Integrated Automated Cell Culture Device

Abstract

The present invention provides an integrated automated cell culture device including a main body, a lid, a stopper, an air supply module, a temperature control module, at least one slide rail section, and a bag clamping part, in which the main body surrounds a culture chamber whose bottom is used to place a culture bag, the lid is installed to cover the opening of the culture chamber, and a stopper for passing a pipeline is installed to be packed into a storage groove of the main body, the air supply module detects the carbon dioxide concentration in the culture chamber and also controls an external gas source to supply air into the culture chamber, the temperature control module detects the temperature in the culture chamber and also selectively heats the culture chamber, the slide rail section is installed at the bottom of the culture chamber, and the bag clamping part is slidably installed on the slide rail section to hold the culture bag in place so as to concentrate the liquid in the culture bag on one side.

Description

[Technical field]

[0001] The present invention relates to a cell culture device, and more particularly to a fully integrated automated cell culture device. [Background technology]

[0002] Currently, in addition to conventional chemotherapy, radiation therapy, or surgical resection, many cancer treatments may also use gene therapy or cell therapy to avoid serious side effects. In cell therapy, the patient's own immune cells do not cause rejection reactions, so they can attack cancer cells more effectively than immune cells provided by others, and they can reduce the burden on the patient's body.

[0003] To perform cell therapy, autologous or allogeneic cells must be cultured and expanded in vitro and then reinjected into the patient's body. Cell culture is usually performed in a soft culture bag, and new culture medium is gradually injected as the number of proliferating cells increases. The culture medium is then placed in a laboratory environment under specified temperature and humidity conditions.

[0004] Among them, in order to check the cell culture status, it is necessary to observe the culture medium, and then manually extract a small amount of the culture medium from the bag many times to calculate the number of cells, and then inject a corresponding amount of culture medium according to the number of cells. In the entire cell culture process, manual observation and cell counting steps must be performed many times, which results in high labor costs, and there is a risk of cell contamination and errors due to manual operation, resulting in unstable quality.

[0005] Therefore, cell culture cannot achieve full automation of the culture process, leading to a situation where the costs are so high that general patients cannot afford it. Summary of the Invention [Problem to be solved by the invention]

[0006] The main object of the present invention is to provide an integrated automated cell culture device that improves the automation level of cell culture by performing observation and culture operations of culture bags in an integrated facility.

[0007] Another object of the present invention is to provide an integrated automated cell culture device that does not require taking out the culture medium for calculation, does not affect the culture environment, and allows the cell density to be known more quickly and the cell density in the culture bag to be easily observed. [Means for solving the problem]

[0008] In order to achieve the above object, the present invention provides an integrated automated cell culture device, including a main body, a cover, a plug, an air supply module, at least one slide rail portion, and a bag clamping part.

[0009] The main body surrounds the culture chamber, has an opening at the top of the main body that connects to the culture chamber, and further has a storage groove on a side of the main body that connects to the culture chamber, and the bottom of the culture chamber is used to place a culture bag. The lid is installed so as to removably cover the opening of the main body in order to close the culture chamber. The plug is installed so as to be filled in the storage groove, and at least one through hole is provided to penetrate the plug, and the at least one through hole is for passing at least one pipe extending to connect the culture chamber to the outside, and one end of the at least one pipe is connected to the culture bag. The air supply module includes a carbon dioxide sensor and an air supply connector, the carbon dioxide sensor is installed in the culture chamber and is electrically connected to the air supply connector, and the air supply connector is installed in the main body so as to be connected to an external gas source to supply gas into the culture chamber. The temperature control module is installed on at least one of the main body and the cover, and includes a heater and a temperature sensor, and the temperature sensor is electrically connected to the heater to detect the temperature in the culture chamber and control the heater to selectively heat the culture chamber. The at least one slide rail part is installed in the culture chamber so as to extend along a sliding direction, and the bag clamping part is slidably installed on the at least one slide rail part, holds the culture bag so that liquid in the culture bag does not flow to one side of the culture bag, and can slide on the culture bag. Preferably, the culture chamber further includes at least one pulling mechanism installed at at least one end of the culture chamber for holding one end of the culture bag.

[0010] Preferably, the device further includes a light irradiation counting module, which includes a light source and a light receiving unit, the light source being installed on one of the culture chamber and the surface of the cover facing the culture chamber so as to be located between the light source and the light receiving unit when the culture bag is placed in the culture chamber, and the light receiving unit being installed on the other of the culture chamber and the surface of the cover facing the culture chamber, the light source emitting a light beam with a wavelength of 400 to 600 nm, a light beam with a wavelength of 400 to 500 nm, or a light beam with a wavelength of 400 nm to the light receiving unit, the light beam passing through the culture bag and being received by the light receiving unit, the light receiving unit calculating the absorbance of the liquid in the culture bag, and thus calculating the cell density in the culture bag.

[0011] In one case, the culture bag has an observation section extending outward in a horizontal direction, liquid and cells in the culture bag can freely enter and exit the observation section, and the positions of the light source and the light receiving section correspond to the observation section so that light emitted from the light source passes through the culture bag via the observation section.

[0012] In one case, an observation window is provided in the center of the lid, heated glass is provided to cover the observation window, and a top-opening lid that can be opened upwards is provided to cover the side of the heated glass opposite the side facing the culture chamber, the top-opening lid selectively shields the heated glass from the outside, the heated glass corresponds to the heater, and the temperature sensor is provided on the surface of the lid facing the culture chamber.

[0013] In some cases, the air supply module and the spigot are located at opposite ends of the incubation chamber.

[0014] In some cases, the device further includes a ventilation stand installed at the bottom of the culture chamber for placing the culture bag. Preferably, a plurality of openwork portions are installed to penetrate the ventilation stand to improve ventilation at the bottom of the culture bag.

[0015] In one case, the device further includes an insulated box and a pump, the insulated box being installed within the main body to accommodate at least one culture medium container, the pump being installed within the main body, and the at least one pipeline being connected to the at least one culture medium container via the pump, thereby supplying liquid into the culture bag. Effect of the Invention

[0016] This allows the environment in the culture chamber to be quickly measured and the environment to be manually or automatically operated without interfering with the cell culture environment. Furthermore, the light irradiation counting module can also measure the cell density in the culture bag, which can be used to judge the cell growth status and provide a reference for adjusting and controlling the growth conditions such as culture medium, gas, temperature, and humidity, thereby achieving stable automated cell culture. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a perspective view of an integrated automated cell culture device of the present invention. [Diagram 2] FIG. 1 is a perspective view of an integrated automated cell culture device of the present invention. [Diagram 3] FIG. 2 is an exploded perspective view of the integrated automated cell culture device of the present invention. [Figure 4] FIG. 2 is a partial cross-sectional view of the integrated automated cell culture device of the present invention. [Diagram 5] FIG. 1 is a front view of the integrated automated cell culture device of the present invention. [Figure 6] FIG. 4 is a diagram showing the relationship between light source wavelength and absorbance in the first embodiment of the present invention. [Figure 7] FIG. 2 is a graph showing the relationship between absorbance and cell density when irradiated with a 290 nm light source in the first embodiment of the present invention. [Figure 8] FIG. 11 is a diagram showing the relationship between light source wavelength and absorbance in the second embodiment of the present invention. [Figure 9] FIG. 11 is a graph showing the relationship between absorbance and cell density when irradiated with a 290 nm light source in a second embodiment of the present invention. [Figure 10]FIG. 13 is a diagram showing the relationship between light source wavelength and absorbance in the third embodiment of the present invention. [Figure 11] FIG. 11 is a graph showing the relationship between absorbance and cell density when irradiated with a 290 nm light source in a third embodiment of the present invention. [Figure 12] FIG. 13 is a diagram showing the relationship between absorbance and cell density when irradiated with light sources of different wavelengths in the third embodiment of the present invention. [Figure 13] 13 is a table showing the relationship between absorbance and cell density when irradiated with a light source of different wavelengths in the third embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] 1 to 5. The present invention provides an integrated automated cell culture device including at least a main body 10, a cover 20, a plug 30, an air supply module 40, a temperature control module, a culture bag positioning structure, and a light irradiation counting module, and preferably further including an aeration stand 70 and a culture medium supply mechanism.

[0019] The main body 10 can be roughly box-shaped, enclosing a culture chamber 11 inside, having an opening connected to the culture chamber 11 at the top of the main body 10, and further having a storage groove 12 connected to the culture chamber 11 on the side of the main body 10. Preferably, the storage groove 12 is cut out and formed near the opening of the main body 10. In addition, the bottom of the culture chamber 11 is used to place a culture bag, and the culture bag is placed horizontally on the bottom of the culture chamber 11.

[0020] The cover 20 is removably installed to cover the opening of the main body 10 in order to close the incubation chamber 11 and maintain a constant and controllable internal environment. Specifically, in this embodiment, corresponding clasps are installed around the periphery of the cover 20 and around the periphery of the opening of the main body 10, so that when the cover 20 is installed to cover the opening of the main body 10, these clasps can be tightly fastened to ensure that the incubation chamber 11 does not communicate with the outside.

[0021] The plug 30 is installed so as to be filled into the receiving groove 12, and at least one through hole 31 is provided so as to penetrate the plug 30, and the at least one through hole 31 is for passing at least one pipe 90 extending so as to connect the culture chamber 11 with the outside, and one end of the at least one pipe 90 is connected to the culture bag. As a result, the pipe is attached to the plug in advance, and then the plug is simply filled into the receiving groove, so that the modularized plug can be installed more easily and quickly than when the pipe is directly inserted into the main body.

[0022] The air supply module 40 includes a carbon dioxide sensor and an air supply connector, the carbon dioxide sensor is installed in the incubation chamber 11 and electrically connected to the air supply connector, and the air supply connector is installed in the main body 10 so as to connect to an external gas source to supply gas into the incubation chamber 11. Preferably, the air supply module 40 is installed on a side of the main body 10. In this embodiment, the air supply module 40 and the stopper 30 are installed at opposite ends of the incubation chamber 11, respectively.

[0023] The temperature control module is installed on at least one of the main body 10 and the cover 20, and includes a heater and a temperature sensor 24. The temperature sensor 24 is electrically connected to the heater, detects the temperature in the culture chamber 11, and controls the heater to selectively heat the culture chamber 11 so as to maintain the temperature in the culture chamber 11 at a constant suitable culture temperature close to the body temperature of the human body. Specifically, in this embodiment, the heater is a heating glass 22, an observation window 21 of a size corresponding to the size of the culture bag is installed in the center of the cover 20, the heating glass 22 is installed to cover the observation window 21, and a top-opening lid 23 that can be opened upward is installed to cover the heating glass 22 on the opposite side to the side facing the culture chamber 11, and the top-opening lid 23 selectively blocks the heating glass 22 from the outside, and the user can open it to observe the internal situation of the culture chamber 11, but in most situations, it can block external light so as not to affect the culture environment of the cells. The temperature sensor 24 can be installed on the surface of the lid 20 facing the culture chamber 11, but is not limited to this. In another possible embodiment, a heater that heats the culture bag from below may be installed at the bottom of the culture chamber.

[0024] The light irradiation counting module includes a light source 51 and a light receiving unit 52, and the light source 51 is installed on one of the surfaces of the culture chamber 11 and the cover body 20 facing the culture chamber 11 so as to be located between the light source 51 and the light receiving unit 52 when the culture bag is placed in the culture chamber 11 (in this embodiment, the light source 51 is installed on the surface of the cover body 20 facing the culture chamber 11), and the light receiving unit 52 is installed on the surface of the culture chamber 11 and the cover body 20 facing the culture chamber 11 so as to be located between the light source 51 and the light receiving unit 52 when the culture bag is placed in the culture chamber 11. The light source 51 emits a light beam with a wavelength of 400 to 600 nm, a light beam with a wavelength of 400 to 500 nm, or a light beam with a wavelength of 400 nm to the light receiving unit 52, and the light beam passes through the culture bag and is received by the light receiving unit 52, whereby the light receiving unit calculates the absorbance of the liquid in the culture bag, and thus calculates the cell density in the culture bag. The conversion between absorbance and cell density will be described in detail in a later paragraph.

[0025] It is particularly noted that the light source and the light receiving unit must be installed at a position where the light can pass through the culture bag, i.e., above and below the culture bag, but the culture bag can further have an observation unit extending outward in a lateral direction, so that the activity and growth of most of the cells in the culture bag are not affected by the light. The observation unit is, for example, a finger-like or ear-like protrusion, and the liquid and cells in the culture bag can freely enter and exit the observation unit, and the positions of the light source and the light receiving unit correspond to the observation unit, and the light passes only through the observation unit, thereby reducing the influence on other parts of the culture bag.

[0026] The culture bag positioning structure includes at least one slide rail portion 61 and a bag clamping part 62, and the at least one slide rail portion 61 is installed in the culture chamber 11 so as to extend along the sliding direction (for example, the slide rail portion may be installed directly on the bottom surface of the culture chamber so as to be directly connected to the bag clamping part, or the slide rail portion may include a smooth surface at the bottom of the culture chamber and a magnet or electromagnet installed below the culture chamber, so that the bag clamping part can be attracted by magnetic force and moved to slide on the smooth surface), and the bag clamping part 62 is slidably installed on the at least one slide rail portion 61, holds the culture bag so that the liquid in the culture bag is concentrated on one side of the culture bag, and can slide on the culture bag. In this embodiment, the culture bag positioning structure includes two slide rail portions 61, and the two slide rail portions 61 are installed on opposite sides of the bottom of the culture chamber 11, respectively, and extend parallel to each other. The bag clamping part 62 includes two horizontal bars spaced apart from each other, each of which has both ends slidably mounted on the two slide rail parts 61. A culture bag extends between the two horizontal bars and is clamped between the two horizontal bars. When the bag clamping part 62 slides along the two slide rail parts, the two horizontal bars slide over the culture bag and push the liquid in the culture bag to one end, so that the liquid in the culture bag is concentrated on one side of the culture bag.

[0027] In addition, the culture bag positioning structure may further include at least one pulling mechanism 63. The at least one pulling mechanism 63 is installed at least at one end of the culture chamber, and releasably fixes one end of the culture bag, thereby pulling the culture bag from the end, thereby preventing the culture bag from being deformed due to the movement of the bag clamping part 62. Specifically, the pulling mechanism 63 is, for example, a clip that clamps and fixes the culture bag from the end, but it can also be arranged as a hook or a stop pin and used in combination with a culture bag having a corresponding hook or hanging hole. Alternatively, the pulling mechanism has a fixed post and a magnet, and the magnet is attracted to the fixed post by magnetic force, and the culture bag can be clamped and fixed between the magnet and the fixed post.

[0028] The ventilation stand 70 is detachably installed at the bottom of the culture chamber 11 to place the culture bag. In order to improve the ventilation of the bottom of the culture bag and ensure that the culture environment at the top and bottom of the culture bag is almost the same, a number of openwork parts are installed to penetrate the ventilation stand 70.

[0029] The culture medium supply mechanism includes at least a cooling box 81 and a pump 82. The cooling box 81 is installed in the main body 10 so as to house at least one culture medium container, thereby preserving the culture medium at a low temperature. The pump 82 is installed in the main body 10, and the at least one pipe 90 is connected to the at least one culture medium container via the pump 82, thereby supplying the culture medium that has been appropriately preserved at a low temperature into the culture bag. In addition, the pump 82 can be further connected to a control mechanism in order to automate the control of the supply of the culture medium and the supply amount.

[0030] According to the above structure, the culture bag containing the culture medium and cells is placed in the culture chamber, connected with a pipe, and the culture chamber is closed with a lid, thereby achieving automation of cell culture. What is important is that the observation and confirmation of cell density required for the culture process can be realized by detecting using each sensor and light receiving unit without destroying the culture environment, and quickly converting from the absorbance, and based on this, the supply of culture medium or gas, or the temperature inside the culture chamber can be adjusted.

[0031] The reason why the above structure can quickly obtain cell density is the linear relationship between absorbance and cell density. In this regard, the present invention conducted experiments on three representative cell lines and proved that there is indeed a linear relationship between absorbance and cell density. According to this linear relationship diagram, it is easy to convert the absorbance into cell density simply by detecting it.

[0032] In the following experiments, cell numbers were calculated as follows: at least 3 × 10 6 100 ml of cells or 7 mL of cell solution were taken, the cells were centrifuged to collect the precipitate and the supernatant, and the cell concentration of the cell solution was adjusted to 3 × 10 6 The total volume required is 1 mL. The cell suspension prepared with the supernatant is serially diluted 3-fold to five concentrations (3 × 10 6 , 1×10 6 , 3.3×l0 5 , 1.1×10 5 , 3.7×l0 4 ) was obtained. 200 μL of the diluted cell solution was taken and placed in a 96-well flat-bottom plate. Each concentration was run three times. In addition, the supernatant collected during the cell centrifugation process was used as a control group, with 200 μL placed in one well and run three times in total. After that, a SpectraMax Paradigm multimode microplate reader was used to scan all wavelengths (230-1000 nm, 10 nm), and the wavelength with the largest difference in absorbance between the highest concentration and the background was determined as the optimal wavelength, and a relationship diagram between absorbance and cell concentration was created.

[0033] First, let us refer to Fig. 6 and Fig. 7 (first embodiment) in which K562 cell line was used as the experimental subject. Fig. 8 and Fig. 9 are for GDT (Gamma delta T) cells (second embodiment), and Fig. 10 and Fig. 11 are for NK (Natural Killer) cells (third embodiment). Fig. 6, Fig. 8, and Fig. 10 show that the absorbance of the three types of cells has an extreme value at a wavelength of 290 nm, and also shows significant absorbance at 400 nm, 500 nm, and 600 nm, and that all of these wavelengths are suitable for detecting cell density. However, as shown in Fig. 12 and Fig. 13, a relatively significant linear relationship was observed with light of 290 nm, so a light source of 290 nm was selected to conduct an experiment on the linear relationship. Fig. 7, Fig. 9, and Fig. 11 show that there is a definite linear relationship between the absorbance of the cell fluid and the actual cell density of the three types of cells under irradiation with light of 290 nm. Therefore, it can be said that using absorbance as a basis for converting cell density is highly reliable.

[0034] The above experiments were mainly carried out at a wavelength of 290 nm. However, considering that high light energy may cause cell damage or adversely affect cell growth, it is preferable to carry out light irradiation counting with light of wavelengths of 400 to 600 nm in practice. As shown in FIG. 12, light of wavelengths of 400 to 600 nm (light of wavelengths of 400 to 500 nm is more preferable, and light of wavelength 400 nm is even more preferable) still has a significant linear relationship between its absorbance and cell density, and the energy of light of long wavelengths is relatively low, and the effect of light energy on cell growth is relatively small, making it suitable for the integrated automated cell culture device of the present invention. Among them, the absorbance of light of wavelength 400 nm is more significant, making it relatively easy to judge and analyze.

[0035] From the above, the integrated automated cell culture device provided by the present invention does not require opening the culture chamber and even the culture bag to take samples, and can easily and quickly detect the environmental factors in the culture chamber and the cell density in the culture bag, which can provide a basis for automating the adjustment of the culture environment, thereby achieving a higher level of automated culture and greatly reducing costs, as well as maintaining a stable culture environment and improving the quality of cell culture, which is what is desired by the relevant industries and the medical industry. Note that the above examples are merely for the purpose of explaining and interpreting the technical content of the present invention. The scope of protection of the present invention is limited by the claims. [Explanation of symbols]

[0036] 10 Main unit 11 Culture room 12 Storage Groove 20 Lid 21 Observation window 22 Heated Glass 23 Top opening lid 24 Temperature Sensor 30 Stopper 31 Through hole 40 Air Supply Module 51 Light source 52 Light receiving part 61 Slide rail part 62 Bag clamping parts 63 Pulling Mechanism 70 Ventilation Stand 81 Cooling Box 82 Pump 90 conduit

Claims

1. An integrated automated cell culture device including a main body, a lid, a plug, an air supply module, a temperature control module, at least one slide rail unit, and a bag clamping part, The main body surrounds the culture chamber, has an opening connected to the culture chamber at an upper portion of the main body, and further has a receiving groove connected to the culture chamber on a side surface of the main body, and the bottom of the culture chamber is used to place a culture bag; The cover is removably installed on the opening of the main body to close the culture chamber, The plug is installed so as to be filled into the accommodation groove, and at least one through hole is provided so as to penetrate the plug, and the at least one through hole is for passing at least one pipe extending to connect the culture chamber with the outside, and one end of the at least one pipe is connected to the culture bag, The air supply module includes a carbon dioxide sensor and an air supply connector, the carbon dioxide sensor is installed in the incubation chamber and electrically connected to the air supply connector, and the air supply connector is installed in the main body so as to connect to an external gas source to supply gas into the incubation chamber; the temperature control module is installed on at least one of the main body and the cover, and includes a heater and a temperature sensor, the temperature sensor is electrically connected to the heater, detects the temperature inside the incubation chamber, and controls the heater to selectively heat the incubation chamber; An integrated automated cell culture device, characterized in that the at least one slide rail portion is installed in the culture chamber so as to extend along a sliding direction, and the bag clamping part is slidably installed on the at least one slide rail portion, holds the culture bag so that liquid in the culture bag does not flow to one side of the culture bag, and can slide over the culture bag.

2. The integrated automated cell culture device according to claim 1 further comprises a light irradiation counting module, the light source being installed on one of the culture chamber and the surface of the lid facing the culture chamber so as to be located between the light source and the light receiving unit when the culture bag is placed in the culture chamber, and the light receiving unit being installed on the other of the culture chamber and the surface of the lid facing the culture chamber, the light source emitting a light beam having a wavelength of 400 to 600 nm to the light receiving unit, the light beam passing through the culture bag and being received by the light receiving unit, and the light receiving unit calculating the absorbance of the liquid in the culture bag, and thereby calculating the cell density in the culture bag.

3. The integrated automated cell culture device of claim 2 further includes a culture bag having an observation section extending outward in a horizontal direction, allowing liquid and cells in the culture bag to freely enter and exit the observation section, and the positions of the light source and the light receiving section correspond to the observation section so that the light beam emitted from the light source passes through the culture bag via the observation section.

4. 3. The integrated automated cell culture device according to claim 2, wherein the light source emits a light beam having a wavelength of 400 nm to the light receiving portion.

5. The integrated automated cell culture device of claim 1, characterized in that an observation window is provided in the center of the lid body, a heating glass is provided to cover the observation window, and a top-opening lid that can be opened upward is provided to cover the heating glass on the opposite side to the side facing the culture chamber, the top-opening lid selectively shields the heating glass from the outside, the heating glass corresponds to the heater, and the temperature sensor is provided on the surface of the lid body facing the culture chamber.

6. The integrated automated cell culture device according to claim 1, further comprising at least one tension mechanism installed at least at one end of the culture chamber for fastening one end of the culture bag.

7. 2. The integrated automated cell culture device according to claim 1, wherein the air supply module and the stopper are installed at opposite ends of the culture chamber, respectively.

8. 2. The integrated automated cell culture device according to claim 1, further comprising an aeration stand installed at the bottom of the culture chamber for placing the culture bag thereon.

9. The integrated automated cell culture device according to claim 8, characterized in that a plurality of openwork portions are installed to penetrate the ventilation stand in order to improve the ventilation of the bottom of the culture bag.

10. 2. The integrated automated cell culture device according to claim 1, further comprising: a cooling box; and a pump, wherein the cooling box is installed in the main body to accommodate at least one culture medium container; the pump is installed in the main body; and the at least one pipeline is connected to the at least one culture medium container via the pump, thereby supplying liquid into the culture bag.

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