CN117384759B

CN117384759B - Micro-needle array-based organoid culture method

Info

Publication number: CN117384759B
Application number: CN202311653619.8A
Authority: CN
Inventors: 柳青; 张学; 王高杰; 张欣怡; 孙晔
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2023-12-05
Filing date: 2023-12-05
Publication date: 2024-03-29
Anticipated expiration: 2043-12-05
Also published as: CN117384759A

Abstract

The present invention relates to the field of culture or maintenance of undifferentiated human, animal or plant cells, such as cell lines, and specifically, to an organoid culture method based on microneedle arrays. The microneedle array-based organoid culture method of the present invention uses an organoid culture device and includes the following steps: 1) Place the organoids in the lower part for culture; 2) Install the middle part above the lower part, and add cells to In the middleware, the cells and culture medium pass through the passage formed by the microneedle array, use gravity to transfer to the organoids located at the end of the microneedle, and diffuse into the organoid tissue through the openings at the end of the microneedle. The present invention is based on microneedle arrays and uses a plug-in combination method to realize co-culture of cell tissues and organoids. It can achieve controllable range and controllable combination of cells and organoids or co-culture between different organoids in vitro. It provides a basis for subsequent research on multi-organ joint treatment and research on human development.

Description

Micro-needle array-based organoid culture method

Technical Field

The present invention relates to the field of culture or maintenance of undifferentiated human, animal or plant cells, such as cell lines, and in particular to a microneedle array based organoid culture method.

Background

In the last decade scientists have taken advantage of the ability of stem cells to self-renew and produce differentiated cells by which all tissues of developing embryos can be produced and tissue homeostasis of adults maintained. These key stem cell features have been exploited to develop "micro-organs", so-called organoids, in vitro. Organoids are 3D cultures derived from stem cells and derived from tissues, whose phenotype cell type composition, structure, and, to some extent, function of different tissues. Multiple organoid models have been developed in vivo, such as brain organoids in different areas (whole brain, forebrain, cerebellum, etc.), kidney organoids, intestine organoids, heart organoids, lung organoids, skin organoids, etc. Organoids have significant advantages in many aspects such as cancer, genetic diseases and the like, and in many aspects such as drug screening.

Traditional organoids were cultured on 6-well plates or 10cm plates, lacking the necessary interconnections. But with the development of micro 3D printing and micro engineering technology, the development of organoid chips is realized. An "organ-chip" is a micro-engineering biomimetic system comprising microfluidic channels lined by living human cells that reproduce the critical functional units of living organs to reconstruct the integrated human organ-level pathophysiology in vitro. These microdevices can be used to test the efficacy and toxicity of drugs and chemicals and create in vitro models of human diseases. Thus, they may represent a low cost alternative to traditional animal models for pharmaceutical, chemical and environmental applications. However, the realization of the whole body on the chip is reported recently.

During organoid culture, multiple organoid fusion culture is difficult to realize due to the problems of organoid central necrosis caused by insufficient supply of central nutrients and oxygen, lack of migration capability of tissue cells and the like, and thus organoids with diversified components are obtained. When the traditional chip is used for performing organoid co-culture, organoids are required to be taken out from a previous culture medium, then different organoids are mixed with cold Matrigel, and then the organoids are placed in the organoid co-culture chip, or the organoids are closely attached to cells with migration capability, and diversified organoids such as vascularized or immunocyte-containing organoids are obtained through the self migration capability of tissue cells, so that the whole operation process is tedious and low-efficiency. There is therefore a need for a method that can achieve rapid, multiple species, reliable delivery of co-cultures at the organoid level, resulting in tissue-diversified organoids.

Disclosure of Invention

The invention aims to provide a micro-needle array-based organoid culture method capable of realizing cell migration.

The invention provides a micro-needle array-based organoid culture method, which uses an organoid culture device, wherein the organoid culture device comprises a lower piece for culturing organoids and a middle piece for culturing cell tissues; the lower part is detachably connected with the middle part, and the cell tissues cultured in the middle part are communicated with the organoids in the lower part through the microneedles; the method comprises the following steps:

1) Placing the organoids in the lower part for culture;

2) The middle piece is arranged above the lower piece, cells are added into the middle piece, and the cells and the culture medium pass through a micro-needle passage, are transferred to the organoid at the tail end of the micro-needle by utilizing gravity, and are diffused into the organoid through the opening at the tail end of the micro-needle.

Further, step 3) is included in which the organoid of the lower member is mixed with the cells, and then the upper member is covered over the middle member, followed by culturing.

Further, the lower part comprises a lower part base and a lower part base platform connected above the lower part base, a culture tank with an opening at the upper end is arranged on the base platform, and at least one organoid placing hole is arranged in the culture tank; a group partition plate is arranged between two adjacent organoid placing holes, the group partition plate is connected with the bottom wall of the culture tank, and a hole is arranged between the group partition plate and the side wall of the culture tank; the culture medium feeding device is characterized in that a lower culture medium feeding groove is arranged on the base, and the lower culture medium feeding groove is positioned on two sides of the culture groove and is communicated with the culture groove through a culture medium inlet.

Further, positioning piles are arranged around the organoid placing holes; one side of the base station is provided with a positioning fillet.

Further, the well piece includes well piece base and connects the well piece base in well piece base top, the position that corresponds with the culture tank of piece down on the well piece base is equipped with well piece U-shaped liquid feeding groove, well piece U-shaped liquid feeding groove (the bottom surface is the arc, and the cell tissue of being convenient for utilizes gravity effect to sink, communicates to organoid, for example the organoid tissue through the microneedle array) in be equipped with at least one well piece cell culture room, the below of well piece cell culture room is connected with the microneedle array, well piece cell culture room is linked together with the microneedle array, and when well piece was linked together with the piece down, the lower extreme of microneedle array inserts in the organoid of cultivating on the organoid placing hole.

Further, the middle piece base is provided with an upward concave groove matched with the edge of the lower piece base, and the side, corresponding to the positioning fillet, of the groove and the lower piece base is provided with an arc inner angle.

Further, the device includes a top piece for the closure device, the top piece being removably connected to the middle piece.

Further, the upper part is a cover body connected above the middle part, and an upper cover supporting block is arranged on the inner surface of the cover body; one side of the middle part base station is a round angle, and one side of the upper part corresponding to the round angle of the middle part base station is provided with an arc inner angle.

Further, the step 1) specifically includes: 11 Input matrigel): firstly, injecting matrigel into an organoid placing hole of a lower piece by using a liquid transfer device;

12 Placement of organoids: carefully placing the organoids in organoid placement holes filled with matrigel;

13 Injection medium: organoid medium was placed into the organoid through the lower feed tank Kong Jiazhu medium.

Further, the step 2) specifically includes:

21 Positioning round corners of the middle piece and the lower piece to be aligned, and slowly pressing the middle piece into the lower piece;

22 Culturing microglial cells in medium: transferring a culture medium of microglial cells into a middle piece U-shaped liquid adding groove, and sequentially inoculating the microglial cells into a middle piece cell culture chamber by using a liquid transfer device;

23 Microglial cell-mixed culture medium is transferred to the organoid at the end of the microneedle through the microneedle channel by gravity and is diffused into the organoid through the opening at the end of the microneedle.

Further, the culturing method in the step 3) is as follows: the remaining amount of medium, if any, of the medium in the daily inspection was removed with a pipette and 4ml of medium was added again; every 3 days, 20ml of medium was changed through the lower piece of the liquid-feeding channel (only through the middle piece). The culture conditions are as follows: placed in 5% CO ₂ Culturing was performed at 37℃in an incubator.

The invention is based on micro-processing technology and micro-needle array, and can realize co-culture of cell tissue and organoid by utilizing a spliced and inserted combination mode, and can perform co-culture of cells and organoid or different organoids in vitro in a controllable range and controllable combination, so as to reproduce and research the reaction of a plurality of organs or tissues to medicines and toxins, and provide a basis for subsequent research of multi-organ combined treatment and research of human development.

Drawings

FIG. 1 is an exploded view of the organoid culture device of the invention.

Fig. 2 is a bottom view of the upper of the present invention.

Fig. 3 is a top view of the middle of the present invention.

Fig. 4 is a bottom view of the middle piece of the present invention.

Fig. 5 is a short side cross-sectional view of the middle piece of the present invention.

Fig. 6 is a perspective view of the lower piece of the present invention.

Fig. 7 is a cross-sectional view of the lower member of the present invention.

FIG. 8 is a graph showing the fluorescence detection results in example 1.

FIG. 9 is a graph (10X) showing the fluorescence detection results of the co-culture of endothelial cells and brainstorming balls in example 2.

FIG. 10 is a graph (100X) showing the fluorescence detection results of the co-culture of endothelial cells and brainstorming balls in example 2.

Reference numerals illustrate: 1. lower part, lower part base, 12, lower part base, 13, culture tank, 14, organoid placement hole, 15, positioning pile, 16, group baffle, 17, lower part culture medium feeding tank, 18, culture medium inlet, 19, positioning fillet, 2, middle part, 21, middle part base, 22, middle part base, 23, middle part U-shaped feeding tank, 24, middle part cell culture chamber, 25, microneedle array, 26, through hole, 27, groove, 3, upper part, 31, cover, 32, upper cover support block.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

As shown in fig. 1 to 7, the present invention provides an organoid culture device comprising a lower member 1 for culturing organoids, a middle member 2 for culturing cellular tissue, and an upper member 3 for closing the device; the lower part 1 is detachably connected with the middle part 2, and the cell tissue cultured in the middle part 2 is communicated with the organoids in the lower part 1 through the microneedle array 25.

The lower part 1 comprises a lower part base 11 and a lower part base 12 connected above the lower part base 11, a culture tank 13 with an opening at the upper end is arranged on the lower part base 12, at least one organoid placing hole 14 is arranged in the culture tank 13, and positioning piles 15 are arranged around the organoid placing hole 14; a group partition plate 16 is arranged between two adjacent organoid placing holes 14, the group partition plate 16 is connected with the bottom wall of the culture tank 13, and a hole is reserved between the group partition plate 16 and the side wall of the culture tank 13 (the hole is smaller than the organoid size, such as the diameter of the organoid); the culture medium feeding grooves 17 are arranged on the base, and the culture medium feeding grooves 17 are positioned on two sides of the culture grooves and are communicated with the culture grooves through culture medium inlet openings 18.

The culture medium inlet 18 is positioned at the bottom of the liquid adding tank, and the height of the culture medium inlet 18 is slightly higher than the bottom of the culture tank, so that the communication and the complete outflow of the liquid in the liquid adding tank are ensured, and the waste is avoided.

One side of the lower piece base 11 is provided with a positioning fillet 19.

The middle part 2 comprises a middle part base 21 and a middle part base 22 connected above the middle part base 21, a middle part U-shaped liquid adding groove 23 is arranged on the middle part base 22 at a position corresponding to the culture groove 13 of the lower part, at least one middle part cell culture chamber 24 is arranged in the middle part U-shaped liquid adding groove 23 (the bottom surface is arc-shaped, so that cell tissues sink under the action of gravity and are communicated into brain-like tissues and other organs through a microneedle array), a microneedle array 25 is connected below the middle part cell culture chamber 24, and the middle part cell culture chamber 24 is communicated with the microneedle array 25; the middle piece base 22 is provided with a through hole 26 at a position corresponding to the lower piece culture medium feeding groove 17, the bottom of the middle piece base 21 is provided with an upward extending groove 27, when the middle piece 2 is connected with the lower piece 1, the lower piece base 12 is inserted into the middle piece groove 27, at this time, the through hole 26 is communicated with the lower piece culture medium feeding groove 17, the middle piece U-shaped feeding groove 23 is positioned right above the culture groove 13, and the lower end of the micro-needle array 25 is inserted into organoid tissues such as brains cultured on the organoid placing hole.

The upper part is a cover body connected above the middle part, an upper cover supporting block is arranged in the cover body, when the upper cover is covered on the middle part, the upper cover supporting block is contacted with the upper surface of the base of the middle part, and a gap for air circulation flows out between the upper cover and the middle part.

The invention also provides a method for culturing the organoid, which uses the organoid culture device and comprises the following steps:

1. placing the organoids in the lower part for culture;

2. mounting the middle piece above the lower piece, adding cells into the middle piece, transferring the cells together with the culture medium to the organoid positioned at the tail end of the microneedle through the microneedle passage by utilizing gravity, and diffusing the cells into the organoid through the opening at the tail end of the microneedle;

3. after mixing the organoids of the lower part with the cells, the upper part is covered over the middle part and then cultured.

The step 1 specifically comprises the following steps: 11 Input matrigel): first, 6ml of cold Matrigel gel (Matrigel) was injected into the organoid placement wells of the lower piece with a pipette, 2ml per well.

13 Injection medium: 30ml of organoid medium was slowly fed through the lower feed tank.

The step 2 specifically comprises the following steps: 21 The middle piece and the lower piece are aligned by positioning fillets, and the middle piece is slowly pressed down into the lower piece 1.

22 Culturing microglial cells in medium: 4ml of microglial culture medium was transferred to a medium U-shaped addition tank, and then microglial cells were sequentially inoculated into 3 cell culture chambers of the medium with a pipette.

23 Microglial cell-mixed culture medium is transferred to the organoid at the end of the microneedle through the microneedle channel by utilizing gravity and is diffused into the organoid tissue through the opening at the end of the microneedle.

In the above devices and methods, the organoid may be a brain-like organ.

The culture method in the step 3) comprises the following steps: the remaining amount of medium, if any, of the medium in the daily inspection was removed with a pipette and 4ml of medium was added again; every 3 days, 20ml of medium was changed through the lower piece of the liquid-feeding channel (only through the middle piece). The culture conditions are as follows: placed in 5% CO ₂ Culturing in an incubator at 37 ℃;

during the incubation, samples can be taken at time nodes of day 7 or even longer, as required by the experiment. During sampling, the upper part is taken off, the medium in the middle part is removed, the middle part is lifted upwards slightly, and at the moment, the brain-like organ is attached to the micro needle at the lower end of the middle part and is taken out together.

Example 1

1. Preparation of materials

1. Preparation of brain-like organs

Ipscs (human induced pluripotent stem cells) were differentiated using a brain-like differentiation kit STEMdiff Cerebral Organoid kit (StemCell Technologies, 08570) according to the procedure of STEMCELL TECHNOLOGIES functional net to obtain brain-like organs.

2. Preparation of microglial cells

Ipscs (human induced pluripotent stem cells) were differentiated into cd34+ and cd45+ hematopoietic progenitor cells using stem diff ™ Hematopoietic Kit. The hematopoietic progenitor cells were further induced to culture using a stem diff ™ Microglia kit to obtain microglial cells.

2. Organoid culture

1. Placing the brain-like organ in the lower part for culture; the method comprises the following steps:

11 Input matrigel): first, 6ml of cold Matrigel gel was injected into the organoid placement wells of the lower piece with a pipette, 2ml each.

12 Placement of brain-like organs: carefully placing the organoids in organoid placement holes filled with matrigel;

The step 2 specifically comprises the following steps: 21 Positioning fillets of the middle piece and the lower piece are aligned, and the middle piece is slowly pressed down to be placed in the lower piece.

2. Mounting the middle piece above the lower piece, adding microglial cells into the middle piece, enabling the cells to be mixed with a culture medium, transferring the culture medium to a brain-like sphere positioned at the tail end of the micro needle by utilizing gravity, and diffusing the culture medium into brain-like tissues through an opening at the tail end of the micro needle; the method comprises the following steps:

21 Positioning fillets of the middle piece and the lower piece are aligned, and the middle piece is slowly pressed down to be placed in the lower piece.

23 Microglial cell mixed culture medium is transferred to the brain-like ball at the tail end of the micro needle by utilizing gravity through a micro needle passage and is diffused into brain-like tissue through an opening at the tail end of the micro needle.

3. After the spheroid of the lower part is mixed with cells, the upper part is covered above the middle part, and then the culture is carried out, wherein the culture method comprises the following steps: the remaining amount of medium, if any, of the medium in the daily inspection was removed with a pipette and 4ml of medium was added again; every 3 days, 20ml of medium was changed through the lower piece of the liquid-feeding channel (only through the middle piece). The culture conditions are as follows: placed in 5% CO ₂ Culturing in an incubator at 37 ℃;

during the incubation, samples can be taken at time nodes of day 7 or even longer, as required by the experiment. When sampling is carried out, the upper part is taken off, the medium in the middle part is removed, the middle part is lifted upwards slightly, and at the moment, the brain-like ball is attached to the micro needle at the lower end of the middle part and is taken out together.

Immunofluorescence detection is carried out on the brain-like tissue cultured on the 7 th day, and the specific method comprises the following steps: sucking out the brain-like ball from the culture device by a 10ml pipette, and performing frozen section with the thickness of 8-10 μm; staining was then performed using IBA1 and DAPI, respectively, and on-machine observations.

The results are shown in FIG. 8. The green substance in the figure is NEUN, which is a marker of neurons in the brain-like, and represents the brain-like; in the figure, the red substance is IBA1, which is a marker of microglia and represents microglia. From the figure, a large number of microglial cells are delivered into the brain-like sphere along the upper microneedle, the fusion state of the microglial cells and the brain-like sphere is good, the deformation and apoptosis of tissues and cells are not seen under the scope, and the brain-like organ with immune cells is formed through continuous fusion.

Example 2: co-culture of endothelial cells and brain-like organs

1. Preparation of endothelial cells:

ipscs were differentiated into endothelial cells using the STEMdiff ™ Endothelial Differentiation Kit (08005, 08007) kit of stemell, and then added to the culture system in the same manner as described above for microglia. Co-culturing for 7 days. Obtaining the mixture of endothelial cells and brain-like cells.

2. The microglial cells in example 1 were replaced with the endothelial cells, and the other steps were the same, and the results are shown in fig. 9 and 10. As can be seen from fig. 9, after the 10-fold mirror is subjected to microneedle insertion co-culture, a large number of endothelial cells enter into the brain-like sphere, and after 7 days of co-culture, the endothelial cells are uniformly distributed at the center and the edge of the brain-like sphere due to the self-migration characteristic of the endothelial cells; it can be seen from fig. 10 that under 100x magnification, endothelial cells have been locally connected together to form a primary-like structure, constituting a vascularized brain-like organ.

In fig. 9 and 10, blue fluorescence stained DAPI nuclei and green stained cd31+ endothelial cells.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. A method for culturing an organoid based on a microneedle array, characterized in that an organoid culturing device is used, wherein the organoid culturing device comprises a lower part for culturing an organoid and a middle part with microneedles for culturing cell tissues; the lower part is detachably connected to the middle part, and the cell tissues cultured in the middle part are connected to the organoids in the lower part through the microneedles; the lower part comprises a lower part base and a lower part base connected above the lower part base, a culture tank with an upper end opening is provided on the base, and at least one organoid placement hole is provided in the culture tank; the lower part base also comprises a lower part culture medium adding tank, the lower part culture medium adding tank is located on both sides of the culture tank, and is connected to the culture tank through a culture medium liquid inlet; the middle part comprises a middle part base and a middle part base connected above the middle part base, a middle part U-shaped adding tank is provided at a position corresponding to the culture tank of the lower part, at least one middle part cell culture chamber is provided in the middle part U-shaped adding tank, a microneedle array is connected below the middle part cell culture chamber, and the middle part cell culture chamber is connected to the microneedle array;

The method includes the following steps:

1) Place the organoids in the lower medium for culture;

2) Install the middle piece above the lower piece. When the middle piece is connected to the lower piece, insert the lower end of the microneedle array into the cultured brain tissue on the organoid placement hole. Add cells to the middle piece and mix the cells with the culture medium. Through the microneedle channel, gravity is used to transfer to the brain-like tissue located at the end of the micro-needle, and diffuse into the brain-like tissue through the opening at the end of the micro-needle.

2. The organoid culture method based on microneedle array according to claim 1, characterized in that, a set of partitions is provided between two adjacent organoid placement holes, and the set partitions are in contact with the bottom wall of the culture tank. There is a hole between the connection and the side wall of the culture tank.

3. The microneedle array-based organoid culture method according to claim 2, characterized in that positioning piles are provided around the organoid placement hole; and a positioning fillet is provided on one side of the base.

4. The organoid culture method based on microneedle array according to claim 3, characterized in that the base of the middle part is provided with an upwardly concave groove that matches the edge of the base of the lower part, and the groove is in line with the edge of the base of the lower part. The side corresponding to the positioning fillet of the lower abutment is provided with an arc inner corner.

5. The microneedle array-based organoid culture method according to claim 1, wherein the device includes an upper part for closing the device, and the upper part is detachably connected to the middle part.

6. The organoid culture method based on microneedle array according to claim 5, characterized in that the upper part is a cover connected above the middle part, and the inner surface of the cover is provided with an upper cover support block. ; One side of the middle piece base is a rounded corner, and the side of the upper piece corresponding to the rounded corner of the middle piece base is provided with an arc inner corner.

7. The organoid culture method based on microneedle array according to any one of claims 1-6, characterized in that the step 1) is specifically:

11) Input Matrigel: First, use a pipette to inject Matrigel into the organoid placement hole of the lower part;

12) Place the organoids: Carefully place the organoids in the wells filled with matrix gel.

13) Inject the culture medium: Pass the organoid culture medium through the lower part of the filling tank, and add the culture medium to the organoid placement hole.

8. The method for culturing organoids based on microneedle arrays according to claim 7, wherein the step 2) is specifically:

21) Align the positioning fillets of the middle piece and the lower piece, and slowly press down to place the middle piece into the lower piece;

22) Culture microglia in the middleware: Transfer the culture medium of microglia to the U-shaped filling tank of the middleware, and then use a pipette to inoculate the microglia into the cell culture medium of the middleware one by one. in the room;

23) Microglia mixed with culture medium pass through the microneedle channel and use gravity to transfer to the organoids located at the end of the microneedle, and spread into the organoid tissue through the openings at the end of the microneedle.

9. The microneedle array-based organoid culture method according to any one of claims 1 to 6, characterized in that the organoids are brain organoids.