Closed-system apparatus and method for gas-permeable cell culture operations

Abstract

To simplify the cell culture and harvesting processes. [Solution] A device for removing culture medium from a cell culture apparatus and concentrating cells, comprising a gas delivery unit that can be connected to a filter connected to a gas-permeable cell culture apparatus, and a fluid detection unit that can send a signal to a fluid flow control unit. The gas delivery unit works to pressurize the internal volume of the cell culture apparatus by delivering gas to the gas-permeable culture apparatus via the filter.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 61 / 838,730, entitled "Closed System Apparatus and Method for Gas Permeable Cell Culture Operations," filed Jun. 24, 2013, which is hereby incorporated by reference in its entirety. Further, co-pending U.S. patent application Ser. No. 10 / 961,814 (hereinafter, "814"), U.S. patent application Ser. No. 11 / 952,848 (hereinafter, "848"), U.S. patent application Ser. No. 12 / 963,597 (hereinafter, "597"), U.S. patent application Ser. No. 13 / 475,700 (hereinafter, "700") and U.S. patent application Ser. No. 13 / 493,768 (hereinafter, "768") are hereby incorporated by reference in their entirety.

[0002] The technical field of the present invention relates to static cell culture methods and apparatuses that enable the efficient growth of cells within a novel gas permeable cell culture apparatus, with little or no risk of contamination, little or no cell loss, and little or no distortion of the gas permeable surface, while allowing the addition or removal of fluid to or from the culture system.

Background Art

[0003] Considerations Regarding the Limitations of the Prior Art Described in the Related Art T cell therapy, adoptive immunotherapy, and adoptive cell therapy involve growing cells of the immune system in vitro and then administering the grown cells into a patient to fight disease, and refer to methods that are effective for treating various diseases. For these forms of therapy to become widespread in society, the growth of the cell population and the recovery of the cells need to be cost-effective, practical, less likely to cause cell loss, and have a minimal or no risk of contamination.

[0004] Today, there is no cost-effective and practical system for preparing and storing cells for T-cell therapy, adoptive immunotherapy, and adoptive cell therapy applications that requires minimal effort to separate cells from the culture medium after production and is free from contamination. A key element in creating a practical T-cell production operation for FDA approval is minimizing the complexity of the operation while minimizing, and even eliminating, opportunities for contamination. In the common vocabulary of this field, a culture operation that is generally closed to contamination is usually referred to as a “closed system.” WAVE Bioreactor®, OriGen PermaLife® bags, and / or VueLife® bags are devices suited to closed-system T-cell production. The bags have an elastic container that can collapse when removing the culture medium and cells, typically by compressing the bag in response to gravity and / or by collecting the culture medium with a peristaltic pump. WAVE Bioreactor® relies on a peristaltic pump to remove the culture medium and cells.

[0005] Recently, G-Rex® cell culture systems have become popular for T cell culture due to numerous advantages over WAVE Bioreactor®, OriGen PermaLife® bags, and VueLife® bags. One of these advantages, described in concurrent 570, is the ability to remove most of the culture medium before harvesting cells, minimizing the time and effort required to separate cells from the medium. However, it has since been discovered that the most advanced techniques for removing medium and cells using the methods described above do not function properly with G-Rex® culture systems. The G-Rex system does not collapse in the same way that the bag collapses when the medium is removed, and by using a peristaltic pump to remove the medium from the G-Rex system, the planar gas-permeable surface moves from its horizontal culture-appropriate position and is drawn into the system's internal volume by the vacuum formed within the system. Attempts to prevent the formation of a vacuum by using larger and wider surface area vent filters have not resulted in a feasible configuration that allows the gas-permeable surface to remain in a planar and / or horizontal position. Pulling the membrane from its planar and / or horizontal position is detrimental to the manufacturing process, creating a leak that may lead to cell loss as the culture volume decreases, or contamination of the culture, and / or potentially exposing workers to a biohazard.

[0006] Therefore, in order to simplify cell retrieval operations in a closed system manner, there is a need to create novel closed-system fluid handling methods for gas-permeable cell culture devices, such as those disclosed in '814, '848, '597, '700, and '768, particularly in the field of T cell therapy. [Overview of the project]

[0007] Summary of the Invention This document discloses a specific embodiment in which a gas allows a portion of the initial volume of culture medium to be transferred to a waste liquid container before the cells and remaining culture medium are transferred to a cell recovery container.

[0008] One such embodiment discloses a device for removing culture medium from a cell culture apparatus, comprising: a gas delivery unit that can be connected to a filter connected to a gas-permeable cell culture apparatus, the gas delivery unit capable of delivering gas into the gas-permeable cell culture apparatus by using the filter; and a first fluid detection unit that can detect when a fluid moving into a culture medium removal conduit connected to the gas-permeable cell culture apparatus changes from liquid to gas, and the first fluid detection unit capable of sending a signal to a first fluid flow control unit that can stop the flow of fluid through the culture medium removal conduit.

[0009] One such embodiment discloses an apparatus comprising a second fluid detection unit capable of detecting when a fluid moving through a cell removal conduit connected to the gas-permeable cell culture apparatus changes from liquid to gas, and a second fluid flow control unit capable of sending a signal to a second fluid flow control unit capable of stopping the flow of fluid through the cell removal conduit.

[0010] One such embodiment discloses a device for reducing the volume of liquid medium in a gas-permeable cell culture apparatus containing cells and culture medium, and for increasing the cell concentration per milliliter of culture medium by connecting the gas delivery component to a filter connected to the gas-permeable cell culture apparatus, wherein the cell culture apparatus includes a culture medium removal conduit, a first fluid detection unit is connected to the culture medium removal conduit, a first fluid flow control unit is connected to the culture medium removal conduit, and gas is delivered from the gas delivery unit, thereby moving the gas to the cell culture apparatus, the gas moving the culture medium from the cell culture apparatus into a culture medium recovery container connected to the culture medium removal conduit, the first fluid detection unit detects when the fluid moving through the culture medium removal conduit changes from liquid to gas, and upon detection, the first fluid detection unit sends a signal to the first fluid flow control unit, and upon receiving the signal, the first fluid flow control unit stops the flow of fluid through the culture medium removal conduit.

[0011] One such embodiment discloses the use of a device for recovering cells, wherein a first fluid control unit stops the flow of fluid through a medium removal conduit, a cell recovery container is connected to the medium removal conduit, the medium removal conduit has a medium removal opening that contacts the medium, the first fluid control unit opens the flow of fluid through the medium removal conduit, gas delivered from the gas delivery unit moves into the cell culture device, the medium and cells move through the medium removal conduit into the cell recovery container, the first fluid detection unit detects when the fluid moving through the medium removal conduit changes from liquid to gas and sends a signal to the first fluid flow control unit, and upon receiving the signal, the first fluid control unit stops the flow of fluid through the medium removal conduit.

[0012] One such embodiment increases the number of cells per milliliter of culture medium in a gas-permeable cell culture apparatus by connecting a gas delivery unit to a filter connected to a gas-permeable cell culture apparatus containing liquid culture medium and cells, wherein at least a portion of the cells are in contact with the culture surface in the cell culture apparatus, the cell culture apparatus includes a culture medium removal conduit and a cell removal conduit, the first fluid detection unit is connected to the culture medium removal conduit, the first fluid flow control unit is connected to the culture medium removal conduit, gas is discharged from the gas delivery unit, thereby moving the gas to the cell culture apparatus, the gas transfers the culture medium from the cell culture apparatus to a culture medium recovery container connected to the culture medium removal conduit, the first fluid detection unit detects when the fluid moving through the culture medium removal conduit changes from liquid to gas, and upon detection, the first fluid detection unit detects the first fluid flow The present invention discloses that a signal is sent to the dynamic control unit, and upon receiving the signal, the first fluid control unit stops the flow of fluid through the culture medium removal conduit, connects the second fluid detection unit to the cell removal conduit, connects the second fluid flow control unit to the cell removal conduit, and starts discharging gas from the gas discharge unit to remove cells from the gas-permeable cell culture apparatus, thereby moving the gas into the cell culture apparatus and transferring the culture medium and cells from the cell culture apparatus to the cell recovery container connected to the cell removal conduit via the cell removal conduit, the second fluid detection unit detects when the fluid moving through the cell removal conduit changes from liquid to gas, and upon detection, the second fluid detection unit sends a signal to the second fluid flow control unit, and upon receiving the signal, the second fluid control unit stops the flow of fluid through the cell removal conduit.

[0013] One such embodiment discloses the recovery of concentrated cells from a gas-permeable cell culture apparatus, wherein a second flow control unit stops the flow of fluid through a cell removal conduit, then the cell culture apparatus is ventilated to the atmosphere, the first flow control unit is opened to allow fluid to flow through the culture medium removal conduit, the culture medium moves into the cell culture apparatus through the culture medium removal conduit, the first flow control unit is closed, the culture medium is agitated to move the cells into the culture medium, the cell culture apparatus is no longer ventilated to the atmosphere, gas is discharged from the gas discharge unit into the cell culture apparatus, the culture medium and cells are transferred from the cell culture apparatus to a cell recovery container connected to the cell removal conduit, and the second fluid detection unit detects when the fluid moving through the cell removal conduit changes from liquid to gas, and upon detection, the second fluid detection unit sends a signal to the second fluid flow control unit, and upon receiving the signal, the second flow control unit stops the flow of fluid through the cell removal conduit.

[0014] One such embodiment increases the number of cells per milliliter of medium in a gas-permeable cell culture device containing a first volume of medium and cells on the culture surface by reducing the medium volume and increasing the cell concentration per milliliter of medium by connecting a gas delivery unit to a filter connected to the cell culture device which includes a medium removal conduit, and by starting the delivery of gas from the gas delivery unit, thereby moving the gas into the cell culture device. The gas transfers the culture medium from the cell culture apparatus to the culture medium recovery container connected to the culture medium removal conduit. The first fluid detection unit detects when the fluid moving through the culture medium removal conduit changes from culture medium to gas. Upon detection, the first fluid detection unit sends a signal to the first fluid flow control unit. Upon receiving the signal, the first fluid flow control unit stops the flow of fluid through the culture medium removal conduit, leaving the remaining culture medium and cells in the cell culture apparatus, and discloses that the remaining amount of culture medium is less than the first volume of the culture medium.

[0015] One such embodiment discloses the concentration of cells in a gas-permeable cell culture apparatus, wherein a first flow control unit stops the flow of fluid through a medium removal conduit, a cell recovery container is connected to the medium removal conduit, the first flow control unit is opened to allow fluid to flow through the medium removal conduit while the medium is in contact with the medium removal opening of the medium removal conduit, the gas discharged from the gas discharge unit moves into the cell culture apparatus, the remaining medium and cells are transferred to the cell recovery container through the medium removal conduit, the first fluid detection unit detects when the fluid moving through the medium removal conduit changes from medium to gas, sends a signal to the first fluid flow control unit, and upon receiving the signal, the first flow control unit stops the flow of fluid through the medium removal conduit.

[0016] One such embodiment involves recovering cells from a gas-permeable cell culture apparatus, the first step of increasing the cell concentration per milliliter by reducing a first volume of culture medium in the gas-permeable cell culture apparatus containing cells by connecting a gas delivery unit to a filter connected to the gas-permeable cell culture apparatus containing culture medium and cells, wherein the cell culture apparatus includes a culture medium removal conduit and a cell removal conduit, a first fluid detection unit is connected to the culture medium removal conduit, a first fluid flow control unit is connected to the culture medium removal conduit, and with at least some cells on the culture surface, gas is discharged from the gas delivery unit, thereby moving the gas into the cell culture apparatus, the gas moving the culture medium from the cell culture apparatus into a culture medium recovery container connected to the culture medium removal conduit, the first fluid detection unit detects when the fluid moving through the culture medium removal conduit has changed from culture medium to gas, and upon detection, the first fluid detection unit sends a signal to the first fluid flow control unit, and the signal is received The disclosure includes the steps of: first, stopping the flow of fluid through the medium removal conduit while leaving a remaining amount of culture medium and cells in the cell culture apparatus, provided that the remaining amount of culture medium is less than a first volume of the culture medium; and second, removing cells from the gas-permeable cell culture apparatus by connecting a second fluid detection unit to the cell removal conduit, connecting a second fluid flow control unit to the cell removal conduit, and starting gas delivery from a gas delivery unit when the cells are distributed throughout the remaining amount of culture medium, thereby moving the gas into the cell culture apparatus and transferring the culture medium and cells from the cell culture apparatus to a cell recovery container connected to the cell removal conduit, and the second fluid detection unit detecting when the fluid moving through the cell removal conduit has changed from culture medium to gas, and when this detection is made, the second fluid detection unit sends a signal to the second fluid flow control unit, and when the signal is received, the second flow control unit stops the flow of fluid through the cell removal conduit.

[0017] One such embodiment discloses a cell recovery method comprising the further step of rinsing the cell culture apparatus to recover any further cells that may remain in the cell culture apparatus and / or the cell removal conduit after the second fluid control unit has stopped the flow of fluid through the cell removal conduit, wherein the cell culture apparatus is ventilated to the atmosphere, the flow of liquid through the medium removal conduit is started, the liquid moves through the medium removal conduit into the cell culture apparatus, the liquid is agitated to move the cells in the cell culture apparatus into the liquid, the gas is started to be discharged from the gas discharge unit into the cell culture apparatus, the gas moves the liquid and cells from the cell culture apparatus into the cell recovery container via the cell removal conduit, the second fluid detection unit detects when the fluid moving through the cell removal conduit has changed from liquid to gas, and when such detection is made, the second fluid detection unit sends a signal to the second fluid flow control unit, and when the signal is received, the second flow control unit stops the flow of fluid through the cell removal conduit. [Brief explanation of the drawing]

[0018] [Figure 1A] A cross-sectional view of a gas-permeable cell culture apparatus is shown. [Figure 1B] This shows a cross-sectional view of a gas-permeable cell culture apparatus equipped with a waste liquid container. [Figure 1C] The image shows a gas-permeable cell culture apparatus equipped with a waste liquid container, a peristaltic pump for moving culture medium into the waste liquid container, and a cross-sectional view of the culture surface that is not in a horizontal position. [Figure 1D] The diagram shows a gas-permeable cell culture apparatus equipped with a waste liquid container, a peristaltic pump for moving culture medium into the waste liquid container, and a cross-sectional view of the culture surface and cells entering the waste liquid container, which are positioned away from the horizontal. [Figure 2A] This shows a cross-sectional view of a gas-permeable cell culture apparatus equipped with a waste liquid container. [Figure 2B] The diagram shows a gas-permeable cell culture apparatus equipped with a waste liquid container, a peristaltic pump for moving the culture medium into the waste liquid container, a culture surface that remains in a horizontal position, and a cross-sectional view of the initial culture medium volume reduced to the amount of culture medium containing cells. [Figure 2C]A cross-sectional view of a gas-permeable cell culture device after it is placed in the direction of cell recovery so that cells and residual medium can be removed. [Figure 2D] A cross-sectional view of a gas-permeable cell culture device showing the direction of cell recovery and after the cells and residual medium have moved into the cell recovery container. [Figure 3] A cross-sectional view of a gas-permeable cell culture device equipped with a medium removal conduit and a medium removal conduit opening. [Figure 4A] A cross-sectional view of a gas-permeable cell culture device with the medium removal conduit and the medium removal conduit opening arranged for medium removal. [Figure 4B] A cross-sectional view of a gas-permeable cell culture device with the medium removal conduit and the medium removal conduit opening arranged for cell removal. [Figure 5A] A cross-sectional view of a gas-permeable cell culture device with the medium removal conduit and the medium removal conduit opening arranged for cell removal. [Figure 5B] A cross-sectional view of a gas-permeable cell culture device with the medium removal conduit and the medium removal conduit opening arranged for cell removal. [Figure 5C] A cross-sectional view of a gas-permeable cell culture device with the medium removal conduit and the medium removal conduit opening arranged for cell removal, where the cell removal conduit opening is within the culture surface pocket. [Figure 6A] A cross-sectional view of a gas-permeable cell culture device equipped with a medium removal conduit and a cell removal conduit. [Figure 6B] A cross-sectional view of a gas-permeable cell culture device after gas has been fed into the device and removed through the medium removal conduit. [Figure 6C] A cross-sectional view of a gas-permeable cell culture device after gas has been fed into the device, the medium has been removed through the medium removal conduit, and has moved into the waste liquid recovery container. [Figure 6D] A cross-sectional view of a gas-permeable cell culture device after gas has been fed into the device and the cells and remaining medium have been removed through the cell removal conduit and moved into the cell recovery container. [Figure 7] A schematic diagram of a gas-permeable cell culture device connected to equipment for automating the removal of the medium and cells. [Figure 8] This shows a cross-sectional view of the culture surface connected to the culture surface support. [Figure 9] This shows a cross-sectional view of the culture surface formed on the culture surface support. [Figure 10] This diagram shows a cross-sectional view of a gas-permeable cell culture apparatus and its operation, in which the culture surface remains in a planar position during the removal of culture medium and cells. [Figure 11] This diagram shows a cross-sectional view of a gas-permeable cell culture apparatus and its operation, in which the culture surface remains in a planar position during the removal of culture medium and cells. [Figure 12A] This diagram shows a cross-sectional view of a gas-permeable cell culture apparatus and its operation, in which the culture surface remains in a planar position during the removal of culture medium and cells. [Figure 12B] This diagram shows a cross-sectional view of a gas-permeable cell culture apparatus and its operation, in which the culture surface remains in a planar position during the removal of culture medium and cells. [Figure 13A] This diagram shows a cross-sectional view of a gas-permeable cell culture apparatus and its operation, in which the culture surface remains in a planar position during the removal of culture medium and cells. [Figure 13B] This diagram shows a cross-sectional view of a gas-permeable cell culture apparatus and its operation, in which the culture surface remains in a planar position during the removal of culture medium and cells. [Figure 13C] This diagram shows a cross-sectional view of a gas-permeable cell culture apparatus and its operation, in which the culture surface remains in a planar position during the removal of culture medium and cells. [Modes for carrying out the invention]

[0019] Throughout this disclosure, unless otherwise specified, the following general considerations apply. When using the apparatus and methods disclosed herein, it is preferable that the cells be uniformly spread on a gas-permeable surface. Those skilled in the art are advised to select a gas-permeable material that is suitable for use in the field of cell culture. The gas-permeable material is preferably liquid-impermeable. Further guidance on the types of gas-permeable surfaces that can be used can also be found in concurrently appended '814, '848, '597, '700 and '768. When any type of non-adherent animal cells are used as the cells to be cultured, the gas-permeable surface is preferably non-porous, liquid-impermeable, and hydrophobic. It is most preferable that this surface be made of silicone and have a thickness of 0.001 to 0.024 inches. Silicone is particularly preferred as the material for T cells. Furthermore, in order to direct the cells toward the gas-permeable material and spread them across the entire surface of the gas-permeable material, the gas-permeable material is preferably in a horizontal position during culture and more preferably has a uniform surface density. Those skilled in the art should recognize that, since gas-permeable materials can move slightly downward in areas where they do not directly contact the support due to the weight of the culture medium, the term “horizontal” throughout the invention includes “substantially” horizontal. The intent of a substantially horizontal orientation is to distribute cells throughout the gas-permeable material. Preferably, a substantially horizontal state of the culture surface is such that the surface does not move out of the surface area or culture surface by more than 20%, more preferably 10%, even more preferably 5%, and most preferably 2.5%.

[0020] If the animal cells to be cultured include adherent cells, the gas-permeable material is preferably hydrophilic and has an easily attachable surface, such as a plasma-charged surface. Throughout this disclosure or any of the concurrently pending specifications '814, '848, '597, '700 and '768, a person skilled in the art should recognize that the term “gas-permeable membrane” is synonymous with and non-limiting to “gas-permeable material.” This is because a person skilled in the art should further understand that the term “membrane” is broadly defined as a gas-permeable material of any form of material known to a person skilled in the art for commonly used in relation to cell culture methods and apparatus, including those described in concurrently pending specifications '814, '848, '597, '700 and '768. Throughout this disclosure, the term “culture medium” is synonymous with a liquid containing any variety of substrates and / or nutrients used for animal cell culture. All materials of equipment that may be exposed to fluids associated with culture operations are preferably suitable for cell culture (e.g., meeting USP VI, non-cytotoxic, acceptable leachable, and particulate standards). Furthermore, cell culture / cell retrieval equipment should preferably allow for visual evaluation of the contents, such as by using optically transparent components, to ensure that it is possible to determine whether cells have been lost during medium removal or to evaluate the contents for other reasons.

[0021] An embodiment of one example of the present invention can be seen in concurrently cited '700' and related drawings, Figure 22B, which is reproduced here as Figure 1A for illustrative purposes, with respect to this figure the item numbers have been changed from the 1000s to the 100s. Figure 1A shows a cell harvesting device 100 in operation at the time when the culture is complete and the cells are about to be harvested. The cells 116 are on the culture surface 106, which forms the bottom of the device and is made of a gas-permeable material with the characteristics described above. The initial medium volume 120A is at the initial medium height 121A, which is the same as the distance from the highest medium surface to the lowest medium surface and preferably exceeds the height of 1.0 cm, which is typical of stationary cell culture bags such as OriGen PermaLife® and VueLife® bags. The initial medium height 121A is preferably greater than 2.0 cm. Any height is possible, but the optimal height will vary depending on the details of the cell culture application. For example, as described in Simultaneous Clause '700, when growing CAR T cells without the need for medium changes, an initial medium height of 10 cm is preferable when attempting to culture from a small number of cells to a much larger number of cells without the need for medium changes. Furthermore, as the medium height increases, it becomes possible to remove more medium before harvesting the cells. We have found that as the medium height decreases, cells that have sunk to the bottom of a medium of an unusual height are not easily distributed into the medium. Those skilled in the art will realize that removing a large volume of medium without disturbing the cells is extremely useful when attempting to perform a medium change (i.e., without the requirement to separate cells from the medium being removed for reintroduction into the apparatus, and / or the requirement to divide the culture into a new apparatus), or when terminating the culture and harvesting the cells (i.e., unlike the use of cumbersome centrifuges, cell separation from the bulk medium is performed in the apparatus).

[0022] Unfortunately, we found that fully utilizing the novel ability to reduce culture medium volume in devices such as those disclosed in concurrent '814, '848, '597, '700 and '768' using standard closed-system fluid handling methods could lead to cell loss and damage to the culture device, thus potentially resulting in high medical costs for T-cell therapy, adoptive immunotherapy, and / or adoptive cell therapy applications. Figures 1B, 1C, and 1D together illustrate examples of problems encountered when attempting to reduce culture medium volume using conventional media handling tools and methods before recovering cells from G-Rex® devices and other gas-permeable devices such as those disclosed in concurrent '814, '848, '597, '700 and '768'. Figure 1B shows the device described in Figure 1A after the waste container 132 has been attached to the culture medium removal conduit 110. As the first step in reducing the amount of initial culture medium volume 120A from which cells 116 must be recovered, a portion of the initial culture medium volume 120A is withdrawn from the cell culture / cell recovery device 100 by pumping it into a waste liquid container 132 via a culture medium removal conduit 110. A common method for pumping the medium is to withdraw it from the culture device using a peristaltic pump 134. As shown in Figure 1C, once a portion of the initial culture medium volume is removed from the cell culture / cell recovery device 100, the pressure drops throughout the sterile vent filter 128, causing the contents 114 to become below atmospheric pressure, and usually the contents 114 of the device to quickly become a vacuum. Since the culture surface 106 preferably contains a gas-permeable material that allows gases to pass through, making the material thin and brittle, once a vacuum is formed, the culture surface 106 is quickly pulled from its desired horizontal plane state into a new non-planar arrangement as shown in the figure. Furthermore, as the rollers 136 of the peristaltic pump rotate, a vacuum is generated within the device due to the gaps between the rollers, causing a pulse that pulses the expanded culture surface 106, removing the cells 116 and dispersing them throughout the culture medium. Subsequently, as shown in Figure 1D, as the culture medium continues to be drawn into the waste liquid container 132, the cells 116 are also drawn into the waste liquid container 132. This series of events makes it highly likely that a considerable number of cells will be distributed into the waste liquid container.Since the course of a patient's illness correlates with the number of cells in the therapeutic dose, the loss of these valuable cells can result in high medical costs. Furthermore, even if cells are not lost, the culture surface can be damaged by being pulled into the medium removal conduit. For example, a hole in the culture surface could expose the contents of the cell culture facility to biohazards, as well as contaminate the culture, rendering it unsuitable for patient use. Even if the culture surface is not damaged by the medium removal conduit, if it is pulled into it, it may become impossible to remove any further culture and cells. In summary, pulling the culture surface from its flat, desirable horizontal position may result in one or all of the following: cell loss, inability to remove cells, damage to the culture surface due to its expansion, damage to the culture surface due to physical contact with the medium removal conduit, contamination of the culture, and biohazard exposure of manufacturing workers. Therefore, a cell recovery method is needed to avoid these risks.

[0023] Generally, after cell culture, when the cells are attracted to the bottom of a device containing a gas-permeable material and the device still holds the culture medium, it is advantageous to move a certain volume of gas into the cell culture / cell recovery device by pressurizing the contents of the device. Preferably, a first volume of gas is moved into the device to move a first volume of culture medium from the device to the waste container. This step is preferably performed in a device positioned so that the culture surface where the cells are located faces horizontally. Once this step is complete, a residual amount of culture medium and cells remain in the device. A second volume of gas is then moved into the device, thereby moving the residual amount of culture medium and cells from the device to the cell recovery container. In this way, the ratio of the volume of culture medium to the number of cells is reduced.

[0024] Figures 2A to 2E provide an example of an embodiment of the present invention, which solves the problem shown in Figures 1B to 1D by forcing gas into a cell culture / cell retrieval device to expel culture medium or culture medium and cells from the device under pressure, unlike the method of extracting culture medium from the device under vacuum. This method minimizes either or all of the potential for cell loss and / or damage to the device by either or all of preventing vacuum formation, preventing upward distortion of the culture surface, and preventing repeated pulses of the culture surface that make it flat or not flat, allowing for rapid cell removal. Figure 2A shows the cell culture orientation of the cell culture / cell retrieval device 1000 and a cross-sectional view of the device in a stationary cell culture state. The cell culture / cell retrieval device 1000 includes contents 1014 separated by an upper end 1012 and a culture surface 1006 located below it, and a culture medium removal conduit 1010 with a vent 1028 and a culture medium removal opening 1008. A culture medium removal conduit clamp 1009 is in a closed position to hold the culture medium in the contents 1014. The upper end 1012 is preferably adjacent to the culture surface 1006 by the side wall 1054, and since the side wall 1054 is perpendicular to the culture surface 1006 and rigid, the inoculated cells can spread uniformly on the culture surface 1006. The initial culture medium volume 1020A is located inside the device. The height of the initial culture medium volume 1020A is the initial culture medium height 1021A within the boundary of the cell culture / cell harvesting device 1000, and this height is equal to the distance from the highest culture medium surface to the lowest culture medium surface. Cells 1016 are also present and are attracted to the culture surface 1006. The culture surface 1006 is preferably planar, placed horizontally during culture, and contains a non-porous, gas-permeable and liquid-impermeable material, and is hydrophobic when culturing non-adherent cells. The culture medium removal opening 1008 is slightly away from the culture surface 1006, and as can be seen in the figure, this distance determines the residual culture medium height 1021B. At some point during cell culture, it is desirable to perform a medium removal procedure to reduce the initial medium volume of 1020A to a smaller residual medium volume, and then either add fresh medium or concentrate the cells to the residual medium volume.

[0025] To remove a portion of the initial culture medium volume 1020A, the cell culture / cell harvesting device 1000 is positioned such that the culture surface 1006 is at the bottom and the upper end 1012 is at the top. In other words, the cell culture / cell harvesting device 1000 is positioned in a location favorable for quiescent cell culture. Cells 1016 are present on the culture surface 1006 at an initial cell density, which is the number of cells 1016 divided by the initial culture medium volume 1020A (e.g., cells / ml). Cells 1016 are also present on the culture surface 1006 at an initial surface density, which is the number of cells 1016 divided by the surface area of ​​the culture surface 1006 on which the cells are located (e.g., cells / cm²). 2). The gas outlet is preferably connected to the vent 1028 and positioned at the top of the device, as is best seen in Figure 2B. Regardless of how the contents of the cell culture / cell harvesting device are aerated, it is preferable that the gas is passed through a gamma-ray stabilized material capable of sterile filtering, such as a 0.2-micron vent filter. In this example, the gas outlet is a diaphragm pump 1050, which is connected to the vent 1028 by a gas conduit 1052. When the gas outlet is actively discharging gas (i.e., in this example, the diaphragm pump 1050 is operating), the gas is pushed into the gas conduit 1052 and enters the cell culture / cell harvesting device 1000 through the vent 1028. The vent 1028 preferably contains a material that can reliably move the gas into the cell culture / cell harvesting device 1000 and is sterile. The vent 1028 is also preferably positioned to minimize the possibility of accumulation and concentration on the filtration surface. In this figure, the filtration surface of the vent 1028 is positioned perpendicular to the culture surface 1006, rather than parallel to the culture surface 1006. When gas is supplied to the cell culture / cell harvesting device 1000 with the medium removal conduit clamp 1009 in the open position, a portion of the initial medium volume 1020A is transferred from the cell culture / cell harvesting device 1000 to the waste liquid container via the medium removal conduit 1010 as it is pushed through the medium removal opening 1008, thereby leaving behind the cells 1016 and the residual medium volume 1020B (present at a residual medium height of 1021B). The waste liquid container does not need to be a housing and does not need to be physically attached to the medium removal conduit 1010. For example, the waste liquid container could be a common one, such as a laboratory sink, but it can also be a sealed container such as a bag, sealed and connected to the culture medium removal conduit 1010 to contain the potential for biohazards in a closed system. Such a sealed container is shown as a sealed waste liquid container 1032 in Figures 2A and 2B, and this waste liquid container 1032 is connected to the culture medium removal conduit 1010.

[0026] During this culture medium removal process, when gas enters the cell culture / cell harvesting device 1000 and the pressure of the contents 1014 increases, it is preferable to prevent the culture surface 1006 from moving from the desired horizontal position. This can be achieved by a culture surface support that holds the culture surface in a horizontal plane, as described in the exemplary embodiment, and prevents damage to the culture surface. Those skilled in the art should refer to concurrently attached '814' and '848 for further guidance regarding the structure of the culture surface support.

[0027] After the culture medium removal process is complete, fresh culture medium can be added by opening the vent 1028 to the ambient air (for example, by cutting or venting the gas conduit 1052) and adding fresh culture medium through the culture medium removal conduit 1010 (or other ports that can be present in the device and can be structured appropriately for that purpose). If it is not necessary to add fresh culture medium to the device and the culture is to be terminated, the cells 1016 can be removed by repositioning the cell culture / cell recovery device 1000 from an orientation preferred for stationary cell culture to an orientation for cell removal (with the culture medium removal opening 1008 located at a lower point in the contents 1014), as shown in Figure 2C. Depending on the specific characteristics of the cells and culture surface, it may be useful to agitate the residual culture medium in the device to remove the cells from the culture surface and suspend them in the residual culture medium before attempting to remove the cells from the device. We have discovered that even when the culture surface is made of a hydrophobic, gas-permeable material such as silicone, non-adherent cells such as T cells tend to remain deposited on the culture surface even when the apparatus is tilted to the point where no more cells have settled in the culture medium. Therefore, to ensure that no cells remain in the apparatus when removing the residual medium, it is preferable to agitate the residual medium before withdrawing it to suspend the cells in it. Those skilled in the art will recognize that there are many ways to suspend cells in residual medium. For example, one can simply move the apparatus to swirl the residual medium around the apparatus and on the culture surface, and visually determine when the cells have been removed from the culture surface and moved into the residual medium. We have found that this method can be carried out smoothly with a cylindrical wall surrounding a circular culture surface. However, this process ends when the cells are removed from the culture surface and dispersed in the residual medium, and cell recovery is performed by conventional methods for recovering the medium and cells (e.g., using a peristaltic pump). However, when the vent contains a sterile filter, a pressure drop occurs throughout the vent, so when cells are extracted by removing the culture medium from the device, a vacuum is formed inside the device, causing the gas-permeable material to move from its planar position. However, this can be done as long as the gas-permeable material remains intact when it moves from its horizontal position to a new position, or as long as the other aspects of the cell culture / cell harvesting device are not affected.However, a preferred method that avoids placing gas-permeable materials or other aspects of the cell culture / cell harvesting apparatus in a situation where there is a risk of damage is to transfer the residual culture medium and cells by introducing gas into the cell culture / cell harvesting apparatus.

[0028] Figure 2D shows how this is done. The cell culture / cell harvesting device 1000 is positioned for cell removal, the medium removal conduit 1010 is connected to the cell harvesting container 1040, which is preferably used instead of a sealed waste liquid container 1032, and a gas outlet, such as a diaphragm pump 1050, is connected to the vent 1028, which is connected to the vent 1028 by a gas conduit 1052. When the gas outlet is actively discharging gas (i.e., in this example, when the diaphragm pump 1050 is activated), the gas is sent into the gas conduit 1052 and enters the cell culture / cell harvesting device 1000 through the vent 1028. When gas is introduced into the cell culture / cell recovery device 1000, the suspended cells 1016 are pushed towards the medium removal openings 1008 extending from one or more side walls 1054, and the residual medium volume 1020B containing the suspended cells 1016 is transferred from the cell culture / cell recovery device 1000 to the cell recovery container 1040 via the medium removal conduit 1010. The cell recovery container 1040 is preferably a sealed container designed to be suitable for any downstream operations such as centrifugation and / or cryopreservation. To prevent damage to the culture surface 1006, it is desirable that a culture surface support is present to keep the culture surface 1006 planar when gas enters the cell culture / cell recovery device 1000.

[0029] Those skilled in the art should recognize that it is advantageous for several reasons to raise the height of the initial culture medium volume above the bottom of the apparatus, including increasing the nutrient source, increasing the size of the cell waste sink, and the ability to remove a larger portion of the initial culture medium volume without cell loss. Although the culture medium height is not limited, it is preferably in the range of 1 cm to 25 cm, more preferably 1 cm to 20 cm, more preferably 1 cm to 15 cm, and even more preferably 2 cm to 11 cm. The residual culture medium height is also not particularly limited, but it is preferably in the range of 0.2 cm to 2.0 cm, more preferably 0.2 cm to 1.0 cm, and even more preferably 0.2 cm to 0.5 cm.

[0030] Figure 3 shows yet another embodiment of the present invention, in which a cross-sectional view of the cell culture / cell retrieval device 2000 includes a cell retrieval opening 2008 located within a medium retrieval conduit 2010. The medium retrieval conduit connects to the contents 2014 without one or more side walls 2054 and moves via the upper end 2012 in this figure. When the cell culture / cell retrieval device 2000 is set up for medium retrieval (i.e., cell culture), the medium can be retrieved as described above, and in this medium retrieval setup, the cells 2016 are located on the culture surface 2006. As shown in Figure 3, a portion of the original medium volume is removed via the medium retrieval conduit 2010. The residual medium 2020B is located at the height of the medium retrieval opening 2008. By simply rearranging the cell culture / cell retrieval device 2000 into a cell retrieval setup, the cells 2016 can be retrieved as described above, and in this cell retrieval setup, the medium retrieval opening 2008 is located at a lower point of the residual medium volume 2020B. Since cells 2016 are suspended in residual medium 2020B, they can be recovered by removing the residual medium volume 2020B via the medium removal conduit 2010.

[0031] Another embodiment of the present invention allows the opening(s) of the conduit to be changed in distance from the culture surface. Figure 4A shows a cross-sectional view of the cell culture / cell harvesting device 3000 in a cell culture configuration and in a state of resting cell culture. The cell culture / cell harvesting device 3000 includes contents 3014 separated at the upper end 3012, a culture surface 3006, a vent 3028, and a culture medium removal conduit 3010 having a culture medium removal opening 3008. The upper end 3012 is preferably adjacent to the culture surface 3006 by one or more side walls 3054, and more preferably one or more side walls 3054 are perpendicular to the culture surface 3006 during the culture operation.

[0032] It is possible to remove the culture medium through the culture medium removal opening 3008 in the manner described above, preferably when the cell culture / cell recovery device 3000 is positioned with the planar culture surface 3006 in a horizontal position and the cells are on the culture surface without being suspended in the culture medium (i.e., not dispersed in the culture medium). After the culture medium removal operation is completed, fresh culture medium can be added by opening the vent 3028 to the ambient atmosphere (e.g., by cutting or venting the gas conduit) and adding fresh culture medium through the culture medium removal conduit 3010 (or other ports present in the device that can be appropriately structured for that purpose). If it is not necessary to add fresh culture medium to the device and the culture is to be terminated, cell removal can be performed. By configuring the cell culture / cell recovery device to include means for changing the distance between the cell removal opening 3008 and the culture surface 3006, the position of the culture medium removal opening 3008 can be changed from a culture medium removal configuration to a cell removal configuration. In this case, the culture medium removal opening can not only operate to reduce the culture medium volume without removing cells, as described above, but also to remove residual culture medium and cells after the culture medium volume has been reduced. Thus, it is also possible to concentrate cells in the residual medium and remove the concentrated cells using just one port. Figure 4B shows an example of how this can be achieved. In this example, one or more side walls 3054 provide a structure that connects the lower end of the contents of the cell culture / cell harvesting device to the upper end. In this figure, the culture surface 3006 is the lower end. One or more side walls 3054 of the cell culture / cell harvesting device 3000 shall include means for adjusting the distance between the medium removal opening 3008 and the culture surface 3006. Those skilled in the art should recognize that there are many ways to achieve this. For example, one or more side walls 3054 may include an elastic material such as silicone and may also be bellows-shaped so that the height of one or more side walls 3054 can be changed. By changing the height of one or more side walls 3054 and moving the medium removal opening 3008 from a medium removal arrangement to a cell removal arrangement as shown, the distance between the medium removal opening 3008 and the culture surface 3006 is reduced so that the medium removal opening 3008 comes into the cell removal arrangement. In this cell removal configuration, the opening is close to the culture surface 3006, allowing residual medium and cells to pass through.In other words, when the cell culture / cell harvesting device 3000 is configured for medium removal, the distance between the medium removal opening 3008 and the culture surface 3006 (in this figure, the lower end where the culture medium can exist when the device is operating in a stationary cell culture state) is greater than the distance between the medium removal opening 3008 and the culture surface 3006 when the device is configured for cell removal. Conversely, when the cell culture / cell harvesting device 3000 is configured for cell harvesting, the distance between the medium removal opening 3008 and the culture surface 3006 is smaller than the distance between the medium removal opening 3008 and the culture surface 3006 when the device is configured for medium removal.

[0033] Those skilled in the art should recognize that there are many different ways to adjust the distance between the culture medium removal opening and the culture surface. For example, there are many ways (including manufacturing them from elastic (bendable) materials) to allow the walls of the apparatus to fold in a piston-like manner, where part of the wall slides liquid-tightly into other parts. In such cases, changing the culture medium removal opening from a culture medium removal configuration to a cell removal configuration reduces the distance between the top of the cell culture / cell harvesting apparatus and the culture surface. However, those skilled in the art should recognize that changing the distance between the top of the cell culture / cell harvesting apparatus and the culture surface is not the only way to move the culture medium removal opening from a culture medium removal configuration to a cell harvesting configuration.

[0034] Figures 5A and 5B show cross-sectional views of the cell culture / cell harvesting device 4000, which is designed to adjust the distance between the medium removal opening 4008 and the culture surface 4006. The elastic side wall 4001 forms a seal with respect to the medium removal conduit 4010 and its upper end 4012. The medium removal conduit 4010 has a sealed connection, which in this example includes a female Luer connector 4011, which interlocks with a male Luer connector 4013, thereby allowing the harvesting container to be easily attached to the medium removal conduit 4010. Those skilled in the art will recognize that there are many ways to make such a connection, including welded connections of sterile tubing. To change the distance between the medium removal opening 4008 and the culture surface 4006, the height of the elastic side wall 4001 is changed. Figure 5B shows the height of the elastic side wall 4001 after applying force to the upper surface of the elastic side wall 4001 to reduce its height (i.e., compressing the bellows portion toward the culture surface 4006), thereby moving the medium removal opening 4008 toward the culture surface 4006 and reducing the distance between the medium removal opening 4008 and the culture surface 4006. Those skilled in the art should recognize that the distance between the medium removal opening 4008 and the culture surface 4006 can be varied without placing the cell culture / cell retrieval device 4000 in a state where contamination is a risk. Preferably, during cell retrieval, the medium removal opening 4008 is adjacent to the culture surface 4006, and the cell culture / cell retrieval device 4000 is in a cell culture configuration (i.e., the culture surface 4006 is in a horizontal position). This facilitates automated removal of the culture medium and cell contents of the cell culture / cell retrieval device without the need to rotate the cell culture / cell retrieval device from its cell culture configuration.

[0035] Before cell retrieval, it may be useful to agitate the apparatus to remove cells from the culture surface and suspend them in the culture medium. We have found that even if the culture surface is a hydrophobic membrane such as silicone, non-adherent cells such as T cells tend to remain deposited on the culture surface when the apparatus is tilted. Therefore, agitation of the apparatus is preferable to ensure that no cells remain when removing residual medium. For example, the residual medium can be simply swirled, and it can be visually determined that the cells have been removed from the culture surface and moved into the residual medium. Once the cells have been removed from the culture surface, cell retrieval can be performed by conventional methods of withdrawing the medium and cells (e.g., using a peristaltic pump). Withdrawing the medium from the apparatus causes the cells to move from a horizontal position due to the creation of a vacuum inside the apparatus. This can be done as long as the membrane remains intact. However, a preferred method that avoids placing the membrane in a situation where it is at risk of damage is to retrieve the cells using the gas transfer method described above.

[0036] Figure 5C shows a cell culture / cell retrieval device 4000, which is further designed to further automate cell retrieval by eliminating the need to tilt the device to position the conduit opening used for cell retrieval at the lowest point within the device. When the culture surface 4006 is in a horizontal plane preferred for cell culture, the cell retrieval pocket 4007 is below the horizontal plane of the culture surface 4006. The medium removal opening 4008 is located within the cell retrieval pocket 4007. At this position, the medium removal opening 4008 is located at the lowest point of the contents 4014 (i.e., the lowest point of the medium when the device is placed in a stationary cell culture state), facilitating the retrieval of the medium and cells. This feature may be present in any embodiment of the cell culture / cell retrieval device.

[0037] Generally, when the culture medium removal opening is in the culture medium removal position, it is preferable that the opening cannot remove all of the contents of the cell culture / cell harvesting device. Even if the gas delivery unit malfunctions or if gas delivery cannot be stopped after all of the culture medium that can move through the culture medium removal opening has moved and gas continues to be delivered into the device, it is desirable that at least a portion of the culture medium and the majority of the cells remain in the device.

[0038] To avoid designing cell culture / cell retrieval devices that require varying the distance between the medium removal opening and the culture surface, or that require significant tilting to retrieve cells, it is easy to create separate medium removal openings and cell removal openings and use the aforementioned operations without risk of cell loss or damage to the culture surface and / or gas-permeable material, thereby simplifying downstream operations. Figures 6A, 6B, 6C, and 6D provide such examples. They show a cross-sectional view of a cell culture / cell retrieval device 5000 in a stationary cell culture configuration, which includes a contents 5014 separated by an upper end 5012 and a lower end (culture surface 5006 in this figure), a medium removal conduit 5010 with a vent 5028 and a medium removal opening 5008, and a cell removal conduit 5004 with a cell removal opening 5002. The upper end 5012 is preferably in contact with the lower end by one or more side walls 5054, and it is more preferable that one or more side walls 5054 are perpendicular to the culture surface. The initial culture medium volume 5020A is located inside the apparatus. The height of the initial culture medium volume 5020A is the same as the initial culture medium height 5021A, which is the distance from the highest to the lowest culture medium surface. During use, the cells 5016 are assumed to be on the culture surface 5006. Generally, those skilled in the art should notice that not all non-adherent cells (i.e., also called suspension cells) are in contact with the culture surface, as non-adherent cells can stack on top of many cells at lower positions that are actually in physical contact with the culture surface. The culture surface 5006 is preferably placed horizontally during culture operations, preferably contains a non-porous, liquid-impermeable material, and further preferably hydrophobic when culturing non-adherent cells. The cells 5016 are assumed to be at the initial cell density, which is the amount of cells 5016 divided by the initial culture medium volume 5020A (e.g., cells / ml). Furthermore, we assume that cells 5016 are also present in the initial surface density, and this initial surface density is defined as the amount of cells 5016 divided by the surface area of ​​the culture surface 5006 where the cells are present (for example, cells / cm²). 2). Furthermore, in the case of non-adherent cells, this shall include the total number of cells that have been attracted to a stationary state and prevented from being further attracted to the culture surface. This also includes cells that are resting on top of other cells (not in direct contact with the culture surface). The medium removal opening 5008 is separated from the culture surface 5006, and as can be seen, the gap constitutes the residual medium height 5021B.

[0039] At some point in the cell culture process, it is desirable to perform a medium removal operation to reduce the initial medium volume 5020A to a smaller residual medium volume, and then either add fresh medium or concentrate the cells to the residual medium volume. To remove a portion of the initial medium volume 5020A, a gas outlet is connected to a vent 5028, as best shown in Figure 6B. In this example, the gas outlet is a diaphragm pump 5050, which is connected to the vent 5028 by a gas conduit 5052. When the diaphragm pump 5050 is activated, it pushes gas into the gas conduit 5052 and into the cell culture / cell harvesting device 5000 via the vent 5028. When the gas is forced into the contents 5014 of the cell culture / cell recovery device 5000, a portion of the initial medium volume 5020A moves into the medium removal opening 5008 and is forced out of the cell culture / cell recovery device 5000 into the waste liquid container via the medium removal conduit 5010, leaving behind the residual medium volume 5020B and cells 5016 as shown in Figure 6C. The height of the residual medium volume 5020B is defined as the residual medium height 5021B, which is the distance between the highest and lowest residual medium surfaces. The cells 5016 are assumed to be at residual cell density, which is the amount of cells 5016 divided by the residual medium volume 5020B (e.g., cells / ml). The cells 5016 are also assumed to be at residual surface density, which is the amount of cells 5016 divided by the surface area of ​​the culture surface 5006 where the cells are located (e.g., cells / cm²). 2) The waste liquid container does not need to be an enclosure, nor does it need to be physically attached to the culture medium removal conduit 5010. For example, the waste liquid container can be a common one, such as a laboratory sink, but it is preferable to use a sealed container such as a bag or centrifuge tube, which will contain the possibility of biohazards in a closed system manner, or be sealed and connected to the culture medium removal conduit 5010 to maintain sterility. Such a sealed container is shown as a sealed waste liquid container 5032 in Figure 6C, and this waste liquid container 5032 is attached to the culture medium removal conduit 5010. When a portion of the initial culture medium volume 5020A is removed from the cell culture / cell harvesting device 5000, the volume of culture medium in the waste liquid container 5032 will be the initial culture medium volume 5020A which is less by the amount of the initial culture medium volume 5020A in the culture medium removal conduit 5010 (if any). During the removal of this culture medium, when gas enters the cell culture / cell harvesting device 5000 and the pressure of the contents 5014 increases, it is preferable to prevent the culture surface 5006 from moving unrestricted in the direction opposite to the upper end 5012. This is done by a culture surface support 5018 that contacts the culture surface 5006 and holds the culture surface 5006 in a substantially planar position, as shown in Figure 6C, thereby preventing damage to the culture surface. The term "substantially" was previously described in relation to the horizontal position, but it also applies to the planar position. Those skilled in the art should refer to concurrently attached '814' and '848 for further guidance on the design of the culture surface support.

[0040] Referring to Figure 6D, the cell recovery container 5040 is attached to the cell removal conduit 5004 of the cell culture / cell recovery device 5000, and the fluid flow is blocked by a medium removal conduit clamp 5009 that closes the medium removal conduit 5010, in order to recover the cells 5016. Blocking the fluid flow in the medium removal conduit 5010 can be done as simply as using hemostatic forceps, or it may be automated as further described in this disclosure. As mentioned above, before cell recovery, it may be useful to agitate the residual medium in the device to remove the cells from the culture surface and suspend them in the residual medium. Cell recovery can be performed by conventional methods for recovering medium and cells (e.g., using a peristaltic pump), but a preferred method is to use the positive pressure provided by the gas transfer method to prevent the culture surface and / or gas-permeable material from moving towards the upper end or becoming distorted in shape during the operation. As shown in Figure 6D, the gas outlet is connected to the vent 5028 via the gas conduit 5052. In this example, the gas delivery unit is a diaphragm pump 5050. When the diaphragm pump 5050 is activated, the gas is forced into the gas conduit 5052 and enters the cell culture / cell recovery device 5000 via the vent 5028. Once the gas is forced into the cell culture / cell recovery device 5000, the residual culture medium 5020B and cells 5016 move into the cell removal opening 5002 and are forced into the cell recovery container 5040 via the cell removal conduit 5004. Subsequently, the cell recovery container 5040 can be sterilized and removed for subsequent processing of the cells 5016 by a preferred sterilization method, such as welding a sterilized tube.

[0041] It is possible to flush out some of the contents of the cell culture / cell harvesting device, which is desirable for recovering any cells that may remain in the device after the initial cell removal process. For example, this can be done without adding a liquid supply that was not in the device at the start of the cell removal operation. This can be done using the culture medium in a sealed waste container as the material to be flushed out. To do this, the waste container is pressurized by opening the medium removal clamp, opening the vent, and simply raising the waste container to the height necessary to allow the medium to flow back into the device. If the waste container is collapsible (e.g., a bag), it is helpful to squeeze it to start the flow into the device. Once the user determines that a sufficient amount of medium has returned to the device, the medium removal conduit clamp can be closed and the cell removal operation can be repeated.

[0042] The previously described process of transferring culture media and / or cells using air can be performed in a simple manner, for example, by opening and closing conduits with hemostatic forceps and switching the gas outlet on and off based on visual judgment of the process. If a large number of cell preparations are desired, automating part of the process may be beneficial. One factor to consider is that the gas outlet may continue to supply gas to the device even after the culture media and / or cells have been displaced into their respective containers. In this case, displacing the gas into the containers and pressurizing them may break the seals on the containers. Furthermore, the gas may cause cell damage because the surface tension of the bubbles and contact between the gas and cells may disrupt the integrity of the cell membranes. Another factor to consider is the pressure that may form inside the cell culture / cell harvesting device itself if the gas outlet is not turned off after the culture media and / or cells have been displaced into their respective containers.

[0043] Figure 7 shows a schematic diagram of an exemplary embodiment of a device designed to solve these problems. Those skilled in the art should recognize that this exemplary embodiment is compatible with cell culture / cell harvesting devices made with only one medium removal conduit, or an adjustable medium removal conduit, or separate medium / cell removal conduits, as shown in Figure 3, etc. Generally, it is preferable for the automated device to recognize the work point at which the medium exiting the cell culture / cell harvesting device via the medium removal conduit or cell removal conduit is replaced with gas. Referring again to Figure 7, the fluid detection units 6044A and 6044B can recognize the work point at which the medium exiting the cell culture / cell harvesting device via the medium removal conduit or cell removal conduit is replaced with gas, and are located close to the medium removal conduit 6010 and the cell removal conduit 6004. In other words, the fluid detection units can detect whether liquid or gas is present in the conduit. The first flow control unit 6046A operates to open and close the culture medium removal conduit 6010, and the second flow control unit 6046B operates to open and close the cell removal conduit 6004. In this example, an electronically operated pinch clamp functions as the flow control unit, and the conduits are made of elastic tubing. The cell culture / cell harvesting device 6000 is preferably a stationary cell culture device in its cell culture configuration (i.e., the culture medium is not subjected to forced mixing, and the culture surface is below the upper end, preferably in a horizontal position). The culture medium 6020 is present in the cell culture / cell harvesting device 6000 at a first medium volume and first medium height, and the cells 6016 are submerged on the culture surface 6006 by gravity. It is preferable to hold the culture surface 6006 horizontally with a culture surface support 6018. The distance from the culture medium removal opening 6008 to the culture surface 6006 is greater than the distance from the cell removal opening 6002 to the culture surface 6006. The cell removal conduit is preferably positioned in contact with the side wall, or in contact with the culture surface, or along an edge connecting one or more side walls 6054 to the culture surface, or in a pocket within the culture surface as described above, thereby positioning the cell removal opening to maximize cell recovery. The gas conduit 6052 has a vent 6028 connected to the gas supply unit 6050. The culture medium removal conduit 6010 is connected to the waste liquid container 6032, and the cell removal conduit 6004 is attached to the cell recovery container 6040.The software algorithm can send out the electronic signals necessary to perform various tasks required to recover cells in a highly concentrated state. To reduce the volume of the culture medium 6020, the flow control unit 6046A allows fluid to flow through the culture medium removal conduit 6010, and the flow control unit 6046B prevents fluid from flowing through the cell removal conduit 6004, activating the gas discharge unit so that gas enters the cell culture / cell recovery device 6000 through the vent 6028, pressurizing the contents 6014, pushing the culture medium 6020 to the culture medium removal opening 6008 and then through the culture medium removal conduit 6010 to the waste liquid container 6032. When the height of the culture medium 6020 reaches just below the culture medium removal opening 6008, gas enters the culture medium removal opening 6008, passes through the culture medium removal conduit 6010, and is detected by the fluid detection unit 6044A. The fluid detection unit 6044A sends a signal to the flow control unit 6046A to stop the fluid flow to the waste liquid container 6032. The gas delivery unit 6038 preferably simultaneously stops the gas delivery and / or opens the pressure safety valve 6056. This step is performed if necessary, but its advantage is to minimize the pressure within the cell culture / cell harvesting device 6000, and those skilled in the art should recognize that there are many ways to achieve this, including the use of a gas delivery unit that prevents gas delivery after a certain pressure threshold is reached, and the use of a pressure safety valve. Once the culture medium 6020 has finished moving to the waste liquid container 6032, the operator can then decide whether to agitate the remaining medium to remove the cells 6016 from the culture surface 6006. Those skilled in the art should recognize that a robust system allows the operator to disable automatic control and stop the movement of the medium to the waste liquid container (for example, to be necessary if cells enter the waste liquid container and are wasted).

[0044] When the operator determines that the cells 6016 are in a proper suspension state within the remaining volume of culture medium 6020 and / or that cell retrieval should be initiated, the user presses a button to resume the operation. The contents 6014 are pressurized, and the fluid control unit 6046B operates to allow fluid to flow into the cell retrieval container 6040. Depending on the operator's preference, or whether the cell culture device is designed to be positioned such that the cell removal conduit is in a position that maximizes the amount of cells to be retrieved when the cell culture device is set up for static cell culture, the cell culture / cell retrieval device 6000 may or may not be positioned so that all the culture medium and cells are concentrated at a low point at this time. Those skilled in the art will recognize that the process of agitating to remove cells and orienting the device to a position that maximizes the amount of cells to be retrieved can also be automatically controlled. In a static cell culture setup, the maximum number of cells to be retrieved can be achieved when the cell removal opening 6002 is lower than the height of the culture surface 6006. For example, this can be achieved by any mechanism that can ensure the cell removal opening is positioned at the recovery location, such as by placing a pocket on the culture surface, a peripheral recess around the culture surface (with its bottom lower than the culture surface), or the culture medium at a lower level than the culture surface, as described above. At some point during the automated cell removal process, gas enters the cell removal opening 6002, passes through the cell removal conduit 6004, and is detected by the fluid detection unit 6044B. At that point, the fluid detection unit 6044B sends a signal to the fluid control unit 6046B to stop the flow of fluid to the cell recovery container 6040. At this point, the container 6040 can be removed, preferably by an aseptic method using a sterile tube welding machine. However, if the operator determines that further washing of the culture membrane would be helpful in recovering more cells that may remain in the device, this operation is considered simple. The fluid control unit 6046A can be left open instead of obstructing the fluid flow through the culture medium removal conduit 6010, and the culture medium 6020 is sent from the waste liquid container 6032 to the cell culture / cell recovery device 6000 by simply lifting or squeezing (preferably the waste liquid container is in the form of a bag). During this process, it is preferable to open the vent 6028 to the atmosphere. Once an appropriate amount of culture medium 6020 has returned to the cell culture / cell recovery device 6000, the cell recovery operation can be repeated.This process of adding or removing culture medium can be repeated as many times as necessary to collect as many cells as the operator deems appropriate.

[0045] The method of using the apparatus in the embodiment shown in Figure 7 involves connecting a gas outlet to a filter, and connecting the filter to a gas-permeable cell culture / cell harvesting device containing culture medium and cells. The cell culture device includes a culture medium removal conduit, a first fluid detection unit is connected to the culture medium removal conduit, and a first fluid flow control unit is connected to the culture medium removal conduit. Gas is then discharged from the gas outlet, thereby initiating any operations or use of equipment that move the gas into the cell culture device. The gas moves the culture medium from the cell culture device to the culture medium harvesting container connected to the culture medium removal conduit. The first fluid detection unit detects when the fluid moving through the culture medium removal conduit changes from liquid to gas, and sends a signal to the first fluid flow control unit. Upon receiving the signal, the first flow control unit stops the flow of fluid through the culture medium removal conduit. After the first flow control unit stops the flow of fluid through the culture medium removal conduit, the culture medium can be stirred as needed to remove cells from the culture surface and send them into the residual culture medium. The cell culture device can also be placed in a new configuration where the cell removal conduit and the culture medium are in contact. However, if the culture surface remains in a horizontal position and the cell removal opening of the cell removal conduit is in contact with or close to the culture surface, there is no need to change the orientation of the device. The cell recovery container is connected to the cell removal conduit, the second fluid control unit opens the fluid flow through the cell removal conduit, the gas from the gas outlet moves into the cell culture device, the culture medium and cells move through the cell removal conduit into the cell recovery container, the second fluid detection unit detects when the fluid moving through the cell removal conduit changes from liquid to gas and sends a signal to the second fluid flow control unit. Upon receiving the signal, the second fluid control unit stops the fluid flow through the cell removal conduit.

[0046] Those skilled in the art will recognize that the apparatus described in the exemplary embodiment of Figure 7 can be simplified by connecting it to a type of cell culture / cell harvesting apparatus having only one culture conduit that acts to reduce the culture medium volume and remove residual culture medium volume and cells. Such apparatuses are described in more detail in Figures 2A-2D, Figure 3, Figures 4A-4B, Figures 5A-5B and related texts. Thus, the apparatus can be simplified by including a gas delivery unit that can be connected to a filter connected to a gas-permeable cell culture apparatus (the gas delivery unit can deliver gas to the gas-permeable cell culture apparatus via the filter) and a first fluid detection unit that can detect when a type of fluid moving through a culture removal conduit connected to a gas-permeable cell culture apparatus changes from liquid to gas (this unit can send a signal to a first flow control unit that can stop the flow of fluid through the culture removal conduit). After the first fluid control unit stops the fluid flow through the culture medium removal conduit, if necessary, it directs the cell culture apparatus to a new position where the culture medium is in contact with the culture medium removal conduit, connects the cell recovery container to the culture medium removal conduit, and the first fluid control unit opens the fluid flow through the culture medium removal conduit, allowing the gas from the gas outlet to move into the cell culture apparatus, the culture medium and cells to move into the cell recovery container through the culture medium removal conduit, and the first flow detection unit detects when the fluid moving through the culture medium removal conduit changes from liquid to gas and sends a signal to the first fluid flow control unit. Upon receiving the signal, the first fluid control unit stops the fluid flow through the culture medium removal conduit.

[0047] Those skilled in the art will recognize that it is beneficial for cells to move out of the cell removal conduit and into the cell recovery container to the maximum extent actually possible. Therefore, when using automatic control as shown in Figure 7, by positioning the fluid detection unit downstream of the flow control mechanism and as close to the container as possible, the volume of culture medium in the conduit when the flow control mechanism stops flowing is limited to what is between the fluid detection unit and the cell recovery container. Furthermore, the number of cells in the conduit can be further reduced by minimizing the inner diameter of the tube. Alternatively, after gas is detected in the conduit, a small amount of additional gas can be carried through one or more conduits to ensure that no liquid is trapped in one or more conduits at the end of the operation. This may be useful in ensuring that all cells are moved to the cell recovery container and / or that sterile tubes can be connected with no liquid in the conduits. Alternatively, by sending gas through the conduits, it is possible to ensure that no liquid is trapped in the conduits at the end of the operation.

[0048] Those skilled in the art should recognize the convenience of using the flow control unit and fluid detection unit, but cell retrieval can also be performed manually. In manual cell retrieval, the operator can cool the clamped flow through the cell removal conduit, open the flow through the culture medium removal conduit, and then inject gas into the apparatus using a syringe until the gas moves into the culture medium removal conduit. The culture medium can then be agitated from a stationary state as needed to remove cells from the culture surface. Closing the culture medium removal conduit and opening the cell removal conduit adds gas to the apparatus, thereby expelling cells and residual culture medium from the apparatus.

[0049] While using gas to move the culture medium is a suitable method to prevent distortion of the culture surface and / or gas-permeable material, if the goal is to withdraw the culture medium and / or cells from the device, as is done with gas movement as described above, it is possible to design the device so that distortion of the culture surface and / or gas-permeable material is minimized. This can be done with the contents of the device at a pressure lower than the external pressure of the device, thereby creating a pressure difference across the entire wall of the device. One method to prevent distortion of the culture surface from its planar state under these conditions is to physically attach the gas-permeable material to a component (e.g., a culture surface support). Figures 8 and 9 provide two such examples. For simplicity, the entire cell culture / cell harvesting device is not shown. In Figure 8, the culture surface 6006 includes a tab 6058 extending from its underside. The tab 6058 engages with a fitting mechanism on the culture surface support 6018. The vertical support 6018A protrudes from the base of the culture surface support 6018 to hold the culture surface 6006 in a horizontal position during quiescent cell culture. The culture surface 6006 preferably contains a gas-permeable material. In the case of silicone, a membrane can be manufactured by liquid injection molding, and the tab 6058 can be made into a membrane. Figure 9 shows another example of maintaining the culture surface in a substantially planar state when the pressure outside the culture surface exceeds the pressure of the contents of the cell culture / cell harvesting device. In this example, the culture surface 7006 contains silicone, which covers the outside of the grid 7060 (i.e., mounts to the grid) and mounts to the culture surface support 7018. The ventilation opening 7018B allows for passive gas exchange of the culture. In other words, ambient gas comes into contact with the culture surface without having to be forced into contact with it. In this way, even if the pressure outside the culture surface 7006 exceeds the pressure of the contents of the cell culture / cell harvesting device, the culture surface 7006 is held in place via the contact points with the culture surface support 7018.

[0050] Another method for maintaining the culture surface in a substantially planar state when withdrawing culture medium and / or fluid from the device is to balance the pressure of the contents of the cell culture / cell harvesting device with the pressure outside the culture surface. Figure 10 shows an example of a method that can achieve this. The cell growth / cell harvesting device 8000 shall include a culture surface support 8018. The culture surface support 8018 includes one or more vent openings 8021. The one or more vent openings allow for the passive movement of ambient gas from the culture surface 8006 to the culture surface 8006, and the vent openings are preferably liquid-impermeable, gas-permeable, and non-porous, and are substantially horizontal during cell culture. In other words, ambient gas comes into contact with the gas-permeable culture surface by passive movement without using any structure that would force the gas to physically come into contact with a gas-permeable material. A culture medium removal conduit 8010 is attached to a waste liquid container 8032 and connected to a peristaltic pump 8030 so that the culture medium 8020 can be drawn out of the cell growth / cell harvesting device 8000 by the peristaltic pump 8030. During culture medium removal, the ability of one or more vent openings 8021 to interact with ambient gas is minimized, and preferably eliminated. In this figure, a liquid removal adapter 8027 is temporarily attached while the culture medium volume is decreasing. The liquid removal adapter 8027 is fitted to a culture surface support 8018 and includes a pressure equilibrium conduit 8033A, and further includes a pressure equilibrium conduit joint 8029, the joint of which can be, for example, Luer-shaped. The pressure equilibrium conduit 8033A can be fitted to a pressure equilibrium conduit 8033B. The pressure balancing conduit 8033B can be attached to the culture medium removal conduit 8010, and when in operation, the peristaltic pump 8030 can draw gas from the space between the liquid removal adapter 8027 and the culture surface support 8018, so that the gas space 8019 is effectively at a lower pressure than the ambient gas. When the pressure balancing conduit 8033B is attached to the culture medium removal conduit 8010, it is preferable that the pressure balancing conduit 8033B includes, if necessary, a check valve 8034 and / or a pressure balancing conduit filter 8035, which can move the gas to the waste liquid container and prevent the culture medium 8020 from moving into the container, while at the same time maintaining the sterility of the device and its fluid connection parts. Those skilled in the art should recognize that it is not necessary to attach the pressure balancing conduit 8033B to the culture medium removal conduit 8010, but it can instead be a separate conduit connected to the peristaltic pump.In this case, a peristaltic pump with multiple channels connected to the culture medium removal conduit and pressure equilibrium conduit is used, if possible. In such a configuration, the peristaltic pump 8030 draws gas from the underside of the culture surface 8006, creating a vacuum on the culture surface 8006, which equilibrium with the vacuum created within the cell growth / cell harvesting device 8000, thereby keeping the culture surface 8006 in a substantially horizontal position, or at least reducing the strain on the culture surface 8006. Those skilled in the art should recognize that any device that draws gas from the pressure equilibrium conduit can satisfy the purpose of preventing strain that could damage the culture surface when removing the culture medium.

[0051] The culture surface support 8018 is designed to connect to the liquid removal adapter 8027, and the culture surface support 8018 is designed to connect to the cell culture / cell harvesting device 8000 so that gas can be removed from the gas space 8019 at a rate such that the pressure outside it adjacent to the culture surface is less than or equal to the pressure of the contents 8014. Those skilled in the art will recognize that the connection does not need to be airtight as long as it controls the pressure in the gas space 8019. However, it is preferable that a seal be provided. Those skilled in the art will also recognize that the liquid removal adapter 8027 does not need to satisfy the purpose of maintaining the culture surface in a substantially horizontal position when the culture medium is withdrawn from the cell culture / cell harvesting device. For example, a pressure equilibrium conduit 8033B can be directly connected to one or more vent openings 8021. In practice, when the connection is disconnected, it is preferable that the liquid removal adapter can be easily connected to and disconnected from the culture surface support, if possible, so that when the connection is disconnected, the ambient gas is forced to passively come into contact with the culture surface and deliver the gas without requiring any mechanism or work. Those skilled in the art should realize that it is not necessary to attach the culture surface support to the cell culture / cell harvesting device in a manner that prevents its removal.

[0052] Figure 11 shows another embodiment of a cell culture / cell harvesting apparatus adapted to allow extraction of culture medium from an apparatus with a culture surface having virtually no distortion. The cell growth / cell harvesting apparatus 9000 includes a culture surface support 9018. The culture surface support 9018 includes one or more vent openings 9021. The one or more vent openings 9021 allow passive movement of ambient gas from the culture surface 9006 to the culture surface 9006, and are preferably gas-permeable, liquid-impermeable, and located in the horizontal plane during cell culture. A vent opening filter 9036 prevents contaminants from approaching the one or more vent openings 9021. Those skilled in the art should recognize that the material selected for the vent opening filter functions as a sterile barrier, allowing gas to passively enter and exit the culture surface at a rate that allows sufficient oxygenation for culture. Preferably, it is a porous material with a pore size of less than 0.45 microns, more preferably less than 0.22 microns. The culture medium removal conduit 9010 is attached to the waste liquid container 9032 and connected to the peristaltic pump 9030 so that the peristaltic pump 9030 can draw the culture medium 9020 from the cell growth / cell harvesting device 9000. The pressure equilibrium conduit 9033 connects the culture medium removal conduit 9010 to the gas space 9019 and may include, if necessary, a check valve 9034 (to prevent the possibility of culture medium entering the gas space) and / or a pressure equilibrium conduit filter 9036 (to prevent contaminants or biohazards from entering the gas space and then passing through the vent opening filter). During culture medium removal, the speed of the peristaltic pump is such that the pressure in the gas space 9019 (i.e., the space between the culture surface 9006 and the vent opening filter 9036) is less than or equal to the pressure of the contents 9014, thereby minimizing or preventing the movement of the culture surface 9006 toward the upper end 9012. Preferably, the pressure balancing conduit is an elastic tube that fits into the tube extending from the vent opening into the gas space, and is used for fitting the welded portion of the sterilized tube.

[0053] Figures 12A and 12B show yet another embodiment of the cell culture / cell harvesting apparatus and operation. In this embodiment, the pressure of the contents of the cell culture / cell harvesting apparatus is lower than the pressure of the ambient environment when the culture medium is withdrawn from the cell culture / cell harvesting apparatus. Figure 12A shows the cell culture / cell harvesting apparatus 10000 in a state of stationary cell culture. Cells 10016 sink by gravity onto a gas-permeable, liquid-impermeable culture surface 10006. In Figure 12B, the ambient gas limiter 10018 is in contact with the cell culture / cell harvesting apparatus 10000. The ambient gas limiter 10018 functions to limit the contact of ambient gas with the culture surface 10006 and functions to create a pressure imbalance across the culture surface 10006 when the liquid is withdrawn from the cell culture / cell harvesting apparatus 10000. In this embodiment, the peristaltic pump 10030 functions to withdraw the culture medium 10020 from the culture medium removal conduit 10010. When the culture medium is withdrawn from the cell culture / cell harvesting device, the pressure decreases if the vent filter 10028 restricts gas from entering the device. Even though the pressure of the contents of the cell culture / cell harvesting device 10000 is lower than that of the ambient environment, the ambient gas limiter 10100 prevents the culture surface 10006 from experiencing relative pressure imbalance. The ambient gas limiter 10100 does not need to be airtightly connected to the cell culture / cell harvesting device 10000, but it is preferable if it is desired to restrict the culture surface from substantially moving toward the upper end 10012. One or more conduits can be used to reduce the culture medium volume before cell harvesting, as described above. Preferably, if a culture surface support 10018 is present, it should be designed to minimize the amount of gas within it.

[0054] Figures 13A, 13B, and 13C show yet another embodiment of the cell culture / cell harvesting apparatus and method. In this method, a predetermined volume of gas is injected into the cell culture / cell harvesting apparatus. Although the method of blowing out the injected gas in this exemplary embodiment is as described, those skilled in the art should recognize that any structure of the blowing portion, such as a bellows-like structure, can distribute a known volume of gas. As shown in Figure 13A, the cell culture / cell harvesting apparatus 11000 is shown in a stationary cell culture state, which includes an initial medium volume 11020A and cells 11016. The piston 11100 is connected to the cell culture / cell harvesting apparatus 11000 via a vent filter 11028. As shown in Figure 3B, when removing waste medium, the piston head 11150 moves a first predetermined distance to deliver a certain volume of gas to the cell culture / cell harvesting apparatus 11000. The gas of the specified volume displaces an equal volume of culture medium from the culture medium removal conduit 11010, which is open to a waste liquid container (not shown for clarity). Once this process is complete, the culture medium removal conduit 11010 is closed to the waste liquid container, and the remaining culture medium volume 11020B remains in the cell culture / cell recovery device 11000 along with the cells 11016. As shown in Figure 13C, when removing the cells 11016 and residual culture medium 11020B, the piston 11100 moves a second predetermined distance to distribute an additional volume of gas to the cell culture / cell recovery device 11000, thereby displacing the cells 11016 and residual culture medium volume 11020B through the open cell removal conduit 11004 to the cell recovery container (not shown for clarity).

[0055] Those skilled in the art will recognize that many modifications can be made to this disclosure without departing from the spirit of the invention. Therefore, there is no intention to limit the scope of the invention to the examples described or explained. Rather, the scope of the invention should be interpreted by the appended claims and their equivalents. All publications, patents, patent applications, and references cited herein are incorporated herein by reference.

Claims

1. A device for removing culture medium from a cell culture apparatus, It comprises a gas delivery unit, a pressure safety valve, and a first fluid detection unit. The gas delivery unit can be connected to a gas filter connected to a gas-permeable cell culture apparatus, and the gas delivery unit can deliver gas to the gas-permeable cell culture apparatus through the filter, and can also be used to pressurize the contents of the cell culture apparatus. The pressure safety valve is located between the gas outlet and the gas filter and can be operated to reduce the pressure of the contents. The first fluid detection unit can detect when a fluid moving through a culture medium removal conduit connected to a gas-permeable cell culture apparatus changes from liquid to gas, and the first fluid detection unit is positioned close to the culture medium removal conduit and can send a signal to a first fluid flow control unit that can stop the flow of fluid through the culture medium removal conduit.

2. The apparatus according to claim 1, comprising a second fluid detection unit, the second fluid detection unit capable of detecting when a fluid moving through a cell removal conduit connected to a gas-permeable cell culture apparatus changes from a liquid to a gas, the second fluid detection unit being positioned in close proximity to the cell removal conduit, and capable of sending a signal to a second fluid flow control unit capable of stopping the flow of fluid through the cell removal conduit.

3. A method for concentrating cells in the apparatus described in claim 1, To increase the cell concentration per milliliter of culture medium, the method includes the step of reducing the volume of liquid culture medium in a gas-permeable cell culture apparatus, which contains cells and culture medium, by connecting the gas outlet to a gas filter connected to the gas-permeable cell culture apparatus. A pressure safety valve is positioned between the gas outlet and the gas filter, the cell culture apparatus includes a culture medium removal conduit, a first fluid detection unit is connected to the culture medium removal conduit, a first fluid flow control unit is connected to the culture medium removal conduit, gas is discharged from the gas outlet, causing the gas to move into the cell culture apparatus, the gas pressurizes the contents of the cell culture apparatus, and the culture medium is transferred from the cell culture apparatus to a culture medium recovery container connected to the culture medium removal conduit, the first fluid detection unit detects when the fluid passing through the culture medium removal conduit changes from liquid to gas, immediately after detection the first fluid detection unit sends a signal to the first fluid flow control unit, and immediately upon receiving the signal the first fluid flow control unit stops the flow of fluid through the culture medium removal conduit, and the pressure safety valve operates to reduce the pressure of the contents.

4. A method for recovering T cells from a gas-permeable cell culture apparatus using a device for increasing the concentration of T cells and recovering them, The first step involves increasing the number of T cells per milliliter of culture medium in a gas-permeable cell culture device by connecting a gas outlet to a gas filter connected to a gas-permeable cell culture device containing a liquid culture medium of a certain medium height. A pressure safety valve is positioned between the gas outlet and the gas filter, the culture medium height is 1 cm to 25 cm before the first step is completed, at least a portion of the non-adherent cells are in contact with the culture surface made of a hydrophobic, gas-permeable material in the cell culture apparatus, the cell culture apparatus includes a culture medium removal conduit and a cell removal conduit, the first fluid detection unit is connected to the culture medium removal conduit, the first fluid flow control unit is connected to the culture medium removal conduit, gas is discharged from the gas outlet, thereby moving the gas into the cell culture apparatus and pressurizing the contents of the cell culture apparatus, the gas transfers the culture medium from the cell culture apparatus to a culture medium recovery container connected to the culture medium removal conduit, the first fluid detection unit detects when the fluid passing through the culture medium removal conduit changes from liquid to gas, immediately after detection, the first fluid detection unit sends a signal to the first fluid flow control unit, and immediately upon receiving the signal, the first fluid flow control unit stops the flow of fluid through the culture medium removal conduit, the culture medium reaches the residual culture medium height, A method comprising a second step of removing T cells from a gas-permeable cell culture apparatus, wherein a second fluid detection unit is connected to a cell removal conduit, a second fluid flow control unit is connected to the cell removal conduit, the cell culture apparatus is agitated to remove cells from the culture surface, gas is discharged from a gas discharge unit so that the gas moves into the cell culture apparatus and pressurizes the cell culture apparatus, the gas transfers the culture medium and cells from the cell culture apparatus to a cell recovery container connected to the cell removal conduit through the cell removal conduit, the second fluid detection unit detects when the fluid passing through the cell removal conduit changes from liquid to gas, and when this is detected, the second fluid detection unit sends a signal to the second fluid flow control unit, and as soon as the second fluid flow control unit receives the signal, it stops the flow of fluid through the cell removal conduit.

5. The method according to claim 4, wherein after the pressure safety valve is activated, the first fluid flow control unit is opened to allow fluid to flow through the culture medium removal conduit, the culture medium moves through the culture medium removal conduit to the cell culture apparatus, the first fluid flow control unit is closed, the culture medium is agitated and cells move into the culture medium, A method comprising: preventing the cell culture apparatus from being ventilated to the atmosphere; supplying gas from a gas outlet to the cell culture apparatus to pressurize it; transferring the culture medium and cells from the cell culture apparatus to a cell recovery container connected to a cell removal conduit; a second fluid detection unit detecting when the fluid passing through the cell removal conduit changes from liquid to gas; immediately after detection, the second fluid detection unit sending a signal to a second fluid flow control unit; and immediately upon receiving the signal, the second fluid flow control unit stopping the flow of fluid through the cell removal conduit.

6. A method for reducing the volume of culture medium in a gas-permeable cell culture apparatus containing cells and culture medium, To increase the cell concentration per milliliter of culture medium, the method includes a step of reducing the volume of culture medium by connecting a gas delivery unit, which plays a role in pressurizing the cell culture device, to a gas-permeable cell culture device containing a first volume of culture medium and cells on the culture surface. A cell culture apparatus includes a culture medium removal conduit, a first fluid detection unit is connected to the culture medium removal conduit, a first fluid flow control unit is connected to the culture medium removal conduit, the discharge of gas from a gas discharge unit is started, thereby moving the gas into the cell culture apparatus and pressurizing the cell culture apparatus, the gas transfers the culture medium from the cell culture apparatus through the culture medium removal conduit to a culture medium recovery container connected to the culture medium removal conduit, the first fluid detection unit detects when the fluid passing through the culture medium removal conduit changes from liquid to gas, immediately after detection the first fluid detection unit sends a signal to the first fluid flow control unit, immediately after the first fluid flow control unit receives the signal the fluid flow through the culture medium removal conduit is stopped, leaving the residual volume of culture medium and cells in the cell culture apparatus, and when the residual volume of culture medium is less than the first volume of culture medium, a pressure safety valve located between the gas discharge unit and the gas filter is activated to reduce the pressure of the contents.

7. A method according to claim 6, comprising the step of recovering cells, A method comprising: stopping the flow of fluid through the culture medium removal conduit; with the culture medium in contact with the culture medium removal opening of the culture medium removal conduit, gas discharged from the gas discharge unit moves into the cell culture apparatus and pressurizes the cell culture apparatus, transferring the residual culture medium and cells to the cell recovery container through the culture medium removal conduit; and stopping the flow of fluid through the culture medium removal conduit when the fluid changes from liquid to gas.

8. The apparatus according to claim 1, wherein the cells include T cells.

9. A method according to claim 4, wherein the T cells include CAR T cells.

10. A method according to claim 4, wherein the gas-permeable material is silicone.

11. A method according to claim 4, wherein the cell culture apparatus includes a culture surface support.

12. A method according to claim 4, wherein the height of the culture medium is 1 cm to 15 cm.

13. A method according to claim 4, wherein the height of the culture medium is 2 cm to 11 cm.

14. A method according to claim 4, wherein the residual culture medium height is 0.2 cm to 2 cm.

15. A method according to claim 4, wherein the residual culture medium height is 0.2 cm to 1.0 cm.

16. A method according to claim 4, wherein the residual culture medium height is 0.2 cm to 0.5 cm.

17. A method according to claim 4, comprising the additional step of rinsing the cell culture apparatus to further collect any cells that may have remained in the cell culture apparatus and / or cell removal conduit after the flow of fluid through the cell removal conduit has been stopped, further comprising the step of starting to flow liquid into the medium removal conduit as the cell culture apparatus is ventilated to the atmosphere, the liquid moving into the cell culture apparatus through the medium removal conduit, and the liquid being agitated to move cells into the liquid within the cell culture apparatus. A method comprising: starting the supply of gas from a gas outlet into a cell culture apparatus; pressurizing the cell culture apparatus with the gas; transferring liquid and cells from the cell culture apparatus to a cell recovery container via a cell removal conduit; and stopping the flow of fluid through the cell removal conduit when the fluid changes from liquid to gas.

Images