Patent Description:
Cell passaging is done when the cultured cells, such as human stem cells, are <NUM>%-<NUM>% confluent. First, an enzyme and related solution(s) are added to the culture vessel to make the cultured cells detach from a surface of the culture vessel, and then the solution with detached cells (hereinafter referred to as "cell solution") are equally dispensed into double centrifuge tubes, and finally the detached cells are separated from the solutions by using a centrifuge. The cells separated by the centrifuge are transferred to different kinds of containers for subsequent cell passaging process or other processes such as cryo-preservation or bottling for shipping.

When manually dispensing the cell solution to the centrifuge tubes, the cell solution must be split equally for the double centrifuge tubes either volume-wise or weight-wise to balance the centrifuge. The existing manual procedure for splitting the cell solution for the double centrifuge tubes is too complicated and time consuming, and therefor is unsuitable and troublesome for directly converting to an automated process.

Peristaltic pumps are generally used to dispense the liquid or cell solution into the containers, such as centrifuge tubes. Though the peristaltic pumps are capable of precisely controlling a flow of the liquid or the cell solution, a part of the cell solution is stranded in the pump due to significant length of the tubing in the pump, and therefore some of the cells are lost each time when the cell solution is dispensed by the pump.

However, a total amount of the cell solution to be transferred for the cell subculture is limited, and each milliliter of the cell solution comprises a significant amount of cells; therefore, using the peristaltic pump to dispense the cell solution results in loss of a great deal of precious cells.

Moreover, in order to dispense the cell solution, the peristaltic pump inevitably has to pressurize the cell solution, and the pressure difference in the peristaltic pump causes damage to the cells. As a result, the use of the peristaltic pump reduces efficiency of the cell passaging and increases cost of cell passaging.

Additionally, several steps need to be performed before transferring the cell solution to different containers in the cell passaging process. For adherent cells, an enzyme and related solution(s) has to be added to make the cells detach from the surface of the culture vessel. However, currently all steps are performed manually, and are therefore time-consuming, difficult to control quality; risk of contamination due to improper handling is also needed to take into consideration.

In view of the problems of the conventional cell passaging, when using automated equipment to transfer a solution with detached cells, the solutions with detached cells from multiple cell culture containers are first collected in a specialized container, and then a gravitational liquid-splitting device is used to split the collected solution with detached cells equally between two centrifuge containers, and then the two centrifuge containers are disposed symmetrically in a centrifuge for centrifugation, thereby ensuring dynamic balance during centrifugation.

<CIT> discloses an apparatus and a method to prepare cell suspensions for clinical use, describing that the cell culture vessel is coupled to the culture medium supply part at a position lower in the direction of gravitational force than the culture medium supply part.

The main objective of the present invention is to provide a gravitational liquid-splitting device, a cell passaging device with the same, and a cell passaging method to mitigate loss of cells when the solution with detached cells is being transferred to different containers.

The gravitational liquid-splitting device has a funnel, a dispensing valve, a liquid-splitting part, and a stirring assembly. The funnel has an inner space and a bottom opening connected to the bottom of the funnel. The dispensing valve is mounted to the funnel and is configured to close or open the bottom opening of the funnel. The liquid-splitting part is connected to a bottom of the funnel and has an input channel and two output channels. The input channel is formed in the liquid-splitting part and extends upward and downward. An upper end of the input channel forms an input opening on a top of the liquid-splitting part and is connected to the bottom opening of the funnel. The two output channels are formed in the liquid-splitting part. Each of the output channels has a first end and a second end. The first end is connected to a lower end of the input channel. The second end extends inclinedly downward and is connected to an exterior of the liquid-splitting part. The stirring assembly has a funnel and a stirrer. The funnel cover detachably covers an upper opening of the funnel. The stirrer is connected to the funnel cover and is configured to stir liquid in the funnel.

The cell passaging device is configured to dispense contents collected from multiple cell culture containers into two centrifuge containers by gravity. The cell passaging device has a base, a container-handling device, and a liquid equal-splitting device. The container-handling device is mounted on the base and has a container-positioning table, multiple container holders, and multiple container-driving assemblies. The container-positioning table is rotatably or movably mounted on the base. The container holders are pivotally mounted on the container-positioning table. Each of the container holders is configured to accommodate one of the cell culture containers or one of the centrifuge containers, and is pivotable to an emptying angle to empty contents in the cell culture container or the centrifuge container. Each of the container-driving assemblies controls an angle of a respective one of the container holders and is configured to rotate the respective one of the container holders to the emptying angle or to sway the respective one of the container holders. The container-positioning table is configured to move each of the container holders to a first injection position. The liquid equal-splitting device is disposed on a side of the container-handling device. The liquid equal-splitting device has a funnel-positioning table and at least one said gravitational liquid-splitting device as mentioned above. The funnel-positioning table is rotatably or movably mounted on the base. The at least one gravitational liquid-splitting device is mounted on the funnel-positioning table. The funnel-positioning table is configured to move the at least one gravitational liquid-splitting device from a position under the first injection position to a position above the two centrifuge containers such that liquid in the funnel of the at least one gravitational liquid-splitting device flows into the two centrifuge containers via the two output channels respectively.

The cell passaging method comprises: using the gravitational liquid-splitting device to split contents collected from the multiple cell culture containers equally between the two centrifuge containers, and then dispose the two centrifuge containers oppositely in a centrifuge and use the centrifuge to perform centrifugation to separate the cells from the solution.

When using the cell passaging device, first add a solution, which can detach cells from the surface of the cell culture container, into the cell culture container. Then, sway the cell culture container using the container-handling device to uniformly distribute the solution in the cell culture container and make the cells detach from the surface of the cell culture container. Then, rotate the culture container to an emptying angle using the container-handling device to make the cell solution in the cell culture container flow into the gravitational liquid-splitting device located under the cell culture container. Subsequently, the cell solution automatically flows downward and is split into two equal portions by the liquid-splitting part due to gravity, and the two equal portions enter the two centrifuge containers respectively.

The advantages of the present invention are as follows:
First, liquid in the funnel automatically flows downward into the centrifuge containers due to gravity because the liquid-splitting part is disposed under the funnel, the input channel extends upward and downward, and the output channels extend inclinedly downward. Therefore, the present invention prevents loss of cells during transferring operation, avoids damage to the cells under pressurization by using pump, and effectively automates the process of collecting solution with detached cells that are already separated from the cell culture containers together and then split the collected solution with detached cells equally between the two centrifuge containers. As a result, the present invention improves efficiency of cell passaging, increases cell survival rate of cell passaging, and reduces cost of cell passaging.

Second, because the stirrer can continuously stir the solution with detached cells in the funnel, the present invention prevents cells in the solution from clustering together or adhering to surfaces of the channels, thereby keeping flow rates in the two output channels remain equal and ensuring the solution with detached cells is split equally between the two centrifuge containers.

Third, the cell passaging device replaces traditional manual operation by automatically swaying the cell culture container to efficiently detach the adherent cells from the surface of the container, transferring the solution with detached cells into the gravitational liquid-splitting device, and splitting the solution with detached cells equally between the two centrifuge containers, thereby reducing labor-intensity in the cell passaging operation. As a result, the present invention has advantages such as high efficiency, stable quality, and reducing risk of contamination of cell passaging processes.

With reference to <FIG> and <FIG>, the cell passaging device in accordance with the present invention is configured to collect contents (i.e. solution with detached cells) from multiple cell culture containers <NUM>, and then split the collected contents between the two centrifuge containers <NUM> for centrifugation. Cells in solution separated by centrifuge can be used for cell subculture with other cell culture devices. The cell passaging is preferably also configured to transfer the separated cells from solution to cryovials (not shown in figures) for cryopreservation, or to cell solution containers (not shown in figures) for shipments.

The cell culture containers <NUM> and the centrifuge containers <NUM> are containers in the same shape preferably. In the preferred embodiment, the culture containers <NUM> are configured to be put in a centrifuge directly for centrifugation, and therefore the centrifuge containers <NUM> can be two of the cell culture containers <NUM> as well.

The cell passaging device has a base <NUM>, a container-handling device <NUM>, and a liquid equal-splitting device <NUM>. In the preferred embodiment, the cell passaging device further has a liquid solution injection device <NUM>, a primary container-moving device <NUM>, an auxiliary container-moving device <NUM>, a centrifuge <NUM>, and an optical inspection device <NUM>.

With reference to <FIG>, the container-handling device <NUM> is mounted on the base <NUM> and has a container-positioning table <NUM>, multiple container holders <NUM>, and multiple container-driving assemblies <NUM>.

The container-positioning table <NUM> is rotatably mounted on the base <NUM>, and is preferably a conventional rotary indexing table. The container-positioning table <NUM> rotates <NUM> degrees each time, thereby defining four stationary positions that are fixed in space. The four stationary positions are respectively a container-receiving position <NUM>, a container output position <NUM>, a first injection position <NUM>, and a second injection position <NUM>, wherein the second injection position <NUM> and the container output position <NUM> are oppositely disposed on the container-handling device <NUM>.

In the terminology of this technical field, the container-positioning table <NUM> has four indexed positions: the aforementioned container-receiving position <NUM>, the container output position <NUM>, the first injection position <NUM>, and the second injection position <NUM> are the four indexed positions of the container-positioning table <NUM>.

A number of the container holders <NUM> is preferably four; the four container holders <NUM> are disposed around the container-positioning table <NUM> at angular intervals of <NUM> degrees. The container-positioning table <NUM> is configured to move each container holder <NUM> cyclically among the container-receiving position <NUM>, the container output position <NUM>, the first injection position <NUM>, and the second injection position <NUM>.

The container-positioning table <NUM> is not limited to rotary motion. In another preferred embodiment (not shown in figures), the container-positioning table <NUM> moves the container holder <NUM> along a straight line or a bent line to move the container holder <NUM> among multiple specific positions. Moreover, the container-positioning table <NUM> is not limited to move each container holder <NUM> to four different positions; in a less-automated embodiment of the cell passaging device, that is, the container-positioning table <NUM> only moves the container holder <NUM> to the first injection position <NUM>.

With reference to <FIG>, <FIG> and <FIG>, each container holder <NUM> is configured to accommodate one of the cell culture containers <NUM> or one of the centrifuge containers <NUM>. Each of the container holders <NUM> is pivotally mounted on the container-positioning table <NUM>, and is pivotable to an emptying angle (as shown in <FIG>) to empty contents in the culture container <NUM> or the centrifuge container <NUM>. To be precise, each container holder <NUM> is mounted on the container-positioning table <NUM> via a shaft <NUM> and is pivotable around the shaft <NUM>. The shaft <NUM> is preferably horizontal.

With reference to <FIG>, <FIG>, and <FIG>, a number of the container-driving assemblies <NUM> equals the number of the container holders <NUM>. Each of the container-driving assemblies <NUM> controls an angle of a respective one of the container holders <NUM> and is configured to rotate the respective one of the container holders <NUM> to the emptying angle or to sway the respective one of the container holders <NUM>.

In the preferred embodiment, each container-driving assembly <NUM> is mounted on the base and has a linear module <NUM>, a motor <NUM>, and one or multiple driving wheels <NUM>. The linear module <NUM> is mounted on the base <NUM>. The motor <NUM> and the driving wheels <NUM> are mounted on a slider of the linear module <NUM>.

A position of said slider is controllable and the slider is configured to move toward the container holder <NUM> to make the driving wheels <NUM> abut against a driven wheel <NUM>. The driven wheel <NUM> is connected to the shaft <NUM> such that the motor <NUM> can control the angle of the corresponding container holder <NUM> via the driving wheels <NUM> and the driven wheel <NUM>. By mounting the container-driving assembly <NUM> on the base <NUM> instead of the container-positioning table <NUM>, makes cabling of the linear module <NUM> and motor <NUM> more simplified.

With reference to <FIG>, <FIG>, and <FIG>, the liquid solution injection device <NUM> is mounted on the base <NUM> and has multiple injection heads <NUM>. Each of the injection heads <NUM> is connected to a container with a kind of liquid solution and is movable to a position above the second injection position <NUM> to inject the liquid solution in a connected container into the cell culture container <NUM> located in the container holder <NUM> at the second injection position <NUM>. The liquid solution injection device <NUM> is preferably disposed on a side, which is toward the second injection position <NUM>, of the container-handling device <NUM>.

In the preferred embodiment, the liquid solution injection device <NUM> is a turret. The injection heads <NUM> are disposed around a center axis of the turret and the turret is configured to rotate a specified injection head <NUM> to the position above the second injection position <NUM> for liquid solution injection. The liquid solutions connected to the injection head <NUM> include kinds of liquid reagents which can make adherent cells detach from a surface of the cell culture container <NUM>, and also include balance reagent which can neutralize the effectiveness of the detaching reagent.

With reference to <FIG>, <FIG>, <FIG> and <FIG>, the liquid equal-splitting device <NUM> is disposed on a side of the container-handling device <NUM>; the liquid equal-splitting device <NUM> has a funnel-positioning table <NUM> and at least one gravitational liquid-splitting device <NUM>. The funnel-positioning table <NUM> is rotatably mounted on the base <NUM>, and is preferably a conventional rotary indexing table.

The funnel-positioning table <NUM> is disposed lower in height than the container-positioning table <NUM>, but disposed higher in height than the position of two centrifuge containers <NUM>. One indexed position of the funnel-positioning table <NUM> is located under the first injection position <NUM> of the container-positioning table <NUM>, and another indexed position of the funnel-positioning table <NUM> is located above the position of two centrifuge containers <NUM>.

With reference to <FIG>, the number of the gravitational liquid-splitting device <NUM> is preferably four, at least one, and the four gravitational liquid-splitting devices <NUM> are disposed around a center axis of the funnel-positioning table <NUM>. Each gravitational liquid-splitting device <NUM> has a funnel <NUM>, a dispensing valve <NUM>, a liquid-splitting part <NUM>, and a stirring assembly <NUM>. In the preferred embodiment, each gravitational liquid-splitting devices <NUM> further has a funnel cover <NUM>. The funnel <NUM> has an inner space and a bottom opening, and the inner space is connected to the bottom opening. A first ferromagnetic part <NUM> of the dispensing valve <NUM> is mounted in the funnel <NUM> and is configured to close or open the bottom opening of the funnel <NUM>. Here "closing the bottom opening" means the dispensing valve <NUM> prevents liquid in the funnel <NUM> from flowing down via the bottom opening, and therefore the funnel <NUM> dispensing valve <NUM> does not have to directly cover the bottom opening.

In the preferred embodiment, the inner space of the funnel <NUM> forms a channel <NUM> (as shown in <FIG>) in the bottom of the funnel <NUM>. The channel <NUM> extends upward and downward, and a lower end of the channel <NUM> forms the bottom opening of the funnel <NUM>. The first ferromagnetic part <NUM> of the dispensing valve <NUM> is disposed in the channel <NUM> and is configured to be controlled to close or open the channel <NUM>, thereby closing or opening the bottom opening of the funnel <NUM>.

With reference to <FIG> and <FIG>, the dispensing valve <NUM> has the first ferromagnetic part <NUM>, a valve actuator <NUM>, and a second ferromagnetic part <NUM>. The first ferromagnetic part <NUM> is movably disposed in the channel <NUM> of the funnel <NUM> and driven by a weight of the first ferromagnetic part <NUM> to move toward the bottom opening of the funnel <NUM> to close the bottom opening (as shown in <FIG>). The first ferromagnetic part <NUM> is preferably a ball with a metal core.

The valve actuator <NUM> is disposed outside of the funnel <NUM> and has a moving end 4222A which is controllable and configured to move toward the first ferromagnetic part <NUM> to an open position (as shown in <FIG>). To be precise, the valve actuator <NUM> is a pneumatic cylinder. The valve actuator <NUM> is mounted on the base <NUM> and can be controlled by pressurized air to make a slider of the valve actuator <NUM> move toward the first ferromagnetic part <NUM>.

The second ferromagnetic part <NUM> is mounted on a moving end 4222A of the valve actuator <NUM>. The second ferromagnetic part <NUM> and the first ferromagnetic part <NUM> magnetically attract or repel each other. When the moving end 4222A of the valve actuator <NUM> is at the open position, the first ferromagnetic part <NUM> is driven by the second ferromagnetic part <NUM> to open the bottom opening of the funnel.

In the preferred embodiment, the second ferromagnetic part <NUM> is a permanent magnet. When the moving end 4222A of the valve actuator <NUM> is at the open position, the first ferromagnetic part <NUM> is magnetically attracted by the second ferromagnetic part <NUM> and opens the channel <NUM>. In another preferred embodiment (not shown in figures), the first ferromagnetic part <NUM> and the second ferromagnetic part <NUM> are permanent magnets which magnetically attract or repel each other, or the second ferromagnetic part <NUM> is a coil which generates magnetic field when powered-on.

With reference to <FIG>, the liquid-splitting part <NUM> is connected to the bottom of the funnel <NUM>. The liquid-splitting part <NUM> has an input channel <NUM>, two output channels <NUM>, and two auxiliary channels <NUM> formed in the liquid-splitting part <NUM>, but the auxiliary channels can be omitted.

The input channel <NUM> extends upward and downward; an upper end of the input channel <NUM> forms an input opening on a top of the liquid-splitting part <NUM> and connected to the bottom opening of the funnel. Each of the output channels <NUM> has a first end and a second end; the first end is connected to a lower end of the input channel <NUM>; the second end extends inclinedly downward and connected to an exterior of the liquid-splitting part.

Liquid entering the input channel <NUM> is split equally between the two output channels <NUM> by gravity and shapes of the output channels <NUM>. In the preferred embodiment, each output channel <NUM> is linear, and its lower end is connected to the exterior of the liquid-splitting part via a vertical channel.

Each auxiliary channel <NUM> has a third end and a fourth end; the third end is connected to one of the two output channels <NUM>; the fourth end extends inclinedly upward and is connected to the exterior of the liquid-splitting part. To be specific, each auxiliary channel <NUM> extends along a same straight line with a respective one of the output channels <NUM>.

Function of the auxiliary channels <NUM> is to balance pressure in the two output channels <NUM>, thereby stabilizing flow speed in the two output channels <NUM>. Therefore, the auxiliary channels <NUM> further ensure the liquid entering the input channel <NUM> is split equally between the two output channels <NUM>. Moreover, each auxiliary channel <NUM> is in line with a respective one of the output channels <NUM> for ease of cleaning. In another preferred embodiment, there is only one auxiliary channel <NUM>, and the third end of the auxiliary channel <NUM> is connected to both of the two output channels <NUM>.

With reference to <FIG>, <FIG>, <FIG>, and <FIG>, the funnel cover <NUM> detachably covers an upper opening of the funnel <NUM>. The stirring assembly <NUM> has a linear module <NUM>, a stirring motor <NUM>, and a stirrer <NUM>. The linear module <NUM> is mounted on the base <NUM> and can be controlled to move a slider 4251A of the linear module <NUM>. The stirring motor <NUM> is mounted on the slider 4251A of the linear module <NUM>. The stirrer <NUM> is rotatably mounted through the funnel cover <NUM> and is configured to stir liquid in the funnel <NUM>.

When the funnel cover <NUM> covers the funnel <NUM>, the linear module <NUM> is configured to move the stirring motor <NUM> toward the stirrer <NUM> (as shown in <FIG>) such that an output axle of the stirring motor <NUM> is connected to an upper end of the stirrer <NUM>, thereby allowing the stirring motor <NUM> to rotate the stirrer <NUM>. Structure of the stirring assembly <NUM> is not limited by the abovementioned as long as there is a stirrer <NUM> configured to stir liquid in the funnel <NUM>.

With reference to <FIG>, <FIG>, <FIG>, and <FIG>, rotation of the funnel-positioning table <NUM> is configured to move each of the gravitational liquid-splitting devices <NUM> from a position under the first injection position <NUM> to a position above the position of two centrifuge containers <NUM> such that liquid in the funnel <NUM> of each gravitational liquid-splitting device <NUM> flows into the two centrifuge containers <NUM> via the two output channels <NUM> respectively.

In the preferred embodiment, the funnel-positioning table <NUM> is both rotatable on a horizontal plane and linearly movable toward or away from the container-handling device <NUM>. The funnel-positioning table <NUM> moves each gravitational liquid-splitting device <NUM> to the position above the two centrifuge containers <NUM> by a combination of said rotation and linear motion.

Operation mode of the funnel-positioning table <NUM> is not limited to rotation. In another preferred embodiment, the funnel-positioning table <NUM> moves the gravitational liquid-splitting devices <NUM> along a straight line or a bent line to move the gravitational liquid-splitting devices <NUM> among multiple specific positions.

With reference to <FIG> and <FIG>, the primary container-moving device <NUM> is configured to move each of the two centrifuge containers <NUM> from the position under the gravitational liquid-splitting device <NUM> to the container holder <NUM> at the container-receiving position <NUM>.

Additionally, the primary container-moving device <NUM> is also configured to move the cell culture container <NUM> in the container holder <NUM> at the container-receiving position <NUM> to the position under the gravitational liquid-splitting devices <NUM> such that liquid in the funnel <NUM> of the corresponding gravitational liquid-splitting device <NUM> flows into the cell culture container <NUM>. Therefore, the primary container-moving device <NUM> allows the present invention to recycle two of the cell culture containers <NUM> so that they can be used again as the two centrifuge containers <NUM>.

In the preferred embodiment, the primary container-moving device <NUM> has a first multi-axis transfer mechanism <NUM>, a container rack <NUM>, a first conveyer <NUM>, a second conveyer <NUM>, a third conveyer <NUM>, a second multi-axis transfer mechanism <NUM>, and a bottling and transferring mechanism <NUM>. With reference to <FIG> and <FIG>, the first multi-axis transfer mechanism <NUM> is preferably a three-axis transfer mechanism which includes three linear modules <NUM>. One of the linear modules <NUM> that extends vertically has a gripper <NUM> mounted on a lower end of said linear module <NUM>. The gripper <NUM> is configured to grip one of the containers (e.g., the cell culture container <NUM> or the centrifuge container <NUM>) accommodated in the container holder <NUM> at the container-receiving position <NUM>, the container rack <NUM>, or the first conveyer <NUM>. The gripper <NUM> is also configured to put the gripped container in any one of the said three components. To be more precise, the gripper <NUM> is configured to rotate <NUM> degrees along a horizontal axis to change an angle of the gripped container.

The container rack <NUM> is configured to accommodate twelve containers to improve operational efficiency. The first conveyer <NUM> includes a translating mechanism and an elevating mechanism such that the first conveyer <NUM> moves containers from a bottom to a top of the first conveyer <NUM> where the first multi-axis transfer mechanism <NUM> can grip the container or put several new containers in the container rack <NUM> one by one.

The second conveyer <NUM> and the third conveyer <NUM> are linear belt conveyors. One end of each conveyor is mounted in a bottom of the first conveyer <NUM>, and another end of each conveyor extends to the second multi-axis transfer mechanism <NUM>.

The second multi-axis transfer mechanism <NUM> is preferably a three-axis transfer mechanism which is configured to move containers among the second conveyer <NUM>, the third conveyer <NUM>, and the bottling and transferring mechanism <NUM>.

With reference to <FIG>, the bottling and transferring mechanism <NUM> is configured to grip the double centrifuge containers <NUM>, double the cell culture containers <NUM>, double cryovials (not shown in figures), double cell solution containers (not shown in figures), or other types of containers. The bottling and transferring mechanism <NUM> is configured to align the two gripped containers with the two output channels <NUM> of one of the gravitational liquid-splitting devices <NUM> such that the liquid in said gravitational liquid-splitting device <NUM> can be transferred to the two gripped containers. After transferring the liquid, the bottling and transferring mechanism <NUM> moves the two gripped containers away from the position under the container-positioning table <NUM> for the second multi-axis transfer mechanism <NUM> to grip for the further operation.

In the preferred embodiment, a cell solution container cap mechanism <NUM> is disposed under the second multi-axis transfer mechanism <NUM>. The cell solution container cap mechanism <NUM> is configured to handle caps of the cell solution containers in collaboration with the second multi-axis transfer mechanism <NUM>.

With reference to <FIG>, the auxiliary container-moving device <NUM>, the centrifuge <NUM>, and the optical inspection device <NUM> are disposed on a same side of the container-handling device <NUM> as where the container output position <NUM> is. The centrifuge <NUM> and the optical inspection device <NUM> are standardized conventional equipment. The optical inspection device <NUM> is configured to measure a number of cells in the culture container <NUM> in order to determine whether a total number of the cells collected in the culture container <NUM> is satisfactory for the further cell passaging process. To be more specific, the optical inspection device <NUM> is configured to calculate automatically the number of detached cells in the cell culture containers <NUM> by a specific AI algorithm.

With reference to <FIG>, the auxiliary container-moving device <NUM> is configured to move the centrifuge container <NUM> or the cell culture container <NUM> in the container holder <NUM> at the container output position <NUM> into the centrifuge <NUM>, and also configured to move the centrifuge container <NUM> or the cell culture container <NUM> in the centrifuge <NUM> back into the container holder <NUM> at the container output position <NUM>.

Additionally, the auxiliary container-moving device <NUM> is configured to move the centrifuge container <NUM> or the cell culture container <NUM> in the container holder <NUM> at the container output position <NUM> into the optical inspection device <NUM>, and also configured to move the centrifuge container <NUM> or the cell culture container <NUM> in the optical inspection device <NUM> back into the container holder <NUM> at the container output position <NUM>. The auxiliary container-moving device <NUM> is preferably a robotic arm.

The cell passaging method in accordance with the present invention is preferably performed using the abovementioned cell passaging device. The cell passaging device is configured to automatically perform various cell passaging methods for cell subculture process. One of the cell passaging methods for subculture of adherent cells, which is in accordance with the present invention, is explained below.

With reference to <FIG>, multiple cell culture containers <NUM> already in the stationary phase are transmitted to the first conveyer <NUM> of the primary container-moving device <NUM> by an external transfer mechanism.

With reference to <FIG>, then, the first multi-axis transfer mechanism <NUM> grips the cell culture containers <NUM> in the first conveyer <NUM> and dispenses the cell culture containers <NUM> into the container holder <NUM> at the container-receiving position <NUM>. The gripper <NUM> of the first multi-axis transfer mechanism <NUM> rotates the gripped cell culture container <NUM> from a horizontal posture to a vertical posture (as shown in <FIG> and <FIG>) before putting the gripped cell culture container <NUM> into the container holder <NUM> for ease of subsequent operation.

With reference to <FIG> and <FIG>, then, the cell culture container <NUM> is moved from the container-receiving position <NUM> to the second injection position <NUM>. A cap mechanism <NUM> moves down to grip a cap of the cell culture container <NUM>.

With reference to <FIG>, then, a cap mechanism <NUM> loosens the cap of the cell culture container <NUM> and moves the cap aside, and the container-driving assembly <NUM> rotates the container holder <NUM> to empty the content (ex. medium) of the cell culture container <NUM>. Then, the injection head <NUM> of the liquid solution injection device <NUM> moves to the position above the second injection position <NUM> and injects the detaching reagent (ex. Trypsin) into the cell culture container <NUM>, wherein the detaching reagent is functioned to make the adherent cells detach from the surface of the cell culture container <NUM>.

With reference to <FIG> and <FIG>, then, the cap mechanism <NUM> puts the cap back onto the cell culture container <NUM> and tightens the cap, and the culture container <NUM> is moved to the first injection position <NUM>. The container-driving assembly <NUM> sways the culture container <NUM> at the first injection position <NUM> to make cells detached from the surface of the cell culture container <NUM>.

With reference to <FIG> and <FIG>, then, the cell culture container <NUM> is moved to the container output position <NUM>, and then the auxiliary container-moving device <NUM> grips the cell culture container <NUM> and dispenses the culture container <NUM> into the optical inspection device <NUM> to calculate automatically the number of cells in the cell culture container <NUM> by using a specific AI algorithm.

When the process of cell counting in the optical inspection device <NUM> has finished, the culture cell container <NUM> is moved back to the container output position <NUM>, and subsequently moved to the second injection position <NUM>. At the second injection position <NUM>, another one of the injection heads <NUM> of the liquid solution injection device <NUM> injects balance reagent into the cell culture container <NUM> to neutralize the effectiveness of detaching reagent.

With reference to <FIG>, <FIG>, and <FIG>, then, the culture cell container <NUM> is moved to the first injection position <NUM>, and the container-driving assembly <NUM> rotates the container holder <NUM> to pour contents (e.g., solution with detached cells) in the cell culture container <NUM> into the funnel <NUM> of the gravitational liquid-splitting device <NUM>. The funnel cover <NUM> on top of the funnel <NUM> has been removed before this step. Then, the above steps are performed again for the rest of the cell culture containers <NUM> until the solutions with detached cells from all culture containers <NUM> are collected in the funnel <NUM>.

Among the cell culture containers <NUM>, the first two cell culture containers <NUM> are moved to the position under one of the gravitational liquid-splitting devices <NUM> after their solutions with detached cells are poured into the funnel <NUM> such that the first two cell culture containers <NUM> serve as the two centrifuge containers <NUM>.

With reference to <FIG> and <FIG>, after the solutions with detached cells from all cell culture containers <NUM> are collected in the funnel <NUM>, the funnel-positioning table <NUM> moves the gravitational liquid-splitting device <NUM> clockwise to the position above the two centrifuge containers <NUM>, and meanwhile the slider 4251A of the stirring assembly <NUM> moves down to connect the stirring motor <NUM> to the upper end of the stirrer <NUM>. The stirring motor <NUM> drives the stirrer <NUM> to keep stirring the solution with detached cells in the funnel <NUM>.

With reference to <FIG> and <FIG>, then, the valve actuator <NUM> of the dispensing valve <NUM> moves the second ferromagnetic part <NUM> toward the first ferromagnetic part <NUM> to the open position to move the first ferromagnetic part <NUM> such that the solution with detached cells in the funnel <NUM> is split equally between the two centrifuge containers <NUM>.

With reference to <FIG>, finally, the two centrifuge containers <NUM> are moved to the container-handling device <NUM> by the primary container-moving device <NUM>, and then moved into the centrifuge <NUM> by the auxiliary container-moving device <NUM> to perform centrifugation. After the centrifugation, the two centrifuge containers <NUM> are ready for subsequent cell passaging operations.

The cell passaging method in accordance with the present invention is not limited to aforementioned as long as contents in the cell culture containers <NUM> are split equally between the two centrifuge containers <NUM> by using the gravitational liquid-splitting device <NUM>, and then the two centrifuge containers <NUM> are disposed oppositely in the centrifuge <NUM> to perform centrifugation to separate the cells from the solution.

In addition to performing the abovementioned cell passaging method, the cell passaging device is also configured to automatically perform various processes related to cell culture processing, such as primary specimen cells extraction process, cell culture container medium exchange process, cell culture container optical inspection process, and some of the cell cryopreservation processes such as cryovial dispensing, partial cell thawing process, and cell solution bottling for shipping process.

In summary, liquid (solution with detached cells) in the funnel <NUM> automatically flows downward into the centrifuge containers <NUM> due to gravity because the liquid-splitting part <NUM> of the gravitational liquid-splitting device <NUM> is disposed under the funnel <NUM>, the input channel <NUM> extends upward and downward, and meanwhile the output channels <NUM> extend inclinedly downward; therefore, no liquid residue is left in the funnel <NUM>, the input channel <NUM>, and the output channel <NUM>.

As a result, the present invention prevents loss of cells during transferring operation, avoids damage to the cells under pressurization by using pump, and effectively automates the process of collecting solution with detached cells together and then split the collected solution with detached cells equally between the two centrifuge containers <NUM>, thereby improving efficiency of cell passaging and reducing cost of cell passaging.

Moreover, with the stirrer <NUM> of the gravitational liquid-splitting device <NUM> continuously stirring the solution with detached cells in the funnel, <NUM>, the present invention prevents cells from clustering together or adhering to surfaces of the funnel <NUM> or channels, thereby keeping flow rates in the two output channels <NUM> remain equal and ensuring the solution with detached cells is split equally between the two centrifuge containers <NUM>.

Claim 1:
A gravitational liquid-splitting device (<NUM>) characterized in that the gravitational liquid-splitting device (<NUM>) comprises:
a funnel (<NUM>) having an inner space and a bottom opening connected to the inner space;
a dispensing valve (<NUM>) mounted to the funnel (<NUM>) and configured to close or open the bottom opening of the funnel (<NUM>);
a liquid-splitting part (<NUM>) connected to a bottom of the funnel (<NUM>) and having:
an input channel (<NUM>) formed in the liquid-splitting part (<NUM>) and extending upward and downward; an upper end of the input channel (<NUM>) forming an input opening on a top of the liquid-splitting part (<NUM>) and connected to the bottom opening of the funnel (<NUM>); and
two output channels (<NUM>) formed in the liquid-splitting part (<NUM>); each of the output channels (<NUM>) having a first end and a second end; the first end connected to a lower end of the input channel (<NUM>); the second end extending inclinedly downward and connected to an exterior of the liquid-splitting part (<NUM>); and
a stirring assembly (<NUM>) having
a funnel (<NUM>) cover detachably covering an upper opening of the funnel (<NUM>); and
a stirrer (<NUM>) connected to the funnel (<NUM>) cover and configured to stir liquid in the funnel (<NUM>).