CN110885780B - Organ chip-based method for simulating human early placenta development microenvironment by hypoxia - Google Patents

Organ chip-based method for simulating human early placenta development microenvironment by hypoxia Download PDF

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CN110885780B
CN110885780B CN201811040903.7A CN201811040903A CN110885780B CN 110885780 B CN110885780 B CN 110885780B CN 201811040903 A CN201811040903 A CN 201811040903A CN 110885780 B CN110885780 B CN 110885780B
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秦建华
王慧
朱玉娟
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention discloses a method for simulating a microenvironment for early placenta development of a human by hypoxia based on an organ chip, which mainly comprises the following steps: the invention utilizes the self-assembly property of the hPSC to form a three-dimensional cavity structure with near physiology in a three-dimensional matrix material, simultaneously inspects the difference of the cell morphology and the function in the early placenta development model established under different oxygen concentrations, and proves that the invasion capacity of trophoblast cells in the placenta generation under the hypoxia condition is obviously enhanced. The invention can provide a new idea and technical platform for exploring HIF signal path and molecular mechanism research in human placenta generation.

Description

Organ chip-based method for simulating microenvironment for early placenta development of human by hypoxia
Technical Field
The invention relates to the field of placenta development, epigenetics, medicine and cell biology, in particular to a method for simulating a human early placenta development microenvironment by hypoxia based on an organ chip.
Background
The incidence of pregnancy abnormalities worldwide is on a rising trend year by year and is caused by a number of factors, of which placental dysplasia is the major factor. Professor of the high-level lifetime researcher, clock south, new york state basic medical research institute, usa indicates that: "more and more studies show that human diseases in childhood and even adulthood are related to the growth and development of placenta. After the human placenta plan is started in the united states, obstetrician experts in china also pay more attention to the research of the placenta, and the research contents mainly relate to related fields of placenta structure, infection and inflammation, genomics and placenta, placenta function, environmental factors and placenta and the like. Because the time node of the placenta is the most transient, modern medicine researches the placenta to a far deeper extent than other organs, which also attracts great attention of researchers.
The development of the placenta, starting with the implantation of the blastocyst, is critical to the growth of the developing fetus by providing effective nutrients and oxygen to the fetus. Early human placental development involves differentiation of precursors of trophoblasts surrounding tooth decay, the appearance of subtypes such as mature to syncytiotrophoblast and invasive trophoblasts, where the supply of oxygen is not a constant value, i.e., the oxygen content varies at different stages during placental development. Between 0 and 13 weeks of gestation, the fertilized blastocyst becomes embedded in the uterine wall and the fetus and placenta begin to develop, at which time the placenta is in a relatively hypoxic environment. However, studies on placental development are still poorly understood due to experimental obstacles.
Human pluripotent stem cells (hPSCs) resemble embryonic cells in their self-renewing ground state, but the suitability of hPSCs in mimicking early placental development is not yet determined. Although the concentration of oxygen in the placenta varies at different stages during pregnancy, the research on the influence of oxygen on the morphology and function of cells in early placenta development, including the differentiation of trophoblast cells, the appearance of confluent trophoblast cells and invasive trophoblast cells, especially under hypoxic conditions, is still rare, and the research on the signal path related to HIF and the molecular mechanism are also involved. These help us to understand more deeply the occurrence of placenta and provide technical support for exploring the aspects of human placental development and embryonic development in terms of cellular level, gene sequencing and molecular mechanisms. Can also help the medical field to better know the nature of life and further develop the research on related life functions.
Disclosure of Invention
The invention aims to provide a method for simulating a microenvironment for early placenta development of a human by hypoxia based on an organ chip, wherein the model simulates the development of the early placenta of the human in a 3D matrix to form a three-dimensional structure with near physiology, and the influence of oxygen concentration on the early placenta development of the human is examined, wherein the influence of oxygen on cell morphology and function is considered. The model can provide a technical platform for exploring cytomorphology research, epigenetics research, molecular mechanism and signal path research in early placenta development of human beings.
The invention relates to a method for simulating a microenvironment for early placenta development of a human by hypoxia based on an organ chip, which adopts an artificial basement membrane (Matrigel) made of a three-dimensional matrix material and comprises the following steps:
(1) Modification of well plate and inoculation culture of human pluripotent stem cells (hPSCs)
The 6-well plate is modified by an artificial basement membrane (Matrigel) and placed in a 4-degree refrigerator, then placed in a 37-degree incubator for 20-50min, digested by cell digestive fluid at room temperature, cell clusters are scraped by a cell scraper, centrifugation is carried out for 600r 1-3min, cells are resuspended by mTeSR1 culture medium containing Y27632 to enable the cells to be small clusters and then inoculated in the modified 6-well plate, and the culture solution is replaced by mTeSR1 culture medium without Y27632 after being cultured for 1-4h at 37 ℃.
The artificial basement membrane modified 6-pore plate specifically comprises the following steps: diluting the artificial basement membrane by 500-1000 times, and putting 1.5ml of diluent in each hole in a 4-degree refrigerator for 4 hours-7 days;
in the mTeSR1 medium containing Y27632, the concentration of Y27632 ranges from 1 to 100mM.
(2) Cells were digested and seeded in a three-dimensional matrix
And (3) carrying out warm digestion by using a digestive juice after the hPSC cells grow to 70-90%, scraping cell masses by using a cell scraper, centrifuging for 600r 1-3min, sucking supernatant fluid, diluting matrigel to a certain proportion by using a culture medium containing BMP4 and inducing hPSC differentiation, then carrying out heavy suspension on the cells to obtain proper inoculation density and inoculating, placing in a 37-DEG culture box for 20-40min to solidify the matrigel in which the cells are suspended, and then replacing the culture medium containing BMP4 and inducing hPSC differentiation.
The matrigel dilution is specifically a culture medium: the volume ratio of matrigel is 0-3 4 -1×10 10 cells/ml, medium components of hPSC differentiation induction medium containing BMP4 were:
96% by volume of DMED/F12, 1% by volume of diabody, 1% by volume of glutamine, 1% by volume of nonessential amino acids (MEMNEAA), 1% by volume of ITS, 100ng/ml of heparin, 10ng/ml of BMP4, and adding 1g of BSA to 50ml of the prepared medium to dissolve the BMP 4-containing medium to induce hPSC differentiation.
(3) Establishment of early placenta development model under different oxygen concentrations
And (3) replacing the culture medium in the step (2) every day, wherein the culture medium is the culture medium containing the BMP4 and inducing the hPSC to differentiate in the step (2), and models with different oxygen concentrations are built for several days.
The oxygen concentration range is 2% -21%, and the model establishing time range is 3-15 days.
(4) Cell morphology and functional characterization
The expression of E-cadherin, F-actin and HIF-1a is detected by adopting a conventional immunofluorescence staining method, and the gene expression related to an HIF channel is detected by adopting a real-time PCR method.
The invention utilizes the self-assembly property of hPSC to form a three-dimensional cavity structure with near physiology in a three-dimensional matrix material, simultaneously inspects the difference of cell morphology and function in an early placenta development model established under different oxygen concentrations, and proves that the invasion capacity of trophoblast cells in the placenta generation is obviously enhanced under the hypoxia condition. The invention can provide a new idea and technical platform for exploring HIF signal path and molecular mechanism research in human placenta generation.
Drawings
FIG. 1 is a schematic diagram of a method for modeling a microenvironment for early placental development in humans based on hypoxia of an organ chip constructed in example 1 of the present invention.
FIG. 2 is a brightfield image of cells seeded in a three-dimensional matrix in example 1 of the invention, including brightfield images on day one and day six, bar:25um.
FIG. 3 is a photograph of immunofluorescent staining of E-cadherin, F-actin, HIF-1a in example 1 of the present invention, bar:25um.
FIG. 4 is a photograph of immunofluorescent staining for E-cadherin, F-actin, HIF-1a in example 2 of the present invention, bar:25um.
FIG. 5 is a graph showing the PCR results of VEGFA, ANKRD37 and SLC2A1 in example 2 of the present invention.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
The present invention uses a three-dimensional matrix material, which is commercially available artificial basement membrane (Matrigel) (ref: 354277, company Corning).
Example 1
The invention relates to a method for simulating a microenvironment for early placenta development of a human by hypoxia based on an organ chip, which adopts an artificial basement membrane (Matrigel) made of a three-dimensional matrix material and comprises the following steps:
(1) Modification of pore plate and inoculation culture of human pluripotent stem cells (hPSC)
The 6-well plate is modified by an artificial basement membrane (Matrigel) and placed in a 4-degree refrigerator, then placed in a 37-degree incubator for 20min, digested by cell digestive fluid at room temperature, cell clusters are scraped by a cell scraper, centrifuged by 600r 2min, cells are resuspended by mTeSR1 culture medium containing Y27632 to form small clusters, then inoculated to the modified 6-well plate, cultured for 1h at 37 ℃, and then the culture fluid is replaced by mTeSR1 culture medium without Y27632 for culture.
The artificial basement membrane modified 6-pore plate specifically comprises the following steps: diluting the artificial basement membrane by 1000 times, wherein each hole is filled with 1.5ml of diluent, and placing the diluted liquid in a 4-degree refrigerator for 7 days;
in the mTeSR1 medium containing Y27632, the concentration of Y27632 was 10mM.
(2) Cells were digested and seeded in a three-dimensional matrix
After the hPSC cells grow to 90%, the cells are digested in a digestion solution at a warm temperature, cell clusters are scraped by a cell scraper, the cells are centrifuged for 600r 2min, supernatant fluid is sucked away, matrigel is diluted to a certain proportion by a culture medium containing BMP4 and inducing hPSC differentiation, the cells are resuspended to obtain a proper inoculation density and inoculated, the matrigel with the suspended cells is placed in a 37-degree culture box for 20min to solidify, and then the culture medium containing BMP4 and inducing hPSC differentiation is replaced.
The matrigel dilution is specifically a culture medium: the volume ratio of matrigel is 0:1, cell seeding density of 2X 10 6 cells/ml, medium components of hPSC differentiation induction medium containing BMP4 were:
96% by volume of DMED/F12, 1% by volume of diabody, 1% by volume of glutamine, 1% by volume of nonessential amino acids (MEMNEAA), 1% by volume of ITS, 100ng/ml of heparin, 10ng/ml of BMP4, and adding 1g of BSA to 50ml of the prepared medium to dissolve the BMP 4-containing medium to induce hPSC differentiation.
The schematic diagram of the method of the placegenesis model established by the above steps is shown in fig. 1.
(3) Establishment of early placenta development model under different oxygen concentrations
And (3) replacing the culture medium in the step (2) every day, wherein the culture medium is the culture medium containing BMP4 for inducing hPSC differentiation in the step (2), the oxygen concentration is maintained at 20%, and the model establishment time is 6 days. The schematic diagram of the in vitro model method for early placenta development of human, which is established according to the above steps, is shown in figure 1. The cell brightfield patterns for the first and sixth days at 20% oxygen concentration are shown in FIG. 2.
(4) Cellular morphological and functional characterization
The expression of E-cadherin, F-actin and HIF-1a is detected by adopting a conventional immunofluorescence staining method, and the gene expression related to the HIF-1a pathway is detected by adopting a real-time PCR method. FIG. 3 is a graph showing the immunofluorescence of the expression of cells E-cadherin, F-actin and HIF-1a in the three-dimensional vacuolar structure at the sixth day of 20% oxygen concentration. As can be seen from the expression graphs of E-cadherin and F-actin, the invasiveness of the cells is weaker in 20% of the conditions, and the HIF-1a signal expression of the cells is basically absent in 20% of the conditions as can be seen from the HIF-1a result graph.
Example 2
The invention relates to a method for simulating a microenvironment for early placenta development of a human by hypoxia based on an organ chip, which adopts a three-dimensional matrix material artificial matrix membrane (Matrigel) and comprises the following steps:
(1) Modification of pore plate and inoculation culture of human pluripotent stem cells (hPSC)
The 6-well plate is modified by artificial basement membrane (Matrigel) and placed in a 4-degree refrigerator, then placed in a 37-degree incubator for 40min, digested by cell digestive fluid at room temperature, cell clusters are scraped by a cell scraper, centrifuged for 600r 3min, cells are resuspended by mTeSR1 culture medium containing Y27632 to form small clusters, then inoculated in the modified 6-well plate, cultured for 3h at 37 ℃, and then the culture fluid is replaced by mTeSR1 culture medium without Y27632 for culture.
The artificial basement membrane modified 6-pore plate specifically comprises the following steps: diluting the artificial basement membrane by 500 times, wherein each hole is filled with 1.5ml of diluent, and the diluent is placed in a 4-degree refrigerator for 4 hours;
the concentration of Y27632 in the mTeSR1 medium containing Y27632 was 10mM.
(2) Cells were digested and seeded in a three-dimensional matrix
After the hPSC cells grow to 90%, the cells are digested in a digestion solution at a warm temperature, cell clusters are scraped by a cell scraper, centrifugation is carried out for 600r 3min, supernatant fluid is sucked away, matrigel is diluted to a certain proportion by a culture medium containing BMP4 and inducing hPSC differentiation, the cells are resuspended to obtain a proper inoculation density and inoculated, the matrigel with the suspended cells is placed in a 37-degree culture box for 30min to be solidified, and then the culture medium containing BMP4 and inducing hPSC differentiation is replaced.
The matrigel dilution is specifically a culture medium: the volume ratio of matrigel is 0:1,
DMED/F12 with a volume concentration of 96%, diabody with a volume concentration of 1%, glutamine with a volume concentration of 1%, non-essential amino acid (MEMNEAA) with a volume concentration of 1%, ITS with a volume concentration of 1%, heparin with a volume concentration of 100ng/ml, BMP4 with a volume concentration of 10ng/ml, and 1g bsa was added to each 50ml of the prepared medium, and the medium containing BMP4 for inducing hPSC differentiation was obtained after dissolution, and a schematic diagram of the method of the placentation model established according to the above steps is shown in fig. 1.
(3) Establishment of early placenta development model under different oxygen concentrations
And (3) changing the culture medium every day in the step (2), wherein the culture medium is the culture medium containing BMP4 for inducing hPSC differentiation in the step (2), the oxygen concentration is maintained at 20%, the model is built for 4 days, the oxygen concentration is maintained at 5%, the model building is continued for 2 days, and the total model building time is 6 days. And establishing an in vitro model of the human early placenta development under the hypoxia condition according to the steps.
(4) Cellular morphological and functional characterization
The expression of E-cadherin, F-actin and HIF-1a is detected by adopting a conventional immunofluorescence staining method, and the gene expression related to an HIF channel is detected by adopting a real-time PCR method. FIG. 4 is a graph showing immunofluorescence plots for the expression of E-cadherin, F-actin and HIF-1a in cells in a three-dimensional vacuolar structure at 20% oxygen concentration for 4 days and at 5% oxygen concentration for 2 days. As can be seen from the E-cadherin and F-actin expression patterns, the invasion ability of the trophoblast cells is significantly enhanced under 5% conditions compared to the case 1 where 20% oxygen concentration is maintained, which means that the invasion ability of the trophoblast cells is enhanced under hypoxic conditions in terms of cell behavior. As can be seen from the results of HIF-1a, the HIF-1a signal expression of the cells in the model constructed in example 2 was very strong compared to the conditions in which 20% of the oxygen concentration was always maintained in example 1, indicating that the behavior of the cells under the hypoxic condition in example 2 is correlated with the HIF-1a signal, as shown in FIG. 5, comparing the expression of the HIF-1 a-associated gene in example 2 with that in case 1, it was revealed from the PCR results of VEGFA, ANKRD37, and SLC2A1 that the hypoxic condition used in case 2 was indeed upregulated with the HIF-1 a-associated gene, which means that the oxygen concentration has a significant effect on the HIF-1a pathway during the development of the early placenta.
Example 3
The invention relates to a method for simulating a microenvironment for early placenta development of a human by hypoxia based on an organ chip, which adopts an artificial basement membrane (Matrigel) made of a three-dimensional matrix material and comprises the following steps:
(1) Modification of pore plate and inoculation culture of human pluripotent stem cells (hPSC)
The 6-well plate is modified by an artificial basement membrane (Matrigel) and placed in a 4-degree refrigerator, then placed in a 37-degree incubator for 50min, digested by cell digestive fluid at room temperature, cell clusters are scraped by a cell scraper, centrifuged by 600r 1min, cells are resuspended by mTeSR1 culture medium containing Y27632 to form small clusters, then inoculated to the modified 6-well plate, cultured for 4h at 37 ℃, and then the culture fluid is replaced by mTeSR1 culture medium without Y27632 for culture.
The artificial basement membrane modified 6-pore plate specifically comprises the following steps: diluting the artificial basement membrane by 800 times, wherein each hole is filled with 1.5ml of diluent, and the diluted liquid is placed in a 4-degree refrigerator for 3 days;
the concentration of Y27632 in the mTeSR1 medium containing Y27632 was 10mM.
(2) Cells were digested and seeded in a three-dimensional matrix
After the hPSC cells grow to 70%, the cells are digested in a digestion solution at a warm temperature, cell clusters are scraped by a cell scraper, the cells are centrifuged for 600r 2min, supernatant fluid is sucked away, matrigel is diluted to a certain proportion by a culture medium containing BMP4 and inducing hPSC differentiation, the cells are resuspended to obtain a proper inoculation density and inoculated, the matrigel with the suspended cells is placed in a 37-degree culture box for 20min to solidify, and then the culture medium containing BMP4 and inducing hPSC differentiation is replaced.
The matrigel dilution is specifically a culture medium: matrigel volume ratio of 3 8 cells/ml, medium components of hPSC differentiation induction medium containing BMP4 were:
96% by volume of DMED/F12, 1% by volume of a double antibody, 1% by volume of glutamine, 1% by volume of an unnecessary amino acid (MEMNEAA), 1% by volume of ITS, 100ng/ml of heparin and 10ng/ml of BMP4, wherein 1g of BSA is added into every 50ml of the prepared culture medium, and the culture medium containing the BMP4 and inducing hPSC differentiation is obtained after dissolution. The schematic diagram of the method of the placegenesis model established by the above steps is shown in fig. 1.
(3) Establishment of early placenta development model under different oxygen concentrations
And (3) replacing the culture medium in the step (2) every day, wherein the culture medium is the culture medium containing the BMP4 and inducing the hPSC differentiation in the step (2), the oxygen concentration is maintained at 20%, the model is built for 4 days, then the oxygen concentration is maintained at 5%, the model is built for 3 days, and the total model building time is 7 days. And establishing an in vitro model of the human early placenta development under the hypoxia condition according to the steps. The in vitro model of the human early placenta development is established according to the steps.
(4) Cell morphology and functional characterization
The expression of E-cadherin, F-actin and HIF-1a is detected by adopting a conventional immunofluorescence staining method, and the gene expression related to the HIF-1a passage is detected by adopting a real-time PCR method. The results show that HIF-1a expression is significantly higher under hypoxic conditions than under normoxic conditions, and PCR results from VEGFA, ANKRD37, SLC2A1 also show that hypoxic conditions are indeed upregulated along with HIF-1 a-related genes, suggesting that oxygen concentration has a significant effect on the hypoxic HIF-1a pathway during development of the early placenta, and the results are similar to FIG. 5.

Claims (1)

1. A hypoxia human early placenta development microenvironment simulation method based on organ chips is characterized in that: a three-dimensional matrix material, namely an artificial matrix membrane Matrigel, is adopted, and the method comprises the following steps:
(1) Modification of orifice plate and inoculation culture of human pluripotent stem cells
Modifying a 6-hole plate by using an artificial basement membrane, placing the modified 6-hole plate in a 4-degree refrigerator for 4 hours to 7 days, then placing the modified 6-hole plate in a 37-degree incubator for 20 to 50 minutes, and carrying out warm digestion by using a cell digestive juice chamber; scraping off cell masses by using a cell scraper, centrifuging for 1-3min at 600r, re-suspending the cells by using a mTeSR1 culture medium containing Y27632 to enable the cells to be small masses, inoculating the small masses into a modified 6-well plate, culturing for 1-4h at 37 ℃, and then replacing a culture solution with the mTeSR1 culture medium containing no Y27632 for culturing;
(2) Cells were digested and seeded in a three-dimensional matrix
Digesting the hPSC cells at a warm temperature by using a digestive juice after the hPSC cells grow to 70-90%, scraping cell masses by using a cell scraper, centrifuging for 600r 1-3min, sucking supernatant, diluting matrigel to a certain proportion by using a culture medium containing BMP4 and inducing hPSC differentiation, then re-suspending the cells to obtain a proper inoculation density and inoculating, placing the matrigel in a 37-degree culture box for 20-40min to solidify the matrigel suspended with the cells, and then replacing the culture medium containing BMP4 and inducing hPSC differentiation;
(3) Establishment of early placenta development model under oxygen concentration
Replacing the culture medium in the step (2) every day, wherein the culture medium is the culture medium containing the BMP4 for inducing the hPSC to differentiate in the step (2), and establishing models with different oxygen concentrations for several days;
(4) Cell morphology and functional characterization
Detecting the expression of E-cadherin, F-actin and HIF-1a by adopting a conventional immunofluorescence staining method, and detecting the gene expression related to an HIF passage by adopting a real-time PCR method; the artificial basement membrane modified 6-well plate in the step (1) specifically comprises the following steps: diluting artificial basement membrane 500-1000 times, and placing 1.5ml of diluent in each hole in a 4-degree refrigerator for 4 hours-7 days;
in the mTeSR1 culture medium containing Y27632, the concentration range of Y27632 is 1-100mM;
the matrigel dilution in the step (2) is specifically a culture medium: matrigel volume ratio of 0-3, cell seeding density of 1 × 10 4 -1×10 10 cells/ml；
The BMP 4-containing culture medium for inducing hPSC differentiation comprises the following components: 96% by volume of DMED/F12, 1% by volume of double antibody, 1% by volume of glutamine, 1% by volume of nonessential amino acid (MEMNAAA), 1% by volume of ITS, 100ng/ml of heparin and 10ng/ml of BMP4, wherein 1g of BSA is added into every 50ml of the prepared culture medium, and the culture medium containing the BMP4 and used for inducing hPSC differentiation is obtained after dissolution;
the oxygen concentration range in the step (3) is 2% -21%, and the model building time is 3-15 days.
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