to lids 1608, 1609, and 1610 on top of the tray assembly 1601 and sealed within a protective overlap 1615. This arrangement allows the end user to receive the tray assembly 1601 pre-packaged within the protective overlap. In some embodiments, the tray assembly 1601 can be sterilized before being placed in the protective overlap. As described herein, the user can then load the desired cells, reagents, cell culture media, etc., into the container and connect the pre-connected lid to the container in a sterile environment. The tray assembly 1601 can then be connected to a base unit and moved to an incubation environment where fluid exchange can be performed to ensure the culture of the desired cells, as described herein.

[0118] The multiport valve 1607 is configured to engage with the valve actuator 1621 of the base unit 1620. In some embodiments, the multiport valve 1607 may include a mounting portion 1616 configured to mate and connect to the valve connector 1622 of the base unit 1620. For example, the mounting portion 1616 may have a shape that allows it to connect to the valve connector 1622 like a puzzle. Examples of such mounting portions and valve connectors are described below with reference to specific embodiments. As shown in Figures 16B and 16C, when the multiport valve 1607 is engaged with the valve actuator 1621 of the base unit 1620, the valve actuator 1621 can actuate the multiport valve 1607 to move to a selected port, enabling selective fluid transfer of various containers and cell culture containers of the tray assembly 1601 (described below). In some embodiments, the multiport valve 1607 may be connected to the valve actuator 1621 while connected to the tray 1602. For example, a valve connector (not shown) connected to a valve actuator 1621 may be located in the base unit 1620 below, where the tray assembly 1602 is detachably connected to the base unit 1620 (for example, as in the base unit 301 or base unit 2120 described herein). In some embodiments, a multiport valve 1607 may be removed from the tray 1602 (while remaining connected to the lid, thereby maintaining the closing system) and mounted to a mating valve connector 1622 of the base unit 1620, for example, as shown in Figures 16B and 16C. Figure 16B shows the connector 1622 without the connected multiport valve 1607, and Figure 16C shows the connector 1622 with the connected multiport valve 1607. In other words, the multiport valve 1607 is detachable from the mating mounting pocket 1618 of the tray 1602 (see Figure 16C) and can be mounted to the valve connector 1622 of the base unit 1620.As described above, the mounting portion 1616 of the valve 1607 mates and engages with the mounting pocket 1618 and the valve connector 1622 of the base unit 1620, shaping to ensure proper positioning and alignment within both the tray assembly 1601 and the base unit 1620. This repositioning of the multiport valve 1607 can be performed while the covers 1608, 1609, and 1610 remain sterilely connected to the multiport valve 1607. By removing the valve 1607 from the tray 1602, the interface between the valve 1607 and the valve actuator 1621 can be fixed, which is well suited to embodiments that include a stirrer to move the tray 1602 relative to the base unit 1620. Similarly, by directly connecting the valve 1607 to the base unit 1620, the interface between the valve 1607 and the valve actuator 1621 is not obstructed by the relative movement between the tray 1601 and the base unit 1620.

[0119] An optional pump holder 1611, also shown in Figure 16A, can be used to hold a port connector 1612 fluid-coupled to the master port of the multiport valve 1607. This port is used to connect a fluid pump 1613 to a tray fluid idix 1602 during the preparation of a tray assembly 1601 for cell culture procedures. The fluid pump 1613 may be used to generate fluid movement in the cell culture system 1600 as described herein. The fluid pump 1613 may be any suitable pump that generates pressure and / or flow within the cell culture system 1600. For example, the fluid pump 1613 may be a syringe comprising a piston rod and a syringe body. The syringe is just one example of the type of fluid pump that can be used in the cell culture system 1600. Various other positive displacement fluid pumps can be used, such as peristaltic pumps. In some embodiments, the pump may be a single-port pump, while in other embodiments, the pump may be a two-port pump as described herein. When using a syringe as the pump 1613, it can be attached to the multi-port valve 1607 and an optional syringe holder 1611 in a sterile environment before cell culture treatment.

[0120] The base unit 1620 (see Figures 16B and 16C) includes a housing 1623 that can support various components of the base unit 1620 and define (or include) a receiving portion 1624 that receives and removably connects the tray assembly 1601. In some embodiments, the receiving portion 1624 may include an opening into which the tray assembly 1601 can be positioned and supported by a tray support (not shown). In some embodiments, the tray assembly 1601 is supported by a support portion of the base unit 1620 such that the tray assembly 1601 is higher than the top surface of the base unit 1620. In some embodiments, the tray assembly 1601 is at least partially supported by engagement with a stirrer (described below) of the base unit 1620. In some embodiments, the tray assembly 1601 may be removably connected to a separate support member that can be connected to the housing 1623 of the base unit 1620. The base unit 1620 may also include one or more transparent sections or openings corresponding to the transparent sections of the tray 1602 so as to be able to acquire images and / or other sensor data related to the contents of the cell culture container.

[0121] The base unit 1620 includes the valve connector 1622 and valve actuator 1621 described above, and also includes a fluid pump section 1627 and a pump actuator 1626. The pump actuator 1626 can be at least partially located within an opening 1625 defined, for example, by the housing 1623. As described above, in some embodiments, the fluid pump 1613 may be a syringe or other type of positive displacement fluid pump that is fluid-coupled to a multiport valve 1607 and then coupled to the fluid pump section 1627 of the base unit 1620. In some embodiments where the syringe is the fluid pump 1613, the fluid pump section 1627 may include a holder (not shown in Figures 16A-16C) that can be used to hold and support the syringe 1613 in the housing 1623. The holder may be a separate component or a component formed integrally or monolithically with the housing 1623. The fluid pump 1613 can be fluid-coupled to the master port of the multiport valve 1607. In this exemplary embodiment, as shown in Figure 16C (showing a tray assembly 1601 connected to a base unit 1620), a multiport valve 1607 is detached from the tray assembly 1601 and connected to a valve connector 1622, and a fluid pump 1613 is connected to a master port by a pipe E. The fluid pump 1613 may include movable members inside the pump body (not shown in Figures 16B and 16C). During operation of the system 1600, the movable members of the fluid pump 1613 (e.g., plungers, rotors) can be actuated to generate suction force to transfer fluid to the pump body, and the movable members can be actuated to push the fluid out of the pump body, as described above for previous embodiments.

[0122] In some embodiments, the base unit 1620 may also include a stirrer 1628. The stirrer 1628 may include, for example, an orbital shaker that moves the tray 1602 in a circular or semicircular motion. The stirrer 1628 may be configured to stir the removable tray assembly 1601 in relation to the housing 1623, as described above for previous embodiments. The stirrer 1628 may stir the tray 1602 in a rocking, vibrating motion, circular swirling motion, or other motion useful for cell culture. In some embodiments, individual cell culture containers / vessels may be stirred independently by a separate stirrer positioned between the cell culture container and the removable tray assembly 1601, as described above. In some embodiments, a stirrer may not be included.

[0123] In some embodiments, the base unit 1620 may also optionally include one or more sensors 1629 (only one shown in Figures 16B and 16C) and an electronic control system 1630 (e.g., valve actuator 1621, pump actuator 1626) for controlling the operation of any component of the cell culture system 1600. The electronic control system 1630 may optionally be incorporated into, connected to, or provided by, a remote computing system, such as within a cloud computing environment. In some embodiments, the sensor(s) 1629 may be mounted on the device to allow the sensor(s) to be movable relative to the housing 1623 of the base unit 1620. Examples of such embodiments are described below with reference to Figures 32-34. The sensor(s) 1629 may include, for example, one or more imaging devices, microscopes, color monitors, or any other type of sensor described herein. Sensors(s) may be used to acquire images or other types of output that can be used to determine that information about the contents of a cell culture container (e.g., 1647, 1648) has been acquired, such as the density of the contents to determine the amount of cells inside the container during a cell culture procedure (e.g., in the case of suspension cells), or the percentage of confluence (i.e., the percentage of the container's surface area covered by cells) in the case of adherent cells. In some embodiments, sensors(s) 1629 may be used to capture images and / or other types of output of a sample portion of the contents of a cell culture container via a cell counting chip 1617. For example, a sample of a fluid mixture in a cell culture container may be extracted to the cell counting chip 1617, and the sensor 1629 may be moved to a position for alignment with the cell counting chip 1617 and used to image or otherwise collect information related to the sample fluid mixture on the cell counting chip 1617. In some embodiments, the sensor(s) 1629 may be operably coupled to or incorporated within the electronic control system 1630.

[0124] As described above, in some embodiments, a light or light source 1682 (see Figures 16B and 16C) can also be provided, for example, which can be used in combination with the imaging device. In some embodiments, the light may be movable relative to the housing of the base unit 1620. For example, the light source may be mounted above the tray assembly 1601 of a movable multi-axis gantry system, thereby allowing it to be controlled to move to the same position as the microscope in the base unit. In some embodiments, the light source may be operably coupled to the same gantry as the imaging device so that the imaging device and the light source can move together. In some embodiments, the system 1600 may include one or more cameras or pairs of LEDs and light sensors for imaging the contents of cell culture vessels.

[0125] In some embodiments, the sensor(s) 1629 may include a valve position sensor configured to generate a valve position signal related to the rotational position of a valve actuator. In this way, the valve position sensor can detect which of the selectable ports is fluid-connected to a master port (e.g., a fluid pump 1613). In some embodiments, the sensor(s) 1629 may include a pump position sensor configured to generate a pump position signal related to the movement of a pump. In this way, the pump position sensor can indicate the movement of the pump and / or the volume of fluid moved by the pump. As described herein, the electronic control system 1630 may determine an estimate of the fluid in (or added to) one of the cell culture vessels based on the pump position signal.

[0126] Figure 17 is a schematic diagram of an electronic control system 1630 that can be used to control the operation of a cell culture system. Components and architecture of the electronic control system 1630 are provided as examples, and in some embodiments, the electronic control system 1630 (or any of the electronic control systems described herein) may include components different from those shown in Figure 17. Furthermore, in some embodiments, the base unit and / or cell culture assembly do not need to include an electronic control system as shown in Figure 17. For example, in some embodiments, the base unit 1620 (or any of the base units described herein) may include the computer system 1500 described herein. In other embodiments, the base unit 1620 does not need to include an electronic control system.

[0127] As shown in Figure 17, the electronic control system 1630 includes one or more processors 1631, one or more memory components 1632, a radio 1633, and various modules, such as an actuator module 1634, a stirring module 1635, a fluid flow module 1636, a valve module 1637, a pump module 1638, a measuring module 1641 (also called a cell sensor module), and / or a network module 1640. Figure 17 shows the electronic control system 1630 located within the base unit 1620, as described above, and the electronic control system 1630 or a part of it may be located outside the base unit 1620 (for example, in a cloud computing environment). The electronic control system 1630 can automatically control the flow of fluids entering and leaving various containers, for example, through the operation of a pump actuator 1626 and a valve actuator 1621. The electronic control system 1630 can also automatically control the operation of a stirrer 1628, a sensor(s) 1629, and a valve actuator 1621. The operation and function of the fluid pump 1613 and valve actuator 1621, the selection of ports on the multiport valve 1607, etc., can be the same as or similar to the operation of these components as described above for the previous embodiments. As described above for the previous embodiments, during operation, the fluid pump, the valve of the multiport valve, the vessel, and the cell culture vessel can be used to transfer liquid between the cell culture vessel and the vessel.

[0128] During preparation for cell culture procedures, the tray assembly 1601 can be placed in a sterile environment (e.g., a laminar flow hood) and the overlap 1615 can be removed. While in a sterile environment (e.g., a laminar flow hood), the cell culture containers or vessels 1617, 1618 can be prepared (e.g., cells and reagents can be added to the vessels), secured to the lid 1608, and placed inside the coupler 1603 on the tray 1602. The cell culture containers 1617, 1618 can be any known type of cell culture container, such as the flasks or dishes described above for previous embodiments. The waste container 1606 and reagent container 1605 can be placed in an upright position within the holder 1604. In other embodiments, the waste container 1606 and / or reagent container 1605 can be placed in any suitable location for transport within the cell culture system 1600.

[0129] Next, the tray assembly 1601 can be connected to the base unit 1620, as shown in Figure 16C. In this embodiment, the multiport valve 1607 remains fluidly connected to the various lids 1608, 1609, and 1610, while being detached from the tray assembly 1601 and connected to the valve actuator 1621. The fluid pump 1613 can be fluidly connected to the multiport valve 1607 via a tube E of a certain length. As described above, in the case of a syringe used as the fluid pump 1613, the syringe can be connected to the multiport valve 1607 inside a sterile environment and can be connected to the tray 1602 before the tray assembly 1601 is connected to the base unit 1620. The syringe 1613 can then be moved to a holder (not shown) on the base unit 1620 and connected to the pump actuator 1626, while remaining fluidly connected to the multiport valve 1607 via the tube. The waste container 1606 and the reagent container 1605 can be removed from the tray 1602 and placed, for example, along or near the tray 1602, and / or inside the incubator or refrigerator. A more detailed description of how to prepare the cell culture system 1600 for use is given below with reference to Figures 21-30. The tray assembly 1601 can be connected to the base unit 1620, either inside or outside a sterile environment. The cell culture system (equipped with the tray assembly 1601 connected to the base unit 1620) can be placed in an incubator ready for cell culture treatment. In some embodiments, the tray assembly 1601 can be connected to the base unit 1620 inside the incubator.

[0130] Any of the base units and / or tray assemblies described herein can be used to perform any of the computer implementation methods described herein. In other words, any of the base units and / or tray assemblies described herein may include (or interface with) an electronic control system for facilitating an automated (or semi-automated) method of culturing cells. As shown in Figure 17, the electronic control system 1630 may communicate with other remote computing devices (e.g., computing device 1643) via a network 1646 (e.g., the Internet), for example, via a service platform 1642 and a cell culture application (i.e., app) 1644. In addition, or instead, the electronic control system 1630 may communicate with remote computing devices via a direct connection, such as a cable connected to the USB port of the base unit 1620. Components, modules, and / or functions described in relation to cell culture system 1600 may be included in any of the cell culture systems described herein. For example, although not shown, cell culture systems 200, 300, and 400 may include an electronic control system similar to or the same as the electronic control system 1630. Furthermore, while the cell culture system 1600 is shown and described as comprising only one connected computing device 1643, in other embodiments the cell culture system 1600 (and any of the cell culture systems described herein) may comprise any number of connected remote computing devices.

[0131] The service platform 1642 may be any suitable computer implementation interface and / or computing entity, such as a server or personal computer, but it is configured to communicate via network 1646 with a remote computing device 1643 and / or any other part of the cell culture system 1600 (e.g., a call center interface, other remote computing devices, etc., not shown). More specifically, the service platform 1642 can receive information from devices within the cell culture system 1600 (e.g., a base unit or a remote computing device), manipulate the information, and generate information for any other device within the cell culture system 1600. For example, in some embodiments, information on cell density or cell confluence related to a tray assembly 1601 may be transmitted from the base unit 1620 to the remote computing device 1643. The remote computing device 1643 can generate notifications to a user via a cell culture application 1644 and can receive input from the user in response to such notifications. The remote computing device 1643 can then transmit input (or commands) to the service platform 1642. Based on user input, the service platform 1642 can send commands to the base unit 1620, which can then execute the commands to perform a desired task (e.g., cell passage). In this way, the service platform 1642 can control and / or manage specific commands, notifications, and / or functions. As similarly stated, in this manner, the service platform 1642 can function as the "backend" of the cell culture system 1600.

[0132] Network 1646 may be a piconet, the Internet, an intranet, a local area network (LAN), a wide area network (WAN), a virtual network, a telecommunications system, any other suitable communication system, and / or a combination of such networks. Network 1646 may be implemented as a wired and / or wireless network. Base unit 1620 and remote computing device 1643 may be connected to the network via any suitable mechanism and / or any protocol. For example, in some embodiments, base unit 1620 may communicate directly with network 1646, remote computing device 1643, and / or service platform 1642 via the LTE Direct protocol or any other suitable protocol (e.g., a 5G mobile radio standard based on the IEEE 802.11ac standard for broadband technology).

[0133] Figure 17 identifies the base unit 1620, but the electronic control system 1630 can be incorporated into (or used together with) any of the base units described herein. As stated above, the base unit 1620 includes or has the electronic control system 1630 mounted thereon. For example, in some embodiments, the electronic control system 1630 may be coupled to and / or located within the housing 1623 and / or any other part of the base unit 1620. As similarly stated, the electronic control system 1630 can be incorporated within the base unit 1620. However, in other embodiments, the electronic control system 1630 may be separated from the base unit 1620 but operably coupled (for example, connected wirelessly or via a wired connection). The electronic control system 1630 is shown to include one or more processors 1631, one or more memory components 1632, a radio 1633, and various modules, such as an actuation module 1634, agitation module 1635, fluid flow module 1636, valve module 1637, pump module 1638, measurement module 1641, and / or network module 1640. In other embodiments, the electronic circuit system does not need to include all (or any) of these modules, but may include any other modules described herein. For example, in some embodiments, the electronic control system may include only a flow module and be configured to perform cell passage and flow methods associated therewith, and does not need to include, for example, an agitation module.

[0134] Processor 1631 and any of the processors described herein may be any suitable processor for performing the methods described herein. In some embodiments, processor 1631 may be configured to perform and / or process application modules, processes, and / or functions related to the cell culture system 1600. For example, processor 1631 may be configured to perform and / or process any of the actuation module 1634, the stirring module 1635, and / or the network module 1640, and / or other modules described herein, and to perform methods related thereto. Processor 1631 may be, for example, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a digital signal processor (DSP), etc. Processor 1631 may be, for example, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a digital signal processor (DSP), etc. Processor 1631 may be configured to retrieve data from memory, for example, memory 1632, and / or write data to memory. As described herein, in some embodiments, the processor 1631 can work in cooperation with the radio 1633 and / or execute instructions from code to provide signals that enable the electronic control system 1630 to communicate via a network such as network 1646 to a computing device 1643 (e.g., via wireless communication) and / or any other computing entity. In some embodiments, the processor 1631 is a Bluetooth® Low Energy (BLE) processor.

[0135] Memory 1632 may be, for example, random access memory (RAM), memory buffer, hard drive, database, erasable programmable read-only memory (EPROM), electrically erasable programmable read-only memory (EEPROM), read-only memory (ROM), flash memory, hard disk, floppy disk, cloud storage, etc. In some embodiments, memory 1632 stores instructions for the processor 1631 to execute modules, processes, and / or functions related to such cell culture system 1600 and / or base unit 1620. For example, memory 1632 may store instructions for the processor 1631 to execute any of the application modules described herein and perform the methods associated therewith.

[0136] As described above, one or more sensors 1629 may be separate and / or included within the electronic control system 1630, and may include, for example, imaging devices, optical sensors, accelerometers, temperature sensors, contact sensors, position sensors and / or any other suitable input devices. In some embodiments, the sensor(s) 1629 may include sensors operable to monitor and / or measure the position (or selection) of the ports of the multiport valve 1607, the position of the fluid pump 1627, temperature, agitation, etc. For example, in some embodiments, the sensor 1629 may include a position sensor operable to detect the position of the multiport valve in the system. In yet another example, the sensor 1629 may include an optical sensor operable to detect the density (or quantity) of cells in a cell culture container connected to the tray 1602. In such embodiments, the optical sensor may detect the attenuation of light (for example, to detect the density of cells in the optical path). Alternatively, the optical sensor may acquire an image (for example, via a photocell, microscope, charge-coupled element, etc.) to determine the quantity of cells in the cell culture container. As yet another example, the sensor 1629 may include an accelerometer that can operate to detect characteristic movement or vibration features of the tray assembly 1601 when the device is being agitated.

[0137] The radio 1633 (also called a receiver, transmitter, and / or transceiver) may be operable to transmit and / or receive signals to radio signals, such as Bluetooth®, ZigBee, Wi-Fi, etc., and 1631 is a Bluetooth® processor, and the radio 1633 may be integrated with processor 1631. In other embodiments, the radio 1633 may include a processor different from processor 1631. The radio 1633 may be operable to connect the electronic control system 1630 to the computing device 1643 and / or any other computing entity via the network 1646 in a communicable manner. The radio 1633 may include or be coupled to ceramic chip antennas, stamp antennas, sintered antennas, PCB conductive trace antennas, and / or any other suitable antennas.

[0138] The measurement module 1641 (also referred to as the cell sensor module in some embodiments) may be a hardware and / or software module (stored in memory 1632 and / or executed on processor 1631). As will be described in more detail herein, in some embodiments, the measurement module 1641 is configured to receive multiple different signals from the sensor 1629 of the electronic control system 1630 and generate information for various other modules within the electronic control system 1630.

[0139] The flow module 1636 may be a hardware and / or software module (stored in memory 1632 and / or executed on processor 1631). As described in more detail herein, the flow module 1636 may be configured to receive indication (e.g., from sensor(s) 1629) and / or transition information related to changes in the state of the multiport valve of the pump or base unit 1620, and to determine, based on the indication or transition information, which valve of the multiport valve 1607 to open or close to allow fluid to enter or leave a particular container of the system 1600.

[0140] The network module 1640 may be a hardware and / or software module (stored in memory 1632 and / or executed on processor 1631). The network module 1640 is configured to exchange information related to the base unit 1620 and the remote computing device 1643 in order to facilitate the communication process. For example, the network module 1640 of the base unit 1620 can cause the remote computing device 1643 and the base unit 1620 to exchange short-term and / or long-term security keys to complete the pairing and bonding process.

[0141] The notification module 1639 may be a hardware and / or software module (stored in memory 1632 and / or executed on processor 1631). The notification module 1639 is configured to generate notifications related to any of the methods and / or application modules described herein. For example, in some embodiments, the notification module 1639 may generate notifications that are transmitted via radio 1633 and received by a notification module on a remote computing device 1643. In this way, the notification module 1639 executed in a cell culture application may generate outputs (e.g., radio communication signals, GUI elements, audible outputs, visual outputs, etc.) to notify a user of an event.

[0142] The agitation module 1635, the valve module 1637, and the pump module 1638 may each be hardware and / or software modules (stored in memory 1632 and / or executed by processor 1631). These modules may be configured to receive indications (e.g., from sensors 1629) and / or transition information related to changes in the state of the pump or multiport valve of the base unit 1620, and to determine what action to take on a particular device (pump, valve, agitator, etc.) based on the indication or transition information. In some embodiments, the valve module 1637 and / or the pump module 1638 may provide information related to the position of the multiport valve 1607 and the pump 1627, respectively. In some embodiments, modules 1637 and 1638 may include (or receive information from) encoders. In some embodiments, the actuator module 1634 may perform some or all of the functions of the agitation module 1635, the valve module 1637, and / or the pump module 1638.

[0143] Computing device 1643 includes, for example, mobile computing entities such as smart phones (e.g., iPhone®, Android® devices, Windows® phones, Blackberry® phones, etc.), tablet computers (e.g., Apple iPad®, Samsung Nexus® devices, Microsoft Surface® devices, etc.), or computers (e.g., laptops, desktops, smart TVs, etc.), and / or any other suitable computing entity. Computing device 1643 may include a processor, memory, user interface 1645, and a wireless device.

[0144] The user interface 1645 of the remote computing device 1643 may be, for example, a monitor or screen that displays visual elements to the user. The user interface 1645 may be a touchscreen (of a smartphone) that can display a set of graphical user interface (GUI) elements (e.g., windows, icons, input prompts, graphical buttons, data displays, notifications, etc.). In some embodiments, the graphical user interface elements (see, for example, GUI elements 1645A, 1645B, and 1645C described with reference to Figures 18-20) are generated by the cell culture application 1644. Furthermore, the user interface may also receive input from the user, for example, input via the touchscreen, input via the microphone, etc.

[0145] The cell culture application 1644 (also called the “Application” or “Cell Culture App”) is configured to communicate with an electronic control system. In some embodiments, the application 1644 can communicate directly with the electronic control system 1630 located in the base unit 1620. In some embodiments, the application 1644 can communicate with the electronic control system 1630 via a computing cloud environment. The application 1644 can be used with the cell culture system 1600 to set up, execute, and monitor various steps of a cell culture procedure. For example, the application 1644 can be used to cause a remote computing device 1643 to generate a series of prompts and information (e.g., via a user interface) to facilitate the cell culture method described herein. Specifically, the cell culture application 1644 can cause the remote computing device 1643 to generate graphical user interface (GUI) elements that can include prompts for inputting various data for a cell culture procedure. Figures 18-20 are sample screenshots showing various GUI elements that can be generated by the remote computing device.

[0146] Figures 21-30 illustrate how to prepare a cell culture system for use in cell culture procedures. The cell culture system 1700 shown in Figures 21-30 may include the same or similar components as other embodiments described herein (for example, cell culture system 1600 or cell culture system 2000), and therefore some details of the cell culture system 1700 are not described in relation to this embodiment.

[0147] The cell culture system 1700 (also referred to herein as the “system”) includes a tray assembly 1701 and a base unit 1720 (see Figures 27–30). For example, as shown in Figure 21, the tray assembly 1701 includes a tray 1702 having the same or similar components as described above for other embodiments (e.g., tray assembly 1601 or tray assembly 2001). For example, the tray assembly 1701 includes a waste container 1706 connected to a lid 1710, a reagent container 1705 connected to a lid 1709, and three lids 1708 (shown in Figures 25–27) each configured to connect to a cell culture container. The lids 1708, 1709, and 1710 may include fluid exchange ports (also referred to as “fluid ports”) and gas exchange ports, as described above for previous embodiments. The tray assembly 1701 also includes a multiport valve 1707 with a master port and several selectable ports, to which lids 1708, 1709, and 1710 can be selectively connected via tubes of a certain length. A waste container 1706 and a reagent container are shown connected horizontally by a holder 1704. The tray assembly 1701 also includes a coupler 1703 to which cell culture containers can be connected as described below. Below where the cell culture containers are placed is a transparent portion (or opening / notch) 1758 of the tray 1702. In this embodiment, a syringe holder 1711 is provided, which holds a syringe port 1712 therein. The syringe port 1712 is also connected to the multiport valve 1707 via a tube T. Figure 22 shows the tray assembly 1701 wrapped inside an overlap 1715 to maintain the sterility of the tray assembly 1701 during transport and storage. This arrangement allows the tray assemblies 1701 to be assembled in a central facility, placed in protective overlaps 1715, and sterilized. Sterilization can be carried out by any suitable method, including radiation sterilization, ethylene oxide (EtO) sterilization, or electron beam sterilization. The pre-packaged and sterilized tray assemblies 1701 can then be stored until required for cell culture treatment.

[0148] The first step in preparing for the cell culture procedure is to prepare the cells and culture medium (e.g., reagents) and the tray assembly 1701, which will be carried out inside a sterile environment (e.g., a laminar flow hood). The cells and culture medium are placed inside the cell culture container or vessel, and in this example, there are positions for three cell culture containers (e.g., 1747, 1748, and 1749 shown in Figures 26-27). The tray assembly 1701 is placed inside a sterile environment (e.g., a hood) and the overlap 1715 is removed. The waste container 1706 and the reagent container 1705 can be moved vertically within the holder 1704 with the lids 1709 and 1710 upright, as shown in Figure 23. In this example, the fluid pump 1713 is a syringe, which can be removed from the outer sterile wrap, and the port 1712 can then be connected to the fluid pump 1713, as shown in Figure 23. Next, the fluid pump 1713 is positioned within the holder 1711, as shown in Figure 24. In some embodiments, the fluid pump 1713 (e.g., a syringe) is not included within the pre-packaged tray assembly 1701, but rather is a separate component. In other embodiments, the fluid pump 1713 (e.g., a syringe) is included within the pre-packaged tray assembly 1701.

[0149] After loading cells and an initial amount of cell culture medium into the cell culture containers, the lids 1708 are secured to the cell culture containers 1747, 1748, and 1749 with the cells and medium inside. The lids 1708 are first removed from the transport support 1795 (see Figure 24) to which they are connected. The transport support 1795 is sized and configured to receive the lids 1708 inside in order to secure them during transport, storage, and initial setup. This arrangement reduces the possibility of undesirable movement during initial setup and the possibility of contamination of the inside of the lids. The lids 1708 are then connected to their respective containers (while remaining fluid-coupled to the multiport valve 1707). The containers 1747, 1748, and 1749 are connected to the coupler 1703 so that the containers are positioned horizontally as shown in Figure 25. In this state, the bottom surfaces of the cell culture containers 1747, 1748, and 1749 are aligned with the transparent portion 1758 of the tray.

[0150] As shown in Figure 26, with the tray assembly 1701 fully assembled, the tray assembly 1701 can be placed on the base unit 1720 as shown in Figure 27. This can be done outside of a sterile environment because the components (e.g., container, lid, valve, syringe) are fluidly connected in a closed system. The tray assembly 1701 should be oriented so that the arrow (labeled and circled) on the tray 1702 points toward the base unit 1720, as shown in Figure 27. As also shown in Figure 27, the base unit 1720 includes a pump actuator 1726, a valve connector 1721, and a valve actuator 1722. In this embodiment, a multiport valve 1707 is removable from the tray 1702 and can be connected to the base unit 1720. More specifically, the mounting portion 1716 of the multiport valve 1707 can be removed from the tray 1702 by removing the fastener 1757 and attaching the mounting portion 1716 to the mating valve connector 1722 of the base unit 1720 with the same or a different fastener 1757, as shown in Figures 28 and 29. The fluid pump 1713 (e.g., syringe) is detached from the tray assembly 1701 and coupled to the holder 1719 of the base unit 1720, as shown in Figure 29. This operation is performed while the fluid pump 1713 remains fluidly coupled to the multiport valve 1707, thereby maintaining the closed system. The holder 1719 may be part of the fluid pump portion (e.g., 1627) of the base unit 1720, as described above for system 1600. As shown in Figure 30, the waste container 1706 and the reagent container 1705 can be removed from the tray 1702 and placed near the base unit 1720 (or in any other suitable location).

[0151] Next, the base unit 1720 and tray assembly 1701 are moved to an incubation environment (for example, an incubator 2275 as shown in Figure 58) so that cell proliferation can be promoted in a temperature-controlled environment when the tray assembly 1701 is connected to the base unit 1720 outside the incubator. In some embodiments, the base unit 1720 is placed inside the incubator when the tray assembly 1701 is connected to it.

[0152] Figure 31 is a flowchart of method 1850 for preparing a cell culture system for use in a cell culture procedure. Method 1850 can be carried out using any of the cell culture systems described herein, for example, cell culture system 1700 described above with reference to Figures 23-30. In 1851, the cell culture tray assembly is removed from the outer protective wrap. The tray assembly may be any of the tray assemblies described herein and includes a tray, a first lid, a second lid, and a multiport valve. The first lid is connected to the tray and configured to be removably connected to a first container, and the second lid is connected to the tray and configured to be removably connected to a second container. The multiport valve is connected to the tray and includes a master port and a number of selectable ports. The first selectable port is sterilely connected to a first fluid exchange port on the first lid, and the second selectable port is sterilely connected to a second fluid exchange port on the second lid. As described herein, pre-connecting the lid to the appropriate port reduces the number of operations performed during initial setup, thereby reducing the possibility of contamination and errors. In 1852, at least one cell sample is added to the first container through the opening of the first container, and in 1853, a certain volume of reagent (e.g., cell culture medium) is added to the first container through the opening of the first container. In 1854, the first lid is connected to the first container to close the opening. In some embodiments, a second lid may optionally be connected to the second container. In 1855, the tray assembly is connected to the base unit. In some embodiments, once the tray assembly is connected to the base unit, the valve actuator of the base unit simultaneously engages with the multiport valve of the tray assembly. In some embodiments, after the tray assembly is connected to the base unit, the valve actuator engages with the multiport valve. In 1856, the fluid pump is connected to the pump actuator of the base unit. For example, the fluid pump may be a syringe or peristaltic pump that can be connected to the base unit. After the preparation of the cell culture assembly, any of the cell culture methods described herein can be carried out.

[0153] As described above, in some embodiments, the automated cell culture system may include an imaging device, which includes a microscope that can be moved relative to the housing of the base unit to image the contents of any cell culture vessel of the automated cell culture system. In some embodiments, the microscope may be mounted on a mechanical system that can move the microscope to align with the cell culture vessel or cell counting chip. The mechanical system may be any suitable assembly for moving the imaging device, such as a two-dimensional or three-dimensional gantry mechanism or a hinged robotic arm mechanism. Figures 32–34 show exemplary embodiments of such optical imaging systems (also called microscope imaging devices). The microscope imaging device 1960 may be mounted within the housing of any base unit of the cell culture system described herein. For example, the microscope imaging device 1960 may be contained within base unit 1720, base unit 2020, or any other base unit described herein. Microscope imaging device 1960 includes imaging device 1962 that can be viewed through a window or transparent portion on the top of the base unit and through notches (or transparent portions) of both the tray (see, for example, transparent portion 1758 described herein) and any shaking platform (e.g., tray support in contact with a stirrer). Thus, using the microscope imaging device 1960, relevant information can be obtained from the contents of the cell culture container and / or within the cell counting chip, as described herein. For example, in some embodiments, the microscope imaging device 1960 can acquire images of the cell culture container and / or cell counting chip during cell culture treatment, and the images can be used to determine, for example, the density of the contents to determine the amount of cells inside the container (e.g., in the case of suspension cells), or the percentage of confluence (i.e., the percentage of the area of ​​the container covered by cells), for example in the case of adherent cells.

[0154] The microscope imaging device 1960 includes a gantry system 1961 that allows the imaging device 1962 to move in multiple directions relative to the housing of a base unit (not shown in Figures 32-34). The gantry 1961 includes a set of rails 1963, 1964, and a cross rail 1965. The cross rail 1965 is mounted relative to rails 1963 and 1964 in the direction of arrow B and can move back and forth relative to rails 1963 and 1964. More specifically, a first motor 1966 can drive a belt 1968 to which the cross rail 1965 is operably connected. The imaging device 1962 is movably mounted to the cross rail 1965 and operably connected to a belt 1969 driven by a second motor 1967, which moves the imaging device 1962 in the direction of arrow B. The imaging device 1962 can be further moved in the direction of arrow C via motor 1973 for focusing on imaging device 1960. Thus, during operation, imaging device 1962 can be positioned in a desired location relative to the cell culture container and / or cell counting chip by moving rail 1965 relative to rails 1963 and 1964 in the direction of arrow A, by moving rail 1966 in the direction of arrow B, and by moving in the direction of arrow C relative to the base of imaging device 1960.

[0155] Lights(s) or light sources(s)(not shown) can be mounted on a separate multi-axis gantry above the system's tray assembly. This allows them to be controlled to move to the same position as the microscope in the base unit. In some embodiments, the light sources can be operably coupled to the same gantry as the microscope (e.g., gantry 1961) so that the microscope 1962 and the light sources can move together. In some embodiments, the microscope imaging device 1960 can be controlled by any electronic control system and according to any of the methods described herein. For example, in some embodiments, the microscope imaging device 1960 (and any associated light sources) can be controlled to automatically image cell culture containers (e.g., to generate sensor outputs related to the cells in the containers). A cell sensor module of an electronic control system (e.g., electronic control system 1630) or any other electronic control system described herein can receive the sensor outputs and generate signals related to the quantity of cells in the container (e.g., cell density or percentage of confluence). Next, based on this information, the electronic control system can generate one or more signals (e.g., valve control signals, pump control signals, stirrer signals, etc.) to cause the movement of cells from one cell culture vessel to another vessel within the system. Similarly, as described, in some embodiments, the microscopy imaging device 1960 can provide input for automated cell passage or cell harvesting operations.

[0156] Figures 35–44 show another embodiment of the cell culture system 2000 for use in cell culture procedures. The cell culture system 2000 may include the same or similar components as other embodiments described herein (including cell culture system 1700) and may have the same or similar functions as the previous embodiments described herein; therefore, some details of the cell culture system 2000 are not described in relation to this embodiment.

[0157] The cell culture system 2000 (also referred to herein as the “System”) includes a tray assembly 2001 (see, for example, Figures 35–37) and a base unit 2020 (see, for example, Figures 38–44). For example, as shown in Figure 35, the tray assembly 2001 includes a tray 2002 with a handle 2014, which has the same or similar components arranged as described above for a prior embodiment (e.g., tray assembly 1601 or 1701). For example, the tray assembly 2001 includes a waste container 2006 connected to a lid 2010, a reagent container 2005 connected to a lid 2009, and three lids 2008 (not shown in Figures 35–44) configured to connect to a cell culture container. Lids 2008, 2009, and 2010 may include fluid exchange ports (also referred to as “fluid ports”) and gas exchange ports, as described above for a previous embodiment. The tray assembly 2001 also includes a multiport valve 2007 with a master port and several selectable ports, to which lids 2008, 2009, and 2010 can be selectively connected via a tube of a certain length (not shown). For example, as described herein, lids 2008, 2009, and 2010 can be pre-assembled and connected to the multiport valve 2007 in overlap. Figures 35–44 do not show the tube and the connections between the various components and the multiport valve 2007 for illustrative purposes. The multiport valve 2007 is connected to the tray 2002 by fitting into a mounting pocket 2018 in a puzzle-like manner and then connecting to the tray 2002 via a fitting mounting portion 2016 therein. For example, the multiport valve 2007 can be connected to the mounting pocket 2018 with a fastener 2057.

[0158] The waste container 2006 and the reagent container 2005 are shown connected horizontally by a holder 2004. The tray assembly 2001 also includes couplers 2003, 2003' which can connect cell culture containers as described herein. Specifically, coupler 2003 is a bracket extending around a first end of a cell culture container (not shown), and coupler 2003' is a pair of tabs that receive a flange portion at a second end of the cell culture container. Coupler 2003' also functions to hold a temporary shipping support 2095 to which the lid 2008 is connected during storage, shipping, and initial setup. Couplers 2003, 2003' hold the cell culture containers in a predetermined fixed position on the tray 2002. Below where the cell culture containers are placed is a transparent portion 2058 of the tray 2002 (see, for example, Figure 36). In this embodiment, as described above for the previous embodiment, a pump holder 2011 is provided that can hold a pump port (not shown). As described above, the tray assembly 2001 is pre-assembled and placed in an overlap (not shown) to maintain the sterility of the tray assembly 2001 during transport and storage. Figure 37 shows the tray assembly 2001 when the overlap is removed (i.e., in a sterile environment), the waste container 2006 and reagent container 2005 are removed, and the fluid pump 2013 is connected to the holder 2011. As shown in Figure 37, in this embodiment the fluid pump 2013 is a syringe.

[0159] As described above for the previous embodiment, a pre-assembled tray assembly 2001 can be removably coupled to a base unit 2020. Figures 38–44 show the base unit 2020. The base unit 2020 includes a housing 2023 and a pump actuator 2026 partially positioned within a recess or pocket 2025 of the housing 2023. The pump actuator 2026 (see, for example, Figures 38–40) includes a pump holder 2019 that can lock a fluid pump 2013 in place and operably connect to the pump actuator 2026. The pump holder 2019 is shown as a syringe flange and a slot member that receives a movable member for securing the syringe flange in place, but in other embodiments, the pump holder 2019 may be any suitable structure or mechanism for securing a pump (which may be any suitable pump) to the pump actuator. The base unit 2020 also includes a valve connector 2022 configured to mate and connect to the multiport valve 2007, and a valve actuator 2021 configured to engage with the multiport pump 2007 when connected thereto. For example, when the tray assembly 2001 is connected to the base unit 2020 as described above, the multiport valve 2007 may be detached from the tray 2002 and connected to the valve connector 2022 of the base unit 2020, so that the multiport valve 2007 operably engages with the valve actuator 2021, as shown in Figures 40 and 41. More specifically, the mounting portion 2016 of the multiport valve 2007 can be detached from the tray 2002 by removing the fastener 2057 and attaching the mounting portion 2016, thereby mating the valve connector 2022 of the base unit 2020 with the same or a different fastener 2057. Figure 41 is a partial exploded view showing the components of the multiport valve 2007 before it is connected to the valve connector 2022.

[0160] In this embodiment, the support plate 2059 is connected to the housing 2023 and has a receiving portion 2024 on which the tray assembly 2001 can be placed. In this embodiment, the support plate 2059 is higher than the top surface of the housing 2023. Figure 42 is a side view showing the support plate 2059 raised. The support plate 2059 is connected to a stirrer 2028 (see Figure 44) located inside the housing 2023. As described above, the stirrer 2028 can be used during cell culture treatment to stir the contents of the tray assembly 2001 and the cell culture container connected thereto.

[0161] Figure 40 shows a base unit 2020 with a syringe 2019 connected to a syringe holder 2019 and a multiport valve 2007 connected to a valve connector 2022. Figure 40 also shows an optional mat 2070 positioned on the upper surface of a support plate 2059. The mat 2070 may be a rubber mat configured, for example, to protect the surface of the support plate 2059 and / or to provide damping when the tray assembly 2001 is agitated by a stirrer 2028. Similarly, in some embodiments, the support plates (or receiving portions) of the base unit may include damping members to dampen any relative movement or contact between the support plates of the containers attached thereto.

[0162] Figures 43 and 44 are opposing side views of the base unit 2020 showing the interior of the housing 2023. Figure 43 shows the valve actuator 2022, and Figure 44 shows the agitator 2028 and pump actuator 2026 in the pocket 2025. The electronic control system 2030 is also shown in Figure 44. The electronic control system 2030 is configured and can function in the same or similar way as the electronic control system 1630 described above. The electronic control system 2030 can optionally communicate with other computing devices and / or communicate within a cloud computing environment and may include some or all of the components and functions described above with respect to Figure 17. Not shown, the system 2000 may also include one or more sensors and / or lights (e.g., microscope, imaging device, etc.), such as the microscope imaging device 1960 described herein.

[0163] Figures 45-51 show another embodiment of a cell culture system that can be used in cell culture procedures. The cell culture system 2100 may include some of the same or similar components as in the other embodiments described herein and may have the same or similar functions as in the previous embodiments described herein; therefore, some details of the cell culture system 2100 are not described in relation to this embodiment. In this embodiment, the cell culture system 2100 does not include a stirrer and includes two multiport valves / valve actuators and two fluid pumps / fluid actuators.

[0164] The cell culture system 2100 (also referred to herein as the “system”) includes a tray assembly 2101 and a base unit 2120. For example, as shown in Figure 45, the tray assembly 2101 includes a tray 2102 with two multiport valves 2107 and 2107', on which four cell culture containers 2147 are shown. The containers 2147 may be pre-assembled on the tray assembly 2101 or added to the tray 2102 immediately before the cell culture procedure. For example, in some embodiments, the tray assembly 2101 is provided inside an overlap, so the containers 2147 are pre-assembled on the tray 2102. The pre-assembled containers can be coupled to or detached from lids 2108 (described below) when placed inside the overlap. During preparation for the cell culture procedure, cells and reagents are fitted into the containers and the lids 2108 are coupled to the containers before the tray assembly 2101 is coupled to the base unit 2120. In some embodiments, the container 2147 is not pre-assembled on the tray 2102 (not provided within the overlap), but rather added to the tray during preparation for the cell culture procedure, as described above. The container is filled with cells and reagents (e.g., cell culture medium), attached to the lid 2108, and added to the tray assembly 2101.

[0165] The lid 2108 can be configured in the same way as the lids described above for previous embodiments, including the lid 803 or lid 2408 of the cell culture container. For example, the lid 2108 may include a fluid exchange port (also called a “fluid port”) and a gas exchange port, the fluid port may be sterilely connected to one of the multiport valves 2107, 2107’ equipped with tubing, as described above for previous embodiments (not shown). For example, two of the containers 2147 / lid 2008 may be fluid-connected to valve 2107, and two of the containers 2147 / lid 2108 may be fluid-connected to valve 2107’. In this embodiment, the multiport valves 2107, 2107’ are fixed to the tray 2102 and remain on the tray 2102 when the tray assembly 2101 is connected to the base unit 2120. Each multiport valve 2107, 2107' may include a master port and a number of selectable ports that can selectively connect a lid 2008 (and / or other lids / containers) via a tube (not shown) of a certain length. The multiport valves 2107, 2107' can be connected to tray 2102 via mounting parts (not shown) that are connected to and fit into mounting pockets 2118 of tray 2102.

[0166] In this embodiment, the base unit 2120 defines a tray receiving portion 2124 and includes a housing 2123 containing two valve actuators 2122, 2122'. Each valve actuator 2122, 2122' includes a valve connector portion 2171, 2171' extending from the top surface of the base unit 2120 inside the receiving portion 2124, as shown in Figure 51. When the tray assembly 2101 is connected to the base unit 2120, the multiport valves 2107, 2107' can be operably engaged with the valve actuators 2122 and 2122' of the base unit 2120 via the valve connector portion 2171, as shown in Figure 47.

[0167] In this embodiment, the base unit 2120 also includes two fluid actuators 2126 and 2126' which can be coupled to fluid pumps 2113 and 2113', respectively. The fluid pumps 2113, 2113' may be, for example, syringes, peristaltic pumps, or other types of positive displacement fluid pumps. Using the two pumps 2113, 2113' and the two valves 2107, separate fluid connections can be provided between the valves 2107, 2107' and various containers of the system, allowing for separate fluid injection and discharge to, for example, a specific container (e.g., container 2147). For example, waste removal from one container can be done in a manner separate from other fresh culture media, ensuring that it does not pass through the same fluid channel. The two pumps can also enable more injection and discharge to the container by duplicating the fluids.

[0168] In this embodiment, system 2100 does not include a stirrer. Although not shown, system 2100 may also include an electronic control system, one or more sensors (e.g., a microscope, an imaging device, etc.). System 2100 may also include various other containers such as waste containers, reagent containers, cell collection containers, etc., each of which may be connectable to one of the multiport valves 2107, 2107'.

[0169] Figures 52–58 show another embodiment of a cell culture system that can be used in cell culture procedures. The cell culture system 2200 may include some of the same or similar components as those in other embodiments described herein and may have the same or similar functions as those in previous embodiments described herein; therefore, some details of the cell culture system 2200 may not be illustrated and are not described in detail with respect to this embodiment. This embodiment shows an exemplary cell culture system including a plurality of separate tray assemblies, each of which may include a lid and / or container that can be fluidly coupled to a separate multiport valve and a separate fluid pump system. In other words, each tray assembly is fluidly coupled to its own multiport valve and fluid pump, but is fluidly isolated from the multiport valve and fluid pump of the other trays. The individual tray assemblies can then be coupled to a single base unit. In some embodiments, each of the separate tray assemblies may be pre-assembled, placed inside a protective overlap, and shipped separately. In some embodiments, the separate tray assemblies may be pre-assembled and shipped together inside a protective overlap. The cell culture system can culture multiple different types of cells without the risk of cross-contamination by maintaining fluidic isolation of each tray assembly from other tray assemblies. For example, each tray assembly can be configured for a different cell type. This embodiment also allows for the culture and incubation of different cell types within a smaller device. For example, in a multi-tray system as described below, the system can be used to grow three types of cells on a single shelf and / or within a single base unit of an incubator without sharing fluids among the three cell types. In some embodiments, if it is desired to grow more of a single type of cell, a single larger tray (two or three times the width of the smaller trays) can be used.

[0170] In this embodiment, the cell culture system 2200 (also referred to herein as the “system”) includes a base unit 2220 and three tray assemblies 2201, 2201', 2201'' which can be connected to the base unit 2220 as described above in the previous embodiment. The three tray assemblies (collectively referred to as tray assembly 2201) and the base unit 2220 may include the same or similar features and components as described above in the previous embodiment. This embodiment also includes three multiport valves 2207, 2207', 2207'' (collectively referred to as multiport valve 2207) and three fluid pumps 2213, 2213', 2213'' (collectively referred to as fluid pump 2213).

[0171] In this embodiment, each tray assembly 2201 may include multiport valves 2207, 2207', 2207'', cell counting chips 2217, 2217', 2217'' (collectively referred to as counting chip 2217), first cell culture containers 2247, 2247', 2247'' (collectively referred to as cell culture container 2247), and trays 2202, 2202', 2202'' (collectively referred to as tray 2202) (see, for example, Figure 55) on which second cell culture containers 2248, 2248', 2248'' (collectively referred to as cell culture container 2248) are placed (see, for example, Figure 55). In this exemplary embodiment, container 2247 is smaller than container 2248. However, it should be understood that the tray assembly 2201 may accommodate containers of other sizes not shown. In some embodiments, one or all of the tray assemblies 2201 may contain the same two containers. Using larger (e.g., 2247) and smaller (e.g., 2248) containers within the same tray assembly 2201 may be desirable for use in, for example, cell proliferation processes. For instance, cells may be placed in the smaller container 2248 to promote better proliferation in the presence of fewer cells, and then the cells may be moved to the larger container as the growth surface of the smaller flask becomes crowded during the expansion process. Using containers of the same size within the same tray assembly 2201 may be desirable for, for example, cell maintenance processes where cell lines are cultured and maintained when needed next.

[0172] Tray 2202 may include transparent or partial cutouts 2258 and 2258', as shown in Figure 57, into which containers 2247 and 2248 can be placed, respectively. As described above for previous embodiments, the transparent or partial cutouts 2258 and 2258' can provide sensor data acquired in relation to the cell culture containers 2247 and 2248. For example, an imaging device or other sensor may be movably positioned within the housing of the base unit 2220 (described below) and moved to a position below the transparent or cutouts 2258 and 2258'. As shown in Figure 57, the transparent or cutout 2258' represents an optional container cradle that can accommodate two different sized containers. Similarly, tray 2202 may also include a transparent or cutout 2268 where the cell counting chip 2217 is placed, in order to provide sensor data acquired in relation to the fluid of the sample placed on the cell counting chip 2217, as described above for previous embodiments.

[0173] Containers 2247 (and 2247', 2247'') and 2248 (and 2248', 2248'') may be pre-assembled on tray 2202 or added to tray 2202 before the cell culture procedure (for example, according to the method described herein). For example, in some embodiments, container 2247 is pre-assembled on tray 2202, and tray assembly 2201 is provided inside an overlap (similar to the overlap described herein, though not shown). The pre-assembled containers may be connected to or detached from a lid 2208 (described below) inside a pre-assembled tray 2202. During preparation for the cell culture procedure, cells and reagents may be added to containers 2247, 2248, and the lid 2208 attached to containers 2247, 2248, before tray assembly 2201 is connected to base unit 2220. In some embodiments, container 2247 is not pre-assembled on tray 2202 (not provided within the overlap), but rather added to tray 2202 during preparation for the cell culture procedure. Containers 2247 and 2248 may be filled with cells and reagents, attached to lids, and added to tray assembly 2201.

[0174] The lid 2208 can be configured in the same way as the lid described above for previous embodiments. For example, the lid 2208 may include a liquid exchange port (also called a “fluid port”) and a gas exchange port. The fluid port may be sterilely connected to one of the multiport valves 2207, 2207', 2207'' equipped with a tube (not shown), as described above for previous embodiments. For example, for each tray assembly 2201, two containers 2247 and 2248 to which the lid 2208 is connected may be fluidly connected to a select port of the valve 2207 of the tray assembly 2201. Each multiport valve 2207 may include a master port and a number of selectable ports to which the lid 2208 (and / or other lids / containers) can be selectively connected. The multiport valve 2207 may be fitted and connected to a mounting pocket (not shown) of the tray 2202 in a puzzle-like manner, as described above for previous embodiments, and connected to the tray 2202 via a fitting mounting portion (not shown) therein.

[0175] In this embodiment, the base unit 2220 includes a housing 2223 that defines a tray receiving portion 2224 capable of receiving each of the three tray assemblies 2201. The housing 2223 also defines a section 2278 which may be a transparent portion or cutout corresponding to a transparent portion 2258 of the tray assembly 2201. The housing 2223 also defines a section 2279 which may be a transparent portion or cutout corresponding to a transparent portion 2268 of the tray assembly 2201 in which the cell counting chip 2217 is placed. As shown in Figures 52-54, the base unit 2220 may also optionally include a plurality of vials or containers 2280 and a plurality of vials or containers 2249. Containers 2280 (2280', 2280'') may, for example, be holders for the associated fluid pump 2213. For example, the fluid pump 2213 could be, for example, a peristaltic pump, and each of the containers 2280 could function as a holder for the pump so that the pump could function similarly to a syringe-type pump. More specifically, container 2280' could be a holder for the fluid pump 2213', and container 2280'' could be a holder for the fluid pump 2213''. The container 2280 can receive a certain volume of fluid from a first location in the system where it is held until the pump operates and moves that volume of fluid to a second location in the system. Containers 2249 (2249', 2249'') can be used to hold various other fluids that can be fluid-coupled to one of separate fluid systems via one of the multiport valves 2207 (2207', 2207''). For example, container 2249 could be used for waste, or to hold a fluid (e.g., a reagent) and warm the fluid after it has been refrigerated. For example, it may be desirable to refrigerate a container (or vessel) to maintain the culture medium inside at a desired temperature (e.g., 4 degrees Celsius). The culture medium can be pumped from the refrigerator into a vessel such as vessel 2249, and as a result, the culture medium can be passively heated to, for example, 37 degrees Celsius due to the temperature of the incubator in which system 2200 is located.

[0176] Each tray assembly 2201 (2201', 2201''), when connected to the base unit 2220, can be fluid-coupled to one of the fluid pumps 2213 (2213', 2213'') to provide a separate, closed fluid flow system. As described above, when the tray assemblies 2201 (2201', 2201'') are connected to the base unit 2220, the multiport valves 2207 (2207', 2207'') can be operably engaged to the valve actuators 2221, 2221', 2221'' (collectively referred to as valve actuators 2221) of the base unit 2220 via valve connector portions 2222, 2222', and 2222'' (collectively referred to as valve connector 2222). More specifically, in this embodiment, the multiport valve 2207 is removably coupled to the tray 2202 and, for example, in the case of the multiport valve 2007, can be coupled to separate valve connectors 2222 (2222', 2222'') (see, for example, Figure 54) and valve actuators 2221 (2221', 2221'') of the base unit 2220 as described above. Each fluid pump 2213 can be fluid-coupled to the master port of the corresponding multiport valve 2207. Each fluid pump 2213 (2213', 2213'') can be coupled to a pump actuator (not shown) that is located in or can be coupled to the housing 2223 of the base unit 2220. Although the fluid pump 2213 is described as a peristaltic pump, the fluid pump 2213 may be other types of fluid pumps, such as a syringe or another type of positive displacement fluid pump.

[0177] As shown in Figure 53, the cell culture system 2200 also includes an imaging device 2260 movably positioned within the housing 2223 so as to be able to move to a position aligned with sections 2278 and 2279. The imaging device 2260 may be, for example, a microscope mounted on a gantry system, resulting in movement of the imaging device in multiple directions (similar to the microscope imaging device 1960 described above). Although not shown in this embodiment, the cell culture system 2200 may also include a stirrer, an electronic control system, and one or more additional sensors (in addition to the imaging device 2260), as described herein.

[0178] In some embodiments, cells on all three tray assemblies 2201 can be imaged using a single imaging device (e.g., 2260) and / or a single stirrer. In some embodiments, separate imaging devices and / or separate stirrers can be used for each tray assembly. The system 2200 may also include various other containers, such as waste containers, reagent containers, and cell collection containers, each of which can be connected to one of the fluid systems via multiport valves 2207, 2207', and 2207''. The cell culture system 2200 may also include various couplers or coupling parts for holding cell culture containers (e.g., 2003, 2103), as well as holders for holding other containers, such as waste and reagent containers (e.g., 2005, 2006).

[0179] Figure 58 shows an example of two incubators 2275 stacked on top of each other, allowing for the placement of multiple cell culture systems 2200 (i.e., trays and base units) for cell culture treatment. As shown in Figure 58, in this embodiment, three cell culture systems 2200 can be placed on shelves inside each incubator 2275.

[0180] Figure 59 is a system diagram showing exemplary fluid flow within the system during cell culture procedures, as well as various containers and other components that can be connected within the cell culture system described herein. Therefore, while the system diagram illustrates various components of the cell culture system 2300, it should be understood that this exemplary diagram is applicable to any of the embodiments described herein.

[0181] Figure 59 shows a tray 2302 with two cell culture containers 2347 and 2348 connected. The cell culture containers 2347 and 2348, as well as the cell counting chip 2317, are each fluid-coupled to the select port of a multiport valve 2307. A fluid pump with a fluid holder 2327 is fluid-coupled to the master port of the multiport valve 2307. Several other containers are also fluid-coupled to the multiport valve 2307, including reagent containers 2305 and 2305', a cell collection container 2374, a waste container 2306, a container 2376 containing cell buffer (e.g., PBS), and a container 2377 containing enzymes (e.g., trypsin).

[0182] During the cell culture procedure, the pump holder holds the fluid solution being pumped from its starting position in the system (e.g., reagent container 2305, 2305'), and valve 2307 selects the desired channel (e.g., one of containers 2347, 2348), after which the solution is pumped to that location. An isotonic, non-toxic buffer (such as PBS) is used to flush out reusable components such as the pump holder. As shown in Supplemental Table 1 in Figure 60, in this example, container 2305 can be initially placed in a refrigerator to maintain the culture medium in container 2305 at the desired temperature (e.g., 4 degrees Celsius). The culture medium from container 2305 can then be pumped to 2305' before the procedure (e.g., 1 hour prior) so that it can be passively heated to approximately 37 degrees Celsius by the temperature in the incubator. To detach the cells, for example, during passaging or harvesting, the culture medium can first be pumped from the cell culture container (2347, 2348) from which the cells will be detached, pushed out, and discarded. A buffer (e.g., 2376) can be added to the culture in combination with any agitation to wash the cells, and then the buffer can be removed from the culture, pushed out, and discarded. An enzyme (e.g., in container 2377) can be pumped into the relevant cell culture container, left for a while with any agitation to aid detachment, then the solution can be diluted with fresh medium (e.g., from 2305') to quench the enzyme, and then the cell suspension can be passaged / harvested using the enzyme diluted in the mixture. Figures 61A and 61B include Table 2, which contains examples of cell passaging procedures for maintaining adherent cell lines, and Table 2 includes a list of each step, the source of the fluid, the destination, the type of fluid, and the volume in each cell culture container during the procedure. While specific procedures are outlined in Figure 61, any method for cell culture described herein can be performed using System 2300 (including the methods described above with reference to Figures 12-14).

[0183] Figures 62A–62C show a container / vessel lid 2408 according to an embodiment. The lid 2408 can be used in any embodiment of the cell culture system described herein. The lid 2408 can be screwed onto the mouth of a cell culture container or other container as described herein, so that the lid 2408 engages with the threads of the mouth of the cell culture container. In this exemplary embodiment, the lid 2408 has a liquid port 2483 and a gas port 2484. The liquid channel 2485 is screw-engaged with the liquid port 2483. The gas filter 2486 (see Figure 62C) is screw-engaged with the gas port 2483. The gas filter 2486 may allow gas exchange in and out of the cell culture container while preventing any microorganisms or pathogens from entering the cell container. In the embodiment, the gas filter 2486 is a 0.22 micron filter.

[0184] Figures 63A–63D show exemplary embodiments of the multiport valve 2407 according to an embodiment. The multiport valve 2407 can be used in any embodiment of the cell culture system described herein. In this embodiment, the multiport valve 2407 includes a valve body 2487 having a master port 2488 on its upper surface, and a plurality of selectable ports 2489 distributed around the valve body 2487 (see, for example, Figures 64A–64C).

[0185] The valve body 2487 has a cylindrical cavity on its underside into which a rotatable cylindrical valve rotor 2490 is inserted. Within the rotatable cylindrical valve rotor 2490 is a fluid channel 2491 (see Figures 65A-65C). Within the valve body 2487 is a fluid channel 2492 which fluidly connects the master port 2488 to the fluid channel 2491 of the valve rotor 2490. The connection between fluid channel 2492 and fluid channel 2491 allows the master port 2488 to be selectively fluidly connected to one of the side ports 2489 via the rotation of the valve rotor 2490 (and thus the fluid channel 2491). The valve rotor 2490 includes a mechanical coupler 2493 (see Figure 65C) configured to mechanically connect to the valve actuator of the system, the valve actuator may have a cavity shaped to receive the mechanical coupler 2493 and transmit rotational mechanical energy to the multiport valve 2407.

[0186] The multiport valve 2407 can be made of any suitable material, and the valve body 2487 and valve rotor 2490 can be made of the same or different materials. Examples of materials that can be used include plastics, TFE-based materials such as polytetrafluoroethylene (PTFE), metals, rubber, or similar materials. In some embodiments, the valve body 2487 and valve rotor 2490 may be machined to fit with very tight tolerances, resulting in a liquid-tight seal between the two components. In some embodiments, additional gaskets, bearings, seals, and / or flanges may be incorporated into the multiport valve 2407 to provide a liquid-tight connection between the valve body 2487 and the valve rotor 2490.

[0187] Figures 66A–66D illustrate schematic diagrams of an automated cell culture system according to another embodiment. This exemplary automated cell culture system 2600 includes a consumable or disposable cell culture tray assembly 2501 (also referred to herein as the “tray assembly,” see Figure 66A) and a reusable base unit 2620 (see Figure 66B). The disposable tray assembly 2601 includes various components described below, some of which are pre-assembled on (or together with) the tray assembly 2601 and sealed within a protective overlap 2615 to maintain the components in a sterile state. Some of the components of the tray assembly 2601 can be added to the tray assembly 2601 in a sterile environment (e.g., a laminar flow hood) before using the tray assembly 2601 in a cell culture procedure. Once the tray assembly 2601 is assembled and ready for use, the tray assembly 2601 can be coupled to the base unit 2620, as described in more detail herein. As shown in Figure 66A, the tray assembly 2601 includes a tray 2602 which can be detachably connected to a base unit 2620 as described herein. In some embodiments, the tray 2602 may include one or more transparent or notched portions so that objects placed on the top surface of the tray 2602 can be seen from the tray 2602. For example, as will be described in detail below, the cell culture system 2600 may optionally include imaging devices and / or other sensors (not shown) placed inside the base unit 2620 and under the tray 2602 when the tray assembly 2601 is connected to the base unit 2620. As will be described in detail below, the transparent portion(s) or notched portion(s) may allow images and / or other data to be acquired through the transparent portion(s) or notched portion, such as the contents of a cell culture container connected to the tray 2602.

[0188] As will be described in detail below, the cell culture system 2600 also includes a cell counting chip 2617 (see Figure 66D). The tray assembly 2601 also includes one or more couplers 2603 that can be used to hold cell culture containers or vessels, as previously described for previous embodiments. The tray 2602 also optionally includes holders 2604 that can be used to removably connect sample containers 2605 and waste containers 2606 to the tray 2602 (for example, to secure containers during transport, initial setup, etc.). Although two couplers 2603 are shown, in other embodiments there may be only one or more than two couplers 2603. For example, in some embodiments the tray assembly 2601 may be configured to support only one cell culture container and therefore include only a single coupler 2603 to maintain the cell culture container in a fixed position on the tray 2602. Similarly, although only one waste container 2606 and one sample container 2605 are shown, in alternative embodiments there may be multiple waste and sample containers. Furthermore, while Figure 66A shows the waste container 2606 and sample container 2605 as part of the tray assembly 2601, in other embodiments, the waste container 2606 and / or sample container 2605 may be separate components within the automated cell culture system 2600 that are not connected to the tray 2602 during use. For example, in some embodiments, the sample container 2605 may be used to contain cell culture medium and may be located in the cooling section (not shown) of the automated cell culture system 2600 or in another cooling location. The coupler 2603 and holder 2604 may be separate components attached to the tray 2602, or they may be components formed integrally or monolithically with the tray 2602. For example, in some embodiments, the coupler 2603 and / or holder 2604 may include a deformable bracket, a movable pin, or any other suitable structure for connecting the container to the tray 2602.In some embodiments, the tray assembly 2602 may optionally include a handle 2614 that a user can use to move and carry the tray assembly 2602. The handle 2614 may be a separate component from the tray 2602, or it may be integrally or monolithically formed with the tray 2602. In some embodiments, the tray assembly 2601 may not include a holder 2604. In this embodiment, the tray assembly 2601 may be pre-assembled with one or more cell culture containers. For example, in some embodiments, the cell culture containers may be provided as part of the consumable tray assembly, but may be removable from the tray if necessary. In some embodiments, the cell culture containers may be provided as part of the consumable tray assembly, but may be fixed or permanently coupled to the tray assembly. For example, the cell culture containers may be integrated with a container lid, fluid piping and / or a multi-port valve, etc.

[0189] As shown in Figures 66A and 66C, the tray assembly 2601 includes two cell culture containers 2647 and 2648. Each of the cell culture containers 2647 and 2648 is connected to a container lid 2608. In this embodiment, the container lid 2608 is connected to the cell culture containers 2647 and 2648. Therefore, the container lid 2608 and the containers 2647 and 2648 can be pre-connected, connected to the tray 2602, and sterilized within the protective overlap 2615. Thus, a pre-sterilized tray assembly with pre-connected and sterilized cell culture containers attached can be used, eliminating the need to connect the cell culture containers to the tray within the flow hood. In this exemplary embodiment, there are two containers 2647 and 2648 and two lids 2608, but of course, a different number of containers and lids 2608 can be provided. For example, referring to Figures 62A to 62C, each of the lids 2608 may include a liquid exchange port (also referred to herein as a "fluid port") and a gas exchange port, as described above (not shown in Figures 66A to 66C, respectively).

[0190] As illustrated, each fluid port is connected to a selectable port of the multiport valve 2607 by piping (see piping A, B, C, and D in Figure 66A). Gas exchange ports allow for the transfer of gas from the cell culture vessel to which it is connected. For example, in some embodiments, the lid 2608 may be similar to the cell culture vessel lid 803 or lid 2408 illustrated and described herein. For example, the lid 2608 may include a gas filter to prevent microorganisms and / or contaminants from entering the cell culture vessel, thereby allowing cell culture and fluid transfer through the lid 2608 while maintaining a closed (and / or sterile) system with other containers in the system (e.g., sample container 2605, waste container 2606, or other containers). In some embodiments, the tray assembly 2601 may optionally include lids 2609 and 2610 connected to the sample container 2603 and waste container 2606, respectively. Lids 2609 and / or 2610 may be similar in structure and function to lids 2608 and / or cell culture container lid 803.

[0191] The tray assembly 2601 also includes a multiport valve 2607 pre-assembled on the tray 2602, as described above and in detail below for previous embodiments. As illustrated, each fluid port is connected to a select port of the multiport valve 2607 by piping (see piping A, B, C, and D in Figure 66A). Thus, the cell culture containers 2647 and 2648, pre-assembled on the tray 2602 and connected to the lid 2608, have permanent fluid communication with the multiport valve 2607. The multiport valve 2607 may include the same or similar components and functions as the multiport valves described above for previous embodiments (for example, the multiport valve 600 or multiport valve 2407 described herein), in the same or similar manner.

[0192] More specifically, the multiport valve 2607 may include a master port 2651 (see Figure 66D) configured to connect to a fluid pump 2613 of the base unit (described below and shown in Figures 66B and 66C), and a number of selectable ports that can be fluid-connected to the fluid exchange ports of lids 2608, 2609, 2610 and / or other components of the cell culture assembly 2600 described herein. For example, as shown in Figure 66D, one of the selectable ports may be sterile and / or fluid-connected to a first fluid exchange port on a first lid 2608 connected to a container 2647, and a second selectable port may be sterile and / or fluid-connected to a second fluid exchange port on a second lid 2608 connected to a container 2648. In some embodiments, the third port of the multiport valve 2607 can be connected to the liquid exchange port of the sample container 2605 (see Figures 66A and 66C), the fourth port can be connected to the liquid exchange port of the waste container 2606 (see Figures 66A and 66C), and the fifth port can be connected to the liquid exchange port of the cell collection container 2652 (see Figure 66D). A schematic diagram of an exemplary system illustrating some other timely connections of the multiport valve is shown in Figure 59. Thus, when driven, the multiport valve 2607 can facilitate the exchange of fluids between various containers in the automated cell culture system 2600. For example, as described herein, the multiport valve 2607 can be driven to facilitate the addition of cell culture medium or reagents to cell culture containers, the removal of cells from cell culture containers (e.g., cell subculturing or cell collection), or any other fluid movement related to cell culture.

[0193] In this embodiment, as shown in Figures 66C and 66D, the cell counting chip 2617 is connected to the master port 2651 of the multiport valve 2607 between the multiport valve 2607 and the fluid pump 2613. As described herein, the cell counting chip 2617 may include a bottom transparent section which can be used to obtain information about the contents of the cell culture container. In some embodiments, instead of pre-assembling the cell counting chip 2617 on the tray assembly 2601, it may be connected to or mounted within the base unit 2620. The cell counting chip 2617 can be positioned upstream of the pump 2613 to allow cells to be counted without losing (or wasting) any cells. For example, cells are pumped from one of the containers 2647, 2648 to the retainer 2688, and the cells are counted as they pass through the cell counting chip 2617. Then the cells are pumped back from the retainer 2688 to the containers 2647, 2648. By having the cell counting chip 2617 in the channel to the master port 2651 (rather than in the channel connected to the waste chamber 2606), cells can be non-destructively counted and / or characterized within the cell counting chip 2617. In other words, depending on the desired method, the counted cells are not destroyed (or sent to the waste container 2606).

[0194] However, in an alternative embodiment, the cell counting chip is connected to one of the other selectable ports of the multiport valve 2607. In such an embodiment, the cell counting chip may be placed on one of the output lines communicating with the waste container 2606 so that the cell sample is flushed into the waste once cells have passed through and been counted.

[0195] As described above for previous embodiments, the tray assembly 2601 can be enclosed within the protective overlap 2615. In some embodiments, the tray assembly 2601 can be sterilized before being placed within the protective overlap. To prepare the cell culture system for use in cell culture, cells to be cultured can be added directly from the seeding container 2653 to the cell culture containers 2647 and 2648 contained within the tray assembly. The seeding container 2653 can be provided separately, or in some embodiments, it can be included within the tray assembly 2601. The seeding container 2653 does not need to be permanently fluidized with the multiport valve 2607; rather, it can be removed, loaded with cells, and reattached to establish fluidized connection with the multiport valve 2607. The seeding container 2653 may have a removable lid, or the fluid lines can be aseptically cut and reconnected. As described herein, the user can load the desired cells, reagents, cell culture media, etc., into containers (e.g., containers 2647, 2648, 2605) in a sterile environment. The tray assembly 2601 can then be connected to the base unit 2620 and moved into an incubation environment where fluid exchange is performed to ensure the desired cell culture, as described herein. The preparation process is simplified by loading cells into containers 2647 and 2648 using a single seeding container 2653 (as opposed to connecting each container to its corresponding lid after loading cells into each container).

[0196] When the tray assembly 2601 is attached to the base unit 2620, the multiport valve 2607 is configured to move away from the tray 2602 and engage with the valve actuator 2621 of the base unit 2620 (see Figures 66B and 66C). In some embodiments, the multiport valve 2607 may include a mounting portion 2616 configured to mate and connect to the valve connector 2622 of the base unit 2620. For example, the mounting portion 2616 may have a shape that allows it to connect to the valve connector 2622 like a puzzle. As shown in Figures 66B and 66C, once the multiport valve 2607 is engaged with the valve actuator 2621 of the base unit 2620, the valve actuator 2621 can drive the multiport valve 2607 to move to a selected port, enabling selective fluid transfer between the various containers and cell culture containers of the tray assembly 2601 (described below). In some embodiments, the multiport valve 2607 may be connected to the valve actuator 2621 while connected to the tray 2602. For example, a valve connector (not shown) connected to a valve actuator 2621 can be positioned on a lower base unit 2620, to which the tray assembly 2602 is removably connected (for example, as in the base unit 301 or base unit 2120 described herein). In some embodiments, a multiport valve 2607 can be removed from the tray 2602 (while remaining connected to the lid and container, thereby maintaining the closing system) and mounted to a mating valve connector 2622 on the base unit 2620, for example, as shown in Figures 66B and 66C. Figure 66B shows the connector 2622 without the multiport valve 2607 connected, and Figure 26C shows the connector 2622 with the multiport valve 2607 connected. In other words, the multiport valve 2607 can be removed from the mating mounting pocket 2618 of the tray 2602 (see Figure 66C) and mounted to the valve connector 2622 on the base unit 2620.As described above, the mounting portion 2616 of the valve 2607 is shaped to ensure proper positioning and alignment within both the tray assembly 2601 and the base unit 2620 by mating and engaging with the mounting pocket 2618 and the valve connector 2622 of the base unit 2620. This repositioning of the multiport valve 2607 can be performed while the covers 2608, 2609, and 2610 remain sterilely connected to the multiport valve 2607. By removing the valve 2607 from the tray 2602, the interface between the valve 2607 and the valve actuator 2621 can be fixed, which is well suited to embodiments that include a stirrer (e.g., the stirrer 2628 described below) that moves the tray 2602 relative to the base unit 2620. Similarly, by directly connecting the valve 2607 to the base unit 2620, the interface between the valve 2607 and the valve actuator 2621 is not obstructed by the relative motion between the tray 2601 and the base unit 2620.

[0197] The base unit 2620 (see Figures 66B and 66C) includes a housing 2623 that supports various components of the base unit 2620 and defines (or includes) a receiving portion 2624 that receives and removably connects the tray assembly 2601. In some embodiments, the receiving portion 6624 may include an opening into which the tray assembly 2601 can be positioned and supported by a tray support (not shown). In some embodiments, the tray assembly 2601 is supported by a support portion of the base unit 2620 such that the tray assembly 2601 is higher than the top surface of the base unit 2620. In some embodiments, the tray assembly 2601 is at least partially supported by engagement with a stirrer (described herein) of the base unit 2620. In some embodiments, the tray assembly 2601 can be removably connected to a separate support member that can be connected to the housing 2623 of the base unit 2620. The base unit 2620 may also include one or more transparent sections or openings corresponding to the transparent sections of the tray 2602 so as to be able to acquire images and / or other sensor data related to the contents of the cell culture container.

[0198] The base unit 2620 includes the aforementioned valve connector 2622 and valve actuator 2621, and also includes a fluid pump section 2627 and a pump actuator 2626. The pump actuator 2626 can be at least partially located within an opening 2625 defined by, for example, the housing 2623. In this embodiment, the fluid pump 2613 is provided with the base unit 2620 and can be coupled to the fluid pump section 2627. For example, the fluid pump can be a peristaltic pump coupled to the base unit 2620. In such an embodiment, when coupling the tray assembly 2601 to the base unit 2620, the user can complete the fluid pump by loading a portion of the piping in the closed system (including the container and valve) from the tray assembly 2601 into the head of the peristaltic pump. When in use, the head of the peristaltic pump includes rollers (or a set of rollers) that deform that portion of the piping to move the fluid (e.g., a cell sample) within the closed system. Therefore, the portion of the piping (that deforms) can be called a fluid pump, and the peristaltic pump head can be called a pump actuator (or part of a pump actuator). In other embodiments, the head of the peristaltic pump can be detachable (and separate) from the pump actuator of the base unit 2620. In such embodiments, the peristaltic pump head can be pre-attached to the piping and included in the tray assembly 2601. Therefore, when installing the tray assembly, the pre-connected pump head (i.e., the pump head including the portion of the piping) can be connected to the pump actuator of the base unit 2620. As described herein, the fluid pump 2613 can be used to generate fluid movement within the cell culture system 2600. The fluid pump 2613 can be any suitable pump that generates pressure and / or flow within the cell culture system 2600. For example, in some embodiments, the fluid pump 2613 can be a syringe including a piston rod and a syringe body. Various other positive displacement fluid pumps can be used.For example, as described herein, in some embodiments the pump may be a single-port pump, while in other embodiments the pump may be a two-port pump. The fluid pump 2613 can be fluidly connected to the master port 2651 of the multi-port valve 2607 by closed piping provided with the tray assembly 2601. In this exemplary embodiment, as shown in Figure 66C (showing the tray assembly 2601 connected to the base unit 2620), the multi-port valve 2607 is detached from the tray assembly 2601 and connected to the valve connector 2622, and the fluid pump 2613 is connected to the master port 2651 by piping E. The fluid pump 2613 may include movable members within the pump body (not shown in Figures 66B and 66C). As described above in relation to previous embodiments, during the operation of the system 2600, the movable members of the fluid pump 2613 (for example, plungers, rotors, or deformable parts of tubes) can be driven to generate suction force to guide the fluid into the pump body, and the movable members can be driven to push the fluid out of the pump body.

[0199] In some embodiments, the base unit 2620 may also include a stirrer 2628. The stirrer 2628 may include, for example, an orbital shaker that moves the tray 2602 in a circular or semicircular motion. As previously described for the earlier embodiments, the stirrer 2628 may be configured to stir the removable tray assembly 2601 relative to the housing 2623. The stirrer 2628 may stir the tray 2602 in a rocking, vibrating motion, circular swirling motion, or other motion useful for cell culture. In some embodiments, as described above, individual cell culture containers / vessels may be stirred independently by separate stirrers that move between the cell culture container and the removable tray assembly 2601. In some embodiments, a stirrer may not be included.

[0200] In some embodiments, the agitator 2628 may include a plate to which the tray assembly 2601 is connected when the tray assembly 2601 is connected to the base unit 2620 (similar to the support plate 2059 described herein). The agitator plate can be connected to an agitator actuator (not shown) by means of a screw coupling or the like. In some embodiments, the agitator 2628 can be detachably connected to the agitator actuator, for example, by a magnetic coupling that allows for easy removal and installation by the user. This arrangement allows the agitator plate to be removed for cleaning, sterilization, etc. In other embodiments, the agitator plate can be detachably connected to the base unit and / or the agitator actuator by any preferred mechanism (e.g., a clip, pin, etc.) that facilitates easy removal.

[0201] In some embodiments, the agitator 2628 can stir in an orbital pattern. In some embodiments, the agitator 2628 can be, for example, figure eight The user can program the system to stir using different patterns, such as specific patterns. Sometimes, depending on the application, certain stirring patterns may be preferred. For example, figure eight The pattern may be desirable for obtaining a uniform distribution of cells (for example, when seeding into a new cell culture vessel) or for mixing the fluid within the vessel. In some embodiments, the stirrer 2628 can be programmed by the user to stir with a windshield wiper (or reciprocating) motion. Such embodiments may be better for detaching cells from the vessel, for example, during subculturing or harvesting of adherent cells.

[0202] In some embodiments, the base unit 2620 may also optionally include one or a sensor 2629 (only one is shown in Figure 66B) and an electronic control system 2630 to control the operation of any of the components of the cell culture system 2600 (e.g., valve actuator 2621, pump actuator 2626). The electronic control system 2630 and sensor 1629 may be the same as, and may function in the same way as, the electronic control systems and sensors described herein for other embodiments. As previously stated, in some embodiments, a light or light source 2682 (see Figures 66B and 66C) may be provided, for example, which can be used in combination with an imaging device.

[0203] While the tray assembly 2601 is illustrated and described as including piping for interconnecting various containers to a multiport valve 2607, in other embodiments the tray assembly may include internally defined fluid passages to minimize (or eliminate) the use of piping. Figures 67A and 67B are a plan view and a schematic side view, respectively, of embodiments of trays that may be included in a consumable tray assembly for a cell culture system described herein. In this embodiment, the tray 2702 includes a fluid channel integrally formed within the tray body. Thus, instead of fluid piping, the fluid path is defined within the material of the tray body itself (e.g., plastic). The fluid path 2754 can be used to load and empty cell culture containers (e.g., cell culture containers, sample containers, waste containers, etc.) (which may also be part of the tray assembly as described above). More specifically, as shown in Figures 67A and 67B, the tray 2702 may include a fluid channel 2754 defined below the upper surface of the tray 2702. The fluid channel 2754 can fluidly communicate with an opening 2755 defined within the upper surface of the tray 2702. The opening 2755 can be positioned to fluidly communicate with, for example, cell culture containers 2747, 2748, and 2749. Thus, for example, cells from seeding containers 2753 can be loaded into the containers from below through the fluid channel 2754 and the opening 2750. The opening 2755 can be driven to open and close (for example, by a diaphragm). Such a tray can simplify the manufacturing and assembly of the tray assembly.

[0204] In some embodiments of the cell culture systems described herein, a multiport valve is driven to pump a fluid from a location in a first vessel (e.g., vessel 2605) to a second vessel (e.g., vessel 2747) so that a master port is connected to port A of the multiport valve, which is connected to the first vessel 2605. The pump draws the solution (e.g., fluid) from vessel 2605 into the master port. The master port transfers the fluid to a retainer immediately after the fluid pump, so that the solution (e.g., nutrient medium) from vessel 2605 is loaded into the retainer. The multiport valve is driven again so that the master port is connected to the second vessel 2647 via port B of the multiport valve. The pump pumps the solution from the retainer into vessel 2647 via port B.

[0205] In an alternative embodiment, the cell culture system can be configured to pump fluid from a first vessel to a second vessel without using the retainer described in the above example. For example, as shown in Figure 68, the cell culture system may include two multiport valves 2507 and 2507' (each connected to a fluid pump 2513). More specifically, the master port 2551 of multiport valve 2507 is fluid-coupled to the master port 2551' of multiport valve 2507' by the fluid pump 2513 between them. Thus, the fluid pump can operate to selectively pump fluid into and out of each of the multiport valves 2507 and 2507'. In such an embodiment, the fluid can be moved, for example, from a vessel 2547 connected to multiport valve 2507 via port A to a vessel 2548 connected to multiport valve 2507' via port B of multiport valve 2507' without the use of a retainer. For example, the multiport valve 2507 is driven to connect the master port 2551 to port A of the multiport valve 2507. The multiport valve 2507' is driven to connect the master port 2551' to port B of the multiport valve 2507'. Next, the pump 2513 is driven to pump the fluid directly from container 2547 to container 2548. In some embodiments, a check valve can be used to optionally deliver the fluid from either container 2547 or 2548 to the retainer as needed. In this embodiment, the number of fluid tubes can be reduced by using an additional multiport valve. In addition, having two multiport valves allows for easier distribution of the fluid. For example, in some embodiments, one valve can be directly connected to all containers located outside the system (e.g., reagent containers and waste containers), and a second valve can be connected to cell culture containers and cell counting chips located inside the system body (e.g., on trays).In some embodiments, one of the multiport valves can be located outside the incubator and connected to all external solutions (e.g., chilled solutions), while the other multiport valves can be located inside the incubator and connected to all containers (e.g., flasks). Thus, only a single tube is required to enter the incubator from outside. Specifically, by connecting all external solutions through a single multiport valve, the input to the tray assembly is formed through a single tube from the master port of the "external" multiport valve to the multiport valve inside the tray assembly. Therefore, this arrangement eliminates the need for multiple tubes (one from each container of external solution) routed from an external location (e.g., a refrigerator) into the tray assembly.

[0206] As previously described with reference to Figures 32-34, the cell culture systems described herein may include an optical imaging system that can be mounted within the housing of any of the base units of the cell culture systems described herein. The aforementioned microscopic imaging device 1960 includes an imaging device 1962 that can be viewed through a window or transparent portion on the top of the base unit and through notches (or transparent portions) in both the tray and an optional shaking platform (e.g., a support for the tray in contact with a stirrer). Thus, the microscopic imaging device 1960 can be used to obtain information relating to the contents of the cell culture container and / or information in the cell counting chip, as described herein. For example, in some embodiments, the microscopic imaging device 1960 can acquire images of the cell culture container and / or the cell counting chip during a cell culture procedure, and the images can be used to determine, for example, the density of the contents to determine the amount of cells in the container (e.g., in the case of suspension cells), or the percentage of confluence (i.e., the percentage of the area of ​​the container covered by cells), for example in the case of adherent cells.

[0207] As previously mentioned, in some embodiments, lights(s) or light sources(s) (not shown) can be mounted above the tray assembly of the system on a separate multi-axis gantry, thereby allowing them to be controlled to move to the same position as the microscope in the base unit. Alternatively, in some embodiments, the light sources can be mounted on the same side as the sample to be imaged. This type of imaging and illumination is called reflected illumination. Figure 69 illustrates an example of reflected illumination for a fluorescence system, although reflected illumination may also be used for bright-field applications. In this fluorescence example, the sample to be imaged is illuminated with light of color A using a light source from below the sample. The sample then emits its own light of color B (e.g., "fluorescence"), which is observed by a sensor. A dichroic mirror is included to stop the reflected light of color A from reaching the sensor but to allow the light of color B to pass through. Such a reflected illumination system can be incorporated into the cell culture systems described herein.

[0208] In such embodiments, the light source can be operably coupled to the same gantry (e.g., gantry 1961 described herein) as the imaging device (e.g., microscope) so that the imaging device and the light source can move together. Alternatively, the light source and the imaging device can be mounted on separate gantry, or in other cases, they can be mounted separately from the imaging device and operated independently. As described above for previous embodiments, the microscope imaging device (e.g., imaging device 1960) and the light source can be controlled by one of the electronic control systems and according to one of the methods described herein. For example, in some embodiments, the microscope imaging device 1960 (and any associated light source) can be controlled to automatically image a cell culture container (e.g., to generate a sensor output related to the cells in the container). A cell sensor module of the electronic control system (e.g., electronic control system 1630) or any other electronic control system described herein can receive the sensor output and generate a signal related to the amount of cells in the container (e.g., cell density or percentage of confluence). Next, based on this information, the electronic control system can generate one or more signals (e.g., valve control signals, pump control signals, stirrer signals, etc.) to initiate the transfer of cells from one cell culture container to another in the system. As similarly described, in some embodiments, a microscopy imaging device (e.g., imaging device 1960) can provide input for automated cell passage or cell harvesting operations.

[0209] In some embodiments of the cell culture system, a machine-readable optical label or barcode, such as a Quick Response Code (“QR code”), is included on the consumable tray assembly. In some embodiments, an imaging device (e.g., the aforementioned 1960) can be used to observe and confirm that the consumable tray assembly is an approved consumable tray assembly. For example, when the tray assembly is connected to the base unit, the imaging device can visually recognize / scan the QR code to confirm that the tray assembly is the approved tray assembly to be used with the base unit.

[0210] As previously mentioned with reference to Figure 17, any of the cell culture systems described herein may include an electronic control system (e.g., electronic control system 1630) that can be used to control the operation of the cell culture system. Referring to Figure 17, the electronic control system 1630 may communicate with other remote computing devices (e.g., computing device 1643) via a network 1646 (e.g., the Internet), for example, through a service platform 1642 and a cell culture application (i.e., app) 1644. In addition, or instead, the electronic control system 1630 may communicate with remote computing devices via a direct connection (e.g., a cable connected to the USB port of the base unit 1620). As shown in Figure 17, the electronic control system 1630 includes a network module 1640, which may be a hardware and / or software module (stored in memory 1632 and / or executed within the processor 1631). The network module 1640 is configured to exchange information related to the base unit 1620 and the remote computing device 1643 to facilitate the communication process. For example, the network module 1640 of the base unit 1620 can allow the remote computing device 1643 and the base unit 1620 to exchange short-term and / or long-term security keys to complete the pairing and bonding process.

[0211] The electronic control system 1630 can also include a notification module 1639 that can be a hardware and / or software module (stored in the memory 1632 and / or executed within the processor 1631). The notification module 1639 is configured to generate notifications related to any of the methods and / or application modules described herein. For example, in some embodiments, the notification module 1639 can generate notifications to be transmitted via the radio 1633 and received by the notification module of the remote computing device 1643. In this way, the notification module 1639 executed in the cell culture application can generate an output (such as a wireless communication signal, a GUI element, an audible output, a visual output, etc.) to notify the user of an event.

[0212] The remote computing device 1643 can generate notifications to the user via the cell culture application 1644 and receive input from the user in response to such notifications. Next, the remote computing device 1643 can send the input (or command) to the service platform 1642. Based on the user input, the service platform 1642 can send the command to the base unit 1620, and the base unit 1620 can then execute the command to perform the desired task (such as cell passage).

[0213] Figures 70-73 illustrate computer implementation methods. Here, the user receives notifications from the notification module described above. Notifications may include, for example, images and suggested actions. The user can review the data in the notification, or otherwise access the received information (for example, a link may take the user to the notification page), and use the data to determine the operation of the cell culture system (for example, whether the system should proceed with its suggested procedure). In some embodiments, the computer implementation method may include receiving user input to stop (or not proceed with) a particular operation. In this way, the user can disable any function regardless of whether the user agrees (or disagrees) with the automatically read values ​​or other information provided by the system. Figure 70 is a flowchart illustrating an exemplary workflow when detaching adherent cells. Figure 71 is a flowchart illustrating a workflow when counting cells. Figure 72 is a flowchart illustrating a workflow when measuring confluence. Figure 73 is a flowchart illustrating a workflow when there is an automated confluence / cell count that exceeds a threshold and triggers a suggested action. As illustrated, in some embodiments, the computer implementation method may include one or more modules that can generate signals and / or allow the system to perform specific tasks without further human intervention. For example, as shown in Figure 71, the method may include performing automated cell counting using an imaging system as described herein. The method may further generate one or more notifications that allow the user to invalidate the automatically generated counts. The method may further continue automated counting if the user does not provide an invalidation value.

[0214] In some embodiments of the cell culture systems described herein, the system includes a tangential flow filtration ("TFF") system (often also called cross-flow filtration or hollow fiber filtration). TFF can be used, for example, for in-line removal of cell dissociation reagents during passage. As described herein, when the number of culture vessels or containers of adherent cells (e.g., cells that adhere to surfaces) increases and the percentage of the container floor covered by cells (% confluence) exceeds a certain threshold, it becomes necessary to detach some cells and transfer them to a new container. As described herein (see, for example, Figure 12), dissociation reagents are enzymes used to detach adherent cells (i.e., cells that adhere to surfaces) from the surface. However, excessive exposure to cell dissociation reagents can be detrimental to the health of the cells. Typically, cell dissociation reagents are removed via a centrifugation process. This requires removing the cells from the system (for centrifugation) and returning them to the system. Repeated handling of cells, especially when they are removed and reintroduced into the system, can lead to cell damage and loss of multipotency; therefore, it is desirable to remove cell dissociation reagents while limiting cell handling. For example, centrifugation can cause cells to aggregate or be damaged due to the forces applied to the cells during the centrifugation process. Furthermore, using TFF may eliminate the need to remove cells by centrifugation (which requires user intervention). In addition, centrifuges can be large and cumbersome machines that are difficult to integrate in-line with cell culture systems. Below, we describe a method for successively and selectively removing dissociation reagents and / or other culture medium components using tangential flow filtration while maintaining cells in the container and / or the entire system. Moreover, such a system can, in some embodiments, adequately remove dissociation reagents from cultured cells. The ability to adequately remove dissociation reagents can be very important for distributing usable cells. For example, in some cases, "adequately removed" means that only trace amounts of dissociation agent remain in the cultured cells.For example, in some embodiments, thoroughly removing the dissociation reagent may include removing at least 99 percent of the dissociation reagent.

[0215] More specifically, adherent cells will behave differently or begin to die if the surface on which they are growing becomes too crowded. To mitigate this, cells are periodically detached by exposure to a cell dissociation reagent, which may be an enzyme or a chemical, and some of the cells are transferred to either waste or a new, empty flask (e.g., a cell culture vessel). For example, by including multiple cell culture vessels on a single tray assembly, the system described herein is well-suited for subculturing cells into a new (empty) cell culture vessel when the first vessel reaches its maximum cell capacity. The most commonly used cell dissociation reagents are a class of enzymes known as proteases (enzymes that break down proteins). These tend to be relatively indiscriminate in which proteins within a cell they digest. Therefore, to avoid compromising the health of the cells, exposure to the cell dissociation reagent should be minimized, and the cells should only be exposed for the length necessary to detach them.

[0216] Typically, in manual cell culture processes, cell dissociation reagents are removed by placing a cell suspension (a mixture of cells, cell dissociation reagent, and culture medium) in a centrifuge tube and rotating it in a centrifuge to force the cells to settle, forming a pellet at the bottom of the tube. The supernatant (the liquid above the cells) is then removed, and the cells are washed with buffer. The centrifugation process is repeated, and optionally, the cells are washed again with buffer to ensure the removal of the dissociation reagents. The cells are then resuspended in fresh culture medium and reintroduced into one or more cell culture vessels to continue culturing. This type of centrifugation process can be undesirable for several reasons. Firstly, performing centrifugation in-line can be very difficult, and therefore, it may be necessary to transfer the sample to a separate centrifuge and then return it to the cell culture vessel. Secondly, centrifuges typically take up a lot of space in an integrated design. Centrifugal force depends on the rotor diameter and rotational speed, so a large rotor or a motor capable of moving the elements at high speed is required. Thirdly, centrifuges can be dangerous if they mechanically fail during operation. For example, centrifuges can potentially aerosolize and disperse dangerous biological agents such as viruses. They can also potentially apply forces to cells that affect their health.

[0217] Some known systems employ standard (i.e., "through-flow") filtration methods. This typically fails to separate cells from dissociation reagents because the filter clogs rapidly, requiring high pressure (or long process times) to force the liquid through. Furthermore, cells can become trapped within the filter and, as a result, remain undetached even after backwashing.

[0218] An alternative to the aforementioned problems of centrifugation and standard filtration methods is to use tangential flow filtration for the removal of dissociation reagents. With tangential flow filtration, the solution flows parallel to the surface of the filter rather than through the filter, so that with each cycle, components smaller than the pore size of the filter membrane can pass through the filter (i.e., liquid and small solutes), while components larger than the pore size of the membrane are retained along with some unpermeated solution. For example, the pressure difference across the filter causes smaller components to pass through the filter, while larger components are retained by the filter and pass along the membrane surface. After a certain number of cycles, the liquid can be completely replaced without losing any solids. This approach reduces filter clogging because the flow through the filter constantly removes solids from the filter's blockage.

[0219] TFF can be used to remove cell dissociation reagents during cell subculturing, so as to be incorporated into the cell culture systems described herein. Figure 74 is a system diagram illustrating exemplary fluid flow within the system during cell culture treatment, various containers and other components that can be connected within the cell culture system, and a TFF system connected to and contained within the fluid flow system. While the system diagram illustrates various components of cell culture system 2800, this exemplary diagram can, of course, be applied to any of the embodiments described herein. In other words, the tangential flow filtration components and methods described for cell culture system 2800 can also be included in any of the cell culture systems described herein (e.g., cell culture systems 100, 200, 400, 1600, 1700, 2000, 2100, 2200, 2300, 2600, 2800, 3600, 4000, etc.).

[0220] Figure 74 illustrates a part of a cell culture system 2800, which includes a multiport valve 2807 connected to two cell culture containers A and B (not shown), and a culture medium container 2805 containing dissociation reagents. A first fluid pump 2813 with a fluid (or pump) holder 2874 is fluid-connected to the master port of the multiport valve 2807. The multiport valve 2807 is also fluid-connected to a TFF holder 2872. The TFF holder 2872 is fluid-connected to a second pump 2813' and a TFF cartridge 2856. The TFF cartridge 2856 is connected to a waste container 2806.

[0221] As shown in Figure 74, flow arrow 1 indicates how fresh culture medium is fluidly connected to the multiport valve. The multiport valve itself is connected to cell culture containers A and B, pump holder 2874, and TFF holder 2872. Flow arrow 2 indicates the fluid flow path from the multiport valve 2807 to cell culture containers A and B. Flow arrow 3 indicates how the cell solution circulates through the TFF cartridge to remove the dissociation reagent. Flow arrow 4 indicates the flow of permeate, which refers to the old culture medium (dissociation reagent and new culture medium that may be unintentionally lost) flowing through the holes in the TFF cartridge 2856 into the waste container 2806.

[0222] In one exemplary method of using TFF in a cell culture system, after adding a dissociation reagent to the cultured cells and after the user confirms through imaging that a set time has elapsed for the cells to adhere or detach of that type of cell, the system can optionally add a dissociation reagent neutralizer to slow down the effect of the dissociation reagent. Then, using TFF, this solution of dissociation reagent and neutralizer is thoroughly removed from the cells while fresh medium is added. The cells are transferred to a new container or harvested as needed. More specifically, referring to Figure 74, first, after the cells have detached, the detached cell solution (including the dissociation reagent) is transferred, for example, from containers A and / or B into the TFF holder 2872. The cell solution is circulated through the TFF cartridge 2856 by pump 2813' and returned to the TFF holder 2872. Periodically (or continuously), fresh medium is replenished in the TFF holder 2872 to replace the permeated old medium that has been removed while flowing through the TFF filter cartridge 2856. The old culture medium, passing through the filter cartridge 2856, is transferred to the waste container 2806. Ultimately, in one example, after approximately 5 cycles, up to 99 units of old culture medium in the cell solution are replaced with fresh culture medium. The cells are pumped from the TFF holder 2872 into a new cell culture container to complete the subculturing. For example, cells can initially be cultured in container A (not shown), and container B (not shown) can be empty. Similarly, container B can be an "expanding container" in which some of the cells can be subculturished to continue culturing after the dissociation reagent has been removed.

[0223] In some embodiments, the TFF cartridge can be included on a tray assembly for a cell culture system. For example, a tray assembly 2801 is illustrated as shown in Figure 75. The tray assembly 2801 includes a tray 2802, two cell culture containers 2847 and 2848, a waste container 2806, a culture medium container 2805, a valve 2807, and a TFF filter cartridge 2856. The TFF filter cartridge 2856 can be fluidly connected to one or more of the containers on the tray 2802 and to the valve 2807. The TFF filter cartridge 2856 can also be connected to a fluid pump as included and described for other embodiments herein.

[0224] Figures 76-80 each illustrate exemplary cell culture systems including a TFF system. Figures 76-80 illustrate various components of the cell culture system to illustrate the components and functions of the TFF system, although some components of the cell culture system are not shown or described. Naturally, any of the cell culture systems shown in Figures 76-80 may include any of the components included in other embodiments described herein. For example, although not necessarily shown in Figures 76-80, each cell culture system may include, as described for other embodiments herein, a multi-port valve or valve system to which the TFF is fluidly connected, various containers (such as cell culture containers, waste, reagents, and culture medium containers), and piping for fluidly connecting various components. Thus, the parts of the cell culture system illustrated and described in Figures 76-80 illustrate only the TFF system portion of the cell culture system. Furthermore, any of the TFF systems described in Figures 76-80 can be incorporated into any of the cell culture systems and / or tray assemblies described herein. For example, in some embodiments, tray assembly 4101 or any of the other tray assemblies described herein can be modified to include any of the TFF systems described in Figures 76-80.

[0225] Figure 76 illustrates a portion of the cell culture system 2900 (also referred to herein as the “System”). This includes a dual-pump TFF system, which includes a TFF cartridge 2956 for filtering cell solutions as described herein. The System 2900 includes a container 2972. The container 2972 ​​can function as a holder for containing a cell sample solution that may contain cells and other fluids (e.g., culture medium and / or reagents). In some embodiments, the container 2972 ​​can be maintained at a controlled temperature to maintain the viability of cells in the cell sample. For example, in some embodiments, the temperature is maintained at about 37 degrees Celsius (e.g., by a water bath). A first fluid pump 2913 is fluid-coupled to the container 2972 ​​and also to the TFF cartridge 2956. The TFF cartridge 2956 is also fluid-coupled to a second pump 2913'. The second pump 2913' is fluid-coupled to a waste container 2906.

[0226] As shown in Figure 76, the flow arrows indicate the flow of the cell sample solution through the TFF system. More specifically, the cell solution flows from container 2972 ​​to the first pump 2913 and then to the inlet of TFF cartridge 2956. The cell solution circulates through TFF cartridge 2956, for example, to remove dissociation reagents. Unpermeated cell solution flows out of the outlet of TFF cartridge 2956 and returns to container 2972. The permeate flow (referring to the old culture medium (dissociation reagents and new culture medium that may be lost unintentionally)) flows through the holes of TFF cartridge 2956 and exits from the outlet of the second TFF cartridge 2956, entering the waste container 2906. In this embodiment, the second pump 2913' helps to draw the permeate out of TFF cartridge 2956 and into the waste container 2906. Similarly, the second pump 2913' can maintain a desired flow rate of permeate out of the TFF cartridge. Specifically, the operating conditions of the second pump 2913' can be adjusted to adapt to potential changes in the TFF filter (e.g., clogging or increased filter load), thereby maintaining the desired outlet flow.

[0227] Figure 77 illustrates a portion of the cell culture system 3000 (also referred to herein as the “System”). This includes a dual-pump TFF system, which includes a TFF cartridge 3056 for filtering cell solutions as described herein. In this embodiment, the TFF system is also illustrated in an example of use for washing cell solutions. The System 3000 includes a container 3072. The container 3072 can function as a holder capable of containing a cell sample solution that may contain cells and other fluids (e.g., culture medium and / or reagents). In some embodiments, the container 3072 can be maintained at a controlled temperature to maintain the viability of cells in the cell sample. For example, in some embodiments, the temperature is maintained at approximately 37 degrees Celsius. A first fluid pump 3013 is fluid-coupled to the container 3072 and also to the TFF cartridge 3056. The TFF cartridge 3056 is also fluid-coupled to a second pump 3013'. The second pump 3013' is fluid-coupled to a waste container 3006 and to a container 3048 with washing reagents.

[0228] As described above, this embodiment illustrates a TFF system used to wash the TFF cartridge by performing a backflushing. For example, if the TFF filter is clogged, the filter is no longer effective. In such cases, the TFF system can be driven to run in a backflow configuration. In some embodiments, filter clogging can be determined by a drop in pressure or an increase in power required to operate the pump in a forward configuration (for example, to wash cells). Running in a backflow configuration allows the cell sample from container 3072 to first be transported into the appropriate container in the system. This allows container 3072 to contain water or other reagents used to facilitate the backflushing operation. A second pump 3013' can pull the washing reagent from container 3048 and pump it into the TFF cartridge 3056. The washing reagent travels backward through the porous filter media, enters the main chamber of the TFF cartridge 3056, and exits through the inlet port of the TFF cartridge 3056. The washing reagent is delivered into container 3072 and mixed with any other fluids inside (e.g., water, other reagents). The liquid from container 3072 flows backward into the impermeable outlet of TFF cartridge 3056. In this operation, culture medium (not cells) can be loaded into container 3072. This flow from container 3072 can be controlled by pump 3013, which in turn can cause the flow in the main chamber of TFF cartridge 3056 to move backward. The two flow processes flow into the main chamber of TFF cartridge 3056 and exit through pump 3013'. The flow path to waste container 3006 can be blocked, and the output fluid flow from TFF cartridge 3056 and pump 3013' can flow into container 3072.

[0229] Another embodiment of the TFF system may include a dual-cartridge filtration system that can enable cell passage and extraction of biological material in a single filtration process. By having a system with two or more TFF filter devices, each having membranes of different pore sizes connected in series, instead of a single TFF cartridge, different components can be separated based on size. In one example, a first TFF cartridge (or the first filter portion of the cartridge) can extract cells, and a second TFF cartridge (or the second filter portion of the cartridge) can remove viruses. In some embodiments, a third TFF cartridge (or the third filter portion of the cartridge) can extract antibodies. Such a TFF system allows for the selective removal of culture medium components. For example, changing cell culture medium can often be harmful to cells, especially if they require autologous growth factors to function. The ability to change the medium without extracting specific components can be useful for certain cell types. Figure 78 illustrates a portion of a cell culture system 3100 (also referred to herein as the “System”) that includes a dual pump and a dual-cartridge TFF system. The TFF system includes a first TFF cartridge 3156 and a second TFF cartridge 3156' for filtering cell solutions as described herein. In this embodiment, the TFF system is illustrated in examples of its use for washing or filtering cell solutions as described above for systems 2900 and 3000, and for filtering and collecting viruses from permeates that would normally be sent to waste, for example. System 3100 includes a container 3172 that can function as a retainer for containing a cell sample solution that may contain cells and other fluids (e.g., culture medium and / or reagents). In some embodiments, the container 3172 can be maintained at a controlled temperature to maintain the viability of cells in the cell sample. For example, in some embodiments, the temperature is maintained at about 37 degrees Celsius (e.g., by a water bath). A first fluid pump 3113 is fluid-coupled to the container 3072 and also to the first TFF cartridge 3156.The first TFF cartridge 3156 is also fluid-connected to the second pump 3113'. The second pump 3113' is fluid-connected to the inlet of the second TFF cartridge 3156'. The second TFF cartridge 3156' is fluid-connected to the waste container 3106 and the collection container 3148.

[0230] As shown in Figure 78, the flow arrows indicate the flow of the cell sample solution through the TFF system. More specifically, the cell solution flows from container 3172 to the first pump 3113 and then to the inlet of the first TFF cartridge 3156. The cell solution circulates through the first TFF cartridge 3156, for example, to remove dissociation reagents. Unpermeated material from the cell solution flows out of the outlet port of the first TFF cartridge 3156 and returns to container 3172. In this embodiment, the permeate flow (referring to the old culture medium (dissociation reagents and new culture medium that may be unintentionally lost)) flows through the holes of the TFF cartridge 3156, exits the second outlet port of the first TFF cartridge 3156, and enters the inlet port of the second TFF cartridge 3156'. In this embodiment, the second pump 3113' can be used to pump the permeate out of the first TFF cartridge 3156 and into the second TFF cartridge 3156'. The permeate from the first TFF cartridge 3156 can be filtered by the second TFF cartridge 3156', and the unpermeated material from the second TFF cartridge 3156' can flow out through the first port of the second TFF cartridge 3156' and enter the collection container 3148. The unpermeated material from the second TFF cartridge 3156' may include viruses or other particles that are desired to be collected from the cell culture. The permeate from the second TFF cartridge flows out through the second outlet port' of the second TFF cartridge 3156 and enters the waste container 3106.

[0231] Figure 79 illustrates a portion of the cell culture system 3200 (also referred to herein as the “System”). This includes a dual-pump TFF system, which includes a TFF cartridge 3256 for filtering cell solutions as described herein. The System 3200 includes a container 3272. The container 3272 can function as a holder for containing a cell sample solution that may contain cells and other fluids (e.g., culture medium and / or reagents). In some embodiments, the container 3272 can be maintained at a controlled temperature to maintain the viability of cells in the cell sample. For example, in some embodiments, the temperature is maintained at approximately 37 degrees Celsius. A first fluid pump 3213 is fluid-coupled to the container 3272 and also to the TFF cartridge 3256. The TFF cartridge 3256 is also fluid-coupled to a second pump 3213'. The second pump 3213' is fluid-coupled to a waste container 3206.

[0232] As shown in Figure 79, the flow arrows indicate the flow of the cell sample solution through the TFF system. More specifically, the cell solution flows from container 3272 to the first pump 3213 and then to the inlet of TFF cartridge 3256. The cell solution is circulated through the cycle TFF cartridge 3256, for example, to remove dissociation reagents. Unpermeated material from the cell solution flows out of the outlet of TFF cartridge 3256 and returns to container 3272. The permeate flow (referring to the old culture medium (dissociation reagents and new culture medium that may be lost unintentionally)) flows through the holes of TFF cartridge 3256 and exits from the outlet of the second TFF cartridge 3256, entering the waste container 3206. In this embodiment, the second pump 3213' helps to draw the permeate out of TFF cartridge 3256 and into the waste container 3206. Similarly, the second pump 3213' can maintain a desired flow rate of permeate out of the TFF cartridge. Specifically, the operating conditions of the second pump 3213' can be adjusted to adapt to potential changes in the TFF filter (e.g., clogging or increased filter load), thereby maintaining the desired outlet flow. In addition, in this embodiment, a pressure sensor 3298 is placed in the fluid line between the second pump 3213' and the TFF cartridge 3256. The pressure sensor 3298 can be used to measure the pressure in the system to obtain an indicator of the overall flow through the system. For example, by measuring the inlet pressure and permeate pressure, some indication of a pressure drop (and therefore a decrease in flow) through the porous membrane of the TFF cartridge 3256 (i.e., the waste flow) can be obtained. The pressure drop can be used to evaluate the waste flow (e.g., whether the filter media is clogged). By using positive displacement fluid pumps (e.g., pumps 3213 and 3213') instead of centrifugal pumps, the pump speed is directly related to the flow rate. Therefore, in some embodiments, the system may not include the illustrated pressure sensor. Instead, the flow rate can be determined based on the pump speed.

[0233] FIG. 80 illustrates a portion of a cell culture system 3300 (also referred to herein as the “system”) that includes a dual pump TFF system and a dual multi-port valve. The TFF system includes a TFF cartridge 3356 for filtering cell solutions as described herein. In this embodiment, a first multi-port valve 3307 and a second multi-port valve 3307' are connected to a first pump 3313. The valves 3307 and 3307' and the first pump 3313 are configured to allow fluid to enter and exit various containers of the system 3300 (e.g., cell culture containers, media containers, sample containers, etc.) and to move between the containers and the TFF cartridge 3356, as described for other embodiments herein. As shown in FIG. 80, the second multi-port valve 3307' is connected to a cell culture container 3347 and a pump reservoir 3374. The system 3300 also includes various other containers, such as the sample container 3305 and the cleaning solution container 3348 shown in FIG. 80. These are connected to the first multi-port valve 3307'. The pump reservoir 3374 can be selectively connected to the outlet of the pump 3313 via the second valve 3307'. The pump reservoir 3374 can be used to hold any fluid within the device. In the case of the TFF process, the pump reservoir 3374 can hold, for example, “warm fresh media”. The pump reservoir 3374 can also be used to receive solution from the permeate output as needed.

[0234] The system 3300 also includes a container 3372 which can function as a holder for containing a cell sample solution that may contain cells and other fluids (e.g., culture medium and / or reagents). In some embodiments, the container 3372 can be maintained at a controlled temperature to maintain the viability of the cells in the cell sample. For example, in some embodiments, the temperature is maintained at approximately 37 degrees Celsius. A second fluid pump 3313' is fluid-coupled to the container 3372 and also to a TFF cartridge 3356. The TFF cartridge 3356 is also fluid-coupled to a second multiport valve 3307. The second multiport valve 3307 is coupled to the first pump 3313. The first pump 3313 is coupled to the first multiport valve 3307 and the waste container 3306. In this embodiment, the first pressure sensor 3398 is located in the fluid line between the TFF cartridge 3356 and the second multiport valve 3307', and the second pressure sensor 3398' is located between the second pump 3313' and the TFF cartridge 3356. Using the first pressure sensor 3398 and the second pressure sensor 3398', the same or similar information as described above can be obtained for the pressure sensor 3298.

[0235] As shown in Figure 80, the flow arrows indicate the flow of the cell sample solution through the TFF system. More specifically, the cell solution flows from container 3372 to the second pump 3313 and then to the inlet of TFF cartridge 3356. The cell solution is circulated through the cycle TFF cartridge 3356, for example, to remove dissociation reagents. Unpermeated cell solution flows out of the outlet of TFF cartridge 3356 and returns to container 3372. The permeate flow (referring to old culture medium (dissociation reagents and new culture medium that may be unintentionally lost)) flows through the holes of TFF cartridge 3356 and exits from the outlet of the second TFF cartridge 3356. In this embodiment, the permeate flows through the first and second multiport valves 3307 and 3307' and the first pump 3313 to the waste container 3306.

[0236] By filtering / removing dissociation reagents using the TFF system, undesirable components can be removed in-line after passage of adherent cells without losing cells during the process. As mentioned above, such cell loss or damage can be achieved by other filtration methods (e.g., standard filtration or centrifugation as described above) or by performing the removal offline. TFF allows for the replacement of degraded media in suspension cell cultures without losing cells or diluting the degraded media with fresh media. Furthermore, cell solutions can be concentrated without losing cells. The TFF system also reduces or eliminates the need to change the medium of the solution to discard debris formed in the cell solution.

[0237] TFF filtration can be performed, for example, following adherent cell passage, to replace the medium containing dissociation reagents in the cell solution with fresh medium. TFF can also be performed following the resuscitation of frozen cell solutions, i.e., to replace the frozen medium in the cell solution with fresh medium. As described herein, TFF can be used to replace degraded medium in suspension cell cultures without losing cells or simply diluting the degraded medium with fresh medium. Another use of TFF involves directly extracting purified biologics (e.g., viruses and proteins) from the medium without simultaneously extracting cells (with the separation of the biologics from downstream cells).

[0238] TFF filtration can also be used when harvesting adherent cells. For example, old culture medium containing dissociation reagents can be removed from the cell solution without losing cells and replaced with fresh medium or another liquid (as needed for the harvested cells' intended use case). TFF can also be used when harvesting adherent cells for collection by concentrating the cell solution to a desired cell density, or when harvesting cells to be frozen by removing, for example, the medium used to grow the cells without losing cells and replacing it with frozen medium.

[0239] In some embodiments, TFF can be used to remove contamination smaller than the cells being cultured (e.g., bacterial contamination) from the cell culture. The detached cell solution is circulated through the TFF, and the contamination is slowly removed with each circulation through the filter. The cell solution is periodically or continuously replenished with fresh, uncontaminated medium.

[0240] In some embodiments, TFFs can be used to completely replace the culture medium in which stem cells are growing with new medium required for specific steps in a differentiation protocol (a protocol to transform stem cells into a particular type of cell), ensuring that chemicals in the old medium do not interfere with the differentiation process. This is done by circulating the detached cell solution through the TFF, while the old medium is lost through the pores, with new medium being added periodically or continuously to the cell solution.

[0241] In some embodiments, the TFF cartridge may eventually become too clogged to be reused. For example, in some cases, the TFF filter may clog if the cell solution loses liquid too quickly. In one embodiment of the TFF system, the cell solution can be passed through the inside and outside of the cartridge to help prevent clogging. When the cell solution is passed through the inside, the old medium seeps out and enters the permeate stream, which is then discarded. New medium is added into the TFF holder. Cells slowly clog the inside of the cartridge over time. When the cell solution is passed through the outside of the cartridge (permeate stream), the old medium seeps in and enters the impermeate stream (solution that passes along the surface of the membrane), which returns to the supply reservoir (e.g., a reservoir with solution directed towards the membrane), which is then discarded. New medium is added into the TFF holder, and as the old medium seeps in, it detaches cells from the inner wall and is discarded. Cells slowly clog the outside of the cartridge.

[0242] In another exemplary embodiment, a flow sensor may be included to prevent clogging of the filter cartridge. For example, in some embodiments, the flow sensor can be used to calibrate the TFF for different types of cells at different densities in the solution. In some embodiments, the flow sensor may be included as part of the consumable tray assembly. In some embodiments, the flow sensor may be placed in the permeate line to determine how much fluid is being removed. This allows the system to replenish the retainer with fresh medium at a similar rate. This can also be inferred by placing the flow sensor on the unpermeate line instead. If the system replenishes too slowly, the cell solution may become too dense, and the filter may clog because there is not enough fluid to move the cells through the filter fibers.

[0243] In some embodiments, the system may include a flow sensor integrated therein, for example, above either the permeate line or the impermeate line. In such embodiments, the flow sensor can be used to determine when sufficient total liquid has been removed, and to confirm that the old medium has been completely or nearly completely removed. At that point, the system can stop filtering the solution. Thus, this arrangement avoids the disposal of new medium, unnecessary handling of cells, and the time-limited but continuous removal and disposal of new medium.

[0244] In one embodiment that does not use a flow sensor, the system can provide a list or table of fluid removal rates for different cell types at different concentrations, allowing the user to select the correct setting for each cell type and density. Furthermore, to remove a large portion of the old culture medium (similar to the amount removed by centrifugation), the system can be calibrated to determine how much of the total fluid needs to be removed (i.e., how long it needs to be circulated through the TFF system) relative to a given total volume of cell solution.

[0245] In yet another exemplary embodiment, the TFF system may include pre-treated membranes or alternative membrane materials to enhance filtration capacity. For example, pore size membranes are typically defined based on exclusion limits, and therefore smaller pores are always present in larger pore membranes. Pre-coating and treatment can improve the uniformity of the filtration process, i.e., by blocking the smallest pores. TFFs can be used to break up large cell aggregates because the cross-sectional area of ​​the filter is limited, and different cell types can be separated based on their tendency to permeate through the filter.

[0246] In some embodiments, a TFF system can be used in any of the systems illustrated and described herein to facilitate a cell harvesting method in which cell density can be adjusted. Specifically, in certain situations when cells are used for testing purposes, it may be desirable to use a solution in which the cell density (or volume) is within a given range. Therefore, if the harvested cell solution is below the desired density, current methods include a further step of taking a portion of the supernatant (e.g., solution) and processing the cells (e.g., via centrifugation) to increase the cell density. However, if the harvested cell solution is above the desired density, current methods include a further step of processing the cells to add more solution. In some embodiments, a TFF system can produce a cell solution for harvesting that is within a desired cell density range. Specifically, after removing potentially harmful dissociation reagents, the cell solution can be measured (e.g., by a counting chip) to determine the current cell density. The cell solution can then be processed through the TFF to remove excess solution (if the cell density is too low) and remeasured. Alternatively, if the cell density is too high, additional cell medium can be added. Thus, cells can be harvested from the system at the desired density. In some embodiments, the user can select a desired cell density (using an electronic control system such as the electronic control system 1630). Thus, the system can be adapted to provide cells at a desired density for various different cell types, use cases, etc.

[0247] The following describes various methods and workflows for culturing and / or processing stem cells, including the use of a TFF system, according to various embodiments. All workflows described below assume the use of any embodiment of the systems and methods described herein (e.g., cell culture system 4000). For simplicity, adding medium, transferring cells, measuring confluence, and removing and adding fresh liquid with TFF may not be described for these workflows without explicit detail. These details can be found herein, as they are described for other similar use cases (e.g., feeding, passaging, removing dissociation reagents, and detecting confluence on adherent cells).

[0248] The described workflow, including the use of TFF, is particularly well-suited for culture operations performed with pluripotent stem cells (PSCs), such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). In one exemplary workflow, feeder-free PSC cultures (cultured as single cells or aggregates) can be maintained on the device. A wide range of dissociation reagents are used to detach cells from the cell culture vessel during the cell culture process. However, these reagents must be removed from the medium after the cells have been detached because they can be harmful to stem cells. Centrifugation is typically used for this purpose. A novel approach involves using TFF to remove the dissociation reagents from the cells. When the medium reaches the desired / appropriate confluence (detected by a microscope combined with either algorithmic or user input), the dissociation reagent is added to detach the cells. The system then neutralizes the dissociation reagent by adding a neutralizing solution as soon as it detects that the cells have been sufficiently detached (detected by a microscope combined with either algorithmic or user input). Next, use TFF to thoroughly remove the dissociation reagent and transfer the cells to a new pre-coated flask.

[0249] Another exemplary workflow involves maintaining 3D aggregates of stem cells, such as embryoid bodies, organoids, and spheroids. 3D structures are most commonly generated from PSCs for three main reasons: i) to test whether the PSCs have retained their ability (i.e., to form many different cell types); ii) to initiate the first step of a differentiation protocol; and iii) to differentiate PSCs into desired lines because 3D media better replicates physiological conditions. In these use cases, these 3D suspension structures need to be supplied with fresh medium, and the depleted medium in which they were cultured needs to be removed. Removing medium from such a medium is time-consuming and difficult because there is a risk that these structures may be damaged or removed from the medium during the medium change. By changing the medium using TFF, this process can be carried out efficiently without losing the 3D structures. The system optionally continuously agitates the flask to prevent the aggregates from sticking together. After aspirating the aggregates, the system circulates the aggregate solution through the TFF. Some old culture medium is usually lost through the filter holes with each circulation, but the aggregates are retained. Regularly or continuously, replenish the aggregate solution with fresh culture medium. Eventually, all old culture medium is replaced with fresh culture medium. The aggregates are either returned to the old flask by the system or seeded into a new flask.

[0250] Another exemplary workflow involves feeder-free culture to maintain PSCs. Here, MEF cells are used to condition the medium. Generally, feeder-free culture is easier to handle but requires more expensive conditioning media. The method combines the best aspects of both approaches. MEF cells are housed in one flask, and these cells secrete chemicals that condition the medium in which they are growing. Periodically, the system pumps some or all of the supernatant from this first flask into a second flask of PSCs on the same machine, supplying it to the second flask. This avoids the need to purchase expensive media for PSCs. Optionally, TFF can be performed on the medium to remove cell debris before supplying it to the second flask (collecting the filtered medium through the permeate).

[0251] Another exemplary workflow involves co-culturing ("feeder" culture) PSCs to maintain them. Often, PSCs grow on a bed of different types of cells. These "feeder" cells secrete chemicals that aid in the growth of PSCs. In this workflow, mitotically inactivated (unable to perform "mitosis," i.e., unable to divide) mouse embryonic fibroblast (MEF) cells are seeded into one flask (coated with gelatin or Matrigel). The MEF cells are maintained for 2-3 days. After this period, PSCs are seeded into the same flask and grown on top of the MEF cells. The cells are then passaged together for 1-2 passages into another coated flask. When it is desirable to harvest only PSCs, one of the following steps is performed. The first option is to repeat the process several times to allow both types of cells to adhere in the coated flask and then (detach) passage them into a new flask. Since MEF cells do not divide but PSCs do, they are diluted after several passages. The second option is to passage them into a gelatin-coated flask. Since MEF cells attach first, if the supernatant is extracted from the flask within 30 minutes (and as soon as MEF cells are found to have attached), the supernatant will contain only PSCs. This supernatant can then be subcultured in a newly coated flask. A third option is to use TFF to size-separate larger MEF cells from smaller pluripotent stem cells. Pluripotent stem cells are collected in the permeate.

[0252] Another workflow involves co-culturing human fibroblasts to reprogram them into iPSCs. This workflow is the same as the one described above, except that human fibroblasts are used instead of MEFs, and a separate set of human fibroblasts to be reprogrammed is used instead of PSCs. Further reagents are needed to bring about the actual reprogramming.

[0253] Another workflow involves reprogramming human fibroblasts into iPSCs using feeder-free culture. Human fibroblasts are used to condition the culture medium. Generally, as mentioned earlier, feeder-free culture is easier to handle but requires more expensive conditioning media. One flask contains human fibroblasts. These cells secrete chemicals that condition the medium they are growing in. Periodically, some or all of the supernatant is taken from this first flask and used on the same machine to feed into a second flask containing human fibroblasts to be reprogrammed. This avoids the need to purchase expensive media for use during reprogramming. Optionally, TFF (transfer of fluid) is performed on the medium to remove cell debris before feeding it into the second flask (the filtered medium is collected via permeate).

[0254] In an alternative workflow, feeder-free culture is used to differentiate PSCs into a different cell type B. The culture medium is conditioned using cell type A. In this method, cell type A cells are contained in a flask, and these cells secrete chemicals that condition the medium in which they are growing. Periodically, some or all of the supernatant is taken from this first flask and used on the same machine to supply a second flask containing PSCs to be differentiated into cell type B. This avoids the need to purchase expensive culture media for use during differentiation. For culture media used to differentiate PSCs into nerve cells, cell type A can be stromal cells or astrocytes. Optionally, TFF is performed on the medium to remove cell debris before supplying it to the second flask (the filtered medium is collected via permeate).

[0255] In another workflow, macrophages are harvested using monocyte culture. Monocytes are a type of cell that differentiates into macrophages. When they divide, one becomes a monocyte and the other a macrophage (so they essentially "produce" macrophages like a factory). Flasks of adherent monocytes divide and differentiate, producing macrophages in the culture (so the monocyte population is almost maintained). Macrophages are suspended in the culture medium. Periodically, the system extracts the medium from the flask to collect the macrophages. The system uses TFF to concentrate the harvested macrophages before they are collected, so the user does not need to remove large amounts of medium. Optionally, cells can also be counted before harvesting so the user knows the density of macrophages in the solution. Optionally, cell counting can be used over time to track macrophage production.

[0256] In an alternative workflow, TFF is used to remove contaminated cells during PSC culture maintenance. During PSC maintenance, contaminated cells are those cells that the user does not want to differentiate (they are the result of unwanted differentiation). In cultures with some undifferentiated PSCs, as well as unwanted differentiated cells (i.e., contaminated cells), all cells can be detached using a dissociation reagent. Using TFF, contaminated cells can be separated from the cells that are to be retained. If the PSCs are smaller than the contaminated cells, they are collected in the permeate, and the stained cells enter the unpermeate. If the PSCs are larger, the reverse is true. Optionally, the PSCs are subculturised into a new flask, in which case the culture is contaminant-free.

[0257] Another workflow involves using TFF to remove contaminated cells during the differentiation of PSC cultures. This workflow is the same as or similar to that described above. An example of the use of such a method is when attempting to form neural stem cells, and neural crest stem cells can also be formed as contaminated cells. In both this method and the previous methods, the contaminated cell workflow can be triggered by the system detecting cells of an unwanted morphology.

[0258] The automated cell culture system 1600 and other systems, as shown in the illustration, include a multiport valve (for example, multiport valve 1607 or multiport valve 2607), but in other embodiments, the automated cell culture system 1600 may include any suitable valve assembly (or set of valves) configured to control the flow into and out of various containers within the system. For example, in some embodiments, the automated cell culture system may include a set of valves (each independently driven) configured to collectively control the flow into and out of containers within the system. Each valve may be driven by a single actuator (for example, an electronic, pneumatic, or hydraulic actuator).

[0259] For example, Figure 81 is a schematic diagram of a cell culture system 3600 according to an embodiment that includes a valve assembly 3607 having a series of individual valves for controlling fluid flow, but without a multiport valve. Similar to the cell culture systems described herein (e.g., systems 1600, 1700, and 2000), the cell culture system 3600 includes a consumable or disposable cell culture tray assembly 3601 (also referred to herein as the “tray assembly”) and a reusable base unit (not shown, but similar to any of the base units described herein). The disposable tray assembly 3601 includes various components described below. Some of these are pre-assembled on (or with) the tray assembly 3601 and sealed within a protective overlap to maintain the components in a sterile state. In some embodiments, some of the components of the tray assembly 3601 can be added to the tray assembly 3601 in a sterile environment (e.g., a laminar flow hood) before using the tray assembly 3601 in a cell culture procedure. Once the tray assembly 3601 is assembled and ready for use, it can be connected to the base unit as described herein.

[0260] A base unit (not shown, but may be, for example, base unit 1620, 1720, or 2020) is a reusable component of the system and includes articles that operate on or interact with the tray assembly 3601 to facilitate the cell culture method described herein. For example, a base unit may include a fluid pump 3613 or pump actuator (not shown) that generates a desired fluid flow within the system 3600, a stirrer (not shown), one or more sensors (not shown) that detect information relating to the contents of a cell culture container, and an electronic control system, or any or all of these.

[0261] Tray assembly 3601 is similar to, and may include, any of the components from the tray assemblies described herein (e.g., tray assembly 1601, tray assembly 1701, or any of the tray assemblies 2001). Therefore, they will not be described in detail below. As shown in the figure, tray assembly 3601 includes tray 3602 which can be detachably connected to a base unit. Tray 3602 includes a set of cell culture containers 3647, 3648, 3649, a waste container 3606, and a sample container 3605. The containers are connected to each other and connected to a pump 3613 via a valve assembly 3607 (including a set of independent valves 3607A-3607G), piping, and a retainer 3674. The retainer 3674 may be a container or vessel with a structure similar to the other containers in the system. In other embodiments, the retainer 3674 may be a manifold structure (e.g., consisting of piping) that facilitates multiple inputs and outputs, as shown in the figure.

[0262] The containers can be connected to the tray 3602 in any preferred manner. For example, in some embodiments, one or all of the containers are not included in the sterile package as part of the tray assembly 3601, but rather are provided separately, such as the cell culture containers 1647 and 1648 of the tray assembly 1601 described above. In such embodiments, the cell culture containers 3647, 3648, 3649, sample container 3605, and waste container 3606 can be prepared (e.g., for seeding cells, loading nutrients, etc.) and connected to the tray 3602 in a sterile environment (e.g., a fluid hood). In other embodiments, one or all of the containers are not included in the sterile package as part of the tray assembly 3601, but can be removed from the tray during use (e.g., for refrigeration) and / or fluidly separated from the system to facilitate the startup procedure. In yet another embodiment, one or all of the containers are not included in the sterile package as part of the tray assembly 3601, but are permanently attached to the tray 3602 and / or are not fluidly separated from the system during use, as are the cell culture containers 2647, 2648 of the tray assembly 2601 described herein. Thus, the tray 3602 may include any suitable mounting clips or structures configured to attach the containers to the tray, as described herein.

[0263] Each container is connected to a container lid having fluid exchange ports and gas exchange ports, similar to lid 803 or lid 2408 as illustrated and described herein. Specifically, cell culture containers 3647, 3648, and 3649 are connected to lid 3608, the sample container to lid 3609, and the waste container to lid 3610. In some embodiments, lids can be detachably connected to their corresponding containers (e.g., to facilitate startup procedures, system maintenance, etc.). In other embodiments (e.g., as described for cell culture system 2600), lids are permanently connected to their corresponding containers. As illustrated, each lid is fluid-connected within system 3600 via piping. In this way, fluids can be transferred between various containers as described herein (e.g., in the case of cell subculturing, cell harvesting, etc.).

[0264] In contrast to system 1600 (which includes a multiport valve that selectively defines fluid paths to move fluid within the system), the cell culture system 3600 includes a valve assembly 3607 (having a set of valves 3607A-3607G, each of which can be driven independently). Valves 3607A-3607G have a single input and a single output, and the flow of fluid through them can be controlled in an "on / off" manner or by throttling the flow (i.e., to control the flow rate through the valve). When used in an "on / off" manner, valves 3607A-3607G can provide a simple control system without requiring valve position sensors or rotary actuators. Since valves 3607A-3607G can each be driven independently, a series of different flow paths can be defined between the vessel, the retainer 3674, and the pump 3613. For example, valve 3607A controls the flow in and out of cell culture container 3647, valve 3607B controls the flow in and out of cell culture container 3648, and valve 3607C controls the flow in and out of cell culture container 3649. Valve 3607D controls the flow between the cell culture container and the retainer 3674, respectively. Valve 3607E controls the flow in and out of waste container 3606, and valve 3607F controls the flow in and out of reagent (or cell nutrient) container 3605. Valve 3607G controls the flow between the fluid pump 3613 and the retainer 3674.

[0265] In one embodiment, the aforementioned on / off valves 3607A-3607G are contained on the tray 3602 and are fitted and engaged by the user with a valve actuator in the base unit. In other embodiments, the valves 3607A-3607G are closed tubular sections located within a fixed pinch valve on the base unit. In some embodiments, there may be as few as three on / off valve / tubular sections located within the valve: one controlling the flow to a first container, one controlling the flow to a second container, and one controlling the flow to a third location (e.g., a container on the tray or another location).

[0266] Although not shown, the system 3600 may include further valves connected to various other components (e.g., cell counting chips, cell collection containers, various reagents and enzyme containers, etc.). In this way, when driven, the various valves in the valve assembly 3607 can facilitate fluid exchange between the various containers in the automated cell culture system 3600. For example, as described herein, the valves can be driven to facilitate the addition of cell culture medium or reagents to cell culture containers, the removal of cells from cell culture containers (e.g., cell passage or cell collection), or any other fluid movement related to cell culture.

[0267] Valves 3607A to 3607G can be any suitable valve having an input port and an outlet port. In some embodiments, one (or all) of valves 3607A to 3607G can be a pinch valve that receives a portion of piping and, when driven, deforms the piping to close off the portion of the piping and prevent fluid flow through it. This arrangement may be advantageous in that there is no fluid connection between the piping and the valve (i.e., the portion of the piping remains intact and is positioned within the cradle or receiving portion of the pinch valve). In other embodiments, one (or all) of valves 3607A to 3607G can be a needle valve, a ball valve, or any other valve mechanism to control the flow through it.

[0268] In some embodiments, valves 3607A-3607G may include integrated valve actuators (e.g., solenoids) contained on the consumable tray assembly 3601. Thus, in contrast to other tray assemblies described herein (e.g., tray assembly 1601), valves 3607A-3607G can be fixedly coupled to tray 3602 and are not removed from the tray to be coupled to actuators in the base unit (e.g., external actuators such as actuator 1621 described above). This arrangement can reduce the required setup time. In some embodiments, as described herein, integrated valves and actuators can be sterilized by a accepted method to facilitate sterilization of the entire tray assembly. For example, in some embodiments, a tray assembly with actuators fixedly coupled to tray 3602 can be sterilized by any preferred low-temperature method (i.e., a method that does not adversely affect the function of the electronics). For example, in some embodiments, a tray assembly can be sterilized by ethylene oxide (EtO), which uses a lower temperature than some other sterilization methods.

[0269] In other embodiments, the system may include one or more non-electronic valve actuators fixed to and coupled to the tray. This allows for the use of a wider range of sterilization methods. For example, by including non-electronic actuators fixed to and coupled to the tray, sterilization methods that are generally compatible with electronics (e.g., steam sterilization) can be used. In some embodiments, any of the tray assemblies or systems described herein may include one or more pressure-driven valves (e.g., pneumatic valves or hydraulic valves). For example, Figures 82A, 82B, 83A, and 83B are schematic diagrams of a portion of a tray assembly 3701 including a pressure-driven valve 3707 according to an embodiment. Figure 82B is a cross-sectional view taken along the line segment XX in Figure 82A, and Figure 83B is a cross-sectional view taken along the line segment XX in Figure 83A. As shown, the tray assembly 3701 includes a container 3747. The container 3747 may be any of the containers described herein (e.g., a cell culture container). The container 3747 can be connected to other containers, pumps, or any other components of the tray assembly 3701 via piping A (which may be similar to the piping illustrated and described in relation to Figures 16A-16C). The pressure-driven valve 3707 is a chamber, container, or other structure that, when driven, applies pressure to contract piping A, thereby preventing flow through it, as shown in Figures 83A and 83B. In some embodiments, the valve 3707 can be driven by applying gas pressure within the valve 3707 to form pressure on piping A. In other embodiments, the valve 3707 can be driven by applying hydraulic pressure within the valve 3707 to form pressure on piping A. In the illustration, the valve 3707 includes a single pressure member that crosses or traverses piping A, but in other embodiments, the valve 3707 may include a series of pressure members that cross (or traverse) piping A.

[0270] Figures 84-801 illustrate another embodiment of the cell culture system 4000 used in cell culture treatment. The cell culture system 4000 may include the same or similar components as other embodiments described herein (including, for example, cell culture systems 1700 and 2000), and may have the same or similar functions as the earlier embodiments described herein. Therefore, some details of the cell culture system 4000 will not be described for this embodiment.

[0271] The cell culture system 4000 (also referred to herein as the “system”) includes a flask tray assembly 4101 (see, for example, Figures 84–90), an input tray assembly 4281 (see, for example, Figures 91–92), and an instrument 4300 (see, for example, Figures 93–101). The flask tray assembly 4101 can be detachably coupled to the instrument 4300 as described below. Similarly, the input tray assembly 4281 can be detachably coupled to the instrument 4300 as described below. The flask tray assembly 4101 can be packaged or stored in a sterile overlap as described above for previous embodiments. In some embodiments, the input tray assembly 4281 is stored in the same overlap as the flask tray assembly 4101. In some embodiments, the flask tray assembly 4101 and the input tray assembly 4281 are wrapped in separate sterile overlaps. In some embodiments, the flask tray assembly 4101 and / or the input tray assembly 4281 are placed together or separately in a double overlap (e.g., bagged twice).

[0272] For example, as shown in Figures 84-86, the flask tray assembly 4101 includes a tray 4102. The tray 4102 has a handle 4114, a valve assembly 4184, a cell counting chip 4117, six containers 4147, and six corresponding lids 4108 arranged on it. The containers 4147 can be, for example, cell culture containers, and can also be made of a transparent material so that the inside of the containers 4147 can be seen. In some embodiments, the containers may be, for example, laboratory flasks or dishes. The containers can hold cell cultures, growth media, and any other additives or reagents related to cell culture. The cell cultures in the containers may be any kind of sticky or suspension cell culture. The containers 4147 can be pre-assembled on the tray 4102 of the tray assembly 4101 and can be provided within the overlap surrounding the tray assembly 4101, as described above for previous embodiments. The pre-assembled container 4147 can be attached to or detached from the lid 4108 when placed within the overlap. Cells and reagents can be added to the container 4147 via the lid 4108 during preparation for the cell culture procedure and before attaching the tray assembly 4101 to the instrument 4300. For example, if the lid 4108 is not already attached to the container 4147 before it is overlapped, the lid 4108 can be attached to the container 4147 to introduce cells and reagents into the container 4147 via the lid 4108. In other embodiments, the lid 4108 is attached to the container 4147 in advance to form a closed system (i.e., substantially isolating the container 4147 from the external environment to limit the entry of microorganisms into the system). Cells and reagents can be introduced into the container 4147 without opening the lid (i.e., while maintaining the closed system). In some embodiments, the container 4147 is not pre-a...

Claims

1. A method for seeding a cell sample into a cell culture container, The connection of a cell culture tray assembly to a support plate of an instrument, wherein the cell culture tray assembly includes a tray, the cell culture container is connected to the tray, a pump and valve assembly is removably connected to the tray, the cell culture container is sterilely connected to the pump and valve assembly to form a closed system, the valve assembly and the pump are each configured to drive to cause the transfer of fluid into and out of the cell culture container, the instrument includes the support plate, valve actuator, pump actuator, and a stirrer assembly, the stirrer assembly is configured to stir the support plate, and the connection is as described above. The seeding container is connected to the cell culture container, the pump, and the valve assembly within the closed system, wherein the seeding container contains the cell sample, and the connection is as follows: To drive at least one of the pump or the valve assembly to transport a portion of the cell sample from the seeding container to the cell culture container and seed the cell sample in the cell culture container, The method comprising driving the agitator assembly to agitate the support plate and the cell culture tray assembly while transferring the portion of the cell sample from the seeding container into the cell culture container.

2. By driving the aforementioned agitator assembly, the support plate and the cell culture tray are agitated in a first agitation pattern, and the method further, The method according to claim 1, further comprising driving the agitator assembly to agitate the support plate and the cell culture tray assembly in a second agitation pattern after a portion of the cell sample has been transported from the seeding container into the cell culture container.

3. The first pattern is an orbital pattern, The method according to claim 2, wherein the second pattern is a reciprocating pattern.

4. The agitator assembly includes a plurality of rotatable coupling elements, The method according to any one of claims 1 to 3, wherein each of the plurality of rotatable coupling elements is connected from a plurality of mounting locations on the support plate to a corresponding mounting location, thereby maintaining the position of the support plate relative to the device in at least two directions.

5. The aforementioned device includes an electronic control system, The electronic control system includes an actuator module implemented in at least one of a memory or processing device, The method according to any one of claims 1 to 3, wherein driving the agitator assembly to agitate the support plate and the cell culture tray assembly while the portion of the cell sample is being transported from the seeding container to the cell culture container comprises generating a agitator signal via the actuator module that rotates a rotatable coupling element to agitate the support plate and the cell culture tray assembly.

6. The apparatus includes an electronic control system, the electronic control system is operably connected to the pump actuator, and the electronic control system includes an actuator module mounted on at least one of a memory or processing device. Driving the pump or at least one of the valve assemblies includes driving the pump, The method according to any one of claims 1 to 5, wherein driving the pump includes generating a pump signal to drive the pump via the actuator module to the pump actuator.

7. Multiple protrusions extend from the support plate, The tray defines a plurality of openings, The method according to claim 1, wherein connecting the cell culture tray assembly to the support plate includes positioning each of the plurality of openings around the corresponding protrusions of the plurality of protrusions.

8. The device includes a base housing having a pump coupling slot, The method according to claim 1, further comprising connecting the pump to the pump actuator by locking a portion of the pump within the pump coupling slot.

9. The method according to claim 1, wherein connecting the seeding containers includes sterilely connecting the seeding containers to the cell culture containers, the pump, and the valve assembly within the closed system.

10. By driving the agitator assembly, the support plate and the cell culture tray assembly are agitated in an orbital pattern. The method according to claim 1.

11. By driving the aforementioned agitator assembly, the support plate and the cell culture tray assembly are agitated in a reciprocating pattern. The method according to claim 1.

12. By driving the agitator assembly, the support plate and the cell culture tray assembly are stirred in a circular or semicircular motion. The method according to claim 1.

13. By driving the aforementioned agitator assembly, the support plate and the cell culture tray assembly are agitated in a figure-eight pattern. The method according to claim 1.

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