CN213739510U - High-oxygen air vent plate device for organoid sphere culture - Google Patents

High-oxygen air vent plate device for organoid sphere culture Download PDF

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CN213739510U
CN213739510U CN202021675457.XU CN202021675457U CN213739510U CN 213739510 U CN213739510 U CN 213739510U CN 202021675457 U CN202021675457 U CN 202021675457U CN 213739510 U CN213739510 U CN 213739510U
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culture
organoid
hole
unit
cavity
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罗国安
马立东
王乙同
王义明
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Zhejiang Hongrui Medical Technology Co.,Ltd.
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罗国安
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Abstract

The utility model discloses a hyperoxia bleeder vent board device for organoid spheroid is cultivateed, including apron (1), cultivation pore unit (2) and orifice plate base (3), be equipped with cavity (30) of taking penetrating bottom outlet (31) on orifice plate base (3), cultivate pore unit (2) embedding and be in cavity (30). Through setting up penetrating bottom outlet as ventilative window, can ensure that the cell culture process has sufficient dissolved oxygen environment, solve the near hole plate bottom insufficient dissolved oxygen problem of organoid cell ball, effectively promote the rapid growth of organoid cell ball, promote cell activity, improve cell culture efficiency, avoid causing the disintegration of organoid cell ball to die because of long-term culture, be expected to provide a new research tool for constructing the three-dimensional organoid cell ball model that the oxygen demand is higher. Compare in existing culture apparatus on the market, the utility model discloses device gas mass transfer efficiency is higher, and the organoid cell ball boundary of cultivation is clear, and the compactness of cell gathering is strong, does not have the phenomenon of disintegrating.

Description

High-oxygen air vent plate device for organoid sphere culture
Technical Field
The utility model relates to a biological cell culture technical field, especially organoid cultivates the field, concretely relates to high oxygen bleeder vent board device for organoid spheroid is cultivateed.
Background
In vitro cell culture is a common and important technical means in cell biology research methods, and is also an important technical means for researching tissue engineering and drug screening. During the cell culture process, the microenvironment in which the cells are cultured plays a key role in cell growth, for example, the gas environment, especially the oxygen concentration, is essential in the process of cell metabolism. Oxygen is involved in the tricarboxylic acid cycle of cells to provide energy for cell survival, metabolism and synthesis. Some cells can obtain energy by glycolysis under the in vitro hypoxia environment, but most normal somatic cells cannot be cultured and applied on a large scale under the in vitro hypoxia environment because the normal physiological metabolic function of the cells is influenced, especially the cells in tissues with large oxygen demand, such as the cells in the tissues of lung, liver, heart, kidney and the like.
At present, organoids, as a multicellular three-dimensional structure with the microdissection characteristics of the source organ, cultivated in vitro, have outstanding advantages in simulating the main structural and functional characteristics of human tissues and organs. In the process of culturing organoid cytospheres with large oxygen demand, the cell proliferation and the close connection among cells obviously improve the requirements on oxygen mass transfer efficiency and dissolved oxygen concentration. However, in the prior art, commercial organoid cell pellet culture pore plates are mostly adopted for culturing organoid cell pellets, and the cover plates of the commercial organoid cell pellet culture pore plates are almost close to and hermetically covered on the culture plates provided with a plurality of culture pores, so that organoid cell pellets immersed in a culture solution grow in an environment where dissolved oxygen is lower than the concentration of oxygen required for cell growth, and particularly in the cell proliferation process, the rate of continuously consuming oxygen in the energy generation process of aerobic respiration of cells through mitochondria exceeds the dissolved oxygen rate of the culture solution, so that the dissolved oxygen concentration of the culture solution is lower than the oxygen concentration in the initial cell culture, the degree of oxygen deficiency of the cells is not constant, and the accuracy of experimental results is further influenced. Meanwhile, the area of the organoid cell ball close to the bottom of the culture plate is far away from the gas-liquid exchange interface and is relatively in a 'full' closed state, so that an anoxic area is easier to form. For organoid cytospheres with large oxygen demand, the hypoxia can cause slow cell growth speed, and the cells can be seriously damaged in a large range, thereby bringing serious loss to experimental research. Therefore, it is necessary to develop a high oxygen permeable orifice plate device especially for the culture of organoid cytospheres with high oxygen demand.
SUMMERY OF THE UTILITY MODEL
In order to solve the above-mentioned problem among the prior art, the utility model provides a high oxygen bleeder vent board device for organoid spheroid is cultivateed can provide good oxygen and carbon dioxide mass transfer efficiency for the growth of organoid cell ball, satisfies the required growing environment of organoid cell ball.
The technical scheme of the utility model is detailed below:
the utility model provides a high oxygen bleeder vent board device for organoid spheroid is cultivateed, includes apron (1), cultivates hole unit (2) and orifice plate base (3), be equipped with cavity (30) of taking penetrating bottom outlet (31) on orifice plate base (3), cultivate hole unit (2) embedding in cavity (30).
The number of the culture well units (2) and the corresponding cavities (30) is not limited, and the culture well units are generally arranged into a plurality of cavities and arranged in an array so as to increase the total culture amount, and for example, the number of the culture well units can be 6, 12, 24, 48, 96 and 384, or can be arbitrarily arranged according to requirements.
The device enables the organoid cytosphere cultured in the corresponding embedded culture hole unit (2) to grow and have good oxygen and carbon dioxide mass transfer efficiency by arranging the ventilation window, namely the through bottom hole (31), on the pore plate base (3).
Optionally or preferably, in the high oxygen permeable plate device for organoid sphere culture, the material of the culture hole unit (2) is an organic silicon compound. The organic silicon compound has the characteristic of high gas permeability, is non-toxic, tasteless and transparent, and is convenient for observing the cell ball.
Optionally or preferably, in the high oxygen permeable plate device for organoid sphere culture, the longitudinal interface structure of the culture well unit (2) is flat-bottom, U-shaped, V-shaped, inverted cone-shaped or micropore array. The micropore array type is that a plurality of small holes are arranged in an array.
Alternatively or preferably, in the above high oxygen permeable plate apparatus for organoid sphere cultivation, the cultivation well unit (2) is provided with a positioning buffer wall surface (211, 221, 231, 241 or 251) which is horizontal or inclined inward. The positioning and stopping of the pipette head can buffer the impact force of the fluid during the liquid feeding process.
Alternatively or preferably, in the high oxygen permeable plate apparatus for organoid sphere cultivation described above, the minimum thickness of the bottom surface of the culture well unit (2) is 0.2-2 mm.
Optionally or preferably, in the high oxygen permeable plate device for organoid sphere culture, the surface of the bottom surface of the culture hole unit (2) contacting organoid cells is in the shape of a smooth circular arc.
Optionally or preferably, in the high oxygen permeable hole plate device for organoid sphere culture, the inner side wall of the cavity (30) and the outer side wall of the culture hole unit (2) are both cylindrical, and the through bottom hole (31) is circular or regular polygon and has the same axis as the cavity (30) and the culture hole unit (2). The cylindrical cavity (30) having a through bottom hole (31) is adapted to the flat bottom type culture well unit (21), the U-shaped culture well unit (22), the V-shaped culture well unit (23), the inverted conical shaped culture well unit (24) and the micro-well array type culture well unit (25).
Optionally or preferably, in the high oxygen permeable hole plate device for organoid sphere culture, the plurality of permeable bottom holes (31) are uniformly distributed and arranged. For example, three or more than three can be arranged, and the two or more than three are evenly distributed around the center of the bottom of the cylindrical cavity (30). The cylindrical cavities (30) of the three or more through bottom holes (31) are suitable for the U-shaped culture hole unit (22), the V-shaped culture hole unit (23) and the inverted cone special-shaped culture hole unit (24).
Alternatively or preferably, in the high oxygen permeable pore plate device for organoid sphere culture, the permeable bottom hole (31) is arranged at the outer edge of the visible observation area (32) of the culture pore unit (2) in the cavity (30). Namely, the through bottom hole (31) is tangent with the circular visual observation area (32). The visual observation region refers to a region that can be directly observed by the naked eye.
Optionally or preferably, in the high oxygen permeable pore plate device for organoid sphere culture, the inner side wall of the cavity (30) is cylindrical, and the maximum inner diameter of the through bottom hole (31) is less than or equal to half of the difference between the inner pore diameter of the cavity (30) and the diameter of the visible observation area (32) of the culture pore unit (2).
Compared with the prior art, the utility model discloses following beneficial effect has:
the utility model discloses a high oxygen bleeder vent board device for organoid spheroid is cultivateed can ensure that the cell culture process has sufficient dissolved oxygen environment, solves the near hole board bottom insufficient dissolved oxygen problem of organoid cell ball, can promote organoid cell ball fast growth, promote cell activity, improve cell culture efficiency effectively, avoids arousing the apoptosis that disintegrates of organoid cell ball owing to cultivate for a long time. Compare in existing culture apparatus on the market, the utility model discloses device gas mass transfer efficiency is higher, and the organoid cell ball boundary of cultivation is clear, and the compactness of cell gathering is strong, does not have the phenomenon of disintegrating.
Drawings
FIG. 1 is a schematic diagram of an overall configuration of an orifice plate assembly according to an embodiment;
FIG. 2 is a central sectional view of 5 different culture well unit structures in different types of well plate devices;
FIG. 3 is a structural diagram (with 1 air hole) of the assembled 5 culture hole units and the hole plate base in FIG. 2;
FIG. 4 is a structural diagram (with 6 air holes) of the assembled culture well unit with the well plate base in 3 different styles;
FIG. 5 is a graph showing the comparison of the dissolved oxygen content of the culture solution in different organoid sphere culture well plates;
FIG. 6 is a bright field diagram of organoid cell spheres in different organoid sphere culture well plates;
FIG. 7 is a graph comparing the cell viability of organoid cell spheres in different organoid sphere culture well plates.
Reference numerals:
1: orifice plate cover, 2: culture well unit, 21: flat bottom type culture well unit, 22: u-shaped culture well unit, 23: v-shaped culture well unit, 24: inverted cone shaped culture well unit, 25: microwell array type well unit, 211, 221, 231, 241, 251, positioning buffer wall, 3: orifice plate base, 30: cavity, 31: through bottom hole, 32: a visual observation area.
Detailed Description
The technical solutions of the present invention will be apparent from and elucidated with reference to the accompanying drawings and specific embodiments, so as to enable a person skilled in the art to better understand the present invention and to practice it.
As shown in fig. 1 to 4, the present embodiment provides a high oxygen permeable pore plate apparatus for organoid sphere culture, comprising a pore plate cover 1, a plurality of culture well units 2, and a pore plate base 3. The well plate base 3 is provided with a plurality of cylindrical cavities 30, and the culture well unit 2 is used for accommodating target cells and culture solution and is approximately in a bowl-shaped structure. The culture well unit 2 may be inserted into the cylindrical cavity 30 with a portion of the bottom of the cylindrical cavity 30 extending inward for holding the culture well unit 2. The bottom of the cavity 30 is provided with a through bottom hole 31. The through-bottomed holes 31 allow the cavities 30 to communicate up and down when not inserted into the culture well unit 2.
The culture hole unit 2 is made of materials such as breathable, transparent and nontoxic high-molecular organic silicon compounds and is used for bearing and culturing organoid cytospheres in the culture hole unit, and external oxygen or carbon dioxide is more easily dissolved into culture solution due to the breathable property of the culture hole unit, so that an adequate dissolved oxygen environment and a stable PH environment are provided for normal growth of the organoid cytospheres.
In a preferred embodiment, as shown in FIG. 2, the culture well unit 2 has various structural configurations, and is classified into a flat-bottom type culture well unit 21, a U-shaped culture well unit 22, a V-shaped culture well unit 23, an inverted conical shaped culture well unit 24, and a micro-well array type culture well unit 25 according to the cross-sectional structure.
In order to facilitate the liquid suction and liquid feeding operation of the pipette head on the culture well units 2 with various configurations, horizontal or inward-inclined positioning buffer wall surfaces 211/221/231/241/251 are arranged in the culture well units 2 with various configurations and are used for positioning and stopping the pipette head and buffering the impact force of fluid in the liquid feeding process.
In order to facilitate the permeation of oxygen or carbon dioxide into the organoid cell sphere culture section, the thickness H of the bottom of the flat-bottom type culture well unit 21, U-shaped culture well unit 22, V-shaped culture well unit 23, inverted conical shaped culture well unit 24, and micro-well array type culture well unit 25, i.e., the height between the organoid cell sphere culture interface and the lower surface thereof, is preferably 0.2 to 2 mm.
Among them, the flat bottom type culture well unit 21 can be used for culturing organoid cells embedded in conventional matrigel, and can also be used for culturing organoid cell balls independent of the scaffold structure. The U-shaped culture hole unit 22, the V-shaped culture hole unit 23, the inverted cone special-shaped culture hole unit 24 and the micropore array type culture hole unit 25 are more suitable for culture of organoid cytospheres independent of a support structure, because the structure at the culture interface of the organoid cytosphere at the bottom is set to be a smooth arc shape, cells are easier to gather and self-assemble into organoid cytospheres.
The cavity 30 is used for accommodating the culture hole unit 2 embedded therein, and the bottom part is provided with a through bottom hole 31 for providing a ventilation window for the culture hole unit 2, so that organoid cytoballs cultured at the bottom part in the culture hole unit 2 have an opportunity to obtain sufficient oxygen and carbon dioxide, and further meet the environment required by the normal growth of organoid cytoballs.
In a preferred embodiment, as shown in fig. 3, the number of the through holes 31 in the cavity 30 is 1, and the through holes are coaxially arranged with the cavity 30 and the culture hole unit 2, so that oxygen or carbon dioxide can directly permeate from the center of the bottom of the culture hole unit 2 to the organoid cell sphere culture area through the 1 through holes 31 without interfering with the light transmission in the center area, thereby facilitating the optical observation of the cell spheres.
The cavity 30 having 1 through-bottomed hole 31 is adapted to the flat-bottom type culture well unit 21, the U-shaped culture well unit 22, the V-shaped culture well unit 23, the inverted conical shaped culture well unit 24 and the micro-well array type culture well unit 25. The planar shape of the through bottom hole 31 is circular or regular polygon, and the maximum aperture distance D at the vertical section is smaller than the inner aperture D of the cavity 30, so that enough solid area for bearing the culture hole unit 2 is reserved at the bottom of the cavity 30.
In another preferred embodiment, as shown in fig. 4, the number of the through bottom holes 31 in the cavity 30 is 3 or more, and the through bottom holes are uniformly distributed around the center of the bottom of the cavity 30, and the arrangement enables oxygen or carbon dioxide to gradually penetrate into the organoid cytosphere culture area from the edge of the center of the bottom of the culture hole unit 2, i.e. from the lateral direction, through the 3 or more through bottom holes 31, which avoids the organoid cytospheres cultured in the organoid cytosphere culture area from being affected by the micro-bubbles generated at the bottom of the air-permeable center of the culture hole unit 2 due to the temperature difference change, and also does not interfere with the light transmission in the center area, thereby facilitating the optical observation of the cytospheres.
The cavity 30 having 3 or more through-bottomed holes 31 is adapted to the U-shaped culture well unit 22, the V-shaped culture well unit 23 and the inverted conical shaped culture well unit 24. The through bottom hole 31 is tangential to the circular viewing area 32. The plan shape of the through bottom hole 31 is also circular or regular polygon, and the maximum aperture distance D in vertical section is less than or equal to 1/2(dw) of the difference between the inner aperture D of the cavity 30 and the diameter W of the circular viewing area 32, again in order to leave enough solid area at the bottom of the cavity 30 for carrying the culture well unit 2.
The number of cavities 30 and culture well units 2 in the well plate base 3 is not limited to 96, and can be any value of 6, 12, 24, 48 and 384, so as to meet the requirements of scientific researchers for different experimental fluxes.
The advantages of the high oxygen permeable pore plate device for organoid sphere culture of the present invention over the existing commercial organoid sphere culture pore plate can be demonstrated by the following embodiments.
Example 1
Human hepatic progenitor cells (HepaRG, purchased from Thermo Fisher) were cultured in medium as specified. According to the hepatocyte suspension for 2000/hole concentration respectively inoculate commercial organoid spheroid culture orifice plate with the utility model orifice plate device A (containing 1 penetrating bottom hole 31) and orifice plate device B (containing 3 or more penetrating bottom holes 31). The inoculated well plates were placed at 37 ℃ in 5% CO2The cell culture box is used for culturing, the culture solution is replaced every two days for the cells, the organoid cell balls are continuously cultured until the 6 th day, and the dissolved oxygen concentration of the cell culture solution is measured and recorded by adopting an oxygen microelectrode detection system.
As shown in FIG. 5, the concentrations of the dissolved oxygen in the culture solution of the well plate device A and the well plate device B of the present invention were 0.168. + -. 0.006mol/m, respectively3And 0.153. + -. 0.007mol/m3The two have no statistical difference, and the dissolved oxygen concentration of the culture solution in the commercial orifice plate is 0.120 +/-0.006 mol/m3Obviously be less than the utility model orifice plate device A and B, and the difference has statistical significance. The result shows that the dissolved oxygen concentration of the culture solution in the orifice plate device A and the orifice plate device B is obviously higher than that of the culture solution in the commercialized orifice plate. Compared with the commercial orifice plate, the orifice plate device A and the orifice plate device B have higher gas mass transfer efficiency and can provide favorable oxygen environment for the growth of organoid cytoballs.
Example 2
Commercial orifice plate after the completion of the measurement of the dissolved oxygen concentration of the culture solution in example 1 and the orifice plate device A and B are taken out from the incubator, and the organoid cytosphere in the orifice plate is photographed and observed and the cell activity is detected respectively. The cell viability was tested using lactate dehydrogenase cytotoxicity assay kit LDH (purchased from bi yun biotechnology limited), and the operation procedures were performed according to the instructions.
The result is shown in fig. 6, the organoid cell balls cultured in the orifice plate devices A and B of the utility model have clear boundaries, strong compactness of cell aggregation and no disintegration phenomenon. However, in commercial well plates, the cell aggregation density decreases, cavitation and disintegration phenomena occur, and a small number of cells are separated from the cell balls and scattered around the cell balls. Cell viability results are shown in fig. 7, the cell viability of organoid cytospheres in well plate devices a and B is (96.97 ± 1.14)% and (95.35 ± 0.85)%, respectively, and there is no statistical difference between them, but the cell viability of organoid cytospheres in well plate devices a and B is significantly higher than the cell viability of organoid cytospheres in commercial well plates (69.53 ± 5.96)%. These results indicate that the low dissolved oxygen concentration in the culture medium in the commercial organoid sphere culture well plate is likely to result in slow cell growth rate and even cell damage. The culture solution in the orifice plate devices A and B has higher dissolved oxygen concentration, which is beneficial to the growth of organoid cytospheres and can effectively improve the structural integrity and the cell activity of the organoid cytospheres.
The inventive concept is explained in detail herein using specific examples, and the above description of the embodiments is only used to help understand the core idea of the present 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 intended to be included within the scope of the present invention.

Claims (10)

1. The high-oxygen air-permeable hole plate device for organoid sphere cultivation is characterized by comprising a hole cover plate (1), a cultivation hole unit (2) and a hole plate base (3), wherein a cavity (30) with a through bottom hole (31) is formed in the hole plate base (3), and the cultivation hole unit (2) is embedded into the cavity (30).
2. The hyperoxia vent plate apparatus for organoid sphere culture according to claim 1, wherein the material of the culture well unit (2) is organic silicon compound.
3. The hyperoxia vent plate apparatus for organoid sphere culture according to claim 1, wherein the longitudinal interface structure of the culture well unit (2) is of flat bottom type, U-type, V-type or micro-well array type.
4. The high oxygen permeable plate device for organoid sphere cultivation according to claim 3, wherein the cultivation well unit (2) is provided with positioning buffer wall surfaces (211, 221, 231, 241, 251) which are horizontal or inclined inward.
5. The high oxygen permeable plate apparatus for organoid sphere cultivation according to claim 1, wherein the minimum thickness of the bottom surface of the cultivation well unit (2) is 0.2-2 mm.
6. The high oxygen permeable plate apparatus for organoid sphere culture according to claim 1, wherein the surface of the bottom surface of the culture well unit (2) contacting organoid cells is in the shape of a smooth circular arc.
7. The hyperoxic air-permeable plate device for organoid sphere culture according to claim 1, wherein the inner side wall of the cavity (30) and the outer side wall of the culture hole unit (2) are both cylindrical, and the through bottom hole (31) is circular or regular polygonal and coaxial with the cavity (30) and the culture hole unit (2).
8. The high oxygen permeable vent plate apparatus for organoid sphere cultivation according to claim 1, wherein the through bottom holes (31) are plural and uniformly distributed.
9. The hyperoxia vent panel apparatus for organoid sphere culture according to claim 8, wherein the through bottom holes (31) are provided at the outer edge of the visual observation area (32) of the culture well unit (2) in the cavity (30).
10. The hyperoxia vent panel apparatus for organoid sphere culture according to claim 8, wherein the inner sidewall of the cavity (30) is cylindrical, and the maximum inner diameter of the through bottom hole (31) is less than or equal to half the difference between the inner diameter of the cavity (30) and the diameter of the visible observation area (32) of the culture well unit (2).
CN202021675457.XU 2020-08-12 2020-08-12 High-oxygen air vent plate device for organoid sphere culture Active CN213739510U (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111793563A (en) * 2020-08-12 2020-10-20 江苏信安佳医疗科技有限公司 High-oxygen air vent plate device for organoid sphere culture

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111793563A (en) * 2020-08-12 2020-10-20 江苏信安佳医疗科技有限公司 High-oxygen air vent plate device for organoid sphere culture

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Effective date of registration: 20210730

Address after: 318000 Room 501, building a, Fangyuan e-commerce Pioneer Park, 155 xitaihe Road, Haimen street, Jiaojiang District, Taizhou City, Zhejiang Province

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Patentee before: Luo Guoan