CN114032174A - 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research - Google Patents
3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research Download PDFInfo
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- CN114032174A CN114032174A CN202111155181.1A CN202111155181A CN114032174A CN 114032174 A CN114032174 A CN 114032174A CN 202111155181 A CN202111155181 A CN 202111155181A CN 114032174 A CN114032174 A CN 114032174A
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Abstract
The invention discloses a 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research. The work designs an open microfluidic control chip with a tree-shaped branch structure, the capillary action of paper-based fibers is utilized to realize the diffusion transportation of fluid, the problem that the traditional microfluidic gradient generator needs external power is solved, and meanwhile, the gradient generator and a cell three-dimensional culture area are integrated on the same platform, so that the drug screening experiment operation with high flux, repeatability and low reagent consumption is realized. Experiments begin with the establishment of an insulin resistance three-dimensional cell model, the reaction degree of insulin resistance of human hepatoma cells HepG2 induced by insulin with different concentrations and the level of glucose consumption of HepG2 cells improved by pioglitazone are discussed, and a stable insulin resistance model and a pioglitazone intervention insulin resistance model are established.
Description
Technical Field
The invention relates to the technical field of medicines and engineering, in particular to a 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research.
Background
Insulin Resistance (IR) is a pathophysiological state in which a target tissue of the body has a lower biological effect than normal insulin, mainly occurs in the liver and peripheral tissues, and is a common pathophysiological basis for common clinical diseases such as type II diabetes, metabolic syndrome, hypertension, obesity, atherosclerosis, and the like. IR is mainly manifested by decreased insulin sensitivity, decreased glucose utilization in peripheral tissues, and failure of liver tissues to effectively inhibit gluconeogenesis and glycogenolysis. Dietary structure, lifestyle habits, genetic and environmental factors can all contribute to insulin resistance. Generally, researchers have studied the pathogenesis of IR and screened effective drugs for treating IR by establishing animal models or two-dimensional interface cell models. However, the difference between the animal and human in physiological biochemistry and anatomical structure exists, and the animal model has the defects of long period and high cost; the traditional two-dimensional culture cell model can only preserve part of original biochemical properties such as cell phenotype, metabolism and gene expression when growing on a two-dimensional interface lacking a three-dimensional microenvironment, and can not simulate a relatively real biological or physiological occurring environment. The three-dimensional cell culture system is a compromise selection of a simple two-dimensional culture model and a complex and expensive animal model, can provide an in-vivo-like microenvironment with dynamic interaction of cells-cells and cell-extracellular matrixes, allows concentration gradients of oxygen, metabolites, protein factors and the like to occur, has the characteristics of short period and relatively low cost, and provides a new direction for constructing a proper IR in-vitro model to research an insulin resistance pathogenesis and an action mechanism of a hypoglycemic drug.
The micro-fluidic chip can realize the accurate control of micro-nano fluid in a channel of micron or nano level, regulate the flow velocity and flow of an inlet sample by elaborately designing a micro-channel network, and form a preset concentration gradient through repeated shunting and confluence in a molecular diffusion leading mode, and is also called a micro-fluidic gradient generator. The microfluidic gradient generator is widely applied to the fields of analytical chemistry, biology, material science and the like due to the advantages of high throughput, low consumption, small volume and the like, and particularly shows unique advantages in the aspect of biology. First, advances in microfabrication technology have made possible the continued miniaturization of channel geometries, with material transport times closer to the physiological range, providing physiologically significant timescales and spatial dimensions. Secondly, the microfluidic chip can integrate a plurality of module designs into the same operation platform, and simultaneously realize gradient generation and three-dimensional cell culture, so that the growth state of cells and the action mode of medicines are closer to the condition in a living body, and a method for controlling the microenvironment of cells in vitro research is changed. Despite the enormous development potential, the large and expensive additional drive pump makes it only feasible for laboratory use and commercial mass production. The dynamic problem of liquid transportation becomes a great obstacle to the micro-fluidic gradient generator to leave the laboratory.
On the basis of the research, the 3D printing concentration gradient chip is constructed by utilizing the characteristics that the 3D printing technology is rapid in forming, a microfluidic platform is easy to integrate, and paper fibers transport liquid by virtue of capillary action, and the 3D printing concentration gradient chip is used for establishing an insulin resistance HepG2 three-dimensional cell model and screening high-throughput medicines. The glucose uptake rate of the cells measured by enzyme-linked immunosorbent assay shows that the cells are 3.334 multiplied by 10-6 μmol·L-1Under the action of insulin, the glucose consumption rate is reduced to about half of that of a normal control group after 24 hours; the pioglitazone dried prognosis, insulin sensitivity of the insulin resistant cell model was improved.
Disclosure of Invention
1. The invention aims to provide a 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research.
The invention aims to realize a 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research, which is characterized in that a chip model with a tree-shaped branch structure is obtained by a 3D printing technology, sodium carboxymethylcellulose (CMCNa) solution is used as an adhesive, and alpha-cellulose is used as a paper-based matrix to construct an open concentration gradient microfluidic chip.
The 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research is characterized in that the chip main body structure of the open concentration gradient microfluidic chip is a tree-shaped branch channel and a circular liquid storage tank, and the circular liquid storage tank in each row can be used for three-dimensional cell culture; all unit channels in the tree-shaped branch channel are horizontally or/and vertically arranged, and each unit channel is 1000 micrometers in width, 500 micrometers in depth and 0.8 cm in length; the diameter of the circular liquid storage pool is 0.8 cm, and the depth is 500 mu m; a cylindrical sample inlet is connected to the unit channel at the top of the tree-shaped branch channel, the diameter of the cylindrical sample inlet is 1 cm, and the height of the cylindrical sample inlet is 1 cm; CMCNa-alpha-cellulose colloidal liquid with the thickness of 500 mu m is uniformly coated on the surfaces of the unit channel and the circular liquid storage tank, and a paper-based medium with a transmission function can be formed after drying.
The 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research is characterized in that the open type concentration gradient microfluidic chip is based on a model channel obtained by 3D printing, and the distribution of material distribution, mixing and concentration gradient is realized by utilizing the capillary action of paper-based fibers.
The 3D printing concentration gradient chip is used for researching the glucose consumption of a pioglitazone improved HepG2 insulin resistance model, and comprises the following steps: (1) three-dimensional culture of cells on an open concentration gradient microfluidic chip: taking cells in logarithmic growth phase for digestion, counting, and diluting to 4 × 105Diluting with the same volume of hydrogel solution to 2 × 105Perml, with 30. mu.L of cell suspension per well, inoculated into an open concentration gradient microfluidic chipIn a circular reservoir, 5 WT% CO at 37 ℃2The incubator is kept still for culture; (2) intervention of insulin and pioglitazone: sucking out culture medium after culturing cells for 24 h, and respectively adding 1 × 10 concentration micro-fluidic chip into the first sample inlet-5And 1X 10-7 μmol·L-1Adding normal cell culture solution into the second sample inlet, co-culturing with cells for 24 h, and sucking 10 mu L of cell culture solution per hole for ELISA experiment; sucking out culture medium after culturing cells for 24 h, and adding 1 × 10 final concentration sample into the first sample inlet of the open type concentration gradient microfluidic chip-4 μmol·L-1Adding normal cell culture solution into the second sample inlet, co-culturing pioglitazone and cells for 6 h, discarding the cell culture solution containing pioglitazone, and adding 9.095 × 10-6 μmol·L-1After 24 hours, 10 mu L of cell culture solution is absorbed by each hole for ELISA experiment; (3) determination of cellular glucose uptake: after intervention of pioglitazone and insulin, the content of the residual glucose in the culture solution was determined by using a commercial ELISA kit, and the glucose content in the culture solutions of the blank control group and the normal control group was simultaneously determined to calculate the change of the glucose uptake rate of the cells.
2. The invention also aims to provide an open concentration gradient chip, which is characterized in that a chip model with a tree-shaped branch structure is taken as a basis, a CMCNa-alpha-cellulose self-made paper-based medium is modified in a channel, and the material shunting, mixing and concentration gradient distribution are realized by utilizing the capillary action of fibers.
3. The third purpose of the invention is to establish an insulin resistance in-vitro three-dimensional cell model and a pioglitazone intervention insulin resistance cell model on the open concentration gradient chip.
4. The technical scheme of the invention comprises the following steps:
(1) three-dimensional culture of cells on concentration gradient chips
Taking cells in logarithmic growth phase for digestion, counting, and diluting to 4 × 105Diluting with the same volume of hydrogel solution to 2 × 105Perml, 30. mu.L of cell suspension per well was inoculated into a circular cell culture cell of a concentration gradient chip at 37 ℃ with 5% CO2The incubator (2) is kept still for culture.
(2) Intervention of insulin and pioglitazone
Sucking out culture medium after culturing cells for 24 h, and respectively adding 1 × 10 concentration solution into the first sample inlet 1 of each chip-5And 1X 10-7 μmol·L-1After the insulin solution and the cells are co-cultured for 24 hours, 10 mu L of cell culture solution is absorbed by each hole for ELISA experiment; sucking out culture medium after culturing cells for 24 hr, and adding the final concentration of 1 × 10 into the second sample inlet 2 of the chip-4 μmol·L-1Co-culturing pioglitazone and cells for 6 h, discarding the cell culture solution containing pioglitazone, and adding the cell culture solution into a circular cell culture pool at the concentration of 9.095 × 10-6 μmol·L-124 h, 10. mu.L of cell culture medium per well was aspirated for ELISA experiments.
(3) Determination of cellular glucose uptake
After intervention of pioglitazone and insulin, the content of the residual glucose in the culture solution was determined by using a commercial ELISA kit, and the glucose content in the culture solutions of the blank control group and the normal control group was simultaneously determined to calculate the change of the glucose uptake rate of the cells.
The invention has the beneficial effects that:
the diffusion transportation of fluid is realized by utilizing the capillary action of alpha-cellulose, the trouble that the traditional microfluidic gradient generator needs external power is eliminated, and meanwhile, the gradient generator and the three-dimensional cell culture area are integrated on the same chip platform, so that the drug screening experiment operation with high flux, repeatability and low reagent consumption is realized.
Drawings
FIG. 1 is a structure diagram of a 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research (in the figure, 1: a sample inlet 1, 2: a sample inlet 2, 3: a flow channel, and 4: a cell culture pool).
Fig. 2 is a test chart of a concentration gradient chip in a 3D printing concentration gradient chip for pioglitazone improved HepG2 insulin resistance model research, in which: a is a colorimetric representation map; and B is a gray distribution diagram.
Fig. 3 is a three-dimensional reconstruction diagram of HepG2 cell-hydrogel confocal structure in a 3D printing concentration gradient chip for pioglitazone-modified HepG2 insulin resistance model research, wherein: a is agarose gel picture; b is HepG2 cell profile; c is agarose gel-HepG 2 cell fusion picture.
FIG. 4 is a glucose consumption level investigation diagram of HepG2 cells in a 3D printing concentration gradient chip for research of pioglitazone improved HepG2 insulin resistance model after insulin drying prognosis.
FIG. 5 is a glucose consumption level graph of a HepG2 islet resistance in vitro cell model in a 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research after the prognosis of pioglitazone.
Detailed Description
In order to make the technical problems, technical solutions and effects to be solved by the present invention clearer, the present invention is described in further detail below with reference to embodiments and drawings. The first and second aspects of the present invention are used for distinguishing purposes only and are not used in a sequential relationship.
As shown in fig. 1, the structure diagram of a 3D printing concentration gradient chip for pioglitazone improved HepG2 insulin resistance model research is as follows: the main structure of a 3D printing concentration gradient chip (also called an open concentration gradient micro-fluidic chip) is a tree-shaped branch channel and a circular liquid storage tank, and the circular liquid storage tank in each row can be used for three-dimensional cell culture; all unit channels in the tree-shaped branch channel are horizontally or/and vertically arranged, and each unit channel is 1000 micrometers in width, 500 micrometers in depth and 0.8 cm in length; the diameter of the circular liquid storage pool is 0.8 cm, and the depth is 500 mu m; a cylindrical sample inlet is connected to the unit channel at the top of the tree-shaped branch channel, the diameter of the cylindrical sample inlet is 1 cm, and the height of the cylindrical sample inlet is 1 cm; CMCNa-alpha-cellulose colloidal liquid with the thickness of 500 mu m is uniformly coated on the surfaces of the unit channel and the circular liquid storage tank, and a paper-based medium with a transmission function can be formed after drying; taking the cells in logarithmic growth phase for digestion and countingDiluting to 4X 105Diluting with the same volume of hydrogel solution to 2 × 105The cell suspension of 30 mul per hole is inoculated into a cell culture pool (also called as a round liquid storage pool) 4 of a concentration gradient chip (also called as an open concentration gradient micro-fluidic chip, and is called as a chip for short) at 37 ℃ and 5 percent CO2The incubator (2) is kept still for culture. Cell culture medium and drug-containing conditioned medium are respectively added into a first sample inlet 1 and a second sample inlet 2 of the chip, and are transported to a cell culture pool (also called a circular liquid storage pool) 4 through the capillary action of fibers in a flow channel (also called a unit channel) 3, so that the concentration gradient distribution of the drugs is realized.
Example 1:
diluting the blue ink stock solution by 200 times, injecting 1 ml of blue ink diluent into a first sample inlet 1 of the chip, simultaneously injecting 1 ml of deionized water into a second sample inlet 2, keeping the pressures at two sides balanced, completely carrying out mixed transmission of the solution by virtue of capillary action, and analyzing and processing the change of color intensity by ImageJ after a stable colorimetric gradient is formed.
As shown in a in fig. 2, the color gradient of the whole chip is very obvious, and at the final outlet, the colors of blue become lighter from left to right in sequence, and the rightmost end is colorless, so that the generation of the concentration gradient is confirmed; because the surface morphology of the alpha-cellulose powder is ordered, the color of the chip is uniform, and no obvious coffee circle reaction exists. The change of the relative gray value is consistent with the color change of the chip, and the digital presentation mode is more favorable for intuitively calculating the specific concentration directly corresponding to the outlet of the chip.
Example 2:
a cell culture method in a 3D printing concentration gradient chip for pioglitazone improved HepG2 insulin resistance model research comprises the following steps:
taking cells in logarithmic growth phase, digesting, counting, and diluting to 4 × 105Diluting with the same volume of hydrogel solution to 2 × 105Perml, 30. mu.L of cell suspension per well was inoculated into a circular cell culture cell 4 of a concentration gradient chip at 37 ℃ with 5% CO2The incubator (2) is kept still for culture.
As shown in FIG. 3, for the confocal three-dimensional characterization reconstruction of the cell cultured in the hydrogel, it can be observed that the green fluorescent protein labeled HepG2 cell is suspended in the middle of the hydrogel medium, the cell and the cell can be in full contact, and signal molecules can be transmitted to distant cells through the hydrogel, the living space of the cell grows exponentially, and the multilayer culture mode is adopted; the cells are uniformly distributed in the horizontal direction, the polarization of the cells is reduced, and synapses are shortened, and the cells are just like being embedded in the hydrogel, but the vitality of the cells is strong enough to indicate that the hydrogel can provide a biocompatible in vivo-simulated microenvironment for cell proliferation.
Example 3:
a method for establishing an insulin resistance cell model in a 3D printing concentration gradient chip for research of a pioglitazone improved HepG2 insulin resistance model comprises the following steps:
taking cells in logarithmic growth phase for digestion, counting, and diluting to 4 × 105Diluting with the same volume of hydrogel solution to 2 × 105Perml, 30. mu.L of cell suspension per well was seeded into the circular cell culture well 4 of the concentration gradient chip. Sucking out culture medium after culturing cells for 24 h, and respectively adding 1 × 10 concentration solution into the first sample inlet 1 of each chip-5And 1X 10-7 μmol·L-1The insulin solution of (1). After the insulin and the cells are co-cultured for 24 h, 10 mu L of cell culture solution is absorbed by each hole for ELISA experiment, and the glucose consumption rate is detected.
As shown in fig. 4, the level of glucose consumed by the cells decreased under the action of insulin in a dose-dependent manner, with increasing concentrations of insulin acting, the cells uptake glucose at lower and lower levels, i.e., with lower and lower insulin sensitivity. The hydrogel three-dimensional scaffold can simulate a complex microenvironment around the liver, small molecular substances such as amino acid, glucose, growth factors, drugs and the like exist in the matrix scaffold in a concentration gradient closer to a physiological microenvironment, the mechanical property and the biochemical property of the hydrogel become unpredictable variables of interaction between cells and the hydrogel, the heterogeneous development of the cells is promoted, the diversified reaction of the cells to stimulation is guided, and the development condition of real insulin resistance in vivo is closer to the development condition.
Example 4:
an intervention method for improving the glucose uptake rate of an insulin resistance cell model by pioglitazone in a 3D printing concentration gradient chip for research of a pioglitazone improvement HepG2 insulin resistance model is as follows:
taking cells in logarithmic growth phase for digestion, counting, and diluting to 4 × 105Diluting with the same volume of hydrogel solution to 2 × 105Perml, 30. mu.L of cell suspension per well was inoculated into a circular culture well of a concentration gradient chip. Sucking out culture medium after culturing cells for 24 h, and adding 1 × 10 final concentration into the first sample inlet 1 of the chip-4 μmol·L-1Co-culturing pioglitazone and cells for 6 h, discarding the cell culture solution containing pioglitazone, and adding the cell culture solution into a circular cell culture pool at the concentration of 9.095 × 10-6 μmol·L-1After 24 hours, 10. mu.L of cell culture solution per well was aspirated for ELISA assay to determine the glucose consumption rate.
As shown in FIG. 5, under the three-dimensional culture condition, the pioglitazone action obviously improves the glucose consumption rate of cells and improves the sensitivity of the cells to insulin. The cells generate different response levels to different concentrations of pioglitazone, and the higher the concentration of pioglitazone is, the more the sensitivity of the cells to insulin is improved, and the more glucose is consumed by the cells. Under the condition of three-dimensional hydrogel scaffold culture, HepG2 cells are more favorable for keeping the original physiological state, namely 1 × 10-4 μmol·L-1Under the intervention of pioglitazone, the glucose uptake rate of cells is improved by about 20 percent.
Claims (4)
1. A3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research is characterized in that a chip model with a tree-shaped branch structure is obtained by a 3D printing technology, sodium carboxymethylcellulose (CMCNa) solution is used as an adhesive, and alpha-cellulose is used as a paper-based matrix to construct an open type concentration gradient microfluidic chip.
2. The 3D printing concentration gradient chip for pioglitazone improvement HepG2 insulin resistance model research of claim 1, wherein the chip main body structure of the open concentration gradient microfluidic chip is a tree branch channel and a circular liquid storage tank, and the circular liquid storage tank of each row can be used for three-dimensional cell culture; all unit channels in the tree-shaped branch channel are horizontally or/and vertically arranged, and each unit channel is 1000 micrometers in width, 500 micrometers in depth and 0.8 cm in length; the diameter of the circular liquid storage pool is 0.8 cm, and the depth is 500 mu m; a cylindrical sample inlet is connected to the unit channel at the top of the tree-shaped branch channel, the diameter of the cylindrical sample inlet is 1 cm, and the height of the cylindrical sample inlet is 1 cm; CMCNa-alpha-cellulose colloidal liquid with the thickness of 500 mu m is uniformly coated on the surfaces of the unit channel and the circular liquid storage tank, and a paper-based medium with a transmission function can be formed after drying.
3. The 3D printing concentration gradient chip for pioglitazone improved HepG2 insulin resistance model research of claim 2, wherein the open type concentration gradient microfluidic chip is based on a model channel obtained by 3D printing and realizes the distribution of material distribution, mixing and concentration gradient by using the capillary action of paper-based fibers.
4. The 3D printing concentration gradient chip as defined in any one of claims 1-3, which is used for research on improvement of glucose consumption of HepG2 insulin resistance model by pioglitazone, and comprises the following steps: (1) three-dimensional culture of cells on an open concentration gradient microfluidic chip: taking cells in logarithmic growth phase for digestion, counting, and diluting to 4 × 105Diluting with the same volume of hydrogel solution to 2 × 105Perml, 30. mu.L of cell suspension per well was inoculated into a circular reservoir of an open concentration gradient microfluidic chip at 37 ℃ with 5 WT% CO2The incubator is kept still for culture; (2) intervention of insulin and pioglitazone: sucking out culture medium after culturing cells for 24 h, and respectively adding 1 × 10 concentration micro-fluidic chip into the first sample inlet-5And 1X 10-7μmol·L-1Adding normal cell culture solution into the second sample inlet, and co-culturing with cells for 24 hThen, 10. mu.L of cell culture solution was aspirated from each well for ELISA assay; sucking out culture medium after culturing cells for 24 h, and adding 1 × 10 final concentration sample into the first sample inlet of the open type concentration gradient microfluidic chip-4 μmol·L-1Adding normal cell culture solution into the second sample inlet, co-culturing pioglitazone and cells for 6 h, discarding the cell culture solution containing pioglitazone, and adding 9.095 × 10-6 μmol·L-1After 24 hours, 10 mu L of cell culture solution is absorbed by each hole for ELISA experiment; (3) determination of cellular glucose uptake: after intervention of pioglitazone and insulin, the content of the residual glucose in the culture solution was determined by using a commercial ELISA kit, and the glucose content in the culture solutions of the blank control group and the normal control group was simultaneously determined to calculate the change of the glucose uptake rate of the cells.
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