CN110029061B

CN110029061B - Stem cell amplification culture system and stem cell amplification culture method

Info

Publication number: CN110029061B
Application number: CN201910357223.6A
Authority: CN
Inventors: 刘利彪; 邓坤学; 袁玉宇
Original assignee: Medprin Regenerative Medical Technologies Co Ltd
Current assignee: Medprin Regenerative Medical Technologies Co Ltd
Priority date: 2019-04-29
Filing date: 2019-04-29
Publication date: 2022-08-02
Anticipated expiration: 2039-04-29
Also published as: CN110029061A

Abstract

Provided are a stem cell expansion culture system and a stem cell expansion culture method. The stem cell expansion culture system comprises: a cell culture cassette for housing a cell culture unit; a cell printing head for printing a biological material containing stem cells to a cell culture unit; a cell culture unit drive means for at least grasping the cell culture unit from the cell culture cassette and/or placing the cell culture unit into the cell culture cassette. The cell printing head and the cell culture unit driving device are matched with each other, and the biological material containing the stem cells is printed on the cell culture unit, so that the stem cells can grow in a three-dimensional external environment, and therefore, the cell culture unit has enough growth space, not only can well reserve multiple differentiation potentials of the stem cells, but also can reduce the frequency of cell passage operation, reduce pollution risks, and realize high-quality and high-efficiency stem cell culture.

Description

Stem cell amplification culture system and stem cell amplification culture method

Technical Field

The invention relates to the technical field of biological medical treatment, and particularly relates to a stem cell amplification culture system and a stem cell amplification culture method.

Background

Stem cells have important applications in modern medical and scientific research, but efficient and high-quality expansion of stem cells is always a difficult problem.

In the traditional cell culture or amplification, cells are planted in culture bottles, culture dishes and the like, and when the cells are proliferated to a certain density, the cells are required to be digested, collected by centrifugation and then re-dispersed into a plurality of culture bottles for continuous culture.

The method is simple and reliable, but has the following defects:

first, culture devices such as culture bottles and culture dishes provide a two-dimensional growth environment for cells, and since the culture environment is different from the three-dimensional environment in an organism, in practical applications, the growth characteristics of the cells change with the increase of the number of culture generations, and particularly, the dryness of stem cells is obviously lost with the increase of the number of culture generations.

Secondly, the stem cells are inhibited in contact after being proliferated to a certain density in a two-dimensional plane, and the stem cells are easy to differentiate due to the fact that the stem cells are too high in concentration, the potential of differentiation is lost, so that efficient stem cell amplification cannot be achieved, and the yield is not high.

Thirdly, in large-scale culture, cells need to be changed every two days, and about four days, the cells need to be passaged, and the daily maintenance operations need to consume a large amount of time and labor cost, so that the labor cost is high.

Fourth, during the long-term culture, the culture flask needs to be opened repeatedly for liquid change and passage operation, which greatly increases the risk of contamination.

Disclosure of Invention

The present invention has been made in view of the state of the art described above. The invention aims to provide a stem cell amplification culture system and a stem cell amplification culture method, which can provide a three-dimensional growth environment for stem cells, better reserve multiple differentiation potentials of the stem cells, reduce the pollution risk of the stem cells and improve the yield of the stem cells.

Provided is a stem cell expansion culture system, comprising:

a cell culture cassette for housing a cell culture unit,

a cell printing head for printing a biological material containing stem cells onto the cell culture unit,

a cell culture unit drive device at least for grasping the cell culture unit from the cell culture cassette and/or placing the cell culture unit into the cell culture cassette for culturing.

In at least one embodiment, the stem cell expansion and culture system further comprises a cell printing head driving device, wherein the cell printing head driving device is used for driving the cell printing head to move, so that the cell printing head prints the biological material containing the stem cells to different positions of the cell culture unit.

In at least one embodiment, the cell culture unit driving means includes a robot arm capable of gripping the cell culture unit from the cell culture cassette and/or putting the cell culture unit into the cell culture cassette, and the robot arm is also capable of driving the cell culture unit to rotate on its own axis,

the cell printing head driving device comprises a linear motion module, the cell printing head is arranged on the linear motion module, and the linear motion module can drive the cell printing head to move along the rotation axis of the cell culture unit.

In at least one embodiment, the cell culture unit is formed as a hollow tube having a wall with a medium passage hole, the outside of the hollow tube being adapted to be combined with the stem cell-containing biomaterial, the hollow tube being capable of allowing the medium inside thereof to permeate through the medium passage hole to the stem cell-containing biomaterial outside of the hollow tube.

In at least one embodiment, the stem cell expansion culture system further comprises an internal circulation system, wherein the internal circulation system is communicated with the cell culture box and is used for introducing fresh culture medium into the interior of the hollow tube so as to circulate the culture medium in the interior of the hollow tube.

In at least one embodiment, the internal circulation system comprises:

an inner loop liquid storage bottle for storing fresh culture medium;

an internal loop driven pump for driving the medium to flow in the internal circulation system and continuously flow through the cell culture cassette;

a gas exchanger for exchanging oxygen and carbon dioxide for the medium flowing into the cell culture cassette;

a monitoring assembly for monitoring a predetermined parameter of the media flowing from an inner loop reservoir into the cell culture cassette,

wherein, in the internal circulation system, the internal loop liquid storage bottle, the internal loop driving pump, the gas exchanger and the monitoring assembly are connected in series.

In at least one embodiment, the stem cell expansion culture system further comprises an external circulation system in communication with the cell culture cassette for circulating a culture medium around the stem cell-containing biomaterial outside of the hollow tube.

In at least one embodiment, the external circulation system comprises:

an external loop drive pump for driving the culture medium surrounding the stem cell-containing biomaterial out of the cell culture cassette;

an external circuit liquid storage bottle for storing a culture medium around the stem cell-containing biomaterial;

an electromagnetic valve for controlling the starting or stopping of the external circulation system,

wherein, in the external circulation system, the external loop liquid storage bottle, the external loop driving pump and the electromagnetic valve are connected in series.

In at least one embodiment, the cell culture box comprises a box body and a box cover which are detachably connected, the box body and the box cover enclose a containing space for containing the hollow tube, the box cover is positioned on a passage of the hollow tube into and out of the cell culture box, and a branch pipeline which is butted with a pipeline of the internal circulation system is arranged in the box cover and the containing space so that the internal circulation system can be communicated with the inside of the hollow tube when the box cover is connected with the box body.

In at least one embodiment, the stem cell expansion and culture system further comprises a box body, wherein a cabinet is installed inside the box body, and a printing space is formed in the box body outside the cabinet;

the cabinet comprises a cell culture cabin, the cell culture boxes are arranged in the cell culture cabin in parallel along the height direction, and the cell printing head and the cell culture unit driving device are positioned in the printing space;

the cell culture unit driving device can grab the cell culture unit from the cell culture box to the printing space so as to enable the cell printing head to print the biological material containing the stem cells on the cell culture unit.

Also provided is a stem cell expansion culture method, which comprises the following steps:

a cell culture unit obtaining step of obtaining a cell culture unit from the cell culture cassette by the cell culture unit driving means;

a stem cell-containing biomaterial forming step of printing the stem cell-containing biomaterial to the cell culture unit by the cell print head;

a cell culture unit loading step, wherein the cell culture unit driving device loads the cell culture unit into the cell culture box;

and a cell culture step of culturing the stem cells in the cell culture box.

In at least one embodiment, in the step of forming the biological material containing stem cells, the cell culture unit is driven to rotate by the cell culture unit driving device, and when the cell culture unit rotates, the cell printing head prints the biological material containing stem cells onto the cell culture unit and moves the cell printing head along the rotation axis of the cell culture unit.

In at least one embodiment, the stem cell-containing biomaterial comprises a hollow fiber having diffusion pores, the hollow fiber being homogeneously mixed with the cells in the stem cell-containing biomaterial. The stem cell amplification culture system and the stem cell amplification culture method adopting the technical scheme can at least obtain the following beneficial effects:

firstly, the cell printing head and the cell culture unit driving device are matched with each other to print the biological material containing the stem cells onto the cell culture unit, so that the stem cells can grow in a three-dimensional external environment, and an enough growth space is provided, thereby not only well retaining multiple differentiation potentials of the stem cells, but also reducing the frequency of cell subculture operation, reducing pollution risks and realizing high-quality and high-efficiency stem cell culture.

Secondly, the invention adopts an internal circulation system to introduce fresh culture medium into the cell culture unit formed into the hollow tube, so that the culture medium in the hollow tube circularly flows, dynamic culture is realized, and the stem cells are prevented from being hindered from growing due to metabolite accumulation in the stem cell culture process; fresh culture medium can diffuse to stem cells through the hollow tube, so that adverse effects of shearing force on the stem cells are avoided, and the efficiency of stem cell amplification is improved.

Thirdly, the invention is provided with an external circulation system, so that the culture medium outside the hollow tube can circularly flow, and the diffusion of nutrient substances and oxygen in the culture medium in the hollow tube is accelerated, thereby improving the survival rate of stem cells and improving the nutrient supply.

Fourthly, the cell printing head driving device, the cell culture unit driving device, the cell culture box, the internal circulation system and the external circulation system are integrated in the box body, so that full-closed and full-automatic operation is realized, frequent contact with the outside is avoided, time and labor are saved, and the pollution risk is reduced.

Drawings

Fig. 1 is a schematic diagram illustrating a three-dimensional structure of one embodiment of a stem cell expansion culture system provided by the present disclosure.

Fig. 2A is a schematic view showing a three-dimensional structure of a culture cassette of the stem cell expansion culture system in fig. 1.

Fig. 2B is another schematic view illustrating a three-dimensional structure of the culture cassette of the stem cell expansion culture system of fig. 1, showing the cassette cover removed from the cassette body.

Fig. 3A is a schematic diagram illustrating the three-dimensional structure of one embodiment of a cell culture unit provided by the present disclosure.

FIG. 3B is an enlarged partial view of the cell culture unit of FIG. 3A.

Fig. 4 is a schematic view illustrating a medium circulation system of the stem cell expansion culture system in fig. 1.

FIG. 5 is a schematic diagram of a stem cell hydrogel system in one embodiment of the cell culture unit in FIGS. 3A and 3B, showing the composition of the stem cell hydrogel system.

Fig. 6 is a schematic diagram of a stem cell hydrogel system in another embodiment of a cell culture unit provided by the present disclosure.

Description of reference numerals:

the robot comprises a box body 1, a mechanical arm 2, a base 21, a mechanical arm body 22, a linear motion module 3, a mounting seat 31, a mounting arm 32 and a cabinet 4;

5 cell culture box, 51 inner loop inlet, 52 inner loop outlet, 53 outer loop inlet, 54 outer loop outlet, 58 box body, 59 box cover, 50 cell culture cabin;

6 cell culture units, 60 joints, 61 hollow tubes, 610 culture medium passing holes, 62 stem cell hydrogel system, 621 hydrogel, 622 cells, 623 hollow fibers;

7 culture medium circulation system, 71 internal circulation system, 711 internal circulation pipeline, 7111, 7112 main pipeline, 7113, 7114 branch pipeline, 713PH meter, 714 oxygen concentration meter, 715 internal loop driving pump, 716 gas exchanger, 719 internal loop liquid storage bottle, 72 external circulation system, 721 external circulation pipeline, 722 external loop driving pump, 724 solenoid valve, 729 external loop liquid storage bottle;

8 cell print head.

Detailed Description

Exemplary embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the detailed description is intended only to teach one skilled in the art how to practice the invention, and is not intended to be exhaustive or to limit the scope of the invention.

The invention provides a stem cell amplification culture system and a stem cell amplification culture method.

As shown in fig. 1 to 4, in one embodiment of the present invention, the stem cell expansion and culture system comprises a box 1, a robot arm 2, a linear motion module 3, a cabinet 4, a cell culture box 5, a culture medium circulation system 7 and a cell printing head 8. The stem cell expansion culture system can be used for culturing stem cells on a plurality of cell culture units 6.

The rack 4 is located the inside of box 1, is formed with the printing space in the box 1 outside the rack 4, and arm 2, cell are beaten printer head 8 and linear motion module 3 and are located the printing space, and the inside of rack 4 forms into the accommodation space, is formed with cell culture cabin 50 and stock solution cabin in the accommodation space, and cell culture box 5 arranges side by side along the direction of height in cell culture cabin 50, and culture medium circulation system 7 installs in thermostatic control's stock solution cabin. In other embodiments of the present invention, the cabinet 4 is further provided with electrical equipment for driving, controlling temperature, and the like.

The box body 1 can ensure that the stem cell amplification culture is carried out in a sterile environment. In one embodiment, the housing 1 may be made of a heat insulating material to form a constant temperature environment inside the housing 1, which is beneficial to ensure a reliable environment for stem cell survival. The case 1 may be, for example, a square body (substantially rectangular parallelepiped, substantially square body, or the like).

The cell culture boxes 5 can be multiple, the multiple cell culture boxes 5 are arranged in parallel in the height direction in the cell culture cabin 50, the inside of the cell culture cabin 50 is divided into multiple mutually independent spaces, each cell culture box 5 can be used for accommodating multiple cell culture units 6, and the cell culture units 6 are used for bearing biological materials containing stem cells, so that the cell culture units 6 of the cell culture boxes 5 can be operated independently, and the flexibility of the system is high.

Of course, the number of the cell culture cassettes 5 may be one.

As shown in fig. 3A and 3B, the cell culture unit 6 may be a hollow tube 61, the wall of the hollow tube 61 may have a plurality of medium passage holes 610, the outside of the hollow tube 61 is used for combining with the biomaterial containing stem cells, and the hollow tube 61 enables the medium inside to permeate the biomaterial containing stem cells outside of the hollow tube 61 through the medium passage holes 610. The medium passing through the holes 610 does not have a specific position requirement as long as it is uniformly distributed for good diffusion of the medium.

In the view of fig. 1, the cabinet 4 may be mounted on one side of the cabinet 1, the robot 2 includes a base 21 and a robot body 22, the base 21 is located at one end (left end) of the printing space away from the cabinet 4, and the robot body 22 is movable in a right space of the base 21.

The linear motion module 3 includes a mounting seat 31 and a mounting arm 32, and the mounting seat 31 may be located on the same side of the cabinet 1 as the base 21. The mounting arm 32 extends from the mounting base 31, for example, in a space on the right side of the mounting base 31, and the mounting arm 32 is located above the robot arm body 22. The mount 31 of the linear motion module 3 may be fixedly mounted relative to the base 21 of the robot arm 2. The other end (right end) of the mounting arm 31 of the linear motion module 3 is mounted at the top of the cabinet 4, so that the cabinet 4 provides support for the linear motion module 3.

The cell printing head 8 is attached to the attachment arm 32 of the linear motion module 3 and can be reciprocated by the drive of the linear motion module 3 along the extending direction (left-right direction) of the attachment arm 32 to perform cell printing at an appropriate position.

In one embodiment of the present invention, the stem cell expansion culture system works as follows: the mechanical arm 2 (mechanical arm body 22) grabs the hollow tube 61 from the cell culture box 5 to the printing space, then drives the hollow tube 61 to rotate, the linear motion module 3 drives the cell printing head 8 to move along the rotation axis of the hollow tube 61, so that the cell printing head 8 can print the stem cell hydrogel system 62 with a spiral path on the hollow tube 61, the stem cell hydrogel system 62 is combined with the hollow tube 61, and the hollow tube 61 is returned to the cell culture box 5 by the mechanical arm 2 for culture after the stem cell hydrogel system 62 is solidified, formed and stabilized.

In other embodiments, the mechanical arm 2 may print the stem cell hydrogel system 62 in a strip-shaped path on the hollow tube 61 by moving the cell printing head 8 along the axial direction of the hollow tube 61 without rotating the hollow tube 61, or the mechanical arm 2 may print the stem cell hydrogel system 62 in a circular path on the hollow tube 61 by rotating the hollow tube 61 while keeping the cell printing head 8 still. Of course, the embodiments of the present invention are not limited to the above several printing modes of the spiral path, the loop path, and the strip path.

It should be understood that the terms "front", "back", "left", "right", "up" and "down" are not intended to limit the disclosure, but merely illustrate the positional relationship of the components of the stem cell expansion and culture system by way of example, and in other embodiments, the orientations may be adjusted according to the specific situation when the stem cell expansion and culture system is used.

As shown in fig. 4, the medium circulation system 7 may include an internal circulation system 71 and an external circulation system 72 provided independently of each other, the internal circulation system 71 communicating with the cell culture cassette 5 for introducing fresh medium into the cell culture cassette 5 and circulating the medium inside the hollow tube 61; the external circulation system 72 is also in communication with the cell culture cassette 5 for circulating the culture medium around the stem cell hydrogel system 62. The inner circulation system 71 and the outer circulation system 72 may both be in a constant temperature environment.

As shown in FIGS. 2A and 2B, the cell culture cassette 5 is provided with an inner circuit inlet 51, an inner circuit outlet 52, an outer circuit inlet 53 and an outer circuit outlet 54.

The internal circulation system 71 includes an internal circuit liquid storage bottle 719, an internal circuit driving pump 715, a monitoring module, and a gas exchanger 716 connected in series via an internal circulation pipe 711. Wherein the inner loop reservoir bottle 719 stores fresh medium and the inner loop reservoir bottle 719 is connected to the inner loop inlet 51 and the inner loop outlet 52 on each cell culture cassette 5 enabling bi-directional fluid communication between the inner loop reservoir bottle 719 and the cell culture cassette 5. The inner loop driving pump 715 is connected to the inner loop reservoir 719 and the cell culture cassette 5, and drives the medium to flow in the inner circulation system 71 and continuously flow through the cell culture cassette 5. The monitoring assembly includes an oxygen concentration meter 714 and a PH meter 713, and the oxygen concentration meter 714 and the PH meter 713 are disposed between the inner loop reservoir 719 and the inner loop inlet 51 of the cell culture cassette 5 to detect the oxygen concentration and PH of the culture medium in real time to provide suitable survival conditions for the cells 622 in the stem cell hydrogel system 62. The gas exchanger 716 is provided between the inner circuit liquid storage bottle 719 and the inner circuit inlet 51 of the cell culture cassette 5, and reduces the carbon dioxide concentration of the medium flowing into the cell culture cassette 5 and supplements oxygen at a predetermined concentration.

The external circulation system 72 includes an external circuit reservoir 729, an external circuit drive pump 722, and a solenoid valve 724 connected in series by an external circulation line 721. Wherein the external loop reservoir bottle 729 is connected to the external loop inlet 53 and the external loop outlet 54 on each cell culture cassette 5, enabling bidirectional fluid communication between the external loop reservoir bottle 729 and the cell culture cassette 5. The external loop driving pump 722 is connected with the external loop liquid storage bottle 729 and the cell culture box 5, and drives the culture medium around the stem cell hydrogel system 62 to flow. Solenoid valve 724 is used to control the start or stop of the external circulation system (72), and the external circulation system 72 can also be controlled to pause through solenoid valve 724 so as to sample stem cells.

As shown in fig. 2A and 2B, the cell culture cassette 5 includes a cassette body 58 and a cassette cover 59 detachably connected, the inside of the cell culture cassette 5 forms an accommodation space, hollow tubes 61 are arranged in parallel in the accommodation space, and the inner circuit inlet 51 and the inner circuit outlet 52 are respectively located on two opposite cassette walls of the cell culture cassette 5 on the end side of the hollow tubes 61. The outer loop inlet 53 and the outer loop outlet 54 are respectively located on the box wall of the cell culture box 5 on the same side of the axis of the hollow tube 61.

The hollow tube 61 may be made of polysulfone, dimethylacetamide, or regenerated cellulose. The hollow tube 61 may have an outer diameter of, for example, 2.0mm to 8.0mm, an inner diameter of, for example, 0.3mm to 6.0mm, and a diameter of the medium passage hole 610 of, for example, 0.01mm to 0.5 mm. The interior of the hollow tube 61 itself may have a support frame which facilitates its maintenance of the shape of the hollow tube 61.

The box cover 59 is located on the path of the hollow tube 61 entering and exiting the cell culture box 5, the box cover 59 is provided with a main pipeline 7112 connected with the inner loop outlet 52 to connect with the circulating system 71 and a plurality of branch pipelines 7114 with one end communicated with the main pipeline 7112, and the other end of each branch pipeline 7114 corresponds to one hollow tube 61 and is communicated with the same.

A main pipeline 7111 communicated with an inlet of the inner loop and a plurality of branch pipelines 7113 with one end communicated with the main pipeline 7111 are arranged at one side of the cell culture box 5 opposite to the box cover 59, and the other end of each branch pipeline 7113 corresponds to and is communicated with one hollow pipe 61. In this way, the internal circulation system 71 is connected to each hollow tube 61 via the

main tubes

7111 and 7112 and the

branch tubes

7113 and 7114 in the cell culture cassette 5, and the medium flows between the

main tubes

7111 and 7112 and the

branch tubes

7113 and 7114, so that the medium is dispersed into the branch tubes 7113 through the main tube 7111 and flows into the hollow tubes 61, and the medium in the hollow tubes 61 flows into the main tube 7112 through the branch tubes 7114 and flows back to the internal circuit liquid storage bottle 719. During this process, nutrients and oxygen in the culture medium diffuse through the culture medium of the hollow tube 61 through the holes 610 to the outside of the hollow tube 61, providing nutrients and oxygen to the stem cells outside the hollow tube 61, mimicking the in vivo aortic blood vessels.

In one embodiment of the present invention, joints 60 may be provided at both ends of the cell culture unit 6, and the

branch lines

7113 and 7114 may be butted against the hollow tube 61 via the joints 60.

In the present invention, the external circulation system 72 is connected between the external loop inlet 53 and the external loop outlet 54 of the cell culture box 5, so that the culture medium around the stem cell hydrogel system 62 outside the hollow tube 61 can flow circularly, and the diffusion of the nutrients and oxygen in the culture medium in the hollow tube is accelerated, thereby increasing the survival rate of the cells and improving the supply of nutrients.

In one embodiment of the present invention, the biomaterial containing stem cells may be, for example, a stem cell hydrogel system 62, as shown in fig. 5, the stem cell hydrogel system 62 may include cells 622 and hydrogel 621, the cells 622 are distributed in the hydrogel 621, and the hydrogel 621 may provide a three-dimensional growth environment simulating extracellular matrix for the cells 622.

The hydrogel 621 may, for example, be a homogeneous mixed solution of multiple biological materials, which may include one or more of the following: for example, a 5% to 20% strength gelatin solution, a 0.2% to 6% sodium alginate solution, a 0.1% to 6% fibrinogen solution, a 0.1% to 10% chitosan solution, a 0.1% to 10% hyaluronic acid solution. Or other biomaterials with good biocompatibility.

The hydrogel 621 may also be Matrigel, which is in a solution state at a low temperature and forms a gel state at 37 ℃ in a culture environment after being extruded from the cell printing head 8.

It is noted that the concentrations referred to herein are percentages of the mass of the solute in grams to a predetermined volume of the solvent, which is 100 ml.

The stem cell hydrogel system 62 may further comprise hollow fibers 623, and the hollow fibers 623 may have diffusion holes to simulate the siphoning effect of capillaries, so as to supply sufficient nutrients and oxygen to the cells 622 inside the stem cell hydrogel system 62 and improve the survival rate of the cells 622. The hollow fibers 623 may also function as a scaffold for the hydrogel 621, facilitating the formation of the stem cell hydrogel system 62.

The cells 622 inside the stem cell hydrogel system 62 may be stem cells, specifically, embryonic stem cells, bone marrow-filled mesenchymal stem cells, adipose-filled mesenchymal stem cells, and the like, and the density thereof may be 1 × 10 ⁶ -0.1×10 ⁶ One per ml.

The stem cell hydrogel system 62 can provide a three-dimensional matrix environment for the cells 622, and various physiological characteristics of the cells 622 in the three-dimensional matrix environment are different from those in the two-dimensional environment.

In addition, the cells 622 grow in the stem cell hydrogel system 62, nutrition and oxygen are gradually diffused to the cells 622 through the hollow tubes 61 of the culture unit and the hollow fibers 623 in the stem cell hydrogel system 62, the hollow tubes 61 simulate an aorta in a body, the hollow fibers 623 simulate capillary vessels, the bionic effect is achieved, and the proliferation efficiency of the cells 622 is obviously improved.

Of course, as shown in fig. 6, the stem cell hydrogel system 62 may not have hollow fibers 623 therein.

The concentration of the hydrogel 621 can be adjusted, for example, decreased, and the porosity inside the hydrogel 621 is obviously increased after the concentration is decreased, which is beneficial to the migration, growth and wide connection of the cells 622.

The hollow fiber 623 can be manufactured based on the coaxial electrospinning principle, two solvents with different volatilization rates are used as electrospinning solutions, for example, polylactic acid (PLA) is dissolved in hexafluoroisopropanol and chloroform, the electrospinning solutions are introduced into outer layer channels (inner layer channels are left empty) of coaxial electrospinning needles, and the hollow fiber 623 is formed by spraying under the action of high-voltage static electricity, and because the two solvents have different volatilization rates, diffusion holes on the hollow fiber 623 are formed after the solvent which volatilizes quickly volatilizes.

When the stem cell expansion culture system provided by the present disclosure is used to culture (e.g., expand) cells, the following steps may be performed:

(1) the stem cell hydrogel system 62 was printed on one hollow tube 61:

uniformly mixing the cell 622 and the hydrogel 621, and placing the mixture into a cell printing head 8;

the mechanical arm 2 grabs and takes out the hollow tube 61 from the cell culture box 5;

when the hollow tube 61 is completely located in the printing space outside the cell culture box 5, the mechanical arm 2 drives the hollow tube 61 to rotate, and at the same time, the linear motion module 3 drives the cell printing head 8 to move (e.g., move at a constant speed) along the rotation axis of the hollow tube 61, so as to print the stem cell hydrogel system 62 on the hollow tube 61 according to, for example, a 3D cell printing process;

after the stem cell hydrogel system 62 is cured and formed, the robotic arm 2 places the hollow tube 61 printed with the stem cell hydrogel system 62 into the cell culture box 5.

(2) The stem cell hydrogel system 62 was printed on the other hollow tubes 61 in the same cell culture cassette 5:

the above-described procedure is performed on the other hollow tubes 61 in the same cell culture cassette 5.

(3) Printing to form the hollow tube 61 inside the other cell culture cassette 5:

the above-described steps are performed one by one on the hollow tubes 61 in the other cell culture cassettes 5.

(4) Starting a culture medium circulating system 7:

the internal circulation system 71 of the medium circulation system 7 circulates the medium around the stem cell hydrogel system 62 outside the hollow tube 61 by introducing fresh medium into the cell culture cassette 5 and circulating the medium inside the hollow tube 61.

Therefore, the stem cell amplification culture system provided by the disclosure can be used for fully automatically culturing (for example, amplifying) stem cells, so that the operations of manual liquid exchange and the like are omitted, and time and labor are saved.

It will be appreciated that the stem cell hydrogel system 62 may also be printed onto the hollow tube 61 by:

firstly, when the robot 2 takes the hollow tube 61 out of the cell culture box 5, the linear motion module 3 drives the cell printing head 8 to move along the axis of the hollow tube 61, and the stem cell hydrogel system 62 is combined with the hollow tube 61 in a strip body extending along the hollow tube 61; or after forming one strip, the robotic arm 2 drives the hollow tube 61 to rotate at an angle so that the stem cell hydrogel system 62 forms another strip juxtaposed to the previous strip.

Secondly, when the mechanical arm 2 takes the hollow tube 61 out of the cell culture box 5, the mechanical arm 2 drives the hollow tube 61 to rotate around the rotation axis, the cell printing head 8 is kept still, and the cell printing head 8 prints out an annular body at a specific position on the axis of the hollow tube 6; alternatively, the linear motion module 3 drives the cell print head 8 to move along the axis of the hollow tube 61, so that the cell print head 8 prints another annular body juxtaposed to the previous annular body at another specific position on the axis of the hollow tube 6.

The robotic arm 2 and the linear motion module 3 may be operated in a variety of ways to cooperate, so that the stem cell hydrogel system 62 is combined with the hollow tube 61 in different shapes.

The present disclosure integrates the cell printing head 8 and the cell culture unit driving device into the stem cell expansion culture system, thereby printing the stem cell hydrogel system 62 onto the hollow tube 61, thereby making the stem cell have a three-dimensional growth environment, thereby making the stem cell have a sufficient growth space and good growth characteristics, reducing the frequency of cell generation operation, reducing the risk of contamination, improving the yield of the stem cell, and being capable of culturing the stem cell with high quality and high efficiency.

The method integrates cell culture and cell printing, can be conveniently applied to industrial production, and has better safety and higher amplification efficiency.

The cell printing process employed in the present disclosure may be based on temperature phase change.

When the hydrogel 621 is the above-mentioned mixed solution, the printing conditions may be: the cylinder temperature of the cell printing head 8 was 15 ℃ to 35 ℃, the temperature of the cell culture case 5 including the hollow tube 61 was 5 ℃, and after adding a calcium chloride solution (1% to 6% concentration) to crosslink and solidify it, the temperature of the cell culture case 5 was returned to 37 ℃.

When the hydrogel 621 is Matrigel as described above, the printing conditions may be: the cartridge temperature of the cell printing head 8 was 2 ℃ to 6 ℃ and the temperature of the cell culture cassette 5 including the hollow tube 61 was 37 ℃.

The environmental humidity of the cultured cells may be a relatively high relative saturation humidity of 90% to 96%.

It should be understood that the above embodiments are only exemplary and are not intended to limit the present invention. Various modifications and alterations of the above-described embodiments may be made by those skilled in the art in light of the teachings of the present invention without departing from the scope of the invention.

(1) And the cabinet 4 is also mounted with electrical equipment for controlling temperature and the like.

(2) The stem cell expansion culture system and the stem cell expansion culture method provided by the present disclosure, and the cell culture unit 6 can also be used for culturing general cells other than stem cells.

(3) The stem cell expansion culture system, the stem cell expansion culture method and the cell culture unit provided by the present disclosure can be used for, but are not limited to, expansion of cells.

(4) The internal circulation pipeline 711 and the external circulation pipeline 721 can adopt medical silicone tubes.

(5) The cell culture unit 6 can be driven by other driving devices (cell culture unit driving devices) which are common in the prior art besides the mechanical arm 2, and the cell print head 8 can be driven by other driving devices (cell culture unit driving devices) which are common in the prior art besides the linear motion module.

Claims

1. A stem cell expansion culture system, comprising:

a cell culture cassette (5), the cell culture cassette (5) being configured to accommodate a cell culture unit (6), the cell culture unit (6) being formed as a hollow tube (61), a wall of the hollow tube (61) having a medium passage hole (610), an exterior of the hollow tube (61) being configured to be coupled to a stem cell-containing hydrogel, the hollow tube (61) being configured to allow a medium therein to permeate the stem cell-containing hydrogel outside the hollow tube (61) through the medium passage hole (610);

a cell printing head (8), the cell printing head (8) being configured to print the hydrogel containing stem cells to the cell culture unit (6);

cell culture unit drive means for at least grabbing the cell culture unit (6) from the cell culture cassette (5) and/or placing the cell culture unit (6) into the cell culture cassette (5) for culturing; and

an internal circulation system (71) and an external circulation system (72).

2. The stem cell expansion and culture system according to claim 1, further comprising a cell print head driving device for driving the cell print head (8) to move so that the cell print head (8) prints the hydrogel containing the stem cells to different positions of the cell culture unit (6).

3. The stem cell expansion culture system according to claim 2,

the cell culture unit driving device comprises a mechanical arm (2), the mechanical arm (2) can grab the cell culture unit (6) from the cell culture box (5) and/or put the cell culture unit (6) into the cell culture box (5), and the mechanical arm (2) can also drive the cell culture unit (6) to rotate,

the cell printing head driving device comprises a linear motion module (3), the cell printing head (8) is arranged on the linear motion module (3), and the linear motion module (3) can drive the cell printing head (8) to move along the rotation axis of the cell culture unit (6).

4. The stem cell expansion culture system according to claim 1, wherein the internal circulation system (71) is in communication with the cell culture cassette (5) for introducing fresh medium into the interior of the hollow tube (61) to circulate the medium in the interior of the hollow tube (61).

5. The stem cell expansion culture system according to claim 4, wherein the internal circulation system (71) comprises:

an inner loop reservoir bottle (719), the inner loop reservoir bottle (719) for storing fresh medium;

an internal loop drive pump (715), the internal loop drive pump (715) being configured to drive the media to flow in the internal circulation system (71) and continuously through the cell culture cassette (5);

a gas exchanger (716), the gas exchanger (716) configured to exchange oxygen and carbon dioxide for the media flowing into the cell culture cassette (5);

a monitoring assembly (714, 713) for monitoring a predetermined parameter of the culture medium flowing from an inner loop reservoir bottle (719) into the cell culture cassette (5),

wherein, in the internal circulation system (71), the internal circuit liquid storage bottle (719), the internal circuit driving pump (715), the gas exchanger (716) and the monitoring assembly (714, 713) are connected in series.

6. The stem cell expansion culture system according to claim 1, wherein the external circulation system (72) is in communication with the cell culture cassette (5) for circulating a culture medium around the stem cell-containing hydrogel outside the hollow tube (61).

7. The stem cell expansion culture system according to claim 6, wherein the external circulation system (72) comprises:

an external circuit driving pump (722), wherein the external circuit driving pump (722) is used for driving the culture medium around the hydrogel containing the stem cells to flow out of the cell culture box (5);

an external circuit reservoir bottle (729) for storing a culture medium around the hydrogel containing stem cells;

a solenoid valve (724) for controlling the start or stop of the external circulation system (72),

wherein, in the external circulation system (72), the external circuit liquid storage bottle (729), the external circuit driving pump (722) and the solenoid valve (724) are connected in series.

8. The stem cell expanding and culturing system according to any one of claims 4 to 7, wherein the cell culturing box (5) comprises a detachably connected box body (58) and a box cover (59), the box body (58) and the box cover (59) enclose a containing space for containing the hollow tube (61), the box cover (59) is positioned on the passage way of the hollow tube (61) to and from the cell culturing box (5), and the box cover (59) and the containing space are internally provided with branch pipelines (7114) which are butted with the pipelines of the internal circulation system (71) so that the internal circulation system (71) can be communicated with the inside of the hollow tube (61) when the box cover (59) is connected with the box body (58).

9. The stem cell expansion and culture system according to claim 6, further comprising a box body (1), wherein a cabinet (4) is installed inside the box body (1), and a printing space is formed in the box body (1) outside the cabinet (4);

the cabinet (4) comprises a cell culture cabin (50), the cell culture boxes (5) are arranged in parallel in the height direction in the cell culture cabin (50), and the cell printing head (8) and the cell culture unit driving device are positioned in the printing space;

the cell culture unit driving device is capable of grasping the cell culture unit (6) from the cell culture cassette (5) to the printing space to cause a cell printing head (8) to print the hydrogel containing stem cells on the cell culture unit (6).

10. A stem cell expansion culture method for culturing cells (622) in the cell culture unit (6) using the stem cell expansion culture system according to any one of claims 1 to 9, the stem cell expansion culture method comprising the steps of:

a cell culture unit (6) obtaining step of obtaining a cell culture unit (6) from the cell culture cassette (5) by the cell culture unit driving means;

a stem cell-containing hydrogel forming step of printing the stem cell-containing hydrogel on the cell culture unit (6) by the cell printing head (8);

a cell culture unit (6) entry step, wherein the cell culture unit driving device places the cell culture unit (6) into the cell culture box (5);

a cell culture step of culturing the stem cells in the cell culture cassette (5).

11. The stem cell expansion culture method according to claim 10, wherein in the stem cell-containing hydrogel formation step, the cell culture unit (6) is driven to rotate by the cell culture unit driving device, and when the cell culture unit (6) rotates, the cell print head (8) prints the stem cell-containing hydrogel on the cell culture unit (6) and moves the cell print head (8) along the rotation axis of the cell culture unit (6).

12. The method for stem cell expansion culture according to claim 10, wherein the hydrogel containing stem cells comprises hollow fibers (623), the hollow fibers (623) have diffusion holes, and the hollow fibers (623) are uniformly mixed with the cells (622) in the hydrogel containing stem cells.