CN110903976A

CN110903976A - A orifice plate device for organoid spheroid is cultivateed

Info

Publication number: CN110903976A
Application number: CN201911327604.6A
Authority: CN
Inventors: 罗国安; 马立东; 王乙同; 王义明
Original assignee: Jiangsu Xin'anjia Medical Technology Co Ltd
Current assignee: Zhejiang Hongrui Medical Technology Co ltd
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2020-03-24
Anticipated expiration: 2039-12-20
Also published as: CN110903976B

Abstract

The invention relates to the technical field of biological cell culture, further relates to the field of organoid culture, and particularly relates to a pore plate device for organoid sphere culture. The invention can realize the safe liquid changing operation of the organoid cytosphere, shorten the balling time of the cytosphere, avoid forming the satellite cytosphere, and is convenient for the later tissue slice analysis of the organoid cytosphere by the excellent microscopic imaging effect and the compatible tissue slice technology, thereby being hopeful to provide a new research tool for constructing a three-dimensional organoid cytosphere model.

Description

A orifice plate device for organoid spheroid is cultivateed

Technical Field

The invention relates to the technical field of biological cell culture, further relates to the field of organoid culture, and in particular relates to a pore plate device for organoid sphere culture.

Background

Organoids (Organoids) are multicellular three-dimensional structures grown in vitro with the microdissection characteristics of the source organ. The organoid tool cells are mainly tissue-specific pluripotent stem cells or precursor cells. To date, organoids from a variety of organs have been marketed, including brain, intestinal tract, stomach, tongue, thyroid, thymus, testis, liver, pancreas, skin, lung, kidney, heart, and retina. In addition to organoids derived from healthy tissue, organoids of a number of disease models (including tumor models) are also emerging. The main features of organoids include directed differentiation based on self-organization and spatial limitation of cell classes, similar to developmental processes in vivo. They contain a variety of organ-specific cells that are spatially organized, arranged, and similar to the organ of origin. Therefore, organoids can be used not only for drug toxicity detection, drug efficacy evaluation, new drug screening, etc., for establishing disease models to study genetic diseases, infectious diseases and cancers, but also for precise medical treatment, studying tissue and organ development, and for transplantation and repair of tissue and organs.

At present, organoid culture techniques are mainly classified into three-dimensional culture techniques relying on an exogenous scaffold and three-dimensional culture techniques not relying on an exogenous scaffold. The three-dimensional culture technology relying on the exogenous scaffold mainly comprises the steps of constructing a porous structure similar to a scaffold for cell attachment and growth in a three-dimensional space, enabling cells to attach to the scaffold for three-dimensional growth, migration and aggregation, and enabling main scaffold materials to comprise extracellular matrixes, various hydrogels and the like. However, space occupation of the scaffold affects the formation of a regular and dense organoid three-dimensional structure by cells, affects the growth and differentiation of the cells, and has a small volume for culturing the organoid, so that the period of the process for forming the organoid three-dimensional structure is overlong, and in addition, the scaffold material of animal origin or human origin has a certain pathogenic risk, and the material may have batch difference. In addition, the three-dimensional culture technology relying on the exogenous scaffold mostly has the problems of complex operation process, inconvenient subsequent detection operation, high cost and the like, and has a larger gap from an ideal organoid culture model. In order to make up for the deficiency of the three-dimensional culture technique relying on the exogenous scaffold, the three-dimensional culture technique not relying on the exogenous scaffold has been gradually emphasized in recent years, and the technique mainly comprises

The cells suspended in the culture medium are gathered to a certain density by a physical method to form compact cell-cell connection, so that the cells are self-assembled into an organoid three-dimensional structure independent of an exogenous scaffold structure. The current technical methods applied to the market mainly include a suspension method, a pendant drop method and a low-adhesion surface method. Among them, the suspension culture method can prepare a large number of organoid cell spheres, but the obtained cell spheres are different in size, and the prepared organoid cell spheres lack controllability and are greatly limited in application. The hanging drop method can well control the distribution and the size of cell balls, but the commercial product has a complex structure and is complex to operate, the transfer process needs to be careful and careful to prevent hanging drops from falling off, and the hanging drop method cannot be prepared in large quantity. The low-adhesion surface method mainly utilizes U-shaped and V-shaped pore plates modified by the low-adhesion surface to promote cells to gather at the bottom of the concave hole to form organoid cell balls, and is adopted by more researchers due to the relative simple structure and convenient operation. However, the current U-and V-well plates for organoid cell sphere culture still have a number of serious drawbacks: firstly, the side wall of a cell culture hole in a current commercial U-shaped and V-shaped pore plate and the center of the concave bottom are both in a smooth arc transition structure, when a culture solution is replaced or a liquid medicine is added, the center of the concave bottom is easily impacted by high-speed fluid due to the flow guide effect of the smooth arc transition structure, wherein the cultured organoid cell balls are often blown out of a specified culture area, or cells on the outer surface of the cell balls are damaged or even necrotized in different degrees due to extremely high fluid shearing force; in addition, the curvature of the semicircular concave hole structure at the bottom of the culture hole of the U-shaped pore plate is too small, so that the formation time of compact organoid cell balls by inoculated cells is too long, and irregular organoid cell balls (for example, a plurality of satellite cell balls are easily formed), although the curvature of the semicircular concave hole structure at the bottom of the culture hole is improved by the V-shaped pore plate, the cell aggregation process is too long compared with the culture of the organoid cell balls with the common diameter of 50-500 μm, and the external contour of the bottom of the culture hole of the current commercial V-shaped pore plate is designed by adopting a radian, so that light is seriously refracted at the interface of external air and the external contour at the bottom of the culture hole, and the micro-imaging effect of the organoid cell balls in the V-shaped pore plate is not good; finally, the tissue slide technique is a main method for observing the physiological and pathological morphological changes of organoid cytospheres, the current U-shaped and V-shaped pore plates are not compatible with the technical requirement, the cell culture pore structures are all integrally designed, so that a sample is not damaged violently after being embedded and is very difficult to take out for subsequent slicing operation, the cytospheres are usually required to be taken out firstly and then embedded and subjected to subsequent slicing operation in order to carry out slide analysis on the cytospheres, the embedding position of the taken-out cytospheres is not fixed, the positioning is difficult during slicing, and the detection operation of researchers on the organoid cytospheres is greatly limited and the large-scale high-throughput detection is difficult to realize.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a pore plate device for organoid sphere culture, which can realize the safe liquid changing operation of organoid cell spheres, shorten the sphere forming time of the cell spheres, avoid the formation of satellite cell spheres, have excellent microscopic imaging effect and be compatible with tissue slice technology so as to facilitate the later tissue slice analysis of organoid cell spheres by detachably installing a plurality of culture pore units with optimized structures.

In order to achieve the above object, the present invention provides a well plate device for organoid sphere culture, comprising a well plate cover, a plurality of culture well units and a well plate base, wherein the culture well units are detachably mounted in the well plate base, and the culture well units comprise a positioning buffer ring, a first flow guide cavity and a second organoid sphere culture cavity from top to bottom.

Further, a plurality of holes are formed in the hole plate base, and the culture hole unit is detachably mounted in the holes of the hole plate base.

Further, the culture well unit is made of a high molecular organic silicon compound or a hydrogel material.

Further, the width of the positioning buffer ring is 0.2-1 mm.

Further, the first flow guide cavity is in an inverted cone shape, and the included angle of the center section of the first flow guide cavity is 60-150 degrees.

Furthermore, the second organoid sphere culture chamber is of an inverted cone shape, and the included angle at the central section of the second organoid sphere culture chamber is 30-120 degrees.

Furthermore, the bottom of the inverted cone shape of the second organoid sphere culture chamber is hemispherical, and the radius of the bottom is 0.2-1.5 mm.

Further, the distance between the bottom of the second organoid sphere culture chamber and the lower surface of the culture well unit is 0.1-1 mm.

Furthermore, the lower opening of the first flow guide cavity is matched with the upper opening of the second organ-like sphere culture cavity to form an integrated special-shaped inverted cone structure.

Further, when the culture well unit is mounted in the well of the well plate base, the bottom contact surface and the outer bottom surface of the culture well unit, which are in contact with the well of the well plate base, are smooth, transparent planes.

The invention provides a technical scheme of a pore plate device for organoid sphere culture, which can better solve the defects of the existing related commercial technology. The invention has the beneficial effects that: compared with the existing commercial organoid cell sphere preparation technology, the method mainly has the following advantages:

(1) the orifice plate device can realize the accurate positioning of the pipette tip through the positioning buffer ring structure in the culture orifice unit, can also be used for buffering high-speed liquid impact and disturbance generated in the liquid changing process of liquid, and avoids that organoid cell balls are blown out of a specified culture area or cells on the outer surface of the cell balls are damaged or even necrotized to different degrees due to extremely high fluid shearing force.

(2) According to the orifice plate device, the double-inverted-cone-shaped structure of the first flow guide cavity and the second organ-like sphere culture cavity is adopted, so that the larger culture hole diameter is rapidly converged into the smaller diameter in a limited space, a compatible standardized 96 or 384 orifice plate structure is realized, the rapid formation of organ-like cell spheres is realized, and the generation of satellite cell spheres is avoided.

(3) According to the orifice plate device, the contact surface and the outer bottom surface of the bottom of the culture orifice unit and the bottom hole of the orifice plate base are smooth and transparent planes, so that the refraction of light generated at the interface between the external air and the external contour of the bottom of the culture orifice is reduced to the maximum extent, and the optimal microscopic imaging effect of the organoid cell balls is realized.

(4) The orifice plate device is convenient for in-situ embedding treatment of organoid cytospheres by arranging the culture orifice unit which is detachably embedded, and simultaneously, the embedded cytospheres are randomly taken out and quickly positioned and sliced without damaging the orifice plate device, so that the difficulty of slice detection operation of researchers on the organoid cytospheres is greatly reduced, and large-scale high-throughput detection is easily realized.

Drawings

FIG. 1 is a schematic diagram of the overall construction of the orifice plate assembly of the present invention;

FIG. 2 is a central sectional view of a culture well unit in the well plate device according to the present invention;

FIG. 3A is a central sectional view of the well plate device of the present invention after the culture well unit and the well plate base are assembled;

FIG. 3B is a top view of the well plate device of the present invention after the culture well unit and the well plate base are assembled;

FIG. 4 is a schematic view showing a manner of taking out a culture well unit from the well plate apparatus according to the present invention;

FIG. 5 is a comparison of cell pellet status in different organoid pellet culture well plates (3 days of culture);

FIG. 6 is a comparison of the balling times of cell spheres in different organoid sphere culture well plates;

FIG. 7 is a graph showing the comparison of cell pellet loss rates in different organoid pellet culture well plates after 5 fluid changes;

FIG. 8 is a drawing showing a structure of a slice of organoid cell balls cultured in the well plate device according to the present invention.

In the figure:

1. the device comprises a hole plate cover, 2 culture hole units, 3 hole plate bases, 2-1 positioning buffer rings, 2-2 first flow guide cavities, 2-3 second organ ball culture cavities, 3-1 bottom contact surfaces and 3-2 outer bottom surfaces.

Detailed Description

The present invention will be described in further detail with reference to specific embodiments in order to make the technical field better understand the scheme of the present invention.

Referring to fig. 1, the well plate device for organoid sphere culture of the present invention comprises a well plate cover 1, a plurality of culture well units 2, and a well plate base 3. The orifice plate base 3 is provided with a plurality of holes, and the culture hole unit 2 is detachably mounted in the holes of the orifice plate base 3.

The well plate base 3 may be a 96-well plate or a 384-well plate compatible with a commercially standard format.

The culture well unit 2 is made of a transparent, nontoxic and elastic polymer organic silicon compound or hydrogel or other materials.

According to the attached figure 2, the culture well unit 2 comprises a positioning buffer ring 2-1, a first diversion cavity 2-2 and a second organ-like sphere culture cavity 2-3. The positioning buffer ring 2-1 is located on the top of the culture well unit 2. The positioning buffer ring 2-1 can realize the accurate positioning of the pipette tip and also can buffer the high-speed liquid impact and disturbance generated in the liquid changing process of the liquid. The first flow guide cavity 2-2 is positioned at the central shaft of the culture hole unit, the cross section of the first flow guide cavity is in an inverted cone shape with openings at the upper and lower parts, and the upper opening of the first flow guide cavity 2-2 is positioned at the upper surface of the culture hole unit 2. The second organoid sphere culture chamber 2-3 is located below the first flow guide chamber 2-2, the cross section of the second organoid sphere culture chamber is in an inverted cone shape with an opening at the upper part, the lower surface of the inverted cone shape is in a hemispherical shape, the upper opening of the second organoid sphere culture chamber 2-3 is connected with the lower opening of the first flow guide chamber 2-2, and the two openings are in the same shape. Through the double-inverted-cone structure of the first flow guide cavity 2-2 and the second organoid sphere culture cavity 2-3, the diameters of larger culture holes can be rapidly converged into smaller diameters in a limited space, so that organoid cell spheres can be rapidly formed, and satellite cell spheres are prevented from being generated.

The width W of the positioning buffer ring 2-1 of the culture hole unit 2 is 0.2-1mm, the included angle theta at the central section of the inverted cone shape of the first diversion cavity 2-2 of the culture hole unit 2 is 60-150 degrees, the included angle α at the central section of the inverted cone shape of the second organoid sphere culture cavity 2-3 of the culture hole unit 2 is 30-120 degrees, the radius R of the bottom hemisphere is 0.2-1.5mm, and the distance H between the bottom of the second organoid sphere culture cavity 2-3 and the lower surface of the culture hole unit 2 is 0.1-1 mm.

Referring to FIGS. 3A-B, the culture well unit 2 is detachably mountable in the well plate base 3, and when the culture well unit 2 is mounted in the well plate base 3, the culture well unit 2 is mounted in the bottom of the well in the well plate base 3 with the side surface contacting the inner side surface of the well; and the contact surface 3-1 of the bottom of the culture hole unit 2 and the hole in the hole plate base 3 and the outer bottom surface 3-2 are smooth and transparent planes. The smooth and transparent plane can reduce the refraction of light at the interface between the external air and the external contour at the bottom of the culture hole to the maximum extent, and the optimal microscopic imaging effect of the organoid cytosphere is realized.

Through cultivateing detachable construction between hole unit 2 and the orifice plate base 3, can be convenient for organoid cell ball's normal position embedding processing, take out the cell ball through the embedding wantonly and carry out the location section to the cell ball fast under not destroying the orifice plate device simultaneously, greatly reduce the researcher to organoid cell ball's section detection operation degree of difficulty and realize extensive high flux easily and detect.

The following examples demonstrate the advantages of the well plate device for organoid sphere culture of the present invention over the existing commercial organoid sphere culture well plates.

Example 1

Organoid cell sphere culture experiments: culturing human liver cancer cell HepG2 in DMEM high-sugar medium (containing 10% fetal calf serum and double antibiotics: penicillin content is 100U/ml and streptomycin content is 100 μ g/ml), culturing in a cell culture box with temperature of 37 ℃ and 5% CO2, periodically observing cell growth state, digesting with pancreatin every 2-3 days and subculturing; subsequently, no more than 10 generations of HepG2 cells were digested and resuspended in cell culture medium, and seeded into commercial organoid sphere culture well plates (i.e., U-well and V-well plates) and the well plate device of the present invention at a density of 1000/well, respectively, and the state of the cells was observed every 2 hours and the time for dense cell spheres to form in each well plate was recorded; and after 3 days, microscopic photographs were taken of the state of the cell balls in 3 kinds of well plates. As shown in fig. 5, the curvature of the semicircular concave hole structure at the bottom of the commercial U-shaped organoid sphere culture well plate is too small, which results in poor cell balling effect, and a large amount of satellite cell spheres with different sizes are formed in the culture well and affect the comparative analysis of cell sphere shapes in different wells; the V-shaped organoid sphere culture well plate improves the cell balling effect, but the external contour of the bottom of the culture well is designed by adopting a radian, so that light is seriously refracted at the interface between external air and the external contour of the bottom of the culture well, and the micro-imaging effect of organoid cell spheres in the V-shaped well plate is poor and the micro-focusing is difficult; the liver cancer organoid cell balls cultured in the orifice plate device can be seen with clear edge profiles under the condition of ensuring better balling effect, thereby achieving good microscopic imaging effect. In addition, as shown in fig. 6, the well plate device of the present invention has a significantly lower culture organoid cell sphere balling time than commercial U-well and V-well plates by providing the culture well unit (2) with an optimized structure. This example further illustrates that the well plate device of the present invention can efficiently culture organoid cell balls and greatly reduce the time cost of researchers.

Example 2

Organoid cytosphere exchange experiment: taking HepG2 cells of not more than 10 generations for digestion and re-suspending in a cell culture medium, respectively inoculating the cells into commercial organoid sphere culture pore plates (namely, U-shaped pores and V-shaped pores) and the pore plate device of the invention for continuous culture for 5 days at the density of 1000/pore, and performing liquid replacement operation on culture liquid in 3 kinds of pore plates by using a liquid transfer gun according to the conventional operation every 24 hours; when the pore plate device is subjected to liquid changing operation, the tip end of a pipette tip needs to abut against the position of a positioning buffer ring (2-1) in the culture pore unit (2); after 5 times of continuous liquid change, as shown in fig. 7, the average loss rate of cell balls in commercial U-shaped and V-shaped organoid ball culture well plates is higher than 30%, which causes serious influence on the subsequent experimental result analysis, especially the loss caused by primary cells or stem cells with difficult and precious sources, which greatly limits the large-scale clinical application of organoid cell balls and puts high requirements on the experimental operation of organoid cell ball culture personnel. The average loss of the cell balls in the orifice plate device is nearly 0 (namely no loss is achieved), the problems of serious cell ball loss and the like in a commercial organoid ball culture orifice plate are successfully solved through the culture orifice unit (2) with an optimized structure and the positioning buffer ring (2-1) arranged in the culture orifice unit, and the large-scale popularization of the organoid cell balls in practical application is facilitated.

Example 3

Organoid cell pellet tissue section experiments: HepG2 cells of not more than 10 generations were digested and resuspended in cell culture medium, and seeded into the well plate device of the present invention at a density of 1000 cells/well for 3 days of continuous culture. Then, reagents are sequentially added into the orifice plate device to carry out processes of dehydration, transparence, wax dipping, embedding and the like of the cell balls in situ, finally paraffin blocks are prepared, the culture orifice unit (2) is integrally taken out through a special taking-out device (4) with a micro barb structure (4-1), and then positioning slicing and conventional HE staining are rapidly carried out, wherein the taking-out mode of the culture orifice unit is shown in figure 4. The final experimental result, as shown in fig. 8, shows that the sliced cell sphere structure is an obvious sphere structure, the cells are concentrated and uniform, and the whole structure is complete. The embodiment shows that the pore plate device is compatible with the conventional tissue slide making technology, has simple and convenient operation, does not need to damage the structure of the pore plate device, does not need special instruments and reagents, is very worthy of being popularized in scientific research and clinic, and is beneficial to realizing large-scale high-throughput detection by means of an automatic manipulator in the later stage.

The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.

Claims

1. The utility model provides a orifice plate device for organoid spheroid is cultivateed, includes orifice plate lid (1), a plurality of cultivation hole unit (2) and orifice plate base (3), its characterized in that, cultivate hole unit (2) detachably and install in orifice plate base (3), cultivation hole unit (2) is by last location buffer ring (2-1), first water conservancy diversion chamber (2-2) and second organoid spheroid cultivation chamber (2-3) down.

2. The well plate device for organoid sphere culture according to claim 1, wherein the well plate base (3) is provided with a plurality of wells, and the culture well unit (2) is detachably installed in the well of the well plate base (3).

3. The well plate device for organoid sphere culture according to claim 1, wherein the culture well unit (2) is made of a high molecular organosilicon compound or a hydrogel material.

4. Well plate device for organoid sphere culture according to claim 1, characterized in that the width (W) of the positioning buffer ring (2-1) is 0.2-1 mm.

5. The well plate device for organoid sphere culture according to claim 1, wherein the first flow directing cavity (2-2) is of an inverted cone shape with an included angle (θ) at its central cross section of 60-150 °.

6. Well plate device for organoid sphere culture according to claim 1, characterized in that said second organoid sphere culture chamber (2-3) is of inverted conical shape with an included angle (α) at its central section of 30-120 °.

7. Well plate device for organoid sphere culture according to claim 6, wherein the inverted conical bottom of the second organoid sphere culture chamber (2-3) is hemispherical with a radius (R) of 0.2-1.5 mm.

8. A well plate device for organoid sphere culture according to claim 1, wherein the distance (H) between the bottom of the second organoid sphere culture chamber (2-3) and the lower surface of the culture well unit (2) is 0.1-1 mm.

9. The well plate device for organoid sphere culture according to claim 1, wherein the lower opening of the first flow guiding chamber (2-2) matches with the upper opening of the second organoid sphere culture chamber (2-3) to form an integral shaped inverted cone structure.

10. The well plate device for organoid sphere culture according to claim 2, wherein the bottom contact surface (3-1) and the outer bottom surface (3-2) of the culture well unit (2) that contact the well of the well plate base (3) are smooth, transparent planes when the culture well unit (2) is mounted in the well of the well plate base (3).