CN108456642B - Three-dimensional pellet-focusing culture plate and method for screening cancer cell chemotherapeutic drugs and concentrations - Google Patents
Three-dimensional pellet-focusing culture plate and method for screening cancer cell chemotherapeutic drugs and concentrations Download PDFInfo
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- CN108456642B CN108456642B CN201810441618.XA CN201810441618A CN108456642B CN 108456642 B CN108456642 B CN 108456642B CN 201810441618 A CN201810441618 A CN 201810441618A CN 108456642 B CN108456642 B CN 108456642B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2531/00—Microcarriers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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Abstract
The invention relates to a three-dimensional ball-collecting culture plate, which comprises a pore plate and a plate cover matched with the pore plate. A plurality of culture holes are arranged on the three-dimensional ball-collecting culture plate, the culture holes comprise a first cavity and a second cavity which are connected through a first diversion hole from top to bottom, and the three-dimensional ball-collecting culture plate further comprises at least one hemispherical third cavity arranged at the bottom of the second cavity, and a first locating ring is arranged at the junction of the first diversion hole and the first cavity. The three-dimensional pellet-gathering culture plate can realize three-dimensional pellet-gathering culture and improve the survival rate of cells. The invention discloses a cancer cell chemotherapeutic drug concentration screening method by adopting a three-dimensional pellet-focusing culture plate, which comprises the following steps: sterilizing the three-dimensional pellet-forming culture plate and coating the three-dimensional pellet-forming culture plate with a surface modifier; inoculating the primary cell suspension to a three-dimensional pellet-gathering culture plate to culture into a cell three-dimensional pellet; after the three-dimensional cell aggregation reaches the requirement, adding a drug to be detected with concentration gradient into the culture hole; after a predetermined period of incubation, the cell activity index is measured to determine the cell chemotherapeutic agent and the chemotherapeutic agent concentration.
Description
Technical Field
The invention mainly relates to the field of biology, in particular to a three-dimensional pellet-gathering culture plate mainly used for cell culture, and a cancer cell chemotherapeutic drug and concentration screening method using the three-dimensional pellet-gathering culture plate.
Background
The Chinese is one of countries with high liver cancer incidence, and the treatment of liver cancer has very important positions and influences. When developing chemotherapeutic drugs, it is often necessary to culture cancer cells, and the culture amount of the cultured liver cancer cells is large. The currently adopted culture apparatus can mainly realize the planar culture of liver cancer cells, and is difficult to simulate the real conditions in vivo. Meanwhile, due to the structure of the culture vessel, when liquid is sucked, liquid such as medium is replaced and medicine is added, the cultured cells are often blown out of a designated culture area due to fluid disturbance, or the cultured cells are damaged or even die to different degrees due to fluid shearing force. In the prior art, under the planarization culture, the liver cancer cells have low cell viability and small cell density, and the operations such as frequent liquid exchange, medicine addition and the like lead to cell death or injury, thereby affecting the simulation effect of in-vitro experiments and further affecting experimental conclusion. Meanwhile, due to planarization and extensive culture of in-vitro experiments, the cultured cells are different from in-vivo states to a certain extent, so that the cultured liver cancer cells cannot simulate the in-vivo states well, and experimental result deviation is caused. The above problems have resulted in the results of drug screening methods using conventional culture vessels and cell culture methods deviating from the actual situation or being severely affected.
Based on the defects and encountered problems in the prior art, the invention aims to provide a three-dimensional pellet-gathering culture plate capable of realizing three-dimensional pellet-gathering culture and a screening method of cancer cell chemotherapeutic drugs and concentrations.
Disclosure of Invention
The invention provides a three-dimensional ball-collecting culture plate which comprises a pore plate and a plate cover matched with the pore plate, wherein a plurality of culture holes are arranged on the three-dimensional ball-collecting culture plate, the culture holes at least comprise a first cavity and a second cavity which are connected through a first diversion hole from top to bottom, the three-dimensional ball-collecting culture plate also comprises at least one hemispherical third cavity arranged at the bottom of the second cavity, and a first positioning ring is arranged at the junction of the first diversion hole and the first cavity.
Preferably, the first deflector aperture is defined by a first annular sidewall having a deflector upper diameter greater than a deflector lower diameter thereof.
More preferably, a first extension of a busbar of the first annular side wall intersects a side wall of the second cavity.
More preferably, the first cavity is formed by expanding upwards from the upper guide edge, and comprises a first upper edge serving as the edge of the culture hole and a first lower edge overlapped with the upper guide edge; the second cavity is formed by expanding downwards from the diversion lower edge, and comprises a second upper edge overlapped with the diversion lower edge and a second lower edge bordered by the cavity bottom.
Further preferably, the second upper edge and the second lower edge intersect perpendicularly with the second extension line at the same time.
Still further preferably, the diameter of the first upper edge is greater than or equal to the diameter of the first lower edge.
Further preferably, the second cavity includes a first transition hole bordering the first deflector hole, and a second deflector hole connecting the first transition hole and the cavity bottom.
Still further preferably, the first transition hole is a cylindrical hole or an inverted conical hole; the second diversion hole is an inverted conical hole; the junction of the first transition hole and the second deflector hole is defined as a second positioning ring.
Preferably, the total volume of the culture wells is greater than or equal to 50 μl; the sum of the volumes of the second cavity and the first diversion hole is equal to the volume of the first cavity.
Preferably, the third cavities are circumferentially arranged and/or equidistantly arranged at the bottom of the second cavity.
Preferably, the diameter of the third cavity is 400 μm; the depth of the third cavity is 400 mu m; the center-to-center distance between adjacent third cavities in the same culture well is 300 μm.
Preferably, the culture wells are arranged in a matrix form on the well plate.
The invention also provides a screening method of the concentration of the cancer cell chemotherapeutic drug. The screening method adopts any one of the three-dimensional pellet-gathering culture plates, and comprises the following steps:
(1) Sterilizing the three-dimensional pellet-forming culture plate, and coating the three-dimensional pellet-forming culture plate with a surface modifier;
(2) Inoculating the primary separated cell suspension into the three-dimensional pellet-forming culture plate for three-dimensional pellet-forming culture to form a cell three-dimensional pellet;
(3) After the three-dimensional cell pellet reaches the test requirement, adding a drug to be tested with a preset concentration gradient into the culture hole;
(4) After co-culturing with the drug to be tested for a predetermined time, determining cell activity index to determine the chemotherapeutic drug to the cells and the sensitive concentration of the chemotherapeutic drug.
Preferably, in the step (1), the surface modifier adopts at least one surface modifier of 0.01-5% of bovine serum albumin, sulfhydryl polyethylene glycol and 2-methacryloyloxyethyl phosphorylcholine; and/or the surface modifier coating treatment time is between 12 and 48 hours.
Preferably, the density of seeded cells in step (2) is from 500 per culture well to 2000 per culture well.
Preferably, in step (2), the medium in the culture wells is refreshed every 24 hours.
More preferably, in step (2), the proportion of medium in each renewal of the culture well is between 40% and 60%.
It is further preferred that the operation is performed with a micropipette against said first positioning ring when the medium is renewed and/or the drug to be tested is added.
Preferably, in step (4), the cell activity index comprises at least one of a cell metabolite concentration index, an apoptosis marker concentration index, and an intracellular enzyme activity index.
The invention provides a three-dimensional pellet-gathering culture plate and a method for screening cancer cell chemotherapeutic drugs and concentrations, which can better solve part of technical problems and have the following advantages:
(1) According to the three-dimensional pellet-gathering culture plate, cells enter a three-dimensional pellet-gathering culture state through the special structure of the third cavity, so that the cells and cell clusters can be in an approximately in-vivo state, the in-vivo state can be better simulated, and a certain foundation is provided for the screening method;
(2) The three-dimensional ball-collecting culture plate is provided with a plurality of flow guide structures and buffer structures, including but not limited to a first flow guide hole, a second flow guide hole and the like, through culture holes in the three-dimensional ball-collecting culture plate, and is used for buffering liquid flow generated during liquid sucking and liquid adding operation, so that the position and the growth state of cells are prevented from being influenced;
(3) The three-dimensional ball-collecting culture plate comprises a first positioning ring and a second positioning ring, wherein the positions of the first positioning ring and the second positioning ring are used for controlling the quantity of liquid sucked each time, so that the proportion of the preset replacement liquid is realized, and the operation of a user is facilitated;
(4) According to the three-dimensional ball-collecting culture plate, the requirements of setting a control group, a blank group and repeated experiment compound holes can be met by setting a large number of culture holes, so that drug screening experiments can be realized on the same culture plate, and confusion is avoided;
(5) The screening method for cancer cell chemotherapeutic drugs and concentration, which is related by the invention, can screen drugs and provide drug sensitivity maps for different donors by applying the three-dimensional pellet-gathering culture plate, can simulate the in-vivo state of the donors to a higher degree by utilizing the three-dimensional pellet-gathering culture of the culture plate, has reliable results and short screening period, and can meet clinical requirements.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a top view of a three-dimensional pellet-harvesting culture plate of the present invention;
FIG. 2 is a cross-sectional view of a single culture well of the three-dimensional pellet-packed culture plate of the invention;
FIG. 3 is a top view of a single culture well of a three-dimensional pellet-focused culture plate according to the invention;
FIG. 4 is a step diagram of a method for screening for chemotherapeutic agents and concentrations in cancer cells according to the present invention;
FIG. 5 is a microscopic magnification of isolated primary hepatoma cells of the method of screening for cancer cell chemotherapeutic agents and concentrations of the present invention;
FIG. 6 is an immunofluorescence staining chart of Alpha Fetoprotein (AFP) of isolated primary hepatoma cells of the method for screening cancer cell chemotherapeutic agent and concentration according to the present invention;
FIG. 7 is a DAPI immunofluorescence staining chart of isolated primary hepatoma cells of the method for screening cancer cell chemotherapeutic drugs and concentrations according to the invention;
FIG. 8 is a microscopic magnification of a single well in a three-dimensional pellet plate used in the method of screening cancer cell chemotherapeutics and concentrations according to the present invention;
FIG. 9 is a microscopic enlarged view of the third chamber of the single culture well of FIG. 8;
FIG. 10 is a graph showing the fit of cisplatin drug concentration to hepatoma cell inhibition ratio in the method for screening cancer cell chemotherapeutic drug and concentration according to the present invention;
FIG. 11 is a graph showing the fit of oxaliplatin concentration to the inhibition of hepatoma cells in the method for screening chemotherapeutic drug and concentration in cancer cells according to the present invention;
FIG. 12 is a graph showing the fit of gemcitabine concentration to hepatoma cell inhibition in a method of screening for chemotherapeutic drug and concentration in cancer cells according to the present invention;
FIG. 13 is a graph showing the fit of doxorubicin drug concentration to hepatoma cell inhibition ratio in the method of screening cancer cell chemotherapeutic drug and concentration according to the present invention;
FIG. 14 is a graph showing the sensitivity of the drug to the concentration of the chemotherapeutic drugs in cancer cells according to the screening method of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
Referring to fig. 1, the present embodiment provides a three-dimensional pellet-culturing plate 1, wherein the three-dimensional pellet-culturing plate 1 includes an orifice plate 11 and a plate cover (not shown) matched with the orifice plate for covering. More specifically, the three-dimensional pellet-collecting culture plate 1 has a plurality of culture wells 12 arranged in a matrix. In this example only, the culture wells were arranged in a matrix of 8 wells per row for a total of 6 rows, with 48 wells. In other possible embodiments, the number and arrangement of the culture holes 12 on the three-dimensional pellet culture plate 1 can be adjusted according to the culture requirements.
The detailed structure of the culture well 12 is shown in fig. 2 and 3. FIG. 2 is a sectional view of the culture well 12 taken longitudinally along the axial center of the culture well 12, and FIG. 3 is a plan view of a single culture well 12.
Referring to fig. 2, the culture hole 12 includes a first cavity 121 and a second cavity 122 from top to bottom, the first cavity 121 and the second cavity 122 are connected by a first deflector hole 124, the culture hole 12 further includes a third cavity 123 disposed at a bottom 122-4 of the second cavity 122, the third cavity 123 is of a hemispherical design, and the shape design of the third cavity 123 facilitates aggregation of the cultured cells during proliferation and growth, and further forms a cell mass with or similar in-vivo functions. The first positioning ring 125 is located at the position where the first diversion hole 124 and the first cavity 121 are adjacent, and the first positioning ring 125 can support against, i.e. provide positioning function, for example, a pipette tip. At the same time, the set position of the first positioning ring 125 determines the amount of liquid sucked at one time.
Specifically, referring to fig. 2 and 3, the first flow guiding hole 124 is formed by a first annular sidewall 124-1, the first annular sidewall 124-1 has a flow guiding upper edge 124-1A and a flow guiding lower edge 124-1B, the flow guiding upper edge 124-1A is an upper limit of the first annular sidewall 124-1, and the flow guiding lower edge 124-1B is a lower limit of the first annular sidewall 124-1. The perimeter of the upper flow guiding edge 124-1A is greater than the perimeter of the lower flow guiding edge 124-1B. In the case where the upper guide edge 124-1A and the lower guide edge 124-1B are circular, the diameter of the upper guide edge 124-1A is greater than the diameter of the lower guide edge 124-1B.
More specifically, with continued reference to FIG. 2, the first annular sidewall 124-1 may be considered to be a side of an inverted circular truncated cone in space, with the first extension 124-1C being substantially an extension of the generatrix of the first annular sidewall 124-1. The first extension line 124-1C intersects the second deflector aperture 122-5 of the second chamber 122 to restrict the first extension line 124-1C from passing through an area of the second chamber 122 outside the chamber bottom 122-4. The purpose of this design is to allow fluid flowing through the first annular sidewall 124-1 to be directed under inertial as well as natural gravity to avoid the area of the cavity floor 122-4. Notably, the first extension 124-1C is an extension of the bus of the first annular sidewall 124-1, and spatially, the area swept by the plurality of first extensions 124-1C should also be outside the area of the cavity bottom 122-4 of the second cavity 122.
More specifically, referring to FIG. 2, the first cavity 121 is formed by expanding the upper guide edge 124-1A upwards, and the first cavity 121 includes a first upper edge 121-1 serving as the edge of the culture hole 12 and a first lower edge 121-2 overlapping with the upper guide edge 124-1A. The area of the first cavity 121 is defined by a first upper edge 121-1 and a first lower edge 121-2.
With continued reference to fig. 2, the second cavity 122 is formed by a downward expansion of the diversion bottom edge 124-1B, and the second cavity 122 includes a second top edge 122-1 coinciding with the diversion bottom edge 124-1B and a second bottom edge 122-2 bordering the cavity bottom 122-4 of the second cavity 122.
More specifically, the second upper edge 122-1 and the second lower edge 122-2 intersect perpendicularly with the second extension line 126 at the same time. The second upper edge 122-1 is of equal circumference or diameter as the second lower edge 122-2.
More specifically, the perimeter of the first upper edge 121-1 is equal to or greater than the perimeter of the first lower edge 121-2, and the diameter of the first upper edge 121-1 is equal to or greater than the diameter of the first lower edge 121-2 when the first upper edge 121-1 and the first lower edge 121-2 are circular. In this embodiment, the diameter of the first upper edge 121-1 is greater than the diameter of the first lower edge 121-2. As can be seen in connection with fig. 2 and 3, in this embodiment, the first cavity 121 defined by the first upper edge 121-1 and the first lower edge 121-2 presents a bottomless, capless, inverted landing profile.
More specifically still, the second cavity 122 includes a first transition aperture 122-3 bordering the first deflector aperture 124 and a second deflector aperture 122-5 connecting the first transition aperture 122-3 and the cavity floor 122-4. The upper edge of the first transition aperture 122-3 is defined by the lower guide edge 124-1B.
Still further specifically, referring to fig. 1, in this embodiment only, the first transition hole 122-3 is a cylindrical hole. In other possible embodiments, the first transition aperture 122-3 may be a reverse tapered aperture (or reverse truncated cone). The second diversion holes 122-5 are reverse tapered holes for facilitating the flow of the liquid and concentrating at the cavity bottom 122-4. The junction of the first transition hole 122-3 and the second deflector hole 122-5 is a second positioning ring 122-6. The second positioning ring 122-6 can be used by an appliance such as a pipette tip to provide a positioning point, and the setting position of the second positioning ring 122-6 also determines the sucked liquid amount.
Specifically, the total volume of the culture well 12 is equal to 50. Mu.L only in the present embodiment, and in other possible implementations, the total volume of the culture well 12 may be set to be greater than 50. Mu.L. The volume of the first cavity 121 is equal to the sum of the volumes of the second cavity 122 and the first deflector hole 124. Taking the pipette to suck liquid as an example, in this embodiment, the tip of the pipette tip abuts against the first positioning ring 125 to suck all the liquid in the first cavity 121, and the liquid below the first positioning ring 125 is retained. Under the design that the volume of the first cavity 121 is equal to the sum of the volumes of the second cavity 122 and the first diversion hole 124, about 50% of the liquid in the culture holes 12 can be sucked each time (the floating range is 40% -60%), and the purpose of half liquid exchange is achieved. The present embodiment is only referred to herein, and the present invention allows for adjusting the position of the first positioning ring 125, i.e. adjusting the ratio between the volume of the first cavity 121 and the sum of the volumes of the second cavity 122 and the first diversion hole 124, so as to adapt to the needs of different liquid exchange amounts.
Specifically, referring to fig. 3, the third cavities 123 are circumferentially arranged at the bottom 122-4 of the second cavity 122. In this embodiment, a total of 6 third cavities 123 are circumferentially arranged and equidistantly arranged at the bottom of the cavity. In other possible embodiments, the third cavities 123 are equally spaced or circumferentially spaced at the bottom 122-4 of the second cavity 122.
More specifically, the diameter of the third cavity 123 is 400 μm, and the depth of the third cavity 123 is 400 μm. The near hemispherical cavity of the third cavity 123 may be decomposed into a cylindrical hole having a diameter equal to that of the hemispherical cavity and a hemispherical cavity connected to the cylindrical hole.
Referring to FIG. 3, in the same culture well 12, the center-to-center spacing between adjacent third cavities 123 is 300. Mu.m.
In other possible embodiments, either side of the three-dimensional sphere-gathering plate 1 is provided with a chamfer for marking or a frosted area that can be marked with a pen. The three-dimensional ball-collecting culture plate 1 is made of nontoxic and harmless organic high polymer materials, glass and the like. In this embodiment, the three-dimensional poly-sphere culture plate 1 is made of Polydimethylsiloxane (PDMS) material. In other possible embodiments, the three-dimensional pellet-forming culture plate 1 is manufactured by using a 3D printing mold and casting using the printed mold. More specifically, the printed mold is made of photosensitive resin. And (3) printing the mold by adopting photosensitive resin through a 3D printing technology according to a design drawing and the like by an operator, filling polydimethylsiloxane into the mold after the mold is molded, and stripping the mold after the mold is molded. The three-dimensional pellet-forming culture plate 1 can be manufactured in a batch manner with high precision by using a 3D printing method and a mold casting method.
Referring to fig. 4, the present embodiment also provides a screening method for the concentration of a chemotherapeutic drug in cancer cells (hereinafter referred to as "screening method"), wherein the screening method uses the three-dimensional pellet culture plate 1. The operation and practical effects of the three-dimensional pellet culture plate 1 and the screening method are explained below by more specific examples of cancer cell chemotherapeutic drugs and concentration screening applications.
The screening method comprises the following steps:
s1: the three-dimensional pellet-forming culture plate 1 is sterilized and treated with a surface modifier coating. Specifically, in S1 of this embodiment, the surface modifier coats the three-dimensional pellet-aggregation culture plate 1 with 3% bovine serum albumin. In other possible embodiments, the surface modifier employs 0.01% to 5% of at least one surface modifier selected from bovine serum albumin, sulfhydryl polyethylene glycol, 2-methacryloyloxyethyl phosphorylcholine, and the like. The surface modifier has the function of modifying the surface inside the three-dimensional pellet-aggregation culture plate 1 and preventing cells from adhering to the wall in the culture process. In other possible embodiments, the surface modification is performed on the inside of the three-dimensional pellet culture plate 1 by a surface modification method based on plasma surface treatment, an electrostatic self-assembly method, ultraviolet light irradiation graft polymerization, surfactant dynamic modification, and the like, so that cell attachment can be further prevented.
Specifically, the treatment event of the surface modifier coating the three-dimensional pellet culture plate 1 was 24 hours. In other possible embodiments, the coating treatment time can be between 24h and 48h according to the material and the surface modifier concentration of the three-dimensional pellet culture plate 1.
In other possible embodiments, the three-dimensional pellet culture plate 1 may be thoroughly cleaned by sterilizing the three-dimensional pellet culture plate 1 with 75% alcohol, and if necessary, by washing with potassium dichromate.
S2, inoculating the primary separated cell suspension which reaches the cell activity rate and the cell density requirement into the three-dimensional pellet culture plate 1 for three-dimensional pellet culture to form the cell three-dimensional pellet. Specifically, in this example S2, the density of inoculated cells was 1000 per culture well. In other possible embodiments, the cell density may be 500 to 2000 per culture well. In this embodiment, in particular, to the operation, the operator may inject the counted cells into the culture well 12 in batches, then settle the cells and remove bubbles by a shaker or a plate centrifuge, so that the cells grow and proliferate in the third chamber 123 and finally the cell aggregation is achieved.
More specifically, in S2 of the present embodiment, the medium in the culture well 12 is refreshed every 24 hours. More specifically, the proportion of the medium in each renewal of the culture well 12 is 50%. In other possible embodiments, the position of the first positioning ring 125 is set differently according to the culture requirement, that is, the ratio of the volumes of the first cavity 121 and the sum of the volumes of the second cavity 122 and the first diversion hole 124 can be adjusted according to the culture requirement, and the ratio of the culture medium in each update culture hole 12 can be changed in a floating manner between 40% and 60%. Since the volume of the third chamber 123 is small in proportion to the whole culture well 12, it can be ignored in rough calculation. When it is necessary to strictly calculate the update ratio, the volume of the third chamber 123 may be calculated into. In other words, considering that the amount of liquid sucked and added is precisely controlled due to the setting position of the first positioning ring 125, the volume of the third chamber 123 may be taken into consideration when the whole design and manufacturing of the culture well 12 or the three-dimensional pellet culture plate 1.
In this example, steps and methods of primary cell isolation are given exemplarily. The primary cell isolation step comprises the following steps before the operation of the donor without any anti-tumor treatment:
(1) Taking 1.0cm under aseptic condition 3 Fresh liver cancer tissue with the size, removing blood dirt and necrotic tissue;
(2) The operator rinsed the treated liver cancer tissue twice with Williams' E medium containing double antibody (100U/mL penicillin mixed with 100. Mu.g/mL streptomycin);
(3) The operator cuts the rinsed liver cancer tissue into about 1mm by using a cutter such as an ophthalmic scissors 3 A tissue block of size;
(4) Adding 0.1% type II collagenase into the sheared tissue blocks, and placing the tissue blocks in a shaking table for digestion at 37 ℃ for 1 hour to prepare cell suspension;
(5) Filtering the digested cell suspension with a 100 mesh screen;
(6) Diluting the filtered cell suspension with Williams' E medium and centrifuging at 50Xg centrifugal force at 4deg.C for 5min;
(7) Removing part of supernatant after centrifugation, taking 5mL of lower layer cells, adding Williams' E culture medium for resuspension, and centrifuging for 5min at 4 ℃ under the centrifugal force of 50 xg;
(8) Discarding all the supernatant after centrifugation, adding 5mL of erythrocyte lysate, and allowing the mixture to act for 5min;
(9) Diluting the reacted erythrocyte lysate with Williams' E medium, and centrifuging at 50Xg centrifugal force at 4 ℃ for 5min;
(10) Discarding all supernatant after centrifugation, and re-suspending cells by using a Williams' E culture medium containing 10% fetal bovine serum, 20 mug/L hepatocyte growth factor, 20 mug/L epidermal growth factor, 100nmol/L dexamethasone, 0.2U/mL insulin, 2mmol/L glutamine and the double antibody to prepare a cell suspension;
(11) Counting the cell viability and the cell density of the liver cancer cell suspension by trypan blue dye exclusion, and if the cell viability is lower than 90%, performing the following steps;
(12) Slowly adding 40% Percoll density gradient separating liquid into the obtained cell suspension along the tube wall, and centrifuging at 4deg.C for 5min with 500xg centrifugal force;
(13) Removing supernatant containing a large amount of dead cells, cell fragments and non-parenchymal cells on the upper layer after centrifugation, adding Williams' E culture medium for dilution, and centrifuging at 4 ℃ for 5min with a centrifugal force of 50 xg;
(14) Discarding all supernatant after centrifugation, repeating the steps (10) and (11) to re-suspend the cells with the complete medium and counting the cell viability and the cell density until the cell viability and the cell density reach the culture requirement.
During the culturing process, it is also necessary to replace the medium with the three-dimensional pellet culture plate 1 (particularly the culture wells 12). When the medium is changed, a micropipette is used. The operator pushes the tip of the micropipette against the first positioning ring 125 to perform half-volume exchange, i.e., one-time exchange of the original medium in 50% of the culture wells 12. In other possible embodiments, the first retaining ring 125 may fulfill other proportions of fluid exchange requirements, depending on the culture requirements.
S3: after the three-dimensional cell pellet reaches the test requirement, adding the drug to be tested with a preset concentration gradient into the culture hole 12. Still more specifically, in step S3, when the culture medium is updated or the drug to be measured is added, the micropipette is operated against the first positioning ring 125. In this embodiment, the operator controls the micropipette to slide from the first upper edge 121-1 of the culture well 12 against the sidewall of the first cavity 121 until the first positioning ring 125 (i.e., the junction between the first lower edge 121-2 and the first deflector hole 12-1A) has a significant angular change in the position of the first positioning ring 125, so as to help the operator identify the position of the first positioning ring 125.
When the liquid is added (including the operations of adding the drug to be tested, refreshing the culture medium, etc.), the area affected by the flow of the liquid is avoided from the third cavity 123 located at the cavity bottom 122-4 under the guidance of the first annular side wall 124-1 of the first deflector hole 124, so that the cells in the third cavity 123 are further protected from the shearing force of the fluid, or the cells or cell clusters cultured in the third cavity 123 are blown out of the third cavity 123 due to the flow of the liquid. More specifically, the present embodiment limits the first extension line 124-1C, which is a corresponding extension line of the bus of the first annular sidewall 124-1, so that the fluid has an initial velocity and an inherent inertia due to being ejected from the micropipette, flows to the second diversion hole 122-5 of the second cavity 122 under the influence of the gravitational field, and slowly flows to the cavity bottom 122-4 of the second cavity 122 under the guidance of the second diversion hole 122-5.
When the liquid is sucked here, the micro-pipette tip abuts against the first positioning ring 125, so that the micro-pipette can only suck the liquid above the first positioning ring 125 and the liquid below the first positioning ring 125 is reserved, and the sucked liquid amount can be controlled by the setting position of the first positioning ring 125. Meanwhile, due to the structure in the culture well 125, particularly the blocking of the first annular sidewall 124-1 in the first deflector hole 124 and the buffering of the first transition hole 122-3, the influence of the liquid disturbance on the cultured cells in the third chamber 123 can be shielded.
Further, the second positioning ring 122-6 is further provided in this embodiment, and the position of the second positioning ring 122-6 can control the liquid sucking in the different amount from the liquid at the first positioning ring 125, that is, different quantitative control is realized in the culture hole. Meanwhile, when the culture hole 12 is cleaned, if the cell aggregation makes the combination of the cells and the third cavity 123 tighter, the micro-pipette tip can be abutted against the second positioning ring 122-6, and the fluid can directly reach the third cavity 123 under the guidance of the side wall of the second diversion hole 122-5, so that the cell aggregation in the third cavity 123 can be flushed out, the cell residue is reduced, and the culture hole 12 is cleaned more thoroughly.
In this example, the test requirement for three-dimensional pellet aggregation of cells was to observe an increase in the diameter of the pellet aggregated into pellets to a relatively constant state. Thereafter, the test agent may be added while a control group and a blank group are provided, each group being provided with a plurality of repeated culture wells 12 to control errors.
In this embodiment, the drug to be tested and different concentration gradients are added: cisplatin (concentration gradient of 30. Mu.g/mL, 15. Mu.g/mL, 7.5. Mu.g/mL, 3.75. Mu.g/mL, 1.875. Mu.g/mL, 0.9375. Mu.g/mL), oxaliplatin (concentration gradient of 50. Mu.g/mL, 25. Mu.g/mL, 12.5. Mu.g/mL, 6.25. Mu.g/mL, 3.125. Mu.g/mL, 1.5625. Mu.g/mL), gemcitabine (concentration gradient of 25. Mu.g/mL, 12.5. Mu.g/mL, 6.25. Mu.g/mL, 3.125. Mu.g/mL, 1.5625. Mu.g/mL, 0.78125. Mu.g/mL), doxorubicin (concentration gradient of 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.3125. Mu.g/mL) were added to the culture well 12, while setting a control group and a blank group, each corresponding to 3-fold-line.
S4, after co-culturing with the drug to be detected for a preset time, measuring cell activity indexes to determine the chemotherapeutic drug aiming at the cells and the sensitive concentration of the chemotherapeutic drug.
Specifically, the cell activity index measured in this example employs a method of indirectly measuring the number of living cells by CCK-8. Under the action of an electron coupling reagent, the 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt can be reduced by dehydrogenase in mitochondria to generate a highly water-soluble orange yellow formazan product (formazan). The shade of color (absorbance) is proportional to the activity of the cell and inversely proportional to cytotoxicity. The absorbance was measured at a wavelength of 450nm using an enzyme-labeled instrument, indirectly reflecting the number of living cells.
In this example, the time of co-culture with the test drug was 48 hours, the solution in each culture well 12 was discarded, the CCK-8 solution was added in an equal amount to make the concentration of CCK-8 in the culture well 12 10%, and the incubation of cells was continued for 4 hours. After the incubation time, the absorbance at 450nm was measured for the liquid in the culture well 12 using a microplate reader. Based on the absorbance data of each group, half inhibition concentration (IC 50) of the corresponding chemotherapeutic drug is calculated and a three-dimensional drug sensitivity map is drawn.
In other possible embodiments, the cellular activity may also be measured directly or indirectly by measuring an index of the concentration of a cellular metabolite, an index of an apoptosis marker, an index of intracellular enzyme activity, staining of a cell, etc.
It should be noted that the micropipette gun and the micropipette gun head are only examples in this embodiment, and other devices capable of achieving the same or similar effects are also applicable to this embodiment and the present invention. Meanwhile, a plurality of pipette guns can be used to adapt to the plurality of culture holes 12 arranged on the three-dimensional pellet-collecting culture plate 1.
In the present practiceIn the example, the microscopic image of the primary liver cancer cells just separated is shown in fig. 5, and the primary liver cancer cells are in a dispersed state, and most of the primary liver cancer cells are polygonal and have clear outlines. FIG. 6 is an immunofluorescence staining chart of Alpha Fetoprotein (AFP) after 1 day of culture of human primary liver cancer cells, wherein immunofluorescence staining shows that Alpha Fetoprotein (AFP) is positively expressed in primary liver cancer cells, and the primary liver cancer cells separated in FIG. 6 show a higher green fluorescence proportion, indicating that the cell density of the primary separated liver cancer cells is higher. FIG. 7 shows an immunofluorescence staining pattern of DAPI or 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole),cell nucleusDAPI was stained blue fluorescence. Fig. 8 is a microscopic enlarged view of the culture well 12 after a certain time of culture, and fig. 9 is a detailed view showing a single third chamber 123 corresponding to fig. 8. In FIG. 8, it can be seen that liver cancer cells basically grow in the third cavity 123, and that most of the cells cultured in the third cavity 123 are similar in size and similar in state. More clearly, referring to fig. 9, fig. 9 shows that the liver cancer cells grow into aggregated cell masses and enter a three-dimensional aggregated cell culture state, and the liver cancer cells are in a good state, which means that the three-dimensional aggregated cell culture plate 1 in this embodiment can effectively make the cells enter the three-dimensional aggregated cell culture state and grow well.
The results of the drug screening experiments of this example are as follows:
FIG. 10 shows the liver cancer cell inhibition rate of cisplatin at different drug concentrations. As can be seen from fig. 10, as the concentration of cisplatin increases, the inhibition rate of hepatoma cells to hepatoma cells increases, and the concentration of cisplatin and the inhibition rate of hepatoma cells show a positive correlation trend, and the curve in fig. 10 is a fitted curve. From the fitted curve, it can be seen that the tendency of increasing the inhibition rate of liver cancer cells is slowed down when the concentration of cisplatin is higher than 5 mug/mL. The drug cisplatin is adopted, and the inhibition rate of liver cancer cells aiming at the donor can reach 69.88 percent at the highest.
FIG. 11 shows the inhibition rate of liver cancer cells by oxaliplatin at various drug concentrations. As can be seen from fig. 11, as the concentration of oxaliplatin increases, the inhibition rate of liver cancer cells increases, and the concentration of oxaliplatin and the inhibition rate of liver cancer cells show a positive correlation trend, and the curve in fig. 11 is a fitted curve. From the fitted curve, it can be seen that the trend of increasing the inhibition rate of liver cancer cells is slowed down when the oxaliplatin concentration is higher than 10 mug/mL. The drug oxaliplatin is adopted, and the inhibition rate of liver cancer cells aiming at the donor can reach 81.95 percent at the highest.
FIG. 12 shows the inhibition of liver cancer cells by gemcitabine at various drug concentrations. As can be seen from fig. 12, as the concentration of gemcitabine increases, the inhibition rate of liver cancer cells increases, and the concentration of gemcitabine and the inhibition rate of liver cancer cells show a positive correlation trend, and the curve in fig. 12 is a fitted curve. From the fitted curve, it can be seen that the tendency of the liver cancer cell inhibition rate to rise is slowed down when the concentration of gemcitabine is higher than 5 mug/mL. The drug gemcitabine is adopted, and the inhibition rate of liver cancer cells aiming at the donor can reach 87.24 percent at the highest.
FIG. 13 shows the inhibition rate of liver cancer cells by doxorubicin at various drug concentrations. As can be seen from fig. 13, as the concentration of doxorubicin increases, the inhibition rate of hepatoma cells increases, and the concentration of doxorubicin and the inhibition rate of hepatoma cells show a positive correlation trend, and the curve in fig. 13 is a fitted curve. From the fitted curve, it can be seen that the trend of increasing the inhibition rate of liver cancer cells is slowed down when the concentration of doxorubicin is higher than 2 mug/mL. The drug doxorubicin is adopted, and the inhibition rate of liver cancer cells aiming at the donor can reach 71.29 percent at the highest.
According to the above experimental results, a drug sensitivity map is drawn by using a certain algorithm as shown in fig. 14, and in this embodiment, SPSS (Statistical Product and Service Solutions, statistical product and service solution) software is exemplarily used for calculation. In FIG. 14, the ordinate indicates the drug concentration (in mg/mL) at half-inhibition time (IC 50) of liver cancer cells, and the abscissa indicates the drug name. As can be seen from FIG. 14, under the condition of half inhibition of liver cancer cells, the half inhibition concentration of cisplatin is highest and reaches 2.48mg/mL; the half inhibition concentration of the liver cancer cells of the gemcitabine is the lowest and is as low as 0.21mg/mL; oxaliplatin half maximal inhibitory concentration was 0.96mg/mL; the half inhibition concentration of the doxorubicin hepatoma cell is 0.42mg/mL. In contrast, in this example, it can be concluded that the liver cancer cells of the donor are most sensitive to gemcitabine, doxorubicin and oxaliplatin, and least sensitive to cisplatin. The drug sensitivity result can guide the drug application and compatibility of the drug pertinently, and the sensitivity result has specificity for the donor based on the screening method and is more pertinently.
It is noted that the above drugs are merely exemplary in this embodiment, and the operator may select other drugs and apply the screening method without departing from the scope of the guiding ideas and technical solutions of the present invention. The invention can be applied to drug screening experiments of other cancer cells or other malignant proliferation cells except liver cancer cells.
The three-dimensional pellet-gathering culture plate 1 can enable cells to enter a three-dimensional pellet-gathering culture state due to the special structure of the third cavity 123, so that the cells and cell clusters can be in an approximately in-vivo state, the in-vivo state can be better simulated, and a certain foundation is provided for the screening method. The culture hole 12 in the three-dimensional ball-collecting culture plate 1 provided by the invention is also provided with a plurality of diversion structures and buffer structures, including but not limited to a first diversion hole 124, a second diversion hole 122-5 and the like, which are used for buffering the liquid flow generated when liquid is sucked and added, so that the position and the growth state of cells are prevented from being influenced. The culture holes 12 in the three-dimensional ball-collecting culture plate 1 of the invention also control the quantity of liquid sucked each time by arranging the positions including but not limited to the first positioning ring 125 and the second positioning ring 122-6, thereby realizing the ratio of scheduled replacement liquid and facilitating the operation. The three-dimensional ball-collecting culture plate 1 adopts a manufacturing method of 3D printing combined with die casting, and the three-dimensional ball-collecting culture plate 1 can be rapidly and batched produced so as to adapt to the culture requirement. The three-dimensional ball-collecting culture plate 1 has a large number of culture holes, can meet the requirements of setting a control group, a blank group and repeated experiment compound holes, and can realize drug screening experiments on the same three-dimensional ball-collecting culture plate 1 to avoid confusion. The screening method provided by the invention is applied to the three-dimensional poly-sphere culture plate 1, and can screen medicines aiming at different donors and provide a medicine sensitivity map. The screening method utilizes the three-dimensional pellet-gathering culture of the three-dimensional pellet-gathering culture plate 1, can simulate the in-vivo state of a donor to a higher degree, has reliable results and short screening period, and can meet the current requirements.
The foregoing is only a partial embodiment of the invention, and it should be noted that it will be apparent to those skilled in the art that modifications and adaptations can be made without departing from the principles of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (15)
1. The utility model provides a three-dimensional ball culture plate that gathers, its includes orifice plate and with the supporting board lid of orifice plate, its characterized in that: the three-dimensional ball-collecting culture plate is provided with a plurality of culture holes, the culture holes at least comprise a first cavity and a second cavity which are connected through a first diversion hole from top to bottom, and also comprise at least one hemispherical third cavity which is arranged at the bottom of the second cavity, a first positioning ring is arranged at the junction of the first diversion hole and the first cavity, and the tip of the liquid-transferring gun is abutted by the first positioning ring;
the first diversion hole is defined by a first annular side wall, and the diversion upper edge diameter of the first annular side wall is larger than the diversion lower edge diameter of the first annular side wall; a first extension line of a bus of the first annular side wall is intersected with the side wall of the second cavity, so that fluid flowing through the first annular side wall avoids the area where the cavity bottom of the second cavity is located under the action of inertia and natural gravity;
the second cavity comprises a first transition hole bordering the first diversion hole and a second diversion hole connecting the first transition hole and the cavity bottom, and the first transition hole is a cylindrical hole or a reverse tapered hole; the second diversion hole is an inverted conical hole; the first transition hole and the second diversion hole are used for slowly draining fluid from the first cavity to the cavity bottom of the second cavity.
2. The three-dimensional sphere-gathered culture plate of claim 1, wherein: the first cavity is formed by expanding upwards from the diversion upper edge, and comprises a first upper edge serving as the edge of the culture hole and a first lower edge overlapped with the diversion upper edge; the second cavity is formed by expanding downwards from the diversion lower edge, and comprises a second upper edge overlapped with the diversion lower edge and a second lower edge bordered by the cavity bottom.
3. The three-dimensional sphere-gathered culture plate of claim 2, wherein: the diameter of the first upper edge is greater than or equal to the diameter of the first lower edge.
4. The three-dimensional sphere-gathered culture plate of claim 1, wherein: the junction of the first transition hole and the second deflector hole is defined as a second positioning ring.
5. The three-dimensional sphere-gathered culture plate of claim 1, wherein: the total volume of the culture wells is greater than or equal to 50 μl; the sum of the volumes of the second cavity and the first diversion hole is equal to the volume of the first cavity.
6. The three-dimensional sphere-gathered culture plate of claim 1, wherein: the third cavities are circumferentially arranged and/or equidistantly arranged at the bottom of the second cavity.
7. The three-dimensional sphere-gathered culture plate of claim 1, wherein: the diameter of the third cavity is 400 mu m; the depth of the third cavity is 400 mu m; the center-to-center distance between adjacent third cavities in the same culture well is 300 μm.
8. The three-dimensional sphere-gathered culture plate of claim 1, wherein: the culture wells are arranged in a matrix on the well plate.
9. A screening method for the concentration of a cancer cell chemotherapeutic drug, which is characterized by comprising the following steps: the screening method adopts the three-dimensional pellet-gathering culture plate as claimed in any one of claims 1 to 8, and comprises the following steps:
(1) Sterilizing the three-dimensional pellet-forming culture plate, and coating the three-dimensional pellet-forming culture plate with a surface modifier;
(2) Inoculating the primary separated cell suspension into the three-dimensional pellet-forming culture plate for three-dimensional pellet-forming culture to form a cell three-dimensional pellet;
(3) After the three-dimensional cell pellet reaches the test requirement, adding a drug to be tested with a preset concentration gradient into the culture hole;
(4) After co-culturing with the drug to be tested for a predetermined time, determining cell activity index to determine the chemotherapeutic drug to the cells and the sensitive concentration of the chemotherapeutic drug.
10. The method of screening for cancer cell chemotherapeutic drug concentration according to claim 9, wherein: in the step (1), the surface modifier adopts at least one surface modifier of 0.01 to 5 percent of bovine serum albumin, sulfhydryl polyethylene glycol and 2-methacryloyloxyethyl phosphorylcholine; and/or the surface modifier coating treatment time is between 12 and 48 hours.
11. The method of screening for cancer cell chemotherapeutic drug concentration according to claim 9, wherein: the density of the inoculated cells in the step (2) is 500 per culture well to 2000 per culture well.
12. The method of screening for cancer cell chemotherapeutic drug concentration according to claim 9, wherein: in step (2), the medium in the culture wells is refreshed every 24 hours.
13. The method of screening for cancer cell chemotherapeutic drug concentration according to claim 12, wherein: in the step (2), the proportion of the culture medium in each updated culture hole is between 40 and 60 percent.
14. The method of screening for chemotherapeutic agents and concentrations in cancer cells according to claim 12 or 13, wherein: when the culture medium is updated and/or the medicine to be detected is added, the micropipette is used for abutting against the first positioning ring for operation.
15. The method for screening cancer cell chemotherapeutic agents and concentrations according to claim 9, wherein: in the step (4), the cell activity index comprises at least one index of a cell metabolite concentration index, an apoptosis marker concentration index and an intracellular enzyme activity index.
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| US20220348852A1 (en) * | 2019-06-28 | 2022-11-03 | Riken | Culture container and method for producing regenerative hair follicle germ using culture container |
| CN110257249A (en) * | 2019-07-19 | 2019-09-20 | 东北大学 | A kind of micro-fluidic chip and administration cultural method for tumour cell dimensional culture |
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