JP2021090394A - Cell culture analyzer, and cell culture analytical method using the same - Google Patents

Abstract

To provide a cell culture analyzer and a cell culture analytical method using the analyzer in which long-term effect consideration by a reagent can be performed.SOLUTION: A cell culture analyzer comprises: an external cylinder 9 which is in a bottomed cylindrical and whose top face is open; an internal cylinder 15 whose top and bottom faces are open and which is arranged freely movable in vertical directions in the external cylinder 9; a glucose sensor 21; a culture solution injection unit 28; a culture solution discharge unit 26; a reagent addition unit 23; a semi-permeable membrane 16 through which nutrient and waste matter can pass; and a push-up plate 19 which is provided for at least one of the internal cylinder 15 and the external cylinder 9. The push-up plate 19 lifts the internal cylinder 15 from a state where a lower end opening part 14 of the internal cylinder 15 contacts a bottom face of the external cylinder 9, and holds the internal cylinder 15 in the state where the lower end opening part 14 is in non-contact with the bottom face of the external cylinder 9.SELECTED DRAWING: Figure 1

Description

The present invention relates to, for example, a cell culture analyzer for floating cancer cells such as leukemia cells and a cell culture analysis method using the same.

In the conventional cell culture analyzer of this type, a culture solution containing floating cancer cells is housed in a culture vessel. A sensor is provided in the culture vessel, and the reagent is supplied into the culture vessel from the reagent supply unit (for example, Patent Document 1 below).
Then, the sensor detects how the state of the culture solution fluctuates. This will evaluate how the reagent acts on planktonic cancer cells.

U.S. Pat. No. 9,170,255

It is extremely important to consider from a long-term perspective how the drugs produced in the future by the reagents will act on such floating cancer cells. Therefore, the cell culture analyzer is required to be able to consider the long-term utility of the reagent.
However, in the conventional example, since nutrients such as glucose are not additionally supplied to the culture solution in the culture vessel, the floating cancer cells and the like die in a short period of time, and as a result, the reagent for the floating cancer cells is used. There was a problem that long-term utility could not be considered.

Therefore, an object of the present invention is to enable long-term utility consideration of reagents.

In order to achieve this purpose, a bottomed tubular outer cylinder with an open upper surface and an inner cylinder with an open upper and lower surface and movably arranged vertically in the outer cylinder to inject a culture solution containing cells. A nutrient detection sensor inserted into the inner cylinder, a culture solution supply unit that supplies the culture solution between the outer cylinder and the inner cylinder, and a culture solution discharge that discharges the culture solution from between the outer cylinder and the inner cylinder. A unit, a reagent supply unit that supplies reagents into the inner cylinder, a semipermeable membrane provided on the outer peripheral surface of the inner cylinder through which nutrients and waste products can pass, and at least one of the inner cylinder and the outer cylinder. It is provided with a holding means provided in the above. The holding means lifts the inner cylinder from a state where the lower surface opening of the inner cylinder is in contact with the bottom surface of the outer cylinder, and the inner cylinder is in a state where the lower surface opening is not in contact with the bottom surface of the outer cylinder. It was configured to hold the cylinder.

Therefore, in the present invention, when a culture solution containing cells is injected into the inner cylinder for cell culture analysis, cell culture is promoted in the inner cylinder. At this time, not only glucose in the inner cylinder but also glucose in the culture solution in the outer cylinder is supplied to the cells in the inner cylinder via the semipermeable membrane, so that the cells may die in a short period of time. Absent.
As a result, the reagent supplied from the reagent supply unit will be considered for long-term utility.

Further, when the cells in the inner cylinder become overcrowded, the inner cylinder is lifted from the state where the lower surface opening of the inner cylinder is in contact with the bottom surface of the outer cylinder by the holding means.
Therefore, the culture solution in the inner cylinder and the culture solution in the outer cylinder are mixed, and the culture area for culturing the cells is expanded. As a result, the appropriate cell density is obtained again, and the cells are continuously cultured.

As a result, the reagent supplied from the reagent supply unit will be considered for long-term utility.

The perspective view which shows the use example of the cell culture analyzer which concerns on one Embodiment of this invention. The perspective view which shows the cell culture analyzer of FIG. FIG. 1 is a perspective view of a main part of the cell culture analyzer of FIG. (A) A cross-sectional view of a main part of the cell culture analyzer of FIG. 1, (b) an enlarged cross-sectional view of a main part of FIG. 4a, and (c) an enlarged cross-sectional view of a main part of FIG. 4a. FIG. 1 is a perspective view of a main part of the cell culture analyzer of FIG. FIG. 3 is a cross-sectional view of a main part of the cell culture analyzer of FIG. FIG. 1 is an exploded perspective view of a main part of the cell culture analyzer of FIG. FIG. 1 is an exploded perspective view of a main part of the cell culture analyzer of FIG. The control block diagram of the cell culture analyzer and the personal computer of FIG. FIG. 3 is a cross-sectional view of a main part of the cell culture analyzer of FIG. The characteristic diagram explaining the operation of the cell culture analyzer of FIG. The characteristic diagram explaining the operation of the cell culture analyzer of FIG. The characteristic diagram explaining the operation of the cell culture analyzer of FIG.

Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.
(Embodiment 1)
FIG. 1 is a perspective view showing an example of use of the cell culture analyzer 1 in the present embodiment. This cell culture analyzer 1 performs cell culture analysis and is utilized in the incubator 2.

The cell culture analyzer 1 in the present embodiment is characterized in that it can continuously perform analysis over a long period of, for example, 8 days. This analysis state is transmitted to the personal computer 3 by radio, for example, and the researcher can confirm the metabolic state of the cells by visually observing the display unit 4 of the personal computer 3.
In the cell culture analyzer 1, as shown in FIG. 2, a dish 6 is arranged in the main body case 5, and the upper surface thereof is covered with a lid 7 so as to be openable and closable.

As shown in FIG. 4, the dish 6 shown in FIG. 3 is integrally provided with a bottomed cylindrical outer cylinder 9 having an opening 8 provided on the upper surface.
Further, as shown in FIG. 3, a plate 10 is arranged on the dish 6 so as to be vertically movable. Specifically, the plate 10 is provided with guide holes 11 at three corners. The dish 6 is provided with guide protrusions 12 projecting upward at three corners on the upper surface. Then, the plate 10 can be moved up and down by passing the guide hole 11 through the guide protrusion 12.

Further, as shown in FIG. 4, the plate 10 is integrally provided with a cylindrical inner cylinder 15 provided with an upper surface opening 13 and a lower end opening 14 (lower surface opening). Further, a semipermeable membrane 16 is provided on the outer peripheral surface of the inner cylinder 15 below the midpoint in the vertical direction. The semipermeable membrane 16 is provided at a position above the lower end opening 14 of the outer cylinder 9 by a predetermined distance.
The semipermeable membrane 16 has innumerable fine pores (not shown). Since the pore size of the semipermeable membrane 16 is smaller than that of cells, cells cannot pass through, but at least glucose (an example of nutrients) and lactic acid (an example of waste products) can pass through the pores.

Further, as shown in FIG. 4B, a recess 17 recessed upward is provided in the lower end opening edge (part A in FIG. 4a) of the inner cylinder 15, and the recess 17 is made of silicon rubber. Packing 18 is provided.
The packing 18 has flexibility, and when the lower end of the inner cylinder 15 is brought into contact with the bottom surface of the outer cylinder 9 as shown in FIGS. 4 (b) to 4 (c), the packing 18 is deformed. Then, most of them are pushed into the recess 17. However, even in this state, the lower end opening 14 of the inner cylinder 15 is closed by the packing 18 and the bottom surface of the outer cylinder 9, and the culture solution in the inner cylinder 15 is closed from the lower end opening 14 of the inner cylinder 15. Does not flow out into the outer cylinder 9. As shown in FIG. 4C, the dish 6 and the plate 10 are fixed by fasteners (not shown) in a state where the lower end of the inner cylinder 15 is in contact with the bottom surface of the outer cylinder 9.

However, as will be described in detail below, when the fastener is removed and the inner cylinder 15 is lifted as shown in FIGS. 4 (c) to 4 (b), the culture solution in the inner cylinder 15 has an opening at the lower end thereof. It will flow out from 14 into the outer cylinder 9.
As shown in FIGS. 3 and 4, in order to hold the inner cylinder 15 in an upwardly lifted state, a push-up plate 19 (holding) that can move inward and outward on the upper surface of the dish 6 constituting the outer cylinder 9, respectively. An example of means) is provided. The push-up plate 19 is provided with an inclined surface 19a that inclines downward from the outside to the inside, and as shown in FIG. 4, the lower surface of the plate 10 constituting the inner cylinder 15 is provided on the inclined surface 19a. Is placed.

Therefore, when the push-up plates 19 on both sides are located outward, the lower end opening 14 of the inner cylinder 15 is in contact with the bottom surface of the outer cylinder 9, but the push-up plate 19 is inward. When pushed from both sides, the plate 10 is pushed upward while resting on the inclined surface 19a. Along with this, the inner cylinder 15 is in a state of being lifted from the state of FIG. 4 (c) to the state of FIG. 4 (b), and in this state, the inner cylinder 15 is held by the holding portion 19b of the push-up plate 19. (See FIG. 10).

As shown in FIG. 4A, a culture solution 20 containing floating cancer cells such as leukemia cells and glucose is injected into the inner cylinder 15. At this time, the lower end opening 14 of the inner cylinder 15 is covered with the packing 18 and the bottom surface of the outer cylinder 9, and the culture solution 20 does not flow out into the outer cylinder 9.
As shown in FIG. 2, the dish 6 in this state is arranged in the main body case 5.

5 and 8 show each functional component inserted into the inner cylinder 15 and the outer cylinder 9. When the dish 6 is arranged in the main body case 5, each of these functional parts is automatically inserted into the inner cylinder 15 and the outer cylinder 9.
Specifically, as shown in FIG. 5, a glucose sensor 21 (an example of a nutrient detection sensor) and a lactic acid sensor 22 (an example of a waste product detection sensor) are formed in the inner cylinder 15 from the upper surface opening 13 of the inner cylinder 15. , The reagent addition unit 23 (an example of the reagent supply unit), and the stirring unit 24 are inserted and arranged. The stirring unit 24 sucks the culture solution 20 into the stirring unit 24 and then discharges it to the outside of the stirring unit 24. As a result, the culture solution 20 is stirred.

As shown in FIG. 6, only the reagent addition unit 23 is arranged on the liquid surface of the culture solution 20 in a state of being isolated for a predetermined period of time, and the lower end of the other units is arranged in the culture solution 20.
Next, as shown in FIG. 5, a culture solution discharge unit 26 (an example of a culture solution discharge unit) is inserted between the outer cylinder 9 and the inner cylinder 15 through the opening 25 of the plate 10, and the plate is also inserted. The culture solution injection unit 28 (an example of the culture solution supply unit) is inserted through the opening 27 of 10.

As shown in FIG. 6, the lower end of the culture solution discharge unit 26 is arranged below the liquid level of the culture solution 29, but the lower end of the culture solution injection unit 28 is below the liquid level of the culture solution 29. Placed above.
That is, with the inner cylinder 15 installed in the outer cylinder 9, the culture solution 29 is injected into the outer cylinder 9 from the culture solution injection unit 28. The culture solution 29 is injected in the same state as the liquid level of the culture solution 20. The culture solution discharge unit 26 discharges the culture solution 29 from between the outer cylinder 9 and the inner cylinder 15.

FIG. 9 shows a control block diagram of the cell culture analyzer 1 and the personal computer 3.
The glucose sensor 21 and the lactic acid sensor 22 are connected to the control unit 31 via the measurement unit 30. The reagent addition unit 23, the stirring unit 24, the culture solution injection unit 28, and the culture solution discharge unit 26 are directly connected to the control unit 31. A power supply 32, a storage unit 33, and a communication unit 34 are also connected to the control unit 31. The communication unit 34 communicates with the communication unit 35 of the personal computer 3.

The communication unit 35 of the personal computer 3 is connected to the control unit 38 together with the display unit 4, the input unit 36, and the storage unit 37.
In the above configuration, in the present embodiment, the researcher first installs the inner cylinder 15 in the outer cylinder 9 and removes the lower end opening 14 of the inner cylinder 15 via the packing 18 as shown in FIG. It is brought into contact with the bottom surface of the cylinder 9. In this state, the outer cylinder 9 and the inner cylinder 15 are fixed by the fastener.

Next, the researcher injects a culture solution 20 containing floating cancer cells such as leukemia cells and glucose into the inner cylinder 15. Then, as shown in FIG. 2, the dish 6 constituting the outer cylinder 9 is installed in the main body case 5, and the lid 7 is closed.
Then, as shown in FIG. 6, the control unit 31 of the cell culture analyzer 1 is provided in the main body case 5, a glucose sensor 21, a lactic acid sensor 22, a reagent addition unit 23, a stirring unit 24, and a culture solution injection unit. 28, the culture solution discharge unit 26, is installed in the inner cylinder 15 and the outer cylinder 9 in the above-mentioned state.

The control unit 31 injects the culture solution 29 into the outer cylinder 9 using the culture solution injection unit 28. After that, the control unit 31 continuously measures the glucose concentration and the lactic acid concentration in the inner cylinder 15 for, for example, 8 days by using the glucose sensor 21 and the lactic acid sensor 22.
Cell culture is started in the inner cylinder 15. The cells consume glucose in the culture medium 20 and produce waste products (for example, lactic acid).

When the cells consume glucose, the glucose of the culture solution 29 in the outer cylinder 9 is supplied into the inner cylinder 15 via the semipermeable membrane 16.
That is, in the cell culture analysis, not only glucose in the inner cylinder 15 but also glucose from the outer cylinder 9 is supplied to the cells in the inner cylinder 15 via the semipermeable membrane 16, so that the cells are cultured. Will continue in the long run.

As a result, the cells are continuously cultured for a long period of time, which makes it possible to consider the long-term effects of the reagents on the cells.
When the cells produce lactic acid, the lactic acid in the inner cylinder 15 passes through the culture solution 29 in the outer cylinder 9 via the semipermeable membrane 16. The cells in the inner cylinder 15 cannot move to the culture solution 29 in the outer cylinder 9 via the semipermeable membrane 16.

In the present embodiment, the inner cylinder 15 has a narrow storage space, and the cell density is suitable for starting cell proliferation.
FIG. 11 shows the relationship between the number of days in which cells are cultured and the amount of glucose consumed by cells.
Floating cancer cells housed in the inner cylinder 15 at an appropriate cell density start to proliferate rapidly, for example, when left to stand for one day. That is, cell proliferation is initiated. After that, the cells obtain glucose in the inner cylinder 15 and glucose supplied from the outer cylinder 9 via the semipermeable membrane 16 and continue to proliferate.

After the cells have started to grow, that is, with the floating cancer cells promoted in the inner cylinder 15, the reagent (for example, glycolytic inhibitor) is placed on the surface of the culture medium 20 from the reagent addition unit 23. ) Is added. The reagent does not pass through the semipermeable membrane 16 and stays in the inner cylinder 15.
The broken line in FIG. 11 shows the behavior (glucose consumption) for 8 days without the reagent added, and the solid line shows the behavior for 8 days (glucose) with the reagent added on the first day as described above. Consumption) is shown.

A few days after the start of cell culture, as a result of the cells consuming glucose, glucose was insufficient in the culture solution 20 in the inner cylinder 15 and the culture solution 29 in the outer cylinder 9, and the pH of the culture solution 29 was adjusted by the lactic acid produced. As it goes down, the culture environment deteriorates.
Specifically, after three and a half days have passed, the control unit 31 discharges the culture solution 29 of the outer cylinder 9 using the culture solution discharge unit 26, and then uses the culture solution injection unit 28 to newly culture. The liquid 29 is supplied. Glucose contained in the newly injected culture solution 29 is provided to the floating cancer cells in the inner cylinder 15 via the semipermeable membrane 16, whereby the floating cancer cells continue to be cultured.

Then, for example, if the cell culture is continued until the 6th day, the number of cells in the inner cylinder 15 is excessively increased and the cells are overcrowded, and the cells may die as they are.
Therefore, in the present embodiment, the inner cylinder 15 is lifted and the culture solution 20 in the inner cylinder 15 and the culture solution 29 in the outer cylinder 9 are mixed in order to expand the culture area for culturing the cells. As a result, the appropriate cell density is obtained again, and the cell culture is continued. As a result, cell culture analysis can be continued.

Specifically, after 6 days, the control unit 31 discharges the culture solution 29 in the outer cylinder 9 using the culture solution discharge unit 26, and supplies a new culture solution 29 using the culture solution injection unit 28. .. That is, lactic acid is discharged and new glucose is introduced.
Then, when the push-up plate 19 is operated after the fastener is removed by the researcher, the inner cylinder 15 is lifted as shown in FIG. 4B, and the culture solution 20 in the inner cylinder 15 is in the outer cylinder 9. It becomes a mixed state with the culture solution 29. The volumes of the culture solution 20 and the culture solution 29 are the same. Therefore, the culture area for culturing cells is doubled.

After that, when the control unit 31 stirs the culture solution 20 and the culture solution 29 using the stirring unit 24, the culture solution 20 and the culture solution 29 are appropriately stirred, and the cells in the inner cylinder 15 are expanded. Appropriately spread in the culture area.
In this way, since the culture area is expanded, the appropriate cell density can be maintained again, and even if the cells proliferate thereafter, the culture can be continued without the cells being compressed. As a result, cell culture analysis can be continued.

Then, as shown in FIG. 11, the planktonic cancer cells will be continuously cultured. When the analysis is continued for such a long period of time, the difference between the case where the reagent is added and the case where the reagent is not added after 8 days may become extremely large. That is, when the reagent is effective, the amount of glucose consumed is extremely small as compared with the case where the reagent is not added. This is because, as is well known, the added reagent is judged to be effective against floating cancer cells.

FIG. 12 is the original data for making FIG. 11 and shows the change in glucose concentration from the start of the culture analysis to the 8th day. This concentration correlates with the amount of glucose consumed, and the amount of glucose consumed can be obtained by continuously measuring the glucose concentration over time, and the accumulated state is the state shown in FIG.
Also in FIG. 12, the broken line shows the behavior (glucose concentration) for 8 days without the reagent added, and the solid line shows the behavior (glucose concentration) for 8 days with the reagent added on the first day as described above. It shows the behavior (glucose concentration).

FIG. 13 shows the lactic acid concentration detected by the lactic acid sensor 22 arranged in the inner cylinder 15. This concentration correlates with the amount of lactic acid produced, and the amount of lactic acid produced can be determined by continuously measuring the concentration of lactic acid over time.
The lactate level can be used not only when examining the culture state of floating cancer cells, but also when evaluating changes in glycolytic metabolism when a glycolytic metabolism inhibitor is added as a reagent.

In the present embodiment, as described above, when the cells in the inner cylinder 15 become overcrowded, the inner cylinder 15 is lifted and the inner cylinder 15 is lifted after exchanging the culture solution 29 in the outer cylinder 9 in order to expand the culture area. The culture solution 20 in the cylinder 15 and the culture solution 29 in the outer cylinder 9 are mixed.
At this time, since the culture area is expanded, the reagent concentration in the culture area is lowered.
Therefore, in the present embodiment, the control unit 31 drives the culture solution discharge unit 26 and the culture solution injection unit 28 to replace the culture solution 29 in the outer cylinder 9, and then when the inner cylinder 15 is lifted, the reagent The reagent was additionally supplied into the inner cylinder 15 using the addition unit 23.

As a result, the reagent concentration in the culture area can be appropriately maintained.

As described above, the present invention is expected to be utilized, for example, as a cell culture analyzer for floating cancer cells such as leukemia cells and a cell culture analysis method using the same.

1 Cell culture analyzer 2 Incubator 3 Personal computer 4 Display 5 Main body case 6 Dish 7 Lid 8 Opening 9 Outer cylinder 10 Plate 11 Guide hole 12 Guide protrusion 13 Top opening 14 Bottom opening (bottom opening)
15 Inner cylinder 16 Semipermeable membrane 17 Recess 18 Packing 19 Push-up plate (example of holding means)
19a Inclined surface 19b Holding part 20 Culture solution 21 Glucose sensor (an example of nutrient detection sensor)
22 Lactic acid sensor (an example of waste detection sensor)
23 Reagent addition unit (an example of reagent supply unit)
24 Stirring unit 25 Opening 26 Culture solution discharge unit (Example of culture solution discharge unit)
27 Opening 28 Culture solution injection unit (example of culture solution supply unit)
29 Culture solution 30 Measuring unit 31 Control unit 32 Power supply 33 Storage unit 34 Communication unit 35 Communication unit 36 Input unit 37 Storage unit 38 Control unit

Claims (7)

A bottomed tubular outer cylinder with an open top surface,
An inner cylinder in which the upper and lower surfaces are opened and arranged so as to be vertically movable in the outer cylinder, and a culture solution containing cells is injected.
The nutrient detection sensor inserted in the inner cylinder and
A culture solution supply unit that supplies the culture solution between the outer cylinder and the inner cylinder,
A culture solution discharge unit that discharges the culture solution from between the outer cylinder and the inner cylinder,
A reagent supply unit that supplies reagents into the inner cylinder,
A semipermeable membrane provided on the outer peripheral surface of the inner cylinder through which at least nutrients and waste products can pass,
A holding means provided on at least one of the inner cylinder and the outer cylinder,
With
The holding means lifts the inner cylinder from a state where the lower surface opening of the inner cylinder is in contact with the bottom surface of the outer cylinder, and the inner cylinder is in a state where the lower surface opening is not in contact with the bottom surface of the outer cylinder. Hold the tube,
Cell culture analyzer. A packing is provided on the lower end opening edge of the inner cylinder.
The cell culture analyzer according to claim 1. The lower end opening edge of the inner cylinder is provided with a recess that is recessed upward.
The packing is provided in the recess.
The cell culture analyzer according to claim 2. A waste detection sensor is arranged in the inner cylinder.
The cell culture analyzer according to any one of claims 1 to 3. A control unit to which the nutrient detection sensor, the culture solution supply unit, and the reagent supply unit are connected is provided.
The control unit drives the culture solution discharge unit and the culture solution supply unit to exchange the culture solution, and then supplies the reagent into the inner cylinder using the reagent supply unit.
The cell culture analyzer according to any one of claims 1 to 4. A cell culture analysis method using the cell culture analyzer according to any one of claims 1 to 5.
The inner cylinder is arranged in the outer cylinder, and the lower surface opening of the inner cylinder is brought into contact with the bottom surface of the outer cylinder.
The culture solution containing cells is supplied into the inner cylinder, and the culture solution containing no cells is supplied between the inner cylinder and the outer cylinder.
The reagent is supplied from the reagent supply unit into the inner cylinder, and the reagent is supplied.
The nutrient in the inner cylinder is detected by the nutrient detection sensor,
The culture solution discharge unit discharges the cell-free culture solution from between the inner cylinder and the outer cylinder to the outside of the outer cylinder.
The culture solution containing no cells is supplied between the inner cylinder and the outer cylinder by the culture solution supply unit.
After that, the inner cylinder is lifted from the state where the lower surface opening of the inner cylinder is in contact with the bottom surface of the outer cylinder by the holding means, and the lower surface opening of the inner cylinder is not attached to the bottom surface of the outer cylinder. The inner cylinder is held in contact with the inner cylinder.
Cell culture analysis method. When the inner cylinder is held in a state where the lower surface opening of the inner cylinder is not in contact with the bottom surface of the outer cylinder, the reagent supply unit additionally supplies the reagent into the inner cylinder.
The cell culture analysis method according to claim 6.

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