CN113278525B - Stem cell ball or tumor ball culture device and culture method - Google Patents

Stem cell ball or tumor ball culture device and culture method Download PDF

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CN113278525B
CN113278525B CN202110567624.1A CN202110567624A CN113278525B CN 113278525 B CN113278525 B CN 113278525B CN 202110567624 A CN202110567624 A CN 202110567624A CN 113278525 B CN113278525 B CN 113278525B
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cell
bottom plate
ball
tumor
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CN113278525A (en
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黄晓文
林惠超
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Shandong Youjian Biotechnology Co ltd
Qilu University of Technology
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Shandong Youjian Biotechnology Co ltd
Qilu University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells

Abstract

The invention discloses a stem cell ball or tumor ball culture device, which comprises a cell culture plate and a micropore culture chip, wherein the cell culture plate consists of a bottom plate and an upper cover, the bottom plate is provided with a plurality of bottom plate holes, and the bottom ends of the bottom plate holes are sealed; the micro-pore culture chip is provided with a plurality of growth holes; the micropore culture chip is made of high molecular polymer materials, such as PDMS, the thickness of the micropore culture chip is 0.5-7 mm, and the diameter of the growth hole is 0.5-2.0 mm; the micro-porous culture chip is positioned between the bottom plate and the upper cover. The invention also discloses a method for culturing the stem cell ball or the tumor ball by using the stem cell ball or the tumor ball culture device. The culture device and the culture method have the advantages of convenient sample adding and liquid changing, and can conveniently and quickly realize cell interaction research. The micro-porous culture chip is made of polymer, and can better form hanging drops. The stem cell ball or tumor ball obtained by the culture device has high survival rate and good balling rate.

Description

Stem cell ball or tumor ball culture device and culture method
Technical Field
The invention relates to a stem cell ball or tumor ball culture device and a culture method, and belongs to the technical field of cell culture.
Background
Stem cells have the ability to self-renew and replicate and are capable of differentiating into a variety of functional cells under certain conditions. The research of stem cells has important significance in the fields of regenerative medicine, life science, oncology, drug screening and the like. However, the proportion of stem cells in tissues is less than 3%, and how to realize the in vitro large-scale expansion of stem cells is a bottleneck restricting the development of stem cell technology on the premise of maintaining the functions of stem cells. Researchers have tried various methods to prepare stem cell spheres having a three-dimensional structure to reduce differentiation of stem cells during in vitro culture, but further improvements are needed to increase the number and function of stem cells in stem cell spheres.
The existing methods for preparing stem cell spheres or tumor spheres comprise a microgravity method, a culture dish surface modification method, a hanging drop culture method and the like.
The microgravity method is to culture cells in microgravity environment, wherein the culture plate or culture column is installed on the base with rotary motor, and the internal tissue, cell or cell mass is kept in suspension state under the dual effects of rotary tangential force and gravity. The rotation speed is adjusted as the cells or tissue grow, and after the cells form clumps, the rotation speed must be increased so that the cells do not settle and touch the bottom. Therefore, the method has the disadvantages of needing special equipment, being expensive and not beneficial to large-scale popularization.
The culture dish surface modification method is to treat the surface of a culture dish by an artificial method to make the culture dish have special performance so as to prepare a stem cell ball, and the common surface treatment method comprises the following steps: 1. coating the culture dish with extracellular matrix such as fibronectin, cohesin, collagen and the like to simulate a stem cell microenvironment and promote the formation of stem cell balls; 2. grafting hydroxyl on the surface of a culture dish; 3. culturing the cells by using a low-adhesion culture plate; 4. the hydrogel is used to coat the porous culture plate, and an inducing liquid is added in the culture process. The surface modification method adds additional operation, which not only is a complicated process, but also is difficult to recycle, and increases the cost suddenly. Meanwhile, the microgravity method and the culture dish surface modification method have loss of stem cell functions and quantity.
The hanging drop culture method was created by Harrison and Carrel in the beginning of the 20 th century and modified from Maximow in 1924 to the double-flap hanging drop culture method. Namely, after dropping cell suspension on the surface of the culture plate, inverting the culture plate, and promoting the cells to form balls under the action of gravity, thereby obtaining the stem cell balls with three-dimensional structures. The method has simple cell planting process, only needs to hang the cell suspension on the surface of the culture plate and invert, but has the defects that: 1. the space for cell growth is narrow, the gas is insufficient and the nutrient content is less; 2. the subsequent operation is difficult, such as the complex process of cell liquid exchange: the liquid changing mode of the upright culture plate easily causes the contact of the cell balls and the culture plate, so that the adherent growth of the cell balls is caused; the liquid changing mode of the inverted culture plate needs to be careful, so that the cell balls are prevented from being sucked by mistake and touching other culture liquid drops of the dense hemp by mistake; 3. in this way, it is difficult to carry out operations such as administration in a culture solution, addition of factors, etc.; 4. a great deal of manpower and time are consumed for carrying out single-hole sequential sample adding operation, if 96 holes are needed, the manual operation is carried out for 96 times, and one-time operation and automatic formation of a high-flux liquid drop array cannot be realized. 5. The droplets are independent of each other, and the research on the cell-sphere interaction cannot be realized at high flux.
Although the stem cell balls or tumor balls with three-dimensional structures can be obtained by the method, the method has the defects of less quantity, higher cost, lower efficiency and the like of the obtained stem cell balls or tumor balls, and cannot meet the requirement of large-scale production.
Disclosure of Invention
Aiming at the prior art, the invention provides a stem cell ball or tumor ball culture device and a method for culturing the stem cell ball or tumor ball by using the device in order to better culture the stem cell ball or tumor ball and research the interaction of the stem cell ball and the tumor ball. The culture device has the advantages of high efficiency, high flux, low cost, reusability, simple operation, suitability for all the existing models and the like. The culture method can form cell spheres with consistent size and uniform shape quickly, stably and in high flux, and can realize reliable culture medium exchange condition and cell interaction research (suspension drop cells form microspheres under the action of gravity, and two or more liquid drops containing the cell spheres are gathered into one liquid drop by utilizing the characteristic that a high polymer film such as a PDMS film is soft and deformable and applying external force lightly, so that the research on the cell sphere interaction influence is realized), and the culture method can be used by laboratories or companies with basic cell operation conditions.
The invention is realized by the following technical scheme:
a stem cell ball or tumor ball culture device comprises a cell culture plate and a micropore culture chip, wherein the cell culture plate consists of a bottom plate and an upper cover, the bottom plate is provided with a plurality of bottom plate holes, and the bottom ends of the bottom plate holes are sealed; the micropore culture chip is provided with a plurality of growth holes; the micropore culture chip is made of high molecular polymer materials, the thickness of the micropore culture chip is 0.5-7 mm, and the diameter of the growth hole is 0.5-2.0 mm; the micro-porous culture chip is positioned between the bottom plate and the upper cover.
Preferably, the high molecular polymer is selected from Polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polystyrene (PS).
Preferably, the growth hole is a through hole which penetrates through the growth hole from top to bottom, is narrow in the middle and wide at two ends, the diameter of the narrowest part of the through hole is 1.2-1.6 mm, and the diameter of the widest part of the through hole is 1.2-2 mm. The reason for this shape of the through-hole is: after the micropore culture chip is punched by a circular puncher, a through hole with a narrow middle, wide two ends and arc-shaped side wall is finally formed due to the soft and variable characteristics of high polymer such as PDMS; the through hole with the shape is more beneficial to the replacement of a culture medium, the addition of an additional growth factor or the addition of a medicament, the culture of the stem cell ball or the tumor ball is facilitated, and the subsequent research and the medicament screening of the stem cell ball or the tumor ball are facilitated.
Preferably, the number of the holes of the bottom plate on the bottom plate is equal to the number of the growth holes on the micro-well culture chip, and the arrangement mode is the same.
Preferably, the number of holes of the bottom plate on the bottom plate is 12, 24, 48, 96, 384 or 1536. Taking a 96-well plate as an example, the bottom plate holes are distributed in an 8 × 12 manner, and each hole is a perfect circle or a square with equal size and shape.
Preferably, the thickness of the micropore culture chip is 1-2 mm.
Further, the stem cell ball or tumor ball culture device further comprises a sample adding channel, wherein the sample adding channel comprises at least one main channel, and the main channel is provided with one or more levels of branch channels (in specific application, the branch channel of which level is selected according to actual requirements such as the number of growing holes, if the number of growing holes is small, the branch channel of one level can be adopted, and if the number of growing holes is large, the branch channel of multiple levels can be adopted); the main channel is provided with a sample inlet, and the branch channel at the last stage is provided with a plurality of sample outlets.
Preferably, the sample-adding channel comprises two main channels, and the two main channels (including the branch channels) are not communicated with each other.
Preferably, the two main channels (including the respective branch channels) are symmetrically arranged (central symmetry), and the respective three-stage branch channels are arranged at intervals.
Preferably, the sample application channel is made of material PDMS.
The culture method for culturing the stem cell balls or the tumor balls by using the culture device comprises the following steps:
(1) Placing the micropore culture chip on a bottom plate, sucking 10 mu L of single cell suspension of stem cells or tumor cells by using a pipette, and injecting the single cell suspension into a growth hole, so that the liquid automatically forms hanging drops at the lower end of the growth hole;
or: placing the micropore culture chip on a bottom plate, placing the sample adding channel on the micropore culture chip, and aligning the sample outlet with the growth hole; single cell suspensions of stem cells or tumor cells are added through the main channels (two different stem cells or tumor cell suspensions can be added through the two main channels), liquid flows into the growth hole after flowing out through the sample outlet, and a hanging drop (the volume is 10 mu L) is automatically formed at the lower end of the growth hole;
(2) Injecting water (making water atmosphere environment, preventing hanging drop evaporation) into the bottom plate hole of the bottom plate, covering the upper cover, and placing CO into the culture device 2 Culturing in a cell culture box (for example, culturing at 37 ℃ for 24-72 h), and aggregating stem cells or tumor cells in the hanging drop into a cluster under the action of gravity, so that the stem cells or the tumor cells grow into three-dimensional pellets with the diameter of 17-100 mu m;
(3) When liquid needs to be changed, a proper amount (for example, 5 mu L) of old culture medium is sucked out from the upper end of the growth hole, then a proper amount of new culture medium is supplemented, and because the cell ball is arranged at the lowest part of the hanging drop, the liquid changing mode can effectively avoid the situation that the cell ball is sucked out and discarded when the liquid is changed;
when administration or addition of growth factors is required, the procedure is as above.
Furthermore, when cell interaction research is needed, because the three-stage branch channels to which the two main channels belong are arranged at intervals, the culture solution components on the two adjacent rows of growth holes are different, and the micro-pore culture chip is repeatedly pressed to deform the micro-pore culture chip, so that the two adjacent liquid drops can be mixed into one liquid drop, the cell factors generated by the two cell balls are communicated, and the cell interaction is researched.
The culture device and the culture method have the advantages of convenient sample adding (batch sample adding can be realized), convenient liquid changing and convenient and rapid cell interaction research. The micropore culture chip is made of high polymer materials such as PDMS, has the characteristic of soft deformation, is easy to punch, is easy to deform, and can better enable adjacent hanging drops to collide and mix (if other materials such as glass are adopted, the deformation is not easy to generate, and then the collision and mixing of the adjacent liquid drops cannot be realized). As the cell culture plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, etc. can be used. The stem cell ball or tumor ball obtained by the culture device has high survival rate and good balling rate. The culture device and the culture method are suitable for culturing stem cell spheres or tumor spheres, such as mesenchymal stem cells and tumor cells. The mesenchymal stem cells comprise umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and placenta mesenchymal stem cells; the source of the fat tissue can be from the placenta or umbilical cord of a newborn after birth or from the fat tissue of an adult, and the fat tissue has wide tissue source and wide application range.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the present invention will be described in connection with the specific meaning of the term.
Drawings
FIG. 1: the structure of the cell culture plate of the stem cell ball or tumor ball culture device is shown schematically.
FIG. 2: photograph of cell culture plate.
FIG. 3: schematic cross-sectional view of the growth well.
FIG. 4: and (5) a picture of the actual hole of the bolt punched by the puncher.
FIG. 5: the structure of the sample loading channel is schematically shown.
FIG. 6: schematic diagram of mesenchymal stem cell three-dimensional sphere.
FIG. 7: schematic representation of live-dead staining (Calcein AM/PI) of mesenchymal stem cells three-dimensional spherocytes.
FIG. 8: schematic representation of adjacent hanging drops before and after mixing.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention generally and/or specifically describes the materials used in the tests, as well as the test methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like in the following examples are all conventional experimental methods, detection methods, and the like in the prior art.
Example 1 culturing of Stem cell spheres or tumor spheres Using Stem cell sphere or tumor sphere culture apparatus
The stem cell ball or tumor ball culture device comprises a cell culture plate, a micropore culture chip and a sample adding channel; the cell culture plate consists of a bottom plate and an upper cover, as shown in fig. 1 and 2, 96 bottom plate holes (namely 96-hole plates, 8 multiplied by 12 are distributed, each hole is a right circle with equal size and shape) are arranged on the bottom plate, and the bottom ends of the bottom plate holes are closed; the micro-pore culture chip is provided with 96 growth holes corresponding to the holes of the bottom plate; the micropore culture chip is made of PDMS, the thickness of the micropore culture chip is 1.5mm, and the diameter of the growth hole is 1.0mm; the micro-porous culture chip is positioned between the bottom plate and the upper cover.
The growth holes are through holes which penetrate through the growth holes from top to bottom, are narrow in the middle and wide at two ends, and as shown in fig. 3, the diameter of the narrowest part of each through hole is 1.2mm, and the diameter of the widest part of each through hole is 2.0mm. The reason for this shape of the through-hole is: after the micro-porous culture chip is punched by a circular puncher, a through hole with a narrow middle, wide two ends and arc-shaped side wall is finally formed because PDMS has the flexible and variable characteristic (a bolt hole punched by the puncher is shown in figure 4); the through hole with the shape is more beneficial to the replacement of a culture medium, the addition of an additional growth factor or the addition of a medicament, and not only is beneficial to the culture of the stem cell ball or the tumor ball, but also is beneficial to the subsequent research and medicament screening of the stem cell ball or the tumor ball.
The sample-adding channel, as shown in fig. 5, includes two main channels: a first main channel and a second main channel; each main channel is provided with three stages of branch channels: two primary branch channels are arranged on the main channel; each primary branch channel is provided with two secondary branch channels; three tertiary branch channels are arranged on the two secondary branch channels belonging to the same stage of branch channel), and six tertiary branch channels are provided. The main channel is provided with a sample inlet, and the three-stage branch channels are respectively provided with 8 sample outlets. There is no communication between the two main channels (including the respective branch channels). The two main channels (including the branch channels) are symmetrically arranged (central symmetry), and the three branch channels are arranged at intervals.
The sample loading channel is made of PDMS.
The micropore culture chip can be prepared by the following method:
(1) Mixing the PDMS prepolymer and a curing agent (medical liquid silica gel pouring sealant RTV 615) according to the weight ratio of 10;
(2) Taking an upper cover of a 96-well plate (the upper cover contains a corresponding number of hole-shaped convex structures), pouring the PDMS mixed solution into the upper cover, keeping the thickness at 1.5mm, vacuumizing to remove air bubbles, and placing the mixture in an oven at 80 ℃ to bake until the PDMS liquid becomes PDMS solid.
(3) And (4) slightly prying the edge by using tweezers, demolding and taking out the PDMS solid layer from the upper cover to obtain the PDMS film with the upper cover hole-type structure.
(4) Four edges of the PDMS film were cut off 0.5-1.5 mm each with a razor blade.
(5) In the center of each hole of the 96-hole PDMS film, a round hole puncher with the diameter of 1.0mm is used for punching the PDMS film, and the pin holes punched by the puncher are shown in FIG. 3, so that the PDMS film with 96 through holes is obtained.
The method for culturing the stem cell balls or the tumor balls by using the culture device comprises the following steps:
(1) The cells to be cultured (umbilical cord mesenchymal stem cells) of the present example were mesenchymal stem cells of P3 generation to P5 generation; digesting mesenchymal stem cells with trypsin digestion solution, adding culture medium to stop digestion, centrifuging to remove supernatant, adding culture medium to adjust cell concentration to 10 4 ～10 5 Per mL, mechanically blown to a single cell suspension. The research adopts serum-free culture solution to culture and amplify the MSCs so as to maintain the low differentiation state of the MSCs, avoid mature differentiation in the proliferation process and maintain the multidirectional differentiation capacity.
(2) Placing the micropore culture chip on a bottom plate, placing the sample adding channel on the micropore culture chip, and aligning the sample outlet with the growth hole; single cell suspension is added through the main channel (two different stem cell suspensions can be added through the two main channels), liquid flows into the growth hole after flowing out through the sample outlet, and hanging drops (with the volume of about 10 mu L) are automatically formed at the lower end of the growth hole;
(3) Injecting water (making water atmosphere environment, preventing hanging drop evaporation) into the bottom plate hole of the bottom plate, covering the upper cover, and placing CO into the culture device 2 Culturing in a cell culture box at 37 ℃ for 24-72 h, and aggregating the stem cells in the hanging drop into clusters under the action of gravity, so as to grow into three-dimensional pellets with the diameter of 100 μm, as shown in figures 6 and 7;
(4) When liquid needs to be changed, a proper amount (for example, 5 mu L) of old culture medium is sucked out from the upper end of the growth hole, then a proper amount of new culture medium is supplemented, and because the cell ball is arranged at the lowest part of the hanging drop, the liquid changing mode can effectively avoid the situation that the cell ball is sucked out and discarded when the liquid is changed;
when administration or addition of growth factors is required, the procedure is the same as above.
(5) When cell interaction research is needed, because the three-stage branch channels to which the two main channels belong are arranged at intervals, the culture solution components on two adjacent rows of growth holes are different, and the microporous culture chip is repeatedly pressed to deform (the PDMS membrane has the characteristic of soft variability), so that two adjacent liquid drops can be mixed into one liquid drop, as shown in figure 8, and further, the cell factors generated by two cell spheres are intercommunicated to research the cell sphere interaction.
The above examples are provided to enable those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Claims (4)

1. A stem cell ball or tumor ball culture device, characterized in that: the cell culture plate comprises a bottom plate and an upper cover, wherein the bottom plate is provided with a plurality of bottom plate holes, and the bottom ends of the bottom plate holes are sealed;
the micro-pore culture chip is provided with a plurality of growth holes, the growth holes are through holes which are vertically penetrated, have narrow middle parts and wide ends, and have arc-shaped side walls, so that suspension drops can be automatically formed at the lower ends of the growth holes after single cell suspension of stem cells or tumor cells is injected into the growth holes; the micropore culture chip is made of high molecular polymers and has the characteristic of softness and deformability;
the thickness of the micropore culture chip is 1-2 mm, and the diameter of the growth hole is 0.5-2.0 mm; the micropore culture chip is positioned between the bottom plate and the upper cover;
the high molecular polymer is selected from polydimethylsiloxane, polymethyl methacrylate and polystyrene;
the stem cell ball or tumor ball culture device also comprises a sample adding channel, wherein the sample adding channel comprises two main channels which are not communicated, and two different stem cell or tumor cell suspensions are added through the two main channels; the main channel is provided with a plurality of stages of branch channels; the main channel is provided with a sample inlet, and the last branch channel is provided with a plurality of sample outlets; the last branch channels of the two main channels are arranged at intervals, and the microporous culture chip is deformed by repeatedly pressing the microporous culture chip, so that two adjacent hanging drops can be collided and mixed into a liquid drop, and the cell factors generated by the two cell balls are communicated.
2. The stem cell sphere or tumor sphere culture apparatus of claim 1, wherein: the number of the holes of the bottom plate on the bottom plate is equal to the number of the growth holes on the micro-pore culture chip, and the arrangement mode is the same.
3. The stem cell sphere or tumor sphere culture apparatus of claim 1, wherein: the number of the holes of the bottom plate arranged on the bottom plate is 12, 24, 48, 96, 384 or 1536.
4. A method for culturing a stem cell pellet using the stem cell pellet or tumor pellet culture apparatus according to any one of claims 1 to 3, comprising the steps of:
(1) Placing the micropore culture chip on a bottom plate, placing the sample adding channel on the micropore culture chip, and aligning the sample outlet with the growth hole; adding single cell suspensions of two different stem cells through the two main channels, enabling the liquid to flow into the growth hole after flowing out through the sample outlet, and automatically forming hanging drops at the lower end of the growth hole;
(2) Injecting water into the bottom plate hole of the bottom plate, covering the upper cover, and putting the culture device into the CO 2 Culturing in a cell culture box, and aggregating the stem cells in the hanging drop into a cluster under the action of gravity to grow into a three-dimensional pellet with the diameter of 17-100 mu m;
(3) When the culture medium needs to be changed, sucking out the old culture medium from the upper end of the growth hole, and then supplementing a proper amount of new culture medium;
when cell interaction research is needed, the micropore culture chip is repeatedly pressed to deform the micropore culture chip, so that two adjacent hanging drops collide and are mixed into a liquid drop, and then cell factors generated by two cell spheres are communicated to research the cell sphere interaction.
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