CN211035951U - Suspension drip chip and cell culture device - Google Patents

Abstract

The utility model relates to a chip and cell culture device are dripped to suspension belongs to biochip technical field. The suspension dripping chip comprises a macroporous layer, a small porous layer and a reinforcing layer which are sequentially stacked; the macroporous layer and the microporous layer are thermoplastic hydrophobic elastic polymer films; the macroporous layer is provided with at least one suspension drip hole, the small pore layer is provided with a liquid supplementing hole with the diameter smaller than that of the suspension drip hole corresponding to the suspension drip hole, and the suspension drip hole and the liquid supplementing hole correspondingly arranged form an open micro-disc structure with a pore diameter difference; and a yielding hole with the diameter larger than that of the liquid supplementing hole is arranged at the position, corresponding to the liquid supplementing hole, on the reinforcing layer. The suspension drop chip has good thermoplasticity, hydrophobicity and biocompatibility, has a liquid exchange function, does not need additional surface modification, is low in cost, simple and convenient to manufacture, can be quickly formed, and can flexibly adjust the size, the specification and the like according to experimental needs.

Description

Suspension drip chip and cell culture device

Technical Field

The utility model belongs to the technical field of biochip, a suspension drips chip and cell culture device is related to.

Background

At present, the three-dimensional cell culture technology is greatly concerned in the field of biological research, and has great potential in the aspects of tumor biological research, tumor stem cell enrichment and drug screening. The three-dimensional tumor sphere is a micro cell aggregate, is applied to the construction of in-vitro culture models of various tumor cells, and can accurately simulate some characteristics of solid tumors. Various three-dimensional tumor sphere culture methods exist in the prior art, such as a scaffold method, a dynamic rotation (or stirring) method, an ultra-low adhesion culture method and a hanging drop culture method. Among them, the hanging drop culture method is the most commonly used culture method for three-dimensional tumor spheres, and is to drop a cell solution on a hydrophobic substrate, turn the hydrophobic substrate over, and form a hanging drop by using surface tension, so that cells are gathered at the bottom of the drop by self gravity to form a tumor cell sphere. Compared with other three-dimensional culture methods, the size of the tumor cell balls formed by the hanging drop culture method is controllable, the uniformity is good, and the influence of unexpected mechanical force is avoided.

The suspension drop chip provides a platform for suspension drop and three-dimensional cell culture, but the existing suspension drop chip also has the following defects: (1) although the suspension drop culture platform constructed by directly utilizing the culture dish is the simplest method, liquid exchange cannot be carried out, and long-time culture is difficult to realize; (2) suspension droplet culture platforms constructed by surface modification require wet chemical processing, potential solvent residues may reduce biocompatibility; (3) the microfluidic suspension droplet chip relates to a complex micro-processing process and a special instrument, has higher cost and needs professional operators; (4) the traditional commercial suspension drop culture plate is expensive and single in specification, and cannot be flexibly adjusted according to experimental needs. These barriers greatly limit the widespread use of suspension culture in the fields of biochemistry, medicine, etc., and in particular in biochemical laboratories with limited resources.

SUMMERY OF THE UTILITY MODEL

In view of this, the utility model discloses a solution exchange can not be realized to the solution adopts ordinary culture dish to carry out suspension formula three-dimensional cell culture, and current micro-fluidic suspension drips the chip and the problem that the culture plate is with high costs is dripped to the commercialization suspension, can not adjust size, specification in a flexible way, provides a suspension and drips chip and culture apparatus, has good thermoplasticity, hydrophobicity and biocompatibility, possesses the liquid exchange function, need not extra surface modification, low cost, the preparation is simple and convenient, take shape fast to can adjust size, specification etc. in a flexible way according to the experiment needs.

In order to achieve the above purpose, the utility model provides a following technical scheme:

a suspension dripping chip comprises a macroporous layer, a small porous layer and a reinforcing layer which are sequentially stacked; the macroporous layer and the microporous layer are thermoplastic elastic hydrophobic polymer films; the macroporous layer is provided with at least one suspension drip hole, the small pore layer is provided with a liquid supplementing hole with the diameter smaller than that of the suspension drip hole corresponding to the suspension drip hole, and the suspension drip hole and the liquid supplementing hole correspondingly arranged form an open micro-disc structure with a pore diameter difference; and a yielding hole with the diameter larger than that of the liquid supplementing hole is arranged at the position, corresponding to the liquid supplementing hole, on the reinforcing layer.

Furthermore, the macroporous layer and the small pore layer are manufactured by adopting a sealing film.

Further, the diameter of the suspension dripping hole is the same as that of the abdicating hole.

Further, the suspension drop chip is square.

Further, the macroporous layer, the small porous layer and the reinforcing layer are stacked through hot plastic packaging.

A suspension drop cell culture device, comprising the suspension drop chip as described in any one of the above, and a support for horizontally supporting the suspension drop chip.

Furthermore, the support is a bolt symmetrically inserted on the suspension drip chip, the head of the bolt is arranged on one side of the reinforcing layer, the screw rod part of the bolt is arranged on one side of the large pore layer and is provided with a nut matched with the bolt, and the suspension drip chip is locked by the nut.

The beneficial effects of the utility model reside in that:

(1) the utility model discloses a suspension drip chip and culture apparatus, the diameter of the suspension drip hole and the diameter of the liquid supplementing hole are different, and an open micro-disc structure is formed due to the difference of the diameters, on one hand, when the solution is dripped into the suspension drip hole, the small hole layer plays a role in supporting and stabilizing the solution; on the other hand, in the cell culture process, operations such as culture medium updating, medicine adding and the like can be performed through the liquid supplementing hole, so that solution exchange is realized, the maintenance of a long-term culture system and the medicine test are facilitated, and the defect that liquid supplementing cannot be performed due to the fact that the bottom of a common culture dish serving as a suspension drop culture platform is completely closed is overcome.

(2) The utility model discloses a suspension drip chip and a culture device, which have simple structure, convenient preparation, rapid forming within ten minutes and low processing cost; the size, specification and aperture size of chip all can be adjusted according to the experiment needs in a flexible way, economical and practical, sexual valence relative altitude.

(3) The utility model discloses a suspension drips chip and culture apparatus chooses for use the hydrophobic elasticity polymer film of thermoplasticity for the first time, especially chooses for use the sealing film to make the chip, has good thermoplasticity, hydrophobicity and biocompatibility, need not to carry out surface modification to the chip again, also can not bring the risk that reduces biocompatibility because of surface modification; the sealing film is a necessary tool for experiments, is easy to obtain and low in price, and reduces the cost of the chip.

Drawings

In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention provides the following drawings for illustration:

FIG. 1 is a schematic diagram of a suspension droplet chip;

FIG. 2 is a schematic structural view of an open microdisk structure;

FIG. 3 is a schematic view showing liquid exchange through a feed hole;

FIG. 4 is a schematic illustration of the effect of the size of the diameter of the drop hole of the test suspension on the shape of the drop;

FIG. 5 is a schematic illustration of the effect of the size of the diameter of the drop hole of the test suspension on the speed of tumor sphere formation;

FIG. 6 is a schematic diagram of the testing of the effect of initial cell concentration on tumor sphere diameter;

FIG. 7 is a schematic diagram of the growth kinetics of tumor spheres on a test suspension drop chip;

FIG. 8 is a schematic illustration of the physiological characteristics of tumor spheres on a test suspension drop chip;

FIG. 9 is a schematic diagram showing the difference in cell viability between three-dimensional culture of the test suspension drop chip and two-dimensional culture of the tissue culture plate.

Reference numerals: the large-hole layer 1, the suspension drip hole 101, the small-hole layer 2, the liquid supplementing hole 201, the reinforcing layer 3 and the abdicating hole 301.

Detailed Description

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

As shown in fig. 1 and 2, the present embodiment provides a suspension droplet chip, which is composed of three square film layers, specifically including a macroporous layer 1, a microporous layer 2 and a reinforcing layer 3 stacked in sequence. The macroporous layer 1 and the small pore layer 2 are made of sealing films, the thermoplasticity and the hydrophobicity of the sealing films are fully utilized, additional surface modification is not needed, and good biocompatibility is guaranteed; meanwhile, the sealing film has the advantages of economy, low price and easy obtainment.

Be equipped with the suspension drip hole 101 that three rows three columns matrix arranged on the macropore layer 1, it is equipped with the fluid infusion hole 201 that the diameter is less than suspension drip hole 101 to correspond suspension drip hole 101 on the micropore layer 2, suspension drip hole 101 and the fluid infusion hole 201 that corresponds the setting constitute the open microdisk structure that has the aperture difference, as shown in fig. 2, in this open microdisk structure, set for fluid infusion hole 201 diameter and be 1.5mm, suspension drip hole 101 diameter can be 3mm, 4mm, 5mm, also can change concrete aperture size according to the experimental needs. The diameter of the corresponding suspension drop hole 101 in the open type micro-disc structure can control the size of the suspension drop; the corresponding small-hole layer 2 plays a role in supporting and stabilizing the solution, and the liquid supplementing hole 201 allows the solution, the medicine and the like to be added in the cell culture process, so that the effect of liquid exchange is achieved. As shown in FIG. 3, the blue dye is used as the tracer solution, and the solution is added into the suspension drop through the solution supplementing hole 201, and the suspension drop presents uniform blue within 5 minutes, which indicates that the solution can rapidly diffuse and equilibrate in the suspension drop, and the uniformity of the culture environment is maintained.

The reinforcing layer 3 is a polyethylene terephthalate film (PET film), and the reinforcing layer 3 is provided to prevent deformation due to the soft texture of the sealing film and ensure that the entire structure of the chip is flat. The reinforcing layer 3 is provided with a yielding hole 301 corresponding to the liquid supplementing hole 201, the diameter of the yielding hole 301 is larger than that of the liquid supplementing hole 201, the liquid supplementing hole 201 cannot be shielded by the yielding hole 301, and the smoothness of liquid supplementing is guaranteed. The diameter of the suspension dripping hole 101 is the same as that of the abdicating hole 201, so that the preparation of the suspension dripping chip is convenient.

Regarding the preparation of the suspension drip chip provided in this embodiment, first, a desktop paper cutter is used to design chip patterns, that is, the shapes of the macroporous layer 1, the microporous layer 2 and the reinforcing layer 3, and the arrangement structure and diameter of the suspension drip holes 101, the fluid infusion holes 201 and the relief holes 301, and the desktop paper cutter is used to carve the chip patterns on the sealing films of the macroporous layer 1 and the microporous layer 2 and the PET film of the reinforcing layer 3; then, the macroporous layer 1, the microporous layer 2 and the reinforcing layer 3 are aligned and stacked in sequence, and then subjected to thermal plastic packaging at a temperature of 80 ℃ and repeated three times. In this embodiment, the diameter of the suspension drip hole 101 is the same as the diameter of the abdicating hole 201, the corresponding patterns of the macroporous layer 1 and the reinforcing layer 3 do not need to be designed respectively, and the sealing films and the PET films for manufacturing the macroporous layer 1 and the reinforcing layer 3 can be stacked and then engraved with the corresponding patterns, so that the time for pattern design and engraving steps can be saved. The preparation of the suspension drop chip is quick and simple, and the size, specification and aperture of the chip can be adjusted according to the actual needs of experiments.

Regarding the application of the suspension drop chip provided by this embodiment in the three-dimensional tumor sphere culture, in the operation process, the suspension drop chip is flatly placed on a horizontal operation table, the macroporous layer 1 faces upward, and a cell solution is dripped into the suspension drop hole; then the suspension drop chip is turned over to enable the macroporous layer 1 to face downwards, the suspension drop chip is horizontally erected on an opening vessel arranged on a horizontal operation platform, the inner cavity of the opening vessel contains suspension drops, the depth of the inner cavity is larger than the height of the suspension drops, sufficient drooping space of the suspension drops is ensured, cell solution forms suspension drops under the action of surface tension, and cells are gathered at the bottom of the suspension drops under the action of self gravity to form tumor balls.

As shown in FIG. 4, the diameter of the fluid infusion hole 201 is set to be 1.5mm, and the diameter of the suspension drip hole 101 is set to be 3mm, 4mm and 5mm, respectively, and the influence of the diameter of the suspension drip hole 101 on the shape (diameter and height) of the suspension is tested by using the suspension drip chip provided by the embodiment. When the diameter of the suspension drop hole 101 is constant, the diameter of the suspension drop is constant, and the height of the suspension drop is increased along with the increase of the volume of the suspension drop; when the volume of the suspension drop is constant, the diameter of the suspension drop increases with the increase of the diameter of the suspension drop hole 101, and the height of the suspension drop decreases with the increase of the volume of the suspension drop.

As shown in FIG. 5, the diameter of the solution supply well 201 was set to 1.5mm, and the diameters of the suspension drop wells 101 were set to 3mm, 4mm, and 5mm, respectively, and the suspension drop chip provided in this example was used under the culture conditions of (1) the volume of the suspension drop was 20. mu. L, and (2) the cell density was 5X10³The cell type (3) is human liver cancer cell (HepG2), and the culture environment (4) is 37 ℃ and 5% CO₂And (5) the culture time is 12 hours, and the influence of the diameter size of the dropping hole 101 of the suspension on the tumor sphere forming speed is tested. According to the test results, the smaller the diameter of the suspension drop hole 101, the faster the tumor sphere formation speed.

As shown in FIG. 6, the diameter of the wells 201 is set to 1.5mm, the diameter of the wells 101 is set to 3mm, and the chip for suspension droplets provided in this example was used under the culture conditions of (1) the volume of the suspension droplets is 20. mu. L, and (2) the cell densities are 1.25, 2.50, 5.00, and 10.0x10, respectively³The cell type (3) is human liver cancer cell (HepG2), and the culture environment (4) is 37 ℃ and 5% CO₂The culture time is 5 days, (6) a fluorescent tracer:

green long-term cell tracers stained the cell membranes, (7) taking 7 tumor cell spheres per concentration and counting the average diameter of the tumor spheres, testing the effect of initial cell concentration on the diameter of the tumor spheres. According to the test results, the size of the tumor sphere is determined by the initial cell density, the larger the tumor sphere diameter.

As shown in FIG. 7, the diameter of the solution supply well 201 was set to 1.5mm, and the diameter of the suspension drop well 101 was set to 3mm, and the suspension drop chip provided in this example was used under the culture conditions of (1) the volume of the suspension drop was 20. mu. L, and (2) the cell density was 10.0X10³Respectively, (3) the cell type is human liver cancer cell (HepG2), (4) the culture environment is 37 ℃, 5% CO2, (5) the culture time is 3 days and 7 days respectively,(6) staining of live and dead cells by fluorescence: Calcein-AM stains live cells, PI stains dead cells, and growth kinetics of tumor balls on the suspension drop chips are tested. According to the test results, when cultured for 3 days, the fluorescence signals representing living cells are strong and uniformly distributed, and the fluorescence intensity of dead cells is negligible. However, after 7 days of culture, the fluorescence intensity of live cells in the center of the tumor sphere was significantly decreased, and the fluorescence intensity of dead cells was correspondingly increased. The change of fluorescence intensity indicates the change and distribution of the ratio of live cells/dead cells, and the change and distribution indicate that the tumor cell balls can simulate the central necrosis characteristics of solid tumors.

As shown in FIG. 8, the diameter of the solution supply well 201 was set to 1.5mm, and the diameter of the suspension drop well 101 was set to 3mm, and the suspension drop chip provided in this example was used under the culture conditions of (1) the volume of the suspension drop was 20. mu. L, and (2) the cell density was 10.0X10³The cell type (3) is human liver cancer cell (HepG2), and the culture environment (4) is 37 ℃ and 5% CO₂And (5) culturing for 0, 3, 5 and 7 days, respectively, and (6) taking 100 tumor cell spheres at each time point to perform experiments to test the physiological characteristics of the tumor spheres on the suspension drop chip. The relative expression quantity of mRNA of liver metabolic markers CYP3A4/CYP1A1 and Albumin (Albumin) of tumor fine balls growing on the suspension drop chip and HePG2 cells growing adherently in a tissue culture plate is measured by adopting real-time fluorescent quantitative PCR, cells cultured in a two-dimensional mode in the tissue culture plate (namely, cells cultured for 0 day by the open suspension drop chip) are used as a control group, and the result shows that compared with the cells growing on the tissue culture plate, the cells in the tumor balls growing on the open suspension drop chip have higher metabolic activity and stronger protein secretion capacity, and can better simulate solid tumors.

As shown in FIG. 9, the diameter of the fluid infusion hole 201 is set to be 1.5mm, the diameter of the suspension drop hole 101 is set to be 3mm, and the suspension drop chip provided in this example is used under the culture conditions of (1) the volume of the suspension drop is 20 μ L, and (2) the cell density is 1.25X10³The cell type (3) is human liver cancer cell (HepG2), and the culture environment (4) is 37 ℃ and 5% CO₂The culture time is 5 days, (6) the adriamycin (dox) drug concentration is 0, 5, 10, 20, 40 and 80 mu M respectively, and after 2 days of drug action, the tumor is treated by pancreatinThe ball is digested and dispersed into single free cells, and the difference of the cell survival rates of the three-dimensional culture of the suspension drop chip and the two-dimensional culture on a tissue culture plate is detected by using CCK-8. The results show that the liver cancer tumor balls formed in the suspension drop chips have stronger tolerance capability to anti-tumor drugs compared with liver cancer cells growing in a monolayer form in a tissue culture plate.

The above experimental results fully show that the suspension drop chip provided by the embodiment can completely meet the requirements of performing three-dimensional cell culture and performing related tests and researches by using a suspension culture method, and can completely obtain accurate experimental results under the condition of reasonable setting of other experimental conditions.

The embodiment also provides a suspension drop cell culture device, four bolts are symmetrically inserted at four corners of the square suspension drop chip, the heads of the bolts are arranged on one surface of the reinforcing layer 3, the screw rod parts of the bolts are arranged on one surface of the macroporous layer 1, a nut is arranged on each bolt from one surface of the macroporous layer 1, and the suspension drop chip is locked by the nuts. The bolt can be used as a support, after liquid is added into the suspension drip hole 101 and the suspension drip chip is turned over, the screw rod part of the bolt is stably placed on the horizontal operating platform, so that the suspension drip has enough drooping space and does not interfere with the horizontal operating platform.

Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the scope of the claims of the present invention.

Claims (7)

1. A suspension dripping chip is characterized by comprising a macroporous layer, a small porous layer and a reinforcing layer which are sequentially stacked; the macroporous layer and the microporous layer are thermoplastic elastic hydrophobic polymer films; the macroporous layer is provided with at least one suspension drip hole, the small pore layer is provided with a liquid supplementing hole with the diameter smaller than that of the suspension drip hole corresponding to the suspension drip hole, and the suspension drip hole and the liquid supplementing hole correspondingly arranged form an open micro-disc structure with a pore diameter difference; and a yielding hole with the diameter larger than that of the liquid supplementing hole is arranged at the position, corresponding to the liquid supplementing hole, on the reinforcing layer.

2. The suspension droplet chip of claim 1, wherein the macroporous and microporous layers are formed using a sealing film.

3. The suspension droplet chip of claim 1, wherein the diameter of the suspension droplet hole is the same as the diameter of the relief hole.

4. The suspension droplet chip of claim 1, wherein the suspension droplet chip is square.

5. The suspension droplet chip of claim 1, wherein the macroporous layer, the microporous layer, and the reinforcement layer are stacked by thermal molding.

6. A suspension drop cell culture device, comprising the suspension drop chip according to any one of claims 1 to 5, and a holder for horizontally supporting the suspension drop chip.

7. The suspension drop cell culture device of claim 6, wherein the support is a bolt symmetrically inserted on the suspension drop chip, the head of the bolt is arranged on one side of the reinforcing layer, the screw part of the bolt is arranged on one side of the macroporous layer, and a nut matched with the bolt is arranged on the bolt and locks the suspension drop chip.

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