TWI745196B - Automatic cell culture device - Google Patents

Abstract

The automatic cell culture device includes a main body and a clamping member. The main body encloses a receiving space and is formed with a platform at a top thereof. The platform has a sliding portion extending along a sliding direction. Two ends of the platform along the sliding direction are defined as a first end and a second end. The platform is adapted for a culture bag to place thereon. The culture bag has at least one inlet near the first end of the platform. An end of the clamping member is slidably arranged on the sliding portion. The clamping member is adapted for pressing the culture bag to prevent the fluid in the culture bag to flow toward the second end. The clamping member is slidable on the culture bag along the sliding direction. Thereby, the culture fluid in the culture bag can be gathered.

Description

Automated cell culture device

The invention provides a cell culture device.

Medical technology is changing with each passing day. Unlike chemical drugs used to treat cancer and other diseases in the past, with the development of regenerative medicine, gene therapy or cell therapy is more commonly used instead of chemical drugs. Taking immune cell therapy in cell therapy as an example, using the patient's own immune cells can more effectively combat cancer cells and avoid damaging the patient's immunity, with lower side effects.

In order to perform cell therapy, autologous or allogeneic cells must be cultured in vitro, proliferated and then injected into the patient's body. The cell culture is usually carried out in a soft culture bag. With the number of cell proliferation, gradually Inject new culture fluid and place the culture fluid in a laboratory environment with a specific temperature and humidity.

Among them, in order to confirm the number of cells, it is necessary to manually extract a small amount of culture fluid in the bag to calculate the number of cells, and then inject the corresponding amount of culture fluid according to the number of cells. In the entire cell culture process, this step must be performed many times. It consumes labor costs, and there is a risk of cell contamination and differences in human operations, resulting in excessively high cost of cell culture, difficult burden for ordinary patients, and unstable quality.

Existing cell culture devices, such as WAVE Bioreactor (Cytiva), use fixed-size culture bags for cell culture. In addition, since the initial cell number is small at the beginning of the culture, the amount of culture medium in the culture bag is not much, and the cells are too dispersed when dispersed in the entire culture bag. It is known that the interaction between cells is helpful for cell growth. During the culture process, if the number of cells per unit area is too low, the interaction will be reduced, and the cell proliferation rate will be slow, or the growth will be uneven. needs to be improved. In addition, in order to solve the problem that the number of cells per unit area is too low due to the small number of starting cells, the past solution was to replace the culture container. It is too scattered, and as the number of cells increases, replace with a larger container at this time. However, the above-mentioned method of replacing the culture container may increase the risk of cell contamination, and it is difficult to achieve the goal of automating the whole process of cell culture, which still needs to be improved.

One of the objectives of the present invention is to provide a cell culture device that can maintain the concentration of the culture medium regardless of the amount of culture medium in the culture bag, so as to ensure the stable quality of cell culture and facilitate automated culture production.

In order to achieve the above-mentioned objective, this creation provides an automated cell culture device, which includes a body and a bag holder.

The body encloses an accommodating space and the top surface forms a platform. The platform has at least one sliding rail portion extending along a sliding direction. The opposite ends of the platform along the sliding direction are respectively defined as a first One end and a second end, the platform is for a culture bag to be placed on, wherein the culture bag has at least one liquid inlet, and the liquid inlet is located at an end of the culture bag closer to the first end; the clip bag The piece is slidably disposed on the at least one slide rail portion, the bag clamping piece is pressed against the culture bag to prevent the liquid in the culture bag from flowing to the side of the culture bag close to the second end of the platform, and the bag clamping piece can Slide along the sliding direction on the culture bag.

As a result, the cell culture device provided by the present invention can maintain the concentration of the culture solution during the sequential addition of the culture solution, avoiding the dispersion of the culture solution in the entire culture bag, which affects the efficiency and quality of cell culture, and achieves the regulation of the culture bag The function of the number of cells per unit area, and can also cooperate with the medium adding mechanism, so as to achieve an automated production process without manual operations.

Please refer to FIG. 1 to FIG. 7, the present invention provides an automated cell culture device, which mainly includes a body 10 and a bag clamping member 20, and may further include at least one stretching mechanism, a moving drive mechanism 40 and a pushing mechanism 50.

The main body 10 encloses an accommodating space and forms a platform 11 on the top surface. The platform 11 has a sliding rail portion 12 on opposite sides, each of the sliding rail portions 12 extends along a sliding direction, and the platform 11 extends along the sliding direction. The opposite ends of is defined as a first end 101 and a second end 102 respectively. The platform 11 is for a culture bag 70 to be placed flat on, wherein the culture bag 70 contains the target cells to be proliferated and the culture solution, and There is at least one liquid inlet 71 for the injection of culture solution, the liquid inlet 71 is located at one end of the culture bag 70 closer to the first end 101 for connecting a culture solution supply source, such as a peristaltic motor; preferably, The platform 11 can be provided with a venting member 60, the surface of the venting member 60 has a concave-convex structure, and the venting member 60 is sandwiched between the platform 11 and the culture bag 70, so that the bottom surface of the culture bag 70 can be breathable. For example, in this embodiment, the ventilation member 60 can be a metal mesh, a hole plate (the surface of the hole plate is regarded as a convex structure, and the perforation of the hole plate is regarded as a concave structure) Or other objects of similar structure.

The opposite ends of the bag clamping member 20 are respectively slidably disposed on the two slide rail portions 12, and the bag clamping member 20 is pressed against the culture bag 70 to prevent the liquid in the culture bag 70 from flowing to the culture bag 70 close to the On one side of the second end 102 of the platform 11, for example, the bag clamping member can directly press against the culture bag in the direction of the platform, or it can be clamped and pressed against the culture bag in other ways to achieve a blocking effect; the bag clamping member 20 It can slide along the sliding direction on the culture bag 70.

The at least one stretching mechanism is provided at least on the second end 102 of the platform 11 for clamping the culture bag 70 near the end of the second end 102 of the platform 11. Preferably, the at least one stretching mechanism can be further Set at the first end of the platform for clamping the culture bag at an end close to the first end of the platform; specifically, the at least one stretching mechanism includes at least two clamping members 30, the two clamping members 30 respectively Are arranged on opposite sides of the main body 10 close to the two slide rails 12 for clamping the culture bag 70 on opposite sides of the end close to the second end 102 of the platform 11, if the at least one stretching mechanism is arranged at The first end of the platform can also be set in the same configuration; more specifically, in this embodiment, each of the clamping members 30 includes two magnetic blocks 31, 32, one of which is fixed on The platform 11 and the other magnetic block 32 are detachably magnetically attracted to the magnetic block 31 fixed on the platform 11, so that one corner of the culture bag 70 is sandwiched between the two magnetic blocks 31, 32, However, each of the clamps can also be other structures, such as conventional clamps. It should be particularly noted that the height of the two magnetic blocks 31 is preferably not lower than the height of the bag clamping member, so that one end of the culture bag corresponding to the second end of the platform is higher than other positions of the culture bag. It is indeed avoided that the culture liquid flows towards the second end due to the influence of gravity and capillary phenomena.

Regarding the bag clamping member 20, in this embodiment, it includes two transverse rods 21, two connecting members 22, and two sliding members 23. The two transverse rods 21 are arranged horizontally perpendicular to the sliding direction. The two cross bars 21 are spaced apart from each other. The distance between the two cross bars 21 and the platform 11 is different, that is, the heights of the two cross bars 21 relative to the platform 11 are different; Two connecting pieces 22, the two connecting pieces 22 are detachably disposed on the two sliding pieces 23, and the two sliding pieces 23 are slidably disposed on the two sliding rail portions 12, for example, each sliding piece 23 may have a groove to For the connecting piece 22 to be embedded.

The moving drive mechanism 40 is arranged in the accommodating space of the main body 10. In this embodiment, it includes a belt set 41 and two magnetic parts 42. The two magnetic parts 42 are arranged on the belt set 41 to be able to Driven by the belt set 41 to move along the sliding direction, each sliding member 23 is provided with a magnetic attraction member 231. The belt set 41 can drive the two sliding parts 23 to move in the sliding direction, so that the platform does not need to be provided with penetrating slide rails, so that the containing space of the body can be kept in a closed state to prevent internal parts from being exposed to external temperature and humidity. Rust due to the influence of gas.

The pushing mechanism 50 is arranged in the accommodating space of the main body 10 at the first end 101 of the main body 10. The pushing mechanism 50 can move up and down to reciprocally lift the culture bag 70 close to the first end 101. And reset downwards, so that the culture solution in the culture bag 70 can be slightly shaken to ensure uniform mixing; in this embodiment, the pushing mechanism 50 may include a rotating device 51 and an eccentric wheel 52 With an ejector plate 53, the rotating device 51 can be a rotating shaft driven by a motor, etc. The eccentric wheel 52 is eccentrically arranged on the rotating device 51 and eccentrically rotates with the rotating device 51, the eccentric wheel 52 The peripheral edge of the eccentric wheel is provided to push against the ejector plate 53, and the ejector plate 53 is provided against the culture bag 70. When the eccentric wheel 52 rotates eccentrically, the height of the top of the eccentric wheel 52 constantly changes, so that the ejector plate 53 is reciprocated and continuously pushed upward and reset downward by the eccentric wheel 52. However, the pushing mechanism is not limited to the above-mentioned mechanism, and other lifting mechanisms may also be used.

In actual use, as shown in FIG. 4, the culture bag 70 passes between the two crossbars 21 toward the first end 101 and then straddles the upper crossbar 21 at one end relative to the liquid inlet 71 , And then continue to extend toward the second end 102 and be fixed at the second end 102 by the two clamping members 30. With the above structure, except that the middle section of the culture bag 70 is wound between the two cross bars 21 It is pressed and clamped, and the end of the culture bag 70 corresponding to the second end 102 will be higher than the end corresponding to the first end 101, so as to further prevent the culture solution from flowing toward the second end 102; It is explained that in this embodiment, the "automated cell culture device" of the present invention refers to a device that automatically moves the bag holder. However, the present invention does not exclude the situation where the bag holder is manually shifted and adjusted manually. , Hexian explained. The culture bag 70 may have a fixing portion 72 corresponding to one end of the first end 101. The fixing portion 72 is, for example, a sampling tube of the culture bag 70. The push plate 53 may have a corresponding fixing member 531. The fixing portion 72 It can be positioned on the fixing member 531 so that the two ends of the culture bag 70 are respectively positioned on the body 10, so that when the bag clamping member 20 moves along the sliding direction, the culture bag 70 can be close to The culture solution on the side of the liquid inlet 71 is pushed toward the direction of the liquid inlet 71 to prevent the culture solution from being dispersed in the entire culture bag 70. Even if the number of starting cells is small, the number of cells per unit area in the culture bag can still be Achieve the amount that contributes to the contact between cells, as shown in Figure 5. In this embodiment, the fixing member 531 is a fastener that can be elastically deformed and buckle the tube body, and is disposed on the push plate 53 and used to position the fixing portion 72 of the culture bag 70. However, if the fixing member 531 is changed to be, for example, Other types of clamps, such as spring clamps, magnetic clamps, etc., are arranged on the push plate 53 or the body 10 adjacent to the first end 101, and can be used to position the culture bag away from the other end of the stretching mechanism, and can achieve the same effect as this embodiment. Similar use effect.

In the process of cell culture, new culture medium is gradually injected into the culture bag 70, the culture medium and the number of cells in the culture bag 70 gradually increase, and the bag clamping member 20 gradually moves toward the second end 102 continuously. , Or move the bag holding member 20 in stages, as shown in FIG. 6, to release the space in the culture bag. The time point, speed and distance of its movement can be determined by the number of cells per unit area to be achieved and The amount of culture medium injected is adjusted and controlled.

In addition, it can be seen from FIGS. 5 to 7 that the pushing mechanism 50 can push and reset the culture bag 70 near one end of the first end 101 reciprocally, thereby slightly shaking the culture solution in the culture bag 70 to Achieve the effect of moderate mixing, promote the uniformity and good efficiency of cell culture.

Regarding the actual culture effect of the cell culture device provided by the present invention, please refer to Figure 8. Natural killer cells isolated from the peripheral blood samples of the same subject were taken and cultured in vitro with three different devices or methods: (1) Use the cell culture device of the present invention, (2) use the commercially available cell culture device WAVE Bioreactor, and (3) manual cell culture (that is, not use the cell culture device). The aforementioned peripheral blood samples were collected from the arm of the subject in accordance with the plan approved by the ethics committee. For the aforementioned cell culture in the three ways, the starting cell number, the medium used, and the number of days of in vitro culture are all the same. It can be seen from Figure 8 that compared to manual culture, the cell culture device of the present invention can be cultured to the 15th day, and the cell expansion ratio can reach 80% of that of manual culture. It can be seen that the cell culture device of the present invention can already be Instead of manual culture, it effectively saves manpower. In addition, the experimental results in Figure 8 also show that compared to manual culture, using the existing commercially available WAVE Bioreactor for cell culture to the 15th day, the cell expansion ratio only reached 26% of that of manual culture. It is significantly lower than the cell expansion multiple using the cell culture device of the present invention, which shows that the cell culture device of the present invention can indeed improve the performance of the existing cell culture device.

With the above structure and operation, the present invention can be connected with the culture fluid supply source, additional temperature, gas, pH value, and humidity monitoring and control mechanism to calculate the injection frequency, speed, and injection volume of the culture fluid or gas with a model. Then set the moving timing and moving speed of the bag clamping piece based on this, and drive the pushing mechanism at the appropriate time and frequency to perform the shaking and mixing of the culture solution, and the whole process can be controlled in an automated manner without manual labor. Injecting and shaking can also ensure that the culture medium is not dispersed, so that the cells can proliferate and grow in a constant state, thereby improving the efficiency and yield of cell culture. Importantly, it can greatly save the cost of manual operation, and Can reduce the error of manual operation.

As a result, the cell culture device provided by the present invention can greatly reduce the required labor cost while maintaining the efficiency and quality of cell culture, thereby reducing the cost of cell culture and facilitating mass production, making the original expensive and difficult-to-load cell therapy The price reduction, the benefit of various patients, and the promotion of medical well-being are indeed expected by the medical industry and the general public. However, the above embodiments are only to illustrate and illustrate the technical content of the present invention. The patent scope of this patent shall be the scope of the patent application described later. allow.

10: body 101: first end 102: second end 11: Platform 12: Slide rail part 20: Clip bag 21: Crossbar 22: Connector 23: Slider 231: Magnetic Parts 30: clamp 31: Magnetic block 32: magnetic block 40: mobile drive mechanism 41: belt set 42: Magnetic part 50: Pushing mechanism 51: Rotating device 52: eccentric wheel 53: Push plate 531: fixed parts 60: Breathable parts 70: culture bag 71: Liquid inlet 72: fixed part

Figure 1 is a perspective view of the present invention. Figure 2 is a schematic diagram of the use of the present invention. Figure 3 is a cross-sectional view of the present invention. Figures 4 to 7 are cross-sectional views of the present invention when in use. Figure 8 is a comparison diagram of the results of cell culture using the present invention and conventional techniques.

10: body

101: first end

102: second end

11: Platform

12: Slide rail part

20: Clip bag

21: Crossbar

22: Connector

23: Slider

231: Magnetic Parts

31: Magnetic block

40: mobile drive mechanism

41: belt set

42: Magnetic part

50: Pushing mechanism

51: Rotating device

52: eccentric wheel

53: Push plate

531: fixed parts

60: Breathable parts

Claims (10)

An automated cell culture device, including: A body, an accommodating space is enclosed and a top surface forms a platform, the platform has at least one slide rail portion, the at least one slide rail portion extends along a sliding direction, and the opposite ends of the platform along the sliding direction are defined as a A first end and a second end, the platform is for a culture bag to be placed flat on, wherein the culture bag has at least one liquid inlet, and the liquid inlet is located at an end of the culture bag closer to the first end; A bag clamping member is slidably disposed on the at least one slide rail portion, and the bag clamping member is pressed against the culture bag to prevent the liquid in the culture bag from flowing to the side of the culture bag close to the second end of the platform, the The bag clamping member can slide along the sliding direction on the culture bag. The automated cell culture device according to claim 1, further comprising at least one stretching mechanism, which is provided at least at the second end of the platform for clamping the culture bag at an end close to the second end of the platform. The automated cell culture device according to claim 2, wherein the at least one stretching mechanism includes at least two clamping members, and the two clamping members are respectively provided on opposite sides of the main body for clamping the culture bag close to the Opposite sides of one end of the second end of the platform. The automated cell culture device according to claim 3, wherein each of the holders includes two magnetic blocks, one of the magnetic blocks is fixed on the platform, and the other magnetic block is detachably magnetically attracted and fixed on the platform The magnetic block is used to sandwich one corner of the culture bag between the two magnetic blocks. The automated cell culture device according to claim 1, wherein the bag holding member includes two cross bars, the two cross bars are respectively perpendicular to the sliding direction, the two cross bars are spaced apart from each other, and the distance between the two cross bars and the platform Different. The automated cell culture device according to claim 5, wherein opposite sides of the platform are respectively provided with the slide rail portion, and the bag holding member further includes two connecting members and two sliding members, and opposite ends of each of the cross bars Are respectively fixed to the two connecting pieces, the two connecting pieces are respectively detachably arranged on the two sliding pieces, and the two sliding pieces are respectively slidably arranged on the two sliding rail parts. The automated cell culture device according to claim 6, further comprising a moving drive mechanism, the moving drive mechanism is arranged in the accommodating space of the main body and comprises a belt set and two magnetic parts, the two magnetic parts are arranged on the The belt set can be driven by the belt set to move along the sliding direction. Each of the sliding parts is provided with a magnetic attraction part. The group can drive the two sliding parts to move along the sliding direction. The automated cell culture device according to claim 1, further comprising a pushing mechanism, the pushing mechanism is arranged in the accommodating space of the body and located at the first end of the body, the pushing mechanism can move up and down to reciprocate One end of the culture bag close to the first end is lifted upward and reset downward. The automated cell culture device according to claim 8, wherein the pushing mechanism comprises a rotating device, an eccentric wheel and a pushing plate, and the eccentric wheel is eccentrically arranged on the rotating device and eccentrically rotates with the rotating device , The peripheral edge of the eccentric wheel is for pushing against the pushing plate, and the pushing plate is for pushing against the culture bag. When the eccentric wheel rotates eccentrically, the pushing plate is reciprocally pushed up and down reset by the eccentric wheel. The automated cell culture device according to claim 1, further comprising a vent, the vent is arranged on the platform and the surface has a concave-convex structure, the vent is sandwiched between the platform and the culture bag, so that The bottom surface of the culture bag can be breathable.

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