CN113150984A

CN113150984A - Perfusion device and perfusion method for culturing embryonic stem cells

Info

Publication number: CN113150984A
Application number: CN202110270074.7A
Authority: CN
Inventors: 姜胜维
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2021-07-23

Abstract

The invention relates to the technical field of stem cells and discloses a perfusion device for culturing embryonic stem cells, which comprises a bracket, a current-limiting component and an extrusion component, wherein a perfusion bottle is fixedly arranged at the top end of the bracket, a feeding pipe is fixedly arranged on the inner wall of the perfusion bottle, and a discharging pipe is fixedly arranged on the inner wall of the perfusion bottle. Thereby limiting the discharge amount of the discharge pipe so as to control the flow rate of the culture medium.

Description

Perfusion device and perfusion method for culturing embryonic stem cells

Technical Field

The invention relates to the technical field of stem cells, in particular to a perfusion device and perfusion method for culturing embryonic stem cells.

Background

The research of stem cells provides guarantee for the health of people, the stem cells as progenitor cells can be differentiated into various cells of human bodies, which has strong treatment guarantee for cancers or various serious diseases, but the stem cells need to be extracted and cultured, and when the cancers or the serious diseases are treated, the required amount of the stem cells is large, so that if the stem cells are not cultured, the loss amount of the stem cells exceeds the extraction amount by a large amount, and the situation of short supply and short demand is formed.

In the stem cell culture process, a perfusion mode is generally adopted, wherein stem cells are placed in a culture container, a culture medium is continuously supplied through a connecting pipe, the culture medium is supplied with stem cell nutrients, and generated waste liquid is discharged from the culture container along with the used culture medium, so that the culture medium is continuously supplied to the stem cells.

Disclosure of Invention

In order to achieve the purpose of adjusting the amount of the culture medium required by different periods, the invention provides the following technical scheme: the utility model provides a perfusion device for embryonic stem cell culture, includes support, current-limiting component and extrusion subassembly, the top fixed mounting of support has the perfusion bottle, the inner wall fixed mounting of perfusion bottle has the inlet pipe, the inner wall fixed mounting of perfusion bottle has the discharging pipe, the one end fixed mounting that the perfusion bottle was kept away from to the discharging pipe has the perfusion case, the bottom fixed mounting of perfusion case has electronic flexible post, the left side fixed mounting of perfusion case has first electromagnetic block, the inner wall swing joint of perfusion bottle has the second electromagnetic block, one side fixed mounting that the perfusion bottle was kept away from to the second electromagnetic block has the connecting rod, the top of connecting rod and current-limiting component's bottom fixed connection, the top of current-limiting component and the bottom fixed connection of extrusion subassembly.

As optimization, the current-limiting component includes the drain pan, the outer wall swing joint of drain pan has the mesochite, the inner wall swing joint of mesochite has the swinging arms, the one end swing joint that the mesochite was kept away from to the swinging arms has the pivot, the top fixed mounting of pivot has the connecting rod, the one end swing joint that the pivot was kept away from to the connecting rod has first inner circle, the inner wall fixed mounting of first inner circle has the bent plate, the front swing joint of bent plate has the guide arm, the back fixed mounting of bent plate has the backup pad.

As optimization, the extrusion subassembly includes the epitheca, the inner wall swing joint of epitheca has the commentaries on classics box, the inner wall swing joint who changes the box has the second inner circle, the inner wall swing joint of second inner circle has the stripper plate, the outer wall swing joint of epitheca has the commentaries on classics tooth, the bottom fixed mounting who changes the tooth has the threaded rod, the outer wall swing joint of threaded rod has the outer tube, the inner wall swing joint of outer tube has the foundation.

As optimization, the number of the swing rods is four, every two swing rods are in one group, and each group of swing rods are symmetrically distributed by using the horizontal central line of the first inner ring, so that the swing rods can be squeezed in the ascending process of the bottom shell, and the first inner ring is driven to rotate.

Preferably, the number of the bent plates and the structures of the bent plates are two, the two bent plates and the structures of the bent plates are uniformly distributed around the center of the discharge pipe, and one end of the guide rod, which is far away from the first inner ring, is fixedly arranged on the outer wall of the discharge pipe, so that the bent plates can be driven to pass through in the rotating process of the first inner ring, and the guide rod extrudes the discharge pipe.

As optimization, the connecting rod constitutes for two parts about, and the partial inner wall in left side of connecting rod and the outer wall swing joint of discharging pipe, the right side part of connecting rod passes through the right side part swing joint of activity telescopic link and connecting rod, and the right side part of connecting rod is the stretching post for in-process that the connecting rod rises can drive the drain pan earlier and rise, when the drain pan rises motionless, can make the partial continuation of connecting rod right side rise, thereby drive the bottom prop and rise.

Preferably, the diameter of the threaded rod is smaller than the inner diameter of the bottom column, and the inner wall of the bottom column is provided with a threaded lug, so that the bottom column can drive the threaded rod to rotate.

As optimization, the bottom of foundation and the right side partial top fixed connection of connecting rod, and the inner wall of changeing the box is provided with the flange, and the stripper plate is close to one side of discharging pipe and the outer wall fixed connection of discharging pipe for the connecting rod can drive the drain pan and rise, and when the limited unable rising of drain pan, when continuing to remove the second electromagnetism piece, the right side partial continuation that can drive the connecting rod rises, thereby promotes the foundation and drives the threaded rod and rotate.

The first embodiment is as follows:

s1, preparing, namely preparing a stem cell culture medium, and cooling the stem cell culture medium to 6-15 ℃;

s2, putting the stem cells into a container and putting the container into a perfusion box;

s3, in the perfusion stage, the stem cell culture medium is communicated with a feed pipe and enters a perfusion tank through a discharge pipe;

s4, starting a peristaltic pump in the perfusion tank, and keeping the temperature at 30-36 ℃;

s5, introducing oxygen, and introducing oxygen to a perfusion bottle to ensure that the content of the culture oxygen reaches 40-50%;

s6, keeping for 5-7 days, and keeping the perfusion speed at 0.3-1.6 RV/day;

s7, releasing, wherein releasing is started in 8-10 days, and the speed is kept at 0.1-1 RV/day;

and S8, finishing releasing, and taking the stem cells out of the perfusion box to finish stem cell culture.

The invention has the beneficial effects that: this a perfusion device and perfusion method for embryonic stem cell culture, use through the cooperation of second electromagnetism piece and drain pan, make at the ascending in-process of electronic flexible post, can drive first electromagnetism piece and rise, first electromagnetism piece rises and drives the second electromagnetism piece and rise, so that the second electromagnetism piece drives the connecting rod and rises, make the drain pan rise, the ascending extrusion swinging arms of drain pan, make the swinging arms drive first inner circle and rotate, thereby drive the bent plate and rotate, make the guide arm receive the bent plate influence, extrude to the discharging pipe, thereby make the diameter of discharging pipe narrow down, thereby restrict the volume of discharging pipe ejection of compact, with this control culture medium flow.

This a perfusion device and perfusion method for embryonic stem cell culture, use through the connecting rod and commentaries on classics box cooperation, make in the in-process that the connecting rod rises, can't continue to rise by the swinging arms influence, then the right side part of connecting rod can drive the sill pillar through movable telescopic link and rise, the sill pillar rises and the threaded rod contact, thereby it rotates to drive the threaded rod, with this threaded rod drive commentaries on classics tooth rotation, it rotates to change the box drive, make the flange extrusion stripper plate who changes the box, make stripper plate extrusion discharging pipe, thereby make the diameter of discharging pipe narrow once more, with the flow of this secondary restriction culture medium, with this required not isovolumetric culture medium in different stem cell stages of realization.

Drawings

FIG. 1 is a schematic sectional elevation view of the structure of the present invention;

FIG. 2 is an enlarged view of the structure at A in FIG. 1 according to the present invention;

FIG. 3 is a front cross-sectional view of the shell structure of the present invention;

FIG. 4 is an enlarged view of the structure at B in FIG. 3 according to the present invention;

FIG. 5 is a schematic top cross-sectional view of the shell structure of the present invention;

FIG. 6 is a schematic top sectional view of the rotating box structure of the present invention.

In the figure: 1. a support; 2. filling a flow bottle; 3. a feed pipe; 4. a discharge pipe; 5. a perfusion tank; 6. an electric telescopic column; 7. a first electromagnetic block; 8. a second electromagnetic block; 9. a connecting rod; 10. a current limiting assembly; 1001. a bottom case; 1002. a middle shell; 1003. a swing lever; 1004. a rotating shaft; 1005. a connecting rod; 1006. a first inner race; 1007. bending a plate; 1008. a guide bar; 1009. a support plate; 11. an extrusion assembly; 1101. a top shell; 1102. box turning; 1103. a second inner race; 1104. a pressing plate; 1105. rotating the teeth; 1106. a threaded rod; 1107. an outer sleeve; 1108. a bottom pillar.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1 to 6, a perfusion apparatus for embryonic stem cell culture includes a frame 1, a flow restriction assembly 10 and a pressing assembly 11, the top end of the bracket 1 is fixedly provided with an perfusion bottle 2, the inner wall of the perfusion bottle 2 is fixedly provided with a feeding pipe 3, a discharge pipe 4 is fixedly arranged on the inner wall of the perfusion bottle 2, a perfusion tank 5 is fixedly arranged at one end of the discharge pipe 4 far away from the perfusion bottle 2, the bottom end of the perfusion box 5 is fixedly provided with an electric telescopic column 6, the left side of the perfusion box 5 is fixedly provided with a first electromagnetic block 7, the inner wall of the perfusion bottle 2 is movably connected with a second electromagnetic block 8, one side of the second electromagnetic block 8 far away from the perfusion bottle 2 is fixedly provided with a connecting rod 9, the top end of the connecting rod 9 is fixedly connected with the bottom end of the flow limiting assembly 10, and the top end of the flow limiting assembly 10 is fixedly connected with the bottom end of the extrusion assembly 11.

Referring to fig. 1, 3, 4-5, the current limiting assembly 10 includes a bottom case 1001, an outer wall of the bottom case 1001 is movably connected to a middle case 1002, an inner wall of the middle case 1002 is movably connected to swing rods 1003, one end of each swing rod 1003 far from the middle case 1002 is movably connected to a rotating shaft 1004, a top end of each rotating shaft 1004 is fixedly installed with a connecting rod 1005, one end of each connecting rod 1005 far from the rotating shaft 1004 is movably connected to a first inner ring 1006, the number of the swing rods 1003 is four, each two swing rods 1003 are in one group, each group of the swing rods 1003 is symmetrically distributed along a horizontal center line of the first inner ring 1006, so that the swing rods 1003 can be pressed to drive the first inner ring 1006 to rotate in a process of the bottom case 1001, a bent plate 1007 is fixedly installed on an inner wall of the first inner ring 1006, a guide rod 1008 is movably connected to a front surface of the bent plate 1007, the bent plate 1007 and the number of structures of the bent plate 1007 is two, and the two bent plates 1007 and the structures of the bent plates 1007 are uniformly distributed by taking the circle center of the discharging pipe 4 as the center, and one end of the guide rod 1008, which is far away from the first inner ring 1006, is fixedly arranged on the outer wall of the discharging pipe 4, so that in the rotating process of the first inner ring 1006, the bent plates 1007 can be driven to extrude the discharging pipe 4 from the guide rod 1008, and a supporting plate 1009 is fixedly arranged on the back surface of the bent plates 1007.

Referring to fig. 3, 4 and 6, the extruding assembly 11 includes a top housing 1101, the inner wall of the top housing 1101 is movably connected with a rotating box 1102, the inner wall of the rotating box 1102 is movably connected with a second inner ring 1103, the inner wall of the second inner ring 1103 is movably connected with an extruding plate 1104, the outer wall of the top housing 1101 is movably connected with a rotating tooth 1105, the bottom end of the rotating tooth 1105 is fixedly installed with a threaded rod 1106, the outer wall of the threaded rod 1106 is movably connected with an outer sleeve 1107, the inner wall of the outer sleeve 1107 is movably connected with a bottom column 1108, the connecting rod 9 is composed of a left part and a right part, the inner wall of the left part of the connecting rod 9 is movably connected with the outer wall of the discharging pipe 4, the right part of the connecting rod 9 is movably connected with the right part of the connecting rod 9 through a movable telescopic rod, the right part of the connecting rod 9 is a pull column, so that the bottom housing 1001 can be driven to ascend first in the ascending process of the connecting rod 9, and when the bottom housing 1001 ascends, can make connecting rod 9 right side part continue to rise, thereby it rises to drive foundation 1108, the bottom of foundation 1108 and the right side part top fixed connection of connecting rod 9, and the inner wall of changeing box 1102 is provided with the flange, and the outer wall fixed connection of one side that extrusion plate 1104 is close to discharging pipe 4 and discharging pipe 4, make connecting rod 9 can drive drain pan 1001 and rise, limited unable rising when drain pan 1001, when continuing to remove second electromagnetism piece 8, can drive the right side part of connecting rod 9 and continue to rise, thereby it drives threaded rod 1106 and rotates to promote foundation 1108, the diameter of threaded rod 1106 is less than the internal diameter of foundation 1108, and the inner wall of foundation 1108 is provided with the screw lug, make foundation 1108 can drive threaded rod 1106 and rotate.

In use, referring to fig. 1-6, when a culture medium is introduced into a feed pipe 3, the culture medium is introduced out of a discharge pipe 4 and enters a perfusion tank 5, when a plurality of cells are cultured, and the amount of the culture medium required is detected to be reduced, an electric telescopic column 6 can be opened, so that a first electromagnetic block 7 is lifted to drive a second electromagnetic block 8 to lift, the second electromagnetic block 8 drives a connecting rod 9 to lift, the connecting rod 9 is lifted to drive a bottom shell 1001 to lift, and a swinging rod 1003 is squeezed, the swinging rod 1003 swings to squeeze a first inner ring 1006, the first inner ring 1006 rotates to drive a bent plate 1007 to rotate, so that a guide rod 1008 is limited to squeeze out a material pipe 4, the diameter of the discharge pipe 4 is narrowed, the discharge amount of the discharge pipe 4 is reduced, a plurality of cells enter another stage, the required culture medium is reduced again, the electric telescopic column 6 is opened again, and the first electromagnetic block 7 is lifted again, drive second electromagnetism piece 8 and rise, second electromagnetism piece 8 drives connecting rod 9 right side part and continues to rise, connecting rod 9 left side part position is unchangeable, make connecting rod 9 right side part drive bottom pillar 1108 rise, make bottom pillar 1108 and threaded rod 1106 contact, thereby threaded rod 1106 rotates and drives commentaries on classics tooth 1105 and rotate, commentaries on classics tooth 1105 drives commentaries on classics box 1102 and rotates, it drives the flange and extrudees board 1104 to change box 1102 and rotate, thereby make extrusion board 1104 continuously extrude material pipe 4, make the diameter of discharging pipe 4 narrow down once more, with this volume that makes culture medium in discharging pipe 4 reduce once more, reach controllable culture medium inflow with this.

The first embodiment is as follows:

a perfusion method for stem cell culture comprises the following specific steps:

s1, preparing, namely preparing a stem cell culture medium, and cooling the stem cell culture medium to 6 ℃;

s2, placing the stem cells into a container and placing the stem cells into a perfusion box 5;

s3, in the perfusion stage, the stem cell culture medium is communicated with a feeding pipe 3 and enters a perfusion box 5 through a discharging pipe 4;

s4, starting a peristaltic pump in the perfusion box 5, and keeping the temperature at 30 ℃;

s5, introducing oxygen, and introducing oxygen to the perfusion bottle 2 to ensure that the content of the culture medium oxygen reaches 40%;

s6, keeping for 5 days, and keeping the perfusion speed at 0.3 RV/day;

s7, releasing, wherein releasing is started at 8 days, and the speed is kept at 0.1 RV/day;

and S8, finishing releasing, and taking out the stem cells from the perfusion box 5 to finish stem cell culture.

Example two:

s1, preparing, namely preparing a stem cell culture medium, and cooling the stem cell culture medium to 10.5 ℃;

s4, starting a peristaltic pump in the perfusion box 5, and keeping the temperature at 33 ℃;

s5, introducing oxygen, and introducing oxygen to the perfusion bottle 2 to ensure that the content of the culture oxygen reaches 45%;

s6, keeping for 6 days, and keeping the perfusion speed at 0.95 RV/day;

s7, releasing, wherein releasing is started when the day is 9 days, and the speed is kept to be 0.55 RV/day;

Example three:

s1, preparing, namely preparing a stem cell culture medium, and cooling the stem cell culture medium to 15 ℃;

s4, starting a peristaltic pump in the perfusion box 5, and keeping the temperature at 36 ℃;

s5, introducing oxygen, and introducing oxygen to the perfusion bottle 2 to ensure that the content of the culture medium oxygen reaches 50%;

s6, keeping for 7 days, and keeping the perfusion speed at 1.6 RV/day;

s7, releasing, wherein releasing is started in 10 days, and the speed is kept at 1 RV/day;

To sum up, the perfusion device and perfusion method for culturing embryonic stem cells, through the cooperation of the second electromagnetic block 8 and the bottom case 1001, in the process of ascending of the electric telescopic column 6, the first electromagnetic block 7 can be driven to ascend, the first electromagnetic block 7 ascends to drive the second electromagnetic block 8 to ascend, so that the second electromagnetic block 8 drives the connecting rod 9 to ascend, the bottom case 1001 ascends to press the swinging rod 1003, the swinging rod 1003 drives the first inner ring 1006 to rotate, thereby driving the bent plate 1007 to rotate, the guide rod 1008 is influenced by the bent plate 1007 to press the discharging pipe 4, thereby the diameter of the discharging pipe 4 is narrowed, thereby the discharging amount of the discharging pipe 4 is limited, so as to control the culture medium flow, in the process of ascending of the connecting rod 9, the swinging rod 1003 can not continue to ascend, the right part of the connecting rod 9 can drive the bottom column 1108 through the movable telescopic rod, the bottom column 1108 ascends to contact with the threaded rod 1106, thereby drive threaded rod 1106 and rotate to this threaded rod 1106 drives commentaries on classics tooth 1105 and rotates, and commentaries on classics tooth 1105 drives commentaries on classics box 1102 and rotates, makes the protruding board extrusion plate 1104 of commentaries on classics box 1102, makes extrusion plate 1104 extrude material pipe 4 to narrow down the diameter of discharging pipe 4 once more, with this secondary restriction culture medium's flow, with this culture medium that realizes the different quantities that different stem cell stages need.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.

Claims

1. A perfusion device for embryonic stem cell culture, comprises a bracket (1), a current limiting assembly (10) and a squeezing assembly (11), and is characterized in that: an irrigation bottle (2) is fixedly arranged at the top end of the bracket (1), a feeding pipe (3) is fixedly arranged on the inner wall of the irrigation bottle (2), a discharge pipe (4) is fixedly arranged on the inner wall of the perfusion bottle (2), a perfusion tank (5) is fixedly arranged at one end of the discharge pipe (4) far away from the perfusion bottle (2), the bottom end of the perfusion box (5) is fixedly provided with an electric telescopic column (6), a first electromagnetic block (7) is fixedly arranged on the left side of the perfusion box (5), the inner wall of the perfusion bottle (2) is movably connected with a second electromagnetic block (8), a connecting rod (9) is fixedly arranged on one side of the second electromagnetic block (8) far away from the perfusion bottle (2), the top end of the connecting rod (9) is fixedly connected with the bottom end of the current limiting component (10), the top end of the flow limiting assembly (10) is fixedly connected with the bottom end of the extrusion assembly (11).

2. A perfusion device for embryonic stem cell culture according to claim 1, wherein: current-limiting subassembly (10) include drain pan (1001), the outer wall swing joint of drain pan (1001) has mesochite (1002), the inner wall swing joint of mesochite (1002) has swinging arms (1003), the one end swing joint that mesochite (1002) were kept away from in swinging arms (1003) has pivot (1004), the top fixed mounting of pivot (1004) has connecting rod (1005), the one end swing joint that pivot (1004) were kept away from in connecting rod (1005) has first inner circle (1006), the inner wall fixed mounting of first inner circle (1006) has bent plate (1007), the front swing joint of bent plate (1007) has guide arm (1008), the back fixed mounting of bent plate (1007) has backup pad (1009).

3. A perfusion device for embryonic stem cell culture according to claim 1, wherein: extrusion subassembly (11) are including top shell (1101), the inner wall swing joint of top shell (1101) has changes box (1102), the inner wall swing joint who changes box (1102) has second inner circle (1103), the inner wall swing joint of second inner circle (1103) has stripper plate (1104), the outer wall swing joint of top shell (1101) has commentaries on classics tooth (1105), the bottom fixed mounting who changes tooth (1105) has threaded rod (1106), the outer wall swing joint of threaded rod (1106) has outer tube (1107), the inner wall swing joint of outer tube (1107) has base pillar (1108).

4. A perfusion device for embryonic stem cell culture according to claim 2, wherein: the number of the oscillating rods (1003) is four, every two oscillating rods (1003) form a group, and each group of oscillating rods (1003) are symmetrically distributed by the horizontal center line of the first inner ring (1006).

5. A perfusion device for embryonic stem cell culture according to claim 2, wherein: the quantity of the bent plates (1007) and the affiliated structures of the bent plates (1007) is two, the affiliated structures of the two bent plates (1007) and the affiliated structures of the bent plates (1007) are uniformly distributed by taking the circle center of the discharge pipe (4) as the center, and one end, far away from the first inner ring (1006), of the guide rod (1008) is fixedly installed on the outer wall of the discharge pipe (4).

6. A perfusion device for embryonic stem cell culture according to claim 1, wherein: the connecting rod (9) is composed of a left part and a right part, the inner wall of the left part of the connecting rod (9) is movably connected with the outer wall of the discharge pipe (4), the right part of the connecting rod (9) is movably connected with the right part of the connecting rod (9) through a movable telescopic rod, and the right part of the connecting rod (9) is a stretching column.

7. A perfusion device for embryonic stem cell culture according to claim 3, wherein: the diameter of the threaded rod (1106) is smaller than the inner diameter of the bottom pillar (1108), and a threaded convex block is arranged on the inner wall of the bottom pillar (1108).

8. A perfusion device for embryonic stem cell culture according to claim 6, wherein: the bottom end of the bottom column (1108) is fixedly connected with the top end of the right side part of the connecting rod (9), a convex plate is arranged on the inner wall of the rotating box (1102), and one side of the extrusion plate (1104), which is close to the discharge pipe (4), is fixedly connected with the outer wall of the discharge pipe (4).

9. A perfusion method for embryonic stem cell culture according to claim 1, which comprises the following steps:

s2, putting the stem cells into a container and putting the stem cells into a perfusion box (5);

s3, in a perfusion stage, the stem cell culture medium is communicated with a feeding pipe (3) and enters a perfusion box (5) through a discharging pipe (4);

s4, starting a peristaltic pump in the perfusion box (5) and keeping the temperature at 30-36 ℃;

s5, introducing oxygen, and introducing oxygen to the perfusion bottle (2) to ensure that the content of the culture oxygen reaches 40-50%;

s6, keeping for 5-7 days, and keeping the perfusion speed at 0.3-1.6 RV/day;

and S8, finishing releasing, and taking out the stem cells from the perfusion box (5) to finish stem cell culture.